Contents Preface ix
11. Plantcell andtissueculture;biological conversions; clonalpropagation j2
Contributors xi
12. Phytochemicalvariationwithin a species 80 Pqrt I Introducfion
13. Deteriorationof storeddrugs 9l I
l. Plantsin medicine:the originsof pharmacognosy 3 2. The scopeandpracticeof pharmacognosy 5 3. Plant nomenclatureand taxonomv 8 Pqrl2 The plont qnd onimql kingdoms os
sourcesof drugs
ll
4. Biologicalandgeographical sourcesof drugs 13 5. A taxonomicapproachto the studyof medicinalplantsandanimal-derived drugs 15 6. Pharmacologicalactivitiesof natural products 4I 7. Synergyin relationto the pharmacological actionof phytomedicinals 49 E. M. Williamszn
14. Quality control 95 Pqrt 4 Some currenl frends
107
15. Plant productsand High Throughput Screening 109 M. J. O'Neill, J. A. Lewis 16. Biologically activecompoundsfrom marine organisms 115 G. Blttnden 17. Traditionalplant medicinesas a sourceof new drugs 125 P. J. Houghton Pqrt 5
Phyrochemisrry 135 18. Generalmethodsassociatedwith the phytochemicalinvestigationof herbal products 137 19. Basicmetabolicpathwaysandthe origin of secondarymetabolites150
Port 3 Pqrt 6 Principlesrelqted to the commerciqt qnd relqted drugs of Phqrmqcopoeiol production, quolity qnd stqndqrdizqtion biologicol origin l7l of nolurql products 55 Introduction 173
8. Commercein crudedruss 5i R. Baker
20. Hydrocarbonsand derivatives 174
9. Productionofcrude drugs 6l
21. Carbohydrates 191
10. Plantgrowthregulators 6l
22. Phenolsand phenolicglycosides 214
Id..:e6Nif 23. Volatile oils and resins 253 24. Saponins,cardioactivedrugsand other steroids 289 25. Miscellaneousisoprenoids 315 26. Cy anageneticglycosides,glucosinolate compoundsand miscellaneous glycosides 321 27. Alkaloids
37. Aspectsof Asian medicineand its practicern the West 461 M. Aslam
38. Chineseherbsin the West 482 S. Y.Mills
39. Plantsin African traditional medicine-an overview 488 A. Sofowora
333
28. Tumour inhibitors from plants 394 P. M. Dewick 29. Antiprotozoalnatural products 401 C. W. Wright 30. An overview of drugs with antihepatotoxic and oral hypoglycaemic activities 414
Port 8 Nonmedicinol toxic plonts ond pesticides 497 40. Hallucinogenic, allergenic,teratogenicand other toxic plants 499 4L. Pesticidesof naturalorigin
509
3L. Vitamins and hormones 42I
33. Colouringandflavouringagents 435
Pcrrt9 Morphologicol qnd microscoPicql exqminqtion of drugs 513
34. Miscellaneousproducts 440
I n t r o d u c t i o n5 1 5
32. Antibacterial and antiviral drugs 429
Porl7 Plqnts in complementqry ond troditionql syslems of medicine 445 Introduction 441 35. Herbal medicine in Britain and Europe: regulation and practice 449 S. Y.Mills 36. Homoeopathicmedicine and aromatherapy 460 M. A. Healy, M. Aslam
42. Plant description,morphology and anatomy 516 43. Cell differentiation and ergasticcell contents 526 44. Techntquesin microscoPY 538 Index
549
E
Plonts in medicine: theorigins of phormocognosy
The universal role of plants in the treatment of diseaseis exemplified by their employment in all the major systemsof medicine irrespective of the underlying philosophical premise. As examples, we have Western medicine with origins in Mesopotamia and Egypt, the Unani (Islamic) and Ayurvedic (Hindu) systemscentred in western Asia and the Indian subcontinent and those of the Orient (China, Japan,Tibet, etc.). How and when such medicinal plants were first used is, in many cases, lost in pre-history, indeed animals, other than man, appear to have their own materia medica. Following the oral transmission of medical information came the use of writing (e.g. the Egyptian Papyrus Ebers c.1600 nc), baked clay tablets(some 660 cuneiform tablets c. 650 ec from Ashurbanipal's library at Nineveh, now in the British Museum, refer to drugs well-known today), parchments and manuscript herbals, printed herbals (invention of printing l44O AD), pharmacopoeias and other works of reference (ftrst London Pharmacopoeia, 1618; first British Pharmacopoeia 1864), and most recently electronic storage of data. Similar records exist for Chinese medicinal plants (texts from the 4th century ec), Ayurvedic medicine (Ayurveda 2500-600 nc) and Unani medicine (Kitab-Al-ShiJa, rhe Magnum Opus of Avicenna,980-1037 ao). In addition to the above recorded information there is a great wealth of knowledge concerning the medicinal, narcotic and other properties ofplants that is still ffansmitted orally from generationto generationby tribal societies,particularly those of tropical Africa, North and South America and the Pacific countries. These are areas containing the world's greatest number of plant species, not found elsewhere, and with the westernization of so many of the peoples of these zones there is a pressing needto record local knowledge before it is lost forever. In addition, with the extermination of plant species progressing at an alarming rate in certain regions, even before plants have been botanically recorded, much less studied chemically and pharmacologically, the need arises for increasedefforts directed towards the conservation of genepools. A complete understandingof medicinal plants involves a number of disciplinesincluding commerce,botany,horticulture, chemistry enzymology, genetics, quality control and pharmacology.Pharmacognosyis not any one of theseper sebut seeksto embracethem in a unified whole for the betterunderstandingand utilization of medicinal plants.Aperusal ofthe monographson crude drugs in a modem pharmacopoeiaat once illusfates the necessityfor a multidisciplinary approach.Unlike those who laid the foundations of pharmacognosy,no one person can now expectto be an expert in all areasand, asis illustratedin the next chapter, pharmacognosycan be independently approachedfrom a number of viewpoints. The word 'pharmacognosy'had its debut in the early 19th century to designatethe discipline related to medicinal plants; it is derived from the Greek pharmakon, 'a drug', and gignosco, 'to acquire a knowledge of' and, as recordedby Dr K. Ganzinger(Sci. Pharm.1982, 50, 351), the terms 'pharmacognosy' and 'pharmacodynamics' were probably first coined by JohannAdam Schmidt (1759-1809) in his hand-written manuscript Lehrbuch der Materia Medica, which was posthumously published in Vienna in 18I 1. Schmidt was, unril his death,professor at the medico-surgical JosephAcademy in Vienna; interestingly he was also Beethoven's physician. Shortly after the above publication, 'pharrnacognosy' appears again in 1815 in a small work by Chr. Aenotheus Seydler entitled Analecta Pharmacognostica. Pharmacognosyis closely related to botany and plant chemistry and, indeed, both originated from the eartier scientific studieson medicinal plants. As late as the beginning of the 20th century, the subject had developed mainly on the botanical side, being concemed with the description and identification of drugs, both in the whole state and in powdeq and with their history, commerce, collection, preparation and
k t -
\
"% '& i'r!-
. d
s. d
$
,.k
j
&{'/d
.d.
ffi
;
-3"*
tu
s
ry
storage. Such branches of pharmacognosy are still of fundamental importance, particularly for pharmacopoeialidentification and quality control purposes, but rapid developments in other areas have enormously expandedthe subject. The use of modern isolation techniquesand pharmacologicaitesting proceduresmeansthat new plant drugs usually find their way into medicine as purified substancesrather than in the fbrm of galenical preparations.Preparationis usually confined to one or a few companieswho process all the raw material; thus, few pharmacists have occasion to handle dried Catharanthus roseils although they are familiar with formulations ofthe isolated alkaloids vinblastine and vincristine. For these new drugs it is important that the pharmacist, rather than being fully conversant with the macroscopical and histological charactersof the dried plant, is able to cary out the chromatographic and other proceduresnecessaryfor the identification and determination of purity of the preparation supplied. Similar remarks apply to such drugs as Rauwolfia, the modem preparationsof ergot, and the cardioactive and purgative drugs. When specific plants,including thoseusedin traditional medicine, suddenly become of interest to the world at large, the local wild sourcessoon become exhausted.This necessitates,as in the caseof Catharanthusroseus,Coleus forskohlii, Arnica montana and Taxus brevfolia, research into the cultivation or a.rtificial propagation by cell culture, etc., of such species.In order to avert the type of supply crisis that arose at the clinical trial stage with the anticancer drug taxol, isolated from 7. brevfolia, the US National Cancer Institute has initiated plans for future action when a similar situation again arises (see G. M. Cragg et al., J. Nat. Prod., 1993, 56, 165'7).
However, it has been repofied that as a result of demandfor the new drug galanthamine(qv) for the treatmentof Alzheimer's disease,the native sourceof Leucojum aestivumis now in danger. The use of single pure compounds,including synthetic drugs, is not without its limitations, and in recent years there has been an immenserevival in interestin the herbal and homoeopathicsystems of medicine, both of which rely heavily on plant sources,At the 9th Congress of the Italian Society of Pharmacognosy(1998) it was stated that the current return of phytotherapy was clearly reflected by the increased market of such products. In 1995 the latter, for Europe, reached a figure of $6 billion, with consumption for Germany $2.5 billion, France $1.6 billion and ltaly 600 million. In the US, where the use ofherbal products has never been as strong as in continental Europe, the increase in recent years has also been unprecedentedwith the market for all herb salesreaching a peak in 1998 approaching$700 million. Again, illustrating the same trend, the editor of Journal of Natural Products, 1999, writes that in response to the increasing prominence of herbal remedies, additional contributions describingscientific investigationsof a rigorous nature are welcomed. Undoubtedly, the plant kingdom still holds many speciesof plants containingsubstances of medicinalvalue which haveyet to be discovered; large numbers of plants are constantly being screenedfor their possiblepharmacological value (particularly for their anti-inflammatory, hypotensive, hypoglycaemic, amoebicidal, anti-fertility, cytotoxic, antibiotic and anti-Parkinsonismproperties). Pharmacognosistswith a multidisciplinary background are able to make valuable contributions to theserapidly developing fields of study.
;i+'i
'r:-#
Thescopeond proctice of phormocognosy
Until relatively recently pharmacognosywas regarded, almost exclusively, as a subject in the pharmaceuticalcurriculum focused on those natural products employed in the allopathic system of medicine. Coincident with the increasing attractivenessof alternative (complementary) therapies and the tremendous range of herbal products now generally available to the public, regulatory requirements covering medicinal herbs are being introduced by many countries in order to control the quality ofthese products.Monographs are now available on a large number of such drugs giving descriptions,testsfor identity and purity and assaysof active constituents.These monographs are being compiled by a number of bodies (see below). In this respect recognition should be given to the pioneering production ofthe Brirlsh Herbal Pharmacopoeia, first produced in 1974with subsequenteditions, and a new one pending. Pharmacognosyis also important in those countries having their own systems of medicine in which plants are important components. Although pharmacognosyis principally concernedwith plant materials, there are a small number of animal products which are traditionally encompassedwithin the subject; these include such items as beeswax,gelatin, woolfat, vitamins, etc. Other natural products such as the antibiotics, hormones and others may or may not be involved, depending on the teaching practice of a particular institution. As is shown in Chapter 16 m4rine organisms,many of the animal kingdom, are receiving increasing attention. Materials having no pharmacological action which are of interest to pharmacognosistsare natural fibres, flavouring and suspendingagents,colourants,disintegrants,stabilizers and filtering and support media. Other areasthat have natural associations with the subject are poisonous and hallucinogenic plants, allergens,herbicides,insecticidesand yrolluscicides. Vegetabledrugs can be arrangetlfor study under the following headings.
t . Alphabetical. Either Latin or vernacular names may be used. This arrangement is employed fol dictionaries, pharmacopoeias, etc. Although suitable for quick referenceit gives no indication ofinterrelationshipsbetween drugs. 2. Taxonomic.On the basis ofan acceptedsystemofbotanical classification (Chapter 3), the drugs are arranged according to the plants from which they are obtained, in classes,orders, families, genera and species.It allows for a precise and ordered arrangement and accommodatesany drug without ambiguity. As the basic botanical knowledge of pharmacy studentsdecreasesover the years this system is becoming less popular for teaching purposes. Morphological. The drugs are divided into groups such as the following: leaves, flowers, fruits, seeds,herbs and entire organisms, woods, barks, rhizomes and roots (known as organized drugs), and dried latices, extracts, gums, resins, oils, fats and waxes (unorganized drugs). These groupings have some advantagesfor the practical study of crude drugs; the identification of powdered drugs (see Chapter 44) is often basedon micro-morphological characters. 4 . Pharmacological or Therapeutic. This classification involves the grouping of drugs according to the pharmacological action of their most important constituent or their therapeutic use. R. Pratt and H. W. Youngken Jr. were, in 1956, the first to use this approach for an English languagetextbook and now, with so many plant materials being screenedfor specific pharmacological activity, this type of listing is found increasingly in the literature. Its use is illustrated in Chapters28-32.However, it is important to appreciatethat the constituents of any one drug may fall into different pharmacological groups. Chemical or Biogenetic. The important constituents,e.g. alkaloids, glycosides, volatile oils, etc., or their biosynthetic pathways, form
\"
4: 1
r'l''
p..
J
$
-q
w** rf,
d."
G ,"-# *
W
**,Y n
"-%*
€ .*x i6.s
,'
%
r
Thescopeond proctice of phormocognosy
Until relatively recently pharmacognosywas regarded,almost exclusively, as a subjectin the pharmaceuticalcumiculumfocusedon those natural products employed in the allopathic system of medicine. Coincident with the increasingattractivenessof alternative(complementary)therapiesand the tremendousrange of herbal productsnow generally available to the public, regulatory requirementscovering medicinal herbs are being introducedby many countries in order to controlthe quality o1'theseproducts.Monographsarenow availablcon a largenurnberof suchdrugsgiving descriptions,testsfor identity and purity and assaysof active constituents.Thesemonographsare being compiled by a number of bodies(seebelow). In this respectrecognition shouldbe given to the pioneeringproductionof theBriti,shHerbal Phormacopoeia,first producedin 1974with subsequent editions,and a new one pending.Pharmacognosyis also importantin thosecountries having their own systemsof medicine in which plants are important components. Although pharmacognosyis principally concemedwith planrmaterials, there are a small number of animal productswhich are traditionally encompassedwithin the subject; these include such items as beeswax,gelatin,woolfat. vitamins,etc.Othernaturalproductssuchas the antibiotics, hormones and others may or may not be involved, dependingon the teaching practice of a particular institution.As is shown in Chapter l6 marine organisms,many of the animal kingdorn, are receiving increasingattention.Materials having no pharmacological actionwhich are of interestto pharmacognosists are natr.rral fibres, llavouring and suspendingagents.colourants.disintegrants,stabilizers ir:r.li" iiiilt;..,,r and filtering and supportmedia.Other areasthat have naturalassocia- ,t:tltiiut:ti : tions with the subjectare poisonousand haliucinogenicplants. aller- ititlu:itau itit.ti]illlrl: gens,herbicides,insecticidesand molluscicides. tit,lri1; Vegetabledrugscan be arrangedfor studyunderthe fbllowing head:tiiril rliiit ings. l . Alphabetical.Either Latin or vernacularnamesmay be used.This
arrangementis employed for dictionaries, pharmacopoeias,etc. Although suitablefbr quick referenceit gives no indicationof interrelationshipsbetweendrugs. Taronomic.On the basisof an acceptedsystemofbotanical classification (Chapter3), the drugs are arrangedaccordingto the plants from which they are obtained,in classes,orders,families, genera and species.It allows for a preciseand ordered arrangementand accommodatesany drug without ambiguity.As the basic botanical knowledgeof pharmacystudentsdecreases over the yearsthis system is becominglesspopuiarfor teachingpurposes. 3 . Morphological. The drugs are divided into groups such as the following: leaves.flowers, fruits, seeds,herbs and entire organisms. woods.barks,rhizomesand roots (known as organizeddrugs).and dried latices,extracts,gums, resins,oi1s,fats and waxes (unorganized drugs).Thesegroupingshave some advantagesfbr the practical study of crude drugs;the identitlcationof powdereddrr.rgs(see Chapterzlzl)is often basedon micro-morphologicalcharacters. 4 . PharmacoktgicaLor Therupeutic.This classificationinvolves the grouping of drugs accordingto the pharmacologicalaction of their most impoftant constituentor their therapeuticuse. R. Pratt and H. W YoungkenJr. were, in 1956.the first to use this approachfor an English languagetextbook and now. with so many plant materials being screenedfbr speciticpharmacologicalactivity.this type of listing is found increasinglyin the literature.Its use is illr-rstrated in Chapters28-32. However,it is importantto appreciatethat the constituentsof any one drug rnay fall into different pharmacological groups. Chemicalor Biogenetic.The importantconstituents,e.g. alkaloids. glycosides.volatile oils, etc., or their biosyntheticpathways.form
IE
INTRODUCTION As rnentioncdpreviously,a nllrnberof bodieshave implernented the basis of classificationof the drugs.This is a popular approach on ncdicinal herbs.The aint has is phytochernicallybiased. researchand publishednlonogrilphs when the teaching of pl.rarmacognosy Anbiguities alise when particulardrugs possess a numberof active been to sct standardsfix' cluality.etlicacy and sat'etyin order that the principlesbelongingto diflerent phiitochenricalgroups.as illustrat- many tl'aditionalherbs mcct legal requirernents.The follou'ing are of note: ed b1,liquorice.ginseng.r'aleliart.ctc. The sclremeis ernployedin drugs. Chaptels20 27 tbr anangin-tthe cstablishedphartnacopoeial 'I'hese 'nvet'c cleveloped German Commission E monographs. 'I'he *'ill fbllou'ing list of works. arrangedin the abcxe five gror.rps. fbr the Gelrnan FccleralHealth Authoritl' betwccn 1978-1994and andalsoproviclea usefullist oftcxtbooksandt'orks serveascxanrples involr,e32.1herbsused in Gelrnantladitionalmedicine.The monoof reftlence:tlroseno longer in print may be fbund in established graphs give sollrces.constitllentsand considerablcphannacological pharnracer,rticirl liblaries. They havc nou' beentranslatedinto English and clinicalintbrrnation. b1'theArnericiinBotanicalCtiuncilin 1999as tr single and pLrblishcd l. Alphobeticol a handbookfbr practiceon a sciBissetN G (cd). Wichtl N'l 1996Herbal cltr.rgs. u'ork. errtific basis.Meclphanr ScientificPublishcrs,StLtttgat'l r . l i t i s h H c r b a lM c d i c i n e B r a c l l e l ' PR ( e d ) 1 9 9 2B r i t i s hh e r b a lc o n t p c n d i u n B A s s o c i a t i o nB. o u r n e m o u t hU. K Press.London Vols I-II Blitish PhalmacopoeiaI 999. Pharrnaceutical B r i t i s hH c r b a lP h ; r r m a c o p o e1i a9 9 6 .B r i t i s hH e r b a lM e c l i c i n eA s s o c i a t i o t r . Exeter.UK Niartindale:the Extr-aPharmacopoeia.32nd ccln. | 999. PharntaccuticalPress. Londou l999 2-llNationalFornrr-rlary LinitedStatesPhamracopoeia Wren R Cl I988 Potter'sneu'c1'clopocidaol botanrcaldrugs and pfeparations. reuritten b1'WilliamsonE NI and Er'ansF J. C W Daniel Co. Safll'orr \\alden. UK Tl-re national pharmacopocias of niarry countries and the Europeau Pharmacopoeia; the relevant crude drug mono-qraphs of the latter are i r r c l u t l e tilr t t h e B l i t i r h P h r r t n l r c o p o e i r
2. Toxonomic . a r i s . 3r ' o l s . a s s o nP P a l i sR R . N I o l ' s eH 1 9 6 5 .1 9 6 7 .1 9 7I M a t i d r cr n d d i c a l eM ThornasH 1929 Handbuchdet'Phalmacie.Urban and Schu arzcnbcrg.Berlin. Band V 2 r'ols, Phannacogrtosr' . t he d n .B a i l l i i r e T i n d a l la n d T r e a s eG E , E r , a n sW C 1 9 7 2P h a r n a c o g n o s l l' O Casscll.London
3. Morphologicol f, .-1 !
/...
E la u
kt
Mauclt'ich.Vienna.Vol I. Barks and Belger F Hanclbuchder Dt'ogenkunclc. f l o u ' e l s .1 9 . 1 9V: o l I I . L e a r e s .1 9 5 0 :V o l I I I . F f u i t sa n d u ' o o d s ,1 9 5 2 1V o l I V . Vol V. Roots. I 960r Vol Vl. Rcsinsetc and seeds.196.{:Vol Herbs. I 9-5,1: V I I . I n d e x .1 9 6 7 JacksonB P. Snou don D W I 990 Atlas o 1'nticrtrscopl'of nredicinalplants. culinar.vherbsand spices.BelhavenPrcss.l-ondon N'lortonJ F 1977Mixor medicinalplants:botany'.culturc and uses.Thomas. S p l i n g f i e l dI.L . U S A 5th edn. Churchill Livingstone. \\allis T E, I 967 Tertbook of Pharmacognosl'. l"ondon
Coopelativefbr Scier.rtific ESCOPmonographs. ESCOP(European Phytotherapy)is an alllliation of Europeanassociatioltswhich has ploducecl60 monographson helbal clrugs.pttblishedin loose-leaffbrm in six thscicr-rle s. harmonizingthe standaldsfbr thesedrugsthror-rghout thc Er.rlopeanUnion. Intbrrration is given on approved therapcutic uses, ancl unlike the ComrnissionE nono-graphs.plovidcs ref'elerrces. AHP monographs. The American Herbal Pharntacopoeiaplanned by the end of 2000.'nvithsorne30 to havepublishedI l- I 3 t.nonographs of American traditional more pending.Thele is naturally a selectiot-t herbs n'ith somc overlap l''ith thc European monographs.The as trncxamplethe St John'sWort monograph tleatmentis exhaustir,e: rvith ovcr publishedin HelbalGraml99T. No .l extendsto -i2 pa-ees andchemicalfbrmttlae. I50 ref'erences. colourphotographs WHO monographs. The World Health Organization published MerlicirutlPluttts irt 1999.It ttrtSeleL'tetl Volunre I of its MottogrtLplt.s contaiusstandardsfbr clualityof dlr.rgstogethelwith a thelapeuticsccin the tion: 3l plant species.the majcx'ityol u'hich at'ealso inclLrded Vcrlume2. containinga further29 rnonoabovelists.are consiclered. graphs.is cluelbr pLrblicationin 2000. is alsoproducUSPmonographs. The United StatesPharr-nacopocia all involving ing helbnl monographs.Elevcn har"ebeertpr"rblished. drugstteateclabove.and twelve more are expecteclduring 2000.
wishing to read ttliginnl resealchwill Current awareness. Str.rdents in this book and shouldlearnhow to find sin-rilar find rnar-ry ret'erences A, Libcrti t- E I 988 Natural ploduct nredicinc.G F Stickley. Del i\'lalclerosian As no onecanhopeto readall the scientificliteraonesfbl themselves. P h i l a d c l p h i aP. A . U S A specialior.rrnllsare clevotedto the publicationof ture that is pLrblished. PrattR, YoungkenH W. Jr 19-56Pharrlacognosy.2nd edn. Lippincott. P h i l a d e l p h i aP.A . U S A brief abstlacts from the oliginal papers. Such abstractsgive the Ross N4S F. Brain K R 1977An intfoductionto phytopharmacy.Pitnlall author's name.the sub.iectoi the rcscarch.the ref'elencencccssaryto N{edical.Tunbridge\\iells a blief outlineof the locatethe papelin the oliginaljoLrrnalanclusr-rally work it contains. Most phat'nracydepartntentlibraries contain 5. Chemicol phy'tochcrnistry'. medicinarlplants.Intefcept BnrnetonJ 1999Pharnracognos-v. Cltenitul Abstrrr't.r'and Biolo,gital Abstrccls,which in the appropriate Scicntific. N'ledicaland TechnicalPublications EVen st'. the :)':tetrllttr scctionscover all aleasof pharntaco-[nosy. Deu,ick P N{ I 997 \Iedicinal naturalproducts.a biosl'nthcticiipproach.John canilselfbe ofthe absuactsto covera broacllielclof intcrests searching W i l e y .C h i c h e s t e r lrtles and publicatictns as and such ClrcntittLl rnost tinre-consunrin-9. 6 t l r i n e g g e l E 1 9 9 9 P h a r m a k o g n o s i c P h y t o p h a r m a z i e . H i i n s eR l . S t i c h c rO . S t e CurrentCottentscan be usedto give a rrore rapidindicationof recent edn. Springer.Berlin (|ir C)t'ntrcnt1 bibRobbersJ E. SpeedicM K. Tl ler V E 1996 Pharmacognosyand pharmacoregularlyincludesa selected publications. Phltothenq1'Re.search b i o t e c h n o l o g lW ' . i l l i a m s& W i l k i n s .B a l t i m o r e lioglaphy relating to plant dlugs. Inlbrntatioti stot'ageanclretrieval is Tauchnitz.Leipz-iglttro editiort.s TschirchA Handbuchder Pl.tilrnako-unosic. occupiedby sucnow itself a scicnce.anc'la glanceat the shelf:space t oltnttt'strltto l 9-l-i1 tutd nunterou.s is sutlicierrtto indicatethatbetbre ceedingyearsof CftcrnlL:trlAb.strtLcts long. if not already'.manual searchesof the litcrature u'ill become With the increase in interest in rnedicinal plants world-wide there are Inevitablyit will bc necessalyto rely on inrpossiblylon-eprocedures. n o \ \ ' n r a n y p u b l i c a t i o n s c o \ ' e f i n g r c g i o n a l a l e a so f t h e g l o b e . T r e a t m e n t databasesfbr litelature scanniug.PltunnucogttostTitlesis a cornputcr of the plants in thcse wolks rna)' be on an1, of the abovc lines. Some researchpublicationsup to 1974 of phytochenrical abstl'act co\rerage cxamples are given fbllowing the Introduction to Paft VII.
4. Phormqcologicol or Theropeutic
j
i.{
t
.J..{#
T H ES C O P EA N D P R A C T I COEF P H A R M A C O G N O S Y tl0 vols) producedundel the directionof Prof-essol N. Farnsr','or.th.as anv book can). Symposiall,hich cor,ervaLiolrsaspectsof pharnraI i n i ' " ' e r s i t y o f I l l i n o i s . S u b s e q u c n t l y .F a r n s w o r t h i n t r . o d u c e c lcognosyarc tiequently held in variouspartsofthe world and scientists \APRALERT. a NaturalProcluctDarabasewhich is rrainly. br.rtnor can easilvbecomeacquninted u'ith othershar,inglike interests. Often cntirely.post-ltl75and is vieu.'ed bv rrany as a logicaland inclispens- the infbn-r-ral discussionswhich invariably ariseat sr.rch meetingscanbe able collectiono1'pharmacognostic information.The NAPRALERT an extremelyuselulnteansof disseminating infbrmation.ln addition, database is availableon a schedulcd-fee basisft) scientists.industrial the lecturesplesentedat such nteetingsare often sr.rbsequentlv pubfirns. -qovemment agenciesand acadenticinstitutions. Among othcr lished in book fbrm. Modern comntunicationsystemsmake u'orlduselirl databaseshaving a reler,'ance to pharntaco-tnosy and published '"videcontactbetweenresealchers much sintpler. on the Web arc MEDLINE. conrpiledby the US NarionalLibrarl,of Now al'ailableto Westernscientists interestcd in orientalrnedicineis Medicineand EMBASE. producedby ExccrptitMedica. the qualtelly joumal Abstnrc:t.s o.l'CltineseMeditirrc, publishedby the S o m e j o u l r r a l s f o r e r a r - n p l e .P l r t r t t u M e d i t ; u . . l o u r n o l o f ' ChineseUniversityol HorrgKong. This givesabstractsin Englishof Ltltrtophunrrut'ologv Plntocltetnisn'y nnd Jountttl of' NtttunLl significantChineseresearch papersfrorn morethanonehundredscienPt'o(luLt.\-periodicallycolttuin reviL-\\s on \oll(. i.rs|ectof medicinal titic journalsnot feadilyar,ailable outsideChina. plants.Otlrel pcliodical publicationsappearingin bor-rndfbr-m are Usefirl dictionaliesto be lbund in most Unir.ersitylibrtrriesinclude devoteclto reviewson certainaspectsof plar-rtconstituclttsand are use- DicticrttLryof Organic Contpountlsconsisting of 7 r'olurnesand 10 ful fbr updating;ofien the rcr,ielvscor,eronly the adrrncesin I parti- sLrpplements (to 1992).Dittiotmry ol Alkaloitl.s12 r,olunies)(1989). culal tlc-lclsincethe pler,iousr,olunte.Examplesare NtfturtLl Product Dictionar-t o.f Tbrpertritls( 19911and Dit:tionurt' of Natuml pnxlucts Reports(six issuespcl vear) .andAlkaloitl.y(AcadenricPrcss).Books (1994) all publisheclbv Chapmanand Hall and also phttot'hemictrl thatarenot partofa seliesbut. like the above,rrultiauthorand dealing Dictiornrt': A Hundbookol Bioactit'eCompountls.frcmplutfi.t ( 1993), \\'ithccrtainspeciaiized areas(c.g.alkaloids.flavonoids.isoprenoids). publishedby Taylol and Francis.Sorneof thesemore expensivevolcorrtinuallyappearand generallvgive up-to-dateinfolmation (in so flr Lrmesare availableon CD-ROM.
z
IE BOTANICAT NOMENCTATURE Beforethe time of Linnaeus( 1707 1778)many plantswere known by a double Latin title; however,it is to this great Swedishbiologist that we owe the generaladoptionof the presentbinon-rialsystem.in which the flrst narrc dcnotcs the genus, while the second(specific) name denotesthe species.All specificnamesmay be written with small initial letters although tirmerly capitals were used where specieswere named after persons. Thus the species of Cinchona named after CharlesLedger,who broughtits seedsliorn Brazil in 1865.is now writtcn Clrrry'rono Ietlgerionaratherthan CinchonuLedgerianu. The specificname is usually chosento indicatesome striking characteristicof the plant fbr example.the hemlock with the spottedstem is namedConiunrntaculuturtt(.mutulutus.-d. -1u11. spotted).Sometimes the reasonfbr the name is not as obvious as in the examplejust mentioned.but onceit is discoveredit will serveas a reminderofa characteristicof the plant-fbr example,Stl c/rnos potatorum (potator,-oris, a drinker) bearsa namewhich is only intelligiblewhen it is known that the seedsof this speciesareusedin India lbr clearingwater. The modernrules governingthe terrninologyof plant taxonomy are laid down in the Internroioncl Codeof BotanicalNomenclature. Unlike the names of chernical substances,which are subject to changeswhich confolm to evolving systemsof nomenclature. systematic plant namesare strictly controlledby ruleswhich give precedence to that name used by the botanist who first described the species. Nevertheless, this seeminglystraightfbrwardapproachcan give rise to variousquilks in spelling.The following are threeexamplesinvolving rnedicinal plants Rauvolfia vis ir vis RauwoLfia;the former name was given to this Apocynaceousgenusby Plumier in 1703,honouringthe botanistLeonard Rauwolf. This spelling ovelsight causednuch contentionover the yearscentringon whetherPlumier's obvious intention shouldbe adoptedin the nameRaLntollia.Both spellingsarecommonly fbund but the rules dictate that Rauvolfia has priority. In anotherexample the downy thornapplemay be encounteredas eilher Datura innoria or Datttra irnxia. The fbrmer. as Datura irutoxia Miller. was used in 1768(Gurd.Dlo.. edn.8, Dutttru no. 5) and this spellingwas invariably employedfor some200 years:howeverin Miller's original description. the plant was characterizedas: 'Datura (lnoxitt) pericarpiisspinosis inoxiis ovatis propendentibusfoliis cordatis pubescentibus'(W. E. Saflbrd,J. \Wrsh.Acud. Sci.. 1921,11,173) and taxonomistsnow considerD. irro-rlaMiller to havepriolity. Both versionsarestill commonly encountered. A third exarnpleconcernsthe genusofthe cocaplantwhich may appearas Er1,1fup.ry1u^, or in older literature as Erythrox:-lon.
Plont nomencloture ondtoxonomy
3. i
r
SUBDIVISIONS OF THEPHYTA The branchesofthe genealogicaltreediffer so much in sizethat it is not easyto decidewhich are of equalsystematicimportance,and what one biologist may consideras a lamily anothermay regardas a subfamily. Similarly. the speciesof one botanistmay be the subspeciesor variety of another.The main hierarchicalsubdivisionsof a division. aranged according to Engler's scheme.may be illustrated by the lollowing exampleshowingthe systematicpositionof peppermint.
h !: I
l
BOTANICAT NOMENCLATURE8 SUBDIVISIONSOF THE PHYLA
8
BOTANICAT SYSTEMS OF CTASSIFICATION9 TAXONOMICCHARACTERS9 PTANTTAXONOMY CHEMICAT
ro
Division Class Subclass Order Suborder Family Subfamily Tribe
Anglospermae Dicotyledoneae Sympetalae Tubiflorae Velbenineae Labiatae (Lamiaceae) Stachydoideae Satureieae
.q.#{
PLANTNOMENCLATURE AND TAXONOMY Genus Species Varieties
Mentha MenthapiperitaLinnaeus(Peppermint) Menthupiperita var.olrtcinalis Sole (White Peppermint) Menthupiperita var.vul,qari,s Sole(Black Peppermint)
It will be notedthat in pharn-racopoeias and in researchpublications botanicalnamesarefbllowed by the nar-nes of personsor their accepted (e.g.Linnaeusand Sole in the caseofpeppermintgiren abbreviations above).Theserefer to the botanistwho first describedthc speciesor variety.Studentsneednot attemptto memorizethesenames.and in the fbllowing pagesthey areusuallyomittedexceptin caseswheredif-fbrent botanicalnameshave at differenttimes beenappliedto the sameplant and thereis possibilityof confusion.The sourceof cloves,fbl example. is now usuallygiven asS,t';.r'glum aromaticltm(L.) Men. et Pen'y:prior to 1980 the B.P. used the name Eugetict <:ortophlllus (Spreng.) Sprague;other synonymswhich may be found in the older literatureale E. caryopltyllatzz Thunb. andE. urontuticc(L.) Baill. Worldwide,not all authorsof researchpapersusethe currentlyacceptednauteso cautionis necessaryand botanicalsollrcesshor-rld be checked.
BOTANICAT SYSTEMS OF CTASSIFICATION Before the widespreadacceptanceof the principle of evolution. biologists,being convincedof the lixity of speciesand lacking much of the infbrmationavailabletoday.confinedthemselvesto more or lessartificial methodsof classification,their systemsbeing fiequently basedon one or a f'ewcharactersinsteadof upon the organismas a whole. These earlier systemsare now mainly of historic interest.but certainof their f'eatures-for example.the large division of seed plants into monocotyledonsand dicotyledonsas used by John Ray (1628-1705) survive today. Linnaeus' Sper:lesPluttnrunt of 1753 is the starting point fbr the rnodeln nomenclatureof plants,althoughhis actual system of classificationis er.rtirelyartificial and of little signilicancetoday. The Prodrontu.r. startedby A. P de Candolle(1778-1841)and completedLrnderthe editorshipof his sonAlphonse( 1806-93).was a massive work of l7 volumes which prof'essedto be an accountof every flowering plant then known. The system of classificationenrployed was a modification and extension of that introduced earlier bv De Jussieu( I 748- I 836) and further demonstratedthe inadequacies of the Linnaean system which were then becorningapparent.Bentham and Hooker's GeneraPlattarum (1862-1883)was pattelnedon the de Candolles'work, eachgenr.rs bein-uredescribedfrom herbariumspecimensand not consistingof a restatementof earliel literature.Although largely artificial, it was convenientto retain this systernas a basisfor collectionssuchas the herbariaof Kew and the British Museurn. Durin-9the last 100 years a considerablenurnber of phylogenetic systemsof classificationhave beenpropounded:thesesystemsan'ange taxa (any groups used for classificationsuch as orders,families, genera, etc.) to indicate the possiblerelationshipof one taxon to another. Such systemsare clearly susceptibleto changewith increasingknowledge, and no final system acceptableto all taxonomistsis in sightl indeed,r-nanytaxonomistsare now more concernedwith the production of sound.workable pheneticsystems.A close examinationof the phylogeneticsystemsreveals that certain taxa lbrm precise group\. othershavelesswell-definedboundariesand other groupsare difllcult to accommodatephylogenetically.The work of Engler ( 182141930) in associationwith other German systematistsis still adheredto in this connection.Engler's scheme of classificationlargely embodied the fundamentalconceptsof Eichler ( 1839-87)and was exemplifiedin the 20-volume work (1887-891 Die natiirlichen Pflun:.enfimilien. by Engler and Prantl.Subsequentto this, thereappcaredmany editionsof Engler's Syllabusder P.flartsnftunilien.the eleventhby Engler and
Diels in 1936.The last versionof the S.t'11aDus. producedby Melchoir as two volumes,was publishedin 196:1;lhe plant lamiliesin Chapter Five of this textbook are anangedin this order.The imntediatepopularity of E,ngier'sworks was due to their applicabilityto plants of the whole world: they aflbrdeda meansof identifying all known genera.A modern replacementfbr this classical wolk is The Funilie.s uncl Generao.fVasculrLr Plants (.K. Kubitzki et a/. (eds)).So far three volumeshavebeenpublished.the lastin 1998. Obviously. large n'orks such as the erboveare not easily cornpiled and many taxonomistshar,'eproducedphylogeneticschcmesdirected at various levels of classificationwithout the completesystematicsof the Engler series.Of the schemes,those of Cronquist(1981) and Takhtajan (1959.)are generally similar whereasthat of Hutchinson (1992) differs in that the dicotyledonsare divided inro two large gfoups-those characterist.ically and primitively woody (Lignosae) and those chalactelisticallyand primitively herbaceous(Herbaceae). Theseschemesincorpolatedataoften not acccssibleto the earliertaxonomists:thus Cronquist,while emphasizin-e classicalrnorphoiogical charactersand following the strobilartheory ofAngiosperm evolution also takes account of micromorphological data (e.g. ernbryology and pollen structure),chemical data (e.g. secondarymetabolitesand serology)and the fbssil record.Dahl-eren's proposals(1983).which involve a taxonomic method telnreclcladistics.demorrstratethe distribLrtionof charactersand his cladogramsof the orders of An-tiospermscan be convenientlyused fbr illustratingthe occunence of secondarymetabolitesthroughoutthe hi-uherplants.
TAXONOMICGHARACTERS All plantspossesshundredsol'chalactersof a morphological.histological, embryological,selological.chenricaland geneticnaturewhich arc potentially availablefor building up a classificationof the plant kingdom. ln the artificial schemesthe charactersemployedwere those that experiencehad showncould be usedto producesuitablegroupsor taxa.The eventualscheme.into which could be insertednew plants as they were discoveredol in which any plant cor"rldeasily be traced. resembleda catalogLlewith a 'telephonedirectot'y' arangentent of plants in which the groupsof individualslisted togetherdid not necessarily haveany phylogeneticrelationship.but rnerelypossessed certain common f-eatures. Phylogeneticclassiflcations.wl-richendeavourto indicate the relationship of one taxon to another.imply the use of charactersthat are capableof showing suchrelationships.Becausesornegroupsof plants are firore primitive than othels on the evolLltionaryscale.certaincharacters will also be primitive. whereas other characterswill have evolvedfrom thenr.Thus, woody plantsaregenerallyregardedas more primitive than herbaceousones and flowers with f-ew parts more advanccdthan thosewith rrranyparts. The difliculties facing the ttronornist lre apprecieble.The appearance of a particular characterin celtain plants does nol necessarily imply a relationshipbetweenthcseplants.becauseat somc tinte in the past. under favourableconditions.whole groups ol unrelatedplanls could have undergonethis change(e.g. the developmentof fuscd corollas from polypetalousflowers; this is known a contergence). Altcrnatively.relatedplantsmay.in somc point of time. havestartcdto diverge in their characteristicsso that the modern phenotypesappear very dissimilar this is divergen<'e. Parallelism ret-ersto tl-resintilar evolution of charactersin related plants or related groups of plants. Having decidedwhich charactersare of value and how many can be used. the taxonomist then has to considel whether each character shouldbe eivenequalvalueor whethera 'wei-ghtin-q'system shouldbe employed. Compr.rtershave an obvious role in dealing with large
r
INTRODUCTION nLlmbelsof charactersappliedto thousandsof plants.not only tl'on'rthc aspectol stolagcand retrievalof infbrnration.but alsofbt'thc sciencc lole in of ttunteric'ulto.totlotit\'.which will probably'plal' an increasin-rr For a fuller discr-rssion of this subject thc dcveloprnentof systematics. the readeris referredto Hevwood'sPIurttTtt.rctrtorrn.
PTANTTAXONOMY CHEMICAT rnLlchattentionand has.attermany This subjecthasrecentll,attlactccl yeals,broughtthc plant cherristback to systematicbotany.The concept that plantscan be classifiedon the basisof their chemicalconstitucntsis not ne\\':lbl examplc.earlyu'orkersclassifiedthealgaeinto grccn.brou'nand fed fbrms.but it is only duringthe last;10yealsthat ha\reled to thc of isolationand characterization moderntechniclues of plant samples.Compared chemical scleeningof rnanl' thor-rsands with molphological chalacters.chemical constitucntsale often more preciselydefinablcanclcan be of more fundamentalsigniticancefbr classificationpurposes.Plant trxononrists.in generrtl.hold the view bc considareyct anothert1'peof charactct'to thntchcmicalcharactels thoscnscdtraditionally. but it cloesnot necessarill'fbleredalon-gside lou'that taxa constmctedon a purell chemicalbasis.if such wele possibleon the clataat pl'esentavailable.woulclnecessalilycoincicle u'ith thosearrivedat by classicalmethods. The characters cmployedin chemicaltaxonomyneedto be thoseof distributionin the plant kingdom.The presenceof sr.rch internrediale ubicpritouscompoundsas the essentialamino tcids and comfiton sugarsis oflittle diagnosticvalueand.at the othcrextreme.the occurof the large in the singlespeciesConrrlrttntLt'ulatun renceof ccrrriine Chalacters is alsoof little taxonomicsigniticance. farnilyUmbellif'elae n-roststudied in this connectionare therelbrc seconclaryrnetabolites (alkaloids.isoprenoids.flavorroids.characteristicglvcosides.etc.). pharmaceutical interest. manyof which areof established rretabolitesmay be subjecttcr Horvever,asdiscussedlater.sccondary' valiationin the living plant.dependingon environmental considerable and ontogeneticfictors. and more stablechemical characteristicsare of'fbled by those closely rssociatcd r.r'ithDNA composition of the species.Increasingly.it is becoming possibleto use DNA hyblidization. serolilxonomyand antino acid sequencingtechniquesfor taxoapplication(Y Mino rurrr1., nonic purposcs.One pharmaco-gnostical of the com1993.33.601) hasbccnthe deterrninatiott Pltrtoclternisn-r plesplctc amino acid sequenccof one of the iron sulphurf'en'edoxins suppollthe view that ent in .,irrieticsof Duturo stromoniunt.The lesr.rlts lbrms are valietiesof a singlespeciesand that the the r"'hiteand pr"u'ple tree datlrras(e.g.D. urboreal ale best regardedas constitutingone section of the gcnus Dcttura and not a separategents (ltlern.. ibitl.. andD..fitstu\991.37..129:1995.'13,I 186):work with D. querci.foliu r.,.rrr also sllggeststhat the amino acid sequencedependsnot on the applied but on the section.Similar studieswele subsequently species. (Y. Mino and K. YasudaPhytoc'lrctnisrrt' 1998,49, 163l). to P/rr'.ia1l.r A secondexample(H. Mizukamiet aL..Biril.Pharnt.BulL..1993.16. 3881shor.vsthat restrictionfi'agmentlcngth polymorphisrns(RFLPs) can be r.rsedas a sirnple and elficient method for distinguishing betweenDuboi.sictleiclthardtii,D. mtoporoide.sand the hybrid of the tu,o specics(RFLPs are producedby digestionof DNA with restriction species and vary in numbcrand sizeaccordingto -9enus. endonucleases
etc.) Ho$'e\,el'. the sanre,rrouptbrlnd (ihid.. 1993.16. 6ll) that the tcchniqucdiclnot clistinguish betweerr the variousgeoglaphical strains of tlre traditiona]Chrnesedrug Gleltniulittoruli.s(Unrbellif'erae) containirr-sdilferent furanocoumarincompositions but did so with B uplettrtrnt.fhlcutttrrt(iltid.. p. 279). The diff-erentiatior.r bctu'ccr-rsamplesof Parttn ginsertg (Oriental (Ar.nericarr ginscn-e). ginsen-u) P. Erirtqrte.fttliurr and adultelants can be clilficLrltbv conventionallncansand F. Ngan et ol. (Plrltochenti.str-t and difl'elentiation. 1999.50. 787) havereportedon the authentication one tl'ornanother.ol si.r speciesof Partu-rand also their adulterants. using RFLPs invol"'ingthe DNA scclucnccs in a sclcctcdlibosomal rcglon. DNA analysishasbeenuse'dto disRandomarnplifiedpoly'rnorphic of Melissao./.ficiuuli.s corlnron tin-suish betweenthe varioussubspecies market.Prcviously.sampleshavc bccn classion tl'rephalmaceutical fied accordingto the distributionpattcrnof compoundsprcscntin the lemonbalnroil (H.-T.Wolf r:ta1..PlantoMetlicu 1999.65.83). studiesof Acrirrrrgr.rmexudateshavedemonstrated Serotaxonorric testsin thechemota-ronomic anall,scs the r,alueof suchimrnunological (T. C. Balihvin at tl.. of these economically impoilant proc'lr.rcts Phvochemi.strt1990.50. 599). (in German) is that of A standardwork on chemotarxonomy Hegnauer(see.Further Reading):it contprisesI I volumespLrblished over neally :10years.A fbur-r'olumework in English is that ol Darnle-v it does not appearto have Gibbs. publishedin 1974;r-rnfbrtr.rnatell'. beenupdatecl. the P G. Waterman(Pht'rotlteni.itnl998. 49. 1175)hasdiscussed indicatir-rg tl-refactorswhich still currentstatusof chemosystematics. contributcto thc peripherallole of the subjectin taxonoury.
Further reoding Generql fqxonomy of cllssitlcation of flou'eringplants. A I 9EI An integfated s)'stern Cronquist Press. NewYork.USA Columbia L,niversrt-v E 195.1. 196.1. Sl,llabus der BnglelA.\{elchoirH. Werclelnrann r Bomtraeger, Berlin.Germanl'. 2 rols Pflanzentar.r.rilien. Gcbriide Arnolcl.Lontlon.UK Hevu'ood V H 1987Planttaxonoml'. 3rclecln.Edu'ard Longman. Harlo*.Esser.UK Holrncs S 1986Outlineof plantclassification. Alnold.London.UK 3rdecln.Edrvard Jeffre-v C 1989Biologicalnomcnclature. anclHall.London. UK MinelliA 1993. Biolo-lrical s)stematics. Chaprnan (ed of planttarononrv to thcprinciples SivalqrnV V 1991Introduction Llrir.ersitv Press. Carnblidge. UK RobsonN K P).2ndedn.Carnbridgc 2nclecln.Carnbticlge StaccC A I 991Planttrxonomyandbiosl'stcrratics. Unrversitl' Prcss.Camblidge, UK Chemicol tqxonomy of lloweringphnts.N4cGill GibbsR Darnley197;1 ChemotaKrnomy .1r'ols N'lontreal. Clanacla. UnilersityPress. Academic Press. Harborne J B, TurnerB L i 98.1.Plantchemosystelnatics. London. UK Recentadvanccs in thcchcmosrstcuratics of Harborne J B. WilliarnsC A 199,1 Phl"tocherrristry 37(1t: 3 I 8 theN4onocotyledons. Prcss. andscrotaxonotry. Acadcrnic Harvkes J G (cd) 1968Chemotaxononrv London. UK Bitkhliuscr. Basle. dcrPt'lanzcn. Hcgnaucr R 1962-l992Chemotaronomie VolsI-X Su'itzerhnd. M H I99;1.1996Chemotaxonouiic dct Pflanzen. He-enauer R. Hcgnauel parts1 ancl2 Birkhrir.rser. Basle.Switzerlancl. XIa andXIb. Legr.rminosae
3t:'S*
PART
2
The plqnt qnd qnimol kingdoms qs sourcesof drugs
d',-*
iJ A
"r* t
"t
E
Biologicol ond geogrophicol sources of drugs
GEOGRAPHICAT SOURCES 1 4
Culrenl estimatesof the numbelof specieso1'llowelingplantsran-ue between200 000 and250 000 in son"re 300 familiesand l0 500 genera. Despitea rapidlyexpandingIitelatureon phytochemisrry. only a small percentageof the total specieshas been examined chcrniczrlly.and thereis a lalge field fbl firtureresearch. However, rlrandid not requirethe modernrnethodsof invcstigation to collect fbr hirnselfa materiamedicaof plantswhich he often usedin conjunction with magictrl and other ritual plactices.SLrchfolk medicines natulallv varied accordingto the plants availablein a particular climatic area and can be slr.rdiedtoday in those rnore or less undisturbedplimitive socictieswhich still exist. It is interestingto reflect that suchcollectionsof herbalntedicinescompiledovel centuriesby trial and error,and plesurrablyusing the patientas thc experirnental animal throughout,ntllst sr.rrelycontain solne material worthy of furthel in',estigation and shouldnot be too readilydiscarded. Is it not also possiblethat thosematerialsproducin-tadlersercactions.one of the principal problernsin the introductionof new drugs, ntight also have beeneliminatedby sucha systemof developrrent'l In the currentsearchtbr new drugshaving.tor exanrple.antitumout or hypotcnsiveactivitythe plantsinvolved,unlike many of the more tladitionalmedicaments, very oftenshowno imrnediateindicationsof pharmacologicalactivity.Investigatorsarethus laced with the problem of making a systerratic investigationiront among the thctusandsof speciesstill unex:urined.One obvious line of approachis to startwith lblk medicinesof the world on the assumptionthat theserraterialshave alreadybeensubjected to somehumanscrcening. howcvercrude.and found acceptableto thoseculturesthat usethem. For many areasof the world. the plants used in folklore have beenadequatelyrecordedlbut for other regions-tbr example.in SouthArnerica.with its vastt-loraof potentially useful plants the art of lblk medicinein aburiginal societiesis in lapid declineowing to a chan-eing modeof lif'eof rhepeople. Ethnobotanistsare cun'ently fighting a battle againsttime to record suchintbrmation before.within a generationor so. it is lost and with it a possible short cut to some rnedicinally useful plant. However. the urgencyis well-recognizedand as describedin Chapter8. the biological and geographicalsourcesof many traditional plant relnediesare being actively researchedand documented.Much recordedintbrmation still requiressifting.An exampleof the employmentof a combination of literaturesurveysand data fiom other sourcesin the searchfbr new drugsis the US NationalCarrcerInstitute'sscreeningof thousands of plant extractsfor antineoplasticand cytotoxic activity. This undertaking involvesthe team-wolk of botanists.phytochenrists. pharrracologists and clinicians. In the absence of such sophisticated collaborat.iorr, rnuch useful researchby one discipline fails to be fbllowed throughto a r-rtilitarian conclusion. An inspectionofthe plant or plant-deriveddrugsinclLrdedin western phalmacopoeiasshowsthem to be composedof thosewhich hat'e sr.rrvived tiom Gleek and Roman eras.inclLrdrngsome spices.thosemore chalacteristicof our own flora (e.g. cligitalis)and introducedat a later date. other pharrlacologicallyactiie drugs (e.g. cinchona-quirrine -and ipecacuanha) addedas a resultof increasedtraveland colonial expansion,drugs (e.g.rauwollia reselpine)long usedin othel systems of medicinebut of rnore recenl introductioninto Westernnredicine. and finally. recently discoveredplant constituentsof therapeutic value (e.9. vinblastineand vincristine trom CathtLrunthu.s roseus)and those semisyntheticproducts(e.g. steloidalhormones)which depend on plant sourcesfor startin-ematerial. An examinationof the list of drugs derivedfrom natural sources.as included in any pharn-racopoeia, soon reveals that the majority are derivedfrom the Spermatophyta-the dominantseedbearingplantsof the land. Within the Spcrmatopl.ryta the numbel of speciesarrd the number of usefirl medicinal plants is divided unevenly between the
fE
T H EP T A N TA N D A N I M A LK I N G D O M SA S S O U R C E O S F DRUGS phyla Gyrnnospermae. which yields some usefuloils. resinsand the alkaloid ephedrine. and rhe Angiospermae.which is divided into Monocotyledons andDicotyledons(bothof theseproviciemany useful drugs but especiallythe Dicotyledons).Of the other divisions of the plant kingdom. the fun-siprovide a number of useful drugs,especially antibiotics.and are importantin pharmacyin a numberof other wavs. The algae are a sourceof a limited number of dr.ugs(e.g. agar and alginic acid), but the flll pharrracologicalimportanceof this large groupof aqr.ratic plantshasstill to be realized.At the nroment.lichens and mossescontribute littlc to nredicine and the Pteridophytesare pharmaceuticallybest-knownfbi' the taenicidalf'ernsand lycopodiurn. Land animalsprovide suchtraditionalpharmaceuticalmatedalsasgelatin. wool tat. beeswaxand cochineal,and are a sourceofhormones. vitaminsand scra.Among the rrany intportantphar.maceutical aspects of the Bacteriophytaare the production of antibiotics. their use in etl'ectingvarious chemical conversionsof added substratesand their employmentin -eeneticengineeringas. for example,in the pr.oduction of human insr.rlinand the transfilrmationof hi-eherplant cells by incorporationof part of thc DNA of a bacter-ial plastidinto the plant genorne.
Andes of South America, were most successfully introduced to Indonesia(particularly Java) and India. These becamerhe principal geographicalsourcesfor the bark and its alkaloids. Java f'ell to the Japanese in 1942and,as India normally consumesmost of its own quinine production,there was a great shortageof this vital alkaloid at a time when large armies were lighting in malarial areas.Expeditions searchedthe original SouthAmerican habitatsand wild treessupplied usefulbut inadequatequantities.Fortunately.very successfulsynthetic anti-malarialswere discoveredat this time and put into large-scaleproduction. However, 50 years on the malarial parasitehas, in certain areas.becomeextremelyresistantto thesedrugsand the useof quinine has again been invoked.There is also a steadydemandfor other cinchona alkaloids. and Zaire and other Aliican states,together with Guatemala.producemost of the world's bark. China has now emergedas a major producerof a number of quality medicinal plant products including coumarin, menthol and oils of eucalyptus.peppermint.spearmint,sassafrasand valerian in addition to its establishedlistings.Other changesevident from market reports include the acceptanceby the Europeanmarket of expensivehighquality Australian coriander.Guatemalaas the principal producerof cardamonsandPodophyllumemodifrom China. GEOGRAPHICAT SOURCES Many countriesproducelimited quantitiesof medicinal plants and spicesfbr domesticconsumptionand theseare not listed on the interTwo tactorswhich determinethe commercialgeographicalsourcesof a nationalmarket. dru-eare the suitabilityof the plant to a particularenvironmentand the Governmentalpolicies on the export of raw materialsmay af-tect economicfactorsassociatedwith the productionof a drug in a particu- geographicalsources.as when the Indian government limited the lar area. export of crr"rderauwolfia root, selling only the more highly priced Many plantsgrow equallywell in nunterouslocalitieshavingsimilar extract. Supplies of the root were subsequentlyobtained from climates;and aseconomicconditionschangein one area,so the collec- Thailand. Changes in the legal cultivation of medicinal opium in tion ol cultivationof a drlg plantmay move in accordance. Developing Turkey must eventr-rallyaffect the geographicalsource of the drug: countriesmay also stafi the cultivationofrledicinal plants.and in this thus,the opium poppy has beencultivatedin Tasmaniaon a largescale respectSouthAmerica. India and S.E. Asian countrieshave beenpar- in recentyearsbut political f'actorsare militating againstthe continuaticularly active. Cinnamon, traditionally produced in Sri Lanka, has tion ofthis source.Political considerationshave also led to changesin beenintroducedto the Seychellesas a commercialcrop. with so much the startingmaterialslbr corticosteroids.Up to 1970the sole intermesuccess that the planthas now becomea weed!Cinnamonalsogrows diate sourcematerialfbr the manufactureof contraceptivesteroidswas well in West Africa bLrtis not commercially utilized ther"e.It must be diosgeninderived from the Mexican yam. Then the Mexican governremembered.however.that a plant may _qrowwell in different situa- ment nationalizeddiosgeninproductionand increasedprices to above tions but fail to producethe sameconstituents(e.g.cinchonasgrowing thosefor total synthesis;as a result,in subsequentyearsmanufacturers at altitudeand in the plains).The cultivation of Duturtt stnmtoniumin have turned to other startingmaterialssuch as hecogenin(trom sisal), En-elandas a half-hardy annual has long become uneconomic and solasodine(.fromSoLanumspp.), microbiologicalproductsfrom chomuch material is now intported fionr easternEurope. Similarly. the lesterol,stigmasterol,sitosteroland squalene,and petroleumproducts. USA. which at one time utilizeddomesticsuppliesof the solanaceous The more recent emergenceof China as a producer of high-quality, drugs and digitalis. now obtains such raw materials fiom fbrmer low-priced diosgeninhas againchangedthe situation. Yugoslavia.Bulgaria and Hungary as well as fiom South America. National and international restrictions on the collection of wild During and after World War II, agar production was initiated around plants have also afTectedthe sourcesof some drugs; the Washington New Zealand.Australiaand SouthAflica. bLrtwith the re-emergence of Convention on InternationalTrade in EndangeredSpecies(CITES) the Japaneseindustrythesesourcesbecamelessimportant.Scarcityof placed all speciesof Aloe exceptA. rera on the pr.otectedlist without acaciafrom the Sudanprornptedthe exploitationof the Nigerian gum. warning;this causedproblemsin the marketingof aloesproducedfiom Pharmacopoeial oncecameexclusivelyfrom Jamaica:a decline the r.rsualspecies.Other medicinal plants which have recently been _uinger in productionin Jarnaicnarrda vastimprovementin the quality of some given CITES listing include Htdrasti.scanadensis(1997) and prLtnus Afiican -eingershas led to the use of these and Chineseginger on a africana ( 1998). large scale.Olllcial storax wzls once derived only from the Turkish A numberof the abovetactorshavegiven addedimpetusto research Lfuuidambarorienruli.s(e.-s.Us'Pfiom 185I ) but limited suppliesled on the applicationof cloning techniquesin cultivationand to the artifito the adrnissioninto many phalmacopoeiasof American storax.l. cial cultureof plant cells and organs(seeChapter12). stvrociflua (e.g. USP from l936). Similarly. a lasting shortageof Rio (Brazilian)ipecacuanha root hrs led to the widespreaduse of the Furtherreoding Artuso A I 997 Drugs of naturalorigin: economicand policy aspectsof discovCartagena.Nicaraguaand Panamavarieties,which areobtainablefiom ery. developmentand marketing.PharmaceuticalProductsPress. a mr.rchwidel areaof Southand CentralArnerica.With the exceptionof Binghampton.NY India. attemptsto cr.rltivatethe drug in other areas(e.g. Malaya) met J o u r n a l o f E t h n o p h a r m a c o l o g y , s p e c i a l i s s u e l 9 9 6 5 l ( l 3).Intellectualproperty with onl1,lirlited success.In contrast.the cinchonas,indigenousto the rights.naturally-derived bioactivecompoundsand resourceconser\xtiun
.;:--lr!F
A toxonomic opprooch to the studyof medicinql plonts ondonimoL
derived drugs
For classificationpurposesthe plant and anirnal kingdorns are each divided into a numberof phyla and in additionto the phyla. the classification includesgroupingsofgradually dirninishingsize,namelydivisions.classes,orders.subordersand families.According to the systent used,thesegroupingsmay, or may not. indicatephylogeneticrelationships. In this chapterthe principalplant lamilies of pharmaceuticalinterest are ananged accordingto the botanicalschene of Engler (q.v.).The chapter is divided into six parts: Thallophytes;Bryophytes and Pteridophytesl Gymnosperms: Angiosperrns (Dicotyledons): Angiosperms(Monocotyledons);The Animal Kingdom. At the beginning ofeach largetaxon ofplants a table is given to clarify the orders and tamilies involved.According to the botanicalsystemof classification considered,schemesvarry!as do the numbels of families. genera and speciescited within an order.
The old term 'thallophyte'includesthoseplantswhich are not ditTerentiated into root, stemand leaves.Englerdividesthem into l3 phyla. They includebacteria.algae,fungi and lichens.The positionsof the main families of pharmaceuticalinterestare indicatedbelow. l . -
r
- - ^ , , -
-
. - - :
- i : . -
- '
BACTERIAAND ALGAE
- . ' , . , i " i ' . " l : 1 . 1. .: , ' 1 . . , , ' . , ' 1
BACTERIOPHYTA
THATLOPHYTES r 5 BACTERIA AND ATGAE l 5 FUNGI t 6 LICHENS
r8
The bacteriaare unicellularorganisms,the great rnajorityof which rangein size fiom 0.75 to 8 ptm.They reproduceby binary fission. Most speciesof bacteriacontain no chlorophyll, althoughthere is one group whose nembers contain a chlorophyll-likepigrnentand photosynthesize. Recent researchhas revealedmuch more of the detailsofcellular structureand it has beenpossibleto distinguishin certain species,in no small detail, the various componentsof the cell. Forexample.in Escherichiacolithe cell wall consistsof afburlayer structure,the inner one being rigid. the three outer onesnonrigid. Within the three-1ayerwall structure lies a protoplast membraneenclosingthe cytoplasm.The protoplastmembraneacts as a pelmeability barrier to all but very large moleculesand contains enzymes concerned with respiration and the active transport of n'retabolities. Bacteriaexist in a numberof characteristicshapes,namely: 1. Rod-shapedor bacillaryfbrms (e.g.Clostridiumyyelchii,Escherichia coli andBacillussubtilis\.
BRYOPHYTES AND PTERIDOPHYTESr 8
r8 PTERIDOPHYTAr 8 BRYOPHYTA
GYMNOSPERMS 1 9 ANGIOSPERMS: DICOTYIE DONS 20 SUBCTASS ARCHICHTAMYDEAE2 l
Phyla
O rders
Familie,s
Bacteriophyta
Eubacteriales
Chrysophyta (Diatomeae)
Di scales Pennatales
Phaeophyta(Brown Algae)
Laminariales Fucales
Rhodophyta(Red Al-sae)
Gelidiales Gigartinales
Rhizobiaceae, Micrococcaceae Actinodiscaceae Fragilariaceae, Naviculariaceae Lamirrariaceae Fucaceae, Sargassaceae Gelidiaceae Gracilariaceae. Gigartinaceae
SUBCIASSSYMPETALAE 3 t ANGIOSPERMS: MONOCOTYTEDONS36 ANIMAT PRODUCTS 39
tE
T H EP L A N TA N D A N I M A LK I N G D O M SA S S O U R C E O S F DRUGS 2. Sphericaior coccal tbrnts. which can occur singly but ar.eusually tbund in characteristica-egregates-i.e.in chains(streptococci).in groups of two (diplococci).four.(tetracocci)or eight (sarcinae). Agglegates of iregr,rlarpatternaresaidto be of staphylococcal fbrm. -1.Twistedor spiriliarformsrvhich.if havinga sinsletwist. belongto the genr-rs VlDrio,.while thosewith rnorethan one twist belongto the genus5prru1ftlrr. zl. Blanched lbrms which sontetimes occur in the genus Myt ttbutteriunt.
PHAEOPHYTA!
IAMINAR|AIES
Lqminoriqceoe The brown algac are r.nainlv nrarine and vary fiom microscopic branchedfllanrentsto leatheryfiond-like fbrms up to 60 rn in length. They or",etheir brown colour to the carotenoidpignient fircoxanthin. Vn'hich rnasksthe otheIpiglnents. Many of the30 species<'tfLutttinuriuareusedin coastalclistricts lbr agricultural purposes. Thev areusedlbr the manulacture ofalginic acid ( q . r .t . n t a n n i t orln J i o t l i n c .
Other in-rportanlrnorphologicalf'eatures which are of value in classif,ving bacteriaare: (l) Thc possession of flagella.thread-likeprocesses FUCATES whosenurnberandpositionarcofieno1'diagnostic importance. (2) The PHAEOPHYTA: fbrnrationof capsr.rles consistingof polysaccharidernaterialwhich is of Fucqceqe ond Sorgossoceoe grcar impol'tancein relation to the imrnunologicalpropertiesof the Exarr-tplcs of theFucaceae (about30 spp.)andpelt,etia;and lu'cFur.u.r orgrnism.(3) The possession 'Ihese of endospores, which arehighly refr.act- of the Sargassaceae about250 speciesof Szligzls,varr. arecollected ir.'ebodiestbrrnedby certainspeciesunder what appearto be adverse on a l:Lrgescalein many partsof the wor.ldfbr the pr.oduction of alginic environmentalconditions.The position of the sporein relation to the acid and its derir,atives.The specieshave been n'ruchinvestigatedfbr rest of the cell is of diagnosticimportance.(:1)Pigrnentationl utuny biologicalfl'activepropefiies E resitulosu_i.tbr example.gives waterbacteriaarecirpableof claboratin_q corrplexcolouringmarrers. solubleextractsthat inhibit the activity of the HIV r.everse-tr.:rnscriptase Bacteriaare able to carr)' out a very wide r-angeof chemical reacenzynre. tions, some of which are usedlbr identificationand clifl'erentiation. in addition to fbrming the basisof n-ranyimportant industritrlptocesses. Bacterialactionis used.fbrcxarnple.in theproductionoir.,inegar. GEUDIAIES acet- RHODOPHYTA: onc. br.rtylalcohol. lactic acid and L-sorbose.Notable examplesof Gelidioceoe reactionswhich are useful fbr characterizingbacteriaar.ethe abilitl, to The red algaeale divided into I I or.dcr.s. The 3000 specicsare mainly fermentcarbohydratesu,ith the fbrmation of acidic and gaseousprodmarine and are particularly abundtrntin the tropics and subtropics. ucts:the abilityto digestplotein.as shou'nby gelatinlicluelactionl the Most are relativelysmall.'I'heirplastidscontain chlorophyll,the red productionof hydro-qensulphidcliom organicsulphr.rr compounds. pi-enrentphycoerythrin(usLrallyin sufTicientcluantityto mask the other Bacteriaare most important in medicine and pharmacyin the fblpl-qrrents). and sontetimcs the bluepignterrtphycocyanin. lowing respects:as disease-producirrg organisms(about l0% of bacImportant generaof thc Gelidiaceaeare Gelicliwn (about :10 spp.) teriaareprobablypathogenic): fbl producingantibiotics(Chapter32): alndPterot:latlia(5 spp.).Many of thesetrreusedin the preparationof lbr eflectingbiochemicalconversions; as agcnrsin the deterioration of agars(q.\'.). clude drugs and medicar-nents (Chaptcr l3)l in geneticengineering involving recombinantDNA (c.-e.the productionof hurnaninsLrlin). Bactcriiralso play a I'ital role in nature-tbr example,in the nitrogen RHODOPHYTA: GIGARTINAIES cycle atmosphericnitrogen is fixed by A:pnfiacter or. symbiotically. qnd Grocilorioceqe Gigortinoceoe by various speciesof RhizobiL.nn. Nitrosonttnnsis able to oxidize Grucilariu (100 spp.l is a sourceof agar.particular-lvG. lit.henoides. zrnrmoniato nitl'ite. while Nitr.tlbaclel.can oxidize nitrite to nitr.ate. found in the Indian Ocean. Ir-rthe GigartinaceaeChondrtts crispu.s Bacteri:rare important in sewagepurification. in the lening of fibres yiefds cara-eeen or Irish moss(q.v.):()igrtrtinu.stelluttt hasbeeninvessuchasjuteand flax, anclin the ripeninuofcheese. tigatedas a sourceol British agar.
CHRYSOPHYIA: DISCALES
FUNGI
Actinodiscoceqe The Chrysophytahasthreeclasses, oneof which is theBaciltar-iophyceae The fungi are saplophytic or parasitic r.nentbers of the Thallophyta. (Diatomeae). containingsome l0 000 speciesof diatom.They areunicelentirell'devoidof chlorophyll.The plantbody is madeup of filaments lular algac.havea silicaskeleton.iind showinfinitevarietvin shapeandin or hyphae,which to-eetherconstitutcthe mycelium. The hyphaentay the sculpturingof the cell wall. Thereare two main types:the subclass be aseptateand coenocytic. but arc ofien septate.the individual pennatae Centricae(in u'hich Discalesis one of the orders)and sr.rbclass segments beinguni-. bi- or multinucleate. In the fbrmationof fr.uiting (which inclLrdes the order Pennatales). The Cenhicaeare centricor.disbodies the hyphaernay becomewoven into densemassesof pser.rclocoid in shapeand -uencllllvma'ine. while the pennataeare pennateor palenchyma(e.g.the sclerotiumof elgot). naviculoicland rrorc olien occurin fiesh water. The plotoplastof fungalcellsconsistsof granularor reticulatecytoAn exampleof the larnily Actinodiscaceae is Aruchnoidi.scrr.r.. found plasn, which in older cells is ofien vacuolated.The nr.rcleus mav shor.v in Japaneseagar'(q.r,.)and other generaare fbund in cliatoniteor a delicatereticulunt and one ol' morc nr"rcleolior its contentsrral' be kieselgLrhr'(q.r,.). condensed into a chromatinbody.The cell wall in manyAr.chirnycctes and somePhycomycetes (Oornycetes) and in the )reasts consistsmainly ofcellulosc. but in other fungi eelluloseis rephceclby the nitrogenCHRYSOPHYTA: PENNATATES ous substance chitin. Frogilorioceoe qnd Nqviculqriqceqe Sexualand asexualreproductionoccur.The characteristicsporesof Species of thesetr,o fantiliesoccurin diatontiteandareillustrated in the sporophytcgenerationare known as oospores(pr.oducedendogenFig.3;1. L ously) or basidiospot'es(produceclexogenously).In the Fungi
..:l-,f##
A TAXONOMICAPPROACH TO THESTUDYOF MEDICINALPLANTS AND ANIMAL.DERIVED DRUGS Irrrpcrl'ecti the sporophytegenerationis missing.The fungi trlsoproJLrcesporcshaving no significancein the altelnationof the sener.rtions:they ale bolne on the gametophyte(Phycolrycetesand (nlsts and some Autobiisidio\scomycetes)or on the spot'ophy'te nr\ cctcs).Theseaccessory sporesofien takethe fbrrn of conidia.nonnrotilespores.hornecxtemallyon conidiophores. The Archinry'cetes arethe simplestfirngi.in which the myceliuntis lubscntol mdimentary.A ntemberof this group causeswilrt diseasein potirtoes.The tirllowing glorrpsand fiimilics are of pharrraceutical i nterest.
Clus.s
Orzler
f utnilie.s
Phyconycetes Ascomycetes
M ucotales Pr"otoascales Plectascales Sphaeriales CLivicipitales Polypolinales Agaricales
Mucolaceae Siiccharomycetaceiie Asper_qillaceae Hypocreaceae C lar,icipitrceae Polyporaceae Tricholometaceae Amanltaceae Agaflcaceae Phallinaceae Dematiaceae
Basidiornycctcs
Fun,qiImperfecti
Phallinales Moniliales
PHYCOMYC ETES: MUCORALES
E
CLAVICIPITATES Clovicipiioceoe Like otherAscomycetes. the ascospores areproducecl in I saeor.ascus. Genera of the Clar,icipitaceaeinclude Clat iceps ( 10 -spp.)and (100 spp.),See'Ergot'.C.purpureuand its lif-ehistory. Corth'ceps
BASIDIOMYCETES: POIYPORINAIES Polyporoceoe The Basidionrvcetes producebasidiospores. borne externallvon the sporemothercellor basiclium. They have septatemyceliawhich produce elaboratefi'uitin-g bodies(e.g. mushlooms).Somentembersare edible.otherspoisonous. T h e P o l y p o r a c e a ei n c l u d e s P o l t p o r u s , P o l t s t i c h u s , f o m e . ; . GanoclerntuandBoletus(200 spp.).PolvltorusrlJicinulis (white agaric) and P .fomenturiuswere fbnlerly nsed in nredicine.Cunodernct lucitlo has long beenused in Chinesernedicineand its biologically active triterpenoidshave attractedrecent attention.IJoletuseduli.sis edible.
AGARICALES Tricholometoceqe An orderof severaltamilies.of u'hichthe Tricholonteraceae conralns (q.r'.).Othcr Clitotybe(80 spp.),speciesof which producer.nuscarine fanrilies crf the order contain Strophurio, Psiloc:tbe and Conoctbe. (q.r'.)such as psilosinarrcl which producehallucinogenicsubstances psilocybin.
Mucorqceqe These fun-ei have an aseptate mycelium: members include Pltttoplttlrorain.festans, which causespotatobli-eht.In the Mucoraceae we haveMucor (40 spp.)and Rhippus (8 spp.),which are amongthe AGARICATES moulds associatedwith badll' stored fbod ploducts. Sonre R/l;o7;a.i Amqniloceqe speciesareusedindustriallyfor the saccharillcation of starchymaterial Thefarnilf inclr-rdes Amunita(-50-60spp.)andPluteus; Amanitutttusandfirr producingo-lacticacid fbm glucose:they areimpoltantin the caritr (t1y agalic ) and A. pdntherino contain rnuscarine (c1.v. ). microbiologicalconversions of steroids(q.r,.1.
ASCOMYCETES:PROTOASCALES
AGARICATES Agoricoceoe
Socchoromycetqceqe This family inc[tdes the common ntushroom.Agtu'itu.s cLuttpL',\tt.i\. This group includesthe yeasts.some 30 speciesof Sacchanttn'ces. The genusAgrtricus(P.sttlliotrr) containsabout30 species. Dlicd yeast(q.v.) is preparedfi'om a strainol S. cereyi.sicte. Amcntber of the lelated lamily Cr'-vptococcaceae ts Cantlitfu arrlrs.which producestorulayeast.a rich sourceof proteinsandvitamins. PHATLINATES
PTECTASCALES Aspergilloceoe In this oldcr the conidial sta-eeis more prominentthan the ascalstage. Penicilliurn(over'100 spp.)l,ieldsimpgrtantantibiotics(q.r'.)suchas penicillin and griseofirlvin:alsothe i rnmunosuppressant ntycophenolic acid.P. i.slcurclioorr fbrms emodin.Ar-nongthe 60 speciesof Aspergillu.; may bc noted A. orr:oe. r-rsedin the manufactureof soya saucc:A. prodLrcing the antibioticfumagillin; andA..flut'ttsproducine .fumigutu.s, allatoxin in poorly storedt'eedingmaterials.
SPHAERIATES Hypocreoceoe (10 spp.)produces Gibberelltt the plantgrowthregulators knownas gibbelellins. filstisolated tron G..fugikunrl. seeChapter: 8.
Phollinoceoe Theordercontains two families.ThePhallinaceae takesits namefrom ( l0 spp.). Plmllu.s Phnllu.s intputlitus is thestinkhorn.
FUNGIIMPERFECT: MONItIAtES Demqtiqceqe The Fungi lmperf-ectiis a group in which sexualsporeshave not been demonstrated.Some membersmav be Ascomyceteswhich have completely lost the ascusstage. The iamily Denratiaceaecontains Helmintho.sltorimn(150-200 spp.),a speciesof which. H. gruntiniutt't,.produces helminthosporin (q.v.).Another tamily of the group, the Tuberculariaceae. contains Fnsaritrrtt(65 spp.).f-usariutrt/irri will transfirrmdigitorigenininro digoxigenin. Fungal sporesale a common sourceof allergens.
T H EP L A NA T N DA N I M A LK I N G D O MASSS O U R C EOSF D R U G S surfacehas been skimmed off. soil may be excavatedin a very pure form. Sphagnummoss. consistingof a mixture of various speciesof Sphugntun,can be collectedin many parts of Britain. It rnay be used A lichenis a symbioticassociation ofan algaand a fungalpartner.Some. (enclosedin muslin bags)as arrabsor.bent dressingor compressedinto particularly in zrctic regions. are used as fbod. The deseft species sheets.making absorbentmattresses. Largequantitieswere usedin this Lectuutra esculentais regardedas the biblical manna.The 'oak moss' way in World War I. used as a llxative in pefumery is the lichen E',,entiaprunastri. Many The pharmacologically activeterpcnoids(sesquiterpenes, diterpenes) lichenscontainderivativesof orcinol. orcellic acid and lecanoricacidl and aromaticcompouncls of the bryophyteshavebeenwell studied.(For thesecompoundsare termeddepsidesand are phenolicacidsfbrmed by a review seeY Asakawa.Proc. Phttothem.Sot'.Eur.. 1990.29.369. the interactionof the carboxyl gloup of one molecule with the hydroxyl This volume (eds. H. D. Zinsmeisterand R. Mues). published by -eroupof another.A classof theseacidstenned depsidones(e.g.norstictic ClarendonPress.Oxford. alsoincludesa further28 review iu-ticlescol, andpsoromicacids)complexwith metalsandareprobablyresponsible for eringthe chemistryandchernicaltaxonomyof the Br-yophytes.) the ability of lichensto flourishon mineral-richsoilsincludingmine tailOf the many bryophyte orders. farnilies and genera.a few which ingsandto accumulate largequantitiesofmetals,suchascopper,zinc etc. have beensubjectsofrecent researchale listed below. Lichen dyes were formerly much used in the textile industry. Litmus. prodr.rced fl'orn certainlichens (e.g. Lecanora,Roccellu spp.) Clctss Order Genera by fermentation.is usedas an indicator. Icelandmoss. Cetroria islandiccr.has been used for disguisingthe Hepaticae Jungerrnaniinales Buzz,ania, SoIeno,stoma, tasteof nauseousmedicinesand with other species(e.g.Clarkmia spp.) Glntnonitrion, for thc treatnrentof cough.It containsthe very bitter depsidone.cetraric Diploph-,'Llunt acid. Many lichens have antibiotic propertiesas illustratedby usnic Jubulineales Luttulariu acid.fbund in Cladoniu and Usneaspp.(seeE. R. Conechdet al. 1998, Musci (336 spp.) Sphagnales Spltugrtunt Fitoteropia. 69, 1931'V. Marcano et al., J. Ethnoplnrmacolog,t 1999, Dicranales I)i crcur urrt t -52 spp.) 66, 343); 29 speciesoflcelandic lichenshaverecentlybeeninvestigated Funariales Funuriu(ll7spp.1 lor their cancer chernopreventiveand cytotoxic activity (K. Ing6lfsd6ttirer al., Pharm.8io1.,2000.38,313). It is possibleto isolateand grow the algaefrom lichens as suspension cultures(seeP Hdrmiiliet al., Fitoterapia, 1992,63.211). PTERIDOPHYTA The accompanyinglist indicatessomeof the families and generaof recentinterest. The Pteridophytaincludesthe Filices (ferns),Articulatae (horsetails) and Lycopsida(club mosses).They show an alternationof-renerations. the sporophytebeing the larger'.A few are of medicalimportance. Order Fttmih Genent Of the many families, subfamiliesand generathe following mav bc noted. Rocceliales Roccellaceae Roccella(3 I spp.) Lecanorales Pertusariaceae Pertusuria(608 spp.) Lecanotaceae Lecanora(1100spp.) Class Order Funtilt G e t t er a (800 spp.) Parmeliaceae ParmeLict Cetraria (62 spp..1 Filices Filicales Polypocliaceae Pohpodium(.abou| Usneaceae U,vrea(500 spp.) (dividedinto 50 spp.) Evemia (8 spp.) many subfanrilies)D rl opteris (about Alectorio (48 spp.) 1 5 0s p p . ) Caloplacales Caloplacaceae Culoplaca(,180spp.) Plerls (280 spp.) Teloschistaceae Xanthnria(21 spp.) Preridium(1 sp.1
LICHENS
Thesetwo phyla areof relativelysmall pharmaceuticalimportance,but h a re : o r r r cp h l t o e h e r n i c a r nl l e r e s t .
BRYOPHYTA The phylum is clir,idedinto two classes,Hepaticae(liverworts) and Musci (mosses).Both show alternationof generations.The more consplcuousgametophytegcnerationis a leat--likethallusin the liverworts and a leafy plant with a stem in the mosses.On the latter is borne the sporoph)le generation u ith sporangium. Peat.longusedasa domesticfuel.consistsofpartly decayedmosses and othel plants.In some areas(c.g. partsof lreland) depositsof bog moss(lar-uelyspeciesof Sphagnuntlare many feet thick. and after the
Articulatae Equisetales Equisetaceae Lycopsida Lycopodiales Lycopodiaceae
Ont'chium(6 spp.) Dennst0edtid (7i) spp.; AdiantLon(.abotl 200 spp.) Athyrimn(180 spp.) Asplenium(about700 spp.) Ecluisetunt(32 spp.) Lt'copodiunt(abouI .100spp.)
Male fern rhizome(q.v.) derivedfrom Drtopteris filir-mas is one of many fernscontainingphloroglucinolderivatives.The insect-rr-roulding hormonesor pterosinsarewidely distributedin f-ernsand attl-actconsiderableresearch. The dried sterilestemsof the horsetail. Ecluiseturn on)enseare used in herbal medicine and ale listed in rhe BHP. Pleparationsare used internally to treat inflammiition and mild infectior"rsof the genito-
A TAXONOMICAPPROACH TO THESTUDYOF MEDICINALPLANTS AND ANIMALDERIVED DRUGS ..:llrrr\ tract and externallyfbr wound healing,fracturesetc. There are .,;.p.rrcntl\'two chemotypesof the specieswith diffelent f'lavonoid -, 'nrpositions. Horsetailsgive a high mineralashcontainingconsider.:irlc'amountsof silica. Correct identificationof the herb is ir-nportant ..cause the relatedspeciesE. paLustreis poisonous. The sporesof lycopodium (.Lt'copodium clavattrm)are usedin quantitatire microscopy(q.v.)and to a limited extentin medicatedsnuffs. Ju:tin-9powdersand lubricants.As a dustingpowder for rubbergloves rt has beenknown to give rise to dermatitisand mild cautionhas been e'\pressedregardingits use as a lubricantnon-stickagentfor condoms rclative to a possiblecauseof granulomas.The lycopodium alkaloids have been extensivelystudied(for a review seeW. A. Ayer and L. S. Trifbnov,Alkaloids, 1991,45, 233). Bracken(Pteridium aquilinum) has been a recentcauseof concern o\\'ing to its carcinogenicpropertiesand known bovine poisoning.The useofthe young shootsfor culinary purposesis discouragedand avoidance of bracken sporesin the atmospheresuggested.The toxic con\tituent is ptaquiloside.an unstable glycoside of an illudane-type rrorsesquiterpene. Other similal compoundsare widely distributedin the genusPte.ridium.
ageing process,the leavesof the ginkgo tlee have been extensively investigated.For furtherdetailssee 'Diterpenoids'.
(ORCONTFERATES) coNTFERAE Pinoceqe All mernbersof the older are trees or shrubs:mostly evergreenwith needle-likeleaves:monoeciousol dioecious.Sporophyllsr.rsually in cones.Resinductsoccurin all parts. The Pinaceaeare trees,rarely shrubs.Important generaare: ADles (,50spp.).Pseudotsugu (7 spp.), Tsuga (l 5 spp.),P i cea (50 spp.).larn (11 spp.),Cedru,s (1spp.).andPinus(.10 100spp.).They areabundant in the northern hemisphereand extend southwaldsto Indonesiaand Central America. Apalt fiorr their great value as timber and papermaking material. many species (e.9. Pinus) yield oleoresin (see 'Colophony Resin and Crude Turpentine'). Other speciesare ADles balsttnea. yielding Canadabalsam: P.seudtxsugu taifolia (Douglas tir).: Picea ables (Norway spruce): and Lari.r eltropoelts(larch). The barks of larch and hen-rlockspr'r.rce ale tanningmatelials.Pirtusltirtea (the umbrellapine) produceslargeedible seeds(pignons).
CONIFERAE The division Gymnospermaecontainsmany fossil members.Of the I I ordersin the Engler classification,it is only necessaryto mentionfive ordels and l0 families: Orders
Families
Cycadales Ginkgoales Coniferae
Cycadaceae Ginkgoaceae Pinaceae,Taxodiaceae. Cupressaceae. Araucariaceae. Podocarpaceae, Cephalotaxaceae Taxaceae Ephedraceae
Taxales Gnetales
The gymnospermsare one of the two great divisions of the seedbearing plants or spermaphyta.They differ from the angiospermsin having ovuleswhich are not enclosedin an ovary.A perianthis absent exceptin the Gnetales.The seedsusually containone matureembryo with from two to 15 cotyledonsembeddedin endosperm.The wood is composedlargelyof tracheids,vesselsbeing absent.
CYCADALES Cycodoceoe The order containsonly l0 generaand about 100 species.The family Cycadaceaecontainsthe single genus C-vcas,with 20 species.A sago (not to be confusedwith that fiom certainpalms) is obtainedfrom the pith of C-r'ca.r circinttlis and C. revolula.
Tqxodiqceqe A snrall family of l0 genera. including Sequoitr, Turodiunt. Cr1'ptomerio,Tetraclini,;, Tain'aniuand Ctuutinghantiu'.| 6 species. The resin sandaracis produced in North Afiica and Spain fiorn Tetraclini,rarticuldtd. Some of the farnily contain antifungal diterpenes.othersalkaloids.
CONIFERAE €upressoceoe A family of 19 generaand 130 speciesol'treesand shrubs. Merrbersdifl'erfiom the Pinaceaein that the lear,esand cone-scales areusnallyoppositeor wholled andthe ovuleserect.The generainclude CttLlirris(16 spp.,Australasia),Thujn (5 spp..China, Japanand North (15-20 spp.),Chumaect'paris(7 spp.),Jwtiperus America). Cupressu,s (60 spp., northern hemisphere).Jtutilterus conrmutis yields juniper berriesand volatile oil (q.v.);J. virginicntu.the red cedal wood r.rsed lbr pencils:andJ. sabina,volatile oil ofsavin: J. oxt'cedrus,by destructive distillation.yields oil of cade,which was formerly much usedin veterinary wolk. This tar-like oil containscadineneand phenols. Various diterpenesand flavonoidsof the lamily havebeenstudied.
CONIFERAE Arqucqriqceoe Two generaand 38 speciesof trees.which sometimeshave pungent leaves. ArarLcaria( I8 spp.)providesuseful timbers; andAgathis (20 spp.), the resins known as copals ol animes. which are used fbr varnish. Manila copal is obtainedfiom the MalaysianAgathis alba; andkauri copal fromA. ctustralis.the kauri pine. in Austlalia and New Zealand. The best copals are usually those for.rndin the ground long aftel the treesare dead.
GINKGOALES Ginkgooceoe With theexceptionof Ginkgobiloba,themaidenhair-tree, theplantsof this orderare foundonly as fossils.In recentyears,owing to their increasing usefor thetreatment of variousdiseases associated with the
CONIFERAE Podocorpoceoe Six generaand 125speciesof treesand shrubs.The largestger-rus, (100spp.),extendsfrorntropicalto tcmperate Podocarpus zonesand
E
T H EP L A N TA N D A N I M A LK I N G D O M SA S S O U R C E O S F DRUGS vicldsvaluabletimbels.A chalacteristic chemictrlf eatureof this genus flowersmay be unisexr.ral (e.g.Salicaceae). bLrtaremoreusuallybisexis the widespreadoccurrcnceof nol'diterpene and bisnorditcrpene ual. The perianth may or may not be difl'erentiatedinto sepalsand clilnctones: thcsecompoundserhibit a r.'arietyof biologicalactivities petals.and the latter rnay be free fiom one anotheror-fused. ( s e cI . K u b oe t a l . . P l t y t o c h e n i s t r t ' . 1 9 9 2 . 3115.4 5 ) . The classificationadoptedin the fbllowing pagesis that of Engler. who divides the dicotyledonsinto two groups.the Archichlamydeae and Syrnpetalae.The Archichlamydeaeare further divided into 37 CONIFERAE ordersand about 226 farr-rilies and the Svmpetalaeinto I I ordersand Cepholotoxoceqe about63 f'amilies. A firnrili, of onc genus(.Caphuloto.rtrs) and scvcn speciesof treesancl The namesof the ordersterminatein '-ales'.subordersin '-neae'. shrubs(plum yews)fbund from thc easternHimalavasto Japan.Thev families usuallyin '-aceae'(Compositae. Gramineaeand Labiataeare havebeenintensivelvstudiedfirl thcir antiturnourconstituents andthe exceptions). and sometimes into subfamilies endingin '-oideae'. alkalcrids(harrin-qtonineand hor-r-roharringtonine) of C. hu rt.ingtoniu The Archichlamydeaecontain those families that in early editions haveshou'nconsiclerable promise(seeChapter28). rvereglouped under Monochlarnydeaeand Dialypetalae.The llowers have either no perianth or a perianththat is difl-erentiated into sepals TAXATES and petals. the latter being free. Engler's classificationof the Dicotyledonsis given in a somewhatabbreviatedfbrm below. Tqxoceoe
f, t:
i,
An order of onlY one tanily. r"'hichincludesthe -genera Zi-rir,i( I 0 spp.). Psetrclota.ru.s, Torreta . Austrotu-ru s andAnertotu.rus. The common yew. Zl.ur.r'bcrct'trtu,produces valr-rablewood. The fruit hasa fleshyred aril.All partsofthe plantarevery poisonous. and cattleandholsescandie ver.vlapidly aftereatinsthe leaves and stems. In additionto alkaloids.a cyanosenetic glycosideandanrirnrnout a-eenr havebeenreportedin the genus. Ta.rrtsbrevirtilia(the Paciticyew). The bark of this speciesyieldsthe prorrisin-t anticancerdlug taxol. a nitro,eenous diterpene.Low yields fiom the bark and thc scarcityof rau'r.naterialleadingto damageto fbrests by. olien illegal.over-collectionharrpeledthe developmentof the drug. The ini estigationof alternatir.e sollrceshasnow led to tissuecultureproceduresfbr the procluction of taxolbr.rttheyieldsarestill low.A pronrising developmentinvolving a renevn,able sourcehas beenthe isolationof l0deacetylbaccatin frornthefreshneeclles of T.bucutu in up to 0.1%yield irndits chen"icalconversiorr to taxol. T.rrrcdia.7.. orspitktu andT.chinensi.r'areotherspeciesinvesligatedtbr taxancalkaloids.For firrtherreading seethe supplernent to issueNo. I of f'rroterzpia1993.vo|64.
FamiLt
Orclt'r'
SubclassArchichlamydeae Juglandales Salicales Fagales Urticales Proteales Santalales Polygonales Centrospermae
Cactales Magnoliales
GNETATES
i t
i. I
Ephedroceoe The order consistsof three families (Gnetaceae. Ephedraceacand WeL"r'itchiirceae). threegencraand aboLrt70-75 species. The Ephedraceae containsthe single genusEphedra (q.v.),about,10 speciesof shrubs.They occur in arid legions of thc subtropicsandtropics. Theil seed.with two cotyledons.is enclosedin a perianthwhich becomeswoocly.Variousspeciesyield the drug ephedra(q.v.) and the aLkaloidenhedrine.
Ranunculales Piperales Aristolochiales Guttif-erales Sarraceniales Papaverales
F.
h E I
I
The angiospern-rs or f-loweringplantsinclr.rde inorethan 250 000 species ol herbs.shrubsand trees.The sporophylls(stamensand carpels)ar.e usr.ralll, aranged with otherleaves(theperianth)to fbnn a 'flou'er'. The ovulcs are enclosedin a chamber(the ovary) fbrrnedfiom the carpels, anda stigmais providedfbr theleceptionand gernrinationof thepollcn. The en-rbryoplant containedin the seedhas one or two seedleavesor cotyledons.The woocl almost invariablycontainstrue vessels.Thc phylun-ris divicledinto rronocotyledonsand dicotylcdons. The dicotyledons are herbs. shrubs of trees, the seedsof which havetwo cotyledons.The leavesareusuallyreticulatelyveinedand the typical stem strLrctlrre is a ring of open vascularbundles.Unlike the monocotyledons,which typically have their floral parts in threes. drcotyledonousllowers are usually pentanerousor tetramerous.The
Rosales
Geraniales
Rutales
Sapindales
Myricaceae.Juglandaceae Salicaceae Betulaceae.Fagaceae Ulmaceae.Moraceae(including Cannabinaceae) and Urticaceae Proteaceae Olacaceae.Santalaceae. Loranthaceae Polygonaceae Phytolaccaceae. Caryophyllaceae. Chenopodiaceae Cactaceae Magnoliaceae.Winteraceae. Annonaceae.Eupomatiaceae. Myri sticaceae.Canellaceae. Schisandlaceae. Illiciaceae, M o n im i u e e a eC. a l v c u n l h a c e a e . Lauraceae,Hernandiaceae Ranunculaceae. Berberidaceae. Meni spermiiceae, Ny mphaeaceae Piperaceae Ari stolochiaceae Paeoniaceae. Dipterocarpaceae. Theaceae.Guttiferae S a n a c e n i a c e aNee. p e n l h a c e u e . Droseraceae Papaveraceae (including Fumariaceae). Capparaceae. Crr.rciferae H a m u m ei d l a c e a eC, r u : s ul l c e a e . Saxifragaceae. Rosaceae. Leguminosae,Krameriaceae Geraniaceae, Zygophyllaceae, Linaceae,Erythroxylaceae, Euphorbiaceae Rutaceae,Simaroubaceae. Burseraceae. Meliaceae, Malpighiaceae, Polygalaceae Anacardiaceae, Aceraceae. Sapindaceae. Hippocastanaceae
\ -1-
, : -;4*:i
::*t*g
A TAXONOMICAPPROACH TO THESTUDYOF MEDICINAT P LANTS ANDANIMAI.DERIVED DRUGS Familv \ubclass Archichlamydeae (continued) ('dIastrales Aquifoliaceae,Celastraceae, Rhumnales \ I a lv a l e s
Thr,melaeales \riolales
Cucurbitales Myrtiflorae
Umbellillorae
SubclassSympetalae Ericales Primulales Plumbaginales Ebenales Oleales Gentianales
Tubiflorae
Plantaginales Dipsacales Campanulales
Buxaceae Rhamnaceae, Vitaceae Elaeocarpaceae, Tiliaceae, Malr uceae.Bombacaceae, Sterculiaceae Thyrnelaeaceae. Elaeagnaceae Flacourtiaceae, Violaceae, Turneraceae, Passifloraceae, Cistaceae,Bixaceae, Tamaricaceae, Caricaceae Cucurbitaceae Lythraceae,Myrtaceae, Punicaceae,Rhizophoraceae, Combretaceae, Onagraceae Alangiaceae,Comaceae, Garryaceae, Araliaceae, Umbelliferae
morerecently,sedative; seeM. Girzuet al., pharm.Biot., 199g.36. 280.
SALICATES Sqlicqceqe The single family of the order containsonly two genera,Sall.r (500 spp.)and Populus(35 spp.).The dioeciousflowers are in catkins. Both generacontainphenolicglycosidessuchas fragilin, salicinand populin.Salicin,formerly usedin medicine,hasbeenreplacedby other drugs. Willow charcoal is the chief kind of wood charcoal used in Britain. osiers (sa1l-r:purpureaands. viminarrs)are used for basketmaking, and S. alba yar. caeruleais usedfor cricket_bats.Speciesof Populuscontainrafflnoseand stachyose.The dried winter buds of var_ ious speciesof poplar constituteBalm of GileadBud BIlp, andthe hive productpropolismay be derivedfrom poplar bud exudates.
FAGALES
Betuloceoe ond Fogoceoe Thesefamilies consistof monoecioustreesand shrubs.Their crassificatlon togetheris confirmedby similaritiesin constituents. The Betulaceaehastwo genera,Alnus (35 spp.) and,Betula (60 spp.). Ericaceae constituentsinclude many phenolicsubstances suchas myricetin. detMyrsinaceae,Primulaceae phinidin and ellagic acid; also terpenoidssuch as lupeol and betulin. Plumbaginaceae The wood of Betula alba is usedfor charcoal. Sapotaceae, Ebenaceae, The Fagaceaehas eight generaand about900 species.Fagus (10 Styracaceae spp.) includes the beech, F. sylvatictt, the nuts of which are Oleaceae expressedto yield o1l Castanea(12 spp.)includesthe sweetchest_ Loganiaceae,Gentianaceae, nuI, C. sath)a, which yields timber and a bark used for tannins. Menyanthaceae, Apocynaceae, Quercus(450 spp.)provides valuabletimber. Differe nt euercus spp. Asclepiadaceae, Rubiaceae containshikimic acid (a cyclitol), methyl salicylateand terpenoids. Polemoniaceae, Convolvulaceae, The cupules and unripe acorns of e. aegelop.s(valonia) are used in Boraginaceae, Verbenaceae, tanning. Q. ilex and Q. robur yield tanning barks and e. tinctoria, a Labiatae,Solanaceae, yellow dye. Q. suber affordsthe commonly usedcork, in an industry B uddlejaceae,Scrophulariaceae, worth (1987) some f 120 million p.a. to portugal's economy; Bignoniaceae,Acanthaceae, becausethere is no plannedre-afforestationthe industry now facesa Pedaliaceae, Gesneriaceae. decline. An extract of Q. stenophylla has been marketed for the Myoporaceae accelerationof the elimination of renal and urethral calculi. Turkish Plantaginaceae galls (q.v.), an important source of tannic acid, are vegetable Caprifoliaceae,Valerianaceae, growths formed on the young twigs of the dyer,s oak, e. infectoria, Dipsacaceae as a result of the activity of a gall-wasp. Similar galls are produced Campanulaceae (including on the English oak, Q. robur. Lobeliaceae),Compositae
URTICATES SUBCIASSARCHICHTAMYDEAE JUGTANDATES
Ulmoceoe, Moroceoe, Cqnnqbinqceoe, Urficqceqe The Cannabinaceae,originally included in the Moraceae. rs now regardedas a separatefamily.
Myricoceoe ond Juglondqceqe l. The Ulmaceaecontains 15 generaand 200 speciesof tropical or The order containsonly thesetwo small families. temperateshrubsand trees.Generainclude ILlmus(45 spp.),Celtis The Myricaceaehas three or four generaof trees and shrubswith (80 spp.) and Trema(30 spp.).Members contain no latex (distinc_ unisexual flowers. Some members contain volatile oil (e.e. Myricct tion from Moraceae).Mucilage is abundantin the barks of ulmus prrle.the bog myrrlel. rubra (.see'SlipperyElm Bark') and U. campesrris;raffinose and The Juglandaceaehas seven or eight genera and the best_known stachyoseoccur in Ulmus,an indole alkaloid occursin Celrls. speciesis the walnut,Juglansregla, which producestimber and edible 2. The Moraceae has about 53 genera and 1400 species.They are nurs.Juglans containsthe naphthoquinonejuglone, the sugarsraffi_ mainly tropical or subtropicalshrubsor treescontaininglatex. The noseand stachyose,flavonoidsand phenolicacids. fruit is often multiple, as in Flcr.rs,the fig. The large genusFlcas The leavesand pericarpof J. regia havelong beenusedasextractsin (about 800 spp.) includes trees and shrubs of very varied habit. traditional medicine and pharmacologicallydemonstratedto be anti_ These include F. benghalensls(banyan),F. elastica (indiarubber helmintic, antidiarrhoeal,antifungal, astringent,hypoglycaemicand, tree) and F. carica (common fig). The latex is often anthelmintic.
THEPLANTAND ANIMAL KINGDOMSA5 SOURCES OF DRUGS owing to the proteolyticenzymef icin. Another genusCastlllorz( l0 spp.), yields. from C. eLastica,Panama rubber or caoutchouc. Among other constituentsrcported in the tamily are cardenolides (in five genera)and pyridine alkaloids(in two genera).Speciesof Morus areusedin orientalmedicine. 3. The Cannabinaceae or Cannabidaceae consistsof the two genera Cannabis and Humulus comprising C. siliva (hemp), H. lupulus (common hop) (q.v.) and H. japonica (Japanesehop). The chemistry of theseplants is a justification for their separationfrom the Moraceae.Connabisproducesthe besthemp when grorvnin a temperateclimate,whilst the more activesamplesof Indian hemp (q.v.) are usually associatedwith warmer climates. 4. The Urticaceaehas45 generaand 550 species;tropicalor temperate herbs or undershrubswithout latex. Some genera have stinging hairs (e.g. Urtica, 50 species of stinging nettle), others lack such hairs (e.9. Pilea, Boehmeriaand Parietaria). Root extractsof the common stinging nettle are used in herbal medicine, often in combinationwith other species,for the treatmentof benignprostate hyperplasia.For a review with 78 referencessee E. Bombardelli and P Morazzoni, Fitoterapia 1997, 68,38'7.
I
ii
r;
l' I r,-
H
i
F-'
Kz
h:
'-"rl
is a one-seeded,usually three-wingednut (e.g. dock and buckwheat). Anthocyaninpigmentsare common; also flavonesand flavonols-for example,buckwheat,Fagopyrumesculentum,is a commercialsource of rutin. Quinones (anthraquinones,phenanthraquinones, anthrones and dianthrones)are tbund in many speciesof Rheum. Rumer and Pol!-gonum.
CENTROSPERMAE
Phytoloccoceoe/ Coryophylloceoe, Chenopodioceoe The order contains13 lamilies which show a passagefrom the monochlamydeoustype of flower (e.g. Phytolaccaceaeand Chenopodiaceae) to the dichlamydeoustype of flower (e.g. Caryophyllaceae). Most tamilies ofthe order,exceptthe Caryophyllaceae,producecharacteristicbetacyaninand betaxanthinpigments.which indicateafTinity with the Cactales(the next order,below). The Phytolaccaceaeis a lamily of 12 genera and 100 species; herbs, shrubs and trees, found particularly in tropical America and S o u t hA f r i c a . P h . t t o l n c c ra3 5 s p p . t i n c l u d e sP h . t t o l a tttt ( u n e r iat n o (Poke root), the leaves and roots of which have been found as an adulterant of belladonna: its berries contain a dyestuff. The roots which contain saponinsare included inthe BHP (1983) for the treatPROTEATES ment of rheumatic diseases;the roots elaborateantihepatotoxicneolignans. Proleoceoe The Caryophyllaceaehas 70 generaand about 1750species,mostly The singlefamily of the order,the Proteaceae, contains62 generaand herbs, and is wide-spread. Genera include Saponarin, Stelluria, 1050 species.Shrubsand treesare particularlyabundantin Australia. Arenaria, Spergularia,Hentiariu, Silene, Lychnis, Gypsophila (125 New Zealand and South Africa. Many specieshave been examined spp.) and Dittnthus (300 spp.). Many of these plants are rich in chemically,and the constituentsreportedinclude cyanogeneticcomsaponins.The root of Saponariaofficinalis hasbeenincludedin many pounds, alkaloids, tannins,leucoanthocyanins, arbutin and the sugar pharmacopoeias. It containsabout 5% of saponinsand is widely used alcohol polygalitol. Genera include Protea (130 spp.), Grevillea, as a domesticdetergent. Persoonia,Hakea andKnightia, The Chenopodiaceae contains 102 generaand 1400 species;most grow naturally in soils containing much sait (halophytes).Genera include Beta (6 spp.'), Chenopodium (100-150 spp.), Salicornia. SANTATALES AtripLer and Anabasis.From the wild Beta vulgaris (sea-beet)have Olococeoe, Sqntqlqceqe, Loronthoceoe been derived garden beetroot, sugar-beetand the mangold-wurzel. The ordercontainssevenfamiliesof which only four needbe men- Chenopoditrmanthelminticumyields the anthelminticMexican tea or 'wormseed' tioned. and its oil of chenopodium(q.v.). The Olacaceaehas about27 generaand 250 species.Few have been examinedchemically.Acetylenic acidsoccur in OLaxstricta. The Santalaceae containsabout 30 generaand 400 species.Of the CACTALES genera,Santafun contains25 speciesandThesiumabout 325 species. Cqctqceqe Monoterpenesand sesquiterpenes occur in severalgenera.The plants The Cactaceaeis the only family of the order and containsfrom 50 to arehemiparasiticherbs.shrubsand small trees.Santalum. aLbumyields 150 genera and about 2000 species.The plants are xerophytesand, sandalwoodand sandalwoodoil (q.v.)and is rich in sesquiterpene alco- with possibly one exception,are all native to the Americas.They will hols. Australian sandalwoodand its oil are obtained fiom another not grow wherethereis vitually no rainfall, but thrive in desertswhere member of the family, Eucarya spicata. there is a reasonablerainfall even if rain occurs very infrequently. The Loranthaceaeis a fairly large family of 36 generaand 1300 Some cacti occur in rain forests,where they are often epiphytes(e.g. species.The genus Visr:an consistsof about 60 speciesof parasitic Epipb'llum). The majority are succulentand storewater in their stems. evergreenshrubs,which often containcyclitols. The dried aerialparts The plant body is usuallyglobularor cylindrical and bearswool, spines of the common mistletoe, Visctnnttlbum, which grows on apple and and flowers, buI rn Epiph,vllum the stems are flattened and consist of other treesare included in the BllP 1983and are usedfbr variouscir- jointed segments,which areoften mistakenfor leaves.Among the genculatory conditions.It containsglycoproteins(the mistletoe lectins), era are Epiphl,llum (21 spp.),Opuntict(250 spp.), Cephalocereus(48 polypeptides(viscotoxins),lignansetc. spp.'),Cereus (50 spp.) and Echinocereus(75 spp.). The leaves of Opuntia and Nopolea provide food for cochineal insects (q.v.). Opuntiaficus-indicct,the prickly-pear,is sometimesgrown as a hedge POTYGONALES but can become a troublesomeweed. Dried cactusllowers (Opuntia Polygonoceoe spp.)areusedas an astringentherbalremedy.Lophophoran'illiamsiiis This one-family order occupiesan isolatedposition. It has about 40 the plant producingpeyote,anhaloniumor 'mescalbuttons';it contains generaand 800 species,mostly herbs.About 29 speciesare indigenous mescaline(q.v.).Severalgeneracontainsimple isoquinolinealkaloids; to Britain. Generainclude Rheum (50 spp.),Rumex(about 180 spp.), cyanogeneticglycosidesare very rare or absent;most speciescontain The fiuit Fagopyrum(15 spp.),Coccoloba(150 spp.) andPoLt'gonum. abundantmucilage.
s::.,{#
A TAXONOMIC TOTHESTUDY APPROACH OFMEDTCTNAL ANDAN|MAr.-DER|VED DRUGS Ed PLANTS
ttAAGNOHAtES Mognolioceoe, Winferoceoe, Annonqceqe, Myristicoceoe, Conellqceoe, Schisondroceoe, llliciqceqe, Monimiqceoe, Louroceoe ond Hernondiqceoe fhis order contains22 families. i The MagnoliaceaecontainsI 2 generaand about230 species,which occur in both temperateand tropical regions. They are trees or shrubswith oil cells in the parenchyma.Generainclude Magnolia t 180 spp.),Michelia (50 spp.) and Liriodendron (l sp.),tulip tree. The remedial propertiesof L. tulipifera, which is reportedto contain alkaloids (e.g. dihydroglaucine) and sesquiterpenes,have been (Crellin,J.K. Pharm.J., 1988.240,29). assessed I The Winteraceaecomprises7 generaand 120 species.Drim.vswinterr (Winter's bark) has beenmuch usedfor its stimulantand tonic propertiesand a number of pharmacologicalinvestigationshave been reported in the literature. The bark contains volatile oil, sesquiterpenes, lactones,flavonoidsand,recentlyreported,polygo1998,62, dial derivatives(V. C. Filho et al., J. EthnopharmacoLogy 223). ,r. The Annonaceaecontains 120 genera and about 2100 species, which are found mainly in the tropical regions of the Old World. Genera include Uvaria (150 spp.), Xybpia (100-150 spp.), Monodora (20 spp.) and Annona (120 spp.). The seeds of Monodora myristica are usedlike nutmegs.Many membersof the family containalkaloidsmainly of the isoquinolinetype.The family is of importancein folk medicine. 1. The Myristicaceaeis a family of 18 generaand 300 species,which are mainly fbund in tropical Asia. Generainclude Myristica (120 spp.),Virola (60 spp.),Horsfieldia (80 spp.) and Knemtt (37 spp.). The flowers are dioeciousand consist of an inconspicuousthreelobedperianthwith 3-18 monadelphousstamensor a solitarycarpel containing a basal anatropousovule. The fruit is a fleshy drupe, which splitsalongboth dorsaland ventralsutures.The singleseedis more or lesscompletelyenvelopedin a lobed aril. The chemistryof the family has not beenthoroughlyinvestigated.Somespecies(see 'Nutmeg' 'Mace') and containvolatile oil and hallucinogenicsubstances. 5. The Canellaceae is a small family of five generaand l6 species; trees with gland-dotted leaves. Fruit is a berry. Genera include Canella, Cinnamodendronand Wttrburgia.Canella bark, a spice, is obtained from Canella alba, grown in the Bahamas and Florida. 6. The Schisandraceae comprisestwo generaand 47 speciesof climbing shrubs..lc/risandrahas25 species;lignansarecommonconstituents. -. The Illiciaceae,sometimesclassifiedunder Magnoliaceae,has the single genus llLicium (42 spp.), fbund in Asia, Atlantic North 'Star-aniseFruit and America and the West Indies.See Oil'. \. The Monimiaceaeincludes20 generaand 150 speciesof treesand shrubs. which often contain volatile oil and resin. In South America there are many traditional uses of plants of this family; G. G. Leitao et aL. (J. Ethnopharmacology1999,65, 87), have reviewed the pharmacology and chemistry. The genera include Hedycarya (25 spp.) and Peumus(one sp.). Peumusboldus has a hard wood, its bark yields a dye and its leavescontain the alkaloid boldine. L). The Lauraceaehas 32 generaand about 2000-2500 species.These are tropical or subtropical trees and shrubswith leathery,evergreen leaves;the flowers are usually bisexual(e.9. Cinnamomum),rarely unisexual(e.9. Laurus). The fruit is a bery or drupe. Alkaloids, volatile oils and fixed oils occur in manv species.Volatile oil cells
occur in the leavesand cortex.Generainclude Persea(150 spp.), Ocotea(.300400 spp.),Cinnttmomum(250 spp.),Anl&a (40 spp.), Litsea (400 spp.),Neolitserz(80 spp.),Lindera (100 spp.),Laarus (2 spp.) and Cry-ptocaryrz (200-250 spp.). The bark of Cnptocarl'a massoiayields an essentialoil that has a coconut-likearomaand is usedas a cosmeticadditive.e.g.in shampoos; C moschatagives the maceof Brazilian nutmeg.The bay laurel, lnurus nobilis, is the only European representativeof the family: 45 constituentsof the essentialoil have been identified, the principalone being I ,8-cineole.Otherconstituents of the leavesare glycosylatedfl avonoids,(-)-epicatechin,(+)-catechin,(+)-epigallocatechinand procyanidins.For recentresearchand other references see C. Fiorini et al., Phytochemistry1998,47,821. For drugs see 'Cinnamon bark','Cassiabark'and'Camphor'. 10. The Hernandiaceae has threegeneraand 54 species.Membersare tropicaltrees,shrubsor lianeswith oil cells. Speciesof Hernandia (24 spp.) contain tumour-inhibitingalkaloids and lignans including podophyllotoxin;they have been used in traditional Samoan medicine.
RANUNCULALES: Rqnunculqceqe, Berberidoceoe, Menispermqceqe qnd Nymphoeoceoe Of the sevenfamilies in this orderthe abovefour areof medicinalinterest.The families show a considerablevariety ofplant constituentsand alkaloids are very common. In the four named families the alkaloids are often basedon benzylisoquinoline,bisbenzylisoquinolineor aporphine. 1. The Ranunculaceae comprises59 generaandabout1900species. The plantsare mostly perennialherbswith a rhizomeor rootstock.They are well represented in Britain. Many members are poisonous. The flowers are bisexual,regular (e.g. Ranunculus)or zygomorphic (.e.g.Aconitum). The perianth is simple or differentiated into calyx and corolla. The stamensare numerousand free. The carpelsare usually numerous in the regular flowers or fewer in the zygomorphic ones.The fruit is an etaerioof achenesor follicles, or a berry. Genera includeHelleborus(20 spp.),Aconitum (300 spp.),Thalictrum(150 (400 spp.), spp.),Clematis(250 spp.),Actttea(lO spp.).Ranunculri.s Anemone(150 spp.),Delphinium (250 spp.).Adonis (20 spp.) and Hepatica ( I 0 spp.).The family has diversechemical constituentsand is of considerablephytochemicaland chemotaxonomicinterest.For basedon sizeand shape,fall into two disexample,the chromosomes, tinct groups, the Ranunculus type (R-type) and the Thalictrumtype (T-type). The glycoside ranunculin has been found only in plants of the R-type. This glycosidehydrolysesto protoanemonin,which is vesicantand accountsfor this property in many species.Isoquinolinederived alkaloids occur in Thalictrum, Aquilegia and H1;drastis; diterpene-derived alkaloidsin Delphinium andAconitum.Saponins, mainly triterpenoid, occur in Ranunculus, TroLlius, Clematis, Anemone and Thalictrum, cyanogeneticglycosidesrn RanuncuLus and Clematis; cardenolidesrn Adonis, bufodienolidestn Helleborus. Black helleborerhizome, from Helleborus niger, containsvery powerful cardiac glycosides but is now little used in medicine. Various aconite roots, containing highly toxic alkaloids (q.v.) have also lost much of their former popularity. Black Cohosh of the BI1P is the dried rhizome of Cimicfugtt racemosa:it containstriterpenoidglycosides structurally related to cycloartenol, also isoflavones including formononetin. The drug contains substanceswith endocrineactivity and is usedin herbal medicine to treat menopausaland other female disorders.and also variousrheumaticconditions.A numberof other
THEPLANTAND ANIMAL KINGDOMSAS SOURCES OF DRUGS species e.g. C. simple,r, are used in Chinese medicine (see, for example,A. Kussanoet al., Chem.Plnrm. Bull., 1999,47, I 175)and to date suchspecieshave beenmore thoroughly examinedchemically than C. racemosa.Recently,however,a new 9,19-cyclolanostanetype triterpenexyloside, and other known relatedxylosideshavebeen characterizedfrom the latter species(E. Bedir and I. A. Khan, Chem. Pharm. Bull.,2000, 48, 425; see also Y. Shao et al., J. Nat. Prod., 2000. 63, 905). The EuropeanC. foetida (bugbane)is a traditional vermin preventative. 2. The Berberidaceaehas 14 generaand about 575 species,they are perennialshrubsusually with spiny leaves.The flowers are hermaphrodite,regular and hypogynous.The perianth is difl'erentiated into calyx and corolla.The stamensare generallyin two whorls and the ovary is composedof one carpel. The fruit is a berry with one to numerousseeds.Generainclude Berberis(450 spp.).Mahonia (70 spp.),Epimeditun.Vancotteria andLeontice. The Berberidaceaecontains alkaloids of the benzylisoquinoline, bisbenzylisoquinolineand aporphine Iypes. Leontice contains an alkaloidal amine and quinolizidine. Lignans such as dehydropodophyllotoxinoccur; also triterpenoidsaponins.The root tubers ol Leonticeleontopetalumcontain saponinand alkaloidsand have been usedfor the treatmentof epilepsy.For other drugs see under 'Hydrastis','Podophyllum' and'IndianPodophyilum'. In some members of the lamily there is a close resemblanceto the Ranunculaceae. Hydrastis,lbr example,is sometimesplacedin the Ranunculaceaein the same tribe as the peony. There are also relationshipswith the Papaveraceae,narcotine being found in both families. 3. The Menispermaceaeis a family of 65 genera and 350 species; mainly tropical twining shrubs,herbs or trees.The plants usually have palmately lobed leaves and dioecious flowers. Anomalous stem structure is frequently found and abnormal secondarygrowth often takesplacein the roots (e.g.of Chondodendrontomentosum'), successive cambiabeing producedto give concentricrings of wood. The broad primary medullary rays found in the stem of Coscinium are a family characteristic. The fruit is a drupe the dorsal side of which developsmore rapidly than the ventral, as in the tish beny Anamirta cocculus.Thesecontainthe highly toxic substancepicro'Calumba' 'Curare'. toxin. For drugs see and The generainclude Chondodendron(8 spp.), Tiliacora (25 spp.), Triclisia (25 spp.),Anamirta (l sp.), Cosclnlun (8 spp.),Tinospora (40 spp.),Jateorhiza(2 spp.),Abuta (35 spp.),Cocculus(11 spp.), (30 spp.) Menispermum(3 spp.),Stephania(40 spp.),Cissctmpelcts and Cyclea(30 spp.). Alkaloids are impoftant constituentsof the family and have recently beenreviewed(J.M. Barbosa-Ftlhoet al., TheAlkaloids2000,54, 1). The authorsreport that in the period 1970-1997,1560 alkaloids have beencited in twenty-twodifferentclassesincludingbenzylisoquinoIines (604), aporphines(303), protoberberines(275) and other smaller citations. Saponinsare presentin many species.Cosciniumfenestra'tree tum ('false calumba', turmeric') stems are widely used in SE Asia and India for the heatmentof a variety of ailments.The principal alkaloid constituentsareberberineandjatrorrhizine. Stephaniapierrii (5. erecta) contains bisbenzylisoquinoline alkaloids and is used in Thai folk medicine as a musclerelaxant. Tinosporacordi.foliars used inAyurvedic medicineand a considerablenumberofpharmacological actions, including immunomodulatory have been demonstratedfor 'Calumba'and'Curare'. the drug.For otherdrugssee 4. The Nymphaeaceaeis a small family of about six generaand 70 species.Speciesof Nymphaea(waterJilies) are widely cultivated. The genera include Nymphaea (50 spp.), Nuphar (25 spp.), Nelumbo (2 spp.) and Ondinea.Alkaloids occur which resemble
thoseof other families of the order but the chemistryof the family requires further research.Nuphar ttariegatum rhizomes contain antibacterialtannins and have long been used in folk medicine, similarly Nymphaen spp..NeLumbon.ucifera(the sacredlotus) and Nymphaealotus (.thewhite lotus) were reveredin the ancientcivilizationsof India, China,Tibet and Egypt.
PIPERATES Piperoceoe Four families are includedin the Piperales;only the Piperaceaeis consideredhere. The Piperaceae(excludingthe Peperomiaceae) consistsoffour genera and about 2000 species.The plants are tropical, mostly climbing shrubsor lianes,with swollen nodesand fleshy spikesof flowers. The Ieavescontain oi1cells. The one-celledovary has a singlebasalovule and developsinto a berry.The seedscontainendospermand abundant perisperm.The four generaare Piper (3bofi 2000 spp.), Trianaeopiper (18 spp.),Ottonia (70 spp.) andPothonorpfte (10 spp.).In additionto the above,Engler includesin the Piperaceaethe Peperomiaceae with its four generaand about 1000 speciesof succulentherbs and subshrubs.AII but 5 of its speciesbelong to the genus Peperomia.The Piperaceaecontainsphenolic estersand ethers;pyrrolidine alkaloids; volatile oils and lignans.The peppers(q.v.) are widely usedas condiments. Cubebs,Piper cubeba,was formerly used in medicine but is now obsolescent. In the SouthPacific islandsan aqueousextractof the roots of P methysticum(kava-kava)is consumedas a dtual stimulant; large dosescauseintoxication.In herbalmedicinethe root is usedas a diuretic, stimulant and tonic. the active principles are pyrone derivatives (kava lactones);a piperidinealkaloid,pipermethysticine,hasalso beenisolated.
ARISTOTOCHIATES Aristolochioceoe The ordercomprisesthreefamilies,of which only the Aristolochiaceae is of importance. The Aristolochiaceaehas seven genera and about 500 species. Members occur in the tropics and warm temperatezones,excluding Australia.Most are herbsor climbing shrubs.Oil-secretingcells occur throughout the family, often forming transparent dots on the leaves. The principalgeneraareAristolochia(350 spp.)andAsarum(70 spp.). Constituentsof the family include alkaloids(aporphineand protoberberine), aristolochic acid, phenolic esters and ethers, volatile oils and flavonoids. Some species show tumour-inhibiting properties. Asarun europaeum,asarabacca root, was formerly used in European medicine.
GUTTIFERALES Pqeoniqceqe, Dipferocorpqceqe, Theoceoe, Guttiferoe Of the 16 families in the order.only the four aboveneedbe noted. 1. The Paeoniaceae containsthe single genusPaeoniawith 33 species which are perennial rhizomatous herbs, occasionally shrubby. Paeonia mosculais one of Britain's rarestwild plants;it grows on the island of Steep Holme, in the Bristol Channel, having been introduced there by monks in the 14th century for medicinal purposes.Peony root is important in Chinese medicine and the speciesused,P. lactiflora, has beenextensivelyinvestigated.It is a constituent of a herbal tea which, in the UK. has come into orominence for the treatment of children's eczema.The active anti-
\ '-'rJ
r'- i.#.:.';
:-;r*{
A TAXONOMICAPPROACH TO THESTUDYOF MEDICINALPLANTS AND ANIMAL-DERIVED DRUGS ::rllammatory ingredient appears to be paeonol, 2'-hydroxy- 1. The Papaveraceae is a family of 26 generaand about 300 species. -:'-nrethoxyacetophenone; other constituentsof the rhizome are The plantsare usuallyherbswith solitary,showy flowers of the flo:r()noterpenoidglycosidesone of which, paeoniflorin,is used as a ral formula K 2 - 3, C 2 + 2 or 2 + 4, A -, G (2 - *). The fruit is gen^r.is fbr the quality control of the drug by HPLC. erally a capsule,with numerous seeds,each containing a small - fhc- Dipterocarpaceae has 15 generaand about 580 species.Many embryo in an oily endosperm.Generainclude Platvstemon(abolt .,rc largetreesyielding usefui timbers.Oleoresinsare a characterof 60 spp.),Romnera(2 spp.),Eschscholtzia(10 spp.),Sanguinaritt(.1 :he thmily. Generainclude Dipterocarpus (76 spp.), Shorea (180 sp.), CheLidonian(1 sp.),Bocconia (10 spp.),Glaucium (25 spp.), 'lp.). Dr)-obalanops (.9 spp.) and Hopea (90 spp.). Products Meconopsis(43 spp.),Argemone(10 spp.)andPapaver(100 spp.). :rrcfude:gurjun balsam from Dipterocarpus turbinal,s; varnish All memberscontainlatex tissue.The latex is sometimesin vessels :c.ins trom speciesof.Sftorea,Hopea andBalanocarpus;an edible which accompanythe vascularsystem(e.g.in Papaver);sometimes irt rr hich can be usedinsteadof cocoabutterin chocolatemanufacin latex sacs (e.g. Sanguinaria).The family is rich in alkaloids. rure. fiom the nuts of Shorca macrophylla; and Borneo camphor, Some, such as the opium alkaloids (q.v.),are of great medicat and t rom D ryobalnnzp,ta romatica. economic importance. Eschscholtzia californica, used by the ' I'hc'Theaceae or Ternstroemiaceae consistsof 16 generaand about Californian Indians as a sedativeand now similarly prescribedin l,x) speciesof tropical and subtropicaltrees and shrubs.Genera Europe,has been validatedexperimentally.Mustard oil glycosides '.'.;lurJe Camellic (82 spp.)andTernstroemla(100 spp.).Among the appear to be absent from the family (compare Capparaceaeand -,rn\tituents are purine basesin Camellia, saponins,tannins and Cruciferae). ::r.-d oils. By far the most important plant commercially is 2. Fumariaceae.Includedin the Papaveraceae by Engler but now genr ttrtelliu sinensis,which yields tea and caffeine.The so-called'tea erally regardedas a separatefamily. It contains16 generaand about .:c-d oil' is an edible oil which is a possibleadulterantof the more 55 species:they contain a watery, not milky, juice. Isoquinoline :rpensive olive oil and is obtainedfrom Camellictsasanqa. alkaloidsare a featureof the family. Fumitory is usedfor liver dis- I hc'Guttiferaecontainsabout40 generaand 1000species.They are orders,seeChapter30. ::ccs. shrubsor lianes,exceptHypericl.rn,which is oiten treatedas 3. Capparaceae. A family of 30 generaand 650 species;usually trees -: :eptrote family, the Hypericaceae. The main genera are or shrubs,often xerophytic.The genusCapparis(250 spp.)includes Htpericum (400 spp.), Kielmeyera (20 spp.), Clusia (145 spp.), Capparis spinosa,the buds of which (capers)are used in flavourGtt rci n ia (400 spp.)andCalophylltua ( I I 2 spp.). Constituentsof rhe ing. Like the Cruciferae,the family has myrosin cells and mustardternily include resins,volatile oils, alkaloids,xanthonesand seed oil glycosidessuch as glucocapparin.The only alkaloid reportedis ,'ils. Products include resin from Caloph,vllumand gamboge, a pyrrolidine in two genera. Cardenolides,which occur in some ioloured gum resin, trom Garcinia. The edible fruit mangosteenis Cruciferae,havenot beenfound in the Capparaceae. zl. Cruciferae.A family of 375 generaand about 3200 species;herbs ,'btainedfrom Garcinia manpostanaand a few undershrubs.The inflorescenceis typically a raceme withoutbracts.The flowersareof thetype,K2 +2,C2 +2, A2 + 4, SARRACENIATES G(2). The stamensare tetradynamousand the ovary is divided into two loculi by a replum uniting the two parietalplacentas.The fruit Sorrqcenioceoe, Nepenfhoceoe, Droseroceoe is called a siliqua when elongated,as in the wallflower and mus.::c threesmall families of insectivorousplantsforming this order are tards; or silicula when almost as broad as long, as in shepherd's : nrinorpharmaceuticalinterest. purse and horseradish.The testas of the seeds often contain The Sarraceniaceae consistsof three generaand 17 speciesof pitmucilage. Genera include Brassica (about 30 spp.), Slnapis (10 -:'.1'r-plants. The Nepenthaceaehas two genera and 68 species,of spp.),Nasturtium(6 spp.),Lepidium (150 spp.),Hesperis(30 spp.), .,.:ich 67 belong to Nepenthes.Intheseplants,which occur mainly in Cheiranthus (10 spp.), Isatis (45 spp.), Erysimum (100 spp.), ::',pical Asia, the leaves are modified into pitchers, which attract Crambe(25 spp.) andLunariu (3 spp.).CultivatedBrassicaspecies ::.c'ctSby their colour and honey-like secretion.The Droseraceaehas include: B. nigra (black mustard); B. oleracea (cabbage,cauli: ,ur seneraand 105 species.Ofthese, 100 belongto Drosera,which is flower, broccoli, etc.); B. campestris,turnip; and B. napus,rape or -:nresentedin Britain by three sundews.In thesecommon bog-plants colza oil. Sinapis alba yields white mustard Nasturtium officinale, ::r!' leaves are covered with glandular hairs which trap and digest water cress;LepidiLLmsativum, garden cress;Isatis tinctoria, Ihe :r'ects.The Europeansundew,Drosera rotundifolia, has long been dyestuff woad, made by grinding and fermenting the leaves(for the ::rployed in folk medicine and has been included in some pharmasignificance of indoxyl derivatives in this connection see T. -,poeias (BHP 1983).In Italy it is an ingredientof a liqueur.It contains Kokubun et al., Phl,"tochemistry 1998,49, 19); Crambe maritimcL, ::rcnaphthoquinone plumbagone,which has antimicrobialactivity. the seakale. Many membersof the Cruciferaecontain mustard-oil glycosidesand in specialmyrosin cells containthe enzymesnecessary for their hydrolysis.Cardiacglycosidesoccur in some genera PAPAVERALES and the seedsusuallycontainmucilageand fixed oil. Popoveroceoe/ Fumqrioceoe, Cqppqroceoe, Cruciferqe ROSALES \n order of sevenfamilies, if the Fumariaceaeis separatedfiom the i'.ipaveraceae. The Papaveraceae belongsto the suborderPapaverineae Homomelidoceqe, Crqssuloceqe, Soxifrogqceqe, .,rJ the Capparaceae and Cruciferaeto the Capparineae. Someworkers Rosoceoe, Leguminosoe, Kromerioceoe :lgard the Papaveraceaeas related to the Ranunculalesand the The order Rosalesconsistsof l9 families divided into four suborders. t'.ipparineaeas derived from the Cistales.Chemical support for this The iamilies to be consideredfall into the subordersas follows: . rcri is that alkaloids of the Papaveraceae are relatedto those of the Hamamelidineae (Hamamelidaceae);Saxifragineae (Crassulaceae, R.rnunculaceae,and that thiogluconates are absent from the Saxifragaceae and Pittosporaceae); Rosineae (Rosaceae); i'rpaveraceaebut presentin the other two families. Leguminosineae (Leguminosae and Krameriaceae).The flowers are
>r-.
E
lm
OF DRUGS THEPLANTAND ANIMAI KINGDOMSAS SOURCES usuallyhermaphrodite(rarelybisexualby abortion:e.g.koussoflowers from Bral,era');hypogynous.perigynousor epigynous.The sepalsand petals are usually free; stamensand carpelsfree or united.
|,,
F l.' t"
A family of 26 genen and 106 speciesof trees 1. Hamamelidaceae. and shrubs,chiefly subtropical.GeneraincludeHamamelis(6 spp.) and Liquidambar (6 spp.). Drugs (q.v.) include hamamelisleaves (Hamamelis virginiana), Levant storax (Liquidambar orientalis) and American storaxor sweetgum (L. s6raciflua). The family contains tannins, balsamic resins,phenolic acids and cyclitols; alkaloids are absent. 2. Crassulaceae. A family of 35 generaand I 500 species;many perennial xerophytes. Genera include Sedum (abort 600 spp.), Sempervivum(25 spp.) and Crassula(about300 spp.).An interesting chemical character of the family is the presence,often in large amounts,of isocitric acid;it was in this xerophyticlamily that a distinctive build-up of malic acid during the hoursof darknesswas first acid metabolism(CAM) given to noticedand the term crassulacean this particularadaptationof the photosyntheticcycle (seeChapter 19). The carbohydrate sedoheptulose occurs in both the and the Saxifragaceae. Cyanogeneticglycosidesand Crassulaceae cardiacglycosidesoccur in some species;tanninsare common but alkaloidsrare.Speciesof Sedumhavebeenusedmedicinallybut the family containsno importantdrugs. is a family of 30 generaand about 580 species; 3. The Saxifragaceae chiefly north temperateherbs.GeneraincludeSaxiJrage(370 spp.), AstiLbe and Ribes. The latter, sometimes separatedas the family includes such well-known fruits as the blackcurGrossulariaceae, rant, redcunant and gooseberry.These are rich in citric and malic acidsand in ascorbicacid.Black Cunant SyrLrpis usedmedicinally. containtanninsand saponins, Somemembersof the Saxifragaceae but alkaloidsare rare. 4. Rosaceae.A family which includes about 100 genera and 2000 speciesof herbs, shrubs and trees. The leaves are simple (e.g. Pntnus) or compound(e.g.Rosa).Considerablevariety existsin the flowers and fruits. There are no anatomicalfeaturescharacteristicof the family as a whole, and the various subfamiliesfrequentlyshow differences in stomatal arangement, origin of cork, etc. Genera (3 spp.),Malus include Spiraea(100 spp.),QuiLlaja(3 spp.), P1'"rus (35 spp.), .Sorbls (100 spp.), Kerria (1 sp.), Rubus (250 spp.), Potentilla (500 spp.), Geum (40 spp.), Alchemilla (250 spp.), Agrimonia (15 spp.),Poterium (25 spp.),Rosa (250 spp.),Prunus, including Laurocerasus (430 spp.) and Crataegus (200 spp.). Constituentsof the Rosaceaeinclude cyanogeneticglycosides, saponins,tannins, seed fats, sugar alcohols, cyclitols, terpenoids and mucilage; alkaloids and coumarins are rare. The secondary metabolites of the family have been the basis for a number of chemosystematicstudies(for such studiesinvolving hydrolysable tannins see T. Okuda et aL., Phytochemistry, 1992, 31, 3091). Impoftant products (q.v.) are oil of rose (Rosa damascena),rose hips (R. canina), wild cherry bark (Prunus serotina), almond oil hawthorn (Pruntrsamygdalus),quillaia bark (Quillaia saponaricL) (Crataegus oxycanthoides).Other products are cherry-laurel leaves (Prwttts laurocerasus'),quince seeds (Pyrus c1'donlc), prunes (Prwtus domestica),raspbery fruits and leaves(RaDasidaeus)and morello cherries(Prunuscerasus). 5. Leguminosae.This is the secondlargestfamily of flowering plants and contains600 generaand about l2 000 species.It includesmore important drugs than any other family. It is divided into three subfamilies-the Papilionaceae, the Mimosoideae and the containing,respectively,about 377, 40 and 133 Caesalpinoideae, genera.Importantcharactersand generain eachsubfamily are:
(1) Papilionaceae:Herbs, shrubsor trees; leaves simple or compound; flowers zygomorphic and papilionaceous(e.g. in broom); stamens 10, monadelphousor diadelphous; fruit a legume.GeneraincludeMyroxylon (2 spp.),Sophora(50 spp.), Crotalaria (550 spp.), Lupinus (200 spp.), C.vtisus(25-30 spp.), Ononis (75 spp.), Medicago (100 spp.), Melilotus (25 spp.'),Trfolium (300 spp.), Psoralea (130 spp.), IndigoJera (700 spp.),Astragalus(2000 spp.),Vicia (150 spp.),Lens (10 spp.), Lathyrus (130 spp.), Pisum (.6 spp.),Abrus (12 spp.), (10 spp.),Ery-thrina(100 spp.),Mucuna (120 spp.), GL1-cine P haseolus (200-240 spp.),A r ac his ( I 5 spp.), Tri g onell a (abotr 100 spp.), Butea (30 spp.), Derris (80 spp.), Lonchocarpus (150 spp.), Copaifera (25 spp.) and Er1-throphleum(17 spp.). Drugs (q.v.) from this subfamily are fenugreekseeds,calabar bean,tonco seed,liquorice root, denis, lonchocarpus,Tolu balsam, Pem balsam, arachis oil and tragacanthgum. Common British plants with poisonous properties include broom (C,yrisusscoparius),laburnum (Cytisus laburnum) and many speciesof Lupinus. Among economicproductsother than drugs may be mentionedsunnhemp (.Crotalariajuncea),lentils (lens esculents),peas(Pisun sotivum),soya bean (Glycine hispida), scarlet runner, French and Lima beans (Phaseolus spp.), groundnut (Arachis hypogaea) and copaiba oleoresin and copals (Copctfera spp.). Species of Indigofera, including L tinctoria, are a source of indigo (for biosynthesisof indigo see Z.-Q. Xia and M. H. Zenk, Phytochemistry, 1992, 31,
269s). (2) Mimosoideae: Most members of this subfamiiy are trees or shrubs;leavesusually bipinnate;flowers regular;calyx usually gamosepalous; stamensequalin numberto the petalsor twice as numerous; fruit a legume. Important genera are Mimosa (450-500 spp.) and Acacia (150-800 spp.). Productsinclude acaciagumsfromAcacia spp.,wattlebarksusedin tanningfrom Acacia spp.and volatile oil suchas oil of cassie(A.Jarnesiana), which are usedin perfumery. (3) Caesalpinoideae: Membersaretreesor shrubs;leavespinnateor bipinnate;flowers zygomorphic;typical floral formula K5, C5, A 5 + 5, G L Drugs derived from this subfamily are sennaleaves and pods,cassiapodsand tamarinds.Otherproductsarethe redwoods logwood, from HaematoxyLon,and sappanwood, from Caesalpinia;sassybark, from Ery-throphleumguineense,and carobbeansfrom Ceratoniasiliqua. Of the threesubfamilies,the Mimosoideaeand Caesalpinoideae are mostly tropicalplants,while the Papilionaceaeoccur in both tropical and temperate regions. Tannin sacs are common, The conparticularlyin the Mimosoideaeand Caesalpinoideae. stituentsof the Leguminosaeshow many similaritiesto thoseof the Rosaceae.Both contain cyanogeneticglycosides,saponins, tannins, mucilage and anthocyanins.Alkaloids, however, are common in the Leguminosaebut rarein the Rosaceae.For a discussionofthe chemotaxonomicrelevanceof seedpolysaccharides and flavonoids in the family see R. Hegnauerand R. J. Grayer-Barkmerjer,Phyrochem., 1993, 34, 3. An important work of referencefor the family is Phytochemical Dictionary of the Leguminosae,Vols I and 2 (1994), compiled by I. W. Southonand publishedby Chapmanand Hall, London. (4) Krameriaceae: This, often includedin the Leguminosae(q.v.),is a family of one genus, Krameria, and 20 species.They are shrubs or herbs found liom the southern USA to the Argentine. but two of the petals The flowers resemblethe Caesalpinoideae, are modified into glands and there are only three stamens.They containphlobatannins;see'Rhatany'.
\ '*-a
-'J^'-irlli'-
.* ;-
::rt6#
A TAXONOMICAPPROACH TO THESTUDYOF MEDICINALPLANTS AND ANIMAL-DERIVED DRUGS
GERANIATES Geronioceo e, Zygophyllqceqe, Linoceoe, Eryf hroxyloceoe, Euphorbiqceoe . ::l order Geranialesconsistsof nine families. fhe Geraniaceaeconsistsof five generaand 750 species;mostly herbs.Members of the two large generalGeranium (2100spp.) and Pelurgonium(250 spp.) are popularly called 'geraniums'and the ;orrrmercialrose geraniumoil is obtainedfroma Pelargoniunt.The tamily containsvolatile oils which are widely used in perfumery: nroreresearchis neededon other constituents. - The Zygophyllaceaeis a family of 25 genera and 240 species; nrostlywoody perennials,tropical and subtropical.Generainclude (100 spp.), Tribulus (20 spp.), Guaiacum (6 spp.), 1,tgoph1:lLum Peganum(5 or 6 spp.) and Balanites(25 spp.).Peganumharmala .rtd Tribulusterrestrisare usedin Indian medicinelthe alkaloidsof the tbrmer (harmine, harmaline and derivatives)have been proJucedin hairy root cuituresand the saponinsof the latterhavebeen r'\tensively studied(seeY.-X. Xl et aL.,Phltochemistr,r1998, 49, 199). Some memberscontain alkaloids, steroidalsaponinsor lignans.For drugssee'GuaiacumResin'. : The Linaceaeis a lamily of l9 generaand about290 species;mostlr herbs or shrubs.About 230 of the speciesbelong to Linum. Constituentsof the family include cyanogeneticglycosides,fixed oils. mucilages,diterpenesand triterpenes.Flax and linseedand its ,ril (q.v.)are obtainedfrom Linum Ltsitatissimum. : The Erythroxylaceaeis closely relatedto the Linaceaeand comprises .ome thtee to four generawith E r!-thro.r-rLum pre-eminent(about 200 .pp). The other small genera including Nectaropetalwn are repre\entedby about l0 species.Ery-throxy-lunt spp.aredistributedwidely throughouttropical and subtropicalregions with large areasof diver'iflcation in South America and Madagascar.Alkaloids have been tirundin most speciesanalysedby modernmethods;theseareprincipally estersof the tropanetype togetherwith biosyntheticallyrelated hasessuch as hygrine, hygroline and cuscohygrine.Nicotine is of linrited occurrence,cocaine and cinnamoylcocaineappear to be restrictedto the cultivatedspeciesE. coca and E. novogranotense. The small treesor shrubsproducehard woods which constitutea rich \ource of diterpenoidcompounds.Quinonesand saponinsprobably contributeto the colour and durability of the wood. Phenoliccon\tituents in the form of aromatic acids such as cafteic and chlorogenicacids,and flavonoidsmay havechemosystematic impoftance. ' The Euphorbiaceaeis a large family of about 300 generaand 6000 or ntore species.Most members are treesor shrubs,a few herbs. Some renera (e.g. Euphorbia) are xerophytic. The flowers are unisexual rnd regularwith usually five perianthleaves;stamensmay be numerous and are free or united.The superiorovary ofthree calpels forms a trilocular capsule.Genera include Euphorbia (about 2000 spp.), Phyllanthus(about 550 spp.) Mallotus (2 spp.), Ricinus (1 sp.), Croton (150 spp.),Hevea (12 spp.),Jatropha (175 spp.),Manihot t 170spp.),Sapium(120 spp.),Poranthera(10 spp.),Securinega(25 .pp.),Aleurite,s(2 spp.)andHippomane(5 spp.).In additionto castor reedsand castoroil (q.v.), productsinclude the dye and taenicide kamala (from Mallotus philippinensis'); rubber, frorn species of Hevea, Manihot and Sapium; manihot or cassava starch, from llunihot esculentus(rzrllis);Chinesetallow, a fat, from Sapiumsebilerum, and a drying otl, from Aleurites moLuccana.Phyllanthus is a ri idespreadtropical genusand has beenmuch employed in traditional medicines. Minor drugs are croton oii, from Croton tigliunt, and cascarillabark, from C. cascariLlaand C. eleuteria.In somecasesthe lirtex is poisonousor irritant (e.g. that of Hippomane), and the resinous product 'euphorbium' was formerly much used as an
ts
antifbulingagentfor ships'hulls.Many speciescontainirritantor piscicidal substances.Some members such as cassavamay contain cyanogeneticglycosides.This plant, Manihot esculentus,occursin two varieties,sweetand bitter, only the latter yielding prussicacid. In addition to the constituentsalready mentioned, some members contain anthraquinones,triterpenoids, fatty acid epoxides, unsaturated fatty acids and antitumour agents.Alkaloids, when present,areusually of the aporphine,pyridine, indole, quinoline or tropanetypes. Latices from Euphorbitt specieshave widely varying biochemical propefiies with respectto their protein and carbohydrateproperties, the type and amountsof enzymespresent,and their haemagglutinar ing activities.For a review of the chemistry,taxonomy,and economic botanyofthe family, seeTheEuphorbiales,S. L. Jury,T. Reynolds, andD. F. Cutler (eds)( 1987),London,UK: AcademicPressandfor a review (293 refs) of the phytoconstituentsof Euphorbia spp. seeA. K. Singlaand K. Pathak,Fitoterpia 1990,61,483.
RUTATES Rutoceoe, Simorouboceoe, Burserqceoe, Melioceoe, Molpighioceqe, Polygoloceoe This order is closely allied to the Geraniales.The chief differenceis that in the Rutales the plants are mainly shrubs or trees. The flowers havea disc betweenthe androeciumand the gynaecium.Oil glandsare of generaloccurrence.The order contains 12 families and is divided into three suborders.Of the families mentionedabove,the Rutaceae, Simaroubaceae, Burseraceae andMeliaceaeall belongto the samesuborder. the Rutineae. l. Rutaceae.The family consistsof about 150 generaand 900 species; mainly shrubsand trees;distributedin both temperateand tropical countries,but particularlyabundantin SouthAfrica and Australia. Oil giandsare presentin the leavesand other parts.The flowers are usuallyin cymeswith 4-5 sepals,4-5 petals,8 or l0 stamensand a superiorovary.The fruits areof varioustypes,but in the orangesubfamily, the Aurantioideae,it is a hesperidium.Fruits include orange and lemon (q.v.), lime (.Citruslimetttt and C. medica var. acidtt), ciIron, bergamot, shaddock and grapefruit. There are 12 different speciesof Citrus. Other generainclude Zanthoxylum(20-30 spp.), Fagara (250 spp.), Choisya (6 spp.),Ruta (7 spp.),Dictamnus (6 spp.),Diosma (15 spp.),Galipea (13 spp.). Cttsparia (Angostura) (30 spp.), Ptelea (3 spp.), Toddalia (l sp.), Skimmia (7-8 spp.), Limonia(1 sp.),Aegle(3 spp.),Moniera (2 spp..),Haplophyllum(70 spp.),Teclea(30 spp.),Esenbeckia(38 spp.)andMurra,-a (12 spp.). Productsnot mentionedabove are buchu leaves (.Barosmaspp.); jaborandi leavesand their alkaloid, pilocarpine(Piktcarpus spp.); Japan pepper (Zantfutx,-lumpiperitum); elephantapple (Limonin acidi ssima); Braziiian angostura(Esenbeckiafeb rifu,ga);and angostura or cusparia (Galipea fficinalis). Speciesof Haplophtllum, Evodia, CLausena,Phellodendron, and ZanthoxyLumhave all been used in traditionai medicine. Bael fruits (Aegle marmelos)are an impofiant Ayurvedic medicine;they containcoumarins,flavonoids (rutin and marmesin)and a glucosylatedpropelargonidincontaining up to five units of pelargonidinwith immunomodulatoryactivity (A. M. Abeysekeraet al., Fitoterapia 1996,67,367). Prickly Ash bark ol the BHP is derived from Zanthoxylumclaya-hercalis;among other constituentsit containsalkaloids e.g. chelerythrineand nitidine, a lignan-asarininand an n-isobutyl polyeneamide.It is used as an antirheumatic.The bis-indolealkaloid yeuhchukenefrom Murraya panicuLatahas been studiedfor its anti-implantation,contraceptive activity; for informationon the uses,activity and phytochemistryof this species,togetherwith a report on the isolation of cinnamates
tu
T H EP L A NA T N DA N I M A LK I N G D O MA SSS O U R C EOSF D R U G S and coumarins from the leaves, see Atta-ur-Rahman et al., P hytochemist ry 1997,44,683. Constituentsof the Rutaceaeinclude a wide variety of alkaloids, volatile oils, rhamno-gh,rcosides, coumarins and terpenoids.Alkaioids include alkaloidal amines, imidazole, indole, isoquinoline,pyridine, pyruolidine,quinazoline and quinoline types.Many of the fruits are rich in citric and other acidsand in vitamin C. A tamily of 20 generaand about I 20 species;trop2, Simaroubaceae. ical and subtropical shrubs and trees. Members differ from the Rutaceaein not containingoil glands.Bitter principlesiue a characteristic of the family. Genera include Quassia (Simarouba) (40 spp.),Picrasma (Aeschrion')(6 spp.),Brucea (10 spp.),Souktmeu (10 spp.),Ailanthus (10 spp.), andPerriera (l sp.).Quassiawoods (q.v.) are used as bitters.Ailanthus glanduLosa,Tree of Heaven.is widely cultivated and its leaveshave been used to adulteratebelladonnaand mint. For a discussionof the quassinoidsof the lamily 'Quassia'. see Burseraceae. A family of about 16 generaand 500 species:tropical in north-eastAfrica, Arabia shrubsand trees.Many representatives and tropical America. The leaves, like the Rutaceae,are glanddotted.Oleoresincanalsare found in the phloem and sometimesin the pith. Genera include Commiphora (185 spp.), Bonvellia (24 spp.),Bursera (80 spp.), and Canaritutt(75 spp.).The best-known product of the family is myrrh (q.v.). Other productsare frankincense (Boswellia spp.), American elemi (.Bursera gummifer), Manila elemi and Javaalmond from Canariumluzonicum. Meliaceae.A family of 50 generaand about 1400 species:treesor shrubs.Someyield timber (e.g.mahoganyfromSwieteniamahagonl); others, seed oils. Generainclude Cedrela, Swietenia,Khat'tt, Carapa, Melia and Azadirachta. Azadirachta lndlczz(Neem) is an important Indian medicinal plant (q.v.); bark, leavesand seedsare used. Significant constituentsof the family are triterpenoidsand limonoids. 5. Malpighiaceae.A family of 60 generaand 800 species;shrubsor small trees.GeneraincludeMalpighia (35 spp.) andBanisteriopsis. Some membershave stinging hatrs.Banisteriop.rlscontainsindole alkaloids,and plantsmay be hallucinogenic. A family of 12 generaand about800 species,of which 6. Polygalaceae. in Britain by some600 speciesbelongto PoLvgala.Itis represented the milkwort, Po$gala vulgaris. Other genera are Mrtnnina, Securidaca and Carpoktbia. Senegaroot (q.v.) is obtained from the North American Polygala senega. Characteristic constituents are triterpenoid saponinsand,in Po\tgala, the sugaralcohol polygalitol. Methyl salicylateis commonto a numberof genera
speciesof Rftls yield Japaneselacquerand the fat knorvn as Japan wax. Mastic resin containstriterpenoidacidsand alcohols. Aceraceae.A family of trees and shrubs.In the genusAcer (100 spp.)is Acer .rrz ccharttm,which yields maple su-9ar. A tamily of about150generaand2000 species:tropical 3 . Sapindaceae. and subtropical.Generainclude Paullinia (180 spp.),Supindus(13 spp.),Curdiospermum(12 spp.'),Eriocoelunt,Blighia andRadlkoferu. The seedsof PuLilliniacupanLtaremade into a pasteand dried to form guarana;this contains cafl-eineand is used as a beverage.Sapindus saponoriahasbeenusedin Brazil and India asa soapand for the treat(q.v.) it containssaponinswith hederagenin ment of severaldiseases; as an aglycone.Constituentsof the family include saponins.cyanogeneticglycosides.cyclitols(e.g.shikimicacid);the seedfats contain a high proporlion of oleic acid.Alkaloids have beenleported in a f'ew species,includingcaffeineandtheobrominein Paulliia. A small family of only two genera and 15 4 . Hippocastanaceae. species;tropical treesand shrubsof southernAfrica. Aesculus(13 spp.) containsthe horse chestnut,A. hippocustanum.Its seedfat, like the Sapindaceae, containsa high proportion,about65-'707o,of oleic acid. Severalof the speciesexaminedcontainphenolicacids, coumarins,cyclitols and saponins;alkaloidsappearto be absent.
CETASTRATES Aquifolioceoe, Celqsfrqceqe, Buxoceqe An order of 13 families;treesand shrubswith simple leaves.
1. Aquifbliaceae.The holly family consistsof two generaand about 400 species,all but one of which belongto the genusl1er.Members are trees and shrubsfound in the temperateand tropical regions. Matd or Paraguaytea is obtained from lle.r paraguariensis and other productis cassina,the leavesof A similarcaffeine-containing species. Ilex cassine. Constituents reporled in the family include caffeine. theobromine,cyclitols (shikimic acid and inositol), triterpenesand triterpenoid saponins. 2. Celastraceae. A family of 55 generaand 850 species:tropical and temperatetrees and shrubs.Generainclude Euont'rltts (176 spp.). Celastrus(39 spp.), Cassine (40 spp.), MuytenLts(225 spp.;. Prionstemrna, Catha, Tripterl,giunt and Peripterygitt. Catha edulis yields the leavesknown as khat or Abyssiniantea:thesecontainthe The root bark of Euorrymus alkaloid cathine (norpseudoephedrine). containscardioactiveglycosidesand is includedin the atropLrrpureLts 811P.Constituentsof the family includealkaloidalamines,alkaloids of the pyridine and purine types,sugaralcohols(dulcitol),saponins, cardenolides,terpenoidsand substanceshaving antitumor.rractivity. 3. Buxaceae.A family of five generaand 100 speciesof tropical and temperate,usually evergreen,shrubs.The generaareBa-rrls(70 spp.), SAPINDATES and Notoburus (7 spp.), Sarcocolla (16-20 spp.), Pachv-,candra Simmondsia.Much researchhas beendone on the steroidalalkaloids Anqcqrdioceqe, Aceroceoe, Sopindqceqe, of the family, includingthe genusBrnus, somespeciesof which find Hippocostonqceqe usein traditionalmedicine;seeAtta-ur-Rahmanet aL.,J. Nat. Prod.. The Sapindalesconsistof 10 families. 1991, 60,976 fbr a study of '8. longifolia. Other constituentsinclude Afamily of60 generaand some600 species;main1. Anacardiaceae. phenolic acids and waxes. The seedsof ,Slnmondsittchinensisyreld ly tropical trees and shrubs.Generainclude MangiJern (40 spp.), the Iiquid wax, jojoba wax, which consistsof straightchain estersof Anacardium (15 spp.), Rhus (250 spp.), Pislacia (10 spp.), 20 1, 22:1,24:I fattyacids(q.v.)andalcohols. Toxicodendntn(15 spp.), Lannea (70 spp.), Cotinus (3 spp.) and Schinus(30 spp.).Productsinclude the mango,Mang(bra indica; cashewnut,Anacardiumoccidentale;sumacleaves,usedin dyeing RHAMNALES and tanning, Rhus coriaria; the Japanesewax tree, Thus sucRhqmnoceqe, Viloceoe cedanea; mastic resin, Pistacia lentiscus; and pistachio nuts, The order containsthreelamilies. Pistacia vera. The family containsdyeing and tanning materials, l. Rhamnaceae.A farnily of 59 genera and about 900 species: and phenolic compoundssome of which causedermatitis(e.g. the cosmopolitan,usually trees or shrubs. Genera include Rhttntntts vesicantconstituentsof the poison ivy. Rhustoxicodendron).Other
J
r-:
--
*.+--31513.1.;:
:'*,;*
--5i)bF
A TAXONOMICAPPROACH TO THESTUDYOF MEDICINAL AND ANIMALDERIVED PLANTS DRUGS I I {) spp.),Zizyphus1I00 spp.),Scutia(9 spp.),Discaria(I0 spp.), (-rtl11p17,r1,rr, (17 spp.),Maesopsisand Hot,enia.British speciesare :he alder buckthorn,Rlumuutsalnus, and the buckthorn,Rhamnu.s arlturtit'ct.These,like R. purshiana,which producescascarabark, .()rrtainquinones.The edible fruts of Zizipltus.jujubct are known as Frenchjujubes and are used in traditional Chinesemedicine as a rlild sedative.The constituentsof the family include purgative .luinones (anthraquinones.anthranols and their glycosides).A1kaloidal peptidesoccur in some genera;also terpenoidsand triterpL'noidsaponins. - \ itaceae.The family contains about 65 spp. of Viri.s(vines). V. . iti.fertL producesgrapes,wine, raisinsand cunants.Therehas been ionsiderablerecentinterestin the beneficialefl-ectofred wine arisrng tiom its antioxidant properties with respect to low-density lipoprotein and protection against atherosclerosisand coronary heart disease;see E. N. Frankel and A. S. Meyer, Pharm. Biol., 1998.36(suppl.),14.The compoundsinvolvedincludeflavonoids, arrthocyanins,flavonols and phenolic acids. For a review of Vitis vitti.t'era with 108references.seeE. Bombardelli and P Morazzoni, f i t r t t c t " P1i .9l 9 56. 6 . 2 9 ' .
gum come from Sterculiaurens.As in the Malvaceae.mucilageis common;pr.rrinebasesoccur in Theobromaand Cola.
THYMELAEATES Thymeloeoceqe, Elqeqgnqceoe An order of five families. l. Thymelaeaceae.A family of 90 genera and 500 species;mostly temperateand tropical shrubs.Generainclude Gnidia (lO0 spp.), Daphne (70 spp.) and Pimelett(80 spp.).Some speciesof Daphne arepoisonousand containvesicantresins.The bark of Daphneme1ereum was formerly official; the floral fragranceis composedof about 957c s-(+)-linalool.Mucilage and coumarinscommon in the f a m i l l: a l k a l o i d a sbrent. 2. Elaeagnaceae.A family of three genera and about 50 species, inciuding Hippophae(3 spp.),Elaeagnus(45 spp.) andShepherdia (3 spp.).Amongthe constituentsare indole alkaloidsand cyclitols.
VIOLATES AAALVAIES Eloeocorpoceqe, Tilioceoe, Mqlvqceqe, Bombocqceqe, Sierculiqceqe \n order of sevenfamilies. Herbs, shrubsor trees;tropical and tem:'.irate.Many speciescontainmucilagelalkaloidsal'erare.
1,
FF
Elaeocarpaceae. A family of 12 generaand 350 speciesof tropical and subtropicaltrees and shrubs.Chief generaElaeocarpus(200 :pp.) and Sloanea (120 spp.). Indolizidine alkaloids occur in Elueocarpus. I Tiliaceae.A family of 50 generaand some450 species;usuallytrees crrshrubs.Generainclude Corchorus( 100 spp.) and Tilia (50 spp.). Jute flbre is obtained from Corcltoruscapsulari.sand C. olitorius. Lime Tree Flower BHP, BP 2000 is the dried inllorescencesof Tilia plary'ph.t'll.os or T. cordata and is used for treatingrespirttory tract infectionsand as a nervine and tonic: constituentsinclude volatile oil (composedof over 90 cornponents),flavonoids and phenolic acids.The American lime yields phloem fibres, usedby gardeners under the name of 'bass'. Cardiac glycosidesare reported from Corchorus'.alkaloidsare absent. i Malvaceae.The family contains75 generaand about 1000 species of herbs,shrubsand trees;tropical and temperate.Generainclude ,llalva (.40 spp.), Goss1pium (20-41 spp., authorities differ), Hibist:us (300 spp.), Althaett (12 spp.), Pavonia (200 spp.) and Thespesia(15 spp.).The cottons,speciesof Gossl-pium,are imponant both ior their seedhairs and seedoil (q.v.).Marshmallow root, lromAlthaea o.fficinali.s, is usedas a demulcent(q.v.).The common hollyhock is Althaea rosea.Few specieshave been studiedchemically, but saponins,tannins.leucoanthocyanins and phenolicacids occur;typical alkaloidsappearto be absent. I. Bombacaceae. A small family of about20 generaand 180 speciesof tropical trees. Genera include Bombax (8 spp.), Ceiba and .\tlunsonia. Kapok, which consists of the lignified, silky hairs n hich line the fruits of various speciesof Bombax and Ceiba, has beenusedfor life-beltsand as a stuffing material. i Sterculiaceae. A tamily of 60 generaand 700 species;mainly tropical. Genera include Sterc'ulio(300 spp.), Theobroma(30 spp.), Cola (125 spp.) and Brachtchiton. Cocoa, oil of theobromaand chocolateare preparedfrom Theobromococao; kola or cola nuts corne from Cola vera and C. acuminata; and sterculiaor karaya
Flqcourtioceoe, Violoceqe, Turneroceqe, Pqssiflorqceqe, Cistoceqe, Bixoceoe, Tomoricoceoe, Coricqceqe An orderof 20 famiiies.It includesherbs,shrubsand trees;tropicaland temperate.Cyanogeneticglycosidesoccur in someof the families (e.g. Flacourtiaceae. Turneraceaeand Passifloraceae), but are absentfrom others(e.g.Violaceae). l. Flacourtiaceae.A family of 93 genera and over i000 species. Generainclude Ervthrospermurz(6 spp.),Htdnocarptrs (40 spp.), Flacourtia (15 spp.) and Homolium (200 spp.).The seedoils have beenparticularlystudied,as speciesof H y-dnocarpus containcyclic, unsaturatedacids which are bactericidaltowards the micrococcus of leprosy.Other constituentsof the family include cyanogenetic glycosides,tanninsand phenolic acids.Alkaloids reportedonly in one genus,Ryctnitt. 2. Violaceae.A family of22 generaand about900 species;cosmopolitan, herbsand shrubs.Generainclude Viola (500 spp.),H v-banthus (150 spp.) and Hymenanthertt(7 spp.).The pansy (Viola tricolor'), sweetviolet (V. odoruila')and dog violet (V c'anina)have beenused medicinally.They contain volatile oil and anthocyanin,flavonoid (rutin) and carotenoid pigments. Hybanthus ipecacuanha has occuned as an adulterantof genuineipecacuanha. 3. Turneraceae.A family of seven genera and i20 speciesof trees, shrubsand herbs. Turnera has 6 1 speciesand T. diffusa is the source of damianaleaves. 4. Passifloraceae. A family of l2 generaand 600 species;tropical and warm temperate;shrubsand trees,often climbers.The main genera are Passiflora (500 spp.),Adenia (92 spp.) and Tetrapathaea,Ihe latter a singlespeciesfrom New Zealand.Someof the fruits are edible (e.g. passion fruit, from Passiflora edulis). The dried aerial pafis of P. incarnata are a popular herbal sedative;they contain flavonoids and tracesof cyanogeneticglycosides,volatile oil and harmane-typealkaloids.Pharmacologicaltestshave supportedthe traditionalusage(R. Soulimaniet al., J. Ethnopharmacologl,1991, 5 7 . 1l ) . 5. Cistaceae.A family of eight generaand 200 species.Members are herbs and shrubs,particularlyfound on chalk or sand.The genera include Helianthemum(100 spp.),Cistus (.20spp.) and Halirnium. Speciesof Clsarsyield the oleo-gum resin ladanum,used in perfumery and for embalming.
A TAXONOMICAPPROACH TO THESTUDYOF MEDICINAL PLANTS AND ANIMAT-DERIVED DRUGS
I
i l{) spp.),ZiTplns (100 spp.),Scutio(9 spp.),Discaria(10 spp.), ( olutttbrina( l7 spp.).Maesopsisand Hoveria. British speciesare the alder buckthorn.Rhamrus al.nus,and the buckthorn,Rhamnus , utlturtica.These.like R. purshiana,which producescascarabark, J()ntainquinones.The edible truts of Ziziphus.jujttbttareknown as l'rcnch jujubes and are used in traditional Chinesemedicine as a rlild sedative.The constituentsof the family include purgative quinones (anthraquinones,anthranolsand their glycosides).Alkaloidal peptidesoccur in some generaialso terpenoidsand triterpenoidsaponins. Vitaceae.The family contains about 65 spp. of Vitls (vines). V. viti.feraprodtces grapes.wine. raisinsand cunants.Therehas been considerablerecentinterestin the benellcialefTectof red wine arisin_sfrom its antioxidant properties with respect to low-density lipoprotein and protection against atherosclerosisand coronary hcart diseaselsee E. N. Frankel and A. S. Meyer, Pharm. Biol., 1998,36(suppl.),1zl.The compoundsinvolvedincludeflavonoids, anthocyanins,flavonols and phenolic acids. For a review of Vitis rittifera with 108references,seeE. Bombardelli and P. Morazzoni, L itrttsl'p1a Iq95.66. 29 | .
MAIVAIES Eloeocorpoceqe, Tiliqceqe, Molvqceoe/ Bombococeoe, Sterculiqceqe \n order of sevenfamilies. Herbs. shrubsor trees:tropical and temIrrrate.Many speciescontainmucilage;alkaloidsarerare. l. Elaeocarpaceae. A family of I 2 generaand 350 speciesof tropical and subtropicaltrees and shrubs.Chief generaElaeocarpus(200 spp.) and Sloanea (120 spp.). Indolizidine alkaloids occur in Elueocarpus. l. Tiliaceae.Afamily of 50 generaand some450 species;usuallyffees crrshrubs.Generainclude Corchorus(100 spp.) and Tilia (50 spp.). Jute fibre is obtainedfrom Corchoruscapsularis and C. olitorius. of Tilia Lime Tree Flower BHP. BP 2000 is the dried inflorescences plattphy-llos or T. cordata and is used for treating respiratory tract int'ectionsand as a nervine and tonic: constituentsinclude volatile oil (composedof over 90 components).flavonoids and phenolic acids.The American lime yields phioem fibres. used by gardeners under the name of'bass'. Cardiac glycosidesare reported from Corchorus.alkaloidsare absent. .1. Malvaceae.The family contains75 generaand about 1000 species of herbs,shrubsand trees;tropical and temperate.Generainclude Malva (.40 spp.). Gossypium (2041 spp., authorities diff'er). Hibiscus (300 spp.), Althaea (12 spp.), Pavonia (200 spp.) and Thespesia(15 spp.).The cottons,speciesof Gossypium,areimportant both for their seedhairs and seedoil (q.v.).Marshmallow root, from AlthaeaolJicinulis,is usedas a demulcent(q.v.).The common hollyhock is Althaea rosea.Few specieshave been studiedchemand phenolic acids ically, but saponins,tannins,leucoanthocyanins occur;typical alkaloidsappearto be absent. -1. Bombacaceae. A small family of about20 generaand 180 speciesof tropical trees. Genera include Bombax (8 spp.), Ceiba and Adansonia. Kapok, which consists of the lignified, silky hairs which line the fruits of various speciesof Bombax and Ceiba,has beenusedfor lif'e-beltsand as a stuffing material. 5. Sterculiaceae. A family of 60 generaand 700 species;mainly tropical. Genera include Sterculia (300 spp.), Theobroma(30 spp.), Cola (.125spp.) and Brachtchiton. Cocoa, oil of theobromaand chocolateare preparedfrom Theobromacacao; kola or cola nuts come from Cola vera and C. acuminata; and sterculiaor karaya
lF
gum come from Sterculiauren.s.As in the Malvaceae,mucilageis common;purine basesoccur in Theobromaand Cola.
THYMETAEATES Thymeloeoceqe, Eloeqgnqceoe An order of five families. l. Thymelaeaceae.A family of 90 genera and 500 species;mostly temperateand tropical shrubs.Generainclude Cnldia (100 spp.), Daphne (70 spp.) and PimeLea(80 spp.).Some speciesof Daphne arepoisonousand containvesicantresins.The bark of Daphneme7.ereum was formerly official; the floral fragranceis composedof about95% s-(+)-linalool.Mucilageand coumarinscommonin the i a m i l y :a l k i r l o i dasb s e n l . 2. Elaeagnaceae.A family of three genera and about 50 species, including Hippophae(3 spp.),Elaeagnus(45 spp.) and Shepherdia (3 spp.).Among the constituentsare indole alkaloidsand cyclitols.
vtotAtEs Flocourtioceoe, Violoceoe, Turnerqceoe, Possifloroceoe, Cistoceqe, Bixoceoe, Tomoricoceoe, Coricoceoe An orderof 20 families.It includesherbs,shrubsandtrees;tropical and temperate.Cyanogeneticglycosidesoccur in someof the families (e.g. Flacourtiaceae. Turneraceaeand Passifloraceae), but are absentfrom others(e.g.Violaceae). l. Flacourtiaceae.A family of 93 genera and over 1000 species. Generainclude Erythrospermurr(6 spp.),Hy-dnocarpus(40 spp.), Flttcourtia (15 spp.) and Homalium (200 spp.).The seedoils have beenparticularlystudied,as speciesof Htdnocarpus containcyclic, unsaturatedacids which are bactericidaltowards the micrococcus of leprosy.Other constituentsof the tamily include cyanogenetic glycosides,tanninsand phenolic acids.Alkaloids reportedonly in one genus,Ryania. 2. Violaceae.A lamily of 22 generaand about900 species;cosmopolitan, herbsand shrubs.Generainclude Viola (500 spp.'),H)tbanthus (150 spp.) and Hymenanthera(1 spp.).The pansy(Vbh tricolttr), sweetviolet (V. odorata) and dog violet (V canina) have beenused medicinally.They contain volatile oi1 and anthocyanin,flavonoid (rutin) and carotenoid pigments. Hybanthus ipecacuanha has occurredas an adulterantof genuineipecacuanha. 3. Turneraceae.A family of seven generaand 120 speciesof trees, shrubsand herbs.Turnerahas61 speciesandT.dffisa is the source of damianaleaves. A family of 12 generaand 600 species;tropical and 4. Passit'loraceae. warm temperate;shrubsand trees,often climbers.The main genera are Passiflora (500 spp.),Adenia (92 spp.) and Tetrapathaea,the latter a singlespeciesfrom New Zealand.Someof the fiuits are edible (e.g. passion fruit, from PassiJktraedulis). The dried aerial parts of P. incarnata are a popular herbal sedative;they contain flavonoids and tracesof cyanogeneticglycosides,volatile oil and harmane-typealkaloids.Pharmacologicaltestshave supportedthe traditionalusage(R. Soulimaniet aL.,J. Ethnopharmacology1997, 5/- tt) .
5. Cistaceae.A family of eight generaand 200 species.Members are herbs and shrubs,particularlyfound on chalk or sand.The genera include Helianthemum(100 spp.),Cistus (20 spp.) and Halimium. Speciesof Cls/us yield the oleo-gum resin ladanum,used in perfumeryand for embalming.
@
T H EP L A NA T N DA N I M A LK I N G D O MASS S O U R C EOSF D R U G S
-,frii;r;ri:r 6. Bixaceae.This consistsof the single genusBlra with fbur species.
i::"i"'fi
Bixa orellancLis cultivated for the colouring matter of its seeds. 'annatto',is usedasan ediblecolourant,see This, underthe nameof Chapter33. 7 . Tamaricaceae.A family of four genera and 120 species;herbs and shrubs. Tamarlx (54 spp.) includes T. mannifera, a plant which when puncturedby scaleinsectsyields the mannaof the Bedouins. 8 . Caricaceae.A family of four genera and 55 species;small trees mainly found in tropical America and Afiica. Carica contatns 45 species,and Carica papaya (papaw) is cultivated for the milky juice which is the sourceof the proteolytic enzyme papain.
CUCURBITATES Cucurbitoceqe An order containing the single family Cucurbitaceae. A family of about 110 generaand 640 species;abundantin the tropics; mostly herbs climbing by tendrils, with abundantsap and very rapid growth. Flowers are generally unisexual, regular and pentamerous,exceptin the gynoecium,which is reducedto three.The fruit is the fleshy type seenin the cucumber.Most membershavebicollateralvascular bundles.Generainclude Cucurbita (5 spp.),Cucumis(25 spp.), Ecballium (l sp.), Citrulfus (3 spp.),Luffa (.6 spp.),Bry-onia(4 spp.) and Momordica (45 spp.), Cucurbita pepo rs vegetable marrow; C. maxima, great pumpkin; Cucumis melo, Ihe melon; C. salivu.r, the cucumber; Ecballium elcLterium,the squirting cucumber,yields the purgative elatertum; Citrullus colocynthis yields colocynth; C. lanatus, the water melon. The vascular network of the pericarp of L.ftl is used as a bath sponge(loofah). The fruits and seedsof L. acutangula contain saponins and are used in Ayurvedic medicine. Bryony root, from Bryonia dioica, was formerly used as a purgative and for the treatment of gout; it containssaponinsand sterols.Br1-oniaalba containsantituThe enzyme elaterasehydrolysesthe bitter glucomour substances. sides of the family to cucurbitacins and glucose.The cucurbitacins are triterpenoidbitter principlesnamedA to Q, and the compoundformeris cucurbitacin-E. ly known as cx,-elaterin
MYRTIFIORAE
guajavcLgives the edible fruit guava. Constituentsof the family other than volatile oils are leucoanthocyanins,cyclitols, tannins (e.g. in eucaiyptuskinos), phenolic acids and esters.Cyanogenetic glycosidesand alkaloidsarerare. Punicaceae.The family containsthe single genusPunictt with two species.The fruit rind of Punica granatum, lhe pomegranate, containstannin; in its stem and root bark tannin is accompaniedby the liquid alkaloids pelletierine and isopelleterine.Pelletierine tannatewas formerly official. A family of 16 generaand 120 species;often trees Rhizophoraceae. of mangrove habit. Rhizophora (7 spp.) yields the tanning material mangrovecutch. Afamily of 20 generaand 600 species;tropical and 5. Combretaceae. subtropicaltreesand shrubs;usually rich in tannin.Generainclude Terminalia(250 spp.),Combretum(250 spp.),Quisqualis(17 spp.) and Anogeissus(l I spp.). Myrobalans, the fruits of Terminalia chebula,arerich in tannin and areusedboth in tanningand in medicinel Combretum butlnrosuntyields a butter-like substance; and AnogeissusLatfolia yields the gum known as ghatti gum. A family of 21 generaand 640 species;temperateand 6. Onagraceae. tropical; mostly perennialherbs,but a few shrubsand trees.Genera include Fuchsia (100 spp.),Oenothera(80 spp.).Clarkia (36 spp.) and Epilobium (215 spp.). Many are cultivated for their flowers. Seeds of Oenothera spp. have become important as sourcesof evening primrose oil (q.v.). Various speciesof Epilobium e.g. E. angustfolium and E. hirsutun are used traditionally for the ffeatment of benign prostatehyperplasia;the active componentsappear to be two macrocyclicellagitanninsoenotheinA & B (B. Ducrey e/ al., Planta Medica 1997, 63, I I 1). Tanninsand a few cyanogenetic plantsarerecorded;alkaloidsare rare or absent.
UMBELTIFTORAE Arolioceoe, Cornoceqe/ Gorryoceoe, Alongioceoe, Umbelliferoe An order of seven families. A11. except the Umbellif'erae and Araliaceae,are small. Acetylenic compoundsoccur throughout the order.
l. Alangiaceae.A family of 2 genera and 20 species,of which 17 belong to A/an gium; rropical trees and shrubs.Alkaloids and triterpenoid saponinsoccur.A. Iamarkii is an Indian medicinalplant. A family of 12 generaand 100 species;treesand shrubs, 2. Cornaceae. rarely herbs.Generainclude Cornus(4 spp.) andAcuba (3 spp.). l. Lythraceae.A famiiy of 25 generaand 550 species;herbs, shrubs and trees. Genera include Rotala (50 spp.), Lythrum (35 spp.), 3. Garryaceae.Contains only Garrya, with 18 species.Shrubs containing alkaloids. Decodon (1 sp.), Lagerstroemia(35 spp.), Heimia (3 spp.) and lawsoneoccursin henna,the 4. Araliaceae.A family of 55 generaand 700 species;mainly tropical Lawsonia( I sp.).The naphthoquinone treesand shrubs,someclimbing (e.g.ivy). GeneraincludePanax (.8 leavesof lzwsonia inermis.Somespeciescontainalkaloids. spp.), Tetrapanc.r(l sp.), Aralia (.35 spp.), Hedera (15 spp.), of ever2. Myrtaceae.A family of about I 00 generaand 3000 species Cussonia (25 spp.), Pseudopanax(6 spp.), Fatsin (l sp.) and in Australia,the EastIndies greenshrubsand trees;well represented Sciadodendron(1 sp.).The best-knowndrug, which has beenused and tropical America. The family is divided into two subfamilies, for many centuries,is ginseng,from Panax schinseng(P. ginseng). the Myrtoideae (fruit a berry or drupe) and the Leptospermoideae Tetrapanaxpapt,rferum is the ricepaper tree, and Hedera helix the include Myrtoideae (fruit a loculicidal capsule). Genera of the common ivy. Resin passagesoccur in the tamily. Constituents Myrtus (100 spp.),Psidium (140 spp.),Pimenta (18 spp.),Eugenia include saponins,a few alkaloids,acetyleniccompoundsand diter(1000 spp.),Pseudocaryophyllusand Syzygium(Jambosa).To the penoidsand tritetpenoids. Leptospermoideae belong Eucalyptus (over 500 spp.), Leptospermum(50 spp.) andMelaleuca (about I 00 spp.).The large 5. Umbeltif'erae.The family contains about 275 genera and 2850 species.Most membersare herbswith furrowed stemsand hollow genusEucalyplzs has presentedtaxonomic problems and following internodes.Some are annuals(e.g. coriander),some biennials(e.g. a recentrevision for Flora of Australia the nine, previouslyestabhemlock) and some perennials(e.g. speciesof Ferula). The three lished, subgeneraare each afforded generic status.Many of the gensubfamiliesand main generaare as follows: (1) Hydrocotyloideae cloves era provide importantvolatile oils and spices-for example, Hrdrocot,nle(100 species);(2) Saniculoideaeincludes includes pimento. Psidium and (q.v.), cajuput oil oil, its oil eucalyptus and
Punicqceoe, Rhizophorqceqe, Lythroceoe, Myrtoceoe, Onogroceoe Combretqceoe, An order of 17 families.Many membersrich in tannins.
'.'-{ll*1
..
.:5*erj
A TAXONOMICAPPROACH TO THESTUDYOF MEDICINAL PLANTS AND ANIMAT-DERIVED DRUGS EnrLgium(230spp.),Aslrantia(10 spp.)andSanicLrLa (37 spp.);(3) ApioideaeincludesChaerophtllum(40 spp.),Coriandrum(2 spp.), Stnlrnium (8 spp.),Conium (4 spp.),Bupleurum(150 spp.).Apiln ll sp.), Petrctselinum(5 spp.). Carum (30 spp.), pimpinella (.150 spp.), Se.rell(80 spp.), Foeniculum (5 spp.), Oenanthe(40 spp.), Ligtrsticum (60 spp.), Angelica (80 spp.), Ferula (133 spp.), Peucedanum(120 spp.),Pastinaca(15 spp.),Laserpitium(35 spp.), Thap.s ia (6 spp.),Dtttrcus(60 spp.),Ammi ( I 0 spp.),H eracleum (70 spp.),Prangos(30 spp.)andAnethum(l sp.).The leavesareusually largeand havea sheathingbaseandmuch-dividedlamina.The flowers are small and arrangedin simpleor compoundumbels.Each has a five-lobed calyx, five petals,five stamens,and an inferior twocelledovary.The fruit is a cremocarp,which is frequentlycrowned with a stigma-bearingdisc known as the stylopodium.When ripe, the two mericarps separatefrom one another but frequently remain attachedto the simple or forked carpophorewhich lies between them. The line separatingthe two mericarpsis known as the commissure.Each mericarp containsa single seedwhich consistsof a largeendosperm,which has a small embryo embeddedin it nearthe apex.Five primary ridgescontainingfibrovascularbundlesrun from baseto apexin the pericarp,and secondaryridges sometimesalternate with these. Between the ridges are schizogenousoleoresin canalsknown as vittae. Members of the family differ in the number and arrangementof the vittae in eachmericarp,but six is common, four on the dorsalsurfaceandtwo on the commissural.Similar ducts occur in the stemand roots and in speciesof Ferula yield the oleogum resinsand asafbetida,ammoniacumand galbanum. The main umbelliferousfruits and their volatile oils used in pharmacy are fennel, Foeniculurn vulgare; caraway, Carum carvi; dill, .\rtethum graveolens; coriander, Coriandrum sativum; aniseed, Pimpinella anisum; and cumin, Cumminumcyminum.Bupleurumfalcetum roots contain oleanenesaponinsand are an important antihepatotoxic drug in oriental medicine. Msnaga, from Ammi visnaga, Among poisonousplants l ields khellin, a dimethoxyfuranochromone. of the family may be mentionedConium maculatum,the sponedhemlock, which containsthe alkaloid coniine; and Oenanthecrocata, the hemlock waterdropwort,which contains oenanthotoxin.Other wellknown plants of the family are celery, Apium graveolersl parsley, Petroselinumcrispum; parsnip,Pastinacasativa; and carrol,Daucus cttrota. Constituentsof the family, other than volatile oils and resins, include coumarins (e.g. umbelliferone), furocoumarins,chromonocoumarins, terpenes and sesquiterpenes, triterpenoid saponins and acetyleniccompounds.Alkaloids occur (e.g.coniine)but arerare.
spp.),Lyonia (30 spp.'), Arbulls (20 spp.) andArctosraphyios(71 spp.). In addition to the well-known gardenplants, the family includes the wintergreen,Gaultheriaprocumbens,which yields natural oil of wintergreen(now generallyreplacedby syntheticmethyl salicylate);and bearberry leaves from Arctostaphl-losuva-ursi, which contain the phenolic glycosidearbutin and are again official. The medicinalproperties of Var:ciniummyrtillus (bilbeny, blueberry, whortleberry) have been utilized since the Middle Ages and have recently beenreviewed (P. Morazzoni and E. Bombardelli, Fitoterapia 1996,67, 3). The family producesphenolicacids,phenolicglycosides(e.g.arbutin),aucubin glycosides,diterpenoids(grayanotoxin),triterpenoids(ursolic acid), cyclitols and leucoanthocyanins.A few species are cyanogenetic; saponinsare absent.
PRIMUTATES Myrsinoceoe, Primuloceoe An order of threefamilies. The Primulaceaeconsistsof 20 generaand about 1000 speciesof herbaceousperennialswith rhizomesor tubers.Especiallycommon in the north temperateregions. Many members are cultivated as garden plants. Genera include Primula (500 spp.), Cyclamen (15 spp.), Anagallis (31 spp.) and Dionysia (41 spp.). The dried flowers of Primula uerls (the cowslip) are used in herbal medicinefor insomnia and as a sedativein combinationwith otherherbs.The flowers areparticularly rich in flavonoids.Saponinsare presentin somespecies;also phenolicestersand ethers.Alkaloids appearto be absent.Anthocyanin pigmentsare common,but not betacyaninsor betaxanthins.
PTUMBAGINATES Plumboginoceoe The Plumbaginaceaeis the only family of the order, and contains l9 genera and about 775 species.Members are herbs or shrubs often found on seacoastsor salt steppes.GeneraincludePlunrbago( l2 spp.) and Ceratostigna. Roots of Plumbago spp. are used in traditional Indian medicine; immunosuppressiveand antitumour activities have beendemonstrated.Constituentsfound in the family include phenolic acids, tannins, anthocyanin pigments and naphthoquinones(e.g. plumbagin).
EBENATES Sopotoceoe, Ebenoceoe, Sfyrococeoe An order of sevenfamilies consistingmainly of treesand shrubs.
SUBCTASSSYTNPETATAE The Sympetalaederive their name fiom the fact that their petals are fused.The subclassconsistsof i I ordersand 63 families. the chief of which have alreadybeentabulated.
ERICATES Ericqceoe An order of five families, including the Pyrolaceae,Epacridaceaeand Ericaceae.Only the last family, the largest,will be described. Ericaceae.A family of about80 generaand 2000 species;pafticularly common on moors and peaty soils. Members are shrubsor small trees.The generainclude Rhododendron(500-600 spp.),Ledum (lO :pp.), Erica (over 500 spp.), Calluna (l sp.). Vaccinium(300-400 (49 spp.),Gaultheria (about 210 spp.),Pieris (lO spp.). Ga,1/ussacia
l. Sapotaceae. A family of some 35-75 ill-defined generaand about 800 species;most are tropical trees, often yielding good timber. Generainclude Mimusops (57 spp.),Madhuca (Bassia)(85 spp.), Achras (70 spp.),Pierreodendron(2 spp.),Palaquium (about 115 spp.)andBu4.'rospermum(l sp.).Latex sacsoccur in the leaves,and in the coftex, phloem and pith of the stems.Gutta-perchais the coagulatedlatex from speciesof Palaquium andpayena. The oily seedsof Burl^rospermum parkii are expressedto yield sheabutter which can be usedas an ointment and creambasefor topical applications.Constituentsinclude latex, seedfats, cyanogeneticglycosides,saponins,tannins,leucoanthocyanins, pyrrolizidine alkaloids and the cyclitol D-quercitol. 2. Ebenaceae. A family of threegeneraand about500 species;tropicai treesand shrubs.The chief genera areDiospt,ros(about 500 spp.) and Euclea (20 spp.). Varieties of ebony are obtained from DiospyrosebenumandEucleapseudebenus . In South-EastAsia the
E
OF DRUGS THT PHNT AND ANIMAL KINGDOMSAS SOURCES fresh unripe fruits of D. mollis have long been used as an anthelmintic (hookworms and tapeworms). Other speciesyield edible fruits, 'date-plums'.For recentphytochemicalresearchon the root- and stem-barksof Diospyros spp. involving the isolation of triterpenoids,diquinonesand napthoquinonesseeM. R. Khan and D. Timi, Fitoterapia 1999,70, 194; 209. Naphthoquinonesare a characteristicof the family. A family of 12 generaand 180 speciesof trees and 3. Styracaceae. shrubs.Generainclude S4rrax(130 spp.) and Halensia. Benzoins (q.v.) are obtainedfrom speciesof Sry^rax. Constituentsof the family include balsamicresins,phenolicacids,tanninsand the benzofuran egonol.
OtEAtES Oleoceqe The Oleaceaeis the only family of the order.It is widely distributed, andcontains29 generaand about600 species.GeneraincludeOlea (20 spp.), Forsythia (7 spp.), FraxintLs (70 spp.), Syringa (30 spp.), Osmanthus(15 spp.), Jasminum (300 spp.) and Ligustrum (.4V50 spp.). Many speciesare grown in Britain (e.g. Syringa vulgaris, lhe iilac). The manna of present-daycommerce is obtained by making incisions in the stem of the manna ash, Fraxinus ornus. Of great economic importanceis the olive, Olea europoea.In addition to olive oil (q.v.),the family producessugaralcohols(e.g.the mannitol of manna), saponins,tannins,coumarinsand iridoid glycosides.Alkaloids arerare.
GENTIANALES Menyonthoceoe, Logonioceoe, Gentionoceoe, Asclepiodoceoe, Rubioceqe Apocynoceoe, An order of sevenfamilies which is of medical and chemicalinterest. All the families contain alkaloids,but importantglycoside-containing generaalso occur. 1. Loganiaceae.A family of 18 generaand 500 species;trees,shrubs and herbs, some lianes. Genera include Strychnos (200 spp.), Logania (25 spp.), Gelsemium (2 spp.), Geniostoma (60 spp.), Anthocleista(14 spp.) and Gardneria (5 spp.).Nux vomica seeds (q.v.),the seedsof Slrycftnos nux-vomicc,arethe principalsourceof 'Curare') owes its strychnine and brucine; calabash-curare(see activity largely to Snl,chnosspecies.The family is rich in alkaloids of the indole and oxindole groups.Other constitutentsare the aucubin glycosideloganin,and iridoids. 2. Gentianaceae.A family of 80 generaand about 1000 species;herbs and a few shrubs widely distributed. The leaves are opposite and decussate.the corolia lobes are contofied in the bud and the axis shows bicollateral bundles and interxylary phloem. Genera include Gentiana (400 spp.),Exacum (.40spp.),Sebaca(100 spp.),Erythraea (.Centaurium)(30 spp.), Chironia (30 spp.), Swertia (100 spp.), Halenia (about 100 spp.)and Enicostema(3 or 4 spp.).Gentianroot (see 'Gentian'), from Gentianalutea, has long been usedin medicine. The herb of Swertia chirata (Chvetta) is describedtnthe BHP (1983);it is usedto stimulatethe appetitein anorexia.S.japonicats used in Japan as a stomachic;hairy root cultures produce xanthone derivatives, the secoiridoids amarogentin and amaroswerin, and phenylglucosides.Some members,on account of their bitter principles, are usedin liqueurs. The family containsalkaloids; iridoid glycosides;flavones,xanthonesand their glycosides;phenolic acids; tannins; and the trisaccharidegentianose. This small family of five generaand 33 speciesis 3. Menyanthaceae. It consistsof aquatic01 sometimesincluded in the Gentianaceae.
marsh herbs such as the British planl Menyanthes trifoliata, the it containsbitter principles but buckbean.Like the Gentianaceae, the leavesinsteadofbeing oppositeand decussateare alternate. 4 . Apocynaceae.A family of about250 generaand 2000 specieswhich Many membersare woody is closely allied to the Asclepiadaceae. climbers found in the tropics and subtropics.The only British speciesare the periwinkles, Vinca major and V.minor. Types of fruit are a pair of follicles, a berry, capsulesor two indehiscentmericarps. The plants contain latex in non-articulated,branched or unbranched laticifers. The vascular bundles are bicollateral. Important genera, arrangedunder two subfamilies, are as follows: Arduina-Carissa(35 spp.),Allamanda(15 spp.), A. Plumieroideae'. Landolphia (55 spp.), Carpodinus (50 spp.), Hancornia (1 sp.), Pleiocarpa (3 spp.), Plumeria (7 spp.), Alstonia (50 spp.), Aspidosperma (80 spp.), Rha4lta (2 spp.), Amsonia (25 spp.), Lochnera-Catharanthus (5 spp.), Vinca (.5spp.), Tabernaemontana (100 spp.), Voacanga(25 spp.),Alyxia (80 spp.),Rauwolfia (100 spp.),Ochrosia (30 spp.) and Cerbera (6 spp.); B. Apocynoideae: Echites (6 spp.), Dipladenia (30 spp.), Odontadenia (30 spp.), Mandevilla (114 spp.), Baissea (40 spp.), Funtumia (3 spp.), Apoctanum (7 spp.), Nerium (3 spp.), Strophanthus(60 spp.), Wrightia (23 spp.), Parsonia (100 spp.), Lyonsia (24 spp.) and Malouetia (25 spp.).Drugs include the Madagascaranperiwinkle, Catharanthus rosers (also known as Lochnera rosea and Vinca rosea); strophanthusseeds;rauwolfia roots; kurchi or holarrhena bark; alstonia barks; and aspidospermabarks. Many speciesof Landolphitt, Carpondinus and Hancornia yield rubber latex. [For a review (148 refs) of Rhazyastricta andR. orientalis,usedin Unani medicine, seeAtta-Ur-Rahmanet al., Fitoterapia, 1989, 60,291. For uses,chemistryand pharmacologyof Malouetia spp.(41 refs) seeN. G. Bisset,/. Ethnopharm,1992,36, 43.l Constituentsof the Plumieroideaeinciude a vast rangeof indoline alkaloids; over 500 rn Alstonia, Aspidospermum,Catharanthus, H unteria, P leiocarpa, Tabernaemontana,Rauwolfia and Voacanga. Steroidal alkaloids occur in Holarrhena and harman-type alkaloids in Amsonia and Aspidosperma. Cardioactive glycosides occur rn Acokanthera, Carissa andMelodinus and in Apocltanum,Nerium andStrophanthas.Other constituentsof the family are cyanogenetic glycosides,leucoanthocyanins,saponins,tannins,coumarins,phenolic acids, cyclitols and triterpenoids.Widely grown omamental plantsof the family includespeciesof Ansonia, Nerium (oleander), Vinca, Plumeria (frangipani), Thevetia (yellow oleander) and M andevilla (Chileanj asmine). A family of 130 generaand 2000 species;tropical Asclepiadaceae. and subtropicalshrubs,often twining, or perennialherbs.Genera include Asclepias(120 spp.), Tylophora(150 spp.),Xysmalobium (l sp.), Cryptostegia (2 spp.), Cynanchum,Marsdenia, Pergularia and Hemidesmrzs.The latex cells usually contain a latex rich in triterpenes.Other constituentsinclude: alkaloids of the indole, phenanthroindolizidineand pyridine groups; cardenolides;cyanogenetic glycosides;saponins,tannins,and cyclitols. Although the family yields no important drugs, many membersare used in folk medicine in their countriesof origin and others as arrow poisons. Among the better-known are Indian sarsaparilla,from Hemidesmus indicus, and condurango bark, from Marsdenia condurango. Cryptostegia grandiflora, the rubber vine or pink allamanda, contains poisonouscardenolideswith some therapeuticpotential; the plant, introduced to Australia as an ornamental, has become an aggressiveweed. 6 . Rubiaceae.A tamily of about500 generaand 6000 species,most of which are tropical treesand shrubs.A few membersare herbs growing in temperateclimates(e.g. speciesof Galium, such as the bed-
5;;i*41
A TAXONOMICAPPROACH TO THESTUDYOF MEDICINALPLANTS AND ANIMAL-DERIVED DRUGS \traws,goosegrass or cleaversofBritain). The first subfamilyis the subcontinentis used in a number of folk remedies:an alcoholic Cinchonoideae,the carpels of which bear numerous ovules and extractofthe plant hashypotensiveand bradycardiaceffects. nhich includes Oldenlandia (300 spp.), Condaminea (3 spp.), Anatomicalcharactersincludethe presenceof latexcells,bicollaterCinchona(40 spp.),Uncaria (60 spp.),Naaclea(35 spp.),Gardenia al bundlesand frequentlyabnormalvasculardevelopmentsuchas is r250 spp.) and Mitragyna (i2 spp.). The second subfamily, found in drugssuchasipomoeaandjalap (q.v.).Brazilian arrowroot Rubioideae,has only one ovule in eachloculus; it includesthe genis the starch obtained from the tubers of the sweet potato, Ipomoea era Coffea (40 spp.), Ixora (400 spp.), P(Netta (400 spp.), batatas. Many speciescontain hallucinogens(q.v.) such as ololiPsychotria(700 spp.),Cephadlis-Uragoga(.780spp.),Mitchella (2 uqui, from Rlvea corymbosa,and morning glory seeds,from species ;pp.), Asperula (200 spp.),Galium (400 spp.) andRubia (60 spp.). of lpomoea. The family containsindole, isoquinoline,pyrrolidine Productsinclude cinchonabarksand their alkaloidssuchas quinine and tropane alkaloids,purgative resins,phenolic acids and tritert q.v.),ipecacuanha root (see'Ipecacuanha'),catechu,from Uncaria penoid saponins.Pynolizidine alkaloidshave recentlybeenreportqambier,and coffee (q.v.),from Coffeaarabica and other species. ed in the family for the first time (seedsof 1. hederifolia). Pst'chotriaviridis is a componentof the hallucinogenicpreparation 3. Boraginaceae.A family of about 100 genera and 2000 species; avahuasca(q.v); other speciessuch as P. brachypoda and P. colmostly herbs,often perennial.Inflorescencea coiled cincinnus.The orota are constituents of traditional painkillers (M. B. Leal and flowers change colour during development. Genera include E. Elisabetsky,Int. J. Pharmacognosj-1996,34,267). Of the many Heliotropium (250 spp.), Cynoglossum(50-60 spp.), Symphytum other alkaloid-containingspecies may be mentioned mitragyna (25 spp.), Borago (3 spp.), Anchusa (50 spp.), Alkanna (25-30 leaves, from species of Mitragyna, and yohimbe bark, from spp.),Pulmonaria (i0 spp.) and Lithospermr.rn(60 spp.).Products Pansinystaliayohimbe. Alkaloids of the indole,oxindole,quinoline include speciesof Lithospermum,which have hormone activity; and purine types are common; anthraquinonesoccur in Morinda, alkanna or anchusaroot, from Alkanna tinctoria. which contains Rubia and Galium, and many have long been used as dyes (e.g. red colouring matters.Both the Lithospermum spectesandAlkanna madder, from Rubia tinctorum'). Anthocyanins occur in Cinchona; contain naphthoquinone derivatives. Formerly official were aucubin glycosides (e.g. asperulin), cyclitols (e.g. quinic acid), Cynoglossumfficinale (hound's tongue),Borago fficinale (borcoumarins,depsidesin Coffea; phlobatannins,catechins(e.g. in age) and Pulmonaria fficinale (lung-wort).The seedoil of borage Uncaria); diterpenoidsand triterpenoidsand iridoid glycosidesin is particularly rich in y-linolenic acid (q.v.) and the plant is now Genipa.Morinda reticuLatais interesting,as it accumulatesselenbeing cultivatedin Englandfor its oil. Naphthoquinones, the ureide ium and is very toxic;M. citrifulia, M. eLliptica andM. Lucidaare all allantoin,pyrrolizidine alkaloids,cyclitols, phenolicacids and tanemployed in traditional medicine, some anthraquinones(e.g. nins, occur in the family. Allantoin is particularly abundant in the alizarin-l-methyl-ether)have been shown to possessantifungal root of comfrey, Symphytumfficinale. The common occurrenceof properties(G. Rath et al., Int. J. Pharmacognosl1995,33, 107). pyrrolizidine alkaloids (Alkana, Borago, Cynoglossum, Heliotropium, Symphytum)has causedconcern regarding the herbal useof a numberof species. TUBIFTORAE 4. Verbenaceae. A family of about 100generaand 3000 species;herbs, shrubs and trees, many lianes. Genera include Tectona(3 spp.), Polemoniqceqe, Convolvuloceoe, Boroginoceoe, Lippia (220 spp.),Verbena(250 spp.),Callicarpa (140 spp.),Vitex Verbenoceoe, Lobiotoe, Solonoceoe, (250 spp.),Nyctanthes,Duranta and Stilbe.Teak is obtainedfrom Scrophulorioceoe/ Bignonioceoe, Aconthoceoe, Tectonagrandis; verbena oil from Lippia citriodora; and vervain, Pedolioceoe, Gesnerioceoe, Myoporqceqe formerly official, from Verbenaofficinalis. Viter agnus-castushas .{n order of six subordersand 26 families containing many important beenusedmedicinally in Europe at least sinceclassicalGreek and drugs.The families consideredbelow fall into the following suborders: Roman times and today is still availableas a herbalpreparationfor l. Convolvulineae:Polemoniaceae and Convolvulaceae. the treatment of female conditions relating to the premenstrualsyn1. Boraginineae:Boraginaceae. drome and the menopause. (For a report of recent research see -1. Verbenineae:Verbenaceaeand Labiatae. Pharm. J. 2001,265, 106.) By GC-MS 34 compoundshave been J. Solanineae:Solanaceae, Scrophulariaceae, Bignoniaceae, identified as componentsof the volatile oil, principaliy mono- and (Zeljan MaleSet al., Planta Medica 1998, 64,286). Acanthaceae, Pedaliaceae and Gesneriaceae. sesquiterpenes 5. Myopineae:Myoporaceae. Labdan diterpenes appear to be partialiy responsible for the dopaminergic activity of the fruits (E. Hoberg et al., Planta Medica Membersof thesefamilies are usuallyherbswith alternateor oppos2000, 66, 352). For an article on the drug seeP Houghton,Pharm. ite simple leaves.The flowers are generallybisexualand often zygoJ., 1994,253,'720.Among the constituentsfound in the family are morphic. They usually have two or four epipetalousstamensand a volatile oils, saponins,tannins,quinones,iridoids, piscicidal subbicarpellary superior ovary. stances;alkaloids appearto be rare. I . Polemoniaceae.A family of I 5 generaand 300 species;herbs which 5. Labiatae (Lamiaceae).A family of about 200 genera and 3300 are often cultivated as border plants. Generainclude PhLox(67 spp.) species;aromatic,annual or perennialherbs or undershrubs.The andPolemoniun (50 spp.).The chemistry of the family is imperfect family is well representedin the Mediterranean area and in Britain. ly known, but constituentsinclude saponinsand tannrns. The flowers are bisexual, zygomorphic, usually have the floral forl. Convolvulaceae.A family of about 55 genera and 1650 species. mula K(5), C(5), A4, G(2), and are arrangedin verticillasters.The Most are annual or perennial herbs, often with twining stems:a few corolla is bilabiate and the stamensdidynamous.With the exception shrubsand trees.Generainclude Convolvulus(250 spp.),Ipomoea of Rosmarinus,the generahave a gynobasic style and the four nut(500 spp.), Pharbitis-lpomoea, Argyreia (91 spp.) and Cuscuta lets have only a small surface of contact with one another. ( 170 spp.).The genusCuscutaconsistsof parasiteswhich aresomeRosemary also differs from most genera in having only two statimes placedin a separatefamily, the Cuscutaceae. Speciesofdodmens. The herbaceousmembersof the family have squarestems. der are parasitic on clover and on flax. C. reflexa found in the Indian The leavesand other aerialpartshave clothing hairs (seeFig. 43.4)
E
THEPLANTAND ANIMAL KINGDOMSAS SOURCES OF DRUGS and glandularhairs (Fig. 43.5), which secretethe volatile oil. The generaincludeAjuga (40 spp.),Teucrium(300 spp.),Rosmarinus(3 spp.),Scutellaria (300 spp.'),Lavandulc (28 spp.),Marrubium (40 spp.),Nepeta (250 spp.),Lamium (40-50 spp.),Ballota (35 spp.), Stnchl's(300 spp.),Salvia (700 spp.).Monnrda (12 spp.),Sature.ja (30 spp.),Origanum ( l5-20 spp.),Th.y-mus (300-,100spp.),Mentha (18 spp. and 13 hybrid spp;),Pogostemon(40 spp.),Ocimum (l5O spp.),Leonuru.s(14 spp.) andLeonotis(41 spp.).Many membersof the family are used as culinary or medicinal herbs, as sourcesof volatile oils and in some casesfor the preparationof constituents of the volatile oils such as menthol and thymol; chemicalracesare common. In addition to rosemary, lavender, peppermint and spearmint, described elsewhere, note pennyroyal (Mentha pulegium'), sweet marjoram (Ori ganum mctrjo rana), patchouli (.Pogostemon patchouli'y,melissa (Melissa fficinalis), germander (.Teucriumchamaedn,s),wood sage(7. scorodonia),wood betony (StachysfficinaLis), sage(Salvia fficinalis), sweetbasil (Ocimwn basilicum), holy basil (O. sanctum),savory (Saturejaspp.), horehotnd (Marrubium spp.) and hyssop (H1-ssopusofficinalis). Also Ihyme (Th,tmusvulgaris) andMonarda punclata, both of which are imporlant sourcesof thymol. In addition to the volatile oils, constituents of the family include diterpenoids and triterpenoids, saponins,a f'ewpyridine and pyrrolidine alkaloids,insect-moulting hormones,polyphenolsand tannins,iridoids and their glycosides, quinones,furanoids,cyclitols, coumarin, and the sugarsraffinose and stachyose. The family has been extensively researchedand the book Advancesin Labiate Science,R. M. Harley and T. Reynolds(eds), 1992,publishedby the Royal Botanic Gardens,Kew, contains38 conferencepaperscoveringall aspectsofthe family. Also from Kew comesthe Lamiales NewsLetter,from February 2000 renamedVitex, which provides a forum for discussionfor scientistsof all disciplines working on the Labiataeand Verbenaceae. (The Lamialesis an order of the Bentham-Hooker system of classification and includesthe abovetwo families.) 6. Solanaceae. A family of about90 generaandover 2000 species;tropical and temperate; herbs, shrubs or small trees. Genera include Nicandra (1 sp.),Lycium (80-90 spp.),Atopa (4 spp.),Hyoscyamus (20 spp.),Phy"salis(100 spp.), Capsicum(about 50 spp.),Solanum including L,-copersicon(1700 spp.),Mandragora (6 spp.),Datura (10 spp.), SoLandra(10 spp.), Cestrum (150 spp.),Nicotiana (66 spp.), Petunia (40 spp.), Salpiglossis(18 spp.), Schizanthus(15 spp.), Scopolia (6 spp.), Withania (10 spp.), Duboisia (2 spp.), Acnistus (50 spp.) and Fabiana (25 spp.). Only five speciesare indigenousto Britain but many others are cultivated. The indigenous ones are Datura stramonium, thomapple; Solanum dulcamara,bittersweetor woody nightshade;Solanumnigntm, black nightshade; Atropa belladonna, deadlynightshade;Hyoscyamusni ger, henbane. Among species commonly cultivated in England are Solanum tuberosum, potato; Solanum lycopersicum, tomato; Nicotiana tabacunt, tobacco; Phr-saLisalkekengi, Chinese lantem; Capsicum spp.,red and green peppers;Solanum p seudocapsicum, winter cherryt Lycium halimfoLium,Duke of Argyll's Tea-plant. The flowers are bisexual and seldom markedly zygomorphic, although the carpelsare placed obliquely (see Fig. 25.6D). They have the floral formula K(5), C(5), A'5, c(2). The ovary is typically bilocular,but frequentlybecomesfalsely three-to five-locular(e.g. Daturct). On the flowering branchesadnation of the leaves with their axillary branchesoften occursand the true origin of the partsis only made out by cutting sections.All membersof the family have intraxylary phloem which is often accompaniedby sclerenchymatous fibres.
Productsof the Solanaceaeinclude: stramoniumleaves:henbane leaves; belladonnaherb and root; capsicums;potato starch fiom Solanum tuberosum; mandrake, from Mandragora fficinarurn; duboisia,liom speciesof Duboisia which are usedfor the manufacture of ffopane alkaloids; scopolia leaves and roots, sourcesof tropanealkaloids;tobaccofrom Nlcotiana tabacunt,the wasteproducts of which are usedfor the extractionof the insecticidenicotine. The family containsa wide range of alkaloids which are of grear taxonomic interest.Types of alkaloid recordedare tropane,alkaloidal amine, indole, isoquinoline, purine, pyrazole, pyridine, pynolidine, quinazolizidine,steroid alkaioids and glycoalkaloids. Other constituentsinclude steroidal saponins, withanoiides, coumarins, cyclitols, pungent principles (e.g. in Capsicum). flavones,carotenoidsand anthraquinones in Fabiana. Buddlejaceae. A family of 6-10 generaare 150 spp.;principal genera areBuddl eja ( I 00 spp.) andN uxia (40 spp.). Although B uddlei a spp. have been used in traditional medicine in many parts of the world it was only relatively recently (e.g. P. J. Houghton, J. Nar. Prod., 1985,48, 1005) that detailedphytochemicalinvestigations were initiated.The genusproducesflavonoids,iridoids and acylated iridoid glycosides,sesquiterpenoids, phenylethanoids,lignans and saponins(saikosaponins).Until the isolation of saikosaponinsby Yamamotoet al. (.Chem.Pharm. Bull. 1991,39,2'764)rhesecompounds were known only in Bupleurum spp. (Umbelliferae). Apparently,the eating habits of some insectsindicate a similarity betweenBuddleia and the Scrophulariaceae. Scrophulariaceae. A tamily of 220 generaand about 3000 species; annualor perennialherbs or undershrubs,a few trees;some semiparasites. Members differ from the Solanaceaein that they have carpelsplaced in an anterior*posteriorplane, in the aestivationof the corolla and in not possessingbicollateralbundles.The flowers are usually zygomorphic and the stamens reduced to four. Anatomicai charactersinclude glandularhairs in which the headis divided by verticalwalls only, and the stomata,which are surrounded by three or more epidermalcells. Calcium oxalateis relatively rare; when present,it occursin small solitary crystals.Generaare Verbascum (306 spp.), Calceolaria (300-400 spp.), Linaria (150 spp.),Antirrhinum (42 spp.), Suophularia (about 300 spp.), Penstemon (250 spp.), Mimulus (100 spp.), Gratiola (20 spp.),Veronica(300 spp.),Digitelis (20-30 spp.),Isoplexis(4 spp.), Melamptrum (35 spp.), Euphrasia (200 spp.), Bartsia (30 spp.), Pedicularis (500 spp.'),Rhirunthr.rs(50 spp.), Odontitis (30 spp.), Chaenorrhinum(20 spp.'),Bacopa (100 spp.) and Gratiola (20 spp.).Important drugs are the leavesof Digitalis purpurea and D. lanata and their cardiac glycosides. Picrorhiza rhizome or 'Indian gentian' from Picrorhiza kurroa containsbiner iridoid glycosides;it is usedin India to treatliver ailments.Verbascumthapsus (mullein), commonly cited as an adulteranrof digitalis,is usedas a herbalmedicinein Europeand India, particularlyfor bronchialconditions.The four speciesof lsoplexls,formerly placedin the genus Digitalis, are found only in the Canary Islands and Madeira. Cardenolideswhich are mono- and diglucosidesonly (cf Digitalis), occur in both genera; the clonal propagation of I. canariensis has beendescribed(F. Schallerand W Kreis, Planta Medica 1996,62, zl50).Other constituentsof the family include: steroidaland triterpenoid saponins;cyanogeneticglycosidesin Linaria spp.;aucubin glycosides; naphthoquinonesand anthraquinones;aurones; iridoids.Alkaloids are not very comfilon, but monoterpenoid,quinazoline and quinolizidinetypesoccur in somespecies. Bignoniaceae.A family of about I 20 generaand 650 species;mainly tropical trees,shrubsor lianes; particularly abundantin Brazil. Genera include Jacaranda (50 spp.), Catalpa (11 spp.), Tecoma
s;r*2fr
A TAXONOMICAPPROACH TO THESTUDYOF MEDICINAT PLANTS AND ANIMAI.-DERIVED DRUGS ( l6 spp.).Mussatia (3 spp.),Amphicome(2 spp.),Kigelia (1 sp.) and Tecomella(1 sp.).Kigelia pinnata bark is used in traditional Nigerian medicine; it containsnaphthoquinoidsand iridoids and has beeninvestigatedfbr antibacterialactivity by p Houghton at King's College, London (Pharm. J., 1993,250, 848). Tecoma stens,a shrubwith bright yellow funnel-shapedflowers is found in British greenhouses; it containsmonoterpenealkaloids,seeA. p. Lins and J. D'A. Felicio, Phy,tochen,1993,34,876. In South America the leavesof Mussatiahtacinthina and other speciesare chewed alone or mixed with coca leaves for their sweetening, euphoric or medicinal effects.The above genera have received considerablephytochemicalattentionand constituentsofthe family include iridoids and iridoid glycosides, saponins, phenylpropanoids,tanninsand quinones;alkaloidsarerare. 10. Acanthaceae.A family of 250 generaand 2500 species.Shrubs and herbs, including some climbing plants and xerophytes. Abundant in the tropics but also found in the Mediterranean, Australia and the USA. Genera include Acanthus (50 spp.), Andrographis(20 spp.),Blepharis (100 spp.),Adhatoda (20 spp.) andBorLeria (230 spp.).The leavesof Adhatoduvaslcaareusedin India for the treatmentof cough and contain the atkaloid vascine (peganine);the antitussiveactivity has been demonstratedpharmacologically (J. N. Dhuley, J. Ethnopharmacolog,t1999, 67, 361). Andrographispaniculata (q.v.) is also an important Indian drug plant. In addition to alkaloids,the family containstannins, diterpenoids,cyanogeneticcompoundsand saponins. I l. Pedaliaceae. A small family of l2 generaand 50 species;tropical herbs,or rarely shrubs;mostly shoreor desertpiants.The genera include Harpagophytun (8 spp.),Pedalium (l sp.) and Sesamum (30 spp.).Sesamumindicum yields the fixed oil sesameor gingili o1l.Harpagophytumprocumbens(Devil's Claw) is found especially in the Kalahari desert and Namibian steppes.Its secondary tuberizedroots aremuch usedin traditionalmedicineand it is now includedin westernmedicine(q.v.).Other constituentsof the family includephenolicsand flavonoids. 12. Gesneriaceae. A family of 120 generaand 2000 species;mainly tropical and subtropicalherbs.The genus Streptocarpushas 132 species.Tannins,naphthoquinones, chalconesand anthraquinones but no alkaloidshavebeenreported. I 3. Myoporaceae.A family of threegeneraand some240 species.The gents Eremkophila (209 spp.) is entirely Australian and has been usedin aboriginalmedicine.Myoporum (c. 30 spp.)occursin the SW Pacific area and the monotypic Bontia is found only in the WestIndies.The family containsa strikingrangeof terpenoidsand also tannins,cyanogeneticglycosidesand furans. (For a review (150 refs) seeE. L. Ghisalberti,Phttochemistn-1994,35,7)
PTANTAGINALES Plontoginoceoe An order containingthe single family Plantaginaceae, which consists of three generaand abofi 270 species;annualor perennialherbs.All but four speciesbelongto the genusPlantago (plantains).The seedsof severalspeciesare usedin medicine;see 'Psyllium'.
DIPSACALES Coprifolioceoe, Volerionoceqe, Dipsococeoe An order of four families; includesherbs,shrubsand small trees. 1. Caprifbliaceae.A family of 12 generaand about450 species;mainly north temperate, herbs and small trees. Important genera are
Viburnum(200 spp.),Lonicera (200 spp.) andSambucus(40 spp.). British members include the common e1de4 Sambucus nigra; honeysuckles,species of Lonicera; the guelder-rose,Viburnum opulus. EIder flowers, usually in the form of an ointment, are used as a domestic remedy and are included in the Bp 2000. The root bark of black haw, Viburnum prunifolium, is official in the BHp (1996). Constituentsreported in the famity are valerianic acid (compareValerianaceae), aucubinglycosides,saponins,coumarins and cyanogeneticglycosides. 2. Valerianaceae. A family of I 3 generaand about360 species.Herbs, rarely shrubs. Genera include Valeriana (over 200 spp.), ValerianeI la (80 spp.),Centranthus ( I 2 spp.) andp atrina (20 spp.). The first three of thesegenera are representedin Britain. The flowers of Valeriana and Valerianella have three stamens. those of Centranthus only one. Centranthus ruber is the common red valerian. The valerian roots of commerce are derived from the European Valerianafficinalis (q.v.) and the Indian valerian,V.wallichii.The family contains esters yielding isovalerianic acid (compare Caprifoliaceae), alkaloids and iridoids; valepotriates are characteristic of the tribe Valerianeaebut are not found in the tribe patrinieae. 3. Dipsacaceae.A family of eight genera and about 150 species. Genera include Scabiosa (100 spp.), Knautia (50 spp.) and Dipsacus (15 spp.). The ripe flower-headsof Dipsacusfullorum (fuller's teasel)are still usedto a limited extent for raising nap on certain cloths. Constituentsreported for the family include aC-glycoside,a few alkaloids,and tannins.
CAMPANULATES Gomponuloceqe, Compositoe An order of eight families. In the families consideredbelow, the anthers are either in contact with one another or fused so as to form a tube into which the pollen is shed. The flowers are hermaphrodite or unisexualby suppression.Mainly herbs with latex vesselsor oil passages. 1. Campanulaceae. A family of about 70 genera and 2000 species. Tropical or subtropical herbs; a few trees or shrubs.The subfamily Campanuloideaecontains Campanula (about 300 spp.) and has regular flowers, generally with free anthers. The subfamily Lobelioideae(which has sometimesbeen consideredas a separate family, the Lobeliaceae) contains Lobelia (200-300 spp.). In this subfamily the flowers are zygomorphic and the anthers syngenesious.Family charactersinclude the presenceof latex vesselsand inulin. Of medicinal importanceis lobelia herb or Indian tobacco, Lo beIi a infl ata, which containsalkaloids (q.v.). Indian lobelia, from L. nicotaniaefolia, also contains lobeline. In addition to the constituentsalready mentioned, members of the family contain phenolic compounds, tannins and triterpenoid glycosides. The piperidine alkaloids found in the Lobelioideae have not beenfound in the other subfamilies, but they are not considered sufficiently significant to justify a separate family. 2. Compositae. The Compositae is the largest family of flowering plants and contains about 900 genera and some 13 000 species. Compared with some other large families such as the Leguminosae, the number of important economic products derived from it is relatively small. Chemical researchin recent years has increasedmedical interest in the family and we now have a better knowledge of many almost-discardedfolk remediesas well as hitherto uninvestigated plants. The latter include some having antitumour or antibacterial activity and others forming commercial sources of rubber latex. The two subfamilies and their main generaare as follows.
'l
",'
;#i
;r{;.'.
;1},'.rlt,
'{,
THEPLANTAND ANIMAT KINGDOMSAS SOURCES OF DRUGS
i.,..' i...t,..':. i.:l,:,,,t", i:,llrlr'''l
(1) Tubuliflorae: In this subfamily latex vesselsare absent. but schizogenous oil ductsarecommon.The corollasof the disc-florets are nonligulate.Genera include Senecio (1300 spp.), Xanthium (30 spp.),Ambrosin (30-40 spp.), Zinnia (20 spp.), As the name indicates, monocotyledonshave an embryo with one Helianthus (1i0 spp.), Dahlia (21 spp.'), Helenium (40 cotyledon. Many members are herbs, usually with parallel-veined spp.), Tageres(50 spp.). Solidctgo(100 spp.), Bel/ls (1-5spp.), leaves.The stelehasscattered,closedvascularbundles;the flowers are Aster (500 spp.), Erigeron (200 spp.), Achillea (200 spp.), usuallytrimerous. As with the dicotyledonsa much abbreviatedform of Engler's clasAnthemis(200 spp.),Chn,santhemum(c.200 spp.),Matricaria (40 spp.),Tanacetum(50-60 spp.),A rtemisia(400 spp.),Blumea sification indicating the main orders and families of pharmaceutical (50 spp.), Inula (200 spp.). Doronicum (35 spp.). CalenduLa and phytochemicalinterestis given below. (20-30 spp.), Euparorium (1200 spp.), Arnica (32 spp.), Vernonia (600 spp.). Echinops (100 spp.), Carlina (20 spp.), Farnilies Orders Arctium (5 spp.), Carduus (100 spp.), Centaurea(600 spp.), (13 (70 spp.) and Gerbera spp.).Omitting the huge Carthanrus Liliif'lorae Liliaceae,Agavaceae.Amaryllidaceae. number of cultivatedgardenplants,note chamomileor Roman Hypoxidaceae,Dioscoreaceae, chamomileflowers, from Anthemis nobilis; Germanchamomile, Iridaceae Matrir:arin chamontilla; insect flowers, Chrysonthemum. Bromeliales Bromeliaceae cinerariffblium; wormseedor santonica,Artemisia cina; arnica Graminales Gramineae flowers and rhizome, Arnica monttzna; calendula tlowers, Principes PaImae Calendula ofJicinalis; yarrow herb, Achillett milLefoLium: Araceae,Lemnaceae Spathiflorae grindelia herb, Grindelia camporum: blessed thistle leaves, Cyperaceae Cyperales Cnic'usbenediclus;coltsfoot leaves,Ia.isi/agofafiara; wornMusaceae,Zingiberaceae,Cannaceae, Scitamineae wood herb, Artemisitt absinthitrm:pellitory or pyrethrum root, Marantaceae Anacycluspyrethrumt elecampaneroot, InLrlahelenium; Ngai Microspermae Orchidaceae camphor, frcm Blumea balsam.fera.The llorets of the saff-lower, Carthamustinctorius,areusedas dye and as a substitutefor saffron. Refined Sunflower OiI EP/BP is expressedfrom the seeds uHtFtoRAE relined. of HelianthusannuLtsand subsequently (2) Liguliflorae: In this subfamily latex vesselsare present but Liliqceqe, Agovoceoe, Hypoxidoceoe, volatile oil is rare.All the flowers haveligulate corollas.Genera Amoryllidoceqe, Dioscoreoceoe, lridoceoe include Cichorium (9 spp.),Crepis(200 spp.),Hieraciurn (over The order is divided into five suborders and 17 families. Some 1000spp.),Taraxacwn(60 spp.),Lttctuca(100 spp.),Scorzonera botanistsfavour further subdivision-for example, the separationliom ( I 50 spp.) andSonchus(50 spp.).Vegetablesobtainedfrom this the Liliaceae of Smilttr and Rrlscls into separate families, the group include lettuce,scorzoneraroot, chicory and endive. Of respectively. Smilaceaeand Ruscaceae members used medicinally may be noted dandeiion root, Many membersof the group areperennialherbshaving a bulb, corm Taraxacumo|ficinaLe;lactucarium or iettuce-opium,Loctuca or rhizome. The flowers are hermaphrodite.regular or zygomorphic, virosa; mouse-earhawkweed,Hi eraciumpiloseI La-which has andtypicallyhavethefloral fbrmulaP3 + 3,A3 + 3 or3 + 0. G(3).The antibiotic activity and has been used for the treatmentof Malta perianthis usually petaloid;the ovary superior(e.g.Liliaceae)or int'efever. Chicory root and dandelion root have been used,particu- rior (e.g.Iridaceae).Fruit a capsuleor berry. larly in war-time Europe, as adulterantsof or substitutesfor cof'fee. A Turkestan dandelion, Taraxacum ktk-sa,qhiz, has since I Liliaceae.Awidely distributedtamily ofabout 250 generaand 3700 species;mostly perennialherbswith a rhizome or bulb. The flowers 1936beenwidely cultivatedin the former USSR as a sourceof usuallyhave six stamensand a superior(rarely semi-inferior)ovary, rubber latex. with numerousanatropousovules attachedto axile placentas.The fruit is a loculicidal or septicidalcapsule,or a beny. As might be expectedfiom its size,the Compositaecontainsa wide Without troubling the readerwith the namesof subfamilies or tribes, variet-vof chemicalconstituentsand the literatureis enormous.Already the numbersinsertedbelow will indicatethe principal generawithin mentionedare the latex of the Ligulillorae and inulin which is often each: l. Vertrtrum(25 spp.), Gloriosa (5 spp.) and Colchicwn (.65 present in very large amounts (e.g. in dahlia tubers). Some of the (12spp.),Bowiea(2spp.), spp.);2. Herreria(8spp.):3.Asphodelus volatile oils fbund in the Tubuliflorae contain acetyleniccompounds. Funkia-Hosta (10 spp.), Kniphofia (75 spp.) and Aloe (about 330 Sesquiterpenes known as azulenesgive the blue colour to fieshly disspp.); 4. Gagea (70 spp.),Allium (450 spp.); 5. Lilitun (80 spp.), lactonesoccur tilled oil of chamomileand yarrow.Many sesquitetpene (85 spp.). TuLipa(lOO spp.); 6. Scl11a(80 spp.), Urginea Fritillaria (e.g. santonin), and are of varying types, including eudesmanolides (100 spp.),Ornirhogttltrm(150 spp.),Hyacinthus(30 spp.),Mu,scari germacranoiides. guaianolidesand pseudoguaianolides; someof these (60 spp.); 7. Asparagus (300 spp.). Pol-tgonatum(50 spp.), have cytotoxic activity. The toxic Senecio alkaloids derived from Convallaria (1 spp.), Trillium (30 spp.), Paris (20 spp.); 8. pyrrolizidine have been much researched.Some causeliver damage Ophiopogon(20 spp.); 9. Aletris (25 spp.); 10. Smilax (about 350 and are therefbredangerousto livestock. Alkaloids of the pyridine, 'Agavaceae'. spp.).For Yucca,Dracaena andAgaveseebelow r.rnder quinoline and diterpenoidtypes also occur in the family. Other conSome members of the family are cultivated for their flowers. stituentsinclude the insecticidalestersof pyrethrum(q.v.) triterpenoid Vegetablesinclude asparagus(Asparagus oflicinalis) and onion, saponinsofgrindelia, cyclitols.coumarinsand flavonols.Steviarebaugarlic, shallot,leek and chives (all from speciesof Allium). Drugs diana, a herb indigenousto Nofih EasternParaguay,is the sourceof include squill, sarsaparilla,veratrum, colchicum seed and corrn, stevioside,an ent-kaureneglycoside used as a sweetenerfor soft aloesand cevadillaseed.Garltc(Alliun sativum).an age-oldremedy drinks.etc.
;:,rifl
A TAXONOMICAPPROACH TO THESTUDYOF M'DICINAt PTANTS AND ANIMAL.DERIVED DRUGS frequently used for the treatment of colds, bronchitis, etc., has recentlyreceivedmuch attentionas a preventativeof heaft disease, as an antibioticand as an anticancerdrug. Many member"sof the family contain alkaloids, which are of the steroidal,isoquinolineor purine types. Other steroidalsubstances includesterols,cardenolides, bufadienolidesand steroidalsaponins. The amino acid azetidine-2-carboxylic acid occursin many genera and is also found in the Agavaceae.Other constituentsinclude quinones (benzoquinones,naphthoquinones,anthraquinonesand anthrones);flavonoids (anthocyaninsand flavonols); rhe y-pyrone chelidonicacid: cyanogeneticsubstances; and fructosan-typecarbohydrates.Somevolatile oils of the family haveantimicrobialproperties. Agavaceae.A family of 20 genera and 670 species.The genera (40 spp.).Agave (300 spp.),Cordyline(15 spp.), include YLLcca (150 spp.),Sansevieria(60 spp.),Phonnium (2 spp.), Drac:cteno Nolina (30 spp.) and Furcraeu (20 spp.).Dragon's blood is a dark red secretionfrom the leavesand trunkof Dracaena draco andD. t'innabctri;the tbrmer is a native of the Canary Islandsand Willis (A Dictionary of the Flowering Plants ttnd Ferns. I 985) citesa tree blown down in 1868.70ft high and 4-511in girth, rhat was supposedto be 6000 yearsold. Steroidalsaponinshave recently been reported as constituents of the resin (Y. Mimaki er al., Phytochemistn' 1999, 50. 805). D. cinnabari was the source of commercialSocotradragon'sblood, the flavonoids of which have beenstudied(M. Masaoudet al., Phytocltemisn.r 1995,38, 745; 751). The resin is traditionally used as an astringentin the treatment of diarrhoea;it alsohashaemostaticand antisepticpropefiies. Note that anotherdragon's blood is obtained from Daemonorops spp.(Palmae)(q.v). Steroidal saponins occur in species of Yucca,Agave and Furcraea; Sansevieria 7e-vlanicayields bow-string hemp and Furcraea giganteu Mauritius hemp. The leavesof Agrzuesisnlana yield the fibre sisalwhich is producedin largequantitiesin Central America and Kenya: the fermented sap of this plant forms the Mexican drink pulque. Speciesof Sansevieriaare common houseplants under the name of 'mother-in-law'stongue'. Alkaloids and cardenolidesappear to be absent from the family but the constituentsotherwiseclosely resemblethoseof the Liliaceae. 3 . Amaryllidaceae.A family of 85 genera and about 1100 species; mostly tropical or subtropical,and often xerophytes.Many have bulbs or rhizomes.Perianthpetaloid.A 3 + 3, G(-3).Fruit a loculicidal capsuleor berry.GeneraincludeGalotthus (20 spp.),Leuco.junt (l2spp.'1.Aman'llis (l sp;),Nerine(30 spp.),Puncratittm(15 spp.), Hy-menocttllis(50 spp.), Narcissus(60 spp.), Ungernia (6 spp.), Hippectstrurn(75 spp.) and Sternbergia(8 spp.).The tamily contains typesof alkaloid unlike thoseof any other family in the order. R. Darnley Glbbs (Chemotaxotlom)' of Flowering Plants,Yol.I, p. 148, 1914) lists nine groups of theseAmaryllidaceousalkaloids. One of these alkaloids, lycorine, has a marked fungicidal action; another,galanthamine.obtainedfrom Leucojumaestivumand other species.is a newly marketeddrug for the treatmentof Alzheimer's disease. Hypoxidaceae.A family of seven generaand 120 species.Herbs from a tuberousrhizome or conn. SeveralHypoxis spp.have found use in traditional medicine as anti-inflammatoryand anti-tumour drugs.A lipophilic extract is marketedin Germany,and the dried corm in South Africa, fbr treatmentof prostatehypertrophy.The genus containsphenolic glycosidesof the norlignan type (see M. Nicoletti et ul., J. Ethrtopharm.,1992,36.95). -5. Dioscoreaceae. A lamily of five generaand about 750 species; tropical or warm temperateclimbing herbs or shrubs.The plants
have fleshy tubers (called yams) or rhizomes. The leaves are often arow-shaped. Flowers dioecious.Fruit a capsuleor berry. The genera are Dioscorea (over 600 spp.), Tamus (5 spp.), Rajann (25 spp.), Stenomeris (2 spp.). Tamus commuLris,the black bryony, is exceptionalin that it grows in Europe.It is common in Englishhedgerowsand its scarletberriesarepoisonous;a number ofphenanthronederivativeshave been reportedfrom the rhizome as well as dioscin and gracillin (see R. Aquino, J. Nat. Prod. 1985, 48, 502,81i). The yams of Dioscorea batatas con' tain abundant starch and are important foodstuffs in the tropics. Steroidal saponinsare presentin many speciesof Dioscorea and Tamusandtheir extractionhas becomean important industry (see Chaprer24). 6. Iridaceae.A iamily of more than 60 generaand 800 species.Found in both tropical and temperateregions.Many perennialherbswith corm (e.g. Crctcus)or rhizome (e.g. lrls). Flowers hermaphrodite, regular or zygomorphic; P3 + 3 petaloid, ,A3, G(3). inferior (or rarely superior).The fiuit is a loculicidal capsulewith numerous seeds.The genera include Crocus (15 spp.),Romulea (90 spp.), Sis,-rinchium(100 spp.), Tritonia (55 spp.),Freesia (20 spp.) and lris (about 300 spp.). Products of this family are saff,ron,from Crocus sativus,and orris root, from speciesof 1rls. Constituents include quinones(naphthoquinones and anthraquinones), aromatic ketones (e.g. in lris), carotenoidpigments (e.g. in saffron), terpenoids (mono-, sesqui-,di-and tetraterpenoids),and flavonoids (anthocyanins,flavones,flavonolsand isoflavones).
BROMELIATES Bromelioceqe The Bromeliaceaeis the only family of the order and containsabout44 generaand 1400 species;mainly tropical and subtropical,xerophytes and epiphytes.Fruit a berry or capsule.Theseinterestingplants vary very much in size and habitat; many are grown as house-plantsor in greenhouses. Generainclude Bromelia (40 spp.) andAnanas (5 spp.). Ananas comosus(syn. A. sativus)is the pineapple,the juice of which containsbromelain,a protein-splittingenzymewhich can be usedas an adjunct in the treatment of trauma and oedema arising from surgery and for soft tissueinflammation.It is cited as an aid to digestionand recentstudies(Gut 1998,43,196) have suggestedit as a possiblecure for travellers' diarrhoea. The chemistry of the family requires much more study,but many speciescontain gums and mucilagesand there arereportsof tannins,phenolicacidsand flavonoids.
GRAMINATES Gromineqe An order of one family. The Gramineae contains about 620 genera and l0 000 species. Mostly herbswith fibrousroots;rarely shrubsor trees.Annuals,biennials and perennialsalmostuniversallydistributedandmany of greateconomic importance.Leavesalternate,with sheath;blade usually long and nalrow; veins normally parallel. Jnflorescenceof numerous spikelets, with scales.Flowers bisexual, protected by palea; stamensusually three;ovary unilocularwith one ovule.Fruit one-seeded, usuallya caryopsis (rarely a nut or berry). Some important genera are Bambusa (70 spp.), Arundinaria (150 spp.), Oryza (25 spp.), Arundo (12 spp.), Triticum (about 20 spp.), Agropyron (100-150 spp.), Hordeum (20 spp.),Secale(4 spp.),Avena (.'70spp.),Sorghum(60 spp.),Zett (l sp.), Saccharum(5 spp.)andAndropogon(113 spp.),Cymbopogon(60 spp.), Pfuilaris (20 spp.)andVetiveria(10 spp.).Productsincludebamboosof
THEPLANTAND ANIMAL KINGDOMSAS SOURCES OF DRUGS many difl'erent sizes, fiom speciesof Bambusa and ArundincLria, rtce from Oryz.asdlir,r, wheat from Triticum, barley from Hordeurn, rye from SecaLe,millet or guinea corn from Sorghumvulgare, maize or Indian com from Zea nnls and sugarcane from Sucr:harumoJJicinarum. The tribe Paniceae, which includes Andropogon, Cymbopogon and Vetiveria,is rich in volatile oils. These grassoils are relatively cheap and are much used in perfumery,especiallyfor scentingsoap.They includecitronellaoils, lemon-grassandpalmarosa(geranium)oils from speciesof Cymbopogon;oil of vetivertfromVetiveria zizanioides.The rootsofthe latterplant,khus-khus,are usedboth in perlumeryand as a drug and are alsowoven into fragranFsmellingmatsand f'ans. The Gramineaecontains a very wide range of constituentsbut a large proportionof the chemicalwork has been devotedto the abovementioned fbodstuffs, starches,sugar and volatile oils. Other constituentsinclude l1 difl'erentclassesof alkaloid (fbr details seeR. D. G1bbs, Chemotaxonom\ of Flon'ering Plants, MonIreal, Canada: McGill University Press),saponins,cyanogeneticsubstances, phenolic acids,flavonoidsand terpenoids.
sonouslatex, the poison being destroyedby heat.The generainclude Acorus (2 spp.), Arum (15 spp.), Monstera (50 spp.), Dracunculn, Amorphophallus and Cnptocoryne. Ca\amls or sweet l1ag rhizome is derivedfrom the pelennialherb Acorus calamus,which is widely distributed in damp situationsin Europe and North America. Arum, also known as lords and ladies,cuckoo-pintand wake robin, is Arummaculutum, a common hedgerowplant in England. lt and other speciesof Arum are poisonous;these plants contain amines and cyanogenetic compounds.Speciesof Monsteraareoften grown as house-plants. The rhizome ol Cryptocorvnespiralis is known as 'Indian ipecacuanha'. Amorphophalluscampanulunrsis the elephant-footyam. Many membersof the family arecyanogenetic.Alkaloids, eitherpyridine or indole types, are reported in a f'ew genera. Other constituents include saponins,tannins.phenolicacids,aminesand terpenoids.
CYPERATES
Cyperoceoe The order containsthe singlefamily. The Cyperaceaehas about 90 generaand 4000 species:widely disPRINCIPES tributedherbs.Generainclude Cl,perus(550 spp.),Scirpus(300 spp.) Polmoe and Carex (150-200 spp.).Formerly oflicial in a number of pharmaThe order containsthe singlefamily Palmae. copoeiaswere the rhizomesof Carex arenaria and Cr-perusrotundus. The Palmaehas 217 generaand about 2500 species.Wideiy distri- Papyrus, used in ancient times as paper, is derived from Cyperus buted in the tropics and subtropics.Mostly treeswith an unbranched pap)-rus.Like some members of the Gramineae.certain speciesof stem bearing a crown of large, often branched leaves.The flowers are Scirpus andAntpektdesmoseryeas host plants for speciesof Claviceps usually unisexualand regular,consistingof two trimerous whorls of and produce ergot-like sclerotia. Other lamily constituentsinclude small perianth leaves, six stamensand three superior carpels. The volatile oils, tannins,phenolicacids,flavonoidsand sesquiterpenoids. carpelsrnay be free or united and develop into a berry. drupe or nut. The seedscontaina very small embryo but abundantendosperm.Important generaare Pfutenir (17 spp.), Sabal (25 spp.').Copernicia (30 spp.), SCITAMINEAE Metroxt'lon (15 spp.), Calamus(375 spp.),Areca (54 spp.),Elaeis (2 Musoceoe, Zingiberoceqe, Cqnnoceqe, Morontoceoe spp.), Cocos (l sp.), Phytelephas(l-5 spp.). Serorcta (1 sp.) and An order of live families. (100 spp.). Of economic interest are the date palm, Dttemonorr.tps I . Musaceae.A family of two generaand 42 species,namelyMLrsa(35 Phoenix dactylfera; sago from the stemsof Metro.nlort rumpii and M. spp.)and Enseta(7 spp.).The plantsaregiant herbswith 'false'aerlaeve; raltan canes from speciesof CaLamus;areca or betel nuts from ial stemsand sheathedleavesarisingfrom a rhizone. Fruit a berry. Areca catechu;palm oil and palm kernel oil from Elaeis gLtineensis; Of economic importance are the fruits of Musa paradisiacu (plancoconut from Cocos nucifera; camaubawax from Copernicia cerifera. tain) and M. sapientum(banana),which are rich in starch.Manila The resin from speciesof Daemorutrop.igives the EastAsian dragon's hemp or abacais derived from Mustt re-rti1ls.Constituentsof the blood and this appears(1997) to be the only available commercial family include starchand fructosans,phenolicacids,anthocyanins, source; the constituentsinvestigatedare of flavonoid origin, some terpenoidsand sterols;only isolated examplesof alkaloids (alkaderived from 5-methoxyflavan-7-oland 5-methoxy-6-methylflavanloidal amine and an indole alkaloid)are reported. 7-o1.Isolatedcompoundshavebeennameddracoflavans(A. Arnone el al., J. Nat. Prod.,1991,60,971).The fruits of Serenoarepens(saw-pal- 2. Zingiberaceae.A family of about 49 genera and 1300 species. Perennialaromaticherbswith fleshy rhizomesand tuberousroots; metto) native to the SE coastal statesof North America are a popular flowers in racemes,headsor cymes. Perianth6-merous,the outer remedy for male impotence and are used in the treatment of the sympcalyx-like, the inner corolla-like.Two of the stamensmodified as a toms of benign prostatehyperplasia.Seedsof PhVelephas spp. (P petaloidlabellum.Fruit a loculicidal capsule.Seedwith perisperm. aequatorialisin Ecuador)are the basisof the vegetableivory industrv Genera include Curcuma (5 spp.), Alpinia (250 spp.), Zingiber (for a reportseeA. S. Barfod et al., Econ.Bot., 1990,44,293).In addi(80-90 spp.),Amomum (150 spp.),Elettaria (7 spp.),Aframomum tion to the fixed oil, carbohydratesand leaf wax mentioned above, the (50 spp.) andHedychium(50 spp.).Cosrls (150 spp.)is includedby family containssaponins,tannins,catechins,llavonoids,terpenoidsand Engler in the Zingiberaceae, but is sometimesseparatedasa distinct ketones.AlkaloidsoccurinAreca, but few havebeenfound in othergenfamily. Its main difference from the other genera listed is that its era.Steroidalsubstances occur(e.g.estronein the pollen of Pfutenix). aedal partsarenot aromatic.Productsof the family includeturmeric, the rhizomesof Curcumalonga; grnger,the rhizome of Zingiber SPATHIFTORAE officinale; cardamom fruits from Elettaria cardamomum; and Arqceqe grainsof paradise,the seedsof Aframomummelegueta.Volatileoiis The order consistsof two families. the Araceaeand the Lemnaceae. and pungentprinciplessuch as are found in ginger (q.v.) are a feaThe latter, which containsonly six generaand 30 speciesof aquatic ture of the family. Other constituentsinclude the colouring matters herbs,is of little importance. known as curcuminoids(see 'Turmeric'), tannins,phenolic acids, The Araceaehas I 15 generaand about2000 species;rnainlyherbsor leucoanthocyanins, flavonoids,ketonesand terpenoids.Only a few climbing shrubsand over 90Vctropical; many memberscontain poiisolatedexamplesof alkaloidshavebeenreported.
;.-ril5
A TAXONOMICAPPROACH TO THESTUDYOF MEDICINALPLANTS AND ANIMAL.DERIVED DRUGS 3. Cannaceae.This iamily containsonly the single genus Canna (55 B) which inhibit tumour promotion.As with the spongesthereis much spp.).The rhizome of Canna edulis yields the starch,Queensland cunent biochemicalinterestin this ohvlum. arTowroot. :1. Marantaceae.A family of 30 generaand zl00speciesof tropical herbaPTATYHEI.JYIINTHES ceous perennials.The genus Maranta (23 spp.) contains Maranta arundinacea,the rhizome of which yields WestIndian arowroor. (Flotworms)
MICROSPERMAE
ThisphylumincludestheTrematodes (theparasiticflukessuchasthe liverfluke andSchistosoma) andtheCestoda (tapeworms).
Orchidoceoe NEMATODA The order containsa singlefamily. The Orchidaceaeis one of the largestlamilies of llowering plants, (Roundworms) with some 735 genera, over 17 000 species and many hybrids. Someareparasitic in manandanimals. Cosmopolitanperennialherbs,some terrestrialbut many tropical epiphytes(e.9. Vanilla).The ovary is inferior and the fruit a capsule;seeds very small and light. GeneraincludeOrchis (35 spp.).Ctpripedium (50 MOTLUSCA spp.),Phalaenopsis(.35 spp.),Dendrobium (1400 spp.'),Liparis (250 Class Gastropoda includesthe snails,slugsand limpets. Some snails spp.),Malaxis (300 spp.), Vanda(60 spp.),Cry.ptosrr-lls (20 spp.) and are vectorsof parasitessuchas Sc/rlslosona. Vanilla(90 spp.).The glycosidesofvanilla, which producevanillin and Class Lamellibranchia includes scallops, mussels, oysters and other aromatic substancesby slow enzymic change,have long been clams. known. More recently, attention has been directed to the numerous Class Cephalopodaincludesthe squids,cuttlefishesand octopuses. alkaloids present in the family. Sorne of these are of an unusual Cuttlefish bone (from Sepia fficinalis) has been employed in dentiindolizidine type, others are derivatives of indole, pynolidine and liices and as an antacid. pyrrolizidine. Other constituents include phenolic acids, tannin, fl avonoids,coumarinsand teruenoids.
ANNE[IDA (Segmented \,vorms) Earthworms,lugworms and leeches(q.v.) belong to this phylum. The potent neurotoxlc agent nereistoxin is obtained from the Japanese As with the Plant Kingdom, animalsare classifiedinto Phyla, Classes, speciesLumbr i conereis heterop oda. Orders,Families,Generaand Species.Although the number of pharmacognosticalproductsderived from animal sourcesis limited there ARTHROPODA hasbeen,in recentyears,an immenseinterestin the chemistryof many marine creaturesas potential sourcesof drugs and biologically active A very large phylum of jointed animals including the crustaceans, materials.In this respectmuch researchhas beenpublishedon the sim- insectsand arachnids. pler marine organisms. Class Crustacea includes the shrimps, crabs, lobsters,centipedes Listed below are selectedanimal phyla which embracespeciesof and millipedes. Of little medicinal significance,neverthelessbrine interest (the many animal products used in traditional medicinesof shrimpsare being increasinglyused in place of higher animalsfor the Africa, India and the Orient are not included). preliminary testingof phytochemicalsfor toxicity. Class Insecta. Of the many ordersof this taxon the following have medicinalinterest:
PROTOZOA
Unicellular microorganisms including parasites causing malaria (Plasmodium), sleeping sickness (Trypanosonttt)and dysentery (Entamoeba). Some dinofl agellates (Pro ro cent r um, D in op ht si s) pr oducepolyethertoxins responsiblefor someshell-fishpoisoning.
PORIFERA (Sponges) Metabolites include bromophenols(antibacterialproperties),cyclic peroxides and peroxyketals (antimicrobial, ichthyotoxic, cytotoxic activities),modified sesquitetpenes (antimalarial,antifungal,antibacterial, anticanceractivities).Siliceousspongespiculesare often found in samplesof kieselguhrand agar(q.v.).
COETENTERATA (Jellflishes, seq onemones, corqls) ThesoftcoralPlexaLtahomomallais a rich sourceof prostaglandin ,{2 andSarcophyton glaucumcontainsditetpenoids(sarcophytols A and
(l) Order Anoplura (Lice): Commonly infest birds and mammals. Important from human standpointts Pediculushumanusencounteredas the body louse(.corporis)and the headlouse(capitis);itts a proven carrier of typhus fever and an indirect transmitter of relapsingfever. (2) Order Hemiptera (Bugs): The cochineal beetle (q.v.) is an lmportant colourant and shellacis a resinoussubstanceprepared from a secretion that encrusts the bodies of a scale insect Laccifer lacc'a. (3) Order Coleoptera(Beetles):Beetlesof the generaCantharis and Mylabris, known as blisteringbeetles,possessvesicantproperties and preparations of C. vesicatoria were at one time used in Westernmedicinein the form of plasters,collodionsetc. asrubefacientsand vesicants.Their use continuesin the traditionalmedicine of EasternAsia (R. W. Pemberton,J. Ethnopharmacology 1999,65, 181).A numberof small beetlesare importantinfestants ofstored drugs(seeChapter13). (4) Order Lepidoptera (Butterflies and moths): Some moths infest storeddrugs (seeChapter l3). Silk has been traditionally used in pharmacyin the form of oiled silk.
THEPLANTAND ANIMAL KINGDOMSAS SOURCES OT DRUGS (5) Order Hymenoptera (Ants, bees, wasps, etc.): Hive products derived from Apis mellifica include honey, beeswax, royal jelly and propolis (q.v.). (6) Order Diptera (Flies, gnats and midges): The successfuluse of maggotsin the treatmentof woundsinfectedwith antibiotic-resistant Staphylococcusaureus has received recent attention.Sterile larvae of the common greenbottleLucilia sericatc are used.For a report seePharm. J . 1999,262, 422. Class Arachnida. Arthropods with two divisions to the body (cephalothoraxand abdomen)including spiders,scorpionsand mites. Order Acarina (Mites): the common housemite(q.v.) is a causeof allergyin humans;other speciesinfest storeddrugs(seeChapter13).
CHORDATA The most important subphylum of the Chordata is the Vertebrata (Craniata)composedof all those animals with backbones;a number of classification schemes will be found in the literature and two major groupings often referred to as superclassesare the Pisces
(aquatic vertebrates)and the Tetrapoda(terrestrialvertebrates)each divided into four classes.The followins four classeshave medicinal significance: 1. Class Osteichthyes (Bony fish). Cod and halibut important for their liver oils which contain vitamins A and D and eicosaoentaenoic acid (dietary supplement). 2. ClassAmphibia (Frogsand toads).Dried and powderedtoadskins contain cardioactive principles and were used for the treatment of dropsy before the widespreadadoption of digitalis. 3 . Class Reptilia (Crocodiles,snakesand lizards).Snakevenomsare important products. 4 . Class Mammalia (Warm-blooded animals which suckle their young). Variouslydivided into subclasses, infraclassesand orders. SubclassEutheria embracesthe placental mammals, e.g. bats, rodents, carnivores, whales, ungulates and primates. Formerly important was the whale-product spermaceti but its collection is now illegal. Other pharmaceuticalsinclude lard, suet, wool fat, wool, gelatin, musk, catgut, insulin, hormones,blood and liver oroducts.vaccinesand sera.
r.'iis
r
As indicated in Chapter 2, a val,id scheme for the study of medicinal plants and their products,and one which emphasizespharmaceutical '!, use, can be based on pharmacologicalaction. The scheme can be extended to include numerous plants which, although eliciting a --,."1i pharmacologicalresponse,arenot, for variedreasons,usedasdrugs.In i !, the latter categorywould be placed hundredsof alkaloid- and glyco4a side-containingplants. Somemajor pharmacologicalgroupingsinvolve drugswhich act on r d the nervous systems,heart and blood vessels,lungs, gastrointestinal tract,kidneys,liver, reproductiveorgans,skin and mucousmembranes. Other categoriesinclude hormones,vitamins and chemotherapeutic lr drugs used for the treatment of infections and malignant diseases. r.it Someplants(e.9.Papaver,ipecacuanhaand liquorice)containa range #i{ 1 , of compounds with differing pharmacologicalproperties. Oliver..'!i. Bever's classicalreview ("/. Ethnopharmacol.,1983, 7, 1) on West '9i African plants which act on the nervous system well illustratesthe problemsof constructinga purely pharmacologicalclassificationfor ! herbal materials. A system based on clinical usage may be more straightforward for the throughly-studied allopathic drugs used in ' 1 Westernmedicine but difficulties can arise for plants used in tradition. al medicinebecauseof the often numerousconditionsfor which anv '.1{i i""' one drug may be employed. I j
Phormocologicol qctivities of noturol t , prooucts
,
"
s
;f
.l
;|
, ''..c
.io" ..,lri ,-
DRUGSACTINGON THENERVOUS SYSTEM 4I THEHEART,CIRCUTATION AND BLOOD 42 ACTIONON THEGASTROINTESTINAT TRACT 44
!61 *
r
The nervous systemcoordinatesand regulatesthe various voluntary and involuntary activitiesof the body and is convenientlyconsidered under two headings-the centralnervoussvstem(CNS) and the autonomic nervoussystem.The two are interlinked and somedrugs which affect the CNS may also produce reactions associatedwith the autonomic system.In the caseof otherswhich act via the autonomicsystem it is sometimesmore convenientto classifythem underother headings appropriateto the organsinvolved; thus,thoseproducingvasoconstriction or vasodilation may appearunder the considerationof the circulatory and respiratorysystems.
THENASATAND RESPIRATORY SYSTEMS 45 THELIVER 45 THEURINARYAND REPRODUCTIVE SYSTEMS 45 THESKIN AND MUCOUSMEMBRANES 45 ACTIONON SUGARMETABOTISM 46 AND ANTI.INFTAMMATORY STEROIDS DRUGS 46 NONSTEROI DAt ANTI.IN FTAMMATORY DRUGS 46 TREATMENT OF INFECTIONS 47 TREATMENT DISEASES 47 OF NAALIGNANT TREATMENT OF ALTERGIES47 THEIMIVTUNESYSTEM 47
vfTA ^tNs 48
THECENTRALNERVOUSSYSTEM The central nervous system comprisesthe brain (cerebrum,cerebellum, medulla oblongata)and the spinal cord. It coordinatesthe voluntary activities of the body and exhibits numerousinteractionswithin the system together with linkages to the autonomic system. Drugs involved with the CNS can be broadly classifiedaccordingto whether they have a general stimulatory or depressant action with further subdivisionregardingspecificactionssuchas anticonvulsantand psychopharmacologicalactivities.Some of the most useful natural drugs of the group arethe narcotic(opioid) analgesics;otherssuchasthe hallucinogenicdrugshave importantsociologicalimplications.SeeTable 6. I for a summary of drugs acting on the central nervous system.
THEAUTONOMTSNERVOUSSYSTEM The autonomic nervous system supplies the smooth muscle tissues and glandsofthe body.Its function is complex,involving gangliasituatedoutsidethe spinal cord; it is composedof two divisions,the sympathetic (thoracolumbaror adrenergic)division, which arises from the thoracic and lumbar regions; and the parasympathetic(craniosacral or cholinergic) division, originating in the brain and in the sacral region. In general,an increasein activity of the sympathetic systemgearsthe body for immediateaction (fight and flight), whereas stimulation of the parasympatheticor vagal systemproduceseffects
'1$i. !;i
;
i
, 1
l
m
OF DRUGS THEPLANTAND ANTMALKINGDOMSAS SOURCES
Toble 6.I
Drugs octing on the cenlrql neryous syslem.
Drugsoffecting mentolactivity Lysergicocid diethylomide Mescoline Connobis Purineboses(e.g.coffeine,theophylline, theobromine) Cocoine I
Ginkgo bilobo Ginseng Golonthqmine Hypericum Soge Reserpine Yohimbine Volerion,Possifloro
fromergotolkoloidsor by ortificiolculture Prepored by pcrtiolsynthesis Hollucinogenic. Hollucinogenic. Obtoinedfrompeyotecoctus contoinedin the resinof Cannobissotivo Hollucinogenic. Activeconstituents of beveroges-coffee,feo, cocoo,kolo, mst6 Stimulotementoloctivity;conslituents oddiction.Contoinedin the leoves One of fhe eorliesidrugsusedos o mentolstimulont.Produces ol Erghroxylumcoca lmprovesshorttermmemory porticulorlyin the elderly lmprovesmentolconcentrotion diseose of Alzheimer's olkoloidfor treofment Promising Amoryllidoceous Populorherbolremedyfor reliefof mild-moderotedepression memoryloss Revivedinferestin its usefor counterocting Depresses mentoloctivity.Usedin psychiohictreoiment.Obtoinedfrom Rouwolfiospp. reoctions ond effecton heortmusclerenderit o[ bul itsontiodrenoline Similoroctionlo reserpine of theApocynoceoe no clinicaluse.Foundin voriousspecies ond improvesleepquolity Sedotiveond hypnotic;oid sleeplessness
of the CNS in Anolepticdrugs(stimulonts odditionto ihe menlolstimulonts indicotedobove) Picrotoxin Lobeline Strychnine Comphor
Anolepticpreviouslyusedin the freotmentof borbiturotepoisoning.Obtoinedfrom berriesof Anomirtococculus Obtoined from Lobelioinflato Obtoinedlrom the seedsof Weok onolepiic;toxic dosesproducespinolconvulsions. Strychnos spp. comphoraond by synthesis Weok onoleptic.Obtoinedlrom Cinnamomum
of motor function Central depressants Troponeolkoloids(e.g.hyoscine, otropine,etc.) root Gelsemium Anolgesicdrugs Morphine Codeine
of Porkinson's diseose. Formerlythe only drugseffectivein the olleviotionof the symptoms ond deliriumtremens Usedin treotmenfof trovelsickness clinicollyowing to high toxicity.Golenicolpreporotionsoccosionollyusedos Rorely-employed oniisposmodics octionon lhe coughond respirotorycentres.The Effective for reliefof severepoin. Depressonl principololkoloidof opium Althoughlessoctivethonmorphineit is o muchsoferdrugfor thereliefof mildpoin ond for use os o cougnsuPpressonl
Many factors affect the complex regulation of the heart and the large group of drugswhich is known to possesscardiovascularactivity is not confinedto actionon the heartmuscleitself. Thus thosedrugspossessing antiarrhythmic, antihypetensive, antihyperlipidaemic,vasoconstrictor, vasodilator,blood anticoagulant,and platelet aggregation activitiesmust also be consideredin this group.As with other importis an activesearchin the plant kingdom for compounds ant areas.,there of new which may also serveaslead compoundsfor the semi-synthesis drugs.For sometherapeuticgroups,the lack of simplereliable screening techniquesis a problem. AND BTOOD THEHEART.CIRCULATION In a review (over 390 refs) E. L. Chisalberti et aL. (.seeFurther Reading)have iisted some447 speciesfrom 109 families having carIn developedcountries,coronary and associatedcirculatory diseases diovascularactivity,togetherwith a compilationof over 700 secondary now constitutethe principal causeof human mortality.Not surprising- plant metaboliteshaving suchactivity. ly, therefore,this is an areaof intensivetesearch,not entirely devoted to treatment, but also to the prevention of these diseases.With increasedpublic awarenessof the importanceof the latter. healthier Cordiooctive glycosides living focusedon diet, supplementaryfood factors,exercise,etc. has A considerablenumber of plants scatteredthroughoutthe plant kingtaken on a more important role, not least in the mind of the commercial dom contain C21or Cz+steroidalglycosideswhich exerta slowing and world where health food storesnow supply many dietary supplements strengtheningeffect on the failing heart. In Western medicine it is the and medicinal plant products which overlap the traditional pharma- glycosidesof variousDigitalis speciesthat are extensivelyemployed. The pharmacologicaleffectivenessof the cardioactiveglycosidesis ceuticalranqe.
more associatedwith those occurring during sleep and with energy conservation.Two important neurotransmittersubstancesof the autonomic nervous system are acetylcholine and noradrenalineand its which either mimic or antagonize derivatives;hence,other substances the action of either of these will produce a marked physiological response.Drugs acting on the autonomicnervoussystemare summarized in Table6.2.
-*t
tfl
PHARMACOLOGICAL ACTIVITIES OF NATURAL PRODUCTS
Toble 6.2
E
Drugs octing on the outonomic nervous system.
AcetylcholineJike drugs Pilocorpine Arecoline Muscorine Physostigmine Antogoni stsof ocelylcholine Troponeesterqlkoloids(e.g.hyoscine, olropine)
Neuromusculor blockingogenls(e.g.tubocurorine) Gonglionblockingogents{e.g.tubocurorine in lorgedoses)(notclinicollyimportont)
Fromleovesol Pilocarpusmicrophyllus Fromseedso[ Arecacalechu FromAmonitospp.ond otherfungi A cholinesterose inhibitorfrom seedsof Physostigno venenosum
ililrl::,,,'
Fromo numberof Solonoceoe{e.g. Duboisio,Atropa,Doturaelc.lTheyhove widespreodusesinvolving theirgostrointeslinol, bronchiol, genito-urinory ond ophfholmiceffectsin odditionto the CNS octivity{q.v.1 Fromleovesond siemsof Chondodrendontomentosum
AdrenalineJikedrugs Ephedrine
Fromstemsof Ephedrospp.; moinlysynthetic
Antogonistsof odrenoline Ergotolkoloids(e.g.ergotomine)
Fromsclerolioof C/ovicepsspp.
No rodrenoline d e pletion Reserpine
Hosontihypertensive effectresultingfromdilotionof heortond circuloloryvessels
Ophtholmic preporations Theeye, being underthe confrolof the outonomic nervoussystem,is offecfedby someof the drugs mentioned obove;theseincludeotropine,hyoscine, physostigmine ond pilocorpine.
dependenton both the aglyconesand the sugarattachments;the inherent activity residesin the aglycones,but the sugarsrendbr the compounds more soluble and increase the power of fixation of the glycosidesto the hearl muscle. The overall action of the digitalis glycosidesis complicatedby the numberof differenteffectsproduced.and their exactmode of actionon myocardialmusclein relationto currentviews on cardiacmusclephysiology is still an areaofinvestigation.Digitalis probably actsin competition with K ions for specificreceptorenzyme(ATPase)sitesin the cell membranesof cardiacmuscleand is particularlysuccessfulduring the depolarizationphaseof the muscle when there is an influx of Na+ ions. The clinical effect in cases of congestiveheart failure is to increasethe force of myocardial contraction (the positive inotropic effect)resultingin a completeemptyingof the ventricles.As a iesult of depressionof conductionin the bundleof His, the atrioventricularconduction time is increased,resultingin an extendedP-R interval on the electrocardiogram. Arising from their vaguseffects,the digitalisglycosidesare also usedto control supraventricular(atrial) cardiacarrhythmias. The diuretic action of digitalis, important in the treatmentof dropsy,arisesfrom the improved circulatoryeffect. However, fbllowing the introductionof saferdiureticsin the 1950s,diuretic therapyfor heart lailure has becomemuch more imporlant and in somecasescan replacedigitalis treatment. Among the many other plant generacontainingcardioactiveglycosidesrelatedto thoseof Digitalis, and usedsimilarly, areStrophanthus, Convallaria, Nerium, Thevetia and Erysimwn For a full account of thesedrugs seeChapter24
Antiorrhyrhmic drugs As mentioned above, the cardiac glycosidescan be used to control supraventricular (atrial) cardiac arrhythmias. There are a number of other drugs such as the alkaloid quinidine (obtainedfrom variouscinchona barks. q.v.) which act on both supraventricularand ventricular
arrhythmias.Quinidine is oflicial in most pharmacopoeias as its salts and finds prophylacticuse in recunentparoxysmaldysrhythmiassuch as affial fibrillation or flutter.Its therapeuticusefor the attemptedconversion of atrial librillation to sinus rhythm has now been largely replacedb1 electricalcardioversion.
Antihypertensive drugs The control of hypertensionis an imporlant element in the management of cardiovasculardisorders.Primary hypertension,as distinct from other specialfbrms which usually requirehospitalization,representsabout 907c of all casesranging from mild conditionswith the occasionalrise in blood pressureto thosewith severeunrelievedhigh pressure.Of the hypotensiveplant drugs rauwolfia and its principal alkaloid reserpinetogetherwith Venfirumextractswere recognizedin allopathic medicine in the early 1950s.However, a number of other plants regularly employed by Western herbal practitionersinclude mistletoe. Crataegus, Yarrow, Tilia and, Fagopt.runt. In Ayurvedic medicinePiper betle,Josminumsabac,Cardiospermumhalicacabum and Tribulus terrestris,used in the treatmentof hypertension,have beenshownto exhibit a high angiotensinconvertingenzymeinhibition suggesting a possible mechanism of action for these drugs (B. Somanadhanet al., J. Ethnophrtrmacology1999,65, 103). Plotelef octivofing foctor (PAF) ontogonists In the circulatorysystemthrombi may be causedon the arterialsideas a result of the adhesionof blood plateletsto one anotherand to the walls of the vessels.This platelet aggregationis triggered by the platelet activating factor which is releasedliom activatedbasophils. PAF from the rabbit was characterizedin the 1970sas a l-O-alkyl-2acetyl-sn-glyceryl-3-phosphorylcholine. A number of prostaglandins and thromboxanesarealso involved in the aggregationmechanismand thromboxane,A2which is synthesizedfrom arachidonicacid (q.v), is particularlypotent.In undamagedvesselsthromboxane'{2 is possibly
aa:,:')::.::.:
I
3
THEPLANTAND ANIMAL KINGDOMSAS SOURCES OF DRUGS balancedby a prostaglandine.g. prostacyclinof the arterial intima which hasdeaggregation properties. For the secondarypreventionof cerebrovascularor cardiovascular disease,aspirin, which irreversibly acetylatesthe platelet enzyme cyclo-oxygenasehas been employedat dosagesof 300 mg daily with useful results.A large number of piants have been screenedfor antiPAF activity. One of the first natural products so identified was the neolignankadsurenoneobtainedfrom Piper futokadsurn,a plant long used in Chinese traditional medicine for allergy treatments.Other plants of traditional medicine reported to have anti-PAF activity include speciesof Forsythia,Arctium, Centipeda,Tussilago,P1-rola, Populus and Peucedanum.The active constituentsinclude lignans, sesquiterpenes, coumarins,pyrocatecholand salicyl alcohol. Extractsof the maidenhairtree,Ginkgobilobahaveprovedespeciaiiy interestingand are commerciallyavailablein Europe for the treatment of variouscirculatorydisorders. Certainfish oils (e.g.cod-liver,halibut-liver)once employedsolely for their vitamin contentshaverecentlyreceiveda resurgencein popularity as dietary supplements.One favourable responseis that they decreasethe ability of plateletsto aggregateby virtue of their high eicosapentaenoic acid content;this acid tendsto favour the biosynthesis of thromboxaneA3, a weakerstimulatorof plateletaggregationthan thromboxaneA.2. Drugs ocfing on blood yessels (Table 6.3) These drugs are essentially either vasoconstdctor or vasodilator substances but their action may originatein a variety of ways (direct, cenffal,peripheralor reflex). Some of the drugs (e.g. ergot,bronchodilators,diuretics),which are particularlyuseful in relation to specific systems,areclassifiedelsewhere.
Warfarin sodium is one of the most widely useddrugs.Plantsusedin herbal medicinewhich containcoumarinderivativesand possessantivitamin K activity include Meliktu.y officinalis, Galium aparine and Ltnandula offic inal is. Other anticoagulantsare heparin.which is given by injection, and hirudin, produced by the leech; hirudin, a polypeptideof 65 arnino acids,can alsobe obtainedfrom geneticallymodified Sctccharon4'ces.
Hypolipidoemicdrugs In recentyearsmuch prominencehas beengiven to the associationof high levels of blood cholesteroland plasmatriglycerideswith atherosclerosisand ischaemicheartdisease.Treatmentof hyperlipidaemiais preferablydietaryaccompaniedby other naturalregimes.Drug therapy is reservedfor the more intractableconditions.Naturalproductshaving a beneficial action include nicotinic acid (Chapter31) and those fish oils containinghigh quantitiesof to-3-marinetriglycerides.The latter involve eicosapentaenoic acid and docasahexaenoic acid which, when colnting from the methyl end,possessthe first doublebond at C-3. I'Qhesuggestedbeneficiatpropertiesof garlic (Altiunt sativum) for the treatmentof variouscardiovascularconditionsremain a subjectof extensiveinvestigation.With ref'erenceto hyperlipidaemicpatients, the majority of publisheddata suppoftsthe hypothesisthat garlic lowers serum total cholesteroland improves the lipid profile. There is a tendencytowardsreductionof low-densitylipoproteinand an increase in high-densitylipoprotein giving a more favourableHDL:LDL ratio. A reduction in the serum levels of total cholesterol. low-density lipoproteincholesteroltogetherwith a lowered atherogenicindex was observedwith mild hypercholesterolicpatients after a three-month courseof psyllium seeds(K. Sagawaet al., Biol. Phurm. Bull. 1998,
21,r84).
Orol onticoogulonts Thesecompoundsinhibit the clotting mechanismof the blood and are ACTIONON THEGASTROINTESTINAT TRACT of value in arterialthrombosis;they haveno effect on plateletaggregation. One group of active drugs constitutesthe 4-hydroxy-coumarins The gastrointestinaltract can be divided into threeregions-the upper which act by antagonizingthe effectsof vitamin K (seeChapter31). (mouth, stomach and upper pofiion of the duodenum),the middle
Ioble 6.3
Drugs ocfing on blood vessels.
Peripheral vo soconstrictor d rug s Ergotomine(torkote)from C/ovicepspurpureo Ergotoxine Ephedrine(synthetic ond from Ephedrospp.) Nicotine
Produces o direciconstrictor effectin vosculorsmoothmuscle;the reversqlof the dilotionof croniolvessels leodsto iis useol fheonsetof clossicol migroineottock Similorto ergotomine Prolonged ociionon bloodpressure-see olso'Autonomic NervousSystem' gonglio,ond by it Vosoconstrictor effectsorisefrom itsoctionon sympothetic promotingreleoseof vosopressin ond odrenoline
Cenlral vo soconstrictordrugs Mostof the drugs(e.g.picrotoxin)which stimulote the centrolnervoussystemolso stimulotethevosomotorcentrein the medullo,producingo risein blood pressure. Althoughot one time usedos respirolorystimulonts, thesedrugshovebeenlorgelyreplocedby mechonicoldevicesfor o*ificiolventilotion of therunos Vosodilotordrugs (onopiumolkoloid) Popoverine
Actsdirectlyon the bloodvessels by cousingreloxotion of smoothmuscle. An introvenous iniectionusedfor theheolment of pulmonory orteriolembolism Xonthinederivotives(coffeine,theobromine, theophylline) As popoverine;they olso hoveo centrolvosoconstrictor octioncounterocting the peripheroleffect.Also diurefic Ergotomine Adrenolineontogonist-see'AutonomicNervousSystem' Reserpine Vosodilototion is producedby o peripherolond centroloction(q.v.) Verotrumolkoloids{fromVerolrurn spp.} Brodycordio ond peripherol vosodilototion by sensilizotion of cordioc,oorticond corotidsinusbororeceotors
wl:- |
PHARMACOLOGICAT ACTIVITIES OF NATURAT PRODUCTS , lo.,r'erhalf of the duodenr.rm to the ileocolic sphincter)and the lower icaecum,colon andrectum).It is the upperandthe lower portionsthat |rrc lnost susceptibleto disorderand are consequentlyassociatedwith thc greatestnumberof drLrgsfbr their treatment(seeTable6.zl).
Toble 6.4
THENASATAND RESPIRATORY SYSTEMS A largenumberof drugsof plant origin areto be found in this group.As infectionsof the respiratorytract are amongstthe most comrnonof illnesses,it is not surprisingthat thereare numerousproprietarypreparations lbr their ffeatment(Table6.5).
Drugs octing on lhe gosfrointestinol trocl.
Bitters Al one lime thesewere extensively usedin liquid medicoments lo stimulote oppetite.Thebitterconstituenls stimulote thegustoiorynervesin themouthond giveriselo on increose in thepsychicsecretion of gostriciuice.Extrocts of thefollowing drugshovebeenso employed: gention,quossio,columbo, cinchono(orquinine),nuxvomico(orstrychnine). Consideroble recentreseorch hosinvolvedthe investigotion of o numberof thesebittercompounds for otherpossibleusesie.g. thebittersof theSimorouboceoe os ontiiumour ond onfimoloriol ogents Anticholinergic drugs In thiscopocityhyoscineond hyoscyomine helpdisturbonces cousedby goskicmobilityond musclesposm porticulorly wilh someulcerpotients Emetics lpecocuonho preporotions, on orol odministrotion, hoveo deloyedemeticoctionproducedby irritotion of themucous (see'Expecloronts'). membrones Picrotoxin stimulotes thevomiting centrethroughitsgeneroleffecton the centrolnervoussystem Anti-emeticsGingerhos receivedscientificopprovolfor the prevention of thesymptoms of trovelsickness. Connobisoffords sickness reliefto potients undergoing chemotheropy. Corminolives Theseore oromoticsubstonces whichossisltheeructotion reflex;theirmodeof octionisobscure.Dilloil is usedfor thereliefof flotulence, especiolly in bobies.Otherplontsor oilsusedos corminotives includecorowoy,fennel,peppermjnt, thyme,nutmeg, colomus, pimento, ginger,clove,cinnomon, chomomile, motricorio. Cholkis usedos on ontocidond chorcoolos on odsorbent. Ulcertheropy Derivoiives of glycyrrhelinicocid (o triterpenoidof liquoriceroot)proveeffectivein the treofmentof pepticulcer. Deglycynhizinized liquoricehosolsobeenemployed. Oiher ontiulcer ogentsincludeolginicocid, morshmollow ond comfrey. Demulcents Thesesootheond orotectthe olimentorvtroctond overlopwith somemoteriols usedin ulcertheropy. lcelondmoss, orrisond elmbork moy be includedhere. Loxatives ond purgotives Purgotives moy be clossedoccordingto theirmodeof oction
THELIVER The liver. the principalorganof metabolismand excretion,is subjectto a nunber of diseaseswhich may be classedas liver cirrhosis (cell destructionand increasein librous tissue).acute or chronic hepatitis (inflammatory disease)and hepatitis (noninflammatoly condition). The most commondrug of plant origin usedin Westernmedicinefbr its antihepatotoxic propefiies is Sily-hunttnaricutum. In Indian and Oriental medicine many plants are so used. For a discussionof this field of currentinterestseeChaoter30.
THEURINARYAND REPRODUCTIVE SYSTEMS A numberof plant materialsareto be found in this _eroup; the examples given in Table6.6 are confinedto Westernusage.
:
.,.
:
a
:,.: .,
..
.
..:l::t:::,,
THESKIN AND MUCOUSMEMBRANES In addition to itcting as covering lbr the body, the skin perfbrms a number of other physiological functions. Drugs affecting the skin may be of an emollient nature or they may act as absorbents,astringents.irritants or antiseptics(seeTable 6.7). A nurnberof substances are easily absorbedthrough the skin; this fact is utilized in transdermal medicationbut mllst alsobe bornein mind with respectto \urious poisons.
Agor,psyllium ond ispoghulo Bron
Hydrophilic colloidswhich funciionos bulk-producing loxotives An indigeslible vegetoble fibrewhich obsorbswoterond providesbulk Senno(leoves ond fruit) Contoinsonthroquinone derivotives which ore hydrolysedin the bowel io stimulote Auerboch's plexusin thewoll Coscoro,rhuborb,oloes As senno Costoroil Contoinsglycerides whichon hydrolysis yieldriconoleic ocid, irritontto thesmollbowel Podophyllum resin, Drosticpurgotives,now little jolopresin,colocynth usedfor thispurpose. Theywere oftenprescribedwiih bellodonnoto reducegriping
Rectaland colonicdrugs Arochisoil, esculin,homomelis,pilewort ond bolsomof Peruore exomples of thisgroup;theyincludethose usedin supposiiories Anfidiorrhoeol drugs Morphineond codeineoct by increosing the smooihmuscletoneof thebowelond by reducingitsmobility. Commonlyprescribed with koolin
Toble 6.5 syslems.
Drugr ccfing on the nosol ond respirqbry
Aromoticinholations
Bronchodilotorsond nosoldecongestonts Expectoronts
Benzoin, cineole,eucolyptus oil, menthol pumilopineoil, bolsom ond peppermint, of Tolu,thymol,turpentine Ephedro,ephedrine, xonthines (theophylline) (insubemetic lpecocuonho doses),senego root,liquoricerool,squillbulb,tolu bolsom,pulmiliopineoil, lobelio, grindelio,ongelicorootond leof,storox, cocillqno,colisfoot,sweetviolet, bloodrool
Antiexpectoronts
a^..1^i^.
Cough depressonts
Morphine,codeine,noscopine, wild cherry
Demulcents
Morshmollow, verboscum, plontogo, lcelondmoss,honey
nrrnnina
E
AS SOURCES OF DRUGS IHE PLANT AND ANIMALKINGDOMS
Drugs octing on lhe urinory ond Toble 6.6 reproduclive systems.
Toble 6.7 Drugs used on the skin ond mucou6 membrcrnes.
Diuretics
Xonthine derivotives os presentin mony beveroges(teo,coffee,etc.)promotedilotion Digitolis of therenolmedullorybloodvessels. glycosides improvethefoilingheorlthereby ond glomerulor increosing renolperfusion originol filtrotion; hence,Withering's of infroduction of digitolisfor thetreotment dropsy.Thereis olsoo smollbut finiteeffecf of sodiumions on tubulorreobsorotion
Emollients ond demulcents Theseincludeo numberof vehicles usedin thepreporotion of ointments, creoms,lotions,etc.,ond includefixed oils {e.g.olive,orochis,coconut, theobromo),fots {wool-fot,lord},woxes spermoceti), of onimolorigin(beeswox, gums(ococio,kogoconlh)ond (psyllium, muciloges elmbork) Absorbents
chorcool Storch,olginotes,
Diureticsond urinory ontiseptics
Buchu,beorberry, iuniper,copoibo.These includedrugsusedfor the treotmentof cystitis ond urethrilis
Astringents
. o n n i oc c i d ) ,k r o m e r i o , T o n n i n(se . g T cotechu,golls,Aspidospermo, rind,kinos homomelis, pomegronote
Drugsocting on the uterus
Preporotions of ergotwere trodilionollyused in childbirihond thenlorgelyreplocedby lhe Administered isolotedolkoloidergomefrine. octionon os ifssoltsit hoso directstimulont the incidence theulerinemuscleond reduces hoemorrhoge. Ergotomine octs of postportum for obstehicuse similorlybul is nofsuitoble becouse of itsmorkedperipherol vosoconstrictor oction Blockhow is c uterinetonicond sedotive usedfor the prevenlionof miscorriogeond for dvsmenorrhoeo ofterchildbi*h Hydrostis is employedfor menorrhogio disorders ond othermenstruol Femole, Mole,gossypol{q.v.) see'Steroids'. plontshovebeen,ond ore Numerous being,testedfor ontifertiliiyoctivity
Counter-irritonts
copsicum, Comphor,furpentine, oconiie,methylsolicylote,mustordseed
Antiseptics
Tors,eucolyptus oil, thymeoil, eugenol, thymol,coiuput
Anti-inflommotoryogents
usedlocolly,motricorio, Corticosieroids ornico
Psoriosisond eczema treotment
ollonfoin, codeoil,evening Comfrey, primroseoil, chrysorobin , Lithospermum, sovin,myrrh,grindelio
Wound coverings
Typeof woundcovering(occlusive, is imporiont nonocclusive, hoemosiotic) in the heolingprocess. Seeolginotes, cotton,etc.
Oral controceptives
Mole impotence
(undercorefulmedicol Popoverine yohimbine(erectile dysfunction) supervision),
Benignprostote hyperplosioIBPH\
ore employed A numberof phytomedicinols (oftenos odmixtures) of to keot ihe symptoms withthe ossocioted BPH.Twometobolites ond conditionore dihydrotestosierone oestrogenwhich requiretwo enzymes ond oromotose)for their {5o+eductose in the body. ll hos beenshown,for synthesis someof the drugsused,thof theyore inhibitorsof thesetwo enzvmes.The followingexomplesore well-estoblished: Cucurbitopepo seeds(pumpkin),Epilobium ongustifolium ond otherspecies,Prunus africano lPygeun africanumlbork, Serenoo repensfruifs(sowpolmeito,sobol),Urlico dioicoond U. urensroot extrocts
ACTION ON SUGARMETABOLISftI Many plants have been used in traditional systems of medicine for the oral treatment of diabetes and it is particularly important that Western practitioners be aware of any patients already taking such medication. Among the plants so used are karela fruit (Momordica charantia), cumin fruit, ginseng, Teucrium oliverianum, neem (Azadirachta indica), onion, Aloe spp., Job's tears (Coix lachryma-jobi) and Galega officinalis. For a discussionof the current position on plant-derivedoral hypoglycaemic substancessee Chaoter30.
AN D ANTI.INFLAM'IilATORY STEROIDS DRUGS Two types of corticosteroidalhormoneare the glucocolticoids,which regulatecarbohydrateand protein metabolismand which also possess a stronganti-inflammatoryaction, and the mineralocorticoids,which influence the electrolyteand water balanceof the body. The clinical indicationsfor systemicffeatmentwith thesedrugs are complex, but include use in replacementtherapy,Addison's disease,reduction of lymphatic tissues(leukaemias),suppressionof lymphopoiesis(lymphomas) and as anti-inflammatory agents (a variety of conditions including rheumatoidarthritis,cerebraloedemaand raisedintracranial pressure). Thesehormonesare producednaturaliy in the adrenalcortex but a wide variety of semi-syntheticdrugs of this type is commonly in use. Theseare synthesizedusing plant steroidsas intermediates;diosgenin and hecogenin being the principal sources.To a lesser extent the steroidalalkaloids of the Solanaceaeare employed.There is a large world demandfor thesecompounds,pafticularly for the synthesisof oral contraceptives,and their distribution in nature and chemistry is consideredin more detail in Chaoter24.
NONSTEROIDAL ANTI' IN FI/AMMATORY DRUGS Aspirin, first synthesizedin 1853by Carl Gerhardt,is still one of the most widely-usedmild analgesicand nonsteroidalanti-inflammatory drugs(NSAID). It had its medicinalorigin in the salicylatesand glyco-
PHARMACOLOGICAL ACTIVITIES OF NATURAL PRODUCTS sidesof willow bark, long usedfor the treatmentof rheumaticdiseases, gout and painful conditionsof all types. In view of the universalrequirementfor NSAIDs very many plants havebeenutilized for the purposein traditionalmedicineand in recent yearsconsiderableresearcheffort has been expendedon their investigation. Enzymes have been used to detect anti-inflammatory activity of plants-galangin, a flavonoid of Alpinia olficinarum (Zingiberaceae; galangal rhizome) was found to be a cyclo-oxygenaseinhibitor. Lipoxygenase inhibitors are present in Spilanthes oleracea (a S. American plant of the Compositaeusedfor the treatmentof rheumatic disorders) and also rn Echinacea purpurea root, also Compositae, which containsa rangeof compoundsincluding isobutylamides. ln a review of plantsexhibiting anti-inflammatoryactivity Handa et al. (1992) cite that speciesof 96 generabelonging to 56 families are ascribedsuchactivity. In addition to the wide range of plants involved there is a similar diversity in the chemicalnatureof the active constituents.Flavonoids constituteone group widely associatedwith anti-infl'ammatoryactivity and are exemplifiedin Ihe BHP ( 1996)by the monographson Balm of Gilead Bud, Cimicifuga Rhizome, Equisetum, Jamaica Dogwood, Marigold, Matricaria Flowers,Meadowsweet,Poke Root, Red Clover Flower and Willow Bark. In the caseof an infusion of matricariaflowerc (Chamomilla recutita, German chamoqrile), used for its antiinflammatory action in the treatmentof acute gastritis, it has been shownby the mouse-eartestthat it is the flavonoidsandnot the volatile oils that are responsiblefor activity; however, for Calendula fficinalis the terpenoidswere found to be activeconstituents(seeAnon., Pharm. J., 1992,249, 474). With liquorice root (.Glycyrrhiz.aglabra) both the triterpenoidsaponinglycynhizin and the flavonoidshave,amongtheir other pharmacologicalactions,anti-inflammatoryactivity. Colchicine, an alkaloid of Colchicum autumnale, is the classical drug fbr the treatment of acute attack of gout. It may act by reducing the inflammatory responsecausedby depositsof urate crystalsin the joint and by reductionofphagocytosisofthe crystals.Its usehas been somewhatreplacedby allopurinol (inhibition ofxanthine oxidase)and by phenylbutazone.Guaiacumresin (q.v) is cited for the ffeatmentof chronicrheumaticconditionsand gout; it containsa mixture of lignans. The dried root of Harpagophl;tum procumbens (Devil's claw) (Pedaliaceae), hasrecentlyreceivedpopularattentionfor the treatment of painful rheumaticconditions;iridoid giycosides,e.g. harpagoside, are the characteristic constituents.The juice of Ananas comosus, the pineapple(Bromeliaceae)containsa mixture of at ieastfive proteolytic enzymescollectively called bromelin or bromelain.In Westernmedicine the enzymehas beenintroducedfor its ability to dissolvefibrin in conditionsof inflammatoryoedema. The action of ginkgolides (C2e terpenesfrom Ginkgo biloba) as potent antagonistsof plateletactivatingfactor has alreadybeen mentioned.
Tcble 6.8
Drug; u:td for the treqtrnent of infeclion!. , :
Antibiotics Mony higherplontspossess constituents hoving ontibocteriol properties; nonehosbeenutilizedclinicolly, moinly becouseof high toxicity.Mouldsond streplomyces ore the principol sources; seeChopler32 lor o considerotion of ontibocteriol ond onliviroldrugs Antimaloriols Untilthe odventof the synthetic oniimoloriols, quinine,isolotedfromthe bork of voriousCinchonospp., constiiuted the mosteffectiveogentfor thetreotment of molorio;it is stillusedin ThirdWorld countries ond is of someresurgenr importonce for combofingmoloriolorgonismsresistoni to other drugs.Artemisinin (Qinghooso), on unusuolsesquiterpene loclone, is the octiveconstiiuent of on oncientChinesedrug derivedfrom Artemisio annuo.ltis effectiveogoinsfchloroquineresistont stroins ol Plosmodiumvivox ond P.falciporumos well os qgoinstcerebrol molorio;seeChopter29 Amoebicides Emetine
Anthelminthics Extrociof molefern Sontonin
An olkoloidof ipecocuonho root,usedos itshydrochloride or bismuthiodide in the ireofmenl of omoebicdysentery. Completeerodicotion of thechronic infectionis difficultond combinedtheropy with otherdrugsis oftennecessory Fortopeworminfections Possesses o powerfuloctionin porolysinground-worms; olthoughonce extensively usedits high toxicityhos led to replocement by piperozine
Oil of Chenopodium
Likesontonin; if hosolsobeen extensively usedin hookwormdiseose but it givesvoriobleresulfs
Thymol
At one fime muchusedin hookworm treotment
Catharanthus andTaxusalkaloids) arein seneraluse.For a fullerdiscussionseechapter28.
IREATMENTOF AIIERGIES A largenumberof materialsgive rise to allergicconditionsin sensitive individuals.Extractscontainingspecificallergiesare availableas diagnostickits or for desensitization. Examplesof plant allergensaregrass, flower and treepollens,dried plantsand moulds(seeChapter40).
I}IE IMMUNE SYSTEfrI TREATMENT OF INFECTIONS For naturalproductsin this category,seeTable6.8.
The last 40 yearshavewitnesseda vast searchof the plant kingdom for substanceswith anticanceractivity; clinical trials are currently being conductedon a number of promising compoundsand others (e.g. the
Drugs aff'ectingthe immune system are termed immunomodulatory or adaptogenic.Somerepressthe systemand areofvalue in, for example, preventingrejectionof transplantedorgansand othersare stimulatory and can be usedto help combatviral infectionssuch asAIDS or assist in the treatment of cancer.Until relatively recently such herbal drugs were largely ignored by Westernorthodox medicine, although they have always featuredin traditional Chinese and Indian medicine in seekingto achievehomoestasiswith regardto bodily functions.Now, however,ginsengleadsthe marketin herbalsalesin Europeand the US andEchinaceaspp.,usedby native N. American Indians,ranks around
@
THEPLANTAND ANIMAL KINGDOMSAS SOURCES OF DRUGS fifth in the US herbmarket salesand is widely usedin Europe,with 800 Further recding preparationsbeing quotedas availablein Germany.For immunomodu- GhisalbertiE L, PennacchioM, AlexanderE 1998A review of drugshaving cardiovascular actrvity (nearb- 400 re/s). PharmaceuticalBiology 36(4): latorsof Chineseorigin seeL.-H. Zhang et al., PhvtotherapyResearch 231-219 1995,9, 315, and for Indian drugs seeA. A. Munganliwar et al., J. Handa S S, Chawla A S, SharmaA K 1992 Plants with anti-inflammatory Ethnopharmat'olo gy 1999,65, 125. activity (a review with 278 re;ferencesand 34 structuralformulae).
.:;nifu.}{s'
Fitoterapia63, 3 Wagner H (ed) 1999 Immunomodulatory agentsfrom plants. Birkhauser Verlag,Basle Williamson E M, Okpako D ! EvansF J (eds) i996 Pharmacologicalmerhods in phytotherapy research,Vol 1. Selection and pharmacological evaluation of plant material. John Wiley, Chichester
,1',...l:....:l',1.1:ll:.
Theseaccessorylbod substances are consideredin Chapter31
Yt'-
|
(
^
'
JynergyIn reotion
Some of the most popular and widely sold phytomedicines,such as those containing St John's wort, echinacea,ginkgo, garlic. kava and valerian. have considerablepharmacological and clinical evidence to supporttheir use,and synergyis generally assumedto play a p:Lrtin their medicinal effects. Attempts are rarely made to isolate a single constituentfrom theseextractsfor reasonswhich will be outlined later. and in Western medical herbalism. traditional Chinese medicine and Ayurveda,combinationsofherbs are fundamentalto their philosophy as well as being due to empirical observationsand historicalusage.This attitude to their formulation and use dilTerentiatesherbal products from conventionalmedicines,eventhosewhich containan isolatedplantconstituent.For example,pharmaceuticalscontaining ephedrine,cafTeineor evenpeppermint oil are rarely consideredas phytomedicinesdespitethe fact that they are obtained from plant sources.Valuable drugs such as digoxin, theVinca alkaloidsand taxol are alsonaturalproducts,but due to their h:ighpotency and toxicity the plants containing them have do not havesucha long historyof usageand haveneverbeenconsideredto be 'herbal medicines'.Modern herbalremediesareusuallyfound as whole or semi-purified exffacts and ideally should be standardizedlbr their activeconstituentswherepossibleto ensureclinical reproducibility.
to the phormocologicol octionof phytomedicinols
ll
.,1!.
,,t
.
WHAT 15SYNERGY? 'working Synergybroadly means together'and occurswhen the combined action of constituentsis greaterthan would be expectedfrom a considerationof individual contributions.In pharmacology,synergy has a specific definition, but the term is often misapplied to describe any kind of interactionbetweenconstituentsof a singleextract,as well as the componentsof a mixture of herbs. These interactionsmay involve a potentiationof therapeuticeffects,or an attenuationof toxicity or side-effects,within the preparation.Whether an effect is truly synergistic,or merely additive, is rarely establishedand evidenceto prove it conclusively is sparse.The opposite,antagonism.meaning 'working against',is much easierto define, being a reduction in the overall expectedeffect.Medical herbalistshaveoften insistedthat better resultsare obtainedwith whole plant exffactsratherthan with isolated compounds, for example the side-effects of ephedrine are not usually found with an extract of the herb Ephedra. If true synergy is occurring in phytomedicines,standardizationbecomes even more important, since the ratio between synergisticagentsis critical, and small changescan make resultsunpredictable.If combinationeffects are merely additive,changesin the ratio are lesscrucial. lt may therefore be useful to define synergypreciselybut routinely use the term 'polyvalent action' when the natureof the interactionis unclear.
E. M. Williamson
WHAT IS SYNERGY? 49 MEASURINGSYNERGY 49 DEMONSTRATING SYNERGYAND POIWAIENT ACTIONIN PHYTOMEDICINES 50 (r ). STNGLE ENHANCEMENT HERB OF ACTTVTTY EXTRACTS 5I (2). MULTTPTE ENHANCEMENT OF ACTTVTTY HERBEXTRACTS 52 SYNERGISTIC OTHERMISCETLANEOUS INTERACTIONS 53 ATTENUATION OF TOXICITY
coNctustoN
54
s3
]i,,.;ir ..; i!ll
.,..:il ',..r irtiltiri
:ltri riit. : -,t ,iait:,i ll,rii!1ir{lil
."
:i*,fir::lrl :r'l:. :;ll!;rrl
t*ir,:ti:i!9r ti:liitiEiiiui tiililtitt;3!t,ii!
MEASURINGSYNERGY Although the idea of synergyis easyto understand,the measurement of it is more problematic.It is fairly straightforwardto identify synergy when one of the agentsis inactive and a combinationof this with an active agentproducesan effect greaterthan that observedfor the active alone,but difficulties in measurementarisewhen more than one (and theremay easilybe several)areactive.The variousmethodsfor calculation are explainedthoroughlyby Berenbaum(see 'FurtherReading') andaresummarized belou'
'
- ,1.:ltii.,r
't.....
Summation of effects. Synergy is deemedpresent if the total effect of a combinationis greaterthan would be expectedfrom the sum of effects.This can be expressedmathematicallyas: E(da,db)=E(da)+E(db) where E = the observedeffect, and da and db are the dosesof agentsa andb.
::
fll
THEPLANT ANDANIMAL KINGDoMS As SoURCEs oF DRUGs Although the validity of this methodseemsevident.it actuallydepends upon the mechanismof action being similar for both agents,and a linearity of response.The method would be valid under theseconditions but they areunlikely to apply to complexherbalmixtures. Measurement of the effect of a fixed dose of one on the doseresponsecurve ofthe other. This methodalsoassumesa knowledge of the dose-response curve of each agent and a linearity of response, and would be almostimpossibiewith a herbalmixture. Comparison of the effect of a combination with that of each of its constituents. This methodwas originally suggestedby Gaddum (Pharmac'olog_r,, Oxford University Press,1940)and synergydeemedpresentif the effect of the combination was greaterthan that of each of the individual agents,(ie da,db> d a. and da,db> db). Although this method is independentof a knowledgeofthe mechanismof action.it can be shownto be flawed by consideringthe caseof a shamcombination(where a and b are the same agent and interaction is therefore impossible). Berenbaum quotes the following argument to demolish it effectively: if two men, working separately,can each cut down 10 trees in a day, but working togetherthey cut down I 5, the effect ofthe combinationcertainlyexceeds that of eachconstituentand would thereforefulfil Gaddum'smathematical criteria for synergy.In reality, this would be a nonsense. The isobole method. Although mathematicallythe most complicated, this is now consideredto be be the methodofchoice sinceit is independent of any knowledge of mechanismsand applies under most conditions.It makesno assumptionsaboutthe behaviourof eachagent and is applicableto multiple components,so can be applied to the analysis of effects in herbal mixtures. An isobole is an 'iso-effect' curve,in which a combinationof ingredients(da, db) is represented by a point on a graph,the axesofwhich arethe dose-axesofthe individual agents(Da and Db). If agentsdo not interact,the isobole(which is the line joining the points representingthe combination,to those on the dose-axesrepresentingthe individual dosesisoeffectivewith the combination) will be a straightline. If synergyis occurring,then smaller amountsare neededto producethe eff'ectand the isobole is said to be 'concave-up'. This is shown in the graph (Fig. 7.1) of actual results (Table 7.1) obtained from investigating synergy between the ginkgolideA and B. The oppositeappliesfor antagonism,producinga
14
12
E1 r- o
io *o
J c
.
0L
0
0.5
1.0 1.5 2.0 2.5 3.0 3.5 B (pM) Ginkgolide
4.0
Fig.7.l lsobolecurvefor 50% inhibition of GinkgolideA + B mixtures. (FromB Steinke. Chemisch-onolytische und Phormokologische Unfersuchungen von Pflonzlichen PAF-Antogonisten und Inhibitoren der Thrombozylenoggregotion. Thesis, MunichI 993, with permission.)
Toble 7.I The lC5s* vqlues of vqrious Ginkgolide A + B mixlures obtoined by on in vitto induced ploteler oggregqlion tesf.
mixture
3 :
2.40
t .
.) .)i
:3 :]0
quontityGinkgolide
B{cB)
lCso
GA:GB [tts/ ^l]
'.2
quontityGinkgolide
A {GAl
1.80 1.55 1.40
r.30
fus/ ml1
tttMl
[ps / nt]
[t'M]
l B0 |.47 0.90
4.41 3.60 2.21 1.27 0 .B 8 0.29
0.60 o.73 0.90 r.03 1.09 I.18
1.42 1.72 2.12 2.43 2.57 2.79
v.J I
0.36 0.12
n= 2-9 * lc5o = 50% Inhibition of GA + GB mixtures
'concave-down' isobole.It is possibleto have synergyat a pafiicular dosecombinationwith antagonismat a differentcombinationand this is reflected in the isobole. Other approachesto defining synergy are describedby Berenbaum,togetherwith examplesand proofs.
DEMONSTRAfl NG SYNERGYAND POLWATENT ACTIONIN PHYTOMEDICINES Proving synergy even within a single herbal extract would require fractionation ofthe extract,testingeachfraction, recombiningand testingthe whole again,and would alsonecessitate tesringpermutationsof the various fractionsto seehow eachis interactingwith the others.To complicate mattersfufther, herbalistsnormally usemixturesof extracts,and many are traditional combinationswhich are not intendedto targeta particularbiochemical system or enzyme (S. Y. Mills and K. Bone, Principles and Practice oJPh.ttotherapy',Churchill Livingstone, 2000), making evaluation of additive or synergisticeffects even more diflicult. Simple examples of this practicewould be the inclusion of laxative herbsin products usedfor haemorrhoids,or cholereticherbsin digestivepreparations.This is not synergy but more a way of approachingffeatment from several anglesconcurrently,and could be describedas 'polyvalent action', a term usedto include the variouseffectsofmultiple activeconstituentsacting in combination, in harmony and possibly in synergy, and therefore overcomes some of the problemsof defining the overall effect as synergistic where it could eveninclude antagonism,where that appliesto a reduction of undesirableeffects.As a preliminary step in looking for synergistic interactions,it is possibleto testthe effect of individual extractssingly and in combination, which will give an indication of synergy or antagonism althoughno real evidenceasto which compoundsareinteracting. In conventionalmedicineit is now common practiceto use several drugs to treat a singie complaint, such as in hypertension,psychoses and especiallycancer,and this applies even more to plant extracts, sincecombinationsare alreadypresentwithin the plant. There may be sound reasonsfor not isolating individual componentsin some herbs such as ginkgo, St John's wort and ginseng,becausethese are traditionally usedas standardized extractsfor which we havepositiveclinical data.Furtherexamplesaregiven in TableT.2. Otherreasonsfor not isolatingcompoundsinclude the situationwhen the active ingredients arenot fully known, or when they aresuspectedof beingunstable,such as with raspberry leaf. If their effects involve synergy the situation is even more complexand bioassay-ledfiactionation,the normal method
s Y N E R GrYN R E L A T T T OO NT H EP H A R M A C O L O G TACCATLT OO N Fp H y T O M E D t C t N A L S r
lable 7.2
Plontsfor which whole extrocts ore normolly used.
Reosonsfor not isolotingindividuol compoundsfrom extracts
Exomplesof herbol productsinvolved
Exhocts knownto contoino rongeof similoroctivecompounds, withwell documented clinicoloctivityfor wholeor semi-purified extroct
Aloe,Arnicq,Bilberry, Blockcohosh,Chomomile, Devil'sclow, Echinoceo, Essentiol oils,Feverfew, Ginger,Ginkgo,Koreonond Siberionginseng,Globeortichoke, Horsechestnuf, Liquorice, Senno ond olheronthroquinone loxotivedrugs,StJohn's wort,Volerion
Unsureof ocliveconstituenf(s) evenif somechemistryknown
Clivers,Chostetree,Fringetree,Howthorn,Kelp,Rospbenyleof, Possionflower,Nettle,Oots,Wild lettuceond monyoihers.
Aclivesknownor suspected to be unstoble
Gorlic,Ginger,Hops,Volerion
Synergyor onlogonism knownor suspecled
Artemisio onnuo,Connobis,Flovonoid confoining drugs,Ginkgo, Kovo,Liquorice, Rosemory, StJohn's wort,Sow polmefto, Volerionond monyothers.
used for identifying actives, and isolating lead compoundslbr drug development,must be usedwith cautioll sincethe existenceof interactions would render it useless(seeP. Houghton. Phl,tother.Res, 2000 14(O:419-123). The main activesofeven such an irnportantherb as the antidepressantSt. John'sworr (.Hypericumperforatum)are stil1underdiscussion, andeveniftrue synergyis occurring,it wouldbe unwiseto exclude,by over-purification,any constituentswhich may contributeto efficacy. There are numerousother herbsin the sameposition.where the phytochemistrymay be well documentedbut the actualcontributionof individual componentsto the overall eff'ect has not been ascertained. Examplesinclude hawthorn(.Crataegusoxl'cantha)as a caldiac tonic; the coneflower, Echinacea spp.. as an immunomodulator; hops (.Humulusluptilus')as a sedative;black cohosh (Cimifuga racemosu) and chasteberry(Vitex agnu,s-castus) as hormone-balancingagentsin women; saw palmetto(Serenoarepens)as an antionandrogenfor proprocumbens)as statichyperplasia,and Devil's claw (Hurpagopl't,*tttm an anti-inflammatoryagent.In othercases,the activesareunstable,and 'protection' of the herb or whole attemptsto remove them from the extractcould renderthem inactive.Here the obviousexamplesare garlic (ALliumsativutn)and valerian \.Valeriantrspp.tiGarlic is often formulated as a product containingthe precursoralliin. and the enzyme alliinase which in solution (i.e. in the stomach)liberatesthe active allicin and other unstable,but still active, decompositionproducts. This is not synergy,but efTectivelya drug delivery systeml-Interactionsin vivo may also occur between combinationswhich enhance or hinder therapeutic activity by affecting absorption, metabolismor excretion.Someof thesecan be seenin vitro. suchasthe complexingof plant polyphenolsand tanninswith many drugs,which This doesnot seemto be could theoreticallyreducetheir efTectiveness. a real problem, otherwisetea drinkers would find many of their prescribedmedicinesinactive. Other interactionswill only be seenclinically, suchas the efl'ectsof cytochromeP450 enzymeinductionwhich are only seenafter a pedod of treatment,as may happenwith concnrrent ingestionof grapefruitjuice. Examplesof synergyor polyvalent actiontakenfrom the literaturewill be dealt with accordingto whether they are thought to enhanceactivity or reduce toxicity.
ENHANCEMENT OF ACTIVITY(l). Single herb extrqcls
is likely that they work together.Recently synergyhas been demonstratedbetweenginkgolidesA and B using a plateletaggregationassay (seeTable 7.1 and Fig. 7.1). Ginkgo is usedfor two main indications: asthmaand cerebralinsufficiency.and thesecan be partly relatedto the differentconstituents.The ginkgolidesarediterpenesand areknown to be plateletactivatingfactor (PAF) antagonists.Numerousstudieshave shown that the ginkgolides antagonizemany of the effects of PAF, including bronchoconstriction,bronchial hyperresponsiveness, plateletaggregation,and allergicresponses. They may contributeto the efficacy of ginkgo in cerebralinsufficiency but their main benefit is probablyin inflammatorydisordersincluding asthma.Ginkgo flavones are also anti-inflarnmatory.the combinationbeing consideredadditive and possibly synergisticin effect as well as increasingblood circulation to the brain, and a total ginkgo extractactsas an antioxidantactivity in brain preparations.Clinical studieshave shown ginkgo to be effectivein improving cognitive function (U. Rigney et al., Ph\tother Res.1999,13 (5) 408-415) as well as the early stagesof dementia;the preparationusedbeing a total extractand notjust the flavonoids.This suggestsa polyvalentas well as synergisticactivity. Kovo Synergyhasbeenproposedas occuring in the effectsof the psychoactive herb kava, Piper ntethysticum,which is used traditionally in the South Pacific islandsand more recently elsewhereas a sedative,anxiolytic and mild hallucinogen.An extractof ground kava root and several isolatedknown sedativecompoundswere testedlbr their effects on the CNS, using a mousemodel of antagonizingclonic strychnineinduced convulsionsand death.The total kava extract was effective, and dihydromethysticin(DHM) determinedto be the most potentcomponent.A reconstitutedmixture of kawain, dihydrokawain,methysticin. dihydromethysticin,yangoninand dihydroyangonin,in the same ratio in which they were fbund in the crudeextract,was then testedand relatedto a potencysimilar to that of DHM. Howeverasthis compound representedonly 5% of the total extract, and the other constituents wereknown to be lesspotentthan DHM, the mixture wasconsideredto be synergistic.This was corroboratedwhen the anticonvulsantaction of yangonin and desmethoxyyangoninwas shown to be increased when given togetherwith otherkava constituents.Furtherdetailsof the complex pharmacologyof kava are availablein the review by Y. N. Singh and M. Blumentha\(HerbaLgrttm,1991, 39:33-56).
Ginkgo
Volerion
fbr their ginkgolide, Most Ginftgobiloba preparationsare standardized flavonoid and sesquiterpene content(for folmulae seeFig. 25.5), and althoughthesegroupsof compoundshave discretemodesof action,it
Severaldifferentcompounds(Fig. 25.2) presentin valerianhave sedative activity.The natureof any interactionsis not understood,but there is circumstantialevidencethat synergismis present.Holzl (pp 55-75
S F DRUGS T H EP L A N TA N D A N I M A LK I N G D O M SA S S O U R C E O
lirl::arl.l tii,uipii itrirrt. i:tri:,ri: :li':i.
uu'i,rli
timesthat expectedfrom a summationolthe individual components.It l' in'. Metlicinal and Arotnotic Plants-lndustrial Pro.files' Vtl is also possible that an unknown compound nray be involved' and Valerian.Harwood, 1997) has reported that an extract of ValeriancL Iruch furthertestingwould be needed.illustratingonce againthe diffiofticirutlisreducesglucoseconsumptionin areasofthe rat brain; howculties in proving synergy.Ginger is yet anotherexample of a cheneverindividuallyvaltrate.didrovaltrate,valerenicacidandvalerenone ically unsttrblelange of compoundsbeing responsiblelbr activity' and showednosuchelTectalthoughtheyareknowntobesedativeinother probably acting synergistically(S. M. Beckstlom-Sternbergand J A' biological systems.This may be an indicationthat synergismis occurHerbs and Edible Fungi I 994' Elsevier)' Duke, pp 210-223 in: STrrccs, ring(orbecausetheresearchelshavenotyetidentifiedtheactualconr. pound responsiblefbr this activity)
ENHANCEMENTOF ACTIVITY(2). Multiple herb
5t John's wort exlrqcls StJohn'swortisoneofthemostimportanthel.bsir-rclinicalusetoday. medicine typesof constituentare implicatedin its action TroditionolChinese Again several
r
SYNERGY IN RELATION TOTHEPHARMACOLOGICAL ACTIONOF PHYTOMEDICINALS n.ttle root. Urticu tlioictt. with pygeum bark, Pt'geurn africttnunt, \onretirneswith otherherbs.This cornbinationhasbeenshownto have :r nergisticactivity in an in vitro system.where its effect againstthe enzymesaromataseand 5-cx-reductase. which are irnplicatedin BHP, qcre tested.Nettle root extractalone was only efl'ectiveagainsteither enzyme at high concentrations.and pygeum bark. although more activeon 5-o-reductase,had only weak activitv againstaromatase. The combinationshoweda potencyequivalentto that of pygeurnbark on -5o-reductzrse. and aromatasewas significantlymore inhibitedby a mirture of the extracts than by each sepalately (R. Hartmann et al.. Plty'tonretlic ine. |996, 3/2: I 2 | - I 28). A mixture of lbur herbs used in Italy to aid digestion.known as Amaro MedicinaleGiuliani.is a classicexampleof herb con.rbining. The preparationhas beenshowr-rtcrbe not only clinically efl-ectivebut the mixture to be more eflicaceousthan the exll'acts.which were tested in pairsconsistingofgentian + rhubalb.and boldo + crscan, rgainst placebo.Althou-ehsynergywas not unequivocallyproven.the ingredients are certainly working together and a case can be made fbr the inclusion of each.Gentian,Gentiutu lutea, and lhubarb, Rheum spp.. both stimLrlategastricsecretion(at high dosesrhubarbis laxative.but in this preparationthe dose is low); boldo leaf. Petunusboltlu.s.ts reputed to increase bile f'low. and cascara, Rhantnus purshiuta, increasescolonic peristalsisby stimulatingthe myoentericplexus.The mixture was shown to increasepancreaticexocrine secretionand to accelerategastrointestinalmotility in humans,to a greaterextentthan the pairs of extractstested.It is therefbrethought to act as a digestive by stimulatingthe appetite,aiding gastric,pancreaticand biliary secretions, and enhancingcolonic transit (G. Blunetti et al.. pp 15-21 in Natural Drugs tutd the DigestiveTract. 1992.EMSI. Rome).
Stimulatin-sCOMplex) r'accines,where they enhancethe antibody responseto an antigen (R. Bomfbrd, Phttother. Re.i, 1988, 2: 159 16,1).At presentthey are rnore widely used in veterinarymedicine. It is likely that other saponin-containingdrugs have a similar effect as well as other.as yet not defined,synergisticactivity.
Drogon's blood The sap of Croton draconoitles and C. lechlerl, also known as Dragon'sblood becauseof its dark red viscousappearance. is usedtopically to aid wound healing.The healingqualitiesarerhoughtto be due to both physical and chemicalproperties.The occlusiveaction of the sap.which dries to tbrm a serni-perrneable covering,helps to prevent penetrationofpathogens.and within this protectivelayer the polyphenols act as antiseptics.Thc anti-inflamrnatoryand antioxidantcompoundsalso presentin the sap are thoughtto contributeto the healing and protective action (Z-P. Chen e/ al.. Pkmrtt Med., 1991, 60 -541-545).In this case.the plant extractis cleally polyvalentin action althoughprobably not exhibiting true synergy.
ATTENUATION OF TOXICITY
Liquorice Liquorice. Glvyrrhiz.a glabrct,is widely used for its antiulcer',antiinflammatory and antihepatotoxicproperties.One of its major constituents,the saponinglycyrrhizin (Fig. 24.12)exhibitsactivity in all of these areas.but so also do the flavones. isoflar,onesand chalcones. Glycyrrhizin is certainlyresponsiblefor the rnore seriousside-eff-ects relatedto its corticosteroid-like activity(suchas Cushing'ssyndrome) associatedwith ingestion of large amountsof liquorice.A small study on the bioavailabilityoi glycyrrhizin when given on its own or as part of a liquorice root extlact showed that absorptionof glycyrrhizin is OTHERMISCETTANEOUS SYNERGISTIC lower when taken as the extract than il given alone (G. CantelliINTERACTIONS Forti et al., Environ. Hedth Perspecr.,1994.102 (Suppl.9):65-68). Flovonoids Monitoring of blood and r.u'ine levels in volunteersafler ingestingglyIn addition to the involvement of flavonoids in the herbs mentioned cyrrhizin eitheraloneor as an equivalentdosein liquoricc showedsigpreviously.other examplesinclude that of the Chineseherb QLringhao, nificantly lower levelsin thosewho had takenthe extract.and although Artenti.siaannua. a source of the antirnalarialsesquiterpenelactone the samplesize was small. when a similar experimentwas caried out artemisinin.which is being usedas a templatefor the developmentof in rats the resultswere in agreement.The authorssuggestthat ditTerothercompoundssuchas artemether. J. D. Phillipson(Phltother Res-. encesaredue to an unspecifiedinteractiontaking placeduring intestin1999,13: l-7) reportedwork caried out by his group on the efTectof al absorption.In TCM. an extractmay be addedto a prescriptionpulely the other constituentsin an extractof the herb, where they discovered to attenllatethe side-eff-ects of another,and an example is the use that the llavonoids present,especiallycasticin and artemitin. sigrrifi- of liquorice as a detoxifier.Many Chinesephysiciansinsisr on using cantly enhancedthe in r''ilroantimalarialactivity of artemisininagainst liqr.roricein conjunctionwith aconiteroot and a preliminary study has Plasmodium.falcipttrLmr. Although not clinical evidence,it doesshow producedsomeevidenccto supportthis. When the amountof liquorice that the synelgisticactivity is not just a questionof enhancedabsorp- in a preparationcontainingfixed amountsof aconiteand gingerextract tion, excretion or bioavailability in vivo. Flavonoids are presentin rvas increased,a conespondingreduction in the amount of aconitine many phyton.redicines as well as lbods. and it is possiblethat they play which could be extracted fiom the decoction was observed (P. a role in clinical efficacy which could be synergisticin nature. In Miaorong and L. Jing, pp 28-30, Prrx'. 10rh Ann. Conf. Beijing Unit. anotherstudy. a seriesof con-rbinations of two flavonoids.including CltineseMed., 1996'1. This study indicatesthat the subjectof detoxifiamong others baicalein. genistein, naringenir-r.hespeletin and cation by use of liquorice deservesfurther attention,and it is quite quercetin,was assayedusing inhibition of ploliferation of a human probablethat many of the principlesof combinationsobservedin TCM breast cancer cell line, MDA-MB-435. Al1 combinationsexcept lbr will be fbund to be basedon similar nhenomena. narigeninand hesperetin,which are fairly closely chemically related. showed synergistic behaviour. with the most potent combinations Connobis being those which included quercetin(So et ttl., Nutr. Canc'er,1996, Althou_ghcannabisis generallytakenfbr its relaxantef-fbcts. it hasbeen 26: 161 181).This ofcourse has significantimplicationsfbr the effect known fbr many yearsthat tetrahydrocannabinol (THC), the main psyof diet as well astreatmentof illnesswith ohvtomedicines. choactiveingredient,can induce anxiety in a way not usually associated with the herb. For examplebatchesof cannabisresin from South Soponins Africa which naturally contain negligible amounts .of the nonSaponinsare surtactantswith biological activity so are likely to have psychoactivecannabidiol(CBD), have causeda psychoticreactionin modifying effects on other agents given concurrently. Quillaja users.It has since been verified that CBD can block the anxiety prosuponariu saponins ale used as adiuvants in ISCOM (lmmr.rno- voked by THC in a doLrble-blind procedureusing normal subjects,and
T H EP L A N TA N D A N I M A LK I N G D O M SA S S O U R C E O S F DRUGS the etl-ectwas considerecl to be a particularfbrm of antagonismbetween ratherthana generalblock ofTHC effects,sinceCBD thecannabinoids typicallyhad someactionswhich u'ereoppositcto thoseof TttC. bLrt not othcrs(Ztardi et ul. Pst'cltopharntucologt1982,76,245).Thcreare othcr indicationsthat CBD modifies the pharmacologyof THC and which support anecdotalreports by cannabisusers.This moditying efl'ectcor-rldbe i"eryimportantif anclwhen cannabisbecomesavailable fbr therapeuticuse and substantiates the theory that cannabisextrtrct may be rnore uselul in the tleatnrentof spasticit),and pain disordels would rendcrit unacceptable. thanTHC. whereanxietyas a side-eft-ect
coNcrusroN Therecan be no dor.rbtthat most herbsrely for their ef'fectson a variety ofconstituents andthe icleaof synergywithin andbetweenthemis also Whetherthey areactingin r 1r'u11 synergisticuay -sainingacceptance. ol by additive ef-tectsis not well documentedbut it is important.both for clevelopingmethodsof standardizationas rvell as to frLrtherour is also more knowledgcof mechanisms of action.Clinical evalLration diflicult without knowing the extent to which synerg,voccurs within the herbalpreparation.and it shouldbe further investigatedfbr all these to showthat synerreasons. In the meantimeevidenceis accunrulating gism doesoccurin extractsand mixturesand that we shouldcontinlle r sinto LlseouLwhole extlactswith confidence.ratherthan :rssruning gle chemicalentity is responsible which shouldbe cxtractedand used
alone.This has been describedrs the 'herbalshotgun'approachas 'silver opposedto the synthetic bullet'.It is hor.vever still vital to ensllre that extlactsare standardizedproperly tbr the activeprinciplesknown at the time and that any knoun srnergisticintt-'r'aetions are takeninto account.Synergy.however.aseviclcncccl in herbalrnedicine.may lend to ir-nprovedproductsn'ith incleasedefficacy.reducedtoxicity and in the caseof antibioticaction.lesslikely to leadto rniclobialresistance. The principlesapply to both plant-bnsecl and conventionalrnedicines. and althoughthis is an olclideairr naturalmedicine.it is relativelynew 'Cocktails'of rnedicines. lo conVentional chenrotherapeulic agentsare no.,vbeing usedto treatAIDS and cancerand historicallynanl' people. even if contrar'1' to rneclicaladvice.have alwaysinstinctivelypractisec'l polypharnracy.whetherherbalor not.
Furtherreoding Berenbaurr1\,1 1989What is s_vnelgl) PharmacologicalRer iews ,1I : 9-l l,l I Duke J A. Bogenschutz-Godu in M J 1999Thc syncrgl'principlc at u'or-kin p l a n t sp . a t h o , u e nisn.s e c t sh. e l b i l o r e sa n c h l u m a n s I. n : K a u l n r a n nP B . C s e k e L J. Warbel A. Dukc J A. BricnrannH L Natural proclnctsflom plants.CRC P r e s sB . o c aR a t o n .F L A . U S A . p p 1 8 3 - 2 0 5 lzacldoostM. RobinsonT 1987 S1'nergyand antaqonisnrin the phanracology o f a l k a l o r d apl l a n t s I. n : C r a k e rL E . S i m o nJ E ,( c d s )H e r b s .s p i c e sa n d nredicinalplants:rccctrtacllanccsin botirnt.horticu]tureanclpharnracology. . h o e n i r .A Z . U S A . V o l 2 . p p 1 3 7 l 5 l l O r r x P r e s sP W a g n e rH 1 9 9 9N c u ' t a r g o t si n t h e p h l t o p h a r m a c o l o goyf p l a n t s .I n : E r n s tE (ed) Herbal medicine.a conciseor,erviewlirr healthcareprofessionals Buttefworth Heinelnann.UK
E PART
3
Principlesrelqred to the commerciqlproducfion, quq lity ond sfqndordizslion of nqturql producls
\ ?' f".
/ t\
x{.
A
E
Commerce in crude drugs
HISTORICAT DEVETOPMENTS5 7 CURRENT ASPECTS 5 8 THEFUTURE 60
,il ,ti rlli'
I l{lstoRlcAt trtvELoFmElNtSl.,..'l.,,'....'.l.:.'.'''.',
R. Baker
CHANGING DEMANDS
Like almostall other basic commodities,the trade in crudedrugs is of great antiquity.The necessityibr goodsto be collected,graded,transported and distributedeffectively has rarely been consideredby the pharmacistas part of his or her remit and thus it has been left to the tradel or merchantto perfbrm this lessscientificbut by the sametoken importantgroup oftasks. The essentialsofthe tradeare still very much the sameas they were 10, 100 or 1000yearsago,though the speedand efficiency with which they can be performedhas improved exponentially with tirne. It is well within quite young living memory that one had to book a telephonecall to one's supplierin Brazil, China or India somehoursor perhapsa day in advance:now communicationsby fax, or increasingly popularly by e-rnail take moments to perfbrm, and repliescome with similar alacrity.
59
The absoluteorigins of the tradein crudedrugsareof courselost in the mists of time. One supposesthe first contract for the collection and supply of a drug with a third party in exchangelbr speciecame about when the physicianfound himself too busy to do this relatively menial task himself. The trust the physician had in the collector must have been remarkableas unadulterateddrug was essentialif only to avoid poisoningthe patient! Ref'erences from antiquity to the drug trade are rare, though somemural inscriptionsfrom Ancient Egypt dating back to 3000 ec evidenceknowiedgeof the effect of medicinal plants,and there are in the British Museum clay tabletsfrom the library of King Ashurbanipal (668-626 ec) of Assyria which evidencethat around 2500 ec the Sumerianshad a form of Herbal. By 660 ec around 250 drugs were recognizedby the Assyriansthemselves,some of which were actively cultivated.Hippocrates(f1.46'7Bc) was well acquainted with a variety of drugs (though it is improbablethat any of the works attributed to him are actually 'of his hand'). Theophrastus,like Alexanderthe Great,was a pupil of Aristotle, and latter becamechief of the Aristotelianschool.He listed some500 plantsknown to him, and distinguishedCinnamon from Cassia (an art which, apparently,is being lost in this day and age.at leastby somemanuf'acturers of foodstuffs!). It is instructive to note that the use of the Mercury's or Hermes' caduceus,nowadaysa widely understoodsymbol for medicine, was originally a symbol fbr commerce.Mohammed was said to be a spice trader,and at that time spice traderswere invariably also concernedwith crude drugs,particularlyas many productswere used for both culinary and medicinalpurposes,asthey aretoday.The adventures of all of the major explorers.such as Marco Polo, Columbus, Henry the Navigatorand the like were undertakenpartly with a view to the sourcingof botanicalcrude drugs.The establishmentof the great National Trading Companies,fbr instanceThe HonourableEast India Company, The Netherlands United East India Company and the DanishAsiatic Companywas undertakenwith a similar view. Prior to modern times. there was no real distinction between the Drug and Spice trades,and thus during London's developmentas an entrepot the Guiid of Pepperers of London (later the Grocers' Company)were chargedwith the overseeingof both trades.The fbundation of the East lndia Company in 1600placeda near monopoly in 'East the import of India Produce'into England,and it was from this that the modernGenelal Producetradeemerged. Initially in ad hoc form, and later by way of the lamous 'Coffee Houses', trade in drugs. even until the SecondWorld War, was conductedin the main by auction.The problemwas that the shippersat origin had no direct representationin the consumingcountries,and thus had to engagethe servicesof a broker.The usual format was for the goods,be theysent'on consignment'(i.e. sentspeculatively to London
''li'1i!
lff,|
l
pRrNcrpLES pRoDUCTroN, pRoDUcTS RELATED To THEcoMMERcTAL ANDsTANDARDTzATToN euALrTy oF NATURAL in the reiisonable hopeola salc)or'ex stock'(beingthe propertyofthe importing merchant)to be put up for show priot to auction.The valious broking firms then attemptedto sell the merchandiseentrustedto them by pLrblic auction (the order in which the vtrrious brokers did so beingdecidedby lot) on the basisofthe -eoods placedon view in the r.varehouse or sarrrplesdrawn there and placedon view in the blokels' ofTices.Towardsthe end of the popularity of auctionsin London tiequentlvno. ol alnrostno. goodswele sold at the auction.the auctioneer"susual comment being'see you after'in order to be able tcr ne-qotiate betweenthe shipperand the potentialclrstomera mutually price. acceptable During the SecondWorld War when lines of communicationwith the variousori-tinswere disrLrpted. a certainamountof regulationwiis irnposcdby the gor,ernnrenl. which sau'an end to both the auctions and. due to licensingcontrols,the supply of goods to London on consignment.The trade then took a rrewfbrm: sampleswere displayedin 'I'he blokers' oflices lbl all to see. traders.blokersandbuyersmet in the (and sometimes Coln Exchangeand dealswere doneby word of mor"rth ir-rcovert wl-rispers) in the best traditionsof the trade.This niethod.by the 1960s with in-rprovir-rg communicationsand increasedvolumes. bccameimpracticaland the tradefinall)' took to conductingmost busin e : sh 1 r l p i e lt c l c c o m r n u n r c a t r o n . Obviously.whilst the tradeamongthe dealersandbrokerswas small and therelorealmost self'-regulating, littlc regulatoryinterferencewas reqr,rired.Horvever.the increasingnumbers of firms involved in the tradereqLrireda systen of settlingdisputesin an inexpensiveand swift lnanner.Thus in 1876the GeneralProduceBrokers'Associationof London was fblmed. This body performed a number of furrctions:a united voice to thoseoutsidethe trade.a meansofregulating the trade by meansof a systemof arbitrationand appealand a fbrum in which to voice concernsof interestto membersin general.The GPBA thrived initially, but sLrff-ered as time went on with variousgroupsof the trade fbrming their own Associations,leaving the GPBA with only the smaller partsof London CornmodityTrading as its remit. More recently broking as part of the London trade becameless relevant and the name was accordingly changed in 198I to the 'General Produce Associationof London' and finally in 1985 the name was again 'lntelnational cl-ranged, this time to the GenelalProduceAssociation'. to reflect the current true nature of the trade. Forms of contract are issuedby the Associationfol the r"rse of membersand others(it is probably true to say thatthe IGPAcontracts.termsand conditionsare those rnostgenerallyusedworld-wide). and thereis a thriving, and lelatively switi and inexpensivesysternof arbitrationand appeal.
ASPECTS CURRENT London's pre-eminenceas the drug-trading centre has diminished sonrervhatover the years.Whilst still Lrndeniably the largestmarketfbr' the trade in essentialoils and aromatic chemicals(a trade misnomer' tiequently nsed to denoteflavour trnd perfumerychemicalsand isolatessuchas menthol.camphor'.piperonal,vanillin and the like. be they naturalor synthetic).its role concerningcrudeherbsand botanicals (sr.rch asrhubarb.ipecacuanha andboldo, for instance)hasdecreasedto Hanrburg'sbenefit.andthe tradingof spiceshasmoved substantiallyto Rotterdarn.Nonetheless,when disputesneedto be settled,most e).es still invariablyturn to London in order to obtain a 'fair deal'. The North Arnericantradeis. asit alwayshasbeen.substantiallydiff'erent.Unlike most of WesternEurope.Canadaand the United States of Americaas well as being largeconsumersof crudedrugsare also largeprodr.rcers. Many pl'oductsregularly tradedin the Europeandrug markets emanatealnrost exclusir"elvfrom North America. There a
number of firms hold stocksof drr,rgs(many of native origin but not exclusively so) speculativell',being almost celtain that there will be demandfbr the productheld on their books in the near term. Glanted. there is a trend tou'ards thc European malket usagesof trade. but noncthelessconsumersin North America shouldbe thankful that due to this patternof br.rsiness it is still usuallypossibleto obtain materialsfor immediatedelivery fiom a trader'sstock.ratherthan waiting tbr shipment fiom oligin. There have recentlybeena number of companiesin the USA who have. lbr one reason or another.withdrawn fiorn the trade,andconsequentlythc NorthAn-rericanmarketis edging.however reluctantly,tolvardsthe Europeanmodel. Consumingpatternsin Europehavealsochanged.Therehas been substantialconsolidationof companiesconcernedwith crude and processedbotanrcaldrugs. particularlybLrtnot exclusivelywithin Gerrnany.to the extent that nowadays one large conglomerate, whilst not controllingthe business.hasgreatinfluence.A numberof recentclosuresand rationalizationhavecausedconcern.particularly on the part of consumingcompanies.What efl-ectall of this will eventr-rallvhave on the wor'ld markets for crude drugs has yet to be seen.but the curlent mood of the n'rarketcould rvell be describedas 'melancholic'. Tradenowadaysl-raschangedsubstantially.In relativelyrecenttimes many drugs r.vereimported speculativelywith a view to selling them 'afloat'(i.e. either oncc confln-nedthe goodswere aboardship) or fiom 'the spot' (i.e. with tl-rcgoods in storein a Eulopeanwarehouse,available fbl irmlediate delivery). With changingrequirernentsof biryers. be it eitherlor the quality ofthe goodsrequiredor with changingtrends in the particulardrugs used.this has becomelesscommon, and nowadaysit is more likely that a customerwill requestof a traderan offer lbr a specificquantityof crudedrug fbr shipmentfronr oligin and delivery afiel saf'earrival of shipment,be that in one delively or parts.either againsta sampleor (more rarely) a mr-rtuallyagreedlevel of quality. Whilst the customeris of courseat liberty to approachorigin direct lbr supplies.tht: ad\rantages of purchasingliom a traderare thoseof transf-erenceof risk. If the quality or condition of goods is sr.rchthat on arlival the goods are either of lower quality than that contractedor damagedin someway, or delir,eryis delayedbeyondthe agreedperiod, recoulse is to the local trader from whom one is far more likeiy to obtain settlementrathel than origin where satisfactionof a claim may be far more difficult or in some casesimpossibleto perfonn, due fbr instanceto local currencyregulations.Also if the traderconcernedsupplies the samematelial to various buyersit can fiequently be possible to re-arrangethe allocationson his book to provide the buyer with an 'emergency' delivery should production demands require this. In leturn lbr this servicethe tradel asks a small prernium over the price paid to origin and absoluteadherenceto the contract terms such as delively datesand termsof payment.for it is on this basisthat the price has beencalculatedand thus. by inf'erence,the margin. Margins in the crudedrug tradearecurrentlyprobablyassmall asthey haveeverbeen. For the customerto delay or cancelan order placed,or to delay payment once goodsare delivered.is unacceptableas would be the trader defaultin-e. deliveringgoodslate or of poor quality.A fallacy still holds in the consumerindustriesthatthe 'rapacious'trader makesvastsums fiom his livelihood.This may oncehavebeenthe case.centuriesrgo. but almost invariably nowadaysmargins are confined to low. single figure percentagelevels:representinga small fraction of thosethat the customer is usually making. This point cannot be overemphasized: contracts(which, atter all are basedon the conceptof'equity' or fairness)work both ways and,in London at leastthe old adage'Verbamea pacta'still holds with the traders:it must also do so with the customer. Is it reasonablethat if a traderhas purchaseda specilic product fbr a customer.for which he has no other potentialoutlet.he shor-rld without
;..yiff'-
C O M M E R C EI N C R U D ED R U G S . , ()Llr\cbe lequired not to cl(]llvcl'goodsbecauseof an erro| on the part : rhacustolner. or on a wl-rirl()f the customer'scustorner'l 'fhe qr,rcstion of quality fr.tlrrltl-retrader'spoint of r reu i: irrpr'or'l. Strongcompetitionat ol'iginhasseena -qcneral increasein quali. .r.ross the boarcl. Many goods are still traded on the olcl :,''efiptionssr-rch asthe 'Comr"non Round'or''Flat'gladesof Chinese 'Mossel ::rrburbor Bny'or' 'Port Elizabeth'aloesflom SouthAfl'ica. l , t n r i s a l w a y ss u l ' et h a t ' M o s s e B a y ' a l o e sw i l l p a s st h er e q u i r e m c n t s i the British Pharmacopoeia. whereas'Port Elizabeth'will rarely.if - . cr. do so. Nonetheless. lrore and nroretradingis being undeltaken :r the basisofpre-shipmentsantples.and shippersat origin teel hap.'rer to do so ltow thatthe *,ot'ld-widenetworksof cor.rrier sen ir:esare :r nl1gg.as a sampleo1'thespecificlot in cluestiorr can be on the desk t thc tradert.nvoor three days aftel it was dispatchedtrom origin. .,thcrthan the two or three weeks it used to take. or two or thlee rr{)llth\befbrethe adventof airntail.Some prodr-rcts are stiil traded ..n()nthe basisofoutturn analysis.therebeingdefineda n0rrinalcon.-'ntoi isolateupon which the price of the contractis calcr.rlated, a :rininrunrlevel below which the parcel is rejectable.and an agreed :\r.()-l'rta fonrula fbr adjustingthe irrvoicevalueonce the qLralityhas '.ecrrindependently established. Thc'r',orld from a commelcialpoint of view haschangeddrarnatical\ ()\er the pastlew yeals.Thc changein EastemEul'opeffom state..rpitalism(lcssnccurately comr.r-rurrism) to free-market economies has .:i.()changcdsupplypatternsti'om thesecountries.Fifieen yearsa-uoas . llil(lcf. ()llc hJd ()tlc ()t l\\(r \otttce CompiLnie\ lrr c()nlijel itt a couttl11.
Be it desirableor ot.herwise. therehas beena move over recentyears lor the goods to be extractedat ori-uinand the isolate rather than the bulk drLrgbeing shipped.At flrst glancethis has many advanta-ees for irll concerned:the lesstechnicaliydemandingwork is pelformedat ori-Qinwherelabor.rrand iacilities arelessexpensiveand wherelocal regulations as legards.say,elfluent are less stringent,which addsvalue to the goods and increasedfbreign exchangeearningsfbr the country of origin. It is of advantageto the trader as there are less fi.eight.wat.ehor.rsing and handlingcostsinvolvedand finally it givesthe customer an (at leastserni-)preparedproductwith which to work thus elirninating the erpensiveand laboriousinitial extlactionprocesses.However, this conceptis a double-edgedsword.One may be temptedto be a little more cLrrsoryas regardsquality control ofthe inward product.particularly atier a long run of -9oodexpcrience.If a parcelof materialis found to bc of inferior quality lbr whateverreason.the lossdue to this tendsto be the -Qreater as concentrationof the product leads frequently to the acquisitionof t-ewer.largel parcelsand (this being the most important) deprivesthe corrsumingcountryor,'ertime of the knowledgeand equipment with which to perform thc completeprocess.In relativelypeaceful times this last considerationma1' be irlelevant; but if lines of communicationwith the tladitional suppliersale disruptedby war or naturaldisaster', theneventhoughit would bc possibleto obtainthe raw rraterial fiou'r elsewherethis wor-rldbe of little use if the know-how and/or equiprrentrequiredtbf thc contpleteextractionprocessis lost. Comnrercially this is of little moment.but fbl thegreater-eoodit is vital not to losethe expcrtisethat hasbeenbuilt up over lnen; centuliesin the consumingcountliesfbl tr smallcostsar,ing.
...hc-reas now thereis a plethoraof suppliers. Rcliabilitl,haschangcdas .' ell: tbnr-rerlycontractswct'esacrosanctbut nowadaysattitu.lesare a rttlcrnorerelaxed.Further.in orderto increaseincome.commercially . .tluablecrudedrugsarebeingproducediu colrntrieswhich pre",ioLrsly CHANGINGDEMANDS :r.r!lnot doneso.Indiais an ercellentcasein point.This countryhadfor' rirn\ \rear\ beena large nett importer of ipccacuanharool (C. ucunri- The patternof demandis changing.Recentinroadsinto the market by ,;/(1 ) 1brbothinternalconsuutption anclre-exportof finishedalkaloids. 'Nutraceuticals'(bio-actiVeproductspresentedas'dietary supple(;r.rnted.thcrehas beena small local cr.rltivatictn of this material.but ments' or similar')have changedthe demand lbr various botanicals. :rc qLralityuntil recently had becn lelativelv poor. Ol'er thc past f'erv Productsof which one would have heard rarely some l0 or 20 years .:.rrs.however.Indiahasbeenableto ofl'eripecacuanha of high qual, ago are now objectsof daily discussion.The lack of licensingof these :r rnd at vely competitivelcr,'els.These developmentsall tenclto productsis a point of major concern.The majority of the botanicals :rtlucethe price of the drugsin question.Br-rtat what point doesthis inl'oh'ed are well documentedand every companythat the writer has ...'!()urcdeleteriousto the market?The answermust bc dilectly related come acrossto date has beenresponsibleand reputable.However.the ',' cconomics.Subsequent to the changesin EastcrnEuropethe prices relatively high malgins that theseprodr.rcts attractwhen ofl'eredto the ,1 \orne drugshar,cfallen consistently. Thus in a placewhere aspir- consumer with this cachet and the comparative lack of technical .,trrrns tbl pelsonalinconrearelising tast (not unreasonablv. considering expeltise neededto manufacturethe product tbr presentationto the :'.i\t historyandcurrentcircurnstances). thc incorrefiom theirproduce public could probablyin time attract(or perhapshas alreadyatrracted) . consistentlyfalling. Pelhapsdue to increasedcompcririon.or cou- r,rndesirable elementsinto the trade.with potentiallydisastrousconse.'r:elv fior.r.rf actorsat the point of consunrption.the plicc falls. The quences.Origins are off-eringprepaled extractsthat need little rnore :rtial eil'ectoflovn,erpricesis in the shortterrnfrequentlyto raisethe than r.r.rixing with an excipientand tabletingor filling into freely avail..Lrelin'of the prodr.rct delivered.with a vieu to securin-e the next order. able capsules.If nutraceuticals,originally and naturally the domain of ilrr\\e\'€t'. if low pricesaresr.rstained theneventuallythe qualityof the the chemist'sshop.the supen'narket or the health tbod storeappearat - r,,pslowlyfallsastheproduceris r"rnable to f'ertilizeor tendthecropto more unlikely venuessuch as open-airmarketsand car boot sales(as rL' r)ptimllm ler,'els.Adulteration either with admixed drug or mole this traderhas seen.on occasion)then the potentialproblemsmultiply. ..rhtlv vn,ithu,ater(i.e. incleasingthe moisturecontent)solnetimes It is almost inconceivablethat well,known brandedproducts rvithin .,kesplace.Finalli' the fatnteror coliectoris presented with the deci- theil statedshelf lif'e would errergeat thesesites,but -eivena substan-:r)nofeithel continuingthc cultivationofthe ploductin theabsence of tial price advantage1othe public. is it not reasonableto speculate.be it ''r,,fit or to changecrop in tavour of one that gives a return. In thc by accidentol desi_en tlrat any problem startingwith a retail outlet of r rr|stcrse sLrpplies of a drug could well ceasefbr lack of protit to the this type attachedto goods of questionableorigin woLrldreflect badly 'r{)ducerwhich.whentakenin thecontextof thepriceat which the fln- on the industry as a whole?Licensingboth ofthe ntanufacturerand the .hcdproductis retailedto thepublic.is alrrostinsignificant. lt may be retailer would minimize this risk. even i1 that licensing wele far less :r.rtthe readeris taken abackby what appcarsto be a hard-nosedtrad- stringentthan that requiled for dispensing.Coincidentally,and as an ,: nrakin-ea piea fbr a fair deal to the ploducer';tl-ris.however,is not the aside,at the time of writing H.M. Governmentissuedan urgent warn-.,.c. The writer would lar prel'erto have a steady.susttrinedbusiness ing regardingthe interactionsof St. John'swort. a common nlltraceut. cr an extendedperiodratherthan a phenomenalshorttrading 'boom' ical, with oral contraceptivesand HIV medication.It is probablyfair to .:r.1 thereafter be out of busrness. expectmore instancesof this type in the future.
E
PRINCIPLES RELATED TO THECOMMERCIALPRODUCTION,QUALITYAND STANDARDIZATION OF NATURALPRODUCTS A relatively new term that has entered the ntarket is 'organic'. Certainly, valerian root and senna pods are amon-qstthose organic pharmaceuticalproductsavailable.In spicesand herbstherehasbeena substantialdemandfbr materialsthat c.ury this cachet.At government and sr,rpra-government level therearein placeregulationson what mily be describedas 'organic'and many organizationsthat monitor prodlrcers, tradersand manufacturerswho handle materialsof this description. Botanical medicinesare perceivedby the public as a pref'erable alternativeto extensivelypreparedor syntheticmedicines.and thus in orderboth to presenta more 'green'imageand to steala marchover the competitor,the conceptof 'organic'productionof pharmaceuticalraw materialswill undoubtedlybecornemore widespread.The problem with this is the size of the martet itself. The cost of employing an inspectorfrom one of the Europeanor Amelican certilicationorganizittions could well be f1000 (USSI600) daily. [f a collectorof. say. yohimbe bark in WestAfrica who blings to market 1000kg per annum is to have his few treescertifiedas 'organic'(whichthey almostcertainly would be) it would cost some years' wofih of income to obtain that certiflcation.hnpractical,yet if the demandfor 'organic'yohirrbe bark were to becomecontnterciallyinterestin_e then a largecommercial concernworlld move in to fill the gap.to the greatdetlimentof that collector'slivelihood.And all because(in thewordsoia well-knowntrader in botanical raw materials): 'the paper's more important than the goods'. We in the West are por-rringvast sums of money into the less well developed parts of the world to enable people such as this hypotheticalcollector to make a reasonablelivin-g,but with the other hand are stoppinghim frorn profiting from our money and his eflblt by what amolrnts to little more than a chan-eein fashion. There also tbllows fiom this point a lLrther concern.Recentlythe sLrpplyof organic nutmeghasbeendiflicult. due to the lact that goodstiee of prohibited levels of aflatoxin could not be obtained. The inabilitv to use
modern techniquesof cultivation (due to the requirementsof the 'organically-rninded' consumer)by direct conseqLlence can causethe ploduction of inferior quality goods: thus one could well ponder.the sanity of paying a substantialpremium (three-tbldin some cases,and at times more) fbr goodswhich, when viewed objectively.are of relatively poor quality.
THEFUTURE In rny f'ewyearsin the tradeI haveseenthe total demiseof the telegram as a tradingtool. and telex is goin-qthe sameway. If the rate of change continuesas it is. by the time this appearsin print the lax will be considered a quaint, slow fbrm of communication. E-mail may well be superseded within a shorttime. The tools of the tradechange.but the methods and principles do not. Trade is based on mutual trr"rst.If the supply of a raw materialneedsto be guaranteed,then thereis only one possiblemethod of obtainin-ethat guaranteedsupply. and that is from one's pret-erredtrader.If the tradertiom whom you obtain yourraw materialssays that the goods will be deliveredat a certain time then,within the tenns of the contract.that traderwill move heavenand eafih to do so.just as the trader'spredecessors did 10, 50 or 100 years ago.Many is the time that I have listenedto the cornplaintthat, 'Origin haslet me down, I need5 tons of XYZ root next week'. If supportis not tbrthcoming in ordinary times. then where is the impetus on the trader's part to plovide supportto those who require it in difTicult tirnes? Perhtrpsaccounting and pr"rrchasingdepartmentsin manufacturing organizationsshouldcarefully considerthis questionwhen looking at the 2c/cor so saving they make (or more often appearto rnake)when purchasingdirect fiom origin. As an insurancepolicy. the tladeris very inexpensive.
EI The crucledrue $'hich re:rchesthe pharntacer-rtical manufactulinglinc r,villhavepassedthroLrgh vadousstages.all of lvhichinfluencethe nature and amountof activeconstituents prescnt.Theseaspectswill be conside r c d u n c l e rt h e h c a d i n g s ' E n r , ' i r o n m e n C t aol r r d i t i o n s ' . ' C r - r l t i v a t e d and Wild Plarrts'.'Collection'. 'Drying'. 'Stora-re'ancl 'European Regulations'.
Production of crude drugs
ENVIRONMENTAT CONDITIONS Plant growth and dcvclopmenl.and often thc nature and quantity o1' secondarymetabolites.are atl-ectcdby tentperatr-rre. rainlhll. aspect. lengthof day (inclLrding the qualityof Iight)and altitude.Suchef-tecrs have been studiedby glou,ingpalticularplantsin difttrent climrtic areasanc'l observingvariations. The lindingsof suchresearch ale illustratedby work on cannabisby El-Kheiret til. in 1986in n'hichsccdsof cannabis.grou,'nin En-elandand rich in CBD and dcvoid o1'THC..,vhen cultivatedirr Suclanstartedto produceTHC in the flrst generationand in the secondgcnerationcontainedup to 3.3% THC r.iith a furlher' dccrease(dorvnto 07c in some plantsl of CBD (scc 'Cirnnabis'fbr cxplanrlionof chemistry).However.it is rmpossiblcto controlall the variablesin sLrchexperiments.and speciallaboratorics(phl,totrons) have beenconstrLlcted in u'hich all the factols are indcpendentlvcontrollable.Even so. a rneanin-etr-rl erpressionof the rcsnltscan ofierr prcscntsorneclifficr-rlty. For-example. a particularfactorma!' leadto the developmcntof a srnallplant which. rvhcnanalvsedon a percentage drv weight basis.indicatcsa high ploportion of ntetabolite.even thoughthe olerall yield per plantcouldbe quite low. Conversely. certain nutricntsnravresultirrthe productionof lrrge plantsrvith a someu'hat low analyticalfigure tbr constituentson I percentagcdr,vrr'eight basis.but yield per plantrrrayexceecl thatofthe control. Temperoture Temperature is a majortactorcontrollingthe developntent and rretabolisrnof plants.Althoughcachspeciesliasbecorneaclaptecl to its orvn naluralenr,ilonment. plantsale freclr-rently able to exist in a corrsidet'able ran,seof temperature. Nlanl tropicnland subtropicalplantsu'ill glow in temperateregionscluring summel utouths.but lack fiost |esistance to withstandthe rvinter.In general.thc highesttenlperatllres are erperiencedncar the Equltor. br,rtas the tempcratllrefalls lbout 1"C fbr every200 rr of elevation.it is possiblein" sa1,. Jan'raica to liave a tropicalclimateon the coastand a tenrpet'ate one in the mountains. The annualvariationsin tenrperttuleirrL-ju\tar irnportantas thc tcntperatureof the hottestmonth.At Sin-eapore thc annualrangeof temperatureis as iittle as 1.5oC.whereasMoscou. with its lrot summers and cold wintcrs.has a rangeo1'29.3"C.In gencral.the fbr"ntation of volatileoils appearsto be enhancecl at hi-shertcnrperatlrres. althou-gh 'fhe very hot claysnray lead to an excessphysicnlloss of oil. mean optimum temperatuletbr nicotincproductionin NicotitutL/ |rr.irrr',r is 2 0 ' C ( l o w e ra t I l - 1 2 ' C a n da t 3 0 " C ) .S c v c r a al u t h o r sh a v ei r r d i c a t c d thattixed oils producecl at low ternperatures containfatty acids$'ith a highercontentof doublebondsthan thosefbmted at higherternpera-
ENVIRONMENTAL CONDITIONS 6 l
tlll'es.
CUTTIVATED AND WIID PLANTS 62
Roinfoll Thc in-rportanteff-ectsof raintall on \,egetatioumust be considcredin relationto the annualrainfall.its distributionthronghoutthe 1,ear.its effect on humidity and its cflbct coupledwith the I'ater-holding propertiesofthe soii. Variablcrcsnltshavebeenreportedlor tl-reproduction of volatileoils underdiff'elentconclitions of lainfall anclmav in some i n s t a n c c sb e c o u p l e c li v i t h t h e d e v e l o p n t c not 1 ' s l a n d u l a rh a i r s . Continuousrain can leaclto a loss ol watcr-solubie-subslances fioni
COLTECTION63 DRYING 64 STORAGE 65 EUROPEAN REGUTATIONS66
PRINCIPLES RELATED TO THECOAI1MERCIAL PRODUCTION,AUALITYAND STANDARDIZATION OF NATURALPRODUCTS lear,esandloots by leaching:this is known to apply to someplantsploducing alkaloids.glycosidesand evenvolatile oils. This could account fbr loll,yields of someactiveconstituents in uet seasons tiom plants whosegeneralcondition appearsto be good. (Tinnevelly senna)it has been shown that With Cas.rlaongustill)liLL short-termdroughtincreasesthe concentrationof sennosides A+B but in the longel term causesloss of leaf biomass(H. Ratnayakaet c1.. PlantaMedit u.1998.64. :l3ll). Doy-length ond rodiotion chorqcteristics Plantsi'rry much in both the amountand intensityof the light which they requile. In the rvilil statethe plant will be found where its shade reqnircmcntsare met. and undel cultivationsimilar shademust be plovidecl. In certain casesresezrrchhas shown that iight is a factor r"'hichhelpsto determinethe amountof glycosidesor alkaloidsproWith belladonna.stramonium r.nd Cinchona ledseriona fLtl\ dr-rcecl. sunslrinegivesa higl-rer contentof alkaloidsthan doesshade.At Gifsur-Yvetteerperimentsindicatedthat with Datura strailtortituut',tr. nttulu ).ongexposureto intenselight brought about a sharpincreasein h1'oscinecontentat the time of flowering. An important in viv'ctrcaction in the fbrmation ol thc antitumour alkaloids of Cctthttranthus roserr,ris exemplified by tl.redimerization of the indole alkaloids catharanthineand vindolinc leading to vinblastine;Hirara et ttl. (.PlrtntaMed.. 1993, 59, 46) demonstratedthat irradiation of intact plants with near ultraviolet light in the ran-ge290-380 nm (peak 370 nm) stimulatesthe synthesisof dirneric alkaloids.probably by inducing catharanthine oxidationas a tliggerrcaction.This observationhas supportfiom ln l'ilrc studies. It hasbeenshownthat underlong-dayconditionspeppermintleaves plants grown contain menthone.menthol and tracesof menthofur"an; undershort-dayconditionscontainmenthofuranas a major component a long photoperiodtbr young leaves of the volatile oil. Fr.rrtherrnore activates the reduction path\vay with conversion of menthone to Inethol.ln studieson the day-night changesin the relative concentrasr'lveslrl.s and other species tions of volatilesfiom flowers of Nicoticutct a rrarked increase(about tenfbld) in aronraticcornpoundsincluding benzyl alcohol was detected at night. whereas no increase in the volatiies(e.g.linalool, caryoph-vllene) originatingfi'om the mevalonic acid pathway(q.r'.)was noted(J. H. I-oughrinet al., Phytochem.,1990, 29.2113\. metabolitesis The daily variation in the proportion of secondeLry plobably light-controlledand is discr-rssed more fully under 'Collection Times'. Many plants initiate flowers oniy in certain day-lengths,and uhere flowering is essentialthis lactor must be carefully considered before plantingin a new region. Presenceor absenceof light. together u ith u avelenqthl'ange.havea markedeffecton the secondarymetabolite prodr-rction of someplantsin tissuecr.rlture. Thc t1'peof radiationwhich plantsreceiveis alsoimportant.With Ocirrtttntbtt.silicum. C. B. Johnsonet ttl (Pltl'tochemistrt'.1999.51. har e fbund. in relationto herbsraisedurrderglassand leceiv507). in-qno UV-B radiation.that supplementary UV-B ladiationincreases lcvcls of both the phenyl-plopanoidsand terpenoidsof the ieaves. Flavonoiclsand anthocvaninsare also known to be int'luenced by UV-B radiation.Depletior-r of the ozonelayer and the consequent elfect of incleasedradirtion at the earth'ssurf'acehasbeena topic of much recentspeculation.Concerningmedicinalplants.R. Karouson et ul. (.Pltvtotlterttistr.t'. 1998, 49, 2273) r'aisedtwo chemotypes of Mentlta .;1tit'utuuhich were subjected to increased UV-B radiationequivalentto a l5% ozone depletionover Patras,Greece. ln one chemotypeessentialoil production was stimulatedby the treatmentwhile a similar non-significanttlend was noted with the other.
Altifude The coconutpalm needsa maritime climate and the sugarcaneis a lowland plant. Conversely, tea. cocoa, coff-ee,medicinal rhubarb, tragacanthand cinchonarequireelevation.In the caseof Cinchona succ'irubrathe plantsgrow well at low levelsbut producepractically no alkaloids. The bitter constituentsof Gentiana lrilea increasewith altitude. whereas the alkaloids of Aconitum napellus and Lobelia influta and the oil content of thyme and peppermint decrease.Other oil-producing plants may reach a maximum at certain altitudes. Pyrethrum -eivesthe best yields of f-lower-headsand pyrethrins at high altitudes on, or near, the Equator. It is therefore produced in East Afiica and north-west South America. However. vegetative growth is more lush under irrigated conditions at lower altitude, so the propagationfarms (fbr the vegetativemultiplicationof plants) are, in Ecuador.situatedat lower levels than the final commercial farms.
AND WIID PLANTS CUTTIVATED Certain drugs are now obtained almost exciusively frorn cultivated plants. These include cardamoms.Indian hemp, ginger, and peppermint and spearmintfbr oil production. Others include Ceylon cinnamon, linseed.fennel.cinchonaand opium. In other casesboth wild and cultivatedplants are used.Someplantshave beencultivatedfrom time immemorial (e.g. l1ax, opium poppy and coca). Others are now grown becausesuppliesof the wild plantsareinsufficientto meetthe demand or because,owing to sparsedistribution or inaccessibility, collectionis difficult. Cultivation is essentialin the case of drugs such as Indian hemp and opium, which are subject to govemment control. and recently for thosewild plants in dangerof over-exploitation and which have now been given CITES (q.v.) listing. In many casescultivationis advisablebecauseof the improvedquality of the drug which it is possibleto produce.The improvementmay be due to the following. (1) The power to confine collectionsto species,varietiesor hybrids which havethe desiredphytochemicalcharacters(e.g.aconite.cinnamon.f'ennel.cinchona,Labiatedrugsand valerian). (2) The better developmentof the plants owing to improved conditions of the soil. pruning.and the control of insectpests,fungi, etc. (3) The better facilities for treatmentafter collection. For example, drying at a correcttemperaturein the casesof digitalis,colchicum, belladonnaand valerian,and the peelingof cinnamonand ginger. For successin cultivation it is necessaryto study the conditions under which the plant flourishesin the wild stateand reproducethese conditionsor improve on them. Small changesin ecology can aff'ect plant products;thus,satisfactoryrubbertreesgrow wild in the Amazon basin but cleared areasconvertedto rubber olantationshave been a lailure.
Soils Dil}'erent plant speciesvary enormously in their soil and nutritive requirements.and this aspecthas receivedconsiderableattentionwith medicinalplants.Threeimportantbasiccharacteristics of soilsaretheir physical.chemicaland microbiologicalproperties. Variationsin particle sizeresult in diff-erentsoils rangingfiom clay, via sand.to gravel.Particlesizeis one factor influencingwater-holding capacity, and some plants (e.g. Althaea o.fficinalisl which produce rnucilage as a water-retainingmaterial contain less mucilage when grown on soil with a high moisture content. Although particular specieshavetheir own soil pH tolerances(Daturu strarnrntitutt6.O-8.2,
P R O D U C T I OO N F C R U D ED R U G S .\lujorana hortensis5.6-6.4), no markedinfluenceof pH value within the tolerancerange has been demonstratedfor essentialoils (.Mentha piperita) and alkaloids(D. stramonium).All plantsrequirecalcium for their normal nutrition but plants known as caliphobousplants (e.g. Pirtttspinuster and Digitalis purpurea) cannot be grown on chalky :oils, probablyowing to the alkalinity.In other casesdifferentvarieties of the same speciesmay grow on dilTerentsoils. For example, in Derbyshire,UK, Valerianafficinttlis var. sambucifollais common on the coal measures,but avoids the limestone,where it is replacedby \'hIeriana officinalis v ar.mikanii. The eff-ectof nitrogen-containingnutrientson alkaloid production has received considerable study (solanaceousdrugs including .\'icotiana,opium); generally nitrogen fertilizers increasethe size of the plantsand the amountsof alkaloidsproducedbut, as indicatedelserihere, the method of expressingthe results of such experimentsis irnpofiant.The effectsof nitrogen on glycosideand essentialoil contentsappearvariable;presumablyin thesecasesthe final result arises fiom the generaleffect of nitrogenon the plant'smetabolism.Nitrogen t'eftilizationhas been shown to increasethe silymarin content of the tiuits of Sl/1bum marianurngrown on reclaimedground.The eft'ectof potassiumon alkaloid production shows no consistenttrend, but an interestingexampleis the increasein putrescineproductionin barley grown on a potassium-deficientmedium, where it is possiblethat the organicbasehas beenformed to act as a substitutefor potassiumions. It has long been maintainedthat trace amountsof manganeseare necessaryfor the successfulproductionof Digitttlis pLrrpureaand more recentlyit was shown that a regimen of manganeseand molybdenum teedingover the two yearsof developmenlof D. grandiJloragives significant increasesin glycosideyield. Little work appearsto have beenperformedon the microbiology of .oil with respectto secondarymetabolism;Hardman and Petropoulos rPluntaMed.,1975,27,53) studiedthe responseof Trigonella.foenum,4roecum(potential source of diosgenin) to field inoculation with Rhiz,obium meliloti,210l in their attemptto establishthis speciesas a temperatecrop. Soil bacteriaof the genusAgnhacteriwn are finding rpplication in the productionof'hairy root'cultures (q.v.). Propogotion from seeds To ensuresuccessthe seedsmust be collectedwhen perfectly ripe. If not plantedimmediately,they shouldnormally be storedin a cooi and drv place and must not be kiln-dried. Some seedssuch as cinnamon. .oca and nutmegsrapidly losetheir power of germinationif allowed to Jry or if storedfor quite shortperiods.Long storageof all seedsusuallr much decreases the percentagewhich germinate. Although seedsare naturally sown at the seasonwhen they ripen, it ir frequentlymore convenient,especiallyin the caseofthe lesshardy crotic species,to defer sowing until the spring. In some cases,howL-\er, immediatesowing of the fresh seedis advisable.For example,it hasbeenshown that ifthe seedsof Colchicumautumnaleare air-dried L'\'enfor a few days,only about5clogerminatein I year and somemay not germinatefor 5 years; whereasif sown as soon as the capsules Jehisce,30Vc will germinatein the first year. In some instances,as ttirh Datura ferox and foxglove. seeds may remain viable in the ground for many years before germinating.With Er-t'throxylumcoca tnd E. novogranatensethe seeds stored at ,l'C for 24 days gave, respectively,297c and 07o germination (8.L. Johnson,PLantaMed., I989, 55, 691). Seedsmay, if slow germinating,be soakedin water or .r 0.27csolution of gibberellicacid for 48 h befbre sowing; more drastic methods,such as soakingin sulphuric acid in the caseofhenbane .eeds,or partial removal of the testaby meansof a file or grindstone, tave also been recommended.With lpontoea purgu Qalap')scailficaiion of the seedshas been the secret of successin obtainins 957c
E
germinationin eightdays (A. Linajeset al., EconomicBotuny,1994, 48, 84). Time of seed-sowingmay afTectthe active constituents,as illustrated by Cl'tamomillarecutita-for 17 cultivars investigatedmost gave a significantlyhigher yield of oil if they were spring-sownrarher than autumn-sownand the oil compositionalso varied (O. Gasicet ul., . i l R e s .1, 9 9 1 , 3 . 2 9 5t.h r o u g hC h e mA J. EssO . bs.116,37955). Propogofion by vegetolive meqns The following examplesof vegetativepropagationmay be mentioned. ( 1) By thedevelopment of bulbs(e.g.squill);conas(e.g.colchicum); tubers(e.9. jalapand aconite).or rhizomes(e.g.ginger). (2) By dit'isior?,a term usually applied to the separationof a plant which has a number of aerial stemsor buds, into separateparts eachhaving roots and a growing point. This methodmay be used lbr althaea.rhubarb,gentianand male f'ern. (.31By runnersor offsets(e.g.chamomileand the mints). (41 By suckersor stolons(e.g.liquorice and valerian). (5) By cuttings or portions of the plant severedfrom the plant and capableof developingroots. Successby this method has been extendedto a large number of plants by the use of rooting hormones(seeChapterl0) and by the employmentof mist propagation. Cuttings may be employed for the propagationof mints, lavender,rosemary,duboisias,tree daturas.coca and vanilla, to mention but a few. (6) By lay'eir.A layer is a branchor shootwhich is inducedto develop roots befbre it is compietely severedlrom the parent plant. This is doneby partly interruptingthe fbod supplyby meansof a cut or ligatureand embeddingthe part.Alternatively the slit portion of the branchis enclosedin moist peat.surroundedby moss, and the whole enclosedin polythene.This methodhas beenused successfullyfor the propagationof cascara. (7) By grafting and budding. Grafting is an operationin which two cut surt'aces,usually of different but closely related plants, are placedso as to unite and grow together.The rootedplant is called the s/ock and the portion cut ofTthe scion or gruft. ln Guatemala young Cinchonaledgerianascionsare graftedon Cinchonasuccirubra root-stocks.eventuallygiving a treewhich producesbark rich in the alkaloid quinidine. Grafting of female scions of Myristica.fragruns on male stocks may be used to increasethe proportionof fruitbearing treesin the plantation.The methodhas beenused considerablyin phytochemicalresearchto study sites of synthesisof metabolitesetc.Budding consistsof the introduction of a piece of bark bearing a bud into a suitablecavity or Tshapedslit madein the bark of the stock.Budding is largely used for Cilrus species,selectedstrainsof sweetorange,fbr example, being buddedon sour stocks. (8) By Jbrmentation.Thisprocessappliesparticularlyto the production of moulds and bacteria,and is extensivelyusedin the manufacture of antibiotics, lysergic acid derivatives and some vitamins. (9) By inocularlon.Specific to ergot whereby the sporesof the fungus are artificially culturedand injectedinto the rye headsby special machines. (10) By cell cuhure followed by difl'erentiationlseeChapter I l.
i':,i:]ii.]i,,]ii:iiii]''i.i..,i]
.l.::.oiiicn]6N..].::.']'.'..''....'..''.' Drugs may be collected from wiid or cultivated plants, and the task may be undertakenby casual.unskilled native labour (e.g. ipecacuan-
PRINCIPLES RELATED TO THECOMMERCIAT PRODUCTION,QUALITYAND STANDARDIZATION OF NATURALPRODUCTS ha) or by skilled workers in a highly scientific manner.(e.g. digitalis. belladonna and cinchona).In the USA the explosivedemandfor some herbs has led to concernover wholesaleuncontrolledcollection. socalled wildcrafting. resultin_u in the or.'er-harvesting of such planfs as Panax quinqueJblium. Polt'gula .tettego. Ecltinat.ea spp. and Cimicifilga racenloso (black cohosh). Elsewhere Prunus afi-it'anrr (pygeumbark) found fiom Nigeria ro Madagascar.RtrulrolJiuserpentina frorn India, and Turnera di.ffusct(.daniana)from Mexico are other e x a m p l eor f o r e r - e x p l o i t n t i o n . A strategyfbr the sr.rstained halvestingof Cumptothecaucuntintttu (Nyssaceae). the sourceof the anticancerdrug camptothecin.has been describedby R. M. Vincenter al.. (J. Nut.Prod.. 1997.60.618).The alkaloid is accumulatedin young leavesand by their repeatedremoval axillarv bud outgrowth is stimulatedgiving an increasedhlrvestable amountof camptothecinin a non-destructivemanner. The seasonat which eachdrug is collectedis usuallya matterofconsiderableimpoftance.as the amount.and sometimesthe nature.of the activeconstituentsis not constantthrou-ehor.rt the 1'ear.This applies.tbr example,to thecollectionof podophyllum.ephedra.rhubalb.wild cher-r.v and aconite.Rhubarbis repor-ted to containno anthraquinone derir,atives in winter but anthranolswhich.on the arrivalof wamer weather.areconvertedby oxidatiorrinto anthraquinones: also the contentsof C-glycosides,O-glycosidesandfreeanthraquinones in thede.,'eloping shootsand leavesof R/ralutuspurshiunafluctuatemarkedlythroughoutthc yeal-. The age of the plant is also of considerableimporttrnceand governs not only the total quantity of active constituentsproducedbr.rtalso the relative proportionsof the componentsof the active mixtur.e.A f'ew examplesaregiven in Table9. I br-rtsomeontogeneticr,'ariatjono1'constituentsmustexistfor all plants. Thereis increasingevidencethat the contpositionof a nr-rmberof secondaryplantmetabolitesvariesappreciablythroughouttheday andnieht. In some cases-fbr example.rvith digitalis and the tropanealkaloidcontainingplantswhich havebeenextensivelystudied-the evidencehas beensomewhatconllictingit-rthis respect.However.this may be largell,' due to the methodsof analysisemployed:thus, throughoutthe day the overaliamountof alkaloidor elycosidemay not charrgeto any extentbut there may be an interconversionof the variousalkaloidsor glycosides present.Daily variationsof the alkaloidsof the poppy,hemlock.lupin. broom, the solauraceous plants and ergot havebeenrepofted,alsowith the steroidalalkaloidsof industrialshoots'of SolanLunluc.irtiuturt?. the cardiac glycosidesof Digitalis purlttuea andD. lanatu. the simptephenolic glycosidesof Saln andthe volatileoil contentof PlnasandSah'la. Generally speaking.leavesare collectedas the flowers are beginning to open. flowers just before they are fully expanded.and underground organsas the aerial partsdie down. Leaves.llowers and fl-uits shouldnot be collectedwhen coveredwith dew or rain. Any which are discolouredor attackedby insectsor slugs should be rejected.Even with hand-picking.it is difiicult, certainlyexpensive.ro ger leaves. flowers or fiuits entirely free fiom other parts of the plant. In cases suchas sennaleafanddigitalisthe ofTlcialmonographs allon'a certain percentageof stalksto be presentor a limited amountol 'foreign matter'(fbr delinition.seeBP. fP and Chapterl4). Similar.ly. with roors and rhizomesa celtain amountof aerial stem is olien coliectediurd is permittedin the caseof senegaroot. The harvestingof umbellif-erous fmits resemblesthat of wheat. Reaping machinesare used. and the plants. after drf ing in shocks. are threshed to separatethe fruits. Specialmachinesale usedto harvestergot and lavenderflowers (illustrationswill be fbund in earlier editions).Barks are usually collected after a period of darrp weather,as they then separatemost readily fi-om the wood. For the collectionof -enms.gum resins,etc..dry weatheris obviouslyindicatedanclcare shouldbe taken to excludevegetable debrisas firr as possible.
Unclelgroundorgans must be fl'eed tiorn soil. Shaking the dru-e befbre.during and after dryin-u.or brushingit. rnaybe suflicientto separatea sandysoil. but in the caseof a clay or other hcavy soil rvashing is necessaly.For example,valelian collectedliont the wild is washed in the streamson the banks of which it usually grows. Before drying, any wormy or diseasedrhizomesor roots shouldbe rejected.Thoseof small size are ofien replanted.In certaincasesthe rootletsare cut off; rhr.rbarb. ginger and malshmalloware usually peeled.All largeorgans, such as calumbaroot and inr-rlarhizorne,shouldbe sliced to tacilitate drying. Befbre gentianroot is dried. it is made into heapsand allowecl to f-ermcnt.Seedssuch as nux vomica and cocoa.which are extracted lionr mucila-tinor.rs fiuits. ale rvashedfiee fiom pLrlpbeforedrying.
DRYING If enzymicactionis to be encouraged. slow drying at a moderatetemperatureis necessary. Examplesof this will befbund under'OrrisRhizome'. 'Vanilla Pods','CocoaSecds'and'GentianRoot'. If enzymicactionis not desired,dlying shoLrlcl takeplaceassoonaspossibleatier collection. Dlugs containingvolatileoils ale liableto losetheir aromaif not driedor if the oil is not distilledfrom them immediately.and all moist dmgs are liable to developmould. For thesereasons.drying apparatusand stills shor-rld be situatedas nearto the growing plantsaspossible.This hasthe furtheradvantagethat freightageis much reduced.as many tiesh dru-qs containa considerable amounl(60-90%) of water. The dr.rationo1'thedrying processvariesfrom a f'ewhours to many weeks.and in the caseof open-airdlying dependsvery lar.gelyon the weather.In sr-ritable climatesopen-airdlying is usedfbr such drugs as clove.colocynth.cardamorrr andcinnamon.Even in warnt anddry climrtes arrangementshave to be made fbr getting the drug under the cover of shedsor tarpaulinsat night or during wet lveather.For drying in sheclsthe drugs may be suspcndedin bundlesfi'om the roof. threaded on strings.as in the caseof Chineserhubarb,or. more comntonly, placedon traysmadcof sackingot'tinnedwire-netting.Papersspread on a wooden framework are also used. palticularly for fr.uits 1'rom rvhichit is desiredto collectthe seeds. Drying by artificial heat is more rapid than open-airdrying and is often rrecessary in tropicalcountries(e.g.WestAfiica. wherethe humiditv is very high. and Hondurastbr drf in-ecardamomfruits). In Europe continllous belt driers are used fbr large crops such as digitalis. Altemativelyheatmay be appliedby meansof opentires (e.g.nutmegs). siovesor hot-waterpipes.In all drying shedstheremust be a spaceofat leastl-5cm betweensr-rperirrposed trays.andair must circulatefreell'. H. N. ElSohly er a!. (Plcnru Metlictt. 1997.63. 83) have studiedthe ef}'ectof drying conditions on the taxane content ol' Tarus needles. Whenthelengthofdrying extendedup to l0 and 15 daysasin a shadehouseor in the laboratorythe recoveryof taxaneswas adver.sely afTected. Drying in a tobaccobarn. -{recnhouseor oven, and freeze-drying was generallvsatisfactor)'fortaxol and cephalomannine recoveriesbut the recoveriesfbr lO-deacctvltaxoland 10-deacetylbaccatin III were only 75-807cof thoseexpectcd. Rapid drying helps flowers and leavesto retain their colour and aromaticdrugs their aroma.but the temperatu|eused in each case must be governedby the constituentsand thc physicalnatureof the dru-e.As a generalrule. leaves.herbs irnd flowers may be dried betrveen20 and '10'C. and balks and roots between30 and 65"C. In the casesof colchicumcorm and di-qitalisleaf it will be notedrhatthe BPC and BP specify the temperaturesat which drying is to be done. For rural tropical areas.solar dryers have some distinct advantages over conventionalartiflcial heat dryers and have beenintroducedinto somecountnes.
PRODUCTION FC R U D D ERUGS
Toble 9.I
Exomples of the ontogeneric voriqrion of some metobolites.
Exomple Volotileoils Mentho piperito M. spicoto Cloves Coriondumsotivum Achilleamillefoltum
Lourusnobilis Volerionaofficinolis Cinnomomum comphoro Diterpenes Taxusbaccato Connobinoids Connobissotiva Cordiooctive glycosides Digitolispurpureo D. lonato Cyonogenetic glycosides Linum u sitotissimumseeds Sferoidol sopogenins Agovesp. Yuccosp. Dioscoreotokoro Alkoloids Popaversomniferum Doturosfromonium Duboisiomyoporoides
Ontogenetic variotion
Relotively highproportion of pulegonein youngplonts:replocedby menthone ond mentholos leovesmoture Progression frompredominonce of corvonein youngplontsto dihydrocorvone in olderones Contoinobovt| 4-21% of oil; mother'blown'clovescontoinvery liitleoil Morked_ chongesin oil composition ot the beginningof floweringond fruiiing Duringflowering,monoterpenes 1,8-cineole} predominote in oilifrom leovesond flowers.Oil {principolly obtoinedduringthevegetotive periodcontoinsprincipolly (92%)withgermocrone sesquiterpenes D the moior comoonent Highestyield:end of August{Portugol), .luly(Chino),Spring(lsroel), coincidingwirhhighesilevelof l,B
lpomoeovio/oceoseeds Steroidol olkoloids Solonum dulcomoro lr uits Solosodine contentfluctuoles duringmoturotion of fruit;tomotidenol predominote ond solodulcidine eveniuolly Cihusglycosides ond limonoidsLimonin ond noringinlevelsin gropefruit follos fruitmotures Furonocoumorins Ammivisnogo Unripefruitsrichestin bothkhellinond visnogin Tonnins Liquidomborformosono seosonoble voriotionof hydrolysoble leoftonnis.mosiropidchongesin thespring Vonillin Vanillaplonifolia Highestroteof vonillinbiosynlhesis occursB monthsofterflowerpollinotion
Exactly how far dlying is to be carr.iedis a matter.for. practical erperience.If leavesand other delicatestructuresare overdried.they becomevery brittle and tend to break in transit.Drugs such as aloes rnd opium may requirelurther drying aftel importation.
STORAGE The lar-ue-scale stora-se of drugs is a considerableundertaking.Except in a f'ew cases,such as cascal'abark, long storage,although ofien unavoidable"is not to be recommended.DrugssuchasIndianhemp and .lrsaparilla deteriorateevenwhen carefr-lllystored.It hasbeenreported
that the contentof taxol in Taxusbttctuta leavesand extractsstoredat room temperature lbr one year decreasedby 3040Va and j0-807a respectively;storagein a freezerand out of direct sunlightproducedno adversedeterioration(B. Das et ul., Plunta Medicct,1998,64,96). Similarly the alkamides of the popular immunostimulant herb Echinatea pLtry)ureadecreaserapidly on storage;N. B. peny e/ a1. (PlcuttaMedica, 2000, 66. -54)have shown thar althoughdrying has liule efl'ecton the quantity of alkamides,storagefbr 64 weeksat 2,lo produces an 80% loss,anda significantlossevenat - I 8 o.Drugs storedin the usual contiliners-sacks. bales, wooden cases,cardboardboxes and paper bags-reabsorb about 10 l2c/c or more of moisture. They are then termed 'air-dry'. Plastic sackswill effectively seal the contents.The
PRINCIPLES RELATED TO THECOMMERCIALPRODUCTION, OUALITYAND STANDARDIZATION OF NATURALPRODUCTS
t I
pemissible moisturecontentsof starch,acaciagum and otherswill be found in the BP and EuropeanPharmacopoeizz. The combinede11'ects of moistureandtemperature water-condenon humidity andthe subsequent sation when the temperaturefalls, must be consideredin drug storage. Drugs such as digitalis and Indian hemp should never be allowed to becorneair-dry or they lose a considerablepart oftheir activity.They may be kept in sealedcontainerswith a dehydratingagent.For largequantities thebottom of a casemay be filled with quicklimeand separated from the drug by a perfbratedgrid or sacking.If the lime becomesmoist, it should berenewed.Volatileoils shouldbe storedin sealed.well-filledcontainers in a cool,dark place.Similarremarksapplyto fixed oils,parlicularlycodliver oil. In the latter casethe air in the containersis sometimesreplaced by an inert gas. Air-dry drugs are aiways susceptibleto the attack of insects and other pests, so they should be examined frequently during storageand any showing mould or worminessshouldbe rejected. In order to reducetrndesirablemicrobial contaminationand to prevent the developmentof other living organisms,some plant materials may requiresterilizationbeforestorage.
To ensurethat satisfactory standardsfor the growing and primary processing of medicinal and aromatic (culinary) herbs are achieved throughout the European Union 'Guidelines for Good Agricultural Practice of Medicinal and Aromatic Plants' was issued as a final Europeanversion in August 1998. It covers seedsand propagation, cultivation. harvesting,primary processing,packaging, storageand transport, personnel and facilities, documentation, education and quality assurance.Details can be found in ICMAP News, No 6, April 1999 (ICMAP = InternationalCouncil for Medicinal and Aromatic Plants).
Furtherreoding Hayward M D, Bosamtrk N O, RomagosaI (eds) 1994Plant breeding.principlesand prospects.Chapmanand Hall, London Hornok L (ed) 1992Cultivation and processingof medicinalplants.J. Wiley and Sons.Chichester,UK
@ ..,ru1ilMt :uat'1149!'i
ripllr
The growth and developmentof plants is regulatedby a nunrberof chemical substanceswhich together exert a complex interaetion to meet the needsof the plant. Five groups of plant hormonesare well established: they arethe auxins,gibberellins. cytokinins,abscisicacid and its derivatives.and ethylene.Thesesubsttincesare of wide distribution and may, in fact, occur in all higher plants.They are spccific in their action, are active in very low concentlations"and regulatecell enlargement,cell di vi sion. cell dif'fbrentiation. or-Qanogenesis. senescence and dormancl'.Their action is probably sequential.Other hormonesconcernedwith flower fbrmation and reproduction.but as yet uncharacterized have also been envisaged.The essentialrole of thcse substances is illustratedby cell andtissuccultures:without the addition of suitablehormonesno developmentor cell division occurs. The eft'ectsofthese very activesubstances on the productionofsecondplantscontainingur ary metabolites.pafticulally with a view to prodr.rcin-e enhancedproportion of active constituent, are of interest to phamaIn sr.rch cognosists. studiesthe mannelin which the resultsarerecordedis all-inpoftant.particularlyas the lreatmentmay alsoinf-lr.rence the sizeof the testplantcomparedwith the controls.For commercialpurposesyield pel hectaleis an obviouscriterion,whereasfor biosyntheticstutlies1'reld per plarrtor per centfreshweight rnaybe of more significance.For final drug evaluationpercentdly weightis themostlikely requirement. In spiteofthe earll'enthusiasm fbr resealchon drugenhrncernent b1 the use of hormonesappliedto medicinal field crops.very little in the way of useful practicalapplicationemergcd;the rcsultsu'ere.hower-er, of interest and selectedexamolesof this older work continue to be retuined in thi\ chilpter.
.,t:iililii''''lrriirllil litrnal tliri:
tiiti:r t{itu
ti;tt;t, t{:i{1u1
u{11t uutalrt t:.{iul uit'uitla t;tlJf irililrl:l ritlt:tlt; .iiutrr uiiilt!l , . :: tri:it!:il
Plont growth regulotors
t.,
,,;,:.'
''.,. ,AuxrNs :
:
These growth-promotin-9substanceswere first studied in 193I by Dutch workers who isolatedtwo growth-regulatingacids (auxin-aand auxin-b,obtainedfiom humanurine and cerealproducts,respectively). They subsequentlynoted that thesehad similar propertiesto indole3-acetic acid (lAA). the compound now consideredto be the major' auxin of plants. and fbund particularly in actively grouing tissues. Severalsimilar acids. potential precursorsof indoleaceticacid. have also been r"eportedas natural products:they include indoleacetaldehyde, indoleacetonitrileand indolepyruvicacid.Thesecompoundsand IAA are all derived,rn the plants,fiom trypbphan. Typical effects of auxins are cell elongationgiving an increasein stern length. inhibition of root growtl-r,adventitiousroot production and fruirsetting in the absenceof pollination.A numberof widely used synthetic auxins include indole-3-butyric acid. naphthalene-1-acetic acid (NAA) and 2,zl-dichlorophenoxyacetic acid (2.4-D).
(Y-T-CH2.cooH \,,/\N/ H lndole-3-aceticacid
o-cH2cooH
AUXTNS 67
I
GIBBERETTINS 68
4-\-cl
V
CEtt DIVISIONHORMONES: CYTOKININS 69
I
GROWTHINHIBITORS 70
cl
ETHYTENE 7 l
acid 2,4-Dichlorophenoxyac€tic
I I
ASTOI'l I j\! t11':qSf Ty
I tIERARY& t N F O i i ; * r r i l i O N S f RVICE t s
Il
PRINCIPLE RS E L A T ETDO T H EC O M M E R C I APL R O D U C T I O NQ,U A L I T Y AND STANDARDIZATION OF NATURAL PRODUCTS ln the plant.oxidativedegladationof IAA to sive a numberof prodGIBBERETLINS ucts is controlledby IAA oxidasc.Sone substancessuch as the orthodiphenols(e._s.cafl'eic and chlorogenic acids and qr.rercetin) inhibit the actionol'the enzyrneand. hence.stimulategrowth therr- This group of plant growth re-qulatorswas discoveled by Japanese 'bakanae'(foolish seedlings) disease of selves.Conr,elsely. r-nonophcnols suchas7.r-courllric acidpromotcthe rvorkersin connectionwith the actionof IAAoxidase anclso inhibit gr'o$'th.IAA rnayalsobc conjr"r- rice. In this. the afl'ectedplantsbecomeexcessivclytall and are r"rnable gatedin the plantrvithasparticacid.-llutamicacid,-qlycine, sugarsand to supportthcmseh'es;through a combinationof the resulting weakthey eventuallydie. The causativeorgantsm cvclitols:suchbound lbrnrsnrayrepresent a detoxicationmechanisnr nessand parasitedan-rage g i kuroi. and in I 926 Kurosawafound that of the discaset s GibbereILa.fu or are inactivc stora-{cforms of the hormone. The main practicalusesof auxinsare (1) in low conceutratir)ns to extl'actsof the fungr-rscould initiate the disease symptoms when appliedto healthyrice plants.Sorrrel0 yearslater.Yabutaand Hayashi accelelatethe looting of woody and herbaceor.rs cuttin-us and (2) in higher concentrations to act as selectileherbicidesor weed-killers. isolatecla crystallinesampleof the active material which they called 'gibberellin'. Pleoccupationwith the auxinsby westernplant physioloPlacccllbr 2.1h in a l:-500000 solutionof NAA. cuttin_ss r.villsubsequenth'clevelop roots.This inclucles cuttingsfiom treessuchas holly. -qists.the existenceof languagebarrielsand the adventof World War II of these which lr'ele fbrmelly i'er'1'diflicultto plopagatcin this way and had to meant thrit a firrthel l0 yearselapsedbefore the sigr-rificance groupsin Britain, outsideJapan.In the 19-50s be raisedtl'onrseedor bl grafiing. Sirnilarly.indolc--3-butyric acid was findingswasappreciated successful vith CiuL:lututL cuttin-qs. savin-q solne2 or 3 yearscompared the USA and Japanfurther investigatedthesecompounds.which were uith grou'th fl'ornseed.Sirnilarresultshave beenobtainedwith cr-rt- shown to har,eamazingefl'ectswhen appliedto plants.lt soonbecame tingsof Curicu,Cuft?a,Pirtt.ts and otherspecies. ln biogeneticstudies. apparentthat a range of gibberellinswas involved. and they are now use has been lnade of auxins to induce rclot forrnation on isolated distinguishedas GA1. GA2, GA3, etc. GA-,. comntonly ref'erredto as leavessuclras thoseof Nicotitutttandl)uturu species. Auxins usedin gibbcrellic acid and ploduced commercially by fungal cultivation. is suitableconcentration(usuallystlongerthan when usedfbr rooting probably the best-knownof the serics:its structurewas finally detercuttings)selectivelydestroysome speciesof plant and leaveothcrs nrined in 1959. The tirst good indications that gibberellinsactually mol'eor lessunaff'ected. They have.therefore,a very irnportantrole as existedin higher plants came with West and Phinney'sobservationin 1956 that the liquid endospermof the wild cucumber (.Echinocytis selectiveweed-killersin horticr.rltr-rre Thus 2.,1-Dis arrdagricultr.rre. particularlytoxic to clicot,vlcdonous plantsr"'hile.in suitableconcentra- rrtucrocur;ta)was particularlyrich in substancespossessinggibtion. lraving little efl'ecton nonocotyledons.It can. therefbrc.be used berellin-like activity and Radley's reporl of a substancefi'om peaacidon paperchromatograrns. Finally" to destroysuchdicotyledonous weedsas dandelionand plantaintion-r shootsbehavinglike -eibbelellic isolatedcrystallineGA1 fiom Phaseolus glass lirwns. (N.B. Certain carbi.urateiLnd urea derivativeshar,e an in 1958MacMillanand SLrter By 1980,58 gibberellinsr'vereknown of which abouthalf oppositeef'l'ectand can be usedto destroygrassu'ithout seliousinjury' rnultif'lorLts. were derivedfiom the Gibberellafungus and half fiorn higher plants. to clicotyledonous clops.) GA117was charactelized from fern gametophytes in 1998(C. Wynne Thele have beenscr,eralrcpol'tson the ellects of anxins on the fbret al.. Plrttochenti.stn'. 1998.49. 1837).Of thesemany GAs rnostare mationol secondary metabolites on medicinalplants. Seedlingsar-rdyoung plantsol-Mentlrupiperitu,uhen treatedwith either deiid-enclrnetabolitesor are intennediatesin the fbrrnation ol only a lirnitednumberhavehormonalactivityp€r derir':rtives of NAA, gave in the mitture plilnts an increasedyield activecompoLrnds: (30-50%) of oil which itself contained4.5-L).0%more mentholthan sa. It is now consideredpossiblethat thesc substancesare pre\ent in tl-recontrols.The study of the efl'ectsof auxins on alkaloid fblrnation nrost.if not all, plants. Gibberellinsare synthesizedin leavesand they acculnulateirr relalras concentratedprincipalll' on the tropane alkaloids ol Duturu species. Molphologicalchanges in theplantswereobserved(2.4-D,fbr tively lalge quantitiesin the imrnatureseedsand fiuits of someplants. exarnple.producedabnorrnaland bizalrefbrms of D. ,stnrntoniurn: an The most dramaticef-fbctof gibberellinscan be seenby their applicarncreasein tlichome production.palticularlf in branchednonglandr"rlar tion to short-nodeplants for example.thoseplantsproducingrosettes lbrmsl smoothfiuits as distinct f'romtl-rosewith spinesland a prolifer- of leaves (Digitulis. Htoscl'antus)-when bolting and flowering is ation of vasculartissue).Genclall.v.wolkcls tbr-rndno markedel-lecton induced; also. dr','alfvalieties of many plants. when treatedwith the alkaloidproductionor on the type of alkaloidsprocLrcecl, althougha horrrone, -qrowto the sameheight as taller varieties.Other irnportant of various Russirnpaperrecoldsthatwith thornapplearrdscopoliatissuecultures aclionsof the gibbelellinsarethe initiationof the synthesis a stimulatingef1'ect on alkaloid production\\'ilsobtainedwith NAA ancl hydrolyticand proteolyticenzymesLrponwhich seedgerminationand seedlingestablishment depencl. an inhibiting effect '"vith 2..1-D: sirnilal resr-rltswere reportecilbr The growtl.retfectof gibberellir.rs arisesby cell elongationin the subIlarnrolfiu :erytentinutissuc cultules r.vith thesc fivo horrnones.An increasedalkaloid production has been repolted fbl submcrgedcul- apical neristem region where young internodesare developin-e.The efttcts of gibberellins and auxins appear complernentarv,the fLrll tules o1'certrinelgd strainswhen tleatedu,ith variousauxins(lAAl NAA: l.'1-D: inckrlepropionic acid: indole butl'ric acidl. r'n,heleas stirrr.rlationof elongatronby eithel hormonenecessitatingan adequate unplcdictablcirregularclurntitrtiveand qurlitativeell'ectson ergoline presenceof the other'. As with auxins,gibberellinsappealalso to occur in plarrtsin deactialkaloicl ploduction were observed with the same hormones in Ilsottroctr.Rllcrr and Argt'reia (Conl'olvulaceae)suspensioncultures. vatecltbmrs: thus. f3-D-glucopyranosylesters of GAl. GA*, GA8. Erpelimentscalriedout in Hungalyinvoh.'ingthc injcctionof IAA into GA-j7and GAjg ale known. As such they rlray servea depot function. poppy'capsulesI anclf davsatter tlowerirrgploduceda relativelyelon- The glucosylesterof GA3 has beenpreparedin severallaboratories. The biogeneticpathwaysof the gibberellinsappearto be similar in alkaloidcontent.In stud-tatedcapsulelbrm rncl.in general.a reducecl ies on anthlacluiuoncproduction b1' cell suspcnsior.r cultures of both higher plantsand Gibberelh. They ariseat the Crq geranylgeranyl level of the isoplenoidr.nevalorric acid pathway(q.v.) Morittdu citriloliu. Zc-nkanclco-u'orkershave shown that cells grown pyrophosphate with cyclizations the C2s tctlacyclic diterpernoid entkauin the presenceof NAA havea substantial productiorr anthlaquinone -eiving but thoseu ith l.-l-D assoleauxindo not.IAA appears to haveno benc- renoic acid. which by a rnultistep ring contraction furnishes the GAtrficial eflecton the productionof sennosides in Ca,rslautgustifoliu. -eibbanering system as exemplilied by the key intern.rediate
PLANTGROWTH REGULATORS been publishedon the efTectsof the hormoneon the morphology and alkaloid contentof treatedplants.H_r,osr.r.omu.s niger is a perennialand the hormoneeffectsincludedstemelongationgiving a two- to threefbld increasein height: a spindly and vine-like growrh; slightly chlororic and narrow leaves;a more rapid onset of flowering; increasesin the stcrndry weights but decreasesin the dry weights of leaves.tops and Gibberellic acid (CA3) roots.The overall yield of alkaloidsin the treatedplants was reduced by about a half and the concentrationof alkaloidsin variousmorphological parts ranged fi'om 13 to 81c/cof that of the controls,with the stemsshowingthe greatestteduction.Subsequentexperimentsshowed that with belladonna.increasedalkaloid yields could be obtainedby adjustingthe doseof GA treanr-rent to favoul overallgrowth in the older plant. Similar resultshavebeenreportedwilh Daturutspp..plants showing GA12aldehyde. Key diversification point for synthesisof GA s the predictablernorphologicaleff'ectsand giving a reducedalkaloid yield. The efl'ectswere not transmittedto the second-generation plants. With NicoticuttttabucnntandDuboisiu hybrids,treatmentagaingavea aldehyde.Severalpathwaysdiverge fiom GA1,-aldehydeto give the generallyreducedalkaloidcontentaccontpaniedby characteristicrnorknown 90 or so gibberellins.All GAs have either the ent-gibberellane phologicalel1'ects. (C19 GAs) (loss of C-20) tC,e GAs) or the ent-20-norgibberellane Other alkaloid-containingplants which have been subjectedto GA carbon skeleton.Both types are rnodified by the position and number treatmentinch-rdeCathnranthusftrJetl.r(generallya lowering of alkao1'OH groups.oxidationstateof C-18 and C-20. lactonefbrrnation. loid contentand somechangein the relative proportionof vinblastine presenceand position of double bonds on ring A, epoxide fbrmation, to other alkaloids');Rauu,olJia.\erpentinu(loweringof alkaloidconcenthe presenceof a carboxylgroup and hydrationof the C I 6-C 17 double tration in the roots,the e1I'ectincreasingwith dose):andTltea sinensis bond. (slight differencein cafl'einecontentof leaves).It worlld appear,thereThe gibberellinshave beenusedto treat rnany plants which contain fore. that with the alkaloid-containingplantsso tar tested.the substanusetul secondarymetabolites.A sumnraryof someof the findings, fbr tial internodal growth produced by GA is oflset by a lower ovelall difterentgroups,is given below. acculnulationof alkaloid. Volotile oils ond ierpenoids Early wolk involving GA treatmentof volatile oil-containing plants n'as corrcernedwith resultantchangesin morphologicalcharactersin generasuchas Citru,s,Eucoh'ptusandFoericulum.GA sprayingof the llowers of Huntulus Lupulusadvancedthe maturity of the hops by 10 days and gave a more evenly developedcrop. Although the conesof treatedf1owerswere more subjectto wind burn than normal.their yield rvasincreasedby about407c.The cr-resincontentof the hops,on which theil commercia]value depends.however.was l.Sclccomparedrvith l0.2Vc for the controlsl the volatile oil corrposition also difl'ered. Several studies have becn made of the ef}'ectsof GA treatment on Mentha piperitu. The detailedresultsvary, but the generalresult is a loweling of the oil content (possibly by reduction of the number of slandularhairs)with little changein the oi1composition.In contrastto the above,Kaul and Kapoor havereportedlavourably on the GA tt'eatrrent of ChenopodituttambrosioitlesandAnethunt.spp. with respectto rolatile oil content.The tbnner affordeda 33% increasein volatile oil with no appreciablechange in ascaridoie content. Wilh AnethLun grttvectlens specificdosesof GA increasedthe oil contentby up to 50% and with A. sol rr (Indiandill) by up to 30%.At the lower dosesof GA trcatmenttherewas no significantchangein the can'onecontentof the oil, but at higher concentl'ations thel'eappealedto be a slight increase over the (then)official limit (53%. BP 1958).With FoenicLrlum vulgare and Cctriandrumsatiyturt Gjerstad found that tblial spral's of 100 palts/I 06 GA, apptiedbi-weekly.gave a diff-erence in caulinelength of 200-3007cbut no dit-ferenccs were detectedin yield of fiuits and quantity andquaiityof volatileoil.
Glycosides In 1959 Sayedand Beal reportedthe efl'ectsof the daili,'GA rreatment of first-year rosetteDlgrtalis purpurea plants. Flowering occurredin the first year with treatedplantsand the leavesbecamelongerand more linear.with an increasein dly weight.An increasein cardioactiveglycosideswas obtained but no incteasein the digitoxose content was observed.Sinrilar experimentsby Burton and Sciuchettiin 1961 involving weekly treatmentsof D. lanuta produced similar results, with 'bolting' afterrhetwelfth week.The total glycosideper shootof the treatedplantswas considerablyincreasedin the first 8 weeks.and at harvest time showedabout 30cloincreasewith the lowcr doseof hormone (10 ptgweek r), and a 507cdecrease with the higher(50 prgweek-l).The authors concluded that the effect of the treatment of glycoside productioncorrelatedmore closely with thc growth responsethan with the e1l-ect on carbohydratefbrmation.With both leaf and root cultures Lui and Staba (1981) found GA to have a positive eff-ecton the productionof digoxin. Applicationof GAto Cassiaangustifoliu(Bhatiaetal.,PlantaMed.. 1978.34.437)appeared to redllcethe sennoside contentofthe leaves at all concentrationsused.but slightly increasedthe dry weight of the shoot.
Fixed oils Gjerstad obtaineclcastor plants (.Ricinuscomntttnis)five times the hei-ehtof controls as a result of GA treatment.No significant differenceswere observedregardingthe quantity and quality of the fixed oil of the seed.
Alkoloids The seeds ol the tropanc alkaloid-producingspeciesol Atropct. CELIDIVISIONHORMONES:CYTOKININS Ht'oscyarttus tnd Dttturtt often exhibit protracteddormancyor en'atic GA treatmentof the seedscan be r.rsed to assistin obtain- Auxins and gibberellinsare concernedlargely with cell enlargement: _uermination; ing unifolm -eelminationiind total cmergence.Considerablework has and although they influence cell-multiplication processes,there are
@
OF NATURALPRODUCTS RELATED TO THECOMMERCIATPRODUCTION,QUALITYAND STANDARDIZATION PRINCIPLES other substanceswhich have a more specific efTecton cell division (cytokinesis).The activity of the latteris not only confinedto cell division in a tissueper se; they also regulatethe patternand frequencyof organ productionas well as position and shape.They have an inhibiThe plesenceof such compoundshas been tory effect on senescence. suspectedfor many years, the German botanist Haberlandtin 1913 having noted that phloem tissuescontainedwater-solublesubstances cells of woundcapableof promoting cell division in parenchymatous ed potato tubers.It was not until many yearslater (1954) that Miller, working at Wisconsinon tissuecultures,discoveredthat agedor autoclaved DNA from herring sperm stimulatedcell division. This active degradationproductwas calledkinetin and in 1955was identifiedas 6furfulylaminopurine (6-furfuryladenine).Kinetin itself has not been isolatedfrom plants,but in 1964,atler the indication of cytokinins in liquid endospermof the coconut and in extractsof maize embryos at the milky stage,an active substancenamed zeatin was isolatedfrom the latter source.Like most other cytokinins,it is a 6-substitutedade1)-aminopurine.It nine derivative, 6-(4-hydroxy-3-methylbut-2-eny has sincebeenshown to be associatedin maize with zeatinriboside(1 B-o-ribofuranose)and with a phosphateester of this compound.The hormonecomplex has been detectedin the cambial region of various and dihydrozeatinare examplesof woody plants.lsopentenyladenine cytokinin isolatedfrom other sources;many more have been detected but not identified.The sidechainof cytokininsis of isoprenoidorigin.
NH-Cl{a-c-
l N
-
f"-f' -cl-l o
r\ t
=*^li) Kinetin
Cytokinins have been much employed in tissue culture work, in which they are usedto promotethe formation of adventitiousbuds and shoots from undifl'erentiatedcells. In cell cultures, they have been shown to promote the biosynthesisof berberine(Thalictrum minus), condensedtannins(Onobrychis viccfo lia')andrhodoxanthin(Rlclnus). A limited study only has been made of the efTectsof cytokinins on secondarymetabolismin intact plants. Concerningplants producing tropanealkaloids.Ambroseand Sciuchettihavecomparedthe actionof . Plantsreceivedweekly dosesof kinetin and GA on Datura meteloides 25 pg kinetin; in relationto the controlsthe following differenceswere noted: Kinetin treatment
Gibberellicacid treatment
Shorterand bushierplants Decreasedgrowth No changein alkaloid contentin plant organs Delayedresponse
Taller and spindly plants Increasedgrowth Decreasedalkaloid production Rapid response
ment was that in field plantsit led to a delayin the usualseasonalfall in alkaloid content(February-April in Australia),thus permitting an economically useful,extendedcollectionperiod.Shahet al. (1990) reported favourableincreasesin growth and alkaloid yield with Hrosct,amus muticustreatedwith kinetin (50 p.p.m.). Verzdr-Petri injected benzyladenineand kinetin separatelyinto developingpoppy capsules:the effectswere similar to thoseobserved with auxins.Leavesof the coffee plant after kinetin treatmentdeveloped a transientincreaseof up to 107cin their caffeine content.The effect was transitoryand passedafter 6-12 days.With Cassiacrngustifolia plants low concentrations of hormone were found to increase slightly the sennosidecontent and to favour an increasein the dry weight of shoots.
NH.cHr-q":i"*t
cH2oH ryA-\ \^-/
H
Zeatin
GROWTHINHIB|rOR5 Natural growth inhibitors are presentin plants and affect bud opening, seedgerminationand developmentof dormancy.One such substance, abscisic acid [3-methyl-5-(1-hydroxy-4-oxo-2,6.6-trimethyl-2-cycloacidl was isolated and charhexane-1-y 1)-cis,trans-2,4-pentadienoic acterizedin 1965;it has also beenisolatedfrom the ftrngusCenospora rosicola. The structuralsimilarity of abscisicacid (ABA) to the carotenoids prompted researchon the relationshipof these two groups of comthat somexanthophylls,parpoundsand it hasnow beendemonstrated ticularly violaxanthin,producea germinationinhibitor on exposureto 'apoin supportof an indirect light. Evidencehas now accumr-rlated carotenoid'pathwayfor ABA biosynthesis,the most likely precleavage precursorsbeing 9'-cis-neoxanthinand 9-cls-violaxanthinwhich fracture acrossthe I 1,12 (1 1'J2) doublebond to producexanthoxinwhich in plant tissuesis readily transfbrmedinto ABA (seeA. D. Parry and R. 815).However,it is alsopossiblethat Horgan,Phytochem.,1991.30, level of the MVA ABA arisesfrom farnesolat the C15sesquiterpenoid pathway (q.v.).The theoreticalpostulationinvolves a number of steps in which, as with the apo-carotenoidpathway,cis-A2-xanthoxincould
Mq. Y" rM" s " :ac:\c,'C--gH
.L-z\tX.*
Eoot
Abscisic acid (ABA) Luanratanaand Griffin (J. Nat. Prod., 1980, 43, 546, 552:.1982, 45, 270) observedthe beneficialeffectsof a commercialseaweedextract containingcytokinin activity on Dtrboisia hybrids grown both hydroponicatly and in a commercial plantation.In the latter there was an I 87cincreasein leaf yield anda 16% increasein hyoscinecontentcomparedwith the controls.A further pointer to the usefulnessof the treat-
cis-A'-Xanthoxin
, PLANTGROWTHREGULATORS also be involved. In accord with isoprenoidbiosynthesis,mevalonic acid (MVA) has been demonstratedto be stereochemicallyincorporated into ABA in higher plants, and likewise labelled acetate in Cenosporarosicola. Other substancesrelated to abscisic acid have been isolated from plants and include vomifoliol (several sources), which lacks the 2,4-pentadieneside-chain;it has the sameactivity as abscisicacid in stomatalclosuretests.Little or no work appearsto have been reported on the effects of abscisic acid on the production of pharmacognostically interestingsubstances. A number of syntheticgrowth inhibitors havebeenstudied;the first to be describedwas maleic hydrazidein 1949.N-Dimethyl-aminosuccinamic acid can be consideredas a hydrazinederivativeand actsas a shootelongationinhibitor by suppressingthe oxidation of tryptamine to IAA. Sciuchettiand colleaguesshowedthat this compoundsprayed onto Datura stramonium and D. innoxia plants reducedthe eventual height of the plants and lowered, overall. their total alkaloid content; however,significantincreaseswerenotedin the concentrations of stem alkaloid (567cincreasein the secondweek and 9O7cinthe fourth week comparedwith the controls).The inhibitor, tributyl 2,4-dichlorobenzylphosphoniumchloride (phosphon)producedsimilar resultswith D. fbror. Trigonelline. an alkaloid of fenugreek seeds,promotes cell arrest in G2 (a specific period precedingmitotic division of the nucleus)in variouslesumes. r ii.,.:prli.r,..r. 1 r.:"':'rl :r:rp.r:tir,.ri:r:r
E]r.H,,]!*g
It has been known for many years that ethylene induces growth responsesin plants,and in 1932it was demonstratedthat the ethylene evolved by stored apples inhibited the growth of potato shoots enclosedwith them; it has a role in fruit ripening. Current thought maintainsthat this simple compound should be included among the natural plant hormones. Ethylene is synthesizedin the plant from S-adenosylmethionine via the intermediate 1-aminocyclopropaneIcarboxylic acid (ACC). The gene fbr ACC synthasehas been cloned irom tomato squash (H. KIee and M. Estelle in Annu. Rev. Plant Phl,siol., 1991, 42, 529). One biochemical action of ethyleneis the stimulationofthe de novo synthesisand secretionofcell-wall dissolving enzvmessuchas cellulaseduring leaf abscissionand fruit ripening.
A compoundwhich givesrise to a typical ethyleneresponsein plantsis (2-chloroethyl)phosphonic acid (ethephon)applied in aqueoussolution in concentrations of the orderof 100-5000p.p.m.In the cell sap,at pH valuesabove4.0 it is broken down to ethyleneand phosphate.lt is marketedas Ethrel. At low concentrationethylenehas beenshown to increasethe sennoside concentrationin Cassia ongustifolia, and applied to tobacco leavesit stimulatesproductionof the stresscompoundsphytuberinand phytuberol (these are compounds produced in responseto tobacco mosaicvirus); with Digitalis lanata tissuecultures,cardenolideaccumulation is decreased.Ethephonis now increasinglyusedas standard practice for enhancing the flow of rubber latex. Sprayed on to the scrapedbark (tapping groove) of the rubber tree it increaseslatex yields by from 36 to 130c/c. Other growrh regulqtors In addition to the well-known plant growth substancesdiscussed above, a very large number of other compoundshave been isolated from naturalsourceswhich in someway influenceplant growth. Some are widely distributed and others are of restricted occurrence. Generallythey have a less specific action than the regulatorsalready mentionedabove.They haveno commonchemicalstmctureand only a few recurrentfunctional groups(e.g. phenolichydroxy groups and cmethylene-Y-butyrolactone moieties). This implies that these substances may be acting at many different sites along the growth regulatoryprocess.Substances involved include aliphaticand aromatic carboxylic acids, phenolic and neutral compounds, salicylate, polyamines, ,5- and N-heterocyclic compounds,including alkaloids and terpenes.Acorus calamus producesa number of sesquitetpenes having the skeletal structuresof cadinane,acoraneand eudesmane which inhibit the germinationof lettuce seeds(K. Nawamaki and M. Kuroyanagi, Ph.vtochemistry,1996, 43, 1175).A new class of plant growth regulatorsknown as brassinosteroids is found in the seeds.pollens, galls, leaves,flower-budsand shootsof a considerabierange of plants. Some 40 of thesecompoundsare known; they stimulatecell enlargementand cell division and influencegeneexpressionand nucleic acid metabolismat the molecular level, see V. A. Khripach et al. (1999),Brassinosterctids. a NeyvClassof Plnnt Hormone, San Diego: AcademicPress.
a
3
rllllrliilr: lt,itlt.tll:
Plont cellondtissue culture; biochem icoI ,,', conversions; clonol propogotion
INDUSTRIATSIGNIFICANCE 72 illll:lt:i:ill:irir:lrl'::l
CUTTIVATION OF PTANTCELLS 73 PRODUCTION OF SECONDARY METABOTITES74 INDUCED SECONDARY METABOTISM IN CEtt
curTuREs75 BIOCHEMICAT CONVERSIONS BY PTANTCELI CULTURES75 IMMOBITIZED PLANTCEtts ORGAN CUTTURE 77 CTONALPROPAGATION 78
76
One of the rapidly expandingareasof phannacognosyhasinvolved the applicationof the artiticial culture of plant cells, tissuesand olgansto the str.rdvof rredicinal plants.Principaltopicsinclr-rde the development of commercialproductionof expensivebiomedicaments. the discoverv of new metabolites,the selection of superior strains of rnedicinal plants.the eh-rcidation of biosyntheticpathwaysof secondarymetabolites with isolationof correspondingenzymes,and the improvementof medicinalplant speciesby geneticengineerin-e.
INDUSTRIAT SIGNIFICANCE A numberof factorsnilitate againstdependence by the pharmaceutical industry on the useofbotanical sourcesofdrugs. and thesehave been. to sonleextent,responsible lbr the reluctance of industlyto inrest in the exploitationof the plant kingdom.Thesefactorsincludethe lollowing. | . At'oilahility o.fravr moterial. Sorneplants.althoughhighly desirable as sourcesof biochenticals,just cannotbe producedin an economically sufticient quantity to satisfy demand. An example is Strophttntltus,\armetltosLts seeds.which, early in the search for corticosteroidprecursors(1950s).were known to conrrin a rery suitable compound, sarmentogenin.conveniently substitutedin the steroidC ring (seeChapter24). But the plants,tlopical lianesexisting as dilTerentchemical races (Chapter 12). are not particularly abundant and are difl-icult to cultivate. More recent examples include the very limited supplyof the Pacificyevr'(7irrrr.i bret,ifolia) the principal sourceof taxol, a diterpenewith considerablepotential as a startingn-raterial fbr the serr-risynthesis of promising anticancer drugs and also Coleu.sJbrskohlii, an Indian speciesnow listed as vulnerableto extinctionin the wild as a resultof indiscriminatecollection tbr the isolationof forskolin. a diterpenoiduscd in the treatment of glaucomaand heartdisease. 2. Fluctttutiorto.f.suppliesuttl qualitv. The productionof crude drugs is subjectto the vagariesofthe climate. to crop disease.to varying methodsof collectionand drying which influencequality.and to the inherentvariation of active constituentsarising fiom plants of the samespecieshaving di1I'erent geneticcharacteristics. 3. Politital considerations.When a new drug has been successfully marketed.its country of origin may seekto exploit the situationby placing an embargoon the expofi ofthe crude drug and upvalueits own exports by processingthe plants fol the actire constitLlents. This occurred with Ruuvolfia serpentinu (lndia) and Dioscorecr spp.(Mexico). Historicallythe productionof many spices,cinchona for extraction.and opiurn hasbeenthe prerogativeof specificcountlies. Even today. the location of opium poppy cultivation in the
ffll.:H"J:il:,::
",rn
bvporiticar considerarions asbveco-
1. Putent rigftts. Generally. it is not possible to patent a naturally occurring plant nretaboliteas such. only a novel method fbr its extractionand isolation.Hence,there is little incentivefor a pharmaceuticalcompanyto sperrdntany yearsand vast sumsof money on launching a new natural product ovel which it has no patent rights. Following from the above.it is not surprisingthat industry is taking a close interestin the corrmercialpossibilitiesof cultivatingparticular speciesof plant cells. under conditionsanalogousto the productionof antibiotics.that will yield biomedicinais.By this means.production could at all tinresbe gearedto dentand.and a productof standardquality assured.Furthermore.a highly sophisticatedand speciticrnethodof p r o t l u c t r ocna nh e p u t e n t e d .
P L A NC T E LA L N DT I S S UCEU L T U RBEI O ; C H E M I CCAO L N V E R S I O NCSL;O N AP LROPAGATION
@
Shikonin. a dye and antibacterial.is commerciallyproducedby the cultivation ol Litho,spermurrplant cells and the productionof the ginsenosidesand antitumour alkaloids of Cathartmthusroseu.sare currently being developed; Japanesepatents exist concerning the manufactureof many secondarymetabolitesincluding the purple pigment from Melissn offic:irurlis,the production of catharanthineand ajmalicineby cell culturesderived from C. rosers anthers,the manuf'actureof an analogueof taxol (q.v.) by callus culturesof la-tus spp., and tropanealkaloidstrom DtLboi.sia tissuecultures.A vast amountof work hasbeenleportedduring the last decadeand the majority of comrnonmedicinalplants,and many Iesscofilmonones"havebeensubjected to cell cultureinvestigation. Nevertheless. in the majorityof cases, yields of metaboliteshave been commerciallydisappointing.Staba.a pioneerin the investigationof medicinal plant cell culture.has stated (J. Nat. Prod. 1985.48,203)'theleare argr.rably as many gravestones as milestonesalong the way in developingplant tissueculture systems fbr the productionof secondarymetabolites'.Ultimately.of course,as Fowler pointed out (Chent.Ind. 1981,229).'no matterhow eiegantthe science,the f'undamentalcriterion has to be price comparabilitycoupled with plofitability'. a fact clearly obviatedby subsequentevents.
Sevelal forms of suspensionculture are commonly utilized, as fol1ows.
l. Btrtcltsuspensiort cultLrres. In this techniquethe cellsnrultiplyin a iiquid mediLrnr whichis beingcontinuously agitatedro brcakup any cell aggregates.Except fbr the circulation of air. the system is 'closed'with respectto additionsor subtractionsfrom the culture. Typically. the oliginal inoculation of cel1sinto the medium is fbllowed by a lag period and then, after increasingin rnass.the cells undergoa period ofexponentialgrowth and division. Finally, a stationary glowth phaseis reached.at which point somecomponentof the medium. essentialfbr growth. has probably been exhausted. Growth will recommencewhen cells aretransf-erred to a freshrnedium or when more medium is addedto the original culture. 2. Senticontintu.ttrs t'tiltures.lnthis instancethe system.an 'open'one. is designedfbr the periodic removal of culture and the addition of fresh medium.by which meansthe growth of the cultr.rre is continuously n-rainttiined. 3. Continuous cultures. Trvo fbrms of this 'open' system are the chemostatand turbostatsystems.in which the volume of cr.rlture remainsconstantand frcsh medium and culture are, respectively, continuouslyadded and withdlawn. The essentialI'eatr-n'e of these two systemsis that cell prolif'erationtakesplaceunderconstantconCULTIVAITION OT PIANT CE1T5 ditions. In the chemostatarrangement.I steadystateis achievedby addingmedium in which a singlenutrienthasbeenadjustedso as to Althou_ehthe f'easibilityof artificially cLrltivatingpiant cells had been be grrntlt-limilirrg; this contrastswith the batch cr-rlture rnethod,in recognizedat the beginningof the century.and White had propagated which the transientconditions in which the cells find themselves isolatedtomatoroots for periodsof over 30 years,it was only somef'ew lead to continuouschangcsin their glowth rate anclrnetabolism. decadesago that modern developmentsin the cultivation of cells of higherplantsasa callus,or asa suspension liquid culture.reallybegan. The ploblems associatedwith plant-cellculture are not completely In this connectionthe publicationof P R. White's Cultivutiortol Plttnt identical with those encounteredin the fermentationof microorganantl Animal Cells in 1954 and H. E. Street'sdevelopmentalwork at isms and fungi. With the former thereis much slowercell prolif'eration L e i c c s t eLr n i v e r ' i t i t l e s e ne m e n t i o n . iind it takes about 3-6 weeks to progressfiom the shake-f-lask level (300 ml) to prodr.rction Culturesof single cells -erowingunder controlledconditionsin a licapacity(20 000 l). Also in plant cell cultures, qr.ridmediurn,or callus culturesconsistingof undifl-erentiated masses large aggregatesof cells may fbrm which then exist under different of cells developing on a semi-solid n'redium.can be initiated from environmental conditions lrorn the suspendedcells. Dispersal of parcnchymatoustissuesof shoots.roots and other plant strllctLlres (see aggregatesby the use of the r.rsualf-ermenterpaddleswas originally Fig. I 1.I ). The maintenance of suchculturesdependson an adequate consideredtoo vigorous fbr fragile plant cells, resulting in their rupsupply of nutrients,including growth factors.and a contlolled sterile tLlre.To overcomethis,varior.rs designsof low-shearfermentersincludenvironment. The cells. although undifl'erentiated,contain all the ing the air-lilt or drum types were designed. However. such geneticinfonnationpresentin the nonnal plant. By suitablernanipula- refinementsare not always necessaryand the shikonin production tion of the hormonecontentof the medium.it is possibleto initiate the mentioned above is. in fact, caffied out in conventionalstirred-tank developmentof roots,shoots,and completeplantsfiom the calluscell vessels.As R. Verpoorleet al. have pointed out ("/. Nat. Prod. 1993. cultureandto encolllagcthe divisionofcells in a suspension culture. 56. 186). for the industlial application of plant cell cultures, the
cullure Suspensron of eilhersrngle,or s m o l lo g g r e g o l e s o { , c el l s
.. 1
Tronsferlo s e m r s- o l r o medrum
S u r f o c es-t e n l i z e d seclronof youngslem wilhochvemenslem
Fig.I l.l O r i g i no f p l o n ct e l lc u l t u r e s
Portronof colluslo | q u r dm e d r u m w t l hs h o k i n g ,, -.i
Developmenl Porllonol of collus collusto horrnone odlusledmedrum
Difterenlrolion of orgons
PRINCIPTES RELATED TO THECOMMERCIALPRODUCTION, QUALI]YAND STANDARDIZATION OF NATURALPRODUCTS recognition that shear-tolerantplant cell cultures exist is important becausethe fermentationindustry at presentusesstiffed tanks almost exclusively.Investmentin new ingeniousbioreactors,as reportedfor experimentalcell cultures in recent years,would place a major constrainton the commercializationof plant cell biotechnology. To maximize on the cell massproduced,the cell suspensionculture eventuailybecomesvery denseand this presentsproblemsofeven aeration. Most pilot studieshave utilized fermentersof 5-15 litres capacitv and the reportedscalingup to 20 000 and more recently75 000 litres in West Germany and Japanpresentschemical engineeringproblemsof considerablecomplexity.
PRODUCTION OF SECONDARY METABOTITES The genetic information required for the manufactureof secondary metabolitesis also presentin the undifferentiatedcells of the species concerned,and when activatedshould lead to the productionof these materials.Much interesthas beenarousedby this aspectof cell culture with the aim of growing particularplant cells on a commercialscalefor the productionof valuablemetabolites. A pioneerin the cell culture of medicinal plants was E. J. Stabaof MinnesotaUniversity,and his group was the first to demonstratethat many medicinal plants did produce in cell cr-rlturetheir characteristic secondarymetabolites,albeit often in low yield. Notable advances were made by Zenk and colleagueswho in 1975demonstrateda l0% (dry weight) productionof anthraquinonederivativesin a Morinda citrifolia culture-at that date the highest yield of secondarymetabolite achievedby cell culture.By 1991 (M. H. Zenk. Phttrx'hemistry1991. 30, 3861) almost I 000 speciesof calluswere depositedin the collection at Braunschweig.Commercially orientatedresearchhas concentrated on those speciesthat produce high-value speciality phytochemicals. Obvious examplesarc Catharanthusroseus(dimeric antitumouralkaloids), ginseng(ginsenosides) and Taxusspecies(taxol). Apart from the generalproblemof low yield of productother factors which needto be addressedwith cell culturesas a sourceofphytopharmaceuticalsare:instabilityof ce11lines.compartmentalization and isolation of the products, and the nature of the metabolitesproduced. Somepoints concerningtheseproblemsare given below. Low production of desired metobolites Knowledge of the enzymology of secondarymetaboliteformation is still incomplete.Secondarymetabolicprocessescompetewith primary metabolism for precursorsand potential bottlenecksfor the former may involve those enzymeslinking the primary and secondarypathways,for example,tryptophandecarboxylase convertingtryptophanto tryptaminein the formation of indole alkaloids and cyclaseenzymes involved in the synthesisof cyclohexanoidmonoterpenestiom geranylpyrophosphate. With cell cultures,as distinct from whole plants, particulargenesmay be repressedand needto be activatedby suitable elicitors,a techniquewhich is currently an important areaof research andis discussed below. The compositions of the media in which culture cells are grown have been extensivelyinvestigatedwith a view to increasingboth the biomassand secondarymetabolites.Often, asreportedwith Dioscorea deltoidea fbr instance, rapidly dividing cells produce little or no metabolitesof interestand a changefrom a growth medium (high biomass) to a production medium is required to effect the necessary biosynthesis.In this connectionZenk's 'alkaloid productionmedium' fbr ajmalicinetn C. roseusmay be noted.
Variationsin the relative hormonal contentsof the growth medium can aiso affect metabolism.In 1986Morris reportedthat reducedconcentrationsof 2, 4-D increasedalkaloidformation in C. rosellscultures and it is claimed (ChemAbs 1988, 109, 53250) rhat abscisicacid and antigibberellin compounds have similar etTects.With Thalictrum nzinas,ethylenehasbeenshownto activatethe productionof berberine in cell culturesfrom the key intermediate(S)-reticuline(Y. Kobayashi et al. Ph,ttochemistn',1991, 30, 3605) and the ethylene-producing reagent 2-chloroethylphosphoricacid stimulatesanthraquinoneproduction in callusculturesof Rheumpalmatum (F. Kurosakiet al. ibid., 1992, 3I, 2735). Conversely,cardenolideaccumulationin Digitalis lanata tissuecultureswas decreasedby ethylene(T. BerglandandA. S. Ohlsson, J. Plant Physiol., 1992, 140,395). Cytokinins have been found to enhancesecondarymetaboliteaccumulationin a number of tissueculture studies-indole alkaloids(C. roseus\.condensedtannin (Onobrychis sp.). coumarins (.Nicotianasp.), rhodozanthin(Ricirzr.rs sp.), berberine(ThaLictruntminus) (seeA. Decendit et al., Plant Cell Rep. 1992,11, 400). A patent application(Chem.Abs. 1983, 118, 123122)coversthe manufactureof ginkgolidesA, B and C by cultivar ing Ginkgo biloba cells in a medium containing naphthalene acetic acid and kinetin. Rhodeset al. (Planta Med.1986,220,226) found a five-fold increasein alkaloid contentof a cultureof CinchonaLedgerian.l occurs when cells are transfenedfrom a 2, 4-D, benzyladenine medium to one containingIAA and zeatinriboside.For informationon plant hormones,seepreviouschapter. Although alkaloids from a wide range of medicinal plants have been producedsatisfactorilyby cell culture in the laboratory.a singular lack of successhas been experiencedin obtaining quinine and quinidine from Cinchona cultures and morphine and codeine from thoseof Papauer somniferumalthough,in both cases,other alkaloids are formed. To some extent the problems with the former are being overcomeby the use of transformedroots (seebelow) but the growthrate is very slow. With morphine biosynthesisit appearsthat lack of developedlaticiferous tissue in the unorganizedcell culture may be responsiblebecausecytodifferentiationleading to latificer-type cells Ieads to morphinan alkaloid production. One problem with Cathuranthuscel1cultureshasbeentheir inability to dimerizethe requisite indole monomersto form the medicinally important anticancer alkaloids vinblastineand vincristine. In a similar way the accumulation of monoterpenesin cel1cultures of some volatile oil-producing plants is severelylimited, probably becauseof the absenceof such storagestructuresas glands, ducts and trichomes. Banthorpe et n/. demonstratedin 1986 that, in Rosa damascenacallus and suspension cultures, negligible amounts of monoterpenes are accumulated, althoughenzymeswith high activity for the conversionof mevalonate and IPP into geraniol and nerol (seeChapter l9) are extractablefrom the apparentlyinactive callus. In this case,noncompartmentalization of the metabolitesprobably leadsto their further metabolism.This is supportedby Lappin et al.'s l98l finding that, when addedto culrures of Lcuandula angustiJolia,the monoterpenoidaldehydes geranial, neral and citronellal are reduced to their correspondingalcohols, geraniol, nerol and cirtronellol which, once formed, disappearfrom the culturesover about 15 h. It has been observed that the origin (stem, root, etc.) of the callus can play an importantpart in determiningthe biochemistryof the subsequentculture. Improved metaboliteproductionmay sometimesbe achievedby the addition of precursors to the culture medium. Thus, addition of coniferin (a phenylpropane)to cell suspensioncultures of PodophJ,llunthexandrumimproved podophyllotoxin production 12.8-fold and an increasein quinoline alkaloids was obtained with Cinchctna ledgeriana cultures fed with r--tryptophan.
;FF
PLANT CELtAND TISSUE CUITURE; BIOCHEMtCAt CONVERSTONS; CLONALpROPAGATION Light intensity and selectivewavelengthsof light have been shown to have a stimulating effect on the production of some secondarymetaboIites in various tissuecultures.Thus, in one repofi (1990) blue light enhanced, whereas red light decreased,diosgenin production in Diosc'oreadehoideacal1uscultures.However. suchbeneflcial treatments aredifficult to accommodatewith conventionalstirred-tankfermentors. The selectionof high-yielding cell lines has been a major factor in counteringlow productivity. Such selection,perhapsinvolving a few plantsfrom severalthousand,has beengreatlyfacilitatedby the useof modernimmunoassays(q.v.).In the caseof Catharanthusroseusclltures,for example,recentresearchhas concentratedon the production of the dimeric alkaloidsvinblastineand vincristine(q.v.),the important anticancerdrugs.The alkaloidsare producedat the end of a complex biogeneticpathwayin which the monomersarelirst produced.The latter,as corynanthe-,strychnos-and aspidosperma-type alkaloidscan all be produced (0.1-1.5E;) in culture using Zenk's alkaloid production medium. Different cell culturesderivedfrom any one speciesof plant may vary enormously in their synthetic capacities,so that, in the above case,distinct high ajmalicine-producingand high serpentineproducingstrainsarepossible. Examplesof other plants for which somaclonalvariation has been exploitedincludeMcotia na rustico(nicotine)(of no commercialintetesI), Coplis japonica (berberine), Anchtrsa officinalis (rosmarinic actd), Lithospennn11 erythrorhllon (shikonin) and Hl.oso.anrus muti r:ns(hyoscine).For Tholictrumninus (berberine)a straingiving a 350fbld increasein alkaloidproductionhasbeenreported.
of Glyc,trrhiz,aechinata,sesquiterpene lactonesfrom Androgrophispaniculata cultureq new minor alkaloidsand anthraquinonesfrom CinchoncL ledgeriana and C. pubescens,and tropane alkaloids, not previously obtainedfrom the species,fiom belladonnaroot-cellsuspension cultures. Recently the novel compound (2-glyceryl)-O-coniferaldehydehas been obtained from cultures of Artemisia annua and Tanacetumparthenium (L. K. Sy and G. D. Brown, P/r,l/ochemistr:,, 1999,50,781) and the quinone-methidetritetpenes,tingenoneand 22-hydroxytingenone,from callus cuitures of Catha edulis (8. Abdet Sattar et a!., Saudi phatm. J., 1998,6, 242). Other plantsyield cultureswhich producea dift'erentspectrum of secondarymetabolitesfrom thosefound in the intact plant. These aspectsof cell culture, althoughgenerallyunhelpful for the promotion of this technique ior industrial purposes,have imporlant implications for other areasof phytochemistry.However, in the case of cell cultures of PctpaversomniferumandP. bracteatumwhich do not producemorphinan alkaloids, sanguinarine,a benzophenanthridinealkaloid of commercial importance, is obtained in yields sulliciently high to allow industrial exploitation.
INDUCEDSECONDARY METABOTISfiI IN CELtCUTTURES
Although the undifferentiatedcells of a plant suspensionculture are generally totipotent, i.e. they possessthe complete genetic make-up of the whole plant, many genes,including thoseinvolved in secondarymetabolism, are repressedwith the consequencethat the yields of desiredcomInstability of cell lines. It is well known that changesin the genetic poundsin suchculturesare disappointinglylow. However,it is becoming characteristics of cells occur within a culture so that callus selectedfor increasingly apparent that a large number of second:ry metabolites specificbiochemicalpropertiesmay needreselectionafter a period of belong to a classof substances temed phytoalexins.Theseare stresstime. In a few cases,for example anthraquinoneformation, selection relatedcompoundsproducedin the normal plant as a resuit of damaging may be achievedon a colour basis but, more usually,assayssuch as stimuli from physical, chemical or microbiological factors. When cell radio-immunoassayare necessary.Gross changes in chromosome cultures are subjected to such elicitors, some genes are derepressed, numbermay occur in culturedcells; thus.Tabataand colleaguesnoted resulting,among other things, in the formation of the secondarymetaboin 1974 that with a particular suspensionculture of Datura innoxia lites which are found in the entire plant. The techniqueis being increascells there was a 327clevel of diploid celis, with the remaindermostly ingly employed in cell-culture studies and examplesgiving a range of at the tetraploidlevel, ranging in constitutionfrom 4n-5 to 4ir+3 (see both abiotic acid and biotic inducersare siven in Table 11. I . Chapter l2 for extra chromosomaltypes).Another straincontainedno diploid cells but cells with 46 or,14 chromosomesoccuring in the propofiions of 79Vcand2l %, respectively.Nevertheless, for alkaloid proBIOCHEMICAL CONVERSIONS BY PIANT duction, Kibler and Neumann in 1979 found that haploid (1n) and CELICUTTURES diploid (2n) cell suspensionculturesof D. innoxia showedno difTerence in tropanealkaloid production (c.f. leavesof 1n and 2n plants; In nuch the sameway that modificationof a particularsubstratecan be Table 12.1), but for protoplast-derivedcell-culture clones of effectedby microbial fermentationso, too, can plant suspensionculHyoscyamusmuticus, Oksman-Caldenteyet al. have found that cul- turesbe employedfor the sameputpose. turesfrom ln plantsare richer in hyoscinethan thosefrom 2n plants. Of possiblecommercialsignificanceis the ability of some cell cultures of Digitalis Lanatato effect glucosylations,hydroxylations,and Isolation of product. For continuouscultivation and production of acetyiations.Reinhardand colleaguesdemonstratedthat their cell cu1active metabolitesit is preferable,for isolation purposes,that the ture, strain 291, cultivatedin air-lift bioreactors,was particularlyeffimetabolitesbe excretedinto the medium ratherthan be retainedwithin cient in the conversionof B-methyldigitoxininto B-methyldigoxin(a the cells. The biomasscan then be separatedfrom the nutrient liquid 12B-hydroxylation).Commercial exploitation of rhis processwould from which the active constituentsare extracted.Two-phaseculture enableutilization ofthe large stocksofdigitoxin which accumulateas systemshavebeendescribed.With thesean immisciblenontoxicliquid a byproduct in the manufacture of digoxin from D. lanata. More phase.e.g. a silicone product, is added to the 1'ermentation tank to recently,Stricker (Planta Med., 1986,418) reportedon a highly efiiextractthe metabolitesand in this way the developmentof the culture cient 128-hydroxylationofdigitoxin itselfusing D. lanata cell suspenis not disturbed.The removal of entrappedmetabolitesfrom immobi- sionswith a two-stageprocessinvolving first, the proliferationof cells lized cells (q.v.) without killing the cells is anotherrecentinnovation. in a growth medium and then a transferto a suitableproductionmedium. With cell culturesof both D. lanata andThevetianeriifolia anumNature of metabolites produced. Sometimescompoundsnot detected ber of new cardenolides have been biosynthesized frorn added in the original plant appearin the cultures; thus, a new coumarin, ruta- precursors.Cell suspensionculturesof Stroplmnthusgratus will effect cultin, hasbeenisolatedfrom suspensioncell culturesofRz/c g raveolens, variousbiochemicalconversionsof digitoxigenin,as will culturedgintwo new chalconeshave been characterizedfrom static (callus) cultures sengcells (K. Kawaguchiet aL.,Ph,-rochemistry,1996,42,66j).
IF''
PRINCIPLES RELATED TO THECOMMERCIALPRODUCTION, QUATITYAND STANDARDIZATION OF NATURALPRODUCTS
Tcble | | . I
Induced produciion of metcbolites
in cell cuhures by vorious elicitors.
EilCIOT
Plont-cell suspen sionculfure
tltecl
Colchicine
Valerionawollichii
Coppersulphote
Lithospermum erythrorhizon VoriousSolonoceoe
Colcium
Alkonnotinctoro
Acetylsolicylic ocid
Cothoronthvsroseus
Methyljosmonote
Sanguinoria conodensis
Sixlyfoldincreose in volepotriotes withsix new compounds {notdueto higherploidylevel} Greotlyincreosed shikoninproduction Inducedformotion of sesquiterpene phytoolexins of lubimintype Stimulotion of songuinorine ond chelerythrine biosynthesis lncreosed production of lumourcellsuspensions (505%),totolphenolics ll587%1, furonocoumorin s (612%1, onthocyonins (1476%) Dihydrobenzophenonthridine oxidoseoctivotedin loststepof songuinorine biogenesis Production of novelonihroquinones (robustoquinones) witho roreoxygenotion potternin ringA Promotes biosynthesis of soponins ond inhibits phytosterol production Stimulqtion of coumorinsynthesis
Cinchonarobusta
Thiosemicorbozide
Panoxginseng
Sterilized fungolmyceliolPythium, Phytophthora, Verticilli un), etc. or extrocts
Pimpinelloonisum Petroselinium crispum Ammi mojus Cotharanthusroseus Cepholotaxus horringtonio Cinchonoledgeriona Gossypiumorboreum
Yeost,yeostextroctsond preporotions corbohydrote
Eschscholtzi o colifornico Thalictrum rugosum Rutagroveolens Orthosiphonoristotus
Produclion of cothoronthine ond othermojorindole olkoloidsstimuloted Dromoticincreose in olkoloidcontent lncreose in onlhroquinone production One hundredfold increose in gossypolofter120 h incubotion Lorgeond ropidincreose of benzophenonthridine olkoloidproduction Up io fourfoldenhoncement of berberine Increosed production of ocridoneexpoxides but not rutocridone Stimulotion of rosmorinic ocid production
Monoterpenebioconversionshave beendentonstlatedwilh Menthct Nicotinna trLbacuntcell cr-rltures can selectivelyhydrolysethc R-conceli lines capableof transformin-qpulegoneto isomethone.and ( )figurational lbrms of monoterpenessuch as bornyl acetate and menthoneto (+)-neomenthol. isobornylacetate. The rue plant (Ruta gnneolens) and its normal tissueculturesconMany other biochenical transfbrmationsby cell cultureshave been tain a numberof constituents.including l'uranocoumarins derivedfrom demonstrated.and include epoxidations.esterfbrmation and saponifi(Fig. 11.2,seriesA). In 197'1. 7-hydloxycoumarin SteckandConsttrbel cation.glycosylation.hydroxylation.isornerization. methylation.and showedthat two chemicalmimics of the 7-hydroxycoumarin precul: demethylationand oxidation. sor',the .l-methyl and 8-methyl derivatives.when fed to the Rrld cell For this techniqueto be commerciallyviable. the product mllst be culture. gave rise to a number of the correspondingunnatural ana- sutliciently important. the substratemust be available in reliable logues(Fig. 11.2.seriesB anclC). amounts,and the reaction should not be one that is more easilv DerPossibilitiesfbr the productionof anticancerdru-esare illustfatedby formed by microorganismsor by chernicalrneans. the biotransfbrmationof syntheticdibenzylbutanolides to lignanssuitable fol conversionto etopside (Chapter 28) by a ser.ni-eonrinuous processinvolvin-eculturesof Podophl'llLun peltotunt(J. P.Kutney el c/. IMMOBITIZEDPIANT CEttS H ete rocycl e,s.1993. 36. l 3). Interestingand potentiallyuscful hydroxylation and oxidzrtionreactionshave also been demonstr-atcd for the Irnn'robilizedplant ceils can be used in the samc way as immobilized biotransfbrmation of podophyllumlignansin cell suspension cultures enzymesto eff-ectchemicallydifficult reactions.Reinhardand coworkof Forst'thia intennetlia (A. J. Broomhead and P M. Dewick. ers have reportedon the irnmobilization()f Digitali,t lunotu cells by Phttochen'Lisrtl', 1991.30. l5 I l). suspendingthem in a sodium alginatesolr.rtion. precipitatingthe algiThe principalalkaloid producedby the cultivationoI Rauw,olfioser- nate plus entrappedcells with calcium chloride solution,pelleting,and pentitlocells is the glucoalkaloidraucaffiicine.However.f'eedingwith allowing the productto harden.The granulescatalysedthe conversion high levelsof ajrnalineleadsto the productionof a new group of alka- of digitoxin to purpureaglycoside A (fbrmula, see Table 21.71 and loids.the raumaclines(S. Endrefset ul. Phytochemistry..1993,32,725). hydroxylated B-methyldigitoxin to give B-n'rethyldigoxin.Although Some biotransfornrations are stereo-specificand have potential for the hydroxylatingactivity of the entrappcdcells was aboLrthalf that of the isolation of optically active compoundsfrom the racemate:thus, suspendedcells. the pelletshad the advantagethat rhe biocatalystwas
P L A NC T E LA L N DT I S S UCEU L T U RBEl o; c H E M t c AcLo N V E R s l o N SC; L o N A Lp R o p A G A T t o N
--l>
otu-u
HO R,|
7- Hydroxycoumorin derivotives
^"/
\
\\ Olw-
OH
orc:o
cHso
o-c:o Psorolenderivotives
SeriesA (Rt = P.= 11; S e r i e sB ( R 1: H ; R . = C H s ) i .=H) S e r i eC s ( R r = C H rR
Rl Herniorin derivotives
re-r.rsable for periodsextendingto over 60 days. Other workers report thc biotransfbrmationof codeinoneto codeineby immobilized cells of Pupuversonuifentm and the releaseof papaverine.codeineand morphine to the mediurn (32,10--,1050Fg 1-l); decarboxylationof rtyrosine and I-DOPA has also been recorded.Japaneseworkers have studied seaweed immobilized cell cr-rltur-es fbr the production of tlavour constituents.Glyoxal cross-linkedpolyacrylantide-hydrazide hasbeenusedas the imrnobilizing agentfor Ment/ra cells which effect the monoterpenereductionsmentioned above and the techniquehas been further developed by arresting unwanted cell division of the entrappedcells by gamma inadiation. Other immobilization supports which havebeenusedincludepolyurethanefbam. acrylantideand xanthan-acrylamide. A techniquewhich has been lbund useful lor the study of alkaloid tbrmirtion in Crtfi?aarabica is to use a membraneof polypropylene sheetingof specified pore size. porosity and thicknesson which to immobilize the cells in a 3-rnm thick layer; the nutrientmedir-rmcir.cr.rlatesbelow the membrane.Ccttharunthusrc.rea.rcel1shave also been considerablystudiedand progressis bein-qmade in developingmethods fbr the releaseof alkaloidssequesffated in the cell vacuoleswithout killing the culture. It has been suggestedthat the immobilization of mycelia-fbrming nricroorganisms(e.g. Clrn,lcepspospalil by alginic acid rright be effective in the biotechnologicalproduction of complex metabolites suchas the ergot alkaloids.
ORGAN CUTTURE In the same way that it is possibleto culture Lrndifl'erentiated plant cells, so asepticsuspensionculturesof leavesand roots can be r.r.riiintained.The organscan be obtainedin the culture either by difTerentiation from callustissueculturesby suitablehormonalmanipulation,or by the use of sterilizedroots or -erowingpoints fiom whole plants or seedlings.Ofien. these cultured organs will synthesizesecondar.y metabolitesu,hichmay be eithernonexistentor in poor yieid in the normal cell culture.Thus. cardenolideproductionin Digitalis lantLtaand. D. purpurea cultures increasesas tissue differentiation proceeds.
F,
v
Rl
M o r m e s i nd e r i v o t i v e s
Fig.| 1.2 Biochemicol conversions involvingRuto g roveolen s cellcultures.
\
Enhancedproductionof alkaloidsoccursvn,hen roots developtr.omthe callusculturesof the tropanealkaloid-producing Solanaceae. In contfast to the latter. in which leaf diffbrcntiation alone prodr.rces little alkaloid. leaf organ cultures of Cutharantltu.\roseu.\ and Ruuv,olfio seryterilittttsynthesizea i,ariety of alkaloids. Dirneric alkaloids have beencletcctecl in or-Qan culturesof C. nrsr:u.r.suggestinethc possibility of an eflicient procluctionsysten'rfbr these valuablc alkaloids. The dimersoccurredonly in thosecultureswhich also containedvindoline and catharanthine.Whereas ccll suspensioncultures of popaver bracteotLullwere found to synthesizeorientalidineand sanguinarine. the root and shootculturesproducedthebaine.For ginsenosideproduction. ginsengroot cultureshavebeengrown in 20 000 I bioreactors. As with cell culture,elicitationcan give increased yieldsof secondary metaboliles.Thus both normal and hairy (q.r,.below) root cultures of Hyosct'arnusmuticLtswhen treatedwith jasmonic acid and its methyl esterat concentrationsof 0.001 to 10 pM prodr"rced large increasesin the levels of methyl putrescineand conju-eated polyatnines.However the increasc of tropane alkaloid prodr-rctionwas not remarkable (S. Biondeet ul.. Pleit CeLlRep..2000.19. 69 I ). Tro nsformed root cu lture-'hq iry rool' c ultu re Certain soil bacteriao1-thegenusAgrobtrtteriun?causea transformation of plant cells by introducinginto their genomerDNA from a bacterial plasrnid. Such transfbrmedroots, produced by inoculating the host plant. when growr.rin a horntone-fieemedium give rise to copious roots referred to as 'transformedroots' or 'hairy roots' (see Chapter l2). On removal of the Agrobacterium the roots continue to develop profusely and fbr sorre plants which normally prodLrcesecondary rnetabolitesthe hairy roots accumulatethesen-retabolites in quantities comparablcto thosefbunclin the norrral intactplant. Agrcbut:teriunt rhi:,ogenesand A. tunteftrciensare the bacterial speciesmost commonly usedto etl-ecttransfbrmation. Comparedwith the few examplesgiven in rhe l3th edition of this book thereis now a considerablebibliographyon transtbrmedroot culturesof medicinalplants.Someexamplesaregivenin Table | 1.2. As with ordinary'cellcr.rltnres and root cr-rltures it is possibleto utilize transfbrmedroots to caffy out biological conversionsnot normally associatecl with the intact plant. The rapid growth-rateof hairy roots
PRINCIPLES RETATED TO THECOMMERCIATPRODUCTION, QUALITYAND STANDARDIZATION OF NATURALPRODUCTS
Toble | 1.2
Metobolites of some lrqnsformed {hoiry) roots.
Alkoloid-contoini ng plonts at,^^^
h^ll^,1^^^^
Colhoronthusroseus Cinchonoledgeriono Doturostromonium 1 1 , , ^ . . , , ^ ^ , , "^ t h , , . H. muticus Solonum lociniatum Flovonoids c1,.,,,,h;.^ ^l^h,^ lridoids Voleriono officinolis Lignons Linum flavum Polyocefylenes Lobelio infloto Quinones Sesomum indicum Steroids Ponox ginseng Trig on el Io fo e n um-gr a ec um
Increose in growthrotecomporedwith untreoted roots Optimizotion of selected linesmoygivesourceof cothoronthine ond vindoline Quinolineolkoloidproduction comporoble with thotof intoctplonls Alkoloidcontentcomporoble with normolrootsof intoctplonfs;increosed by useof voriouselicitors e.g. mefhyljosmonote New piperidone olkoloidobloinedtogether withtroponeolkoloids Culturetreotedwithchitosonoccumuloted hyoscyomine 2.5-to3-foldcomporedwith untreoted hoiryroots yieldincreosed Solosodine oboutfourfold Knownond new flovonoids produced;olsoo new prenyloted biourone Yieldof volepotriotes fourtimesthotfoundin normol9-month-old roots 5-Methoxypodophyllotoxin production 2-5 timesthotof untronsformed roots;5-,l2 timeshigherthoncell suspension culiure;comporoble with noturolroots Polyoceiylenes produced,somehovingo gentiobiose moiety.Thesecompounds hoveonticoncer octivify Yieldof ontimicrobiol nophthoquinone increosed by morethon5O timesthotfoundin intoctplont potentlor production produced.Production of ginsenosides; diol ond trioltypeginsenosides more Joponese thonwithordinoryroolcultures. effective By optimizofion of theculturolconditions, up to threetimeslhe production of diosgenincomporedwith nonelicitedroots
hairy root cnltures oft'ersthe possibilityof lapid conversions. Ginsen-e hal'e beenshownto convertdigitoxigenin(the aglyconeof a nur-nbelof Digittlis cardiacglycosidesq.v.)into ner.r, compoundsbir esterification at palnritateand rrvristate.and by the formationolgenC-3 with stearate, tiobiosicles andsophorosides. Palr e1n1.(Pht'tocherrti.sttr . I99 I . 30, 2dl7 ) in studieson the biosynthesisof tropanealkaloidst'edthe S-analogue of tropinone(8-thiabicyclo[ 3.2.1loctan-3-one ) to translbnnedroot cnltures ol Duturtr strunoniun'tand obtainedthe S-analogucof tlopine,together with thc 3-O-acet,vl ester. PapuverSonCell culturesderiVedlionr A. r'lri.:r,gc,/rd,f-tftrnstru'med niJerLrnttissue have been studied lbr alkaloid production (R. D. Willianrsand B. E. Ellis.Phytoclterr.iln.1993,32.1I9). As with nor mal cultures,rrornorphinan alkaloidswele prodr,rced but lalgeamounts of sanguinarine were.
CTONALPROPAGATION As noted earlier (Fig. I I . 1), by adjustnrentof the plant glowth legulators irr the cell culturemedir.rmit is possibleto promoteditl-erentiation of olgans fronr callus tissuesand to carry these lbrwtrrd to produce entireplants.As all cellsof the callusarederivedtlon a singlemerisplantsshouldbe -eenetically tem.all legenerated identical.This tact has in a shortpcliod obviouscomrrercialimplicationsfbr the productiorr" of time, of unifbrm crops derived fiom a small number o1'desirable plants(or cvcn a singleindividual). processwere presented The practicalapplications of the scaling-Lrp at a synposiurnin Knoxville,Tennessee. 1980,at which. ol pharrnaceutical interest.Levy leported from Ecuadoron the llrst lar-ue-scale commercialapplicationol in t'itro culture lbr the masspl'opagationof clonesofpyrethrum plants.The scaleof suchplojects is evidentfrom the originalobjectiie.which u,rs to produce.from 57 superiorclones isolatedover a 10-yearresearchperiodup to 1976, l2 million plants
pel'year to leacl.ra plantationtargetof the numberrequiredfor 1000ha of land over '1 1'ears. Four stagesare involved:Stagel. cultureinitiation: Stage2. subculturetbr shoot multiplication: Stage3, r'ootin_q of plantlets: Stage :1. hardening otJ for tlansplanting into field. 81, enrployinga 2,1-hshifi systemfbl the manipulationof the cultures, alter abor.rt 8 monthsthe operationreachedits targetlelel of production of 62 000 plantspt:r month. Each batchof 62 000 plants moveson to a 'propagationtarm'. at a considerablylower altitr-rde than that at which the final commercialI'almsare situated,and is kept undel irrigation fbr iLrst'1-6months.During this tirre the young plantsundergoa rapid vegetativedeVeloprnent for a final vegetatir,'e propagaand are sr.ritable tion step which increasestheir number l-5-fbldto give the rcquircd 930 000 plants per n-ronth.Thc bnild-up rate of the number of young plantsfbl transplantation to the comnrercialfalm is crucialbecausein the EcuadorianAndesthe plantin-eseasonis restlictedto the 90 daysol' Octoberto Decelnberduring which weatherconditionsare suitable. Thus.theoverallbeneficialresultsof tissuecr.rlture propa_eation fbl the pyrethruurindustryof Ecuaclorale: ( 1) the ability to introduceselected high-yieldingstrainson a commercialsclle in a shorttime: (2) the creationofimprovedgrowthchalactclistics; and(3) a greatereaseofplantation planning.A similal techniquelvasdevelopedfbr thc commercial prodr,rction. ol'er a numberof yeals.of DutLtrusurtglineu.a sourceof h-voscine. Clonal propagationis a potentially valuable method tbr ploducing hi,eh-yieldin-e cropsof specieswhich tend to be variablewhen gron'n fror-nseed.Fennel (Foellculurn vulgure). for instance.is genetically heterozygonsand produceswide variationsin oil yicld and composition.In 1987Mir-rra e/ al. (PlctntaMed..1981.53, 921recordeda 2-year tdal ini,olvingthe productionof unifbrni clonal plantsderivedtiorr somaticembryoidsof suitableparentplants.In the normal sexually propagatedplar-rts the anetholcontentof fiuits varied flom 0 to 12.9% (mean2.82clc). whereasin theclonalplantsit showeda narrowerdistributionof 0.7-3.0%(rneanl.92ct).
P L A N TC E L IA N D T I S S U C E U L T U RBEI ;O C H E M I CCAO L N V E R S I O NCSL;O N AP LROPAGATION Genefic homogeneity Further reoding ln practice.genetic changescan occut' in cells during theil artificiLrl Charluood B V. RhodesM J C (eds.)1990Secondaryproductstiom plant tissueculture.Proceedin-qs of a PhytochcmicalSocietl of Europe culture.andcarefulregulationofthe rnediunris necessary. However.it Slrnposium.ClarendonPless.Oxfbrd. LrK hasbeerrshown,at leastftrr Datara innoriu, that on difl'erentiationit is VcrpoorteR. Hcijden R van der. SchripsentaJ 1993Plant cell biotechnolo,uy the normal 2n plants. as distinct fronr the abnormal chromosontal fbr thc productionof alkaloids:prescntstatusanclprospects(.retit'tt' ytith I 30 tvpes. that are fnvoured. Clonally propagatedspeciesfbr which the rql:!).Journalof Natural Products56: I 86 homogeneityof the secondarymetaboliteshas been shown include Z a l a r R . A e r i V . D a r t aA 1 9 9 2A p p l i c a t i o no f p l a n t r i s s u ea n d c e l l c u l t u r ef o r p r o d u c t i o no f s e c o n d a r ym e t a b o l i t e s( r e y ' i e t v i t h 8 0 r e . f s 1F.i t o t c r a p i a Ac ortitunt carmiclutel i i, Artgelictt ocutilobo, Bupleurmn .folcutum. 63: 3-l Gent iattu st:trb r o, Rehmonn itt,q.l uti rn sa trnd,llel'Iarelurubi ana.
Phytochemicol voriotion withino ,:,. sPectes
As wasindicatedin Chapter3, the speciesrepresents theunit ofplant classification;it may constitutea homogeneoustaxon of plantswith little variationtiom one specimento anotheror it may includevmious vuieties or raceswhich eachhave somedistinctivef'eatLrre(s). Thus rvith Daluiz sfruntr.tnittnt lour varietiesare described:var.,stramonium-whiteflowers.thornycapsules;var. inernis-white t'lowers.bald capsules:\/'ar.tutula lilac flowers,thomy capsule s andvar.godronii-lilac flowers,bald capsules.Often sucl-rvarietiesfepresentsingle gene mutations(in the abor.'ecasetwo independentgenesare involved) and are morphologically recognizable.In other instancesthe mutationgives rise to a varianthaving a diff'erentsecondarymetaboliteprofile-not necessarilydiscemiblein themorpholo-qical fbrm; thesearetermedchemicalracesor chemodemes. The mlltationrray involvethe presence or absenceof a singlecomponent or. if acting at an early stage of the biosynthetic route. may involve a whole seriesof compounds. Knowledge of the existenceof such chemicalracesis not new and A. Tschirch(18-56-1939)rnhrs Handbuch tler Phormakognosiedeals 'physiological with the various lbrms'of Varilla pktnifoliu, Cincltona spp.. t-ennel,Piper nigrunt and the balsam-conttriningtrees.Also, 60 years ago A. R. Penfbld and co-workers were describing chernical races of various Australian oil-producing speciesof Leptospermunt. Boronio andEucalvptus. point mutaIn additionto the abovewhich involve submicroscopic tions associatedwith an alterationin the DNA chrorrosomalmaterial. there are other genetic variations which rnay affect chemical constituentsof the species.Theseinclude (a), the existenceof polyploids in which chromosomesetsbecolnedoubled,tripled, quadrLrpled etc., (b), the additionof one or a few chromosomesabovethe normal complerrrent(extrachromosorr-ral types), (c). gross structuralchangesto a chromosome,(d), artificially producedtransgenicplants. Two consequences of the aboveare,on the positive side,it provides the possibility tbr the selectionand establishmentof superiorstrains with respectto the chernicalconstituentsas describedin this chapter. The negativeaspectconcernsthe arrival on the drug marketof material This. togetherwith other factors, which is low in active constitr.rents. necessitates the need fbr strict quality control (Chapter l4) which is by variouslegislativebodies(Chapter'2). being vigorouslyaddressed Transgeniccrops presenttheir own problems.
CHEMICALRACES,CHEMODE'I/TES
RACES, CHEMICAT CHEMODEMES 80 NUMBER 84 CHANGESIN CHROMOSOME PRODUCTION ARTIFICIAT OF MUTATIONS 86 HYBRIDIZATION 8 7 MEDICINATPTANTS 89 TRANSGENIC
The plant kingdom hasbeensubjectedto an extensive.but not exhaustive, chemicalinvestigation.Thousandsof sampleshave beenscreened for sr.rbstances of medicinal value or lbr suitableprecursorsof therapeutically active compounds.Many other plants have been studied plant resistance chemicallyliom the viewpointsof manuraltreatrnents. and biosynthesisof active constituents.From such observationshas e m e r g e d e v i d e n c e f o r t h e e x i s t e n c eo l ' c h e m i c a l r a c e s ' o r 'chernodemes'. Theseare definedas chemicallydistinctpopulations within a speciesand have sirnilar phenotypesbut different genotypes and as such are identical in external appearancebut difI'er in their chen-rical constituents. Beforethe existenceof a chemicalracecanbe established. cerlainfirnarenecessary. A chemicalanalysisof a numberof damentalobservations random samplesof a particul:Lrspeciesmay show a variationbetween the samplesbut would be insufTicientto demonstrateany geneticaldift-erences. since tactorssuch as age. climate and soil can all exert profound eff'ectson the resultof the ultimateanalysis.Samplesof seed.or clonesl'romditferentplants,mustbe raisedtogetherunderuniform conditions.andto excludehybrids,which do not breedtrue,cultivationfor a numberof generationsis desirable.It may then be possibleto demon-
P H Y T O C H E M I CV AA L RIATIOW N I T H I NA S P E C I E S \tratethatdiff-erences occurin eitherthe natureor quantityof a particular constituentandthat thesedifl'erences areof a hereditarynature. Suchobservations necessitate numerousassaysandprecisehorticultural work. to which must be added the difficulties of dealing with plantswhich may take )rearsto mature.Furthermore.many of the more in-rportant vegetabledrugscannotbe successfullycultivatedin temperatc climates,so that it is not surprisingthat the numbel of rnedicinal plantsfully investi-qated underideal conditionsis still limited. Fixed oils. Agriculturally,the cultivationof seedoil plantsis second only in importanceto that of cereals.Most of the llxed oil producedis usedby the fbod industry but there are also impoltant industrial and other.including phannaceutical.uses.It is not surpdsingtherefbrethat sustainedbreeding programmesfor the irnprovementof yields and quality of oi1have beenin progressover many years.Normal rapeseed oil contains,as an acylglycerol.20-4oc/a of erucic acid. an acid havin-e an extra long carbonchain (C22)and one doublebond. Its plesencein quantityrendersthe oil unsuitablefor ediblepurposesbut valietiesare now extensivelygrown which containno erucic acid.The value of the crop has been lurther enhancedby coupling low erucic acid content ivith one giving low glucosinolatesin the protein rnealthus improving the animal feed properties.However,erucic acid is industriallyirnportant for the manuiactureof lubricants,artificial fibres and plasticizers. so that varietiesof rapedevelopedfor their high erucic acid contentare alsoimportantagriculturalcrops. The production of oil liom sunflower seedhas been improved by varietiesthat yield linoleic acid-enrichedoil and which are more convenientfor harvestingby having a largesingleflower headand no sideshoots.Groundnuts.the sourceofArachis Oil BP. exist as rurious strainswith difl-erentrelativeproportionsof fatty acids. Safflowerconstitutesan importantoil-seedcrop and its geneticvariability hasfacilitatedthe breedingof varietieswith widely differing oil constitutions.High oleic varietiesare usedfor oil fbr humanconsumption and high linoleic varietiesare important for oils usedas industrial coatingsand lubricants. The above exan'rplesinvolve plants with a short lif'e-spanso that breeding by classical methods is a relatively rapid procedure. However.this is not so with plantssuchas the coconutpaln-r,olive and cocoaso that in thesecasesmoderntechniquesinvolving genetransf'er ri ould havean obviousadvantagefor the introductionof new or modil'iedoil characteristics. Cyanogenetic glycosides. A well-known chemical race in the c1'anogenetic seriesis the almond.There are many I'alietiesof PrunLt.s torrununisshowing difl-erentmorphologicalforms. with and withor-rt amygdalin,but some varietieshave similar charactersand difTel only in the presenceor absenceof the glycoside.Also in this group the clovers,especiallyTriibliLonrepen.s,have beenextensivelystudiedand Lirturiu has been shown by Dillemann to producea chemicalrace by introgressivehybridization. L. striatu contains cyanogeneticglycosides.and.crossedwith the nonactiveL. yulgnris.givesriseto hybrids nhich. on repeatedback-crossingwilh L. tulgarris.give some plants diflicult to distinguishfrom L. vulgttri.sbut which contain the cyanor c n e t i cp r i n C i p l eorf L . . s / r ' i r r l r r . Afkaloids. The Duboisia speciesform an impofi.at conlnercial source of the tropanealkaloidsand havebeenextensivelystudiedby Austrnlian rr orkers.With both D. mtoporoides andD. leichlrurdtii. tlees fiom natural standsin various locations were examined and their progeny were raiscd side by side in experimentalplantations.The trees produce hr oscine,hyoscyarnine. norhyoscyamine. tigloidineandvaleroidine,and thc proportion of any onc alkaloid to total alkaloid may vary greatly.
It was shown not only that seasonaland environmentalt-actorsare involvedin this variation.but alsothatwithin a speciesthereexistsa wide rangeof alkaloidgenotypes. Othervarietiescorrtainingnicotineand nornicotinewerealsorepofted.Interspecifichybridsbetweenthetwo species were studiedand for-n'hybridcloneswere selectedtbr possibleexploitalion ashigh alkaloidyieldingstrains.Thus.in this genuswe havethepossibility of two distincttypesof chemicalrace- differentalkaloid types within a speciesandditl-erentalkaloidtypesamonghybrid phenotypes. An exarnple of the intprovement of the ntorphine content of opium poppicsby genealogical selectionis firmishedby the work of Lecat.The original seed gave capsuleshaving an averagemorphine content of 0.385c/c. From this heterogeneous populationwere selectedsix individuals whose capsulesanalysedabout 0.7% morphine.The seedsof these plantsformedthe headsof the linescultivatedin successive years.during which the best plantswere collectedand all thosecontaininglessth:ur 0.7% moryhinewererejected.The harr,'cst of 1955gavecapsuleswith an averagemorphinecontentof 0.165Vc,thus doubling the original morphinecontentof the popuiation.Sucha methodof breedingdoesnot produce a raceof plantssurpassingindividual morphinecontentstrom the original heterogeneous population:it melely producesa homogeneous raceof the alkaloid-richplants. In 198l/3 Phillipsonand colleagues(PlanraMed.. 1981.41. 105: 1983. 48. 187) reported at least three difl'erent chernical races of Papuver.firyaxandP.armeniat:un in which either ( l) l-benzyltetrahydloquinoline, proaporphine,aporphine.(2) morphinaneor (3) rhoeadine types are the major alkaloids: there are at least two difTerent cherr-rical strainsofP tuuricola containingeitherthe first or third types of the above. Thlee diflerent isoquinoline alkaloid chemotypesof ThaLictnntmintt,yhavebeenreportedfrom Bulgaria.Pultaverbrttr:teutLnnis a speciesexhibiting raceswith respectto thebaine. From ClayicepspurpLtreaa numberof raceshavebeenisolatedcontaining difl'erent -qroupsof ergot alkaloids and these have obvious irnplicationsfor the commercialproductionof alkaloids. One foddercrop in which the presenceof alkaloidsis undesirableis Iupin seed.Ordinaly wild fbrms ale bitter and contain alkaloids of the lupinaneseliesbut over the yearsa number of sweetlbrms have been developedfbr conrmercial pr-uposesin Europe. The strains depend fbr' their low alkaloidcontenton the presenceofii particularrecessiregene. However.asa nunrberof suchgenesexist.cross-ferlilization betweentwo difl-erentsweetstrainswill againgive bitter pfogeny.To avoid this happening.considerable careis necessary in regionswheredifl'erentstlains aregrown sideby side.Otherplantslbr which thereis evidenceof alkaloid varietiesincludeEphedro distachyaand the Lt r:r2odittm species. Chemicalracesappearto be lacking in the phannaceuticallyimportant indole alkaloid-containinggenertr Stryclutos.Rctuv'olfia and Cutharuthus. Anthraquinones. The pr.rrgativeanthraquinonedrugs owe their activity to complex mixtures of the l,S-dihydroxy derivatives of anthranols.their glycosidesand free anthraquinones. The relativeproportionsof the constituentsof thc mixture. which greatlyinf'luencethe pharmacologicalactivity,dependnot or-rlyon time of collection.ageof plant. drying conditionsand geographicalsource,br.rtalsoon genetical tactols.In a programmeinvolving Rheumpulntatwn, yan Os produced races varying in their rhein/chrysophanolratio and other hereditary strainsfor high- and low-yielding total anthraquinones. The analysisof individual Cas,sittungustifolictplants has indicated that selectionof individualsfor high sennosideB-yielding strainsis a possibility. 'With Cardiac glycosides. Digirtlis purpltrea the ptoperty of high glycosideconteutis hereditaly.The proportionof glycosidesderivedliom digitoxinandgitoxin is alsovery difl'erentin plantsofdiff'erentorigin and
r
PRINCIPIES RELATED TO THECOMMERCIALPRODUCTION,QUALITYAND STANDARDIZATION OF NATURALPRODUCTS remainsso during subsequent cultivatioltunderstandardized conc'litions. The strainswere distin-euished chemicallyas digipurpurin.strospcside and digitoxin types. It now remainsto pl'ovethat thesecharactersarc independent of the phenotype(i.e.that they arenot inseparablyassociated with other charactersof thc parent plant). One race. 'Cantbridge'. u'hichis relativelyrich in digitoxin.is easyto distinguish:the otherdigitoxin race fbund in the Vosgesdifl'crs little fiorn the other selections. V:Lriationin the propoltion and qr.rantity of glycosidestn D. kumta has alsobeennotedin rlixed populationsand.by the selfingof selectcdindividuals, strainsrich in a particularglycosidehavc bcen prodLrced. the inherited chalacterbein-estrongly developed.Vrluable physiological fbrms could thus be producedand Ligeti has recommendedthat these strainsbe designatedby suchnamesas 'D. larttrttrElrrh.chemo-r'arieties A andC'. dependingon therespectivepl'edominance of lanatosides A and C. It appealsthat with such in-brecllines continuousselectionis still requiredto pleventreversionto the normalcharacterlevel of the species. (q.v.)fbr thc lapid selecThe gleat valueof the laclioimmunoassay tion of high-yieldingstrainsof Digitalislunutuhtrsbeendemonstrated by Weiler and Westekemper.After two selectioustepsinvolving the arralysis of over l0 000 individualplants.the averagedigoxin content of the plants could be raisedtrvo to threcfbld and severalstrainsrvith averagedigoxin concentrations in the leaf of 0.67r wcle isolated. lndividual plants wele tbund with 0.9-1.0c/(digoxin content.As is usualu"itlrthis type of selection.no plantsbetterthan the f'ewbestof the originalselectionwereobtained. Followingintensi"'echemicalinvestigat.ion of the genusSu'ophonthus. Reichsteinandhis colleagues difl-erentiatcd four chcmicalvalietiesof the
polvrnorphous S. .sarntentosus fiom differentgeographical sources.They are salverogenin-.samentogenin-and samrutogenin-ploducing types with glycosidesof these.and a fourth fi'rrmwhich has a low glycosidal content(Fig. 12.I ). Althoughthelocalityof growthmay producequantitative diff'erences in the constituents ofthe variousraces.the overalltype is geneticallycontrolled.Among thoseplantswhich yield steloidalsaponins. severalthousandof which. fl'om diff-elentlocalitieshavebeenscreenedfor their sapogcnincontent.a similarvariationntayexist. Withanolides. The plant Wirhuriasonmifera(Solanaceae). in addition to producingalkaloids.containssteroidallactones.Investigntionsover the years.carriedout on varioussourcest'rfplantmaterial.andconceming the non-alkaloidalconstituents. hi,rdgiven difl'eringresr-rlts. rvhich were explainedby the work ofAblaham er al. (1968)on Israeliplants.Three chenrotypes werediscoveredamong2'1populationsof W.srnnniferacollectedin variouspartsof the country.ChemotypeI containedpredominantly withaferin A (0.27cof the dry weight). which is the principle responsiblefbr the plant's bacteriostaticand antitumor properties. ChernotypeII containsa cornpoundof similar structrlre.and chemotype III a mixture of relatedcornpclunds comprisinga new groupof steroidal lactones the withanolides(Fi,e.12.1).The only morphologicaldifference obsen'edbetrveenthe chemotypeswas a difTerencein llowering time (12 daysearly) fbr chemotypeIII. Sincethen.other chemotypesof W somnitbrahavebeenreportedfror-nlndia and SouthAfiica. Steroidalalkaloids. Sokuuuttspp.(Solanaceae) containsteroidalglycosidic alkaloidssomeof which havebeeninvestigatedas potentialinter-
Sarverogenin(hypotheticalformula)
Sarmentogenin
Sarmutogenin
H
Fig.l2.l Steroidol constituents of chemicolrocesof Strophonthus ond W ithonio.
\
cH2oH
o Withaferin A
A withanolidc
P H Y T O C H E M IV CA R T I A T I OWNI T H I NA S P E C I E S
Toble 12. I
E
Chemicol roces of Solanum dulcamsra.
Aglycone
Sugors
Glycoside
S o l o d u l i c iidn e
Golotose(1 mol) Glucose(2 mol) Xylose{l mol}
Solodulcidine+etrooside
{2sD}
Solosodine
(25D)
Goloctose(l mol) Glucose(1 mol) R h o m n o s{ e 1mol)
Solosonine
(1 mol) Goloctose (1mol) Glucose R h o m n o s( e1 m o l )
tv.-Solomorine
HO
.\5-Tomotidenol
(2sL)
(the wood-vnight rnecliates in corlicosteroidsynthesis.ht S. tlLtlcrurrurti rhade)Sandcrhasdistinguisheda u,'estEuropeantomatidcnolgroupancl rn eastEuropcansoladulcidine-solitsodine group(Table12.l).Although polyploidfbrms do occur in the genr-rs. thesechemicalvalieticsall l-rad lt = 24 chronrosomesand were geneticallystable.Recentwolt has :hown thatthe ditl'erentchemot)'pcscan occur in the sarnelocalit)'.With the conrmercialspecies.ahout 3-500individual 6-month-oldSolunun luciniaturrtand .S.rn,ftrr1ru"\\'ereanitl),secl (q.r,.) by raclioimmunoassay lurrdfbundto containaveragcleal concentrltiorrs of I .6- L7% solasodine: 1l'omthesea ttr.vindividualswereselectedfor futurebreeclinswol'k.
Eutul:'ptr.t.s r/ilcs containspiperitoneas the chief constitLlent of the oil. but other l'acesal'eknown which prodr"rce plincipally phellandreneor cineole.rvhilestill othclsproduceoils intcrntc.diate in composition. There ale three racesof Melaleucubrooeetd ploducing volatile oils containing chiefly melhyl eugenol. rnethyl iso-eusenoland elemicin.respectively. They can be trzinsfbrmecl one into the otherby simple chemical stcps.rvhich suggeststhat one of the contpounds (e.g. methyl eugenol.)occurs in all the races.With the applopliate enzyme.methyl iso-elr-qenol could be formed by tr simple doublcbond shifi and elernicinbv the additionof a hydroxyl -eroupand subs e q L l e n rt n e t h y l a t i o n .B e c a u s eo f t h i s a o n e - g e n e - o n e - e n z v m e Essentialoils. The biochcmicalgroupof plantsotferinger,idence o1' h1'pothesissuggestsitself and it would be possibleto test this by the lar-sest numberof chemicalracesis that containinsvolatileoils. breedingexperiments. Hele.again.man),ofthe dif-fbrences within a specieswhich havebeen In the Amelican turpentineindustrJ.bleedin-einvestigittionslravcreportedmay be due to facl-orsother than geneticoncs.Australinoff-ers shownthatoleoresinyieldsin pinesareinhelited.Two chernotypes dilleruniqueopportunities fbr the inr esti,sirtion of this problernasthe flora is ing in theil Al-carenecontentof the oil havebeenrecognizcdfor Plirr.r rich in oil-bearin-qplants. As an example. the cornrnonfbrm of .rr'1r'c.rlri"r.
?",
?'*,
fto.', Y
v
Methyl eugenol
Methyliso-eugenol
CH1'CH:CH1
fr.focHr CH:CH CHT
Elcmicin
PRINCIPLES RELATED TO THECOMMERCIALPRODUCTION, QUALITYAND STANDARDIZATION OF NATURALPRODUCTS
Tqble t2.2
Oif
Roce
Oil content
Composition of oil (%)
\t^. .^^^h^,^t^
0.285 0.248 0.236 0.212 0.267
'l Comphor23, cineole 3, pinene10, estrogol40, sesquiterpene 10 Estrogol 73, linolool9, onethole 5, sesquiterpene 5. limonene1 B 'I0, Anethole39, estrogol31, limonene linolool9, sesquiferpene 1.1 Citrol56, estrogol20, terpeneolcoholI0, terpene(oslimonene) 9, sesquiterpene 5 Comphor7, cineole3, eshogol5B
Yor. estragolato Vor. onisoio Yor. citrota Yor. intermedia (befween comphorota ond estrogolotol
Plantsof the Labiataehavelong beencultivatedfor their volatile oils and many varietiesofa singlespeciesmay exist: theseare not. however. necessafilytrue chemicalracesbecausemolphologicaldiffelences may also be involved.Chemicalracesin the generaOcim.um.Meli.s.sa, havebeenstudied.As one example.Rovesti's Micromeria anclThvnu,s observationson the Ethiopian planl Oc'iutrnlmenthoell)liurr are shown and exhibited in Table 12.2.Theseforms occurredat difl'erentaltitLrdes a correlationbetweenhumidity of atmosphereand the constituentsbut cultivationof all foul typesat Asmara showedtlre chemicalracesto be stable and not transitory phenotypesvarying with the environment. Similar wide variationsof constituenlsof Meli.sstto.fticinalishavebeen noted. Sixteen genotypes cultii'ated in the same licld in fbrmcr Yugoslavialbr 2 yearscontained0.046 0.246c/.oil. The constituents were the same for al1 oils but there was wide variation in relative amountsbetweenthe genotypes.namely cit|onellal2.111-12.141%. l i n a l o o l 1 . 5 0 1 - 6 . 3 8 0 7 r c, a r y o p h y l l e n e1 . 2 1 0 - 1 9 . 0 7 3 % g, e r a n y l acetate+ citronellol9.11.026.913 (thlough Chent.Abs.. 1991.114, 58920). In Spain a 5-year selectionand improvententprograntmefor M. offic:inulisraised the essentialoil contentii'om 0.2-0.3clcto more than 0.57c(T. Adzet et al., Plarta Med. 1992,58,558). Cinnanutmuntcumphora (Lauraceae)exists as various chemical raceswhich vary in their volatile oil corrrposition.and a sin-rilarsituation prevailsfor C. z,etlanicum(C. t.'erunt).lrrthe samefat'nilyOt:otea pretissagives oils of the sassafiastype n'hich may or may ntrt contain camphor. Chemical races appearto occur very widely in some Cornpositae. For TanucetumvLig,are(Tansy) ten difl'erentchernotypeshave been reported for Finland with others fbr Piedmont (Italy) and Central Europe.Someprincipalcomponentsof thesefbrrnsarethujone.isothr.rjone, camphor,chrysanthenylacetateand sabinene.AchiLleanillefolinrr (Yarrow) occurs as various chernical races and Artemisia tlrttcunculus(Tarragon)has yielded. fi'om plants originating mainly in France,an oil containingestragol.whereasfrom plantsof Germany and Russia,sabinene.elemicin and t'an.r-isoelemicinare the principal components.Wormwood EPIBP (A. ubsinthiunt) has a number of chemotypeswith respect to its volatile oil content which may contain over 40% of any one of p-thujone, azns-sabinyl acetate
cis-epoxyocimene or chrysanthenylacetate.A cloneof Artemisiaannuct yield a high of the importantantimalarialafiemisininhasrecently -eiving 1993). beenrecorded(D. C. Jainer al., Phvtochemisrty,1996.43,
Nliscellaneous. Other groups of active compoundswhich exist as chemicalracesarethe phloroglucinolderivativesof Drt,opteri,s. cannabinoids in cannabis.the bitter principles(e.g. amaragentin,sugarsand volatileoil) of Gentiunttlutea andthe glycosidesof S'a/Lr. The existence of two discrete chemotypes of Equisetum orvense with respect to flavonoid contentis noted in the British Herbul CompendiunYol. l. Artemisia annrut planls raised in Holland fiom seedsobtained from a numberof countriesgave plantsexhibitingdistinctgeographicchemotypes(T. E,.Whllaarler a 1.,PLantaMedicu.2000,66,57). Theseexamplesserveto show that the occunenceof chemicalraces in plants,whetherthey be of naturalorigin or producedby plant breeding, can ofler considerablescopelbr the improvementof the therapeutic value ofthe drug eitherby adjustmentofthe individual constituents ol by increasein the overall yield.
CHANGESIN CH,ROIfiOSOME NUffIgTR
Polyploidy, ln some organismsthe chromosomescan be glor.rped, not in pairs but in threes,fours or higher numbers;theseare polyploid polyploidscanbe individuals-triploid, tetraploid,octaploid,etc. Sr-rch delived by the multiplication of the chlomosomesof a single species (crutoploids) or as a resultof the multiplicationof the chromosomestbllowing hybridizationbetweentwo species(allopbids). The latter case furnishesa mechanismwherebya hyblid of two species,itself inf'ertile. rnay give rise to a constantf'ertiletype by polyploid formation. In the new polyploid form. pairing of the chromosomesat meiosisis possible which was probably not so in the original hyblid. Types such as this formed as above from the infertile arise naturally: PrimLtlakevt'ensis hybrid P verticillata x P.floribunda was llrst recordedin 1912;since then many new specieshave beensynthesizedas well as someLinnean species(e.g.the productionof the hemp nelt\e,Galeopsistetrahit Ltnn. interestis the from G. pubescensx G. speciosa).Of pharmacognostical
NH'COCH!
c ll .{ "4 r1,z-ocx, ocHl Colchicine
rA
,/. t\:1/
o Isocolchicine
Colchiceine
P H Y T O C H E M I CV AA L RIATIOW N I T H I NA S P E C I E S
Toble I2.3
Influence of chromosome number on constifuents of medicinol plonts. Form
rtonl
Constituents
2n
Atropo bellodonno Doturoinnoxia
o.21 0.03
Dolurostromonium
Totoltroponeolkoloids Hyoscine, dry weight{%} Ahopine,dry weight{%} Totolhoponeolkoloids
Hyoscyomusniger
Totoltroponeolkoloids
Cinchonosuccirubro r)^i,,- ^^^^,,
Quinine,dry weight(%) Morphine y i e l dp e ru n i lo r e o
0.53
Lobelioinfloto
Alkoloidcontent: dry weight(%)per plont Volotileoil contenl(%l
0.25
Acoruscolomus
4n
l)tners
lncreoseof obout 687" over2n 0 . 1 4 ,0 . 11 ( 1n ) 0 . 0 1 ,0 . 0 1( 1 n ) lncreoses of obout60-l 50% over2n lncreose ol 22.5% over2n 1.12
0.32-0.46 52-1 52% thotof 2 n 2 . 1{ l i g h t o ni lo, 6. B (yellow-brown viscous detectoble oil.2-8% B-osorone)
MoinlyBn increose of obout 347. over2n 0 . 2 7l l n J Increoses of up to 100%: 3n plontsespeciolly high
3 . 1 { 3 n )( y e l l o w oil, 0.3% B-osorone)
R-ncnr^^^l
Achilleomillefoliun complex Corumcarvi Menthospicata Fenugreek seeds Digitalispurpureo Digitalislonoto
Urgineoindico l^^^-;^,,^ uuyJ,LU",
J" ^y .
Connobis sotivo
Azulenein volotileoil
Veryvorioble
Mostpromising source
Volotileoil content{%} Volotileoil content{%) Diosgenin Totolglycosides {%) Totolglycosides{%)
6.0 0.48 0.68
r 0.0 0.05 0.60
Proscilloridin A ond scilloren {%} Ascorbicocid {%) Rotiosof morihuono-like octivity{toxicityio fish)
0.004-0.26
0.02-0.4s
0.04-0.09 1.4
0.04*0. r5 2.6
Loweror someos in 2n Loweror someos in 2n. Relotively highcontentof lonotosides A ond B
stabilizationof the (f'ertile)F1 hybrid of Dduru Jero.rx D. stnnnniunt by polyploid formation as indicated later in this chapter.In some speciesthe somatic chromosome numbel varies irregularly within wide limits (e.g. the blue grassPoa praten.si.s fiom 20 to over 100). Such plants. termed aneuploirll',do not breed true and ofien exhibit apogametic(asexr.ral) reproduction. Amon-qthe natural polyploids of medicinal interestmay be mentioned the nints and valerian: many of the former are alloploids. ValerianoccursacrossEurope in a variety of fbrms (2n.,1nand 8n); it is possiblethat the wide variability in pharmacologicalactionof different samplesof ValerianaeRadix is associatedwith these difl'elent fbrms and a rnore rigid detinition of the botanical source rni-ehtbe desirable. Polyploidy can be artificially inducedin many plantsby suitable treatmentwith the alkaloid colchicine. The cytological eff-ectof colchicineon clividingcells was reportedby Dustin, Havasand Lits in 1931 and in the same year used practicallyby Blakesleein his Dutura stLldies. In the presenceof colchicine,chromosomesin a cell undergoingmitosiswill continueto divide without the fbrrr.ration of a mitotic spindlefigure. Sistercells thelefbleare not fbrmed,and in the growing root tips of onion (.2n = 16), a 72-h treatmentwith colchicinesolutionhas given lise to cells containingas many as 256 chromosomes. This 'C-mitotic' activity of colchicinemay alisefiom its interactionwith thc disulphidebonds of the spindleprotein and by inhibition of the conversion of globular proteins to fibrous
No ozulene(8n)
3 . 0( 3 n ) 0.04-0.02{3n}
proteins.On cessationof treatment,the spindlefigureagainforms in the normal way. C-mitotic trctivity is greatly influenced bv modifications of the colchicinemolecule.Thus.colchicineis 100timesmoreactivethanits isomerisocolchicine andcolchiceineis viltLrallyinactire. Modification of substituentsin other dngs may not have such a markedefI-ecton activity-colcemid. which possesses a methylamino substitr-rent in placeof the acetylamidogroup of colchicine.is reported to havee1I'ects the sameas colchicinebLrtwith toxicity to animal cells. Plant materialscan be treatedwith colchicine in a numberof ways. Seeds are fiequently soaked in an aqLreoussoh"rtionof colchicine (0.2-2.1Vcsolutionfor l-4 days)befbreplantin-e.and seedlingscan be inverted onto filter paper soakedin the solution so that the growing points are not damaged.Alternatively. the soil around the roots of young seedlingscan be moistenedwith thc alkaloid solution. Young buds and shootscan be treatedby immersion.and lanolin pastesand agargels are usefulfbr generalapplicationto tissues. Newly lbrrned polyploidsusuallyreqr.rire a numberof generationsto stabilizethemselvesand treatmentsof the abovetype often tail to give a uniform plant regardin-echromosomenumber: such mixochimeric conditions may involve difl'erentchromosomenumbers in the three germ layels of the plant. Typical efI'ects of polyploidy compared with the diploid state are lalger flowers. pollerrgrains and stomata.The influence of polyploidy on the constitr"rents of a number of dru-eplants is indicatedin
PRINCIPLES RELATED TO THECOMMERCIALPRODUCTION,QUALITYAND STANDARDIZATION OF NATURALPRODUCTS Table 12.3;some figures quoted are tilken fronr extensivestudiesarrd are given as an approxirnateindicationof thc diffcrcncesobtained.As can bc seen.the elfectsof polyploicll,ale not genelally predictableand eachspeciesmust be examinedindividually.Caremust be takenthat the methodusedto exprcssthe resultsdoesno1give a c'leceptive effect. Thus,'"vithlobelia.tetraploidplantsare srlallel than diploid ones.so that. in spite of an incrcaseclpercentageol alkaloid in .ln plants expressedon a drv weight basis.the total alkaloiclcontcntper plant may not exceedthat of the 2r plants.A similar sitr.ration cxists with tetraploid Arterrti.sict t:Lnrtutr with respect to arternisinincuntent (T. Wallaartet al, PlurttctMediL:tr.1999,65.728). With tetraploid carawayplants.notwithstandinga l3% snrallercrop of fiuits fi'om one plant,the totalvolatileoil contentof this plantn,asincreased by 100%; the.l/l cara\\'aywas alsofbund to be perennial(2n = biennial)and to possessan increasedti'ostresistance. In somespeciespol.vploidydoesnot afl'ectthe relativeproportionsof the indir,idual constituents-fbr cxamplc. solanacconshelbs prodr.rce increasedquantiliesof tropanealkaloidsin the 'lrr statc anclredr.rced amorurtsas haploids br-rtthe propoltion of hyoscineto hyoscyanine lernairrsr-rnaltered; the proportionof carr,or.rc in oil of carawayclelived florn 4n plantsis alsounchanged. Hou'cr,cr.ltt Digitalis lruiclo is repofied to containa relativelyhigh plopor-tionof lanatosides A and B comparedrvith the 2n fblm. HaploiclD. luucttuphnls laisedfi'om androgenic cell cultr.rres arereportedlysmallerthanthe 2n tbrrn.havesomcmorpl-rologically abnormalflowersand sho\\,r'en variablecaldenolidccontents (B. Diettrich et al.. PIantu Medi<-ct.2000. 66. 237). The sesquitcrpene lactonesof Ambrusiudutnoso.lar-nilyCornpositae.exl-ribitmalked dif: ftrencesbetn'eenthe diploid and polyploidfblms. (.Pltt'tocherni.rar'. V. Lebot and J. Levesclue 1996.43. 397) record that sonre100tons of kava root (Piper nteth'stit:unt)(q. v.) are ir-nport(2ri= I 0 -r The plantsareall stelileclecaploids ed annuallyinto Eurc4re. = 130)and are raisedby smallholclers throlrghoutthe PacitlcIslands. Unfbltunately the yielclsof kar,alactonesfrom dif-lerentsoLrl'ces vary enormouslyand thcre is a rcal neecltbr clonal selectionfbr genetic improvement.The abor,cautholshavc cxamilrcdbv HPLC the chemicrl cor-nposition fiom -5I Paciliclslands. of l2l cr.rltivars nriginatirrg
hyoscinethan hyoscyaminein the leavesof matlrreplants.whereasin diploid strainsthe revelseis true. Mechlel and Haun have reportedon the total alkaloid contentof other 2n + I types.inciuding somesecondary types.Their abstractedlesultsare -qivenin Table 12..1.
ARTIFICIAT PRODUCTION OF MUTATIONS
The n-rutagenicpl'opertiesof X-lays and radiunt ernissions were erploitedas early as l92l by Blakesleeat the commencernent of his classicalstudieson the geneticsof the genusDatura. Sinccthen.all typesof ionizingradiation(a-particles, B-rays.y-rnys,thermalandlast neLrtlons) havebeenextensivelystudiedin this respectand a numberof new valieties of crop plants have been prodr-rced (barley,peas,soya beans, mustard and rape). Li balley approxinratelyone-fifih of all viable mlrtations prodr"rcedby ionizing radiations are of the 'erectoides' (densespike. stiff straw) type: in othel clops increasedyields. carl-vrraturity trndmildew resistancehave beenachier"ed. Subscquentto a small number of investigationson chemicalmutagensdatirr_e fiom 1910.the avalanchcofresearchon this subiectstarted duling and after World War II. following observationsby Auerbach and Robsonon the productionof mutationsin Drutsophilu(fruit fly) by mustardgas.Furtherstimuluswas given to this work by the discovery that many chemicalmutagensalsopossessctucinogenicor anticercinogenic properties.Thesesubstances vary enormouslyin their molecular complexity and chemical propelties.and it is only more recently that theil nrodeof actionhasbeenelucidated. Collectively,ionizingladiationsandchemicalswill producea mutation spectrumwhich covcrsall of the groupslisted earlier.The fbrmer. however.producein the chromosomcsaberrationsof a nrorerandom natllrethan do chemicals,which often act principally at celtain loci particularlyat thoseareasof the chromosomewhich staindifTerentlyat (heterochrornatin). r-r-ritosis Also. the clistributionof elfects between nuclei is more randomwith X-r'aysthan with chemicals. Mutagenic agentsact at various stagesof nuclerr organiz:rtiou. (r-rondividing) Thus. at that stageof the ir-rterphase nucleuswhen DNA synthesisis taking place. abelrationsinvolr'ing chron-ratidexchanges and isochrornatidbreaksoccr.rr. Theseefl-ectsdo not becomeirnmediExtrachromosomal t1'pes. Sometrnresplants occnl w'ith one or ately (0-8 h) manif-estin the cell but appcal as delayedeffects8-48 h more cl-romosomesextrl to the sonraticnurnberanclthesearc known after treatnrent.Ionizing radiationsand most chemicalsproduceaberas ertrachromosorral types.Thev uere filst noticedby Blakeslee's rations of this type. Clearly. breaks which occul in the intelphase groupin 1915, althoughtht'il gcrt-tic(-ou\titr.rtion $ r\ not immediately nucleus chromosomcsbefore DNA synthesisoccurs (chromosomes apparent.when they sporadicallyappearedin pure line cr.rltures of I)atunL struntonurnr.Snchplants rvclc latcl shourr to possess25 chrorlrosomes in the sornaticcell arrdwith Datura (n = l2). twclr,e2r + I tvpesare possible.eachone corrtaininga dif-fbrent extrachromosome. Ioble 12.4 Alkqloids of some primory ond secondory Thc chromosomes\\'ere designatedby nr.rrnbering their halves (or types of Dslvra stramonium\. ends).so thatthe largestchronosomeis 1.2andthe smallest23.24.All l 2 typcseventutrllv irr Blakeslee's appeared culturesanclwereoliginalTyp" Alkoloid content, compared with lr named accordingto somc obvious charirctcristicof the plant (e.g. controls, al vegelotive stote Clobe. Rollecl.IIex. etc.) althou-shthc cnd-numbclingsyster-n can alscr (colculoted on o dry weight bosis) b e u s e ct o l i d e n t i f lt h e m ;t h u s . C l o b e = 2 t t + 2.l 2 2 .O t h e r 2 r r +I t y p e s KOileO 2 n+ 1 . 2 I 36% increose are rlso prodr-rced and are telmed secondalies. tertiatiesand compen'l 2 n+ 9 . 1 0 ECnrnus 43% increose sating.Scconclar)'types have the ertra chromosomemade up of two 'Glossy' 2n+ 3.4 \ (e.g.2n + I .l ) and in teltiuly typesit idcnticalhalvcsof a chromosome 'Stro*berry' I is composedo1'two halvesof difterentclrronrosomes. Compcnsating 2 n + 5 . 5 'Wedqe' 2 n + 1 1 .11 ) I 55-227% increoses types lack onc of the normal chror.nosor-nes. whiclr is compensatedfbr 'o*oif ' 2 n+ 1 7 . 1 7 by two otherseachcarrl,ittga differenthalf of the missingone (e.g.2n ' D i v e r g e n t ' /I 2n+ 19.19 - 1.2+ 1.9+ 2.-5t. At nierosis2n + I typesproducea mixtulc of rr rnd n 'Thistle' 2 n+ 1 0 . 1 0 'Microcoroic' ) + I ganrelesand so clo not breed true: they hal'e proved palticularly 2n+ 13.14 ) 35-40% decreoses ' 'Globe' uscfulto geneticists lirl genelocation. ) 2 n + 2 1. 2 2 In 1963.Starl'reportecl the analvsisof the primarvtypesPoinsettia *FromMechlerond Houn lPlontoMed., 1981,42, 102) (.2n+ 17.18)andGlobe(2rt+ 21.22) andshowedtherr to posse ssmore
P H Y T O C H E M I CV AA L RIATIOW N I T H I NA S P E C I E S r.plit) woulclbe of the chromosometype and theseale inducedby ru\ treatmentand by a few chemicals(e.g.ethoxycafl'eine and strep,niqrin).Othermutationsmay be inducedduring the DNA post-syn:.'tic sta-qeof the intcrphasenucleusand dr.u'ingmitosis itself as in ' rr prodr.rction of polyploids by colchicine and in the inducenent of - rnucleate or polynuclcate conditionsdueto inhibitionof cell platefbr'' .rtionbv cyclic organiccompounds(e.g.halogenated derivativesof 'Jnrene and toluene. hydrazinotroponecompounds. aminopyline). \1()\t mlltagensproduce more than one type of flugDrentationor \.hlnqe effect {e.g.ethorycafTeineand strcptonigrin.besidesproduc:rr chromosome exchanges, alsoinducesub-chromatid exchanges). A '- rr of the manyknown mutagensaregiven in Table 12.5. Factors which nay influence the elfect of nrulagenic treahnent ':llude oxygen tension within the tissues.temperatureand pH. t hc'mical mutagenscanbe appliedin a similarway to colchicine(q.v.). :.'cr,ls.whole plants.isolatedofgans.growing points,etc., are suitable ,r-directirradiation.In order to obtain singlemutationsin a plant. irrii:::rtionof pollen. which is subsequently used to f'ertilizea normal '.,,uer. is often advantageous. lt is unlikely that a pollen -grainwill '- t.rinits viability if it undergoesmorc than one mutationalchange. .\nrong plants of medicinal interest. the production of poli'ploid ' 'r'nrshas already been discussecl.Blakeslee's radiation work on ;),itLrt'ostr(ltnonlunlresulted in the production of many single gene 'rutation types (e.g. Zigzag. Quercina, Bunchl'. Equisetum narles :.'rir ed fiom some characteristicaspectof the plant). These mutants .:L'not isolatedindividualsbut are producedlegularly by radiation ':J;.ltrnent. Someforms such as 'pale' (chlorophyll-deficient)are more ':ctluentthanothers.In manycasesBlakesleewasableto map theposi' of the genesresponsiblefor thesee1l-ects. Other mutantsobtained 'r"nthcsestudieswere of the extra-chromosomal type (q.v.). Severalworkershavestudied.without a full geneticanalysis.alka,rd production in various speciesof DatLtra raised flom irradiated . -'cr1s: typeshavebeenproducedwhich show diffelencesin the relative ':r)portionsof the alkaloids synthesizedbut no new alkaloids have --'.n detectedby this treatment. Ilr the irradiationof poppl seetlsw ith b"Co a nurlber of mutations .r\L-beenproduced.includingonesproducingplantswith an increased '\
Toble I2.5
morphine content;theseincreaseswere maintainedin the X2 generation with an a\rerage morphinecontentof 0.52% comparedwith 0.327c fbr the controls. As mentionedearlier,racesof sweet lupins (ahnost lree of bitter'ness)can be obtainedby selection.More recently.bitter lupin seedsof an X-ray-induced early-maturingmutant of LupirtLtsdigitatus were treatedwith ethyln'rethanesulphonate solution,and,of the zl'10progeny. I I weremutantswhich could be classedas sweet.Four of thesewere of normal vigriur and near-nomal f'ertility. Breedingexperimentshave beenperformedwith irradiatedMentha piperitu in thc USA in an endeavourto producea dominant mutation (bud sport) for Verticilliunr(wilt) resistance.a diseaseto which mints areparticularlyprone:a successfulstrain.Totkl'sMitcham Peppermint, is now cultivated.A radiation-indr.rced mutant of Scotch peppermint (Mertha x gracilis) has been shown to produce an oil typical of the ordinaly peppenr-rint(C-3 oxygenatedmonoterpenes)insteadof the C-6 oxygenatedmonoterpenescharacteristicof spearmint(see Fig. 23.4 for tbrmulae).The resultshave given further insight into the biogenesisof thesecompounds(R. Croteauet al.. Plant Pb'siol., 1991, 96.141). In India mutant strainsof Capsicunt(utt7Lu1t11 witl-rincreasedyields (20-6OE()of capsaicinhavebeenisoiatedfiorn Mj and M4 generations originating tiom seed treated with sodium azide and ethylmethanesulphonate. ln the future.haploidplantswill undoubtedlyfind increasingr-rse fbr the studyof inducedmutations:in many caseswhole plantscan now be regeireratedtiorn haploid tissueculturesof pollen. Such material has the advantagethat induceclrecessivemutations.which in the diploid organismrequile subsequentbreedingexpedmentslbr their study.are immediatelyapparentin the phenotype.
HYBRIDIZATION In plant breedinghybridizationlblms a possiblerneansof combining in a singlevariety the desirablecharlctersof two or more lines, varietiesor species.and occasionallyof producingnew iind desir-
Cytologicolqction of some chemicolmutogens.
arOUp
Exomples
Cytologicoleffect
) N A p r e c u r s o r so n d r e l o t e dc o m p o u n d s
Adenine
Inhibition of eorlyslogesof DNA synthesis, purine formotion Inhibition of deoxyribonucleotide synthesis Complexformotion with inhibition of the DNA-polymerose reoction, degrodotion ond denoturotion of DNA Production of obnormolDNA
Deoxyodenosi ne Ethoxycoffei ne
)NA bose onologues Aniibiotics r k y l o t i n go g e n i s \ i t r o s o c o m p o u n d so n d c h e l o t i n go g e n t s ' . 4i s c e l l on e o u s
T h y m i d i noen o l o g u e 5-bromodeoxyuridine Strepiomycin Actinomycin Nitrogenmustord Diepoxypropylether Mony compounds {e.g.cupferron B-hydroxy-quinoli ne) Inorgonic cyonides Moleichydrozide Hydroxylomine
As ethoxycoffeine As elhoxycoffeine Production of obnormolDNA Possibly combinewith sometroceheovymetols (Fe,Cu)whichmoy be boundwith theDNA molecule Inhibiiion of cytochrome oxidosewith resultont peroxideformotion Reoclion groups with sulphydryl withcytosine Combinqtion component of DNA
PRINCIPLES RELATED TO THECOMMERCIALPRODUCTION, QUALITYAND STANDARDIZATION OF NATURALPRODUCTS ablecharactersnot found in eitherparent.Hlrbridization.particularly betweenhomozygonsstrainswhich harr,e beeninbred fbl a nunrber of generations, introduces a clegree of l-reteloz.vgosityu'ith resultanthybrid vigour (heterosis)often manif-est irr the dirnensions and other characteristics of the plant. Severalmethodso1'breeding cropsby the use of sexr.ral hybridizationare availableand lbr these the readeris referredto standardworks on the subject.Although this chapteris devotedprincipally to chenical I'ariantsof a particular speciesit is convenientto include here. in additionto intenarietal hybridization.interspecifichybridizationin which hybrid vigour is also apparent. The hybrid natureof a nuntberof drugs(e.9.cinchona.q.v.)is welt known. The corlmercial mints are hyblids anclmust therefbrebe propagatedvegetatively.as the plants will not bleecltrue and the pro-qeny vary in their oil composition.Hegnauerconsidersthat with the spearmint-typeoil (high carvonecontent)genesof Menlln kngifolicL or of M. rotundifolia seemto be necessary. The cultivatedpeppermint (M. piperito) is probablya hybrid derivedfiom M. oqucttiut itndM. spicttta and it is the fbrmer that contributesthe menthofulancharacteristics. Hybrids between various speciesof Mentha have been used to stlrdythe inheritanceof a nr.rmberof essentialoil components.including menthol.carvoneand pulegone. Hybrids of speciesproducingtropanealkaloidshave receivedconsiderablestudy. In the genus Daturtt Ihe effect of hybridization on chemicalconstituentsis illustratedby the crossD..l'erotx D. stt.dnloniun. The aerialorgansof the latter normally containhvoscyamineand hyoscine(2:l ratio) at the tlowering peliod: and those of the fornter. hyoscinewith somemeteloidine. Thc F, of the crossconsistsof plants largerthan eithel ol the parents.and containinghyoscineas the principa1alkaloid with only small amountsof otherbases.In the Fr. segregation occurs as re-{ardsboth morphological charactersand alkaloid constituents.With D. leichhurdtii andD. innoriu the former plant produces hyoscyaminearrdhyoscine(2: I ) anclthe latter speciesusuallv mainly hyoscine but sometimes,accordingto conditions of growth. appleciablequantitiesof h1'oscyamine.In this instancethe F1 hybrid containsa hyoscyarnine:hyoscine ratio intcrmediatebetweenthat of the two parents. Various hybrids of tree daturas have also qiven favourablealkaloid yields and have been subjectedto field tr-ialsin Ecr.rador. Theseplantsare self-sterilebut can be propagatedby cloning (Chapter1l). In experimentsperlbrmedinAustralia on hybridsof DLtbctisiu leic.hhctrdtii and D. mto1toroidesno uniform dominanceof eithcr hyoscine or hyosciiminein the F1 was obtained.although the ratio genertrlly favoured hyoscyamineeven in geographicalareasconducive to the ploduction of hyoscine. Investi_qations of this type on Duboi.sirLare complicatedby the occurrenceof chemical racesand by the rnarked susceptibilityof alkaloid contentto environment.However. single desirable hybrids can bc vegetatively propagated and l-ravenow becomeestablished as commercialcrops(q.v.).Experimentsin Japan on artificial crossesof DubolslrrproducedsomeF1 hybrids u'hich contained more than twice the amount of alkaloids (hyoscine and hyoscyamine)than that containedby the parents.Cleatest inr;reascs were fbr-rndwhen D. nn'tryxtroideswas the female parent.Naturally occuring intergenelic hybrids involving Duboisia spp. and other Anthocercideaehave been analysed (El-lmam et ul.. Int. J. Plnrntnt:o9..1991.29.263)andthe alkaloidrnetabolism in callr-rs and regeneratingshootculturesof the commercialhybrid D. leichhunltii x D. ntoltoroidas studied (Glitsanapanand Griflln. Phytochernisn-t. 1 9 9 23 . r. 3069). Nk otinrtotabacmt (Solanaceae). asnorvcultivated,musthavebeen delived liom at least two diff-erentparcnt species,and 'synthetic' tobaccoscan be preparedby using suspectedspeciesIr palents.
Althonghit hasnot beenpossibleto producein this way speciescxactly cornparablcto N. tultutttrn-suchsynthcticplantsare rnostuseful for the stud1,of alkaloid inheritanccchiiracteristics. This is intpor.trntin the comrnercialproductiorrof tobacco.in rvhich both the quantitl' and the naturcof the alkaloidproducedare-important.Demethylationof nicotine may take place in the leavcsof sotne species.and b,vhi'br-id studiesthis reactionhas becn shou'nto bc dr.re.in thc troups of plants stLrdied. to eitherone pair o1'dominanttactorsor trvo pairs of don-rinarrt and indepcndentfactors. The inheritanceof the clpiumalkaloids(morphine.codeine.thebainc.narcotineanclpapaverine)hasbeenstudiedin the crossPtrpat'er .sontniJbrum x P. .setigenunA hetelotic increasein codeine and thebainewas fbund in diff'erentF, plarrts.and in tlre Fl plants.with the exceptionof codcine.sonreinclcasein alkaloidcontentwas noted.An abscnceof narcotine was gencrally dominant orer its ple\cnce (Khannaet ul., PloiltuMetl.. 1986.p. I 57).A continuation of this work to the Fg generation(Shuklaet ul.. Irtt..l. Plturntuc'ognosr.. 1995.33. 228.)r'esulted in a populationthat rvascorrrpletely diploid but'"vhich showed considcrablediversity rvith re-eardto tlre opiurn contentsof rlorphine. narcotineand papaverinc.The patternoi-alkaloidswas closer to that of P. .sontniferulr/ than to thilt of P. setigerwnwith rnorphine curtents rangingti'om 8.0 to 30.0%. It is enl,isa-eed b1'the authorsthat a suitablebreeding pro-qlamrnecould resr,rltin opium with a higher level of rnorphine than that nornallv encountered.F, hybrids of P. brocrc(rtunrandP. orientalc containeda lowel thebainecontentand higher oripar,inecontentthan in eithet parent.a reslrltwhicl-rpror.idecl gcneticevidencefbr the biosynthcticlinkageberweenthesealkaloicls ( F i g .2 7 .l 5 ) . In a continr.ration of thc Israeli work on withanolidcsEastrvooder a1. reportedthat a crossof a Sor.rthAfiican chemotvpeof Withtttiu sontnilero with the Islaeli chcn-rotype II producedthree new r.vithanolides not detectedin eithel parent.The outstandingf'eaturewas the presence ofa new oridizing systemin the hybridwhich apparentll, oxidizesr.ing A to a diketone in the presenceof either the saturatedor the unstturatcdlactone(Fig. 12.2).A furthelnotablef'eatnlewasthe isolationof a compoundhavin-9a saturatcclC2-C3 link. a leaturervhich had only beennoticedin a nrinor constituentof chemotypeI. In Duturu. Ioo. hybrids ma-vcontl.iindifl'elcntwithanoliclesfrorn thoseof eitherparent and involve differentoxidation statesof ring C. Solasodineoccursin Solanwn int'ununtup to thc extentof 1.8% whcreasS. melougenu contairrs only tlacesof steroidalalkaloids.Work
HO Withoferin A HO Chorocferisliccomponents of chemotypehybrid Fig.12.2 Withonolideoxidotions ond reductions chorocteristic ol o Withonio somnifero chemotype hybrid(forcomplete structure of withoferin A see 'Witho nolides') .
-;
P H Y T O C H E M I CV AA T RIATIOW N I T H I NA S P E C I E S .l.rnrnru. India.has shorl'nthe F1 hyblid of thesespeciesto be much itrr)' charactersof an organism.this led the way fbr the altitlcial trans,rL'Irrolificin trLrit-bearin-e thaneitherolits parents.with a contentof f'erof a particr-rlar chalacterfrom one organismto another. i i solasodine.The divcrsit,vof glycoalkaloidsfbund in Soltuturr.t Nature itself does efl-ectcenetic transf-ers betweenvery dissimilar .:--iiesis. in palt.tl-reresultof thc independent inheritance ofthe agll'- organisrls as evidencedby the wcll-known crown gall and hairy root - nc and glycosidicntoietiesand e-rplainsthe occurrenceof a new diseasesof plants.Both are causedby bacterialinf-ectionof the plant ..rloicl.sisuinc.fbLrndin thehyblid of S.u<'nule andS.alanliuiri. This anclinvolve. respecti.n'ely. the soil organismsAgrobucterituntumzfu-:,,Lrpof hyblid cloncsis cultivatedbv the Ayntarapeopleof Western clerr.sand A. rhilogene.s.Crown gall diseaseis essentiall)ra plant can: i , , l i ri a . cer and hair1,root diseaseis alsoa prolif'erationmanif'estation in which In 196l. Calcandiand coworkersdemonstlated (Ttrble12.6)that rn thereis an excessivedevelopmentof the loot system.A turther charac:r hr blidizationof Digitalisspecies. lanatoside foln-ration is dorninant teristicassociatedwith both diseasesis tl-reploduction.by the host.of a purpureaglycosidefbrnration:in 1984 these obselrarionsu'ere group of substances ca1ledopines,the actualcompoundssynthesized ,rgc'lr contirned lbl some crossesanclextendedwith ntore detailed being speciflctbr the particuiarparasiteinvolved.About 20 opinesare :r.rlrsis b1,wichtl and MangkLrdidjoio (Pharrn.Zeir. 1984129.686). cun'ently known: many are dicarboxylic acids, the majority being : , 'r the glycosidecontcntof otherDigirulisF1 hybridsincludirrgthose formed by condensationof an amino acid (lysine, arginine.ornithine. '. D..ferruginetranclD. turiensis. see Fingerhut et ul.. Plunttt Med., histidine)with either pyruvateor cx,-ketoglutarate: othersinvolve sim' ) 9l . 5 7 ( S L r p pN l . o . 2 ) ,A 7 0 l A 7 L ple sugalssuchasmannoseandsucrose. seeFig. I2.3. (Forareviewon PrcliminarvstLrdies by Cornishet al. in 1983indicatedthat tbenr-r- opines( 129refi) seeY. Dessauxet al., Pln'toc'ltemistry,. 1993,34, 3 | .) -r'!'.k seed,a potentialsourceof diosgenin(Chapter2'1).is capableof Another importantf'eatureconcerningthesediseases.first noted in the -Jneticimprovenientregardingthe monoh)rdloxysapogenin yield by late f 960s, was that Agrobacteriun?strainswor.rlddegradeonly those :rrb;ifli2n1i611 of variousracesof Trigottellct.lbermnl-gr(1ecutn. opines fol whose occurrencein the host ttrey rvere responsible.Thus Prrethrurr hybrids havc been usedin Kerryafbr pyrethrin prodr.rc- opines,of no value to the plant cell. constitutean exclusivelbod sub::(jn (\ee Chapter'11)lthesehybridsare prodr.rced eitherbv crossins stratefbr the particularbacterialstrain.When. however,the bacterium ' .,.() clonesassumedto be self:sterileor b1'plantin_e nur.nber a of desir'- is eliminatedfrom the host plant (e g . by treatmentwith an antibiotic) .,i.lecloncstogetl-rer and bLrlkingthe secd.The air-r-r is to obtainproger-rv the characteristicmorphologicalf'eaturesand the productionof opines .i Irich.owing to h,vbridvigour.will increasepyfethrinyield per acre. continueto be maintained. This is becausegeneticmaterial(T-DNA) l. nlirrtunately,it appearsthat increasecl ploduction of pyrethrinsis not of the bacteriumcarryingthe geneswhich expressthe diseasehasbeen .,lsavs associatedwith hybrid vigour. Researchat the Pyrethrr.rrn transportedto, and integratedinto, the plant DNA. Thesegenesarecar\lrrkctir-rgBoard.Kenya,hasindicatedthat clonalselection.basedon ried on large plasmidswhich have been nantedTi (Tuntour-inducin_e) . isour and individual pyfethrin content of hybrids raised tiorn seecl. and Ri (Root-inducing) plasmids.Suchtransgenic plant materialis of :,rllovn cd bv vegetalivepropa-sation. would -9il'ebetterlesults. pal'ticularrnedicinaland phytochemicalinterestwhen grown as transformed cell cultures or hairy loot cultures in studies involving the
TRANSGENICMtDlCl NAt PL/ANTS NHa I ntil lccently the transf-er ofgenetic materialfiont one plant to another ,b-Nx-1cxr)3-cH-cooH . ..t! | .rls dependenton hybridizationas has beendiscr-rssed aboveand its HN llx I .rpplication is limited to thoserelativelyt-ewspeciesuhich ale interMe-CH-COOH ,()mpatibleandgive f'ertileF1 hybrids. Octopine Dcveloprrents in rnoleculalbiologyandchernicalgeneticshavepro,.idr-dnew vistasfor geneticsandit is nou'possibleto explain.in tenns HOCH?-(CHOH).-gHz , \l the DNA nolecule. chromosomeduplicationand the structureof the :ene itself nl'o mysteliesinvolving the natule of lif'e which intrigued Hooc-6H-(cHz)r-coNH, .rndbaffled scientistslbr rlany yelrs. From sr.rchfr-rndamcntal studies :lr.-scienceof geneticengineelingexplosivelyemelgedin 1973.when Mannopine rt becamepossible.by the formation of recombinantDNA. to transf'er .ertain DNA sequencesfrorn the chromosomesof one organism to F i g .1 2 . 3 rhoseof another.SinceDNA assernblages represent thegenesor hered- E x o m p loefso p i n e s .
rlx
Tobfe | 2.6 r\
-r*
Lonotoside formotion an Digitalis hybrids. Glycosidesof the F 1
Porenls
d
D. purpureo
D. lanato
D. purpurea D. luteo
D. luteo D. lonoto
Lonotoside A (principol glycoside). Alsolonotosides B ond E. Smollomountof glucogitoloxin. No lonotoside C or purpureoglycoside A Similorto theobove Somecomposition os theporentplontswhichresemble one onother. Lonotoside A the principolglycosidebut lonotoside C, whichoccursin Iorge omountsin D. lonoto,couldnot be detected
PRINCIPLES RELATED TO THECOMMERCIALPRODUCTION,QUALITYAND STANDARDIZATION OF NATURALPRODUCTS productionof secondarymetabolitesand has alreadybeendiscussedin somedetailin ChapterI l. A further developmenthasbeento utiliseA,grzDuctcriurtt in a vecttl' systemlbr transferringgenesfiont one speciesof higher plantto another. Sr.rch transf'elsr"rsually involvejust a singleenzymesystemlrlultiple enzyme systemsinvolved in secondarymetabolism and originatin,r fi'orn difl'erent chromosonresobviously present great ploblems. Of pharmacognosticalsignificanceis the work of Hashinroto'sgroup ilt Kyoto University on Atroptt belladonna. the conversionof hyoscl'amine to hyoscine(Chapter271 in the plant involves the enzyme hyoscyan-rine-6-hydroxylase and in the norntal belladonnaplant the dominant. -qeneis not strongly expressedand plantsare hyoscyan-rine Conr.ersely,althoirghHrostlutltu.strger containslessalkaloid (% dry weight) than belladonnait containsa higher proportion of hyoscine. The hi,droxylasegeneof H. niger u'as placedunderthe control of the cauliflower mosaicvirus 35S promoterand introducedto A. belludonr?.rloots by a binary vector systemusing AgrtrDacteriLun rhi?,ogenes (.Pht'tochemistn. 1993.32.113).Thc belltrdonna hairyrootsproduced showed increasedamounts.and irrcreasedenzymc activities. of the hydroxylase and contained up to fivefold higher concentrationsof hyoscinethan the wild-type hairy roots. A secondpaper(Prctc:.Nutl. ActLtl.Sci.USA,1992.89,I 1799)reportsthatrvholeplantsreqenerated flom the tlansfbrmedroots showed.in the first tlarrstbrmantand its self-edprogeny,an alkaloid compositionof the leavesand stentsthat was alnost exclusivelyhyoscine.Sucha plant,if developedcommercially. would be a valuabletemperate-region sourceof hyoscine. In the sameareaK. Jouhikainenet al. (PlanrcL,1999, 208. -5'15) have demonstratedenhtrncernent of scopolamineploductionin Hlost: .t'untus nrLfti(Lts hairy root cnltures by introducing thc above 3-5S-ft6fttrans(EC -qenethat codes lbr the enzyme hyoscvarnine-6B-hydroxylase
I
1.14.1l.l | 1. H. mutir:Lts is a speciesproducingup Io 6c/cdryweightof tropane alkaloids, normally hyoscyamine: the introdr.rctionof the abovetransgenemoved alkaloid productiontowardsscopolamine(but not entirely) and indicatedthe potentialfbr greatel conversionbv the useof more etTicienlpromotersystens. With belladonna.the first successfulapplicationof transferringan agronomically uscful trait to a mcdicinal plant has been reported (K. Saitoer al., PlanrCell Rep..1992.11.219).It involvedthe prodr.rction of transgenicfertile A. bellotbrtncLplants integratedwith a herbicide-resistantbrlr gcne bv nreansof Agrobacterlun Ri vector'.The re-eenerated plants$'erc producedvia hairy roots and Ihe bor trait was transf'erred to progeny,which showedresistanceto bialaphosand phosphinothlicin. An exampleof the possiblepotentialfor increasingalkaloid yield in Cilrcltonais illustratedby the successfulintegrationof two genesfi'om Ccrthoranthusraseu,r'into the hairy roots of Cinchonu rfficirtttlis (A. Geerlingset al.. Plunt Cell Re;torts.1999.19. I 9 I ). Thesctwo genes, lesponsiblefor the respectiveproductionof the enzymestlyptophan decarboxylase and strictosodinesynthase.are crucialto the synthesisof both the cinchonaand catharanthus alkaloids(Fi-q.27.34).As a resultof the transferboth the quininetrnclcluinidinelcvels were raisedabovethe norrral fbr C. officirutlishairy r'oots:in this instancehowever.after one ycar.the rootshad lost their capacityto producealkaloids. Transgenicplants of Panar ginsengwhich develop abundantroots havebeenprodrlcedand could therefbrebe of commercialsignificance (D.-C.YangandY.-E.Choi.Plant Cell Reports.2000.19.491). Further reoding LindseyK (ed)2000Transgenic plantresearch. HarwoodAcademic. Amsterdan
r The factorswhich mr-rstbe considercdin relationto clrugdeterioration are moisturecontent.tenpcratul'e.light and the prcsenceof oxygenl (bacteria. whentheseconc'litions aresuitablc.living orgarrisrns moulds. rrites anclinsects)r.i'il1rapidly rnultiply. using the drr.rgas a soulce of nutrient.Dlugs afl'ectcdin this u'ay are exclndcdby nationalpharrnacopoeias.
Deteriorotion of stored drugs
Primory fqctors As indicatedin Chapter9, air'-dr'.v dlugs contain abor-rt10 l2c/col rnoisture.and in somcinstances(e.g.cligitalis)this may be sufficient to acti\,ateenzvtrlesprcscntin the leaies and bring aboutdecornposition o1'the-tlycosides. Othel drugs.suchas powderedsquill.which contain mr.rcilagecluickly absorb moisture and become a sticky mass.The containerizedshiprrentof drr-rsswhich is now corllnol-r practicccan lead to spoila-ee clueto excessivecondensation of moistllre on the inner metal r,valls.It is a palticularproblemwith cargoes in transit fion-r humid moist climates to temperatcregions. An increasein tempclaturc.in combinationrl,ithrlclisture.may accelera t ee n z y m ea c t i v i t y ; al a r g et e r n p e r a t u rl ies e ' " v i 1o1b v i o u s l y] e a dt o a loss of r,olatilc constituents(e.g. essentialoils fiom dried plant lnaterial)and in thc cascof absorbentcotton-woolcausea reorienlation of the small amollnt of fatt1,'materialpresentleading to nonirbsolbency ol lorvel absorbency. Direct sunli-eht can cause decompositiorr of celtainconstitue nts (e.g.r,itnrninsin cod-lil'eroil) as well as proclucinga bleaching of leaves and flou'ers. Oxy-ecn assistsin the resinilicationof volatile oils and in the rancidification of fixed oils. Mould ond bocteriql ottock The moulds fbund in deteliolatin_q drugs are usually the same.rsthose associatedwith poolly storcd fbod products. Speciesof Rhiir4tus, Mucor, Penicillirnt anclEurotiuut are common.Their presenccis indicatedby a massof hvphaewhich bind the particlesof drug and by a chalacteristicsnrell. Deteriolationof clru-usis only one aspectof the impoltancc of rnoulds in phamrncognosy-see Chapter'32. Bacterial attack of crude drugs is less obr,ionsunlesschromogeniespeciesare involr'edor efl'ectsploducedsuchasdustinessin cotton-woolby attack on the tibres.Although not a causeof cleterioration. certainpatho-eenic bacteriasuchassah.nonellae and Escltericltiut'oli aretestcdftrl in sorne crucledrugstaken internally(digitalis.stcrculitr.tra-sacanth. gelatin). Also. as plant mitlerialsrvhichhave beendried unclcrnormal conditionscontainviablebacteriaandmould sporcsin varirblearrrounts. the phamacopoeiassetlimits fbr the total r'iableaerobiccount per glam ol' drLrg(seeQualityControl.Chapter1.1t.
PRIMARYFACTORS 9I MOUTDAND BACTERIAT ATTACK 9 1 COTEOPTERA OR BEETTES91 TEPIDOPTERA92 ARACHNIDA
92
CONTROLOF INFESTATION 93 SPOILAGE BY RODENTS 94
F
Coleoptero or beetles Beetlesale insectsand constitutethe lar'-eest ordcl of the animal kingdom, comprisingsome250 000 species,of r"'hichabout600 have been firund associatedwith stolcd tbocl procluctsor dlr.rgs.Not all of these utilize the storec'l prodrlctitself but may bc tbunclin tl-rewood of packin-e-cases or livin-epredaciously. Beetleshavc a bodl' which is divided into heacl.thorax and abdonren.To the lower side of thc tholax are attachedthlcc pairs of legs.while the r.rppersurfaceusually bcarstwo mcmblanous hind-wings which are tblcled beneathhorny elytra (fbrewings).They show completenretamorphosis of egg. larval. pupal and adult stages.and those which constitutepests in storcd products cause damage both as adr-rltsand as larvae. Among the characters which distingLrishthe lan,ae of most spccicswhich attack foodstuffs and vegetabledrugs ale tl-rewell-clevelopedbiting mouth-partsand a headwhich is dalkel in colour tl.ranthe restof the bocly.Table 13.1lists beetlescommonly fbund in storeddrugs.
PRINCIPLES RELATED TO THECOMMERCIALPRODUCTION,QUALITYAND STANDARDIZATION OF NATURALPRODUCTS
Ioble l3.l
Beeflescommonly found in s?oreddrugs. Description
Fomily Nitidulidoe (sopJeedingbeetles) Corpophilusspp.,e.g. C. hemipterus (driedfruit beetles)
Fomily Silvonidoe Oryzoephilusmercotor(merchont groin beetle)
Obovoteor oblong2-4.5 mm long; I 1-segmented ontennoe witho compocl club.Elytrosomewhot shortened exposingtwo-threeopicolobdominol segments. In thiscountryoboutthreespecies ore foundin gronories, foodstoresond worehouses Dorkbrown,norrow,distinctly flottened beetles, obout3 mm long.Clubbed onlennoe. Attocknutsond driedfruits
(sow{oothedgroin beetle) O. surinomensis Fomily €urculionidoe {weevils) Colandrogronorio{gronoryweevil)
Colondrooryzoe(riceweevil) Fomily Anobiidqe {'furniture beetles'} SfegobiumponiceumlSitodrepoponiceo) Anobium poniceum (drugroombeetle) Anobiumpunctolum(commonfurniturebeetle)
Losiodermo serricorne{tobocco,cigor or cigorettebeetle) Fomily Ptinidoe ('spiderbeetles') Ptinusfur {white-morked spiderbeetle) P. tectus(Austrolion spiderbeeile) P.hirtellus{brownspiderbeetle) Trigonogenius globuIus Niptushololeucus beeile,clothbug) {goldenspider Gibbium psylloides Fomily Tenebrionidoe Triboliumconfusum(confused flour beetle) T.costoneum{rustred flour beetle)
I
Dorkbrownto blockinsects, obout3-4 mmlong.Hind-wings obsent, chorocteristic snoutond ontennoe. Boreintoseedsond fruitsond loy on egg in thecovityby meonsof theovipositor. Lorvoedevelopond pupotewithin the seeds Similor,hindwingspresent. 2.3-4.5 mm long Polereddish-brown in colour,greyishhoirs,2-3 mm long.Antennoe I 1-segmented, with threeterminolsegments formingo looseclub.Common in monystoredvegetoble drugs,formerlyfrequentin ships'biscuits Similorto Stegobium poniceum,3-5mm long.Viewedloterolly, theprothorox exhibitso distincthump.Doesnotottockdrugsbut moyoccurin wood of pocking-coses, floors,etc. Reddishcolour,2-2.5mm long.Foundin monystoredproducts, including gingerond liquorice All rothersimilor,somewhot resembling spiders, with long legsond oniennoe, stoutbodiesond hoirycovering,2-4 mm long.Somespecies(e.g.Niptus hololeucusl ore denselycoveredwith hoirs;others(e.g.Gibbiumspp.)ore globrouswitho shiningcuticle.Of wide occurrence in storedproductsfood,spices,cocoo,cereols,olmonds,copsicum, ginger,nutmegs, etc.
Reddish-brown beetles, 2-4 mm long.Foundin monyfoodstuffs includingflour ond nuts.Infested flourhoslingeringpungentodour.Reported os becoming morecommonin crudedrugse.g. rhuborb
Lepidoptero The Lepidopterainclude the moths and butterflies.and although the number of speciesis large, only a relatively small number of moths causeinjury to drugs.As in the caseof the clothesmoth, the damageis causedby the lalva and not by the mature insect:but since moths are r,erv nrobile and lay eggs, infestationtends to spreadrapidly. Moths reportedin storeddrugsare listedin Table I 3.2. Epltestiakuehniella.the Mediterraneanflour mothl E. crntella.Ihe fig moth: E. elutellct.the cocoa moth. and Plotlia interpunctellu,the Indian rncal moth, all belong to the same family, the Phycitidae. Various membels of the tamily Tineidae, which cause dama-eeto clothes.carpets.etc..ale alsofound in drugs. Ephestitrkueltrtiellais about25 mm long. It has dar"kbrownish-grey scalyforewingsanddirty-whitehindwings.The larvaearewhitishexcept fbr the brownishanteriorandthe dark hairson eachsegment:when fully grown, they remain in the fbod material.where they overwinter and lbrm pupae.In contrast,the -srubsof E. elutella ntgrate away from the food and in sodoingoftenleavethe fbodcontainers cornpletely'webbed'withtheir silky threads:theyentercracksin walls,etc..wherethey spincocoonsand remainuntil they pupatein the springto finally emergeasadultsin May.
Arochnido
The arachnidsor 'mites' difI'erfrom the true insectsin that the mature fblrns have eight legs but possessno antennae.The mer-r-rbers of the Tyroglyphidae (e.g. Ilro.qlyphus dimidittttts, the cheese-miteyare much smallerthan the insects.and individualscan only be seenwith a
Ioble | 3.2
Moths of stored drugs.
Moth
Productsottocked
Ephestio kuehniello
Almond,copsicum, cocoo.cottonseed, g round-nut
E. cautello E. elutello
T^^.^ h.^^
Plodio interpunctello Tineopellionello
.^.^^
Cocoo, tobocco, rose petols, pomegronote root bork C i n n o m o n b o r k o n d y e o s tc o k e A c o n i l e r o o t , o l m o n d s , c o p s i c u m s ,m u s t o r d s e e d , g i n g e r , l i n s e e d ,o r r i s , s o f f r o no n d tobocco
DETERIORATION OF STOREDDRUGS
......3
.w
-u"
Fig.l3.l Insectpestsfoundin crudedrugs. A, Colondrogronario;B, Ptinusspp;C, Gibbtumspp.:{ollx I O).D, Stegobiumponiceum(x I 2). (Photos: Dr M. E. Brown)
lens.If the suspected'dust' is scrapedinto a pile. it will be seento rrove and will gladually becomeflat if rnites arc plesentin considerrble numbers. Tl rogLl'phus.fttrinae (Acarus.ilro) (Fig. 13.2,{.E). the flour ol rneal mite.is 0.zl 0.7 n-rmlong.The body is oval in outline.with a truncated posterior.and is clearly divided into two regiorrs*the anteriorpropodosomaand the posteriorhysterosoma.lt is conrmon in cerealproducts.oil-seedcakesand many other commodities. The Tyroglyphidaemay themselvesbe attackedby other mites such as Cheyletus (Fig. 13.2B). entditLL.s A number of difl'erentmites comrnonly attack cantharides,causing corrsiderable damage.Ergot. quince and linseedarc other drLrgswhich seemvery liable to attack. Mites commonly exist in oldinary house dust as tound under carpets, in mattresses,etc.l and theseinclude Dernntoplutgoidespterorrlss/nrr.r,D. culinue, Ghctphngu.r donrestit'trs(the house rnitc.;and T'rogh'phus.fitrinae(the flour mite). It has beenshown.comparatir,ely
Fig. | 3.2 Arochnids.A, Tyroglyphus forinae,femole, ventrolview,x 40. B, Cheyletus eruditus, femole,dorsolview, x 40. C, Dermafophogoides culinoe,femole,ventrolview. D, D culinoe,mole, ventrolview.E, Electron microgroph of flourmite (bycourtesy of MAFFSloughLoborotory, Crowncopyright).
b,
recently.that thesentites are the aller-eens responsibletbr house dust sensitivity.an allergy fiom which rnany peoplesufl'er.lt is now possible to dinsnosethis conditionby prick testswhich utilize an extract preparedlrom Derntakryltagoitles culinue.This mite (Fi-e.13.2C)does not shorrn, the markeddivision of the body into two palts exhibited by Tr'rcglr'7rftrrs spp. Control of infestotion Thc detection.preventionand eradicationof mite and insectirrf'estation is an importanthl,gienicand economicconsideration for all who have occasionto store and Llsecrude drugs. Effective preventivemeasures involve gor.tdhrgiene in thc warehouse(remolal of spillages.old debris and packa-ringmaterialslclimination of sourcesof inf-ections such as tloor clacks and crevices), eftective .stockc'ontrol (regular inspcction.rotationof stock.early recognitionof inf-estation). ctlttintunt storuge t'ortditiorts(maintenanceof cool. dry environmcnt)and goo11 ltuckugirtg(wol'en sacksand bags.multi-pLypapersacksstitcheclat the
p
E
OF NATURALPRODUCTS PRODUCTION,QUALITYAND STANDARDIZATION TO THECOMMERCIAT RELATED PRINCIPLES seams,paper,polythene film, flimsy cardboardare all penetrableby insectsand mites) If materialbecomesinfestedit may be wiser to sacrifice a small consignmentratherthan to risk contaminationof other materials.After a contaminateddrug hasbeenremoved.pallets,shelves,walls and floors should be thoroughly cleanedand sprayedwilh a contact insecticide or pirimiphos-methyl.Weekly air-spraysuchaschlorpyr"iphos-methyl ing with pyrethrins or" synthetic pyrethroids. and the use of slowrelease dichlorvos strips, control air-borne insects. Various dust formulationsmay be usedin cracks. Fumigationis the only practicalmeansof killing insectsand rnites in bulk consignments.Methyl bromide and ethylene oxide have been commonly used and the treatment needs to be applied under gas-proof sheetsor in other suitable gas-tight enclosut'esor chambers. However in Europe the use of ethyleneoxide has now been prohibited. Owing to the possiblehazardsinvolved, the work should be performed by professionaloperatorsin compliancewith the Health and Saf'etyat WorkActs. Beforedrugsare subsequentlyusedthe fumigants must be completelyremovedas, if they aLeconsumed.they may constitutea healthhazard. Low-temperaturestorageappearsto offer great possibilities.It not only reducesinsectattack.but also will. if a sufTicientlylow temperature be employed,graduallydestroyinsects.larvaeand eggs.The eggs of Ephestiaelutella andE. kuehnielLrare lapidly destroyedat -15'C and more slowly at ratherhigher temperatures. The eggsof the flour mite can withstandexposureto - l0'C for up to 12 days or OoCfbr severalmonths;developmentis possiblewithin the range2.5-30'C, dependingon humidity. Studieson the eff-ectsof ionizing radiations(e.g.liom a 60Cosource.) on cerealpestssuchas Tlrogll'phusmites andvariousbeetlesshow that
small dosesinhibit reploductive ability and larger ones destroy both mitesandtheireggs. The quantitativedeterminationof insect inf'estationin powdered vegetable drugs is of some significance. Melville (J. Pharm. 1, 649; 195l. 3. 926) developeda methodbasedon Pharnncol.,19,+9, the acetolysisof the weighed samplefor the quantitativeisolation of the insectfiagmentspresent.Thesefragmentsar"esuspendedin a suitable medir.rmwith a weighedquantityof lycopodiumspores.Using the per elyffon is characteristicfor fllct that the nunrberof strial punctLlres a particlllarbeetle.one can.by countingthe strial puncturesevidenton the tiagmentsof elytra presentunderthe microscope,arrive at a figure for the number of beetles present.The use of lycopodium spores (94 000 sporesmg-l ) eliminatesthe necessityfor countingall the strial puncturespresentin a particularvolume (or weight) of the suspension; the use of lycopodium powder in this respect is described under 'QuantitativeMicroscopy' (Chapter,l4). Spoiloge by rodents Rodent faecesusually contain the animal's hairs. so that drugs which on microscopicalexaminationshow the presenceof thesehails should be rejected.
Furtherreoding t e s t so f D a l e sM J 1 9 9 6A r e v i e wo f p l a n tm a t e r i a l su s e df b r c o n t r o l l i n gi n s e c p storedproducts.Bulletin 6-5.Natural ResourcesInstitute,Chatham'UK FreemanP I 980 Common insectpestsof storedfbod products.6th edn l i s t o r y ) ,L o n d o n .U K ( E c o n o m i cS e r i e sN o . l 5 ) . B r i t i s hM u s e u m( N a t u r a H HughcsA M 1976The mites of storeclfood and houses.2nd edn. HMSO' London. UK MAAF 198I Mites in storedcommodities.leaflet489. HMSO. Alnrvick, UK MAAF 1986Insectand t.nitepestsin fbod storcs,leafletP483.HMSO, Alnwick. UK
E
QuoliVcontrol
of analysiscontinuouslyimproving, Over the years.and with metl-rods thequalitycontrolofplant drugsusedin the allopathicsysternofmedicine has becomevely well establishedand standardscovering authenticity. generalquality and purity. and the assayof activeconstituents may be lound in all the principal phalmacopoeias.Usersof this book u'ill plobably have greatestrecourseto the British PltctnntLcopoeia (BP) 2000. the Eurr4tean Plnnrtacopoeia (EP). the United Stotes Phurnncopoeia-The Nutionul Forntulan' and other official National publications. Most of the crude drugs official in the EP rre also tlcscribedin the BP. In the pastthe sitr.ration regardingthe quality of the numelousherbal drugs used by rnanufactulelsor sold directly to the public was by no meansso well established. Now however,asa resultof Europeanlegislation which has accompaniedthe enormous demand lbr such products. there are standeLrds available for a large number of the more commonly used dnrgs. They are to be found in the Britisft Herbal Plrtrnttctcopoeicr 1996 (a new edition to be published shorrly). the Brirish Herbtl Conqtendiurn.Vol. l, 1993 (ed. Peter R. Bradley), Ihe United States Herbul Pharmctt'oltoeia cun'entlybeing rclcasedas monographsand the numerollsmonographsof ESCOP.Gernan CorrmissionE and theWHO already rnentionedin Chapter 2. The 1990 edition of rhe Chines, hasmonographscoveling509 drugs.fol many of which Phurnutcopoeir,r high standardsare imposedincluding modernmethodsof anelysis{see reportof lecturegivenby J. D. Phillipson.Plnnn. J.. 1993,25f , 30). One possible problern in devisin-qstandardsfbr crude drugs concelns the requirementfbr an assayof the active constituent\u hen the preciselyascertained. Fufihermore.one of the lattermay not have beer-r tenetsof herbalmedicineis that the maxitttumeffectivenessof the drug extractratherthan fiorn isolatderivesfi'om the whole drug or its crr.rde In caseswhere an assayis lacking it is thelefble of ed con-rponents. paramountimportancethat the crudedrug is properlyauthcnticated,its generalqLralityverified and all fbrmulations of it pleparedin i.lecordancewith good rnanufacturingpractice.Attention shouldalsobe paid to the shelt'-lifeof the crudedlug and its pleparations, Although ofticial standardsare necessaryto control the quality of drr.rgstheir use doesraisecertainproblems.Of necessity.to accr)mrrodatethe considerablevariationthat occursbetweenditl'erentbatchesof which a natural product it is necessaryto set relatively low standarcis allow the useof commercialmaterialavailablein any season.This has resultedin the tendencylbr producersor manufacturersto reduceall of their material to the lowest requirement:fbl exarnple.in a good year the majority of the alkaloid-rich leaves of belladonnaherb rray be removed and used fbr the economicalmanufactuleof galenicalsand the residueof the herb, containing much stem.used to give the powdered drug. Similarly, high-quality volatile oils may be mixed with lower gradesand still remain within ofTiciallimits.
DRUGS APPTICABTE TO CRUDE STANDARDSI There are a number of standards,numelical in nature,which can be drugseitherin thc whole or the powappliedto the evaluationof crr-rde deredcondition.
TO CRUDE APPTICABLE STANDARDS DRUGS 95 AND TO VOLATITE APPTICABTE STANDARDS
FrxED Olrs roo ASSAYS lOr
Er,
Sompling Before a consignmentof a drug can be evaluated,a sample must be drawn fbr analysis;considerablecare mllst be exercisedto ensurethat With largequantitiesof bulky drugs this sampleis truly representative. a difl-erentmethodof samplingis requiled from that involving broken or powdeled drugs.The ,BPgives no specific instructionsfbl this but the methodsof samplingusedin the USA are fully describedin older editionsof the USP.
",,.
:: t:,
,:1" .rrli' : ,t,,
:: ,::
",, :
f,,,:,,,, llli:il.il ;llllllll;
.:l,l,.,. l:llllli':
:,- .":' .
PRINCIPLES RELATED TO THECOMMERCIALPRODUCTION,QUALITYAND STANDARDIZATION OF NATURALPRODUCTS Preliminory exqminqtion In the caseof whole drLrgsthe macroscopicaland sensorycharactersare usually sLrfficientto enable the drug to be identifled. The general appearance of the samplewill often indicatewhetherit is likely to corrply with such standaldsas percentagcof seedin colocynth.of ash in valerianor of natter insolublein alcoholin asafbetida.However.drugs may comply with the descripticrtsgiven in the phaln'racopoeias and yet be unsatisfactory. as it is often difTicultspecificallyto describedeteriorationof drugsowing to ltrulty han esting,shipmentor stolageor deteriorationdueto age.In suchcasesthetlainedworker will be ableto infer much of the histoly of the samplefrom its appearance. The fbllowing exampleswill seli,eto indicatethe typc of evidenceto look lbr. Ifleaves and similar structuresarebaledbeforebein-uproperlydlied. nrr.rchdiscolor-rred matcrial may be lbund in the middle of the balc. Overdrying, on the other hand. nakes iear,esverl' brittle and causes them to break in transit.If starch-containingdrugsbreak with zihorny fracture.it may usually be infelred that the tentperatureof drying has beentoo high and that the starchhas beengelatinized.A pale color.rrin the caseof chamomilesindicatesthat the drug lrasbeencollectedin dry weatherand carefulll,dried.while the colour of the fracturedsultaceof gentian is a good indication as to whethcr it has been con'ectly fermented.Some drr.rgsare particularlyliablc to deteriorationif, during shipmentor stora-qe.they become damp (c.g. cilscara).Under rroist condition moulds readily establishthemselr,eson drugs having a high mucilagecontent(e.g.psylliurn,linseecl. squillandcvdonia).Er,idence of insectattackmust alsobe lookcd fbr. The price of certaindrugsdependslalgely on suchfactorsas sizeand colour. which are not necessarilyrelated to therapeuticvalue. This applies to such important drugs as senna leaf'lets.senna pods. chamomileflowers.ginger',nutrregsand rhubarb.
berries, starch. aloes, libres) i'u'hichcontain little volatile material. direct dryin,q(100 l0-5"C) to constantweight can be cn.rployed.The moistulebalancecombinesboth the dryin,qprocessand weight recording: it is suitablewhere large numbelsof samplesare handledand where a continuousrecordof loss in weight with time is required.For. materialssuch as balsar-ns which contain a considerableproportionof volatile matcrial. the drl,ing may be accorrplishedby spreadingthin layersof the rvei-shed clrugover'-e1ass platesand placing in a desiccator over phosphorus pentoxide.Vacuumdrying over an absorbentmay be utilized, possibll,at a specifiedtemperature.
Separation and measurement of moisture. The 'loss on drying' methods carrbe made more specificfbr tlie detemination of water by separating ander,'aluating thewaterobtainedflom a sample.This canbe achieved b1'passinga dly inerl eastl-rroughthe heatedsampleand r.rsingm absorption train (specificfbl water) to collect the water carriedfbnvard; sr-rch methodscln be extlemely accr.u'ate, as shown in their uselbr the determinationofhydrogenin organiccollrpoundsby cornbustionanalysis. Methods basedon distillationhave been widcly Lrsedlbr moisture detemination.The sarnpleto be analysedis placedin a f-lasktogether u'ith a suitable\vater-saturated immisciblesolvent(toluene.xylene.carbon tctrachloride)and piccesofporous pot and is distilled.The waterin the samplehasa considelablepartialpressureandco-distilswith the solvent.condensin-e in the distillateasarrimmisciblelayer.A simpleapparatLrs(Fig 1:l.lA) ori,rinallydcvisedby Deanand Starkpennitsthe direct measllrement of the waler obtainedand the lessdensesolvent(toluene. xylcne) is continuousll'returnedto the distillationflask. The methodis employed in the [/5P anclin the BP anc]EP tor somevolatile oil-containing drugs(Romiincharnomilef'lorvers, lovageroot. peppennintand sage leaves).To acconrnodatethe lossof waterdueto solubilityin the solvent the BP specifiesa preliminarydistillationof the solventwith addedwater(abor-rt Foreign mqtter 2 nl.): the exactI'olune of watel separatingas a layer is readoff The difTicultyof obtainingvegetabledmgsin m entilelypureconditionis and then the drug (sufljcient to give a flrther 2-3 ml water) Lrddedto the lully reco-enized. and pharmlr'opoeius fiont the drug is calculated containstatements as to the per- f'laskanddistillationresumed.Waterscparated centageof otherpartsof theplantol of otherorganicmattelu/hichmay be tiom the combirredfinal volume.Heavicr'-than-water solventsrequirethe permitted.Table l4.l gives examplesof variousollicial types of lintit receivershownin Fig. 1,1.I B. The methodis readilvapplicablero crude dmgs ancltixrd n-raterials applicableto specificdlugs. Drugs containingappreciablequantitiesol but has the disadvantage that relativelylarge potentfbreignmatter'.animalexcreta,insectsor mould shor.rlcl. (5-10 g) may be required. of the san.rple however. quarrtities Gas-chlomatoglaphic be rejectedeventhoughthe percentage methodshave become increasinglyimpor-tant of suchsnbstances be insufllcient for moisturedetermination to causethe rejectionof the drug on thepercentage becauscof theirspecificityandefliciency.The of foreigrrmattcr. In the caseof whole drugs a weighedquantity ( I 00-500 g. iiccord- u'ater in the weighed. powdered sample can be extractedwith dry ing to the type of drug). of a calef'ullytaken sanrpleis spreadin a thin methanoland an aliquot submittedto chromatography on a colllmn on layer on paper.It is examinedat x6 ma-enification and the fbrci-Qnmat- either10%carbowaxon Fluolopak80 ol Polapak,a commercialpolymer suitableibr gas-liquid clrronarographv(CLC). The water separated ter is picked out and weighedand the percentagcrecorded.Detailswill by this means is readily detemincd liom the resulting chromatogram. be found in the -BP2000.AppenclixXID. Fol fblei-enolgrnic nrartL.rin powdereddrugs.see'Quantitative Teflon-6coatcdwitl.rl07c polyethyleneglycol 1500,with n-propanolas Microscopy'. an intemal standardhas also been ernployedfbr the determinationof moisturein crudedrugs. Moisture contenf Not only is thepurchase of drugs(e.g.aloes.gelatin.gums)which contalnexccsswater.uneconomical, br-rt alsoin conjunctionwith a suitable Chemical methods. The ntost extensively employed chemical temperaturemoisturewill lead to the activationof enzyntesand. given method fbr watel determinationis probably the Karl Fischerprocedsuitableconditions.to the proliferationof living organisms. but also As most ure. which finds exterrsiveuse not or-rlyin the phan-r-raceutical. vegettrbledrugscontainall the essentialfood reqr-rirernents It is usedin the BP for moulds. in the food, chemicaland petrochemicalindr.rstlies. insectsand mites. deteriorationcan be very rapid once infestationhas and is palticularly applicablefor expensivedrugs and chemicalsconttrinirr-esmall quantities of moisture. Dry extracts of alkaloidtakenplace. A large number of rnethodsare now availablelbr moisture deter.- containingdrugs.alginic acid. alginatesand iixed oils (e.g. arachis. castor.olive and sesameoils fbr BP parenteraluse) may usefully be mination.manybeingemployedin industriesunrelatedto pharmacy. evaluated. For crude drugs such as digitalis and ipecacuanhathe Loss on drying. This is ernployedin the EP, BP, and L/SP.Although powdercdmaterialcan first be exhar-rsted of watet'with a suitablear-rhythe loss in weight. in the sarnplesso tested,principally is due to water. drous solvent(dioxan)and an aliquot takentbr titration. small amounts of other volatile materialswill also contribute to the The reagentcorrsistsof a solutionof iodine. sulphurdioxide and weight Ioss.For matelials (digitalis.hamarrelisleaf'.yarrow.hawtholn pyridine in dry rnethanol.This is titrated againsta samplecontaining
QUALITYCONTROL
Toble | 4.1
Exomples of BP limits for foreign moiter.
Drugs leovesond herbs Beorberry leof Birchleof Wormwood
Foreign matfer limits
)B% Foreignmotterof which>5% stemsond *3% ofherforeignmoiter.Leoves of different colourto officioldescription F I0% }'3% Frogments of femolecotkins,#3% otherforeignmotter }5% Stemswith diometergreolerthon4 mm,*27" otherforeignmotter
Fruilsond seeds Howthornberries Psyllium seeds
)2% Foreignmoiter,}'5% deterioroted folsefruiis } 1% Foreignmotterincludinggreenish unripefruits.No seedsof otherPlontogospp.
Inflorescences Colenduloflowers Elderflowers Limeflowers
*5% Brocts,*2'/" otherforeignmotter #B% Frogments of coorsepedicelsond otherforeignmoiter,> l5% discoloured brownflowers *2% Foreignmotter, obsenceof otherlilio spp.
Rhizomes ond roots Couchgrossrhizome Morshmollow root Volerionroot
> 15%Greyish-block piecesof rhizomein cutdrug l2?oBrown deteriorotedroot,*27" cork in peeledroot |5% Stemboses,-|2% otherforeignmotter
Borks Q u i l l o i ob o r k Coscorobork
|2% Foreignmotter )'1% Foreignmotter
Fig. l4.l A , A p p o r o t u sf o r t h e d e t e r m i n o t i o no f m o i s t u r ei n c r u d e d r u g s b y d i s t i l l o t i o no n d f o r v o l o t i l eo i l s h e o v i e rl h o n w o t e r ; B , r e c e i v e ro f o p p o r o t u sf o r t h e d e t e r m i n o t i o no f w o t e r i n c r u d e d r u g s ( h e o v ye n t r o i n m e n to) n d f o r v o l o t i l eo i l s i n d r u g s ; C , r e c e i v e rf o r d e t e r m i n o t i o no f v o l o t i l eo i l i n d r u g s o s ,1980; u s e db y i h e B P D , r e c e i v e rf o r d e t e r m i n o t i o no f v o l o t i l eo i l i n d r u g s o s u s e d b y b o t h t h e E Po n d t h e B P .
\\'ater.which causesa loss of the dark brown colollr.At the end-point u,henno wateris available.the colour ofthe reagentpersists.The basic reactionis a reductionof ioclineby sulphurdioxide in the plesenceof ri ater.The reactiongoesto completionby the removalof sulphurtlioxide as pyridine sulphur trioride, which in turns reacts with the methanolto lbnn the pyridine salt of methyl sulphate. In the absenceof n-rethanol. the pyridine sulphurtrioxide reactswith anothefmoleculeof water'.The rcagenlrequilesstandardization immcdiately befole use and this can bc done b;' employmentof a standard solution of water in methanol or by use of a hydrated sait-for exarnple.sodium tatlate (Na2C4H1O6.2HrO). To eliminateinterf'erence trorn atmosphelicmoisture,the titration is carlied out underan atmosphereof dry nitrogen.the end-pointbeing recordedamperometfically. Equipmentis now availablefbl a completelyautomateddetermination. thus eliminatingthe manualaspectsof santplehandlingand weighing.
F
H2O + 12+ SO,
=+
2Hl + SO3
lt * ,/,\i l | - ' r,,\ \N7 \*-dI
sor+t1+H2O
n \ *z
,o/Jo
r\ ll
\*z
lf cH,ox+
so1-\o
rr\ i l \*? H
H2O:1,
l SOI CHr
PRINCIPLES RETATED TO THECOMMERCIAT PRODUCTION,QUALITYAND STANDARDIZATION OF NATURALPRODUCTS introduction to the Karl Fischer cell. titration and data completion. Although the BP Kall Fischerreagentcontainspyridine. as above.the latter has been replacedby other basesin sorle commercialreagents (for an articie on Karl Fischer, and the nethod, see R. Steiner Intenntional LaborrLton,1992,22,311fbr a discussion on the coulometric titration plocedureseeE. Scholz,ihid.. 1989.19, 16). The principaldrawbacksof the Karl Fischermethodarethe instability of the reagentand the possibilityof substances in the sample.other than water,which may reactwith the reagent. Otherchemicalmethodsfbr waterdeterminationincludetreatingthe samplewith variouscarbides.nitridesand hydridesand measuringthe -uasevolved; -qaschromatographyhas been employedfor the analysis ofthe liberatedgas. Spectroscopicmethods. Water will absorbenergy:rt various wavelengthsthroughoutthe electromagneticspectrumand this fact can be madea basisfor its quantitativedetermination(seelater in this chapter for a generaldiscussionon spectroscopy).Measurementscan be made in both the infraredand ultravioletregions:interfeling substances must be absent.The r-nethod is particulally suitabletor very small qr.rantities of water (e.9. trace qr.rantities in gases).Nuclear magneticresonance (NMR) spectroscopyhas been employed fbr the determinationof moisturein starch.cotton and other plant prodllcts. Electrometric methods. Conductivity, dielectric and coulonretric methodshaveall beenutilized for moisturedeterminationbut har,enot. as yet. found extensiveapplicationto phalmaceuticalproducts. Extrqctive vqlues The detenninationof water-solubleor ethanol-solubleextlactiveis usedas a meansof evaluatingdmgsthe constituents of which arenot readilyestimatedby othermeans.But as suitableassaysbecomeavailable(e.g.with the antluaquinone-containingdrugs). the extractive tests are no longer reqr,riredas ph:umacopoeialstandards.In certain casesextraction of the
drug is by n-raceration. in othersby a continuousexfaction process.For the Iatter the Soxhlet extractoris panicularly useful imd has been in use for many years,not only fbr the determinationof extractives(e.g.tixed oil in seeds)but alsofor smalf scaleisolations(Fig. 1,1.2). A development of the Soxhlettechniqueis alsoshownin Fig. 14.2;in this apparatus exrractionis by boiling solventfollowedby percolation;finally,evaporation yieldsthe extractand the recoveredsolventready for the next sample.Somecxamplesof the typesof extractiveusedaregivenin TableI 4.2. Ash vqlues When vegetabledrugs are incinerated.they leave an inorganic ash which in the caseof many drugs(e.g.rhubarb)valieswithin f airly wide limits and is therefbreof little vah-re lbr purposesof evaluation.In other cases(e.g. peeled and unpeeledliquorice) the total a.s/rfigure is of importanceand indicatesto someextentthe amountof caretakenin the preparationof the dlug. In the determinationof total ash r,aluesthe carbon must be removedat as low a temperature(,150'C)as possible
Toble 14.2
ExlrEcfives employed for drug evsluofion.
Drug
Methodof evoluotion
Genlion,liquorice, monydrugsof BHP Quilloio Volerion,cocillono, osofoetido Ginseng,hop strobiole Ginger,ipomoeoond iolop Benzoin, cotechuond Tolubolsom,myrrh Colocynth crushedlinseed
Percentogeof woter-solubleextroctive Percentogeof ethonol {45%) extroctive Percentogeof ethonol (60%) extroctive Percentogeof ethonol {ZO%}extroctive Percentogeof ethonol (90%) extroctive L i m i t so f e t h o n o l - i n s o l u b l m e oner L i m i to f l i g h t p e k o l e u m e x t r o c t i v e Percentogeof ether-solubleextroctive
tl
1,h,1
I Fig.14.2 l, Soxhletcontinuous extroction opporotus. A, powdereddrugfor extroction in thimbleond pluggedwith suitoblefibree.g. defottedtow or cotton wool; solventrefluxes intothimbleond syphonsintofloskB, conioiningboilingsolvent, whenreceiveris full.ll, Three-stoge continuous extroction ond solventrecovery: left,extroction by boilingwith solvent; centre,percolotion stoge;right,removolof solvent. A, somplefor extroction; A1, exhousted drug;B, solvent; B1,recovered solvent; C, solventcontoining solubleplontconstituents; D, finolextroct.(Soxtec System, Tecotor Ltd.)
QUALITYCONTROL becausealkali chlorides.which may be volatile at high temperatures. would otherwisebe 1ost.If carbonis still presentafterheatingat a moderate temperatrlre.the water-solubleash may be separatedand the residueagain ignited as describedin the BP. or the ash may be broken up, with the additionofalcohol, and againignited.The total ashusually consistsmainly of carbonates,phosphates,silicatesand silica. To producea mole consistentash a sulphutetlaslr, which involves treatment of the drug with dilute sulphuricacid beforeignition. is used. [n this all oxides and carbonateserreconvertedto sulphatesand thc ignition is carriedout at a higher temperature(600'C). Ifthe total ash be treatedwith dilute hydrochloricacid. the percentage ol ucid-insolubleush may be deterrnined.This usually consists mainly of silica.and a high acid-insoluble ashin drugssuchas senna. cloves,liquorice,valerianand tlagacanthindicatescontaminationwith carthy material.Sennaleaf, which may be used directly as the powdered drug, is required to have a low acid-insoiuble ash (.2.5c/a); hyoscyamus,however,which unavoidablyattractsgl'it on to its sticky trichomes,is allowed a higher value ( l2%). Equisetum811P,the dried sterilestemsof Equisetumat1'ense, hasa naturalcontentof silicaand is allowed a limit of 107c.In the caseof ginger a minimum percentageof vater-solubleusi is demanded.this being designedto detectthe presenceof exhaustedginger.
contentsof powdereddrugs are lower than thoselor the corresponding whole drugs. Tqnnin content A f'ew drugs (Harnamelis,Rhatany and Alchernilla) are assayedlbr their tannincontent(BP 2000).The methodrefersto thosepolyphenols adsorbedby hide powder and giving a colour reaction with sodium phosphomolybdotungstate reagent.Seeindividual drugs. Bitferness vqlue This standardis relevantto Centaury,Gentian and Wormwood of the BP. Thesedrugs are usedtbr their bitter eff-ectand specificdirections for the determinationof the standardare given undereachmonograph. The bitternessvalue is deterrninedorganolepticallyby comparison with a quinine hydrochloridesolutionwhich actsas the standard.
Swelling index This is definedin the BP as the volumein millilitres occupiedby I g of a drug, including any adheringmucilage,after it has swollen in an aqueousliquid for 4 h. The drug is treatedwith 1.0 ml ethanol (96%) and 25 ml water in a graduatedcylinder, shakenevery l0 min for I h and allowed to stand as specified.In some instances,as with linseed and psyllium seedwhere the mucilage is in a layer near the surfaceof Crudefibre the drug, the standardcan be determinedon the whole drug; in other The preparationof a crude flbre is a meansof concentratingthe more casessuch as marshmallowroot where the mucilageis distributed resistantcellular materialof drugslbl microscopicalexarnination.It is throughout the tissues,the powdered drug is used. Examples are: particularlyr.rsefullbr rhizomessuch as ginger which containrelative- Agar dl0. Cetrarra{4.5, Fenugreek{6. Fucus d6.0. Ispaghula ly largeamountsofoleoresin and starch.The techniqueinvolvesdefat- husk {zl0 (determinedon 0.1 g powder),Ispaghulaseed{9, Linseed ting the powder and boiling in turn with standardacid and alkali with d4 (whole drug) and d21.5 (powdered drug), Marshmallow suitable washing of the insoluble residue obtained at the diff'erent root < 10. stages(see Chapter'44). The crude fibre so obtained can also be Some variationsin the method of determinationhave given rise to enployed quantitativelyto assaythe fibre contentof foods and animal other terminology.Thus Skyrme and Wallis in their original work on f-eedstuffsand also to detect excessof certain materialsin powdered seedsof Plantago spp. in 1936 used the term swellingfactor (24-h drugs,e.g. clove stalk in clove. For further detailsseethe lzlth edition standingperiod) and the BP (1993) ciles swellingpower in respectof of this book. Ispaghulahusk (variationin shakingprocedureand standingtime). Yet again, for Iceland moss the BP 2000 cites swelling valtre. Determinqtion of volotile oil Minimr-rmstandardsfbr the pelcentageof volatile oil presentin a Rp volues numberof drugsareprescribedby many pharmacopoeias. A distilla- Pharmacopoeiasare increasingly employing thin-layer chromatotion methodis usually employed.and the apparatustlrst described graphy as a meansfor assessingquality and purity. For a discussion by Meek and Salvin in 1937is still widely usedin laboratolies;the ol chromatographicanalysis,see Chapter 18. It suffices to mention receiverfbr this apparatusis very similarto that for waterestimation hele that the Rp value (rate of flow. i.e., distance moved by solute by heavl'entrainment(Fig. I ,1.I B). The weigheddrug is placedin a divided by distancemoved by solvent front) of a compound, deterdistillationflask with water or a mixture of water and glycerin and mined under specific conditions,is characteristicand can be used as connectedto the receiver (cleanedwilh chromic acid), which is an aid to identity.RF valuesvary liom 0.0 to 1.00;the hRF (RF x filled with water and connectedto a condenser.On distillation.the 100) values are in the range 0-100. Quantitative extracts of crude oil and water condenseand the volatile oil which collects in the drugs are preparedand comparedchromatographicallywith standard graduatedreceiveras a layer on top of the water is measrlred.For referencesolutionsof the known constituents.Intensitiesof the visuoils with relative densitiesaround or greaterthan 1.00, separation alized chromatographic spots can be visually compared and the from the water is assistedby pltrcinga known volume of xylene in method can be usedto eliminate inf'erioror adulterateddrugs. In this t h e r e c e i v e r a n d r e a d i n g o f f t h e c o m b i n e d o i l a n d x y l e n e . way semiquantitativetests for the principles of drugs (peppermint. Alternatively.for oils with relativedensitiesgreaterthan water (e.g. saffron.German chamomile,digitalis) not rapidly evaluatedby other c l o v e o i l , 1 . 0 5 ) ,a r e c e i v e rs i m i l a rt o t h e t y p e s h o w ni n F i g . l 4 . l A meanshavebeendeveloped. can be usedand no xylene is necessary. The BP 1980employedthe In an analogousmanner,gas chromatoglaphicretentiontimes and apparatusillustrated in Fig. l,1.lC; it dif}'ersfiorn the above in that peak areascan be employed lbr the examinationof volatile oils and the distillatepassesthroughthe condenserand so is coolerthan with othermixtures. the leflux type.The EP andBP 1993employ an apparatlrs similar"to that shownin Fig. 14.lD. The time takento completethe distillation Microbiol contominotion of the oil varieswith the natureof the drug and its stateof comminu- The BP requiresa number of drugs (e.g. acacia,agar,sterculia,tragation but about4 h is usually sufficient.Solutionof the volatile oil in canth, powdeled digitalis etc.) to be free of Escherichiu coli in the a fixed oil (e.g. in powdered fruits of the Umbelliferae) may retard quantity of material statediothers (e.g. alginic acid, cochineal,tragadistillation.Note that the oharrracoooeialstandardslbr volatile oii canth) are also testedtbr the absenceof Salmonelkl.Upper limits for
E
QUALITYCONTROL ;.c'calrse alkali chlorides,which may be volatile at high temperatures, ri ould otherwisebe lost.If carbonis still presentafierheatingat a mod.rate temperature,the water-solubleash may be separatedand the rcsicir.re againignited as describedin the BP, or the ash may be broken Lrp.u'itlr the additionof alcohol,and againignited.The total ashusuallr consistsrnainly of carbonates.phosphates,silicatesand silica. To producea more consistentash a sulltltutetlasft. which involves treatnrentof the drug with dilute sLrlphuricacid befbreignition, is used.ln this all oxides and carbonatesal'econvertedto suiphatesand thc ignition is carriedout at a higher temperature1600"C1. If the total ashbe treatedwith dilute l.rydrochloricacid, the percenttgc of acitl-insoluhlensft may be detennined.This usually consists rnainlyof silica,and a high acid-insoluble ashin drugssuchas senna. cloves,liquorice,valerianand tragacanthindicatescontaminationwith carthy matelial. Sennaleaf, which may be used directly as the powdcred drug, is required to have a low acid-insoluble ash (.2.57c); hvoscyamus,however.which unavoidablyattractsglit on to its sticky tlichomes.is allowed a higher value ( l2%). Equisetum811P,the dried sterilestemsof Equisetuntarren,te.hasa naturalcontentof silica and is alloweda limit of l)c/c.ln the caseof gingera ninimum percentage of vtrter-solubLe asl is demanded,this being designedto detectthe presenceof exhaustedginger.
Crudefibre The preparationof a crude fibre is a meansof concentratingthe more resistantcellular materialof dlugs lbr microscopicalexarnination.It is particularlyLrsefulfbr lhizomes such as girrgerlr'hich containrelatively largeamountsof oleoresinand starch.The techniqueinvolvesdefatting the powder and boiling in tr.rrnwith standardacid and alkali with suitable washing of the insoluble residue obtained at the difl'erent stages(see Chapter 44). The crude libre so obtained can also be employedquantitativelyto assaythe fibre contentof fbods and animal feedstuffi and also to detect excessof certain materialsin powdered drugs,e.g. clove stalk in clove. For further detailsseethe lzlth edition of this book. Determinqtion of volotile oil Minimum standardsfor the percentageof volatile oil presentin a numberof drugsareprescribedby many pharmacopoeias. A distillation method is usually employed.and the apparatustirst described by Meek and Salvin in 1937is still widely usedin laboratolies;the receiverfor this apparatusis very sirnilarto that for waterestimation by heavy entrainment(Fig. l4.lB). The weigheddrug is placedin a distillationtlask with water or a mixture of water and glycerin and connectedto the receiver (cleanedwith chromic acid), which is filled with water and connectedto a condenser.On distillation.the oil and water condenseand the volatile oil which collects in the graduatedreceiveras a layer on top of the water is measirred.For oils with relative densitiesaround or greaterthan 1.00. separation from the water is assistedby pltrcinga known volume of xylene in the receiver and reading off the con-rbinedoil and xylene. Alternatively,for oils with relativedensitiesgreaterthan water (e.g. c l o v e o i l , 1 . 0 5 ) ,a r e c e i v e rs i m i l a rt o t h e t y p e s h o w ni n F i g . l 4 . l A can be usedand no xylene is necessary. The BP 1980employedthe apparatlrsillustratedin Fig. lzl.lC; it dilTersfi'ornthe abovein that the distillatepassesthrou-9hthe condenseranclso is coolerthan with the reflux type. The EP and BP 1993employ an apparatlrssimilar to that shown in Fig. l,1.lD. The time takento completethe distillation of the oil varies with the natureof the drug and its stateof comminuzl h is usuallysufficient.Solutionof the volatile oil in tion but abor-rt a fixed oi1 (e.g. in powderedfruits of the Umbelliferae)may retard distillation.Note that the pharmacoooeialstandardsfbr volatile oil
Fr,
contentsof powdereddrugs are lower than thosefor the corresponding whole drugs. Tqnnin content A f'ew drugs (Hamamelis,Rhatany and Alchemilla) are assayedfor their tannincontent(BP 2000).The methodrefersto thosepolyphenols adsorbedby hide powder and giving a colour reaction with sodium phosphomolybdotungstate reagent.Seeindividual drugs. Bifterness vqlue This standardis relevantto Centaury,Gentian and Wormwood of the BP. Thesedrugs are usedfbr their bitter effect and specificdirections fbl the determinationof the standardare given undereachmonograph. The bitternessvalue is determinedorganolepticallyby comparison with a quininehydrochloridesolutionwhich actsas the standard. Swelling index This is defined in the BP as the volume in millilitles occupiedby I g of a drug. including any adheringmucilage, afier it has swollen in an aqueousliquid lbr zl h. The drr.rgis treatedwith 1.0 ml ethanol (96clo) and 25 ml water in a graduatedcylinder. shakenevery 10 min for' t h and allowed to standas specified.In some instances.as with linseed and psylliurnseedwherethe mucilageis in a layernearthe surfaceof the drug. the standardcan be determinedon the whole drug; in other casessuch as marshmallow root where the mucilage is distributed thror.rghoutthe tissues,the powdered drug is irsed. Examples are: Agar dl0, Cetraria{4.-5, Fenugleek{6. Fucus {6.0, Ispaghula husk {zl0 (determinedon 0.1 g powder),Ispaghulaseedd9, Linseed 44 (whole drug) and {4.5 (powdered drug), Marshmallow root{10. Some variationsin the method of detenninationhave given rise to other terminology.Thus Skyrme and Wallis in their original work on seedsof Plantago spp. in 1936 used the Ierm swellingfactor (24-h standingperiod) and the BP (1993) ciles .sv,ellingpower in respectof Ispaghulahusk (variationin shakingprocedureand standingtime). Yet again.for Icelandmossthe BP 2000 citesswellingvaLtre. Rp volues Phannacopoeiasare increasingly employing thin-layer chromatography as a meansfbr assessingquality and purity. For a discussion of chromatographic analysis,seeChapter18. It sufficesto mention here that the Rp value (rate of flow. i.e., distancemoved by solute divided by distancernoved by solvent front) of a compound, determined under specific conditions,is characteristicand can be usedas an aid to identity.RF valuesvary from 0.0 to 1.00;the hRp (Rp x 100) valuesare in the range 0-100. Quantitativeextractsof crude drugs are preparedand comparedchromatographicallywith standard referencesolutionsof the known constituents.lntensitiesof the visualized chlomatographic spots can be visuaily compared and the method can be usedto eliminate infer"iorol adulterateddrugs. In this way semiquantitativetests for the principles of dlugs (peppermint, saffron,German chamomile,digitalis) not rapidly evaluatedby other meanshavebeendeveloped. In an analogousmanner.gas chromatographicretention times and peak aleas can be employed for the examinationof volatile oils and othermixtures. Microbiol contominotion The BP requiresa number of drugs (e.g. acacia,agar,sterculia,tragacanth, powdered digitalis etc.) to be free of Escherichia colr in the quantity of material stated;others (e.g. alginic acid, cochineal,tlagacanth) are also testedlbr the absenceof Salmonellu.Upper limits for
@
PRINCIPLES RETATED TO THECOMMERCIALPRODUCTION,QUALITYAND STANDARDIZATION OF NATURALPRODUCTS totalviableaerobiccount.commonly103.104nricroor_ganisms -e-1.are being increasinglyapplied to clude dlugs including the eums, A-ear. Tragacanth,Acacia. Gual and Guar Galactomannan.Xanthan gum (q.v.) producedby t-ermcntationhtrslimits of 103for bacteriaand 102 fbr firngi. g-1. Generalll'. r.nanufhcturers will ensure that. for crude drugsto be ti,rkenintelnally.the limits for bacterialand mould contamination as appliedto fboclstufl.s are adheredto. In an investi-eation by Lutornski and Kedzia (.plttnuLMetl..1980.4l), 212) of rnould contaminationof crudedru-gs.246 samples\vereexaminedand 247cwerecontaminated at thc levelof 10 000 organismsg l. Flom 50 crudc drugs. 15 Aspergill.usand 28 pcnit.illiutn stratns were isolated;othel'common generawereMuc'or,Rltippt.ts andThamnicliunt.There was no evidenceto show that moulds rvere proclucing stronglytoxic substances in the cmde dr.ugand her-balprepar.ations, in contlastto the embryotoxic,teratogenic.iluta-Qenicand carcinogenic substancesproducedby sonte speciesof the above in peanuts.corn. wheat and rice (see Chapter:10.).However. in a seriesof papersRoy (.lnt.l. CrudeDrug Res..1990.28.l5l: Int. J. phcLrnruandcolleagues cogttos\'.1991. 29. 197 and ref'crencescited therein)have shown that roots (e.g. At-orLts<:alunus.Picrutrrhi
Toxic residues
I
These mav arisein crude drugs as a result of pesticideapplication during cr.rltivationof the dlug and at a later.stagefrom fumi-eationof the storedproduct.The problemsand the natureof the toxic residues ale essentiallythoseencountered in the tbod industr.yand in a number of countriesregulationsexist to cover limits of theseresiduesin fbods. cosnretics.drr.rgsand spices.Appendir XI L of the Ap 2000 givesthe requirements relatingto pesticideresiduesfbr herbaldrugsl specificdirectionsfor the samplin-q of bulk materialsare given and the varior-rs insecticidesand their assavslisted.In certuininstancesit may be necessary to test for aflatoxinsand radioactiveintpLu.itics. It hasbeenreportedthat many spices.chamornileand valerianobtained comrnerciallycontninpesticideresidues.but ntainlv within acceptablelevels. Thin-layer chronratography(TLC) and gas chromatographic methodsareavailablefor the determinationof or-sanochlor.ine and urea derivatives. enzymatic methods 1br organophosphorr.rs compounds. cololimetric methodsfbr urea derivatives.and spectroscopic techniquesfor paraquat.triazinesand heal'y metals. Toxic residucsmav be substantiallyreducedor elin-rinated by the use of infusionsof the drieclplant rrraterialand by the extractionof the useful plantconstituents. Storageat 30"C hasbeenshownto reducerapidlv the ethvleneoridc residuesin sennapodsto toler.able levels.Much researcl-r has been devoted to tl-reharmful effects of ethylene oxide which is a very effective insecticideand acar.icide:fbr an in-depth reportseeGolberg( 1986).H a: onl A s.s es.s ntent o.l'Etht Iene Or i d e. B oca Raton.FL.. USA: CRC Press.
APPUCABLE rO vOrAflrEAND 9I_4!!D4BDS FIXEDOIIS
Certainstandardsarc particularlyappropriateto volatile oils and fixed oils.
Refrqctive index The reliactive index of a substanceis the ratio betweenthe velocity of light in air and the velocity in the sr-rbstance under tesr. For light of a grvenwavelength(it is lrsualto employtheD line of sodium.whichhas a doubletof linesat 589.0nm and 589.6nm), the refractiveindexof a nraterialis -uivenby the sine of the angle of incidencedivideclby the sineof the angleof refiaction.The reliactiveindex varieswith the temperature,and pharmacopoeialdeterminationsare madeat 20oC. A convenientinstrument is the Abbi retiactometer.in which the angle measuredis the 'critical angle'for total ref'lectionbetween -elass ol high refiactive index and the substanceto be examined.By this means,and by selectinga particularwavelengthof li-ehtat which to make the measurements.it is possible to calibrate the instrument directly in terms of refiactive index. It is the entergentbeam that is viewed in the instruntent.and the critical an-eleis indicatedby the eclge of the clarkpart of the field of view. In this instrumentthe needfor a monochromaticlight sor-rrce is eliminatedby the inclLrsionof a dispersion 'compensator'placed at the base of the telescopetube of the retiactometer.This consistsof two direct-visionprismsmacleaccurately dilect fbr the D sodium line; the prisms can be made to rotate in opposite directions. The system of variable dispersion which these prisms folm can be madeto counterbalance the resultantdispersionof thc refractometelprism and the matelialbeing examir-red. The temperatr,rreof the sampleis adjustedby a waterjacket. Automatic refiactometerssuch as the Leica Auto Abbd refiactometerare now available.Advantagesover the traditional,visr.ral'transmitted light instrumentsare: (l) they measurerefiactive index with precisionto the tlfth decin-ral place.comparedwith fbur decimalplaces fbr the visual refiactometer(however.cunent pharmucopoeias require only threedecin.ralplacesin standardslor volatile oils): (2) a reflected light principle is employed meaning that light is not rransmittecl throughthe san'rpleand henceploblernswith dark or colouredsamples are avoided:(3) the shadow-linelocation is deterrninedby the instrurrent software.eliminatins variationsin readingscausedby individual sr.rb.jectir,e interpretationsof thc placementof the shadowlineborderon :r crosswtrel(4) no mechanicalcomponentsareinvolved.thusreducing wear with time. Hower.,er. if the increasedsensitivityof suchinstrumentsis to be lully utilizedthenadditionalcareandconsideration must be given to the measurementof temperatureand to the correctionfor eff-ectof temperatureon refiactive index (for further cletailsseeT. E. Ryan.Inl. L4BMATE. 199,1, 19(5).l0). Measurcmentsof rcfiactive index areparticular.lyvaluablefbr purity assessments of volatileandfixed oils.and manyvaluescanbe fbundin the EP. BP, BPC and othcr pharmacopoeias. Oits of cussia,einnanton and cinnamonleaf haverefi'activeindicesof about 1.61.1.573-1.600 and about I .53.respectively.making possiblethe difl'erentiationof the oils. The refractiveindex of lemon oil is l.zl74-l.1i6.and that for terpeneless lemonoil 1.475-1.;185.
Opticol rototion The optical rotation of a liquid is the anglethr.oughwhich the plane of polalization of light is rotated when the polarized light is passed througha sanrpleof the liquid: this rotationrnay be eitherclockwiseor anticlockwise.Along with the fundamentaleffectsof the moleculesof liqLriclunder in'".estigation. the obselvedrotation is dependenton the thicknessof the layer examined.its temperatureand the natureof the light ernployed.The ^BPuses sodium light, a layer I dm thick and a temperatLrre of 20'C. With modern 'half'-shadow'or 'triple-shadow, polarimetersin which the two or threefields ar.eviewed simultaneously and matchedto the sameintensity.r'otationscan be measuredwith an accuracyof atleast+ 0.01degree.
QUALITYCONTROL Most volatile oils containoptically activecomponentsand the direction of the rotation, and its magnitude,is a useful criterion of purity. Examplesto illustr"atethe rangeof valuesfound are carawayoil. +721. to +80o:lemon oil. +57o to +70o:terpeneless lemon oil. -5o to +2.1 cinnamonoil. 0o to -2o: citronellaoil, Java.--5oto +2o:citronellaoil. Ceylon,-9o to -l8o; peppermintoil. -10'to -30o; spearmintoil. -45. to -60o. The BP distinguishesbetween nutmeg oils from the East lndies(+10'to +25') andthe WestIndies(+25" ro 45.). Many solid materialsof natural origin ale optically active and the rotationof their solutions(water.ethanoland chlorotbrm are common solvents)can be measuredin a similar way to the above.The specific rotationofthe solid is given by Angr-rlarrotationper dm of solution Glams of optically activesubstanceper rnl of solution l00cr lc
l00cr ldn
where o is the observedrotation in degrees.1 is the length of the observedlayer in dm, c is the numberof glams of substancecontained in 100 ml solution,d is the densityandp is the nun'rberof gramsof substancecontainedin I00 g of solr,rtion. The record of the specificrotation of a compound should include the solvent used and the concentration.in additionto the type of light employed(sodiunrD line of 589.3 nm or mercury greenline of 5,16.1nm)-for example.[u] $ t 2 . 0 ' i i n c t h a n o l=r - l 5 o . For examplesof the use of specificrotation as a physical constant. studentscan consult the BP or other pharmacopoeialmonographson alkaloidalsalts. The visual polarimeterrequiresthe r,rseof solr-rtions which are not highly coloured or to any extent opaque.and sometirnes.with plant extracts,suchsolr.rtions aredifficr-rltto obtain.Becauseangularrotation talls off linearly, whereas absorption does so exponentially with decreasein path length. the use of short sampletubeshas arr obvious advantageif it is possibleto measureaccuratelythe correspondingly small rotations.Automatic polarimetelsemploy a sampletube about l/lOth the len-qthof that requiredby a visualpolarimeter.Measur.ement of small rotationsis made possibleby Lrtilizationof the Faradayelec-
Toble 14.3
Quontitotive chemicql tesfs A number of quantitativechemicaltests-acid value, iodine value. saponificationvalue. ester va1ue, unsaponifiablematter, acetyl value, volatile acidity-are mainly applicableto fixed oils and are mentionedin Chapter20. Some of thesetestsare also useful in the evaluationof resins(acidvalue.sulphatedash).balsams(acid value, ester \ralLre.saponificationvalue), volatile oils (acid value, acetyl value. estervalue)and gurns(methoxyldetermination, volatileacidItY)
ASSAYS A crude drug may be assayedfbr a particulargroup of constituentsfbr examplc.the total alkaloidsin belladonnaor rhe total glycosidesof digitalis. Alternatively, it may be necessaryto evaluare specific components fbr exantple,the reserpinecontent.as distinct fi.om the totaf alkaloid content.of Rutnt.olfiaspp.Biological assays,which can be time-consuming.were at one time employedfbr the assayof those potent drugs (e.g. digitalis) fbr which no other satisfactoryassaywas available.In pharmacopoeias thesehave now been largelyreplacedby chemicalandphysicalassaysfor routine standardization. However,the biological assayrcr.nainsimportant fbr screeningplant materialsand their liactionatedextractsin the searchfor new drugs. In this respect there is a role for sirnplebiological assays(e.g. brine shrimp toxicity) which can be carried out by the phytochemistwithout the specialist procedulesusedby pharmacologists.Some types of assaycommonly employed are given in Table 14.3. Often a preliminary purification or fiactionation of the active constituentsof the drug is required and fbr this chromatoglaphyis finding increasinguse. Examples of
Types of qssoy employed for crude drugr.
lypes of assoy
Exomples
Seporotion ond weighingof octiveconstiiuents
Colchicine in colchicum cormond seed.Resins of podophyllum ond of the Convolvuloceoe. Crudefilicinin molefern.Totolbolsomicesters of Perubolsom 'Totol olkoloids'of monydrugs{e.g.ocid-bosetitrotions}. Nonoqueous titrotions of olkoloidsolts.Skychnine in nuxvomico.Morphinein opium.Cinnomicoldehyde in oil of cinnomon.Freeolcoholsin peppermint oil. Corvonein oil of corowoy Cineolein eucolyptus oil (f.p.of ocresolcomplex) Most groupsof ocliveconstituents Cordiooctive drugs,ontibiotics, viiomins,toenicides, onthroquinone derivotives, mydrioticdrugs,soponins, ontitumour drugs,ontiomoebic drugs,ginkgolides (onti-PAF oclivil.y) Hesperidin, limoninond noringin(Cifrus), (Drgitolis cordenolides /onofo),logonin (plontcellcultures), sennosides troponeolkoloids(medicinol {Cossioongustifo/io), Solonoceoe), morphineond relotedolkoloids{poppycopsules}, lysergic ocid derivotives plonttissues), oimoline(Rouwo/fio spp.}, {ergot),quinine{cultured vincristine ond reloledolkoloids(Cothoronthus roseus), (So/onurn solosodine spp.) Quossin,neoquossin, 1B-hydroxyquossi n (Quossio ond Picrosmo spp.), podophyllotoxin, troponeolkoloids(Solonoceoe), ortemisinin in Artemisio annuo pyrrolizidine olkoloids, ergotolkoloids, golonthomine in Leucoium oesl'tvum, soikosoponino in Bupleurum, ginsenosides
Chemicol
Physicol Speckometric, includingcolorimekic ond fluorescence Biologicol
Rodioimmunoossoy {RlA)
Enzyme-im munoossoy
h
tro-optic efI'ectlthis involves the rottrtionof the plane of a polarized berm of light in a magneticfleld. the degreeoi rotation being proportional to the field strength.The instrumentis zeroedby meansof two solenoidsthroughwhich passesthe poladzedbeam.The insertionof etn optically active sollrtion betweenthe solenoidsaffords a recordable signal,either (-) or'(+). which is generatedby a photornultiplierat rhe end of the light path.
OF NATURALPRODUCTS RELATED TO THECOMMERCIALPRODUCTION,OUALITYAND STANDARDIZATION PRINCIPTES chromatographicsystemsemployed are listed in Table 14.21and are mole fully explainedin Chapter18. Spectrometricmethods.particularly in conjunctionwith chromatography.are finding increasinguse and are dealti.vithmore fully below.
onolysis Spectroscopic The electromagneticvibrationsutilized in spectroscopicanalysiscan be roughly divided. according to wavelength, into the ultraviolet (780 3000 ( 185-380nm). the visible(380-780nm), the near-infiared nm) and the infrared (3 ;t0 ptm)regions.In spectroscopicanalysiswe are concernedwith thc capacityof certain moleculesto absorbviblations at specitic wavelengths.Thus, the butenolideside-chainof cardiac glycosidesis responsiblefbr a sffon-qabsorptionat 215-220 nm, the conjugateddouble bonds of lycopene(a pigment of tomatoesand other fruits) give rise to the absorptionof light at a wavelengthof470 nm, thus -qivinga red colour. and the carbonylgroup of ketones,carboxylic acids and estersis responsiblefbr a stron-eabsorptionin the infrared at about 5.7-6. I ptm.In the ultravioletand visible regionsthe characteristicabsorption spectrum of a molecule is produced by changesin the electronicenergy levels associatedwith various chromophoric groups within the molecule. These changes involve the absorptionof relatively high amountsof energy (in precisequanta), and they are alsoacconpaniedby changesin vibrationaland rotational energychangeswithin the molecule.The resultis a bandedabsorption spectrum showing no sharply defined peaks. By cornparison.the absorptionspectrumof a moleculein the infraredlegion is much mote complex. becausehere the energiesinvolved are too small to bring about electronic transitionsbut large enor.rghto ploduce numerous vibrational and associatedrotational energy changes.Each of these changesis associatedwith a characteristicwavelengthand the spectrum shows a much finel structurethiin in the visible or ultraviolet regions.The inliared spectrumof a moleculecan be divided into the 'fingerprint' region (7 1l pm.),which is characteristicof the molecule underexaminationbut in which it is dillicult to assignpeaksto specific viblations,and the remainderof the spectrum.in which many firnctional groups can be recognized.The BP employs the comparisonof infrared spectraof phytochemicals(pilocarpine,physostigmine,etc.) w'irh European Pharmocopoeia Chenical ReJerenceStrbstances (EPCRS)as a test of identity. The BP uses ultraviolet absorptioncharacteristicsas standardsfot' benzylpenicillin,lanatosideC and a number of alkaloids-fbr examand tubocurainechloride. ple, morphine,reserpine,cocaine.colchicir-re If li-ehtof a particulal wavelengthis passedthrough a solution of a substance,the transmissionT= I/lo, where /iyis a measureof the light
Ioble 14.4
I
reachingthe detector(a photoelectriccell) when solventalone is used in the lighrpath and -l is the light reachingthe detectorwhen a solution of the substanceunder investigationis examined.f is measuredin experimentsbut the mostusefuivalue is log10(ry1),the decirnaloptical density or simply the optical density (E). The optical density.but not the transmission.is proportionalto the nurnberol absorbingunits in the light-path.For solutionsthis is Beer's law. The absorptionspectrumof a pure substanceunder detinedconditionsof solvent and temperature wavelengthsin a solutionof is a setof valuesof E observedat difl'erer-rt unit concentration(1 mol 1 1) when the thicknessofthe layer traversed b1'the tight is I cm. Alternatively.any other solutionof known strength nraybe used.Fol example,Ior a lo/cw/v solutionwith a layer thickness hy 6i.i ,. Su.t"rabsorptionspecof I cm the optical densityis inclicutecl tra are r,aluablefbr the identilication. determinationof the structure and purity and analysisof compounds.Soine substancesrvill absolb ultravioletlight of a certainwavelengthand duling the period of excitation re-emit light of a longer wavelength and often in the visible region.This is fluorescence,and the fluorescencespectrtlmis chalacThe appliwhich exhibit the phenot.nenon. teristic for thosesubstances analysisare discussedbelow. cationsof f-luorescence For the quantitativeevaluatiottof a substance,a standardcurve is the optical densitiesof a seriesof standard first preparedby measr.rring solutions of the pure compound by use of light of a suitable wavelength, usually that at which the compoundgives an absorptionmaximum. The solntionsmustbe sutTicientlydilute to obey Beer'slaw.The optical densityof the solutionto be evaluatedis thendetenninedand its compositionascertainedfrom the standardcurve. Individual componentsof a mixture can be determinedby ultravioletabsorption,provided that the difl'erent compounds exhibit di1l-erentabsorption tnarima. Thus, for strychnineand brucinethe reportedEffi" valuesat the wavelengths(),) indicatedare:
- l7c tl cnt
Stn'chnine 262 nm 300 nm
-1lZ
5.r6
Bnrcine
3\2 216
By measulementof the extinctionsof the mixed alkaloid solutionat assayis availthe abovewavelengths,a two-point spectrophotometric able lbr the determinationof both alkaloids.This rnethod,official in the BP (1980) fbr the assayof nux vomica seedsand preparations,
Chromologrophic systems employed in the onolysis of drugs.
Type
Employment
Liquidchromotogrophy; BPusessloinless steelcolumns of voryingsize;typicolpocking rophicoctodecylsilyl is chromotog silicogel Gos chromotogrophy
oil),Gorlic,Opium, sesome of fixedoils(e.g.Refined Devil'sclow,triglycerides Arnico flower
rophy ThinJoyerchromotog
profilesfor somedrugse.g. to oid distinciion Volotileoils;BPgiveschromoiogrophic ond thot lrom Pimpinelloonisum.Seporotionof foty beiweenAniseedoil from stor-onise ocidsderivedfromfixedoils testondtestforpurity.Seporoted Extensively usedin BPondBHPoson identificotion ond delerminedspectrometricolly con be removedfromchromofogrom constituents
a&"
- €nF'
QUALITY CONTROL replaced the older'. chemical method. A sirnilar type ol assay is employcdby the EP lor qLrinine-type alkaloidsand cinchonine-type alkaloidsin cinchonabark: nreasulernents aremadeat 3 I 6 and 3:18nrn. Occasionallfit is usefulto errmine the ultravioletspectnlnrof a more complexrrixture:thr-rs. the USPincludcsan ultlavioletabsorbance test for the absenceof fbrei-enoils in oils of lenronand orangeand the BP an extinctionlinrit testbetween268 and 270 nm fbr castoloil. In most casesit is essentialthat no ir-rterf'ering sllbstances arepresent duling thc measurements: thesecan be particr-rlarl,v troublesomcin the ultrar"ioletre-eion.particnlarll'with materialsextractedflorn thin-la1'cr and paper chromilto-{rams.For this leasor-r.if pure solutiousal'e not available ftrr anall,sis.sorre form of colorinetric anall'sis is olien pref'erable.particularly if the reaction used to producc the colour is highly specificfor the corrpoundr.rrrder considelation. Colorimetlicanalyses can be carriedout with a suitablespectrophotomet(]r-most instl'umentswhich operatein the ultlaviolet fange.also have tacilitics fbr work in the visible region-brlt lnr.rclrsirnplercolorineters in which suitablefilters are usedto selecttlre correct war,elengths ol light lequiled are quite satisfactolyfbr nrost purposes.In theseinstrumcntsa simplelighrsourceis usedand.betu'eenthe larnp and the solution to be anirlysecl. a filter is placedwhich tlansrrits that range of wavclcngths absolbed bv the conipor"rndunder test (i.e. a colourcomplementary to thatof the solutionundertest).The transmitted light is recordedby a photoelectliccell and the cornpositior.r of the solr-rtion detennineciby lef-crenccto a standardcurve. Characteristicabso'ption marima from the mole complex infialed spectracan alsobe utilizedin quar-rtitative analysisin the samerl'ayas ultravioletand visible absorptions.Mixtures of substances can also be evaluatedlthLrs.by measurements at 9.80,9.15and 9.00 ptmit is possible to evaluate separatelythe 258-and 25c-cpirr-relicsteroidal sapogeni nspresentin plantextracts. A few of the manyexamplesof thc applicationof spectrometric analysisto constitLlents of dlu-usaregiven in Table1.1.5.
For examination. so1lr1.s may be placed dircctly under the lamp. u'heleirslitluitls rnay be exantinedin non-fluorescentdishes or testtubcs ol after spottin-eon to tilter papcr.Nlany alkaloiclsin the solid state shou, distinct colours-tbr exarnple,aconitine (light blue). berberine(yellow) and emetine (orange).Piecesof cinchonabark when placeclunderthe lamp show a numberol lr-rnlinous yellow patclies turda f-ewlight blue ones.If the inncr surfaceof the balk is touched with dilutesLrlphuric acidthe spotimmedialelyturnsblue.Ipecacuanha root has a brightly luminous appearancewherever the wood is exposed.r'n,hile the u'oodof hydrasticsrhizomeshinesgoldcnyeliow. Areca nuts u,hencut sho$'a lisht bluc endospelnt.Slicesof calumba appearintcnsell'1'ellou,. with the carnbiuntand phloerndistinguished bi'their dark-greencolour'.Precipitatedand preparedchalks ntay readil1' be distin-uuishcd fi'ornone another. Most r.rl1^1, /ats and rlrr,resshow some fluorescencewhcn examined in frlteled ultlar,iolct light. In general.fixed oils and lats fluoresce least.waxesmore strongly,and mineral oils (pai'afTins) most of all. Pottder.srnay be examinedmacloscopicallyas abore or miurtrscopicallyby neansof a fluorescencc microscope. In connectionwith po'"vdereddlu-usrnay be nrentionecl the detectionofergot in flour. ofcocoa shellsin por.vdeled cocoa,and of rumex in powdercdgentian.Diffbrent r,arietiesol'rhubalb may be distinguished from one another.The BP 1973 rncludeda lluorescencetest on the entire or powdereddrug fbr the detectionof lhapontic rhubarb but this is now replacedby a TLC ICSt,
The location of separatedcompoundson papel ar-rdthin-layerchromatogralnsby tl-reuse of ultraviolet li-sht has bccn extensivell' emploled. With plant extractsit is often worthwhile to examine the chromato-eranin ultraviolet light even if the constitr.rents that one is inl'estigatingare not themselvesfluorescent.ln this way the presence of fluolescentinrpr-rrities rray be revealedn'hich,if otherwiseLrndetected. could interfele wilh a subseqr.rent absorptionanalysis.Sometimes fluorescence-qucnching can be employeclto locate non-fluorescent substanceson thin-layer chromatograms.Fol this, ln ultraviolet flr-rorescentback-{roundis proclucedby thc incorporationol a small amount Fluorescence qnqlysis of inorganicflnolescentmaterialinto the thin layer.Tl-resepalatedsubMany substancesfbr exarnple.quininein solutionin dilutesulphulic stancescausca local clucnchingof the back-9r'ound f-luorescence and acid when suitablyilluminated.emit light of eidift-erent wavelength the.vthcrefbreappearas dark spotson a colouredbackground. or colour fiom that which talls on them. This enrittedlisht (flLrorescence)ceasesr"'henthe exciting light is ren-roved. r.rtilizes the flrQuantitative fluorescenceanalysis, This techniqr.re Anall,ticaltestsbasedorr fluorescencein darlight are not nuch orescenceprodr.rced in ultravioletlight fbr quantitative by a compor.rnd used.asthey areusuallyunreliable.owing to the weaknessofthc fluorevaluation.The instrumentemployedis a fluorirneterand consistsof a escenteffect.An exceptionto this is the well known urrbelliferone test. suitableultrar"ioletsourceand a photoelectriccell to measurethe intenwhich can bc applied to ammoniacum.galbanum and nsalbetida.A sity of the crnittcdfluorescentlight. Within certainlirrits of concentrastron-{lyfluorescentsolutionof umbellif'cronccan be prcpalcdb-vboiltion the intensityof the f-luorescence tbr a givcn materialis relatedto its ing galbanumwith acid and filtering into an excessof alcoholicamrr-ro- concentration. It is usualto selecta narrowrangeof wavelengthsfor nia. Other fluolescentsolntionsare thosc of cluininc(in drlr-rteacid;. measLrrement by inselting a filter betrvecnthc fluorescingsolutionand aesculin(by infusinghorsechestrrr-rt bark),chlorophyll(from nettleor the photoelectliccell. The concentration of a substance in solutionis parsleyleaves).B-naphthol(dissolvedin alkali)andaqueoussolutions obtainedby ref-erer-rce to a standardcurve preparedb1'sLrbjecting standof the dyeseosin and flnorcscern. ard solutions to the fluorimetric procedure.With plant extraetsit is A l'erv importantgeneralizationmade by Stokesin 18-52statcdthat ir-nportant to asceltainthat (l) the substance being detclminedis the 'in fluorescence f-lr.rorescent light is alu'aysof greaterwavelength onlv one in the solutionproducinga fluorescenceat the mcasured the thantheexcitinglight'. Light ricl-rin shorlu"avelengths is r er'1actire in wal'elength and (2) therc are no substancesin the solr.rtionr,vhich producin,qfluorescenceand for this reason stlorrg r.rltravioletlight absolbIight at the war,clcngthof the fluorescence.Refinedinstrurnents (suchas can be obtainedfrom a tungstenal'cor Inercuryvapour lamp) arc now availablein which the fluorescencespectrumis automatically producesfluorescence in many substances which do not visibly fluor- analysedand in rvhichthc u'avelengthof the incidentradiationcan also escein daylight. Fluorescencelamps are usually tlttcd \\,ith a suitable be varied. filter which eliminatesvisibleradiationfiorrr the lamp and transnits Quinine can be convenientlvassayedby the rreasulementof the (366 nm) producedby irradiationofthe alkaloidin a ultravioletradiationof the desiredrvavelength. Convenientlong- and blue fluorescence short-waveultraviolet hand lamps are a'naihblefor chrorr-ratographic dilute sulphuricacid solutionat about450 nm. The methodcan be r.rsed obsen,ations;it is most important that the el'es are properly prolbl the assayof quininein the prcscnceof othel alkaloids(e.g.strychtected in the presenceof ultraviolet radiation. nine). Alcxandrian senna has been assal,edby measr.rrement of the
Fp
,ff,
E
PRINCIPLES RELATED TO THECOMMERCIALPRODUCTION,QUALITYAND STANDARDIZATION OF NATURALPRODUCTS
Table 14.5
Some excmples of the opplicotion of speciromefrir onolysis to the constituents of drugs.
Regionof spectrum
Constituents
Ultroviolet
Alkoloids: Lobeline Reserpine Vinblostine Vincristine Tubocurorine chloride Morphine Colchicine Cordiooctive glycosides with butenolide side-choin Soponins-g lycyrrhizinic ocid lridoids-horpogoside in Devil'sclow Quossinoids Cossiooil-oldehydecontent Bergomot oil-bergoptencontenl Flovospidic ocid frommolelern Copsoicin Vonillin Allicin-gorlic VitominA (cod-liver oil)
Visible
Alkoloids: Ergot{totololkoloids) Morphine Reserpine Tropicocid estersof hydroxytropones Anihroqui nones
C o p s o i c i inn c o p s i c u m
Infrored
Cordiooctive glycosides: bosedon digitoxose-moiety bosedon loctonering Ouoboin Cyonogenetic glycosides: {cyonidedeierminotion) Tonnins: Rhotony, homomelis leof (oscyonidinchloride) Procyonidins in howthornberries Vololileoils: Menlholfrompeppermint oil P r o o z u l e n(eossc h o m o z u l e n e ) in yorrow Flovonoids: Birchleof,colendulcflowers (hyperoside), elderflowers {isoquercikin} Alkoloids: Quinineond shychnine mixtures Steroidol sopogenins Volotileoils: oMethoxycinnomoldehyde in cossio il Woter
Wovelengthfor measurement of opticol density
249 nm 268nm 267 nm 297 nm 280 nm 286nm 350 nm 2 1 7n m 250 nm 278 nm 254 nm 286 nm 3 , l 3n m 290 nm 248 ond 296 nm 30,l nm 254 nm 328nm 550 nm by the useof pdimethylominobenzoldehyde reogent;532 nm by the reoctionwithvonillinin concentroted hydrochloric ocid 442 nn by the nitrosoreoction 390 nm by thetreotment of olkoloidwifh sodiumnitritein diluteocid 555 nm by heotment of olkoloidwiih fumingnitricocid followedby evoporotion to drynessond odditionof methonolic potossium hydroxidesolution to qn ocetonesolution of thenitrotedresidue(Vitoli-Morin reoction) 500 nm ofterheotment with olkoli(see'Senno leofB.P.'for thedeterminotion o[ sennoside; olsooloes{512 nm},coscoroond rhuborb{515 nm));530 nm for thecochineol colourvolue 230 nm ofterreoctionwith phosphomolybdic ocid ond sodiumhydroxide solution; 505 nm oftertreotment withdiozobenzene-sulphonic ocid in lO% sodiumcorbonotesolution 590 nm by Keller-Kilioni reoction(seep. 3OB) 620 nm by reoctionwith m-dinitrobenzene 495 nm by reoclionwith olkolinesodiumpicrofe 630 nm by thepyridine-porozolone colourreoction 7 15 nm usingphosphotungstic ocid ond sodiumcorbonotesolution(seeBP) 545 nm
500-579 nm (greenfilter)by useof pdimethylominobenzoldehyde reogenr 608 nm meosured on the blueoil obtoinedby distillotion
425 nm witholuminium chlorideond glocioloceticocid
6.2 ond6.06 pm 'l ,l0.85 pm; seefext 1.1 1 pm ond Meosurements ot 2.1B pm ond7 .62 trrmto distinguish borkoil fromleof ond twig oils 1.9 prm;foirly specificfor woterwilhoutinterference from other-OH groups
r
i*-
:V
QUALITYCONTROL producedin the Borntriigerreactionunderspeciliedcont-luorescence ditions.The hydrastinecontentof hydrastisroot may be determinedby oxidizing an extract of the drug with nitric acid and measuringthe fluorescenceof the hydrastinineproduced;by this n'rethodberberine and canadine,other alkaloidsof hydrastis.areexcludedfiom the assay. Emetineand papaverinemay be deterrninedfluorimetrically after oxidationwith acid permanganate and noscapineafter oxidationwith per.sulphate. Fluorimetric rnethods have also been published fol the estimationof the ergot and rauwolfia alkaloids. umbellif'erone,aflatoxin and a numberof drugsin body fluids. NMR spectroscopy Although this techniqueis usually associatedwith structure-determinations of organic compoundsthe use of lH-NMR spectloscopyhas beendescribedfor the assayof atropineandhyoscinein extractsofbell a d o n n ah. 1 o s ca1n t u sa n d\ l r a m o n i u m . lmmunoossoys Such assaysare highly sensitiveand usually very specilic and have beendevelopedas a powerful analyticaltool fbr the quantitativedetermination of many compoundsin biological fluids. Radioimmunoassays (RIA). The assaydependson the highly specilic reactionof antibodiesto certainanti-qens. Thereare variousmodilications ofthe techniqueand it is the saturationmethodthat has been developedfor phytoanalysis.Usually the relatively small molecr.rles (below MW 1000) constituting the secondaryplant metabolitesare not involved in suchimmunoresponses, but when bound covalentlyto protein carriers,as haptens,they do becomeimmunogenic.(Haptens are moleculeswhich combine with anti-bodiesbut do not stimulate their production unlesslinked to a carrier molecule.)If such a hapten is preparedin the labelledcondition(e.g.3H-or125l-labelled) with a known specificactivity. mixed with an unknown amountof unlabelled hapten and added to a limited amount of antibody in the fbrm of a serum,then there will be competition betweenthe labelled and unlabelled antigen for the restrictednumber ol binding sites available. This results in some bound and some unbound hapten;these can be separatedand a determinationof the radioactivity in either fraction. with ref'erenceto a standardcurve. enablesthe amount of unlabelled antigen to be calculated.The antiserumis raised in suitable animals (e.g.rabbits). Following the rapid developmentof RIA proceduresin clinical analyses,and largelyowing to the work of Weiier,Zenk and colleagues in Germanysince 1976,the methodhasbeensatisfactorilyappliedto a rangeofplant medicinalsas illustratedin Table 14.3. RIA has the advantagethat only small amountsof plant materialare required;it is usually specificfor a single,or small rangeof metabolites; relatively crude, unprocessedplant extractscan usually be used; and the processcan be mechanized.Thus. it is an efficient tool for the screeninsof large numbersof plants,sorne200-800 specimensbeing assayedin I day. For the applicationof the method to the selectionof high-yielding strainsof Digitali.r and Solanum,seeChapter 12. Wirh herbariummaterial,assayscan be performedon quantitiesof sample ranging fiom 0.5 mg to a few milligrams and in the examinationof
tsp
individual plants. strlrcturesas small as antherfilaments (e.g. in digitalis) can be accommodated. Possibledisadvantages of the method are the considerablespecialized expertisereqrliredto setup the iissaysand the possibilityofcrossreactionswith contponentsof the plant extractother than thoseunder investigation.Problemsarising from the latter needto be ascertained befbrethe assay.The RIA for hyoscine,fbr example.is highly specific but norhyoscinewill reacteven mole strongly;the cross-reactionwith 6-hydroxyhyoscyamine is considerablyless,and with hyoscyamine, very much less. Similarly. in the assayfbr solasodinc,tomatidine,if present.will cross-react. Enzyme-linked immunosorbent assays(ELISA). In this method. cornpetitionfor an imrnobilized antibodytakesplace with a modified fbrrn of the compoundunder analysisthat has an enzymebound to it. Releaseof the compound-enzymecomplex from the binding site and detenninationofthe enzymeactivity enablesthe original solutionto be qLrantilled. Examples of applicationsto medicinal plants are given in Table 14.3. As with RIAs, the n.rethodis very sensitive; thus, for the pylrolizidine alkaloid retronecineit can be measuredin the palts per billion range and one sclerotium of ergot is detectablein 20 kg of wheat. Tqndem moss spectroscopy (MS-MS) In phytochemistryto date. massspccrroscopyis usually associated with the structureelucidationof compoundsratherthan with their assay.However. by the simultaneousr.rseof two mass spectrometers in seriesit is possibleto determinequantitativelythe amount of a particular tafgetedcompound in conrplex mixtures, plant extractsor even in dried plant material.Plattnerand Powell in their report on maytansinoididentification(J. Nut. Prod.. 1986.49.475)refer ro it as an irnportantanalyticaltool for'needle-in-a-haystack' analytical ploblems. Sensitivityto picograntsof targetedcompoundscan be achievedwith high specilicity and nearly instantaneous response; for sensitivityit compareswith RIA but is much more rapidly perfbrmed. The rnethod has been used fol the analysis of cocaine in plant materials.pyrrolizidinein Senecioand other genera.taxanes from single needlesof Ta.rLrs cuspitlutu, aflatoxin B1 in peanut butter, xanthones.steroidsand antibiotics.Hoke et al. (J. Nat. Prod., 1991,57, 277) considerit the best overallmethodfor the determination of taxol, cephalomannineand baccatinin T. breti.folia bark and needle extracts.The chemotaxonomyof the Cactaceaehas been investigatcdby this method. microscopy Quonfitolive Powdereddrugs or adr.rlterants which contain a constantnumber, areaol length of characteristic particles/mg(e.g. starchgrains,epidermis.trichomeribs respectively)can be determinedquantitatively by rnicroscopyusin-qlycopodiumsporesas an indicatordiluent.The method, formerly official in the BP, is describedin Chapter4zl. Further reoding WagnerH, Bladt S 1996Plant drug analysis.2nd edn. Springer.Berlin
rllr :lli:lrllll'r,l:l:.ir tlllillulll r.llllilil u:iut,; .llll:lrlill j:..,.t::.,.,,:,1],.,.,::l,,at:,), a,t::..,i
Plont products ond HighThroughput Screen ing M. J. O'l{eill and J. A. Lewis
TRADITIONAT AND ORTHODOXMEDICINE Tladitionalremediesi rrl'ariablf involr,ecrudeplant extractscontaining multiple chemicalconstituents, which vary in potencyfi.om highly active(e.g.Digitulis leaf) to very weak (c.-9.cinnantonbark).In contrast,ofihodox rredicine reliesheavily on single(or a very small number of) chemically well-characterized active ingredientsexhibiting selectiveactivitiesat. in many cases.well-established biologicaltargets.Thesemedicinesale generallyvery potentand many exhibit fair-11'nan'ow windows betweenan efl-ectiveancla toxic dose. Orthodox peclicinesare fbnlrulateclinto dosesthat arecarefully standardizedfbr. bioavailability. Amongst our ntost invaluable orthodox medicines derived from cornpoundsin higher plants are analgesicagents(e.g. morphine and codeine),antimalarialtreatments(e.g.quinine).antitur.nour drugs(e.g. vincristineandtaxol)andasthmatherapies (e.g.cromoglycate). An analysisof the top-sellingpharmaceuticals fbr 1999showsthat l3 of the top-selling30 nredicines, with combinedsalesof over $26 billion.haveactiveingredients whoseclrernicalstructurcis basedon a compor.rndlbund originally in Nature (Wood Muckenzie's PlturntuQuaril,2000).In somecases.naturalInaterialscontinueto be the only viablecommercialsourceof the activecompound.For exanrple. Glaxo Wellcomeharvestsup to l0 000 n'retlictonsdry weight of poppy capsulepef yearto providea sourceof opiatealkaloidsandits contractors producearound170r.netrictons/annunDiqitLtli.tfulrrrrra leaf. asthe sourceof digoxin.
HIGH THROUGHPUT SCREENING A5 A ROUTETO DISCOVERING NEW MEDICINES IN THEPHARMACEUTICAT INDUSTRY In order to discovernew medicineswhich ofI'el significantadvantages over existin-9therapies,a key starting point is to identifi, nor,el biological targetsthat have a critical role in controlling diseaseprocesses. The next stepinvolves creatingbiological assayscapableof cietecting substanceswhich car-rmodily the activity of the tar.get.These 'lead chemicals'are compoundswhich are amenableto chemicalmanipulation in order to optimizetheir bioactivity and bioavaiiabilityprofile, or.
Toble l5.l
TRADITIONAL AND ORTHODOXMEDICINE
r09 HIGHTHROUGHPUT SCREENING AS A ROUTE TO DISCOVERING NEW MEDICINES IN THE PHARMACEUTICATINDUSTRY I09 ACCESS TO PTANTSAND OTHERNATURAT S O U R C E M A T E R I A LISI I DEREPTICATION AND ISOIATIONOF ACTIVE COMPONENTS I I3
-F"
Top-sellingmedicineslor 1999 in gbillion.
Omeprozole Simvostotin Atorvostotin Erythropoetin Pravostatin Amlodipine Fluoxetine Lorof idine Enolopril Sertroline Poroxetine Olonzopine Coniugotedoestrogens lnnc^^.^r^lo Amox-clov
5 .8 5 4.37 3.68 3.68 3.26 2.98 2.93 2.82 2.24 2.09 2.01 83 B3 80 69
Ciprofloxocin Risperidone taxot Celecoxib Cyclosporin Losorton Clorithromycin Hum.lnsulin CSF Azithromycin Ceftriaxone Lisinoprol Sumohipton lnterferon Nifedipine
64 47 44 43
1.42 36 32 30 2l |.21 .20 .09 .09 .09
Thosein itolicshaveo structurebqsedon o compounddiscoveredin nolure
,:.ll11 ,l.1.,: r]l]'t,l.ltl
itrl,tl:,ri.li , i l , i lili ' i , l ' a
irr::itliltllr
rl:rl:ii.lr.riill
ilE
S O M EC U R R E NTTR E N D S much more rarely,arecompoundswhich aredevelopableascandiclates in their own right. 'High The ThroughputScreening'(HTS)processis now perhaps the principalstrategyfor the cliscoveryof ner.r' lead chcr-nicals in the pharmaceutical industr'.v. HTS usesminiiilurizedassayfbrntats.usually rniclotitle platcs in which. lor example.38.1ditltrent samples can be assayed.at less than 50,uL total assayvolulne. ln onc rlln. Using sophisticatcdautornrtiorrecprpnrent.tlpicully. hundredsof thousandsof sarnplcsare screeneda-gainst each biologicaltargetof interest:the final numberslbr cach usually being dictatedb1' the overall cost of the assay.u,hich can vary flont <0.1 p per well to > f 1 . 0 0p e r w el l . HTS is often portrayedb1,'people n,ho knorv little about it. as an activityrequilingvely little intellectualirrput.The lealityis thatHTS is a complexprocesswhich demandsan understanding ofthe rolc ofspecitic biological talgets in diseaseprogression.the der,elopr-nent of bioassayscapableof discor,eringmodulatorsof the target.the clcsign. nriniaturizationand automationof bioassavswhich ule iiutonationfriendly,an understandingof the macro-and rnicro-stmctnreof the biological target so that the sample selectionstrategyis optimized. the en-einccringof custom-built robots capableof skrrase.retrieval and bioassayof millions of samplesper annum and the developmentof sofiwaresystenswhich can enablescientistsinvolvedto nake scnse of the massof datathat emcrges.
I I
Biologicol qssqys ond High Throughput Screening ldcal biologicallssaysfor screeningit1'e thosewhich enablciclcntilicationof compoundsactingon specilicbiologicaltargets.involve a nrinimun number of reagentaddition stcps,pcrform reliably and predictably.alc easily amenableto miniaturizationand automation. and involve low-cost ingredientsancl detection technology. Biochemical targets of interest in pharmaccuticallead discovery range liom enzymesto receptors(nucleal and transmcmblane)to ion channelsar-rd. in the caseof infectiousdisease.to whole microorganismcells. An exantpleof a biologicalassayu'hichhasthecharacteristics needed in a good screenis the squalenesynthaseenzymeassav.which wirs developecl to look fbr inhibitor: of squrlr-ncsvntlrr-\i\.a potentii-iltal'get for the identilicationofnovel cholestcrolIowcringagcnts(Tait. 1992). Using either [1-14C] isopcntcnylcliphosphatcas a prccursorfbr squalcne or 12-l.lCl falnesyl diphosphateas a direct substrateof squalcnc synthase.the procluctionof ladiolabelledsqualeneis clctclrrir-red after adsorptionof assaymixtlres onto silicagel thin-1a1 er chlontatoglaphy precursors sheetsand selectiveelutionof the diphosphate into a solution of sodium dodecyl sullateat alkalinc pH. Thc use of [2- l 4Cl farnesylcliphosphate. and of an endogenous oxygenconsunrltion\) \tenl (ascorbatc/ascorbate oxidase)to preventfurthel metabolismof sclualene.allows the methodto be appliedas a dedicatedassayfor sqr.ralene synthaseactivity.The assaycan be reaclilyoperatedin microtitrc plate fbrmat.rr'l-rich allo*'s 96 or'38;1sarnplesto be screenedper plate.It may be dcploycdcithcrin a quautitilti\e.low-throughput modeor in a qualitative. hi-eh-throughputrrode. which has prorecl to be resistantto interf'erence b1'cornpoundsother than selectil'einhibitols. The endogenousneulopeptidebradykinin (BK) is irnplicatedin the rnediationof various typcs of pain in thc mammaliancentral nervous system.Antagonismof bradykinin to its rcccptorsis a potentialtarget for the developrrentof new analgesicagents.An assa.vhas been devised to detect compoundsrvhich antagonizebinding of racliolabelledbladvkinin to BKll receptorsexpressedin Chinesehamster' ovary (CHO) cells (Sarnpson et ul .,20001.Componndsuncleltestare added to the wells of micrditre plates to which CHO cells har"e adhered.Afier incubatiorrr.r'ithradiolabelledbraclvkinin.the excess
labelledligandis rerrovedby washing.The platesarethencountedin a scintillationcounterso as to assessbindingof labelledbradykininto the receptcxsexpressedon the sulfacesof the cells. This particular sct'censuf'fersinterf'elencetiom compoundswhich possL-s\cyt()toxicity thlough a variet,vof mechanisms.It is thcreforeessentialto run fbllow-np controlassaysa-qainst other ccll tvpesin order to distinguish falsepositives. In the infectiousdiseasearenait is still comntonto run high-throu-qhput. w'hole-cellantifungal or antibacterialassays.in order to detcct samplesrvhich inhibit growth of the designatedstlain. e.g. Candidtt alhicuns. Stapltrlocot:cusolrreus.Optical density or colour changes using a ledox indicator are the most liequently used assaytechnologies. Assays in this ther"apeutic area may be ntechanisrrbased.For example. a Ctuulitla ull.titcu't.s cell-fiee translation s1stem using polyurethane as a synthetictemplate.has beenestablished in order"to searchlbr compoundswhich inhibit fungal protein synthesis(Kinsn'ran et al.. 1998't. Somple ovoilobility for HTS Dr.u'in-e the 1980sanclearly 1990s.natural prodr,rctsampleswel'e the mainstayof HTS pro-qrammes within the pharmaceuticalindustry.due at leastin palt to the lack of availability of lalge numbersof synthetically derivedchemicals.Over recentyearsthis sitr.ration has changcd dramaticalll'.The highly competitivearenaof drug discoyelyproyides pharmaceulicalcompanieswith a clearincentiveto be first to discover and patent new lcad molecr.rles. Thus. a range of technologieshas evolved to facilitateever-increasingnumbels of samplesto be rapidly gcncratedtlnd evaluated. phalmaceutical Most lar'-ec houseshar,ebuilt up a compoundbank containinghundredsof thor.rsands of chemicalclasseswhich leflect the chcmistlies of earlier medicinesdevelopedby the company. Chemical dir,crsity in these collectionscan be supplementedby acqr.risition of new compoundtypesfi'om growing ranksof specialist compor.rnd vendors.Cornputational modellingtechniqr.res ale utilized to generatesets of specitic interest fbr -riven biological targets. Methods are available fbl electror-ricallyfiltering out 'undesirable' compoundsand techniclues suchas pharrnacophore analysis.2- or 3dimensionalstmcturesearchir-rg or chemicalclusteringcanbc usedto delive setsofthe requiredsize.Readyaccessto thesecompoundcollections arc now facilitated by use of lobotic storage and retrieval ftrcilities which can present the sampiesin ibrmats applopliate for' HTS bioassays. Cornbinatorialchemistryis now widely iipplied in the drug discor'ery processespeciallyfor generatingliirge numbersof cornpoundsftrr lead discovery and in the optimization of lead compounds.Llsing robotic systems.tens of thousandsof compor.rnds can be synthesized fiom a small number of rea_qents in a f-ewdai,s.To date. however.it appearsthat the most successfulof theselibrar"ies. in terms of yielding interestin-e bioactive molecules.have utilized focused chemistry basedon strlrctLrral knovn,ledge of the biological targetand the pharmacophoricf'eatures requiledto effect it. In oldcr to supplementthe chemical diversity of the compound banks and the chemically-fbcusedcombinatoriallibralies. a number of pharnaceutical companies continue to scleen natural extracts. Historically. large collectionsof microbial olganisms (notably lungi arrd lilamentous bacteria)were built up fiorn a divelsity of environmentalnichesandemphasiswasplacedon the development of a range of f'elmentationconditionscapableof elicitin-ethe microbesto produce a variety of secondaryrnetabolites.The extractsgeneratedfbr HTS tiom this sourcecan be reproducedon demand.shoLrldfurther stuclies on bioactivity of interest be required. In particular.indLrstrylbund miclobial f-ermentations to be a prolitic sourceof antibiotics.More
P L A N TP R O D U C TASN D H I G HT H R O U G H P USTC R E E N I N G recentiy. f}om the same source.r'aluable immunosuppressant drugs and lipid lowering agentshave beenacldedto the medicinechest. Plant samplesalso f'eaturein the HTS programmesof a nuntberof pharmaccutical discoveryorganizations. The feasibilityof usingplants in a drug discoverypro-qrammedependsupon ensuringthat efl'cctive procuremcntstrategiesare in placeto sollrceboth the pdlDaryrraterial and additionalsuppliesshouldthesebe required.
might addresssuch issues.The policy recognizesthe importanceof mattersconsideledat Rio and subseqr.rent meetingsof the Congressof the Particsand goeson to statethat Glaxo Wellcomewill only collaborate with organizatiorrs who can demonstrateboth the expertiseand the authority to supply natural materials.Only lelatively small quantities of plant materialare collected.fiom sustainablesources.Endangered specieswill not be accessed knowingly.A materialstransferagreement is drawn up which ensurescollectorsarereimbursedfbr cost of collection and fbr their expcrtise.but u'hich also specifiesa financial benefit irr the event of commercialization.The agreententlequires that a significant poltion ofthis royalty paymentis returnedto the sollrcecountry to slrpporttrainin-tand edr.rcation at the community level. Milestone paymentsat valious stagesof the drug developrnentprocessmay also be incorporated.
Selecting somples for screening Advances in screening technology have increasedthe throughput capacityof an averageHTS fiorn tensof thousandsof samplesto hundredsof thousands of samplesover the lastdecade.Even so.the availability of so many sarnplesfol HTS meansthat choicesmay needto be rnadeaboutthe most appropriatesub-setof samplesfbr eachpalticular' target.The sampleselectionstrategymay thenbe 'diversity-biised'. i.e. samplesare chosento representas u'ide a spcctl'umof chemicaldiver- Strotegies for the selection of plont mqteriql for HTS sity aspossible.or 'fbcussed'.i.e.the sarnples representspecificchem- Befbre a decisionis macleon what naturalmaterialswill be evaluated ical typesonly. in a given screen.it is essentialto gathersomeinformationon whether Both strategiesarc likely to play a role in a pharmaceutical cont- a targetis indeedappropriatefbr input of naturalproduct extracts.For pany's n-rethodology. Diversity scleeningmay yield arr unexpccted examplc,ifthe biologicaltalget is verirhighly tractable.ifthere are siginteractionbetweena compoundand a biologicaltarget.althoughthe nificanttime constraints and costof goodsissues,trndif datasuggests questionof what constitlltes'representative' chernicaldiversityin the it is likcly to be relatively straightforwardto obtain synthetically vast areaof potentialchemicalspaceremainsnnanswered.Focused delived. small moleculelead compoundsthen it rnay be inappropriate screeningrequiresa large amountof priot'intbrmation about a target. to screennatufal productsagainstthat target.However,if a targetis of and this may not always be available.A cornbinationof both a classn'hele it is difficult to find sn-rallmolecr.rle hits, e.g.pr()tein-proapploachesrnay be adopted. Computational methodologiestbr hit tein interactions.or if thereis a strongprecedentor rationalefor niitural idcntification are continuously being devcloped. For example. procluctderived actives.then natural product input should be considcompound databasesenabling 3-dirnensionalchemical strLlctLlre ered.The latter may be exernplifiedby. for example,the antimicrobial searchingare often used.If there are known li-gandsfbr a targetthese area.wheLethe tlack recordof dlug discoveryfrom microbial sources can be usedto constructa pharntacophore. which can thenbe utilized is beyonddispute.The samerationalewould apply to the superbtrack to search flrther chenrical databasesand select molecules with recordof plantspecicsin yieldin-qanalgesic medicines. desiredfeatures.Chemicalclusteringcan be usedto derivesetsofthe Ifthe targetappearsto be suitabletbr naturalproductinput, the samrequired size. In the ctrse of combinatorial chemistry-derived ple selectiontbr the screenneedsto be considered.There are various libraries. targeted sets can be generatedwith desired chemical strategieswhich may be adopted.dependingon the extentof the availproperties.by Lrsingappropriatelyselectedchemicalbuilding blocks. able naturalmaterialscollections.and the capacityand 'roblrstness'of Naturalproductsoffer a potentiallyinfinite sourceof chemicaldiver'- the targetitself. sity r.rnmatched by syntheticor combinatoriallvderivedcompound Many companieshave accessto large and diversenaturalmatelials collections(Strohl. 2000). thus makin-ethem a desirabletool for collections.Suchcollectionsare likely to includesamplesacquiredin diversitybasedscreening.If a focusedstrategyis adopted,however. order to add diversity to the potential collection (and inherent in the it is necessaryto develop difI'erenttechniquestor natulal product dcsile fol taxonomic diversity is the assumptionthat this will be sample selectionin order that the rrost appropriatesamplesale ref-lectedin chemical diversity of extracts subsequentlygenerated). accessed for relevanttargets. Most collectit'rnswill also include samplesparticularly selectedlor various reasons.e.g. ii rrricrobialproducel of a given compound or a plant r-rsedethnomedicalli'fol a given condition. These lar-eecollecACCESS TO PTANTSAND OTHERNATURAT tions plobably still only reflecta fiaction of the world's potentialbiodiSOURCEMATERIATS versity. It has been estirratedthat only around 70.000 fungal species are known out of an estimatedI .500.000(Hawksworth,1991).More The United NationsConventionon Biodiversity(CBD) in 1992has recently.it has beenestimatedthat only about 17cof microbial biodinow beenlatitied by 17,5countries(Ten Kate and Laird. 1999).The Versit), actually comprises 'culturable' organisms (An-rmanet 41.. key objectivesofthe Conventionale to ensulethe conservation ofbioI 995). Thus. a huge numberof strainsmay not be amenableto convenlogical diversity.the sustainablcuse of its conponents and to imple- tional isolation and cultivation methodologies.and many groups are ment tair and equitablesharingof benefitsalising f}om its use.Within now working on applying cloning techniquesin ordel to harnessthe the frameworkof thc Conventionarethe conceptsof the sovereigntyof potentialchemicaldiversityof theseorganisms. statesover geneticresourcesancltheir obligation to tacilitate access. A diversity-basedapproach requires acquisition of pre-selected The contractingpartiesale expectedto esttrblishmeaslrreslbl benefit taxonomic -qroups.The stlategymiiy utilize the assumptionthat taxosharingin theeventof commercialutilization.This involvescollaborii- nornic diversitywill inherentlybe reflectedin the chemicaldiversityof tion betweenthe collector.the sourcecountry and the industrialpar! the extractssubseqr.rently preparcdand screened.Various techniques ner. It is now nolmal practiceto draw up a le-ealagreementto cover can be emplol,edto analysethe taxonomic spreadof a plant collection theseissuesand r-nan1' companieshaye issuedpolicy statelents relat- and then rnake ellbrts to fill gaps in order that the collection more ing to this area. completelyreflectsavailablediversity. As an example.the Glaxo Wellcomc policy fol the acquisitionof A more focr-rsedapproach depends on having prior knowledge natural source matclials illr.rstrates how a pharmacer.rtical conpany aboutsclectedsarrples.which rnay suggestthat they containparticular
-,
r
S O M EC U R R E NTTR E N D S chenricalclassesol'interestor that rhey possessclesirable biological properties.This stlatcgl''can be consideredunder two headin-es 'chen'rical targeting'and'biologicaltargeting'. 'Chemical targeting'utilizesnaturtl ntaterials as sourcesof specific compor-rnds of intcrest 1o a particlllar diseasearea. or as sctuLces of chernicalclasses deerned.to havcsuitablepharmacophores. In this uay. cher-r-rical typeswhich are under-r'eplesented in an existing samplecollection can be identifled. Plant-der"ivedchemicals can provide an efl'ectii'emeansof tilling an1, 'gaps'. thereby enhancingthe or..elall diversity of available chemistrl'. It mav not alwavs be necesslrryto expendresoulcetlkin-ethisprocessto full isolationandstructure elucidation.ln somecasesa set of plantscan be selectedon the basisthat they are reportedto producea genelal chemical class of interestand appropliatecrudeor semi-purifiedextractscan be preparedin older to enrich thc extractwith the desiredchemicalt1,pes. 'Biological talgeting' adoptswhat rnay be rhought of as a diseasedrivenprocess.Plantsarnples canbe selected fbr biologicalevaluation usin-qsome t-{pe of infbrmation associatedwith thcnt that sllggcsts their relevancefbr evaluationin a given thelapeutictal'get.Pcrhapsthe nroststliking observationsavailableareethnobotanicalreportsof traditional medicinal uses of plants.A nurrber of orthodox medicines availablecommerciallytoday were discoveredby tbllowing leadspro(Cox, I99;1). videdtl'omindi-genous krrou'ledge
I
pl'ocessof de-r'eplicationand compound idc.ntil'ication(see later). In addition.searches rnavbc carriedout on a rangeofother fieldssuchas nroleculalweight-also useful in compoundidentiflcationfbllowing identitication of a molecularion by massspectrometrv analvsis. reported usesof the nirturalpl'oduct.literaturclel-erences. log P.etc.The databasc comprisesnot just plant metabolitesbut contpt'runds fiom all natural sources.anclrvhilst tl-redata is not corrplete. it ciin representa valuablestafiingpoint by r.vhichto build chemicallv-focused sersof srnrplesf or scleening. In the caseof preparir-rg sampleswhich might be expectedto contain spccific chemical entities.phvtochemicalprocedut'esreportedin the literatr.femay be used in ordcr to qenerateserri-puritied extracts.or extractsenrichedin the compoundor chemicalclassof interest.ln the caseof plants r.vhichl-nve arr ethnontedicaluse. it r.r.rav be possibleto prepareextractsusing methodologyas recommended1br use in traditionalmedicine. These'targeted'approaches arelikely to involvesmallernurrbersof plant sau-rples than a higl-rthloLrghput.randont screeniugpr(rgranlne. Hou'ever.the actual nuntbersof plants selectedand sct'eenedcan be tailoledby the scientist.by makingthe selectioncriteriarnoreor less stringcnt.For example.a selectionprocessmay resultin a smallnumber of plants reportedto ploduce specificcompoundsof interestbr.rtif the assayis capableof screeningmuch largernnmbersof sarnples.the set can be extcndedto include all thoseplants reportedto produce Process for identificotion of plonts for forgeted sets metabolites of themuchbroaclelchemicalclass.Similarly.a setmay be A varietv of approachesare adopteclin assintilatingthe infbrn-ration extendedtaxonornicallyto an optimum size by including plantsof neededto selectplants of particularlelevancefbr a given diseasetar'- relatedspeciesor genera.on thebasisthattheyntay alsoploducerelatget. Some rcsealch gror.rpslely on der,elopinga network of ethno- ed chenristry. Making the selectioncliteriaslightll,rrore'fuzzy'in this botanistsu'ho wolk closely with indigenouscolleagr,res and traditional manner also allows a greirterrole fbl the element of luck-always doctolsin variouscountries.The outcomeo1'thisapproachis a low importantin the drug discoveryproccssl numberof plant samplesidentitiedfbl evalnationin tl-relaboratoryand a great deal of intbnnation on their r-rse. This is the type of stlategy Somple prepqrqtion adoptedby. for exarnple.ShamanPharntaceuticals. u'hose stafThave Pleparationmethodsiu'etailored towat'dsthe typc of natural material worked alongsidcindigenousancllocal peoplesin tropical r'ainforests. bein-eprocessedand the strate-sylbr anall'sisbeing undertaken.For Shamanhave developedvilri()r-rs rer-iprocitvpfograntmesin order to plant sarnples.the standardapproachis to acquileand storedried plant acknowledgethe intellectualproperty rights ol the peoplewith whom material.In lale cases.fol example if an ethnomcdicalrepolt dictates theywork (King andCarlson.1995). useof fl'eshmatel'ial.then this may be undertaken.but would not be the Some pharmaceuticalcompaniespref-elonly to use infbrrnation nonl. Although a lery small amountof plant material-less than half a which is alreadyin the public domain.Variousjournalsand bookshold gram-may provide sLrfficientextract to allow testing in many huna significantamountof infbrmationrelatingto the ethnobotanicusesof dredsof bioassays,it is only sensibleto collect a larger antountof r,aLionsplants,fbr examplethereare many publicationsdescribingthe material.For naturalproductsamplesto remair.rcornpetiti\iesourcesof propefiiesof plants usedin ChineseTraditionalMedicine (e.g. Chang lead compor.rnds. it is necessaryto be able to very rapidly fbllow up any and Btrt. 1986).Perhapsthe mosl time-ef1-ective way of searchingfor' active extracts.For this reason.collection of a few hr"rndred grams of infbnlation relating to reportedbiological or chemical propefiiesof dry materialis more typical. Plant materialis finely ground r"rsirrg techplantsis to useelectronicdatastoredin a rangeofdatabases. One key niquesrangingtiorn pestleand mortar to industrialglinding apparatus exampleof this is the NAPRALERT (NaturalProductsAlert) database as appropriate. (Loub cl a1..198-5). This svstemwas initiatedand is maintainedat the Microbial stlain collections at'e maintair-red either as fieeze dried Univcrsityof Illinoisat Chicago.It containsa wealthof intbrmationin culturesor are preservedby low tentperaturestorage.Requiredstrains the tbrm of a hu-{enurnbelof ref'erences lelating to reportsof biologic- are -eenerallyrevived by inoculatior-r onto a-qarand/or growth media, al actrvityin thc scientificliteraturc.ethnobotanical reportsarrdphyto- and then ale cultivated 06 3 mscliunl-or. rrrore ofteu. a range of chemicaldata. media-which have bcen cler,eloped u'ith the aim of prontorirrgseeFor cherlical infbrmation,databasessuch trstl-reChapmanand Hall ondary metaboliteploduction. Factorslikc incubationtimes and temDictionar'1' of NaturalProductscan be usefultools (Chaprnanand Hall. perature.media composition.rgitatitrn rates. ctc. can all have a 2000). This databasecontainsinftrrmationon well over 100 0(X)nat- signilicantefl'ecton glorvth and n-retabolite synthesis. ural proclucts.often including the speciesfi'om which the compou-rd The next step is to generatean extract fbr antrlvsisin a range o1' oliginates.Sealchesrnay be carriedout on the basisofchemical struc- bioactivity screens.Ofien. there will be no pliol kr-rowledge of rvhich ture. sub-stnlcture.stluctural similarity, presenceof particular func- chemicaltypesmay be presentin the samples. andwhich will be active tional gror.rps.etc. in ordel to build a set of organismsreported to in any -eivenbioassay.Techniqueswill thelefbrebe r-rsed aimcd at soluproducethesecomponnds. bilizing as wide a rangeof compor.rnds as possible.typicallyusingan Altematively. the databasemay be sealchedon the basisof species alcoholicsolventsuclras nethanol or ethanol.or an aqueousalcoholic or genus!in order to build a list of compoundsreportedto derive fl'or-n solvent. Extraction mair take the forrn of a cold infLrsronor it mal particular organism groups. This can be particularly uselul in the involve hot solventextractionusing a Soxhletapparatus (Silva er a1..
& n-'
P L A N TP R O D U C TASN D H I G HT H R O U G H P USTC R E E N I N G 1998). If the natulalsourceIrlaterialis undel investigationbecauseit preparativeHPLC. An isolationmethodu,'ouldnon.rully'involve solhas beenreportedto containa desireclcontpctlrud or chc'micalclass. vent partitioning.fbllowed by a crude chromatographystcp such as a thena bespokcextractionproceclule llill be aclopted. plobablyusin_u a silica colr-rmnor coLtnter-cu1'rent chromatograph.vn'ith a rclativel;litelatr.rerepo't. lf the titfget rnoleculeis less specific.fbl example if small nurnberof fl'actionsbasedon polarity.tblloweclby final puriticathe ainris to accessan.valkaloidnrolecules u,hichmay be plesent.then tion througlr a high-resolutionsepalatioustep such as HPLC. At each alkaloid-enricheclcrtracts nray be generatcclb1, following a mctl-rod sta-ue of the purification.the activecompoundis trackeclbv bioassal,of r u c l ta : r e i d - l r i l \ ep i r t t i t i o l l i n S . the fractions.The onlv sLrre\\,ay'toidentifl the structLrre of a bioactive Arr alternativeapproachrnay favour thc del'elopntentol fraction- metaboliteis to demonstrate activityusingthe isolatedcompoundand irtion methods.ir.rolder thirt severalfi'actionsoriginatingh'orna singlc then to determineits stlr-rcture bI NMR and NtlS.Until a cornpoundis prior to biological testing.Althou-9hmore labor.u' isolated.it is alsoin-rpossible samplcare -eenerated to deterinineits concentration andhence. intensir,c.this can have the benefitof eliminatingrnan1,'ofthe prob- its potencyin a givcn assay.lt is alsopludcntto checkthatthe concenlems r,vhichrnay be secn when testing crucleextractsin sor-nebioas- trationofthe metabolitein the extractand the activity in the Lrnpurified says-e.-9.fiecluentactivescausedby cornmonlyoccun'ingintel'fel'illg extlact tally. so that a nrinor but .tctive component does not go compoundssuchastannins.saponins. llavonoicls. etc.Suchactir,.es can unaccollntedfbr. Seconclarvtesting r.r,illthen be undertakenon the take a significanttime to dercplicateanrdeliminate,anclin somecir- active compound to deternrinethe mechanismand the selectivity culrstancesit ma1'be desirableto reducethis resoulceby spenclin-e of action and evcntually.to el'aluateul r,rlo actir,ity.The cherrrical tirr-rcbelbre biological testingto do sorneserli-puriticationof extracts. structureof the componndrvill also be evahtatc-d to give sor-neindicaTechniques usednrayincluclesolr,entpartitioningor chromatographic tion of tl-reclassical'drug-like'clLralities of thc molecule.Thcse are flactionation. to sonreextent subjectivebLrtinclude consicleration of rvhetherthe compoundis of suflicientlvlow molecularu'eight to allow ready chcmictrl nrodification.and not pre\rentclrue upltrke:of whcther the DEREPLICATION AND ISOTATIONOF compoundis likell' to be stableu'ith respectto oral uptake.whetherit ACTIVECOfVIPONENTS has sitesthat rnay be suitablefbr modification.u,hetherit is likcly to hnve a suitable lo-s P. and so on. In fact. sor.ncclassesof natural Biologicalanalysisis likell'kr f ield a nur.nber of actire c\tri-lcts.as productsare not suitableas dmg candidatesby thcsecritelia, as thc1,' definedbv showir-rg a cerlain level ol activity in a -uivenscreen.A ale too bis and compler or possess unsuitableledor properties.I1-the processthen needsto beginrvhichwill eitherleadto a full idcntifica- molecule is cleened to be of interest.related ntetabolitesfl'om the tion of a tirlly chalactelized. actil'ccompound.or partialidentiflcation sample.or feliitedspecies.mav be accessed lbr stmctureactivitv detcl'of actir"ityto the levelof a famill' of knou'nconrpounds. rnination.Er,enif the structureofthe ntetabolite is not novel.this does Befbrecommencin-sfllll bioassiryguided fiactionationof the active not precludeit becominga leaclmolecule.particularlyif the mode of salnplesit is necessarv to revie'nv thetoleranceof a gir,enassavto crude actiOnis novcl. or serni-pulifiedextrrictsof natr-rralrnatcrials.l'he airl o1'evaluating such samplcsin a biologicalassayis to identify conrpoundswhich irrteractu,itha particularbiologicaltafget.e.g.iln enzvmeol'rcceptor. A recent exqmple of the success of High Throughput Horvever.in practicc.mostassa.vs Lrtilizea measurable svstemsuchas Screening of plonts for new leod compounds colour.light or ladioactivityenablinga hi-ehthroughputof sarnples. The discovery of a seriesof novel and highll, potent euphanetliterThis lcadsto the possibilityof detectionof non-spccificinteractions. penesillr-rstrates thc potentialof plant extractsto genel'ateusefirlclremwhich are particulafly problernaticwhen inr,cstigatincrrLrlticotn- ical leadsin a High ThroughputScreeningprogramme. ponent.uncharactelizecl extractsasopposedto singlecl-rer-nical entities. D r l r i n g a ' r a n d o n r ' s c r e e n i n g p r o g r a m m et o s e a r c hf o l n o r , e l proclr.rcts Examplesof natr.u'al which genelatesuch effects are deter- inhibitots of human thrornbin which were capable of blocking the rvhich dislupt cell membranes. polyphenolics. tbrmation of blood clots and hcnce could be of value in treating and -sent-likecon'rpounds. which fblrn compleres rvith a wide arlav of proteins.antioxidantsarrd preventin-qdeep r,ein thlor-r-rbosis. some 150 000 samplcs.including UV quenchers.It is vital to be trble to detect.and elinrinate.such syntheticcor-r-rpounds and bacterial.fungal andplant extractswere cvali'rctives as rapidly as possible(VanMiddlcsvn'orth and Cnnnell.1998). uated. Methanolic extractsof Lutturttt cdtnurd (Verbcnaceae) leaves. This plocesscan be speecledLrpby testing a standardset of known obtainedfiorl a UK galden.were fbund to displaypotentrcri\ it). interf'ering conrpounds and e\traut\il un ursul plior to the firll screen. Large-sca1eextraction and bioassav-guideclchrontatographic This provides clala to inclicatethe tolcrance of the assay 1o snch fractiorrationleclto the identificationo1'aseriesof novel compounds samples. andmay at timesleadto a decisionnot to proceed $,ithtestins which werecharactcrizcd by NMR andMS irs5, 5-trans-fusecl cyclic of crudeextractsagainstthat target.The phl,sico-chemical properties l a c t o n e - c o n t a i n i negr - r p h a nt lei t e r p e n e s( O ' N c i l l e r a 1 . . 1 9 9 8 ) .T h e of a corrpoundcan give uscfulcluesas to its identity.The rlost com- compounds showeclIC51yvalnes of the orclel of 50 nrl against monly usedpropertiesincludeHPLC lctcntiontime and UV spectlathrorrbin. data that are leadily accluiredthrough stanclardanalyticaltechniques. After the initial activity was detected.literaturesealcheson Luntunu By cornpaling these data with that of knou'n cornpounds.it rnay be (unuro rer"ealedthis plant speciesto be lepolted to be toxic to grazin.'u possiblcto chalactelizethe componentsof a ntixtule lvithout the r-reed animalswhich. on ingestionof the lear,es. dcr,elophepatotoxicity ancl fbr firll isolatiim or at least.it rral'be possibleto narrowthc possibil- photosensitization (Shannaand Sharma.1989).These toxic eff-ects ities. If a librar-vof suchdata florr ir lelatir"elylargenumber of natural hal'e been attributedto the lantaclerres. a selies of pentacyclictriter-proclr.rcts is usecl.this can be r,eryeflectivein identityingthosecom- penes.A furtherstudyof haematological changcsin sheeplbllowing poundsthat are presentin more than or.rcorganism. Lurtttutct poisoningdemonstfated a significantincrease in bloodcoa-tnIf lull isolationof the activt'componr.nt is u rrlrnted. r,ariousrneth- lation time and plothrorr-rbin tirre, with an associatedclecrease in blood ods may bc adopted.Thereis no singlebestisolationtechniqnc.nor is sedimentation late. total plasntaproteinand fibrinogen(Uppal rt a/.. there any single.colrcct rrethod lirr arrvgiven compound.Most separ- 1982).This observationrnay be associatecl with the thrombininhibiation methods inr,olr.c some form of chromato-eraphy-typically tory translactone-corrtaining euphanetriterpenesclescribedabove.
Fr*
"{t'F-
S O M E C U R R E N TT R E N D S
The biological activity of these compounds has been reported in detail (Weir et ul., 1998). Their mechanisrl of action as inhibitors of blood clotting is via acylation of the active site Ser 195 residue of thrombin. This acylating activity has been found to be qenenc rsainst other serine protease enzymes and this finding fon-ns the basis fbr exploitation in drug discovery. References Amman R L. Ludwig W. Schleitcr K H 1995 Phllogcneticidentitlcatronand in situ detectionof indir,idualmicrobial cells without cultivatron. N { i c r o b i o l o g i c aRl e v i e w s5 9 : 1 . 1 3 - 6 l9 Chang H-M. But P-H l9E6 Pharmacologyarrdapplicationsof Chinesernateria medica.World ScientificPublishingCo. Singapore Cox P 199,1The ethnobotanicalapproachto drug disco\,ery:strengthsand limitations.In: Ethnobotanyand the searchfbr nc* chugs.Wilcy, Chichester' (Ciba FounclationSyl'rposiunrI tt-5).pp 25 .11 Dictronaryo1'naturalproducts,5th edn, 2000. Chapmanand Ha11/CRCPress F a r n s w o r t hN R . A k e r e l eO , B i n g e lA S . S o e j a r t oD D . G u o Z 1 9 8 5N l e d i c i n a l plantsin therapy,Bulletin of the World Hcalth Organization63: 865 98 1 Hawksworth D L l99l The fungal dinrensionof biodiversit,v-.; mirgnitude. s i g n i f i c a n ca e n dc o n s e r v a t i o nM. y c o l o g i c a R l c s c a r c h9 - 5 : . { . 1 -I 1 5 2 K i n g S . C a r l s o nT J 1 9 9 5I n t e r s c i e n c i2a0 ( 3 ) : 1 3 , +1 3 9 Kinsman O S, Chalk P A. JacksonH C et al I 998 Isolationand chalactcrisation of an antitingal antibiotic (GR135402)with protein s1'nthesis inhibition. J o u r n ao l f A n t i b i o t i c s5 1 ( l ) : . l l - . 1 9 Loub W D. FamsworthN R. SoejartoD D. Quinn M L 1985NAPRALERT: Computerhandlingof naturalproductrescarchdata.Journalof Chemical I n f b r m a t i o na n dC o m p u t r n gS c i e n c e 2 s 5:99*103
O ' N c - i l lM J . L e u ' i s J A . N o b l e H l v 1e t a l 1 9 9 8l s o l a t i o no f t r a n s l a c t o n c c o n t a i n i n gt r i t e r p e n e sv u ' i t ht h l o n r b i r ri n h i b i t o r l a c t i v i t ) l ' r o mt l - r e l e a v e so f L r i l l r i r a ( u t n a r ( r .J o u r n a lo f N a t u r a l P r o c l u c t 6 s I(1I): ti2ti l33l S a m p s o nJ H . P h i l l i p s o nJ D . B o w e r yN . O ' N e i l l M J . H o u s t o nJ G . L e u , i sJ A 2000 Ethnomedicallvselectedplantsas sourcesof potentialanalgesic c o n r p o u n c l isn: d i c a t i o n o s f i u v i t r o b i o l o g i c aa l c t i v i t yi n r e c e p t o r b i n d i n g assal's.Phytotherapl'Research11: 21-29 SharnraO N,lP. ShamraP D I 989 Natural productsof thc lantanaplant the presentand pfospects.Journalof Scientific IndustrialResearch.l8: 17 | -17E Silva G L. Lee I-S. Kinghorn A D I 998 Spccialproblemsrvith the ertractionof plants.In: ClannellR J P (ed) Natural productsisolation.HumanaPress, Totou'a,NJ. pp 3.13 363 StrohlW R 2000 The role of naturalproductsin a nroderndlug cliscovcry program.Drug Discovely Toda1"5(2): 39J1 Tait R M I 992 Developnrcntof a ratliometricspot-washassal'tbr squalene s y n t h a s eA. n a l y t i c a lB i o c h e m i s t l y1 0 3 ( 2 ) :3 l 0 - 3 l 6 Ten Katc K. Laird S A 1999The cornmercialuse o1'biodiversity, Earth Scan P u b l i c a t i o n sU. K Uppal R P.Paul B S I982 Hematolo-eical changesin experirlcntallantana poisoningin sheep.Indian Veterinary, Journal I [3 2,1 VanMiddlesworthF, Cannell R J P 1998Dcreplicationand paltial identificationofNatural Products.In: CannellR J P (ed) Natural products i s o l a t i o nH . u m a n aP r e s sT. o t o \ \ ' aN . J . p p 3 . 1 33 6 3 Wcil M P. Bethell S S, CleasbyA et al 1998Nor el naturalproduct-5. inhibitols of human a-thrombin:mechanisnrof actionand 5-trirns-lactone structuralstudies.Biochemistr-v37: 66,+5-6657 Wood Mackenzie'sPharnraQuant 2000
E INTRODUCTION
Bioogicolly octive compounds from monneorgontsms G. Blunden
INTRODUCTION I I5
crAssEsoF AcTtvEcoMPouNDS I | 5 ANTIVIRATSUBSTANCESI I5 CYTOTOXTC COMPOUNDS 12r ANTIPARASITIC COMPOUN DS
r22
ANTICOAGUTANTS r22 ANTIMICROBIATAGENTS122 ANTI.INFTAMMATORY COMPOUNDS 122 PROSTAGTANDINSr23
When cornparedto land plantsand animals.the r.rse ol'mar-ineorganisms in fblk rnedicineis r,ery restricted.particularlyoLrtsidcAsia. Much usc has been made of mlu'ineplants and animals for fbod bLrt. probablybccauseof thcir inaccessibility. seriousconsideration of these organismsas sourccso1'biologically activecompoundshasbeenconfinecl to thc last'10 ycars or so. There arc severalr'",ell-knownmarine productsthathavebeerrin usefbr many years.suchascod andhalibut li'n'er oils.spel'ntaceti. protaminesulphateandthe seaweed polysaccharides agar.can'ageenan and alginic acid: all of theseare coveredelsewhere in this book. Marine macloalgae.ol seau'eeds as they are more gener.allyknown. have becn usedas crudecl'ugsin thc treatnentof iodine deficiency states.such as goitre.Bascdorv'sdiscaseand hypothyr-oidism. Some seavn'eeds have alsobeerrr-rtilizedas soul'cesof aclditionalvitamins and in the tleatment of anaemiadurirrg prcr:lnancy. They have also been taken firr thc treatmentof r,lrious ir-rtestinal disorders,as vermifuges. ancl as hypocholesterolaenicancl hvpo-rlycacnticagents.This last property has bcen clairred lbr the senveeds Ct,stoseirttburbata. Surgasstun<:onfusLu'tt andJuniu ruberts.but the mechanismsinvolved in lou'eringblood sugarlevelsby the uscof thesealgaearenot known. Seau'eedshiive been erlployed as dlcssings.ointnlentsand in eynaecoloey.For example.Porlthlru utr(4)L!rpureo has been used in Hau,aiito dresswourrdsand burns andDurt'illaeu antttrctic'arvasused by the Maoris of New Zeab,nd to treat scabies.prepared.sterilized stipesoi Lantirurriu digit(ltd havc been utilized. in conjr.rnctionwith prostaglanclins. to dilate the cervix. as the stipcssr,vellto severaltimes their originaldiameteln'hen moisrenecl (B. Rubin.Et'ott.Bot.. l9jl , 3 1 .6 6 ) . During the past'10years.numel'ousnovel cornpoundsha.n,e beenisolltecl fi'om marinc organismsand manv of thesesnbstances have been demonstrated to possess interestin-e biologicalactivities.To appreciate the rangc of conpounds feportcd.lhe ler..iewsby Faulkner should be consulted(.Nur.Prod.Rep.(R. Cltetn.Soc.\,16.I l3 andearlierreviews cited therein).However.much of this rvorkhasbeenmotivatedby an intelest in thc cherrristryof the nor,cl r-netabolites rathcr.than in their biologicalacti\,ities. Ottenthe compoundshar.ebeenisolatedin small alnounts.which resn'ictsthe lange of pharrracologicalevllr-ration.In somecascsI rnuchnroreextensileran-{eof activitiesmav havebeen testedtbl thanthosepublished.but becanseof their potentialeconomic r.'alue.the clatuhar,cnot beerrrcoorted.
CIASSESOF ACTIVECOMPOUNDS It is the intentionin this chapterro ilhrstriltethe ranceof metabolites isolatedfi'om marine olganisms.bltt concentl'atingon cornpoundsof rncdical,biomedical.phalmaceuticaland agliculturalinterest.The numbersin parcntheses fbllowing the nanresof the compoundsref'erto the relevrntchcmicll structures in Fis. 16.l.
PROTEINS 123 AGROCHEMICAT USAGE r23 DINOFIAGEttATE. AND DIATOM.DERIVED sHEtrFrsHPotsoNlNG 123
TOXTNS r24
coNcLusroNsr24
-l
'ffi''
ANTIVIRATSUBSTANCES Drr.rgsacti'n,e againsthur-nanvimses are in great demandand phar.rnaceuticalcor.nprnies. rcscarchinstitutesanclacademicgroupshave set r-rpscreeningprocedr-rres in the sealchfbl sr-rchdrugs. This work has beenhighli-ehtedin the popular pressby rhe worldwide AIDS epidemic. Not surprisingly.r-nanvrnarineorganisrnshave bcen screcnedfbr the prescnceof compouncls with antiviralactivity.but the searchhas beendisappoirrtin-t and.to datc.only onecompoundl-ras beensho\\'nto
E
TT RENDS S O M EC U R R E N NHr
t -
IjT\ -*^T ^ l HOHTCYUj \F,llOH
Hd
M e O
(.\n-'A M".. 1 a k) I ' b oH
ra!
Me
A R A - A( 1 )
OMe DidemniB n (2) n
HO
\_ ( \ \ \ /
)-\ \_/
a-\^,
LJ
r
lrila
.^';xlve l A (3) Eudistomin
l
u
Lrl-l
(-i*'"
l
l M e Me
Me
Avarone(5)
Avarol(4)
Me OH
O
\-1--i
\-f.-.2 l M e
Me
at--\ i rr/lo
OH
HO Me Me
Me N
s{rLu C h e m i c oslt r u c t u r oe fs Fig. l6.I b i o l o g i c o l ol yc t i v ec o m p o u n df sr o m m o r i n eo r g o n i s m s .
Me B (6) Patellazole
-
S9,c
.FrF
-
FROMMARINE BIOLOGICALLY ACTIVE ORGANISMS COMPOUNDS Fig. I6.l (continued)
(7) Bryostatin-1
ry*" t")-r"
n I N
'";-^*rl} M e
O
H O
(B) Dolastatin-10
Me OH a -
-''--\-tn
= Dolastatin-H (9)
-{'n
= lsodolastatin-H (10) LOH
Me Me
Me
= Aulrttt.ne-A (11) R = COCH2CHMe2 (12) R = COCH(Me)EI= Auripyrone-B
S O M EC U R R E N TR T ENDS Fig. l6.l (continued) HO
MeO
O-:. AcO
Me
.N_,, -\ H
Ho
ofl Niphatesine D (14)
o Ecteinascidin 743 (13)
Globostellatic AcidA(16)
'"L Me
E p i n a r d iA n (15)
v
/-\
(17) Suberosenone
OMe CoHrs (19) R = Ac, Clavepictine-A (20) R = H, Clavepictine-B NHz
P o l y c a r p i n e( 2 1 )
/
^\ - c o o H
Me----<' - '
/ \ \ttAcooH
cooH
'\"oo*
Me
I H
N I H
s-Kainic acid (22)
cooH
Domoicacid(23)
F.;{I*$e
j-J'r:'
BIOLOGICALLY ACTIVE FROMMARINE COMPOUNDS ORGANISMS Fig. l6.l
(conrinued)
BengamideF (24)
-
W
l
l onY
"";" v-/
)-o b I onY o dV-1 -o3so=- " \ " 0HO-
OH
1 /v
F (25) Cucumechinoside
HO'\
'"/_b, o
o\
A'A;.o'"
-lo" CH2OCOMe
t i
R,,/\ '/
I-T-Y
--+ra.; HO2CCH(CH2)3COHN NHz
0H2NH2
lstamycinA Rl = H, R2= NH2(27)
Cephalosporin C (26)
lstamycinB R1= NHz,R2= H (28)
(30) Palauolol
ocH3 (+)-7'-Methoxy-2,3,5,5'-tetraH-indole(29) bromo-3,4'-bi-1
HOOC. >----
Me
Me
\W furan(31) Sesquiterpene
r
TT RENDS S O M EC U R R E N Fig. l6.l (continued)
cooH
cooH
Me
Me OH OH E2 (PGE2)(32) Prostaglandin (33) F2" (PGF2q) Prostaglandin Me \+ Me-N-
S-S
YI N
Mel
(CH2)n-COOH2N\*.'o tl
Md 'Me
H-N
(35) n = I = glycinebetaine
Pfrs,
acidbetaine(36) n - 2 = y-aminobutyric Nereistoxin(34)
H H , H
. rN'
,rfiA*
N-,,
r -OH OH
acidbetaine(37) n = 3 = 6-aminovaleric
Saxitoxin(38)
OH H
cHo
Me .H HH
o
H
BrevetoxinA (39)
HOOC Me OH
Me
1 (40) Dinophysistoxin-
Me
cHo
t"-*
Y" -Y
-
H
I
N
OH
o
;=^ (41) Lophotoxin
(42) Lyngbyatoxin
-
|F-
BIOLOGICATLY ACTIVE COMPOUNDS FROMMARINE ORGANISMS Fig. l6.I
(confinued)
Debromoaplysiatoxin (43)
O
M
e
H
A^-]{>
H
t"-rt"o t Mu-N
Il
O
i
O
l
l^l*--r O
M
HM"
e
l
O
(44) Majusculamide
havesignificanttherapeuticactivity.This is ara-A (1), which is a semisvntheticsubstancebasedon the arabinosylnucleosidesisolatedfrom the sponge Tethya cn,pta. Severalcompoundsisolated from marine organismshave been shown to have pronouncedin yitro activity. but only the didemnins(2) have producedactivity in yivo',theseare cyclic depsipeptidesisolatedfrom Trididemnnmspecies(tunicates).In addition to antiviral properties,some of the didemninsexhibit antitumour activity. Other interestingcompoundshave been isolated,particulariy from tunicates,spongesand gorgonians.The eudistomins(3), first isolated from Eudistontnolivaceunt(family Polycitoridae).form a secondfamily of compoundsfrom tunicateswith potent antiviral activity; these are B-carbolines. The two compounds,avarol (4) and avarone(5), extractedfrom the sponge Disizleo ayara, have been reported to inhibit immunodeficiency virus, have high therapeuticindices and the ability to crossthe blood-brain barrier.These compoundswere thus proposedas having potential use in the treatmentof AIDS (P. S. L. Sarin e/ nl., J. Natl. ConcerInst.,1987,78, 663). Very potent in.vitro actlity againstherpessimplex viruseswas displayed by patellazoleB (6), isolated from the tunicate Lissoclinum patella. A review of the range of antiviral substancesreportedfrom rrarine organismshas been published by K. L. Rinehart er al. (tn .l'larine Biotechnologt, 1993, Vol. l, Phannar:euticalttnd Bioactive .\{utLtralProdLrcts(edsD. H. Attaway and O. R. Zaborsky),New York: PlenumPress,p. 309).
CYTOTOXTC COMPOUNDS \ot surprisingly,many marineorganismshavebeentestedfbr cytotoxicity in the searchlbr compoundsactive againstcallcerand numerous \ubstanceshavebeenshownto possesssuchproperties.It would not be
appropriateto review this work here, but a few selectedcompounds have been chosen to illustrate the range of materialsreported.(For reviews see M. H. G. Munro, R. T. Luibrand and J. W. Blunt. in Bioorganic Marine Chemistry. 1987, Vol. 1 (ed. P. J. Scheur),New p. 93. and F. J. Schmitz,B. F. Bowden and S. I. York: Springer-Verlag, Toth, in Marine Biote(:llnologt,Vol. 1. PhurntaceLfiical and Biocrc'tive Nattrral ProdtLcts,1993(edsD. H. Attaway and O. R. Zaborsky).New York: PlenumPress,p. 197). Probably the best known of the compounds are the group of macrolidesknown as bryostatins.which have been isolatedprimarily tiom the bryozoan Bugula neritina, although some have also been extractedfrom collectionsof spongesand tunicates.Many bryostatins havebeenisolatedand characterizedand shownto possesspronounced biological activity. The potential use of bryostatinsfor treating neoplasticbone-marrow-f'ailure stateshasbeensuggestedas a resultof the compounds stimulating human haemopoieticcells. Bryostatin-l (7) triggers activation and diffelentiation of peripheralblood cells fiom lymphocyticleukaemiapatients.It alsodisplaysother activitiessuchas the activationof protein kinaseC (PKC) and arachidonicacid metabolite release.Both bryostatin-1and -2 enhancethe efficiency of interleukin-2 in initiating development ol in vivo primed cytotoxic T lymphocytes.Bryostatin-I has undergonephase2 clinical trials. Many other bryostatinshave been isolatedand bryostatins-16, -17 and -18 havebeenreportedto have antileukaemicactivity. Cell growth inhibitory activity in vitro has been demonstratedby a group of cyclic and linear peptidesand depsipeptidesknown collectively as dolastatins.These were lirst isolated frorn the sea hare Dolabellaauriculttria. AtIhe time of publication,dolastatin-10(8) was reported to be the most active antineoplastic substance known (G. R. Pettiter al., J. Am. Chem.Soc.,1987,109.6883).More recently. other dolastatinshave been isolated and both dolastatin-H (9) and isodolastatin-H(10) have beenshown to be highly cytotoxic (H. Sone
E
S O M EC U R R E NTTR E N D S et al., J. Am. Chem. Soc, 1996, 118, 1874). Also cytotoxic are the polypropionates. auripyrone-A(11) and -B(12),which havealsobeen isolatedfrorn D. aLtriculuria(K. Suenaga,H. Kigoshi and K. Yamada, TetrahedronLett., 1996.37,-5l5l ). Extracts of the tunicate Ec'teinascidiaturbinatu were shown to increasedramaticallythe life-spanof rnice inoculatedwith P388 cells. The active compoundswere characterizedas a group of complicated alkaloids,which were calledecteinascidins. Ecteinascidin743 (13) has undergonephaseI clinical trials as an anticanceragent. Spongeshavebeena rich sourceof cytotoxiccompounds.As well as the bryostatins,many of the other active compoundsare macrolides. Exarnplesof other cytotoxic compoundsreportedlbr spongesare the antineoplasticalkaloid niphatesineD (14), which was extractedfiom a Ni)hates species(J. Kobayashiet al., J. Chem.Soc.,Perkin Trans, l, 1990.3301); the epinardins(15), isolatedfrom an unidentifiedspecies (M. D'Ambrosio et al., Tetrahedron.1996, 52, 8899); and the globostellatic acids (16), which are isomalabaracanetriterpene constituents oI StelLetta globostelluta (G. Ryu, S. Matsunaga and N. Fusetani. J. Nat Prod., 1996,59. 512). Cytotoxic compounds isolated from coelenteratesinclude the sesquiterpene,suberosenone(17). extracted from Subergorgia strberosa(H. R. Bokesch et al., TetrahedronLett., 1996,37 3259), the diterpene sarcoglane(18), trom Surcophyton glaucum (E. Fridkovsky et al., TetrahedronLett., 1996, 37,6909) and the cembranoids trom Sintularia gibberosa (C-Y Duh and R-S Hou, J. Nar. Prod., 1996,59,595). Exarnplesof activecompoundsfrom tunicares are the alkaloids clavepictine-A(19) and -B (20) isolated fiom Clayelinapicta (M. F. Raub er al.. J. Am. Chem.Soc., 1991, 113, 3178) and the dimeric disulphidealkaloid polycarpine(21) from Polycarpa (layata (H. Kang and W. Fenical, Tetrahedron Lett., 1996.37.2369\. The didemnins,mentionedearlier r.rnderantiviral substances. have alsobeendemonstratedto havepronouncedantitumourpropertiesand didemninB hasundergoneclinical trials.
ANTIPARASITIC COMPOUNDS
laminarioidesrvas recorded.Similar effects were demonstratedwith caffageenansfrom ChondrtrscrispLts,Euchemaspinosum andPol r-ides rcttundus. It was later shownthat carrageenans had a directefiect on the in vitro inacllation of thrombin. The anticoagulantefl'ectsof isolatedfractionsfrom the brown alga Fucus vesiculosashave been studiedand an antithrombineffect was attributedto fucoidan.Evidencehas beenprovided that the antithrombin effect of fucoidan is mediated through heparin cofactor Il. Antithrombin activity associatedwith an unknown plasmatactor has been reported lbr crude, aqueous extracts of the green alga Codiwn .fragile ssp. tomentosoides.Another subspeciesof C. .fragile (ssp. yielded high molecularweight proteoglycanswhich poscttLanticum) sessedanticoagulantactivity.
ANTIMICROBIAT AGENTS Marine microorganismshave been studiedas a sourceof biologically activesubstances and severalcompoundshavebeenisolatedand characterizedwhich have been shown to possessantimicrobial activity. Of particular note is the fungus Cephalosporiumacremonium,from which cephalosporinC (26) was isolated,a semi-syntheticderivative of which, cephalothinsodium, is widely used as an antibiotic drug. Other examples of antimicrobial compounds isolated fi'om marine microorganismsare istamycinsA (27) and B (28), which were producedby fermentationof the marine streptomycete,Streptomtcestenjimariensis SS-939.In vitro activity is observedagainstGram-negative and Gram-positivebacteria,including thosewith known resistanceto t h ea m i n o g l y c o s i daen t i b i o t i c s . A leatureof many metaboiitesof marineorganismsis the occurrence of halogens,in particularbromine. Many of thesecompoundsexhibit antimicrobialactivity, but are either not sufficiently potent or are too toxic, or both, to renderthem of clinical value.
ANTI.INFTAMMATORY COMPOUNDS
The sulphated polysaccharidecarrageenanis used in several tests The red alga. Digenitt sintplex, has been used as a vermifuge for devised for the screening of anti-inflammatory drugs. The carhundredsof years.The active substanceis cr-kainicacid (22), which rageenan-induced,non-immune inflammatory response, rat paw has been marketedfor many years as a bload spectrumanthelmintic. oedemaassayis a standardmodel systemfor this purpose.Other test It is effective againstthe parasitic round worm, the whip worm and systems involving the use of carrageenanhave been developed, the tape worm. Takeda PharmaceuticalIndustries markets various including the infusion of the polysaccharideinto the lungs of either preparationsof the compound. A chemically related compound, mice or rats to produce a pleurisy-like reaction. Potential antidomoic acid (23), also has anthelminticpropertieslthis has been inflammatory drugs are used in this system to determinethe extent isolated from the red algae Chondria armata and Alsidium coralLi- to which they alleviate the condition. Carrageenanhas also been mLLtn. injected into the synovial fluid of animalsto producean arthritis-like Studiesof marine animalsas a sourceof antiparasiticagentsled to model which can be used to test arthritis-specificanti-inflammatory the isolation and characterizationof anthelmintic compoundsfrom drugs. sponges,a nudibranchand a zoanthid.Interestingsubstancesinclude A seriesof novel bi-indoleswas isolatedfrom the marinecyanobacbengamideF (24), which has been demonstratedto be effective ln terium, Rlyrl/aria firma. One of the major compounds was (+)-7'yitro. Another is cucumechinoside(25), isolatedfrom a seacucumber, methoxy-2,3, 5, 5'-tetrablomo-3,4'-bi- I H-indole (29), which is active which has antiplotozoalactivity.For more informationon theseagents in both the canageenan-induced rat paw oedemaand kaolin rat paw from marine animals seethe review by P. Crews and L. M. Hunter in oedematestsand alsoin centralnervoussystemtests.Therewas promMarine Biotechnology,1993, Vol. l, Pharmaceuticaland Bioactive ise in the isolatedcompoundfor developmentas a medicinally-useful Natural Prodtrcts(edsD. H. Attaway and O. R. Zaborsky),New York: product, but the company that was responsiblefor this work did not PlenumPress.p. 343. proceedwith this compound. In more recent years, other compounds with anti-inflammatory activity have been reported for marine organisms. Examples are the ANTICOAGUTANTS palauolol (30), from the sponge Fascaplysinopsissp. sesterterpene Anticoagulant activity associatedwith polysaccharidesfrom marine (8. W. Schmidt and D. J. Faulkner,TetrahedrcnLett., 1996,37, 3951) algaehasbeenknown lbr many years.The earliestreport is lrom 1936 and a sesquiterpene furan (31) from the coelenterate, Sinularia sp.;this when an anticoagulanteffect of a sulphatedgalactan from lridaecr has subsequentlybeensynthesized.
*.|&.
TU
FROMMARINE BIOLOGICATLY ACTIVE COMPOUNDS ORGANISMS dried plant material rvith either water or aqueousalkali, whereasthe suspensionsare produced from fresh algae. The most widely used Prostaglandins of constitutea group of biologically potent substances speciesin the preparationof the products areAscopltylluntnodostun. wide spectrumactivity. Although first recognizedin the 1930s,they Ecklonia maxintn and Dun'illaea pot(fiorum. Many diff'erent eff'ects remained of little interest until the structuresof two of them were have been reportedas a result of the usageof theseseaweedextracts deterrninedtn 1962. Structurally. all the compounds are based on and suspensions, including increasedcrop yields, increasedresistance prostanoicacid,which is itself inactive.Six series(A-E) ariseby mod- oftreatedplantsto stressconditions,increaseduptakeof nutrientsfrom ification of the cyclopentanering and theseare now availableas syn- the soil, reducedstoragelossesof fruit and vegetables,and improved thetic products. However, prior to synthetic cornpounds being seedgermination.The active compoundspresentin the productshave available.researchon prostaglandinswas restrictedby inadequatesup- not beenfully elucidated,but cytokininsand betaineshavebeenimpliplies of the two activecompounds,PGE2(32) and PGFI^ (33).The dis- cated (see G. Blunden in Setuteed resoltrcesin Europe: usesand covery that the soft coral,Plexaura homomulla,was a rich sourceof potential. 1991 (eds M. D. Guiry and G. Blunden), Chichester:John lS-epi-PGA2 and its acetate,methyl ester derivative, was a major Wiley & Sons, p. 65). ln recent years several publications have breakthroughas the Upjohn Company developeda syntheticpathway appearedshowing that seaweedextract treatedplants have increased to converttheseinactivecompoundsinto the requiredactiveones.The resistanceto pathogenattack.In particuiar,significantlyreducedlevels syntheticroutes to the requiredcompoundssavedthe possiblewide- of inf-estation by root-knot nematodes(Meloidog1nespecies)has been spreadcollectionof Pleraura. demonstratedand the role of the betainescontainedin the extractsin Prostaglandinshave also beenisolatedfrom red algaebelongingto bringing about these effects has been clearly demonstrated.In the the genusGrucilaria. ruttlosum,glycinebetaine(35), extracts preparedfrom A.scopli1'llunt y-aminobutyricacid betaine(36), and 6-aminovalericacid betaine(37) havebeenisolated. PROTEINS Betainesare not restrictedto the seaweedspeciesusedin the prepaThe detectionof endotoxinsin, for example,pharmaceuticalprepara- ration of these extracts,but either betainesor a tertiary sulphonium tions and water,is basedon the clotting of amoebocytelysateobtained analoguehave been found in all marine algae,except one, testedfor from the horseshoecrab,Limulus polyphenus. in the presenceof the thesecompoundsto date. found on the cndotoxins.Endotoxinsarecomplexlipopolysaccharides, outer cell wall of Gram-negativebacteria.which are relatively heatDINOFIAGEILATE. AND DIATOM.DERIVED stable. POISONING SHELLFISH good group proteins has received a deal of The of known as lectins attention in recent years. Lectins are carbohydrate-bindingproteins Large concentrationsof dinoflagellatesoccur periodically in the sea and are found in a wide variety of life forms. including marine organ- which, becauseof theil pigmentation,give the water a brown to red isrns.Probably the best known lectins from a marine sourceare the coloration.The 'blooms' areknown as red tides.Certaindinoflagellate potenthaemagglutininsobtainedfrom the haemolymphof L. poll'phe- speciescontaintoxins which, when consumedby filter feeders,suchas rirr,r.One of the lectins,limulin, has been isolatedand shown to be a shelllish.are concentratedin the flesh of the animals.When blooms of large, l8-subunit protein with a molecular massof 350 kDa. Limulin dinoflagellatesoccur they can prove hazardous to human health .hows specificity for binding to sialic acid, although other sffuctures becauseconsumptionof contaminatedbivalves by man can result in .rrerecognized.Another lectin that shows specificity to sialic acid is severetoxic effects,including death.The toxins are usually classified that obtainedfrom the haemolymph of the lobster,Hontarus anteri- as eitherparalyticshellfishpoisonsor diarrheticshellfishpoisons.The
PROSIAGLANDINS
( (lllvs.
former can prove fatal, but the latter,althoughthey can producehighly unpleasanteffects.arenot tatal. The best known paralytic shelllish toxin is saxitoxin (38), although other relatedcompoundshavebeenreported,suchas neosaxitoxin,the 1l-cx and ll-B-O-sulphatesof saxitoxinand neosaxitoxin,and carbamoyl derivativesof saxitoxin and neosaxitoxin.Another group of paralytic shellfishtoxins are known as brevetoxins,the most potentof which is brevetoxinA (39). The name is derived from G.tmnodiniun't brevis),which is responsiblefor the red tides breve(.nowPt ,nchodiscus of Florida and the Gulf of Mexico. The bestknown ofthe diarrheticshellfishtoxins are the dinophysistoxins, suchas dinophysistoxin-1(40), which are producedby species of Dinophysis. Other important diarrhetic shellfish toxins are the pectenotoxrns. Paralytic and diarrhetic shelltish poisonings represent potential health hazardsand, as a result, monitoring programmeson shellfish AGROCHEMICAT USAGE toxicity havebeenintroducedin many countries.For paralyticshellfish fhe marine annelid,Lumbriconerisheteropoda,has long beenknown poisons,the methodsinclude mice testsas well as high pelformance :', be toxic to insects.This activity was shown to be due to nereistoxin liquid chromatographicprocedures,and for diarrheticshellfishtoxins the methods include bioassays,immunoassaysand physicochemical -1-li.which has rapid anaestheticpropertieson insectsand is toxic to :r'h and mammals,affectingthe nervoussystemand heaft. Studieson methods. In addition to dinoflagellate-derivedshellfish poisoning. ::ri' compoundled to the introductionof the syntheticpesticidePadan. "evere Both extractsand suspensions ofbrown algaeareusedin agriculture toxic effects can result fiom the ingestion of shellfish contaminated with diatoms. The earliestreoortedincident occurred in 1987 when .,:rJhorticulture.The fbrmer productsarepreparedby extractionofthe
Marine algaehavebeen studiedextensivelyas a sourceof lectinsin rccentyearsand severalarenow marketed.Much ofthis work hasbeen conductedby Rogersand co-workers.They showedthat the red alga, Prilota plumosa, contained a lectin which reacts specilically with the cr-(1,3)-linked-D-galactose unit and hence preferentiallyagglutinates group B human erythrocytes.Extracts of another red alga, \olieria chordalis, have anti-sialic acid specificity, and those from the green alga Codiumfrnglle ssp.atlanticLonreactpreferentiallywith \ -acetyl-D-galactosamine. The Iectinsfiom al1threespecieshavebeen nartially characterized.The occurrenceand propertiesof lectins tiom nrarinealgae were reviewed in 1991 (D. J. Rogers and B. C. Fish in LtLtin Reviews,I99I (edsE. van DriesscheandT. C. Bog-Hansen), St l-ouis:SigmaChemicalCompany.p. 129).
fFFFr*.
.drt(-",
E
S O M EC U R R E NTTR E N D S over 11 casesofpoisoning were recordedin easternCanadaas a result Another interestingtoxin is lophotoxin(41), a diterpenelactone,isoof eatinginf'ectedmussels.Threefatalitiesresulted.The toxic compon- Iated from the gorgonian corals ofthe genusLophogorgful.Lophotoxin ent was identilied as domoic acid (23), previously isolated fiom producesan irreversiblepostsynapticblockageat neuromuscularjuncspeciesof red algae(seesectionon Antiparasiticcompoundsearlierin tions. this chapter).The organism responsiblefor this compound was the The contactdermatitisknown as 'swimmers' itch'was shown to be diaLomPseutlonit:.schiapLtngensforma multiseries. The toxic effects causedby the indole alkaloid, lyngbyatoxin (42), produced by the produced are known as amnesicshellllsh poisoning. In 1991, many marine cyanobacteriumLl,ngbiamajttscula.This speciesis of interest brown pelicansand cormorantsdied as a resultof domoic acid poison- becauseits constituentsvary considerablydependingon the place of ing causedby P austraLis.Since then, domoic acid has been isolated collection.A totally unrelatedskin irritant was isolatedfrom a different liom other Pseudonitzschlaspecies,including P. seriata fiom Danish collection of L. majuscula, the compound responsiblebeing debrowaters. moaplysiatoxin(43); this compound has also been reportedto have The efTectsof domoic acid poisoninginclLrdegastrointestinalsymp- antineoplasticactivity.MajuscLrlamide C (44) was isolatedfiom a deep toms such as nausea.vomiting, anorexia,gastric bleeding,diarrhoea water variety of the same species.This compound, a depsipeptide, and abdominal cramps. followed by neurologicaldisorderssuch as inhibits the growth of fungal pathogens. confusion, disorientation.loss of shofi-term memory and coma. In Phloroglucinoland its derivativeshave a widespreaddistributionin view of thesesevereefTects,the needto monitor seafbodfor the pres- the brown algae; many of these compounds have large molecular ence of domoic acid is self evident (N. Lundholm, J. Skov, R. weights. These polyphenolscan cause considerableproblems when Pocklingtonand O. Moestrup,Phvcologia, 1991,33.475). investigatingbiological activity of brown algae.For example,the compoundsbind to proteinsand thus inhibit enzymeactivity.When brown algae are processedand used in animal f'eeds,the ability of the TOXTNS polyphenolsto form insolublecomplexeswith protein make the latter Many compoundsisolatedliom marine organismsposepotentialhaz- unavailablefor dietary use. Severalpotentially interestingbiological ardsto humanhealth.Examplesof thesesubstances are the dinoflagel- activities recorded lbr extracts of brown algae have been shown. late toxins,responsiblelbr paralyticand diarrheticshellfishpoisoning. on further investigation,to be due to the non-specificpropertiesof and domoic acid. responsiblefor amnesicshellfish poisoning, all of polyphenols.For example,extractsof certain speciesof brown algae, which havebeencover"ed in this chapter.Another exampleis the potent when mixed with human erythrocytes,causedagglutination.Extracts neurotoxin,tetrodotoxinwhich, until recently,was thought to be pro- treatedwith polyvinylpynolidone did not producethis effectand it was duced by the pufferfish (family Tetraodontidae).ln Japan,this fish is thus consideredthat the activity was the result of polyphenolspresent consideredto be a great delicacy,which necessitates great care in its in the original extracts. The active compounds were isolated and preparationfor culinary use.Tetrodotoxinhas also beenisolatedfrom provedto be polyphenools. other animalsincluding crabs.and thusthe origin ofthe toxin becamea matterof considerabledebate.The idea that the toxin has a microbial sourcewas proposedand many marinebacteriahave beensubsequen! ly shown to producethis dangeroustoxin. Recentwork also strongly suggeststhat the paralytic shellfish toxin, saxitoxin, is produced by During the past 40 years, numerous novel compounds have been bacteriaand not by dinoflagellates. isolated from marine organismsand many of these substanceshave Ciguatoxin:isa toxin responsiblefor ciguaterafish poisoning,which been shown to have pronouncedbiological activity. In this chapter, is perhapsthe biggestsingleproblem associatedwith the utilizationof exampleshavebeengiven to illustratethe array of chemicalstructures tropical fish lesources.The toxin has been isolatedand characterized and activitiesrecorded;many otherscould easily have been included. and shownto originatefrom the dinoflagellateGambierclisctts ktricus. However, the conclusionhas to be drawn that, despitethe large efTort Ciguatoxin is inotropic and depolarizes excitable membranes. investedin marine natural productsresearch,the impact on the pharHowever, the many symptoms resulting fiom ciguatoxin ingestion, maceuticalindustry,at leastto date,is very limited, althougha few of including neurologicalsymptoms.suggestthat other active sites are the cytotoxic compoundshave undergoneclinical trials as potential involved. G. toxictrsis also the sourceof maitotoxin.a powerful calci- anticancerdrugs.Moreover,the difficuities encounteredin the collecum channelactivator.Numerouspharmacologicalefl'ectsareproduced tion and cultureof largequantitiesof marineorganismswould discourby maitotoxinin concentrationsin the pico- to nano-molarrange. age pharmaceuticalcompaniesdevelopinga drug that was dependent The predatorycone shellsof the -eenusConlls contain small, phar- on thesenaturalproductsunlessthe compoundcould be obtainedreadmacologically-activepeptides,which are targetedto variousion chan- ily and economicallyby a syntheticroute.Becauseof this, biologically nels and receptors.Becauseof their ability to select liom closely activesubstances from the seashouldperhapsbe seenas valuablenew relatedreceptorsubtypes.the conotoxinshavebecomevaluablefor use 'lead' compoundswhich, if appropriate,can then be synthesizedand in neuroscience research. modified to producethe desiredmaterialat an economicprice.
coNcLUstoNs
f-ql;llF
f-!!Dn
E - - 1 1
rlirtritr r,tr. ;li:lllr: riii'l:lii
- : -
: -
DEFINITION
,rrilfir
it];ltiil ,il!:li]' ,rillliil:il
Troditionol plont medicines oso source of newdrugs P.J. Houghton
DEFINITION 125 HISTORICATDIMENSION 125 THEPROCESS OF MODERNDRUGDISCOVERY USINGETHNOPHARMACOTOGY127
.
'
,
The scientitjcstudyoftraditional plant medicinescan be consideredas a major part of ethnopharmacology, a term introclllcedonly in 1967but which describesan approachto the discovery of single biologicallyactive moleculeswhich has been usedever sincethe first compounds were isolatedfron-rplant material.Ethnopharmacologycan be defined as 'the scientific study of materialsusedby ethnic and cultural groups as medicines'and in most instancesthis is synonymouswith the study of traditionalmedicines.In most casestheseare the flowering plants, and so in most instancesethnophal'macology can be consideredas a branchof ethnobotany,the study of the usesof plantsby ethnic groups. However.it shouldbe notedthat somecultures.e.g. Chinesetraditional medicine. also make use of animal and mineral matter and so ethnopharmacology would alsoencompassthe study of these. Somediscussionhastakenplaceconcerningthe boundariesof what is meantby 'ethnic'. Somewould incl-rdea1lethnically-basedsystems outsideWesternscientific medicine but the balanceof opinion probably rests on a more restrictivedetinition which includes only those bodiesof knowledgewhich arerestrictedto a group which has lived in a locality for a long period of time but which doesnot have a sophisticatedtheoreticalframework.folmal educationand a documentedwritten history. This more rigid definition excludesthe medical systems which have developedover thousandsof years in cultures based in China and the Indian sub-continentand emphasizessmall ethnic groups where there is a threat of the rapid loss of knowledge due to globalization,loss of habitat,migration and other factorswhich might leadto lossof culturaldistinctiveness. Some ethnographerswould distinguish between folklore and ethnopharmacology, claiming that the former is commonknowledgein the populationas a whole, largelyconcerningremediesfbr minor conditions,basedon relativelyinnocuousmaterial.Ethnopharmacologyis more concernedwith the knowledge of a few specialistswho are regardedby the societyas able to correctly diagnoseand treat disease states,generallyusing more potent products.In many situationsthese specialistsare also linked with the leligious practicesof the society. It shouldalso be notedthat the discoveryof new drugsmight derive from a wider use of plants than for strictly medical purposesalone. Thus materialsusedas poisons,in pest control, in agriculture.as cosmetics,in fermentationprocesses and for religiouspurposesmight also yield active substances which can be exploitedas leadsfor drug development.It can thus be seenthat ethnopharmacology is a very interdisciplinary subjectand any thoroughinvestigationwill probablyneedthe input of a variety of specialistssr,rchas anthropologists,botanists, c h e m i s la s n dp h a r m a c o l o g i s t s .
SOMEMODERNEXAMPTES OF DRUG DISCOVERY BASEDON THE ETHNOPHARMACOTOGICAT APPROAC H
lH]6ToHtALD|.filENgloN. r32
THEVATUEOF THE ETHNOPHARMACOTOGICAT APPROACH I33 PROBTEMS WITH THE ETHNOPHARMACOTOGICAT APPROACH
r33
coNcrusroN r34
The isolation of some of the opium alkaloids in the early nineteenth century was a key event in the developmentof modern pharmacy.It showed that isolated compoundshad much the same activity as the existingethnopharmacological materialand so pavedthe way ior current orthodoxWesternmedicinewhich usespure compoundsfor treatment. Since then. a vast amount of money has been spent on the synthesisof novel compounds,but also on the isolation of molecules from naturalsourcesandtheir developmentinto medicines.The contribution of traditional plant medicinesto this processhas been signiticant and somenotableexamplesare shown in Table 17.1. It shouldalsobe rememberedthat the activemoleculesisolatedliom traditionalmedicinalplantsmight not only provide valuabledrugsbut
S O M EC U R R E NTTR E N D S
Tqble l7.l
Some common drugs originoting from trqditionol medicinol plonts.
Phormoceutical
Structure
Plontsource
Troditionaluse
Atropine
CHr
Deodlynightsho de Akopa bellodonnoond other soronoceous orugs
H o l l u c i n o g e n i cb r e w s i n E u r o p e , p u p i l e n l o r g e m e n ti n e y e
Scopolomine (Hyoscine)
CHr
MondrokeMondrogoro officinolisond os for otropine
Pre-operotive sedoiiveond o n o l g e s i ch,o l l u c i n o g e n i c preporotions in monyports of the world
CoffeeCoffeo orobico, TeoIheo sinensis
Stimulont drinkor postein Ethiopio(coffee), drinkin Chino
FoxglovesDigitolisspp
D. purpureoleovesusedin Englondond Wolesfor oedemo dueto congestive heortfoilure.
\--'-^ H\...cH2oH
*\
"l-h o
Coffeine CH?
il
CHa -n,-^..-.-
'i I
N
tl
I
\-/
I
//) N
o4N4 I
cHs Digoxin
CHs
H:g\^{t-o Ho. ioHo Ephedrine
uof
CH:r I
..,ru-ctrs
Ephedrosinicoond otherspp. In Chinofor respirotory comploints
H
Ergometrine
ErgotC/ovicepspurpureo
s'{R*@
ln centrolEuropeto oid childbirth
' F a
PLANTMEDICINESAS A SOURCEOF NEW DRUGS TRADITIONAL
Toble | 7.1 {continued}
Sorne common drugs originoting from trtrditionol medicinol plonts.
JoborondileovesPilocorpus In Brozilfo induces w e o t r n g joborandi
Pilocorpine
.-CHs
CHs
,N-ir/--fl,
( 'N-/l l
l\O F o
Rouwolfiaserpentino
R e s e r pni e
Extensively in Ayurvedic medicine{lndio}for mentol illness
H
ocH3
'lead molecules'which might be modified chernare also valuableas ically, or serve as a template fbr the design of synthetic molecules incorporating the pharmacophoreresponsible fbr the activity. Examplesof drugshavingthis origin areshownin Table 17.2. 'drug discovery' is generallyuseclto However, although the telm ref'erto the isolationof nroleculeswith activity,it shoLrldalsobe remenrbeledthat thereis increasinginterestand rccognitionthat a'drug tl'eatnrent'may consistof a mixtureof compountls.This hasalwaysbeenthe which concaselbr plant extracts(and most other naturalsubstances) 'active ingledients'.It shouldbe noted that such extracts. tain several usuallybasedon a reputedtraditionaluse somewherein the world. are beingintroducedand increasinglyusedas a complementarytherapeutic apploachin the West.A selectionof common ones.togetherwith their roots,is shownin Table17.3. ethnopharmacological has increasedover the last Scientific interestin ethnopharmacology f'ew years and this is reflected in the formatictnof the International Societytbr Ethnopharmacologyin 1990and the EuropeanSocietyfor at aboutthe sametime. Both of thesegloups hold Ethnopharmacology regularmeetings.Severalscientificjournalsalsopublishpaperson this topic. notably the JournalofEthnopharmacology.fbundedin 1979. The scientific interestis reflectedby widel Westernsocietywith its tascinationwith a much wider rangeof aspectsof other cultures(e.g. dress.music.fbod, philosophy.as well as medicines)and this hasbeen catalysedby large population migr"ationsto the West and the relative easeof exposureto exotic cultureswhich hasbeen facilitatedby largescaleinternationaltravel. as Severalrecentsurveyshaveshownthat usingethnopharmacology a basis of selecting species for screening t'esultsin a significant 'hit rate' for the discoveryof novel active compounds increasein the conrparedwith random collectionof samples.With a very large nutnber of living organismsstill awaiting scientific investigation(about 907cofthe estimated250 000 speciesofflowering plants,probablythe most studied part of the biosphere).ethnopharmacologyappearsto oft-era reasonableselectivestratcgyto be consideredin decidingwhich orsanismsto studv.
IF-rh'*'i*F**
DRUG OF NilODERN THEPROCESS DISCOVERY USING ETHNOPHARMACOLOGY of severalstages.The first stage The cliscoveryprocessis cot-nposed nraterialfor somepurrrust be the l'epolteduseof a nitturallt,-occttrrirrg posewhich canbe relatedto a nreclicitluse.Considerationol'the cultr-rral with it is importantin decidingpossiblebasesof practice-s associated thenthe the reputedactivity.Ilthe-reis an indicationofa genuineefl-ect, accordingto scientific materialneedsto bc identifiedand characterized nomenclature.It can t.henbe collectecllbl expcrimetttalstudies.r-rsually comprisingsometeslsfor relevantbiolo-eicalactir,itylinkeclrvith isolapresentu'hich might tion and structuledeterminationof any chet-nicals 'active' cornpouncls by setare usr-rally discovet'ccl be lesponsible.The ofthe extractlinkedu'ith lestinglbl'activity eralcyclcsoffl'actionation areisolirtedfiorn the activefracof eachtlaction.until pure contpouncls These comll'actionaticln. tions. a processknown as bioassay-guicled pounds. once their activity is proven and their ntolecr-rlarstl'tlcture ascertained,serve as the leads fbr cleveloprnentof clinically-useftrl in dctail below'. products.Thesevariousstagesarc cliscussecl sources lnformqtion The most reliabletype of infbrrnationarisesflonr in-depthstudiescarof a par'ried out by field workers.living in the partictrlat'contr.ttunit-v ticularethnicgroup.ou the useofthe local plantsand othernlaterials. This usuallycomplisesfrequentcontlrunicationrviththe local population. pref'erablyin theil own langttage.In shouldbe noted.ltowevcr. that an extensiveknowledgeof traditionalmedicinesmay residewith only a few people and a fbctrs on this gt'oup wor.rldyield greater results.However.many suchpeoplearc often reluctuntto gir-r'it$u1 'protccted'insotnewa1,.and this is knowledgcwhich is rcgardedas by concernthat sucl-tknowledgemay be exploitedby clrug exacerbated of this companieswith linle or no rcturn to the oliginal possessot's knowledge.
E/
SOMECURRENT TRENDS
'.c -ol
-D
c c)
U
o E s o .l
I
;.
-q
! ? o
,l
E
c
c) C
' 7 , - 6 o E > .
S q ,
{.=
!t= a 7
+ 9 l s q
;
(
:
:
h
C
x ' ; . q . :
tr !
.3:
u
d
=
o ) g I 5 ! 9 c < + ! ; , i > V Y c
5 : E ;
€ . X
h I o c ' N i E : x o v * L . - = c o
c h
i
o=
!.i
c Y s =
F
s E i Y i E
d- =
72
e u cg
a o .E ,9 'u
[|
U a)
E o n
E E c
.c
,o
s o_ -E
.c o s :z
L
.9
-
I
! E E !
c t
€
ul E' t o
I
o
o E
E o
n
L
!
r
Y
^
/-v
I
o
!,
o o
(
o o ? a
o
5 'g
c
+ (E
z(\
E l
q
F
.E o
E o
o
e{ \
E o
o
E
a
ta
o
.l -o
U)
\ / \
/ \
O
g o o.
,
zFO
\ -\ -/ /
\_/ / F
o
I () o
0 -o f
E o o T X c :
o E
o -o '6 o_ o U
TRADITIONAL PLANTMEDICINESAS A SOURCEOF NEW DRUGS
o
o
o .2 O
E C o-Y
y
f
c
J
- .ctr Y
f . Y
E X o RFfi
- o -o-9
3 E
9 <
c
E ; ; J -.9-o
.2 o o
.e-)
-o .Y o o -:? ^
o o-
E
F
a
o-o
*
o
^
o
c
6
E
v s
;
Eh<
()
:
'; V
o o o >ry
=
Q O
o ;-r
- t ; . :
o o9
o
o
o
!
6
O
Y R E . X
:
n
;sF
o<
^v;
E . :
x o) Y
o
o
|
/
-Z
/ 1
,
I V _p\.,,\--o
I,{ v
\
/
!
I
o
7
/,/-\ \ \ / / \- ^ / \o
(
,
t
-v
6
I t\
.-E
o .c E
o .c t o-
E
o) U
< L
I
\ I
-
o
v
o
f,z-(
I v
\ /
O
/,/-\ \ \ \_____-/ \ / I
Q-Z
\
o
a-z
L ^
o
-
z
//-\ \ \ \\ \ ,//
Q-Z +\
-
I
\
/
F\)
z
^
/ /--v I
I
N
I
c o s
c) . r :
r' .:l : >-x 9r : = ; i
9 u x o _12 ;d J t
--l
,l*
CJ .c
E
= a)
z
o
o c
E
.9) 6 o
I
o E x
o
o .c
:9 4
o
L
E
TT RENDS S O M EC U R R E N
Toble17.3 Herbol
Erhnophormocologicol origins(otherlhen Europe)of some commonherbol 'medicines'
'medicine'
ond botanicol source
AfriconPruneBorkPygeumofriconum BlockCohoshCimicifugorocemosorools
Devil'sclow Horpogophytu m procumbensrool DongQuoi Angelicosinensisroot Eleutherococcus, Siberion GinsengEleutherococcu s roots senlicosus Ginkgo Ginkgobilobo leoves GinsengPonoxginsengrool GoldenSeolHydrastisconodensisroots Gotu Kolo, HydrocoiyleCentella osioticoherb GuoronoPoullinia cupanoseedkernels Kovo Pipermethysticum rools l^^^.h^
p ^ , ,n ' 4 . . ^
Sow polmetlo Serenoo repens fruits
Currentuse
Geogrophicol source
Toreducebenignprosfotic hyperplosio Depression ossocioled with menstruol cycleond menopouse Rheumotism
CentrolAfriconhighlonds
Menopousol symptoms
Lntno
Reliefof fotigue,generol heolth
Troditionoluse (if different)
NorthAmerico
Arthritis, neurolgio; mensfruotion d isorders
Southwest Africo (Kolohori desert)
Siberio
Purgotive ond for treotingulcers ond boils lrregulormenslruotion, blood deficiency Tohelpcope with slress
To reduceCNS effects of ogeing Reliefof fotigue,generol heolth Cotorrh,oppetiteond digestion stimulont Woundheolingond skin conditions Tonic Anxiolytic ond tronquillizer
LNINO
Forbronchitis
S t i m u l o t i o nf i m m u n e systemto prevent infections Toreducebenignproslotic hyperplosio
TropicolSouthAmerico
thesituationis that Althoughthc in-depthapproachis rnostr"aluable. mostof the dlugs rvhichhavebeendevelopedhavearisenfrom lessrigThns orousobservations as a by-productof conquestor ccllonization. the more enlightened members of the Spanish t:onquistudoresoI CentralarrdSouthArnericanotedthe practicesof the valious Native American groups. and members of the Blitish and French colonial administrtrtions^ together with non-governmentalgroups such as Christianmissionaries,cataloguedthe usesof plantsin Afiica. Nlost of thesc observationscannot rro*' be checkedin any way at first-hand.sincethe authorsare long dcad,br-rttheir recorcls,booksand have becn lefi as sollrcesof infbrmation.This also other docunrents appliesto cultureswhich havelett sometype of writtcnrccordsso that irrl'olmation ou materials used in rnedicine in ancient E-eypt, Babl'lonia.IndiaandChinais available. Bcibre such knou'ledgecan be investiglted scicntiflcally,thc infbrmation providedq'ill often needclarificationand transiationinto scientific terrrs. Of particul:rrimportanceis the correctidentificationof the speciesusedr,''hichcan be vcry ditlicult due to a lack of. or poor quality, data on the part illustrationsas vn'cllas languagedifficulties.Howe'n'er. used.time of collection.methodsof preparation.fbrmulationand application are also nccessarysincethey all affect the natureand anrountof any biologically activeconrpounds.Any restrictionson usedue to tinie of year may be importantsincethey rnay indicatelo'nvlevels(leadingto risk of toxicity)of active ineflicacy)or high levels(with concornitant from being cornpounds. Similarly.any typesof individualsexclr-rcied lreatedrnayindicategloupsat risk clueto age,genderor occupation. Definition of the diseasestatein Westernn-redicaltelms may also not be easy if the intbrrnationis derivedfrom a culturewhere concepts of diseasecause and symptomsarc ver)'different. In many
^1
.
Chino,Koreo NorthAmerico
Goskointestinol ond skindisoroers
lndio
Rheumotism ond skinconditions
NorthernAmozonforest T o n g oF, i i i
Stimulont drink Socioldrinkto oid reloxotion, treotment for skinconditions Generoltonic
SouiheoslUSA
lecords the condition treatedis describedby a symptom which might be due to a numberof diseasestates,e.g.a headachemight be due to stress.tiredness,migraine attackor a brain tumour. Conversely,a parof symptoms ticular diseasestalemight be characterizedby a nurr-rber which all have to be addressedwhen searchinglbr possibleleads to tleating that illness.Thus, as an example,when searchingan inventory of plants with a view to selectingthose used fbr diabetes,those usedtbr treatingexcessurination,weaknessandulcersshouldbe considered.especiallyif diabetesis not recognizedas a distinct illness diagnosedby sweet-tastingurine. Unfbrtunately,all thesef'actorsare 'plant often ignored in current researchwhen often a staternentthat speciesX is usedto treatillnessY by peopleliving in Z'is considered to provide adequateethnopharmacologicalintbrmation. Such vague statementsdo not take into accountall the possiblesourcesof variation of biologicalactivitywhich mustbe consideledbeforeany investigationproceeds. 5cientific investigotion Extraction. The extlact used for testing should approximate as closely as possibleto that obtained by the traditional processused. Much researchwhich hasbeenpublishedon the chemistryand activity of medicinalspeciesis not very relevantto the traditionalusesbecause it has concentratedon extractsmade with non-polar solventssuch as ethel or chloroform whereaspolar solventsare most commonly used. ln rntrnycasesthesewill be simple extractionswith hot water but a variety of other solventsmay be used as well as various additivesor treatmentof the materialbeforeuse (seeTable i7.4). In most instances however. it is likely that fairly polar compoundswill be extracted.
T R A D I T I O N APLL A N TM E D I C I N E A SS A S O U R C E OF NEW DRUGS
Tqble | 7.4
Effect of pre-lreotment
freatment
Beforeextrocfion Roosting Sookingin woter Sioroge
Exkoctionprocess Woter + oshes
qnd exlrqction
processes on plonf consliluenls.
EftCCI
Equivalent scientificprocess
Exomplefrom troditionol medicines
Deslruction of unwonted componenrS Hydrolysis of glycosldes
Heotingol reguloted temperoture
Enzymotic ociivityleodingto formotion of desired compounds or removolor unwontedcompounds
lncubotion with purifiedenzymes understondordized conditions
R e m o v o lo f t o x i c p r o t e i n sf r o m s e e d so f Cossia occidentolis {West Africo} R e m o v o lo f c y o n o g e n i c g l y c o s i d e sf r o m Cossovo F o r m o i i o no f v o n i l l o f l o v o u r R e m o v o lo f t o x i c o n t h r o n e sf r o m C o s c o r o
Useof ommonioor diluteolkoli
'quid' 'chewing' Troditionol usedfor coco
Useof diluteocids Diluteolcoholexlroction
Mony troditionol tinctures
Alkolinemediumfovours exiroction of phenolsond ocids,slowsreleoseof olkoloids Woler+ ocidicfruitiuice Fovoursexlroctionof olkoloids Locololcoholicbeveroges Extroction of lesspolor compounds Animolfotsor plontoils Lipophilic compounds
Boiling
Petrolof chloroformextroction
althoughthe solubility of less polar substancesmay be elevatedconsiderablydueto solubilizingcompoundse.g.saponins, alsobeingpresent. Thus, an aqueous extract of the antimalarial plant. Artentisitt turtrtuu,conlainsappreciablelevelsof the majol ar-rtin-ralarial sesquiterpeneartemisinin, which on its own hasvery low solubilityin water.
Hypericumperforotumoil exhoctfor heotingburns
ing causesof the disease,e.g. testslbr the efficacl' of a preparationfbr an inflammatorydiseasesuchas arthritisshouidencompasskey mechanismsassociatedwith the lbmration of the variousmediatorsinvolved such as tl-re lipoxygenasesand cyclo-
'illDt-
known conpound.
TT RENDS S O M EC U R R E N '
.
i
': ' :' :,',:"Flovopiridol 30l,i= ffiODERNEXAMPIESOFIDRU6 The bark of the Indian tree D,-sox),lummalabaricunt(Meliaceae)was .. ] , , ' . DISCOVE.RY BASEDON THE traditionallyusedto treatarthritisand investigationsinto the relatedD. E?HNOPHARNfiACOTOGIC*I. APPROAEH.,. binectarfbrum provided rohitukine, a chromone alkaloid which exAlthough the list of eristin-edrugsintroducedas a result of ethnopharmacologicalleadsis impressive,sornenewerinterestingexamplesof con-rpounds arising from this approachhave appealedin recentyears. Relevantformulaeareillustratedin Fig. 17.1. Prostrqtin An ethnobotanicalsr.rrveyo1'Samoacan'iedout by the ArnericanPaul Cox in the late 1980srevealedthe traditional use of a hot water infusion of the stemwood of Homalarthusnutansfbr the viral diseaseyellow fever. Work carried out in conjunctionwith the National Cancer Institutein the USA resultedin the isolationof a phorbolnamedprostratin which was for-rndto be effectiveagainstthe killing of humanhost cells by HIV. Prostratinappearedto stimulateproteinkinaseC and this was a novel mode of action for an anti-HIV drug. Although extensive trials were conrmenced,plostratin has since been dropped from the anti-HIV drug developmentprogrammedLreto sometoxic effects.
hibited anti-inflammatoryactivity in laboratoryanimals.Studiesinto the mechanismof action found that it was different fiom many other natural anti-intlammatory compounds in that it inhibited tyrosine kinase.A seriesof analogouscompoundswas developedto optimize the effect and the flavonoid derivativeflavopiridol was shorvnto have superioractivity.Sincenumerousoncogenesencodefor the production of protein with such kinase activity, inhibition may decreasetumour growth and this was found to be the casewith flavopiridol, which is now in advancedstagesof clinical testingas an anticanceragent.
HuperzineA A tea fi'om Huperiin serrata, a club moss,was a traditionaldrink for elderly people in several areas of China. During the last 20 years huperzineA, an alkaloid with cholinesterase inhibitory properties.has beenisolatedand shownto have significantbeneficialeffectson memoly in patients.Since memory appearsto be impaired in patientswith 1owlevelsof acetylcholine(ACh) in the brain,inhibition of the enzyme
HO HO
o
OH CHs Prostratin R =
-)'
A
Rohitukine
Flavopiridol
cl
CHs Huperzine A
Galanthamine
"9
Fig. l7.l by t h e Structures of somemoderndrugsdiscovered e t h n o p h o r m o c o l o goi cpoplr o o c h .
o Artemisinin
T R A D I T I O N APLL A N TM E D I C I N E A S S A S O U R C EO F N E W D R U G S which degradesACh will have the net efl'ectof raisingACh levels and therebyimproving memory.HuperzineA is in clinical usein China fbr treatmentof elderly patientsshowinglossof memory and clinical trials in other countriesarebeing planned.
The possiblevalidationoftheir traditionalremediesby the scientific ethnopharmacological approachis also valuablein helping small ethnic groupsretain or recovertheir senseof identity and value, especially when threatenedby globalizationand cultural imperialismby more politically or commerciallypowerful groups.
Golonthomine Galanthamine.originally isolatedfrom a speciesof Galanthus(snowPROBLEMS WITH THE drop) found in the Balkans and used by the local people for muscle ETH NOPHARMACOTOGICAT APPROACH weakness,has also been found to have cholinesteraseinhibitory efTects.After large-scaleproductionof galanthamineby extractionof Reliobilityof informotion daf1bdilbulbs (Narcissls spp.).successfulclinical studieshave 1edto Someof the difficulties of obtainingaccurateand reliable infbrmation the licensingof this compoundin Europe as a symptomatictreatment have been discussedabove. It should also be noted that the use of a for the early stagesofAlzheimer's disease. plant may not be due to rationalobservationor experimentalevidence. but may be based on a subjectiveconsideration.Probabty the most Artemisinin notoriousexampleof an irrational approachis the 'doctrine of signaArtemisia annun ts a plant which was recordedin old Chinesemanu- tures'wherebysomesensoryaspectof the plant was thoughtto indicate scripts as being useful fbr treating f'eversand malaria. Teststo identity its medicinal usefulness.e.g. a plant with yellow juice may be conthe antimalarial compound present resulted in the isolation of sideredfor treatingjaundice.one with heart-shaped leavesfor cardiac artemisinin. a novel sesquiterpenewith an endoperoxide group. complaints,etc. Although, in some cases,relevant activity has been Artemisinin actsagainstthe malaria parasitein a very dilTerentway liom fbund for plants identifiedin this way. theseare usuallythe exceptions quinine and most of the syntheticquinolineantimalarials.SeverallaLrge ratherthan the rule. trials studieshave shown the efficacy of arlemisinin but the more soluble analoguesartemetherand artesunatesare now widely Lrsedand are rec- Biologicol vqriotion ommendedby WHO as antirnalarials in chloroquine-fesistant areas. On a scientificlevel the chiefconcernsarethc difficulty ofknowing the chemicalcorr-rposition of a plant or its extractdue to its complexity and also dne to the variation causedby geneticor environmentalfactors. THEVATUEOF THEETHNOPHARMACOTOGICAT This complicateselucidationof the activecompoundsand also makes APPROACH the activity of different batchesless reliable. Indigenousknowledge Efficocyond sofety often has subtleways of distinguishing'best' plant speciesor r arieties The argumentmost often used to suppofi the ethnopharmacological and only thoror.rgh investigationwill uncoverthe criteriaused. approachto drug discovery is the fact that a plant material has been used fbr generationsin a particular culture. Lack of technoiogical Lossof species sophisticationis not synonymouswith a lack of appreciationof effiIt is estimatedthat 25% of the flowering plant speciesof the world cacy or safetyand so it is likely that therewould be no seriousadverse might become extinct within the first 25 years of the 2lst century. effectsassociatedwith the regularuse of the material.In many casesit Clirnate change,increasingpopulation nraking denrandson land and has been shown that potential harm is minimized through a seiected resources,as well as commercialexploitationof the environrrent all method of preparationof the material. by its administrationbeing play a part in this and result in a loss of habitats.It is not unusualto restrictedto trainedpersonnel(most commonly the 'medicinemen (or record the presenceof an interestingspeciesin one locality only to women)' of the community),or by its being usedin specialways, par- return a few monthslater to find that the whole areahas beencleared ticularly by the additionof other materialswhich may decreasethe tox- for somecommercialactivity.Although ethnicgroupsin all partsof the icity by counteringunwantedpharmacologicaleffects or by altering world are affected.and most have a rich fund of knowled-eeabout the the bioavailabilityor metabolismof the material.The additionof alka- medicinal usesof local organisms,it is probably thosesmall groupsin line ashto cocaleavesin the traditionalmethodof 'chewing'them is a the centresof biodiversity,especiallytr"opicalrainfor.ests, who aremost good exampleof this. The high pH favoursthe lesswater-solubleform vulnerableto irretrievableloss of species.The imminenr extinction or of cocaine,so aflecting its releaseinto the saliva and uptake into the increasingrarity of speciesare often promoted as a loss of potential bloodstream.and possiblyreducingthe addictivepotential. new drugs fbr the developedworld; it should be rememberedthat the local inhabitantsmay lose many of their basic affordableor available Economic ond sociopoliticol considerqtions medicines. Although not closely relatedto the processof'drug discovery'by the In light of this, many medicinally-importantspecieshave beensubindustrially-developedcountries,the scientific validation of a local jected to some lbrnt of cr.rltivationand in severalplaces gardensof remedy from a developingcountry may encourageits r.rseand intro- medicinalplants,formerly collectedfiom the wild. are now being culductioninto therapyin its original habitat,or its growth and adoptionas tivatedto provide local healerswith speciesthat they formerly collecttherapyin areaswith similar growing conditions.Cultivation and pro- ed from the wild. duction of extractsfrom such plants might be a substitutefor more expensiveWesterndrugsand medicines.especiallyin countrieswhere Loss of knowledge healthcareresourcesare stretched. The current opportunity to exploit ethnopharmacologymay not last A notable example of this is Plantasdo Nordeste,a collaboration long. Whilst much concernhas been quite rightly expressedover the betweenthe Royal Botanic GardensKew. UK and a consoftiumof sci- disappearance of the world's biodiversity and habitats.Iess attention entistsand agriculturistsin the impoverishedregion of north-eastern has beenpaid to the threatto the immensewealth of knowledgeabout Brazil. The project has evaluatedsome of the local medicinal plants the medicinal usesof the local flora which exists amongstthreatened and promotedthe cultivation of them acrossthe region as an aspectol ethnic groups.The irretrievableloss of large amountsof this knowhealthcare.togetherwith the training ofpersonnelin their use. ledgeis possibie.due not only to the extinctionofplant speciescaused
S O M EC U R R E NTTR E N D S by clirnatechange,urbanerpansionand destluctionofhabitat. but nlso by the breakdownin traditionalsocietalstructuresof the transmissiorl of such krrowledge.The threat is especiallyacute in cultures whele intorrration is oral rather than written and is exacerbatedby urban drift, particulally of young people.neglectof local materials.adoption of Western globalized products and health strltcturesand by war. famine and other causesof miglation and consequentdisruption of traditionalsociety.
and returns1i'ominvestmenthas been consideredclosely by pharmaceutical companies and some have decided not to take the risks involved. Some other companies,however,have been willing to sign agreementsaimedat sharingprofits directly or making substantialpaymentsto countriesin exchangefbr accessto their flora for testingpurposes.
coNctustoN
The need for dereplicotion Extractslrorn plants may shttw an interestingactivity and conventionfractionally this is fbllowed by labolious,prolongedbioassay-guided such However. responsible. the compotinds isolate ation to considerableefTortmay result in the isolationof known compoundsor suchaspolyphenols, the activity rnay be fbund to be due to substances, which have a generalinhibitory activitl on lnarly enzymes.This problern may be ovelcome by the use of dereplicationprocedureswhere suspectedknown activecompoundtypesare removedby fractionation and pulification procedures.Alternatively.activeextractsand tiactions can be examined by techniques,such as chromatographylinked to massspectrometry,which can aftbrd subst.rntialamountsof information about each componentand data fiom the peaks obtainedcan be screenedagainsta datalibrary of known compounds.
The study of plants used in traditionalmedicinehas receivednew impetuswith the introductionof a wide valiety of small-scalebioassay methodsand improvedmethodsfor fiactionation,isolationand characterizationof conpounds. In some cases,individual compoundsresponsiblefor the reputedactivity have been isolatedand used as lead molecules.However.it is also apparentthat, in many cases,the observedefI'ectsseenin patientsor animalsare due to a mixture of constituents,perhaps each contributing a difl'erent biological eftbct to the overall activity.Thus it is likely that not only wiII tladitionai medicinescontinue to provide new moleculesfbr drug discovery,but also may form the basis lbl the wider acceptance of crude extracts. in a standardizedform, as another type of medicine in orthodoxpractice.
Intellectuol property righrs issues
Furtherreoding
approachhave The commercial aspectsof the ethnopharmacological arousedmuch controversyin recent yearswith regardto the intellectual propertyrights of the groupshaving the knowledge.Severalinternationalagreements,partictllarlythe Rio Declarationand Convention of Biological Diversity (CBD) of 1992 have concentratedon sharing with the solrrcecountriesthe benefits and profits which might arise frorn the developmentof new drugs basedon ethnopharmacological leads.The etl'ectthat such measuresmight have on their patentrights
Balick M J, Cox PA | 997 Plants.peopleand culture:the scienceof ethnobotany.Freernan.London Chadwick D J. Marsh J (eds) 199'1Ethnobotanyand the searchlbr new drugs Ciba FoundationSyn.rposiun185.Wiley. Chichester.UK Pliillipson J D. AndersonL A 1989Ethnopharmacologyand western medicine. Journalof Ethnopharmacology25: 6l Proceedingsof the Filst lntemationalConglessof Ethnopharmacology, I992. 32: 1-23-5 Strasbourg.| 990. Journalof Ethnopharn.racology, Proceedingsof the SecondInternationalCongressof Ethnopharn'racology. Uppsala.1992.Journalof Ethnopharnracology1993.38: 89-225
{n-*,
E
PART
5
Phytochemistry
I |[
{ ) <7
{.,
m
-*4 t
h
{
Jl,r.. I
ft
rE
PART
5
Phyfochemistry
I t"
{
d,.
t
-r*
..,rtia&i
rll*rt
.,. i
""'.' iffi . r ,
ffi
Generol methods ossocioted withthe phytochemicol investigotion of herbolproducts
EXTRACTION OF PLANTMATERIAT I38 SEPARATION AND ISOLATIONOF : {
CONSTITUENTSI39 CHARACTERIZATION OF ISOTATED
coMPouNDs r46 BIOGENETICINVESTIGATIONS 146 TRACER TECHNIQUES 146 ISOLATED ORGANS,TISSUES AND
cE[s
148
GRAFTS 149 MUTANTSTRAINS 149
Befbre about 1800only slow progresswas madein phytochemistry.A few compoundssuch as cane-sugar,starch,camphorand benzoicacid had long beenknown, as their preparationwas extremelysimple; also complex mixtures such as fats, fixed oils, volatile oi1s,tars and resins had beenpreparedand used,althoughvirtually nothing was known of their composition.The early scientific workers in the phytochemical field failed to appreciatethe extremecomplexity of the materialsthey were trying to investigateand almost entirely lacked the techniques necessaryfor real progress.Many hundredsof plants were burnt to yield ashesand theseearly investigatorswere disappointedto find no significant differencesbetween the ashesof poisonousand those of non-poisonousplants.Expression.aqueousextractionand evaporation had long been used for the preparationof sugarfiom sugar-caneand the FrenchapothecaryNicholasLemdry (1645-1715)extendedrhe use of extractionprocessesand made use of alcohol as a solvent.Robert Boyle (1627-9 1) disposedofthe ancienttheory ofAristotle that matter was composedof four elements,and although he never isolated an alkaloid,he was obviously moving in the right directionwhen he trea! ed opium with potassiumcarbonateand alcohol. In 1747sucrosewas isolatedliom many plants,including sugarbeet,by the GermanapothecaryA. S. Marggraf (1709-80). K. W. Scheele(1142-86) was highly successfulin the phytochemicalfield and isolatedcitric. gallic, malic. oxalic, tartaricand prussicacids. In the nineteenthcenturyprogressbecamemore rapid. In 1803narcotine, the first alkaloid, was isolated;morphine.strychnine,emetine and many othersfollowed rapidly. Between l8l3 and 1823 Chevreul elucidatedthe chemicalnatureof fats and fixed oils. Until well into the middle of the twentiethcentury the main emphasisin natural-product chemistryremainedthe isolationand structuredeterminationof a wide variety of compounds.At this point it becameapparentthat the principal structuraltypes commonly found in plants had beenlargely elucidated. Indeed, by this time the attentionof natural-productchemists was turning to the elucidation of the actual biosyntheticpathways found in the plant. Suchstudiesweremadepossibleby the introduction of new techniquesof separationand analysis.This emphasishas continued until today,when most of the major pathways,including stereochemical aspects,have been studied in some depth. There has also developeda renewed interest in the patternsof occurrenceof compoundsin plants(comparativephytochemistry). Not all the chemical compoundselaboratedby plants are ol equal interestto the pharmacognosist. Until relatively recentlythe so-called 'active'principles were frequentlyalkaloidsor specificglycosidesusually with pronounced pharmacologicalproperties; these therefore receivedspecialattention,and in large measureconstitutedthe principal plant drugs ofthe allopathicsystemof medicine.It is now realized that many other constituentsof plants, particularly those associated with herbal medicine, have medicinal propertieswhich manifest themselvesin more subtle and less dramatic ways than the obviously poisonous plants. This has considerably widened the scope of plant metabolitesconsideredworthy of mole detailed investigation.Other groups such as carbohydrates, fats and proteinsare of dietetic importance,and many such as starchesand gums are used in pharmacybut lack any markedpharmacologicalaction.Substances, such as calcium oxalate,silica,Iignin and colouringmatters,may be of assistance in the identificationof drugsand the detectionof adulteration. As a result of the recentinterestin the plant kingdom as a potential sourceof new drugs, strategiesfor the fractionationof plant extracts basedon biological activity rather than on a particularclass of compound, have been developed.The chemicalexaminationfollows after the isolationolthe acrivefracrion. The phytochemicalinvestigationof a plant may thus involve the fbllowing: authenticationand extractionof the plant material;separation
ti,
PHYTOCHEMISTRY in the many common organic solventsand leavesno solvent residr"res product. It also allows a low temperatllreprocessand has proved of value fbr the extractionof labile expensivefragrancesand medicinal phytochemicals.To render it nore polar a smal1amount of modifier. e.g.methanol.may be addedto the carbondioxide.The high pressures' the high temperatures.involr'ed in supercritiand fbr some substances of the technique' principaldisadvantages the are extraction tluicl cal MATERIAT EXTRACTIONOF PLANT Pioneerwork on medicinalplantswas carriedout by Stahl andcoworkcitedtherein).They studied ers(PlanruMed. 1980,40, 12,andreferences as much time All plant materialusedshouldbe properly authenticated. and liquefied nitlous dioxide carbon supercritical and of liquefied the use and money can be wastedon the examinationof maferialof doubtful including various plant constituents' various of the extractioll tbr oxicle of the the nature on procedure depends origin. The choiceof extraction of chamomile.With thecomponents pyrethrins and the alkaloids. of types are materials Dded be isolated. to components and the plant material pyrethrumtlower extract,the contentof pylethrins is substantiallyhigher usuallypowderedbefbreextraction,whereasfiesh plants(leaves.etc ) (up to 50c/c)than in commercially availablepetroleumetherextracts.By a The as alcohol can be homogenizedol maceratedwith a solventsuch two-step precipitation the active ingredientscan be raised to up to 607c dropping by leaves liesh latteris alsoparticultrrlyuselul fbr stabilizing withor( decompositionof the thermolabilepyrethrins. for many general solvent a is Alcohol solvent. boiling them into the Fulther examplesinvolving the extractionof phytochemicalswith plantconstituents(mostfixed oils excepted)andas suchmay give probcarbondioxide follow: supercritical lems in the subsequentelimination of pignlents.resins, etc Water(essential and petroleum immiscible solventsare widely used-light Decall-einationof greencoffee (industr"i(l) Alkaloids: fixed oils. steroids).ether and chlorofbrm (alkaloids'quinones)'The al application) basificaextractionof organicbases(e.g.alkaloids)usuallynecessitates Isolation of vindoline from CatharttnthtLs tion of the plant materialif a water-imrnisciblesolventis to be used:fbr roseu.\ aromatic acids anclphenolsacidificationmay be required Extraction (2) Diterpene: Extractionoftaxol from lrrras brevfolia itself may be perfbrmedby repeatedmacerationwith agitation.pelcola(extractionmore selectivethan convention or by continuousextraction(e.g.in a Soxhletextractor,Fig' lzl'2)' tional ethanolextraction) Specialmethoclsfor volatile oils. such as rhe enfleurtlsaprocess'are also from T. t:ttspidata consicleredin Chapter 23. Uitrasound may enhancethe extraction (3) Fixed oils: E r t r u c t i o no l ' o i l l r o n re r e n i n cp r i m r o s e processtor someplant materialsand the BP usesthis in the preparation l s u b t l es h i f t i n t r i g l y c e r i dceo m p o : i t i o n : of a 50% ethanolicsolutionof opium for the assayof alkaloids' oxidation of y-linolenic acid during extractionreduced) Spouted bed extroction Extractionof annattoseeds (4) Pigments: of annattopowder from the In cerlain instances.as in the prodr-rction In conjunctionwith gas chromatography lactones (5) Sesquiterpene of Bl-rrrorellaru. the physicalremovalof the pigmentlayer of the seecls for the isolation of Parthenolide seed-coatcan yield a lessimpaired productthan that ploducedby solfrom feverl-ew.Addition of methanol vent extraction.Such methodscan involve the use of a ball rnill or a or methyl cyanide as CO2 modifiers spouteclbed unit. A developmentof the latter.the conical spoutedbed gave higher yields but produced coextractor.has been investigatedfor annattoproduction' Basically it extractlves. consistsof a cylinder taperedat both endsand containingthe seedsat methanolin COl for the The useof 1oa/o the lower end through which ajet ofhot air is forced. Seedsand pigextraction of trilactones fiom ginkgo ment-loadedfine particles are propelled into the space above fiom could be of commercialsignificance. whencethe see
of the isoland isolationof the constituentsof interest:characterization atedcompounds;investigationof the biosyntheticpathwaysto partlcular compounds:and quantitativeevaluations.Parallelto this may be the of the separatedcornponents. pharmacologicalassessment
.
aD'
GENERAL METHODS ASSOCIATED WITHTHEPHYTOCHEMICAL INVESTIGATION OF HERBAL PRODUCTS phase microextractiontibres, has extractedthe volatiie oil from the headspaceabove fiesh cut eucalyptusleaves(37o lbr l0 min.). The fibres were then desorbedat 200' fbr capillary gas chromatographyof the oil. Not surprisingly.the oil compositiondiiTersfr.omrharof steamdistilledoils.
SEPARATION AND ISOTATIONOF CONSTITUENTS As the instrumentationfor the structureelucidationof organic compoundsbecomesever rnoreeffective,and allows the useof increasingly small amountsof material.the most difficult operationin phytocherrical researchbecomesthat of the isolationand puiification of plant constituents.Although the chemical propertiesof functional groups and moietiescontainedin compoundssuchas aldehydes.phenolsand alkaloids can be exploited for their separationfrom other materials,such methodsmight not fiactionatecomponentsof the sameclass;it is in this latterareathat new techniquesareconstantlybeingdeveloped. Sublimotion Sublimationmay sometimesbe possibleon the whole drug, as in the isolationof cafl'einefrom tea or for the purificationof rr-raterials present in a crude extrzrct.Modern equipmentemploys low pressureswith a strict control of temperature. Disfillotion Fractionaldistillation has beentraditionally usedfor the separationof the componentsof volatile mixtures: in phytochemistryit has been widely usedfbr the isolation of the componentsof volatile oils. On a laboratory scale it is not easy by this method to separateminor componentsof a mixture in a pure state and gas chromatographyis now routinelyused(q.v.). Steam distillation is much used to isolatevolatile oils and hydrocyanic acid from plant material. The TAS oven (see 'Thin-layer Chromatography')involves steam distillation on a semi-micro scale for the direct transf'erof volatile materialsfiom a powdereddrug to a thin-layerplate. Frqctionol liberotion Some groups of compoundslend themselvesto fractional liberation fiom a mixture. As an example.a mixture of alkaloid saltsin aqueous solution.when treatedwith aliqr.rots of alkali. wilt give first the weakest basein the fiee statefollowed by baseliberationin ascendingorder of basicity.If the mixture is shakenwith an olganic solventafter each addition,then a fractionatedseriesof baseswill be obtained.A similar schemecan be usedfor organicacidssolublein water-immisciblesolvents;in this case,startingwith a mixtureof the acidsalts,it is possible to fractionallyliberatethe acidsby additionof miner.alacids. Frqcfionql crystollizotion A method much usedin traditionalisolationsand still valuablefor the resolutionof oflen otherwiseintractablemixtures.The rnethodexploits the differencesin solubility of the componentsof a mixture in a particularsolvent.Frequently,derivativesof the particularcomponents areemployed(picratesof alkaloids,osazonesof sugars).
Adsorption chromotogrophy Of the variousmethodsof separating and isolatingplantconstituents. the 'chr omatographicprocedure'originatedby Tswett is one of the most usefll techniquesof generalapplication.The use of charcoalfbr the decolorization and clarification of solutionsis well known: colouredimourities
rl-3,
areaclsorbedbythecharcoalanda colourlesssolutionresultson flltration. All finely divided solidshave the power to adsorbother substances on their surfacesto a greateror lesserextent:similarly,all sr-rbstances are capableof bein-eadsorbed,some much rnore readily than others.This phenomenonof selectiveadsorptionis the fundamentalprinciple ol adsorptionchromatography.the general process of which may be describedwith referenceto one of Tswett'soriginalexperimcnts. A light petroleurr extract of green leavesis allowed to percolate slowly througha column ofpowdered calcium carbonatecontainedin a vertical glass tube. The pigmented contents of the solution are adsorbedon the substanceof the column and undergo separationas percolationproceeds.The more stfongly adsorbedpigments.xanthophyll and the chlorophylls, accumulatein distinct. characteristically coloured bands near the top of the colurnn, while the less strongly adsorbedpigments,the carotenes.accumulatelower down. Frequently.completeseparationof all the constituentsinto distinct bandsdoesnot result dLrringthe first 'adsorptionstage'.but the bands remain crowded togethernear the top of the column. Such a column may be developedby allowing more of the pure solvent to percolate through the column when the adsorbedmaterialsslowly pass downwards and the separatebandsbecome wiclerapart.In many casesthe processmay be renderedmore efficient by the use of a difTerentsolvent, one from which the substancesare less strongly adsorbed.If, fbr example. light petroleLrmcontaining a little alcohol is percolated through the chromatogram obtained in the experiment described above.the bandsbecomewider apart and passdown the column more rapidly than when pure light petroleum is used.As percolationcontinues,the lower bandsreachthe bottom of the column and disappear; the pigment is then obtainedin the solution leaving the bottom of the column. This processof desorptionis termedel.utionand the solution obtainedisthe eluate. It was fiom suchclassicexperimentsof Tswetton the separationof colouredcompoundsthat the term 'chromatography'aroseand it has remainedto describethis methodof fiactionationalthou-ehits application to colourlesssubstances is now universal. Substancesare more readily adsorbedliom non-polarsolventssr-rch as light petroleumand benzene,while polar solvents-alcohol, water and pyridine. for example-are useful eluting media:many substances are adsorbedat one pH and elutedat another. Various substancesrnay be used as adsorbingmaterials:alumina is the most common and other materials include kaolin. magnesium oxide, calcium carbonate.charcoaland sugars. When colourless substancesare chromatographed.the zones of adsorbedmaterialarenot visible to the eye,althoughthey rray, in some cases.be renderedappalentas fluorescentzoneswhen the column is examinedunder ultraviolet light. Failing this. it becomesnecessaryto divide the chromatograminto discrete portions and elute or extract eachportion separately.Sometimesit is more convenientto collect the eluatefrom the whole column in fl'actionsfbr individual exanination. The apparatusrequiredis simplc and consistsessentiallyof a vertical glasstubeinto which the adsorbenthasbeenpacked:a small plug of glasswool or a sinteredglassdisc, at the baseofthe tube, supportsthe column. With volatile developingsolventsit is irsually preferableto use a positivepressureat the headof the column. Numerousmodifications of the apparatusare usedfbr large-scaleoperations.fbr use with heatedsolventsand ibr chromatographyin the absenceof air or oxygen. Adsorptionchromatographyhas proved particularlyvaluablein the isolationand purification of vitamins.hormones,rnany alkaloids.car.diac glycosides.anthraquinones.etc. It is commonly ernployedas a 'clean-up' techniquefor the removal of unrvantedmaterialsliom plant extractsorior to assav.
PHYTOCHEMISTRY Thin-layer chromatographywith adsorbentssuch as alumina is an adaptationof the methodand is discussedseparatelyin this chapter. Portition chromotogrophy was introducedby Martin and Syngein l94l Partitionchrornatography for the separationof acetylatedamino acidsand was first appliedto the separationof alkaloids by Evans and Partridgein 1948.The method by the more sophisticatedHPLC (see has now beenlargelv superseded beiow) but it retains the advantageof being inexpensiveto set up and operate.The separationof the componentsof a mixture is, as in counter-cunentextraction,dependenton difTerencesin the partition coefficientsof the componentsbetweenan aqueousand an irnmiscible organicliquid. The aqueousphaseis usually the stationaryphaseand is intimately 'carrier' mixed with a suitable suchas silica gel, purilied kieselguhror powderedglass and packed in a column as in adsorptionchromatography.The mixture to be fractionatedis introducedon the column, in a small r,olumeof organic solvent,and the chromatogramis developed with more solventor successivelywith differentsolventsof increasing undergoeluting power.When wateris the stationaryphase,the solLrtes speedsdependingon ing separationtravel down the column at di1I'erent their partition coefficientbetweenthe two liquid phases;the use of a as bufTersolutionas aqueousphasewidens the scopeof the techniqLre. ionizationconstantsand partitioncoefllcientsareexploitedin effecting separatron. The separatedzones may be located by methods similar to those employed in adsorptionchromatography.With water as the aqueous phase,the positions of separatedzones of acids ol alkalis may be shown by ernpioyinga suitableindicator dissolvedin the water.This method is clearly not applicable to bufl'er-, acid- or alkali-loaded columns,and in thesecasescompleteelution (elutiondevelopment)of The eluateis collectedin aliquot the separatedzone is often necessary. portionsand estimatedchemicallyor physicallyfbr dissolvedsolute.A graphofthe analyticalfigure (titration,opticalrotation.opticaldensity, refractiveindex, etc.) for eachfraction of eluatemay then be plottedto show the degreeof separationof the solutes. The fractionationsobtainedin partition chromatographyare influenced to a considerabledegree by the displacementefTectof one solute on another and advantageis taken of this in displacement development,in which the chromatogramis developedwith a solution of an acid or a basethat is strongerthan any in the mixture to be separated.The effect is for the strongeracids or basesto displacethe weaker ones, resulting in a rapid clear-cut separationof the constituents.For the elution developmentof these separatedzones it is essentialthat there is no distortion of the zones.sincethe front of one band follows immediately on the tail of the precedingless acidic or lessbasiccomponent. There have been several theoreticaltreatmentsof partition chromatography,all involving certain approximations,since a theory taking into accountall known variableswould be extremelycomplicated. For general purposes,one of the most satisfactorytreatmentsof columns loadedwith water is that of Martin and Synge,in which the theoreticalplateconceptof fractionaldistillationis appliedto partition chromatography.In this theory,diffusion from one plate to anotheris taken as negligible and the pafiition of soiute betweentwo phasesis independentof concentrationand the presenceof other solutes.
thin-layerchromatography(TLC), it remainsthe methodof choicefor the tiactionationof somegroupsof substances. The solLrtionofcomponentsto be separatedis appliedas a spotnear one end of a preparedfilter-paperstrip.The paperis then supportedin an airtight chamberwhich has an atmospheresaturatedwith soivent and water.and a supplyof the water-saturated solvent.The most satisfactory solventsare those which are partially miscible with water, such as phenol,n-butanoland amyl alcohol.Either the papermay be dippedin the solvent mixture so that the solvent front travels up the paper (ascendingtechnique)or the trough of solventmay be supportedat the top of the chamber, in which case the solvent ffavels down the paper (descendingtechnique).The BP 2000 givesdetailsof both methods.As the solventmoves,the componentsalso move along the paperat varying rates.dependingmainly on the differencesin their partitioncoefficients between the aqueous(hydration shell of cellulosefibres) and organic phases.After the filter-paperstrips have been dried, the poscomponentscanbe revealedby the useof suitable itions of the separated developingagents:ninhydrin solutionfor amino acids;iodine solution (or vapour) or a modified Dragendorff's reagent for alkaloids; ferric derivatives.antichloridesolutionfor phenols;alkali lbr anthraquinone mony trichloride in chloroform lbr steroidsand some componentsof volatile oils; anilinehydrogenphthalatereagentfor sugars.The relative positionsof the componentsand the size of the spotsdependupon the solvent.and this shouldbe selectedto give good separationof the componentswith welfdefined. compactspots.Improvedseparationof mixturescan often be obtainedby adjustingthe acidity of the solventwith ammonia, acetic acid or hydrochloric acid or by impregnating the paper with a buffer solutionor formamidesolution. For the separationof some substancesit is necessaryto use a twodimensionalchromatogram:first one solvent is run in one direction, then, atter drying ofthe paper,a secondsolventis run in a directionat right anglesto the first-this is particularlyapplicableto mixtures of aminoacids. The ratio betweenthe distancetravelledon the paperby a component of the test solution and the distancetravelled by the solvent is termed the Rp value and, under standardconditions,this is a constant for the particular compound.However, in practice, variationsof Rp otien occur and it is desirableto run referencecompoundsalongside unknown mixtures. The quantity of substancepresentdeterminesthe size of the spot with any one solventand can be made the basisof quantitativeevaluation. Also, the separatedcomponentsol the original mixture can be separatelyeluted from the chromatogram,by ffeating the cut-out spots with a suitable solvent, and then determinedquantitativelyby some suitable method-for example,fluorescenceanalysis,colorimetry or ultraviolet adsorption. Drugs so evaluated include aloes, digitalis, ergot, hemlock, lobelia, nux vomica. opium, rauwolfia, rhubarb, herbsand volatile oils. broom" solanaceous
liquid chromotogrophy High-performqnce (HPtC)/High-speed LC HPLC is a liquid column chromatographysystemwhich employsrelatively narrow columns (about 5 mm diameter fbr analytical work) operatingat ambienttemperatureor up to about200'C at pressuresup to 200 atm (20 000 kPa). The columnsare costly and it is usualto employ a small precolumn containing a cartridge of packing material to remove adventitious materialswhich might otherwise damagethe main column. Normal Portition chromqtogrophy on poper ln l9ll Consden,Gordon and Martin introduceda methodof partition flow rates of eluate are 2-5 ml min-l but can be up to 10ml min-l. 'carriers'for the analysis dependingon the diameterof the column and the appliedpressure.The chromatographyusing stripsoffilter paperas of amino acid n-rixtures.The techniquewas extendedto all classes apparatusis suitablefor all types of liquid chromatographycolumns (adsorption,partition by the use of bonded liquid phases,reversed of natural products, and although to a large measurerepiaced by
GENERAT METHODSASSOCIATED WITH THEPHYTOCHEMICAL INVESTIGATION OF HERBAL PRODUCTS Solvent
Solvent
Precolumn Pumps
Fig. I 8. I Schemotic representotion of opporotus for high-performononce liquidchromotogrophy utilizingtwo solvenls.
Injection
Pressuregouge
Frocfioncollector
oH
CtrH"r
\ . / si . / \ -o o'
l
HO-
l
-si-o-si-o-siI t
oH
l
l
I
Si -
_ o
I I
o-si
oH
-o-
I
To reduce tailing efTectswhich might be causedby the remaining liee hydroxyl groups the latter can be masked by treatment with a short-chainsilane,usuallytrimethylchlorosilane. Silica-basedcolumns are restrictedto usein the rangepH 3-8 and to overcomethis polymer
---''qF,
Column
Volves
phase,gel filtration, ion exchangeand affinity). The arrangementof suchan apparatus,suitablefor usewith two solventsand giving gracled elution, is illustrated in Fig. 18.1.Detection of the ofren very small quantitiesof solutein the eluateis possibleby continuousmonitoring of ultravioletabsorption,massspectrum,refractiveindex, fluorescence and electrical conductance;nuclear magnetic resonancecan now be addedto this list. To improve detection,solutesmay be either.derivatized befbre chromatography(this technique can also be used to improve separations)or treatedwith reagentsafter separation(postcolumn derivatization).A transportsystemfor monitoring is commercially available; in this a moving wire passesthrough the flowing eluate(coatingblock) and the dissolvedsolute,depositedon the wire. is pyrolysed and its quantity automaticallyrecorded.It will be noted that, tbr any particularfractionation,some detectorsystemswould be selectivelbr certaingroupsof compoundsand otherswould be universal. HPLC can give much improvedand more rapid separations than can be obtained with the older liquid chromatographymethods and it is therefore linding increasing use in numerous areas.As with GLC apparatus,it is availablefrom many manufacturersand can be completely automated. Many stationaryphasesare available,the mosr widely used being silica based.In these,which consist of porous particles5-10 prm in diameter,the silanol groups (Si-OH) afford a polar surfacewhich can be exploitedin separationsusing an organicmobile phaseas in ordinary adsorption chromatography. Reversed-phase packing material(SphelisorbODS) is producedby the bonding of octadecylsilylgroups (Ct8H37Si-)to silica gel. In the commercialmaterial there appearsto be a considerableproportion of residual silanol-OH groups, so that adsorptionand partition effects may operate during separation.The hydrocarbon chains pr.obably allow nonpolar interactionto take place.The structureof the packing materialmight be representedas: creHrz
- confrolled Temperofure oven
phasesoperatingat pH I 13 are available;these.however.have the disadvantageof exhibiting a high column back-pressure. Of relatively recentintroductionare chiral stationar.y phaseswhich are utilized to separatethe enantiomersof racemic mixtures. These have great potential fbr the study of natur.alproducts,many of which are optically active, and for isolating the pharmacologicallyactive enantiomer fiom the racemic mixture of a synthetic drug. Phalmacognostical examplesinclude,amongmany others,the assayof partially racemizedhyoscineextractedfiom plants.the separationof aromaticsand the resolutionof mixtures of (+)- and (-)-epicatechin and other proanthocyanidinenantiomersof Casslrr fistula and C. javanica. The mode of action of chiral stationaryphasesis not fully understood;typical materialsale cyclodextrins,cellulose-and amino acid-derivativessuitably bonded.Two productsare sorbentsnade of sphericalsilica gel particlesto which B-cyclodextlin(ChiraDex@)or y-cyclodextrin (Chira-Dex@GAMMA) are covalently bonded via a carbamatebond and long spacer. For a review on the rapid detection and subsequentisolation of bioactiveconstituentsof crudeplant extractswith schemesfbr LC/UY LCIMS, LCIMS/MS and LC/NMR see K. Hostettmanet al.. Platfio Medicu, 1997,63,2. Counfer-current extrqction This is a liquid-liquid extractionprocessand the principles involved are similar to thoseof partition chrornatography. Developedby Craig in 1911,the extractionmachineused(for that time) small amountsof extract and overcamethe tedious multiple extraction processesthen employed.Now, however,for researchpurposes.only the smallestof samplesfor fractionationand analysisare required.making the use of the cumbersomecounter-currentapparatuslargelyunnecessary. Briefly, a lower, stationaryphaseis containedin a seriesof tubesand an upper, moving immiscible liquid is transf-erred from tube to tube along the series,the immiscible liquids being shakenand allowed to separatebetweeneachffansf'erence. The mixture to be fractionatedis placedin the first tube containingthe immiscibleliquids and the apparatusis agitatedand the layersare allowed to separate.The components of the mixture will be distributedbetweenthe two layersaccordingto their partition coefficients.The upperphaseis moved along to the second tube containing lower phaseand more moving phaseis brought into contactwith the lower phaseof tube 1. Shakingand transf'erence again takesplace and continuesalong a sullicient number of tubesto g i v ea f r a c t i o n a t i oonf l h e m i x r u r e . The distributionof eachslrbstanceover a given numberof tubescan be ascertainedby the terms of the binomial expansion.If, for the substance,after an equilibration,a fraction p appearsin the upper phase
PHYTOCHEMISTRY and a liaction q, i.e. (l p). in the lower. then atier threeequilibrations the distributionir-rthe upperandthe lo$'erphasesis:
tr74tr If bufl'ersolutionsare usedas the stationaryphase.this exploits dit'f'erences in ionizationconstantsofthe varioussolutes.In someinstances the efJ'ectiveness ofthe separationcan be improvedby using a seriesof bufl'er solutionsof gradedpH value as the lower phase.Solutionsof acidsand alkalisalsofind sorneapplicationas lower phases. A developnrentof the extractor is the Steady-StateDistribution Machine. This allows true counter-currentextractionand can be programmedso that both solventsmove in oppositedirections.The mixture to be fiactionatedis f'edcontinuouslyinto the centreof the train of cells and, accordingto the solvents,the solutesmay move in either direction.This machineallows the concentrationof minor components from a mixture and the continuousfractionationof mixtures. The applicationsof counter-currentextractioncor eled n-ranyfields of plant chemistry.including alkaloids.amino acids,antibiotics.phenols including anthraquinonederivatives,cardiacglycosides,essential oils. tatty acids.prostaglandins.steroidsand vitamins. High-speedcounter-current chromatography (planet coil centrifugal CCC). This is a methodwherebya spool wound with PTFE tubing is roratedat about800 r.p.m.in a planetarymotion arounda central axis. In such a system.heavier and lighter immiscible solventswill move in difTerentdirectionsand it can be arlangedso that one phase remains stationaryand the other passesthrough the column from a pumped supply. Coils of tubing with an internal diametel of 1.6 2.6 mm andvolumesof 300-380 ml havebeenusedto good effect. Mixtures of the endoperoxidesartemisininand artemisitine.not separable by partition chromatographyor fiactional crystallization,have beeneificiently separatedby this method.as have crude tanninsand a commercialextractof Ginftgo biloba" Droplet counter-current chromatography (DCCC). This technique.first describedby Tanimuraand colleaguesin 1970,is an extension of counter-currentextractionand to achieveliactionation utilizes the passageof dropletsof mobile phasethrough an immiscible, liquid stationaryphaseheld in narow-bore colLlmns. Conditions that must be fulfilled for satisfactoryoperationof the processincl-rde:the retentionof discretedropletsduring their passage thror-rgh the columnsand the nonmovementof the stationaryphaseinto the capillaries.Critical for this are the intemal bore of the columns (belolv 1.0 mm ploducesdisplacementof the stationaryphaseand too wide a bore leadsto ineflicient partitioningofthe solutes)and the correctchoiceof solventsystems(someof thosecommonlyusedin counter-culrent extraction are troublesome; satisfactory ones include CHCIj/MeOH/H,O and CH2CI2/MeOH/H2O). Factorsthat give DCCC an advantageover other specifictechniques are: quantitiesof mixture to be fractionatedfrom 1 mg to 4 g can be accommodated; thereis no solid supportand therelbreno lossof solute by adsorption;the quantitiesof solvent used are minimal compared 'Counter-current with most other preparativemethods:no shaking(cf. produce means not extraction') that saporrinsiu tr-oublesome foams and unwantedoxidation changesare avoided;little preliminarypurili-
cation of the plant extractsis required;and the apparatusis simple to operateand clean.The disadvantages arethe relativelylong separation times (up to several days) during which unstable compounds may decompose,and a resolutionwhich doesnot comparewith HPLC. The methodhasbeenusedfor the satisfactoryseparationof many groupsof compoundsincluding saponins(triterpenoid,spirostanol,cardioactive, ginsenoside,Bupleurunr (saikosaponins)),iridoid and secoiridoid glycosides,polyphenolsincluding anthraquinoneglycosides,alkaloids and sugars.The method is illustratedand describedin more detail in previouseditionsof this book. A further developmenteff'ectingmore rapid separationsis centifugol droplet CCC.
Thin-loyerchromotogrophy In 1958Stahl demonstratedthe wide applicabilityof TLC, a technique which had been knorvn in principle for many yearsbut was never developed.It has now achievedremarkablesuccessin the separationof mixturesofall classesofnatural productsand is establishedas an analytrcaltool in modernpharmacopoeias. In outlinethe methodconsistsofpreparing,on a suitableglassplate. a thin layer of material,the sorbent,which may be either an adsorbent as used in column adsorption chromatographyor an inert support which holds an aqueousphaseas in column partition chromatography. The mixtures to be resolved are dissolved in a suitable solvent and placedas a seriesof spotson the film towardsone end of the plate;this end is then dippedin a suitablesolventmixture and the whole enclosed in an airtight container.The solvent fiont travels up the film and after a suitabletime the plateis removed,the solventfront is marked,the solvent is allowed to evaporateand the positions of the separatedcompoundsare determinedby suitablemeans. TLC has certain advantages over paper chromatography. Fractionationscan be effectedmore rapidly with smallerquantitiesof the mixture: the separatedspots are usuallv more compact and more clearly demarcatedfron-rone another;and the natureof the film is often such that drasticreagents.such as concentratedsulphuricacid, which would destroya paperchromatogram,can be usedfor the location of separatedsubstances. With adsorptionTLC varioussubstances exhibit differentadsorptive capacitiesand any one materialmay vary in its activity accordingto the pretreatment.The adsorbentmust be chosenin relation to the properties of the solventand the mixture to be fractionated.In general,for a given substance,if a highly active adsorbentis used,then a solvent with a correspondinglyhigh power of elution for this substanceis required.Alumina (acid, basic and neutral)of different activity grades is very commonly employed.In order to producea lilm with reasonable handlingproperties,tbe adsorbentmay be mixed with about 727o of its weight of calcium sulphate(CaSOa.1/2H2O)to act as a binder. Ready-mixedpowdersare obtainablecommercially;they requiremixing with a given quantity of water and the slurry needsto be spreadby a mechanicaldeviceor with a glassrod on to glassplates.The film sets within a few minutesand is then activatedby heatingat a suitabletemperature (105'C for 30 min is common). Comrnercial ready-spread plates are available.The thicknessof the film is characteristicallyof the order of 250 pm, but for preparativework layers of up to several millimetresthicknessare employed.The thick films must be carefully dried to avoid cracks. Solutionsof substancesto be examinedareapplied to the film with the aid of capillary tubes or',for quantitative work, with microsyringes and micrometerpipetteswhich permit volumesto be readoff to +0.05 LLl.A useful innovation for applying steam-volatilecomponentsof a powdered drug directly to a thin-layer plate is the Stahl TAS oven. The drug sampleis placedin a cartridgetogetherwith a suitablepropellant(e.g.
ffi
GENERALMETHODSASSOCIATED WITH THEPHYTOCHEMICAL INVESTIGATION OF HERBAT PRODUCTS hydratedsilicagel) and inserledin the TAS oven maintiiinedat a predeterminedtemperature. The taperedexit ofthe cartridgeis situateda short distancefrom thebase-lineof a TLC plateand 'steam-distilled' con.rponents from the drug are depositedready tbr irnmediatedevelopment(for. illustration see 12th edition). Stahl (.Plant(lMecl., 1916, 29, 1) has describeda developmentof this apparatuswhereby l8 samplescan be simultaneouslydepositedon a plate under uniform conditions.A more recentapplicationhasinvolvedthe studyoftannin-containingdrugs. The solventsusedfor running the chromatoglammust be pure, and common ones are methanol,ethanol and other alcohols,chloroform, ether.ethyl acetate,n-hexane,cyclohexane.petroleumspirit and rnixturesof these.For routine assaysautomaticmultiple developmentwith polarity graduationof the developin-esolvent can be used. It must be rememberedthat chloroform ordinarily containsup Io lVc of ethanol, which gives it quite different elution propertiesliom those of pure chloroform. Benzene,formerly liequently usedas a componentof the rnobilephase,hasnow for healthreasonsbeenroutinely replacedwith other nonpolar solvents.Similarly, inhalation of chlorofbrm-containing mixturesshouldbe avoided. TLC, which involves the patition of a substancebetween two immiscible phases,is againanalogousto the column procedureand to paperchromatography. In the latter,the hydrationshell of the cellulose fibres fbrms the stationaryphaseand thin-layerchromatogramsutilizing powdeled cellulosegive comparableresults.Kieselguhrand silica gel are also commonly employed, and their propertiesas thin layers can be modified by the inclusion of acids,basesand buffer solutions. The thin layersmay also include a substancelluorescentin ultraviolet light and this facilitatesthe detectionof soluteswhich causea quenching of the backgroundfluorescence.Much used is Kieselgel GF2ro 'Merck' which gives a backgroundgreenfluorescencewhen irradiated with ultraviolet light of wavelength 254 nrn. Small quantities of ammonia solution,diethylamine,aceticacid, dirnethylfolmamideand pyridine areoften presentasconstituentsofthe developingsolventstbr silica piates.Other layersusedincludepolyamide,which is particularly suitablefbr phenoliccompounds,'Sephadex'(Pharmacia,Uppsala), a cross-Iinkeddextranusedasa molecularsieve(gel filtration), andion exchangers.'Reversed-phase' plates are now availablecommelcially and are statedto be Lrsefulin that they will indicatethe type of separation which a mixture would undergoby reversed-phase HPLC (q.v.). As with paperchromatography,the methodcan be extendedto twodimensionalchromatography, to electrophoreticseparatiorls. to quantitative eval-rationsand to work involving radioactivesubstances. Compoundsresolvedon the TLC plate are visualizedusing either' generalor specificmethods;thus,ultravioletlight will indicatefluorescent compounds(theseshouldbe examinedin both long- (r:. 365 nm) and short- (c. 263 nm) wavelength ultraviolet light. Fluorescence-
quenchingcompounds(a very largenumber)ale detectedby the useof impregnatedsorbents(see above).Iodine and Dragendorff'sreagents are used in the form of sprays for the general detection of alkaloids althoughthey (iodinein particular)are not absolurelyspecificfor alkaloids.For indolealkaloids,the reagentp-dirnethylaminobenzaldehvde is useful for ergot and a phosphomolybdicacid reagent for others. Antimony trichloride in chlolofbrm is used as a spray reagent for steroidalcompoundsandotherterpenoids.similarlyanisaldehyde in sulphuric acid; both theserequire the sprayedchromatogramsto be heated at 100'C lbr varying times (5 20 min) in order to developthe colours. Ammonia vapour can be used tbr free anthraquinonecompounds and FastBlue Salt B 'Merck'for cannabinoidsand phloroglucides.Sugars separated by TLC usingphosphatebufl'eredaminolayers(e.g.precoared platesof silica gel-Merck NH2) can be locatedby in-sitLrthermalreaction (150"C for 3-zl min.) and fluorescence monitoring. The EttropeanandBritish Phannacopoeia.iemploy thin-layerchromatographictestsfol most vegetabledrugs. Preparative TLC. As mentioned above.thicker layers of sorbentare employedfor preparativework and the sep:Lrated bandsof compoundsare scrapedfrom the plate and subjectedto solvent extraction.With modern spectrometricmethods for structure-determinationavailable, this techniquegenerates quantitiesof materialsufficientfbr a completeanalysis. To speedup separationsand to make them on-line for continuous recording,viLriousmodificationsof preparativeTLC have been developed. These inchde centr ifugall r- trcceIeratetl la ver cIuomatog roph,r and overpressttrelaler chromatograpltr,.The latter involves the completecoveringof the sorbentlayerwith an elasticmembraneunderexternal pressurethuseliminatingthe vapourphasefron thechromatographic plate.The mobile phaseis forced up the sorbentlayer througha special inlet. This method,introducedby Tyih6k er al. in 1979-80,was subsequentiy adaptedIo on-line overpresslffelayer chromatogru4lrl'forthe preparativeseparationof a number of natural products (the isolation of liangula-emodin; noscapineand papaverinefractionation;furocoumarin isomers of Heracleum sphondt'litmt (Umbelliferae); the preparationof the secoiridoidglycosidesof Genticnapurpureti).SeeJ. PothiereI a/. (Fitorerapio,1997 isolationofthe alkal,68.12) for the semi-preparative oids of Slrycftnos rttu-yontictt, opiJm. DatLLrastr(rmoniutll andLrqtinus.
Gos-liquid chromotogrophy The use of a liquid stationaryphase and a mobile gaseousphase in chromatographywas lirst suggestedby Martin and Syngein 19,11and developedby Jamesand Martin in 1952for the separationof the lower fatty acids.Gas-liquid chromatographyis now extensivelyused in all branchesof analytical chernistry.Many commercial instrurnentsare available,the more sophisticatedbeing corrpletely automated.For a schematicdiagramseeFig. 18.2.
Somple injeclion
Corrier gos.-
( A+ B )
meosurements
Fig. | 8.2 Schemotic diogrom of gos-liquid chromotogroph wifhoncillory ottochmenls.
-.*,
IE
PHYTOCHEMISTRY The emp6 columns are made of glass or metal and are either straight,otien up to about I.3 m in length.or coiledand up to 16 m in length. Jamesand Martin used a tube of zl mm internal diameter.The liclttid .stotionartphase is held on an inert nutteriol. commonly par'tially fusecldiatomite. A unifbrrn particle size, with a minimum of dust. is essential,lbr the inelt support and the particlesshould be as small as possibleto give a large surfaceareabut sufficiently large to allow even packingof the column.The choiceof stationaryphaseis governedby the temperatureat which the column is to operateand the natureofthe materialto be fractionated:it shouldbe nonvolatileat the operatingtemperaturear-rdshould not react with either the stationary and rnobile phasesor the solutes.Some materials commonly used, with their recommendedtemperatureof operation,include (l) nonpolar compounds-silicone oils 200 250"C. apiezonoils and greases 275-300'C, silicone gum rubber 400'C, high-boiling-point paraffins such:rsmineraloil 100'C, squalene75"C; (2) moderatelypolar compounds-high boiling point aicoholsand their esters100-225'C; (3) strongly polar compounds-polypropylene glycols and their esters 225'C. Up to 25c/cby weight of stationaryphase is commonly employedon columns;one method of dispersingthe stationaryphase over the inert support is to dissolve it in a low boiling point solvent such as ether. mix thoroughly with the suppofi and spread out the powder to allow the solvent to evaporate.The powder is then packed into the empty column a little at a time and as evenly as possibleand then enclosedin a unilbrmly heatedoven. A cornbined support and stationaryphase which can replace the above is furnished by cross-linkedpolymers of specified pore size. These are produced as beads fiom styrene-likecompoundsand are 'Porapak' marketedr-rnder the tradename . Advantagesof suchcolumns are that they remove undesirableadsorptionsitespresentin diatomite. bleedingof the column (gradualleakageof stationaryphasefiom the column) is reduced,and the lapid elution of water and othel highly polal moleculesis achievedwith little or no tailing. The operating temperutureof the column is critical. Mixtures of low-boiling-point substancescan be liactionatedat low temperatures; someeihers,fbr example,can be dealtwith at room temperature.Other materialsrequiremuch higher temperatures-volatileoils I 50-300'C, steroids250'C and pesticides400'C. Modern instrumentscan be temperature-prog1'ammed so that the column temperalureincreasesas chromatographyproceeds.This has the advantagethat good separations of mixtures containingcompoundswith widely dilTerentproper"tiescan be obtained in one operation and long waits for the emergenceof the more stronglyretainedfiactions.with correspondingly lessresolution,are lessened. The mobile phase is a gas which is inert in so far as the other componentsof the chromatograma1'econcerned.The choice of gas is dependenton the detectol system (see below), and gasescommonly usedarehydrogen,nitlogen.heliurnand argon.The/or.r.,raleof the gas is important;too high a flow rate will give incompleteseparationsand too slow a ratewill give high retentiontimes and diffusepeaks.Typical tlow ratesfbr shortcolumnsare 10-50 ml min 1. injectiondevice.the sampleto be analysedis By rneansof a sr,ritable introducedon to the top of the column; 1.0-5.0 ptl is a typical volume but with somedetectorsit can be considerablyless.The measurement of such small volumesis difficult, especiallyif quantitativeresultsare required,and ofien the sampleis dissolvedin a low-boiling-pointsolvent such as ether: the ether passesrapidly through the column and emerges with the gas fiont. The mixture to be analysed should volatilize immediatelyit comesinto contactwith the stationaryphase. Some compounds.not themselvesvolatile. may be converted into volatile derivativesbefbre chromatography.Thus, sugars.flavonoids including anthocyanins,morphine.codeineand the cardioactiveglyco-
sides and aglyconescan be chromatographedas their trimethylsiloxy derivatives.which are tbrmed as below 2ROH + (CH3)3SiNHSi(CHj)j-+ 2ROSi(CH3),+ NH3 Nonvolatile plant acidscan first be convertedto their methyl estersby treatmentwith diazomethane. Along with the analyticalcolumnsof the abovetype, largerpreparative columnscan be usedfor the isolationof the separatedcomponents in quantities sufficiently large for subsequentexamination.Sample sizesof 0.1-20 ml and column lengthsof up to 60 m and internaldiametersof l-2 cm illustratethe dimensionsinvolved. The detector.r.r's/enr analysesthe effluent gas from the column. It may be of the integral type, in which some property-fbr example. titration value of the eluateis recordedor it may be of the difl'erential type. in which somepropertyof the effluent gas is comparedwith that of a ref'erencegas,often the mobile phase.The latter type is the nrost commonly used and examplesare the katharometer,gas density balance.flame ionization.B-argonray and electron-capture detectors.For details of thesedetectorsthe studentis referredto one of the several standardbooks on gaschromatography. All thesedifferentialdetectors give an electrical signal which is recordedgraphically by a suitable recorder.Becausenot all detectorsgive the samerelative responseto given compoundsunderthe sameconditions,somecolumns are fitted with a doubledetectorsystem. The volume of gas that emergesfrom the column befbrethe arrival of the gasfront into which the samplewas introducedat the headof the column is termed the 'hold-up' volume and it is dependenton the capacityof the column. It is obtainedby multiplying the time which the gas front takesto passthroughthe column by the flow rate: the arrival of the front is often indicatedon the recorderby a negativepeakcaused by the small amount of air injected with the sample.The observed retentionvolume Vp.o5..of a componentis calculatedfrom its retention 'hotd-up' time, and the Vp.n1r._ lessthe volume is the adjustedretention volume (Vn.",r.).By taking into accountthe pressuredrop along the length of the column the net retentionvolume (Vn n.J is obtained.This volume is characteristiclbr a given compoundunder the definedconditions of the amount of stationaryphaseand temperature.Of more universalvalue is the specific retentionvolume (Vn.rp),which is the Vp.n.,r'educedto OoCg-l of stationaryphase: vR.rp= V*.,,.,x 27-3 weight of stationaryphaseon column x temperature(K) of column Ref-erence compoundsare usedto aid the identificationof components of a mixture. For quantitativework with differential detectors, the areas enclosed by the peaks are proportional to the quantities of compoundswhich they represent.To obtain the percentagecomposition of componentswithin a mixture. without the necessityof placing known amountsof sample on the column, internal standardscan be used.These are pule substances,mixed in known propoltion with a sampleof materialto be analysed,which give sharppeakson the chromatogramnot overlappingthose of the mixture. Befbre use, internal standardsmust be calibratedfbr detectorresponseagainstindividual c o m p o n e n losf l h e m i x t u r e . Sophisticatedattachmentsare available lbr some equiprnent.It is thus possibleto record the radioactivitiesand massspectraof the separatedcomponentsof a mixture as they emergefrom the column. Such instrumentshave immensepotential in biological research.Data storage and retrievalunits are now availableas standardaccessories. examplesof the applicationsof gas chroSome pharmacognostical matographyinclude the examinationof many volatile oils (see. for
',4ln
,!:lr{
GENERAT METHODSASSOCIATED WITHTHEPHYTOCHEMICAT INVESTIGATION OF HERBAL PRODUCTS examplethe BP assayof Clove Oil). camphor.plant acids,somealkaloids (opium, tobacco and Conium and tropanederivatives)"the resins of the Convolvulaceaeandof Cannabis,and steroidalcompoundssuch as the sapogeninsand cardioactiveglycosidesand aglycones.The BP testfor fbreign oils in fixed oils involvesthe gas-chromatographic separationof the methyl estersof the fatty acidsproducedby hydrolysisof the sample.The detectionand estimationof cocaineandits metabolites in the body is an important forensic application.The estimation of pesticideresidueson cropsis ofutmost importance,and herethe sensitivity of detectorsystems,such as the electron capturedetector,has made possiblethe determinationof the chlorinatedpesticidesdown to the parts-per-billionrange.
25:l with the smallerportion passingto the colurnn) or.are of the socalled splitless-injectortype which are able to accommodatethe relatively large volume of solvent and deliver the dissolvedsolute to the column. As with other"chromatographic techniques(seeHPLC) the introduction of chiral stationaryphaseshas given an addeddirnensionto gas chrornatography.Examples include the separationof the two enantiomersof linalool enablingthe detectionof reconstitutedbergarnotoil in the genuineoil (A. Cotroneoet al.. Flat,otrrFrag. J., 1992,7,15) and the detectionof addedreconstitutedlemon oil in the genuinecoldpressedessential oil (G. Dugo et al., J. Essenr.Oil Res.,1993.5, 21).
Gel filrrotion (moleculor sieves) Copillory-column gos chromotogrophy As the name implies, capillary bore columns are used rather than the standardcolumnsdescribedabove.They afford markedimprovements in resolvingpower and in speedof analysis. The internaldiametersof the columnsrangefrom about0.15 mm to about0.53 mm and the columnscan be I to 60 m in length.They were originally made of stainlesssteel and then glass, but fused-silica columns are now consideredthe obvious choice as they are strong, easyto use,highly inert and give excellentperformance.They can be convenientlyusedin the coiled condition,held, for example,in a 150 mm diametercage.Such columnshold the stationaryphasein a number of ways. ( I ), Wall-coatedopen tubular (WCOT) columnshave the inner wall ofthe tube coatedwith stationaryphaseup to about I prmin thickness.Greaterthicknessleadsto column bleedingin which the stationary phasemoves down the column and eventuallyleaks into the detector.Thicker layers,and hence increasedsamplecapacity,can be achievedwith silica columnshaving speciallybondedphases.WCOT columns have the highest efTiciencybut a low sample capacity.(2), Support-coatedopen tubular (SCOT) columns have the inner wall lined with a thin layer of supporlmaterialcoatedwith immobile phase. This has the effect of increasingthe availableareaof immobile phase. affording the column a greater load capacity.The efficiency, white lower than that of the WCOT columns is much higher than that for' packedcolumns. (3) Micropacked columns involve a coatedsupport packed into narrow-bore columns. In all ways they represent a compromise,being more efficient than the normal packedcolumnsbut having the sameproblemin that column lengthis restrictedby the high back-pressure. Another differencebetweencapillary column chromatographyand standardgas chromatographyconcernsthe method of introducingthe sampleto the column. The volume of sampledissolvedin solventfor analysisby the capillary methodis too sma1lfor a microsyringeand so specialinjectionheadsare necessarywhich eithersplit the sample(e.g.
Hydrophilic gels such as thosepreparedfiom starch,agar,agarose(a component of agar), polyacrylarnide,polyvinylcarbitol and crosslinked dextranshave been usedlbr the tiactionation of ploteins.peptides.amino acidsand polysaccharides. The particlesof thesegels possesspores fornted by the molecular structureof the gel, and when packed into a column and percolated with a solution.they permit large moleculesof solute,which do not enter the pores,to passrapidly down the column with the solvent via the intergranularinterstices.Conversely.small moleculeswhich are able to enterthe ge1poresbecomeevenly distributed(on equilibrium) acrossthe column and passmore slowly dorvnits length.Thin layersof the sorbentcan be r.rsed as in TLC. The dextrangels (Sephadex)are fbrmed by cross-linkingdextrans (polymersof glucosein which linkagesare almostentirelyof the I,6-cr type) with o,-epichlorohydrin(Fig. 18.3). lndividual pore sizes are determinedby the distanceapart of the cross-links,and gels coveringa molecularweight rangeof up to about 200 000 areproduced.Eachgel type possesses a rangeof pore sizes,so that below the size limit of completeexclusionof the large molecules, dilTerentsizedsolutemoleculeswill enterthe gel to a greateror lesser extent and so will vary in their elution rates. Similar principles are involved in the use of controlled-poreglass as sorbent;this gives a rigid column with continuousuniform pores.ObvioLrsly,the methodis most applicableto mixturescontaininglargemoleculesof varioussizes and to the separationof large moleculesfiom small ones(as in desalr ing operationson partially hydrolysedproteins). Electroc hromotog ro phy For the electrophoreticseparationof mixtures. a filter-papersrrip is impregnatedwith a solutionof an electrolyte(usuallya br-rf-fer solution) and supportedin the centre;its two ends are dipped into solutionsin which electrodesare immersed.A spot of the materialto be fiactionated is placed on the paper,the whole apparatussealedand a potential
-Gluc.-Gluc.-Gluc.-
-Gluc.-Gluc.-Gluc.-
9Hr
l ) o
+cH' I
cH2cl
-Gluc.-Gluc.-Gluc.Fig. | 8.3 Formotion of dextron gels
rrr-h
Dextranunits
I o I 9Hz I H-C--OH
I 9Ht I o I
-Gluc.-Gluc.-Gluc.cEpichlorohydrin
of gel Cross-link
E
PHYTOCHEMISTRY differenceof about 2- I 0 volts per centimetreappliedalong the paper. saponinmixture of the Chineseand Indian drugTribuLustetestris, see Some separationsare carried out at much higher voltages than the S. Fang et al., PLantaMedica, 1999,65. 68. Many problemsof strucabove.According to the natureof the chargeon the ions of the solute ture elucidationrvhich 30 years ago were incapableof investigation, mixture.the soluteswill move towalds eitherthe anodeor the cathode. either throughpaucity of materialor throughlack of suitablechernical Thus, the amino acidscan be separatedeither into groups(acid group. methods,can now readily be solvedin a standardresearchlaboratory. neutralgroup, basic group) or into individual amino acids.The migra- The routine analyticalapplicationof someof thesetechniquesto plant tion velocity for a given substancedependson the magnitudeof the drug analysishasbeenconsideredin Chapter1,1. ionic charge and the size and shape of the paticular molecule. If preferable.paper can be replacedby thin layers of the gels described under'Gel filtration'. above.Many alkaloidalmixtureshavebeenseparatedby this method and also plant acids,the componentsugarsof cardiacglycosidesand anthraquinonederivatives. The living material Lrsedin biochemicalresearchis extremelyvaried. A developrnentwhich combines the advantagesof both ge1filtration Somework is possiblewhich r-rtilizesthe whole organismwith a minandelectrophoresis is thatofpolyacrylamidegradientgel electrophoresis. imum of distr-rrbance-forexample.bacteria,yeastsand mouldscan be It is a two-din-rensional electrophoresissystemwhich separatesaccording cultivatedand investigatedbiochemically,and with animals,test subto mobility of solutesin onedirectionand accordingto sizein the other. stancescan be addedto the fbod and the blood and excretaanalysed. Capillar,,"electropltoresi.iis a techniqueof relatively recent intro- With intacthigherplants,however.the ultimatedestructionof the plant dr-rction and can give separationefficienciesof the older of 4 r I 05 the- for analysisis usually necessary.Minces. breis and homogenatesare oretical plates. It provides a more rapid analysis than gel examplesof preparationsin which the tissuesand the cell wall strucelectrophoresisand with detector systemssuch as the laser-induced tures have been destroyedbut in which the intracellularparticlesare lluorescencedetector combines a high resolution with a 500-fold still intact. The componentsof such a mixture can be isolatedby cenincreasein sensitivityover UV detection.The method has been used trifugation and the biological activity of each fraction can be tested. The penultimate stage in a biogenetic study is the isolation of the tbr the analysisof flavonoids. enzymesinvolved in the pathwaysunderconsiderationand the in vitro Aff inity chromotogro phy demonstrationof their properties.Finally, it is becoming increasingly possibleto locateand clone the generesponsiblefbr the synthesisof a This methodhasbeendevelopedlargely for the resolutionof protein mixtures.and it dependson the specific,reversiblebinding of individual pro- particularenzyme.Now that the principal overall pathwaysassociated teinswith a particularligand suchasan enzymesubstrateor inhibitor. The with secondarymetabolism have been largely established,it is the ligands are coupled with a suitable carrier (cellulose, beaded agarose. enzymic studiesthat currentlyreceiveconsiderableattention. glass,polyacrylamideor cross-linkeddextrans),possibly The techniquesdiscussedbelow havebeenusedfor the studyof seccontrolled-pore with the introduction of a spacer-a suitablechemical moiety such as a ondary metabolismand their applicationdatesfrom the middle of the hydrocarbonchain-between the ligand and matrix. Excessligand is last century.It must be rememberedhoweverthat many of the primary removed by washing and the material is packed in a column. A plotein metabolic pathways, e.g. the Krebs (TCA) cycle, were established mixture in a suitablebuffer solution is passeddown the column and a pro- using classicalbiochemicalmethods. tein with sufficient affinity for the bound ligand is retardedand may later be elutedin a purifiedstateby a changein ionic strengthor pH of the colTRACER TECHNIQUES umn bufl-er.The methodhasthe advantageof preparingin one stepa parTracer technology.now widely employed in ali branchesof science, ticular componentin a high stateof purity. had its origin in the early part of the last century,when it was realAfTinity chromatographyhas been applied to the purification of enzymesfbr potential clinical application,for the isolation of certain ized that elementsexistedwith identical chemicalpropertiesbut with antibodiesand for the specific fractionationof different types of cells difl'erentatomic weights.Such isotopesmay be stable(2H, l3C, lsN, l8O), or the nucleus may be unstable(1H. l1C) and decay with the (e.g.erythrocytesand lymphocytes). emissionof radiation.If it is possibleto detecttheseisotopesby suitablemeans.then they can be incorporatedinto presumedprecursorsof ': : : plant constituentsand usedas markersin biogeneticexperiments.
gtogENEitclNvfgr-icArt'oNs,,, r
Or,lgowED cHARAaTs[iZlnoN COMPOUNDS.' ' r
l,'
"
",
It is outsidethe scopeof this book to considerin any detailthe structure elucidationof naturalproducts.It is sufficientto statethat althoughstill chemistsarecomutilizingclassicalchemicalmethodsof degradation, ing to rely more and more on the useof physicaltechniquesto establish structuresof new compoundsand to identily known compoundsin plant sources.Ultraviolet. infiared, massand nuclearmagneticresonance spectroscopytogether with X-ray crystallographicand optical rotatory dispersionrnethodshave all played a significantrole in these developments.Varior.rs modificationsof massspectrometly(MS) have become of increasingimportance fbr the structuralcharacterization and determinationof the active constituentsof plants; these include electronionizationMS. chemicalionizationMS, field desorptionMS, fast atom bombardmentMS and electrosprayionization MS. For an exampleof the applicationof electrosprayMS combinedwith sequential tandern mass spectrometryto the investigationof the steroidal
Radioactive tracers. In biological investigationsthe use ofradioactive carbonand hydrogen,and to a lesserextentand for more specific purposessulphur,phosphorusand the alkali and alkaline-earthmetals, enablesthe metabolism of compoundsto be followed in the living organism.For studieson proteins,alkaloidsand amino acidsa labelled nitrogenatom may give more specificinformationthan a labelledcarbon, but the two availableisotopesof nitrogenareboth stable,necessitating the use of a massspectrometerlbr their useas tracers. two stableisotopeswith massnumbersl2 Naturalcarbonpossesses and 13, the latter having an abundanceof 1.10 atoms per cent. Radioactiveisotopesof carbon have massnumbersof 10, I I and 14. l0C has a half-lif'eof 8.8 s and 11Ca half-life of 20 min. which limits their usefulnessin biological research.However, IaC has an estimated half-life of over 5000 yearsand in the atomic pile it may be produced by the bombardmentof l'+N with slow neutrons,the target material usuallybeing aluminium or beryllium nitride.
GENERAL METHODS ASSOCIATED WITHTHEPHYTOCHEMICAL INVESTIGATION OF HERBAL PRODUCTS The immense possibilities in biological researchfor the use of organiccompoundswith specificcarbonatomslabelledled to the synthesisof many compoundsfi'om the inorganiccarboncompoundsproduced in the pile by routes not before commerciallyutilized. In these synthesesthe purity ofthe productis ofgreat importance,sincea small proportionof a stronglyradioactiveimpurity might seriouslyjeopardize the resultsof any subsequentexperiments. Many compoundswhich are most convenientlypreparedfrom natural sources(e.g.certainamino acidsby the hydrolysisofproteins) are producedby growing Chlorello in an atmospherecontaining l4CO2. A11the carboncompoundsof the organismthus becomelabelled,each compoundpossessinga uniform labellingof its carbonatoms. Many tritium (3H)-labelledcompoundsare commerciallyavailable. Tritium labelling is eff'ectedby catalytic exchange(platinum catalyst) in aqueousmedia, by irradiation of organic compoundswith tritium gas and by hydrogenationof unsaturatedcompoundswith tritium gas. Tritium is a pure B-emitterof low toxicity, half-life I2.213years,with a radiationenergylower than that of laC.
ner. The resulting autoradiographgives the distributionpatternol the radioactivesubstances in the specimen.The methodcan be appliedto whole morphologicalparts (e.9.leaves)or to histologicalsections.fbr which the resultingnegativeis viewed undera microscope.In a similar manner',radioactive compounds on paper and thin-layer chromatogramscan alsobe detectedand the relativeamountsof radioactivity in differentspotsdeterminedby densitymeasurements or by the use of calibratedfilms.
Precursor-product sequence. For the elucidationof biosyntheticpathways in plantsby meansof labelledcompounds,the precursorproduct sequence is commonlyinl'oked.In this a presumedprecursorof the constituentunderinvestigation.in a labelledfonn, is f'edto the plant and after a suitabletime the constituentis isolatedandpurifredandits radioactivity is determined.If specific atoms of the precursorare labelled, it rr-raybe possibleto degradethe isolatedmetaboliteand ascerrain whetherthe distributionof radioactivitywithin the moleculeis in accordancewith the hypothesisundertest.Radioactivityofthe isolatedcompoundaloneis not usuallysufficientevidencethattheparlrcr.rlar compoundf'edis a directpreDetection and assay of radioactively-labelled compounds. When cursor,becausesubstancesmay enter the generalmetabolicpathwaysof radioactivetracersareusedin biogeneticstudies,adequatemethodsfor the plant and from therebecomerandomll'distributedthrougha'"vhole the detectionand estimationof the label are essential.For the soft and rangeof products.If this happens.degradation of the isolatedconstitutent easily absorbedradiation fiom 3H-and l4c-labelled compoundsthe and the deteminationof the activity of the liagmentswould pr.obably instrumentof choice is the liquid scintillation counter.It dependson show that the labelling was random throu-lhout the molecule and not the conversionof the kinetic energyof a particleinto a fleetingpulseof indicativeofa specificincorporationofthe plecursor.Fuftherevidencefbr light as the result of its penetratinga suitableluminescentsubstance. thenatureof thebiochemicalincorporatioltof precursors alisesfrom douRutherford successfullyused this method in his early studies on ble- and triple-labellingexperiments;eithel diff'ercnrisoropes.or specific radioactivityand he countedthe flashesof light producedby bombard- labellingby one isotopeat two or more positionsin the rnolecules.are ment with cr-particleson a fluorescentscreenpreparedfiom zinc sul- employed.The methodhasbeenappliedextensivelyto thc biogenesisof phide.The usefulnessof the detectorwas tremendouslyheightenedby manyplantsecondary metabolites. Leete,in his classicalcrperiment.used the development of the photomultiplier tube, which replaced the two doubly labelled lysines to determinewhich hydrogen of the lysine human eye in recording the scintillation. Liquid scintillation media, moleculewasinvolvedin thefbrmationof thepiperidinerine of anabasine consistingof a solventin which the excitationoccursand a fluorescent in Nicotituto glauca. His f-eedingexperimentsgave the incorporations solute which emits the light to actuatethe photomultiplier,have also shownin Fig. t 8.,1,indicatingthe terminalN to be involved. beendevisedfor the purposeof enablingthe sampleto be incorporated It will be appreciatedthat with the abovelysine precursorsir is not in the samesolute,and,hence,attainoptimum geometrybetweensam- necessarytohave individaal moleculeslabelledwith both l-+Cand l5N ple and scintillator. but that the same result is obtained by using standardmixtures of Moderninstrumentsarefully automatic(e.g.for 100samplesat a time) specificallylabelledf laCllysineand f l5Nllysine.(Note: l-5Nis a stable and will alsomeasuremixed radiationssuchas 3H and 1'1C(thisis possi- isomer'.)Extensiveuse has beenmade of 3H/l'1Cratios in the study of b1ebecause,although both are B-emitters,they have different radiation stereospecilichydrogeneliminationreactions. energies.With all counters, the instrument is connectedto a sr.ritable Competitive feeding. lf incorporationis obtained.it is still necesratemeterwhich recordsthe countsover a given time. With l4C, because sary to considerwhetherthis is in tact the normal rt'ruteof synthesisin of its long half'-life, no decay correctionsare necessatyfor normal biogeneticexperiments.However the half-life is important in carbon dating Hr of old materials.With 3H-labelled material some correction lbr decay may be necessaryif samplesare storedfor any length of time. \ -./ The traditional unit of radioactivity has been Ihe curie, defined as F", !x' that quantityof any radioactivenuclidein which the numberof disinteN.glow grationsper secondis 3.7 x 1010.Subunitsarethe millicurie(3.7x 107 + Hrri'/cHr HrN^ disintegrationsper second)andthe microcurie(3.7 x 104d.p.s.).The SI cooH unit now nsedfor radioactivedisintegrationrate is the bet:querel(Bq), which has a disintegrationrate of I s-1, and its multiples include the L y s i n e - 2r-' C . € - I 5 N Anabasine gigabecquerel(GBq), at 10s c-1 (27.027 millicuriesl, and the megaHt becquerel(MBq), at 106s-l (27.027microcuries). For infbrmation on the theoreticalbasisof radioactiveisotopeutil-zt\ 'cl+ c-H. ization and the regulationsgoverningthe useofradioactive substances N.glauca l' lin universitiesand researchestablishments the readeris refered to the l l standardworks and ofTicialpublicationson thesesubjects. . iitl -cHz HrN-
t
HrN-
Autoradiography. A techniqueusedfbr the location of radioactive isotopesin biological and other materialis autoradiography. In this the specimenis placedin contactwith a suitableemulsion(e.g.X-ray sensitive film) and after exposurethe latter is developedin the usualman-
\
l
cooH
Lysine-2-raC.a-r!N
Anabasine
Fig. | 8.4 lncorporolion of doubly-lobelled lysines intoonobosine.
@
GENERAL METHODS ASSOCIATED WITHTHEPHYTOCHEMICAL INVESTIGATION OF HERBAL PRODUCTS The immense possibilities in biological researchfbr the r-rseof organiccompoundswith speciliccarbonatomslabelledled to the synthesisof many compoundsfrom the inorganiccarboncompoundsproduced in the pile by routesnot betbre commerciallyutilized. In these synthesesthe purity ofthe productis ofgreat importance,sincea small propofiion of a stronglyradioactiveimpurity might seriouslyjeopardize the resultsof any subsequentexperiments. Many compoundswhich are most convenientlypreparedfiom na! ural sources(e.g.certainamino acidsby the hydrolysisofproteins) are producedby growing Chlorella in an atmospherecontaining l4COr. All the carboncompoundsof the organismthus becomelabelled.each compoundpossessinga uniform labelling of its carbonatoms. Many tritium (3H)-labelledcompoundsare commerciallyavailable. Tritium labelling is effectedby catalyticexchange(platinum catalyst) in aqueousmedia, by irradiation of organic compoundswith tritium gas and by hydrogenationof unsaturatedcompoundswith tritium gas. Tritium is a pure B-emitterof tow toxicity, half'-tife 12.43years,with a redialion e n e r g yl o w e rt h a nr h a to l ' l 4 C .
ner. The resulting autoradiographgives the distributionpatternof the radioactivesubstances in the specimen.The methodcan be appliedto whole morphological parts(e.g.leaves)or to histologicalsections,fbr which the resuitingnegativeis viewed undera microscope.In a sin-rilar manner. radioactive compounds on paper and thin-layer chromatogramscan alsobe detectedand the relativeamountsof radioactivity in diff'erentspotsdeterminedby densitylneasurements or by the use of calibratedfi1ms.
Precursor-product sequence. For the elucidationof biosyntheticpathways in plants by meansof labelled compounds.the precr"rrsor-product sequenceis commonly inr.oked.In this a presumedprecursorof the constituentunderinvestigation, in a labclledform, is f'edto theplantandatter a sr.ritable time the constituentis isolatedandpurifiedandits radioactivity is determined.lf specific atonts of the precursorare labelled. it may be possibleto degradethe isolatedrretabolite and asceftainwhetherthe distributionof radioactivitywithin the moleculeis in accordancewith the hypothesisundertest.Radioactivityol-theisolatedconpoundaloneis not usuallysullicientevidencethattheparticr-rlar cornpoundf'edis a directpreDetection and assayof radioactively-labelled compounds, When cursot becausesubstancesmay enter the -rcneralmetabolicpathwaysof radioactivetracersareusedin biogeneticstudies,adequatemethodsfor the plant and from therebecomerandonrll distribr-rted througha whole the detectionand estimationof the label are essential.For the soft and rangeof products.lf this happens.degradation of the isolatedconstitutent easily absorbedradiation from 3H-and I4c-labelled compoundsthe and the detemination of the activitv of tht- fiaqnrents would probably insffumentof choice is the liquid scintillation counter.It dependson show that the labelling was randont throughout the molecule and not the conversionof the kinetic energyof a particleinto a fleetingpulseof indicative of a specifici ncorporationof the prccursor.Fuftherevidencelbr light as the result of its penetratinga suitableluminescentsubstance. the natureof the biochernicalincorporationof prccursorsarisesfrom douRutherford successfully used this method in his early studies on ble- and triple-labellingexperiments; eithel difl-erentisotopes.or specific radioactivityand he countedthe flashesof light producedby bombard- labellingby one isotopeat two or more positionsin the molecules.are rnent with o-particleson a fluorescentscreenpreparedfrom zinc sul- employed.The methodhasbeenappliedextensivellto the biogenesisof phide.The usefulnessof the detectorwas tremendouslyheightenedby manyplantsecondary metabolites. Leete,in his classicalerperiment.used the development of the photomultiplier tube. which replaced the two doubly labelledlysinesto determinewhich hvdrogcnol the lysine human eye in recording the scintillation.Liquid scintillation media, moleculewasinvolvedin thefomation of thepipcridinerine o1'anabasine consistingof a solventin which the excitationoccursand a fluorescent in Nicotiana gkutca. His feeding experirnentsgar.e the ireolporations solute which emits the light to actuatethe photomultiplier,have also shownin Fig. 18.21, indicatingtheterminalN to be invoh ed. beendevisedfor the purposeof enablingthe sampleto be incorporated It will be appreciatedthat with the abovelysine precur.sors it is not in the samesolute,and,hence,attainoptimum geometrybetweensam- necessarytohave individlal moleculeslabelledwith both I-tCand l5N ple and scintiilator. but that the same result is obtained by using stanclardnrixtures of Modern instrumentsarefully automatic(e.g.fbr 100samplesat a rime) specificallylabelledIl4C]lysineand IlsNllysine.(Nore:l5N is a stable and will alsomeasuremixed radiationssuchas 3H and l4C (thisis possi- isomer.)Extensiveuse has beenmade of 3H/l'lC rariosin thc study of ble because,although both are B-emitters,they have different radiation stereospecific hydrogeneliminationreactions. energies.With all counters, the instrument is connectedto a suitable Competitive feeding. If incorporationis obtained.it is still necesratemeterwhich recordsthe countsover a given time. With l4C, because sary to considerwhetherthis is in fact the normal loute of synthesisin of its long half-life, no decay correctionsare necessarJfor normal biogeneticexperiments.However the half'-lif'eis important in carbon dating Hr of old materials. With 3H-labelled material some coffection for decay c may be necessaryil samplesare storedfor any length of time. The traditional unit of radioactivity has been rhe curie, defined as cH, iH, that quantityof any radioactivenuclidein which the numberof disinteI I N.glow grationsper secondis 3.7 x 1010.Subunitsarethe millicurie (3.7 x 107 + Hrli'/cHr H:N'lr\ disintegrationsper second)andthe microcurie(3.7 x 104d.p.s.).The SI cooH unit now usedfor radioactivedisintegrationrate is the becquereL (Bq), which has a disintegrationrate of I s 1, and its multiples include the L y s i n e - 2r-' C . e - r r N Anabasine gigabecquerel(GBq), at 10es-l (27.027 millicuries), and the megaHr becquerel(MBq), at 106s-l (27.027microcuries). For information on the theoreticalbasisof radioactiveisotopeutiltttt\ cl-ll cHr ization and the regulationsgoverningthe useofradioactive substances N.sluca | | in universitiesand researchestablishments the readeris referredto the t t standardworks and oflicial publicationson thesesubjects. . )ts -cHz
t '
t '
HIN-
Autoradiography. A techniqueused for the location of radioactive isotopesin biological and other materialis autoradiography. In this the specimenis placedin contactwith a suitableemulsion(e.g.X-ray sensitive film) and after exposurethe latter is developedin the usualman-
-s,
\
HrN-
cooH Lysine-2-raC.a-rrN
Anabasine
Fig. | 8.4 Incorporotion of doublyJobelled lysines intoonobosine.
@
PHYTOCHEMISTRY the plant and not a subsidiarypathway,invoked as a result ofthe atypical availability to the plant of the administered compound. Competitivefeedingexperimentscanbe of valuein determiningwhich of two possibleintermediatesis normally usedby the plant. In its simplestfbrm, without taking into accounta numberof other factors.competitive feeding could distinguish whether B or B' was the normal intern-rediate in the formation of C fiom A as below:
-
A ---------)
B,
r
'
C
Inactive B and B' are f'edwith labelledA to separategroupsof plants and a control is perfbrmedby feedinglabelledA only to anothergroup. If the incorporationof activity into C is inhibitedin the plantsreceiving in the group receiving B', then we may conclude B, but is unaff-ected pathway fiom A to C probablyproceedsvia B. In suchexperithe that ments involving intact plants,the biological variationbetweennormal plantsis often so greatthat it is diflicult to perfbrm a controlledinvestigation. In comparative studles on the rates of demethylation of codeine and unnatural codeine derivatives in Papaver orientale, Kirkby and colleaguesin 1912 overcamethis problem by using the asthe control and as the testplant.They did sameplant simultaneor.rsly to the plantI nrixtureoflH-lebelledcodeineand this by administering l'1C-labelledunnaturalcodeinederivative.The productsofthe conversion of both could be independentlyfbllowed by their characteristic radiations,evenwhen the metabolitesproducedwere chemicallyidentical. (Fol the use of competitive feeding in the elucidation of the biogenesisof tropane alkaloids, see Beresfbrd and Woolley. Pfi-r'toclrcmistrl', 1975, 11, 2209.) Administration of precursors. Negativeresultsarising from feeding experimentsmust alsobe interpretedwith caution:thus,the administered precursor may never have reached the necessary site of synthesisin the plant, or the plant may not, at the time of the experiments,havebeensynthesizingthe constituentunderinvestigation.Two examplesof the latter situationwhich, until discovered,were the cause of n'risleadingresultsin alkaloid studieswere the cessationol hordenine productionin barley seedlingsl5-20 days after germinationand the restrictedsynthesisof hyoscine.as distinct fiom hyoscyarnine.in old plantsof Daturu strannotunm. Often, the actual weight of labelled material fed to the plant is extremelysmall and contaminationof the solutionby microorganisms. during infiltration. can lead to a loss, or even the completedisappearance.of the original compound.This situationis very likely to arise with infiltrations into nonsterileroots-it can. in some instances,be controlledby the useof broad-spectrumantibacterialagents. A number of methodscan be employed to introducelabelled substancesinto plants.Root feeding is particuiarlysuitedto plants which can be grown in hydroponicculture solution and which synthesizethe compoundsunderinvestigationin the roots.Direct injectionof precursor solutionsis sometimespossible:this is particularly applicableto plants with hollow stems (Umbelliferae) and to capsules (opium p o p p y ) . F o r t h e i n t r o d u c t i o no f ! - l 3 C l g t u c o s e s o l u t i o n i n t o 1998.48, chamornilef'lowelsK.-P Adam andJ.Zapp(Pltl'rochemistry'. 953) used a microsyringe.injected into the hollow receptacleof the inflorescence.With rigid tissuesit is dillicult to avoid a lossof solution liom the site of injection by this method.Infiltrationscan be madeinto rigid stemsby using a wick consistingof a threaddrawn through the stemand dipping into the labelledsolution;alternatively,a flap can be cut in the stemand this dippedinto the solutionto be inflitrated.
Sequentialanalysis. A secondmethodof investigationwith laC is to grow plantsin an atmosphereof laCO2and,by analysisof the plantsat given time intervals,to obtain the sequencein which various related compounds become labelled. From the results obtained, certain biosynthetic routes may become apparentand others rejected.Here, again.degladationof the isolatedradioactivecompoundsis important, becausesomeunits of the moleculemay becomelabelledmore rapidly than others.This methodhas beenvery successfullyusedin the elucidation of the path of carbonin photosynthesisand alsofbr determining the sequentialformation of the opium, hemlock and tobaccoalkaloids. Exposureperiodsto laCO2as short as 5 min have beenusedto obtain evidenceof the biosyntheticsequencepiperitone-+ (-)-menthone -+ (-)-rnenthol in Menthapiperita.In a numberof instancesthe pathways suggestedby these experimentshave been at variance with those obtainedby f'eedinglabelledintermediates;it would seemthat the latter are thereforeexamplesof non-obligatoryintermediates. Use of stable isotopes. The stableisotopes2H, l3C, l5N, and 18O, which have a 1ownatural occurrence,can be usedin the sameway as radioactiveelementsfor labelling compoundsto be used as possible intermediatesin biosyntheticpathways.The usual methodsof detection are mass spectroscopy(l5N and 18O)and nuclear magnetic resonance(NMR) spectroscopy(lH and l3C). It is the latter which is becomingof increasingsignificancefor biosyntheticstudies(theuseof mass spectroscopyand NMR spectroscopyin biogenetic studies shouldnot be confusedwith their extensiveusein structuraianalysisof organiccompounds).
AND CEttS ISOLATED ORGANS,TISSUES The cultivationof isolatedorgansand tissuesof plantseliminatesinterferencefiom other parts of the piant which may produce secondary changesin the metaboiites.It can be used for feeding experimentsin conjunctionwith labelledcompoundsand is also useful for the determination of the site of synthesisof particularcompounds. Isolated shootsof plants, when placed in a suitable solution or in water, will usually remain turgid fbr some days and during this time presumablyhave a normal metabolism;soon,however,a pathological metabolismcommences.The techniquecan be refined by aseptically connectingthe cut end of the shoot to a teservoir of suitable sterile nutrient,when the shootswill remain normal for much longer periods. Such shootsoften developroots at the cut ends-a factor which could invalidatethe resultsof an experiment.lsolatedleavescan be similarly maintained.Rootedleaveshavebeenusedin studieson Nicotiana and Datura. By this methoda largequantity of root is obtainedwith a relatively small amountof aedalparts.It hasthe advantagethat the nutrient solutionrecluiresno sugar,as suflicient starchis synthesizedin the leaf and consequentlybacterialand fungal growth in the nutrientsolutionis rninimized. Petal cliscshave been used in the investigation of the biosynthesisof oil of rose.Isolated roots have been extensivelyused. Surface-sterilizedseedsare germinatedunder asepticconditions,and the root is severedand transferredto a sterilenutrient solution.Under suitableconditionsa rapid growth is obtainedand subculturesmay be producedas necessary.In this way strainsof tomato roots have been maintainedfor many years.For biogeneticand growth studies,selected compounds can be added to the culture as necessary.It has been demonstratedby this methodthat tropanealkaloidsare lbrmed in the Also, the incorporationof a number roots of a number of Solanaceae. of precursorsinto the alkaloidshasbeenfollowed. The techniquecan be extendedto the cultivation of isolatedtissues ttnd cell,s(seeChapterI 1),the useof which affordsconsiderablepotential in the investigationof biogeneticpathways.They ofl'erthe prospect
GENERALMETHODSASSOCIATED WITH THEPHYTOCHEMICAL INVESTIGATION OF HERBAL PRODUCTS of absolutely unifbrm plant matelial, obtainable at all rimes and nanageable under regulated and reproducible conditions-factors rarely possiblein work with entireliving plants.Furthermore.potenrial precursorsof the metaboliteunderinvestigationcan easilybe addedto the systemand samplescan be takenlepeatedlyfor analysis.The aseptic natureof the culture meansthat bacterialand ltngal modifications of the precursorare eliminated.
ism being blocked at a pafiicular stage.Such an orgauisnrmir) accumulate the intermediatecompoundinmediately before the block and for its survival may requirean artificial supplyof anotherintermediate which ariscs afier the block. Such organisms are obvior"rslyuseful materialsin biosyntheticstudiesand have proved of major rnrportance in sonreinvestigations. A mutantof LrzctohaciLlus acidophilus,by its ability to utilize a constituentof 'brewer's solubles'but not acetate.led to the isolation of mevalonicacid.an importantintermediateof the isoprenoidcompound GRAFTS pathway. Grafting techniqueshave considerableuse in biosyntheticstudies,parUltraviolet-inducedmutantsof ergot auxotrophicwith respectto a ticularly lbr the determinationof the sites of prirnary and secondary number of amino acids have been produced:cultures of these have metabolismof somesecondaryplant products. been used to inoculaterye and the resulting alkaloid contentsof the Alkaloid fbrmationby graftedplantshas beenextensivelystudiedin sclerotiahavebeeninvestigated.Gibberellamutantshavebeenr.rsed to Nicotiana and the tropane alkaloid-producingSolanaceae.Thus. to- obtain novel isoprenoidcompounds.With higher plants, in spite of mato scionsgraftedon lo Duluru stocksaccumulatetropanealkaloids, repeatedattemptswith chemicalsand irradiation.lesssuccesshasbeen whereasDctturctscronson tomato stockscontainonly a srnallamount obtained in producing mlrtantsuseful for the study of biogenesisof of tropanealkaloids.This suggeststhe main site of alkaloid synthesis therapeuticallyactive compounds;the production of mature haploid Iobelhe Daturu roots.However.interspecilicgraftsinvolving D.ferot plants from cell culturesof pollcn (seeChapter 12) may offel a rrore andD. strctmonlrrrdemonstratethat secondarymodificationsof alkal- satisfactorystartingmaterialfor futurc work. oids do occur in the aerialparts of theseplants.This has beendemonstrated conclusively by 1-eedingalkaloid-fiee scions of D. .feror on Furtherreoding Cyphontandrabetaceastocksrvith hyoscyamine;on subsequentana- Bevan C D, Marshall P S 1994The useof supcrcriticalfluids in the isolationof naturalproducts.Natural ProductReports I 1(.5):.l-51-.166 lysis, hyoscinewas isolatedfiorn the leaves.More detailsof this conBraithwaiteA. Smith F J 1995Chromatographicmethods.Chapmanand Hall v e r s i o na r eg i v e ni n t h eu p p r o p r i a tsee c t i o n . (now under KluwerAcademic Publishersimprint), Dordrccht.Nl In other generait hasbeenshownby grafis involving tomatothat the HarborneJ B 1998Phytochemicalmethods-a guidc to modern techniques. alkaloid anabasineis producedin the leavesof Nicotiuta gkttrr:u.nicoChapmanand Hall (now under Kluwer AcadeuticPublishersirnprintl. Dordrecht.Nl tine in the roots of N. tabacLuttand the pungentprinciple of capsicum, HoughtonP J, RamanA I 99ll Laboratoryhandbookfor the fl'actionationof capsaicin,in the developingfruits. Reciprocalgrafts of high and low naturalproducts.Chapmanand Hall (now under KluwerAcademic resin-yieldingstrainsof Cannabishave shown this biochemicalcharPublishersimprint), Dordrecht,Nl acterto be determinedby the aerialparts. Khaledi M G I998 High performancecapillary electrophorcsis. J. Wiley. New
MUTANTSTRAINS Large numbers of mutant strainsof microorganismshave been produced which lack a particularenzyme.which resultsin their metabol-
York. NY King M B" BottT R (eds) 1993Extractionofnatural productsusing near-criticalsolvents.Blackie Academic and Professional.Glasgow.UK Meyer V I 999 Practicalhigh-performanceliquid chromatography. 3rd edn. J. Wilcy. Chichester.UK TaylorL T 1996Supercriticalfluid extraction.J. Wiley and Sons,Neu York.NY
,t ,1. I
.t' .,i
11
'r.
The biosynthesisofboth primary and secondarymetabolitesis dependent on the highly organized structureof the plant and animal cell. Unlike animal cells, those of plants possessa rigid cell wall and are separatedone from anotherby an intercellular stmcture,the middle larnella.Direct connectionbetweenadjacentcells is maintainedby primary pit lields throughwhich passthe plasmodesmata. Within the cell wall is the protoplastconsisting of cytoplasm, nucleus and various organelles. The light microscopeshows the nucleus to contain various inclusions such as nucleoli. chromosomes(stainableduring cell division) and the nuclearsap. The nucleusappearsto be suspendedwithin the cell by the cytoplasm,in which there may be large vacuoleswith their own characteristiccontents(crystals,aleuronegrains,etc.).Othercytoplasmic inclusions are mitochondria. Golgi bodies, lysosomesand plastids(chloroplasts,chromoplasts,leucoplasts),but their structureis not resolvable,becauseof their small size.Electronmicroscopyshows a number of the subcellularorganellesto have a highly organizedfine structuresuited to the many and varied biochemicalprocesseswhich they perform. Although, becauseof varied form and function, it is not possibleto illustratea 'typical' plant cell Fig. 19.lA shows diagrammaticallythe structuresthat might be expectedin an unspecializedyoung root cell. Such a cell possesses a rigid wall, which immediatelydistinguishesit fiom an animal cell. but no chloroplastsand only small vacuolesare present.In a greenplantcell (Fig. 19.I B) the samecomponentsarepresent but the large vacuole has oppressedthe nucleus and cytoplasm towardsthe wall: greenplastidsotien with starchgranulesarecommon. The organellesallow the creation of different chemical environmentswithin one cell, and furthermore,by their structurethey increase the area available for surface reactions which are all-important in biological systems.A descriptionof the variousorganellesis given in the Iastedition of this book and Fig. 19.1C illustratessomeaspectsof their interdependence in the nolmal functioning of the cell. The molecular strulcturesof thesebodies have been extensively studied and details will be found in standardbotanicaltexts. Some basic metabolicpathwaysappearto be similar in both plants and animals, whereasothers are more restricted in their occurrence.It is to the secondaryplant products(i.e.thosenot necessarilyinvolvedin the essentialmetabolism of the cell) that the majority of vegetable drugs owe their therapeuticactivity and so it is in thesethat pharmacognosistsare particularly interested.However, as illustratedin Fig. 19.2.the productionofthese secondarymetabolitesis dependenton the fundamentalmetaboliccyclesof the living tissueand so a brief indication of the latterwill alsobe given; for fuller accountsof these.the student shouldconsulta standardwork on plant biochemistry.
.,tlutitlltlatll .;itlilr,,iiuiitti r" :tiit:tiit'i
itlt:iti itltitlii; ;ta,rl:l :tiiititi:
lllfilti
..,utitit:it: t:uitilt:1it,tii:tf
.tll
.
,:illiil
Bosicmetobolic l' l,pothwoys ondthe originof secondory metobolites
ENZYMES I50
ENZYJ-YIE3
PHOTOSYNTHESISt 5 4 CARBOHYDRATEUTITIZATION I55 GTYCOSTDESr 56 FATSAND FATTYACIDS
157
AROMATICBIOSYNTHESIS r 5 8 AM|NO AC|DS r60 PEPTIDES AND PROTEINS T63 ISOPRENOIDCOMPOUNDSI63 167 SECONDARYMETABOTITES
Many r"eactions occurringin the cell areenzyme-dependent, and before it was anythingwas known of the chemicalnatureof thesesubstances recognizedthat they were organic catalystsproducedby animal and vegetablecells. Their wide distribution and the delicacy of their operationhaslong beenappreciated. An enzymeusuallyactson one substance or classof substances, since it is specific for a parlicular atomic group or linkage. Specificity, however, variesl lipases are, in general, not highly specific, whereas fumarase acts only upon L-malate and fumarate. while o-malate is a competitive inhibitor of fumarase.Enzymes are also stereo-and regiospecific in their actionsand, as it becomespossibleto preparemore rare examples,organicchemistsarebecomingincreasinglyawareof enzyme potential for carrying out single-step transformations with complete
BASICMETABOLIC PATHWAYS AND THEORIGINOF SECONDARYMETABOLITES
CELLWALL Deposition of cell wallmaterials
/\ U
^^, urJLur
vtr-rultr
TCA cycle Cytochrome system
GOLGIBODY Glvcolvsis
.-co{
I Anarero ]
I Hexoses, Pyruvate] I othermonosaccharidesI Phosphate I CYTOPLASIV acceprorsl
CHLOROPLAST
q+6rada
ATP
carbohydrates
NHs
DNA
'Lt H,r"..\s",nNn
NUCLEUS
oorrort,6.--Aminoacids/ CULUM
I{IIHI(
{
Fattyacidsynthesis and degradation
RIBOSOMES
Proteins
Storageand waste products; secondary metabolites
Fig. l9.l Theplontcell.A, diogrommotic representotion of on undifferentioted cell:c, cytoplosm; reticulum; g, Golgi opporotus; e.r,endoplosmic l, lysosome; m , m i t o c h o n d r i omn.;l ,m i d d l el o m e l l on; , n u c l e u sn;l ,n u c l e o l uns .; m ,n u c l e om r e m b r o n ep;, p i t i n w o l l ;r , r i b o s o m ew s , p r i m o r yc e l lw o l l ;v , v o c u o l e B, greenplontcell:cp, chloroplost; m, mitochondrion, n, nucleus; v, vocuole.C, Flowchortof somecellmetobolites.
stereochemical exactitude,an aspectimportantin the synthesisof many drugs.An enzymewill conveftmany thousandtimesits own weight.and the gradualdiminution in activity which takesplaceis probablydue to secondaryreactionswhich bring aboutdestructionof the enzyme. The enzymologyof the secondarymetabolicpathwaysin plantshas still been little investigatedbut progressis being rnadeand here again cell cultureshaveprovedusefulin that they areoften a bettersourcefor the isoiationof enzymesthan is the difl'erentiatedplant.In somecases, e.g. cell culturesof volatile oil-containingplants.little or no oil accumulatesin the culture owing to the absenceof storagereceptaclesbut the relevantenzymesfor terpenoidsynthesisare still manutacturedand preparationsof them can be made. New horizonsfbr the study of the enzymologyof secondarymetabolism have now openedup as a result of advancesin genetechnology. ln suitable instances. by cloning. the cDNA responsible fbr an enzyme's synthesiscan be expressedin anotherorganism such as a bacterium and large amounts of enzyme prepared.By conventional
nrethods only very small amounts of purilied enzyme could be obtained fiom the original plant material. Of particular interesthas beenthe isolation.characterization andcloningofthe enzymestrictosidine synthase.This govemsthe key reactit'rnlbr the commencementof the biosynthesisof the very man)' monoterpenoidindole alkaloids, namely the condensationof tryptamine and secologerninto give 3s(S)-strictosidine(seeChapter21): for a review (126 references)on this enzymeseeT. M. Kutchan,Plntocltentistrl'.1993.32. 193. By meansof relativelynew technology.enzymescanbe immobilized on a suitablecarriereitherin whole plantcellsor asthe isolatedenzyme. In this way thesebiocatalystscan be repeatedlyusedin analyticaland clinical chemistry.or to etTectspecilicchernicaltransformations. Like othercatalysts.enzymesinf-lirence the rateof a reactionwithout changingthe point of equilibrium. For example,lipasecatalyseseither the synthesisof glyceridesfi'om glycerol and tatty acidsor the hydrolysis of glycerides,the final point of equilibrium beingthe samein either case. Similarly, B-glucosidase(prunase)has been used fbr both the
E
PHYTOCHEMISTRY
Coz G l v c o s i d feo r m a t i o n -. ^ . ,/ blarcn oentosans. / Glucose ascorbic ac,td / A m i n oa c i d s
A m i n oa c i d s
Aromatic compounds Aromatic a m i n oa c i d s Phenylpropane derivatives
Acetyl-coA Malonvl I CoA
l\,4alonyl-CoA Polyketides Anthraquinones
ll
//
/
+ Lionin
AromaticcompoundsoI m i x e do r i g i n( e . 9 f. l a v o n o i d s ) A m i n oa c i d s
F i g .1 9 . 2 metobolites in Origins of somesecondory pothwoysof relotionto thebosicmetobolic plonts.DOX=deoxyxylulose; IPP=isopentenyl MVA=mevolonic diphosphote; ocid.
\
/
Proteins Alkaloids lsothiocyanates Cyanogenetic compounds Various heterocyclic nitrogen compounds
Porphyrins(chlorophyll) Cytochromesystems givingATP
In plantssuchreversible synthesis and the hydrolysisof B-glucosides. reactionsmay proceedin one directionor the other underdifferentconditions. often resultingin daily and seasonalvariationsin the accumulation of n-retabolites. Enzymes are colloidal in nature and consist of protein or contain protein as an essentialpart. They may be partly puified. and in some casesisolated.by the nrethodsof protein chemistry(i.e. by fiactional precipitation.dialysis and. more recently,gel and aftinity chromatography,seeChapter l8). Most enzymesare solubleeitherin irater or in dilute salt solntionsand areprecipitatedby alcoholor acetone(acetone powders)and by high concentrationsof salts.They are inactivatedby heat. ultraviolet light and X-rays or by any treatmentwhich brings aboutdenaturationof proterns. The activity of enzyrnesis markedly affectedby the reactionol the medium and the presenceof substances suchas sa1ts.It is well known. lbr exarnple,that pepsinworks only in an acid medium and trypsin in havepH optimaof 3.8-7.5 an alkalineone.In general.carbohydrases and lipasesoptimaof pH 5-8, while enzymeswhich act on basesal1 haveoptima rnorealkalinethan pH 7. The effect of heat on enzymesis of considerableimportancein the drying of dru-es.At low temperaturesenzymic changesare not usually marked. although the proteolytic or protein-splittingenzymesin cod livers do bling about some hydrolysis at ternperaturesapproaching zero. The optimum working temperaturesof different enzymesvary. but they usually lie between35 and 50'C. At temperaturesof about 60oC destructionof the enzymesis usually fairly rapid, althoughconsiderable loss mtry take place below this temperature.When dry, enzymesshow increasedresistanceto heat;thus,zymase,which in the presenceof moisture is rapidly inactivatedat 50'C, will, when dry, resista temperature of 85oC.
Chemicol nqture Followingthe isolationof ureasein crystallinefblm by Sumnerin 1926.rnanyotherenzymes havebeenprepaledin a crystallinefbrm
HHHH-
I O=P-OH I
o
I HO-P-OH tl
o
and their protein naturehas beenestablished.Their moleculal weights are high. but vary fiorn about 9000 (hydrogenase)to about 1 000 000. In many casesthe component amino acids are known and in some cases(e.9.ribonuclease)the amino acid sequencewithin the molecule has been determined.During the isolation of an enzynre.the purified protein (apoenzyme)rnay be inactive but regains its activity in the presenceof an essentialcoenzymeor activator',which may be organic or inorganic. Coenzymes Some enzymeswhich require the presenceof smaller organic molecules,called coenzymes,befbre they can function are of very common occurrence and participate in a large number of important biochemicalreactions.One group of coenzymesconsistsof estersof phosphoricacid and vadous nucleosides.The adenosineand uridine
BASICMETABOLIC PATHWAYS AND THEORIG{NOF SECONDARY METABOLITES phosphatescontain one basicunit each(mononr.rcleotides); they serve to transportenergy in the form of high-energyphosphatebonds and this energyis made availablefol biochemicalreactionsin the presence of the appropliateenzymeby hydrolysisof the bond. Thus. the telminal phosphatebond of the adenosinetriphosphate(ATP) on hydrolvsis to adenosinediphosphate(ADP) affords50 000 J mol l. Uridine triphosphate(UTP) is involved in the synthesisof sucrose via diphosphateglucosewhich is also associatedwith the formation of u r o n i cr c i d sr n t l c e l l r r l o s e . Nicotinamide-adeninedinucleotide (NAD) and nicotinamideadeninedinr.rcleotide phosphate(NADP) contain two basic units each and are termed dinucleotides.Thev function in oxidation-reduction systemswith appropliate enzymesl the oxidized forms ale uritten NAD+ and NADP+ and the reduced folms NADH and NADPII resoectivelv.
*?-t
I
H - c - o H 6 "-?-t H-c-oR6l H-C-OHl | |
ticipate in enzymatic reactionswhich involve one-carbonfi'agment transt-ers. A seriesof quinonese.g.plastoquinone and ubiquinoneare widely distributedin plants.animalsand microorganismsand function in biological electrontransferprocesses. Clossificqtion of enzymes As more enzymesare isolated.it becomesimpofiant to have a precise scheme of classification and nomenclatule. Many long-established namessuch as pepsin.prunase,diastaseand narnesfor enzyme mixtures.suchasemulsinand zymase.continuein use.A steptowal'dsLlnifornr nomenclaturewas madewhen they were denotedby the nameof the substrateand the termination'-ase'. Classesof enzynreswere named sinrilarly.Thus, the genelaltenn 'esterase'includes lipases, which hydlolyse fats: chlorophyllasehydrolyseschlorophylll etc. The 196l Report of the Cotnrnissiottort Erta)'nrcs" rnadethe recomntendation that the chemicalreactionscatalyscdbe generallyadoptedfbr classilicationtrndnomenclature. This. ofcoursc.presupposes that the exact chemicalreactionis known: seealso the 198;1InternationalUnion of Biochemistry publication (London. UK: Academic Press) Enzlme NomenclatLtreand any later repol'ts.Enzymes are classifiedinto six main groups(Table19.1).
H-C-OHll | llRibose
Oxidoreductases. As any oxidationimplies r sirnultaneous reduction, the names'oxidase'and 'reductase'rna-vbe appliedto a sin-eletype of enzyme. Eleven different groups of oxidoreductasesafe reco-enized. Many oxidases(that is. enzymeswhich utilize molcculaloxygen as o-P-o-P-o ) acceptor)convert phenolic substancesto quinones.They act on guaii l t l o o acum resin to producea blue colour which is used as ii test fbr their detection.Oxidasesinclude laccase(1.10.3.2),presentin lac. and N A D ' :R - H ascorbateoxidase(1.10.3.3),which is widely distributedin plants. NADP': n. PiOH -oH Oxalateoxidase(1.2.3.4).a flavo-proteinpresentin ntossesand the il o leavesof higher plants. oxidizes oxalic acid into carbon dioxide and (1.11)are distinguishedby thc tact hydrogenperoxide.Peroxidases Another importantcoenzymeis coenzymeA (CoA), which contains that they usehydrogenperoxideand not oxygenasthe hydrogenaccepthe Lrnits adenosine-3.5-diphosphate. pantothenic acid-4-phosphate tor'.Catalase(1.11)mustbe regardedas an exception.sinceit cirtalyses rnd thioethanolamine.It participatesin the transf-erof acetyl and acyl the decompositionof hydrogen peroxide.An oxidoreductaseof the -sroups.acetyl-CoA (active acetate)havin-ea centralrole in plant and morphine biosyntheticpathway (Chapter27) has recenlly been purilunimalmetabolism. fied and characterized;it catalysesthe stereoselectivereduction of salutaridineto 7(S)-salutaridinolusingNADPH as cosnbstrate. N=C-NHa H o-c-N-cH2-cH2-sH Hydrolases. These include many different types. ol which the folHC C-N. lowing are someof pharmaceuticalimportance.
H-c-, "-??H.?H ?" ?".
ll ll )x
?Fh
N-C-l.r
I H_C_r l H-C-OH r H-C-OR
$
I I
9l-1, t NH l O=C l H-C-OH
t o
|
H-6 t
u"-J-u" l
oH cHz cH2oH t - t I t o-P-o-P-o i
o
I I
l
t
l
o
'=i{: o
1. Hydrolysingesters.Theseincludelipases(3.1;. rvhich may be of vegetable or animal origin and which h1'drolyscglycerides. Mammalian lipasesalsohydrolyseotheresters.suchas phenyl salicylate. acetylcholineand atropine.Other csterasesare chlorophyll a s e ( 3 . 1 . 1 . i 4 ) , w h i c h h y d r o l y s e sc h l o r o p h y l l , a n d t a n n a s e (3.1.I.20). which hydrolysesesterlinks in tannins.Otheresterases probably occur in many drugs w,hichcontainestersin their volatile oils (e.g.valerian). 2. Hydrolysing sugarsand glycosides.To the impofiant group of glycosidehydrolases(3.2.I ) belongall thoseenzymeswhich hydrolyse
Coenzyme A
I
F,,
Riboflavine(Fig. 31.2)is a componentof the two coenzymesflavin rrononucleotide(FMN) and flavin adeninedinucleotide(FAD). They .:rrticipatein the biological oxidation-reduction system, and FAD .Lcilitatesthe transfer of H+ ions from NADH to the oxidized \ t()chromesystem. Other coenzynes are the decarboxylationcoenzymes thiamine. .1()1in andpyr'doxine(Fi-e.31.2).Folic acid(Fig. 31.2)derivatives par-
* The above reporthas a nurnbercclclassiflcationfbr eachenzyme.Fol exanrple. thc enzyme presentin garlic. alliine-lyase.is nunrbered,l.,1.l..1. The first number denotesthe tburth of the sir nrain groups alreadymentionedand the other numbersfurther subdivisions.Thus: .l = Iyases :1..{ = carbon-sulphurlyascs ,1.,1.1= In this examplea firrthersubdivrsionis not required. 4 . 4 . 1 . 4= t h e f o u r t h e n z y m e l i s t e d i n t h e g l o u p . n a m e l v a l l i i n c - l v a s c (lbrmerly known as alliinase)or ally1sulphinate-lyase.
E
PHYTOCHEMISTRY
Toble I9.I
Clossificotionof some enzymes.
Group
Triviol nome
Syslematicname
l. Oxidoreductoses
Glucosedehydrogenose pDiphenyloxidose(loctose) Peroxidose Cotolose a-Gluconphosphorylose Lipose Chlorophyllose Tonnose o-Amylose lnulose Aldolyose Moleoteisomerose Asporoginesynthetose
p-o-Glucose: NAD(P)-oxidoreductose pDiphenol:02 oxidoreductose Donor:H202 oxidoreductose H20 2: H20 2 o rthoreduclose cr-1,4-Glucon : orthophosphote glucosyl-tronsferose Glycerolesterhydrolose Chlorophyll chlorophyllidohydrolyose Tonninocyl-hydrolose 'l,4-Glucon cr4-glucono-hydrolyose Inulin'lJruclonohydrolose KetoseJ-phosphote o ldehydelyose Moleotecis-frons-isomerose L-Asportote: ommonioligose(ADP)
2. Tronsferose ? H.^.^t^.^.
4. Lyoses 5. lsomeroses 6. Ligoses(Synthetoses)
sugars,carbohydratesand glycosides.Those hydrolysing sugars (sucraseor invertase).lactase,malinclude B-fiuctofuranosidase tase,gentiobiaseand trehalase.Polysaccharideenzymesare representedby o-amylase (-1.2.1.1),B-ainylase.cellulase,lichenase. inulase.Among the glycoside-hydrolysingenzymesare B-glucosidase or B-D-glucosideglucohydralase(.3.2.L20), which has a More specific glucowide specificity fbr B-D-glucopyranosides. side-hydrolysingenzymesare those acting on saiicin, amygdalin. sinigrin and cardiacglycosides. 3. Hydrolysingthe C-N linkage.Many enzymesact on peptidebonds. To this group belong the well-known animal enzymespepsin.rennin, trypsin, thrombin and plasmin, and the vegetableenzymes papain(from Caricu papat'a)and ficin (frorn speciesof fig). A11the above belong to the group 3.11.21. Other enzymesacting on linear' arnides (3.5.1) are asparaginase(presentin liquorice and many otherplants)and urease.Cyclic amidesareactedon by penicillinase (3.5.2.6). Isomerases. Two enzymesimportantin pathwaysleadingto medicinally importantmetabolitesare isopentenyldiphosphate(lPP) isom(EC 5.5.1.6).The formeris erase(EC 5.3.3.2)amd chalconeisomerase involved in the isomerizationol TPPand dimethylallyl diphosphate (Fig. 19.18),an essentialstep in the fbrmation of terpenoids.and the latteris a key enzymecatalysingthe isomerizationof chalconesto their flavanones(Fig. I 9. I I ). corresponding
thesecontain,in additionto chlorophyll asthe principalpigment,other tetrapyrrolederivatives-the phycobilins. The light microscopereveals no definite internal strLrctureof the chloloplasts,but electron microscopy shows these bodies to have a highly organized structure in which the chlorophyll molecules are arrangedwithin orderly structures(grana), each granum being connectedwith othersby a network of libres or membranes.According to one theory, the flat chlorophyll rnoleculesthemselvesare orientated betweenlayersof protein and lipid moleculesso that the whole chloroplast can be looked upon as a battery containing several cells (the grana), each celi possessinglayers of plates (the chlorophyll molecules). Two fundamentalprocesseswhich take place in photosynthesis, both of which require light, are the production of adenosinetriphosphate(ATP) from adenosinediphosphate(ADP) and phosphateand the light-energizeddecompositionof water (the Hill reaction;namedafter the EnglishbiochemistRobertHill, 1899-1991): H2o-r-->
2tHl+]02
ATP is a coenzymeand the high energy of the terminal phosphate bond is availableto the organismfbr the supplyof the energynecessary fbr endergonicreactions.The Hill reactionproducesfree oxygen and hydrogenions which bring aboutthe conversionof the electroncarrier, 'Coenzymes'). NADP, to its reducedform NADPH (see In this complicatedprocess,two systems,PhotosystenrI and PhotosystemIl (alsoknown as pigment systemsI and lI) arecommonPHOTOSYNTHESIS ly ref-enedto; they involve two chlorophyll complexeswhich absorb light at diff-erent wavelengths (above and below )" = 685 nm). Phuosynthesis,by which the carbondioxide of the trtn'rosphere is con- PhotosystemII producesAIP and PhotosystemI supplies all the vertedinto sugarsby the greenplant. is one of the fundamentalcycles reducedNADP and someAIP. In theselight reactionsthe chlorophyll on rvhich life on Earth, as we know it, depends.Until 1940. when molecule captures solar enelgy and electrons become excited and investigationsinvolving isotopeswereundertaken,the detailedmecha- move to higher energy levels; on returning to the normal low-energy 'carbon nisrnofthis reduction'wasunknown.althoushthe basicover- state.the electronsgive up their excessenergy,which is passedthrough all reiiction. a seriesof carriers (including in the case of PhotosystemII plastolirht quinone and several cytochromes)to generateATP PhotosystemI - rcH.or+ 02 co, + H.o involves an electron acceptorand the subsequentreduction of feredoxin in the production of NADPH. Ref'erenceto the current literature had beenacceptedlbr many years. Photosynthesisoccurs in the chloroplasts:green, disc-shaped indicatesthat the natureand organizationof the photosystemsremains organellesof the cytoplasrnwhich areboundedby a definite membrane a very active resealcharea.Studentswill have observedthat an alcoand which are autoreproductive. Separatedfrom the rest ofthe cell, the holic solution of chlorophyli possesses,in sunlight, a red fluoreschloroplastscan carry out the complete processof photosynthesis. cence-no carriersare availableto utilize the capturedenergyand it is Bodies with similal propertiesare fbund in cells of the red algaebut re-emiftedas light. Hill first demonstratedrn 1931that isolatedchloro-
BASICMETABOTIC PATHWAYS AND THEORIGIN OF SECONDARY METABOLITES plasts,when exposedto light, were capableofproducing oxygen,provided that a suitablehydrogen acceptorwas present.Work with isotopeshas sinceproved that the oxygen liberatedduring photosynthesis is derivedtiom water and not liom carbondioxide. Following the light reactions,a seriesof dark reactionsthen utilize NADPH in the reduction of carbon dioxide to carbohydrate. Current researchsuggeststhat terrestrialplants can be classifiedas C:, C+, intermediateCj-Ca and CAM plants in relation to photosynthesis.
cular bundle cells, where it is oxidatively decarboxylatedto pyruvate again,carbondioxide and NADPH, which areusedin the Calvin cycle. Pyruvatepresumablyreturnsto the mesophyllcells. Plantspossessing this facility exhibit two types of photosyntheticcells which difl'er in their chloroplasttype.IntermediateCj-C.1 pathwaysare alsoknolvn.
CAM plonts This term standslbr 'crassulacean acid metabolism'.so calledbecause it was in the Crassulaceaefamily that the distinctive characterof a build-up of malic acid during hours of darknesswas first observed. C3 plonts Other large families, including the Liliaceae, Cactaceaeand possessmembersexhibiting a similar biochemistry.As The elucidationof the carbon reduction cycle (Fig. 19.3), largely by Euphorbiaceae. Calvin and his colleagues,was in large measuredeterminedby meth- with Ca plants, this is an adaptationof the photosyntheticcycle of ods dependenton exposing living plants (Chlorella) to 1'+C-labelled plants which can exist under drought conditions.When water is not carbon dioxide for precise periods of time, some very short and available,respiratorycarbon dioxide is recycled,under conditionsof amountingto a liaction of a second.The radioactivecornpoundspro- darkness.with the formation of rnalic acid as an intermediate.Carbon ducedwere then isolatedand identified. In this way a sequencefor the dioxide and water loss to the atmospherear"eeliminated,a condition formation of compoundswas obtained.3-Phosphoglycericacid, a C3 which would be fatal for normal Cj plants. compound,was the compoundfirst fbrmed in a iabelledcondition but From fi-nctose,producedin the Calvin cycle, other important conit was only later in the investigation.after a number of 4-, 5-, 6- and stituents,suchas glucose.sucroseand starch,irrederived:erythroseis a was precursor in the synthesisof some aromatic compounds.Glucose7-carbonsystemshad beenisolated,that ribulose-1,5-diphosphate shownto be the moleculewith which carbondioxide first reactsto give 6-phosphateis amongthe early productsfbrmed by photosynthesis and it is an importantintermediatein the oxidativepentosephosphatecycle two moleculesof phosphoglycericacid. An unstableintermediatein this reactionis 2-carboxy-3-ketopentinol. and for conversionto glucose-I -phosphatein polysaccharidesynthesis.
Caplonts Some plants which grow in semi-aridregionsin a high light intensity possessan additionalcarbon-fixationsystemwhich, althoughlesseffiin its use of carcient in terms of energyutilization, is more e1I'ective bon dioxide, so cutting down on photorespirationand ioss of water. Such plants are known as C4 plants.becausethey synthesize,in the presenceof light, oxaloaceticand other C4 acids.Carbonassimilation is basedon a modified leaf anatomyand biochemisffy.Its presencein plantsappearshaphazardand hasbeenlinked with the so-calledKranz syndrome of associatedanatomicalfeatures.In the mesophyll cells pyruvateis convertedvia oxaloacetate to malate,utilizing carbondioxide, and the rnalate,or in somecasesaspafiate,is transportedto the vas-
UTiLtl{IlON,, ,],,CARBOHYDI{TE Storagecarbohydratesuch as the starchof plants or the glycogen of animals is made availablefor energy production by a process'"vhich involves conversion to pyruvate and then acetate,actually acetylcoenzymeA, the latter then passinginto the tricarboxylic acid cycle (Fig. 19.a).As a result of this. the energy-richcarbohydrateis eventually oxidized to carbon dioxide and water. During the process,the hydrogen atoms liberated are carried by coenzymes into the cytochromesystem,in which energyis releasedin stages,with the possibleformation of ATP from ADP and inorganicphosphate.Eventually the hydrogencombineswith oxygento form water. A number of pathways for the initial metabolism of glucose are known for various Iiving tissues.One involves compoundswhich are found in the photosyntheticcycle, and it appealsas a reversalof this cycle but the mechanismis apparentlyquite different.Another pathway is the Embden-Meyerhoffschemeof glycolysis(Fig. 19.5).
Acetyl-CoA *^
-/"roroloocetore
Citrote
Mobte
^"o1 Erythrose4-P
,.oI__-^_ tumorore
-'"'Yi"o
CisoC6nibte
^ Y'*
Succirrfe
lsocitrote
,A
Orobswcinote
),.. @"'
Fig. 19.3 (ofter theformuloe Thepothof corbonin photosynthesis Colvin); of the involved 21. sugors oregivenin Chopter
Fp'
:":h:^
Srccjnrl CoA
M4z,
\o-oxogbto.r"7@2
F i g .1 9 . 4 Thetricorboxylic cycle. ocidcycle(TCA) or Krebs
E
PHYTOCHEMISTRY
cHo
cHo
I H-C-OH I H O- C - H I H-c-oH I H- C - O H
I
H-c-oH I HO-C-H I
ATP llerolinose
--
Phosphogh,cose iso|nero3e +
H-c-oH I H-c-oH
-
I CH2OP Glucosc-6-P
I I
CH2OH 0- Glucose
cHroP t C:O I HO-C-H I H-C-OH I H-C-OH I CH2OP Fruclosc| '6-diP
tnor*gtycero- CH2OP IHrOP CHOH '-* CHOH =i-t l
cooP
cooH
1'3-OiPhosPhoglycericocid
3-Phosphoglycericbcid
1l tl
cl'lzoPOlHz 3Glyceroldehyde
It
cH2oH I
F i g .1 9 . 5 Outline of theEmbden-Meyerhoff scheme of glycolysis.
CH"OH t C=O I HO- C-H I H-C-OH I H-C-OH I CH2OP Fructose6- P
ph06ghole
-Hao
CHOP =!ttl"*I c@H 2- Ph6phoglyc.ricocid
The kinetics and enzymesystemsinvolved in the abovepathwayshave beenstudiedextensively. It will be notedthatonemoleculeofglucosecan give rise to two moleculesof pyruvate,each of which is convertedto acetate,and one molecule of carbon dioxide. One tur"nof the TCA cycle represents the oxidationof one acetateto two moleculesof carbondioxide. giving rise to twelve moleculesof ATP The overall reaction fbr the metabolismof one moleculeof glucosein termsof ADP andAIP is C6HI2O6+ 6CO2 + 38ADP + 38P(inorganic) ---> 6H2O + 6COr + 38AIP The above schemes,given in barest outline, are fundamentalnot only fbr the building up and breakingdown of reservefoodstufl\. but also in that the intermediatesare availablefor the biosynthesisof all other groupsof compoundsfound in plants.The levels at which some of thc importantgroupsariseare indicatedin Fig. 19.2.
i". dop I
Pyiuvole
--- limse
coo|'l
io' i" ----'ito P co
cooH Pyruric
PhosPhocnolpyruvicocid
l-aoo AcclylcocnzymcA
The fbrmation and hydrolysisof an O-glycosidesuchas salicinmay be represented:
EFE
" Sugar
o.x.+ R.ox+Hzo Aglycone ot
Glycoside
!:= C6H'q.O.C6H6.CH2OH C6H11O5OH + FlO.C6H..Cl-'lzOH + l-l2O Salicin Glucosc Salicylalcobol (saligenin)
It will be notedthat suchreactionsarereversibleand in plantsglycosidesareboth synthesizedand hydrolysedunderthe influenceof more or less specific enzymes.While glycosidesdo not themselvesreduce Fehling'ssoh.rtion. the simplesugarswhich they produceon hydrolysis will do so with precipitationof red cr.rprous oxide. The sugarsfound in glycosidesmay be monosaccharides such as glucose,rhamnoseand fucoseor, more rarely.deoxysugarssuchas the ' , GIYCOSIDES i cymarosefound in cardiacglycosides.More than one moleculeof such sugarsmay be attachedto the aglycone eithel by separatelinkages, Glycosiclesare tblrned in nature by the interactionof the nucleotide which is rare.or, more commonly,as a di-. tri- or tetrasaccharide. Such glycosides-fbl erample. Lrridine diphosphate glucose (UDP- complex glycosidesare fbrmed by the stepwiseaddition of sugarsto glucose)-with thc alcoholicor phenolicgroupof a secondcompound. the aglyconemo1ecule. Such glycosides.sometimescalled O-glycosides.are the most numerSince sugarsexist in isomerica- and B-forms,both types are theoous onesfound in natr.rre. Other glycosidesdo, however.occurin which retically possible.Practicallyall naturalglycosides,however,areof the the linkage is through sulphur(S-glycosides).nitrogen (N-glycosides) B-type, although the o-linkage is lbund in nature in some carbohyor carbon( C-glycosides). drates such as sucrose,glycogen and starch.In ft-strophanthoside. a
BASICMETABOLIC PATHWAYS AND THEORIGINOF SECONDARY METABOLITES glycoside formed from the aglycone strophanthidin and strophanthotriose(cymarose+ glucose+ glucose)the outer glucosemolecule has the cr-linkageand the inner glucosethe B-linkage.Isomericglycosides may be preparedsynthetically;for example,from glucose and methyl alcohol one obtains both the cr- and B-methyl glucosidesby introducing methyl groups into the OH groups printed in heavy type in the formulae of glucose below.
Hoa
H O H
rH
?-r cH.oH HO.CH
I
O
lr.or Il t
cH-r I cH2oH
p-Glycosideforming structur€
c--r t
l
HO.CH
O
cH.oH I
6r,o, Il r cH---r
cruoH tr-Gtycoside formrng structure
The term 'glycoside'is a very generalone which embracesall the many and varied combinationsof sugarsand aglycones.More precise terms are availableto describepafiicular classes.Someof theseterms refer to the sugar part of the molecule, others to the aglycone, while othersindicate some well-defined physical or pharmacologicalproperty. Thus, a gLucosideis a glycosidehaving glucoseas its sole sugar component; a pentoside yields a sugar such as arabinose;rharnnosicles yield the methyl-pentoserhamnose;and rhamnoglttcosides yield both rhamnoseand glucose.Terms used for aglyconesare generally selfexplanatory(e.g., phenol, anthraquinoneand sterol glycosides).The names'saponin'(soap-like),'cyanogenetic' (producinghydrocyanic acid) and 'cardiac'(havingan action on the heart),althoughappliedto thesesubstanceswhen little was known about them, are useful terms which do in fact bring together glycosides of similar chemical structure. The older systemof naming glycosidesusing the termination '-in' (e.g. senegin,salicin, aloin, sffophanthin)is too well establishedto be easily changed.It is not ideal, however, since many non-glycosidal (e.g. inulin and pectin)have the sametermination.Modern substances workers more frequentlyuse the termination '-oside' (e.g. sennoside) and attempt to drop the older forms by writing sinigroside for sinigrin, salicosidefor salicin and so on. Although glycosides form a natural group in that they all contain a sugar unit, the aglyconesare of such varied nature and complexity that glycosidesvary very much in their physical and chemical propertiesand in their pharmacological action. In Part 6 they are classilled according to that aglyconefragment with which they often occur in the plant.
anda thiolysisIiberatinga two-carbonunit of acetyl-CoA.A11thesereactionscan be shownby isolatedenzymestudiesto be reversible.anduntil about 1953it was generally assumedthat the biosynthetic roLrteof fafty acids operatedin the reversedirection, stafting from acetyl-CoA. This, however,is not preciselyso,asindicatedbelow.
Biosynthesisof soturoted fotty ocids From the aboveit was generallyassumedthat the biosyntheticroutefor fatty acidsoperatedin the reversedirectionto the B-oxidativesequence for the degradation of these acids and starred wirh acetyl-CoA. However,the unfavourableequilibrium of the initial thiolasereaction in sucha pathway:
o
o
o
l l l l l 2CH3-C-SCoA r= CH3-C-CH2-C-SCoA
+ CoA
&o=1'6x10-5 and the poor yield of long-chain fatty acids obtained with purified enzymesof the B-oxidationpathway togetherwith other observations suggestedthe possibility that this was nor necessarilythe principal biosyntheticpathway.It was then shown,with animaltissues,that malonyl coenzyme A was necessaryfor the initial condensationwith acetate.In fact, in palmitic acid (C16,)only one of the eight C7 units (i.e.the C15 + Cl6 carbons)is deriveddirectlyfiom acetyl-CoA;the other sevenC, units are attachedby malonl'l-CoA. which is lbrmed by carboxylationof acetyl-CoA. One featureof this anabolicpathwayis the involvementof the acyi canier protein (ACP) to producefatty acyl thioestersof ACP.This conversionserves(1) to activatethe relativelyunreactivefatty acid and (2) to provide a carrier for the fatty acid acyl group. Thus. theseACP thioestersappearto be obligatoryintermediatesin latty acid synthesis, whereasit is the fatty acid thioestersof coenzymeA which operatein the catabolic oxidation pathway. The synthesisis explained by the reactionsshown in Fig. 19.7.Reactions4-7 arethen repeated,lengthening the fatty acid chain by two carbons (derived from malonylS-ACP) at a time. Eight enzymes have been identified as being involved in the synthesisof palmitoyl-ACP and stearoyl-ACPfiom acetyl-CoA. In purified enzyme systems,when propionyl-CoA replacedacetylCoA, the productwas a C17acid and, similarly,butyryl-CoA gaveprimadly a C1s (stearic)acid. Thus, the formation of theseC16,C17and C13 acids may well dependon the availability of acetyl-, propionyland butyryl-CoA. The system of biosynthesis of fatty acids in plants appears to operatein the sameway as in animaltissues,but whereasin the latF.t +
Fattyecid
-CFfr-Clt-Cl{2-CO.SCoA
Fats,consideredin more detail in Chapter20, are triglyceridesinvolving long-chainsaturatedor unsaturatedacidsand,as such,constitutean important food reservefor animals and plants (particularlyin seeds). Relatedto the simple fats are the complex lipids, most of which are diesters of orthophosphoric acid; they have the status of fundamental cellular constituents. The fatty acids,on liberation from the fat, are availablefor the production of acetyl-CoA by the removal of C2 units. The elucidation of this spiral (Fig. 19.6) owes much to the work of Lynen and it was primarily investigatedwith animal tissues.In this B-oxidationsequenceone tum of Fig. 19.6 the spiral involves four reactions-two dehydrogenations,one hydration Degrodotion of fottyocids.Reoctions involved in oneturnof thespirol.
rfrFrn
ffi*
PHYTOCHEMISTRY
HOOCCH2CO'$CoA+ ADP + Pi ".ffi; acetyl-SACP + CoASH acetyl-S'CoA + ACP-SH-) malonyl-$CoA + ACP-SH------r malonyl-S'ACP + CoASH
CHgCO-S-CoA+ CO2 + lrf
1.
2. 3. 4. 5. Fig. r9.7 o[ in thebiosynthesis stoges Initiol fottyocids.
malonyl-9AcP + acetyl-SAcP ---') + NADPH* H* ---+ acetoacetyl-S-ACP
6. 7.
acetoacetyl-S-AcP+ CO, + ACP'SH + NADP* o(-)-3-hydroxybutyryl-9ACP
c rotonyl-SACP + HOH o(- )-3-hydroxybutyryl-S-ACP----------+ butyryl-S-ACP+ NADP* crotonyl-S-AcP + NADPH * H* --"----------+
ter the enzymesarelocatedin the cytoplasm,in plantsthey aretbund oration of Ll-l4Clacetateinto 6-methylsalicylicacid by Penicillium in the rnitochondria and chloroplasts. The mitochondria derived griseofulvumtakesplaceas below: from the mesocarpof the avocado fruit have been used to demoncHl strate the incorporationof Il4Clacetateinto esterifiedlong-chain fatty acids.
Biosynthesisof unsoturoted fotty ocids As a numberof unsaturatedfatty acidshave a particularsignificancein pharmaceuticalproducts, a treatment of their formation is deferred until Chapter20.
.cooH
!", z{-€oox
cHr-rcooH --------+ll
cHl
I .cooH
I
\.4o"
tcooH Hr
and the production of the mould anthraquinonemetabolite endocrocin, from eight C2 units, is representedin Fig. 19.9.Decarboxylationof endocrocin affords emodin. The original chain lengthening,which can The shikimicocid pothwoy be representedas a condensationof acetateunits, is the sameas in fatty This appears to be an inlportant route from carbohydrate fbr the acidproduction(q.v.above)but doesnot requirethe reductionof =CO to biosynthesisof the C6-Cj units (phenylpropanederivatives).of which =CH:. Thus, malonic acid plays a similar role in this aromatic synthesis phenylalanineand tyrosineareboth examples.A schemeof biogenesis to that in latty acid formation and the chain is built up from the combinfor thesearomaticamino acids,as elucidatedin various organisms.is ation of malonyl units with a terminal acetyl (the starter)unit. Sometimes given in Fig. 19.8;for higher plants,the presenceof the enzymesystem the stafterunit is not acetate.as indicatedwith the formation of the tetraresponsiblefor the synthesisof shikimic acid has beenconfirmed.An cycline antibioticsfrom nine units; here malonamide-SCoAis the starter important branchingpoint arisesat chorismic acidl anthranilatesyn- unit and by invoking standard biochemical reactions tetracycline is thaseuseschorismic acid as a substrateto give anthranilicacid which formed (Fig. 19.10).Higher plantsalsor"rtilize the polyacetate-malonate is a precursorof tryptophan.The synthesisis controlledby the latter pathway for the biosynthesisof emodin-typeanthraquinones,suchcomacting as a feedbackinhibitor: chorismatemutaseconvertschorismic poundsall having substituentson both outer rings. acid to prephenate,the preculsorof phenylalanineand tyrosine,and a listed in Chapter22, it will be seenthat the From the anthraquinones varietyof controlmechanismsappearto operateat the branchingpoint. stluctures of some anthraquinones are not fully explained by the The opium alkaloidsare synthesizedvia this pathway(Fig. 27. I 5) and acetate-malonatepathway and these are discussedbelow. However, two isofbrms of chorismatemutasehave beenisolatedand character- thereappearto be many exceptionsto the rules in applyingthe general ized liom poppy seedlings (M. Benesova and R. Bode, P/t1'to- pathwaysto specil'icanthrrquinones. 3f , 2983). clrcmistry'.1992. Although there are only smali difl'erencesin the sequenceof reac- Compounds confoining qromqtic rings of different tions lbr the shikimatepathwayin bacteria.fungi and plants there are origin considerabledifferencesin the molecularorganizationof the pathway. The structuresof anthraquinones suchas dlizarin,rubiadin,pseudopur(For a review (123 ref.s),see J. Schmid and N. Amrhetn' Phyto- purin and morindadiol,pigmentsof the Rubiaceaeand other families, chemistrv,1995.39, 13'7.) cannotbe readily explainedon the acetatehypothesis. The shikimic acid pathway is also important in the genesisof the o o g O H aromaticbuilding blocks of lignin and in the formation of some tanZr.-A.r.-S #o' Z\,;{r'\G and coumarins nins, vanillin and phenylpropaneunits of the 1-lavones f' ll rl I l' ll ll I l^ll.ll.l 'FurtherReading'' \,.\ufcoo" \-/W\cn, \-Al^V/'* (seeChapter22). For a teview see
AROMATICBIOSYNTHESIS
nlo*,""'
i"""ll
p*uoao*ilurin
The qcetote hypothesis Circumstantialevidence that these compounds might be formed The centralposition oi iicetatein relationto the generalmetabolismof with the participationof mevalonicacid (a key plantshas alreadybeenindicatedand it is possibleto devisemany pos- from naphthoquinones of isopentenylunits, q'v.) was provided by vxrformation gl\e a precursor in the to occur could sible routesby which acetatecondensation haviety of aromatic compounds.The generalvalidity of the mechanism their co-occurrencein teak and otherplantswith naphthoquinones hasbeenestablishedby the useof labelledcompoundsbut the detaiied ing an isopentenyl residue (e.g. formula below). That mevalonate stepsfor many compoundsremain to be established.Thus. the incotp- (itself derived from acetate)is involved in this formation has been
bASICMETABOLIC PATHWAYS ANDTHEORIGINOF SECONDARY METABOLITES @oH
cooH
I
I
C=O
Q-O-POsFh
Qru
cooH
CHOH
-L
I
tl CHa Phosphoenolpyruvic acid + -
I
I
I H-C-OH I H-C-OH I
cHo I
H-C-OH I H-C-OH I cH?oPo3H2 Erythrose-4-phosphate
t o
E ' " OH acid 3-Dehydroquinic
cruoPo3H2
2-Keto 3-deoxy-7-phosphoo-glucoheptonicacid
l ^oH ; "
6n
3-Dehydroshikimicacid
cooH I
co I
cooH
Ho@aoocHe
fn
i l t l \,/
cooH
Shikimic acid-3-phosphate
ry;"
9H' I t
t
l
Y
Anthranilic acid I RtroeptraiUosyt lrrytahosPhat€
I
Phenylpyruvic acid
OH p Hydroxyphenyl pyruvicacid
n"oorirr" "rin"tionf J cooH cooH I
cH-NH2
?Fh h, \./ Phenylalanine
I
cH-NH2
_
OH
Chorismicacid
I C=O I
?*.
I
H2o3Pos'YoH -
cooH
I C=O I
\,,/
fit.
OH
Prephenic acid
(\
+'.:i.:coo" -Hzo a
?o't-c@H :
fz
H O H
cooH
oPosHa
I
Is",in"
I
rfut-cH2-cH-cooH Nllz \Ap,c-*
H Tryptophan
?"'
h
I OH Tyrosine
Fig. I9.8 vio theshikimic Biosynthesis of oromoticcompounds ocid pothwoy.
shown by tracerexperimentswith Rubia tinctorum;ring C and carbon side-chain of pseudopurpurin and rubiadin are derived from mevalonate, and in the sameplant shikimic acid has been shown to be incorporatedinto ring A of alizarin.
The aromaticrings of somecompoundscan be derived from both the shikimic acid andthe aceticacid pathways.Thus,a phenylpropaneformed by the former route may undergo chain lengtheningby the addition of 'Fatty acetateunits (via malonyl; see Acids', above)to give a polyketide andthen,by ring closure,give a flavonoidderivative(Fig. 19.11). Isoflavones are formed in the samemanner,with a rearrangementof the aryl-B ring in relation to the three carbons (Fig. 19.11). The flavonoids,in addition to their importancein plants as pigments,have interestingmedicinal propertiesand are discussedin more detail in Chapter 22.
PHYTOCHEM!STRY
-
.{. .1. .f.
Cl-l;CoOH CooH CO0HCOOH
fH. ,.r, f^,
HOOC-CH3COOHCOOHCOOH
i i
,rc{t,rc{t rc{' cH3-co co co P Enzsoc-\ )\" z"^" )* co co co
Hsc HOOC O H O O H Endocrocin
A I I I I
<----l
Fig. 19.9 Biogenesis of endocrocin.
OH
CO.NHz I
o
OH
^ CH.CO.SCoA + . ' 1
in.co.scoa - SCoA Molonomide
coao
------+ NHz
MolonylCoA
CO.SCoA A polyketide
co.NHz OH Fig.I9.lO Formotionof tetrocycline.
Telroc line Telrocyc
Rotenoids(insecticidesof Derris and Lonchocttrpa.rspp.) show a structuralrelationshipto the isoflavones;seeChapter41. Stilbenes(q.v.) are also compoundscontaining aromatic units of differentbiogeneticorigin.
AftIINO ACIDS Amino acidsoccur in plantsboth in the free stateand asthe basicunits ofproteins and other metabolites.They are compoundscontainingone or more amino groupsand one or more carboxylicacid groups.Most ol those found in nature are cr-amino acids with an asymmetric carbon atom and the general formula R-CH(NH2)COOH. Some 20 different ones have been isolatedfrom proteins,all having an r--configuration. Other amino acids occur in the free state and some having the oconfiguration have been isolated from plants and microorganisms, where they may form antibiotic polypeptides. Many amino acidscontainonly carbon,hydrogen,oxygenand nitrogen,but other atomsmay be present(e.g.sulphurin cystine,and iodine in thyroxin).As alreadymentioned,more than one amino group may be present (e.g. Iysine, diaminocaproicacid) and more than one carboxylic acid group (e.g. asparticor aminosuccinicacid). Some amino acidsare aromaticsuch as phenylalanine,or heterocyclicsuch as proline (pyrolidine nucleus),tryptophan (indole nucleus) and histidine (imidazolenucleus).
Amino acidsare generallysolublein water but only slightly solublein alcohol.A generaltest is to warm with ninhydrin, when, with the exception of proline, which gives a yellow, they give a pink, blue or violet colour.Amino acids do not respondto the biuret test (comparepolypeptides and proteins). Certain amino acids are detectedby more specihc tests(e.g.histidine gives colour reactionswith diazonium salts). Amino ocids found in proteins Theseinclude o-alanine;arginine;asparagine(amideof asparticacid), abundantin many plants,particularlyetiolatedseedlings;asparticacid; aminosuccinicacid, involved in the biosynthesisof purines;cysteine, which contains sulphur; cystine or dicysteine (in hair and insulin); 3,5-di-iodotyrosine (in thyroid); glutamic acid (a component of the folic acid vitamins); glutamine (free in animals and plants, e.g. sugarbeet);glycine (aminoaceticacid); histidine;6-hydroxylysine(in gelatin); hydroxyproline (in gelatin); leucine (cx-aminocaproic acid); isoleucine; lysine; methionine (contains a sulphur atom); 3-monoiodotyrosine(in thyroid); phenylalanine;proline; serine (in phosphoproteinssuch as casein);threonine(in casein);thyroxin (the iodine-containingthyroid-hormone);3,5,3ltriiodothyronine (in thyroid); tryptophan;tyrosine;and valine. Amino ocids found free qnd not occurring in proteins Following the pioneer researchof Fowden, severalhundred of these amino acids have been characterized and only a few (e.g. 1-
ili
'*Tlrtl
BASICMETABOLIC PATHWAYS AND THEORIGIN OF SECONDARY METABOLITES
osAzo
Me
Hooc tcooH rl," -= ,e . "fi..{r\* rg : 6" ,6t COS.CoA
-
/-
t"^ l[9*
I
O O Polytetidestructure
pCoumaryl-SCoA
I OH
AtYl migratiot
o H o
o H o
Isoflavanonestructure
Chdcone structure
\
Fig. I9.l I Originof flovonones, isoflovonones ond flovones.
Flavanonestructure
aminobutyric acid, o-aminoadipic acid, pipecolic acid and 6acetylornithine) are of wide occurrence.Seedsor fleshy organs of plants are the principal sites for the accumulation of these compounds where they may serve as a nitrogen reserve.Other examples in this class are: B-alanine (B-aminopropionic acid); citrulline (an intermediatein the cycle of urea synthesis);creatine;ergothioneine (a sulphur-containingconstituentof some animal tissues and of ergot); and taurine (a component of bile acids).A number have teratogenic properties (see Chapter zl0). 3-N-Oxalyl-t--2,3-diaminopropanoic acid is a neurotoxin present in the seeds of Lathyrus sativus, the grass pea; it is the causal agent of human neurolathyrism, an irreversible paralysisof the lower limbs, which occurs in some drought prone areasof Ethiopia, India and Bangiadesh(see Yu-Haey Kuo el aL., Phytochemistr;,,1994,35, 9l I for references and biosynthetic studies). Other nonprotein amino acids provide plant protection from insects. Nonprotein amino acids,consideredoddities of plant biosynthesis 3 5 y e a r sa g o .n o w c o n s t i t u t ea g r o u pr e c e i v i n gi n c r e a s i n a gttention becauseof their possiblephysiological and ecological signilicance.
Flavone structure
Glutamic acid, in particular,appearsto be a centralproductin aminoacid metabolismand glutamicacid dehydrogenase hasbeenreportedin a number of plant tissues.The enzyme functions in conjunctionwith NAD: u-ketoglutaricacid + NH3 + NADPH #glutamic
acid + NAD
That the nitrogenof ammoniafirst appearsin the dicarboxylicarnino acidsand is later transf'erred to other nitrogencompoundsr'"'asdemonstratedin someof the earliestplant biochemistryexperiments(around 1940),which utilized l5N.
Proline, hydroxyproline, ornithine qnd orginine Theseamino acids are of importancein the secondarymetabolismof some plants in that they are precursorsof a number of alkaloids. They are metabolicallyconnectedto glutamicacid (Fig. 19.13)and their formation in plant cells is complex in that the reactions are strictly compartmentalized.The enzymesinvolved have been characterized,and for the formation of ornithine it is the ly'-acetylderivatives which are involved. Arginine appearsto be synthesizedfiom ornithine in all organismsvia the reactionsof the urea cycle. (For a review giving the role ofthe mitochondria,cytoplasmand plastidsin Biosynthesis of qmino ocids As amino acidsare alsothe precursorsof somesecondarymetabolites, thesereactions,seeP. D. Shargool et al., Pht,tochentistr,-,1988, 27, their biosyntheseswill be consideredbelow. They arise at various 1 5 7 1 . levelsof the glycolytic and TCA systems. Nitrogen appearsto enterthe metabolismof the organismby reduc- Serine ond glycine tive aminationof q-keto acids;pyruvic, oxalaceticand cx-ketoglutaric Togetherwith cysteineand cystine,theseamino acidsariseat the triose acids give alanine,asparticacid and glutamic acid, respectively(Fig. level of metabolism.Preparationsfiom rat liver use the pathwayindicatedin Fig. 19.14for the formation of serine. 19.r2). In animaltissuesit hasbeenshownthat tetrahydrofolicacid (THFA) By transaminationreactionswith other appropriateacids, alanine, is responsible for the removal of the hydroxymethyl group of serine to aspartic acid and glutamic acid serve as o-amino donors in the formaform hydroxymethyltetrafolic acid. As this compound serves as a tion of other amino acids.The generaltransaminationreactionmay be sourceof formate and methyl groupsin many reactions,the B-carbon written: of serinemay be their original source;this appliesto the formation of -====> R-CH(NH)-COOH + R'-CO-COOH methionine(Fig. 19.15),itself an importantmethyl donor in plant bio+ R'-CH(NH")-COOH chemistry. R-CO-COOH
E
PHYTOCHEMISTRY ,
CH3-CO-COOH Pvruvicacid
*t',
cH!-cH(NH2FcooH Alanine HOOC-CO-CHTCOOH
HOOC-CH2-CH2-CO-COOH acid c-Kctoglutaric
Oxalacetic acid
,| I
^ I
I**'
I NHr
+
+
HOOC-C H2-C Hl-C H(N HTFCOO H
HOOC-CH2-CH(NHdFCOOH AsParticacid
Glutamicacid
A
t
I I NHr
I *",
T
I Y
F i g .1 9 . 1 2 R e d u c t i v eo- n d t r o n s - o m i n o t i o ni n t h e f o r m o t i o no f o m i n o o c i d s .
H2N-OC-C HrCH (N Hil--COO H
H
H2-CH (N Ht-coo
H2N-OC-CH2-C
AsParagine
Glutamine
HrNr
)c=rux
NH
cooH I
CH.NH,
cH.NH2
CH.NH,
fr'
?"' --t*' ?" f''
?" ?"'
cooH
cooH
Glutamic acid
Ornithine
+
(1.oo" F i g .1 9 . I 3 hydroxyproline, Originof proline, o r n i t h i noen do r g i n i n e .
I
\"/_coox
cH.NH2
cooH Arginine
HO-r--r |
\ I H
I H A'-Pyrroline-5-carboxylic acid
|
>-cooH
Hydroxyproline
Proline
COOH-CHOH-CH2OPO3Hz
.--------+COOH--CO-CH2OPO3H2
3-Phosphoglyceric acid
3-Phosphohydroxypyruvic acid
I COOH-CH.NH2-CH2OH +--+
coo H-c H.NH2*cH 2oPo3H2 3-Phosphoserine
Serine Serineand glycineare readilyinterconvertible: F i g .1 9 . 1 4 Formotion ondglycine of serine
-_
CH,OH-CH.NHr-COOH -r HCHO + CH2.NH2-COOH
rrr!-rls
"
PATHWAYS ANDTHEORIG|NOF SECONDARY METABOLTTESEg BASTC METABOLTC
fH'1oH
f"
cH2sH I +HCNHl
HCNH2
I cooH
+
i'-*f" HfNHr
ir'
COOH
HCNH2
cooH
cooH Homoscrinc Cystein€ (or relatcd 4-C compound)
Cystathioninc
n*,* It Plruvate
cHl
I
I
s I
9x, I 9Hr I
HCNH2
cHz-sH N5-methyl THFA
I
f"
HCNH2
cooH
I
cooH Methionine
Homocystcine
Fig. I 9.15 Originof methionine.
ical. chemical and pharmacologicalproperties.The lowest membersare derivedfrom only two moleculesof aminoacid.but highermembershave many amino-acidunits and lbnn eitherpeptides,simpleproteins(albumins, globulins,prolamines.glutalins,etc.) or more complex proteins. conjugatedproteins.in which other groupingsform parl of the molecule-fbr example,carbohydratein mucoproteins,the very cornplex phosphorus-containchlorophyllmoleculein the proteinof chloroplasts, ing proteinssuch as casein,nucleoploteins,in which proteinsarc combined with nucleic acid, and the lipoproteinsof the cytoplasm,in which protein is con'rbinedwith lipids.Among such substances with relatively 1owmolecularweight aresomeantibioticswhich havea cyclic polypep(e.g.gramicidin,bacitracinand polymyxin); peptidehortide str-ucture mones such as oxytocin and vasopressinfiom the posteriorpituitary gland;andglutathione,which is foundin nearlyall living cells. All thesemore or less complex compoundshave two or more mofrom leculesof amino acid united by a peptide linkage which resr"rlts the eliminationof water,an OH colning from one amino acid and an H fiom the other. Thus, a dipeptideis formed: R-CH(NH2)COOH+ R'-CH(NH2)COOH Amino acid
Amim acid
R-CH(NF|2)CO-NH-CH-R' + HzO Die€ptide
Alonine, voline ond leucine Studieswith microorganismsand yeastshave showntheseamino acids to be derived from pyruvate.There is evidencethat o-ketoisovaleric acid is aminatedto form valine and that it can also condensewith acetateto form an intermediatewhich on decarboxylationand amination affordsleucine(Fig. 19.16).
cooH
plant Blighictsupidu has hypoglyA dipeptideof the Sapindaceous caemic propertiesand although nore con-rplex.penicillin also has a dipeptidestructLlre. Tripeptideshavethreeamino-acidcomponentsand polypeptidesfrom ten upwards.Peptidesare usually defined as prohaving molecularweightsbelow I 0 000. In typical tein-like substances proteinsthe molecularweight is higher,ranging flom abor.rt30 000 to 50 000 in the relatively simple prolaminesand glutelins and reaching lsoleucine very high values,sometimesseveralmillion, in the complcx proteins This amino acid suchas thosein sheep'swool. Protein synthesistakes place in associationwith the ribosomes, cH^'cx-tx-coox T"t which are small bodiesfound in the cytoplasmand particularlyin the endoplasmic reticulum area (see Fig. 19.l). The amino acids are cHt-c+{ broughtto the ribosomesassociatedwith a transfer-RNAmoleculeand by the action of the ribosomes,using a sequencedictatedby a particular messenger-RNAmolecule,are linked to folm the peptidechainsof valine comtnencing series of reactions to but is formed by a similar the particularprotein.Although not directly relevantto most pharmaacid insteadof cx,-acetolactic acid. with o-aceto-cr-hydroxypropionic cognosticalstudies,the story ofthe nucleic acidsand their vital roie in the control ofcell metabolismis a fascinatingone which it is suggested Lysine studentsstudy liorn a standardwork on biochemistry. Lysine, H2N-(CH)4-CH(NH2)-COOH. is derived, in plants, from aspartateinvolving a pathway utilizing 2,3-dihydropicolinicacid and diaminopimelicacid.It is the precursorof somealkaloidsof Nicotiana, ISOPRENOID CON,IPOUNDS Lupinus andPunica. For a review of the biosynthesisand metabolism of aspartateStudieson the pyrogenicdecompositionof rubber led workers in the derived amino acids (lysine, threonine, methionine, S-adenosyl latter half of the nineteenthcentury to believe that isoprenecould be 1991,46,395. methionine)seeR. A. Azevedoet al., Phytochemistrl,-, regardedas a fundamentalbuilding block for this materiai.As a result of the extensivepioneeringinvestigationsinto plant terpenestructures. Aromqtic omino ocids 'biogeneticisoprenerule', which indiRuzickapublishedin 1953his Thesehave alreadybeenmentionedin the discussionof the biosynthecatedthat the appositionof isoprenoidunits could be usedto explain sisof aromaticcompounds. not only the formation of rubber and the monotelpenes.but also many other natulal products,including some,suchas sterolsand triterpenes, with complex constitutions.The value of the rule lay in its broad unilying concept,which allowed the postulationof a rationalsequence of events which might occur in the biogenesisof these otherwise 'peptide' includesa wide rangeof compoundsvarying from low unrelatedcompounds.Examplesof variousstructuresto which the rule The term to very high molecularweights and showing markeddifferencesin phys- can be appliedare indicatedin Fig. 19.17.
PHYTOCHEMISTRY
9Ht
cHr
6:O
c:o I
I
I cooH
cHr-c--oH I
c:o I cooH
cH3-c--oH I
cooH Pyruvicacid
cHr I cH3-c-oH I
9x, I
H-C-OH
I cooH apDihydroxyisovaleric acid
a.Keto-/-hydroxy isovaleric acid
cAcetolactic acid
II
J CHr
I
CHI
cH3-c-H
I
CH3-C-H
I C:O
+-
I
I cooH
H-C-NH2
I cooH
aKetoisovalericacid
Valinc
| "."."."
I condengation
+ \
cHj
/
i
cHr-cooH
,, ? n3L\
cH-cooH
6H3r
l
{_-
cH-c-cooH
/ Hlc'
I
cH_c_cooH l oH
p-Hydroxy-P-carboxYisocaProic acid
Dimethylcitraconic acid I
I * cH.oH-cooH I
cHr, \
cHrr
fo-cooH
- - - . } c H _ c\ H _ lc o o H / cHj
l
cH-cH-cooH
cHi
a-Hydroxy-pcarboxyisocaproic acid
cK eto-B-carboxyisocaproic acid
I
+ cHr\
a"r\
f*'
cH-cH2-cH-cooH Fig.I9.16 Formotion of volineond leucine
F-
cHt/ Leucine
The task set biochemistswas to investigatethe validity of the rule, and the work on this subjectconstitutesa brilliant exampleof modern biochemistrylhowever,as will be seenbelow, this chapterof research is still unfinished.By 195I it had beenestablishedthat aceticacid was intimately involved in the synthesisof cholesterol,squalene,yeast sterolsandlubber.The useof methyl- andcarboxyl-labelledaceticacid with animal tissuesindicated that the methyl and carboxyl carbons alternatedin the skeletonof choiesterolor squaleneand that the lateral carbon atoms all arosefrom the methyl group of aceticacid. The discovery,in 1950,of acetyl-coenzymeA, the so-called'active acetate', gavefurther suppoftto the role of acetatein biosyntheticprocesses.
*---ruD
/CH-CH2-CO-COOH CHz' acid oKetoisocaproic
The mevalonic acid pathway. The next major advancein the elucidation of the isoprenoidbiosyntheticroute was the discoveryin 1956 of mevalonicacid and the demonstrationof its incorporation,by living tissues,into those compounds to which the isoprene rule applied. Mevalonic acid (3,5-dihydroxy-3-methylvaleric acid) is a C6 acid and, as such,is not the 'active isoprene'unitwhich forms the basicbuilding block of the isoprenoidcompounds.During the next four years, by researchinvolving the use of tracertechniques,inhibitor studies,cellfree extracts,partition chromatographyand ionophoresisas well as syntheticorganic chemistry,it was establishedthat the C5 compound for which biochemists had been seeking so long was isopentenyl
d!-lr{Fr-"
1Is1Gll
BASICMETABOLIC PATHWAYS AND THEORIGINOF SECONDARYMETABOLITES
A ^n"' Vq;J
t:t:l'rr'i',, rlt. 'l::i":'l'' ..iiu,iall , )::a:1.:':'):
:.' :a:'
/\ Limoncnc Camphor --*-\rMonoterPenes
r-\
-,1[v
\
M L l l t' \
li]ltrt ":::,t ::,,1:l':'
fil:l l:11 lia:::i.....
T
,. L\,/-. 1
Rubber
I
x,c.
, ./ Tritcrpenes
T
.l J.
FCarotene
t"J'-E*' ,/
/IsoPrcne \
carotenoids
\
|
\.4.
Glycyrrhetinic acid Sesquiterpenes
Steroids
A't Fig.19.17 Applicotion of theisoprene rule illuslroting incorporotion of C5 uniis.
pyrophosphate; it is derived from mevalonic acid pyrophosphate by decarboxylation and dehydration. Isoprenoid synthesis then proceeds by the condensationof isopentenylpyrophosphatewith the isomeric dimethylallyl pyrophosphateto yield geranyl pyrophosphate.Further C5 units are addedby the addition of more isopentenyl pyrophosphate. Thesepreliminary stagesin the biosynthesisof isoprenoidcompounds areshownin Fig. 19.18. From geranyl and farnesyl pyrophosphatesvarious structurescan be built up (seeFig. 19.19). Studies, parlicularly by Cornforth and Popjdk, involving the use of stereospecifically3H- and laClabelled mevalonic acid, have demonsffated the stereochemicalmechanism of the initial stages of isoprenoid formation. Only the (R)-form of mevalonicacid gives rise to the terpenoids,the (S)-form appearingto be metabolically inactive. In the formation of isopentenyl pyrophosphate, the elimination is trans and the elimination of the proton in the isomerization to the dimethylallyl pyrophosphateis also stereospecific(Fig. 19.204').
E
) * Zngiberenc
In the subsequentadditions of the C5 isopentenylpyrophosphate units to form the terpenoids the elimination of hydrogen rs trans. Figure 19.208 shows the stereochemistryof the addition of one isopentenylpyrophosphateunit. In the biogenesisof rubber, however, the hydrogen elimination produces a cls double bond (Fig. 19.20C). It is consideredthat a simple changein orientation ofthe isopentenyl pyrophosphateon the enzyme surfacecould produce this changewithout altering the reaction mechanism. In neither rubber nor gutta are hybrid molecules containing both types of bond detectable.The first direct evidence for the presenceof isopentenyl diphosphateisomerase in rubber latex was reported in 1996 (T. Koyama et al., Phytochemistn, 1996, 43, 7 69). In recent years the enzymology of the isoprenoid pathway has been extensively studied and for details the reader is referred to a standard text on plant biochemistry.One key regulatory enzyme is hydroxymethylglutaryl-CoA reductase(EC 1.1.1.34,mevalonatekinase); it
:11:iilll:..ll
:::rlrt
PHYIOCHEMISTRY
OH ---)
CH3-COSCoA
2NADPH>
-ooc-cH+-cH2-coScoA
Acetyl-CoA
ix,
+
H ydroxymethylglutarate
CHTCOCH2COSCoA Acctoacetyl-CoA
oH I
HOCH2-CH2-C,-CH2_COOI cHr Mcvalonate
I ArP I
l OH
I - - -o5,P2ocHz-cHrt-cH2coo-
OH
I - -o3PocH2-cH2-c-cH2-coo-
ATP f--
I
I
cHr
CHr 5 Pyrophosphomevalonate
5-Phosphomevalonate
^rP - cor J - - -O6,P2OCH2-CH2-C:CH2 cHl Isopentenylpyrophosphatc
1l t l
--* a"t-i
t,
- cH3-c:cH-cH2oP2o6I cHr pyrophosphate Dimethylallyl
CH-CH2-CH2-C:CH-CH2OP2O6cHr
cHr Geranylpyrophosphatc
CH3-C:CH-CH2-CH2-C:CH-CH2-CHrC:CH-4 t t cHt CHI
| +l.op"nt"nyl lrroehosehate J
H2OP2O6,-- l CHI
Farnesylpyrophosphate Fig.I9.18 Preliminory stogesin the biosynthesis of isoprenoid compounds.
hasbeenextensivelystudiedin animalsand more recentlyin plants.As with many enzymesthe situation is complicatedby the existenceof more than one speciesof enzyme and a plant may possessmultiple lbrms each having a separatesubcellularlocation associatedwith the biosynthesisof different classesof terpenoids.For a review of the functions and propertiesof the important isomeraseenzyme isopentenyl diphosphateisomerasesee 'Further Reading'. It shouldbe notedthat somemetabolitesof mixed biogeneticorigin involve the mevalonicacid pathway;prenylationfor exampleis common, with Cs, Cro and C15 units associatedwith flavonoids, coumarins,benzoquinones, cannabinoids,alkaloids,etc. The validity of the mevalonatepathway in the formation of all the major groupsnotedin Fig. 19.I t hasbeenshown,and until recentlyno other biosyntheticroute to isoprenoidshad beendiscovered.
precursorfor all isoprenoidsyntheses.However, in 1993M. Rohmer et al., (.Biochem.J., 1993,295,517) showed that a non mevalonate pathway existed for the formation of hopane-typetriterpenoidsin bacteria. The novel putative precursor was identified as 1deoxy-o-xylulose-5-phosphate, formed from glucose via condensation of pyruvate and glyceraldehyde-3-phosphate. Subsequentsteps including a skeletal reaffangement afford isopentenyl pyrophosphate-the same methyl-branched isoprenoid building block as formed by the MVA route. It was soondemonstratedthat this novel route to IPP was also operative in the formation of monoterpenes(Mentha piperita, Thymus vulgcrls), diteryenes (Ginkgo biloba, Taxus chinensis) and carotenoids (Daucus carota). This raised the question of to what extent the two pathwaysco-existedin the plant and it was hypothesizedthat the classical acetate/mevalonatepathway was a feature of cytoplasmic reacThe 1-deoxy-o-xylulose(triose/pyruvate) pathway. Followingits tions whereas the triose/pyruvate sequencewas a characteristic of the discoveryin 1956mevalonicacid cameto be consideredthe essential plastids.This did not exclude either the movementof plastid-synthe-
h.
r;itc-.j!l.
4CJ'
"tI[
BASICMETABOLIC PATHWAYS AND THEORIGINOF SECONDARY METABOLITES A Me. -gH
c"""nyr!|fprrospr,"e -/ Monoterpenes, C16 (straightchain, cyclic and bicyclic)
|
Isopentenyl PYroPhosPhate
,--
TiXi'"" MVA-SPP
I Crspp Farnesyl pyrophoephatc
-/ / Sasquiterpenes,Cl5 / (openchain, / monocyclic / ,Z and bicyclic) / t caoPP
/
\ \
r \ Triterpencs Steroids
IPP
-\L*
Y" u"z\,cFLoen il.
Y \
DMAPP
\ C2oPP
/
|
"
t',,"*"*',
tim" sq,*J.", o,H,:"i", l,r
/ \
,df:{ftoPP H.' -F{o
,/
#a
H";\o;*'one
rf H".
i"
Ircpentenyr pyrophosphate
\
T
Me
Al
t""\
l t Carotenoids Gutta
,r,
FL. (.u. .)"Td.r."r{2oPP_
"' l,i. ^/r^'H"
x"^Tu cf .ue lc-cl^,cxropp
- "-fr'""ioi
--Ff,'
I
PP.J
x. $
Fig.19.19 Biosynthesis of isoprenoid compounds.
Me
Jk\.cHppp \H["
c sized IPP and DMAPP from the organelle to the cytoplasm or the translocationof a suitableCs-acceptorto the plastid. Evidenceaccumulating indicates a cooperative involvement of both pathways. Indeed recent work on the biosynthesisof the isoprene units of (K.-P.Adam and J. Zapp, Phttochemistry, chamomile sesquiterpenes 1998,48, 953) has shown that for the threeC5 units ofboth bisaboloxide A and chamazulene,two were mainly formed by the nonmevalonatepathwayand the third was of mixed origin. The deoxyxylulose(DOX) pathway has helped explain the previously reported rather poor incorporations of MVA into certain isoprenoids.Thus V. Stanjeket al. (Phytochemistry,l999, 50, 1l4l) have obtained a good incorporationof labelled deoxy-o-xyluloseinto the prenylated segment of furanocoumarins of Apium graveolens leaves, suggestingthis to be the preferred intermediate. Fig. 19.21illustrateshow [1-t:C]-*lucose, when fed to plants,can be used to differentiate IPP and subsequentmetabolites,formed either by the MVA pathway or the DOX route.
Hb.
/rx \.Me
"t"fc;d;,
e1/ ucH"oPe
@
x.jr cf..ue ;c-c.^.Hc
,
).1r"
Ft "e cl+oPP
I
t
PPOl"rHl
x".f ^..ue n-c"-\--s
['t'"
s;;t
Fig. 19.2O Stereospecific reoclions in lerpenoidbiogenesis. A, Formotion from mevolonicocid (MVA)of isopentenyl pyrophosphote {lPP)by trons eliminotion; isomerizotion to dimethylollyl (DMAPP). pyrophosphole B, Associolion of thetwo 5-C unitswilh ironseliminotion of hydrogen. C, As B but involvingformotion of cisdoublebondsos foundin rubber.
convertedto male pheromones.The literature concerning chemical ecology is regularly reviewed (J. B. Harborne.Nat. Prod. Rep., 1993, As indicatedearlier in Fig. 19.2,the basic metabolicpathwaysconsti- 10,327: 1997,14,83 1999,16, 509) and the volatile isoprenoidsthat tute the origins of secondaryplant metabolismand give rise to a vast control insectbehaviourand developmenthavebeenreportedon (J.A. array of compounds;someof theseare responsiblefor the characteris- Pickett, Nat. Prod. Rep., 1999, f6, 39). The enzymology associated tic odours,pungenciei and colours of plants, others give a particular with secondarymetabolism is now receiving considerableattention plant its culinary, medicinal or poisonousvirtues and by far the greatest and, with respectto alkaloid formation, a number of enzymesassocinumberare,on currentknowledge,of obscurevalue to the plant (andto ated with the biosynthesisof the tropane, isoquinoline and indole the human race). However, a number of modern authors suggestthat groupshave beenprepared.The biosyntheticorigins of thosemetabosecondary metabolites, rather than constituting waste products of lites of medicinalinterestareconsideredin more detail in Part 6. which metabolism,arebiosynthesizedto aid the producer'ssurvival.The pos- is arrangedprincipally accordingto biogeneticgroups. sible functions in the plant of one large group of secondary metaAlthough a number of the biogenetic groups are characterizedby bolites,alkaloids,are discussedin Chapter27. particularskeletalsffuctures,the actualchemicalpropertiesofparticuRecently, considerable attention has been directed to the possible lar compoundsare determinedby the acquisitionof functionalgroups. ecologicalimplicationsof secondarymetabolitesnot only in relationto Thus, terpenesmay occur as alcohols (menthol), ethers (cineole), plant-plant interaction but also concerning the inteffelationshipof ketones(carvone),etc.,and as suchhave similar chemicalpropefiiesto plants and animals.Various insects sequesterspecific alkaloids, irinonteryenoidcompoundspossessingthe samegroup; aldehydes,as an doids, lactonesand flavonoidswhich serveas defensiveagentsor are exampleof a functionalgroup, may be of aliphaticorigin (citronellal),
PHY1OCHTMISTRY
ro:
Ho-)!\-oH nv
oH
-Gtucose 11-13c1
\\ 'CHq l
^-n v-v
-
I SCoA Acetyl-CoA
II
t
CH3-CO-_CHz -CO-SCoA
cHo
i-
3oo-
Glyceraldehyde-3-P
\ \ \
Pyruvate
/ /
Thiaminediphosphateparticipation
\ ,\.// I Y -cH. t nn
Acetylacetyl-CoA
CHe l -CH2 HOOC C -.CH2- CH2OH
? *. CO
HO-CH I .cH2oP
I H-C-OH I HO_CH I -cH20P
1-Deoxyxylulose-5-P
I
OH Mevalonic acid
HaC ' \
//,c-cH2-cH2oPP
Fig. 19.21 Theincorporotion into of It-13C1-nlucose isopentenyl diphosphote: left,vio the mevolonic ocid pothwoy;right,vio the pothwoy. deoxyxyulose
HeC
HzC
.c-cH2-cH2oPP HzC
lsopentenyldiphosphate
lsopentenyldiphosphate
aromatic (cinnamic aldehyde), steroidal (some cardioactive glycosides);and resultingfrom the introductionof a heterocyclicsystemone biogenetic group may possess some of the chemical properties of another(e.g.steroidaialkaloids). A particular group of compounds may also involve different biogenetic entities; thus, the complex indole alkaloids contain moieties derived from both the shikimate and isoprenoid pathways. In contrast, the same structure,as it occurs in different compounds,may arise from different pathways, as has been previously indicated with the formation of aromaticsystems.
Stresscompounds These are compounds which accumulate in the plant to a higher than normal level as a result of some form of injury, or disturbance to the metabolism; they may be products of either primary or secondary metabolism. Common reactions involved in their formation are the polymerization, oxidation or hydrolysis of naturally occurring substances;many however, are entirely secondary in their formation. A
,ry
number of environmental and biological factors promote the synthesis of stresscompoundsand these include mechanicalwounding of the plant, exposure to frost, ultraviolet irradiation, dehydration, treatment with chemicals,and microbial infection (seephytoalexinsbelow). The productionof suchcompoundshas also beenobservedin cell cultures subjectedto antibiotic treatmentand in cells immobilized or brought into contact with calcium alginate. Examples of the latter include the formation of acridone alkaloid epoxides by Ruta graveolens, and the increasedproduction of echinatin and the novel formation of a prenylated compound by Glycyrrhiza echinata cultures. Stresscompoundsare of pharmaceuticalinterest in that they may be involved in various crude drugs formed pathologically (e.g. some gums and oleoresins)and potential drugs (gossypol);they are implicated in the toxicity of some diseasedfoodstuffs and they play a role in the defensive mechanismof the plant. In the latter area,the phytoalexins have received considerableattention in recent years and can be regarded as antifungal compounds synthesized by a plant in greatly increasedamountsafter infection. The antifunsal isoflavonoid otero-
[email protected]
BASICMETABOLIC PATHWAYS AND THEORIGINOF SECONDARY METABOLITES carpansproducedby many speciesof the Leguminosaeare well known. Other phytoalexins produced in the same famity are hydroxyflavanones,stilbenoids,benzofurans,chromonesand furanoacetylenes. Sesquiterpenephytoalexins have been isolated from infected U/nus glabra and Gossypium.In the vine (Vitis vinfera) the fungus Botntis cinerea acts as an elicitor for the production ofthe stilbenesresveratrol (q.v.) and pterostilbene. Chemically, stresscompounds are, in general,of extreme variability and include phenols, resins, carbohydrates,hydroxycinnamic acid derivatives, coumarins, bicyclic sesquiterpenes,triterpenes and steroidalcompounds.For the promotion of stresscompoundsin cell culturesseeChapterI 1 and Table 11.1.
Furtherreoding Cordell G A 1995Changingstrategiesin naturalproductschemistry. Phytochemistry40: 1585-16 12 Dey P M, Harbome J B (eds) 1997Plant Biochemistry.AcademicPress, London
GraysonD H 2000 Monoterpenoids(a review coveringmid- 1997 to mid-1999).Natural ProductReporrs17(4):385 Ikan R (ed) 1999Naturally occurringglycosides.JohnWiley, Chichester,UK KnaggsA R I 999, 2000 The biosynthesisof shikimatemetabolites.Natural ProductReports 16(4):525-560; 11(3): 269-292 Kruger N T, Hill S A, Ratcliffe R G (eds) 1999 Regulation of primary metabolic pathways in plants. Kluwer, Dordrecht, Netherlands Ramos-Valdivia A C, Van der Heijden R, Verpoorte R 1997 Isopentenyl diphosphateisomerase:a core enzymein isoprenoidbiosynthesis.Natural ProductReports14(6):591-604 RawlingsB J 1998Biosynthesisof fatty acidsand reiatedmetabolites.Natural ProductReports15(3):275-308 Rohmer M 1999 The discovery of a mevalonate-independentpathway for isoprenoidbiosynthesisin bacteria,algaeand higher plants.Natural Producl Reports l6(5): 565-57,1 SeiglerD S 1998Plant secondarymetabolism.Kluwer, Dordrecht, Netherlands Singh B K 1999(ed) Plant amino acids-biochemistry and biotechnology. Marcel Dekker Inc., New York Wink M (ed) 1999Biochemistryof plant secondarymetabolism.Sheffield AcademicPress
E PART
6
Phqrmqcopoeiolq n d relqted drugs of biologicql orrgrn o
o
I !.
(
f ' i {,.
I
t
\
\ {
I
p<" I
I
E I t
Introduction
Drugs of the Europeanand British Pharmacopoeias b,setheru ith scrnre relatedplant materialsareconsideredin somedetail in Chaptcrs10-27. They are arrangedaccordingto the biosyntheticorigins of thcir.principal constituents. An indicationof how thesedr-r-rgs can also be erouped on a pharmacological/clinical usagebasisis given in Chaptcr6. A lalge number of plant species.particularlythose r"rsed in various traditionalmedicines,have been,and still are being. screenedfor specific pharrnacologicalactivities.Overviews involving some areasof this research.with its notablesuccesses, are given in Chapters2g-30 and illustratein conjuctionwith Chapters15-17 the current approach to the searchfbr 'new'drugs ofpotential I'alueto the allopathicsystent of medicine. Someproductssuchas vitanrins.antibiotics, hormonesandflavourrngs are conveniently treated as hetel-ogeneous biochemical groups (Chapters3 I -34). In Part 6, to help distinguishrhe mono-eraphs on drugsof the Bp ancl EP liom thoseof non-official drr-rgs. the usualcorrventionsrelating to hierarchicalheadingshave not beenstrictlv observed;headingsfor the former are printed in bold capitals *,hereasthe latter are in smaller typesize.
lli..,,]llr'
,, .iilr.. ir
.:., ,:,:,:' ::L:: r:,l.l;tl. . ll,,lii.,,'li
l
I
:ir::l,rii.
.
I
l
l
'
. 'uu;u:tl :
illlrill:'
'rilllrl:.
,'uiiuitil .,,alllll:lilrlr ,u:.it::rt ltlllttl! 11111!rt:r ,;.11i:1u'
,u'
.,,J{l,
Hydrocorbons ond derivotives
Hydrocarbonscontain carbon and hydrogenonly and, from these,by the addition of functional groups and by interaction,all other natr-rra compoundscan theoreticallybe derived. In a particularclassof compoundssuch as volatile oils, the componentsof any one membermay be biosyntheticallyrelated (e.g. menthol and menthonein oi1 of peppermint) although because of their difTerent functional groups they may undergo dilTerentsetsof chemical reactions and possessdiff'erent pharmacologicalproperties. Among the most common functional groupsare carboxylicacids,alcohols,ketones,aldehydesand phenols; biochemicalinteractionsproduceesters,lactonesetc. In this book most examplesof medicinalplantscontainingthe above are consideredundertheir respectivebiogeneticgroupingsand in this chapterthe detaileddescriptionof drugsis restrictedto thoseexamples in which simple acids, alcohols and esters comprise the principal medicinalcomDonents.
;l:rf'.ir:
':iii
Although not featuring strongly in the pharmaceuticalarmamentarium, hydrocarbons are important in nature as components of cuticular waxes. The majority of these are odd-numberedlong-chain alkanes within the rangeC75 35formed by decarboxylationof the next higher, even-numbered, free fatty acid.In recentyearsthe long-chainpolyenes of the Compositaehave been systematicallyinvestigatedin relationto their chemotaxonomicimportance.Isoprene(CsHs), the unsaturate hydrocarbon moiety from which the terpenoids (isoprenoids) can be constructed(Fig. 19.17),has not to date been found free in nature.A number of cyclic terpenoidhydrocarbonsincluding limonene.pinene phellandreneand cadineneare componentsof essentialoils. Rubber guttaand the carotenesare polyunsaturated terpenoids.
lil.
il:llar.:,.,r ''111. 'r1.,,,,.. 1l
il\.;r.: "
:
,,i l
:rl " ;*iiltit.t'lilll:l:,.'11rr.. 1:-l -r " I '
I
-4".
::::"
!:
" "."1" , " " :t- " " -t - t l
Organic acids possessone or more carboxyl groups and a monobasic acid may be representedas RCOOH. The very high frequencyof the biochemical occurrenceof the carboxyl group means that acids are found in all living organisms and as derivatives of all the major metabolic groups. They participate in essentialmetabolism and in this capacity range from the simple acids of the respiratory sequenceto the complex deoxyribonucleicacidsassociatedwith the storageand transmissionofhereditarycharacters.In the metaboliccyclesthey liequently function in associationwith coenzymes,and may accumulateas simple salts, estersand amides, or less frequently in the free state Amino acidsare discussedin Chaoter19.
HYDROCARBONS 1 7 4 MONOBASICACIDS 1 7 4 DIBASICAND TRIBASIC ACIDS 178
ArcoHors 179 ESTERS r 8 0
1i:r91,1t,
tt
-,,.,;tl'
DRUGSCONTAININGACIDs,ALCOHOLS
ocids C r -{c Monocqrboxylic A number of theseacids togetherwith hydroxy- and keto-derivative are intermediatesin the early stagesof the biosynthesisof fats, isoprenoid compoundsand various amino acids (q.v.). In the free state they are not found abundantly in naturebut occur scatteredthroughout the plant kingdom in the esterified form as a feature of some volatile oils. resins.fats.coumarinderivativesand alkaloids. Somecommon acidsare illustratedin Table20.1.
AND ESTERS 182 PHARMACEUTICAT FIXEDOILSAND FAIS ; l i ,,1'
li
r85
wAxEs r90
Fotty ocids Theseacids are important as componentsof plant oils (acyl lipids) in which they occur as esterswith the trihydric alcohoiglycerol.They are also componentsof the resinsof the Convolvulaceaeand of waxes in which they areesterifiedwith long-chainalcohols.Most areC I 9 to Crg
HYDROCARBONS AND DERIVATIVES
Tqble 2O.I
Exomples of C1-C6 monocorboxylic ocids.
Nome
Structure
F o r m i co c i d
Aceficocid
Propionic ocid
H@OH
n-Voleric ocid iso-Voleric ocid
Nome derivesfrom itsfirstisolotionfromthe onl, Formicorufo.A decomposition productof monyvegefoble moteriols. Occursfreein ihe hoirs of thestingingnefle;combinedin lhe gitoloxigenin seriesof cordiooctive glycosides.NJormyl-L-methionine is involvedin the initiotionof protein synthesis on ribosomes
MoCOOH
An essentiol primorymetobolite, porticulorly os ocetyl-CoA. Commonin the esterifiedform
MeCfl2C@H
Producedin the fottyocid oxidotivecyclewhen on ocyl-CoAwith on odd numberof corbonotomsis involved.Esterified os o lroponeolkoloid
Me(CI-I.).COOH n-Butyric ocid iso-Butyric ocid
Comments
Mt-cH-M" I c@H
Me(CF{2ICOOH Me.,""-M" I
?Fr,
Occursin trocesin monyfois Occursfree in corob beons{Ceroioniasiliqua)ond os itsethylesterin croton oil. Component of resinsof theConvolvuloceoe ond minortroponeolkoloids. Intermediote in themetobolism of voline Nol common;component ol Convolvuloceous resins Freeond esterified in Voleriana spp.Combinedin somehoponeolkoloids (e.g.voleroidine) ond in thepyronocoumorin, dihydrosomidin. Intermediofe in the metobolism of leucine
cooH 2-Methylbuiyric ocid
Me I
Componentof sometroponeond Veratrum olkoloids,Convolvuloceous glycosides ond thepyronocoumorin visnodin
?Fr,
CHMe I
cooH a^^,^i.
^.iA
Crotonicocid (tronsbutenoicocid)
Me(CH2).COOH
M"-c.H
Occursin trocesin monyfots Constituent of crotonoil
tl
H'c-cooH Tiglicocid
Me-"-H tl Me-c-@oH
Angelic ocid
H-"'Me il
M"-c-cooH S e n e c i o i co c i d
Me-"'Me t1 Htc-c@H
Occursin crofonoil {glycoside)from Crotontigliun.Ihe ocid of monyminor troponeolkoloids, e.g. tigloidine. Component of Convolvuloceous resins. Biosyntheficolly derivedfrom isoleucine
Occursin the rhizomeo[ Angelico.Esterifying ocid of the Schizonthus olkoloidschizonlhine X ond of somevolotileoils,e.g. chomomile oils. Component of theCevodilloseedolkoloidcevodine Firstisolotedfrom o speciesof Senecio(Compositoe). Occursos the esterifyingocid of someolkoloidsol Dioscoreoond Schizonthus. Componeni of the pyronocoumorin somidin
straight-chainmonocarboxylicacids with an even number of carbon atoms.Over 200 havebeenisolatedfrom naturalsourcesbut relatively few are ubiquitous in their occurrence.They may be saturated(e.g. palmitic and stearicacids)or unsaturated(e.g. oleic acid). The double bonds,with a few minor exceptionssuch as the seedoil of pomegranate,are cls. Less commonly they are cyclic compoundssuch as hydnocarpic acid and the prostaglandins.The latter are a group of physiologically activeessentialfatty acidsfound in most body tissuesand are involved in the platelet-aggregation and inflammatoryprocesses. They promote smooth muscle contractionmaking thern of clinical use as effective abortifacients and for inducing labour. All the active natural
prostaglandinsare derivativesof prostanoicacid (seeTable 20.4). A rich source of prostaglandin,A2 (PGA.) is the soft coral Plexaura homomalla.Although recognizedin the 1930s,and their structures determinedrn 1962, it was not until 1988 that prostaglandinswere unequivocallyestablishedas componentsof somehigher plants (cambial zonesand budsof Larir and Populusspp.) The characteristicacid of castor oil. ricinoleic acid (hydroxyoleic acid) has both a hydroxyl group and an unsaturateddouble bond. A range of acetylenicfatty acids occurs throughoutthe plant kingdom and someof them possessantifungaland antibacterialproperties.The biogeneticrelationshipbetweenthese,the olefinic fatty acids and the saturatedfatty acidsis outlinedlater in this chapter.
E
P H A R M A C O P O E IA N L D R E L A T EDDR U G SO F B I O L O G I C AO LR I G I N Examples of fatty acids arc listed in Tables 20.2-20.4. It will be valuc is the numberofpalts ofiodine absorbedby 100 partsby rveight notedthat somehavemore than one unsaturatedbond.the bondsbeing of the substance.Near-infiaredspectloscopycan alsobe usedto deterinterspersedby methylenc groups.Tl-resepolyr.rrrsaturated acids have mine this value as it is directly relatedto the HC=CH stretchbandsat received much attention in recent yeals both le-tardingtheil role in 2 I 30 nm in the spcctrum.Iodine valuesare useful constantsfor acids. dietaryfats and as medicinals.All the conmon acidshavetrivial names fixed oils, lats and waxcs.and help to indicatethe compositionof cornbut in order to indicatemore preciselytheir structureswithout recoLlrse plex mixturesas well as pure substances. to the fLll systcmaticchernicalnameeachcan be lepresentedby a symbol. Thus ct-linolenic.systematicname all-cl,i-A9.12.15-sg1n4ssntrienoic acicl.has l8 carbons and three doublc bonds which can be Ioble 2O.4 Cyclic unsoturqted ocids. represented b1'I 8:3.The position of the doublebondsis thenindicated by the rr-,rconventionwhere ri = numbel of carbonatomsin the molCommon nome Structurolformulo ecule and -r is the number of inclusive carbon atoms frorn the methyl (co)end to the first carbonof the lirst doublebond, in this case3. so that CH:CH Hydnocorpic thc symbolfor q.-linolenicacid is 18:3(n-3).The positionsof the two lx1oq1,ocoox | cft-cH2 remainingdouble bonds are dedr-rced by the lact that they will follow CH:CH on fiom each othel being separatedonly by one methylene (-CH2-) Choulmoogric !r,t"r,.t,r"oo,l | group. In this areastudentsmay find the literaturesituatior-r sor-newhat c&*crq confirsingbecausein some texts the acids rnay be syrnbolizedon the CH:CH L,ofl rc basisof conventionalchemicalsystematicr-rurrbeling-fbr latt1 acids ix1c,.H".1cooH I q,-linolenic the carboxyl carbonbeing C1. Fol acid this is lepresented cru-cFh as 18:3(9c.12c.15c), indicating c a cls-bond.The advanta-qe ofthe lirst Prostonoic systemis that it indicatesany bioequivalenceof the double bonds in an-.','"":..a'4'.cG .4,4'4'4'/ acidsofdifferent chain-length,bearingin rnind that chainelongationin liyo proceedsat the carboxylend of the moleculeby the additionof 2C PGA2 units. Thus it can be seenliom Table 20.3 that y-linolenic acid and arachidonicacid both fall into the biochemicalco-6lamily of unsaturatedfatty acidsand their respectivesymbolsl8:3(n-6)and 20:4(ir-6) reflect this whereassymbolsbasedon chemicalnomenclaturefbl these acidsyl; 18:3(6c,9t:,12c) and 20:4(5c,8c,llc,l4t:1 do not. A comparison of symbolsfor somecolnnon Lmsatruatcd acidsis shown in Table 20.5. Under certain conditions,which are specifiedin pharmacopoeias, Toble 2O.5 Comporiron of symbols sscibed to iodine or its equivalentis taken up at thesedouble bonds and the sounsoluroled fotty ocids. called iodine value is thus a measureof unsaturatedness. The iodine Common name Symbolemploying Symbolbosed of ocid biochemicol on chemicol equivolenceof nomencloture lable 20.2 Stroight-choin sqturoted ocids. doublebonds Common nome
Systematicname
Structurolformulo
Coprylic
n-Octonoic n-Deconoic n-Dodeconoic n'Tetrodeconoic n-Hexodeconoic n-Octodeconoic n-Eicosonoic
cH3{cH2)6cooH cH3{cH2)8cooH cH3{cH2)locooH cH3{cH2)r2cooH cH3{cH2)r4cooH cH3(cH2)r6cooH cH3(cH2)rBcooH
Polmitoleic C anrie
Louric Myristic Polmitic Sleoric Arochidic
Toble 2O.3 Common nome
Petroselinic Ricinoleic Erucic Linoleic Eicosodienoic
16:1ln-71 I B : 1{ n - 9 ) I 8 : 1( n - 1 2 ) 18:'1(n-9) ot n-Z) {hydroxy 22:1 ln-91 1B:2ln-61 2O:2ln-6)
l6:1 {9c) I 8:1 {9c) I B : 1( 6 c ) D ( + )1 2 h18 : 1{ 9 c } (h = hydroxy) 22:1 113cl 1B:219c,12c) 2 O : 2] , 1 1 c , l 4 c )
Stroighr-chnin unsoturoted
Polmitoleic (Jterc P e k o s e l i ni c Ricinoleic Er u c i c Linolenic s-Linoleic y-Linolenic Arochidonic
\_,le Ic
2 3 3 A
Structurolformulo
cH3(cH2)scH=cH(cH2)7cooH cH3(cH2)7cH=cH(cH2)7cooH cH3(cH2) I ocH=cH(cH2)4cooH cH3(cH2)scH{oH}cH2cH=cH-(cH2)zcooH c H 3 ( c H 2 ) 7 c H = c H {IcIH c o2o) H cH3(cH2)4cH=cHcH2cH=cH-(cH2)7cooH c H3cH2cH=c HcH2cH=c HcH2-cH=cH(cH2)7cooH c H 3( c H 2 ) 4 c H = c H c H 2 c H = c H c H 2 c H = c H ( c H 2 ) 4 c o o H cH3(cH2)4cH=cHcH2cH=cHcH2-cH=cHcH2cH=cH(cH2)3co
H Y D R O C A R B O NASN D D E R I V A T I V E S Biosynthesisofsaturated falty acids. SeeChapter 19.
ed in Fig. 20.l. turtherdoublebondsmay be similirr'lr'intlodr.rccd to give linoleicand linolenicacid. Biosvnthesisof unsaturated fatty acids. Befbre the elucidationof Unsatlrratedtatty acidscan alsobe formed in plantsb1'clon_lation of the overall chemistryof fblrnation of polyunsaturatedlatty acids such ir medium-chain-lengthunsaturatedacid. This appearsto occur bv the as linoleic in the eally 1960sby Bloch, knowledgeconcerningthe formation of an intelmediate.B, y-unsaturatedacid rather than thc biosynthesis of thesecompoundslaggedbchind that oi the satulatcd ct,B-unsaturated acid normally producedin saturatedfafty acid biosl,nacids.Recentprogresshtis beenmuch morc rapid and. in general.it thesis:the B.7-bondis not redLrcedand more C2 units are addedin the now appearsthat in aerobicorgal]isms. monoenoicacidswith the douusualway (FiS.20.2). ble bond in the 9. I 0-positionariscby directdehydrogenation of satuSterculic acid. tr conponent of seed oils o1' the Malvaceae and rated acids. In higher plar-rts,for this reaction. cocnzymc A rnay be Stcrculiaceae.is a c1'clopropcneand is also delived frorr oleic acid. replacedby the acyl carrier protein (ACP). and Bloch has demonstratwith methionine supplyin-ethe extra calborr atom to give. first. ed that stearoyl-S-AcPis an efl'ectiveenzyme substrateof the destrtthe cyclopropane.Ricinoleic aciclis a hydroxy latty acid fbund in casLlrasesystemof isolatedplant leaf chloroplasts.The reducedforms of tor oil seedsand is againbiosvnthesized fionl oleic ncid(Fig. 20.3). nicotinan.ride adeninedinucleotide(NADH) or nicotinamideadenine Some of the natural acetl,lenes and acetylenic fatty acidshave obr.idinucieotide phosphate(NADPH) and molecular oxygen are cofacous structuralsimilariticsto the more comnon fatty acids.The h1'pothtors. esis that tliple bonds are fbrned 1r'orncloublebonds by a mechanism analogousto that fbl the tbrmation of doirble bonds and involving CHr(CH2)'6CO--S-ACP + O, + NADPH -> structurallyand stereochernically spccific enzyr.nes has now received (stearo) I-S-ACP) experimental support.By this means(Fig.20..+) therangeof acetylenes CH3(CHr)7CH:CH(CH,)7-CO-S-ACP + HzO + NADP lbund in Basidiomycetesand in the Contpositae.Araliaceae and Urnbelliferaecan be derivedtiom linolcic acid via its acetylenic12,I 3loleoyl-S-ACP.1 dehydloderivative, crepenynicacid. an acid first isolatedfrom seeds The positionofthe introduceddoLrblebond in respectto thc carboxyl oils of CreTrrs spp. group is governedby the enzyme;hence.chain length ofthe substrate acid is most important.The hydrogeneliminationis specilicallycis but Aromotic ocids a ferv unusualfatty acids such as that in the seedoll of PLutir-tt gruno- Two commonaromaticacidsare benzoicacid and cinnamicacid tum with the structurel8:3 (9c.I lt.l3c) hayetrartsbonds.As illr.rstrat- (unsaturated side-chain), whicharcwiclclidistribLrted in natureand
Acetrte + malonate
I I
Y
CHI(CHilTTCOOH
Palmitic
II
+ CH3(CHilt6COOH
Stearic
I l-2H
Y
cH3(cHrTcHlcxlcxrlrcoox oleic l-2H CH3(CHjaCHacx-cxz-cHS Fig.20.l S e q u e n c eo f f o r m o t i o no f o l e f i n i c fottyocids in plonts.
6g16gt7cooH
Linoteic
I l-2H cH!-cH2-cHgcH-cH2-cHgcH-cH2-cHgcH(cHJTcooH
OH C H 3 ( C H 2h C H 2 - C H - C H 2 C O- S - C o A Intemediolep-hydrcry ocid of fotly ocid synthesis
/
./-,,o\ -
cH3(cHahCHzCH:CHCO-S-CoA
Fig.2O.2 Alternotive pothwoys for synthesis of unsoluroted fottyocids.
I n"ar'i- onc
lfurlherodditio.t of 5 r cr units { Sleoricocid
\ CH3(CH2l7Cx:CHCH2CO-S-CoA | *on,on o,
I3rczunirs Oleicocid
Linotenic
@
PHARMACOPOEIAL AND RELATED DRUGS OF BIOTOGICAT ORIGIN CHr(CH2}CH:CH(CHtTCOOH
Oleicacid
I I
Mcthionine
cH3(cHt5cH(oH)cHr-cH:cH(cHtTcooH Ricinoleic acid
, [H3C-S-CH2-CH2-CH(NHdFCOOH]
I
CH3(c Hj7-C\H -c H-( CH|TCOOH Dihydrosrerculic acid V cHz | -.*
c Hr(cHzh_\:t{cxrhcoox Fig.2O.3 Oleicocidostheprecursor of
Srcrculicacid
V
cHr
r i c i n o l e i co n d s t e r c u l i co c i d s .
CHr(CHJiCH:CH-CH2-CH:CH(CHjTCOOH
Linoleicacid
| _,* CH3(CH2)aC-C-CH2-CH:CH(CHTTCOOH
Crepenynicacid
l'" CH3-CH2-CH2-C
H:CH-C:C-C
Hz-CH:
C H(CHTTCOOH
I I
D e h y d r o c r e p e n y n iacc i d
Range of rcet)rlenet formcd by furthcr introduction of acetylenic bonds et the 'distal'
Fis.20.4
of morecules and bv chainshortenins in lr'l,fi:.l:Jl;1.,ti:Jr.crrboxvl sroup)
Formotion of ocetulenic fottuocids. oflen occur liee and combinedin considerableamountsin drugs such as balsams.Truxillic acid. a polymer of cinnamic acid. occursin coca leaves. Other related acids of fairly common occllrrenceare those having phenolic or other groupings in addition to a carboxyl group; suchare: salicyclic acid (o-hydroxybenzoicacid),protocatechuicacid (3,4-dihydroxybenzoic acid), veratric acid (3.,1-dimethoxybenzoic acid), gallic acid (3,4,5-trihydroxybenzoic acid) and 3,,1.5trimethoxybenzoicacid. Similarly. derived from cinnamic acid, one finds p-coumaric acid Q;-hydroxycinnamicacid), ferulic acid (hydroxymethoxycinnamic acid), catTeic acid (hydroxycinnamic acid) and 3,4,5-trimethoxycinnamicacid. Unbelliferone, which occurs in combination in asafbetida,is the lactone of dihydroxycinnamic acid. Acids having an alcoholgroup arequinic acid (tetrahydroxyhexahydrobenzoicacid), which occurs in cinchonabark and in some gymnosperms:shikimic acid. which is lound in Japanesestar-aniseand is an important intermediate metabolite; and mandelic acid, C6HsCHOHCOOH,which occursin combinationin cyanogeneticglycosidessnch as thoseof bitter almondsand other speciesof Prunus. Tropic acid and phenyllactic acid are two aromatic hydroxy acids which occur as estersin tropanealkaloids (q.v.).For examplesof the aboveseeFig. 20.5. Chlorogenicor cafleotannicacid is a condensationproductof caffeic acid and quinic acid. It occurs in matd, coffee, elder flowers, lime flowers. hops and nux vomica and is converted into a green compound, which serves for its detection, when an aqueousextract is treated with ammonia and exposedto air. See also 'Pseudotannins' (Chapter22). The biogenesis of the aromatic ring has been discussedin Chapter19.
DlBASlc AND rii,6As'ie'.l,Aci,ni.,'..'',.,,'..'.ll:'..,,..',l... Oxalic acid, (COOH)2,forms the first of a seriesof dicarboxylicacids which includes malonic acid, CH2(COOH)1,and sr.rccinicacid, (CH2)2(COOH)2.Closely related to malonic acid is the unsaturated acid fumaric acid. COOH-CH=CH-COOH. Malic acid contains an alcohol group and has the forrnula COOH CH2-CHOH-COOH. It is found in fruits suchas applesand tamarinds.A high percentageoitartaric acid, COOH-(CHOH)2-COOH. and its potassiumsalt occursin tamarindsand other fruits. The tribasic acids, citric, isocitric and aconitic are closely related to one another.The Krebs'citric acid cycle,which is discussedin Chapter 19,is very important.Citric acid is abundantin fruitjuices, and aconitic acid,which occursin Aconitumspp..is anhydrocitricacid.It forms part of the Krebs'cycle andthe glyoxalatecycle in microorganisms.
cH2.cooH I
I
HOC.COOH
I
cH2.cooH Gric acid
H.C.COOH -H,O
--€
tl il +H,O c'cooH -+ II cH2.cooH
cis -Aconitic acid
HO.C.COOH
I I cH2.cooH
H.C.COOH
lsocitric acid
Of recentinterestareopines.a group of substances formed by a host plant after infection with Agrobacterium spp.; a number of these compoundsaredi- and tri-carboxylicacids.For furlher detailsseeChapters 1 1a n d 1 2 .
HYDROCARBONS ANDDERIVATIVES @
cooH
I
I
I \
Z
t i l Y\ot"
Yot
I OMe
I OH
\-)
Veratric acid
Protocatechuic acrd
Benzoic acrd
Lower members of the series are found principally combined as esters e.g. methyl salicylate in oil of wintergreen and methyl and ethyl estersresponsiblefor some liuit aromas.Esterified long-chain alcoholsareconstituents of somepharmaceutically importantanimal waxes and include cetyl alcohol (Ct6Hi3OH), ceryl alcohol (C26H51OH)and myricyl alcohol (Cr0H61OH).Such alcoholsalso participate in the forn-rationof esterswhich are constituentsof leaf cuticular waxes: an example is CarnaubaWax BP which contains myricyl cerotate.
z\
t t l
<'\
Monohydric oliphotic olcohols
cooH
cooH
cooH
z\I
t l l
<-\'
I OH
\-/
Gallicacid
I
Monohydric terpene olcohols These are alcohols associatedwith that large group of compounds which arise from mevalonic acid and have isoprene as a fundamental structuralunit. Pharmacognosticallythey are particularly evident as constituentsof volatile oils namely: (l) noncyclicterpenealcohols occur in many volatile oils tbr example,geraniol in otto of rose.its isomernerol in oils of oran-ecand bergamotand linalol both free and combined as linalyl acetatein oils of lavenderand rosemary; (2) monocyclicterpenealcoholsare represented by terpineol and its acetate in oil of neroli and menthol and its acetate in oil of peppermint;(3) dicyciic terpenealcoholsare particularlyabundant in the Coniferae(e.g. sabinoland its acetatein JunipertrssabincL).ln the dicotyledonsoil of rosemar\,containsborneol and its esters.
I
nolAoH
^u-
-cooH
CH:CH
Cinnamicacid
cH-cooH
z\
t t l )'
OMe OH ecoumaric acid
Ferulicacid
CH:CH-COOH
H
OH H O H
H OH
OH
Quinicacid
Caffeicacid
""H
Shikimicacid
(Yo"
A 9o-?-\"\AoH : o
H
^ u
Chlorogenic acid Fi9.20.5 Aromotic ondothercyclicocids.
Monohydric qromqfic olcohols Benzyl alcohol, C6H'CH2OH, and cinnamvl a1coho1, C6H5CH=CHCH2OH,occurboth liee and ascstersof benzoicand cinnamic acids in balsamssuch as Tolu and Peru. The latter balsam is sometimesadulteratedwith cheapsyntheticbenzvl benzoate. Includedin this classis coniferyl alcohol.r'u'hichfornts an inportant component of the lignin molecule. Lignins are e\trL-ntcl\ complex phenylpropanepolymerslthey form an importantstren_qthcning material ofplant cell walls and vary in compositionaccordingro their source, seeChapter22, sectionon 'Lignans and Lignin'.
Dihydricolcohols Dihydric alcohols or gLycolsare compoundscontainin-9two hydroxyl groups;they are found naturallyin manl stnrctlrralclassesof compounds. The bicyclic amino alcohol 3.6-dih1'droxytropane occursfree and as estersin a number of speciesof the Solanaceae and Erythroxylaceae, the dihydric alcohol panaracliolis a component of someginsengsteroids,and oenanthotorin.the poisonousconstituent of the hemlock water dropuorts lOenanthe spp.), is a polyene diol.
Trihydric olcohols
Alcohols possessone or more hydroxyl groups and exist naturally in eitherthe free stateor combinedas esters.Like phenolsthey generally have common namesending in 'ol' (e.g. ethanol,glycerol and mannitol). They can be classedaccordingto the number of hydroxyl groups present:monohydric alcohols-onehydroxyl, dihydric-two, trihydricthree and polyhydric-four or more. Furthermore each hydroxyl group may be classedas primary-'.-CH2OH (e.g. ethanol), secondary'. *CHOH- (e.g. isopropanol) or tertiary: =COH (e.9. t-butanol). The remainder of the molecule may be saturated or unsaturated, aliphatic or aromatic. Numerous examples will be encountered throughoutthe text.
As with the glycols these compoundsoccur in a range of structural types.An importantexampleis glyccrol (propan-1,2.3-triol). an essential componentof fixed oils and f atswhich arediscussedin more detail below.
Polyhydricoliphoticolcohols The following arealcoholswith eitherfour or six hydroxyl groups.The meso form of erythritol. CHTOHCHOHCHOHCH2OH, is lbund in seaweedsand cerlain lichens both liee and combined with lecanoric acid. The hexahydricsugaralcohols(e.g. sorbitol.mannitol and dulcitol) are formed in natureby the reductionof either an aldehydegroup of an aldoseor the keto groups of a ketose.Sorbitol is abundantin many rosaceousfruits, mannitol in manna and dulcitol in speciesol Euonttnus.
ORIGIN DRUGS OF BIOLOGICAL AND RELATED PHARMACOPOEIAL referred to as triglycerides,although with current nomenclaturetriacylglycerolsis preferred.The prefix.rn is now employeclto denotethe nuntberingof the ntolecule. stereospecific
ESTERS Estersarisefroln the union of an alcohclland an acid with lossof water: + HoH
RrCHroH + RzCooH +RrCH:ocoR2
The reaction is reversibleand in plants esteraseenzymescontrol the reactlon. Many dilferent types of estersare known, and those formed by an acetylationol an alcoholic group are very common and are found in rnany biosyntheticgroups of metabolitesincluding volatile oils, e.g. linalyl acetatein lavender'.Esterswhic}r involve aromaticacidssuchas benzoic and cinnamic acids with con'espondingalcohols are sometimes lbund associatedwith free acids.other volatile metabolitesand resins.in such productsas balsams(seedrugs describedat the end of this chapter).A numbel of alkaloids (e.g. atropineand reserpine)are CStCIS.
A particularly important group of estersfrom the pharmaceutical viewpoint is that comprisingthe lipids ol tatty esters.Theseinvolve a long-chainfatty acid of the type describedearlierand alcoholssuchas glycerol and the hi-ehermonohydricalcohols. 'lipid' includesnot only fixed oils, fats andwaxes(simple The term lipids), but also phosphatidesand lecithins (complex lipids), which may containphosphorusand nitrogenin addition to cat'bon.hydrogen are widely distributedin both the vegeand oxygen.Thesesubstances table and animal kingdoms. and in plants they are particularly abundant in fruits and seeds.In animalsthe depotlats resemblethosefound in plants,while the complexiipids occur mainly in the trloreacti\-etissuessuchasthe brain and liYer.The lattergroupplays an inlportantrole in the structureof cellular membranes.the hydrophobicnatureof the latty acidsbeing all-irnportantto their biological role. The lecithinsare estersof glycerophosphoricacid in which the two free hydroxyls of the glycerol are esterifledwith fatty acids while one of the two remaininggroupsof the phosphoricacid is esterifiedto an alcohol (choline, ethanolamine,serine,glycerol or inositol). Because plantshave no mechanismfor controlling their temperature,they must possessmembranelipids that remain n-robileat relatively low temperatures.This property is conferredby the methylene-linkedcls double bonds of the polyunsaturatedacids bound as esters with the polar lipids. Conversely.in simple lipids, al1 three hydroxyl groups are esterifiedwith fatty acidsand thesecompoundshavebeentraditionally
cH2oH I
HOCH
I
cH2oH
cH2ocoRl
R2ococH ?
+
cHzoPocH2cH2N(cHr)3
Glycerol
o-
A complexlipid ine) (a'1,2-diacyl-sn-glycero-3-phosphorylchol
cH2ocoqsH3l
I I
H3Tq5OCOCH cH2ocoqsHll A simple lipid or triacylSlycerol (rripalmitin)
Fots ond fixed oils As agriculturalcrops.seedsusedfor the extractionof fixed oils rate in importancesecondonly to cereals.Over the last 50 yearsthe production of oils for the fbod indLrstryhas increasedenormously.whereas consumption by industrial and other usels has remained relatively staticbut, in the pharrnaceuticalindustry at least,not without interesting developrnents.Fixed oils are also obtained from fruit pericarps and in some instancessuch as the palm. Elaeisguineensls(Palmae), two oils diff'ering in properties and chemical cotnposition are obtained-the pleasantly flavoured palm kernel oil from the endospermand palm oil frorn the orange-yellowfleshy pericarp.Oil seedcrops are particularly advantageouscommercially as following the expressionof the oil a valuablehigh protein cattle feed remains. Also, such crops have benefitedfrom plant bleeding both regarding the yield and natule of the oil pt'oduced,and the morphology of the plant itself(seeChapter12). A naturallyoccurringmixtureof lipids suchas olive oil or oil of theo'oil' and 'fat' have, broma may be either liquid or solid and the terms oil andchaulmoograoil, therefbre.no very precisesignificance.Coconr"rt for example.leave the tropics as an oil and anive in WesternEurope as a solid.Even an oil suchasolive oil will largelysolidily in cold weather.In general,acylglycerols involving saturatedtatty acids are solid and those acidsareliquids.When both typesarepresent,as in crude of unsaturated cod-liveroil. coolinglesultsin the depositionof saturatedacylglycerols suchas stearin.In most medicinalcodlivel oils thesesolid materialsare removed by freezing and filtration. Acylglycerols are representedby the generalformula given below and can be hydrolysedby heatingwith causticalkali to form soapsand glycerin. If the latty acidsrepresentedby Rl, R2 and R3 are the same,the triacylglycerol is known as a simple triacylglycerol-for example.tdpalmitin on hydrolysis yields three molecules of palmatic acid. ln nature,however.Rl, R2 and R3 are usually different and the esteris known as a mixed triacylglycerol.On hydrolysisthey frequentlyyield both saturatedand unsaturatedacids (Fig. 20.6); there is a strongtendency for unsaturatedacids,particular'lythe C1s olefinic acids,to be linked to the secondaryhydroxyl. Biogenesis. Acylglycerols are formed from the fatty acyl-CoA or, more probably,the fatty acyl cariel protein (ACP) and l-cx-glycerophosphate,as indicatedin Fig. 20.7. Extraction. Most commercialoils are derived liom either seedsor fruits and nowadaysare mostly extractedby the producingcountry and exported as the crude oil. Sophisticatedderivatizationsof oils are mainly caried out by the importing countries. The initial treatment before extraction dependson the botanical structure-for exampie,American cotton seedsrequire delinting and castor seedsand ground nuts require decorticating.Specialmachines are availablefor thesepufposes.Removal of the oil may take the tbrm of cold or hot expression,centrifuging or solvent extraction. again dependingon the commodity.W:ith seedsthe remaining cake usually forms a valuablecattlefeed and for this reasoncompleteremovalof the The crudeoil requiresrefining.Cold-drawn oil is not alwaysnecessary. oils usually require nothing further than filtration: castoroil requires steamingto inactivatelipase;the addition of a determinedamount of alkali may be required to remove free acid; and washing and decolorization rnay be perfbrmed. Specific points concerningpreparrtion are mentioned r.rnderthe individual oils describedat the end of the chaoter.
r
HYDROCARBONS ANDDERIVATIVES cH2ocoRl Fig.20.6 Hydrolysis of o mixed triocylg lycerol.
I HOCH
+ 3KoH
R2ococH
|
^
HO-C*H
or FoflyACP
cHz_oo L-a - Phospholidic ocid //..
L-o-Lysophospholidic ocid
ct?o- coRl/'
I
R2CO-O-C-H
------->
-O -CH RzCO
ix.-o @
cHzo- coRl
R uC O- 0 - C -
+
H
I
CH2O- COR3
CH2OH
Triocylglycerol
Quantitative tests. A number of quantitativetests are commonly usedto evaluatefixed oils and fats.Acid value refersto the numberof mg ofpotassium hydroxide requiredto neutralizethe free acids in 1 g of the oij; high acid values arise in rancidified ol7s.Saponificcrtion value: the hydrolysis reactionof Iipids (above)can be used to delermine the saponificationvalue ofthe oil and is expressedas the number of mg of potassiumhydroxide requiredto neutlalizethe free acids in, and to hydrolysethe estersin. I g of the substance.Ester value is the diff'erencebetween the saponificationand acid values.Iodine vttlue (see 'Fatty acids') gives a measureof the unsaturationof the oil. Oils which partially resinily on exposureto air:Lreknown as semidryingor dryingoils.Suchoils (e.g.linseedoil) havehigh iodinevalues.In some cases,particularlyfor animal fats such as butter,the determinationof volatile aciditf is useful.sincethe lower fatty acidssuchasbutyric acid are volatile in steamand this may be usedfbr their separationand estimation. It is frequently Lrselulto determine tnsaponifiable mcttter. which consists of compounds sr-rchas sterols which remain after saponificationof the acylglycerolsand removal of the glycerol and soapsby meansof solvents.Another useful standardis acen,l value, which is the number of milligrams of potassiumhydroxidereqr-rired to
Toble 2O.6
- coRr cH20
c H zo - c o R l
L-a- Glyceroghosphole
o n d R 3m o y o r m o y n o t b e t h e s o m e ocyl groups.
R3cooK
cH2oH
FotiyocyltoA >
cHz-o (B)
Fig.2O.7 Formotion of ocylglycerols; Rl, R2
R2cooK
+
I
lr,o.o*' cH2-oH _C_X XO
Rt cooK
cH2oH
1,2-Diocylglycerol
neutralizethe aceticacidfleedby the hr drolr rrr ol I g of the acetylated fat. The oil is tirst acetylatedwith acetic.rr)h\ilride. r.vhichcombines with any hydroxyl groupspresent.Becau:ethe:c iirr-absentlronr most fatty acids.the smallacetylvaluesLrsuall)ohlilinedlrc clueto relatively small amountsof sterols.In an oil suchas e.r\t()r()il. honcvcr.the acetyl value is high (146-150),ouing to thc lirfrc rinrountsof the hydroxy acid ricinoleic acid. Certainphysicalconstants of flxed oils and fut' urc'significant:specific gravity,melting point, refractiveinder and .onrctinre:optical rotation(e.g.castoroil). Table20.6 showshorr chcnricul.tilndlrrd\are relatedto chemicalcomposition.The gas chrunrat,rslilnirie.c'prlation and quantiticationof the acidsproducedby thc hrdlolr.i. ot :pc'cific fixed oils is an ollicial methodfor their identitlcationrindrlualitr control; type chromatogramsare included in the BP. Sonrc eranrplesof this applicationarementionedunderthe oils dcscribedbclou. Wqxes The term'wax', althoughsometimesapplicdto thc'hrclroearbon nrixturehardparaflin,is bestcontlnedto thosenaturalnrirturc'\e()ntuining appreciablequantitiesof estersderivedtiorn hichcr nronohrdlic alco-
Numericol prcperties of fixed oils. Approximotefo\ composition
Fotor oil
Meltingpoint, "C
Soponification volue
lodine value
Soturoted(%)
Almond Costor
-tB -tB
I 83-208 1Z 5 - t8 3
99-l 03 B4
0.3-2.s
t
I B s - t9 6 18 8 - r9 6 250-264 190-r 98 248 19 3 - t 9 5 192-198 I B0-190
79_BB 86-t 06 7-1 1 1 0 9 - tt 6 r3 . 5
^l \JIIVC
Arochis Coconut Coffonseed rolm
Theobromo Lord Cod liver
-5 to +3 23-26 0 (Before winterizing) 30 31-34 34-41 ^l
L l e o r O TU
*Ol. = oleic;Ln.= linoleic;tdepositspolmitinot 2'C.
aa
ta
s0-66 I 55-tB0
t l
7-20 IB 92 27 50 59 60 U n d elr5
(%) Unsoturoted B BO l . L n . 88-94 Ricinoleic; 5-,l5 Ol. Ln. 75-93 Ol. Ln. 82 Ol. Ln. B Ol. 2 3 L n .O l . 50 Ol. Ln. 4l Ol.Ln. 40 01. Over 85 with up to six unsoturoted bonds
LR I G I N P H A R M A C O P O E IA N L D R E L A T EDDR U G SO F B I O L O G I C AO hols of the methyl alcohol seriescombined with fatty acids. In this seriesof alcoholsthe uembers chan-eefr"omliquids to solids.become less soluble in water and have highel ntelting points with increasein molecular wei-qht.The fi'st solid of the series is dodecyl alcohol, Cl2HrsOH. Waxes include vegetableproductssuch as carnaubawax 'wooland animalproductssuchas spermaceti.beeswaxand so-called fat'. Although waxesareabundantin nature(e.g.on epidermalsurfaces). a limited numberonly areof commercialimportance:someof the bestknown and theil chief alcoholsare given at the end of the chapter. An impoltant practicaldiff-erenccbetweenfats and waxesis that fats may be saponifiedby meansof either aqueousor alcoholic aikali but waxesare only saponifiedby alcoholic alkali. This fact is usedfor the detectionof fats when addedas adulterantsto waxes(e.g.fbr detecting 'Japanwax' as an adulterantin beeswax).Saponificationof the the fat wax estercetyl pahnitatemay be representedas: * alcoholicKOH --> Cl6HrsOH C15H.1.COOCrr,H3-l CetYlalcohol Cetyl palmitate + cl5Hl3.cooK Potassiumpalmitate While fats consist almost entirely of esters.waxes. in addition to estersof the cetyl palmitatetype. often contain appreciablequantities of fiee acids.hydrocarbons,fiee alcoholsand sterols.The hydrocarbons and sterolsare unsaponifiableand both spermacetiand rvool fat. of these,have high saponificawhich contain considerableqr.rantities tion values.If analyticaldata for fats and waxes are cornpared,it will of waxestend to be higher-for example. be notedthat the acid vah-res beeswaxcontainsabout l57r of fi"eecerotic acid. C26H5jCOOH.In most waxes,iodine valuesare relativelylorv and unsaponifiablematter is hish (Table20.7).
N IN G ACIDS, AtC O HO t5 P FU_G_S__C_O_NTAI AND ESTERS Tomorindpulp The drug consists of the fruit of the tree Tamarindus indica (Leguminosae)deprived of the blittle. outer part of the pericarp and preservedwith sugar'.The fiuits are about 5-15 cm long. They have a brittle ep:icarp.a pulpy mesocarp.through which run from the stalk aboutfive to nine blanchedfibres. and a leatheryendocarp.The latter forms from four to twelve chambers.in eachof which is a singleseed. ln the WestIndies the fiuits ripen in June,July and August.The epicarpsale removed,the fiuits arepackedin layersin barrels.andboiling syrup is poured over thern; alternatively,each layer of fiuits is sprinkled with powderedsugar.
Toble 2O.7
Tamarind pulp occurs as a reddish-brown.moist. sticky mass, in which the yellowish-brownfibres mentionedabove are readily seen. Odour'.pleasantand fmity; taste.sweetand acid. The seeds,eachenclosedin a leatheryendocarp,are obscurelyfbursided or ovate and abor-rt15 mm long. They have a rich brown testa marked with a large patch or oreole. Within the testa,which is very thick and hard. lies the embryo. The large cotyledonsare composed very largely of hemicellulosewhich stainsblue with iodine. The pulp containsfiee organic acids (about l}c/c of tartaric,citric and rnalic),their salts(about8% ofpotassiumhydrogentartrate).a litof invert sugar.It is reportedthat t1enicotinic acid and about 30--:l0clc the tartaric acid is synthesizedin the actively metabolizingleavesof the plant and then translocatedto the fruits as they develop.The addition of sugarto the manufacturedpulp, to act as a preservatlve,somewhat lowersthe naturalproportionof acids. Flavonoid C-glycosides (vitexin, isovitexin, orientin and isoorientin) occur in the leaves.The fixed oil ofthe seedscontainsa mixture of glvceridesol saturatedand unsaturated(oleic, linoleic) acids. Tamarind pulp is a mild laxative and was lormerly used in Conf'ectionof Senna:it has tladitional medicinalusesin the W Indies and in China and the leaves have been suggestedas a commercial sourceof tartaricacid. Mqnnq 'manna' is applied to a number of different plant products. The name The biblical mannawas probablythe lichen Lecarutraesculenta,which can be carriedlong distancesby wind. The only mannaof commercial importance is ash manna, derived frorn Fraxinus ornus (Oleaceae) The drug is collectedin Sicily. When the treesare about l0 yearsold. tlansversecuts are made in the trunk. A sugaryexudationtakesplace and when sufficiently dried is picked off (f1akemanna)or is collected on leavesor tiles. Manna occursin yellowish-whitepiecesup to 15 cm long and 2 cm wide or in agglutinatedmassesof brokenf'lakes,with a pleasantodour and sweettaste.It containsabout557cof the hexahydricalcohol mannitol, relatively small amountsof hexosesugarsbut larger amountsof the more complex sugarsmannotrioseand mannotetrose(stachyose). The tliose on hydrolysisyields glucose(1 mol) and galactose(2 mol). while the tetroseyields glucose( I mol), fructose( I mol) and galactose (2 mol). Manna has a mild laxativeaction.
BENZOIN Of the two commercial varieties of benzoir-r-Siam Benzoin and SumatraBenzoin-only the lattet is now includedin the BP. Sumatra Benzoin (Gum Benjamin)is a balsamicresin obtainedfrom the incised poralleloneurtrsPerkins stem of StyraxbenzoirtDryand, and Sry'ra-:r (Styracaceae). It is producedalmost exclusivelyfrom cultivatedtrees grown in Sumatra.althoughthe tree is also native to Javaand Borneo Siam benzoin.derived from Slr,rzr tonkinensis,is producedin a rela-
Chemicol ctandords of woxes.
Wox
Acid volue
Soponificotion volue
lodine value
Importontconstituents
Spermoceti Beeswox
Belowl t8-24
12 0 - l 3 6 volue) 70-80 (ester
below )
Cetylpolmitoteond cetylmyristote 72o/oesters,moinlymyricylpolmitote;free ceroficocid; sterylesfers
Cornoubo Wool fqt
4-7 Belowl
79-95 90-t 06
t0-14 r B-32
B-tI
Sterylesters,estersof otheroliphoticolcohols, fottyocidsond hydrocorbons
H Y D R O C A R B O NASN D D E R I V A T I V E S tively small areain the Thai provinceof Luang Probangand is mainly usedin perfumery. History. The drug was noted by Ibn BatLrta.who visited Sumatrain the fburteenthcentury,but was not regulally in-rported into Eulope until the sixteenthcentury. Collection and preparation. Sumatrabenzoinis a purely pathological product and there is sonte evidenceto show that its fbrmation is brought about not only b1' the incisions made. but also by lungi (see 'Stress Cornpounds'.Chapterl9). In Sumatrathe seedsare sou,nin rice fields,the rice shadingthe young treesduring theil first year.After the harvestingof the rice the treesare allowed to grow until they are about7 yearsold. Tapping, The rather complicatedprocessconsistsof making in eachtrunk threelines of incisionswhich aregradLralli'lengthened.The first triangular wounds are made in a vertical row about 40 cm apalt. the bark between the wounds being then sclaped smooth. The first secretionis verv sticky and is rejected.After rnakingfurther cuts.each about 4 cm above the precedingones,a hardel secretionis obtained. Further incisions are made at 3-rnonthly intervals and the secretion insteadof being amorphousbecontescrystalline.About 6 weeks after each fiesh tapping the product is scrapedofT, the outer layer (flnest quality) being kept separatefr"omthe next layer (intermediatequality). About 2 weeks later the strip is scrapedagain, giving a lower quality darker in color.rrand containingfragmentsof bark. Freshincisionsare then made and the above processis repeated.After a time the line of incisionsis continuedfurther up the tlunk. Grades. The above three qualities are not sold as such but ale blendedin Palembangto give the benzoin gradesof comr.nerce. The best grade containsthe most 'almonds' and the worst containsa few almondsbut abr.rndant resinousmatrix. The blendingis doneby breaking up the drug, mixing difl'erentproportionsof the threequalitiesand softeningin the sun. It was fbrn-rerlyerported aftel stampinginto tins but now the commelcial drug arrivesin plaited containerswith a plastic wrapping. Characters. Sumotrubenzoinoccursin brittle massesconsistingof opaque,whitish or reddish tearsembeddedin a tlanslucent.reddishbrown or greyish-brown,resinousmatrix. Odour. agreeableand balsamic but not very rnarked; taste, slightly acrid. Sianresebenz.oin occursin tearsor in blocks.The tearsare ofvariable sizeand flattened: they are yellowish-brown or reddish-brown externally, but milkywhite and opaque internally.The block folm consistsof snrall tears ernbeddedin a somewhatglassy,reddish-brown,resinousrnatrix.It has a vanilla-like odour and a balsamictaste. When graduallyheated,benzoinevolveswhite tumes of cinnamic and benzoic acidswhich readily condenseon a cool surfaceas a crystalline sublimate.On warming a little powderedbenzoinwith solr:tion of potassium permanganate,a faint odour of benzalclehycleis notedwith Sumatrabenzoinbut not with the Siamese.When an alcoholic solution of f-erricchloride is added to an alcoholic extract of Siamesebenzoin,a greencolour is produced.Sumatrabenzoindoes not give this test. The BP inclLrdesa TLC test tbl the absenceof Dammar gum.
Up to about 20c/rof fiee acids n-raybe plescnt. High-glrde nraterial Irom S. purullelorrcLtrwncontainsbenzoic acid3%, r.anillin 0.-5(Zancl cinnanricacict20-30%. The more expensiveSiamesebenzoindifl'ersfront the above irr that it containsinsufTicientcinnamicaciclto give an odour of benzaldehl,de rvhenwarmeclwith potassiurrlpermanganatesolution.The mnjor corrstitLrent(aboLrt7-5%) secms 1() be the ester coniferyl benzoate. Conif'erylalcohol.3-metl.roxy-4-hydroxycinnamyl alcohol.is fbund in the carnbial strp of both gvmnospermsand an-tiosperms.Other constituentsare fice benzoic acid. tritcrpenoid acids arrd vanillin. Sou're rccent (20001parcels of Siam benzoirremanatinglionr Hong Kong have been fbund to be heavily adrllteratedrvith Surnatlarnaterialand applicationof thepotassiumpermanganate test(seeabove)is essential. Alfied drug. Pulenthcutgben:.oitt.an infelior vadety ploduced in Sumatra.rnay be collectedfiom isolatecltreesfiom which the resinhas not beenstrippedfbr sometime. It is easiiydrstinguished, beingvery light in weight and breaking u,ith an irregulal porous fracture.It consistsalmostentirelyof reddish-broun re-sin.with only a f-ewvely small tearsenbeddcdin it. Palembang bcnzoinis r.rsed as a soufceof natural benzoicacid. Uses. Benzoin. r'n'hentaken intelnalll,. acts as an expectorantand antiseptic.It is mainly useclas an ingledient of friar's balsam.or as a cosmeticlotion preparedliom a simple tincture. It finds considerablc useworld-wiciein the fbod. drinks.perfiulelv rnd toiletrv industries:it is a componentof incense.
TOIU BAISAM Tolr.rBirlsamis obtainedby incision flom the trunk o1'Mlro,rtlon bulsomLrnt (L.) Hanr-rs. (a widely sprcacl polvurorphic species) (Lcguminosae),a large tree which cliltersbut little f}om that yielding balsamof Peru.Wild treesoccurin ColombiaandVenezuela irndin the former countly lalge quantitiesofbalsant lvereproducedin the neighbourhoodof the Magdalenaand Caucl rivers.The treesare cultivated in the West Indies.particularlyin CLrba. History. Balsamof Tolu i,vasdescribedbv Monardesin l-574and its collectionwasobserve clb-vWeir in 1863. Collection. The drug is collectedby making V-shapcdincisionsirr the bark. the secletionbeing reccived in a calabashplaced in the angleof the V. Many suchreceiversare tixed on eachtree.rhr')'ield per tree bcing B-10 k-q.Periodically.the balsarnis transf'erredto larger containers.It is exportedin tins fiont Cartagc.na. Sabanillaand Sta.Marta. Characters. When fieshly imported. tolu is a soti. yellow sernisolid. On keepingit trults to a bro$'lt. brittle solid. Jt sottenson warming. and if a little is then plessed betr.r'eentwo glass slides, microscopicalexaminationshou'scr'1-stals ol cinnamic acid. arnorphous resin and vegetabledebris.Oclouris arorratic and fra-erant:taste. aromaticlthe drug forms a plasticmasswhen chewecl. It is almostentirely solublein alu^olrol. the solutionbeing acid to litmus. and giving a greencolour with lerric chloride (the latter possibly owing to the presenceof resinotannol). Like other drugs containing cinnamic acid, it yields an odour of benzaldehydewhen a filtered decoctionis oxidizedwith potnrsiurnpennanganrtL' solution.
Constituents. Sumatra benzoin contains fiee balsamic acids (cinnamic and benzoic) and estersderir,'edfiom them. Also presentare triterpenoidacidssuch as siaresinolicacid (19-hydroxyoleanolicacid.) Constituents. Tolu containsabor-rt80% of resin derived from resin and sumaresinolicacid (6-hydroxyoleanolicacid). For the formula of alcoholscombinedwith cinnamicand benzoicacids.The drug is rich oleanolicacid seeunder 'TriterpenoidSaponins'.The contentof total in free aromaticacidsand containsabout l2-157c of fi"eecinnamicand balsamicacids (calculatedas cinnamic acid) is at least 20%. and the about 8clcof free benzoic acid (acid value fiom l 00- I 60). Other conarnountof cinnamic acid is usuallv about double that of benzoicacid. stituentsareesterssuchasbenzylbenzoateand benzylcinnamateand a
E
LR I G I N P H A R M A C O P O E IA N L D R E L A T EDDR U G SO F B I O L O G I C AO little vanillin. Recentinvestigationshavc shown the presenceof other cally. The chief balsamic estcrs present are benzyl cinnamate(cinesters.styrene,eugenol.vanillin. l'erulic acid. 1,2-diphenylethane.namein) C6H5CH=CHCOOCH2C6H5(sap. value 23zl). benzyl benhydrocarbonsand alcohols.The balsamalso zoate(sap.value 264.3)and cinamyl cinnamate(styracin).The drug mono- and sesquiterpene Tolu containsfi'om 3-5to 50% of total alsocontainsabout287cof resin,which is saidto consistof perulesinotritcrpenoids. containsnllmerous tannolcombinedwith cinnamicandbenzoicacids.alcohois(nelolidol. balsamicacidscalculatedon the dry alcohol-solublematter. thrnesoland benzyl alcohol) and small quantitiesof vanillin and frcc Uses. GenuineTolu balsarnis difTicultto obtain and the BP now uses cinnarnicacid. Tolu-flavourSolution.containingfive aromatics,fbr the preparationof Work on the isoflavonoidscontainedin the trunk-wood has indiTolu Syrup.Balsarnof Tolu has antisepticproperties.It was a common cated that considerablechemical diff'erencescharacterizethe various ingr"edientof cou-ehmixtures, to which it was added in the fbrrn of gtt:tup. fbrms or speciesof the M. bcLlsantLun syftlpor tlncture. Uses. Balsam of Peru is used as an antisepticdressingfbr"wounds and as a parasiticide.Now that it is no longer usedin Tr-rllegras dressPERUBAIsAM ings, it is of less currentinterestin Westerrrmedicine.Taken internally it is used to treat catarrh and diarrhoea.Allelgic responsesrre balsantumvar. of M.r'rc-n'lon frorn the trunk is obtained of Peru Balsarn pereirae (Leguminosae),after it has been beatenand scorched.The drug is ploduced in Central Arnerica (San Salvador,Honduras and Guaterrala)and is now includedin the EtrropettnPharnurct4toeiaand the BP (2000). History. The drug clerivesits name fiom the tact that when first imported into Spain it came via Callao in Peru. It was known to Monardes and the nethod of preparationwas describedas early as 1576. although aftelwards fbrgotten. In 1860 the coilection was describedand illustratedby Dorat.
possible. Prepored sforox PreparedStorax is a balsam obtained from the wor-rndedtrunk of purified. and sr.rbsequently Licluidamburorientalis (Hamamelidaceae) fbund srnall tlee fi'om a and is obtained Levant storax This is known as in the south-westof Turkey.
Collection and preparation, In the early summerthe bark is injured by bruising or by making incisions.After a time the outerbark may be pared olT, or the whole bark may be lefi until the autumn.when it is Collection and preparation. ln Novernber ol December strips of removed.The piecesof bark are pressedin horse-hairbags.lirst in the bark, nreasuringabout 30 x l5 cm. are beatenwith the back of an axe cold and againafter steepingin hot water.Sometimesthe bark is boiled or other blunt instrument.The bark soon cracksand may be pulled off in the Eastfor with water and againpressed.The exhaustedbark is r-rsed aiter 2 weeks.As in the caseof Tolu balsam.the secretionis purely fumigation.The crudeor liquid storaxis exportedin casksfiom Izmir. pathologicalin origin and r,ery little balsan can be obtainedfrom the Storaxis obtainedby dissolvingthe crudebalsarnin alcohol,filterbark unlessit is chan'edu'ith a torch about 1 week after the beating. ing and recoveringthe solventat as low a temperatureas possibleso as The balsarnproducedin the bark is obtainedby boiling the bark in not to lose any of the volatile constituents.The alcohol-insolublemat(preparedwithout fire) ot halstuttct water and is known as t(lcLtusottte ter consistsof vegetabledebrisand a resin. (balsamof the bark). de cctst:ard The greater part of the balsam. ho*ever. is prepared, after the Characters. Crtrdestorttr ts a gt'eyish,viscousliquid with a pleasant removal ofthe bark, by the secondmethod.The balsamwhich exudes odour and bitter taste. It usually contains about 20-30% of water. is soakedup with rags.which. aftel somedays. are cleanedby gently About 82 879t is alcohol soluble. Purified.s/orz,rfbrms a brown. viscous.semisolidmasswhich loses boiling u'ith water and squeezingin a rope press.The balsarnsinks to (.balsamo not more than5c/aof its weight when dried on a water-bathfor t h. It is the balsam decanled. been watel having and, the the bottom completelysolublein alcohol and partially in either.It has a characterde trapo) is pouredoffand strained. and istic balsamicodour and taste. Less destructivemethodsof preparationhave beeninvesti-qated replacement of include the removal of narrow strips of bark and the Constituents. Storax is very rich in free and combined cinnamic recovthe tree With treatment this of a hot iron. with the use scorching acid.After purilicationit yields 30-,47Vcof total balsamicacids. els in 6 months, comparedwith 8 years aller the drastic traditional By steam distiltation storax yields an oily liquid containing method.The drug is chielly exportedfrornAcajutla (SanSalvador)and pbenylethylene(styrene),C6-H5CH=CHr.cinnamicestels,vanillin and Belize (British Honduras)in tin canistersholding about27 kg. portion of the drug consistsof resin fiee cinnamic acid. The resit.tot-ts with cinnamicacid.The presand combined present both fiee alcohols Characters. Balsam of Peru is a viscid liquid of a somewhatoily is shown by the odour of benzaldeir-r thc drug acid of cinnamic it is ence seen in bulk. When stringiness. nature,but fiee fi'om stickinessand when the drug is mixed with sandand warmed dark blown or nearly black in colour. but in thin layers it is reddish- hyde which is prodr.rced The original containershave a u'hitish scum on with a solutionof potassiumpermanganate. blown and transparent. Recent researchcarried out in Turkey has shown the presenceof the surface.The balsam has a pleasant,somewhatvanilla-like odour compounds not previously reported; these include monotermany taste. and an acrid, slightly bitter penes.phenylpropanesand aliphaticacids. The drug is almostinsolublein water.It is solublein one I'olume of alcohol (90%), but the solutionbecomesturbid on the addition of furAlfied drug. American storax obtainedfrom L. s1'rociJlua,alarge ther solvent.The relativedensity,l.14-l.l'7, is a good indicationof tree found near the Atlantic coast from Centlal America to purity, and if abnorrnalindicatesadulterationwith fixed oils, alcohol, Connecticut.is also used in the USA. This balsarn resemblesthe kerosene.etc. The BP includestestsfor the absenceof artificial bal- Levant storaxin constituents.Thirty-six comporLnds havebeenidentiin petroleumspirit). fixed oils (solubilsams(solubility characteristics fied in the leaf'-oil of the plant and tannins and related phenolics ity in chloral hydratesolution)and turpentine(odourtest). obtainedfiorn cell cultures. Constituents. The ofllcial drug is requiredto contain not less than Uses. Storax is chiefly used in the preparationof friars' balsamand '70Vc wlw of esters,assayedgravimetri- benzoininhalation. 45.jVawlw and not more than
H Y D R O C A R B O NASN D D E R I V A T I V E S
PHARMACEUTICATFIXEDOILS AND FATS ATMONDOIt Alrnond oil is a llxed oil obtained by expressionfiom the seedsof (Rosaceae)yar.tlulcis (sweetalmonds),or P all.1gPnuuts turtygdalrr^s drtlus var. unnru (.biIteralmonds).The oil is mainly ploduced liom almonds grown in the cor.rntriesbordering the Mediterranean(ltaly, France,Spainand North Africa). Characters of plants and seeds. Al-r-rondtrees are about 5 m in height and the varieties,exceptlbr difI'erencesin the seeds,are ahrost indistinguishable.The young fiuits have a soft, t-elrlike pelicarp, the inner part of which gradually becomessclerenchymatous as the tiuit ripensto forn'ra pitted endocarpor"shell.The shells,consistin,emainlv of sclerenchymatous cells. are sometimesground and usedtt'radulteratepowdereddr-r,rgs. The sweetalmondis 2-3 cnr in len-qth.roundedat one end and pointed at the ofher.The bitter almonclis 1.5-2 cm in length but of similar breadthto the sweet almond. Both varietieshave a tl-rin.cinnamonbrown testa which is easily removed after soaking in warm water. a processwhich is known as blanching.The oily kernelconsistsof two large. oily planoconvexcotyledons.and a small plumule and radicle, the latter lying at the pointed end of the seed.Sorre almonds have cotyledonsof unequalsizesand are iffegularly tblded. Bitter almonds al'e sometimesfbund in samplesof su'eetalmonds.particularlythose ofAfrican origin: their plesencemay be detectedby the sodiumpicrate test fbr cyanogeneticglycosides. Constituents. Both varietiesof almondcontain40 55c/cof fixed oil. about207cof proteins,mucilageand emulsin.The bitter almondscontain in addition2.5-1.}c/cof the colourless.crystalline.cyrnocenetic -qlycosideamygdalin(seeChapter'26). Alnrond oil is obtainedby grinding the seedsand expressingthernin canvasbags betweensli-ehtlyheatediron plates.They are bometilnes blanchedbefore-erindir-rg, but this doesnot appearto be of any particlllar advantage.The oil is clarified by subsidenceand filtration. It is a pale yellow liquid with a slight odoLu'andbland,nutty taste.It contains a considerableamount of olein. with smaller quantitiesof the glycosidesof linoleic and other acids.The usualstandardsfbr fixed oils (glc of fatty acids)are includedin the -BPto-qether u'ith testsfbr absenceof variousother oils and sterols. EssentiaLor yolatile oil o.falmotds is obtainedfron'rthe cake left afier expressingbitter ahnonds.This is maceratedwith water for some hor.rrsto allow hydrolysis of the amygdalin to take place. The benzaldehydeand hydrocyanicacid are then separatedby steamdistillatlon. Bitter ahnond oil containsbenzaldehydeand 21% of hydrocyanic acid. Purified volatile oil ofbitter almondshas had all its hydrocyanic acid removedand thereforeconsistsmainly of benzaldehyde. Ht'drogenatedAlmond Ol1is alsoincludedin the ,BP Uses. Almond oil is used in the preparationof rnany toilet articles and as a vehicle fbr oily injections. When taken internally. it has a mild, laxativeaction.The volatilealmondoils are usedas flavouring agents.
ARACHISOIL Arachis oil is obtained by expressionfiom the seeds of Arachi.s Itt'pctguea(Legun-rinosae)(earth-nut, g,rrnnd-nut.peanut) a small annualplant cultivatedthroughouttropicalAfrica and in India, Brazil, southernUSA and Australia.Various senotvDesexist which show dif-
f-erenccsin the relative amounts of fatty acids contained in the oil. Enormousqr.rantities of the fiuits and seedsare shippedto Marseilles and other Er.rropean ports lbr expression.Ground-nutsare the w'orlcl's fourth iargestsor.rrce of fixed oi1. Preparation, During ripening the fruits buly themselves in thc sandy soil in which the plants grow. Each fruit containsflom one to three reddish-brownseeds.The fruits are shelledby a machine.The kernelscontain-10-.50%of oil. Owing to the high oil contenrthe seeds. when clushed. are somcwhat difllcult to express.Atter the initial 'cooking'.part ofthe oil is lcmovedin a low-pressure expellerandthe cake is solvcnt extncted. The two oil fiactions are then rnixed before purification.The presscake firrrnsan excellentcattlefbod. The ground per"icarps havebeenuscclrs ur.tadr-rlterant of powdeleddru-us. Constituents. Arachisoil consistsofthe glycelidesofoleic. linoleic, palmitic,arachidic,steanc.lignocerrcanclothel acids.When saponified with alcoholicpotassiunr hr droricle.crt'stalsof impurepotassitur arachidateseparateon standing.-\rachis oil is one of the most likely adulterants of otherfixed oils (e.g.olivc oil). The BP examinationof the oil is similarto that mentione(lunclcr'C)livcOil' below: the temperatureat which the cooling.h1'dlolrsecl oil be-contes cloLrdyshould not be below 36"C. As with olive oil nrore strinsentstandardsare requiredfor oil to be usedparenterally. The hydrogenatedoi1is alsoofTicial. Uses. Arachisoil hassimilarpropertiesto olir e oil. It is rn insredient of camphoratedoil but is usedmairrlf in the-produetion of ntargarine. cookingfats,etc.
CASTOROtt Castoroil (c'old-drawncastoroil) is a fixed oil obtainetltronr 1l.rc sceds of RicinLtsconununis (Euphorbiaceae).Thc ll'r-ritir r rhrcc ccllecl thornycapsule.The castoris a nativeof India:thc prineipalpnrducing countries are Brazil. India. China. the fbrnrcr Sor iet L'niorr and Thailand.There ale about l 7 varieties.which nrar be rouqhlr _llouped into shrubsand treesproducinglargeseeds.and annuulhcrbsproducing smallerseeds.It is mainlv the smallervarietie:thul .rrc.nou cultivated and these have been developed. br br-ecdtng.to give high-yieldingseed plants.Mechanicalharrestingrs norr replacing hand-picking. Characters of seeds. The seedsshou consiclcrable clifflrencesin sizeand colour.They areova1.somewhatconrprL-\\cd. 8 l8 mm long and4 12 mm broad.The testais vcrl'sntooth. thin and brittle.The colourmay be a more or lessunifbrm grL-\.[]r'o\\n ol black.or may be variously mottled with brown or black. .\ small. ofien yellowish. caruncleis usuallypresentat one cnd. lronr nlrich luns the rapheto terminatein a slightly raisedchalazaat thL-oppositeend of the seed. The testa is easily remor,edto disclosethe papery remainsof the nucellussurroundinga largeoill'cndosperm.Within the latterlies the embryo,with two thin. flat cotl'leclons and a radicledirectedtowards the caruncle.Castor seeds,if in sood condition, have ver:ylittle odour: taste,somewhatacrid. ll'the testasare broken.ranciditvwill develop. Preparation and characters of oil. Ninety per cent of the wolld's castoroil is extractedin Blazil nnd India. Relativelysmall amountsof the whole seedsale now exported.The various pl'ocesses involved in the preparationof castor oil are describedin the 8th edltion of this book. Briefly, the seedsare deprived of their testasand the kernels cold-expressedin suitable hydraulic presses.The oil is refined by stearning,filtration and bleaching.Cold expressionyields about 337c
E
P H A R M A C O P O E IA N L D R E L A T EDDR U G SO F B I O L O G I C AO LR I G I N of r-r-redicinal oil and t'urthcrquantiiiesof oil of lorver quality may be obtainedby other methods Medicinal castoroil is a colourlessor pale yellow liquid. with a slightodourandtaintll,acridtaste.For its chenricalandphysrcalconstants.see the phalrnacopoeias. The acid value increasessomewhat with age and an initiall,v high r,alue indicates the use of darr.raged seedsor carelessextractionor storage.Castoroil has an extremely high viscosity. Constituents. Castorseedscontain46-53c/cof lixed oil. which consists of thc glycosides of ricinoleic, isoticinoleic. stearic and dihydroxystearic acids.Thc purgativeactionofthe oil is saidto be due lo free ricinoleic acid and its stereoisor-ner, which are produced by h1'drolysis in the duodenum.Theseacidshavethe formula.
cHrlcH,lscH(oH)cH.cH:cH IcH l,lTcooH For thebiogenesis ofricinoleicacid.seeFig. 20.3.Castoroil andthe oil from Ricinu,s:.cutT.ibariuus are remarkablelbr their hi-ghricinoleic acid content,which is about887cand92'/c,respectively. The cake lelt afier expressioncontainsextremelypoisonoustoxins knou'n asricins,which rnakeit unfit fbl useasa cattlefood. ln the body they prodrlcean antitoxin (antiricin).Ricin D is a sugarprotein wtth a stronglethal toxicity; it contains.193arnino acids and 23 sr.rgars. Two otherricins.acidicricin and basicricin, havesimilarproperties. Ricin 'Abrr-rs and abrin (see Seeds'below)exhibit antitumou properties.The seedsalso contain lipasesand a crystallinealkaloid.r'icinine,which is not rnarkedlytoxic and is stluctr.rallyrelatedto nicotinamide. Uses. Castoroil. oncervidell'usedasa domesticpurgatire. is noir more restrictedto hospital use lbr adninistration after fbod poisoning and as a prelirninary to intestinalexamination.Owing to the presenceof ricin, the seedshiive a nruch more violent action than the oil and ale not usedas a purgativein the West. The oil is a constituentof llexible collodion. HydlogenatedCastorOil USP/NF (19951is usedas a stifl'eningagent. Undecylenicacid.which is prepaledlrom castoroil, is usedin lungistatic preparations. (polyethoxylated asis alsoits zinc salt.Nonionicsurfactants castoroils) of variablecompositionareprodr.rced by thereactionof castor oil with etl-rylene oxide and are useclin certainintravenouspreparations whicl-rcontaindrugswith low aqr.reous solubility.The oil and its derivatives flnd many nonpharnraceutical usesinchrdingthe nanufactureof plasticizers TurkeyRed O11.soaps,paints.r,'iunishes. and lubricants. Allied drugs. Crctut .seetlsare obtained fron Croton tiglitun (ELrphorbiaceae), a small trce producing similar capsulesto those of castorbu1devoid of spines.The seedsresemblecastorsecdsin sizeand shapebut have a dull. cinnarron-browncolour and readily lose their carunclcs.They contain about 50% of fixed oil which uontainscloton resin;also 'crotin', ii mixtureof croton-globulinand croton-albumin compalablewith ricin. The oil also containsdiestersof the tetracyclic diterpenephorbol (esterifyingacid at Rl and Rl. Rl = H in the fbrmula below):acidsinvolvedareaceticas a short-chain acid.andcapric,lauric and palmiticas long-chainacids.Thesecompoundsarecocarcinogens and :rlso possessinflammatory ancl vesicant properties (see 'Ditelpenes'). Also presentare phorbol-12.13,20-triesters 1Rl. Rr and R3 are all acyl groups in the fbrmula shown).These are 'cryptic irritants', so callcd becausethey are not biologicallyactiveas suchbut becomeso by removal of the C-20 acyl group by hydrolysis.Rotation locular counter-cuffentchromatography(q.v.) has been used to separate thesetwo groLlpsof esters.A number of the phorbol estershave been tested for anti-HIV-I activity (S. El-Mekkawy et al., Phytochemistry'.2000. 53. .157).The plant also contains alkaloids. Croton oil shor-rlcl be handlcd with extremecaution: it is not used in Westernmeclicine.but if takeninten-rally,it actsas a violent cathartic.
Phorbolestersof croton oil Plttsic nttts or Purging nuts Llrethe seedsof Jatropha cLtrcos,another menrberof the Eupholbiaceere. The seedsare black, oval and 15-20 mm in length.They contain about 40% offixed oil and a substancecompalable with ricin, called culcin. Both seedsand oil ale powerful purgatives. (prayerbeads)are the attractivered and black. but poiAhrus .seed.s sonous,seedsof Abrus precaktrils (Leguminosae).They contain a toxic glycoprotein(abrin) resemblingricin togetherwith anothernontoxic peptidehaving haemagglutinatingproperties.Various alkaloids (abrine.hyaphorine.precatorine)of the indole type havebeenreported. also various sterolsand lectins.The seedshave been r.rsedin folklore medicinein Asia.Africa and S. America to treatmany ailments,alsoto procureabortionand to hastenlabour.In lndia they areemployedas an oral contraceptiveand asthey areremarkablyuniform. and eachweighs about I carat(c. 200 rrg), havebeenusedtraditionallyas weights.
ouvEorr Olive oil (sulad oil, n'eet oil) is a fixed oil which is expressedfrom the ripe lruits oI OIea eurq)oeo (Oleaceae).The olive is an evergreentree, which lives to a greatagebut seldomexceeds12 m in height.It produces drupaceousfruits about 2-3 crn in length. The var. ltttifolitt bearsIarger f'ruitsthan the var.ktngifolln, but the latteris saidto yield the bestoi1.The oil is expressedin all the Mediten'aneancountriesand in Califbrnia. haly. Spain,France,GreeceandTunisiaproduce90clcofthe world's production. Olive oil has been ranked sixth in the world's production of vegetable oils (F. D. Gr.rnstone et ul.. 1994,The Lipid Handbooft,Chapman andHall). History. The olive appearsto be a native of Palestine.It was known in Egypt in the seventeenthcenturyBC, and was introducedinto Spain at an early period. Collection and preparation. The methodsusedforthe preparationof the oil naturally vary somewhataccordingto local conditions.In the modern factorieshydraulic pressesare widely used but in the more remotedistrictstheprocedureis cssentiallythat which hasbeenfollowcd for hundredsof yealsandis describedin earliereditionsof this book.The first oil to be expressed from thegror.rnd fruitsis known asvirgin oil; subsequentlythe marc may be solvent extractedto obtain the lower quality oil. The superiorgradesof oil areextravirgin. virgin andpure or reflned. Characters. Olive oil is a pale yellow liquid, which sometimeshasa greenishtint. The amountof color-rring matterpresent,whetherchlorophyll or carotene,appearsto detenninethe naturalfluorescenceof the oil in ultravioletlight. The oil has a slight odour and a bland taste.If the fruits used have beenallowed to ferment.the acid value of the oil will be higher than is officially permitted. It should comply with the tests for absenceof arachisoil, cotton-seedoil. sesameoil and tea-seedoil. The latter oil, which is obtainedfrom China. is not from the ordinarv tea plant but liom a tree, Cantelliuso.t(tnqua.
HYDROCARBONS AND DERIVATIVES Constituents. Olive oils fiom different sourcesdiffer somewhatin composition.This may be due eitherto the useof the dilferent varieties of olive or to climatic differences.Two types of oil may be distinguished:(a) that producedin Italy, Spain,Asia Minor and California. which containsmore olein and lesslinolein than type (b), producedin the Dodecaneseand Tunisia.Typical analysesofthese typesare:
Oleic acid Linoleic acid Palmitic acid'l Stearicacid J
Tj'pe(a)
T'pe (b)
BP limits
Vc
Vc
ch
78-86 o-1
65-'70 10-15
9-12
1)
56-85 3.520.0 [7.s-20.0
io.s-s.o
triglyceridescontainingonly the shortand mediumchain-lengthfatty acids(e.9.octanoic,decanoic;seeTable20.2).It maintainsits lor.',, r'iscosity until near the solidillcation point (about 0'C) and is a useful nonaqucousmedium fol the oral adrninistrationof somemedicaments. Metliurtt-chairtTrigbceritles BP, EP, svnonymouswith the abovc, may also be obtainedflorn the dried endospermof Elaeis guineen.si.s (Palmae).The fatt1'acidcornpositionof the hydrolysedoil, determined by GC, has the fbllou'ing specifications:caproic acid ), 2c/c,caprylic acid 50-80%, caplic acid 20-5OC/o,lauric acid > 3.\ch, n.ryristic a c i d) 1 . 0 % .
COD.LIVER OIt AND HAIIBUT.IIVER OIt SeeChapter3 I
COTTONSEED OIt
The characteristicodour of olive oil, particularlythe virgin oils. arises from the presenceof volatile C,.. alcohols (hexanol. E-2-hexenol, Z-3-hexenol),C6 aldehydesand acetylatedesters.Oils arising from different chemotypescan be distinguishedby headspacegas-liquid chromatography(M. Williams et al., P ht,tochemi str l-, 1998,47, 1253). The dehydrogenaseswhich produce the unsaturatedalcohois have beenstudiedin the pulp of developingolive pericalps(J. J. Salasand J. Sdnchez,Pht'tochemistn',1998, 48, 35). The BP examinationof the oil includesa TLC testfol identity and.to detect foreign oils, the GC determinationof the individual methyl estersof the acidsproducedby hydrolysis.Limits of the principal acids are given above: other percentagelimits, which exclude foreign oils, include saturatedfatty acids of chain length less than C16(#0.1), linolenic(> 1.2),arachidic(>0.7). behenic(>0.2). Therearealsolimits for a numberof sterols.The USP/NF ( I 995) includesspecifictests for the absenceof sesameoil (furfural test),cottonseedoi1,peanutoil and teaseedoil. For the biosynthesisof the acids.seethe introductionto this chapter. The saturatedglyceridestend to separatefrom the oil in cold weather; at I OoCthe oil beginsto becomecloudy and at aboutOoCforms a soft mass.
Cottonseedoil is expressedfr-unr the seeds of varior.rsspecies of (Malvaceae)in Anicrica and Europe.In the UK, Egyptian Gossy1tium and Indian cottonseed.which do not rccluilc delinting on arrival, are largelyused.Seeunder 'Cotton'. The preparationof cottonseedoil i s one of hot crpressionand a pressureof about 10 000 kPa is used.The cnrdeoil is thick and tulbid and is retinedin variousways.that knour.ras 'uinter blcached'beingthe bestofthe refinedgrades.Cottonseed oil is a sentidr1,'ing oil andhasa fairly high iodine value.When usedto adulterateother oils its presence may be detectedby the test for sernidrl,ingoils describedin the ,BP monoglaphfor ArachisOil. The hydrogenatedoil. only, is of1icial.
Uses. Olive oil is usedin the preparationof soaps.plasters,etc., and is widely employedas a saladoil. Oil for usein the manufactureof parenteralpreparationsis requiredto havea lower acid value andperoxide value than that normally required, and to be almost fiee of water' (0.1%) as determinedby the specifiedKarl Fischerrnethod. Recent researchhas suggestedthat olive oil may protect against colonic carcinogenesisby virtue of its action on prostaglandins:rals fed on a diet containingolive oil, as distinct from thosereceiving safflower oil. were protected(R. Bartoli et ttl., Gut,2000, 46, 191).
Macroscopical characters. The seeds are o\ate. flattened and obliquely pointedat one end; about4-6 mm lor.rgand I 1.5 mrn broad. The testais brown, glossyand finely pitted.OdoLrrless: tastc.mucilaginous and oily. If cruciferousseedsare present.a pungentodour and taste may develop on crushing and moistening.A transvelsesection showsa narow endospermand two iar-ec.planoconrex cotyledons.
cocoNuTott The expressedoil of the dried solid part of the endospermof the coconut, Cocos nucifera L. (Palmae)is a semisolid.melting at about 24'C and consistingof the triglyceridesof mainly lauric and myristic acids, together with smaller quantities of caproic. caprylic. oleic, palmitic and stearicacids.This constitutiongives it a very low iodine value (7.0-1 1.0)and a high saponificationvalue. The particularly high proportion of medium chain-length acids means that the oil is easily absorbedliom the gastrointestinaltract, which rnakesit of value to patientswith fat absorptionproblems. Fractionated coconut oil. Fractionatedand purified endospermoil of the coconutC. nut:ifern,or Thin VegetableOil of the BPC, contains
E
LINSEED AND LINSEED OIt Linseed (flaxseed)is the dlied ripe seed of Littttnt Lr.:ir,tti.:sirnutt (Linaceae),an annualherb abotrt0.7 m high qith blue lloriers and a globular capsule.The flax has long been cr-rltivatcdlbr its pericyclic fibres and seeds.Suppliesof the latterarederivedflonr SouthArnerica. India, the USA and Canada.Large quantitiesof oil are erpressedin England.particularlyat Hull. and on the Continent.
Microscopical characters. Microscopicalexaminationof the testa showsa mucilage-containing outer epidcrnris:one or two layersofcollenchyrna or 'round cells': a single la1'erof longitudinally directed elongatedsclerenchyma; the hyalinelal"ersor'cross-cells'composed in the ripe seedof partially or conpletelv obliteratedparenchymatous cells with their long axis at right anglesto thoseof the sclerenchymatous layer; and an innermostlayer of pigment ceils. The outer epidermis is composedof cells, rectangularor five-sided in surfaceview, which swell up in water and becorne mucilaginous.The outer cell walls, when swollen in water. show an or-rtersolid stratifiedlayer and an inner part yielding mucilage. itself faintly stratified. The radial layersor 'round cells'are c.vlindricalin shapeand show distinct triangular intercellularair spaces.The sclerenchyrnatous layer is composed of elongatedcells, up to 250 tim in length,with lignified pitted walls. The hyaline layersoften remain attachedto portionsof the sclerenchymatouslayer in the powdcreddrug (Fig. 12.71).Thepignrentlayer is composedof cells with thickenedpitted walls and containing arnor-
P H A R M A C O P O E IA N L D R E L A T EDDR U G SO F B I O L O G I C AO LR I G I N phous reddish-brown contents (Fig. zl2.7H). The ce1ls of the endospermand cotyledonsare polygonal with somewhat thickened walls, and containnumerousaleuronegrainsand -globulesof fixed oil. Starchis presentin unripe scedsonly.
RAPESEED OIt
Refined RapeseedOil EP, BP is obtainedby mechanicalexpressionor by extractionliom the seedsof Bzrssicanapus andB. canpestris. The crop is now extensivelycultivatedin Europeand oils with various propertiesdependingon glyceridecornpositionare commercially Constituents. Linseed containsabout 30-40c/cof fixed oi]r.6Vc of mucilage(^BPswellingindextbr whole seeds< 4.0),257cof proteinand available.As was indicatedon p.8l not all varietiesyield an oil suitsmall quantitiesof the cyanogeneticglucosideslinamarin and lotaus- able fbr medicinalpurposesso that the specifiedpharmacopoeiallimits tralin.Otherconstituents arephenylpropanoid glycosides(L. Luyengi er of the various esterityingacids are important standardsas are relative ul..J. Nat.Protl..1993.56.2012). flavonoids. thelignan(-)-pinoresinol density(c. 0.917)andrefractiveindex (r.. L473). As with someother oils, the additionof a suitableantioxidantis perdiglucoside(a tetrahydrofurofuran-type lignan-see Table22.7) andthe cancer chemoprotectivemammalian lignan precursor secoisolarici- mitted,the nameand concentrationof which must be statedon the label together with a statementas to whether the oil was obtained by (S.-X.Qil et ol., Phamt.Biol..1999,37.l). resinoldiglucoside Cell culturesof Linun albtonareableto synthesize andaccumulate the m e c h a n i c ae l\ p r e \ s i o no r b 1 e x t r a c t i o n . (T. Smollnyara1., lignanspodophyllotoxinand5-methylpodophyllotoxin Pht'tochenisrn,1998.48. 975). SESAME OIt Sesameotl(Gingelly'oil, Teeloil) is obtainedby refining the expressed or extractedoil from the seedsof Sesantumindicum (Pedaliaceae), a herb which is widely cultivatedin India, China.Japanand many tropical countries.The oil is otficial in the EP and BP. The seedscontain about 507c of lixed oil which closely resembles olive oil in its propertiesand which it has, in somemeasure.replaced. It is a pale yellow. bland oil which on cooling to about-zl'C solidifies to a buttery mass:it hasa saponificationvaluethe sameasthat tbr olive oil and a somewhathigher iodine value ( I 04-120). Principal componentsofthe oil are the glyceridesofoleic and linoleic acids with small proportions of palmitic, stearic and arachidic acids.It alsocontainsabott lVc of the lignan sesamin.and the related sesamolin.The characteristic phenoliccomponentis the basisof the BP test for identity and also the test fol the detectionof sesameoi1 in other oils. The originaltest involvedthe productionof a pink colour when the oil was shaken with half its volume of concentrated Uses. Crushedlinseed is used in the folm of a poultice and whole hydrochloric acid containing l7c of sucrose (Baudouin's test). seedsare employedto make demulcentpreparations.The oil is usedin However, some commerciallylefined oils may not give a positive liniments.and researchhas suggested that hydrolysedlinseedoil has Baudouin's test. With the current BP test for the absenceof sesame potentially useful antibacterialpropertiesas a topical preparationin oil in other oils. e.g. olive oil, the reagentsare aceticanhydride,a that it is efl'ectiveagainstSlapftt'lococt:tts drrclr strainsresisrantio solution of furfuraldehydeand sulphuric acid; a bluish-greencolour antibiotics.Linseedcake is a valuablecattlefood. is a positiveresult.The compositionof triglyceridesin sesameoil is determinedin the BP by liquid chr"omatography. those triglycerides having as acid radicalsoleic I part and linoleic 2 parts.and thosehavPAIM OIL AND PALMKERNET OIL ing oieic 2 parts and linoleic I part being among the most predomiPalm oil is obtainedby steamingand expressionof the mesocatpof the nant. fruits of E/aelsguine?nsis(Palmae).World prodLrction amountsto over As statedabove,sesameseedsand oil also containlignans:these I I .5 rnillion tonnes.over half of which is producedin Malaysia.In terms are antioxidants of the tetrahydrofurofuran-type(Table 22.7) and of world consumptionit hasnow overtakensunflowerandrapeseedoils. include sesamin. sesamolinol and sesamolin. Another lignan, Pahn oil is yellowish-brown in colour. of a buttery consistency sesaminol.is formed during industrialbleachingof the oil. The bio(m.p. 30'C) and of agreeabieodour. Palmitic and oleic acids are the logical activities of these compoundsinclr-rdereduction in selum principalestedfyingacids. cholesterollevelsand increasedvitamin E activities.For the biogenPalm kernel oil. Pahn kerneloil is obtainedby heatingthe separated e s i so f s u c hl i g n a n ss e eM . J . K a t o e t a l . , P h y t o c h e n i s t r y . 1 9 9 8 , 4 7 . seedsfbr 4 6 houls to shrink the shell. which is then crackedand the 5 8 3 . kernelsremovedwhole. The oil is then obtainedby expression.It diff'erschemicallyliom palm oil (above)in containinga high proportion SOYAOIL (50%) of the triglyceridesof lauric acid, a saturated.medir-rmchainlength fatty acid (Table 20.2). The mixture of other acids resembles Soya oil is derived from the seedsof Glycine na,r (Leguminosae). that found in coconnt oil. The oi1 is a source of Medium-chain After refining. it is deodorizedand clarified by filtration at about0oC. It shouldremainbright when kept at OoCfor l6 h. The principal esteriTriglyceridesEPIBP. fying fatty acids are iinoleic (41-62%), oleic (19-307c), palmitic Fractionated Palm Kernel Oil BP is pahn oi1which has undergone ('7 14%),linolenic(1-11%)and stearic(1.1-5.55i). selectivesolventfiactionationand hydrogenation.It is a white, brittle Thereis an official limit test for the amounrpf 1[e ,\s.22-24B-methyl solid,odourlessor almostso. with m.p. 31'-36'C making it suitable plant sterol. brassicasterol,which as the name implies occurs in for useas a suppositorybase. Brassicaspp.including B. raptr (c1.v.). Linseed oil. The extractionof linseedoil is one of hot expressionof a linseed meal and the pressis adjustedto leave sufficient oil in the caketo make it suitableas a cattlefood. Linseed oil of BP quality is a yellowish-brown dryin-e oil with a characteristicodou' and bland tastel much commercial oil has a markedodour and act'idtaste.On exposureto air it graduallythickens and torms a hard varnish.It hasa high iodinevalue({ 175)as it contains considerablequantitiesof the glycosidesof unsaturatedacids. Analysesshow linolenicacid,CITH,eCOOH(36-50c/o). linoleic acid (23-21c/().oleic acidClTHr3CooH (10-18%).togethCTTH3TCOOH er with some saturatedacids-myristic, stearicand palmitic (5-ll%). For the fonnation of the unsaturatedacids.seeFigs. 20.1 and20.2. For usein paint.linseedoil was boiledwith 'driers'sr.rch as lithargeor rnanganese resinatewhicl-r.by fonning metaliic salts.causedthe oil to dry morerapidly.Such 'boiledoils'must not be usedfbr medicinalpurposes.
HYDROCARBONS AND DERIVATIVES
THEOBROMA OIt Oil of theobromaor cocoabuttermay be obtainedfrorn the groundkernels of Theobromacocao (Sterculiaceae) by hot expression.The oil is filtered and allowed to set in moulds. Much is refined in Holland. Cocoa butter.as it is commonly termed.consistsof the glyceridesof steadc,palmitic. arachidic,oleic and other acids.Theseacidsare combined with glycerol partly in the usual way as triglyceridesand partly as mixed glyceridesin which the glycerol is attachedto more than one of the acids.It is the most expensiveof the commercialfixed oils and rnay be adr.rlterated with waxes, stearin(e.g. coconut stearin).animal tallows or vegetabletallows (e.g. from seedsof Barsia Longfolia and Stillingitt sebiJera).For the characterand testsfbr purity of oil of theobroma, see the pharmacopoeias. Its melting point (31-3zl') makes it ideal fbr the preparationof suppositories.
The oil should be protectedliom light and oxygen and srorcdar a temperaturenot exceeding25oC.
Eveningprimrose oil The fixed oil frorn the seedsof Oenotheruspp. (Onagraceae) contains substantial amounts of esterified y-linolenic aeid (GLA). a C 1s6,9.12-triene.
ueffioox acid {GLAI 7-Linolenic
cooH Me
WOOL FAT Wool fat (anhydrottslanolin) is a purified fat-like substanceprepared fiom the wool of the sheep,Oulsarles (Bovidae). Raw wool containsconsiderablequantitiesof 'woo1 grease'or crude lanolin. the potassium salts of fatty acids and earthy matter. Raw lanolin is separatedby 'cracking' with sulphuric acid fiom the washingsof the scouringprocessand purilied to fit it lbr medicinal use. Purification may be done by centrifuging with water and by bleaching. Wool fat is a pale yellow, tenacionssubstancewith a faint but characteristicodour.It is insolublein water and a high proportion of water may be incorporatedwith it by melting (m.p. 36-42'C) and stirring. Soluble in ether and chloroform. Like other waxes, it is not readily saponifiedby aqueousalkali,but an alcoholicsolutionofalkali causes saponification. Saponilicationvalue 90-105: iodine value 18-32; acid valuenot morethan l. Hydrouswool fat or lanolincontains25% water. The chief constituentsof wool fat arecholesteroland isocholesterol. unsaturatedmonohydricalcoholsof the formula C27H.+5OH. both tiee and combined with lanoceric,lanopalmitic.carnaubicand other fatty acids.Wool fat also containsaliphaticalcoholssuchas cetyl, ceryl and carnaubl,lalcohols.BLrtylatedhydroxytoluene,up to 200 p.p.m., may be addedas an antioxidant, WoolAlcohoLsEP, BP are preparedby the saponificationof crude lanolin and the separationof the alcohol fraction.The productconsists of steroidand triterpenealcohols,including cholesterol(not less than 30%) and isocholestelol.As for wool fat, an antioxidantmay be added. To testfor cholesteroldissolve0.5 g in 5 ml of chloroform,add I ml aceticanhydrideand two dropsof sulphuricacidl a deep-greencolour is produced. Hy'drogeruttedWooLFat EP, BP is obtained by the high pressure/ high temperaturehydrogenationof anhydrouswool f-at.It containsa mixture of higher aliphaticalcoholsand sterols. Wool fat is usedas an emollientbasefbr creamsand ointments.
Arachidonicacid
Ez(PGEz) E Prostaglandin z(PGEz)
The principal speciescultivateclin thr-UK is O. bieruis which yields an oil containingl-9c/aGLA. althoughrnorclecent work showshigher yields for the oils of someother species.nantelv O. acen,iphilla novct (15.68%),O. porttdo-ra(.l4.4lVc)anclan ccotvpeof O. rubricaulis (13.15Vc).Researchhas involved breedingneu varietiesfor high yields of oil and reducingthe lif'ecyclcof the plant f rorn 14 to 7 months (Pltarm. J., 1994, 252. 189). The sequencefor the formation of such acids in the plant via cislinoleicacidhasalreadybeenindicatedin Fig. 10.l. In animaltissuesit appearsthat the prostaglandinsareformed from dictarl' linoleic acid by conversionto GLA which undergoesCr addition and further desaturation to give acids such as arachidonicacid an inrmediateprecursorof someprostaglandins. The beneficialeffectsof eveningprimroseoil ntar nell be relatedto affording a precunor of the prostaglandinsfbr those individr,ralswhose enzymicconversionof linoleic acid to GLA is delicient.Thc oil is norv widely marketedasa dietarysupplement.for cosnreticpurposes.andmore specifically for the treatment of atopic eczemil and premcnstrualsyndrome(prostaglandin E may be depletedin this condition).Furlherpossibilitiesincludeits usein diabeticneuroDathv andrheumatoidafthritis. Other sourcesof GLA. A number of other seedoil: contain appreciable quantitiesof GLA; theseinclude thoseof Ribesnigrunt andR. rubrum (the black and red currant).Borago of]it'irnli.s(starflower)and S1-mphytum oJlicinale(comfrey).B. offitirtulis is now cultivatedcommercially in the UK to yield an oil containing 25c/cGLA; details of commercial seed production. plant breeding programmesand husbandry will be found in a generalarticle on the crop by A. Fieldsend (Biolo gisr, 1995,42, 203).
Hydnocorpusoil Corn oil
This is the fixed oil obtainedby cold expressionfrom the fresh ripe Corn oil USP/NF (1995),or maize oil, is obtainedby expressionof seedsof Hl,dnocarpuswightiuttt, H. anthelmintica,H. heterophylla the fixed oil from the embryos oI Zea mays L. (Graminae) and and other speciesof Hy,dnocarpus.and also ol TttrtLktogenos kurz,ii. refined. These plants are lbund in India. Bulma. Siam and Indo-China and For the separationof the embryos from maize see 'Preparationof belongto the Flacourtiaceae. Maize Starch'. The refined light golden yellow oil consistslargely of The oil of H. wightianT contains hydnocarpic acid (about 2187c), triglyceridesof the unsaturatedoleic and linoleic acids with smaller chaulmoogricacid (about 21c/c),gorlic and other acids (formulae.see proportionsofpalmitic and steadcacids.Becauseofthe high unsatura- Table 20.4); the structuresof severalnew cyclopentenylfatty acids tion (iodinevalue I l0-128), it is regardedasofvalue in dietsdesigned haverecentlybeenelucidated.Theseacidsdo not appearto be fbrmed to limit blood cholesterollevels. from straightchain acids and they accumulateduring the last 3-4
P H A R M A C O P O E IA N L D R E L A T EDDR U G SO F B I O L O G I C AO LR I G I N months of matul'ation of the fluit. They are strongly bactericidal towardsthe leprosymicrococclrs.but the oil has now to a largeextent beenreplacedby the ethyl esterstrndsaltsof hydnocarpicand chaulmoogric acid. The esterifiedoil of H. n'ig,htittna is plefelableto that of other species.in that it yields when fiactionatedalmost pure ethyl hydnocarpate. This was includedin the BPC ( 1965) but has now been deleted.as mol'cef1'e ctivc rcmediesale availablc. Lord Lard (.preparedlartll is the purilied internal fat of the hog, Sus scrofc t o r J e l U n - r : t r l l rS t ru. i t l a rc. For medicinal purposeslard is preparedfi'om the abdominal fat known as 'flare'.from which it is obtainedby treatmentwith hot water at a temperaturenot exceeding57'C. Lard is a soft. white tat with a nonrancidodour.Acid v:rluenot more than I .2.Lard hasa lowermeltingpoint (3,1.11"C) anda higheriodine value (52*66) than suet.Saponification value 192-198.It shouldbe lree fiom moisture.beef'-lat.sesame-seed and cotton-seedoils. alkalis andchlorides. Larcl contains approximately 40c/t.of solid glycerides such as myristin. stearin and palmitin. and 60%, of mixed liquid glycerides such as olein. These fiactions arc somewhatseparatedby pressureat 'stearin'and'lard OoCand soldas oil'respectively.Lard is usedas an ointmentbasebut is no longer of1icialin Britain. It is sornewhatliable to becomerancid. but this may be retardedby benzoination,Siamese benzoinbeing more efl'ectivethan the Sumatravariety.
slow bleachingactionof light. air and moisture.In the lattermethod the melted wax is allowed to tall on a revolving cylinder which is kept moist. Ribbon-like str"ipsof wax are thus formed which are exposedon clothsto the actionof light and air. being moistenedand turned at intervals until the outer surface is bleached.The whole proccssis repeatcdat least once, and the wax is linally cast into circular cakes. Characters. Beeswax is a yellowish-brown or yellowish-white solid. It breakswith a granularfractureand has a characteristicodour. It is insolublein water and sparinglysoluble in cold alcohol,but dissolvesin chlorofbnn and in warm fixed and volatile oils (e.s.oil ofturpentlne). Constituents. Beeswax is a true wax. consistingof about 80% of myricyl pahnitate(myricin),CtsH3ICOOC39H6,. with possiblya little myricyl stearate.It also contains about I5Vc of free cerotic acid, C.6H5rCOOH, an aromatic substancecerolein, hydrocarbons,lactones.cholesterylestersand po1lenpigments. 'drop point' Standards. T'heseincludea of 61-65' for both the white 'ester and yellow wax, value' (q.v.) and 'ratio number' (ratio of the ester value to the acid value) which fbr both waxes lies between 3.3 4.3.
Adulterants, The most likely adulterantsandtheir methodsof detection will be lbund rnlhe Pfurrnrucopoeia. Japanwax, which is there mentioned.is not a true wax but a fat and may be saponifiedby means Suet. Sr.retis the purified intelnal fat of the abdomenof the sheep. ofboiling aqueoussodiumhydroxide.Waxesare saponifiedby strong by aqueousalkali. Ovi.suries.It containsabout 50 60% of solid glyceridesand melts at alcolnLic potash, but are practically r-rnaffected about zl,5"C.It is used as an ointment basc in tropical and subtropical Japan wax is preparedfi'om the tiuits of various speciesof R/rls (Anacardiaceae). countnes. Uses. Beeswaxis usedin the preparationof plasters.ointmentsand polishes.
YELLOWBEESWAX,WHITEBEESWAX Bees',vaxis obtainedby rneltingand puritying the honeycornbof Api,i mellifica and other bees.The wax is imported from the West Indies, Califbrnia. Chile. Africa. Madagascarand India. The EP and BP include separatemono-eraphs for the yellow and the white wax. Preparation, Wax is secretedby worker beesin cells on tht: ventral surfaceof the last four segmentsof their abdomen.The wax passesout throughporesin the chitinousplatesofthe sternumand is used.particularly by the yor.rngworkers.to fbrm the comb. Yellon'beeswa,ris prepared.after removal of the honey.by melting the comb underwater (residualhoney dissolvin-gin the water and solid irnpuritiessinking).straining.and allowing the wax to solidify in suitablernor.rlds. Wltite beesv,uris preparedfrom the above by treatmentwith charcoal.potassiumpermanganate. chromicacid.chlorine.ctc..or by the
CARNAUBAWAX Carnar.rba wax, included tn the EP/BP (2000) and USP/NF (1995), is derived from the leaves of Copernicia cerifera (Palmae). It is removed from the leavesby shaking and purified to remove foreign mattel'. The wax is hard.light brown to paleyellow in colour and is supplied as a moderatelycoarsepowder.as f'lakesor irregularlumps; it is usually tastelesswith a slight characteristicodour liee from rancidity. Esters,chietly myricyl cerotate,are the principal components,with somefree alcoholsand other minor constituents.The acid value is low (BP, USP/NF.2-'7)" Ihe saponificationvalue 78-95. lt has an iodine value of 7-14. Carnaubawax is Lrsedin pharmacyas a tablet-coating agentand in otherindustriesfor the manufactureofcandlesand leather polish. It has been suggestedas a replacementfor beeswax in the p r e p a r r l i oo nf p h l t o c o s r n c t i c s .
PHARMACOPOEIAL AND RELATED DRUGS OF BIOLOGICAT ORIGIN sucrose;it is formed in photosynthesis by the reactionof UDPG with (Fig. 21.3).Control mechanismsfor the build-up fructose-6-phosphate of sucrosein leaves,and its breakdown for tlanspofi to storageorgans, areachievedby metaboliteeffectorcontrolof the appropriateenzymes. The reverse process, hydrolysis. is brought about by suitable enzymes or by boiling with dilute acid. The same sugars may be linked to one anotherin variousways.Thus,the disaccharides maltose, cellobiose,sophoroseand trehaloseareall composedof two molecules of glucosejoined by c,-1,,1-,B-1,4-, B-1,2- and cr,cr-1,1-(nonreducing) linkages,respectively.
HO.^rH
cHo
Y-t
I
HcoH I
HCOH I
HOCH I HCOH
HOCH O
xEox Il t
I HCOH I
CH I
cl-tzoH
cH2oH
Aldohexosc (uglucose)
Aldohcxose (F-p-glucose,showing pyranose ring and the OH taking part in glycoside formrtion)
cH2oH I C=O
Polysocchorides By condensationinvolving sugar phosphatesand sugar nucleotides, polysaccharides are derivedfrom monosaccharides in an exactly similar mannerto the formation of di-. tri-and tetrasaccharides. The name 'oligosaccharide'(Greek oligo, tew) is often applied to saccharides containing from two to l0 units. In polysaccharidesthe number of sugar r.rnitsis much larger and the number lbrming the molecule is often only approximatelyknown. The hydrolysis of polysaccharides, by enzymesor reagents,ofien resultsin a successionof cleavages,but the final productsarehexosesor pentosesor their derivatives.The term 'polysaccharide'may usefullybe takento includepolysaccharidecomplexes which yield in addition to monosaccharidestheir sulphate esters,uronic acidsor amino sugars. Table21.2 indicatesthe characterof someofthe polysaccharides. In addition to the well-establishedpolysaccharide-containing pharmaceuticalmaterialsdescribedlater in this chapterthere is now considerable interest in a number of polysaccharideswith other pharmacologicalactivities.These include immuno-modulating,antitumour, anti-inflammatory,anticoagulant,hypoglycaemicand antiviral properties.Specificexamplesare the glycyrrhizansof Gl.1'cyrrlti:a uralensis and G. gkhra and the glycans of ginseng and Eleutherococurs(q.v.).In generalpolysaccharides from lungi exhibit antitumour activity, those from higher plants are immunostimulatory
6
I
H?O3POCH2^
HOCH I HCOH I
OH
t-l"\^
ffi'"-
HCOH I
cH2oH
O H H uFructose-6-phosphate (furanose ring)
Kctohexose (o-fructose)
_TTN{V* c.H2oH
cr-D-Glucosc (pyranosering)
P->Glucose
OH p-o-Glucose (anotherreprosentation)
F i g .2 l . l Hexose structuresond representotion
cHo
cHo F-oH
cHo Hoi
l-or OH
t -or
-
o-Ribose (pentose)
cHo
Fo'
io* cH2oH
fon
l
f--oH
D-Xylose (pentose)
o-Ribulose (pentose)
H O--l
-i
HO
CHg
Hamamelose r-Rhamnose pentose) (hydroxymethylribose) (methyl
fo' -ot cH2oH o-Mannose (hexose)
D-Xylulose (penrose)
CHeOH I -
cHo
HO
HO
Fig.21,2 Exomples of Cato Cz monosocchorides structures). {Fischer
l-oH
I cH2oH
cHo -.1
cHo
uon,c-f oH
Ho--] I -ot cH2oH
r-oH
cH2oH
o-Arabinose (pentose)
n-n
I
l -ot
cH2oH
CH,OH t -
CH,OH l -
Ho--]
l-o*
t l-on
cH2oH D-Erythrose (tetrose)
cHo lot
HO
l-o'
lI
HO
l-ot
CH2OH o-Galactose (hexose)
Ho---] I -I o H
l-ot -ot
cH20H o-Sedoheptulose (heptose)
CARBOHYDRATES Toble 2l. I
Typ" Di-
Telro'
Some di-, tri- ond ietrosocchorides. Nome
Productsof hydrolysis
Occurrence
Sucrose Glucose,fructose Sugorcone,sugorbeet,efc. Moltose Glucose,glucose Eniymichydrofisisof ,rorch Loctose Glucose,goloctose Mil[ Cellobiose Glucose,glucose Enzymicbreokdownof cellulose Treholose Glucose,glucose Ergot,Rhodophy."o",y"oro Sophorose Glucose,glucose SJphoroiaplnico,6vil.fvri, of srevioside Primeverose Glucose,xylose Fitipendu'to'ulmrrr. fiyarjyri, of spiroein Gentionose Glucose,glucose,fruclose Genfionospp. Melezitose Glucose,fructose,glucose Monno from lorx Plonteose Glucose,fruclose,goloctose Seedsof psylliun spp. Roffinose Goloctose,glucoselfrucfose Mony seeds(e.g.cotton_seed) Monneotriose Goloctose,goloctose,glucose Monno of orh, Fror.irrcornm Rhomninose Rhomnose, rhomnose, goloctose Rhomnusinfectorio Sciliotriose Rhomnose, glucose,glucose Glycosideof squill .. other exomplesof irisocchorides ore omongthe glycosidesol DigitolisondstropAonthus (q.v.) Stochyose or monneoietrose Goloctose,goloctose,glucose,fiuctose Tub-ers'of'Stochys ioponicoondmonnoof rroxrnuSornus Olher exomplesof tekosocchorides ore omongthe glycosidesof Drgilol,s{q.v.)
Uridinc diphosphate glucose (UDp-glucose)
pouredin to form a layerbelow the aqueoussolution.With an insol_ uble carbohydratesuch as cotton-wool (celluiose)the colour will not appearuntil the acid layer is shakento bring it in contactwith phosphstc synlhase I Sucrose the material. (ECz.l.t.tl) [ 3. OsaT.one formation. Osazonesare sugarderivativesfbrmed by heat_ ing a sugar solution with phenylhydrazinehydrochloride,sodium SucroseGphosphate acetateand aceticacid.If the yellow crystalswhich form areexam_ ined under the microscopethey are sutTicientlycharacteristicfor I phosphaore(EC 3.1.3.2a) lSrcrosc certain sugarsto be identified. It should be noted that glucoseand t fructoseform the sameosazone(glucosazone.m.p. 205oC).Before melting points are taken,osazonesshouldbe purified by recrystal_ lization from alcohol.Sucrosedoesnot form an osazone.but under the conditionsof the abovetest sufficienthydrolysistakesplacefbr Fig. 2 | .3 the productionof glucosazone. Biosynthesis of sucrose. 4. Resorcinoltestfor ketones.This is known as Selivanoff's test. A crystal of resorcinol is added to the soiution and warmed on a water-bathwith an equalvolume of concentratedhydrochloricacid. and the algal polysaccharides, which often contain sulphate,are good A rosecolour is producedif a ketoneis present(e.g.fructose,honey anticoagulants. or hydrolysedinulin). 5. Test.forpenloses.Heat a solutionof the substancein a test-tubewith Testsfor corbohydrotes an equal volume ofhydrochloric acid containinga little phloroglu_ The following are some of the more useful testsfor sugarsand other cinol. Formationof a red colour indicatespentoses. carbohydrates. 6. Keller-Kiliani testfor deontszgars.Deoxysugarsare found in car_ 7. Reductiond Fehling's solution. To a heatedsolution of the sub_ diac glycosides such as those of Digitalis and Strophantlra.sspp. stanceadd drop by drop a mixture of equal partsof Fehling,ssolu_ (seeChapter24).The sugaris dissolvedin aceticacid containinga tion No. I and No. 2. In certaincasesreductiontakesplacenearthe trace of ferric chloride and transferred to the surface of concen_ boiling point and is shown by a brick-red precipitateof cuprous tratedsulphuricacid.At thejunction ofthe liquids a reddish_brown oxide. Reducing sugarsinclude all monosaccharides, many disaccolour is producedwhich graduallybecomesblue. charides(e.g. lactose,maltose,cellobioseand gentiobiose).Non_ 1. Enzyme reactions.Since certain carbohydratereactionsare only reducing substancesinclude some disaccharides(sucrose and brought about by certain specific enzymes,such enzymesmay be trehalose,the latter a sugarfound in somefungi) and polysacchar_ usedfor identification. ides. Non-reducing carbohydrateswill on boiling with acids be 8. Chromntograpfty.Chromatographic methodsare particularly suitedto convertedinto reducing sugars,but studentsare remindedto neu_ the examination of drug extracts, which may contain a number of tralize any acid used for hydrolysis befbre testing with Fehling's carbohydratesoften in very small amounts.Not only are they applica_ solution,or cuprousoxide will fail to precipitate. ble to carbohydratesoriginally present in the plant, but also 2. Molisch's te.st.All carbohydratesgive a purple colour when treated they may be used to study the products of hydrolysis of polysaccha_ with o-naphthol and concentratedsulphuric acid. With a soluble ride complexessuchasgums and mucilages.As standardsfor compar_ carbohydratethis appearsas a ring if the sulphuric acid is gently ison many pure sugars,uronic acids and other sugar derivatives are
Fructose Gphosnnate !
N uop I
l\- n su.L."
ORIGIN OF BIOLOGICAL DRUGS AND RELATED PHARMACOPOEIAL
Tqble 2I.2
The
€ontoining only monosocchoride units or o-omYlose 1. Amylopectin
hosbronchedchoins of mostsiorches Themoinconstituent {overBO%).Themolecule Severolhundredof these of 20-26 o.-l,4linkedglucoseresidues. eochconsisting choinsgivingo bondsto neighbouring choinsore linkld by a-1,6glycosidic ,o*" 50 000 glycosylunits.Thebronchingpotternthroughout contoining molecule omorphous in someoreosthotore opporently is notuniform,resulting the molecule (llneorchoins ond othersproboblycryslolline (highdegreeof bronching) predominote with littlebronching) 6
qHroH H I
H
t"
Hr
2. Amyloseor B-omylose
of lineor obsent.Consistsessentiolly Moststorchescontoinup to2Q%,but sometimes o constitute glucose units thousond Severol glucoseresidues. choinsof cx-,l,4-linked to thotthereis o verylimitedbronching{o-'l,6-linkoges) choin.lt is now recognized per molecule theextentof 2-8 bronches 6
CH H
3. Glycogen or onimol slorch
thotof Moleculeresembles of onimoltissues. lmporlontreservecorbohydrote omvlopeciin
4. Cellulose
glucose of plontcellwolls.Lineorchoinsof p-1.4-linked Chiefpolysocchoride residues
H
5. lnulin
ond goloctons 6. Xylons,monnons
7. Hemicelluloses
O
H
H
O
H
Lineorchoinsof up obundontin theCompositoe. porticulorly corbohydrote A reserve unit,formulo glucose o single by terminofed units to !O p-t,2-linkedfructofuronose p.204 Theyore difficultto wiih one onotherond with cellulose. Theseore oftenossocioted ond goloctose, monnose xylose, lheyyield isolotein o pureform.On hydrolysis respeclively The ond pecticsubstonces. occurin thecellwoll with cellulose Thesepolvsocchorides not ore hemicelluloses becouse is deceptive nomen.loirr",dotingfrom IB9l, butore formedmoinlyfromhexoseond pentoseunits. of cellulose componenls os xylons.monnons vory occordingto sourceond con be clossified Hemicelluloses occordingto theirprincipolcomponents ond goloctons
CARBOHYDRATES
Tobfe2l12 {continuad} A polysocchoride foundin lichens. Resembles cellulose but molecule contoins obout 25% of g-1,3glucosidic linkoges
8 . L i c h e n i no r l i c h e ns t o r c h
Polysocchoride complexes conloining uronic ocid or other units l. Peclins Theseoccurin the middlelomelloeof cellwollsond ore obundontin fruits{e.g.oppiesioronges, ond roots{beetsond gention}. Theporenlsubstonce protopectin is insoluble but is eosilyconverted by reshicted hydrolysis intopectinicocids(pectins). Pecfins fromdifferent sources vory in their complexconslitution, theprincipolcomponents beingblocksof o-golocturonic ocid residues linked by o.'1,4-glycosidiclinkoges ond interspersed with rhomnose uniis;someof thecorboxylgroups ore methyloted. Thesemolecules ore occomponied bv smollomounts of neutrolorobrnons (bronched polymers of a-'l,5-linked t-orobofuronose unifs)ond golocions(lorgelylineorchoinsof units) B-1,4-linked>goloctopyronose
t-
coc[J a4
H O H
Repeatingunits of D-galacturonicacid 2 . A l g i no r o l g i n i co c i d
Alginicocid is theprincipolconstituenl of thecellwollsof the brownolgoe.lt wos discovereo oy Stonfordin I 880 ond is nowwidelyusedfor the monufocture of olginotesoltsond fibres{q.v.). Thecomposition voriesoccordingto the biologicolsource,thusprovidingo rongeof properties whichore exploitedcommerciolly. lf is o heteropolyuronide consisting of choinsof B-1,4-linked D-monnuronic ocid unitsinterspersed with lengfhsof cr-l,4"linked rguluronicocid unitstogether with sections in whichthetwo monouronide unitsore regulorlyinlerspersed. In olginicocidsfrom d i f f e r e n t s o u r c e s t h e r o t i tohseotfw o u r o n i c o c i d s v o r y l r o m 2t :o1l : 2 . T h e c h o i n l e n g t h v o r i e s withthe melhodof preporotion ond moleculor weight,ond viscosiiy meosuTements suggest molecules of lron220 to 860 units
.l [
.oo"
cooH
|ry1 ; H
p-1,4-linked-n-mannuronic acidunits
Polysocchorides wilh sulphuric ocid eslers
4. Chiiin
#-q cooH
I"{-ki"l]$
I I
{
H
H
H
c- 1,4-linked l-guluronic acid units
J"
Certoinolgoe,includingthoseyieldingogor ond corrogeen, contoino mixtureof polysocchorides. Agor, for exomple,contoinso bioseformedfrom o-ond ,.goloctose but olsoo morecomprex formedfromgoloctose ogoropectin ond uronicocid unitsportlyesterified withsulphuric ocid. Corrogeenhoso similorcomposilion Thisis foundin someof the lowerplonts,in insecfs ond in crusioceons. Themolecule consists of lineorchoinsof B-1,4-linked N-ocetyl-o-glycosomine residues. lfsinclusion in themicrofibrillor component of thefungolcellwoll is onologous to thoiof thecellulose microfibril
NH.CO CHT
5 . G u m so n d m u c i l o g e s
I
H
l-
H
NH.CO.CH.;
Gumssuchos ococioond trogoconth ond muciloges, suchos thosefoundin linseed,psyllium seedsond morshmollow root,ore foundin monyplonts,wheretheyore usuollyformedfromthe cellwoll or deposiledon ii in loyers.Theyore essentiolly polyuronides consisting of sugorond uronicocid units.Somegumshovemethoxyl groups(e.g.trogoconih); in otherstheocidiccomplex is unitedwith mefols(e.g.ococio)
PHARMACOPOEIAL DRUGSOF BIOLOGICAL AND RELATED ORIGIN commercially available.Experimentaldetails and Rf values of sugars in different systemsareto be found in standardbooks on chromatography. The carbohydratespotsobtainedafter separationareidentified by their positions and by reagents.lt may be useful to examine them in ultravioletlight.A non-specificrea-eent for reducingsugarsis a freshly prepiued ammoniacal silver nitrate solution. More specific reagents giving coloured spots with diff-erentsugarsinclude aniline hydrogen phthalate(in water-saturated ir-butanol)and naphthoresorcinol(in acetone, water and phosphoric acid). These are applied to the chromatogramu'ith a spray.Although sugarsare nonvolatile,it is possible by suitabletreatmentto renderthem satisfactoryfor gas chromatography (q.i'.)
Furtherreoding Srir astavaR. KulshreshthaD K I 989 Bioactivepolysaccharides from plants. Ph1'tochemistry 28(1I ): 2877,2833 ]i'i'...i..]':.i]].'.]ii'...']'i',il.]...::....::..:.'....::.)......
COrtll,tERC|At:PlAl{T'DiRwiO, FIBRES AND
PRoDucT5.':.l:.'li':..'l'..::'],''.]:''.'1''.'.'.'...'l..
are usedlor thesetwo types of yarn. which are known as combedand carded, respectively.The cotton-combingmachine separatesall the shorterlibres and a threadis spun consistingof long, well-paralleled, uniform fibres. The short fibres of comber waste areusedfor making the bestgradesofcotton wool. The cardingmachineusesfibres which are shorterand less uniform in length. and the absenceof combing is shownin the yarn by the irregulararrangementof the fibres,the endsof which often projectfrom the surface. Microscopy of unbleached cotton. Cotton consistsof unicellular hairsthe appearance of which hasbeenlikenedto that of empty,twisted fire-hoses.Their length is up to about5 cm, diameter9-24 pm, and the numberof twistsvariesfrom about75 cm-l in the Indian to 150cm-l in the SeaIsland.Piecesof'shell'or seedcoat.which can often be picked from samplesof raw cotton,show hair basesfitting betweenthe thickwalled epidermalcells.The apex is roundedand solid. The cotton hair is cylindrical when young but becomesflattened and twisted as it matures,the large lumen, which containsthe remains of protoplasm being much elongatedin transversesection.The cellulosewall of the hair is coveredwith a waxy cuticlewhich rendersit non-absorbent. The cuticle may be stainedwith rutheniumred. Bleachedcotton yam and absorbentcotton wool (seebelow) arereadily wettedby water.
The biological origin and the structureof plant fibres is discussedin Chapter43; many have importantcommercialusesand for a review on their botany, chemistry and processingsee McDougall et ttl., J. Sci. Tests. The fbllowing testsare applicableto cotton. Food,Agrir:., 1993,62. 1. l. On ignition, which should be done both by advancingthe fibre A numberof vegetablefibres haveimportancein pharmacy.particutowards a flame and by heatingon porcelain,cotton burns with a larly as componentsof surgicaldressingsand for the manuf'actureof flame, gives very little odour or fumes, does not producea bead. artificial fibres and haemostaticdressings.The subject of surgical and leavesa small white ash;distinctionfrom acetaterayon.algindressingswas, and in many Schools still is, regardedas pharmacogateyarn (alsowool, silk, nylon). nosy-related.However with the more recentadvancesin the manage2. Moisten with N/50 iodine and, when nearly dry, add 80% w/w sulment and conceptol wound-healing,many materiaisof non-vegetable phuric acid. A blue colour is produced;distinction from acetate origin are now used which, for an in-depth coverage,bring the topic yarn,jute. hemp (alsowool, silk, nylon). rayon, alginate outsidethe scopeof this book. Describedbelow arethe more important With 3. ammoniacal copperoxide solution,raw cottondissolveswith primary carbohydratematerialsinvolved. ballooning, leaving a few fragmentsof cuticle; absorbentcotton dissolves completely with uniform swelling; distinction from COTTON,RAW COTTON acetaterayon.jute (alsowool, nylon). Cotton consistsof the epidermal trichomes of the seedsof Gosstpium 4. In cold sulphuricacid 80% w/w cotton dissolves:distinctionfrom herbuceumand other cultivated speciesof Gossyytium(Malvaceae).The oxidizedceliulose,jute, hemp (alsowool). plants are shrubsor small treeswhich producetlx'ee-to five-celled cap5. In cold sulphuricactd607cw/w insoluble;distinctionfrom cellusules containing numerousseeds.The USA producesabout half of the lose wadding and rayons. world's cotton, other impoftant sourcesbeing Egypt, India and South 6. In warm (40'C) hydrochloric acid BP insoluble;distinctionfrom America.The chiefAmericancottonsarederivedfrorn G. barbadense(Sea acetaterayon (also silk, nylon). Islandcotton) andG. herbacern (Upland,Texasor New Orleanscotton). 7. Insoluble in 5% potassiumhydroxide solution: distinction from The hairs of the different speciesvary in length or 'staple'. The oxidizedcellulose(alsowool, silk). staplesof impoftant commercialvarietiesof cotton are as follows: ( I ) 8. Treat with cold ShirlastainA for 1 min and wash out; gives shades Sealsland,up to 54.5 mm; (2) Egyptian,31-38 mm; (3) Brazilianand of blue, lilac or purple; distinction from viscose and acetate Peruvian, 29-30 mm; (4) American Upland, about 25.9 mm; (5) rayons,alginateyarn (alsowool, silk, nylon). Inclian.2 1.zl-29.2mm. 9. Treat with cold ShirlastainC for 5 min and wash out: raw cotton givesa mauveto leddish-browncolour and absorbentcottona pink Preparation. When ripe the bolls are collected,dried and subjected one; distinctionfrom flax, jute. hemp.The Shirlastainsmay be useprocessto separatethe hairsfrom the seed.The gin, which to a -einnin-e lully appliedto a small piece of the whole fabric underinvestigamay be of a roller or a pneumatictype, is designedto pull the hairs tion to indicate the distribution of more than one type of yaln. througha narrow spacewhich is too small to allow the seedto pass.In 10. Gives no red stainwith phloroglucinoland hydrochloricacid: disordinary Anrerican or Upland cotton the gin leavesthe seedswith a tinction ftom jute, hemp and kapok. coatingof shorthiiirs which haveto be removedby a secondtype of gin I l. Insoluble in formic acid 907c or phenol 907c (w/w); distinction known as a'linter'. These short hairs are used for making the lower from acetaterayon (alsonylon). gradesof cotton wool and rayons.The seedsare used for the prepar- 12. Insolublein acetone(distinctiontiom acetaterayon). ation ofcottonseedoil (q.v.)and cattlecake.Raw cotton containsvariABSORBENT COTTONWOOL, ABSORBENT ous impurities, such as immatureand broken seeds,fragmentsof leaf, etc.,most of which are removedduring the manufactureof yarn. For spinningvery fine yarnsSeaIslandcotton is used.but for coars- Cotton Wool is mainly preparedfrom linters. cnrd strips.card fly and er yarnsit is possibleto use shorterstaplecottons.Different machines comber waste.Balesof theseshort-fibredcotton wastesnassliom the
woot
CARBOHYDRATES yam manutacturersto the makersof cotton wool. For besfquality cotton wool the comber waste of Amelican and Egyptian cottonsis preferred. In this the fibres are reasonablylong and twisted and thus suitablefbr producinga cotton wool having an averagestapleas specitied in the BP, which will offer appreciableresistancewhen pulled and not sheda significantquality of dust when shakengently. The preparationmay be outlined as follows. The comber waste (which anives in bales)is loosenedby machineryand then heatedwith dilute caustic soda and soda ash solntion at a pressureof l-3 atmospheresfor 10- I 5 h. This removesmuch of the latty cuticle and renders the trichome wall absorbent.It is then well washed with water. bleachedwith dilute sodium hypochlorite solution and treatedwith The biosynthesisof cellulosein the cotton trichomewould appearto very dilr.rtehydrochloricacid.After washingand dryin-eit is in a rnatted involve UDP-glucoseoliginating from sucrose. 'scutched': conditionand is thereforeopenedup by machinesand then that is, it is convertedinto a continuoussheetof fairly even thickness Jute with the tibres loosenedready for the carding machine.The carding Jute consistsof the strandsof phlocrr-rfibres fiom the stem bark of machineeffectsa combing operationand forms a thin continuouslllm Corch.ontscapsularis, C. olitrtrius and other speciesol Corchonts of cotton wool. Severalsuch films are superimposedon one another. (Tiliaceae).Theseare annualphnts about 3 ;1ni high which are cultiinterleavedwith papel and packagedin rolls. vated in Bengal. in the delta region of the Gangesand Brahmaputra rivers,andin Assam,Bihal and Orissa. 'Cotton, Tests. For testson cotton wool, seeunder Raw Cotton'. The flbres are separatcdfrom the othcr plant materialby retting and then spun into yarn which can be made up into hessianand sacking. Structure offibre. Like the cell wall in genelal.that ofconon consists Short llbres left over from the preparatioltof the yarns and lopes conof a prirr-ralyand secondarywall. The fbrmation of the former is modifled stitutelor.r,and in phalmacythe terrn 'tori,'ref'ersto jute. althoughit can to embracethe enormouslongitudinaldevelopmentof the cell, somethoualso be applied to hemp and flax. The commercialstrandsare l-3 m sandsof times greater than the width. The microtlbrils themselvesare long and about30-l40pim in dianteter.Each consistsofa bundleof unexpzurdableand are initially laid down in hoops around the fibre. phloemfibrescomposedof lignocellulose. The heavilylignifiedmidrestrictingits lateral growth. However. becauseof their diff'erentorientadle larnellais destroyedby oxidizingagenrsla mixtureof nitric acid tion at the fibre end, longitudinal extensioncan take place. Owing to the and potassiumchloratemay thereforebe uscd to disintegratethe br-rnbuild-upofpressureliom successive layersofcellulosethe originaloriendles.the individual fibres bein-ethen teasedout and sketched.Prepared tation becomeslost as the flbre matules.Maffix polysaccharidesand protransversesectionsshouldalso be cranrinecland comoaredwith those teinsderivedfrom the Golgi apparatusarealsopresentin the primary wall. of hemp and flax. The secondary wall. some5-10 pm thick andconsistingalmostentirelyof pure cellulose.constitutesthe main bulk of the maturecotton fibre. Flox Flax is preparedfrom the pericyclic libres of rhe stem of Linum usiChemical nature. Raw cotton consistsof cellulose approximately tutissi mLtm (Linaceae). 90c/candmoisture'lc/a,Iheremainderbeing wax. f'at,remainsof protoThe commercialfibres show fine transverscinjLrriesreceivedduring plasm and ash. the preparation.Good-qualityllax fibre is nonlignifieclexceptfbr the Absorbentcottonis a very purefrom of celluloseandits chemicaland nriddlelarnella.Li-qnificationof the secondarr*all. howcr,er.takes physicalpropefiieshave beenextensivelystudied.The cellulosemoleplace as the stemmatures,startingat thc basc.and if the stemsare too cule is built up ofglucoseresiduesunitedby 1.4-B-glucosidic (conlinks old befbreretting.the libre is coarseand harshin terture. trast sffch). The wall of the cotton fible, like that of plant cells in general. shows anisotlopic properlies.When swollen in water, the Hemp swellingis in a directionat right anglesto the long axis.In the direction Hemp is preparedfrorr the pericyclic tibres o1'the stem of Connabis of the long axis it shows considerabletensile strength.Examined in salll'a (Cannabinaceae). The fibre is composeclchiefly of cellulose.but pol:Lrizedlight. it showsbirefiingence.the valueof the doublerefiaction somelignification has usually takenplacc and the percentageofcelludependingon the liquid in which the llbre is immersed.This phenomeIoseis lower thanin flax. non,characteristic of mixed bodieswith rod-likestmcturalelements.has The fibre ends. in contrast to those of flax, are bluntly rounded. beentermedrodlet-doublelefiaction.Stainedwith chlor-zinc-iodineand Someof the fibre endsarefbrked. this bifurcationarisingfrom injuries examinednicroscopically in polarized light (analyserremoved).the to the stem.The lumen of the hemp f ibre is flattenedor oval, in contrast fibre showsgreaterabsolptionwhen orientatedwith its long axisparallel to the small round lr.rmenof flax. Transversestriationsseenin commerto the plane of polarizationthan when orientatedwith the long axis at cial fibres ariseliom beating.the tibres being preparedby partial retright angles(dichroism).Thesephysicalpropefiiessuggestthatthe fibre ting. wall is built up of elongatedstructuralunits orientatedin somedefinite Jute,hemp and f'laxfibres are comparedin Table21.3. manner.The study of the cotton fibre by X-ray analysishas confirmed this andhas shownthat its cell wall is conposedof elongatedchain-like molecules(built up ofrepeatingunits 1.03nm long) and orientatedin a spiralmanner,the spiralmakingan angleof 30owith the long axisof the REGENERATE D CARBOHYDRATE MATERIAT fibre. The lengthof the repeatingunit of structurecorresponds AND CHE'I/IICAILY to that of MODIFIEDFIBRES two giucoseresiduesfLrllyextended.This unit is the 'cellobioseunit', many of which are united in the polysaccharide moleculeof cellulose Regeneratedtibres are thoseproducediiom naturallyoccurring,long(Table21.2). chain moleculeswhich havebeenisolated,controlledand,if necessary,
E
P H A R M A C O P O EAIN AD TR E L A T E DD R U GO S F B I O T O G I CO AR L IGIN
Toble 2l .3
Chor
Hemp (Connobissotiva)
Flox (Linum u sitotissimum)
Apex Woll
Bluntlypointedor rounded Withoutmorkings; lumen voryingin size
Shorplypointed Thickwoll with finecrosslinessome intersecling; lumennorrow
Tronsverse section Length Diometer Phloroglucinol test l o d i n eo n d s u l p h u r o i cc i d
Polygonol, shorpongles;lumen ovolor circulor 0.8-5 mm 10-25 pm Deepred Yellowthroughout
C hlor-zinc-iod ine
Yellow
Mosilybluntond sometimes forked Morkedstriotions, crossfissures, ond swellings; lumenlorgeond uniform Roughly3-to6-sidedwith rounded corners;lumencleftor bronched 35-40 mm 16-50 pm Slightlyred Innerwoll blue;middlelomello yellow Purpleto yellow
rnodifiedto give a suitablefibre fbrm. The term 'rayon', as in viscose, acetateand cuprammoniumrayon, is appliedto thosederivedfiom the polysaccharidecellulose.The term 'artificial silk' is now out of date. Also in this classis alginatefibre, derivedliorn alginic acid (q.v.). Viscose (regeneroted cellulose, royon) This has been developedf}om a processintroducedby three Blitish chemists(Beadle,Bevan and Closs) in 1892, and accountsfor the bulk of the wolld rayon outputtoday.It is alsothe principaltype usedin surgicaldressings. The startingmaterialis a cellulosepreparedeither from coniferous wood. particularlyspruce,or scouredand bleachedcotton linters.The wood is usually delignified at source(Canada.Scandinavia,etc.) by a processsimilar to that usedtbr cellulosewadding.It reachesthe rayon manufacturers asboardsof white pulp. containing80-90% of cellulose and some henricellulosefnainly pentosans).The latter,being alkalisoluble,are removedin the lirst stageofthe process,which consistsof steepingin sodiLrmhydroxidesolntion.After mostof the excessalkaline liquor has been pressed out, alkali-cellulose (sodium cellulosate) remains.This is dissolved by treatmentwith carbon disulphide and sodium hydroxide solution to give a viscous (whencethe name 'viscose') solutionof sodiumcellulosexanthate.After 'ripening'and filtering. the solutionis fbrced through a spinneret.a jet with fine nozzles, immersedin a bath which includesdilute sulphuricacidand sodiumsulphate.when the celluloseis regenerated as continuousfilaments.These are drawn togetheras a yarn. which is twisted fbr strength,desulphurizedby removingfiee sulphurwith sodiumsulphide,bleached,washed, dried and conditionedto a moisturecontentof ljc/c. The viscoseyam may be left as such(i.e. continLtous.fiLanTent ra),ott) tbr usein suchthingsas blousefabrics,or it may be cut up to givestaple ('Fibro') offixed lengthliom I to 8 in. That usedin surgicaldressrznz.,rr in-esanclrnanvother fabricsis madeto resemblecotton in dimensions. Suitablespinnerets areusedto give a diameterof 15 20 ;rm andthe fibre is cut into lengthsusually of 21.8cm. This staplecan be processedon typesof spinningand weavingmachinesusedlbr cottondlessingsor it may be lefi in a loosefibre fbrm as ylscosera1,onabsorbentwool. Viscosera1'onis a very pure form ofcellulose.It yields a traceofash which containssulphur.The cellulosemoleculesof the original natural material, whether u,ood or cotton, becomemore separatedfrom one anotherin the viscosesolutionthan in the vegetablematerialand in the regeneratedfibre are still lessclosely packed.Radiographyhas shown that the side-to-sideag-qregation of the long-chainmoleculesis different liom that in natulal celluloses.The size of the moleculesis also
5 or 6 skoightsides;point-like tumen 25-30 mmoveroge,butup to I 20 mm 12-30 ;rm Colourless or slightpink Blueor violet Purpleto yellow
reduced,wood cellulosehavingmoleculesofthe orderof9000 glucose residueunits, while thoseof viscoserayon have only about450. Viscose rayon gauze and other rayon dressingshave the advantage over cottondressingsin thatthey showno lossof absorbencyon storage. Macroscopical characters. As normally produced, this rayon is a white, highly lustrous fibre Qtatural or g/ossy'viscose).Its tensile strengthvaries from two-thirds to one-and-a-halftimes that of cotton. When wettedit losesabout60% of its tensilestrength,a proportionarely greaterloss than is fbund with cotton. Where more than a certain amountofrayon is usedin a dressing,the labric may be requiredto be renderedwater-repellent(e.9.cotton cr6pebandage). Microscopical characters. The fibres are solid and transparent and 15-20 trrm in diameter. They have a slight twist, and show grooves along their length which ale principally causedby the spinneretsbeing immersedin the regeneratingsolution (comparenylon). The grooves give a characteristicappearanceto the transversesection. The ends of the fibres are abrupt and characteristic.The fibres are clearly seenin chloral hydrate solution or in lactophenol,but are almost invisible in cresol (having the samerefiactive index of 1.53). They appearbright in polarizedlight with crossedNicols. Chemical tests l. The fibres give the generaltestslbr vegetableand regeneratedcarbohydrate libres. 2. On ignition they behavelike cotton; distinctionfrom acetaterayon and alginateyarn (alsowool, silk, nylon and glass). 3. With N/50 iodine and sulphuricacid, 807c,they give a blue colour similar to that given by cotton;distinctionfrom acetaterayon,alginate yarn,jute, hemp (alsowool, silk, nylon). 4. With ammoniacalcopper oxide they behavelike absorbentcotton; distinctionfrom acetaterayon.jute (alsowool, nylon). 5. Cold sulphuricacrd,607ow/w, dissolvesthe fibre; distinctionfrom cotton,oxidizedcellulose,alginateyarn,1lax,jute, hemp (alsowool). 6. Warm (40'C) Hydrochloric Acid BP does not causesolution; distinction from acetaterayon (also silk, nylon). 7. Boiling potassiumhydroxide solution, 5clc,insoluble; distinction from oxidized cellulose(alsowool. silk). 8. ShirlastainA producesa bright pink: distinction from cotron, oxidized cellulose,acetate,rayon (alsowoo1,silk, nylon). 9. Phloroglucinoland hydrochloricacid produceno red stain;distinction from jute, hemp and kapok.
CARBOHYDRATES 10. The iibres, like cotton, are insoh"rble in acetone.formic acid 907c or phenol907c;distinctionfrom acetaterayon (alsonylon). Delustring and dyeing of fibres. Rayon and other artillcial fibres with a naturallustrousappearance may be delustredby additionof the white pigment titanium oxide to the solution (e.g. viscose)or to the melt (e.g.nylon) beforeextrusionof the filaments.ln this way the pigment is evenly distributedinside each filament and delustringis permanent.These fibres may be similarly 'spun-dyed'by addition of an appropriatedye insteadof the titanium oxide. The methodresultsin an exceptionaldegreeof colour fastness. Matt Viscose(delustratetlviscosera-ton)is the form nornally used in the manufactureof surgicaldressings;hence,in generalappearancethese are very similar to those manulacturedfiom cotton. The individual filaments have the appearancealready described,except lbr the matt white colour and on microscopical examination the pigment particles, which appearblack by transmitted light and are scatteredthroughout the filament. The amount of pigment is controlled by the ash value. Titanium is detectedin the ash by dissolving in sulphuric acid, diluting and adding hydrogenperoxide,3c/c, when a yellow colouris produced. Cellulose ethers. These are preparedliom purified alkali cellulose derived from cotton linters or delignified wood pulp by the action of causticsoda,as in the initial stagesolthe productionof viscoserayon. Meth,-LceLlulose EP/BP is a whitish, fibrous powderpreparedby the action ol methyl chloride under pressureon an alkali cellulose,when hydroxyl groups becomemethylated.A useful grade is that in which two of the threehydroxyl groupsof the glucoseresidueunits of the cellulose chain are methylated,and this has the optimum solubility in water. In pharmacy a grade giving a low viscosity is used both to increasethe viscosity and to stabilizelotions, suspensions, pastesand sorneointmentsand ophthalmicpreparations;one giving a high viscosity is used as a tablet disintegrant. In medicine it is used as a hydrophiiic colloid laxativein chronic constipationand can be usedin obesepersonsto curb the appetite,becauseit gives a feeling of fullness.Ethylcelluloseis similarly preparedand has like applications. Cttrmellose Sodium EP/BP (sodium carboxyrnethylcellulose)is an odourlessand tastelesswhite hygroscopic powder or granulesprepared by the action of monochloroaceticacid on alkali celluloseand removal of the byproductsalts.Substitutionof hydroxyl groupsby carboxymethyl groupsoccursover a rangedependingon theconditionsandthecellulose used; there are prescribedlimits for the sodium content. It is water-soluble,and a gradegiving a mediumviscositycontains0.7 carboxymethyl groupsper glucoseresidueunit. It is insolublein organicsolvents.Its pharmaceutical and medicalusesaresimilarto thoseof methylcellulose, but as well asbeingusedas a laxativeit is a usefulantacid. Carmelktsecalcium is alsoofTicial.
Oxidized cellulose Oxidizedcelluloseoriginatedin theUSA asa resultof the * ork publi:hc.d by YackelandKenyonin 1942.Cottonwool or gauzeis treatcds irh nirro gendioxide until the numberofcarboxyl groupslblmed by theoridation ol the primary alcohol groups of the glucoseresidueunits of the cellulose molecules reaches16 227o.T-heoriginal cellulosenow hasglucuronicircicl residueunits(comparealginicacid)aswell assomeglucosercsidueunits. Appearance. Gar.rze, lint or knitted material,very similar to norntal cotton but with an off-white colour, a harshertexture,charredodour and an acid taste.It does not go pasty on chewing.The wool tendstcr disintegrateon handling. In microscopicalappearancethe fibres are very similar to thoseof absorbentcotton. Tests 1. Does not give the tests for animal fibres and animal sourcehaemostatics. 2. On ignition it behaveslike normal cotton. 3. With iodine and sulphuric acid or ammoniacalcopper oxide solution it behaveslike absorbentcotton. 4. Siowly solublein 807csulphuricacid. 5. Insolublein warm hydrochloricacid BP. 6. Solublein the cold in 5% potassiumhldroxide solution. Completesolubility in aqueousalkali is madethe basisof a test for absenceof unchangedcotton and fbrei-unparticles.The solution in alkali gives with excessacid a white flocculentprecipitate(fbrmer BP testfor identity). 7 . I t r e d u c eF r e h l i n g 's o l u t i o n . 8. Shirlastain A givesa paleblue to nauve colour. 9. ShirlastainC gives a brown to olive -grecn. Uses. It is used as an absorbablehacntostaticin many types of surgery,but is incompatiblewith pencillin. dclavsbonerepair and cannot be sterilizedby heat.It hasfbund sorre applicationin chromatography.
Alginote fibres
Theseoriginatedabout I 938 in Britain and u,erefurtherdevelopedduring World War II. The fibres arepreparedby a processsinrilarto that for viscoserayon.An aqueoussolution of sodium alginate(seethis chapter)is pumped through a spinneretimmersedin a bath of calcium chloride solution(acidilied with hydrochloric acid). when water-insolublecalcium alignateis precipitated ascontinuousfilarnents.Thesearecollected.washedand dried. For usein surgicaldressingsiurd bacteriologicalswabsthey are reducedto a staple form which may then be processedto a t uk:ium alginate yyoolor a fabric (e.g.gauze)in the samemillner asusedfbr viscosestapleor cotton. Pyroxylin BP (€ellulose nitrote) As indicatedin Table 21.2, alginic acid is composedof polymersof Pyroxylin is preparedby the action of nitric and sulphuric acids on both mannuronicand guluronic acids.The properties of the two are variwood pulp or cotton linters that have been freed from fatty materials. able and alginates of different origin have different compositions and When dry it is explosiveand must be carefully stored,dampenedwith properlies.This is illustratedby the two commercial haemostaticdressnot lessthan 25a/cits weight of isopropyl alcohol or industrialmethyl- ings-Kalostat (Britcair Limited) and Sorbsan(Steriseal-Pharmaplast atedspirits.It is usedfor making Flexible Collodion BP. Limited). The former is derivedfrom the seaweedLan inariu htperborea collectedoff the Norwegian coastand yields an alginatewith a guluronAbsorboble hoemosfqtic dressings ic:mannuronic ratio of 2:1; the latter is prepared from Inminaria and The control of bleedingis of vital concemin surgery,and the greatdisad- Ascophyllumspeciescollectedoffthe west coastof Scotlandand gives an vantageofthe old-type dressingsuchasa cotton gauzeplug is that it hasto alginatewith a guluronic:mannuronic acidratio of about1:2.On a wound be removedafter bleedinghas beencheckedwith a consequentdangerof surfacethe c-linkages ofthe guluronic acid polymer arenot easilybroken a recurrenceofthe haemorrhage.Gelatin sponge,oxidized celluloseand so that libre strengthis retainedand a stronggel is fbnned on contactwith alginatedressingsovercomethis. in that there is no needto remove them the woundexudate.A high ratioof mannuronicacidpolymer( B-linkages) after thebleedinghasbeenchecked,sincethey areabsorbedby the tissues. yields a productgiving a weakergel and lessretentionof fibre strength.In
E
ORIGIN DRUGSOF BIOLOGICAL AND RELATED PHARMACOPOEIAL practice this meansthat the Kaiostat dressingcan be removed from the wound with forcepsand Sorbsanis removedby irrigation with, tbr example, sodiumcitratesolution. Calcium alginatefibres of commercc contain substantiaitracesof substancesused to inhibit mould and bacterialgrowth in the sodium alginatespinning solution.Spinning lubricantssuch as lauryl or cetyl pyridinium bromide (antibacterial)are also applied to the filaments. Thesesubstancesmllst not be usedol must be removedin the caseof calcir,rmalginatestapletbr use in, for exatnple,bacteriologicalswabs. Beforeuseas an absorbablehaemostaticdressingsornecalcium alginatedressingsmustbe irnmersedin sodiumchlorideto give a fibre of the calcium alginatecoveredby sodiumalginate.The degreeofconversion the desiredrate of absorptionwhen in use; the is conditioned10 ,qi,,,e greaterthe proponionof sodiumalginatethe fasterthe absorptionrate. Alginate filaments are composedof salts of the long-chain moleculcsof alginic acid (seeTable 21.2) and there is little cross-linking the chainsin the fibre. bct'nveen Appearance. Fairly lustrous,pale cream-colouredfihres which in microscopicalappearanceare very similiu'to those of viscoserayon, dressing('Calgitex')is almost beingsolidgroovedrods.The haemostatic to touch.The gauzeis usuallya harsh and rather odourless and tasteless knitted tabric and haslittle sheen.That with a fast rate of absorptionwhen chewedreadilyassumesa pastyform somewhatlike thatof mashedpotato. That with a slow rate of absorptionremains smoothly coarserin the easilyon handling.Thesepointsand sametime.They do not disintegrate the testsbelow will serveto distinguishalginatehaemostaticdressings from thoseof oxidizedcellulose.First-aiddressingsfrequentlyembody an alginategauzeimpregnatedrvith a local anaesthetic. Tests. Theserefer to calcium alginatefibre or the rnixed sodiumand calcium salt fibre. They give the generaltestsfor vegetableand regeneratedcarbohydratefibres. For distinctionsfrom rayonsand oxidized celluloseseeearlier. 1. Smouldersin a flame and goesout when removedfiom flame. 2. With (N/50) iodine and sulphuric acid, a brownish-redcolour is produced.the filaments swell and dissolve to leave a strand of i n s o l u b l ca l c i n i cu u i d . 3 . In ammoniacalcoppernitratesolutionthey swell and dissolve. 4 . lnsolublein 60clcw/rv sulphuricacid. 5 . Insolublein warm (zlO'C)hydrochloricacid BP. 6 . Insolublein boiling 5% KOH (swel1and acquirea yellow tint). 1 . Solublein 57c sodiumcitratesolution. 8 . Fibre,0.1 g, boiled with -5ml of water remainsinsolublebut dissolves when I ml 2OVcwlv sodium carbonatesolution is addedand boiled fbr I min. Awhite precipitateof calcium carbonateis fbrmed' depending on the propoftion of original calcium alginatepresent.When centlifuged and the clear supernatantacidified,a gelatinousprecipitateof alginic aciclis produced.The precipitatewill give a purplecolour after solution in NaOH and addition of an acid solutionof ferric sulphate. 9 . ShirlirstainA gives a reddish-browncolour. 1 0 .Alginate haemostaticfibres are invisible in polarized light with crossedNicols.
arest bleeding and form a protective dressingwhich may be left or later removedin a mannerappropriateto the type of dressingemployed (seeabove).Protectivefilms of calcium alginatemay also be usedby painting the injured surfacewith sodium alginate solution and then sprayingit with calcium chloride solution. Calcium alginatewool as a swab fbr pathologicalwork or bacterial examinationof such things as food processingequipmentand tableware hasthe greatadvantageover cotton wool in that it pennitsrelease of all the organismsby disintegrationand solution of the swab in, for example,Ringer's solutioncontainingsodiumhexametaphosphate' In fabrics the calcium alginatefibres wouid disintegratein alkaline solutions (laundering),but this advantageis turned to a commercial virtue by the use of the yarn as a scaffoldingthreadto suppofi yarns normally too fine to survive the weaving process.The scafToldis removedby an alkalinebath to leavea lightweight fabnc'
Cellulosewodding Celh.rlosewadding was official in the BP 1989. It is preparedfrom high-gradebleachedsulphitewood pulp which is receivedby the manufacturer in the form of boards about 0.75 m squareand I mm thick. These are packedin bales containingabout 180kg pulp. The pulp is put in a'beater', where it is mixed with about 20 times its weight of water and the mixture circulatesbetweena power-dliven roll and the 'beater'.The effect of this is to break up the pulp into bed-plateof the separatefibres.When this processis complete,the contentsof the beater aremixed with a further quantityof water and then allowed to run in 'wire' ol the paper machines.This 'wire' is a a steadyflow on to the very fine wire gauzethrough which water runs. leaving a fine web of 'wire'. This web is then dried and crdpedto give a fibres on top of the thin, soft,absorbentsheet.About 30 ofthese thin sheetsarelaid together to form cellulosewadding. When examinedmicroscopically,chemicalwood pLrlpsor cellulose waddingshow characteristicwoody elements.which, however.give no lignin reaction (distinction from mechanicalwood pulp) Tracheids with borderedpits and characteristicmedullary ray cells are usually observed.The cellulosenatureof the walls is shownby the blue colour' obtainedwith iodine followed by 80c/csulphuricacid and by their soiubility in an ammoniacalsolutionof copperoxide.
STARCHES Starch constitutesthe principal form of carbohydratereservein the green plant and is to be fbund especiallyin seedsand underground organs.The green parts of plants exposedto sllnlight contain small During granulesoftransitionalstalchwhich arisefrom photosynthesis. the hours of darknesstheseare removedto the storageorgans.Starch occurs in the lbrm of granules(starch grains) the shapeand size of which are characteristicof the speciesas is alsothe ratio of the content of the principal constituents,amyloseand amylopectin. A number of starchesare recognizedfor pharmaceuticaluse.They include maize (Zea mays L.). rice (Oryzo satird L.), wheal (Tt'iticunt aesti\tLrmL.) and potato (SolanttmtuberosumL.) (EP, BP). Maize' wheat and potato starchesare official in the USNF (1990).Tapiocaor cassavastarch(Manihot utilissinra)may be usedin placeof the above in tropical and subtropicalcountries. The more importantcomrnercialstarchesarelisted in Table2l.zl.
Uses. The alginate absorbablehaemostaticdressingsare non-toxlc and non-irritant.Thc-vhave advantagesover oxidized cellulose,which include selectiverate of absorption.sterilization(and resteriiization) by autoclavingor dry heat and compatibility with antibioticssuch as Preporotion of stqrches penicillin. They n'raybe used internally in neurosurgery,endauraland Commercialstarches,the preparationof which is describedbelow' are dental surgeryto be subsequentlyabsorbed.Externally,they may be not chemically pure and contain small amounts of nitrogenousand inorganicmatter. used(e.g.fbr burns or sitesfrom which skin graftshave beentaken)to
CARBOHYDRATES
Ioble 21.4 Fomily Cycodoceoe Gromineoe
Plont
Economic product
Zamia floridono Zeo moys /'\-,,-^ vr yzu
-^tt.,^ Juil Yu
Triticumoestivum Avenasotiva H^,4-,,^.^ Secolecereole Polmoe Metroxylon rumphii Musoceoe Musospp. Zingiberoceoe Zingiber officinale Curcurnospp. Connoceoe
Connoedulis
Polygonoceoe Polygonumfogopyrum Euphorbioceoe Monihotutilissimo Leguminosoe
considerableflexibility in drying time is requiredto irccornnrodate the variousmodilled starcheswhich are now produced.
€ommerciol slqrches.
Phaseolus vulgoris Ervumlens Pisumsotivum Convolvuloceoe lpomoeobototos Solonoceoe Solonumtuberosum
Floridoorrowroot Moizeor corn Rice Wheot Oots Borley Ry" Sogo Bononos ond plontoins Ginger EostIndionorrowrool ond turmeric Queenslond orrowroot or touslesmois Buckwheot Monihotor cossovo slorchond topioco Beonflour Lenlilflour Peoflour Sweetpoloto Pofoto
Many patentedprocessesare in use for particularstarches,and the procedureadopteddependson the degreeof purity desired and the natureof the compoundsfiom which the starchhas to be fieed. Cereal starches.for example,have to be freed from cell debris,oil, soluble protein matter and the abundantinsolubleproteins(glutelinsand prolamins)known as 'gluten'. Potatostarch,on the otherhand,is associated with vegetabletissue,mineral saltsand solubleproteins. Wheat and similar starcheswere at one time preparedby kneading the ground material in a stream of water, the gluten remaining rs a sticky mass. while the starch separatedon standingfrom the milky washings.The following methodsare now employed. Preparation of maize starch. The gr"ainis first sofiened by soaking at 50'C for about 2 days in a 0.2clcsolution of sulphurousacid. This assistsdisintegration,enablingthe embryo or genn to be easily liberated intact and permitting the starch to be readily freed from fibre. During this time lactic acid bacteriaare active and metabolizesoluble sugarsextractedfrom the maize.The grain, in water, is then distintegratedby attrition mills; thesedo not breakthe liberatedoil-containing embryos,which, in the oider process,were skimmed off. Nowadays the germs are continuouslyseparatedfrom the suspensionby liquid cyclones (hydroclones)which operatein batteriesof about 12. The germsare usedfor the preparationof germ oils. which arean important sourceof vitamins.The remainderof the grain is ground wet and the starchand gluten separatedfrom fibrous material in rotating, slightly inclined stainlesssteelreelscoveredwith perforatedmetal sheets.The retainedfibre is washed and the total mixture of starch and protein (mil1 starch)is fractionatedinto gluten and starchby the useof special starch pllrification centrifuges;separationdependson the fact that gluten is lighter than starch. In older processesthis separationwas accomplishedby repeated 'tabling', in which the suspensionwas allowed to flow very slowly through troughs about 40 m long and 0.7 m wide, when the heavier starch was depositedfirst. The starch suspensionfrom the centrifugeis lurther purified in other centrifuges and hydroclones.which reduces the protein level. The subsequent drying process may involve flash dryers or a moving-belt dryer:
Preparation of rice starch. Rice is soakedin successire quxntitie\ of 0.47ccausticsodauntil the materialcan be easilydisintegrated.The softenedgrain is ground (the compound grains separatinginto their components).made into a dilute suspensionwhich is repeatedly screened,and the starchseparatedby standingor by meansof a centrifuge. The damp starchis next cut into blocks and dried at 50-60"C fbr 2 days.The brown outerlayer which forms is then scrapedliom the blocks and drying is continued at a lower temperaturefor about 1;l days,during which time the blocks graduallycrack into iregular masses.For pharmaceuticalusethis 'crystal' starchis powdered. Preparation ofpotato starch. The potatoesarewashedand reducedto a fine pulp in a raspingmachineor in a disintegratorof the hammer-mill type. Much of the cell debris is removed fiom the pulp by roraly sieves andthe milky liquid which passesthroughthe sievecontainssrarch.soluble proteinsand salts,and somecell debris.On standing.the stalch separatesmore rapidiy than the other,insoluble.matterand in older processes was purified by techniquesresemblin,uthe 'tabling' describedabove: again high-speedcentrifugal separators.tbr use with potato starch (and cassavastarch),arenow employedfor separationand washing.At two or threepointsduringthe isolation.sulphurdioxideis addedto preventdiscolorationof theproductby the actionof oxidativeenzymes.The washed starchis collected,dried to contain about I 891rnoistureand packaged. Macroscopical characters. Starchoccursin irregular.angularmasses or as a white powder.It is insoluble in cold water but forms a colloidal solution on boiling with about l5 tirnesits weight of water, the solution fbrming a translucentjelly on coolin,s.A starch mucilage is coloureddeepblue with solutionof iodine. the colour disappearingon heatingto 93'C but reappearingon coolin-e.When starchesare heated with water, the granulesfirst swell and then undergo gelatinization. The temperaturesat which thesechangescomnrcnceand ale complete vary with difl-erentstarches.Pregelatinizcdllaize StarchBP. tJSPis employed as a tablet excipient. Starch granules also undergo gelatinizationwhen treatedwith causticpotash.concentratedsolutionsof calcium or zinc chlorides.or concenffatedsolutionof chloral hydrate. Maize starch is neutral. but other commcrcial starchesfiequently show an acid (wheat and potato) or alkaline (rice) reaction.The LtSP givesmicrobial limit testsfor Sulmonellctspp.and for E.schericJtia t:oli. Microscopical characters. Starchescan be identifiedby microscopical examination.They shouldbe mounted in r",ateror Smith's starchreagent (equalpafts of water,glycerin and 50% aceticacid). The size, shapeand structu.eofthe starchgranulesfrom any particularplant only vary within definitelimits. so that it is possibleto distinguishbetweenthe starches derivedfiom differentspecies.Starchgranulesmay be simple or compolrnd,andthe descriptionof a starchgranuleas2-, 3-, 4- or 5-compound refers to the number of component -qranulespresentin the compound granule.In some casesthe compound granuleis formed by the aggregation of a largenumberof sirnplegranules(e.g.rice andcardamoms). The startingpoint of formation of the granulein the amyloplastis markedby the hilum. which may be centralor eccentric.Granuleswith an eccentrichilum are usually longer than bload. On drying, fissures often appearin the granuleand are seento originatefrom the hilum. On microscopicalexamination,the hilum takesthe form of a roundeddot or of a simple,curvedor multiple cleft. The starchgranuleis built up by the depositionof successivelayers around the hilum, and concentricrings or striationsare often clearly visible in larger granules.e.g. potato. The striationsprobably arise
E
DRUGSOF BIOLOGICAL PHARMACOPOEIAT AND RELATED ORIGIN from the diurnal depositionof the starchgiving variationsin refiactive objectsmarkedby a dark cross.the barsof which intersectat the hilum. index. densityand crystallinity.The position andform of the hilum and The starch behavesIike a sphaero-crystal:knowledge of the actual structureof the granuleis much more limited than that of the structures the presenceor absenceof well-deflinedstriationsare of importancein of its components. the characterization of starches. Someof the more importantmicroscopiccharactersof the principal Chemicol compos:tion of storch starches aresetout in Table2 1.5andFigure21.4. Starch granules show double reflaction when examined between Starch granules usually contain two carbohydrates,arnylopectin crossedNicols. the granulesappearingin the dark lield as ill-rminated (a-amylose)and amylose(B-amylose)lthe former constitutesover 807cof
Ioble 21.5
Microscopy of storches. Size (ytm)
Voriety
Form
Moize
Gronulesfromtheouterhorny muller-shoped endosperm Gronuleslromthe innermeoly polyhedrol or endosperm subsphericol. In commerciol storcholl thegronules ore simple smollerones Lorgergronules lenticulor, globulor.A few compoundgronules which,if with2-4 components, seporoted, ore polyhedrol wilh on ongulor Compoundgronules outlineond lrom2 to oboutI 50 components. gronulespolyhedrol, with Component snorpongles Mostlysimplegronules,hotchei-,wedge-, A few compound or mussel-shoped. firmly gronules of 2 or 3 components fusedtogether mullerMosilysimple,subsphericol, shopedor roundpolyhedrol
Wheot
Rice
Pototo
Topioco
Hilum and striotions
5mo/l
Medium
Lorge
t0
15-25
30
10-30
35
30-40
45
Hilumo centrolpoint,seldomcleft.Concentric but rotherfointshiotions
4-6
t0
Hilumo centrolpoinf.No striotions
I 10
Hilumin lhe formof o poinf;eccenhicobout 1/3 to 1/4. Concentricsfriotions somerings,however,more well-morked; distinctihonofhers or cleft.Concentric skiqtions Hilumpunclote
2-9
2
5-',I0
45-65
20-35
Hilumo cenfroltriongulor or 2- to 5-stellote cleft.No striotions
-oo F5 -o9"?o" J ffil P i."offi
3"Si;*"'""""""ryi
ffi-ffif,t ffiwffi s,eX@o%b^opd ;65%:@\.cxqd @6ruK ffiffii^Pffi ffiRs$klH esffs"8ffiflg n&?:?cY'ole""; S66o.'3;
Fig.21.4 Allx20O Phormocopoeiol storches.
Rice
CARBOHYDRATES most starches.Fractionationof the two componentscan be achievedby selectiveprecipitationinvolving the fbmation of an insolublecomplex of amylosewith suchpolar organicsubstances asbutanolor thymol. As indicatedin Table2 1.2,B-amyloseconsistsessentiallyof linearchains;these have a helical anangementwith each tum comprising six glucosyl units and giving a diameterof 1.3 nm. Conversely,amylopectinhas a branched structure;these differencesgive the two substancesdi11'erent propefiies and it is their variation in proportion that contributestowardsthe distinctive characteristicsof a starchfrom a panicularbiological source. Amylose. althoughwater-soluble,gives an unstablesolution.which ireversibly precipitates.It is mainly responsiblefor the deepblue coloration(),nr^c.660nm) given by starchand iodine in which the latteras 15becomestrappedas an inclusioncomplex in the amylosehelix. The strongaffinity of amyloselbr iodine meansthat it will takeup to l9% of its weight of iodine and this iigure can be usedin the determinationof amylosein starch.Dilute solutionsin water or alkali have an appreciable vicosity and the moleculeis extensivelydegradedby B-amylaseto maltose.The courseof the hydrolytic reactionmay be followed: ( 1) by treatingwith iodine and observingthe colour changes(starchgiving a blue: dextrins purple to reddish-brown:maltose,and glucose.if acid hydrolysis,no colour);(2) by testingpoftionsat intervalswith Fehling's sollltion (the amountof reductionincreaseswith the amountsof sugar formed); or (3) by successivemeasrtrements of viscosity (viscosity decreases ashydrolysisproceeds).On the otherhand,solutionsof amylopectin are relatively stable,the colour given with iodine is purple ()"-u*c.5zl0nm), and the iodine-bindingis low. B-Amylasecan only attack the outer linear chains,not being able to bypassthe l-6 interchain links; as a consequence, amylopectinis hydrolysedto the extent ol 50-60Vconly by the enzyme: completehydrolysis is achievedby mineralacidsand otherenzymes. A very small amountof covalentlybound phosphateappearsto be a nonnal componentof starch;its exact location within the moleculeis uncertainbut may representthe phosphorylationof some I in 300 giucose molecules.Cerealstarchesalso contain about 17cof lioid which occupiesthe samehelix spaceas doesaddediodine. Biosynthesisofstarch. The linal stagesofthe synthesisofstarch are associatedwith amyloplasts-double membrane organelles which develop,like chloroplasts.from protoplasts.Sucroseappearsto be the primary substratewhich by the mediationof the reversiblesucrosesynthaseand other enzymesis convertedto fructose,glucose-l-phosphate and glucose-6-phosphate in the cytosol.The precisepathwayinvoived and the specificsubstratewhich passesinto the amyloplastfor the final stagesof synthesisare a cuffent areaof study.One problemis the difficulty of isolatingintact amyloplastsfor biochemicalstudy.a-Amylase activity in barley has beenextensivelystudied;the endospermof germinating maize seedscontainsfbur isozymesof d-amylases(a-amylase-l to -,1) and one isozyme of B-amylase(K. B. Subbaraoet ol.. Phytochemistry,1998, 49,651). The probablefinal reactionsare indi-
catedin Fig. 21.5.(For a review with 8l refs.seeA. M. Smith and K. Denyer.N ew Phttol ctgi st, 1992.122, 21.) Mutant varieties. A number of mutant vuieties of maize and other cropsproduceabnormalstarchgrainssomeof which havecommcrcialuse and possibilities.Thus 'waxy' maize starchcontainsprincipall-vamylopectinproducinga tapioca-likestarch.It derivesits namefiom the shiny appearance of the brokenendosperm.Another mutant, 'amyloseextender' is deficient in one of the enzymesresponsiblefor producingthe branching of the amylopectinmolecule.At least six specificenzymedeficiencies have beenidentified asassociatedwith abnormalrnaizestarchmutants. Uses. Starch linds extensiveuse in dusting powders. in which its absorbentpropertiesareimportant.In mucilageform it is usedas a skin emollient, as a basis fbr sorre enemasand as an antidotein the treatment of iodine poisoning.Starchis also used as a tablet disintegrant. For the US tbod and drinks industrylargequantitiesof maizestarchare convertedto high-fructosecom syrup by a processinvolving hydrolysis (glucoseproducing)and subsequentisomerization.Starchhas also provided the plasticsindustry with a numberof new products.including biodegradablepolyvinylchlorideand polyethyleneplastics. SterilizableMaiTe Starch BP is used as a lubricant for surgeons' gloves;it is maize starchsubjectedto physicaland chemicaltreatments so that it doesnot gelatinizeon exposureto moistureof steamsterilization. Unlike talc, it is compietelyabsorbedby body tissues. Brazilian arrowroot. This is the starchobtainedfrom the tubersof the sweetpoIalo,Ipomoeabatatos(Convolvulaceae). The granulesare rounded,polyhedralor muller-shaped,the largeronesbeing 25-55 prm in diameter. Portfand arrowroot, The English hedgerow plant, ArLun ntaculcrtunt (Araceae)is known by a numberof names,includingLords andLadies. Cuckoo Pint and Wake Robin. The tuberousrootstockis rich in starch and was formerly extractedto give Portlandanowroot. Usedfbr starching Elizabethanruffs. it was a causeof dermatitisamon-qlaundrvmaids.
Modified stsrches As with cellulose,the starchmoleculecan be considerablymodified by chemicaltreatmentsand someof the productshave a use in the paper food. textile,adhesiveand other industries.Treatmentsincludeacetylation, hydroxyethylation,phosphorylation,inor-eanicesteliflcationand cross-linking.For pharmaceutical purposesmarantitstarch(St. Vincent Arrowroot). which is no longer commelcially available. has been replacedby an esterstarchofcassavawhich has similar propefties. Soluble starch. Soluble starch is plepareclby treating commercial potato starch with hydrochloric acid until. aftel' washing. it forms a limpid. almost clear solution in hot watcr. A soluble starch solution
*o3tt',":Tl*" cvo."r GtucoseI -phosphatc firucose-6phosphate (E,C5.1.2.2',)
tI
F i g .2 1 . 5 Proboble finolstoges in the biosynthesis of storch.
,fmfbnfasr I I
I
L
I ADP-tlucosephosphorylase(EC 2.7.7.27) I
I ADP-glucose
I
Strrdr synhase(EC 2.4.l.2l ) (threeisoformsknorn) I I I Branchingenzyme(EC 2.4.1.1t) I Nccessry for lhe fcmation o( amylopectin(peeembryo coldns two fotms yhich rre enco&d on different genes) I I Starch
LRIGIN P H A R M A C O P O E IA N L D R E L A T EDDR U G SO F B I O L O G I C AO should show little reductionwith Fehling's solution and gives a deep blue colour with iodine.On heatingwith -5cZpotassiumhydroxidesolution, it gives a canary-yellowcolour: no colour is aflbrdedby ordinary starchand the dextrinsgive a blorvn colour when similarly treated.
renderthe moleculesmuch lessrigid than either thoseof celluloseor starch.In any sampleof inulin thereis a mixture of molecularspecies the smallermoleculesbeing probably intermediatesin the polymerizing chain.
Commercial dextrins. High-gradedextrinsare preparedby heating starchwhich has bccn moistenedwith a small qlrantityof dilute nitric I acid and dried.at ll0-115"C. The productis known as white dextrin. lnferior dextrins.r"'hichhaver yellow or brown colour,arepreparedby roastingstarchat I 50-250"C without the additionof acid. White dcxtrins may containup to l5% of solublestarch,the rentainlargelyof erythrodextrin.Yellow dextrinsaremore comder consistin,g pletell' h1'drolysedand. unlike the white variety. contain appreciable BP testsinclude a thin-layer chromatographyexanination; clarity. quantitresof rnaltose.which may be detectedand estimatedby means colour and specilic optical rotation (-36.5'to -40.5'. 2% solution)of solution. of Fchlin-s's solutionsand limits for acidity. sulphatedash (0. l%). heavy metals, oxalateetc. Inulin is not metabolizedby the body and is excretedunchanged.As FRUCTANS Inulin lnjection it is usedfor the measurementof glomerularfiltration Fructansare o-fructosepolymerseachchain being terminatedby a sin- rate. gle o-glucosyl residue.They are lbund in nature as oligosaccharides with up to 50units. The Dondelion root with up to l0units and as polysacchalides pharmaceutically,is The root of the dandelion(Thra-racumofficinale) is an importantdrug the most important fiuctan. and best-known inulin, a reservecarbohydratefbund in many roots of membersof the ofherbal medicine.Anrong other constituentsit containsup to zl0%of Compositaeand Campanulaceae.The tubers of the Jer"usalemarti- carbohydrates,particularlyinulin, in the autumn and about 2c/ctnulin choke(Helianthustuberosus)andloots ol chicory (Ciclnrinrn in'.',bLts) in the spring. The fiuctose contentreachesabout 18c/cin the spring. The drug is desclibedin Chapter'30. are particularly rich sources.Other fiuctans are the phleins fbund in grassese.g. rn Phleumpralen.te,agropyrenein couch grass(q.v.).and sinistrina componentof Urgineo ntaritima (q.v.). ALGATGELTINGAGENTS Unlike the biosynthesisof starchand cellulose.that of fructan does units but identical monosaccharide of by the conjugation not originate in all casesstartswith a molecule of sucrose(glucose+ ftuctose) to The two most important phannaceuticalproductsin this classare the A furalginatesand agar. which is successivelyaddedfurthermoleculesof furanofi'uctose. ther distinction fi'om starch biosynthesisis that no monosaccharide nucleotide(cf. glucoseadeninediphosphate)is involved in the addition ATGINICACID of the fiuctose units. Various trisaccharidescontposedof one glucose and two fructosemoleculesoccur in natureand the modeof the linkage Large quantitiesof brown seaweedsare collectedfi'om many of the ofthe secondfiuctosyl unit to the sucloseandthe extentofthe addition colderwatersof the world. Principalproducers,approximatelyin order of quantity.are the USA (Califomia), Norway, Chile, China, Canada of more fiuctosyl units to the tlisaccharidedeterminethe propertiesof the final polymer.Thus. inulin biosynthesisploceedsvia the enzymat- (Nova Scotia), Irish Republic, Australia (Tasmania),Iceland. UK (Scotland), South Afi"ica. Some years ago it was repofied that the ic tlansf-erof a fructosyl group from sucroseto anothermolecule of sucrosegiving the trisaccharide1-ketoseand free glucose.A second Chinesehad developedstrainsof brown seaweedwhich would flourish in the warmerEastand SouthChina Seas. then mediatesthe addition of enzyme (a fructan fi'uctosyltransf'erase) The North Atlantic rockweeds (littoral types) (e.g. Ascophyllum further fiuctosyl units fiom other oligomeric fiuctans.Thus, the linal moleculeis terlninatedat one end by a glucosylunit. However.fol the nodosLun)are cut either by hand with sickles or by meansof various 'combineharvesters'.The remainingworld total is trisacchar- designsof floating fblrnationof someother fructansdifterentfructosylsucrose ides are involved and elongationof the polymer chain may occur at mainly storm-cast.After collection.the raw, wet seaweedmay either either end of the trisaccharideso that the final fructan moleculehas a go immediately fbr processingor be fuel- or sun-dried to 12-11% moisturecontent.in which fbrm it has an indefinitestoragelife. glLrcosylresiduesituatedtowardsthe middle of the chain. Alginic acid, a hard, horny polysaccharide,was first isolatedby the English chemist Stanfbrd(1883.)and in Britain was fit'st marketedin INUTIN 1910; the 1976 estimatedworld production of alginate was 19 507 Inulin BP is obtainedfiorn the tubersof Duhliu variobilis. Helianthus tonnes.New methodsof extractionare continually being patentedbut the pattern of Stanlbrd's processis still much fbliowed. The dried tLrberosLts and other generaof the Compositae;it derivesits namefiom in the 19th milled seaweedis maceratedwith dilute sodiumcarbonatesolutionand it was first isolated from which Inulu helenitutr. the dahlia, century.It occurseither in solution in the cel1sap (cf. starchgranules the resultingpastymassdilutedwith sufficientsofi waterto make pracmaterialas sphae- ticable the separation of the insoluble matter by modern superwhich areformed in plastids)or in alcohol-preserved masses(Fi-e.43.1.6). It is sparinglysolublein cold water decantersor continuous-settlingdevices. Soft water is essentialto rocrystalline but readily dissolvesat around70'C without gelatinizing.It is neither avoid the precipitation of insoluble alginates.The resulting clear liquor, which containsmost of the alginate originally presentin the stainedby iodine solutionnol hydrolysedby mammalianenzymes. Chemicallyinulin consistsof a chainof 35-50 I.2-linkedfiuctofura- algae,may now be treatedin one of two ways: (1) it is poured into noseunits tenninatedby one glucoseunit. The furanosering systems dilute sulphuric acid or dilute calcium chloride solution. when the
I
I
CARBOHYDRATES insolublealginic acid or its salt.calciumalginate,is precipitated as a bu1ky.heavily hydratedgel. from which liquor is rentoveclby roller- or expeller-presses. The product obtained looks and handleslike wood pulp.By moving the calciurnalginatewith constanlagitatronagrinstrt stream of hydrochloric acid. the calcium is removed and the highly swollenpulp of alginic acid is roller-pressedand then neutlalizedwith sodium carbonateto give sodir:malginate.(2) The clear liquor can be made to precipitatesodium alginateof high purity by the addition of ethyl alcoholdirectly or after partial evaporation. Alginic acid (folmula, Table 21.2) is composedof residuesof D-mannuronicand t,-gr.rluronicacids: the chain len-qthis long and varies(mol wt. fiom 35 000 to 1.5x 106)with the methodof isolation and the sourceofthe algae.The degreeofpolymerizationcan be varied to meetthe propertiesrequired.Note the ollicial requirementsconcerning microbial contamination. Alginic acid is insolublein cold water (but swells and absorbsmany times its own weight) and slightly solublein hot water.It is insolublein most or-9anicsolvents.It liberatescarbon dioxide from carbonates. With compoundscontaining ions of alkali metals, or amrnonium or magnesium.it reactsto give salts (alginates)which are water-soluble and fbrm viscoussolutionstypical ofhydrophilic colloids.The saltsof most other metalsare water-insoluble. The alginates,particularlythe sodiumsalt,have,becauseof their -ereater chemicalreactivity.cerlain advantagesover agar.starch,pectin,vegetable gums amdgelatin.Alginatesfind applicationsas stabilizing.thickening. emulsifying.dellocculating.gelling andfilm- and filamentfbrming agents in the rubber, paint, textile, dental.fbod (including ice-cream),cosnetic and pharmaceuticalindustries.The fbrmulation of creams. ointments, jellies andtabletsareexamplesin thelast-named pastes, industry.Alginic acid is alsousedin tabletand liquid preparationsfbr the control of gastrooesophagealreflux. Alginate textile fibres and their uses.fbr example,as absorbablehaemostaticdressinsshavebeendiscussedeallier.
AGAR Agar (JapaneseIsirtglttss)is the dried colloidal concentrateliom a decoction of various red algae, particularly species of Gelidiunt. Ptercclatlia (both Celidaceae, order Gelidiales), and Grut:ilttria (Gracilariaceae.order Cigartinales).Agar is obtained h'om Japan (Geliditun amansii), Korea. South Aflica, both Atlantic and Pacific Coastsof the USA, Chile. Spain and Portugal.Some 6500 tonnesale produced annually.of which about one-third originatesliorn Japan. The genusGelidium plovides abor-rt 357cof the total sourcematerial. Collection and preparation. On the Japanesecoast the algae are largely cultivated in special areas.poles bein-eplanted in the sea to ibrm supportson which they develop.Flom time to time the poles are withdrawn and the algae strippedotT. Some ilre also collected front small boatsby meansof rakesor shovels,or evenby diving. The algae
Fig. 2 | .6 Diotomsossocioied withJoponese (diometer ogor.A, Arochnoidiscus i 3 0 p m ) ;B , s p e c i eosf Grommotophoro; C, Cocconeis;D, Compyloneis; E, oiherdioloms.
are takcn ashore and dried: beatenand shakento l'ento\c sancland shclls:bleachedby wateringandexposureto sunlight.theu asl.rin-q also servingto remove salt.They are then boiled with acidr.rlated \\iltcr 1bf severalhout's(aboutI palt ofdry algaeto 55 or 60 partstll'\\ater).and the muciiaginous decoctior-r tiltered,while hot. throughlincn.On cooling. a jelly is prodr.rced which is cut into bars.thesebein-ral'ter*ards forced through wire netting to fbrn'r strips. The ntanuf-acturc ol agar takes placc only in winter (November to Febrr.rar)).and moistr.rreis removed by successivelylreezin-q.thawing and drying at about 35oC. In Japanthe algaearecollectedfrom May to October. Characters. AgiLroccursin two lbrms: ( I ) bundlesof sorlrewhatagglutinated,tlanslucent,yellowish-whitestrips,thesebeingaboutthethickne-.sof leaf gelatin,,1mm wide andabout60 cr-nlong: (2) coarsepowdel ol f'lakes. Ag:Lrswellsin cold $'aterbut only a small fractiondissolves.A 17r solution may be made by boiting and a stiff jelly sepalatesliom this on cooling. WhenJapanese agaris not used,jelliesof similarstiffnessmay be obtained by using0.7% New Zealand,17cSouthAfiican or 2% Austraiianagar. A nearly boiling 0.27csolutiongives no precipitatewith an aqueous solutionof tarrnicacid (distinctionfiom gelatin).Agar also differs from gelatin in that it contains no nitrogen and therefore gives no amutonia whenheatedwith sodalime.Whenhydroll'sedbi' boiling with diluteacid. galactoseand sulphateions areproduced,the former reducingFehling's solution and the latter precipitatin-ewith bariurr-rchloride. If agaris ashed end the residue,after treatrrent with dilutc hydrochloric acid, examined microscopically. the silicaskeletorrs of diatomsand spongespicLrles will be found. More perf'ectdiatomscan often be isolatedby centrifirginga 0.57csolution.The laryediscoiddiatomAmclutoidis<:u,i. which is about 0.1-{.3 rnm in diarneter,speciesof Grunmatophora and Cocconeis,ltnd (seeFig. spongcspiculesarereadilydiscemiblein theashofJapanese a-ear 2 1.6).Powderedagaris distinguishedfrom powderedacaciaand tragacanthby giving a deepcrimsonto brown coloul with 0.05 r,riodineandby stainingpink whenmountedin a solutionof ruthenir.rm red. Agar BP is leqLriredto comply with tests fbr the absence of Esclterit:hiacoll arrdSalnonella. and genelalmicrobial contamination shouldnot exceeda levelof 103microorganisms per g-l asdetermined by a platecolrnt.It hasa swelling index (q.v.) of not lessthan I 0 and the determinedvalue must be quotedon the prodllct label. Constituents, Agar has long been known to yield on hydrolysis n- and l-galactoseand sulphateions. It is now known to be a heterogeneous polysaccharidethe two principal constituentsof which ale agaroseand agaropectin.Agaroseis a neutralgalactosepolyrner (free tiom sulphate)which is principally responsiblelbl the gel strengthof agar. It consistsof alternateresidr"res of 3.6-anhydro-L-galactose and -D--qalactose (the disacchalideknown as agarobiose).The structureof agaropectin,responsiblefol the viscosityof agar solutions.is lesswell established, but it appearsto be a sulphorrated polysaccharidein which galactoseand uronic acid units arepartly esterifiedwith sulphuricacid.
'm.ffi ffi //
li tt
\\-
\llE
V W
DRUGSOF BIOLOGICAL ORIGIN PHARMACOPOEIAL AND RELATED Pure agaroseis commerciallyavailableand its gels are lecommended fbr the electrophoresis of, fbr exanrple,proteins. Uses, Agar is usedin the preparationof culturemedia.as an emulsifying agentand in the treatmentofchronic constipation.Both agarand agarosefind extensiveuse in al'l'initychrornatography(q.v.).
1992,31,531). The drug Marsr.rhiro and C. C. Urzua, Phytochemistry^. is rich in halogensalts,and, accordingto Schulzen(1964) the extract di1I'ersf}om that of agar in that it has a higher sulphateand ash content. Uses. Chondrusis usedas an emulsifying agentfor cod-liver oil and other oils. as a gelling agent,and as a bindel in toothpastes.Its many technicalusesinvolve mainlv the food industrv.
lrish Moss 'l.'. 'ill.,r,] i.r.r'r.r:I]I' . . ..a. is obtainedfrom the variablered alga Chondrus Chondrus(Curnt,qeert) crus7rrr.r and to somc cxtcnt tiom Gigartina stellatu (Gigartinaceae). Comrnercialsupplicsarc delived frorn the north and north-westcoast Gums and mucilageshavesimilar constitutionsand on hydrolysisyield of Ireland. tiorr Brittany and from the Massachusettscoast south of a mixture of sugarsand uronic acids.Gums areconsideredto be pathoBoston. logical products formed upon injury of the plant or owing to Collection and preparation. The algae grow on rocks just below unfavourableconditions,suchasdrought.by a breakdownof cell walls lou -u ater mark, being coveredby about5 or 7 n of water at high tide. (extracellularlbrmation; gummosis).Conversely,mucilagesaregenerIn Irelandcollectiontakesplaceduring the autumn:in America,during ally normal productsof rnetabolismlbrmed within the cell (intracelluthe summer.The collectorsput out in smallboatsat abouthalf+ide and. 1ar formation) and may representstorage material. a water-storage after detachinga load of algaefrorn the rocks by meansof long lakes. reservoiror a protectionfor germinatingseeds.They areoften found in returnwith them at half-flood.Canageenis bleachedby spreadingit on quantity in the epidermalcells of leaves.e.g. senna,in seedcoats(linit tbr someweeksto the actionof sun and dew seed,psyllium etc.),roots (marshmallow)and barks (slipperyelm). the shoreand submittir-rg with aboLrtfour or five soakings in seawaterat suitable intervals. Bleachingby sulphurdioxide has not proved particularlysatisfactory. TRAGACANTH After drying in sheds,the drug is packedin balesweighingup to 300 kg. 'the air-hardened gummy exudate, World productionof Irish mossis estimatedto be around20 000 tonnes. The EP/BP define Tragacanth as flowing naturally or obtainedby incision,from the trunk and branches Carageenan USP/NF (1995) is obtained trom red seaweedsby extractionwith water or aqueousalkali and recoveredby alcoholicpre- oI Astrag,alusgummffbr Labillardibre and certain other species of Astragalus from WesternAsia'. The genus (Leguminosae)contains cipitation.drLrmdrying or by fieezing. some 2000 speciesand thosethat yield gum are chiefly thorny shrubs found in the mountainousdistrictsof Anatolia, Syria.Iraq, Iran and the Characters. Chondrusr,vhenfiesh variesin colour from putplish-redto and former USSR. So-called Persian tragacanthhas been traditionally purplish-brown, but thebleacheddrugis yellowish-white.translucent employedin the UK, with Anatolian tragacanthfinding a considerable homy. It consistsof cornplete,dichotomouslybranchedthalli about5-15 cm long and of very variableform, somethalli having broad fan-like seg- marketon the continentof Europe.The term Persiantragacanthis used ments,others having ribbon-like ones.Many sampiesof Chondruscon- by pharmaciststo denotethe better gradesof tragacanthproducedin I r a na n dT u l k i s hK u r d i s t a r . . tain large quantitiesof the related alga Gigartina ntatttillostt,the mixture being officially sanctionedin many pharmacopoeias.ln some districts (e.g. south of Boston) almost pure Clnntlrus crl,lpls may be collected, Formation. The mode of formation of tragacanthis entirely different while in others(e.g.nofth of Boston)it is ahnostinvariablycloselyasso- liom that of acacia,the gum exuding immediately after injury and thereciatedwith G. mamilbsa. Thesealgaemay be distinguishedfl"om one fbre being preformedin the plant, whereasacaciais slowly producedafter anotherby the lbnn of their large compound cystocarps.which contain injury. A sectionof a tragacanthstem showsthat the cell walls of the pith caryospoles.Chondrushas oval cystocarpsabout2 mm long which are and medullary rays are graduallytransformedinto gum, the changebeing 'gummosis'. The gum absorbswater and a considerablepressure termed sunk in the tha1lus,while G. mtunillosa has peg-like ones about 2-5 mm is set up within the stem. Hanbury, having cut off branchesof living long. asalsohasG. pistilkttu.The latterspeciesis rareroundthe coastof from the centrea streamof soft. plants,stated,'thereimmediately exr,rdes Britain.andits presencewould indicatea drug of Frenchorigin. matter which efl'ersolid tragacanth,pushing itself out like a worm. to the length of threeChondrusis sometimescoveredwith calcareous vesceswith hydrochioric acid. The drug has a slight odour, and a quartersof an inch,sometimesin the courseof half an hour'. mucilaginousand salinetaste. Chondrusswells in cold water, about4'7c/cslowly dissolving.while Botanical sources, The requirementfor precisebotanicalspecificaon boiling aboutl5% passesinto solution.A 57c decoctionforms a tions and satisfactoryanalyticalproceduresfor tragacanth,necessitated jelly on cooling.Acooled 0.3% solutiongivesno precipitate with solu- by the legal aspectscoveringits useas a food additive,hasrenderedthe tion of tannic acid (distinctionfrom gelatin).and gives no blue colour above BP definition somewhatinadequate.A survey of the Turkish gum-producingspecieshas indicatedtharA. microcephalusis the prinwith iodine (distinctionfrom lcelandmossand absenceof starch). cipal speciesernployedwith smaller amountsof A. gutnmfer and A. Constituents. The constituentsof chondrusresemblethose of agar. kurdicusbeingcollected.Also, in an investigationof tragacanthproducAt least five galactans,known as carrageenans.are present.Three tion in Iran in 1957,Gentry (.Et'on.B ot., I 957, ll. 40) repoftedA. echidrmi s, A. gossyp inus andA. microcep hal us ro be importantspecies. important ones are r-, t- and )"-carageenans,which differ in the nae;t'o they containand in the numberand The approximatedistributionof a number of gum-producingspecies amountof 3,6-anhydro-o-galactose position of the ester sulphate groups. Like other members of the lbund in the areaswheretragacanthis collectedis shownin Table21.6. in the nvo GigartinaceaeC. crisltus producesdi1I'erentcarrageenans predominatesin the gametophyte Collection. Most of the plants fiom which tragacanthis collected phasesof its lit-ecycle; K-carrageenan generation grow at an altitudeof 1000 3000 m. The shrubsare very thorny; each generationand )..-carrageenan in the diploid tetrasporophyte (fbr researchon the heterogeneityof both types of plants see B. of their compoundleaveshas a stout,shalplypointedrachiswhich per-
GUMSAND MUCITNGI5,.,,,
CARBOHYDRATES
Distribution of sum-vieldinsAstrosolus $:5"?]'t Species
Geogrop hicoI d i strib ution
A. gummifer A. kurdicus A. brochycolyx A. eriosfylus A. verus A- leioclados A. echidnaeformis A. gossypinus A. microcepholus {syn.A pycnocladusl A. odscendens A. strobiliferus A. heratensis
Anotolioond Syrio Northernlroq, Turkeyond Syrro Westernond S.W.lron S.W.lron Westernlron Westernond centrollron lsfohonregionof lron lsfohonregionof lron Shirozond Kermonregionsof lron, Turkey Southwesternond southern rron Eostern lron Khoroson to Afghoniston
sistsafter the iall of the leatlets.The mode of collection variessornewhat in difl'erentdistricts.but the lbllowing detailsof collectionin the provinceofFars are typical. Gum can be obtainedfiom the plants in theil first year but is then said to be of poor quality and Llnfitfbr commercialuse.The plantsare thereforetappedin the secondyear.The earth is taken away from the baseto a depth of 5 cm and the exposedpart is incised with a sharp knife having a thin cuttingedge.A wedge-shaped pieceof wood is used by the collector to force open the incision so that the gum will exude more tieely. The wedge is generallyleft in the cut lbr some l2-21h befbre being withdrawn.The gum exudesand is collected2 days after the incision. Some of the plants are burned at the top alter having had the incision made.The plant then sickensand gives ofTa greaterquantity of gum. However,this practiceis not universal.as many plantscannot recover their strength and are killed by the burning. The gum obtainedaftel burningis ol lower quality than that obrainedby incision only. and is reddishand dirty looking. The crop becomesavailablein August-September. Grades. Tragacanthis gradedinto severalqualitiesby the exporter or middle man. The bestgradesform the of1icialdrug, while the lower gradesare used in the food, textile and other industries;their approximaterelativevaluesare listed in Table2 t.7.
Tcble 21.7 Grode
Grades of trogq(onfh. '
Ribbon No. I No. 2 No. 3 No. 4 No. 5 No. 26 No. 27 No. 28 No. 55
Description
Fineflotdruggists' ribbon White flotdruggists' ribbon Lightcreom curlyribbon Mid-creom flotribbon Pinkish mixedriooon Mid-creom thinfloke Amberthickfloke Amber-brown thickfloke Reddish-brown mixed hoggy floke
Tragacanth is an expensive commodity;not only hasrhesr-rpplr situation increasedthe price.but alsothe extratreatmcntand tcstsreqr,rired to meet the EP/BP microbial requirementshave addedto thc cost. Characters. The official Persiantr"agacanth occursin flattenedribbonsup to 25 mnt long and 12 mni wide. The surfaceshowsa nurnber ofridges which indicatesthe successive,temporarystoppagesof tlow fiom the incision. Fine furows parallel to the margin of the 1'lakeare produccdby the unevenedgesof the incision.The gum is white or ver-y pale yellowish-whitein colour.translucentand horny. It breakswith a sholt fracture.is odor:rlessand has little taste. Tragacanthswells into ii gelatinousrrasswhen placedin water.but only a smallportiondissolves.On theadditionof a dilutesolutionof iodineto a fragmentpreviouslysoakedin water.relatively few blue points arevisible (distinction from Smyma tragacanth.which containsmore starch).With strongeriodinesolutionthegum acquiresa greenishcolour(cf.'Agar' ). Constituents. Tragacanthconsists of a water-solublefraction known as tragacanthinand a water-insolublefiaction known as bassorin; they have molecularweights of the older of 8210000. Both are insoluble in alcohol. Tragacanthinand bassorinmay be separatedby ordinaryfiltration of an extremelydilute mucilageand the tragacanthin may be estimatedby the evaporationof an aliquot portion of the filtrate.Testsshow that the bestgradesof gum containthe leasttragacanthin. If the tragacanthincontentand moisturecontent are known. the amountof bassorinmay be calculated. Like othergums,tragacanthis composedof sugaland uronic acid units. Among the productsof hydrolysis galacturonicacid. o-galactopyranose. r--arabinotulanose and o-xylopyranosehavebeenidentified.The BP thinlayer chromatographytest for identification is basedon the products of acid hydrolysis of the gum. Von Fellenberg( I 9 | 8) pointed out thar in rhe tragacanthinfraction there are nclmethoxyl groups.but that the bassorin fractioncontainedabout 5.387cmethoxyl.Rowson (1937) showedthat gums with high methoxylcontentsand high bassorincontentsgave the most viscousmucilages.Heating or fine powderingproducesdemethylation with loss of viscosity.Andelson and Bridgen-ran(Phytochenistn, i985. 24. 2301) showedthat the gum-exudatesof the three principal speciesof Turkish gum-producersare proteinaceouspolysaccharidesand representa protein contentof about3-47c. involving 18 arrino acids.The relativeamino-acidproportionsdift-eredin the thu'eegums. The BP includesa test fbr minimunt apparentviscositl,,of the powderedgum, a flow tinte for gum to be usedin the preparationof emulsions, and a microbial limit test. Peroxidaseenz)'mes are usually consideredto be absent,but their presencehas been dctectedin commercialsamplesof genuinedrug. The presenceof peroxidaseenzymes appearsto be relatedto a high starchcontcnt: both disappearas gummosisproceeds.
Relotive volue
i00 93 B2 62 4l 3B 34 29 26
Theribbongrodesore stotedto complywiththe BPrequirements; it is principolly thefloke126onddown)fromTurkeythotis ovoiloblein quontity.
Uses. Tragacanthis used in pharn-rac1' as a suspendingagent fbr insolublepowders,etc..or as a bindin-gagentin pills and tablets.As substitutes becomeavailable,its usein thc food industrvis declinins.
Allied drugs Nortphttrmaceuticalgrades o.f tnLgncartlt.Large quantitiesof tragacanth of the lowergrades are imported and usedin the textile industry and pickle manufacture.The piecesvary in shapeand are tiom a 1,e1low ivory colour to almostblack. The lower gradesare much contaminated with ealth, and their ashesgive a strong reactionlbr iron. The viscosity of mucilagespreparedfiom thesegradesol tragacanthfalls rapidly fiom the No. I to the No. 55, the nrarkeddifferencein price being fully justified. The lower gradesof tragacanthare known as ftog gum or hog trugucarth.
@
PHARMACOPOE AD LR E L A T E AIN DD R U GO S FB I O L O G I C O AT RIGIN Chitral gum of Indian commerce is said to be obtained fl'orn Astragulus strrh i li fer u.s. Stercttliugunt (Kunnu,qrun) is itsell'animportantarticleof commerceandis described gum is detectbclou. Its prcscncein tragacanth ed by the gel fbrrrationin alcoholicsolutionandby an aciditytest. InsolubleShinq grrntis a gr,urol doubtlul botanicalorigin imported fiom lran. When of good cpalitr'.it resemblcsa ntixtureof bleachedand natural Kordotirn acacia.It mar, bc distinguishedtiont tragacanthby tl-re factthatit containsno starchandthatit givesa rcactionfor oxiclase enzvme.
Uses. The granulargradesare usedas a bulk laxative.being second only to psylliumseedin usein this respect.The powderedgum is used in lozenges.pastesand dentr-rre tixative powders,arrdit hasprovedparticularly uscful as an adhesivefbr stomaappliances.As a bulk laxative anclstimulantit is available.with fransula.as granules.
ACACIAGUM
Acacia(GranA rubic) is a driedgr-rmobtainedfrom the stemandbranches of Acacla .senegulWild. and of sone other speciesof Actrticr (Leguminosae). A. senegalis a treeabout6 m high,which is abundantin STERCUTIA GUM the Sudan.pafiicularll,in the provinceof Kordofarr.in centralAfiica and Stercrrfia (Kunrya Gum. Itulitur Trugot:untlt,Buslora Tragut:unth')is in West Atiica. The trec is known in Koldofan as Httshub and \n the dried gunrnry exudate obtained fiom the tree Sterculia urens Seneganrbia as Verek.The bestgum is that producedin Koldofirn liorn (Stcrculiaceae). It is producedin India.Pakistanand.to a snrallextent. tappedtrees.bLrtsome Senegaland Nigerian gum is of good quality. in Airica. The guln is of relativelyrecentintroduction.bein-egenelally Apart fi'om 'acaciagardens'.wild, self'-sownplantsare the main source rc_galded durir-rgthe early part of the last century as an adulterantand of the gur-n. inl'eriorsubstitulefor tlagacanth.Now. howevcr.havingbeenshownto be superiorto othel'gumsin eertuinrespeets. it constitutes an integral History. Gum was brought from the Gulf of Aden to E-eyptin the part of the gum industryand is oflicial in the BP. seventeenth century BC. and in the works ofTheophrastLrs it is spoken of trsa productof Upper Egypt. The WestAliican productwas importCollection and preparation. In central and northem lndia (Andhra ed by the Poltugr.rese in the fifieenth century.Until quite recently.conrPradesh.MadhyaPradesh.Rajasthanand Uttar Pradesh)two collections merce in the Sudanwas in the handsof a number of local merchants. are madeeachyear.befbreand alter the monsoonseason:in April-June but it is rrowentirelycontl'olledby the 'Gum Alabic CompanyLtd', a andin Septenrber. rcspectively. The lirst collectiongivesa gum atTording concessionalcompany set Llp b)' the Sr.rdanese Government. This thehighestviscosity.Blazes,up to aboutone squarefbot in area.arenrade Companyaloneproclucesabout;10000 tonnesper annum. in the larger trees(smallertreesare tapped)and the gr.rmimrnediately startsto exude;the tlow is greatestduringthe first 24 h and continuesfbr Collection and preparation. Sornegunrexudesfiom treesas a result severaldays.The dried,inegularmasses.weighingup to severalpounds. of thc crackingof the bark, but the most esteemed.Kordotan variety.is arepicked ofTand transportedto villagc ccntresfbr pulchaseby Bombay obtainedfrom trees.about6 yearsold. tappedin FebruaryandMarch, or merchants.The Indian merchantsrelnoveexcessbark and roughly sort earlier.in Septemberafier the rains. when the leavesfirll. The tapper. the gum into two grades;it is fiuther -gradedin Europe and the USA l",ith a blow liorn a srnallaxe. makesa transverseincision in a branch accordingto colourandpresence of foreignorganicmatter'(mainlybark). and so twists the axe that the balk is loosened.strips of it bein-ethen It is tinally sold as a granulated(crirstal).or fir.rell'powdered,product. pulledoff aboveandbelow the cut.The portion of branchsobaredto the Unfbrlunately,in some areasover-productionand destructivetapping cambiummeaslrres aboLrt0.5- I .0 m long and 5 7.5 cm wide. This cammethodshave led to a seriousdeclinein the naturaltree populationand bium producesnew phlocm and in about 20-30 days the tearsof gum havenecessitated the introductionof l0-yearbanson collection.This has which have fbrmed on the surfncemay be picked off. The gum is colstimulatedresearchon ir lllro propagationusingseedlingexplants.nodal lected in leatherbags and is conveyedin sacksto El Obeid and other (S. D. Purohitand A. Dave. Plant centres.r-nostlylocatcdalong the railway. Here the gLrmis garbledto explantsand somaticembryogenesis 1996.15.704:V G. Sunnichanet ul., ibid.. 1998.17.95 l). Cell Reporr.s. fiee it fiorn sandand vegetabledebris.and is sorted.Other acaciagums suchas talka -gurn.the productobtainedfiomA. ^rerzrl(the talka of the Characters. Good-qualitygum occu$ in irregularalmostcolourless, Arabs).are also separated. At one time someof the gum was 'ripened' translucent.striatedmassesweighing up to 25 g or more. Mediurn grades by erposuleto the sun,when it becamebleachedand dried.developing have a nrarkedpinkish tinge. while the lower gradesare very dark and numerollsclacks.Duling this process.rvhichtook 3-4 months,the gum containa considel'able amountof balk. Karayagum hasa markedodourof lost about 30% of its weight. During the last few yearsthis bleached aceticacid.andwhenhydrolysedwith 5% phosphoricacid.hasa volatile gum hasbecomeunobtainable. aciclity(BP) of not lessthan llc/a (tragacanth, about2-3%). Accordingto From El Obeid the drug is serrtby rail to Port Sudan,whencelarge JanotandGounard( I 938).sterculiahasa methoxylvalueof0 (tragacanth quantitiesare shippedto London. the USA and other countries.In the 30--+0).Wren boiledwith solutionof potash.it becomessiightlybrown- London Market Reportsthreegradesare usuallyquoted.namelyhandish (tra_sacanth. canary yellow). Karaya also difl-erstr"orr tragacanth.in picked selected(h.p.s.)Koldofan, cieanedKordofan and talka. Gum thatit contairrsno starchandstainspink with solutionof rutheniumred. alabicis soldin onecurrencyonly ($US).sothatcunencychangesalso In \\'ater.sterculia-qumhas low solubilitybut swellsto many tirrres afl'ect the price. The Senegal acacia -eum is largely used for its ori-cinalvolurnc.This meansthat the processingof the gum influ- pharmaceuticalpurposeson the Continentand is shippedto Marseilles encesthc linal product-the coarser-eranulated give a discon- and Bordeaux.This alsooccursin threegrades.namely 'gornmedu bas -Qrades tinuous grainv dispersiou,wheleas the fine powder afTordsan du fleuve'. 'gommedu hautdu fleuve'and'gommefriable'. apprrentlh l o r n o g c n e od t ri:: p e r r i o n . Spray-dried acacia, produced by the importers. is becorning of increasingimportanceand is included as a monographin the BP. In Constituents. Partialacid hydrolysisof sterculiayields o-galactose. addition to its genelalusefulness.it has tl-refurther advantageof a lou' L-r"hamnose, o-galactr.rronic acid. aldobiouronicacids. an acid trisac- viablebacterialcount(seebelow). charideand aceticacid: the galacturonicacid and rhamnoseunits are the branchingpoints within the molecule.Uronic acid residuesrepre- Characters. Bleached Kordofan acacia.when available.occurs in sentabout377cof the gum ror.rnded or ovoid tearsup to about 3 cm diameter.or in angularftag-
CARBOHYDRATES ments.The oLltersllrfacebealsnumerousfine crackswhich lbrrr dur- Hairl' root cultures, These prodr.rcea mucila-re rvith a dill'erent 'ripening' ing the and make the tearsopaque.The gurn is white or verv polysaccharide compositionto thatofthe parentplant. pale yellow in colour'.The tearsbreak rapidly rvith a somewhatglassy fracture.and much ofthc drug cousistsof srnallpieces.It is odourless Uses, As a generalstabilizerin emulsionsand as ii pharntaccutical a n dh a : a h l r r n du r r rnl t u c i l u g i n o ut r: s t e . necessityin lozenges.etc.Its dcmulcentpropertiesarc employeclin r,arCleanedand h.p.s.Kordolan gum difters fiom the above in htrvirrg ious cough.diarrhoeaandthroatprepalationsbut it is incompatiblc\\,ith f'ewercrackswhich causesit to be more transparent.and in being r-r-rore readily oxidizeclrraterialssuch as phenols.and the vitamin A of codyellowish or pinkish in colour. The tears are usuall.vof less unifbrm liver oil. It haswiclespread use in the food. drinks and otherindustlics. size. some bein,equite small. while othershave a diameterof '1 cm or morc. The better clualities of Senegal gurr-rclosely resemble the Allied drugs. Talkugunt is LrsLrally much brokenand of very variable Kordofan.but someo1'thetearsare vermilbrm in shapeand. speaking composition.someof thete:u'sbeingalmostcoloullessandothersbrown. the gun'ris rathermore yellowish in cololrr. GhtLtti or Indian gurrt is derived frorr Arlr-rgels sus latfolict -qenerally. (Combretaceae). It is producedin ntuchthe samelocaiitiesas sterculia Tests. Acacia is almost completelysolLrblein an equal weight ol gum. and is harvestedand preparedin a sin-rilarmanner.It resembles water.solutiontaking placerathel slowly. Tl.resolutionis slightly acid talka in possessingtears ol various colours. Sorne of the tears are and becomesmore so on keeping. especiallyif hot water is uscd to vernilbrm in shapeand their surlaceshowsf'ervercracksthan eventhe makethe solr.rtion. It is viscid. but not glairy, and when diluted doesnot naturalacacia.Aqueousdispersions of the gurnhavea r iscositf interdepositon standing. mediatebetweenthoseof acacil and sterculiagums. A 107caqueoussolntionis laevolotatory(BP requirement).gives n
This gum is producedartiticirtll by the pure cultute f'ermentationof the bactedumXtuttlton'Lortus t:urnpe.stris on glucose.It, like cellulose, consistsof l.'1-B-glycosidicalty linked chainsof glucosewith, additionally. trisaccharideside-chainson altematinganhydro-elucose units. Thescside-chains are comDosed of two mannoseunits which encom-
P H A R M A C O P O E IA N L D R E L A T EDDR U G SO F B I O T O G I C AOL R I G I N passa glucuronicacid unit. Pyruvategl'oupsare attachedto most of the terrrinal units with acetylgloupsat the C-6 of a numberof the mannose moietiesnext to the glucosechain. The EPIBP ass'ayis basedon the amountof the pyrur"icacid prodncedb1"the acid hydrolysiso1'thegum andshor.rld corespondto a contentol + 1.57c. Othertestsincludea limit fbr 2-propanol(750 ppm asdeterminedby gas chromatography).lbreign polysaccharides, a limit of 103bacteria and 102fungi per -eram.ancla total-ashrangeof 6.5-16.0%consistent with xanthangurn occullin-eas the sodillm.potassiumor calcium salt. Xanthangurr is usedas a pharmaceutical aid. and alsofinds use in t h e f o o dr r r Ji , ' . r r r e l i ' :i n d u s t r i e s .
Seedsof P ma.jor arereportedly used as a medicinal substitutefor P ot'uta;Iheseedoil containsan unusualhydroxyolefinicacid.For a review of the traditionaluses,chemicalconstitutents and biologicalactivitiesof theP.nnior plantseeA. B. Sal'ruelsen, 71. 1. "/.Ethnophann.,2000. Uses. Plantagoseedsare usedas dernulcentsand in the treatmentof chronic constipation.Ispaghulahusk is used for similar purposesbut has a higher swelling lactor (zl0-90).
MARSHMATTOWROOT
Marshmallowroot is derived from Altlnea rffichalis (Malvaceae),a pelennialherbwhich is fbundwild in moistsituationsin southemEngland and Europe. In generalappearanceit closely resemblesthe common holIyhock, Althuea rosea.Theplant has a woody rootstockfrom which :Lrise nulnerousroots Lrpto 30 cm in length.The drug is chiefly collectedon the Continentfrom cultivatedplants at least2 yearsold. The roots are dug up in the auturnn,scrapedfiee from cork and dried, either entire or after slicPSYTUUM ing. The EP/BP drng may be peeledor unpeeled,whole or sliced. (Fleo Seed) The dug occursin whitish,fibrouspiecesaboLrt15-20 cm long and l-2 The dried. r'ipeseedsof Pluttngo rqfiu (.P.pstlliuntl, P. indita(.P. are- cm in diameter,or in small transverseslices.Odour. slight; taste.sweetish naria) and P. ovatu (Plantaginaceae) are used in medicine. The US and mucilaginous.A transversesectionshowsa bark about 1-2 mm thick Ntrtiortttl Forntulury includes all three species under the name which is separatedby a greyish.sinuatecambium from the white. radiate 'Plantago Seed'. The BP desclibesthe seedsofthe first two species wood.The sectionshowsnumerollsmucilagecells.the contentsof which r.rnder the title 'Psyllium'and the hllsks of seedsof P. ovata are includ- are coloureda deepyellow by a solution ofsodium hydroxide. Structures 'Ispaghula ed under Husk'. The lattel consistsof the epidermisand col- to be seen in the powdered drug include: variously thickened vessels; lapsedadjacentlayersremovedfrorn the ripe seeds. fibres; calcium oxalateas clustercrystals20-25-30 35 pm in diameter: The seedsof P. afra andP.indictLareknown in commerceas Spanish starchgranules,mainlysingle.3-25 prmin size;thin-walledcork cells. or French psyllium, while thoseof P. oviltt are known as blond psylMiLrshmallowroot containsabor.rt10% of mucilage,the amountbeing lium. ispaghula.spogelseedsor Indian planta-ee seeds. season-dependent: it containsa polysaccharide givingon hydrolysisgalactose,rhamnose.galacturonicacid and glucuronic acid. Other components Characters. Some of the more inportant charactersof theseseeds of the mucilageare glucansand an arabinan.The mucilagecontentof the aregivenin Table21.8. drug is indicatedby the swelling index which, fbr BP purposesshouldbe not lessthan 10.The upperlimit fbr the total ashof the peeledroot is 6.04/c Constituents. All the seedscontairrmucilagein the epidermisof the andthat fcrrthe unpeeled8.07r.Starch.pectin and sugars,and about27aof testa. The seeds may be evaluated by measuring the volume of asparagineare also plesent. The latter. which is the amide of asparlic mucilageproducedafter shakingthe seedswith water and allowing to (amino-succinic)acid. is alsotbund in asparagus. potatoes,liquorice,etc. stand(seeswelLinginder.Chapter l4). Marshmallowroot, and alsothe leaves,are usedas demulcents.parTwo fiactionshavebeenseparatedfiom the mucilage;one is soluble ticularly fbr irritablecoughsand throatand gastricinflammation. in cold water,and the other in hot water giving a highly viscoussolution which gels on cooling. On hydrolysisfiactionsyield o-xylose, COUCHGRASSRHIZOME t--arabinoseand aldobir.u'orric acid. The seedsalso contain fixed oi1. aucubinglycoside,variousbases,sugars.sterolsandprotein.The aucu- Couch GrassRhizome EPlBP. BI1P consistsof the washedand dried bin contentdiff-ersappreciablyin different seedsamplesand species. rhizomes of Agropyron repens (Ehnus repens),family Gramineae. sliced or whole with most of the adventitious roots and leaves Alfied drugs. Wild seedsof P. ot,rttt and relatedspeciesare reportedto removed. containlessmucilagethan the cultivatedvariety.P usitrtittt.a species The plant. althoughan invasiveand troublesomeweed (twitch), has uscd in Chinesernedicine,containsmucilagethe backbonechain of a long history of nredicinaluse: it was known to Dioscoridesand has r''hich is cornposedof B-1.4-linkedo-xylopyranoseresidueshavingthree subsequentlybeen included in many herbalsand pharmacopoeias. In kindsof branches. Britain it was last included in the BP 1914 but was. in 1999, Dextran, This is anothermicrobialproduct,producedby speciesof Leucottostt.tt.Klebsiella. Acetobttcter and Streptocot-cls.It is an ctl. 6--rlr-rcan and is usedas a replacementlbr blood plasmaand as an absorbentin biochemicalanalvsis.
Tqble 2I.8
Chorqcterr oI Psyllium seeds.
Origin LOtOUT
Shope Length Weightof 100 seeds Swellingindex
r. otrd
r. tndtco
P. ovoto
Fronce,Spoin,Cubo Glossy;deep brown BooFshoped; ouiline elongoiedovole 2 . 0 - 3 . 0m m 0 . 0 9 - 0 .1 0 g dl0
MediterroneonEurope,Egypi Dull;blockish-brown Boot-shoped; outlineellipticol 2 . 0 - 3 . 0m m 0 . 12 - 0 . 14 g dl0
Pokiston, Indio Dull;pinkishgrey-brown Boot-shoped; outlineovole ,l.5-3.5 mm 0 . 15 - 0 . 19 s d9; husk,<40
CARBOHYDRATES returned as a result of its EP status.SE Europe (Hungary) supplies most of the commercialmaterial.
gel.ThLlsanthraquinones havebeenreportedas constituents ol the gel but their presencemay,or may not be, due to someadmirture rr ith the aloeticjuice fiom the pericyclic region of the leaf. Also. r'ariarionsin Characters. The dried, rigid rhizome is pale yellow. shiny.2-3 mm ctrrbohydratecompositionarise due to varietal ditl'elenceslr'ithin the thick andusuallycut into piecesup to 6 mm in length.It is stronglyfurspeciesand to seasonal.climatic and soil factors. rowed longitudinally and, except at the nodes,hollow. The drug is Glucolnannans constitutea principalcomponentofthe gel; sonieconodourlesswith a slightlv sweettaste.Featuresof the powder include: sist of gh"rcose and mannoseonly or glucose,mannoseand glr-rcuronic epidermalcells of two types-one. naffow elongatedwith wavy and acid,othersareacetylated. Otherpolysaccharides aregalactogalactr.rans pitted, lignified walls-the second, alternating with the first type. (galactose + galacturonic acid) and galactoglucoarabinomannans. somewhatrounded and unlignified; endodermalcells with U-shaped Molecularweightsrangefiorn 200 000-450 000.An acetylatedmannan thickenings;tibres: pitted vesselswith annular or spiral thickenings, is availablecommerciallyand has a rangeof reportedbiologicalactivipitted parenchyma.Calcillm oxalateand starchare absent. ties. Glycoproteinliactions of the gel which may also influence the immune systemhavebeenshorvn1ohai,eprolif'eration-promoting rctivConstituents. The rhizornescontain about l0% mucilageand up to ity on human and hamstercells lrr lilro (A. Yagi et ul.. Planta Medir:a. 87c of the fructosanpolysaccharidetriticin. Unlike the inulins (B 2-1 1997,63, 18: 2000,66, 180).In a seriesof papersN. Okamulaar r1. Iinkages)the fl'uctosansof the Grarnineae,termedlevans,arefructofu- (Ph1'toclrcnistry. 1996,43.495r 1997.45. 1511;1998,49, 219) have ranose units linked by B,2-6 glycosidic linkages and, as with the reportedon the isolationof somc clcvcn new chromones.Pectic subinulins. terminatedwith a sucroseunit. Other constituentsare2-3Vcof stances.the triterpenoidlupeol, plant sterols(cholesterol.campestrol. sugaralcohols(mannitol,inositol),0.01-0.05%volatileoil containing ftsitosterol),possibleprostanoidsand otherolsanic tlnd inorganiccomthe polyacetylene agropyrene and other volatiles, vanillin and its poundshavealsobeenidentifiedin the gel. monoglucoside,phenoliccarboxylicacidsand silicates. The intenseinterestin aloe vera productsboth tiom the commercial and scientificviewpointshas made this a topic for very active research.For an extensivereview updateon the subject(over 300 \\ //-\.n2-u:u-L,:\,-\,n3 refs) see T. Reynolds and A. C. Dweck. J. Etlurophonnucology,, \__/ 1 9 9 9 ,6 8 , 3 . Agropyrene Gels from other speciesof Aloe. e.g.A. arboresceri.r. A. .feror.have alsobeenexaminedlbr their polysaccharidecon'rpositiun. Tests. The EPIBP specify a rvater-soh-rble extractiveof 425Va and tbreign matter consistingof greyish-blackpiecesof rhizome 4157c. Quince seeds Tests.dependingon the presenceof starchand thickeningof the endo- Quince seedsare obtained from Cydonictoblonga (Rosacele)"a tree dennic cells are given to detect C),nodondactylon (Bermuda grass), cultivated in South Afi'ica, Central Europe and the Middlc East. h'an fbund on sandysoils in warmertemperateregionsof the world includ- suppliesabout757cofthe total world production. ing the shoresof SW England,andIntperatacylindrictt,a troublesome The seedsare separatedfrom the apple- or pear-shapedfiuits and weed of subtropicalregions. dried. They resembleapple pips and frequently adhere togethel in masses,owing to their surfacecoating of dlied mucilage.The latter is Uses. Couchgrassis usedas a demulcentand as a diureticfor bladder derived liom the outer epiderntisof the testa. Quince seedscontain u n dk i d n e yc o m p l a i n t .c. ) s t i t i \ .g ( ) u te. t c . about 207c of mucilage,conrposedol units of alabinose.xylose and uronic acid derivatives.I 5% of lixed oi1and a small qLrantityof cyano'Aloe vero' products genetic glycoside and an enzyme which efI'ectsits hydrolysis. The Aloe vera productsarederivedfrom the mucilagelocatedin the p:Lrenchy- seedsare used as a demulcent.as an emulsiff ing agent and in the matouscellsof theAbe veraleaf andshouldnot be confusedwith aloes. preparationof hair-fixing lotions. describedin Chapler22, which originatefrom the aioeticjuice ofthe pericyclic region. The mucilaginousgel has been used tiom early times fbr Slipperyelm bork the treatmentof numerousconditions but in recent years its use in the Slippery elm bark is obtainedfrom Ulmus rurDruMr-rhlenberg (.Ulntus herbal and cosmeticindustrieshas becomevery big businessin the USA, fi1r,a Michaux) (Ulmaceae),a tree l5 20 m in height $'hich is widely Europeand elsewhere.Raw materialsareobtainedfrom plantationsin the distributedin the USA and in Canada.In the sprin_efairly old bark is southem statesof the USA, SouthAmerica and elsewhere.Exaggerated strippedfrom the trees.The outerpart of the bark is then removed.only claims have inevitably brought scepticismaboutthe true usetulnessof the the inner part.which forms the commercialdrug.bein,edlied. Aftel this productswhich featureas suntanlotions,tonics and food additives. has been sawn into convenientlengths.it is bound into bundleswith Researchover the last 10 yearshas,however.largely upheld a number wlre. of the therapeuticpropertiesascribedto the gel. These include the antiThe drug occursin broad.f'lat stripsabout-50-100crn in length and inflammatory properties (wound healing, burn healing. fiostbite). gas- lrom 1 to 4 mm in thickness.A feu, reddish-brownpatches of the trointestinal activity (peptic ulcer), antidiabetic activity. anticancer imperfectly removed rhytidome may be sccn but the remainderconactivity (principally animal tests).antibacterialactivity and radiobiologi- sistsonly of secondaryphloem. The outer surtaceis brownish-yellow cal protection.Not all theseproperlieshavebeenunequivocallyaccepted. and striated.the inner sr"rrtace 1'ellorvish-whiteand finely ridged. The The complex chemistry of the gel makesthe attribution of the various bark is easilyidentifiedby the charactelistic,f'enugreek-like odour.the activitiesto specificcompoundsdifficult. Indeedbeneficialclinical results stronglyfibrousfractureand the lact that it yieldsmucilagewhen moisfor a particularcondition may arisefrom more than one component.Thus tened. wound healing benefits may derive frorn the anti-inflatrrnatory fibroThe chief constituentof the bark is mucilage.which is a mixture of blast-stimulating,antibacterial:rndhydrophilic propertiesof the gel. two or more polyuronides.The mucilagehasdemulcent,ernollientand Someof the conflictingresultsof testsmay be due,in somemeasure. nutlitive properties.A poLrlticeof the powdered bark is sometimes to the differentmethodsof collectingand subsequentprocessingof the used.
r
LR I G I N P H A R M A C O P O E IA N L D R E L A T EDDR U G SO F B I O L O G I C AO medicinql plonts Other muciloge'contoining the dliecl leavesof Tussilugo.farftu'tr' oi consists 1983 Coltsfoot BHP It is used(and also the llouers) as a helbal cxpectorant Compositae. andcontainsup to l07r mucilageri hich on hydrolysisyicldsa number of sugarsand ttronicacids.Thc leart-salsocontaintanninand a small percentageof pyrrolizidine alkirloids.e.g. scnkirkine.which ctrnprove hepatotoxicin sr.rlficientcloses.Probablyfbr this reasonthe herb is no longel inclr.rdcclin reccnt editions of the BI1P; the German CommissionE sctslintits lbr the daily doseof thesecompounds'as occurringin coltsloot.Ftrrthcrinfolmation on the drug u'ill be tbund in a reviewb1 NI. Berrr'(Plttrntt.J ., 1996,256,234) For other drugs containing appreciablemucila-{e see fenugt'eek nncllinsced. seccls
ILANEOUSCARBOHYDRATE. MISCE CONTAININGDRUGS HONEY PURIFIED
Uses. Hor-reyis chiefly uscdin phalmacyas a componentof linctuses and cough mixturesand for Oxyn-reland Squill Oxyn'rel.
FIGS Fig BP is the sun-driedsttcculentfruits of Ficr'r'rcaric'ttL' (Moraceae)' It is r','iclelyproduced in the Mediterraneancountries, particularll Turkey (Smyrnafigs). Greeceand Spain. The fruit is producedby the union of the c-vmoseinflorescenceto form a hoilow, fleshy axis bearingthe flowers on its inner surlace The young fruit is rich in latex,but when mature.no latex is firr'rndand the fleshy axis contains much sugar.Figs contain about 50% of sugars (chiefly glucose).appreciablequantitiesof vitaminsA and C. smaller amountsof vitamins B and D. and enzymes(protease.lipaseand diastase).Thereis an official requirementfor a water-solubleextractiveof not less than 60.0%. Figs are used by the BP and BPC in laxative (e.g.CompoundFig Elixir). preparations
FUCUS
''\l'rr. Fucusor bladderwrackconsistsof the driedthallusof Frrtrrsl'('si(-lrl( admits BP/EP The Fucaceae. tamily Honey is a saccharinesubstancedepositedby the hive bee, Apismellif- F. serratusor Ascoph.t'llwttnodctsrorl. is restrictedto F. vesicultr etr (order Hymenoptera,Apidae), and other speciesof Apls, in thc all thlee specieswhereasthe BHP rnonograph a whitishcoating'is having sometimes thallus, grey to black the chief dried but sas. The England. in cells of the honeycomb.Honey is produced mucilaginous' to become in water sofiens readily parts but brittle various harc'l and Chile . Califblnia. Indies. West the sourcesol supply are air vesiclesmay.or may not bepresent' Dependingon the species. of Africa. Australiaand New Zealand. Bladderwrack contains mucilaginous polysaccharidessuch as Puritled Honey BP is preparedby melting honey at a moderatetemand standing. acid. tbr the extractionof which it may be used(q.v.).It is also on alginic collcct which perature.skimming otT any impurities I at 20oC' elementsand iodine.The latter is in the form of inorganic in trace rich g ml of 1.35-1.36 weight to a water clilutingwith salts.bound to protein.and asa constituentof iodo-aminoacidssuchas polyphenols consisting of Collection and preparation. The worker bees'by meansof a long' di-iodotyrosine. Other constituents ale lipids. and complex sterols the units, fiom phloroglucinol nectar take labiurn. hollow tube formed fiom the maxillaeand Note the ofllcial lirnit testsfor heavy metiils and arsenic,the high total flowers they visit and passit through the oesophagusinto the honeyashvalue (> 21.0%)(mainly solublein dilute hydrochloric acid) and loss sacor crop' The nectar''which consistslargell' of suclose'is n-rixedwith the swelling index ({ 6.0) and salivary secretioncontaining the enzyme invertaseand u'hile in the on drying (* | 5.0%). Other standardsare (sodium titration)' (0.03 thiosulphate 0.2%) bee content at the hive the iodine total On arrival honey-sacis hydrolysedinto invert sugar. has, in the past, been and propenies has thyroactive prevla in Bladderwrack them deposits and the honey-sac of brings back the contents with a more preparations other employedin iodine therapyl however ousll preprretluell of the honcycotnb from its arising preferred. Again now are content heather' iodine consistent The besthoney is that derivedfiom f-lowelssuchasclover and as a slimming in teas promoted been has bladderwrack cut fiom the content, iodine and separated obtainedfrom hives that havenever swarmed. agent.In suitabledosesit servesas a dietary supplementfor traceelecornbeitherby drainingor by meansof a centrifuge.Honey obtainedby with the wax.The nectarof certain mentsand as a bulk laxative. expressionis liableto be contatninated may give the honey a flowers (e.g.of speciesof Eucabptusor Bcrnksia) Dutunt slt.tt]lottilult, from nectar Odourand taste; somewlratUnpleasant CETRARIA poisonous honey' give to known spp. are ragwofis andRhododendl?/r is a foliaCetraliaor Iceland moss,Cetruria islandica(Parmeliaceae). Siberia Europe, grass in central and moss growing amidst Characters. Honey, when tieshly prepared,is a c1ear,syrLrpyliquid ceouslichen central of slopes mountain lower the on and to crysAmerica. it tends North and keeping. of a pale yellow or reddish-browncolour.On collected in it is usually purposes medicinal For Spain. depend and and taste Europe odour The granular. and opaque tallize lnd become l u to P e . S c r n d i n a v iaan de e n t r r E vcrv lar'-telyon the llowers usedin its preparation The BP/EP drug consistsof irregularly lobed, leafy thalli' about 5-10 crn long and about 0.5 mm thick. The upper surfaceis greenishConstituents. Honey consistsmainly of invert sugar and water' It oii, volatile anclsometimescovered with reddish points, while the lower brown acid, fbrmic dextrin, of sucrose. quantities containssmall markedwith white. irreguwax and pollcn grains.Micloscopical examinationof the latter atlbrd surfaceis pale brown or greyish-greenand margin of the thallus are the along intervals frequent At The most likely adulterantsale artiti- lar spots. valuableer idcnceof the sour.ce. but becomescartilaginous is brittle drug The dried tot' plojections. The tests glucose. rninute liquid cial invcrt sLlgar.sucroseand commercial mucilaginousand bitter' taste, slightl Odour. water. with tor The limit tests moistening on be noted. should purified honey purity of the BP is stainedblue by which cooling. chloride and sulphateare important, as starch and sucrosemay he A 5clcdecoction tbms a ielly on swelling value of has an offlcial It (distinction carrageen). fi'om hydrolysedwith acidsto give commercialliquid glucoseand artilicial iodine drug reveal the of the drug. Sections powdered on the furfiLral, determinecl contains 4.5 sugar invert sugar. respectively.Artificial invert alga unicellular cells ofthe rounded plant. sn-rall the but this ofthe acid, nature clual hydrochlolic in resorcinol r.r'ith red colour which gives a closely or less rlore the by hunticolctbeing enclosed C1'stococ'cLts may be formed in genuinehoney b.vprolongedheatingor lengthystorwoven hyphaeof the tungus. age.
CARBOHYDRATES The drug containscarbohydrates, known as licheninand isolichenin. Licheninis only solublein hot waterand is nor colouredblue by iodine, while isolicheninis solublein cold water and gives a blue colour with iodine. Both on hydrolysisgive glucose.Cetrariaalso containsa very bitter depsidone,cetraricacid, and other acidssuch as lichestearicand usnic acids.The latter compound is an antibiotic which may not be unimportantin contributingto the efficacyof Icelandmossas a demulcentfbr the treatmentof cough involving throatirritation.Icelandmoss has beenusedas a bitter tonic and for disguisingthe tasteof nauseous medicines.
Prunes Prunes(Rosaceae)are dried plums derived from prunus domestir:u L. Of the many cultivated forms, yar. Juliana de Candolle, which is
largely -urown in France. produces those commonll, uscd in Europeanmedicine.Prunesof excellentquality are alsoproducedin Calilbrnia. The appearance ofprunes requiresno description.The pulp contains about4'1%of suglrs (rnainly glucose),malic acid and water.The kernel contains-1591of flxed oil. and small quantitiesof amygclalinancl benzoicacid. Prunesareusedin Confectionof Senna. Further reoding Weymouth-Wilson A C I 997Theroleof carbohydrates in biologically active naturalproducls. NalurrlProductReports1.1(2): 99-l l0 Whistler R L, BeMillerJ N (edsr I 991Indusrriai gums:polysaccharides and theirderivatives. 3rdedn.Acadentic Press. London, UK WhistlerR L. BeMillcrJ N 1997Carbohvdrate chcr.nistry tbr foodscientists. EaganPress. StPaul.Minncsota
.,
ait.
Phenols probabli, constitute the lalgest gloup of plant secondarr rnetabolites.Widespreadin Nature.and to be for-urdin most classcso1' ruaturalcor.r'rpourrds having aromaticrnoieties.they lange tiorn simplc structureswith one aronatic ring to highly complexpolymericsubstancessuchastanninsand lignins.Phenolsareimpoltantconstituents of some medicinal plants and in the fbod industr-vthey are utilized as colouringagents.flavourings.aromatizelsand antioxidants.This chapter mainlydealswith thosephenolicclasses of pharmaceutical interest. (2) tannins.(3) coumalins namely:(l) simple phenoliccompouncls, (.1)anlhraquinones (-5)naphandtheil gll,cosides, ancitheirglycctsides, (6) flavoncand lciatcdflavonoidglycosides.(7) anthothoquinones. (8) lignansand lignin. The biosynthetic cyanidinsand anthocyanins, origin of someof thesecompoundsinvolr,ingthe shikimicacid pathr v a v i s s h o n ' ni n F i g . 2 2 . l . P h e n o l sr n a y a l s o h a v e a r o m r t i er i n g s (Fi-q.19.9.). derivedby acetateconderrsation
Phenols ond phenolic Alycosides
SIMPLEPHENOLICCOMPOUNDS Catechol (o-dihydroxybenzene) occurs ftee in kola seedsancl in thc leaveso1'Carrltheriaspp.and its deri'n'ativcs arc the urushiolphenolso1' the poison oak ancl poison ivv (q.v.). Dcrivativcs of rcsorcinol (rn constitutcthc nalcoticprinciplcsofcannabisandthc dihydroxybenzene) glucosidearbr"rtin involr,csquinol (hl,droquinone.p-dihydroxybenzene). The taenicidalconstitucntsof malef'ern.the bitterprinciplesof hopsancl thc lipophilic components ol hypelicurn (q.r'.) are phloro-elr.rcinol derivatives.
ol{
OH I a/Y-" I
OH
I
d'
<\
tl
\,/
cooH
HO,,,vOH Phloroglucinol
Catechol
Salicylic acid
frt' CH I
OH
9H' I
n \4ou" OH Eugenol
SIMPTE PHENOLIC COMPOUNDS 2 1 4
I (\
Y
OH Hydroquinone
TANNINS 221 COUMARINSAND GTYCOSIDES2 2 7 ANTHRAGIUINONES AND GTYCOSIDES229 NAPHTHOQUINONES AND GTYCOSIDES244 AND XANIHONES CHROMONES
245
The phenoliccompoundsin this group ofien also possessalcoholic. they,include aldehydicandcarboxylicacicl-9ror.rps: eugenol(a phenolic phenylplopanel.r'anillin (a pl.rer.rolic aldehyde)and various phenolic acids,snchas salicylic.f'erulicand cafl'cicacids.Glycosidefbrmationis common. and the widcly distlibuted glycosidc conif'erin and other derivatives ofphenoliccinnamicalcoholsaleprecursors ollignin. Sor-ne of the bestknown sirnplephenolicglycosicles arelistedin Table22.L
FTAVONOID FLAVONEAND RETATED GTYCOSTDES246 AND GTYCOSIDES2so ANTHOCYANIDINS STILBENES252 TIGNANSAND TIGNIN 252
MALEFERN Male f'ern(Filir Mus) of the BPC (1988)consistsof the rhizome.tiond basesandapicalbud of l)n'opreris.filix-mus(Polypodiaceae). Thc taxonomy of the genusis complicated:Drropteris.fili-r-rna.r aggresateis conrposedof a complexof threerelatedspecies-D. f'ili.r-nuts(L.) Schott.s. str.. D. borrei Nervm. and D. ctbbreviata(Larrr ancl D.-C.) Newm.
PHENOLS AND PHENOLIC GLYCOSIDES
OH
Ho-'Av,oH Dehydtoshikimicacid -
ll
|
'Y
'--'-
tannins
| Shikimic acid
Hydrolysable
COOH Callic acid
I Chorismicacid -
Somcnaphthoquinones and anthraquinones
I
Prephenicacid Somebcnzoquinones-
_ p-Hydroxyphcnylpyruvicacid
Phenylpyruvicacidr'
andnaphthoquinones I
I
Phenyllacticacid
pHydroxyphcnyllactic acid
l
z
lrans-Cinnamicacid
c
o
u
r
n
r
i
n
s
-
\
r
2
"'
ro-(-\>cH=cH-cooH
F
Ho
,/
/
i
unitof flavonoids ----ce-Q Xanthones
\ HO{
Condensedtannrns
,/
\FCH=CH-COOH
\--
E/
p-Counraricacid (p-Hydroxycinnamicacid)
Jome Denzoqurnones and naphthoquinones
,o4cH=cH-cHzoH \:./
Lignin of Monocotyledons
p-Coumarylalcohol caffeic acid
I Hol
CH=CH-Cl.laOH-
F"r=.r-cooH
Y
MeO
Coniferyl alcohol
Fcrulicacid
,o/\* 5-Hydroxyferulic acid
\
\
J
u"J
Ho-!
MeO
V"r="*-cooH
Sinapicacid
LigninofDicotYledons
./
,/
MeO
\_./
\ f
Vanillin
MeO \-
Ugninof Gymnosperms Lignans (e.g. Podophyllotoxi n,
CH=CH-CHzOH
Sinapylalcohol
tig.22.l P h e n o cl ioc m p o u nodrsi g i n o t i fnrgo ms h i k i moi cc i d( s e e F i g .l 9 . Bf o rd e t o i losf s h i k i moi cc i dp o t h w o y )
Hybridizationreadilv occuls andD. .fili.r-ntasitself. with 16,1chromosonres.is an allotetraploidr.i'ithhalf its chromosomcsderivedliom D. ultbreviutttand the othersfiom an unkrrownsource.Twelve varietiesof D. lnrreri.have beendcsclibed.Mlle f-ernis du-eup in the late aLrtumn. divestedof most of its roots and deadportiollsLrnddried. It must have retairredits greencolourintemally.Althoughcommonin Blitain it is collectedin largequantitiesin the Harz andThuringianmountarns.
History. The vermifuge properties of I'elns were known to the ancicnts.their use beins rnentionedin the works of Dioskurides, Theophrastus. Galenand Plinl'.After lapsinginto disuse,r-nalef-ernwas reintroduced by a Flenchphvsician.Jobert.in I 869. Characters. The drug occursin piecesabout7-25 cm in length.consisting of a rhizorre abor.rt2 cm diametersurounded by fiond bases
LR I G I N P H A R M A C O P O E IA N L D R E L A T EDDR U G SO F B I O L O G I C AO rl ()r 5 crn Sonreof charactcristictwo-celledmar,qinalprojections(Fig. 22 2;'The interwhich bring the total diametelof the piecesto about 'Ihe fiond basesare nal glandular hairs have been studied by electron mieroscopy and the larger pieces are sliced to tacilitate drying' compoundsdescribedbelow' they are shownto biosynthesizelhe antheln-rinthic internally brown externallyand denselycoveredwith ramenta: rnelipale ellou to nine six fiom 1 green.and show in transversesection male f'ernare phlorogluciun.filix-foemirid).Tlie rhizomeis bt'own- Constituents. The active constituentsof steles(distinctionfrom A//rr,r.l bi-, tri-and tetracyclic commono-. as occur which nol clerivatives ish extelnally and yellowish-greenintemally. On long storage thc rnonocyclicderivatives the simple of molecules more ol pounds. Two longer is no drug and the interior becomesbrown. the activity decreases butanonemay condenseto tilicinyl and acid tllicinic aspiclinol. such as rnerislarge to nirle six from shows fit for use.A sectionof the r-hizorne compoundssuchasalbaspidinand llavaspidicacid,or tritelesarrangedin a difluse cycle and extemalto thosethc smallcrmeris- give bicyclic asfilicic acid(Fig. 22.3). such ones telesrunning from the fronds.The drug has little odour.The tastels at cyclic of compoundsis greatly extendedby variations range possible The The nauseons. first sweetish.aftelwardsbccomingbittel and extlemely patterns of the units and by the presenceof methoxylation in the l)' fiom that resembles closely f/l-rdrug derived frorn D. mttrginalis asfilihomologuesin the acylfilicinicacid series.In compoundssr'rch nlqs. a butyryl always is apparently unit phloroglucinol the central acicl Characteristicfeatr-rlesas seen under the microscope at'e stat'cb- cic in one spacesof which show clerivativebut the other two units show great variabilityand, containingparenchymacells.the intet'cellular combinations possible the not be identical.Many of shortly stalked internal secretory glands' The ramcnta have very molecule.may
Ioble 22.1
Exomplesof phenolic glYcosides.
Nome Solicin P o p u l i n( b e n z o y l - s o l i c i n ) Arbutin rnlor|ozrn Trilobotin Lonrlenn Goultherin Syringin Glucovqnillin Gein Glucogollin Homomelitonnin
of sources Exomples
Productsof hydrolysis
So/ixond Populusspp. Viburnumprunifolium Populvsfremula ond Rosoceoe Ericoceoe includingspp.of Molus Rosoceoe, Molus,Spiroeo Coniferoe Goultherio,Betu/oond MonotroPo in Oleoceoe Porticulorly Vonil/ospp.ond someGromineoe Geumspp. Rheumspp. Homomelisvirginiono
Solicylolcohol,glucose Solicylolcohol,benzoicocid,glucose glucose Hydroquinone, giucose Phloretin, glucose Phloretin, Coniferylolcohol,glucose Methylsolicylote,primeverose olcohol,glucose Methoxyconiferyl V o n i l l i ng,l u c o s e (glucose + orobinose) vicionose Eugenol, Gollicocid,glucose Gollicocid (2 mols),homomelose
kc AH
ffi ?
-'6'N?
8i9.22,2 D, edgeof some(>50); E' frondboses(x1);C, entireromentum; sectionof rhizomeond surrounding Mole fern.A, Whole drug (r0.5);B, tronsverse g't' o, storchgronules; filixioemino. Athyrium of bose leofy oio section (x65); F, tronsverse glonds sectionthroughrhizomerr,.*i.g'i.i.r."llulor m, meristele. glondulortrichome;
PHENOTA SND PHENOLIC GLYCOSIDES
HrC
HOYYOH l
-
r
HrcQ
CH.
xo-))<]ox
oH
l t i l
i
coc3H7
fcoc,",
,A-
Rco
./
Y
o
OH
Butyrylphloroglucinol
-c tl
Aspidinol
cHr
o
Acylfilicinic acid (R = CHrl C,Hrl CrH')
-CrHr ll
Albaspidin
"{{"" ".,-xl;H
.,",-fi\|.",-\f-.", Fig.22,3 Phloroglucinol derivotives of molefern
o
o
have been detectedas constituentsol the various Drtrtlttt:r'i.sspecies examined. The monomersdo not appearto be plescntto any extentin the lir.,ing plant. and the often reportedprcsenceof aspidinol and desaspidinol results from the breakdown of larger molecules,particulaliy in the presenceof alkali. The drug and its extract are assayedtbr 'filicin', 'crude filicin'or 'crude fllicic acid'. a mixture of the ether-soluble acidic (phenolic) substancesmentioned above. This assaydoes not necessarilyassessthe true phannacologicalactivity of the sanrple,and variousbiological methodshavebeenemployed. The phloroglucidesof male fern are fbrmed via the 'acetate'pathway. with methionineacting as a donor lbr the rnethoxyand methylene bridge-eroups. Monomeric conrpounds(e.g.aspidinol)injectedinto D. ntarginalis have been shown to be incorporatedinto dimeric compoundssuchas desaspidin. Substitutes and adulterants. Many ferns yield substantial amounts of extract. which when assayedmeet pharmaceuticiil requirements. Work by Mittal showedthat of l8lndian ferns examined l-5 contained1.5% or rnore of filicin. Some Europeanf'erns, besidesmale f'ern.contain adecluate amountsof filicin. as also the Americiin D. marsinulis. The increasingtendency to import extract rather than crude drug has to a large extenttransf'erredthe r"esponsibility lbr the colrect identification of the raw materialto the exportingproducersof the extract. The fbllowing f'ernshave beenfbund in samplesoff-eredas male t-ern: Atht'rium fili.r-.fbemina,the lr,rdyf-ern.which may be distinguished by the two dumb-bell-shaped stelesin the tiond basesand by the entire marginsof the ramenta. Drropteris spinulosu,the shieldfeln. which has srrall glandson the marginsof the ramenta. Uses. Male f'ern,usually in the lblrn of the oleoresin,is used as a taenicide.Its userequirescare.as caseshaveoccurredin which it has beenabsorbedand resultedin blindness.Saf'eldrugs are now ar.ailable.
OH
ClHr
o
Filicic acid
HOP STROBIOTE Hops are the dried strobilesoI Humulus lupulus (Cannabinaceae). Only the pistillateplants are cultivated.large quantitiesbeing prodr.rcedin EnglLrnd(particularlyKer-rt).Germany.Belgiun. France. Russiaand Califbrnia.The strobilesare collected.dried in kilns and pressedinto baicsknown as 'pockets'.They are sometimesexposed to the fumes of burning sulphur. which modifies the sulphur componentsalreadyin the hops but which is said to stabilizethe aromaand colour. Hops are included in the EP, BP, BHP and in monographsof the Briti.shHerbal Cornpentlium.ESCOPand GermanCommissionE. The hop strobiole consistsof external and internal sessilebracts which overlapone anotherand enclosethe ovary.Togetherthey lbrm a petiolategreenish-yellowinflolescence2-5 cm in length.The odour is characteristicallyaromatic. On the fruits and basesof the bracts are numerolrsshining glands. These. when sepalated,constitutethe dlug lupulin. The commercial productis generallyvery impure,owing to the fact that it is obtainedby sieving the sweepingsof the hop room floors. lt occursas a granular. reddish-brownpowder with a charactelisticodour and bitter aromatic taste. The bractsand stipulesof the hop containtanninbut the odourand taste of the dru-eare mainly due to the very complex secretioncontainedin the lupulin glands.On distillationthe fiuits yield 0.30-1.07c of an oil composedof well over 100 componentsand containingterpenes.sesquiterpenes includinghumulene,Fig.22.4,and compounds suchas 2-methyl-but-3-ene-2-ol and 3-rrethylbutanoic acid.The two latter.and relatedsubstances. increasesignificantlyduring processing of the freshhops.The bitternessis due to crystallinephlorogluctinol derivativesknown as g-acids (e.g. hurnulone),B-acids (e.g. lupulone) and also about 107c of resins.2.3.4-Trithiapentane, S-pentanoate methylthio-2-methylbutanoate, S-rnethylthio-4-methyl and,l.5-epithiocaryophyllene havebeenisolatedflon'rthe volatileoil of unsulphuratedhops. The valerian-like odour lesults liom decompositionof the oil and one of the resins.
P H A R M A C O P O FAIN AD LRELATE DD R U GO S FB I O L O G I CO AL RIGIN
Humulone
LUpUrOne
Fig.22.4 a^^"rir,,^-r"
^f L^^" v r rr u P J .
Humulene
Hops have thc propertiesofan aromaticbitter and ale said to have a sedativeaction(duein partto 2-methyl-3-buten-2-ol). They arcmainly usedin the preparationof beer.
afier anothcr.Clonal propagationof the r,anillaplanthasbeendescribed togetherwith ilr lirro multiplication r.rsingaxillarv bud explants(P. S. Georgeand G. A. Ravishankar.Plunt Cell Rep.. l9c)1.16. .190).
Kqmolq Karrala consistsof the trichomesand glandsseparatedtiom the fiuits of Mtrllotus philippinettsis (Euphorbiaceae).a tree lbund in India. Pakistanand the East lndies. It occr-rsas a dull leddish-brownpowder without odour or taste.Under the microsct'rpeit is seento consist of very characteristicglobular glandscontainingred resin. and radiating -eloupsof unicellular curved trichomes.It containsthe anthelninthic phloroglucinolderivativesrottlerin and isorottlerin,resinsand wax. It is usedin India for the treatmentof tapewormint'estation;alsofbr treating poultry.
Collection and curing. The fiuits are collectedwhen the Llpperpart of the pod changesin colour flom -ereento yellow. The characteristic colour and odour of the commercialdrug areonly developedas a result of enzyme action during the curing. The details of the lattel pl'ocess 'n'arysonrewhatin differentcountries.but frequentlyit consistsof slow drying in shedswhich ale kept at carefirllyregr.rlated temperatures.
VANIIIA AND VANIIIIN
Characters. Vanilla pods are 15 25 cm long. 8-10 rnm diameter and somewhatflattened.The sulface is longitudinally wrinkled. dark brown to violet-black in colour. and fiequently coveredwith needlelike crystalsofvanillin ('frosted').The fiuits arevery pliableandhave a very characteristicodour and taste.
Packing and grading. Beforegrading,anypodsshowinga tendencl'tcr mouldarepickedor-rt. The remaindcraresonedto sizeandpackedin bundles of 50 pods.Traditionally.thcsc were packedin tin casesor boxes holding about l0-l2kg. solderedup and packedin woodencases.On arrivalin Londonthe tins wereopenedand the podsu'ereexamined.UK Furtherreoding GhisalbertiE L 1995Acylphloroglucinolderivativesfrom EucaLt'ptus suppliesnow arrivevia Franceor Gerrnany.with somefrom Madagascar. During stora-ee cr1'stals fiequentlydevelopon the sudaceof thc pods. spp.Phvtocltetni st1' 1l( 1): 1-22
Yanilla (VtLnillaPods) consists of the carefirlly cured fully grown but unripe fiuits of VanillcrJragran,s(Salis.) Ames (s_r'ri.V planifolitr (Mexicanor Bor.rrbon Andrews)(Orchidaceae) vanilla)andof V tahitensls (Tnhiti vanilla). The fruits of other species.suchas V.pontpona (West Indianvanilla),arealsousedbut to a much more limited extent. Vanill.a.fragransis grown. in a serr-ri-wildstate.in the woods of eastem Mexici'r,its naturalhome.Vanillais cultivatedin Riunion (or Bourbon). Mauritius. Seychelles.Madagascar,Java, Ceylon, Tahiti. Guadeloupe. MarliniqueandIndonesia.Chinaand India arenow majorproducersand dr.reto oversupplypriceshave fallen drarr-ratically over the pastf-ewyears. History. Vanilltrwas found in Mexico by the Spaniards.where it was used fbr flavouring chocolate.a use to which it is still put. It fbund a placein the London Pharmacopoeiaof 1721. Cultivation. Vanilla requires a warm and failly moist climate. Propagationis simple: cllttings 1 3 rn long are attachedto trees(e.g. Casuarinctequisetffblia),where they soonstrikeroots on the bark. The plant is an epiphyte.lt flowers at the end of 2 or 3 yearsandcontinuesto produce fruit fbr 30-40 years.The flowers are r"rsual1y pollinated by women and children,a pointedstick being introducedinto one flower
Constituents. Green vanilla contains glycosides. nanrely glucovanillin (vanilloside) and glucovanillic alcohol. Durin-e the curing theseare actedupon by an oxidizing and a hydrolysingenzyrnewhich occurin all partsofthe plant.Glucovanillicalcoholyieldson hydlolysis glucoseand vanillic alcoholl the latter compor.rnd is then by oxidation convertedinto vanillic aldehyde(vanillin).Glucovanillin,as its yieldson hydloll'sis-qlucose nameir.t.tplies, andvanillin (Fig. 22.5). The three speciesgiven abovedifl-erin their lelative contentsof anisyl (alsoanisylethersandanisicacidestels).piperonal alcohol.anisaldehyde andp-hydroxybenzoicacid.Thesemint'rlcomponents, togetherwith the two diastereoison-reric vitispiranes. trddto the flavourof the pods. Vanillin BP. Vanillin BP is the aldehydecorrespondingto methylprotocatechuicacid and has been synthesizedin a number of ways. Large quantitiesof it are preparedfiom eugenol isolatedfrorn oil of cloves (q.v.) or from guaiacol (methyl catechol).It can also be producedby rnicrobialoxidation of eugenol.In the plant glucovanillin is
P H E N O LA SN D P H E N O L , 3C- \ l 3 S ) t S
OMe
OH OMe
H-C=O Vanillin
P1o H-C =O Piperonal
I
a\ Y
cH2oH
Anisylalcohol
0{>" Vitispirane
Fig,22.5 Constituento s f vonillo.
lra f'erulicacid (seeFig.22.l). Synthesisbeginswhen biosynthesized elongationof the tiuit ceases.rvhich is about 8 months after pollination; befbrethis. other pherrolicglycosidespredorninate. Adulteration. Extracts of Mexican origin may be adulteratedby coumarin.probablyarisingfiom the useof tonkabeans(q.v.).A capillary GC assayhasbeenclescribedfor suchproducts(seeR. J. Marles e/ Bot., 1981,41,4l ). al., Ecortontit' Uses. Vanilla pods are widely usecl in conf'ectionelyand in perfirmer5r. They havebeenreplacedto someextent.but by tto meanscompletely. by synthetic vanillin. About 0.07 parts o1' vanillin are approximatelyequivalentto I part o1 the bean,but an esscnccso prepaled fails to representthe odour and flavour of the whole pods.
:r, li,.::-.J:: arbutin.gallicacidandhr,lr,,.lLrrn, identitydistinguishes is diurctic and astringentand during excretionit crert. .1:r.:r::.J:': action on the urinary tract.
Tor (Pix Liquidal h\ th( Wood tar is known in corrrrerce as Stockhoimtar.It is prcparL.d ln a,j.l destluctivedistillationof varioustreesof the farnily Pinacc-ac-. ition to thc tar.an aqueousdistillateis obtainedfiom which aceticdiiJ. methyl alcohol and acetoneare prepared.A residueof wood chareorl remainsin the retorts.Wood tar is a blackishserniliquidwith a charrc' teristicodour and tiiste. The constituentsincludethe fbllowing phenolsand phenoliccthcrs: phenol.C6H5OH;cresols,C6H1(CH3)OH;methyl cresolslcatecholor pyrocatechin.C6Ha(OH)1;guaiacol (methyl catechol)and its homologues. Also the hydrocarbonsbenzene,toluene (rnethylbenzene). (trimethylrresityleneand pseudocumene xylenes(dimethylbenzenes), benzenes),styrene(phenylethylene).naphthalene(CroHs), retene(rzchrysene(C rsHI z) and paraffins. methylisopropylphenanthrene), Pine tar is characterizedby the large amount of guaiacol and its homologueswhich are present.Other tars. such as those of the birch in composition.Wood tal'is difl'erences and beech.show considerable acid in reaction.whereascoal tar, which is alsoofficial, is alkaline and in light petroleum gives a blue fluorescence.Creosoteis obtained fiom wood tar by distillation. Tar is mainly used externally, in the fbrm of ointment or tar parogen,as a stimulatingantisepticin certain skin diseases.
Willow borks ond solicin
Willow barks,particularly Salix.fragilisand S. purpurea (Salicaceae). are a sourceof salicin (Table22. I ) a glycosidenow seldom used,but generallyregardedasthe naturalforerunnerof aspirin.Salicin,however, is easyto prepareand is a suitablecompoundwith which to introduce studentsto the classof glycosides.The glucosideand tannin are extractedfrom the bark by maceratingit in hot water for somehours. The liquid is filtered, concentratedin vac'uoand treated with lead TEAVES BEARBERRY acetate.After filtration excessof lead is removed by treatment with hydrogen sulphicle.The filtered liquid, which is now fiee liom tan(Uvo ursf then allowedto cool nin, is neutralizedwith ammonia.concentrated, Bealbelry lefi EPIBPIBHP consists of tl.re dried leaves of Thesecrystalsmay be pr.rrifiedby A rctostupItyIos Ln'u-ursi. ElicaceaelESCOPand GemranCol'nmission when crystalsof salicinseparate. dissolvingthem again,filteling throughanimalcharcoaland recrysE nonographson the dru-qare alsoavailable. A. uvu-ursiis ii small evcrgreenshrublbund in centraland northerr tallizing. Willow bark is an ancientremedy,much esteemedfor its analgesic, Europeand in North America.The leavesare dark greento brownishgraduallynarowing to a very anti-inflammatoly and febriluge properties. Considerableresearch gleen.2-3 cm lon-e.obovateor spathulate. shorrpetiole.apexobtuseor retuse.They iue coriaceousin textule and continuesto be publishedon the disffibutionof glycosidesand tannins almostglabrous.The uppersurfhceis shinyandmarkedwith sunkenvein- in variousspeciesand on the effectsofthe drying ofthe bark on these lets:the lower suftaceis lightel and nrarkedwith a networkof dark r,ein- constrtuents. lets.The drug is odourlessbut hasan astringentandsomewhatbittertaste. (Filipendvla ulmoriq, Rosoceoe) Microscopical ftatr-rresinclude: an upper epidermis of polygonal Meodowsweer cells with a thick cuticle;lon'el epidernriswith anomocyticstomata Meadowsweet.a well-establishedEuropeanherbal remedy,contains cellslscarsoftrichomebases,occa- salicylaldehydeas the principal componentof the volatile oil as well by,5 1l subsidiary andsulrounded For a gen+ primeverose). as the glycosidespiraein(salicylaldehyde sionalconicaltrichomcs.clystal tibres. Bealberry containsthe glycosidesalbutin (Tablc 22. l ) and methyl- eral article on this drug see H. Zeylstra, British J. Phl'totherapr arbutin,about 6 7% of tannin.(+)-catechol,ursoneand the flavone 1 9 9 8 / 9 . 58.. derivative quercetin.Sorne 1.1phenolic acid constituents,including Propolisor bee glue gallic and ellagic acids,havebeeurecorded. This is the materialwith which the honeybeesealscracksandcrevices. The pharmacopoeialdrug is required to contain at least 8.07c of hydroquinonederivativescalculatedas arbutin.These are assayedby and varnishessurfaceswithin the hive. Like honey.its cotttpo:itiort reactingan aqueousextractof the leaveswith solutionsof aminopyra- variesaccoldingto geographicalsource. Over 160 conrpoundshave been shown to be involved and one ferricyanideandtransferzolone,ammoniunrhydroxidcand potassir.rnt ring the colouredproductto methylenechloride:the absorbanceof the analysisgavephenolics(587c),beeswax(24Vc).flavonoicls(6ti t. tere.g. Mn. Cr.r.Zn penes(0.57c).lipids and wax (8clc)and bioelements, test for solution is measuredat 455 nm. The official cht'omatographic
E
PHARMACOPOEIAL AND RELATED DRUGS OF BIOLOGICAL OR}GIN (0.5Vc).In temperateregionsof Europethe resinouscoatingof poplar buds (Pqzrlus nigra, P. italica. P. trentula)fbrms a major coliection sourcefbr the bees and thc naturalphenolic contentof the resin, e.g. estersof cafl'eicand ferulic acids. r,anillin, eugenol,f'lavonoids,etc., can be usedto idenlity the rraturalsource. Latterly there have been numerousrepofis concerningthe analysis and biological activity ofpropolis originatingfiom variousregionsand especiallytiom Latin American countries.In these areasspeciesof Arattc:ariu (Araucarizrceae). Baccharis (Compositae) and Clusiu (Guttiferae)have beenestablishedas biological sources.In additionto the constituentslisted previor-rsly. prenylatedcinnamic acid and chlomane derivatives.diteqpenoidacids.lignans and some57 corrponents of the volatile oil havebeenidentified. Notwithstandingthe diff'erencesin chemical compositionof propolis depending on geographicalsource, a pronouncedantibacterial ploperty is commonto all. In temperateregionsflavonoid and phenolic estershave been shown to exefi bacterialactivity but as theseare not presentin tropical samplesother constituentsmust be involved. Propolisis still usedmedicinally in sometraditionalsystemsand in apitherapy-an old tradition which has experienceda recentrevivalit hasbeenshownto exhibit antibacterial,antiviral.anti-inflammatory. local anaestheticand other properties.It has now becorre a popular constituentofhealth foods anclcosmeticpreparations. Readersrequiring further information on this interestingsubstance can refer to the following: V. S. Bankoya et al.. Phltochentistrt, 1998, 49, 1411:Fitoterupia,1999.70, 190;A. H. Banskotaet ul., J. Nut. Prod., 1998,61, 896: A. Kujumgiev et ol., J. Erhnopharmacologt, 1999,64.235;S. Tazawaet al.. Chent.Phurm.Bull., 1999.47,1388.
calicesand pedicelsis officialty limited to 3TobuLsamplesvary. The pericarpis glabrous.shrivelledand orange-red;the Sierra Leone and Zambianchillies usuallyhavea bettercolour than thosefrom Zanzlbar. Internallythe fruits are divided into two cells by a membranousdissepimentto which the seedswere originally attached.The latter,usually about 10-20 in eachf'rlit, are of a f'lattenedreniform shapeand are about 3-4 mm long. Like other solanaceousseeds,they have a coiled embryo and oily endosperm.African chillies are very sternutatoryand have an intenselypungenttaste. Constituents. In 1876, Thresh extractedthe drug with petroleum. treatedthe extractwith aqueousalkali, and by passingcarbondioxide throughthe alkalineliquid precipitatedcrystalsof an intenselypungent compound.capsaicin.As may be inferred from the method of preparation,capsaicinis ol phenolicnature.
cH.o " \-/ / \ HO1
\-./
r
CHTNHCOICH2]aCH=CHCH(CH3)2
Capsaicin(Vanillyl amideof isodecenoic acid)
The pungentphenolicfractionof capsicumalsocontainsa proporrion of 6.7-dihydrocapsaicin. The capsaicincontentof f'ruitsvariesappreciably in a rangeup to L57c and is much influencedby environmentalconditions and ageof the fruit. It occursprincipally in the dissepimentsof the fruitsfbr example,entire fruit 0.49, pericarp0. I 0, dissepiment1.79,seed0.07. The pungencyofcapsicum is not destroyedby trearmentwith alkalis (distinction fiom gingerol, which also contains the vanillyl group) but is destroyed by oxidation with potassium dichromate or permanganate. Block how bork Chilliesalsocontainascorbicacid (0.l-0.57c),thiamine,red carotenoids The root bark of Viburrumtprunifoliwn (Caprifoliaceae) was formerly such as caps:mthinand capsorubinand fixed oil (about 1-167c). They official in most pharmacopoeias, but its useibr dysrnenorhoea.thr.eatyield about20-257c of alcoholic extract(capsicin)and about57o(official ened abortion and asthmahas gradLrallydecreased.It containsabout limit 8%) of ash. Hungarian capsicumsor 'Paprika' are derived from a 0.2% of salicin,volatile oil and isovalericacid.tannin and resin. mild race of C. annuum and are a convenient source of ascorbicacid. Oil of wintergreen According to Bennett and Kirby, the pungentprinciple of C. annuumis Natural oil of wintergreen was formerly obtained from the ieaves of composedof capsaicin69%, dihydrocapsaictn227o,nordihydrocapsaicin procumbens(Ericaceae), GaLrltheria but is now distilledfrom thebark of 7%, homocapsaicin(C11 acid) 1Vcand homodihydrocapsaicin 1%. A Benlu lenta(Betulaceae). Gaultheriaoil otthe Indian Pharmacopoeiais numberofminor components ofthis classhavebeenrecorded. obtainedfrom the fresh plant of GaultheriaJi'cLgrutissimaand contains In a study of the water-solubleconstituentsof the fruits of threevarnot lessthan 98% of esterscalculatedasrnethylsalicylate. ieties of C. unnLrunt,lzumitani et ul. (.Chem.Pharm. Bull., 1990,38, 1299)isolatedtwelve novel acyclic glycosides(geranyllinaloolderivaGAPSTCUM tives) named capsianosides A-F (dirneric estersof acyclic diterpene glycosides)and capsianosides I-V (monomericcompoundsof acyclic The BPC (1988) dru-e(Chillies; Red Peppers)consistsof the dried, diterpeneglycosides). ripe fruits of Capsic'umannuum vur. minintunt (Miller) Heiser, and A numberof colorimetricassayscanbe usedfor thequantitativedetersrrraf l-fi'uited varietiesof C. frutescensL. (Solanaceae). In commerce mination of capsaicin(see Tabie 121.5); the BPC utilizes ultra-violet thc dcscliption given appliesto variousAfiican commercialvarieties absorptionat 248 and 296 nm lbr the ointment and oleoresin. (principally from Zimbabwe and Malawi) and these are sold in En,glandas chillies. whiie the larger but less pungent Bornbay and Biogenesis of capsaicin. Work by Leete and Louden on C. \atal fl'uits are known as capsicums.Very large CapsicLunfrtits, Jrutescensand by Bennettand Kirby on C. annuumdemonstratedthat rcsernblingtomatoesin textureand practicallyltonpungent,are widely phenylalanineis incorporatedinto the C6-C vanillyl unit of capsaicin, 1 the C-3 of phenylalaninegiving the methylenegroup of the vanillyl-9roun in southernEuropeas vegetables. amine residues;the incorporationprobably proceedsvia cinnamic,pHistorl. Capsicr.rrns appearto be of Amedcanorigin and wererefened coumaric,cafl-eicand protocatechuicacids.Tyrosinedid not appearto to in 1:191bl Chanca.a physicianwho accompaniedColumbuson his be a probable precursor.Leete's f'eedingexperimentswith [U-l4CJsecondvol,ageto the WcstIndies.The plantswereintroducedinto India valine gave incorporationsconsistentwith the hypothesisthat the C,u at a very earll'date. possiblyby the Portuguese.'Ginnie Pepper'was isodecanoicacid is fbrmed from isobutyryl coenzymeA and three well known in En-ulandin 1597andwas grown by Gerarde. acetateunits. More recentwork showedthat the homo derivatives(C,, acid) are fbrmed from leucineand isoleucine. Macroscopical characters. AJricun Chillie.sare oblong-conicalin The ontogeneticformation of capsaicinoidsin thefruits of C.J'rutescen.r shape,12 25 mrr long and up to 7 mm wide. The five-toothedcalyx involves a prior active accumulationofp-coumaroyl, caffeoyl and 3.4and straightpedicelare togetherabout20-30 mm long. The amountof dimethoxycinnamoyl glycosides,3-O-rhamnosylquercetin and 7-O-glu-
P H E N O LASN D: - : ' . :
: ctyCOStDES
cosyllut3olin.when the fruit ceasesto incrcasein rcngththe am.unt of theseccxnpoundsfalls and capsaicinoidsynthesiscoutIrrences to-qether TANNINS with that of the glycosidesof r,anillicacicland p_hyclroxybenzataehi,ae (seeN. SukrasnoandM. M. yeoman.plntot.hetnistn,,1993, g39). The term 'tannin'u'asfir-stappliedby Seguinin l-vr. . 32. l\ )i!- \Ubstances prescntin plantextractswhich wereableto cot|.r::- .,trhpro_ Cell cultures. The biogeneticpotential for capsaicinproduction is tein o1'anirralhides.pre'ent their pun'eraction and conreii ::rJ.. irto reported(1991)as 10 tirnesgreaterin immobilizedcell cultLrres (alei_ leather.On this basisa tanninis a substance rvhichis detcitc.i.,...,lrrrrnateentrapment)than in control suspensioncultures. tively by a tanningtest (the -roldbeater.s skin test)rntl is d..rrr;. ..1 quantitati'clyby its adsorption on standard hicrepowder.This dclrrr;rr,,rr Affied drugs. JctpcuteseChitties are probably derived lrorn C. cxcludessimplerphenolicsubstanccs. ofien pr.esent wrth t:utninr.,u.it lrutescensand are about 3-4 cm long. They possessaboutone_quarter as gallic acicl.catechinsand chloroge'ic acid. arthoughthey mar under of the pungencyof the Afiican Chillies. but are now no longer com- certainconditionsgi'e precipitates with gelatin and be partly reraincd n r e r c i a l l r5e l e r n t . by hiclcpowder.Suchsubstanccs of rerativelylou' morecr.rlar weight arc BornbttyCaltsic.unsare ascribed tr.tC. ttnnr.tLutt L. The pericarp is callcd 'pseudo-tannins'. Most true tanninshave molecularweightsot. thicker and tougher than in the chillies. and thc pedicel is trequently trom about 1000 to -5000. To bc efl'ectivefbr tannagethe polyphenol bent.They aremuch lesspungentthanAfrican chillies. molecr-rle mostbe neitrre'solargeas to be unabreto enterthe interstices Natal Capsicurlsare larger than the Bombay variety.being up to g bet*een the collagenfibrirs of the animal skin nor so small that it is cm long. They have a very bright red. transparentpericar.p.They ar.e unableto cross-linkberwcenthe p'otein moleculesofacrjacentfibrirs at muchlesspungentthanchillies. se'eral points.Ma'y tanninsare glycosicres. The definition of a tannin as gi'en aboveis an old. essentiallypracticaronc which r.navbe ourer' Uses. Capsicumsare usedas a condimentunderthe nameof Cayenne fortuitousand. in the light of further research.could prove misleadinrr pepper.The drugis givenintemallyin atonicdyspepsia andflatulence.It is fiom the point of view of plant metabolisrnand plant biochemistrli usedexternallyasa counter'-initant.in the fbrm of ointrnent.plaster,rredIndeed,r.nodernauthorsofien tr.eattanninsnot as a specificphytochent_ icatedwool, etc., for the relief of rheumatism,lr-urbago.etc. Capsaicin ical groLrpbut asexamples of polyphenolsillustratingparticularaspects creamsare availablelbr the relief of pain in osteoarlhritis. post-heryetic ofgallic acid andfla'an-3-ol phytochemistry. The characteristic proper neuralgiaandpainfuldiabeticneuropathy(pharnt.J.. I 99g, 260.6g2J. ties.f tanninsderi'e ti.' the accr,mulatio'withina moderatehrizetr moleculeof a substantial nuntber([-2 pcr 100mol. wt.) of phcnolie -qroupsmany of which are associatedwith o_dihydroxyand (/_rrih\ METISSALEAF droxy orientationrvithin a phenylring. Lemon balm (MelisscL olficinuris.famiry Labiatae)has rong beenextoilecl The abovetannin-protein co-precipitation is imponantn()t()nt\ rn in traditionalmedicine.The dried reavesare incrudedin the Ep and t h el e u t h ei rn d u : r l yb r r ta l r oi n r e l r t i o nt o t h ep h 1 : i o l u s i e :rrritr t r . r ,. r describedin the BP 2000; theBHp r996 chugconsisrsof the dried reaves herbalrnedici'es.taste of fbocrstufr.s anclbe'erages.ancrin lhc .rr. andfloweringtops.The plantis extensivelycultivatecl in galdensanclcom_ tional 'alue of f'eecls fbr herbir"ores. Environmental1uct.r, ir1l..r,! mercialsLrpplies originaternainlyfrom Bulgaria,RoumaniaandSpain. this processhave been studiedby H. Kawamotoantl F. \.rk.rr.rrb,, The macroscopicand microscopicchar.acters are tl-rosetbuna typ_ (.Phtto<'hentistn,, I 997, 16,ljg). ically in the Labiatae.Constituentsof interestinclude a volatile Two main gloups of tanninsare usuallyrecogr.rizctr: oil lnc\. .lr.ellrc (0.06-0.357.) containingmore than 70 identifleclcomponents includ_ h y d r o l y s a b ltea n n i n sa n dt h ec o n c l e n s et a d n n i u s( i l . o i t n r h (\r..r l l J i n . ) . rng geranial,citronella]and nerarwhich give the leafand oil its lemonlike odour. The f'lavonoidcomponentinvol'es glycosidesof luteoli'. Hydrolysoble tqnnins quercetin, apigenin and kaempf'er.ol. Hydroxycinnanric acid deriva_ Thesemay be hydrolysedby acidsor enzynte\ \u!h i1,r.,nn.r\..I hc\ tives include caffeic, chlorogenic.ancl characteristically.rosmar.inic are fbnned liom severalmoleculesof phenolicri.itl. .Lr.lr.,. -lallic acid (caffeicacid esterifiedwith 3,zl-clihydroxyphenyllacric acid).The andhexahydloxydiphenic acidswhich arc uniletlt.r e.rerln)kegc\to a BP drug should contain not less than 4.0c/cof total hydroxycinnamic c e n l r ugl l u c o r e m o l e e L rA l c s. i r n p l e t l n n i r ri j l t r . t r . , t , rt ,1.,_, t. , , , i nrt, . , n e acid derivativesexpressedas rosmarinic acid; theseare determinecl delived from a speciesof surnrc(R/rirr). rr rrh ., 1.,,.i.11.13 .,ru.,ur. o, s h o w ni n F i g . 2 2 . 6 L . i k e - s a l l i ca c i r ,t lh c i r ' \ r , r . l r t , rr-t r r . b l u cr-i i t h i r o n HO salts.They were fbrmerly known as p\ fogrrllL,lrurrrrirtr. becauseon \ H dry distillationgallic acid and sinrilarc()nrl)(Jnclrr\ , / \ l lr.c convertecl into HO_ar /rcH2-c_cooH pyrogallol.Two principaltypesol' Itr clr.olr ,ul.lc runninsare gallitan_ \---l ninsandellagitannins which are.r.cspce E-"o-"r:"r-(-\-on rirclr. uollp()secl of gallic acid and hexahvdroxy-diphenic acid unir,. F_lil.riclcitl (the depsideof g u l l i cr c i d t c i t nu r i s eh 1 l 1 c r , , r r i z . r t,i , r, lr rr c r . , j r . r J r i r r 1 6 l O ; - . , . ' n 1 . OH . O duringchernicalhydrolysisol' rltt.l.r.. r. : rhLrr.thc rerrnellagitanninis Rosmarinic acid a mlsnomel. (measurementof absorbanceat -505nrn) by reactionof a prant extract Ellagitanninsfbund in pJ,rr: .l 'cdi!i.al interest.and fbr which with a sodium nifiite and sodium molybdate solntion. Other con_ strllctures have been elucidatctl rnclLrde_qeraniin(Herb Roben ancl stituentsof the leaf are monoterpenoidand phenylpropanoidglyco_ American cranesbill)and tc-llinrasmninsI ancl 2 (Oak bark and sides,and triterpenesincluding ursolic and oleanolicactds. Meadowsweet): Fig. 22.6. Lemon balm preparationsare prescribeclfor their sedative.soas_ Modern methodsof anarrsis h.r e .r.racle conside'abreadvancesin the molytic and antibacterialproper.tiesinclLrdingincligestionassociated study of tanninchemistr'or'.cdicinal plantsaseviclencecl by the work with nervoustension.Externallyit is usefurlbr the treatmentof heroes of okuda on orientalclrugs.Ir r982 agromoniin.thefirst of a new class and insectbites.The oil. which is sometimesaclurterated r.rith cheaner- of olig.ntcric /t:tlr.h.suble tururittswas isolatecl from Agtrtnt.nia. lemon-t1,pe oils, is useclin aromatherapyand as an inseet repellent. Thesetanninsare cornposedof two. threeor four monor.nencunits.To
r:<.
PHARMACOPOEIAL AND RELATED DRUGS OF BIOTOGICAL ORIGIN OH
OH
OH
I Hovv,oH t t l
O-G-G_G
\1I
coR
OH Ellagic acid
Gallicacidl R-OH
OH llexahydroxydiphenicacid
H"c! e-c-o*^o^r-o-e G'" b-e
Gallitanninof Rils sp.
0-o-Glucogallin; flocH2
n=xg${$,,-,oOH
{' r o l
HO
I
ryl
HO HO
o_ G
o
o
c-o-cH2
g-,'N+;'
o
bo Co
HO OH l; R=OH Tellimagrandin Tellimagrandin2' P = 1P)-OG
Geraniin
G ;n = l G-G; n=2 G-G-G: n=3
Fig.22.6 Exomples tonninsond theircomponent ocids. of hydrolysoble date,somethingless than 20 of theseunits including geraniinand tellimagrandinsI and 2 areknown to be involvedin the productionof over 150 compounds. As an example,many plantsof the Onagraceaee.g. Oenotheraspp. containin additionto tellimagrandin,its dimer oenotheinB and trimer oenotheinA: thesemacrocyclicellagitanninsare also producedin callus culturesof O. lacinata and are of interestfor their anticancerand polygalacturonase-inhibitingproperties (S. TanigLrchi et al.. Phttochemistn',1998.48. 981). C-glut'osidic ellagitunnins are common in a number of families Punicaceaeand Rosaceae including the Myrtaceae.Hamamelidaceae, and several have also been recorded as moieties of more than l0 oligomericellagitannins. For an article on the classificationof oligomeric hydrolysabletannins and the speciticity of their occurence in plants seeOkuda et a1.. I 993.32. 507. Pht,tochenr'.stt't. Researchon the pathwaysleading to the more complex hydrolysable tanninmoleculesis still at its earlystagebut in a seriesofenzymaticstudies Grossandcolleaguesindicatedthe centralpositionof B-o-glucogallin in the early stagesof tanninsynthesistn Quercusroburleaves.This compound appearsto act asboth donor and acceptolof the galloyl group in the
enzymaticformationof 1,6-digalloyl-o-glucose; the responsibleenzyme is B-glucogallin:B-glucogallin6-O-galloyl+ransferase. The presurred immediate precursor of the two subclassesof hydrolysabletannins (gallotanninsand ellagitannins)is 1.2.3,4,6pentagalloylglucose, and in a continr-ration of theil enzyme studies the above group have purified (x500) the enzyme responsiblefbr the conversionof the precursorto the gallotannin3-O- digalloyl-I ,2,4.6The sourceof the enzyme was R/rus tetra-O-galloyl-B-o-glucose. typhina (staghorn sumac) and its designtrtionis B-glucogallin: (R. Niemetz galloyl-transl'erase 1,2.4,6-pentagalloyl-B-o-glucose and G. G. Gross.P/r1'rochenistrl',1998, 49, 321). Condensed tqnnins (proonthocyonidins) Unlike hydrolysabletannins.theseare not readily hydrolysedto simpler moleculesandthey do not containa sugurmoiety.They arerelated to the flavonoidpigmentsand havepolymelic f-lavan-3-ol structures. (leuCatechins,which alsooccur with the tanninsand f-lavan-3,4-diols coanthocyanidins)are intermediatesin the biosynthesisof the polymeric molecules. Stereochemicalvaliations add to the variety of possible structures.Monomeric and dimeric forms are illustrated in Fig. 22.7. Work by Japanesephytochemistshas exploited modern
PHENOLA SND PHENOLIC GTYCOSIDES
fi,ATOH
"oYYoJ",*o"
OH
A.o^
V
\sox
OH (+)-Catechin
OH I
ft* OH (-)-Epicatechin
OH OH Flavan-3.4-diolstructurc
OH A dimeric procyanidin
tig.22.7 Slructures ossocioted with condensed tonnins. techniquesfbr separatingand detennining the structuresof these oligomersand polymersincluding thoseof cassiabark (p. 2731,Cassitt .fistula.cinchona.Quercusand rhubarb. On treatmentwith acidsor enzymesconclensed tannisare converted into red insolublecompoundsknown as phlobaphenes.Phlobaphenes -{ive the characteristicred colour to many drugs such as red cinchona bark, which containthesephlobatanninsand their decompositionproducts.On dry distillationthey yield catecholand thesetanninsaretherefbre sometimescalled catechol tannins. Like catechol itself. their solutionsturn greenwith f-erricchloride. 'Complex
tqnnins' This term has been appliedby Okuda to a newly-discoveredgroup of tannins which are biosynthesizedfiom both a hydrolysable tannin (mostly a C-glucosideellagitannin)and a condensedtannin.The union occursthlougha C C bond betweenthe C-1 ofthe glucoseunit ofthe ellagitannin and the C-8 or C-6 of the flavan-3-o1derivative. The monomersarealsoinvolvedin olisomerformation.
Ellagitcmnins: pomegranaterind. pomegrzrnatebark, myrobalans, eucalyptusleaves,kousso.someAustraliankinos.chcstnut(Castanea spp.)and oak bark. Condensed tqnnins (proonthocyonidins) Somedrugs(e.g.tea.hamamelisleavesand hamamelisbark) contain both hydrolysableand condensedtannins.The following are rich in condensedtannins. (l) Barks: cinnamon,wild cherry.cinchona.willow. acacia(wattlc. mimosa).oak and hamamelis (2) Rootsand rhizomes:krameria(rhatany)and male fern (3) Flowers:lime andhawthorn (4) Seeds:cocoa.guarana,kola and areca (5) Fruits: cranberries.grapes(red wines),hawthorn (6) Leaves:hamamelis,hawthornand tea,especiallygreentea (7) Extractsand dried juices: catechu,acaciaand rrangrove cutches, East Indian kino, buteagurn and eucalyptuskino. 'Complex
Pseudotqnnins As alreadymentioned,pseudotannins arecompoundsof lower molecuIar weight than true tanninsand they do not respondto the goldbeater's skin test. Tanninsare of wide occurence in plantsand are usuallyfbund in greatestquantity in dead or dying cells. They exert an inhibitory efTecton many enzymesdue to proreinprecipirarionand.hence,they may contribute a protective fr-rnctionin barks and heartwoods. Commercialtannins,as used in the leather indLrstry.are obtained from quebracho, wattle, chestnut and myrobalans tl.ees. Pharmaceutical tannin is preparedfrom oak galls (q.v.) and yields glucoseand gallic acid on hydrolysis:many commercialsamples containsomeflee gallic acid. Someplants(clove.cinnanton,etc.) containtanninin additionto the principal thelapeutic constituents.This may complicate extraction or produceincompatibilities with otherdrugs(manyalkaloids,fbr example. areprecipitatedby tannins). Examplesof drugsin this group are given below. Hydrolysoble tqnnins Gcrllitannins: rhubarb, cloves. red rose petals. bearberry leaves. Chinesegalls.Turkish galls.hamamelis.chestnr.rt and maple.
lqnnins' To date, complex tannins have not great relevanceto mainstream pharmacognosy; monomershavebeenisolatedfrom the Combretaceae, (.Querc'us, Casttuted). Myrtaceae.Polygonaceae Fa-gaceae (Rheunl and Theaceae(Cantellia).It is anticipatedthat many more compoundsof this group will be discovered. Pseudotqnnins (1) Callic acid: rhubarband most materialswhich conraingallitannins (2) Catechins:catechu.acaciacutch. many Australian kinos. cocoa, guaranaand many other drugscontainingcondensedtannins (31 Chlorogenicacid: matd, cofTee(particularly unroasted)and nux vomica (a small quantityonly) (.1) Ipecucuanhicctcid:ipecacr.ranha Properties and tests. Tannins are soluble in water. dilute alkalis. alcohol, glycerol and acetone,but generallyonly sparinglysolublein other organic solvents.Solutionsprecipitateheavy metals,alkaloids. glycosidesand gelatin.With f'erricsalts,gallitanninsand ella-eitannins give blue-black precipitatesand condensedtannins brownish-green ones.If a very dilute f'erricchloride solution is gradualiy addedto an aqueousextract of hamamelisleaves (which containsboth types of
@
P H A R M A C O P O E IA N L D R E L A T EDDR U G SO F B I O L O G I C AO TR I G I N tannin).a blue coloul is ploducedvn'hichchansesto olive-grecnas more feric chloricleis addecl.Other r-rscfirl tcstsarc thc fbllowing.
H e n r i n g u , aR v W . L a k sP E ( e d s )1 9 9 2P l a n tp o l y p h e n o l ss:y n t h e s i sp.r o p e r tics. significance.Reportof 2ncl.North AmericanTanninConf'erence.I 991. P l e n u mP l e s s .N e u Y o l k . U S A Goldbeater'sskirttest.Soaka smallpieceof goldbcatcr's skin in 2% WatermrnP G and N{ole S 199.1Analysisoi phenolicplant metabolites. Methods in ecolog-v. tslacknell ScientiflcPublications.Oxford, UK hydlochloricacid: rinser"ith distilledwaterand placein the solr-r-
tion to be testedfbr .5rr-rir-r. Washwith distilleclwater and transfir to a l 7c solutionof felroussulphate.A brown or black colour on thc skin denotesthe plesenceof tannins.Goldbeater''s slin is u mcnrbraneprepared1'romthe intestineof the ox and belravessimilarly to an nntannedhide (a retelencesampleofhide powder.asusedin the BP assayof the tanninsof RhatanyRoot. may be obtainedfrom the EuropeanPharmacopoeia Secretaliat. Strasbour_t). Geluritt..,.rr.Solutionsof tannins(about0.5-l%) precipitatea l7c solutionof gelatincontainin-q l0% sodiumcholoride.Gallic acid and othel pseudotanninsalso plecipitategelatin if the solutionsare sufliciently concentrated. Phenu:rne 1e-!1. To abollt 5 rnl of an aqueollsextlact of the drug add 0.5 g of sodiumacid phosphate,warn. cool anclfilter'.To the filtrate All tanninsareprecipitated. add2c/csolutionof phenazorre. the prccipitatebeing bulky and oftcn colourcd. 4 . T e s t . f o r c a t e c h i n . C a t e c h i n so n h e a t i n g n ' i t h a c i d s f o r m phloroglucinoland they can. therefbre.be detectedby a modil'ication of the wcll-known tcst for lignin. Dip a matchstickin the plant extract,dry. moisten with concentlatedhydlochloric acid and warm near a flame. The phloroglucinolproducedturns the wood pink or red. 5 . Te.stfor chlorogcnic ttcid. An extract containingclrlorogenicacid when treatedwith aqueousammonia and exposedto air -eraduallv developsa greencolour.
GAttS AND TANNICACID Turkishgalls(Irrlccl Gull.s;GaLLa) arevegetablegrowthsfbrmedon the yotrngtrvigsof thc dycr's ottk.Quercu.s infectoriu(Fagaceae). as a result of the depositionof the e-rgsof the gall-rvaspArl/eriagallaetinctoriae. Tl-redyer's oak is a small treeol shrubabout2 m high which is for.rnd in Tu*ey. Sirria.Persia.Cyprusand Greece.Abnormal deveiopmentof Vegetabletissucround the larva is due to an enzyme-containing secretion. proclucedbir the young insectafter it l-rasenrergedfiom the egg. which by the rapid conversionof starchinto sugarstimulatescell divisiorr. As starch disappealsfi'om the neighbourhoodof the insect, shrirrkageoccurs and a central cavitf is lbrmed in which the insect passesthrou-shthe la|viil and pupal stages.Finally. if the galls are not previouslycollcctedand dried.the matureinsector imago boresits way olrt ofthe gall and escapes. Duling thesechangesthe colourofthe gall passesfiom a bluish-greythrou-eholive-greento almostwhite. Galls are collectedby the peasantsofTurkey and Syria.After drying the)' are -sradedaccordingto colour into three grades.blue, greenand white. which ale fbund on the London market. History. Galls wele rvell knou'n to the ancient writers and Pliny recordsthe use of their infusion as a test fbr sulphateof iron in verdigris. possibly the earliestmention of an attemptto detectadulteration by cherricalmeans.
Characters. Aleppogallsaregloblrlarin shapeandfiom l0 to 25 mm in Medicinal and biological properties. Iannin-containingdrugs rvill diameter.They havc a sholt,basalstalk and numerousroundedprojections precipitate protein and have been used tladitionally as stypics and on the surface.Galls arehald and heavy,usually sinking in water.The sointernally fol the protectionof inf-larrred surfacesof mouth and throat. callecl'blue' variety ale actually of a -ereyor brownish-greycolour.These. 'green' galls. They act as antidiarrhoealsand hiive been emploved as antidotesin and to a lesserextent the olive-green are preferredto the poisoningb1'heavy metals.alkaloidsand glycosicles. ln Westelnrnedi- 'white' variety,in wl-richthetanninis saidto havebeenp:u1lydecomposed. cine their use declined after World War II when it was tbund that Whitc galls also diff'er fiom the other gradesin having a circular tlrnnel absorbedtannic acid can callse severecentral necrosisof the liver. thlough which the insecthas emerged.Galls without the openinghave Reccntstudieshave concentratedon the antitllmoul activit)' of tannins inscctremainsir-rthesmallcentralcavity.Gallshavea veryastringent taste. (M. Ken-ichier ul., Biol. Pharnt.Bull.. 1993,16.379)and it hasbeen Sectionsthrough a gall show a l'ely lalge outer zone of thin-walled shown that. to exhibit a strong activity. ellagitannin nronourerunits parenchyma.a ring of sclercnchymatous ce1ls.and a small, inner zone having galloyl groupsat the O-2 and 0-3 positionson the -Qlucose of ratherthick-wa1ledparenchymasurroundingthe centralcavity.The core("!),as in the tellimagrandins(Fig. 22.6.) are required.Anti-HIV parenchymatoustissues contain abundant starch. massesof tannin, activity has alsobeendemonstrated. rosettesand prisms of calcir"rmoxalate. and the rounded so-called Proanthocyanidins(condensedtannins) are associatedr.vithtl-rebene- 'lignin bodies', which give a red colour with phloroglucinol and ficial eff'ectsofvariousherbsandintusionsproducecl from thern.The anti- hydrochloricacid. tumouractivitlrof greenandblackteahasbeenextensivelyrescarched in reccntyearswith positivetindings.Of thc componcntsof tca.epigallocar Constituents. Galls contain 50 1O'/r:of the tannin known as galechin-3-gallate. specifically,has beenshownto ple\ientlngiogr'nesisin lotannic acid (Tannic Acid BPIEP 2000): this is a complex mixture of mice.Cranberryjuice haslong beenusedfor reducingbacteriali nfections phenolicacid glycosides\rarvinggreatly in cornposition.It is prepared of the bladderand theseclaimshave now beensupportedby a random- by f'ennentingthe galls and extractingwith water-saturated ether.Galls izcd. doublc-blind.placebo-controlled trial can'iedout on 153 elderly also contain gallic acid (about 2 4%), ellagic acid. sitosterol,methyl w'ornen(J. Avom et ul.. J. Anter Med Assot..1991.271.751).Fmctose betulate.methyl oleanolate.starch and calcium oxalate. Nyctanthic, has beenimplicateclin tl.risactivity but recently.proanthoc)'aniciins pre- roburic and syringic acids have nrore recently been identified and paredfiom cranberies by reverse-phase ancladsorptionchromatography sydngic acid has been identified as the CNs-active componentof the were shownto inhibit the adherence of P-finbriatedE. r'oli to uroepithc- methanolicextractof -9alls.(For the isolationof llavonoidsof oak galls lial-cell sr-rrfaces: othel lZrcclnlrlr/i spp..includingbluebenieshad similar' seeM. Ahr.ned et ul., Firoterapia),1991.62.283.) bioactivity.sug-sestin-s theircontributicrn to the salutaryeffectsin r.rrinary Tannicacid is a hydrolysablctannin (sce above)yielding gallic acid tractinf'ections(A. Hou,cllet ul.. NettEttgl..1.Metl., 1998,339. 1085). and glucoseand havingthe minimum complexityof pentadigalloylglucose.Solutionsof tannicacidtendto decomposeon keepingwith formation of gallic acid.a substilncewhich is alsofound in many commercial Furtherreoding samplesof tannicacid.It may be detectedby thepink colourproducedon HaslamE, Cai Y 199.1Plantpollphenols (r'egetablerannins):gallic acid m e t a b o l i s mN. a t u r a lP r o d u c tR e n o r t s1 1 ( l ) : ' 1 1 - 6 6 the additionof a 57csolutiorrofDotassiumcvanide.
P H E N O LA SN D P H E N O L I C GLYCOSIDES Allied drugs. Many ditterentkinds of galls areknorvn.Thev aregenVolatileconpounds.althoughpresentin small amountsonlv. have erallyproducedon plants.but sometimes on animals.ln additionto the been studiedbi" GC-MS analysis.Sonte 175 compouncls have been large number producedby insects.particularlv of the gcnertrC,t,rrryr.i distinguished andclassil'ied ashomologousseriesof alkanes.alkenes. andAphis. someale ploducedby lungi. aliphaticalcohols.r'clatedaldehycles. kebnes and fatty acid esters: Chineseuttl Joporcsegulls areof considerablecornmercialimpol'tance. clistinctirerrronotelpenoids were eviclent(R. Engel et ul., Plrutto They areproducedby an aphis,Schletrerttluliuclinen.sis.on the petiolesof M e d i t t t . 1 9 9 8 6. 4 . 2 51 ) . the leaves of RhLtst'ltinettsi.s(Anacardiaceae).These galls. r.vhichthe A proccclurefbl the iclentificationand assa1, involving TLC. HPLC. 'wu-pei-tzu', Chinesecall neaning 'five knots'.ale in'egulalin shapeancl plate densitonietlyand spectrophotometrytbr the proanthocyanidins. parlly coveredwith a grei,.velvety down. the removalof which disclosesa phenoliclcids and flavonoidsin leaf ertractshas beendescribed(B. reddish-brownsurface.They breakeasilyand show a large.ine-9r.rlar car,i- VennataI rr1..Pltarnt.Acttt Helv.. 1992.67.ll). ty containing insectrernains.They contain 51-71ct of tannin and have Af lied drugs. H untuttelisbrirk occursin cun,edor channelled pieces beenval-redin China asastringentsand srypticsfor at least 1250 i,eals. which seldomexceecll0 cnt long or 2 cnr wicle.The bark is silvery CrownedAleppr.tgull,sare sometimeslound in samplesof ordinary grey and smooth.or dark srcv anclscaly.The innel surthceis pinkish Aleppo galls. They are about the size of a pea. ale stalked.and bear a and otten beals fiagmentsof uhitish n,ood.Sectionsshow a cofiex crown of projections near the apex. The insect ploducing thent is containingprismaticcrystalsof calcium oxalate.a completering of Cy'nipspohceru. sclerenchymatous cells.rnd sroup\ ol phkrernfibres.The bark conHungarittngrrllsarc prodLrced by Ctnips lignicolu on Quercusrobur tains a mixtule of haminelitannin and condensed tannin:the former gr"owingin tbrmel Yugoslavia.They are usedin tanning.English ouk has recentlybeendemonstrated tO be it potcntoxvgenscavenger(H. grzl1s,formed by Atlleritt kolluri on Quercu.srubar. contain about Masaki et al., Pltytor'ltenti.stn.1991. 37. 337). Three separate l5-20c/( of tannin. harnamelitannins, o-. B- anclir-. ale nou kno*n. The lrost important, is fbrn-red fronr tuo sallic acid moleculesand one B-hanramelitannin. Uses. Galls are usedas a sourceof tannic acid. fbr tanning ancldyemolecr-rle of the sugarhamamelose. Nr-\\cr qalloylhamameloses and ing. and in the manulactureofinks. Tannicaciclis usedas an astringcnt proanthocyanidinshavc nor.vbeen identifie-cltC. Haberland and H. and styptic. Kolodziej.Plcutru Meclictt 1994.60.-16-l:C. HartischandJ. Kolodziej. Phtrocltenti,st rt. 1996.42. 19I).
HAMAMETISIEAF HamarrrelisLeaf (witch lta:,el leaye.s)consistsof thc dlied leavesof HumameLi.s virginiunu (Harnamelidaceae). a shrubor small tree 2-5 ni high, which is widcly distributedin Canadaand the USA. It is oftlcial in the BP and is the subjectof an ESCOPnrono-eraph. Macroscopical characters. The leaves ale shortly petiolate.7 15 cnt long. md broadlyoval to ovatein shape;baseasymmetricallycordate.apex acute.The lamina is dark brownish-green to green in cololn'and very paperyin textule.The venationis pinnateandthe mar-eincrenateor sinuatedentate. The veinsiu'everyconspicuous on thelou'ersufaceltheyleale the mid-rib at an acuteangleand run straightto the rnlugin.where thev telrlinatein a marginalcrenation.Odour,slight:taste.astringentar"rdbitter. The BP drug is requiredto contain not more thanic/c of stemsand not more Ihan2Tcof other tbreign matter.
Uses. Hamamelisowes its astringentand hue-lt.rostatic propertiesto the tannins.Hamanrelitannin and thc gallorlatedproanthocyanidins isolated f'rom H. rirginiana are rcported to be potent inhibitols of 5lipo-oxygenase. supportingthc anti-inllanintatorv action of the dlug (C. Hartischet ol.. Plonta Mediccr.l997. 61. 106).The abovecompounclsare presumablynot presentin HantantelisWater or Distilled Witch Hazel. which is, however. wiciely usr-d as an application to sprains.bruisesanclsuperficialwoundsanclas un ingredientof eye lotions.It containssafioleandothefvolatrlecomponcnts.
HAWTHORN
The leaves. 1'lowersand t-alsefiuits are all nreclicinallyuseful, the leavcsand flowers being usedprincipally fol the preparationof infusions,etc. with thc lruits ernployedin the nranulncture of prepared rredicaments.The dliecl fhlse frLritsof Crutoegu.srttortogtttttand C. Microscopical characters. The dlug has very distinetir e microscop- laevigttta.family Rosaceae.togetheru'ith theil hlblids are ofTicialin ical characters.These include chalacteristicstomata presenton thc the EP. BP and BHP: similally the leaf and llor.i.'er. fbr which there is lower surfaceonly: vely large lignified idioblasts.crystalcells accom- also an ESCOP rnonograph.The drug has recentll"been exhaustively panying the pericyclicllbres. tannin-containingcells and. especiallyin treatedin an American Herbal Pharmacopoe ia publication. youngleaves,stellatehairs.The calciumoxalateis in monoclinicprisrns The thorny. deciduor.rs t[ees :rre native to Europe and have a long l0 35 ptm long. The stellatehairs (Fig. 43.1H) consistof .1 l2 cells medical and etl-urobotanical l'ristory.Cornntcrcialsuppliesof the dried fruits.requiredto containnot lessthan 1.0,qiprocyanidins.originate unitedat the base.Eachcell is thick-walledand Lrpto -500pin long. from EasternEnropc. Constituents. Hamamelis corrtainsgallitannins,ellagitannins.flec gallic acid, proanthocyanidins,bitter principles and tlaces of volatile Characters. Characteristicof tr number of genera of the fiimily oil. With ferric chloridesolntionthe gallitanninsandthe tiee gallic acid Rosaccae. so-calledhawthornbeniesarelalscfiuits (pontes.andnot in the give a blue color.rand the ellagitannins._qreen. stlict botanicalsenseberries)in which the carpelsbecomeadherentto the The phalmacopoeia(BP/EP 2000) r'equiresthe leavesto contarnnot hollow.fleshvreceptacle andthe sepals.petalsandstiunensbecomesitua! less than 307ctannins;thesetanninsfepresentthe dilTelencebetween ed at the upperend of the fluit (Fig. 42.51.The carpelsbecomestonyso the total polyphenolcontentof the leaf and the polyphenolcontentnot thatthepomecomesratherto resemblea drupe(Ch.42).The talsefrlits of absorbed by hide powder. Reagents employed in the assay are C. rnonogvtttwith one ciupel containa singlestony true fruit whereas Phosphomolybdotungstic reagentand sodiurncarbonatesolr.rtionwith thoseof C. laet,ig,utawith two or thlee carpelscontaintwo or threefruits. absorbancemeasufements r.nadeat 76[Jnnr: pyrogallol is usedas a test The dried reddish-brorvnto dark red fruits have a slight odour and solution. The leaves appear to contain no hamamelitannin(see mucilaginous. slightlyacid taste;with C. tnonog\n(lthey areup to l0 'Hamanteli: B a r k ' ,b e l o u1 . mm in length and slightly larger fol C. luevigntn.At the uppel end of
P H A R M A C O P O E IA N L D R E L A T EDDR U G SO F B I O L O G I C AO LR I G I N the false fruit are the remains of the flve reflexed sepalswhich surround a shallow depressionfiom the baseof which arisestifTlignitled tufts of trichomesand the rcmainsof thc style (two styleswithC. lttevigttta).The baseof the fi'uit may bc eitherattachedto a pedicelor show the scarof attachmerrtof the latter. In additionto the long. lignified. taperingclothing trichomesof the innersurfaceof the receptacle othermicroscopical1-eatures include:cells of the outerreceptacleu'ith red pigmentationlsclereids;calciumoxalate as clustersand in flles of cells as prisms: seedfiagments showing a mucilaginoustestaandembryocellscontainingaleuroneglainsandlixed oil. A rnoredctailcddcscriptionwill be fbund in thepharmacopoeias. Constituents. The fi'uits contain l-37c oligomeric procyanidins,the strLrctures of which appearto be only paltially ascertainedtogether with flalonoids.principallyhyperosideabout l7c. The leavesin contrastcontainlesshyperosideand mole vitexinrhamnoside. Thin layer chromatographyof a methanolicextract of the drug and lluorescencevisualizationat 365 nm is usedas a test for identit1. Procyanidinsare evaluatedby acid hydrolysisof an alcoholic extract followed by absorbancemeasurementsat 5;15nrr of the procyanidinsproduced. butanol-soluble Allied species. Adulterationof the readily-availableproduct is rare: other speciesoI Cratuegusmay be detectedby their having more than threeseeds.C. pinnatifitlu fluits areusedin Chinesemedicine.
The bark readily separates from the wood. The fbrmer is astringentbr-rt the latter almosttasteless. The tanninsof kranleriaroot (kameda-tannicacid)areentirelyof the condenscd(proanthocyanidin)type having a 'polymeric' flavin-3-ol structure.In this instancethele is a procyanidin:propelalgonidin ratio of 3-5:65asdetenninedby acidhydrolysis.Astringencyof the root is due to compor.rnds with a degreeof polyrnerizationof more than tive. (For further detailsseeE. Scholzand H. Rimpler.Planto Med., 1989,55,379). Aphlobaphene(krameria-red),starchand calciumoxal:rtearealsopresent. Stahl and Ittel (1981) reportedthe isolation of two benzoftran derivatives,ratanhiaphenols I andII. from theloot. Both compoundsare effectiveu.v. light filters and coLrldbe usetulin sun-protectionpreparations.The BP and EP includean assayfbr tannins(pollrphenols)of not lessthan 5.0clcbasedon the cololrr reactioninvolving alkalinesodiunl phosphomolybdotungstate(absorbance measured at 760nm). Polyphenolsnot adsorbedby hide powder, also determinedwith the samereagent,areexcludedfiom the calculation. The drug is used as an astringentand the significant antimicrobial activity of the extract gives rational supportfbr its use in mouth and throatinf'ections.
\7\A-y-<'RI l l t t \rAoA1A
l t
t
l
.A.Aon2 Uses. Hawthorn is widely used as a mild cardiac tonic particularly for patients of advancing age. It does not have the toxic ef-tectsof Digitalis and can usef'ullybe employedbeforerecourseis rnadeto the digitalis cardioactiveglycosides.
AtCHEMIttA The flowering and aerial parts of the lady's mantle,Alchenillct xtutaredescribedin thochlora(A. vulgoris.ren.su latiore),lamily Rosaceae, the BP 2000 andBHP 1996.The plant is widespreadin Europe.North Americii and Asia; commercialsuppliesare obtainedprincipally tiom EasternEurope. In addition to the identificationby macroscopicand microscopic charactersthe pharmacopoeiasinclude thin-layer chrozones. matographictestsproviding characteristicf-luorescent The -BP drug is required to contain not less Ihan 7.5Vcof tannins expressedas pyrogallol when determinedby the oflicial method (cf. Hamamelisand Rhatany).The characterizedellagitanninsare pedunculagin and the dimeric alchemillin.Other constituentsare flavonoids, qr.rercetin3-O-p-o-glucosidehaving been isolated as the major flavonoidin leavesof Frenchorigin. Alchemillaactsasan astringentagainstbleedinganddiarrhoeaandhas a long traditionofuse for gynaecological conditionssuchasmenorrhagia.
RHATANY Rhatanyof the BP and EP (Krcunerialis the dried root ol Krarneria trianr/rz (Krameriaceae.a small family related to the Leguminosae),a small shrubwith dccumbentbranchesabout I m long. The drug is collectedin Bolivia and Peru and is known in commerceas Peruvianrhatany. The root has a knotty crown severalcentimetresin diameter and gives ofT numerousbranchroots some ol which attain a length of 60 cm. The roots are nearly cylindrical and are coveredwith a reddishbrown colk, which is scaly exceptin rery young roots.A transverse sectionshowsa reddish-brownbark which occupiesaboutone-thirdof the radius and enclosesa yellowish. finely radiate wood. A small, deeplycolouredheartwoodis sometimespresentin the larger species.
RatanhiaphcnolI: Rl = H; R2 = Me; R3= OH II: Rl = Me; R2 = R3 = H Ratanhiaphenol Allied species. The roots of several other species are occasionally encounteredin commerce,but the Pem'ian dn-rgis the only one generally available.Krumeria cJ',rtisoide,\ of Mexican oligin hasindigenousmedicinal uses.It containsover20 compoLrnds of the ligniur,neolignanandnorneolignantype.Similarconstituents arereportedfor K. lanceolctla; seeH. Achenbach etcI.,Phytctchemisln, I987.26, I|59'. 1989,28. I959. Pomegronote rind This consistsof the dried pericalp of the fruit of Punicu gronatltnl (Punicaceae). It occursin thin. curvedpiecesabout 1.5 mm tl-rick.some of which bearthe remainsof the woody calyx or a scar'left by the stalk. The outer surfaceis blownish-yellow or reddish.The inner surface bearsimpressionsleft by the seeds.Pomegranaterind. usedin India as a herbalremedy for nonspecificdiarrhoea.is very astringentand contains about 28c/r:of tannin (ellagitannins)and colouring matters. It shouldbe distinguishedfrom the root bark. which containsalkaloids. Aspidospermq bqrks The bark of Aspidospermatluebruclurblnnr:o(Apocynaceae), which is used as a tanning material, was lbrrnerly ollicial in severalpharmacopoeias.
Myrobolons Myrobalansarethe dried fiuits of Terntinoliachebulu(Combretaceae), a treecommon in India. The immaturefruits areblack.ovoid and about 1-3 cm long. They contain about 20-zl07c of tannin, B-sitosterol. anthraquinones and a flxed oil containingprincipally estersof palmitic. oleic and linoleic acids.The tannin and anthraqr.rinone constituents make the drug both astringentand catharticin action.Microbiological testssupportthe Indian use of an aqueousextractof the fruit as an anticariesagent(A. G. Jagtapand S. G. Karkera,J. Ethnophurnutcologt', 1999.68.299\.
PHENOLA SND PHENOTIG C LYCOSIDES
CAIECHU Garnbil or pale catechuof the BP 19891'BP (Vetelinary),2000 is a dried, aqueor"rs extractpreparedfiom the leavesand young twigs of a clirnbing shrtb, Uncuria ganrbir (RLrbiaceae). It must be carefully distinguishedfrom black catechuor cutch.The plant is a native of Malaya and it is largely cultivatedlor the prodr.rction of the drug in Indonesia and Malaya fbr rnarketingthrou-ghSingapore.
@
by lossof the elementsof water.It readily yieldsthe phlobaphenecatechu-red.If the drug is caretullyprepared,it r.villcontaina high proportion of catechinand conespondinglysrnalleramountsof catechutannic acid and catechu-red.
Aflied drug. CLrtchor black catechu is an extract preparedfrom the heartwoodof Acacicrcatechu(Leguminosae).Cutch occurs in black, somewhat porous masses.The taste resemblesthat of gambir. Microscopicalexaminationof the water-solubleresidueshows wood History. The catechr-r describedby Barbosa(1514) was black cat- tlbres and large vesselsand sometimesfragments derived fiom the echu or cutch. and the first accountof gambir appearsto be that of a leaveson which the drug is spread. Dutch traderin 1780.ln trdditionto the cubegarnbirusedin pharmacy, Cutch contains 2 12Vc of catechins. 25-33c/a of phlobatannin, lalge blocks of the extlact are importedfbr use in dyeing and tanning. 20-3OVoof gummy matter,quercitrin.quercitin. moisture,etc. lt yields Other fbrrnsareusedin the Eastfol chewingwith betel leaf. 2-3c/col ash. The catechin(acacatechin)is not identical with that in gambir.The drug may be distinguishedfrom gambir asit gives no reacPreparation. The preparation of catechu in Johole diff'ers only tion for gambir-fluorescin. slightly fiom the procedureadoptedin Indonesia.It consistsof extracting the leaves and young twigs with boiling water, evaporatin-qthe Uses. Catechuis usedin r-nedicine as an astrinsent. extractto a pastyconsistencyand dividing it into cubes.which are then sun-dried.Fuller detailsof the preparationaregiven in the I Othedition. Kinos Many difl'elentfbrms of catechuare used in the East,and the drug The name'kino'hasbeenappliedto a nun.rber of driedjuices,rich in phiofor the Easternmarketfiequently has 20 50rlcof fine rice husksadded batanninszurdformerly usedfbr their astringentpropenies.They include as the liquid coagulatesin the tubs. Such catechuis. o1'course.r.rnoftlMalabarkino from Pterocarpusmarsupiurn(Legunrinosae). buteagum cial. andcontainsstarch. or Bengal kino from Butea.frondosa(.8. morutspennn)(Legurninosae) and eucalyptuskino or red gr-rmfrom EucalyptLtstostroto ( Myrtaceae). Macroscopical characters. Catechuoccursin cubes.which are very tiiable and may be brokenup in transitor, if incompletelydried,may be Crolon lechleri more or less agglutinated.Of the samples available, those from Indonesiameasurell-22 nn and have a reddish-brownsurface.olten The b:irk of this and relatedeuphorbiaceoustreesyield, when slashed.a stampedwith a maker'smark. while thoseliom Johoremeasure21-29 blood-red sap commonly known in South American folk medicine as mrr andhavea blackishextedorandthe lacesof the cubearedepressed. Sangrede Grado, Sangrede Drace,or dragon'sblood (not to be confused Internally.both varietiesare cinnamon-brownand porous.Odourless; with the dragon's blood obtained from speciesof Daemonoropspalms. antitumourandwoundhealing taste.very astringenttrndat first somewhatbitter,afterwardssweetish. q.v.).It is usedlocallyfor its anti-int-ective, propefiies.Cai er al. (Phytochemi.sttl'. 1991,30, 2033; 1993,32. 155) (c'.90c/r) to be the principalconstituents Microscopical characters, When mounted in wateq catechu shows have shownproanthocyanidins which vary fiom polyphenols monomers to heptamers. These possess minute.acicularcrystalsof catechin.many of which are branchedand oxygenfree-radicalscavengingactivityand may assistin woundhealing interlacing. They dissolve on warming and a considerableamount of 1997,58, 103).Minor vegetabledebrisis left. The leaves.particularlythe stipules,bearsimple, (C. Desmarchelieret al., J. Ethnophnrmacology. unicellularhairsup to about3,50ptmlong. with sn.rooth.moderatelythick. componentsisolatedarephenols,alcohols,sterolsandfour diterpenoids, lignified walls. The twigs have lignified pericyclicfibres.wood fibres, two of the latterbeing of the clerodanetype.An alkaloid,tapsine,hasbeen grainsarecommonly ascribedas the wound healing constituent;it could also accountfor the and spiral,annularandpittedvessels.Minute sftLrch present,pafticularlyin the lndonesiandrug, but the amountshould be antitumour activity claimed for the latex (2. Chen et al., Pltutta Medica, 1994.60.541). strictlylimited.Rice huskshavebeenobservedin somesamples. Chemical tests (l) For gambir-.fluorestitt. Extracta little of thepowdereddm-9with alcohol and filter. To the hltrate add solution of sodium hydroxide.After shaking,adda f-ewmillilitres of light petroleum.shakeagainand allow to stand.The petroleumspiritlayershowsa sffonggreenfluorescence. (2) For c'atechin.This is a rnodificationof the usual test fol lignin. Phlorogiucinolis lbrmed fiom catechinand with hydrochloricacid turns a matchstickred.
'
i t
. : . .
. : . : . a . . . a ' . . . . . :
: : :
: :
. l . . l i
. r ' , 1 : . i i . ' . r : . , . 1
AND,GIYCOS|DES,,, rcoumARNSr
Derivativesof benzo-o-pyronesuch as coumarin (the lactone of Ohydroxycinnan-ric acid), aesculetin.umbelliferoneand scopoletinare common in plantsboth in the free stateand asglycosides.Not all arephenolic br.rtthey are included here with the phenolic derivatives for convenience.Some 1000 naturalcoumarinshave beenisolated.Coumarin itself hasbeenfound in about I 50 speciesbelongingto over 30 diff'erent families,althoughit is probablypresentin the undamagedplant as/rrarsConstituents. Gambir contains abou 7 .33% of catechins.22-50c/o O-glucosyloxycinnamicacid. Enzyme activity in the damagedtissue leads to a loss of glucose and a trans -+ cis isomerization foliowed by of catechutannicacid, catechured. quercitinand gambir-f'luorescin. BP (Vet.) 2000 standardsinclr-rdea loss on drying of not more than ring closure. Coumarin gives a characteristicodour of new-mown hay 15.0%and a water-insolublematterof not more than 33.07cwith ref'er- andoccursin many Leguminosaesuchassweetcloveqmelitotandtonco beans:the lattercontainabout l-37c of coumarin.It is also recordedin enceto the dried material. Catechintblms white. acicularcrystalswhich aresolublein hot water u'oodruff. A.rper Ltla odoruta (Rubiaceae). In ammoniacalsolutionthesecompoundshave a blue, blue-greenor and alcoholand give a greencolour with ttnic salts.Catechutannic acid is an amorphousphlobatanninwhich appearsto be fomed fiom catechin violet fluorescence,which has long been usedas a qualitativetest for
P H A R M A C O P O EAIN AD TR E L A T E DD R U GO S FB I O L O G I CO AL RIGIN
m \Ao-.o a-Pyrone
Coumarin
7 )
-j
\i
\ i
cHidn, Archangelicin: Rr = R2 = angeloyl Apterin: Rr = H; 3z = gluc. ( C o u m a r i n+ C s - u n i t )
certainr-rrnbellif'elous resinssuchas asalbetidaand galbanur-n. The fluorescenceis. of coulse. more marked if examineclin filtcred Lrltraviolet light iind is uscd tbr the chlomatographic visualizationof the compounds. The sr-rbstitution patternsof sonte common hydroxy and methoxy corLmalinsare given in Table 22.2. Strr,rctr.rrally more cornplex coumarinssuch as the calanolidesand inophyllurrshave received recentattentionaspotentHIV- l-RT inhibitors(seeChapter32). TheJuranocotuntu'ins arecloselyrelatcdto the irboveandoccurparticularly in the Rr-rtaceae and Untbellileare.Fol cxantple.celervfl'uitscontain rutaretinand its dehydratecl delivativeapiut'netin. Bergaptenoccurs in berganrotoil and in the Chineseroot-drugderivedtrorn PeucetLuttutt (Umbellif-erae) clecursivtun. which also contairrsthe less-commonp\.runot:oLuttcu'it'ts. Marmesinderivatives(Fig. 11.2)and archangelicin havea reduceclfuranocor-rmarin struclureconsistingof coulnarinand a C5 subunit. Otherprenvlatedcompor.rnds arethe3-iso-prenl,lcor.rmalins, asillustratcd by rutamarin of the genus Rura: fbl recent researchon R. graveolen.s seeS. D. Srivastavaet ul.. Fitoteruplzi.199{i.69. 80. A wide rangeof biologicalactivitieshasbeendemonstrated fbr thesemetabolites (seeR. H. Galln et al..Plttttx'hentistrt'. 1990.29.2053) Furanocoumarins areresponsibleat leastin part fbr the unpreclictable and ', ariableeff'ectson dru-gavailabilitylesultingfrorr the consumption
loble 22.2
Hydroxy
Bergapten (a furanocoumarin)
O -o-methylbutyroyl Visnadin ( C o u m a r i n+ C s - u n i t )
juice. Two componentsof thej Lrice(6'. 7'-dihydroxyberg of grapeflLrit amottin and FC26) inactivatecytochromeP450 enzymes(specificallr CYP3A4 and CYP3A5) resultingin an increasedoral bioavailabilitvot' variousdrugsr.rsed to treatcancer.hypertension.heafi diseaseand allergies. However. unnamedconstituentsof the juice have recently been shownto i,rctivate the efllux pump controllin-qP-glycoprotein-mediatecl drug transportwhich secretesabsorbeddrugsback into the gut.Irt vitro stndies har"e demonstratedleduced absorption of vinblastine. cyclosporin.losartan.digoxin and fexof'enadine.The two eff-ectsarc thereforeantagonisticand explainthe unpredictableactionofgrapefiuit juice on dru-ebioavailability. Fol reports on this researchsee l/ic Ltttu'et.1999,353, 1335:Pharm.J.,1.999.262,513HerbalGram.199[i. No.213.22. Arnrll speciescontainluranornethoxycoumarins br.rtaremore imptrr' (q.v.under''FIavones'). tant fbr their contentoffllranobenzo-1-pyrones Bicoumarinsare fbrmed from two coumarin ntoietiesand the link age may occur in a number of ways. Dicoumarol is lblmed at C3-C3' througha methylenegroup and was,in 1941.the first of the seriesto bc isolated.It is a constituentof f'ermentinghay and is formed br microbial action of coumarin. It is a powerful anticoagulantand haemorrhagicand can cause the death of animals consuminsthe spoiledfbdder.
ond methoxy coumcrins.
Compound
Additionolgroupings
Occurrence
Umbelliferone
HO ot Z (obove)
H e r ni o r i n
C H 3 Oo i Z
Aesculetin
H O o t6 , H O o t 7
S.^^^l^r;^
C H 3 Oo t 6 , H O o t 7
Froxin Chicorin
CH3Oot 6, HO ot 7, Oglucoseot 8 CH3Oot 6, Oglucoseot Z
Bellodonno ond stromonium Dophnemezereum {Solonoceoe}: Ferulospecies yieldingosofoetido ond {Thymelioceoe); golbonum,ond monyotherUmbelliferoe, chicoryleoves (Compositoe) Lovondulospico{Lobiotoel,Rutagroveolens(Umbelliferoe} ond certoinCompositoe (Hippocostonoceoe), Horse-chestnut certoinRosoceoe ond Froxinus{OIeoceoe) Rootsof gelsemium, oot, iolop,scommony, scopolioond bellodonno; leovesof tobocco,slromonium, chicoryond monyothers) Froxinus spp.{Oleoceoe} Cichoriumintybusherb
PHENOLA SND PHENOLIC GLYCOSIDES
Furfher reoding E s t 6 r ' e z - B r a u n A - G o n z i JR e zG 1 9 9 7C o u r n a l i l r (s1 9 9 5 1 9 9 6 ) .N a t u r a l ProductReports | .1(5): :165-.176 Murray R D H I 995 Couuurins ( I 9t3ll I 99-1).Natnral Ploduct Reports 12(5.): 177 506
Tonco seed Tonco seedor Tonquin beansare the drieclseedsof Diptert,r otktratu Willd. lCr.,anruroutu{ otlorotu Ar-rbl..1 and Diptert.r opposit|fblia (Legurninosae). The lbrmerplantis a natir,eof GuianaanclBlazil and is extensively culti'n'atedin Venezuela,rvhile the liltter is lor.rrrdin Gr-riana and northernBrazil. Both arc largetleesbearingsingle-seeded fiuits about3 5 cm long. The fiuits are collectedvn,hen lipc (Ma1'and June).thcy are opened and the seedsare dded in the sun. If sold without lurther treatmcnt. 'black'beans. they are known as The sccdsploduccd in Vcneznclaand near its border are lar-9elthan those producedin northeln Blazil and partsof Gr.riana. The fbrrner.which are more hi-ehlyvaluecl.are knoln 'An-uostura' 'Para' as and the latteras beans.Largequantitiesof both Angosturaand Parabcansare sentto Trinidad. wherc thc\, trrcr.n'rcer'ated fbr 24 h in lum and dried in the open air. This trcatmcntcausesa crystallinedepositof coumarinto be lblmed on the testaand the sccds 'fiosted'. are said to be Angosturaand Para bcansthus occllr ln co[rnrerceboth black and fiosted. Tonco beansare Lrpto'l0mrn long. l0mm wicleancl5 mrr thick. They ale ror-rnded at orreencland blLrntlypointeclat the other.The surfaceis black and deepl1'wrinkledlongitudinally. a crystallineencrustation being presentin the ftosted variety.A transversesectionshowsa very thin. black testa and two vellowish-brown,planoconvex.oilv cotyledons.Odour.vely tia-erant:taste.aromaticand bitter. Tonco beanscontain l-37c of coumarin.25(/cof fat (containing and a larger alrount of unsaponifiablesitosterinand stigrnaslerin) starch.Ash about3.5%.Toncobeansare usediu tobaccomanufacture and in pelfurnery.Syntheticcoumarinh:rs.to somccxtent.replaccdthe natulal product.
-!r
w
tor elltet: anclarecalciunrantagonists. In herbalmedicinethe root is inclicutecl ilr thc tlcatmentof bronchitisassociated with vasculardcficiencr.
NONESAND GTYCOSI ANTHRAGIUI DTs l-ong belbre an\thins riu: knor,u of their chemistry.rhubarb,aloes, senrriland cascririlrrcrc rccognizcclas fbrming a natural group of purgati\edtugs..\l:o errteirrrcgctableand anirnaldyestuffssuchas maddcrand cochincal\\L'r'er)l r:rerteconomicimpoltancebefbrethe introductionof s1nthctierhcstLrl'l's. Later the chemicalsirnilarityof tl'rcscpulgativcdru-lsaurltlre':tLrlls bccirnrerpparent,as il]ustratedby the tbrrnulaefil' enroclin(thc riqlreLrncof a numberof purgativeglyco(thc uslyconeof a dyestr.rtTof sidcsof Rlrlruir,r spp.)lutl ulizrLr-rrr the maddelnlant).
Emodin
Alizarin
S u b s t a n c eosf t h e a n t h l a q u i n o nter p c \ \ . r t t h e t i r . t l o b g ; g g 1 ; o n i z e c lb, o t h i n t h e f r e es t a t ea n d r s g l r c o s i . i c . .F L r r t h c\r\ ( ) r k. h ( ) \ \c d t h a t n a t u r a l p r o d l l c t sa l s o c o n t a i n e dr e d u e c t l. i t r i r r t i r e . ( ) t t h L . r .rnJ !()lr)(oxanthrones.ilnthrtltol\ antl lrnthr,,ne nnthlaquinclnes pounds fbl-ned by thc union of tu,o anthrollL'nr(Jlc.uli. i.c. the dianthloncs). Becausg e l i , c o s i d easr eo f i e ne a s i l l ' h 1c h o l r : c t l t. h . ' c i . l r l i .\f\ ( ) r k . r \ 1 "11!11rathcrthrll lhc 1.ri111.11 tendedto isolateploductsof hy'clroll'sis s i d e sT . h e f o l l o w i n ga g l y c o n c h s a v c l o n g h c ' c nc ' t l l . l i . h c . l :. h n : o Cefery frvit. Apium; Apii Fruclus phanolor chlysophanicacid f}om lhubarbantl erL..ura:rl().-enrodin The drug cor.rsistsof the clried ripe ti'uits of ATtiuntgruveolett.s from rhr.rbllband senna:rhein from lhuhlLrhirnli \.nnir: clrodin or (Umbellif'erac). frangula-er-nodin from rhubarb ancl clt:curu. lnrpr\)\ad c\traction The cremocarpis brown. subspherical and about 1 1.5rnm long.The methods,cleveloped by Stoll trncihis colleaguc\.lcJ ttr thc i:olationol rnericarpsare nrostlyseparatein the dlr.rgand eachshowsfive straight t h e m a i n s e n n a - e l y c o s i c l esse.n r r o s i d e si.r\n r l[ J . i n l 9 - l l . S i n c ct h i s pdmary ridges.A transverse sectionis almostpentagonaland shor"'s6-9 date manl' new glycosidcs inch-rdingC Slr crr'iJc's and r arious vittae.two on the cornnrissural surfacearrdfbur to sevenin the grooves stcrcoisomcls havc beenisolatedanclthcir \trLre turr'\ dctcrmined. of the dorsal surface.Odour and taste.alomatic.Celcry fi'uits contain In monocotyledons. clerir.rtir e: ure filrnd only in the anlhraclr.rinone 2 3c/c of oil consisting of terpcncs with srnallcr qr-lantitiesof thc Liliaceae.in the forrn of the unusual(:::lreo:ide barbaloin.Among anhydrideof sedanonicacicl.the lactoneof sedanolicaciclanclphenols. dicotyledons Polygonaceae, theyoccurin thc Rubilcr-lc.L!'SrLnrinosae. The fiuits also contain a numbel of cour.narins. firranocoumarinsand Rhamrraceae, Ericaceae.Er.rphorbilceric.l-\ thmceae.Saxifragaceae. coumalinglycosides. The'r appclr to be absentfrom the Scrophularilceaennd Verbcnaccac. Celeryfiuits areofllcial tnthe BHP and havea long-standing useirr Bryoph-vta.Ptcridophytaand Clnrno\p.rnr: but occnr in certainfirngi the treatmentof rheunraticdiseases: the theraper.rtic a(jti(rnrppear'\t() and lichens.Thc fungal anthraquinon!pisnrentsare nearly all chryso(q.v.). be potentiated by Taraxacurn phanolor emodinderivatives. As indicatedin Chaptel 19. naturulanthraquinones are synthesized Angelico either r,ia the acetate malonatc piith\\'av or they are derived fiom The leaf and foot of this plantilfe both ofTicialinthe BHP and alsohar,e shikimate and nrer"alonatc. Thr- rncdicinally important purgative in the USA and in China.The Er-rropean drug is derii'ed anthrzrquinones.are traditionalr.rse fbrmeclbr the ltrrmerroute and all have a 1,8(Umbelliferae). frontAngeLicuarcltungelica the Nofth Amclicanfronr dihydroxy substitution.Convclscll".compounds such as alizarin A. atntpurltureatrnd the Chir-rese liom a nnmbcr of speciesundcr the u'hich har,eone of thc lings r-rnsr,rbstituted arise by the secondpathnamerlilt-nlr or tLulgkltei.Considerablerecentrvork orr the genushas wav.The lelationshipsbetr,reen the oxidizedandreducedforns of the resulted in the isolation of a numbel of fulanocoumarinsand their anthraquinure n in Fig. 22.8. nucleusaresho'nr glycosideslthe folrnr"rlaeof belgapten.alchan-relicin(a diester) and Moderrr reseiirchindicatcsthilt the 1.8-dihydroxyanthraquinone apterinare given aboveand thoseol' marmesintrnd psoralcnin Fig. d e r i v a t i v e s f r e q r . r e n t l l o ' ccur with 1,8-dihydroxynaphthalene 11.2.Thesecompoundsare leportedto have potentcoronaryvasodila- g l v c o s i d e s .
PHARMACOPOEIAL AND RELATED DRUGS OF BIOLOGICAL ORIGIN Anthraquinone derivatives are ofien orange-red compounds. which may sometimesbe observedin situ (e.g.in the medullaryrays of rhubarb and cascara).They are usually soluble in hot water or dilLrtealcohol.Borntriiger'stestis otten usedlbr their detection.The powdereddrug is maceratedwith an imnriscibleorganic solvent. etheris recommended, and afterfiltrltion aqueousamlronraor caustic soda is added,when a pink. red or violet colour in the aqr"reous layer after shaking indicatesthe plesenceof free anthraquinone dcrivativcs.lf glycosidesonly arc prescnt.the test shouldbe rnodified by first hydrolysrngwith alcoholicpotassiumhydloxidesolution or'2 rt acid. When alknli is addedto powdereddrugss1 to seetions. the red colonr produced servesto locate the anthraquinonederivatives in the tissues(e.g. in the rnedullaryrays of cascal'abark). If the drug being tested contains either very stable anthraquinoneglycosidesor reducedderivativesof the anthranoltype, Borntriiger'stest will be negative. Anthraquinones containinga freecarboxylicacid group(e.g.rhein) can be separatedfl'om other trnthraquinonesby extraction fiom an orsanicsolutionwith sodir.rnr bicarbonate solution.
OH
ZYY\
t
\\/\\
I
t
i
-\-t
l
l
I
H Anthranol
lt 2
4H
o Anthraquinone
Anthrone
II
I
|-rr
o
lrr
I
I
o
Oxanthrone
Dianthrone
Fig,22,8 I n t e r r e l o t i o n s h iopf o n t h r o q u i n o n ed e r i v o l i v e s .
Anthroquinones The derivativesof anthraquinonepresentin purgutivedrugs may be dihydroxy phenolssuch as chrysophanol.trihydroxy phenols such as emodinor tetrahydroxyphenolssuchascarminicacid.OthergroLlpsare often present,lbl example,methyl in chrysophanol,hydroxymethylin aloe-emodinand ciLrboxylin rhein and carminic acid. When such substancesoccur as glycosides.the sugarmay be attachedin variouspositions. See lbrmulae for carminic acid and glucofrangulin. Some derivativesaregiven in Table22.3. examplesof anthraquinone
Toble 22.3
Anthrqnols qnd qnfhrones These reduced anthraquinonederivatives occur either liee or comThey are isomericand one may be partiallyconbinedas -elycosides. velted to the other in solution.The parentsubstance, anthfone.is a pale yellow, nonfluorescentsubstancewhich is insoluble in alkali; its isomer,anthranol.is brownish-yelloirand ftrrmsa stronglyfluorescent solution in alkali. Anthlanol derivatives.sr.rchas are found in aloes, have similar properties, and the strong green fluorescence which aloesgive in boraxor otheralkalinesolutionhaslong beenused as a test fbr its identiflcation.Anthranols and anthronesare the main prepared by constituentsof chrysalobin. a mixtule of sr.rbstances benzeneextraction 1i'om the material (araroba)fbr-rndin the trunk cavitiesof the treeAndi ra a rarobo. lf a little chrysarobinis tleatedon a white tile with a drop of fuming nitlic acid. the anthranolsare converted into anthraquinones.A drop of zunmoniaallowed to mix gradually u'ith the acid liquid produces a violet colour. This modification of Bolntldger's test had been used as a test for identity befbrethe underlyingchemistlywas known.
Anthroquinone glycosides ond oglycones. Aglycone
Glycoside P,,h..r/h.i. ^.i.1
Aglycone
Alizorin Rubiodinprimeveroside R u b i o d i n R u b i o d ignl u c o s i d e Rubiodin Chrysophonein Chrysophonol Rheochrysin Physcion Rhein Glucorhein Aloe-emodin Glucooloe-emodin Glucochrysoron Chrysorone f mootn Glucofrongulin A Frongulin Emodin Morindin Morindone lslondicin L O r m r n rocc r o
Sugor Primeverose Primeverose Glucose ^1 \rlUCOSe
Glucose Glucose btucose Glucose G l u c o s e ,r h o m n o s e Rhomnose Primeverose
:l,lucose
OH Groups
l ?
I,B I,B 1,8 1,8 1,2,7 1,6,8 1,6,8 1,5,6 1,5,8 | , 3, 4 , 6
Othergroups
Occurrence
2-methyl 2-methyl 3-methyl 3-methyl6-methoxy 3-corboxylic ocid 3-hydroxymethyl 6-methyl 3-methyl 3-methyl 2-methyl 6-methyl 5-corboxylicocid 8-methyl
Rubiotinctorum Rubiotinctorum Rubiotinctorum Rheumond Rumexspp. Rheumspp. Rheum,Rumexond Cossiospp. Rheumond Cossiospp. Rheumrhaponticum Rhomnus spp. Rhornnus spp. Morindospp.(Rubioceoe) Penicillium islondicum Cochineol
PHENOLS AND PHENOLIC GTYCOSIDES Oxqnthrones The formula given showsthat theseare intermediateproductsbetween anthraquinones and anthranols.They give anthlaquinoneson oxidation and Fairbairn'srnodiflcationof the Borntr:igertest accomplishesthis by meansof hydrogenperoxide.An oxanthronehas beenreportedas a c o n s t i t u e notl c r s e r r ab a r k . Diqnfhrones These are compoundsderived fl'om two anthronemolecules.which may be identicalor dilTerent;they readily fbrm as a resultof rnild oxidation of the anthroneor mixed trnthrones(e.g. a solution in acetone and presenceof atmosphericoxygen).They are irnportantaglyconesin speciesof Cassia,Rlteunt and Rhantnusiin this group the sennidins. aglyconesof the sennosides(seetbrmula). are among the best-known examples.ReidinA. B and C which occur in sennaand rhLrbarbareheterodianthrones.i.e. composedof unlike anthrones.and inl'olve aloeemodin.r'hein.chryophanolor physcion. It will be notedthat two chiral centres(at C- l0 and C- I 0') are present in the dianthrones,and fbr a compound having two identical anthronemoieties.e.g.sennidinA, two forms (the 10S,l0'S and l0R. 10'R) are possibletogether with the neso lbrm (sennidin B). These compoundsalsooccurin the plantastheir 1,1'-diglucosides.
Aloin-fype or C-glycosides The aloin obtained fiom speciesof Aloe, although one of the first glycosidesto be isolated,was a problem for investigatorsfbr a long time. lt is stronglyresistantto normal acid hydrolysisbut rnay be oxidized with ferric chloride. A study of its degradationproducts and inflared spectrumindicateda sugar-likechain and the structureshown. in which the sugaris joined to the aglyconewith a direct C-C linkage (a C-glycoside).Two aloins (A and B) are known and arise liom the chiralcentreat C-10.
ale small shrubsof the lamily Leguminosae.about 1 m high. with paripinnatecompoundleaves.C. sennais indigenousto tropicalAfrica and is cultivatedin thc Sudan(Kordofan.Sennar).C. tutgustifoliuis indigenousto Sornaliland. Arabia.Sind and the Punjab.and is cultivatedin South India (Tinnevelly).The botanicalvalidity for distinguishing betweenthe abovetr,r,oplantshasbeencalledin question(Brenan,Kex Aa1l.,l9-58.l-ll ). but Fairbairnand Shlestha(Llot'dia,1967.30. 61) reinvestigatcdthe *ell-establishedcharacterdifferencesbetweenthe two commercraltlpcs (seehelow) and concludedthat the distinction remains valid: anr further investigationor.rthe two vrrieties growu underidenticalconditionsdocsnot appearto havebeenreported. History. Sennaappcarsto hare becnusedsincethe ninth or tenthcentury. its introdr.rction into ntcdicinebein-udue to the Arabianphysicians. who used both the leares anrl the pods. It was tbrmerly exported through Alexandria, fiorn uhcrc thc nanle of the Sudanesedrug is derived. Collection and preparation. .\lcranrlliarr \cnnil is collectedmainly in September.fiom both wild and cultir utetlpJants.The branchesbearing leavesand podsare dried in tltt-sun irndcon\c\ed to Omdurman. Here the podsandlargestalksarelirrt :c-;rrilured br nrr-rnsof sieves(see 'Senna Fruit').Thatwhichhaspassecl throirrhthe:icr cs is then'tossed' in shallowtrays,the leavesworking to thL.\Lulrrecarrdhervier stalk fragmentsand sandto the bottont.The lcarc' lu. lltcn -r:radr-d. partlv b)' meansof sievesand partlyby hand-pickinginlLrr I r \\h()lc lr-a\c\.(l) whole leavesandhalf'-leaves nrixecl.and (-lr'iliin!.. I'hc uhole lcarcs arethoseusuallysoldto the public.*hile thc otlter -trritlc-. i.rfcu\cd ti)r makinggalenicals. The drr.rg is packed.sontethutl()()\cl\.in hllc-.and sentby rail to Port Sudan,liorn where it is erpon.'.l. Tinnevelly senna is obtained fiont cLrltirrtc'd pl;1p1.rrl (-rr.r.rir ongustifoliagrown in SouthIndia.N.\\'. Paki:tun.rll(l.l]nlntu.\\ hr-rc theplantsaremore luxuriantthanthosetounclu iltl in.\rahrl. lr ntar be
Phqrmocologicol oction The action of the anthraquinonelaxatives is lestlicted to the large bowel; hence their efl'ect is delayed for up to 6 h or longer. The natureof the peristalticinitiation is not known fbr certainbut it has been suggestedthat the common anthraquinoneand anthranol derivativesinfluencethe ion transportacrosscolon cells by inhibition of Cl- channels(J. H6nig er al.. PlantttMed.. 1992.58(Supp]. I ) .A 5 8 6 ) .
SENNA LEAF
c1{2oH CH-r t HO-l
Senna(Serenae FoLiutn)consistsof the dried leafletsof Cnssiasenna L. (C. acutifolic Delile), which are known in commerce as Alexandrianor Khartoum senna.and of Ca.ssiaongustifoliuVahl. which areknown in commerceasTinnevellysenna.The sennaplants
O H O O H Sennidin A (10S,l0'D
SennidinA (10R,l0?)
l
r o ' Ol l
l
ror I cH-J I cH2oH Aloin
O H O O H SennidinB Qneso)
E
P H A R M A C O P O E IA N L D R E L A T EDDR U G SO F B I O L O G I C AO LR I G I N grown eitheron dry landor in wettel'conditions as a successor to rice. Beinga legume.it usefullyaddsnittrgento thesoil.Or.vingto the careful way in which the dru_sis collectecl lncl compressed into balcs,f'ew leafletsarc usuallybrokcn. Macroscopical characters. Scnnalciillcts beal stoutpetiolLrles. The lamina has an entile rrar_rlin. rn acllte apex. and a more or less asyrnmetricbase.Thc surtlccsarc pubcscent. Odour.slightbut charactelistic: tastc.mucilaginous. bittclishand unpleasant. Typicalsennaleatletsare shownin Fig. 22.9.The main difl'erences betweerr the tu o r arietiesaregivenin Table22.4. Nlicroscopical characters. Senna leaflels have an isobilateral stmctLrrc The epidermalcellshavestraightwalls.and lscc Fig. -12.'1). manr contain mucilage.Both surtacesbear scattcred.unicellular. n o n l i _ s n i f i cwda r t y h a i r s u p t o 2 6 0 p m l o n g ( F i g . 2 2 . 9 D . G ) . T h e \tomatahar,etwo cells with thcir long axcs parallelto the pore and \omctimesa third or fburth subsidiarycell (Fig. 22.9E,F). The rnesophvll. consistingof uppel and lower palisadelayers and median spon-qy mesophyll.containsclustercrystalsabout15 20 pm in diameter.The midrib is biconvex.Below the rnidrib bundle is a zone of collenchyma.The midrib bundle and larger vcins arc almost surrounded by a zone of lignifiecl pericyclic fibrcs and a sheathof parenchymatous cells containingplisms of calcium oxalate 10-20 ptmlong (Fig. 22.9C). Vein-isletnumbersand stomatalindices can be usedto distinguish stomatalindex. the two species(seeTable22.r1)andthe BP r,rtilizes
coseand tl-reaglyconessennidinA and B. SennidinA is dextrorotatorv (p. 235). and B is its mesolbrrnfbrmedby intramolecularcompensation The activity of sennawas still not fully explainedby the isolationot thesc constituents.and later work. notably by Fairbairn. Friedrich. Friedmann.Lemli and their associatesdemonstratedthe presenceof many othcr (some pharmacologically active) components. These inch-rde:sennosidesC and D. which are the glycosidesof heterodianthroncs involving rhein and aloe-emodin; palmidin A (see 'Rhubarb'): aloe-emodin dianthrone-diglycoside,rhein-anthrone-8glycoside. rhein-8-diglucoside. aloe-emodin-8-glucoside,aloepossiblyrhein-I -,qlucose, emodin-anthrone-diglucoside, and a primarr glycosidehaving greaterpotencythan sennosidesA and B and distinguishedfiorn them by the addition of two glucosemolecules.A neu anthraquinoneglycoside, ernodin-8-O-sophoroside(a diglucoside). has beenisolatedin0.002lcl yield fiom dried Indian sennaleaves(J. Kinio ara1..Phyrot:hemistn, 1994,37,1685). Two naphthaleneglycosidesisolatedtiom senna leaves and pods (Lernli et al.. PlctnraMed., 1981,43, ll) are 6-hydroxymusizinglucosideandtinnevellinglucoside.The formeris found in Alexandriansenna and the latterin Indian senna:this dilTerencehas beenusedas a distin-
R3o
oRl
/,.rAr'o'*'
n2o.\r\Z-cHr
6-Hydroxymusizinglucoside: Rl = R2= H; R3= p-o-glucopyranosyl Tinncvcllin slucoside: R3= Me Rl = H; R2= p-o--glucopyranosyl;
Constituents. Since Tutin lirst isoiated aloe-emodinand rhein in I 9 I 3, many other compoundsbasedon thcsetwo have beenobtained. Stoll et rr1.(19:11)isolatedtwo activecrystalline-elycosides. sennoside guishing f'eatureof the comrnercialvarieties.seeTable 22.4. Sennaalso A and sennosideB. They both hydrolyseto gii,e two moleculesof -elu- containsthe yellow flavonol colouring matterskaempf'erol(3.4',5,7-
u.e u.p lt .nr
Le
A, Indion senno;B, Alexondrion Fig. 22.9 Sennoleoflets. senno {boihx 1); C, tronsverse sectionof leoflet (xB0);D-H, elements of thepowder(oll x20O);D, leofletfrogmentin tronsverse section; E, F,epidermolfrogments in surfoceview;G, isoloted trichomes; H, portionof fibregroupwithcrystol typerespectively; f, fibregroups;l.e. lower sheoth,c, collenchymo; cic, cicotrix;crl, cr2tcolciumoxolotecrystols of theclusterond prismotic muc,muciloge;m.o.mucronote iype);u.e.upper epidermis; l.p. lowerpolisodeloyer;m, mesophyll; opex;p.mi pressmork,s, stomo(porocytic e p i d e r m i su;. p .u p p e rp o l i s o d leo y e r x; y ,x y l e m .
PHENOLS AND PHENOLIC GLYCOSIDES
loble 22.4
E
€omporison of Alexondrion qnd lndion sennq leoves.
Alexandrian senno
Mocroscopicol choroclers Seldomexceed40 mm in lenoth Greyish-green More osymmelricot bose Rothermorebrokenond curledol theedoes Fewpressmorkings Microscopicol
Tinnevellysenno
S e l d o me x c e e d 5 0 m m i n l e n g t h Yellowish-green L e s so s y m m e t r i co t b o s e S e l d o m b r o k e n o n d u s u o l l yf l o t o w i n g t o c o m p r e s s i o n O f t e n s h o w s i m p r e s s i o n sd u e t o t h e m i d v e i n o f o t h e r l e o f l e f s H o i r s l e s sn u m e r o u s it h e o v e r o g e d i s i o n c e b e t w e e n e o c h b e i n g o b o u t s i x e p i d e r m o lc e l l s T h e s t o m o t oh o v i n g t w o o r f h r e e s u b s i d i o r yc e l l s r e s p e c t i v e l yo r e i n t h e r o t i o (
l
O TO D O U T , / : J
veln-lslet number la-Lv.5
,l0.0-,l5.0, S l o m o t oi nl d e x u s u o l l yl 2 . 5 €hemicol tests* Etherexlrociof hydrolysedocid solutionof drug giveswith methonolic mognesium ocetotesolution: o pinkcolourin doylight, o polegreenish-oronge in filteredultroviolet light fLC test for distinctive nophtholene derivoiivesf 6-Hydroxymusizin glycosidepresent
v e l n - l s tneut m o e rt Y . J - z z . J ,l4.0-20.0, S t o m o t oi nl d e x u s u o l i yl Z 5
Thesometest: qn orongecolourin doylight, o yellowish-green in filteredultroviolet light Tinnevellin glycosidepresent
*Forfulldeloils,seeNondyond Sontro, lnd. Phorn.Monuf., 1968,6,235 J. o ed.,1983,49,36. t S e eL e m lei t o l . ,P l a n t M tetrahydroxyflavone).its glucoside(kaempf'erin) andisorhamnetin;also a sterolandits glucoside,mucilage,calciumoxalateandresin.Thc strucpolysaccharides turesof water-soluble and a lignanhar,ebeenreported. Although sennais not notedfor its volatile components.Tutin in his l9l3 publicationhad observedthe 'strongly aromatic dart-coloured essentialoil'. Over 80 yearslaterW. Schulzet al. (PlantaMedica. 1996, 62,510) have again examinedthe volatilesof sennaleaf and lecorded (GC-MS) more than 200 componentsaffbrdedby aqueousdistillation. 122 constituentswere identified including monoterpenes. phenylpropanes.fatty acidsandesters.etc.Hexadecanoic acidwas a significant componentin addition to many of the more comlnon constituentsof volatileoils. Formatiot'rand distribution of anthrttquinonetlerivtttites.In youn-9 sennaseedlingschrysophanolis the first anthraquinonefbrnled, then aloe-emodinappearsand finally rhein: this ontogeneticsequenceis in keepingwith the expectedbiogeneticorder,which involvesthe successive oxidation of the 3-methyl group of chrysophanol(Table22.3). In the presenceof light glycosylationfollows and later the glycosidcsare translocatedto the leavesand flowels. DLrringfruit developmentthe amountsof aloe-emodinglycosideand rhein glycosidelall rnarkedly. and sennosidesaccumulatein the pericarp. Lemli and Cuveele(PluntuMed.. 1918.34. 311) consideredthat fresh leavesof Cassfulsernzzcontainanthroneglycosidesonly. By drying between 20 and 50'C these are enzymatically convelted to dianthroneforms (sennosides). However,Zenk and coworkers(Plutta Med., 1981,41. 1) rnaintaimed that sennosideformation is not entirely an artefactarising through drying but that thesecompoundstogether' with the monoanthrones, andtheil'oxidizedforms (anthraquinones), are part of a redox systemof possiblesignificanceto the living ceil. The distributionof sennosideB (deterrninedby Zenk and coworkers plant (sample dried at by immunoassay)was fbr a C. ang,Lrstifolia 60'C): flowerszl.3%,leaves2.8%. pericarp2.4clc,stems0.2%. r'oots 0.057c.Within the flowers the anthersand filamentscontained7.27.. carpelsand ovaries5.87c,petals5.27c,sepals4.77cand llower stalks 3.2%,.
Evaluation. It is difficult to renro\eall fntgnt.'nt.ol r'.rihi..fctiolc andstalkfton-rthedrug,bttttheamor.rntot'thc:c\tnr!tLrrL'\i'linritcdbr the BP to 3%. In the wholedru-{the perccntagc ol'thc.c i. ,lclcrntinc-rl by hand-pickingand wcighing.but $,ith the lro\\(l!'fcrltlruL rceour:e h i l \ t ( )b e r r l r d et o q u a n t i t x t i \ e nricro.e,'pr. C. senntt is cultivated in Rr-rssiairnd thc learc. ;.rrchlirrcrtc'd mechanically;this leads to unavoidablcr.nirtureuith peliolc'.and stemsbul, becausethe activeconstituents alc sintilelin .rll lrert.ot tl.rrplant,this doesnot afl'ectthe qualityof the glr co.irlulc\rrir!r\. Lack of knowled-Qe of the preciseactire prineiplc\ !rl \cnnrrc()upled with the sl,nergisticactionof varior:scornpotrrLl\hurrrnl'1s'j thc tlerelopmentof a satisfactory chemicalassay, lbr tlreclrug.Thc 1JPtlcternrinesthe totalsennaleaf glycosides in termsof senno.itl.'Ll rnot le':sthan2.5%.1. This involvesextractionof the -ulycoside:antl ll'e'crLntirlaquinones fiom the leaves.removalof the tiee agll,concsandhr .hrrlr .i: undoridationof the remainingsennosides and othergllco:idc'.to sirc rheinand some aloe-emodin.which are then cleternrinetlrpcctrophotontetrically. Chrornatographic testsfor thc lcaf arc_sircn in the,9Pand[P. The leavesare required(BP) to -qi\e iin ecicl-insoluble ash of not more than 2.5tlc. Alfied drugs. Bontbav, Meccu ulrcl.\rubiurt .Scnrrrr.r are obtained fi'om wild plants of C. nngu.sti.litliu-gro\\n in Arabia. Some of the leatletsare shippedto Port Suclananclule cradedlike the Alexandrian drug. while somearc sentto Bor.r.rbur und l'r'equently arrive in England with shiprrentsof theTinnerellr. The leatletsresemblethosr-ol 1-inncrclll' sennabut are sornewhat more elongatedand narroucr. and ol a brownish or brownish-green colour.Levin (1929)statesthat thc)'rna1'bcdistinguished microscopically from othel senn:ls br theirvein isletnumber. Dog senno, a varietl' ltrrmerly much esteemedand still used in France,is derived fiom Crrs.rlaoltovato. The plant is indigenousto Upper Egypt. but was cultiVatedin Italy in the sixteenthcentury.The leavesare obovateanclcluitcdifl'elentin appearancefrom the oflicial leaflets.When in powder they may be distinguishedby the papillose
P H A R M A C O P O E IA N L D R E L A T EDDR U G SO F B I O T O G I C AOL R I G I N cells of the lower epiderrris.Marlrin tbund thenrto contain | .0-1.15% of anthraquinone derivatives. Pulthl serma,derivedfrotn C(/\.\i(/Ltutitul(tt(t. has been tbund in Indian senna.It may be distin-quished by the long hairs,the crimson colour given when boiled r.r,ithchloral hydratesolutionor treatedwith 80% sulphuricacidanclthe abscnceof anthraquinone derivatives. The leafletsof other speciesof Crr.s.irrr havealsobecnirnported,but may be distinguished fiorn the gcnuinedrugby the charactels givenabove. For Nigcria. the leavesof the local CassiapotktcarTrahavebeensuggested asa substitr.rte firr thc olllcial senna:bioassays havegivenan equivalent activitv(A.A. ElujobaandG. O. Iweibo.PltmraMetl.,1988,54.372).
Tinnevelly sennapods, respectively.Both have separatemonograph: in the EP and BP. Collection. The pods are collected with the leaves and dried as describedabove.After separationfrorn the leavesthey arehand-picked into various qualities.the finer being sold in cartonsand the inf'erior onesusedfol makinggalenicals.
Characters. The characteristicsizesand shapesof the two varieties are shown in Fig. 22.10.The Tinnevelly pods are longer and narro\\'er than the Alexandrian and the brown areaof pericarp surroundingthc seedsis greater.The remainsofthe style are distinct in the Tinnevellr Substitute. i\rgel lettt,es,which are derived from Solenostemma but not in the Alexandrian. (Asclepiadaceae), ai,g/rc1 wereat onetime regularlymixed in a definite After soaking in water the pods are readily openedand about sir proportionwith Alexandriansenna.The plant occursin the Sudan,but wedge-shapedseedsare disclosed,eachattachedto the dorsal surf'ace the leavesare now seldom seen in commerce.If used to adulterate of thepod by a thin lunicle (Fig.22.l0C). Undera lensrhetestasof rhe scnnapowder.it may be distinguishedby the two- or three-celledhairs. Tinnevellyshow a generalreticLllation and wavy, transverseridges. cachof which is surroundedby aboutfive subsidiarycells. while the Alexandlianshow a generalreticulationonly (Fig. 22.l0D. E). The pericarp of thc pod bears unicellular hairs and stomataof a SENNAFRUIT type similal to thosefbund on sennaleaves;portionsof the fibrous Sennirpods (SennaeFrudus) are the dried. ripe fruits oI C. sennu and layer of the endocarparevery evidentin the powder(Fig. 22.I 0K. L. C. angustifolia (Legurninosae).which are known as Alerandrian and M ) .
p.m s.p
A
Wffiq:Q m e.t
s.p p.l
Fig.22.rO Sennofruits.A, Tinnevelly fruit;B,Alexondrion fruit;C, Alexondrion pod openedto showseeds{ollx1); D, seedof Alexondrion fruit;E,seedof Tinnevelly fruit;F,tronsverse section of seed;G, isoloted embryowithonecotyledon removed(ollx4); H, stemwithTinnevelly fruitottoched (x1);l, tronsverse section of pericorp(x90;)l, tronsverse sectionof seedcoot;K, frogments of epidermiswith stomoto;L,frogmentof epidermiswith trichome;M, fibrousloyersfrom endocorpin surfoce view{ollx200J;o, brownoreosof pericorpcovering seeds;c, cotyledons; e, endosperm; e.f,fibrousendocorp; ep, epicorpjf, funiculus; m, mesocorp; p, plumule; pl, plocento; p.l,porenchymotous loyersof testo;p.mipressmorksfromotherpods;r, rodicle;s, seed;sf,stolk;s.p,(J) subepidermol polisode; s p, (A),stylorpoint;s.r,spothole ridge;tr,trichome; v.b,vosculor bundleportiolly enclosed by fibres.
PHENOLA SND PHENOLIC GLYCOSIDES Constituents. The active constituentsof the pods are locatedin the pericarp;they are similar to those of the leaves.togetherwith sennosideA. r.vhichconstitutesabout 1.5%,o1'the sennosidemixture.The seeds contain very little sennosidebut Zenk's grorlp reported the cotyledonsof 3-day-oldseedlingsto containamountsequivalentto in the leaves.Sennoside tl-rose contentvariesfrom about 1.2It..2.57cin thc Tinnevelly and florn about 2.-5 to 1.5Vr'in the Alexanddan. Preparationsof the powderedpericarp.e.g. SennaTabletsBP standardizedin termsof sennoside B. arenow commonlvnrescribed. lJses. The useof laxativesis increasingand sennaconstitutes a useful purgativefbr eitherhabitualconstipationol occasionaluse. It lacks the astlin-eent after-efl'ectof rhubarb.Despitethe availabilityof a number of synthetics.sennosidepreparationsremain among the most i m p o r t r n pt h a r m a c e u t i cl au lx a t i r e s .
E
and rnorcrecentlv(T. Bhaktaer ul.. Pharnt.Biol.. 1998.36. ll0) its antitussireactivitvhasbeenderronstrated.
CASCARABARK Oflicial cascarlsasrada(SucredBark, ChittentBarkl is the driedbark of Rltctntntts DC. (Frangulttpurshiurut(DC) A. Gray ex J. C. lnr.sltiutttr.s Cooper)(Rhamnaceae). The halk is collectedfrom wild trees.which are 6-18 m high. groning r)n tlie Pacificcoastoi North America(British Columbia.WiishingtonanclOregon).Depletedwild US sourcesencoLrragedcultivationof the trcc in \\esternCanada.the USA and Kenya but theseefTorlsdo not appcarto be cunrpletelysuccessful.
History. Cascarais a drr-rsof cornplrltir,ely recentintroductioninto modern medicine.According t() tflrdition. a cascara,probably R. californica, wasknown to earl\ \lcrican anclSpanishpriestsof Califolnia: Cossiopods Rhomntrspurshianrts.hor."ercr.\\a\ not describedr-rntil1805and its (Leguminosere). Cassiapods arethe diled lipe fiuits oI Cu,ssiu.fistulrr a bark was not introducedinto nrcdicincuntil 1877. largetree thoughtto be indigenousto India but now widely cLrltivated The common,Europeanbuckthornrius uell known to the Angloin the tlopics. The drug is chiefly obtained flom the West Indies Saxons:its berrieswereofllcial in the1-orirlr,rr Plktnnu(oDoeia of 1650. (Dominica and Martinique) and Indor.resia. The fl'uit is a cylindrical indehiscentpod about 25-30 cm long and Collection and storage. The bark i: cLrllce tctl frorl mid-April to the 20-25 mm diameter.It is dark chocolatebrown to black in colour and end of August, when it separatesreadrlr tionr thc soocl. l-ongitudinal containsfrom 25 to 100 oval. reddish-brownseedsseparatedby mem- incisionsabout 5-10 cm apart are first n.rr.id.' in thr rnrnk and thc bark branous dissepiments.In the fiesh pods the seeds are completely removed.The tree is then usuallyfellecland the'bnLnehbark :cparatcd. embeddedin blackpulp,which.however.graduallydrieson the septa. The piecesaredriedin the shadewith the cork uppcrrlro.t.Suehntrterial For this reasonpods which do not rattle when shakenare usr.rallypre- is referredto as 'natural'cascara but comme|cial:irpplie'..ilc n\)\\er)ntf'erred.The pulp has a prune-likeodour and a sweetishtaste. minutedto give smallevenfiagmentsknorin a: 'ercnizctl'.'fir1)!L'\\ad' The pulp is dissolvedfrom the cmshedfruit by percolationwith water. or'compact'cascara. Dudng preparation anclsto|asethc hurk rnu't he The pelcolateis stlaineclandevaporatedto a soft extract.The most irnpor- protectedfrom rain and damp. or parlial cxtractionol thc eirrr.tituc-r.rts tant anthraquinonederivativesofthe pulp appearto be rhein andcombined may occur or the bark may becomemouldl. That thc herk nrLr.tbc kcpt semidin-likecompounds. Anthraquinones havebeendetectedin non-dif- fbr at least I year betbreuse is no longera RP rccluilcntcnrhur thc bark ferentiatingcalluscultures.Cassiapulp alsocontainsabout507cofsugars. appearsto incleasein medicinalvalue and price until it i' abourJ rears colouringmatteranda traceof volatileoil. The leavesof C.fstlla contain old. Many companiespref'erto use bark qhich lta' bccn .rored titr fiee and combincd rhein. sennidinsand sennosidesA and B: thesecom- considerablymorethan I year.To reducefi'cightandhantllingeharsc:or.r poundsexhibit a markedseasonabletluctuation.The hearlwoodis repor! the bark.largequantitiesofthe extractareno\\ intportcdtlircetlr. ed to containbarbaloinand rhein togetherrvith a leucoanthocyanidin. Cassiapulp was fbrmerly usedin the fbrm of Confectionof Senna. Macroscopicalcharacters. The bark occursin qLrill'.or channe llcd In Ayurvedicrnedicinethe plant is usedto treata varietyof ailments.Its or nearlyflat pieces.All of thesetomrs mar rttain l() cnt rn lcn,qthand antifungal.antibacteilaland laxativepropertieshave beenestablished are I -,1 mm thick. the thinnerbark being nroste\tccntcd.The ll at strips
C 6 H 1 1 O 5O
OH
C 6 H 1 1 OO5
OH R1
R2
10-10'
cooH cooH cHroH Sennoside C cH,oH D Sennoside Aloe-emodin-dianthrone-diglucosidecH,oH
cHroH
meso trans
Aloe-emodrn-dianthrone-diglucosrdeCH,OH
cHroH
meso
Sennoside A
COOH
trans
B Sennoside
cooH cooH
meso
COOH
trans
PHARMACOPOE AD LR F L A T E AIN DD R U GO S FB I O L O G I C O AL RIGIN
Fig.22.r1 Coscorobork.A, singlequill(x0.66);B, generoldiogromof tronsverse sectionof bork {x2O);C, tronsverse sectionof outertissues; D, dittoof phloem (bothx 200); E, corkcellsin surfoceview;F,frogmentof phloem{rompowder{bothx 250); c. collenchymo; ck, cork;c.p, corticolporenchymo; l, lenticel; li, lichenpotch;l.f, longitudinol furrows;m, moss;m.r,medullory roy; m.s,mussel scole;oxl, ox2,prismotic ond clustercrystols of colcium oxoloterespectively; p.f, pericyclic fibres;ph.f,phloemfibres;ph.p,phloemporenchymo; s, sclereids; s.p,sieveplote;s.t,sievetube;t, scorof iwig. tiom the trunk are usually much wider (up to l0 cm) than the c1uillsor channelledpieces(about5 20 mrn) obtainedfrorn the blanchcs. Cascala(Fig. 22.llA) beirs a somewhatpatchy.silvefy-greycoat of Iichens.Piecesbearingmossarealsocluitccomrron.Betweenthepatchcs of lichenmay be seena smooth.dark purplish-browncork rnalkcclr,vith lighter-coloured. transverselyelongatedlenticels.On scrapirrgthe colk. no bright purple inner cork is disclosed(distinctionliom R. rilna,r).The inner sufice is dull purplish-brownto black. stliatedlonginrclinally ancl somewhatcorlugatedtransversely. The fractuleis shortand granulalin the oLrterpart. but somewhatfibrous in the phloem. ln the lellowishbro'nvncortex and phloemlighter groupsof sclcrenchymatous cells and phloemfibresn-raybe seenwith a 1ens.They ruay be ntaclentoredistinct by staininguith phloro-elLrcinol and hydlochloric acid. The rnedullary rays, which tend to curve togethelin gror-rps. ale wcll seeuin scctiols mountedin potash.Odour.slightbut charactedstic: tastebittcr. Microscopical characters. A transversesection of cascarabark (Fig. 22.11B-D) showsa partialcoat of whitish lichen,some 10-15 layersof flattenedcotk cells with rcddish-browncontentsand a corter composedof collenchyma.palench-vmnand groups of sclereids.Thc
collcnchymatous cellsshon'thickencd pittedwallsandcontainchloloplastsfilled r.r'ithstarch.Someof the parenchymatous cells alsocontain cl-rloroplasts and starch:many of them contain a yellow substance color.rred violet by alkalisand rosettecrystalsofcalciunt oxalater.rsuallr, 6-10 ;.tmcliamctcrbut occasionallyLrp1o,15prmdiameter'. The p:Lrenchymatolrscellsabuttingon thc groupsof sclereidscontainplismsof calcium oxalatc.Tl-resclereidsareire-uularor ovoid in shape.arevariablein size. ancl havc thick lignified q'alls sornetimesshowing stratificationand pits.A pericycleis not clearlydelimitecl.but traversedby funnel-shaped the zone irnrnediatel.voutside the phloem in which sclereids and occasionalfibres occr-r is regardcdas lepresentingthis region. The phloemis composedof zonesof tangentiallyelongatedgroupsof phloerr fibles.enclosedin a sheathof p:u'enchymatous cellscontainingprismsof calciumoxalatewhich altematewith sievetubesandphlocmparenchyma (Fi-s.22.I I F). The individLralflbresareyellow in colour.are 8-15 prn in diarneter.and har,ethick ligniiied walls showing stratificationand pit canals.The sievetubesshow sieveplates.eachwith severalsievefields. on the radialwalls.The sieveplatcsareusuallycoveredwith a depositof callr.rsand can be identifred after stainiug with alkaline solution of corallin.The phloemparenchyr.na resernbles thatofthe cortex.contiiining
) H E N O I SA N D P H E N O L I C GLYCOSIDES plastids,starch,materialcolouredviolet by alkali.androsettesof calcium oxalate.The medLrllzu'y raysare l-5 cellswide and 15 25 cellsdeep.The medullaryray cells are piLrenchyrnatous. somewhatradially elongated and with similal contentsto those of the parcr-rchyma: theh content of materialstainedviolet by alkali is often hi-eh.Fragmentsof mossleaves and li'n'erwoftsareusually found in the powder.
bark to contairrnot lessthan 8.0% of hydroxyanthraccne glvcosidcsof which ntx lessthan 607cconsistsof cascarosides. calculatedas cilsc:lroside A. A tno-point spectro-photometric assay is emplovcd uith itbsorbance nreasurentents at -515 nm and4zl0nm. Experinrcntsby,Bettsand Fairbairnin 1964,althoughbasedon a single.lir.r/iplant.sr,rceested that fiee anthraquinones arelbrmed by the leavesand that thev ale storcdin the bark rnainlyas C-glycosides. the Constituents. It has long been recognizedthat cascarabark stored older bark containin-sthc niost C-glycosides.Rhumnuspurshianuscell fbr at least 1 year gave galenicalswhich wele better toleratcdbut as suspension culturesriill produceanthracene derivativesin which the eff-ectiveas thosepreparedfrorr more recently collectedbark. This is accumulationof theseconrpour.rds. particularlyemodin.is significantpresr.rmably due to hydrolysisol other changesduring storage.It was ly laisedby a I 2 h li_sht/dar-k cvclc: continuousillurninationof the culalsofound at an early datethat the very bitter tasteof cascarais redr.rced tufessuppfesses anthraqulnolte Iorrnatlon. by treatingextractswith alkalis, alkaline earthsor magnesiumoxicle. Proprietaryextracts of this type became vely popular and pharma- Substitutes. Severalspecicsol R/rallltr.shavea similar geo,qraphical copoeiasfbllowed the sameidea to producesuchpreparat.ions. distribLrtion to thatof R. put sltiurttrs. TheseincludeR. dniJblia, which Cascaracontainsabout6-99i anthracenederir.ativeswhich alc pres- is too rare to be a likely substirurr:R. trrxeu. whosebark bearslittle ent both as normal O-glycosidesand as C-glycosides.The fbilowing rescmblanceto the official clrug.untl R. Lttlilltnicu Esch.The latter.is groupsof constituentsare now manif'est. so closely[elatedto R. pur.sltituttrs that \olIe botanistsdo not diVide therr into seprritte plant species. The app.lu'sto havc a much more (l) Fourprimaryglycosidesor cascarosides A. B. C and D: thcy consoutherlydistributiontl-ranthe tr picril R. ltLusltittrtLt.s and is therefore tain both O- and C-glycosidiclinkages.Their structureswere eluciunlikelyto occurin bark ofCanadianorigin.l1ha: a rnoreuniformcoat dated in 1974 by Wa-eneret ul. as the C-10 isomers of the of lichens and wider medullarv rii\\ rhln thc oll'icial spccies.but 8-O-B-o-glucopyranosides ofaloin andchrysophanol. A. Griflini el rescrrbles the latterin having sclcrcnchr nrirtrLr:cells.Thc bark of R. al. (PlantttMed., 1992.58,Suppl. l. A-593)describecl the isolation has been recordcdas a cascaril\ub.rirutc.l:uropeanfiangula ItLllur of the pLrrecascarosides by silica-gelchromatographyand HPLC. bark.distingLrishecl by theBP TLC tcst.is tlc'rurihe.tl belou. The completeassignrnents of lH-and lIC-NMR signalsfor thcse cascarosides were recordedbi' Manitto et ul. (J. Chem.Soc..Perk. Uses. Cascarais a pur-gative \clltl in it. uction. Jt is rescntblin-g TrcmsI, 1993, 1511)and the samegrolrp (J. Nut.Prod.. 1995.38. Inainlyusedin the tbnn of liquid extractor clirir trr a. ruhler.nrL.par!.d 419) have now isolatedtwo analogousglycosides(cascarosides E liorn a dly ext.ract. Itis alsousedin r,eterrniurnork. and F) derivedliom emoclin(Tablc22.3).
OH
FRANGUTABARK
Frangulabark. alderbuckthorn,is obtirinecl lront R/r,lrirrr' tt.tirtqtrla L . ( F - r a u g u l ratl r r r l sM i l l ) ( R h a m n a c e a ea) . s h r u L .r1, : n r h i s h a n d 10 - - 4 f o u n d i n B r i t a i n a n d E u r o p e .C o m m e r i c u lr u p p l r c . i r r c i i \ r i l i . r b l e cH2R fiom Balkan countriesancla little frorl Ru'riu. The.pllnr diftlrs frorr the common birckthorn,R. t:uthurtitu.in ther it tloc. n1y1 p(1.OH sessthorns:it bearsdark-purpleberriestr ho... ntcJi!inril ptr)pcrtics € H / have long been accepted.AlthoLrghntuch Lrredirr Ln-:rlilnd.the HO derrrancl dccreasedwith the increasedpopnlaritr ot e.rseura: on the OH Continent.particularlyin France.cascill'uhri: ntrt r'.-pluced it to thc sameextent. Cascarosides of Rhamnuspurshianus, The balk. includedin the EP arrcl1JP.i. r'cquircdtr)c()ntainnot less = = Configurations; = Cascaroside A I 0p, R OH; B 10o,R = OH; than7.07cglucofran-eulins calculatecl asglucoll'ansulin A. C = 1 0 F , R = HD ; =104,R=H. The bark occursin sin-elcor double qLrill. shich arc usually of smallersizethan thoseof cascal'a rncl ahout0.-5I nrntthick. It has a (2) Two aloins.barbaloinderivedti'om the aloe-enodinanthronesand purylishcork and transversely rr lritishlenticcls.On removeloncatccl. chrysaloinderivedfrorn chrysophanolanthrone.TheseC-glycosicles ing the outer cork cells by scraping.a tllirk crintsoninner cork is areprobablybreakdownproductsfiom ( 1).Also I O-hydroxyaloins A exposed.The transverse sectionclo\!-l\ r'c:entbles that of cascarabut andB (H. W. Rarlwaleder ul., Z. Nctturfrtrst:h,Teil B. 1991.46, 55 l). groupsof sclerenchymatous cellsarc ilb\L'nl. (3) A nurnberof O-glycosidesderivedfrorr emodin oxanthrone.aloeFrangulacontainsantht'aclt-linone clelirutivcs presentmainly in the emodin and chrysophanol. folrrrof glycosides. The rharrnosicle llangulosidc. or fiangulin.wasiso(4) Variousdianthrones.including thosc of emodin, aloe-emodinand latedin 1857.This is r-rovn knoun to colrsistof two isomers.fi'angulochrysophanoland the heterodianthrones pah.r.ridin A. B and C (see sides A and B. fbrmed bl particrl hrdr.olysisof the corresponding 'Rhubarb'). These dimers ale also fbrnted dLrrin-g the preparatioll rhlmnoglucosides, glucofrangulins A rnd B (Table22.3).The fresh and conservationof elixirs and may constituteup to 207c of the balk also contains anthlanols and rnthrones. which are r.rnstable and total anthraceneglycosides(see P. de Witte eI al.. Pluntu Med.. readily oxidize to the colresponcling (1965. anthraquinones; Lemli r99r, s7.440). 1966) detectedemoclin-clirnthrone. palmidin C (see 'Rhubalb'), (5) Aloe-ernodin.chrysophanoland cmodin in the fiee srate. palmidin C monorhanrnosiclc and ernodin-diantl-rrone monorhamnoThe primaryglycosidesaremoreactivethanthealoinswhereasthefiee side.Wagnerct a/. chaflctelizedfi'angulinB as 6-O-(o-apiofirranosyl)anthraquinones anddirnershavelittle purgativeactivity.The cascarosides 1.6.8-trihydroxy-3-methylanthraquinone and more lecently reported havea sweetand morepleasanttastethanthe aloins.The BP requiresthe thenervglycosideerrodin-8-O-B-gentiobioside.
' ) \ -
i-=
P H A R M A C O P O E IA N L D R E L A T EDDR U G SO F B I O T O G I C AOL R I G I N Aflied drugs. The common bucktholn. Rlnntttus t'utharticu. has a glossyreddish-orgreenish-brou,n cork and cloesnot possess scleleids. It containsfrangula-ernodin ancl a gli,coside.rhamnicoside.which yields on hydrolysisrhamnicogcnol(an anthraquinone derivative). glucoseand xylose.Rnuuald and Just (PluntuMed.. 1981, 42,241) glycosidcalatelninl 1.2.6.8reportedtheisolationof thc anthrirquinonc phvscion; chrysophanol:and tetlahydroxy-3-meth)'l-anthraquinone: fiangula-err-rodin. Thc bark rlso containsa numbel of blue-fluorescent substancesr.r'hichin the chromatogramsproducedby the BP and EP testsot'identitl ltrr CascarlandFrangulaservelo distinguishthis adulterant.Thc fluolcsccntsubstances have recentlybeen identifiedas glrcosicles naphtholicle of the sorigenintype.as belor.i..
oR2 oH
R1 glycoside; Rl = OCH: ft2 = glucose cr-Sorigenin o-Sorigenin-primeveroside: R' = OCHr R'= glucose-xylose p-sorigenin-primeveroside; R2-glucose-xylose Rl=H The bark of R. t:aniolicrr has a dull leddish cork and difl'cls fiorr fiangula bark in that it possesses sclerenchymatous cells anclhas wider medullary rays. In recent years the barks of a numbel of Turkish speciesof Rhamnushave been systematicallyexamined firl their anthracluinoneand f-lavonoidcontents (see M. Koskun. 1rrt. -/. Phurmotognos.r.1992.30. l-5I zrndref'crences cited thelein).
RHUBARB
obtainrhubarbfiorn Chinawhich rleets BP reqr.rirernents fbr hydtoxvan thraquinonederivatives.Denrandtbr the betterglades(CR & Flat 80 & 90%) is now almostexclusivelyfor the bevera-qe industry. History. Chineserhubarb has a long histoly. lt is mentioned in l herbalof about2700 BC and subsequentlylbnncd an importantarticle of conrnterceorrthe Chirresetraderoutesto Europe.Today it still holdr a placein medicine.The first internationalsymposiumon the drug rr ar held in Chengde.C,'hina in 1990underthe title 'Rhubarb90'. Collectinn and preparation. Provided that the oldel accountsare still substantilllycorrect.the rhizomesare grown at a high altitLrdc (over3000 m) dug up in autulnnor springwhen abor.rt 6-10 yearsold. dccorticatedand dried. Thc dccorticatcdrhizomes are when whole roughly cylindrical ('ror-rncls') or if cut longitudinally arc in planoconvex pieces('flats'). Piecesusedoften to show a holc indicatingthat they had beenthreadedon cordsfbr drying. The drLrgis exportedfrom Shanghaito Tientsirr.often via Hong Kong. Tlic bcttcrqualitiesare packedin tin-lincclwoodcncascscontainin-e eitlrer280 lb or 50 kg, and inferiorqlralityin hessianbags. Botanicaf characters of rhizome. The Ihizomesof R.pultn(ttwna:ld R. o.fticinuleare similirr in structureexceptfbr the sizeand distribr,rtiun 'star of the abnom-ralvascularbunclles, spots', of the pith. Transverse scctionsof both. after peeling.show phloerr-r on thc outsidc.cambir,rnr. 'star radiatewood anda pith with sports'(Fig.22.121'.8).InR. puhrtu (about2.5 mrn) and mostof thernare tunt Ihelatterarerelativelysn-rall arlangedin zrcontinuousringl in R. tlfticinolethc 'star spots'arc larger (about.1mm) and ale irregr-rlarl1, scattered.
Nlacroscopicalcharacters. Despitethe lalge numberofcorlnrelcial Rhubarb(ChineseRhubarb)consistsof the dried undergroundpalts of grades.it is convenientto describethe variousrhubarbtypes r.rncler RheurLltulnrtturnL. (Polygonaceae) or R. fficirtcile or hybridsof these threeheadings. two species.or mixturesof these.The drLrgappearsstill to be obtained 'fhis l. High-grtule.Chingltaior Slten.si-ttpe. dru-soccr.u's in roundsor from both wild and cultivatedplantsgrown on the high platenuxof Asia llats weighin-eup to about 200 g and up to about 15 crn lon-1. fiorn Tibet to south-eastChina.The BP/EP drug is reqr.riredto containnot althoughr"rsually srnaller.Mr.rchof the present-daydlug tendsto bc lessthan2.2% of hydroxyanthraquinone derivativescalculatedaslhein. of srnall size and may theretbrebe obtainedf}om younger plant' than was fornrelly the case. Species and commercial grades. The genus R/reral compriret The drug has a firnr textute.nonshrunken appearance and a bri-uht about-50species.which may be classifiedinto two sections.the first yellow surfhceshowingwhitish lcticulations.Thesereticulationsarc including R. ltulntatunt and R. officinale. and the secondR. rlrcLponcnt endsof the closelyarranged dr.reto tl-refusifbnnor lozen-re-shaped tit'unt,R. LuttlulcttananclR. emodi.A systematicstudyis madeunusualmedullarylays (whichareleddish-brown)seenagainstthewhitebackly difficult by geographyand by the tendencyof cultivated plants to ground of the phloem pafenchyma. ln the pcltnatrrn type the tblrn lrybrids such as R. pttlnrutuntx R. untlulatuurand R. palntatumx medullaryrays are only about6 cells deep,but in the oJJicinaLe type R. emodi. The exact morphologicaland chemical charactersof such theymay be asmucl.ras200 cellsdcep.This dift'erence accountsfbr thc hybrid rhizones appearnot to have beendescribed.Forrnerlymost of fact thatthe sr"rrlace of theofficiuuletype -qivesthe apperance of para]the drLrg was delivecl fiom R. pctlnrututttL. var. tarrgutitrrrtt lel red and whitc lines rathcr than a rcticLllation.ln both speciesthe N{aximowicz anclR. officinule H. Br. and was traditionally known in appearance of the transversesurfacevadesaccordingto the deptho1' conr'lrerceas Shensi,Cantonand high-driedlhubarb.R. ltttlnnLtwrtand peeling,which may extendinto the radiatewood or eveninto thepith. R. pulnttttrtnt\,at'.tutlglrticloi,now appearto be the chief sources.The The best rhubalb breaks with a rralbled or 'nutmeg' tlacture. the bestgradeof the present-davdrug correspondsto that fbrmerly known fi'eshlybrokerrsurl'ace showinga brightpink colour-this is one charas Shensi rhubarb. Another present-daygrade is similal to the old acter used in grading-see above.Such dru-egives the blight 1'ellou Canton. In addition. some inf'erior drug is exported.mrlch of u'hich powdertavouredby buyers.Paticularattentionis paidby thebuyernot fails to give the pink fiacturecharacteristicof good-qualityrhr-rbalb. only to the colour of the fi'acture,but also to absenceof signsof decar In practice the grading systemis more complex than the above might or insectattack.Ociour,au'on'ratic; taste,bitter and slightly astringent. imply, and culently abouta dozengradesof rhubarbare recognizedby merchants.The gradescommonly listed al'e: 'flat'. 'cornmon round'. 2. Metliton-grucleor Cunton-trpe. This has the generalcharactersol 'smallround'. 'extrasmalllound'. 'sticks', 'third grade'andlowel qualithe drug describedabovebut hasbeenlcsscarefullyprepaled.Sonre piecesale badly trinimed. greyish patchesbeing leti on the outer ties.The flat androundarefurthercategorized on a percentage basis(e.g. 'tlat, 'common round surf'ace.The surface reticr"rlationsare less distinct. the fiacturc 9OC/c' or 80%'). dependingon the pinknessand granularlather than malbled and the tieshly fracturedsurf-ace of n quaiity of the fi'actr.rre. How'ever,not all of thesegrades are necessarily palerpink colour. availableat any one time. Currently (2000) it is alrnostirnpossibleto
PHENOLS AND PHENOLIC GLYCOSIDES
f
@
/
wv#, o l l' '
o'
o
F (/{.'..
, / -' . " r . o ' l G L /'. a U\. ic-_*s i J U\,i"--'---c
t.d./'t
\^\S'**
/f{:/ H.;
NC SK
N\}b
_ 8^YO or)^-mr O O o-9
^okE"o * eil(7 (9 '9s\
e o\:.(y
Fig.22,r2 Chinese rhuborb. A, common round{x0.5};B,storspotin tronsverse (x20);C-E,frogments section of thepowder(.200) C DC.'3ns of reticuloie v e s s e l s ;D , c o l c i u m o x o l o t e c r y s t o l s ;E , s t o r c h ;c , c o m b i u m ; c r , c r y s t o l s ;f , f o c e t sp r o d u c e d b y p e e l i n g ; m . r , m e d u l o r y r o y , p i . . f c e m s f s t o r s p o t ; v , v e s s e l ;x . p , x y l e m p o r e n c h y m o .
3. Tltird grtttle.This ti'eclucntlyconsistsof srnallerpiecesthan in the studied(WS.Chauhanetal..lttt..l.PfldnttLLtt)':n','r.1.),)1.-10.9-lr. higher glades. Only a srnall percentageof the fractureclsnrfaces 3 . E n g l i s h r h u b a r b s . B o t h R . o f J i c i t t u l t ' i , r r r rRi . r / r r , i) / , / l 1 r , r / ) r\ \ c r c l o l show a good pink tiacture and the remainder a -qre)',mauve or m e r l y g r o w n a s d l u g s b u t c r - r l t i r a t i o r t u p | g ; 1 1' l ( ) h l \ c l c r r \ c d . brown one.Thesediffererrcesareshownin the colour of the oorvclel. G a r d e r rr h u b a r b f b r t a b l e u s e i s d e r i r e d t i o n r / i . t - i t;,y , , 1' ,11 , ,1111 Nlicroscopy of powder. Powderedrhr.rbarb (Fig. 22.12) is easily identified.It shorvsabr.rndant calcium oxalaterosettesLrpto 200 $m in diameter:simpletwo to five compoundstarch-qrainslreticulatelessels and other wood elementswhich give no reactiontbr lignin. The yellow contentsofthe rnedullalyray cells (anthraquinone derivatil'es)become leddish-pinkwith anmonia solutionand deepred with causticalkalis. Other rhubarbs. l. Chinese rltuporttic.This is known commercially as 'Chinese Rhapontica'buthasbeenoff'eredunderthe namesof 'Tai-Hwang'or 'Tze-Hwan-{'without indicationthat it is a rhapontictype.It consists of untrirnmedpiecessometimessplit longitudinally.The transverse surfaceshowsa radiatestructure,with concentricrings of palel and darkercolour,and a diflirsering of starspots.The centlernaybe hollow. The odour. wl.rich is sweetish.difl'ers fiorn tl.rtrtof ofTicial rhr"rbarb. Rhaponticlhubarb,like the offlcial. givesa positivetesttbr' anthraquinoneclelivatives.When the test for absenceof rhapontic rhubarb(seebelow) is applied,it gives a distinct blue fluorescence. which rnay be furtl-rerintensifiedby exposureto ammoniavilpour. 2. Irtdi rut rhubarb. Indranrhubarbconsistsof the dried rhizome and roots of R. Lustrole(fonlerly calledR. ernotli)andR. yt'ebbiaum.Itis fbund in Pakistan.Kashrnir,Nepal and eastemIndia. SinceWorld War II. when therewas a shortageof Chineserhr.rbarb, largequantitiesof the lndian drLr-q havebeenexpofted.lt occulsin unpeeledor partlypeeled pieces,which arebarrel-shaped or planoconvex.shrunkenandlight in weight. The fieshly flactLrredslrrtaceis dull olange to yellowishbrown. Cork cells are easily tbund in the powder. It contains anthraquinone derivativesand in ultravioletlight showsa deepviolet fluorescence. The anti-inflarnmatoryactivity of the roots has been
1 . J t t p u r c s a r J t L t b t L r bA. h y b r i d o f R . c o r c u r t L r n lr . r t r i R . i ) , i l t ] t t i i u n t . h c o n t a i n s a n t h r a q u i n o n ed e r i v a t i v e s . n u p h t h r i l c n er I r i , r ' i J c . . i r n i l e r t o t h o s e i l l u s t r a t e d f o r s e n n a .s t i l b e n c q l r c o r i t l c . i i n ( l , . | ! . r l c c h i n . Constituents.
As with other anthraquiuonL'e()ntiLiltill-!th'ur.. rhc chcnt-
ical corrplexity of rhubalb was not fulll' appreeirrtcdlr\ tltc c.rllicl rc:carch worters. Free anthraquinones were thc llrst \Ltb\tlll!r\ trr bc i:Lrlutcd: cluysophanol. aloe-errrodin.rhein. cmodin und cnrt,.lin nl(Jn()nlcth\l!'ther o r p h y s c i o n ( 18 4 4 1 9 0 5 ) .G l v c o s i d e so f s o r n co l t l t c r i t r r rc \ \ . r c a l \ o s c p a r a t e d . T h e s e s u b s t a n c e s d i d n o t a c c o u n t r a t i s l u e t o r rtli)r l t l t c e c t i o n o l - t h e dtug, and modem methods of inr.'estigationhrrrc' t..t.rbli'hcrl tlte presence of the fbllowing types of anthraquinoncr ir.rrtLrbrLll.. L A n t h r a q u i n o n e s w i t h o u t a c a r b o x \ I g r o l r p r s . q . c h l r s o p h a r r o l ,a l o e ernodin. emodin and physcion). .\l:o thcir glr cosidcs (e.g. chrysoplranein and glucoaloe-errodin ). Kopn anrl cou orkers (Plttnta M e d . . 1 9 8 3 , 4 8 . 3 4 a n d r e t ' c r c r . r c cesi t e t l t r r o l a t e d a p h y s c i o n d i g l u c o s i d e a n d i t s 8 - O - B - D - g e r r t i o b i o : i t l eI-' r ' t t nR t . ltulttttrtr?; other cytotoxic anthraquinone gllcosicies har!' fcccntly been isolated i n c l u d i n g p a h n a t i n ( 1 , 8 - d i h r d r L r r r - i - r n e t h v l - a n t h r a q u i n o n e -I - O , 1, 1063 B - 7 o - g l L r c o s i d e ;s e e K u b o e t t r l . , f ' l t t r t t r ' l t a t t t i . s t t l ' , 1 9 9 2 3
with corrigendumon rnisspellirrc t)f nilmcsp. .1399). 2 Anthraquinones with a car-horr1 uloup(c.g.r'heinand its glycoside, giucorhein). Anthronesor dianthronesof chllsophanol,or emodin or aloeemodin.or physcion.The clianthloneglucosidesof lhein (sennosidesA and B) and the oxalatcsofthese(sennosides E andF). have beenisolated( 197't)b)' Japanese workers.Sennosides A and B havc beenidentilled in callusculturesof a R. pttlntutttnihybrid. Heterodianthrones dcrived ll'om two different anthronemolecr.rles. For example.palmidinA fionr aloe-emodinanthroneand emodin
LR I G I N P H A R M A C O P O E IA N L D R E L A T EDDR U G SO F B I O L O G I C AO '
anthrone: palmidin B fiorn aloc-ernoclinanthrone and chrysophanol anthlone: and palrnidin C fiorn ernodin anthrone and chrysophanolanthrone.Rhein anthroneoccllrs combined with aloe-emodinanthrone(scnniclinC and as sennosideC), chrysophanolanthrone(reidinB ) andphyscionanthrone(reidinC). These may'be oriclizcdinto their two componentsby means dianthrones of t-erricchlolicle.
compounds.rhubarbcontainsastrinIn additionto thc abovepur'-lativc gentcompoun(lssuchas-rlucogallin.fiee gallic acid,(-)-epicatechingalof-callicacidincludeglycelolgallate. laternclcatechin.Othr-r'dcrivatives gallatesandisolindleyin(a methylp-hydroxyphenylgallicacid-llucosiclc has A new classof gallototannins propronate clerivative of a glycogallate). a sucrosecorc and chromoneglucosideshave also been identified(Y. 29, 1007). 1988.27,1169:.1990. Kashirirda et ul..Pltvtochenrlsln'. also containsstarchand calcium oxalate.The total ash is Rhr-rbarb ven rariablc. as the amountof calcir-rmoxalatevariesfionr abor.rt5 to BP assayfbr l0ci. Thc acid-insoluble ash shouldnot exceed7c/a.The methodandreplaces anthraquinone deriVativesis a spectrophotometdc the fbrrnerstandardfbr alcohol-solubleextraclive.
drop of the tiltrate on a filter paper.When examinedin ultraviolet li-eht.the spot showsno blue colour with ofllcial rhubalb but a distinct blue fluorescenceif rhaponticrhubarbis present.The colour is intensifiedby exposureto ammonia vapour.The BP test is more specilic and employs TLC with ultraviolet light and phosphomolybdic acid spray fbr visualization of the chromatogramof a methanolicextractwith rhaoonticinas a standard. is used as a bitter stomachicand in the treatmentof Uses. Rhubr,rrb diarrhoea,purgationbeing followed by an astringenteffect.The drug is suitableas an occasionalaperientbut not for the treatmentof chronic constipation.
Furtherreoding Foust C M 1992Rhubarb-the wondrousdrug. PrincetonUniversity Press.Princeton,NJ. Zftis brnk givesthe history o.flhe commerce. botanyand ntedicirnl aspectso.fthe drug.
ATOES Ontogenetic production of anthraquinone derivatives. As the BP seavariationsin constitllentsarisingfior-r-r drug is collectcdin ar.rtuntn. considerable presentno problem.Nevertheless. sonablechangesshor.rld researchhasbeendevotedto this aspectover many years.Work by Lemli and colleagues(1982) indicated that oxidized compounds.the tttixtttrein are the major cornponentsof the anthrrlcene anthraquinones. the summermonthsand the reducedfbt'ms,thc anthtones,in wintcr.The occur within a tine lapseof about3 weeks.andjust befole conversior.rs markedly.Experiments contentincreases each,the anthronedi-elycoside showedthat the anthraquinone) anthroncconversioncould be artifiEarlierlepo'ts by cially inducedby decreasingthe ambicnttemperature. Schmid (1951) suggestedthat the trgeof the rhizome also affectedthe ratio of reduced:oxidizedglycosides. Chincse workers have also addressed the problem(Chern.Abs.,1992,117,66652:66653). Constituents of rhapontic rhubarb. Rhaponticrhubarb containsa derivaglycoside.rhaponticin.which is a stilbene(diphenylethylene) tive of the formula
HO
J ca=cH{
\:./
c6H11os-o
OH
Vou"
Aloes is the solid residue obtainedby evaporatingthe liquid which drains from the transverselycut leaves of various speciesof A/oe (Liliaceae).The juice is usually concentratedby boiling and solidifies on cooling. The oflicial (BP. EP, USP) varieties of aloes ale the Cape fiom Sor.rthAfiica and Kenya, and the Barbados(Curaqao)fiom the West Indian lslandsof Curagao.Aruba andBonaire.Thereare separatepharmacopoeialmonographsfor each type. Socotrineand Zanzibar varietiesare no longer official. Plants. Of about 180krown speciesofA/oe. the drug is mainly obtained tionr the following: Capevariety from ALoe.ferorandits hybrids; Curaqao variety from Aloe barbudenrls; Socotrine and Zanzrbar varieties liom Aloe pen1i. The genus A/oe includes herbs, shrubs and trees, bearin-u spikes of white. yellow or red flowers. Aloe ferox is an example of the arborescenttype and A. barbutlensisof the herbaceoustype. Aloe leaves arefleshy,arestronglycuticularizedand:re usuallyprickly at the margins. It has beensuggestedthat if natural stocksofA. /ero.r becameexhausted thenA. d tts.seni i andA. t trrkanensis would be preferablefor cultivation becausechemical raceswould not be a problem and their production of sideshootswould make vegetative propagationeasier.However. some problems have arisen conceming the commerce in the African aloes becausethe WashingtonConferenceof the Convention on Intemational Species(CITES)placedall speciesot Aloe, with the Tradein Endangered exceptionof A. vera (a cultivar of A. barbadensi s). on the protectedI i st.
(glycosideof 3.5-dihydroxyThis substance and desoxyrhaponticin -l'-rrethoxystilbene)account for the diffelence in fluorescence Leaf structure. Transversesectionsol an Aloe leaf usually show the betr'"ccrrofflcial and rhaponticrhubarbs.Rhnponticrhubalb doesconlbllowing zones:(l) a stronglycuticularizedepidermiswith numerous tain anthraquinonederivatives,althoughthesedifl'er florn thosein the slomata on both surfaces;(2) a region of parenchymacontaining (seeTable22.3). otlicial drug.One is the glucosideglucochrysaron chlorophyll. starch and occasionalbundles ol needles of calcium oxalate; (3) a central region which fiequently occupiesabout thleelilths of the diameterof the leaf, consistingof large.mucilage-contain Chemical tests. cells; (4) a doublerow ofvascular bundleswhich with I0 ing parenchymatous powder is shaken A little l. Te,st lor utthnrquinonetleriv'atit'es. ml of f'en'icchloride solution mixcd with 5 ml of hydrochlolic acid lie at the jr,rnctionof the two previouszonesand have a well-marked andhcatedon a x'ater-batlrfor 10min. After filtrationandcoolingthe pericycleand endodermis.The aloeticjuice from which the drug is prefiltrate is extrnctedr.vithl0 ml of carbontctrachlolide.The organic pared is containedin the large, pericyclic cells and sometimesin the adjacentparenchyma.When the leates are cut, the aloeticjuice flows u'ashedwith 5 ml of waterarrdshakenwith 5 ml of Iayeris separated. dilute solution of ammonia.Oflicial rhubalb gives a rose-pinkto out. No pressureshould be applied or the aloeswill be contaminated with mucilage.The mucilage,containedin zone 3 as above is used in cherry-redcolour.Rhaporrticrhubarbalsogivcsthis test. 'aloe vera' preparations(sec the cosmetic and herbal industriesin 2. Te.st t'or rhaltonlic rhubtrrb.Macerate0.5 g of powder with l0 ml of 45% alcohol for 20 rnin. shakingoccasionally.Filter and place one Chapter2l ).
PHENOLS AND PHENOLIC GLYCOSIDES History. Accolding to legend,Socotrinealoeswasknown to the Greeks as early as the lburth century BC: the Greek colonists were sent to the island by Alexander the Great solely to preserveand cultivate the aloe plant.The drug was apparentlyknown in Englandin the tenthcentury. and fi"om the seventeenthcentury recordsof the East India Company it would appearthat they frequentlypurchasedthe whole stockof aloesof 'King the of Socotra'. Socotrine andZanzlbar aloe were fbr many years the only otTicialaloes,but they have now been replacedby the Cape and varieties.Cape aioeswas first exportedabout 1780 and became Cr"rraEao ofTicialin Britain in 1932.Barbadosaloeswasproducedfrom about1650 and lapsedaboutthe beginningofthe presentcentury.The productionof Curagao(also called Barbados)aloeswas starledby the Dutch in the Aruba and BonaireaboutI 8171recentlyaloesof simIslandsof Curac;ao. ilal type hasbeenexporledirom nearbyVenezuela. Preparation of Cape aloes. Capealoesis preparedf}om wild plants of A. ferox andits hybrids.The leavesarecut transverselynearthe base and about200 of thernarearanged round a shallowhole in the ground. which is lined with plasticsheetingor more traditionailya pieceof canvas or a goatskin.The leavesare arrangedso that the cut endsoverlap and dlain fieely into the canvas.After about 6 h all the juice has been collectedand it is transferredto a drum or paraffin tin in which it is boiled for about 4 h on an open fire. The product is poured while hot into tins. eachholding 25 kg, where it solidifies.For export the tins ale placedin casesholding eithertwo, four or eight tins. Preparation ofBarbados (Curagao) aloes. Curagaoaloesis produced from cultivated plantsofA. barbadensis.The cut leavesare stackedin Vshapedtroughs,arrangedon a slope so that thejuice flows from a hole at one end of the trough into a collectingvessel.When sufficientjuice has usedis in a coppervessel.The temperature beencollected,it is evaporated generally lower than in the caseof Cape aloes and the product is. theremay be fbre, usually opaque,although some which is semi-transpeLrent producedand is known in commerceas 'Capey Barbados'.Originally Barbadosand CuraEaoaloeswere packedin gourds.now seenonly in lnuseums.The present-daydrug is exportedin caseseachholding about 58.5kg. Characters of Cape aloes. The drug occursin dark-brownor greenish-brown,glassymasses.Thin fragmentshavea deepolive colour and The powder is greenish-yellowand when pieces are semitransparent. of the drug have rubbed againstone another,patchesof powder are fbund on the surface.The drug has a very characteristic.sour odour (the so-called rhubarb or apple-tar1odour'), which is particularly noticeableif one breatheson the drug beforesmelling.Taste,nauseous and bitter. The powder when examinedunder the microscopein lactophenolis usually amorphous.
Chemical tests 1. Gcneral.For the fbllowing testsboil I g of drLrgr.r'ith100ml ol u,atcr.add a little kieselguhrand filter. Use separateportionsof the llltrate firt the tbllowing tests. (l\ Bont.rrcoctiort. To 5 ml of solutiorrof aloesadd0.2 -sof borax anclheatuntil dissoli,ed. Pour a f'ewdropsof the liquid into a test-tLtbe nearll'ful1of water.A greenfluorescenceis produced the origin ol ri hiclris discLrssed below. (2) Bntrttirtt'tc.rt.To I u-rlof solutionof aloesadd 2 ml of freshly preparedsolution of bromine. A pale yellow precipitateof is nloducecl. This testis not soecificfbr aloes. tetrabronraloin 2. Special. -lb5ml ( l ) N r f r t a c i t lt e s t . o l s o l u t i o n o fa l o e s a d d 2 m lo f n i t l i c acid. Capc alocs qirc-: a bronnish colour rapidly changingto grcenlBarbadosa decpbron nish-rcdlSocotrinea pale brownish-yellow:Zutzibtr a r cllon ish-browncolour.Nitric acidrnay be applieddilect to the porrclelecldrugsu'rth similarresults. (2) Nitrous acitl tcst. To an uqueou: solution of aloesadd a few smallclystalsof sodiunrnitritt'unrla little aceticacid.A rich pink to carmineis -unenbr Brrhirckrsand a lesserpink by :horr little chan,ee Cape:SocotrineandZanziburin colour. (3) Klunge'sisobarbaloirt/e.\/.To l0 nrl of an aclueolrs I in 200 cLrpper sulphatesolusolr.rtion of aloesadda dropol'\atuflrtcri ehlolitlcand l0 rnl of alcotion, following by I g o1'sotliurn hol 90c/c. With Barbados alot. rL u inc-recl colour is developed. which persiststor at lcr.t l l ir. \\ ith Crpc alocsa lessercolorationrray dei elop.s hich.htrucr cr. rapidlr lacies to yellow.Zanzlbarand Socotrinculoc':qirc no colour.The br lr alnring. appearance of the red colourmav bc l.trste'nttl (1) Modified Borntriiger',s te.\t. As nrcntioned under 'Constituents', smallquantitiesof aloc crnotiinnr.rroccurir.r to tbnn aloes.but the amountsare nsuallt tOo:rrlill 1or1[.','r.r a nroriitictlBorntniser's the basisofa reliabletest.Therefirre. test which ernploysferric chloricleand tlilutc hrdrochloric acid to bring about o-ylddlilt,/ilrlrrA.sn. een be used. The anthraquinones liberatedare extractecln rth c;.irbontctrachloride andgivea rose-pinkto cherrl-recicolorirs hcntheirsolution is shakenwith diluteamnronia. in the RP trrgether r.r'ithassaysfor Achromatographictestis inclr-rded hydroxyanthracenederivatives.Of thc iattcr Blrbrdos aloes should containnot lessthan287candCapealocsnot lessthan I 8% calculated asbarbaloin.
Constituents. Aloes containC-glvcosiclc:and resins.The crystalline glycosidesknown as 'aloin' were first prcperer,l by T. and H. Smith of Edinburgh, alot-sin lfi5 I :.\loin (BP. 1988)contains UK, fiom Barbados not lessthanTOC/c anhydrousbarbaloin.The rnaincrystallineglycoside, barbaloin.is tbundin all the commcrcinlrlrir-tics(Leger,1907).Leger Characters of Barbados aloes. Typical Barbadosaloes varies in thaton heatingto about 160'C barbaloinis partly convefiedinto colour from yellowish-brownto chocolate-brown,but poorerqualities sl-rowed that have been overheatedmay be almost black. The drug is opaque amorphousB-barbaloin.This substanceis said to be absentfrom the 'Capey Barbadosvariety.but presentto the e\tent of abor.rt 8% in the Cape. and breaks with a waxy fracture. The semi-transparent derivativeof aloeBarbaioinis a C-glycoside-a I 0-glucopyranosyl Barbados'becomesmore opaqueon keeping.Curagaohas a nauseous Unlike O-glycosides.it is r.rothydrolysedby heating and bitter tasteand a characteristicodour recallingiodoform. Mounted emodin-anthrone. with dilute acidsor alkalis. It can. horvever,be decomposedbyo.xidain lactophenol.it showssmall acicularcrystals. tive hl,drohsis,with reagentssucl.r:rs ferric chloride. when it yields Substitutes and adulterants. Socotdne and Zanztbar aloes are now glucose.aloe-emodintlnthloncrnd a little aloe-emodin.It will be seen is possibleat C- I 0; rare in the British market. and Natal aloes frorn A. carulelabrumis no fiom the formula ol barbalointhat stereoisomerism iongerimported.Socotrineis yellowish-brownto blackish-brown,opaque rn 1979both isomersu'ere obtainedby HPLC of a methanolicextract fractve.Zanztbar is similar but hasa wexy fiacof Aloe .t'brotand in 1980ALrterhoffeI a/. separatedcommercialaloin and bre:ks with a poror.rs 'monkey-skin into its stereoisomers. The absoluteconfigurationof the two aloinswas ture and may be packed with leaves or skins (so-called from official aloesby chemicaltests. independentlyel-rcidatedby Rauwald et ul. (Angew.Chem.,It'rt.Ed. aloes').AII may be distinguished
PHARMACOPOE AD LR E L A T E AIN DD R U GO S FB I O L O G I CO AL RIGIN
i--
cHroH Gluc
).-H
"_i;l
HO-C-H
t
H-c-oH
t H-C I
cHroH
O
"- \ -f
l
I
't
:./z
Aloesin (Aloeresin B)
AloeninA: R, = H; R2 = glucosyr AloeninB: Ft = glucosyl; fl2 = glucosyl.2.p.coumarate
Barbaloin
Irrg1. I 989.28. I 528) andManittoet al. (.J.Chent.Sor'.,Perk. Trurts.I. 'Aloe verq' products See Chapter 21. 1990.1297):iiloin A is (10S)-barbaloin anclaloin B the ( l0R)-epimer. Thc trr o are interconvertiblevia the correspondinganthranolfbrnt. All Chrysorobin rarietiesof aloesgive a stron-9greenishfluolescencen'ith borlx. a Chlysalobin is a mixtr.rreof substancesobtainedfiom ararobaor Goa characteristicof anthranols.which are readily fbrmed f}om anthrones powdel by extractionwith hot benzene.Araroba is extractedfrom car by'isomericchiinge.This haslon-ubeenusedas a genertrltestfbr aloes. ities in the n'unk of Arrlll tt ururoltu (Legr.rn'rinosae). Chrysalobincon Small quantitiesof aloe-emodinare sonretimespresentin aloes.and tains chrvsophanolanthranol.the correspondin-tanthroneand othcr Capealoesalsocontainsaloinosides A nnd B. which :re O-glvcosidesof similrr constituents: it gives a stronggreenlluorescence in alkalinrbarbaloin;aloinosideB hasthamnoseattachedvia an oxvnethyl glor,rpat solntion.Chrysarobin rvaslorrnerlyrnuchusedfbr skin diseases and is C-3. In A. burbudensisfi'eeand estcrified7-hydroxyaloinsA nnd B are still occasionallv prescribed. chiu'acteristic l0-C-glucosyl-anthrones. Thesccompor.rnds arercsponsible fbr the i,iolet-purplecoloursgiven in i'iuions speciflcteststbr Barbados Modder aloes(seeH. W. Rauwaldet ul.. Pluta Med.. 1991.57.Suppl.2. A 129). The loot of Rrfuicrtiilctorum (RLrbiaceae) was fbrmerly ,qrownin large The resinofaloes.reputedto havea plrrgativeaction.hasbeenperi- quantitiesas ii dyestufT.but has been alntost completely replacedbV odically investigatedfi'om thc end ofthe nirreteenth centuryonn arcls.ht syntheticdyes.It containsseveralanthraquinone the chiel -ulycosicles. SouthAfiican spp.(e.g.A. t'ero,r)aloesin(now often ref'erredto ns aloe- of which. ruberythricacid (Table22.3) yields on hydrolysisalizalin resinB) wasidentilledin 1970by Hayneser al..andwasthefirst C-glLr- and primeverose.Twenty compoundshavebeenisolatedfront the roots cosyl-chromoneto be clescribed.Other 5-mcthvlchlomonesisolated ancl their mr.rtagenicitystLrdied(Y. Kawasaki et ul.. Chern.Phanrr. fiom Cape aloes include aloeresinA and C rvhich are 7r-coumaroyl Bttll.. 1992.40. 1504).and three hydroxymethylanrhraqr-rinone glycoderivativeslinked via a hycl'oxyl of the _slucose. Tu'o non-glr.rcosylated sidcs have been recently described(N. A. El-Emary and E. Y. presentin sntalleramountsthan the aloesinswere Birckheet.Phyrochemist rt', 1998.49. 21I 5-methylchrornones ). reportedin 1991.A glycosidic6-phenylpyran-2-one derivative(aloenin A) was isolatedand charactedzedfiom A. urbore.scens leavesin 1974 HYPERICUM-ST JOHN'S WORT by Japaneseworkers.Akrenin B has now been obtainedfiom Kenva (scc (For aloes forrrulae). researchon theseirndrelatedconstituentssee Hypericum consistsofthe dried aerialpartst>fHtltericurn perforatrutt. G. Speranza et al.. Phttochernistt-t'. 1993.33, 175:J. Nut.Prod.. 1992. family Hypericaceae(Clusiaceae)-qathercdusually at the tirne ol' 55.723; 1993.56.1089;1997.60,692:.T. Reynolds,Bor.J. Linn. Soc.. flowering or shortly befbre.Commercial extractsare standardizcdon 1985,90, l5l; 119.)Threenew naphtho[2.3-clfuran content.expressedas hypericin. delivativeshave theil naphthodianthrone The plantis abundantthroughoutEuropeirr grassland. recently been isolatedfiom a cornmcrcial santpleof Capc aloes (J. woodlandsancl hed-res, extendin-q Koyanraet al.. Phyrocltemist rv, | 994.37. lI17 ). to the HirnalayasandCentralandRussianAsia.except As with otheranthraquinone-producing plants.in A/oe speciesthecon- in Arctic regions.It was introducedinto NE Americaand Ar-rstralia at an of colonizationwhereit hassincebecomea noxior-rs tent of anthraquinones is subjectto seasonalvariation.and theseconr- earlysl-aee weed.It is perennial.usuallv lbrmin-ea colony with a spreadingloot l.roundsare implicateclin the active metabolismof the plant.McCarthy a herbaceous andcou orkersin SouthAliica haveshownthattheanthraquinorre deriva- systen.The bright yellow flon,crsarein handsometerminalcorymbs. tivcsale confinedto theleafjuicesandthataloinreachesa mlxirnurn coucentrationin the dried leaf .juicesof A. .fero.rand A. ntarlotlrl in the History. The plantwasknown in ancicntGreecefbr its medicinalattributes and since the Middle Ages has been used for lts anti-inflammatoryand slulnrcr(21.1'7in Noverrrber) andis lowcstin winter( 14.87c in July). healingproperries.It alsobecamehighly regardedfor thetreatntentof mental Uses. Aloesis ernploycdaspurgative.It is seldomprescribedalone.and illness.The genericnamederivesfrom the Gteek /l-rper-above, and ic.r/? its activityis increascd whenit is adlninistered with smallquantitiesof soap (eikon)-picture. ref-erringto the ancientpracticeof hangingtheplantabove or"alkaline salts.r'n,hilc canninatives moderate its tcndenc)'to car-r\e griping. religiouspicturesto ward off evil spirits.The commonnameSt John'swon is attribr-rted (Friars'Balsam). to the fact. among others.that it comesinto flower ar.oundSt It is an ingredientof CompoundBenzoinTinctr.u'e There appcalsto be little variation of the major constituentsof the John'sDay (June24th). The clrug is now inclr.rdedin the BP 2000, a number of European leaf exudatcof A..ftr'o.rdeperrdingon geographicallocationof the plant but selectionof high-vieldingstlainsgiving a high productionof aloin pharmacopcreias, the Bntl.t'ft Herbal Pharntacopoeiu.the Atnericatt (2-5%)is recommended fbr commercialcultivation(B.-E.van Wyk r:r Herbul Pharmacoltoeia. and as monogl'aphs fbl the Gernan CommissionE and ESCOP al.. PlarttaMedica. 199-5. 61. 250).
P H E N O L SA N D P H E N O L I CG L Y C O S I D F S
Collection. Collection is from wild ancl cultivated plants and increased demandhasmernt thlt thrnrersin the US irndAustraliawho battledto eradicateit as a weed now halvest it as a r.'iablccrop. Care hasbecn sl-rould be takenduringcollcctingas contactphotosensitivity reported.Dlying at 70" fol l0 hoursis recommended.
f o r i n c o n t e n t . T h e s e p h l o r o g l u c i n o l s c o n s t i t r - l t et h c p r i n c i p a l c o m p o n entsof the lipophilic extract of the plant. Flurottoitl.s. These include f-lal'onols sr-rchas kacmpf'erol. luteolin ancl q u e r c e t i n .t h r ' l l i l r i l n o l g l y c o s i d e s q u e r c i t r i n . i s o q u e r c i t r i n a n d h y p e r o s i d c . T h c b i l l l r o n o i c la m e n t o f l a v o n e( F i S . 2 2 . l 5 ) i s c o n f i n e d p r i n c i p a l I y t o t h c f l o u c r s l A . L . n r e ke t a l . . P l a n r o M e t l i c o , l 9 9 9 . 6 5 . 3 8 8 ) .
Nlacroscopy, The drug consistsof green leaf fragmentsand sterrrs. S e l e c t e c fl t r r n t u l u t -l o r t h e a b o v e a r e s h o w n i n F i g s 2 2 . 1 3 a n d 2 2 . 1 - 5 . r.rnopened V t l u t i l e o r 1 . L p t o 0 . . 1 5 ( l c o n s i s t i n _ sp r i n c i p a l l y o f s a t u r a t e dh y d r o br.rds andvellou'flowers.Oil glandsarevisiblein the leavesas transparentareas.hence the specific n'ameperforaturtt.anclas snrall c a r b o n s i n c l u c l i n g u l k a n e s a n c la l k a n o l s i n t h e r a n g e C t o C : s . black dus on tlre lower surfnce.Tl-reopposite.sessileleavesare 1.5 .:1.0 O t l t e r c u r . s t i t r r t ' 1 1 .\rl .a r t r o t h e r c o m p o n e n t s o f h y p e r i c u m h a v e b e e n crn in length.elliptictrlto ovatein outline. glabrousrvith i.urentile rnargin. r c p o r t c d i r - r c l r . r c l i nrgu r i o u : p l r r . r t a c i c l s ( c a f f e i c . c h l o r o g e n i c . e t c . ) . Pieceso1'hollowsten arecylindricalvn,ith two faintlibs on eitherside. a m i n o a c i d s . r , i t a n r i n C . t u n n i n r a n c lc a r o t e n o i d s . The odouris clistinctanclthe tasteslightlys\\'eetand aslringent. Many reports harc ap|eurctl eonc!-r'ningthe distribution of the above constituents in different or!lins o1'the plant ancl generally on a weight fbr
Microscopy. The uppelepidemal cellsof theleafaresinuonsin outline possL'\sc\anolnocvtic with beadedanticlinalwalls: the lowel epidelr-r-ris oil and paracyticstomata.The mesophyllhas largchypericin-containin-e glands.somewith reclcontents,and thesearc alsotbunclirr the petalsand sepals.Pollen glains are ellipsoidal.20-2-5pm in diameter.with three polcsanda smontherine.Trichomesandcalciumoxalateareabsent. with bioConstituents. Hypelicurncontainsa varictyof constitr.rents logicalacti'"'ity. Principalli"hirpclicin anclpseuclohypericin: also Anthrutluinone.i. iso-h-vpcricinand emodin-anthrone.The RPIEP recluiresnot lessthan as hypericincalculatedwith reler0.8% of total hypericinsexplessecl by absorpenceto thedrieddrug.The extlactedhypericinsareassayed tion measurcment at 590 nm. P rent Iut etl p h Io rog I uci n oI tle rivutiv e.s. Hypedbri n (2.0-1.5c/c). adhyperfbrinanclfurohyperfbrin(L. Verottaet al., J. Nat. Procl..l99L). of aboutfii"epercentof thehyper62,170),the latterat concentrations
rveight basis it is the f'krrrer.. palticularlr tlle petals. that possessthe highe s t c o n c e n t r a l i o n sA . c c o r t l i n - t 1 0 r i n . \ u : t r - u l i a nr e p o r l ( I . A . S o u t h w e l l e l u l . . P h t t o c l t e n u . i t n .1 9 9l . 3 0 . - l l 5 t i t i s t h r ' n u r r o u , - l e a v e d v a r i e t i e s . b o t h in Eulope and Australia.thilt po\\e\\ r fr'lilti\r'l\ high proporfion of oil g l a n d s a n d g i v e a h i g h e r y i . ' l d r r l h r p e l i . i n e o r n p a r c - dw i t h t h e b r o a d lcaved fbrms. Hypelicin conc!-r)trrti()r'l\tlctcrnrinccl on twelve salnples of hcrb collected thloughout Orcqon ruric'd sidr'lr (0.01-0.38%) (G. H. C o n s t a n t i n c a n d J . K a r c h e s l ' . P l t u n n . 1 J r o 11. .9 9 t . - 1 6 . . 1 6 . 5 )B. . B L r t e r e / d / . ( Plmtu Medicu. 1998. 64. ;13l ) hlr c \Lr,q,qc\t!'\i thut r ker llictor for succ e s s f u l f r - r t u r el i e l d p r o d u c t i o n u i l l r c " t i r t t h c ' . e ' l c ' c t i o no l ' g e n e t i c a l l y s u p e r i o r s t l a i n s g i v i n g i n c r e a s c c l \ ! - a ( ) n ( l i i r ' \n r L ' t i . r b ( ) l i tper o d u c t i o n . t o g e t h e l w i t h i n t p r o v e m e n t si n a - g r o t e c h n o l o L i i : inl r c t h o t l ' . Cell culture of Htpericuttt spp. untl thcif .hcnl()t\pc. hil\ pro\L-d e x t r e m e l y v a r i a b l e i n n a p h t h a d i a n t h r o t r rr- i c l t l ' r i r t h l . . c u t l r r h rp e r i c i n production exceeding that of hlpericin rl.
cH2R CHe
OH Hypericin, R= H Pseudohypericin, R = OH
tig,22.13 H y p e r i c icnnsdp h l o r o g l u c i n oo f ls hypericum.
Klflnr!
tI Lii.. l)lttrttrt
M e t l i c t r . 1 9 9 6 . 6 2 . - 5l ) . F l a v o n o i d s . c o n r p l c t . ' 1 1t l i l l c l c n t t o t i r r r : c o l t h e i n t a c t p l a n t a n d i n c l u d i n g t h e n e u c o n r p ( ) u n r l( r - ( ' | 1 . ' 1 1 l1l u t c r r l i n . h a r c
Hyperforin R = CH(CHs)z Adhyperforin R = CH(CHe)CHzCHs
Furohyperf orin
@
P H A R M A C O P O E IA N L D R E L A T EDDR U G SO F B I O L O G I C AO LR I G I N beenidentifiedin callusculturesof H. ltelbrat urr (A. P.C. Diaset rr1.. Phytochenisttl'.1998.48. 1165). Other HypericrJrnspp. The genuscontainssornezl00 spp.: rnostof the more comrlon oneshure lorler or nil hypericincontentsanclare in the clriedconditionby the natureofthe rid,ees ollen distinguishable (lnrperateSt John'swort) is similarin conon the stem.H. rttuctrlutLrnt stituents ro H. pefibruturirbut containsless:it may be distinguished by the slightl.v quaclrangularstem and larger leaves. H. hirsLttLtnt.H. u/ areother common Europeanspecres. tetropterrunttndH. rrtorttut
I
a)r^l
o l
H
N\
\A/ r rl
I I OH Oglucosyl
OH O Juglone
4$ o-Glucoside of c-hydrojuglone
o
o l
o
w w a\^aa
4-r/-n"
Action and uses, The currentexplosionin the populalityof St John's o H o o H o r.',ofirelatesto its unregulatedavailabilityfor the treatmentof mild tcr Plumbagin Lawsone lnoderatedepression. In the USA, for the lirst eight monthsof 1999,it rankedsecondto ginkgo as the best sellingproductof thc helbal r-r-rain(Ebenaceae)napthoquinones occur as monomcr:. streanrnrarket.with retail sales valued at over $78 million (M. e.g. Diospyrosspp. complex and trimers. In addition to timber dimers usage(ebony)manr Blunrenthal.HerbalGram,1999.No. 11,64).In Germanyit represcntcd species of Dioq)l'r?r are used world-wide in the traditional medicineot' (M. Gleeson,l-ecturegivento the prescriptions 2-5-ci of all antidepressant r countries where they gtou'. Plmnbttgo ..ellanica (Plumbaginaceae Societl-of Pharmaceutical Medicine,JurreI 998).lt hasbeendescribedls grows throughouttropicalAl'ricaandAsia and the loot is usedin Indian 'Nature's side-eff'ects of thc lattcr'. Prozac'without the disadvantageous in additionto pentacyclic triterpenes. However,a cautionarywarninghas recentlybcen struckby two reports rredicine:it containsjLrglone Naphthoquinones have shown to be biosynthesized via a varibeen (S.Piscitelli etal.,krncet,2000,355.5'l7lF.Rnschitzkaetul..ibid.,518). ety of pathwa)rsincluding acetateand malonate (plumbagin ot' In thefirst, St John'swofi wasobservedto lowcr plasmaconcentrations of Plumbago spp.). shikimate/succinylCoA cornbined pathway (lauthe proteaseinhibitor indinavir. In thc second repofi. hearl transplant combinedpathway(alkannin). rejection.aserresultof theloweringof cyclosporinplasmauoncentrations sone)and shikirnate/mevalonate below therapeuticlevels.followed St John'swort ther"apy. Obviouslythe questionof co-medicationof hypericLrmvn'ithothel impoltant medica- Further reoding A K. RaoY R 1998Pharmacology Mallavadhani U V. Panda and mentsrequiresimmediateappraisalandsuggests the needfor sone regu901 951.Ret,r'arllt/r chenrotaronontyofDloqlrzs.Phytochernistry'{9(.1): latory control on the availability of the herbal products.The UK 27I refs metabolites Mcdcntscv A G.AkimenkoV K 1998Naphthoquinone of the Comnittee on Safetyof Merllcine.rhas issuedadviceon this aspect. .lT(6):9-35959.Reviet'n ith 112refs firngi. Phytochernistry recently the hypericins were regarded as the sole antidepresUntil sant principles of hypericum but in a numbet of papels (e.g. S. S. (Suppl. l), 7) it has Hennq ChatterjeeeI a1.,Pharntttcopsycltiatry.1998,31 inerntis(Lythraceae). a been demonstratedthat the hypeforins also have this ploperty. Hennaconsistsof the dried leavesof Lnr'r,.ionia in north Africa including Egypt, lndia The shrub cultivated and Ceylon. fbrthcorning. further investigations will undoubtcdly be Considerable leavcsare grecnish-brownto brown and about2.5-5 cm long. The apcr is muclonate,the mzLrginentire and revolLlte,and venation pinnutL'. Furtherreoding Hennacontainsa colouringmatter.lawson(a h1'droxynaphthoquinonc t. American Herbal Phzirmacopoeia and ThelapelrticCompendium(Jul1,1997). St John's wort. Htpericum petforulunt,S2pp. Puhli.sltetlusu .sepurate various phenolic glycosides.coumalins,xanthones,quinoids, B-sitos tnonogruphrr HerbalGram l99T, No. .10 terol glucoside,flar,onoidsincludin-eluteolinand its 7-O-glucoside. tat:. BornbardelliE, Morazzoni P 7995Htparictnt perlbrattrnt.Fitoterapia6l( l): resin and henna-tannin.(For a lepolt on ncw glucosidesseeY Takeda 43 68. An ctll-embrat'ingreviev'v,ith 96 ref.s Hennais commonlr and M. O. Fatope.J. Not.Procl.,1988,51,72-5.) Lcc M R 1999 Saint John's vort (HypericLunpe(it utLutt1.Abaln for hurt minds'l Edinburgh29: 2-53257 Proceedings ofthe Royal Collcgcof Physicians. usedasa dye for thehair andwool washedin a dilute solutionof ammonia and boiledin a decoctionof the drug shoLrld be dyedTitian red. Cochineol The astringentstenr-barkof L. inernrisis traditionally usedin Indra Cochinealis an importantcolourantandindicatorirndconsistsof thedded lbr the treatmentof jaundice.enlargement of the liver and spleen.and t'enraleinsects.Dactl'lopiuscoccrrs.contlining eggsand larvae.It con- fbr various skin diseases.Isoplumbagin.exhibiting signilicant antitainsabout l07c of carminicacid which is a C-glycosideanthraquinone inflamrnatoryactivity. has been isolatedfrom the bark in 0.057cyield (Table22.3).The insectsaredescribedin detailin Chapter33. (M. Ali and M. R. Grevcr.Fitoteropitt,1998,69. 181).For a reccnt dcrivatir,'c report on the hepatoprotective activity of the bark seeS. Ahmed et al.. 2000.69. 157. J. Ethnophurmucologr.
NAPHTHOQUINONESAND GIYCOSIDES Lithospermums was indicateclon p. 1591the-vare proThe natureof thesecompoLrnds and exhibit a bload ducedbl hiehel plants.fungi and actinomycetes insectirangeof biokrsicalactionsincludingfungicidal.antibacterial. In plantsthey comcidal.phytotoxic.cytostaticand anticarcinogenic. monly occur in the reducedand glycosidic forms as illustratedby the 4B-o-glucosideof a-hydroju-elone,a constitlrentof walnut tLeelelves (JtLgktns rcgia, Juglandaceae). On extractionand u'ork-up.or in the soil. the compoundsarc oxidaIn sorneheart-woods, tively convefiedto the colourednaphthoquinone.
(60 spp.) (Boraginaceae)contains plants The genus Lithospernt.Lun with hormonal activity. The seeds of the European L. ofticirtulc (gromwell) wele tbnnerly official in severalpharmacopoeias. The reportedconstituentsofthe helb trreshikonin,a naphthoquinone cafdedvative;scyllitol,a cyclitolla cyanoglucoside-lithospermocidel f-eic. chlorogenic and ellagic acids: and catechin-typetannins. Shikonin, the enantiomerof alkannin (tbund in Anchusa Root, see below) is alsoa constituenrof L. en'throrhiaonroot andis producedfbr the cosmeticand phannaceuticalindustdesin Japanby cell culture of
P H E N O LASN DP H E N O L G I CL Y C O S I D E S the plant. Among the many publications on this subject.Tani et al. (Ph1-tochemistn.1992,3f, 690) reported on the strucrure of an endogenouslyproducedoligogalacturonidenecessaryfbr the induction of shikonin biosynthesisin the culture. For investigationsrelating to the biosynthesisol shikonin from p-hydroxybenzoicacid and geranyl pyrophosphate in l,. er)'throrlti?,ot1see T. Okamolo et al., Phytochemistrl,,1995.38, 83. In the Far East.preparationsof the purple roots havelong beenused fbr the treatment of bulns, inflammations. wounds and ulcers. In Europe,alkannaroot hasbeensimilarly employedand it has now been shown in laboratoryteststhat shikonin and alkannin have no significant differencein anti-inflammatoryactivity. The occunenceof these naphthoquinonesis of interest.since similar compoundsoccur in the related families Rubiaceae (Galiun, Rubicr\. Verbenaceaeand Bignoniaceae.Litl'tospermumarvenseis used as an oral contraceptive in Central Europe, as it suppressesthe oestrus cycle. The North American Litho,spennLnt ruderalehas similar hormonalactivity. Alkqnnq root Alkanet or Anchusae Radix is the dried root of Alkanna tinctorio (Boraginaceae), a herb fbund in Hungary.southernEuropeand Turkey. It consistsof reddish-purpleroots about 10-15 cm long and l-2 cm diameter near the crown. The surfaceis deeply fissured and readily exfoliates.Attached to the crown are the remains of leaves having whitish, bristly hairs.Alkanna is usedfor colouringoils and tars and in the form of a tincture for the microscopicaldetectionof oils and fats. The pigmentsarenaphthoquinonederivativesof the lbrmulae below. Alkannin itself rnay be an artefactarisingfrom variousesters.Most of the pigment compoundsproduced in cell culture appear to give alkannin on KOH hydrolysis (TLC, Rlvalues) and root culturesgive pigmentsidenticalto thoseextractedfrom normal roots (G. Mita et al., Planr Cell Rep., 1991,13. 406).
a
-O-
t- -l Y t t l o Bcnzo-1-pyrone (Chromone)
Eugenin
.z\./-\./\. t i l i !-
rt
-l
tt
\Z
l
l
Y: o
Furanobenzo-1-pyrone (Furanochrornone)
o IJ
a/Y t
-
4t"o;rz1a'oue t i l i l
\1\
i
v
l
i
l
Xanthone
_
l
^tY
l
Fto
o o H
v
Benzophenone
Gentisin
Mangiferin
Fig.22.14 C h r o m o n e so n d x o n t h o n e s
(w^ \ry o H a
A l k a n n a n :R = - C H ' - C H r - . - r - { : :
^'."'o'
importantstepin their biosynthesisappearsto bc rheorirlarirc coupling of hydroxylatedbenzophenones. Sintplc orr genlrcd derivatives, such as gentisin which contriblltesto thc rellorr colour of fermentedGentianRoot (q.v.),are fbund in horh rhc Genrianaceae o H o -cHr-ct=\r. R= / Shikonin: and Guttiferae.More highly oxygenatedconrpound:and O--elycosylxanthonesare found in the former lantilr uherc-asprenylated Other rnembersof the Boraginaceae-for example, Mctcrotomia xanthones,severalofwhich haveantinticrobialproperties.arewidecep halo t es (SyrianAlkanet)-produce similarred naphthoquinones. ly distributedin the latter.For studieson rhe irnrifungalxanthones from the roots of Mariltt lariflortr. Guttilerae. see J.-R. Ioset. P h a r n t .B i o l . , 1 9 9 8 , 3 6 , 1 0 3 .T h e C ' - g l r c u r r lr a n t h o n em a n g i f e r i n (Ftg. 22.14'7is found in several spr-ciesof Htpericum and in CHROIT/IONES AND XANTHONES Cratoxylem p r uniJTo rum and Chiretta(.lrr'clrrat;h i ruta). Mangiferin These compounds are structural derivatives of benzo-y-pyroneand has anti-inflammatory,antihepatotoxicand antiviral properties.In althoughnot of greatpharmaceuticalimporlancea 1'ewcompoundsare contrast to its CNs-stimulant propefriesother xanthonesexhibit CNS depressivepropertiesin rats and ntice. worthy of mention. The mycotoxin pigmentsof CluvitttTt.r purpurea (ergot)are complex Chromonesare isomericwith the coumarins.A simple derivativeis eugenin(Fig. 22.14) found in the clove plant, S1i;.glaraLrromuticLtm. xanthonescalled secalonicacids.Thev contribute,with the ergot alkalMore complex are the furanochromones. the active constituentsof the oids.to the toxic propertiesof the u hole drug. frnits of Arzrzi visnaga(.q.v.). Xanthones are found mainly in the Gentianaceaeand Guttiferae, Further reoding otherwise scatteredsporadicallythroughout the plant kingdorn as in frornGuttiferae. Phytochemistry 28(4): the Moraceaeand Polygalaceae.The characteristicoxygenationpat- BennettG J, LeeH-H 1989Xanthones 961-998.165rel's tern of these compounds derived from higher plants indicated that PeresV. NagemT J 1997Triorrgenated naturally occuningxanthones. they were of mixed shikimate-acetate origin whereas xanthones zt'1(2): Phytochemistry l9l 211.Ret'ien'u;ith262 refsand l8l li.sted enb pourtcls derived from fungi show a charactelisticacetatederivation.An incltrding55.fungal andliclrcnmetabolites R = /-\CHr-CH=lHt Alkannin:
"oo,!^
):l:
AND RELATED DRUGS OF BIOLOGICAL PHARMACOPOEIAL ORIGIN
'
FLAVONOID FLAVONTAND RELATED GTYCOSIDTS
The llavonoidsu,hich occul both in thc fiee stateand as glycosidcsare the largestgroup of naturallv occurring phenols.More than 2000 of
thesecompoundsarenow known, with nearly500 occurringin the frec state. They are formed from three acetateunits and a phenylpropanc' unit as hasalreadybeenoutlined(Fig. 19.11)and are typedaccordinr to the stateof oxygenationof the Cj unit. i.e. C-2,3,4(seeFig.22.l-s and Table 22.-5).Examplesgiven in this section all have a T-pyronc moiety with the exceptionof the chalcones.which althoughnot strictlr
Z"',to\
CoHs
| i l i l l - - \
S-\11-{ i
l
:
)
lsoflavone (3-phenyl-y-chromone)
Flavone (2-phenyl-y-chromone)
Flavonol
,-\.-oH
r.1-.oMe
w
w
o H o
o H o
Naringenin; R=H R = rhamno-glucosyl Naringin: (Flavanones)
Hesperetin; R= H Hesperidin; R = rhamno-glucosyl (Flavanones)
fi-Yo* '"(T").V-",
LYo' '"yy"f*",
)-T* O
Flavanone
*o)o'-1,-o.-1A/-orl
*o--<--y"-D
I
CoHs
OH
H
O
O
R= H Quercetin; Hyperoside; R = galactosyl
)-Y H
Luteolin (Flavone)
lsoquercitrin; R = glucosyl Rutin;R = rhamno-glucosyl (Flavonols) OH
HO HO
o H o Amentoflavone
Robustaflavone (Biflavonols)
tig.22.15 typesof flovonoids, Structurol oglycones ond glycosides
Chalcone
PHENOLS AND PHENOLIC GLYCOSIDFS
Toble 22.5
Flovonoid fypes ond exomples.
Typ"
Compound
Nolure
Occurrence
Flovone
Chrysin
Dihydroxy 5,2
Flovone Flovone Flqvone
Bulin Apigenin Luteolin
Irihydroxy7 ,3',4' Trihydroxy5,7,4' Tetrohydroxy 5,7,3',4'
Flovone Flovonol
Fisetin Quercetin
Tekohydroxy 3,7,3',4' Pentohydroxy 3,5,7,3',4'
Flovonol Flovonone Flovonone Cholcones Xonthone lsoflovone
Koempferol Tehohydroxy 3,5,7,4' Eriodictyol Tekohydroxy5,7 ,3',4' Liquiritigenin Dihydroxy7,4' Unsiobleisomersof flovonones Gentisin 1,7-Dihydroxy-3-mefhoxy Formononefi n Dihydroxy7,4' Genisiein f rihydroxy5,7 ,4' Amentoflovone Hexohydroxy2x {5,7,4'l
Prunus,Populus(heortwoodond Bolmof GileodBud) Bufeomonosperrno seeds{onlifertiIity octivity) Porsley, Romonchomomile ftower Resedolufeoloond os glycosidesin celery, peppermint, wild corrotetc. Yellowcedorwood ond the dyestuffsumoc As the rhomnoglucoside rutinond os mony otherglycosides Senno Yerbosonto(Hydrophylloceoe) Liquorice F o m i l yR u t o c e ooen d l i q u o r i c e Gentionoond Swertlospp. Cimicifugorhizome,red cloverflower Redcloverflower,os glucoside in Genisto Rhusspp. Ginkgo,hypericum,
Biflovone
flavonoidsarebiosyntheticallyrelated.The anthocyaninsaredescribed later. The flavonesand their closerelationsareoften yellow (Latinf/auas. yellow). They are widely distributedin naturebut a1'emore common ln the higher plantsand in young tissues,wherethey occur in the ce1lsap. They havebeenusedextensivelyas chemotaxonomicmarkersand are abundantin the Polygonaceae.Rutaceae.Leguminosae,Umbellit'erae and Compositae(seeTable22.5). They occur both in the free stateand as glycosidesimost ire O-glycosides but a considerablenumber of flavonoid C-glycosidesare known. Dimeric compoundswith. for erample. a .518-carbon--carbon linkage are also known (biflavonoids).The glycosidesare generallysolublein water and alcohol.but insolublein organicsolvents;the geninsareonly sparingly solublein waterbut are solublein ether'.Flavonoidsdissolvein alkalis. giving yellow solutionswhich on the additionof acidbecomecolourless. Although the original high hopes for the therapeuticusefulnessof flavonoids were not immediately realized, recent researcheshave demonstrated their involvernentin the medicinalactionof drugssuchas liquoriceroot, Romanchamomileand gingko. It is very probablethat a numberof herbalremedies,whoseconstituentsareasyet unknown.will be shownto containactiveflavonoids.Of the 8.1drugsdescribedin the BHP,Yol1. 1991,some36 containf'lavonoidsbut not necessarilyasthe active constituents. A number of flavonoid-containingherbshave now been includedin the EP and BP; examplesare Birch Leaf. Calendula Flower,Elder Flower and Lirne Flower.The groupis known for its antiinflammatory and anti-allergic effects, for antithrombitic and vasoprotective properties, for inhibition of tumour promotion and as a protectivefor the gastricmucosa.Theseeffectshavebeenattributedto the influence of flavonoids on arachidonicacid rnetabolism.Many flavonoid-containingplants are diuretic (e.g. buchu and broom) or antispasmodic(e.g. liquorice and parsley). Some flavonoids have antitumour, antibacterialor antifungal properties.E.-A. Bae et al. (Plann Medica, 1999.65, zt42)have recentlyinvestigatedIhe in yitro ptlori activity of a numberof flavonoids(hesperidin. anli-Helicobac'ter l-resperetin, naringin,naringenin,diosrnin,diosmetin)and suggestthat even if not potentinhibitorsof. they may contributeto the preventionof gastritis.Others.e.g. tustic (from the wood of Morus tirtt'toria) and sumac(leavesof Rhu,sspp.)arecolouringand tanninmaterials.
Pure flavone.which is colourless.occr-ll'\()n the.urfacr-ol somc speciesof Prirnula.As shownin Tablell.-5. ntanr Ilrironesarephenolic or methoxyi derivativesand form sap-solublc!lreos1lg1.l5e intensityof their yellow colourincreascs rl ith thc nunrhc'r ot-hr drorr I groupsand with increaseof pH. Isoflavonoids. Isoflavonesare found in the ht-artso()d()l .pccic\ of Prurtusand in speciesof 1ris, and are particularlruhuntllnt in the '[-hc (e.g.in dyer'sbroom,Gerrlstutirtt'trtrittt. latt.-rconLeguminosae tainsgenistin(not to be confusedwith the gentisinol gcrrtianr.thc 7glucosideof genistein.Rotonenonecontrincd in the root. o1'1)cllrs andLoru:hocurpas species(seeChapter;11 ) is an irollar onLridin ri hich the 2.3 doublebond of an isoflavoneis redr,rct-d. Phyto-oestrogens. Isoflavones. along qith counre:tuns (alscr flavonoids)andlignans(q.v.),belon-u to a classof:uhstancesknuu n as non-steroidalphyto-oestrogens. Both struclurallr and tirnctionally they aresimiiarto oestradiol(Fi-e.24.-ltirncl relaterl\c\ hormonesand exert weak oestrogeniceffects.Thel u.reprL'\!-ntin certain fbods and herbalremediesand are well-docunrentccl il\ producinginfertility in animalsas. for example,clover diseasein :heep glazing on clovers containinga high phyto-oestrogen contcnt. Studiesprovokingmuch nredicaland g!'neralprcssattentionhave centredon the role of phyto-oestrolL-n\as clietart'constituentshaving positive eff-ectsin the prevention ot canccr. heart diseaseand postmenopar.rsal symptoms. Foods containingappreciablequantitiesof isotlavonesare soya beans.soy productsand other legumecrops:they arealsopresentin the herbs Red Clover Flowet BHP ('li'ifolirtnt protense')and broomtops (Cl'tl.russcoporius).In the plant thcv occur fiee or in the glycosidic fbrm, in the lattercasebein-eh1'drollscd by colonic bacteriato give the activeaglycone;genisteinand daidzeinare the principal examples,the latterbeing formed flom forrnononetin(Table22.5). A new class of non-steroidrl phyto-oestrogensare the prenylated flavonoids.Many of thesecompoundsareknown but it is only recently (M. that this activity has been clescribedfor S-isopentcnylnaringenin Krtaokaer al.. Pltutta Medicu.1998,64, 5 i 1l M. Miyamoto et al., ibid.. 516).
AND RELATED DRUGSOF BIOLOGICAL PHARMACOPOEIAL ORlGIN Biflavonoids. The first biflavonoid to be isolated was gingetin, in CAIENDUIAFTOWER 1929.Now more than 100are known rvith a varietyof biological activities being reported.Amentoflavoneis of wide distribution.e.g.species Source, Calendula flower derives from the marigold Calendulu of Ginkgo, Hy'pericuttt,Rlus and together with robustaflavonehas rfficinalis, family Compositae, and is not to be confused with 'marigold'referring Io Tagetesspp. The EP andBP specify the wholc recentiybeenshown to have activity againstinfluenzaA virus, HSV- 1 or cut, dried. and lully openedflowers detachedfrom the receptaclc -M. andHSV-2viruses(Y. Lin er al.. PlantaMedica,1999,65, 120). and obtainedfrom cultivatedvarieties.The BHP allows alsothe whole compositeflowers which includesthe involucreof bracts. Furtherreoding C. o|:ficinalis is a native of Central, Eastern and Southern Europe BarronD. lbrahinrR K 1996Isoprenylated tlavonoids-a survey.Phytochemistry . 1 3 ( 5 ) : 9 2 91 8 1 and commercialsuppliesareobtainedlargelyfiom EastelnEuropeand Bohm B A 1998 Introductionto flavonoids.Vol. 2 of Chemistryand biochemEgypt. istrv of organicand naturalproducts.Hiuwood Academic,Amsterdam Donnelll D NI X. Boland G M 1998Isoflavonoidsand relatedcompounds. Natural ProductReports l5(3): 211-260 Knight D C. Eden J A 1995Phytoestrogens-a shortreview.Maturitas22: 167-175 PiettaP-G 1000 Flavonoidsas antioxidants.JournalofNatural Products63(7): 1035 10.12
BIRCHIEAF
I
Birch leaf is official in the EP and is now describedin the BP. The drug has been included in several European pharmacopoeiasfbr many years.It consistsof the dried whole or brokenleavesof Betulapendula andior B. pube.\censcontaining not less than |.5c/cflavonoids calculated as hyperoside.B. pendula (silver birch) is common throughout (downy or white birch) is Eurasianin origin. Europe andB. pubescerz.i Cornmercialsuppliesderive largely from EasternEurope, China and the former USSR. Leavesfrom B. pendula are 2.5-6.0 cm in length and 2-5 cm wide, glabrous,green on the upper surface,lighter green on the lower: in shaperhomboidal.triangularor ovoid in outline with a broadly tapering or cuneatebaseattachedto a long petiole.The margin is biserrate, the apex long and acuminate.Larger veins are pinnatewith an overall reticulation. B. pubescenshas similar, somewhat smaller, more rounded and often slightly pubescentIeaves.The apexis neitherlong nor acuminate and the marginalteethare smallerand lessobviously apically directed than with B. pendula. The microscopicalcharacteristicsof both speciesare similar, the lower epidenni having, in surface view, straight-walledcells and numerous anomocytic stomata with four to eight subsidiary cells. Calcium oxalatecrystalsoccur as clustersin the mesophyllcells and as crystal fibres near the larger veins. Peltatesessileglands situatedin shallow depressionsare numerous on both surfaces.B. pubescens possessesunlignified unicellular thick-wailed covering trichomes 80-100-200-600 prmin length. The flavonoid compositionsof both .8.pendula andB. pubescensare similal with total flavonoids (about 37o) including hyperoside(up to 0.87c).quercitrin (up to 0.14Va),myricetin galactoside(up Io 0.37%) and other glycosidesof quercetin (e.g. the arabinosideaviculzLrin), kaempf'eroland myricetin. For a detailed analysisof 14 batchesof B. pendula and threebatchesof B. pubescensseeA. Carnatet al., Ann. Phurnt.Frunc.. 1996,54,231.This groupalsofound the flavonoidlevel of older leavesof B. penduLato be lower than that of young leaves.The small proportion(0.lVo)of essentialoil containssesquiterpene oxides. Other constituentsof the leavesare (+)-catechin,monoterpeneglycosides,triterpene alcohols and estersof the dammarenetype. The mineral content(c.4a/o)ts pafticularlyrich in potassium. Preparationsof the leaf are used as an irrigant of the urinary tract, especiallyin casesof inflammationand renal gravel.Birch Ieaf is also an antirheumaticand has beenemployedto treatgout; as an astringent it is usedas a mouthwash.
Characters. Detaileddescriptionsfor the ligulatefloretsandthe composite flower headswill be found in the -BPandBHP respectively.Diagnostic featuresare the molphology of the ligulate and tubular florets, various biseriateclothing and glandulartrichomes(seee.g.Fig. 213.4), pollen grains with a spiny exine,and corolla liagmentswith yellow oil-droplets,calciunr oxalateandfairly largeanomocyticstomata. Constituents. Flavonoids,triterpenoids,essentialoil and polysac charidesare the principal constituentsof calendulaflowers.All groups have been shown to exhibit pharmacologicalactivity and serve to illustratethe difficulty of devising an assaywhich representsthe true therapeutic activity of the drug. The EP and BP determine the flavonoid content, expressedas hyperoside (not less than 0.4%i. utilizing the same method as for Birch Leaf. Other assaysbasedon triterpenoidassessment havebeendescribed. The flavonoid mixture involves quercetin and isorhamnetin derivatives. Triterpenoid saponins (calendulosidesA-F, see Table 22t.5)aLc glycosidesbasedon oleanolicacid-3-O-po-glucuronide and are present in variable proportions (2-l0Ed depending on time of harvesting and chemotype.The roots area richer sourcethan the flowers. Thesesaponin: have haemoiyticand anti-inflammatoryactivity (R. Della Loggia. Pkrrrr Medica, 1994, 60, 5 16). Polysaccharidesinclude a rhamnoarabinogalactan (Mr 15 000; rhamnose24.8%,, arabinose31.2qc,galaclrlse41.07c)and two arabinogalactanswith Mr's of 25 000 and 35 000 (J. Varljen er a1.. Plrytochemistn',1989,28, 2379).Antitumourand phagocytosisstimulation propeties havebeenrepoftedfor the polysaccharidefraction.At leasr I 5 compoundshavebeenidentified in the essentialoil. Other constituentsof the flowers are triterpenealcohols(e.g. a- and and carotenoids. B-amyrin,calenduladiol,etc.),sesquiterpenes Uses. Calendulais usedintemallyfor the alleviationof gastrointestinal disordersand extemally,as an ointmentor lotion, fbr the treatmentol minor wounds and rashes.
ETDER FLOWER Elder flower consists of the dried llowers of Sambucusrrigra, fan"rih Caprifoliaceae.This shrub or small tree is native throughoutEr"rropeand Westernand CentralAsia; commercialsuppliesof the flowers come principally from EasternEurope.small quantitiesare collectedin the UK. Characters. The elderinflorescenceconsistsof small regularl1osers arrangedin compoundumbel-likecymes;calyx superior,5-toothed: corolla flat, rotate,deeply 5-lobed,creamywhite with 5 stamensinserted in the tube; anthersyellow. The flowers have a slightly bitter tasteand a sweet,not altogetheragreeableodour. The microscopicalcharactersof the corolla include numeroussntall oil globules and an upper epidermiswith cells having slightly thickened,beadedwalls and a striatedcuticle. Epidermalcells of the calr r also have striatedwalls and those at the basalend exhibit unicellulrr marginalteeth.Calcium oxalateis seenas idioblastsof sandycrystal:.
P H E N O LASN DP H E N O L G I CL Y C O S I D E S There are numerouspollen grains about 30 pm in diameter(as measured in a chloralhydratemountant)with a faintly pitted exine and three germinalporesandfumous. Constituents. The drug containsa small proportion (up to c.0.2Vc) of a semi-solidvolatile oil consistingof free acids,principally palmitic acid.and Ct+-C:r n-alkanes.By 1985over 80 componentshad been identifiedin the oil. Flavonoids (up to 3.0%) are predominantly flavonols and their glycosides:rutin predominateswith smallerquantitiesof isoquercetrin, astragalirrand hyperosidetogetherwith the aglyconesquercetinand kaempferol. Other constituentsare triterpenes(a- and B-amyrin principally as esters of fatty acids), triterpene acids (ursolic, oleanolic and 2OBhydroxyursolic acids), various other plant acids (chlorogenic, pcoumaric,caffeic and ferulic acids.seep. 179.and their B-glucosides), sterols.mucilage,tannin and tracesof sambunigrin(Table26.1).
OH glucose
k a e m pef r o l
t
I
acio r-coumaric
_
lrlrrosrde
galactomannans. anda smallploportionof volatileoil (c. 0.02-0.I 7c) containing farnesol.farnes\l itcetitte.-geranioland eugenol gives the dlug its characteristiclaint odor-rr.more pronouncedwith the liesh flowers.Phenolicacidsand proanthocvanidins are alsopresent. The official upper lirnit tbr fbrcign matter in Lime Flower is2.)c/o 'f. with observationsfor the ubsenceof utneri<:onc(basswood)and il tomentosabasedon flower structurc.\o lr:slrr,is given but methods Standards. Thele are EP/BP limits for discoloured. brown flowers havebeenpublishedin the Gcrmrn literature. (15%) and for fiagments of coarsepedicelsand other foreign matter As with most herbalremedicslime llrrrrer: havr.a rnultiplicityof (87c). Thin-layer chromatographyis employed as a test for identity applications. In actionthey are diaphor..tie. lnti\pasmodicand expecwith further modificationto detectadulterationwith Sambucusebuhrs. torantand as suchareused.oftenin con.lunctiLrn s ith otherherbs.as a Flavonoids,calculatedas isoquercetrinare determinedby absorbance nerve tonic. for the treatmentof catarrh rLndin,:iilrcrtit)n.irnd fbr the measurements at 425 nm. alleviationof headaches. Allied species, Sambucttsebulus (danewort) is a perennial,foetid glabrous herb with a creeping rhizome and updght little-branched PASSIFTORA stems.It occurs throughout Europe and apart from habit. is distinguished from ,5. nigra by obvious ovate stipules. S. cttnadensis, Passiflora(PassionFlower)consistsof thedriedrcrill pun. ot Ptrr.rr/lrrlrr American elder,is a somewhatsmallertree than S. nigra and is widely incarnatacollectedduringthe floweringand il'uitinqpcrirrtl.Thc dnro is describedin the BP, EP, BHP, the Briti.rltHerltul Crnnt,urrilirl. \ol. I spreadthroughoutNorth America; it is usedsimilarly Io S. nigra. andin anESCOPmonograph;it is alsoofllcial in tltc li'cneh. Clcrntanand Swisspharmacopoeias. The genusis natire to South..\rrre.rica and :pecies Uses, Elder t'lowers are administeredprincipally as an infusion or herbal tea fbr the treatmentof feverish conditions and the common are widely cultivatedas omamentals.P. irttttnufitri. intporlcdlhrnt the cold; it acts as a diaphoretic but the mechanism and constituents USA. India andto someextentfrom the \\tst lnthe:. By the natureof its definition, the macroscopicaland nricro:eopical involved are unclear.The llowers alsohave diureticproperties. of the drug arenumerousand for thc-'cthc rcaderrhould It may be noted that the sialic acid-bindinglectin presentin elder characteristics stem-barkextractsfinds considerablecurrent use in cefiain biochem- consultone of the sourcesmentionedabore. ical procedures.
LIMEFLOWER Lime Flower EPIBP consistsof the dried inflorescences of TiLiacordara (small-leaved lime). T. plan'phylLos (broad-leavedlime), I. x vulgaris or a mixture of these,lamily Tiliaceae.The treesare native throughoutEurope and extensivelyplanted. Commercial suppliesof the flowels come from China, the Balkans, Turkey and Hungary, the latterexporting(1997) over 100 tonnes. The inf'lorescences consistofpendulouslong-peduncledcymesconsisting of yellowish-greenflowers, the pe
HO
n Vitexin
Constituentsof the genusinclr.rdeflavonoids,mainly C-glycosides of apigenin and luteolin such as vitexin. isovitexin, orientin, isoorientin and their 2"-B-o-glucosidcs.The BP drug is requiredto contain not less than 1.5% total f-lavonoidscalculated as vitexin (measurement at 401 nm after treatmentof a dry extractwith methanol in glacial acetic acid follon'ed by boric acid and oxalic acid in anhydrous formic acid. Other constituentsinclude traces of volatile oil. cyanogeneticglycosidesand possiblytracesof alkaloidsof the harman type. Speciesother than D. iltcannta (P. coertilea,P . eduli.s)al'eeliminatedby thin-layerchromatography.
DRUGSOF BIOLOGICAL ORIGIN AND RELATED PHARMACOPOEIAT Passiflora hassedativeactions:it is a popularingredientofherbalpreparestlessness andinitability. rationsdesignedto counteractsleeplessness. Hesperidin qnd rutin usedin suchashesperidinandrutin zLre Althoughllavonoid preparations medicine.they cionot appearto hai'ejustilied the high hopeswhich fol'citrin'(sometimes lowed the work of Szent-Gy'orgfi in 1935 on the known as vitan'rinP1of paprikaand lemon peel.Citrus and other fruits have long beenincludedin the human diet and, in additionto ascorbic acid and otherconrpounds.provideflavonoidswhich decreasecapillary fi'agility and aretherefirrecrnployedin casesof hypertensionand radia'citrin'is now krown to formerly known as tion injurics.The sr.rbstance of eriodictyol (a tetrahydroxybe a mixture of the rhamnoglucosides Among the commercially flaronc) and n.rethyleriodictyol(hesperetin). availablcproductsofthis type are someproducedby the citrus industry of rutin,therharnnoglucoside A similarglycosicie, containinghesperidin. qucrcetin.is tbund in mar-ryplants,and commercialsuppliesare made ti'onr tobacco residues, Sophora anrJ Eucalyptus spp. or buckwheat (L ttEioptrurnesculentum), which yieldsabout3-4%. Hairy root cultures c>fF. esculentur?are reported to give a higher tlavonol production than 1993,32,929). normal root cultures(F. Trotin et al.. Phvktchemi,strl-, nigra (.elder)havelong beenusedin domestic The flowersof Sambuctt,s and veterinarymedicine,particularlyin the fbnr-rof ointment.They containp-coumalicacid,rutin andkaempferol. Rutin occurs as a yellow crystalline powder, soluble in alkali but only slightly soluble in water. Rutin on hydrolysis yields quelcetin. rhamnoseand glucose,while hesperidinyields hesperetin(or rnethyl eriodictyol),rhamnoseand glucose.
cH2R2 Khellin: Rr = OCH:, R2 = H Visnagin: Rl = R2 = H Khellol glucoside: Rl = H, R2 = O-Glucoside
DINS AND GTYCOSIDES ANTHOCYANI Anthocyanidins are llavonoids structurally related to the flavones. Their glycosidesare known as anthocyanins.Thesenamesare derived lionr the Greek untho-, flower, and kyanos,blue. They are sap pigmentsand the actualcolour of the plant organis determinedby the pH of the sap.For example,the blue colour of the cornflower and the red of roses is due to the same glycosidesand both of these plants on hydrolysiswith hydrochloricacid yield cyanidinhydrochloride.
of{ structure A nthocyanidin cl
Silybin ond silymorin
I
ol flavonolignans-for example,silybin and silymarin(a 1,zlA nun-rber dioxanproducedby the oxidativecombinationof taxifblin and conif'eryl properties,and extractsof plantsconalcohol)-have anti-hepatotoxic taining then-r-fbr example, Sil.-bum trutrianwn (.Curuluus mariun tts), Compositae-are widely usedin Germanyfbr the treatmentof liver ailments.The fruits of S. marinnLntcontain silybin. silydianin and silychristin.For furlher detailsand structureof silybin seeChapter28.
to\y'-r-9.--.lt \ ' r _ _i _ , . _l C . O lH I
OH
F
Cvanidinchloride
Table22.6 gives a f'ewexamplesof thesenumerousand very widely distributedcompounds.The most common anthocyanidin,cyanidin, occurs in about 807cof permanentlypigmentedleaves,69olcof fruits and 50% of llowers. Cyanidin is followed in order of frequencyby delVisnogo phinidin and pelargonidin. The drug consists of the dried ripe fruits of Ammi visnagtt Anthocyanidinsareprecipitatedfrom aqueoussolutionsas lead salts (Umbelliferae).an annual plant about l-1.5 m high. It grows in the or as picrates.Alter hydrolysis with 207c hydrochloric acid. anthoMiddle Eastand is collected,pafticularlyin Egypt. The greyish-brown cyanidin hydrochlorides,being only slightly soluble.often crystallize mericarpsare usually separatebut are sometimesattachedto the carout. Chromatographicmethodsare widely usedfor the separationand pophore.Each mericarpis broadly ovoid and about0.5 mm long. It has identificationof both the aglyconesand slrgars. five plominent primary lidges and six vittae.Odour, slightly aromatic; taste.very bitter. Khellin, the most importantactiveconstituent,is crystallineand has It occurs It is 2-rnethyl-5,8-dimethoxyfuranochrome. beensynthesized. loble 22.6 Common onthocyonidins. beingreportedin the to the extentof about I 7c,the highestconcentration R' Occvrrenceof glycosides Anthocyonidin R immature truits. and is accornpaniedby two other crystallinecompounds,visnagin(about0. I o/c)and khellol glucoside(about0.37c).The FlowersoI Pelorgonium n Pelorgonidin H fruit
