Advances in Phytomedicine Series, Volume Three Editors: Mahendra Rai and Marı´a Cecilia Carpinella
Naturally Occurring Bioactive Compounds
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Advances in Phytomedicine 3
Naturally Occurring Bioactive Compounds Mahendra Rai Department of Biotechnology, SGB Amravati University, Amravati, Maharashtra, India
Marı´a Cecilia Carpinella Laboratoria Quı´mica Fina y Productos Naturales, Facultad de Ciencias Quı´micas, Universidad Cato´lica de Co´rdoba, Co´rdoba, Argentina
Amsterdam – Boston – Heidelberg – London – New York – Oxford – Paris San Diego – San Francisco – Singapore – Sydney – Tokyo iii
Elsevier Radarweg 29, PO Box 211, 1000 AE Amsterdam, The Netherlands The Boulevard, Langford Lane, Kidlington, Oxford OX5 1GB, UK First edition 2006 Copyright r 2006 Elsevier B.V. All rights reserved No part of this publication may be reproduced, stored in a retrieval system or transmitted in any form or by any means electronic, mechanical, photocopying, recording or otherwise without the prior written permission of the publisher Permissions may be sought directly from Elsevier’s Science & Technology Rights Department in Oxford, UK: phone (+44) (0) 1865 843830; fax (+44) (0) 1865 853333; email:
[email protected]. Alternatively you can submit your request online by visiting the Elsevier web site at http://elsevier.com/locate/permissions, and selecting Obtaining permission to use Elsevier material Notice No responsibility is assumed by the publisher for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions or ideas contained in the material herein. Because of rapid advances in the medical sciences, in particular, independent verification of diagnoses and drug dosages should be made Library of Congress Cataloging-in-Publication Data A catalog record for this book is available from the Library of Congress British Library Cataloguing in Publication Data A catalogue record for this book is available from the British Library ISBN-13: 978-0-444-52241-2 ISBN-10: 0-444-52241-7 ISSN: 1572-557X (series)
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Contents Preface to the Series
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Preface
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1. Natural compounds as antioxidant and molting inhibitors can play a role as a model for search of new botanical pesticides Carlos L. Ce´spedes A., J. Guillermo Avila, J. Camilo Marin, Mariana Domı´nguez L., Patricio Torres and Eduardo Aranda
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2. Pesticides based on plant essential oils: from traditional practice to commercialization Murray B. Isman and Cristina M. Machial
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3. Natural substrates and inhibitors of multidrug resistant pumps (MDRs) redefine the plant antimicrobials George P. Tegos
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4. New concept to search for alternate insect control agents from plants Isao Kubo
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5. Role of Melia azedarach L. (Meliaceae) for the control of insects and acari: present status and future prospects Marı´a C. Carpinella, Marı´a T. Defago´, Graciela Valladares and Sara M. Palacios
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6. Bioactivity of fabaceous plants against food-borne and plant pathogens: potentials and limitations Deepak Acharya, Aniket Gade and Mahendra Rai
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7. Screening of plants against fungi affecting crops and stored foods Olı´via C. Matos and Caˆndido P. Ricardo
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8. Opportunities and potentials of botanical extracts and products for management of insect pests in cruciferous vegetables Tong-Xian Liu, Han-Hong Xu and Wan-Chun Luo
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9. The potential for using neem (Azadirachta indica A. Juss) extracts for pine weevil management in temperate forestry Jonathan R.M. Thacker, Wendy J. Bryan, Robin H.C. Strang and Stuart Heritage
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10. Plant allelochemicals in thrips control strategies Elisabeth H. Koschier
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11. Importance of plant secondary metabolites for protection against insects and microbial infections Michael Wink
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12. Naturally occurring house dust mites control agents: development and commercialization Young-Joon Ahn, Soon-Il Kim, Hyun-Kyung Kim and Jun-Hyung Tak
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13. The search for plant-derived compounds with antifeedant activity Monique S.J. Simmonds
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14. An overview of the antimicrobial properties of Mexican medicinal plants Diana Jasso de Rodrı´guez, Jose´ Luis Angulo-Sa´nchez and Francisco Daniel Herna´ndez-Castillo
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15. Promissory botanical repellents/deterrents for managing two key tropical insect pests, the whitefly Bemisia tabaci and the mahogany shootborer Hypsipyla grandella Luko Hilje and Gerardo A. Mora
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16. Naturally occurring anti-insect proteins: current status and future aspects Tzi Bun Ng 405 17. Antifungal natural products: assays and applications Doris Engelmeier and Franz Hadacek
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Contributors
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Subject Index
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Preface to the Series The systematic study of herbal medicinal products and the investigation of the biologically active principles of phytomedicines, including their clinical applications, standardization, quality control, mode of action and potential drug interactions have emerged as one of the most exciting developments in modern therapeutics and medicine. Studies in phytomedicine have moved from purely descriptive analytical studies to conceptual inquiries on the pharmacodynamic advantages and limitations of plant medicines for the treatment of moderate or moderately severe diseases and prevention. Healthcare practitioners and medical scientists have come to accept herbal medicinal products as drugs that are different from the pharmacologically active molecules that they may contain. Several comparative clinical studies have been published to show that these plant medicines could have full therapeutic equivalence with chemotherapeutic agents, while retaining the simultaneous advantage of being devoid of serious adverse effects. Developments in molecular biology and information technology have now made it possible for us to begin to understand the mechanism of action of many herbal drugs and the associated phytomedicines, which differ in many respects from that of synthetic drugs or single chemical entities. Herbal medicinal products are now generally available in both industrialized countries and traditional societies. With the current lack of standardization and regulation of herbal products, it is important to develop common criteria for judging safety and efficacy of phytotherapeutic agents. This ‘new’ science demands different approaches to the classical methods of drug analysis, dosage formulations, manufacturing and claims substantiation. The therapeutic response observed with most herbs and phytomedicines are often not fully explainable using the currently available methods. Their activity usually characterized as polyvalent and interpreted as an aggregate or additive outcome of several constituents in the plant medicines are subjects of intense pharmacological studies. In most cases, a rationale does not even exist for the observed pharmacodynamic effects of very low doses of phytomedicines after prolonged or long-term application. The public press is replete with lay information and claims about the use of herbal remedies, however, there is scarcity of scientifically accurate reviews and guides on the efficacy and safety of plant medicines. The time therefore seemed ripe to broaden the communication on the use and benefits of phytomedicines as safe and useful natural health care products aimed at the health professionals and scientists. It is also important to provide a broader dissemination of the extant scientific literature on phytomedicines, to enable the conventional medical community to fully appreciate the fact that plant extracts specifically, and natural products generally, offer valuable and needed benefits in the treatment and prevention of diseases, especially for conditions where there is no effective or generally acceptable drugs. Although there are many papers published yearly on the use and analysis of plants as sources of biologically active molecules and some published materials on the use of plants as medicinal substances, volumes specifically addressing the needs of vii
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scientists and clinicians in the use of herbal products and standardized extracts as medicinal agents are far-in-between. We considered it therefore appropriate to fill this gap by producing an entirely new multi-volume series on the sourcing, selection, standardization, safety and clinical application of herbal medicinal products. As the name of the series implies, emphasis will be on those herbal medicinal products that are well characterized, standardized and substantiated as phytomedicines. The series will also provide timely reviews on the industrial production, regulatory and policy issues related to the use of phytomedicines. Although the literature in this field is evolving so rapidly that books on the subject become obsolete soon after they leave the press, the volumes in this series will aim at capturing the fundamental framework of each topic while remaining thoroughly up-to-date and comprehensive in scope. The aim of this series is to present to the scientists and clinicians the state of current knowledge in various fields of phytomedicine research, development and use. The approach is to provide the historical background to each topic, discuss methodological issues and illustrate current trends with case studies and critical examples, and when ever possible, the authors will indicate those plant medicines that are available for immediate use in clinical settings. The series is not intended to serve or replace the many excellent journals in this field but it will rather attempt to distill information from primary references and introduce elements of medicinal plants research and development that are in transition from speculative knowledge to standard practice. Advances in Phytomedicine is also not meant to be a textbook of the various topics covered in the series. It will, however, provide a guide to specialized articles and books on the topics that are relevant to scientific research and development of plant medicines, as well as information on the regulatory issues, clinical trials and application of phytomedicines for healthcare. Advances in Phytomedicine will therefore serve as a platform for reviewing recent developments in the use of herbal medicinal products. The coverage will include reviews of studies and use of all plant medicines, phytotherapeutic agents, nutraceuticals, plant cosmetics and therapeutically important molecules derived from these plant medicines. The first volume in this series has been devoted to exploring the ethnomedical approaches to drug discovery. It is indeed a very important starting point in addressing the relationship between plants and human health. Subsequent volumes will deal with other aspects of the use of herbal medicinal products. Selection of subjects will be through consultations with experts in the various fields of interest. We shall be guided by the principles of the so-called 6S, that is herb selection, sourcing, structure, standardization, safety and substantiation. Acknowledged experts and authorities in the various aspects of phytomedicine will be invited to assemble and edit specific volumes in the series. The series is the outcome of extensive consultations among several biomedical scientists and clinicians who participated in the workshops and conferences organized by the Bioresources Development and Conservation Program (BDCP) on related subjects. I am immensely grateful to these colleagues for their support in developing the original concept. Many thanks go also to Ms. Kim Briggs and Ms. Joke Zwetsloot of Elsevier Science for their suggestions and help in producing this series. I am indebted to my colleagues at BDCP and the International Centre for Ethnomedicine and Drug Development for their contribution; and to my wife, Kate for her love and support. I acknowledge the International Cooperative Biodiversity
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Group (ICBG) of the Fogarty International Centre, United States National Institutes of Health for providing financial support to my research group at the Walter Reed Army Institute of Research and BDCP. Maurice M. Iwu M.Pharm., Ph.D. Se´ries Editor
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Preface There has been a long history of success in discovering drugs from natural sources, particularly in tropical countries like Japan, China, India and Nepal. Unfortunately, natural products have fallen out of favour in current high-throughput screening. However, the diversity of three-dimensional shapes of natural molecules still surpasses that from synthetic compounds, and this ensures that natural products will continue to be important for drug discovery. Owing to the excessive use of the chemicals, the environment has been polluted. Consequently, there is a pressing need of search for new and natural chemicals for the sustainability of the environment. The plant-based drugs provide the natural and harmless system for the cure of various diseases. Majority of the drugs are prepared from the plants or natural products. There are a number of new and emerging diseases and new spectrum of microbes which has warranted the scientists to go for new natural drugs. Since time immemorial, herbal drugs have been in use all over the world in general and tropical countries like Japan, China and India, also Mexico and South Africa, in particular. Natural dyes and colours are health-friendly and responsible for maintaining the sanctity of the environment. The medicinal use of plant parts and extracts has been traditionally practiced since ancient times. However, a scientific approach to understand the relation between active chemicals and cure of diseases is recent. The use of plants in medicine has evolved because, from the biochemical point of view, they produce different chemical compounds against bacteria and fungi, the number of chemical structures is large and only a few are well known. Secondary metabolites obtained from plants have diverse functions into the plant such as defensive and protective process. The phytochemical studies are biodirected with an aim to find botanical origin biopesticides. Diterpenes, limonoids, triterpenes, sesquiterpene lactones, coumarins and flavonoids from Agavaceae, Asteraceae, Cactaceae, Celastraceae, Meliaceae and Zygophyllaceae families and also some chemical derivatives from them are important. These compounds possess antioxidant, antifungal, insect growth regulator (IGR) or insecticidal activities. The acetylated and carbonyl a-b-unsaturated derivatives showed mainly a low activity, while those that contain hydroxyl, carbonyl and oxy groups are more active as well as antifungal, IGR or insecticides. The pests and diseases of plants as well as animals have developed resistance as a result of which the existing antibiotics do not give the expected results. Therefore, there is a great need to develop the natural product-based drugs. The book will not only describe the effects of natural extracts and/or their isolated compounds, but also an update of its use and commercialisation. In some cases, the results related to the effects are new or they are an update of known data. The book will also help to make the use of effective and environmentally safe botanical pesticides, a new weapon to control these negative pests and diseases, which means a considerable alternative to the dangerous and excessive use of synxi
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thetic pesticides. Although there are several books on natural drugs, the present book covers multiple aspects of natural products, which is the need of the hour. We are indebted to Professor R.C. Rajak, director of Basic Sciences, Bundelkhand University, Jhansi, Uttar Pradesh; and to Professor S.C. Agrawal, head, Department of Applied Microbiology and Biotechnology, Dr. H.S. Gaur University, Sagar, Madhya Pradesh, who graciously offered suggestions for this book. We thank all the contributors for submitting their valuable manuscripts. Thanks are also due to my colleagues – Drs. Anita Patil, N.J. Chikhale, P.A. Wadegaonkar, P.V. Thakare, Aniket Gade and S.D. Kove. MKR thanks Deepak Acharya and Ravindra Ade, who checked manuscripts and helped in type-setting. Our special thanks to Drs. Susanna Zacchino, Donatella Mares, Michael Wink, George Tegos, N.P. Manandhar and Shubhangi Ingole, who went through the chapters and offered helpful suggestions. We are grateful to Dr. Kamal Singh, Vice Chancellor, SGB Amravati University, Amravati, for inspiration. Finally, and most importantly, MKR thanks Shivangi, Shivani and Aditya for their unconditional love, support and patience during the editing process. Mahendra Rai, Maria Cecilia Carpinella
Rai and Carpinella (eds.) Naturally Occurring Bioactive Compounds r 2006 Elsevier B.V. All rights reserved.
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CHAPTER 1
Natural compounds as antioxidant and molting inhibitors can play a role as a model for search of new botanical pesticides CARLOS L CE´SPEDES A, J GUILLERMO AVILA, J CAMILO MARIN, MARIANA DOMI´NGUEZ L, PATRICIO TORRES, EDUARDO ARANDA
Introduction Nowadays, pesticides of synthetic origin have been widely used, producing a strong impact on the environment with the emergence of resistant strains of microbes and insects to these types of compounds. New plant protection chemicals are needed for modern pest management due to insect resistance and ecological disorders associated with numerous currently used pesticides (Castillo et al., 1998). One area of investigation that is not so much an innovation as it is a return to an old approach with new technology is the characterization of secondary plant products (Berenbaum, 1989, 2002), and this can help in plant defense through new biotechnological approaches like metabolomic, genomic, and proteomic (Berenbaum, 1995, 2002; Kessler and Baldwin, 2002; Sumner et al., 2003). Most of them have potent effects on insect pests, low mammalian toxicity, lack of neurotoxic activity, low persistence in the environment, and biodegradability (Jacobson, 1989; Singh et al., 1997). Thus, organic molecules of botanical origin may offer a safe source of compounds for pest management, being environmentally friendly, and an excellent alternative to persistent synthetic insecticides (Berenbaum, 1989; Miyazawa et al., 1997; Castillo et al., 1998; Crowley et al., 1998). Plants in general produce a great variety of secondary metabolites that do not have apparent function in physiological or biochemical processes; these compounds (or allelochemicals) are important in mediating interactions between plants and their biotic environment (Berenbaum, 1989, 1991, 1995, 2002; Kessler and Baldwin, 2002). There is a widespread effort to find new pesticides, and currently it is focused on natural compounds such as flavonoids, coumarins, terpenoids, and phenolics from diverse botanical families from arid and semi-arid lands of Mexico and Americas. Additional experimental work has been carried out with natural products, which are potential models for defensive substances against insect and fungal predators (Kubo et al., 1994, 1995; Crombie, 1999; Kubo et al., 2000) and as enzyme inhibitors,
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tyrosinase or acetylcholinesterase (AChE) for instance (Kubo, 1997; Keane and Ryan, 1999; Ortego et al., 1999; Kubo et al., 2000; Ce´spedes et al., 2001a, 2001b; Kubo et al., 2003a, 2003b). The increasing interest in the possible application of secondary metabolites for pest management has directed the investigation towards search for new sources of biologically active natural products, with new mode, sites, and mechanisms of action, selectivity, and specific action (Jacobson, 1989; Gonza´lezColoma et al., 1997; Singh et al., 1997; Conner et al., 2000; Eisner et al., 2000; Eisner and Meinwald, 1995); these characteristics may enhance their value as commercial pesticides (Gonza´lez et al., 1992; Isman et al., 1995, 1996; Valladares et al., 1997; Gonza´lez and Este´vez-Braun, 1998; Gonza´lez et al., 2000). Recent studies have demonstrated that many plant species produce and accumulate a large variety of secondary metabolites that provide defense against insect predators (Berenbaum, 1989; Guella et al., 1996; Marvier, 1996; Berenbaum, 2002). Among several efforts to find new pesticides, current research is focused on limonoids from the Meliaceae family due to their potent effects on insect pests and their low toxicity to non-target organisms (Koul and Isman, 1992; Kumar and Parmar, 1996; Singh et al., 1997). Some examples are Azadirachta indica (Meliaceae) and Derris elliptica (Fabaceae), which produce very well known insecticides azadirachtin and rotenone, respectively (Gomes et al., 1981; Kraus et al., 1993, 1995). The main characteristics that account for the successful use of these secondary metabolites as natural insecticides are mentioned above, which make them lesser aggressive to the environment than the synthetic ones (Camps, 1988; Berenbaum, 1989; Castillo et al., 1998). Although the members of the family Meliaceae are widely distributed in the world, only Melia, Toona, Cedrela, and Swietenia have been studied (Arnason et al., 1987; Champagne et al., 1992; Kubo, 1992; Arnason et al., 1993; Kraus et al., 1993; Kubo, 1993; Govindachari et al., 1995; Chan and Taylor, 1966; Ce´spedes et al., 2000), and have afforded some limonoids such as azadirachtin, gedunin, toosendanin, cedrelanolide, mexicanolide, odoratol, anthothecol, nomilin, bussein, entandrophragmin, among others. Azadirachtin is the best known example of these limonoids (Champagne et al., 1989; Ramji et al., 1996). This compound and their analogues are potent insect antifeedant and ecdysis inhibitors (Govindachari et al., 1995; Kraus, 1995). However, the structural complexity of this compound precludes its synthesis on a commercial scale (Isman et al., 1996). These facts have led us to search for new simple secondary metabolites with insecticidal activity from other families including plants of Agavaceae, Asteraceae, and Meliaceae, such as Yucca spp., Parthenium spp., Roldana spp., Tagetes spp., and Cedrela spp., respectively, specially from tropical and subtropical lands of Mexico. As mentioned earlier, tyrosinase also known as polyphenol oxidase (PPO) (Meyer, 1987) is a copper containing enzyme widely distributed in microorganisms, animals, and plants. It catalyzes two distinct reactions of melanin synthesis (Robb, 1984), the hydroxylation of a monophenol (monophenolase activity) and the conversion of an o-diphenol to the corresponding o-quinone (diphenolase activity). Tyrosinase is responsible for browning in plants and is considered to be deleterious to the color quality of plant-derived foods and beverages. In addition, sclerotization and molting regulation processes in insects show that tyrosinase is one of the key enzymes in the insect metamorphoses process (Andersen, 1990).
Natural compounds as antioxidant and molting inhibitors
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AChE, which is the enzyme contained in nerve tissues, plays an exceedingly important role in the transmission of a nerve impulse. The free acetylcholine in the inactive form bound to proteins accumulates in the ending of a nerve in vesicles. The consumed acetylcholine is constantly replenished by its synthesis (by the acetylation of choline). All these processes are occurring when an impulse is transmitted through a cholinergic synapse. Thus, the process of synaptic transmission is an involved biochemical cycle of acetylcholine exchange. AChE has a key role in this cycle because the inhibition of activity leads to the accumulation of free acetylcholine in the synaptic cleft, producing the disruption of nerve impulses, then convulsive activity of the muscles can be transformed into paralysis, and other features of selfpoisoning by surplus acetylcholine appear. It occurs when AChE is inhibited by some terpenoids (Ryan and Byrne, 1988; Miyazawa et al., 1997). On other hand, modifications to AChE can confer resistance to insecticides (Fournier et al., 1992). In addition to many chemicals (flavonoids, stilbenoids, phenylpropanoids, and phenolics, among others) that show tyrosinase inhibitory activity, these compounds also show a strong antioxidant (AOX) activity against diverse reagents as model of AOX measurements (i.e., DPPH, ABTS, TROLOX, TRAP, ORAC, etc.). Mainly the activity is due to the presence of diverse moieties of the chemical structure of the molecules, for instance, orcinol or catechol groups, or hydroxyl bonded to an aromatic system (Gallic acid, Gallates in general, resveratrol, quercetin, etc.); in these cases, it is possible to correlate the AOX activity with tyrosinase inhibition and insect growth regulatory (IGR) activity. All these data are important for allelopathic, antifungal, IGR, and insecticidal studies, and are being accepted as direct or indirect measures of different physiological processes affected by the assayed chemicals (Baldwin et al., 2001; Schultz, 2002; Kubo et al., 2003a, 2003b; Torres et al., 2003). Some investigations on sites and mechanism of action of insecticidal or IGR report that different phenolic compounds are enzymatic and metabolic inhibitors (Klocke and Kubo, 1982; Kubo and Klocke, 1986; Kubo et al., 1994, 1995; Hammond and Kubo, 1999; Kubo and Kinst-Hori, 1999a, 1999b; Kubo et al., 2000; Shimizu et al., 2000; Caldero´n et al., 2001; Panzuto et al., 2002; Kubo et al., 2003a, 2003b). In addition, many of these compounds are polyphenolic secondary compounds that are ubiquitous in angiosperms and that have antifeedant effects on phytophagous insects (Feeny, 1968, 1976; Rhoades and Cates, 1976; Swain, 1979; Champagne et al., 1989, 1992; Simmonds, 2003). It has been assumed that phenols bind to proteins, acting as nutritional protein precipitating agents, thus reducing their digestibility (Feeny, 1976; Rhoades, 1979). We have previously demonstrated that diverse secondary metabolites have different sites of action and different molecular targets when they interact with enzymes and metamorphosis processes (Ce´spedes et al., 1999, 2000; Caldero´n et al., 2001; Ce´spedes et al., 2001a, 2001b; Ce´spedes et al., 2002; Torres et al., 2003; Ce´spedes et al., 2004). Our field observations indicate that these botanical species from arid and semiarid lands possess a strong resistance to the insect attack. The aim of this work is to correlate this phenomenon between the phytochemical composition with the inhibitory behavior on growth and development of Spodoptera frugiperda J.E. Smith (Lepidoptera: Noctuidae). The role of these compounds as ‘‘chemical messengers’’ has proven important to our understanding of many ecological problems and has led to the development of ‘‘chemical ecology’’ (Seigler, 1997).
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Aspects such as insecticide and growth regulatory activities, rate of development, pupation time, adult emergence, and deformity were evaluated and compared with those of gallic acid 10, gedunin 19, toosendanin 21 (Chen et al., 1995), anisic acid 38, and Cedrela-MeOH extract known growth inhibitors of S. frugiperda and tyrosinase, respectively (Ce´spedes et al., 2000; Caldero´n et al., 2001; Ce´spedes et al., 2001a, 2001b; Kubo et al., 2003a, 2003b). S. frugiperda is a very common insect pest in North- and South-American crops. These bioassays were used as model systems of insect pests. Our data also indicate that it is possible to correlate some AOX activities (i.e., crocin, 2,2-diphenyl-1-pycrilhydrazyl (DPPH)) with growth and development of fungus and insects; these data are important for allelopathic and chemical ecology studies (Ce´spedes et al., 2002). In addition to gedunin 19, toosendanin 21 (Figure 3), parthenolide 1, santamarin 2, 20 ,40 ,60 -trihidroxy acetophenone 3, rutin 4, and (7)-naringenin 5 were used as natural pattern substances and internal standard, together with THQ 6, BHA 7, quercetin 8, caffeic acid 9, gallic acid 10, and tocopherol 11 (Figure 1) in tests concerned with the reduction of DPPH and other AOX measurements. In the present chapter, we present a review of extracts and bioactive compounds isolated by our group from selected endemic botanical species, and their AOX and biological activities. The chapter includes antifeedant activities shown by assayed compounds, i.e., insecticidal or IGR activities and their activities on some enzymes.
Insecticidal or IGR (insect growth regulator) activity Agavaceae Yucca periculosa F. Baker, known commonly as ‘‘palmitos’’ or ‘‘izote’’ is a tree, endemic to Mexico, that grows in the semi-arid regions of Tehuacan–Cuicatlan Valley, Puebla–Oaxaca States. In the wild, these plants survive under different environmental stress conditions (Casas et al., 2001), with a lifespan of up to 100 years or so. Our field observations indicate that this specie has a strong resistance to insect attack. From methanol extract were isolated 4,40 -dihydroxstilbene 12, resveratrol 13, and 3,30 ,5,50 -tetrahydroxy-4-methoxystilbene 14 (Figure 2). The most active compound was stilbene 14 which showed the highest larval mortality together with MeOH extract of Y. periculosa (Me-Yuc), producing 100% of larval mortality at concentrations greater than 25.0 and 50 ppm, respectively, being noteworthy insecticidal. Thus, compound 14 and the Me-Yuc extract showed relative growth index (RGI) values of 0.25 and 0.45 at 10 and 15 ppm, respectively (Table 1). In addition, this compound was more active than gedunin and the MeOHCedrela extract used as positive control. The LD50 values for 14 and Me-Yuc extract were 5.4 and 7.18 ppm, respectively. In addition, compounds 12, 13, 14, gedunin 19, and the Me-Yuc and Me-Ced extracts inhibited each larval stage and diminished considerably the percentage of larvae that reached pupation; 14 and Me-Yuc showed the highest larval growth inhibitory activity, significant delay in pupation, and reduction of the percentage of emergence (Torres et al., 2003).
Natural compounds as antioxidant and molting inhibitors
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OH
O HO
OH
CH 2
O O
CH 2
H O
O
(1)
OH
O
(2)
(3)
OH OH HO
O OH O
HO
OH
O
O
CH 3 O
O OH
OH
O
OH
OH
OH
OH
O
OH
(5)
(4)
OH OH
O HO
HO
OH
O OH
HO
OH
OH O
OCH 3
(6)
(7)
OH
HO
O
HO
O
(8)
O HO
OH
HO
OH HO
(9)
(10)
CH 3 HO H H3C
O CH 3
CH 3
H
CH3
CH 3 CH 3
CH 3
(11)
Fig. 1. Chemical structures of pattern and standard compounds used for antioxidant and bioassays.
In order to establish correlation between IGR and acute toxicity activities with the AOX properties of these phenolic compounds, crocin and DPPH radical scavenging tests of these stilbenes were carried out. In 14 the presence of a methoxyl group increases the strength of these compounds upon inhibition of DPPH. We suggest that insect growth inhibitory activity of Me-Yuc extract could be caused by a
Naturally occurring bioactive compounds
6 H HO
C
C
OH
H
12 HO H C
C
OH
H HO
13 HO
OH H C
CH3O
C H
HO
14
OH
Fig. 2. Stilbenes from Y. periculosa.
synergistic effect of the stilbene composition. The inhibition of DPPH activity by stilbenes is well known (Burns et al., 2000, 2002; Rimando et al., 2002). Therefore, the plant stilbenes may be considered as efficient IGR and radical scavengers (Stivala et al., 2001; Kim et al., 2002; Rimando et al., 2002). The AOX activity of these compounds was also evaluated spectrophotometrically on the bleaching of the H2Osoluble crocin (Bors et al., 1992). Compounds 12, 13, 14, and Me-Yuc extract were all active, with activities comparable to gallic acid (positive control). Resveratrol 13 and the methoxylated stilbene 14 had more potent insecticidal inhibitory activity. This shows that nature of the substituent at C-3 plays an important role for the insecticidal activity (Torres et al., 2003). Meliaceae Cedrela is a genus belonging to the Meliaceae family. This genus includes several species like C. odorata, C. oaxacensis, C. dugessi, and C. salvadorensis, among others. Particularly we were interested in these species, because it is known that several of these species contain limonoids of gedunin-type (i.e., gedunin, photogedunin, cedrelanolide, mexicanolide, etc.), which have been isolated from Mexican species of the Genus Cedrela, and limonoids are noted because of their potent insect antifeedant characteristics and its potential for the development of ‘‘green pesticides’’ in insecticidal and chemical ecology studies. C. salvadorensis Standley is a small tree that grows on the dry pacific slope ranging from Jalisco to Chiapas in Mexico, through Central America, and up to the north of Panama (Rzedowski, 1972; Huerta, 1981; Rzedowski, 1991, 1993). Previously, we reported the isolation of photogedunin epimers 15, 17 as a mixture from a CH2Cl2 extract of this plant and the synthesis of its acetate derivatives 16 and 18 (Figure 3) (Ce´spedes et al., 1998).
Natural compounds as antioxidant and molting inhibitors
7
C. dugessi is also a small tree that grows on the dry pacific slope ranging from Michoacan to Oaxaca states in Mexico, in a similar form to C. salvadorensis. C. odorata is a tree of 4–5 m height that grows on the cost of Gulf of Mexico, throughout Veracruz and Tabasco states. C. oaxacensis is a tree of 4–5 m height that grows on the slope of Sierra Madre Mixteca, throughout Michoacan, Oaxaca, Veracruz, and Tabasco states (Rzedowski, 1972; Huerta, 1981; Rzedowski, 1991, 1993). Limonoids, photogedunins 15, 17, gedunin 19, cedrelanolide 20, and photogedunin epimeric mixture (15+17) were isolated from young trees of C. salvadorensis and C. dugessi (Ce´spedes et al., 1998, 2000). Photogedunin epimeric acetate mixture (16+18) showed the highest insecticidal activity at 10.0 ppm with 17% survival; the photogedunin epimeric mixture (15+17) with 50% survival at 10.0 ppm in a similar form to toosendanin 21 was next; gedunin 19 showed this effect at 39.0 ppm. Cedrelanolide 20 induced only moderate larval mortalities (o40%) while photogedunin epimeric mixture (15+17), gedunin 19, photogedunin acetates (16+18), and toosendanin 21 generally produced higher mortalities (>45%). The surviving larvae produce deformed pupae, which did not survive (Ce´spedes et al., 2000). Table 1 Insect growth regulatory activity of the isolated compounds, mixtures, and extracts, isolated from mentioned plants against S. frugiperda larvae in a no-choice bioassaya 7 Days Treatment 12 13 14 Epimers (15+17) Epimers (16+18) Gedunin 19 Cedrelanolide 20 Toosendanin 21 Agarofuran 22 Agarofuran 23 24 25 26 27 28 29 Argentatin A 30 Argentatin B 31 32 33 34 35 36 37
21 Days
Pupation
GWI50b
GLI50c
LD50d
GI50b
EI50b
pI50e
26.54 8.75 6.97 5.0 7.5 2.87 25.0 1.75 3.84 7.55 9.7 13.5 30.3 N.D. 3.1 4.0 o1.0 >5.0 5.1 24.7 5.9 6.8 31.7 57.8
32.74 11.6 7.24 10.0 15.0 5.53 75.0 29.0 o6.5 o12.0 5.28 27.3 8.14 N.D. 3.1 8.36 17.8 36.1 21.3 20.6 5.6 5.8 14.7 N.D.
27.6 6.4 5.4 10.0 8.0 30.08 N.D. 8.5 7.5 8.2 3.9 N.D. 27.8 N.D. 10.7 3.46 21.3 37.0 19.12 20.76 33.31 5.77 62.02 81.81
9.24 5.94 3.45 o5.0 o4.0 1.90 >5.0 2.68 0.37 0.43 o1.0 6.5 6.5 9.5 o1.0 >2.0 o5.0 5.0 8.07 14.65 4.20 4.90 18.08 24.41
N.D. 12.29 4.83 N.D. N.D. 3.95 N.D. N.D. o1.5 o2.5 0.55 4.68 4.46 6.91 0.74 3.05 o15.0 o15.0 4.32 22.60 4.59 10.54 39.91 7.07
0.96 0.77 0.54 N.D. N.D. 0.40 N.D. N.D. N.D. N.D. 0.26 0.67 0.64 0.84 0.13 0.48 N.D. N.D. 0.90 1.16 0.62 0.69 1.26 1.39
PI50f 38.26 7.19 9.02 10.0 o5.0 14.01 >60.0 o5.0 o1.5 o2.5 3.46 N.D. N.D. N.D. 2.11 4.62 o15.0 o5.0 3.84 24.27 6.04 14.21 23.01 7.61 (Continued )
Naturally occurring bioactive compounds
8 Table 1 (continued ) 7 Days
21 Days
Pupation
Treatment
GWI50b
GLI50c
LD50d
GI50b
EI50b
pI50e
PI50f
M Ma Me-Yuc Hex-G. microcephala Me-G. microcephala Me-Ced Me-P. argentatum Me-Maytenus Hex-Maytenus
4.1 19.2 14.99 3.2 5.5 3.56 o10.0 14.0 7.3
33.4 33.2 13.22 12.82 14.45 10.87 6.4 13.5 7.5
17.76 27.51 7.18 7.5 7.95 8.22 6.9 9.4 6.0
2.44 5.17 5.13 10.0 12.0 14.17 7.5 4.8 0.37
8.92 3.60 5.79 10.37 13.85 1.43 3.5 o5.0 o5.0
0.39 0.71 0.78 1.01 1.14 0.99 N.D. N.D. N.D.
13.90 4.78 18.82 5.91 12.4 3.84 o0.5 o5.0 o5.0
Notes: M and Ma correspond to a mixture of the compounds 32, 35, and 38 and its acetylated form, respectively. a The parameters are in ppm values. b The GWI50 and GI50 correspond to the inhibitory concentrations for reduction of 50% of larval growth in weight at 7 and 21 days, respectively, in ‘‘no-choice’’ test (po0.05) and EI50 correspond to concentration producing 50% of emergence and were calculated as the dose corresponding to midpoint between complete inhibition (100% of control) and no effect by PROBIT analysis and ANOVA (po0.05) corresponding to the growth inhibition at 7 and 21 days, respectively, under Microcal Origin 6.0. c GLI50 correspond to the growth inhibition in length at 7 days, and was calculated as the dose corresponding to midpoint between complete inhibition (100% of control) and no effect by PROBIT analysis and ANOVA (po0.05) under Microcal Origin 5.1. d LD50 is the concentration producing 50% of lethal mortality at 7 days in ‘‘no-choice’’ test calculated by PROBIT analysis and ANOVA (po0.05). e pI50 correspond to log GI50. f PI50 correspond to concentration producing 50% of pupation, and was calculated as the dose corresponding to midpoint between complete inhibition (100% of control) and no effect by the computer program ANOVA (po0.05) under Microcal Origin 6.0.
We do not ignore the fact that these compounds could act as antifeedant because we did not do the election test, but as pupation did not take place (100% of inhibition above 52 ppm), we suggest that the mechanism by which these compounds act may be due to physiological effects in a similar form to other limonoids (Nihei et al., 2002). We are investigating to elucidate the target, mechanism, and mode of action of these compounds and probably corresponds to a combination of antifeedant action and/or postdigestive toxicity, as found for other limonoids (Champagne et al., 1992; Isman et al., 1995). Celastraceae Celastraceae family embraces shrubs and trees of moderate height occurring in southern Chile. Plants from this family belonging to Maytenus genus have antifeedant and anti-inflammatory effects. These species show high allelopathic and antifeedant effects on plant and insect species in their habitat, respectively (Gonza´lez et al., 2000; Ce´spedes et al., 2001a). Maytenus genus is characterized by the occurrence of different bioactive compounds, especially b-dihydroagarofurans (Itokawa et
Natural compounds as antioxidant and molting inhibitors
9
OR
OR
O
O
O
O
O O
O O
O
O
O O
O
O
O
OAc
O
OAc
15 R = H 16 R = Ac
O
OAc
17 R = H 18 R = Ac
19
O OAc
H O O
O
OH
OAc COEt OHC
O O
O AcO
O
OH
O CO2Me
20
HO H
21
Fig. 3. Limonoids from Cedrela spp., Meliaceae.
al., 1993; Backhouse et al., 1994; Shirota et al., 1994). Until now, we focused mainly on two South-American species of this genus, Maytenus disticha (Hook) Urban and M. boaria. The former, commonly known as ‘‘maitencito’’ or ‘‘romasillo,’’ is a small tree that grows in rainfall forests in the South Pacific slope ranging from Araucanian Region to ‘‘Tierra del Fuego’’ in the Patagonian Region in Chile. M. boaria, the sole tree among the Celastraceae family in Chile, usually grows in the arid climate in the Slope Mountains. Considering that in Celastraceae family the main biologically active compounds are b-agarofuran type sesquiterpenoids (Figure 4), we selected these plants due to their high resistance to insect attack and no insecticidal work has been carried out on this plant. Moreover, we investigated the inhibition of AChE, a key enzyme in the insect nervous system in which the colynergic system is essential. From the aerial parts of M. disticha we isolated six agarofurans (Alarco´n et al., 1991, 1995, 1998) and from the seeds of M. boaria, four b-agarofuran polyesters were obtained and chemically characterized (Alarco´n et al., 1991; Ce´spedes et al., 1999). Insecticidal bioassays showed that a hex/EtOAc (HE) extract produced the highest mortality (79.1%) at 12.0 ppm, similar to 22, 23, and MeOH extract (>65%) and at 15.0 ppm, 23 and HE extract showed the highest larval mortality (100%). When larvae were fed with a diet containing 25.0 ppm or higher doses of all extracts and compounds tested, all the larvae died. Both b-agarofurans 22 and 23 inhibited larval growth after 7 days, and showed a significant growth reduction after 23 days. Compound 23 was more active than the positive control (toosendanin) and
Naturally occurring bioactive compounds
10 CH2OAc
OAc
OBz
OAc AcO
OFu OAc
OAc
O
O OAc 22
OAc 23
Fig. 4. b-Agarofuran type sesquiterpenoids from Maytenus spps.
compound 22, decreasing larval growth, total length of the larvae, and percentage of larvae that reached pupation and emergence. It is noteworthy that b-agarofuran 22, although was not so effective as 23, showed an appreciable reduction on larval length growth compared with the control, for this reason the activity of these compounds is comparable to the commercial insecticide toosendanin 21 (Chen et al., 1995). In all cases the adult weight was clearly reduced. Moreover, these compounds and extracts showed dose-response dependent activity. In order to determine the site of action of compounds and extracts from both plants, we carried out an AChE activity assay. MeOH and HE extracts, and 22 and 23 showed inhibitory AChE activity in a dose-dependent manner confirming that these compounds are active inhibitors of AChE in Maytenus spp. The activity of extracts may be due to a synergistic effect more than an activity of a single compound, and the presence of a furanoxy ring substituent increases the strength of these compounds on inhibition of AChE (Ce´spedes et al., 2001a). Asteraceae Our interest is centered on the study of possible insecticides of desert shrubs belonging to the Asteraceae family, due to their strong resistance against insect attack observed in nature. We have worked on four species of this family – Gutierrezia microcephala, Parthenium argentatum, Roldana barba-johannis, and Tagetes lucida – and evaluated their insecticidal and IGR activities on FAW. Gutierrezia microcephala G. microcephala A. Gray is commonly known as broom wood and grows in arid regions of the Central and North of Me´xico and in the southwestern region of the United States (Roitman and James, 1985). The plant collected in Saltillo, Coahuila State, is rich in flavonoids (Wollenweber et al., 1997). Four flavones 24–27 were obtained from the acetonic and methanolic extracts that were isolated previously by Fang et al. (1985, 1986). n-Hexane extract afforded bacchabolivic acid 28 (Zdero et al., 1989, 1992), a new ent-clerodane reported on this plant, which was lately submitted to esterification to yield the methyl ester 29. Insecticidal effects of 24–29
Natural compounds as antioxidant and molting inhibitors HO
11 OH
OMe OMe
OMe HO
HO
O
O
OMe OMe
MeO OH
MeO
OMe OH
O 24
O 25
OMe HO OH
OMe
OMe
OMe MeO HO
O
O
OMe OH OMe
MeO
MeO
OMe OH
OH
O
O 26
27
O CO2R
28 R = H 29 R = Me
Fig. 5. Flavonoids and clerodane from G. microcephala.
(Figure 5), and hexane and methanol extracts against larvae were evaluated (Caldero´n et al., 2001). Although 24, 28, and MeOH and Hex extracts produced significant larval mortalities (>49%) at a low concentration (10 ppm). Compound 29 produces higher mortality (81%). It is relevant that 29, 24, hexane, MeOH extracts, and 28 were more active than control (gedunin) with MC50 (50% lethal concentration of larvae at 7 days) of 3.46, 3.9, 7.5, 7.95, and 10.7 ppm, respectively. Compounds 24, 26, 28, 29, and hexane and MeOH extracts inhibited specifically each larval stage. Compounds 28, 29, and hexane and MeOH extracts produced higher inhibition (upto 90% of weight). Clerodane 28 showed the highest inhibition (100% of length and weight) at 50.0 ppm and flavonoids had lower larval inhibition at the same concentration. Growth reduction at 21 days was produced mainly by 24 and 28. Although 28 produced a significant delay in pupation, most important effect was observed by 24, 28, and hex extract. Significant delays in pupation time were observed at 10.0, 15.0, and 50.0 ppm for 28, 29, and hexane extract. Furthermore, hexane extract
12
Naturally occurring bioactive compounds
significantly reduced pupae weights at 50.0 ppm. Related to percentage of emergence, all the compounds and extract tested showed further reductions. Nevertheless, 24, 29, and hexane extract drastically reduced the percentage of adult emergence to 0% at 25.0, 15.0, and 50.0 ppm. In many of the treatments, mean adult weight was significantly delayed in the average time to reach the adult stage relative to control larvae. Growth index (GI) and RGI clearly showed that the stronger effect was shown by 28, 29, and hexane extract. It is possible to infer that the substitution of polymethoxy flavones induces an increase on the activity of these flavones and a furan ring seems to be necessary for insecticidal activity of ent-clerodane type diterpenes. In order to determine the site of inhibition on the IGR activity and the acute toxicity of the compounds and extracts, the effect on AChE activity was studied. In a similar form to the acute toxicity, 28, 29, MeOH, and hexane extracts showed the greatest inhibitory effect at 50.0 ppm and flavonoids 24–27 displayed a lower inhibitory effects. At minor concentration (25.0 ppm), 28 and hexane extract showed stronger activity level than MeOH extract and 29. In the same way the last two were more active than flavonoids. In addition, both extracts and ent-clerodanes 28 and 29 inhibited AChE activity in a dose-dependent manner (Caldero´n et al., 2001). In addition, the presence or absence of a methyl ester group increases or decreases, respectively, the strength of these compounds on inhibition of AChE in clerodanes. It is obvious that the nature of the ester substituent at C-8 plays an important role for the insecticidal activity of ent-clerodanes. The most active compound 28 contained a small and relative hydrophilic acid group at C-8, whereas 29 with a bulky and more lipophilic ester group exhibited a lower activity level. This is according to previously reported QSAR analysis performed by Rodriguez et al. (1999), where they proposed that antifeedant activity of the respective natural products depends on the polarity of ring B and the size of the ester substituents. With respect to the flavonoids, the presence of an extra methoxyl substituent in the A ring seems to be the cause of growth inhibitory activity (length and weight) shown by 24 and 25. In a similar form to polymethylated flavonoids where the introduction of a methyl ether excluding the B-ring in the flavonoids structure increased the antifeedant activity (Morimoto et al., 2000). Parthenium argentatum P. argentatum, commonly known as ‘‘guayule,’’ is a shrub endemic to the Chihuahua desert and is used as an important source of natural rubber. Plant was collected in Matehuala, State of San Luis Potosi, Me´xico. After collection, aerial parts were milled and extracted exhaustively with methanol and partitioned with n-hexane and ethyl acetate. Preliminary assays showed that MeOH extract had a considerable insecticidal activity. Chromatography purification afforded mainly two cycloarten-type triterpenes named Argentatin A and B (Rodrı´ guez-Hahn et al., 1970; Matsubara and Romo de Vivar, 1985; Martı´ nez-Va´zquez et al., 1994). As both compounds were purified in sufficient amount these were used in bioassays. Insecticidal effects of the Argentatin A 30, B 31, toosendanin 21 as a control, and methanolic extract against larvae of first instar of S. frugiperda were performed. Argentatin A produced higher mortality (80.2%) at 35 ppm; however, MeOH extract
Natural compounds as antioxidant and molting inhibitors
13
shows the highest insecticidal activity (96.5%) at a lower concentration (10.0 ppm). It is important to note that when larvae were fed in a diet containing 15.0 ppm or higher doses of methanolic extract all larvae died. Argentatin A and B inhibited specifically each larval stage. At 23 days this growth reduction was clearly significant between 25.0 and 50.0 ppm. However, only toosendanin 21 showed the highest larval growth inhibition at the same concentrations. All tested compounds in comparison to control decreased the percentage of larvae that reached pupation. Although compounds 30 and 31 showed significant delay in survival after pupation, pupation time, and percentage of emergence, the most important effect was observed by MeOH extract and the control (Figure 6). Furthermore, 30 and MeOH extract significantly reduced pupae weights, but toosendanin 21 showed the greatest effect (Ce´spedes et al., 2001b). GI and RGI clearly showed that the stronger effect was shown by MeOH extract and toosendanin. These parameters together with LD50 values corroborate the highest effect that showed methanolic extract. Compounds 30 and 31 showed moderate acute toxicity, nonetheless toosendanin and MeOH extract showed a potent acute toxicity on larvae of last stage at 35.0 and 15.0 ppm, respectively. The LD50 values of 21 and MeOH extract were 1.5 and 3.1 ppm, respectively. In order to determine the site of inhibition on the IGR activity and the acute toxicity of compounds and extracts tested, AChE activity was studied. Argentatin B showed a moderate AChE inhibition activity, unlike Argentatin A, MeOH extract, and toosendanin that are strong inhibitors of AChE activity in a dose-dependent manner, confirming that these compounds are the active inhibitors of AChE. The presence of a ring with seven members, as in Argentatin B, decreases the strength of this compound on inhibition of AChE, opposite to the presence of a furan ring. Strong activity showed by MeOH extract may be due to a synergistic effect (Ce´spedes et al., 2001b). Roldana barba-johannis The methanol extract from the aerial parts of R. barba-johannis (Asteraceae) afforded sargachromenol, sargahydroquinoic acid, and sargaquinoic acid. These natural products and the corresponding acetylated and methylated derivatives showed
OH O OH OH
O
O
O
31
30
Fig. 6. Triterpenes from P. argentatum.
Naturally occurring bioactive compounds
14
insecticidal and IGR activities against FAW. The most active compounds were sargachromenol 32, and its acetylated derivative 34; sargahydroquinoic acid 35 and its acetylated derivative 37; and a mixture of sargachromenol 32, sargahydroquinoic acid 35, and sargaquinoic acid 38 (6:3:1) and the acetylated form of this mixture; 33 and 36 showed a minor activity. All these compounds and mixtures had significant effects between 5.0 and 20.0 ppm in diets. Most compounds were insecticidal to larvae, with lethal doses between 20 and 35 ppm. In addition, these substances also demonstrated scavenging properties toward 2,2-diphenyl-1-picrylhydrazyl radical (DPPH) in TLC autographic and spectrophotometric assays. These compounds appear to have selective effects on the pre-emergence metabolism of the insect (Figure 7). The results from these compounds were fully comparable in activity to those known natural insect growth inhibitors such as gedunin and methanol extracts of C. salvadorensis and Y. periculosa (Ce´spedes et al., 2004). Tagetes lucida T. lucida known as ‘‘perico´n,’’ is a medicinal plant used from pre-Columbian times by Aztecs and other Mesoamericans. This specie is distributed from Northern Me´xico to Northern Nicaragua (Rzedowski, 1972, 1991). Their leaves and inflorescence are used for stomachache and for the treatment of diverse anti-inflammatory ailments. From a dichloromethane extract, we isolated several coumarins: scopoletin
O CO2R
R'O
32 R, R' = H 33 R = CH3 R' = H 34 R = H R' = Ac
CO2R
OR'
35 R, R' = H 36 R = CH3 R' = H 37 R = H R' = Ac OR'
O
CO2R
38
O M = 32 + 35 + 38 Ma = M methylated
Fig. 7. Plastoquinones and tocotrienols from R. barba-johannis.
Natural compounds as antioxidant and molting inhibitors CH3O
15 HO
HO
HO
O
O
HO
39
O
O
40
CH3O
O 42
O
O
O
O
O
41
AcO
CH3O
CH3O
CH3O
AcO
O
O
43
AcO 44
Fig. 8. Coumarins from T. Lucida.
(6-methoxy-7-hydroxycoumarin) 39, esculetin (6,7-dihydroxycoumarin) 40, 7-methoxy-6-hydroxycoumarin 41, scoparone (6,7-dimethoxy-coumarin) 42, and their derivatives 6,7-diacetoxy coumarin 43 and 6-methoxy-7-acetoxycoumarin 44 (Figure 8). The compounds 39, 40, 41, and dichloromethane extract showed high mortality effects between 5.0 and 15.0 ppm and an IGR activity between 1.0 and 5.0 ppm; 40 being the most active compound. The compounds 42, 43, and 44 showed IGR activity at concentrations higher than 70.0 ppm. A complete inhibition of the pupation and emergence above 70.0 ppm was observed. In the literature there are few information about insecticidal effects of coumarins. Nagaiah et al. (1992) reported about the IGR and antifeedant effects of some coumarins from the bark of Xeromphis uliginea on the growth of Spodoptera litura. These compounds are formed by the action of phenylalanine ammonia lyase (PAL) and are then converted to ortho-coumaric acid by the action of cinnamic acid o-hydrolase (Seigler, 1997). Coumarins are located within specialized structures such as secretory ducts called vittae (Berenbaum, 1991). Araucariaceae In the continuation with our search for natural products with possible insecticide activity, we have studied trees of the rain forest of southern Chile, due to their strong resistance against insect attack observed in nature. We have evaluated the insecticidal activity of Araucaria araucana and IGR activities on FAW. Araucaria araucana (Mol.) K. Koch This conifer is endemic to rain forest of southern Chile and Argentina. It has high commercial, ethnobotanical, taxonomic, and ecological value derived from its long biogeographical and remote occurrence. These characteristics offer a unique opportunity for the search of secondary metabolites, especially those that have a defensive role against pathological and phytofagous pests. Five lignans (secoisolariciresinol 45, lariciresinol 46, pinoresinol 47, eudesmin 49, and methyl-pinoresinol 48) were
16
Naturally occurring bioactive compounds
isolated from MeOH extracts of bark and wood of A. araucana and their structures were determined with spectroscopic methods. In addition to antifeedant, insecticidal, and IGR activities against S. frugiperda (FAW), the antibacterial activity of these compounds was determined against Gram (+) and Gram () bacteria and the antifungal activity against Fusarium moniliforme, Aspergillus niger, Trichophyton mentagrophytes, Mucor miehei, Paecilomyces variotii, Ceratocystis pirifera, Trametes versicolor, and Penicillium notatum. These lignans exhibited antifungal and antibacterial activities (Ce´spedes et al., 2006a) and antifeedant and IGR activities on FAW (Ce´spedes et al., 2006b), in the range between 1.0 and 50.0 ppm. Against FAW the strongest compound was pinoresinol 47 with an LD50 19.0 ppm, the MeOH extract from wood shows an LD50 of 400.5 ppm, and the MeOH extract from bark shows an LD50 of 92.1 ppm, these values show the antifeedant action of A. araucana against a Lepidoptera as FAW. In addition, these results show that the larval mortality of all compounds – below 50 ppm – is stronger than the bark MeOH extract and this suggests that this effect could be attributed to the lignin occurrence in this extract, since these lignans were isolated from this extract and all these measurements were twice lower than natural positive control – MeOH extract from bark of Y. periculosa – that showed a 98% of mortality at 50 ppm, as was previously observed (Torres et al., 2003; Ce´spedes et al., 2004). In addition to the above effects these lignans showed a strong AOX activity against DPPH radical reduction, with very low concentrations, between 1 and 10 ppm; almost all lignans showed an I50 inhibition at around 4.5 ppm, except methyl pinoresinol 48 and eudesmin 49, which did not show inhibition (Figure 9).
Experimental part Bioassays with fall armyworm (FAW), Spodoptera frugiperda Larvae used for the experiments were obtained from the culture at the Centro de Investigacio´n en Biotecnologı´ a at the Universidad Auto´noma del Estado de Morelos, Cuernavaca, Morelos, Me´xico, maintained under previously described conditions (Aranda et al., 1996; Caldero´n et al., 2001; Ce´spedes et al., 2000, 2001a, 2001b, 2004; Torres et al., 2003). Acute toxicity on FAW Acute toxicity was elucidated by topical application to last stage larvae of S. frugiperda. The larvae of S. frugiperda were iced to stop their movement and treated on their abdomens with each one of the test compounds. The solvent used was 10.5 ml of acetone in topical form with 50 ml microsyringe, and control was only treated with 10.5 ml of acetone. After 24 h, survival was recorded. Five larvae were used for each concentration, respectively (Torres et al., 2003; Ce´spedes et al., 2004). Relative growth index and growth index The RGI and GI were calculated according to Zhang et al. (1993).
Natural compounds as antioxidant and molting inhibitors
17 OCH3 OH
OH
HO
O H 3CO
OCH3
HO
OH
45
H3CO
O
46
R3 COOH
O O
R1
OH
HO
R4
O O OCH3 R2
O
50 47 R1=R3=CH3; R2=R4=H 48 R1=R3=R4=CH3; R2=H 49 R1=R2=R3=R4=CH3
Fig. 9. Lignans from A. araucana and anisol from other sources.
Reduction of 2,2-diphenyl-1-picrylhydrazyl [ ¼ 2,2-diphenyl-1-(2,4,6-trinitrophenyl) hydrazyl; DPPH] radical TLC autographic assay After developing and drying, TLC plates were sprayed with a 0.2% DPPH solution in MeOH. The plates were examined 30 min after spraying. Active compounds appear as yellow spots against a purple background. In a similar form, TLC plates were sprayed with 0.05% b-carotene solution in CHCl3. The plates were examined under UV254 light until the background had become discolored (bleached). Active compounds appeared as pale yellow spots against a white background. Spectrophotometric assay (Bors et al., 1992; Cuendet et al., 1997, 2000): A solution (50 ml) containing the compound to be tested was added to 5 ml of a 0.004% MeOH solution of DPPH. The measurement was done after 30 min, and the percent of activity was calculated (Torres et al., 2003). Inhibition of acetylcholinesterase An enzyme extract containing AChE was obtained according to the method of Grundy and Still (1985). Inhibition of AChE was determined according to the Ellman’s procedure (colorimetric method) (Ellman et al., 1961) using both the control (MeOH) and test solutions (compounds and extracts). All AChE inhibition was carried out according to procedures described previously in Ce´spedes et al. (2001a, 2001b).
18
Naturally occurring bioactive compounds
Discussion The sites and mode of action of these compounds is being investigated and probably correspond to a combination of diverse antifeedant actions as midgut esterase inhibition and postdigestive toxicity, as found for other limonoids (Champagne et al., 1992; Nakatani et al., 1994; Isman et al., 1996) and extracts (Feng et al., 1995) or as enzyme inhibitors such as estearases, proteases, tyrosinase, or AChE (Miyazawa et al., 1997; Ortego et al., 1999; Kubo et al., 2003a, 2003b). There are mainly three types of tyrosinase inhibitors based on their inhibition mechanisms. First, the inhibitors directly interact with the binuclear copper active site of the enzyme, known as either copper chelators or substrate analogues. The inhibitors belonging to this category are mostly aromatic compounds and usually act as competitive inhibitors; the inhibitors disrupt the tertiary structure of the enzyme rather than directly interact with the active site. These inhibitors are not only aromatic but are also non-aromatic compounds, and generally act as noncompetitive inhibitors. Second, some molecules have a hydrophobic part and this section interacts with the hydrophobic domain in the enzyme. These subunits associate in a geometrically specific manner, known as a quaternary structure. The hydrophobic moiety which is exposed on the other side of the molecule may disrupt tyrosinase’s quaternary structure. The low conformational stabilities of native proteins make them easily susceptible to denaturation by altering the balance of the weak non bonding forces that maintain the native conformation (Kubo et al., 2003c). Third, the inhibitors do not interact with the enzyme directly or indirectly but act as reducing agents for dopaquinone. In addition to these three main types, there are some additional types of inhibitors. For example, inactivators of the enzyme and some tannins belong to this type of inhibitors. Hence, tyrosinase inhibitors might ultimately provide clues to control insect pests by inhibiting tyrosinase, resulting in incomplete cuticle hardening and darkening (Kubo, 1997, 2000; Kubo et al., 2002). In the case of AChE, the action is directly related with the interaction between the inhibitors and acetylcholine, the inhibition of the enzyme occurs with detachment of the acid residue of the toxic compound, and the nature of acid residue does not affect the structure of the inhibited enzyme, but acts strongly on the process of its inhibition. Natural compounds carrying a charge in an acid residue are very active inhibitors of cholinesterase, such as alkylammonio, alcohols (Brestkin et al., 1992), alkaloids (physostigmine, phenserine, tolserine, among others) (Yu et al., 2002), some terpenoids (Ortego et al., 1999; Caldero´n et al., 2001; Ce´spedes et al., 2001a, 2001b), and monoterpenoids (Gracza, 1985; Grundy and Still, 1985; Miyazawa et al., 1997). Some investigations on sites and mechanism of insecticidal or IGR action report that different phenolic compounds are enzyme and metabolism inhibitors (Hammond and Kubo, 1999; Kubo and Kinst-Hori, 1999a; Kubo, 2000; Kubo et al., 2000; Shimizu et al., 2000; Caldero´n et al., 2001; Panzuto et al., 2002). It is important to note that similar IGR activity on S. litura (Common cutworm) was studied by Morimoto et al. (2000). These authors reported that flavonoids of Gnaphalium affine (Asteraceae) have insect antifeedant properties against this insect. It is possible to infer that the substitution pattern of flavonoids induce an increase in the activity
Natural compounds as antioxidant and molting inhibitors
19
of those phenolic compounds (Caldero´n et al., 2001). Until our report (Torres et al., 2003), there were no reports about insecticidal activity of stilbenes, but only those having tyrosinase inhibitory (Shimizu et al., 2000), and antifeedant activities of similar phenolic compounds (Kubo and Kinst-Hori, 1999a; Kubo, 2000; Kubo et al., 2000, 2003a, 2003b). The presence of hydroxyl, methoxyl, and furan moieties seems to be necessary for insecticidal activity as in limonoids containing several of these chemical groups (Nakatani et al., 1994; Ce´spedes et al., 2000, 2001a, 2001b; Caldero´n et al., 2001) and in other phenolic compounds as alkanols, tannic, gallic, and anisic acid, respectively (Hammond and Kubo, 1999; Panzuto et al., 2002; Kubo et al., 2003a, 2003b). It is worth mentioning that resveratrol 13, the methoxylated stilbene 14, photogedunin epimeric mixture (16+18), toosendanin 21, agarofurans 22 and 23, the flavonoid 24, the diterpenes 28 and 29, sargahydroquinoic acid 35, 6,7-dihydroxycoumarin 40, and pinoresinol 47 showed the most potent insecticidal activity. It is obvious that the nature of the substituents as well as hydroxyl and methoxyl groups in the aromatic ring plays an important role for the insecticidal activity. These results prove previous findings on the quantitative structure-activity relationship of stilbene derivatives for instance, namely that the inhibitory tyrosinase activity of the respective natural products depends on the polarity of ring A and on the size of the substituent (Gorham et al., 1995; Shimizu et al., 2000). These facts show that the antifungal activity showed by stilbenoids (Schultz et al., 1990, 1992; Pacher et al., 2002), acute toxicity, and growth inhibition may be due to the inhibition of tyrosinase, and this target was demonstrated also for other stilbenes and phenolics from natural sources, such as gallic 10 and anisic acid 50 (Shimizu et al., 2000; Gilly et al., 2001; Kim et al., 2002; Kubo et al., 2003a, 2003b). In addition to the enzymatic activities, IGR correlate very well with AOX measurements of the reduction of DPPH radical, in many of our cases mainly when the phenolic compounds show AOX activity they also show IGR activity. Our phenolic compounds could be considered as efficient IGR and radical scavengers (Ce´spedes et al., 2000, 2001a, 2002, 2003, 2004; Stivala et al., 2001; Kim et al., 2002; Rimando et al., 2002). Thus, it is possible to infer that when some compounds show AOX properties they can also show IGR or tyrosinase inhibitory activity. In our case the compounds that showed these activities were stilbenes, sargachromene and sargahydroquinoic acid, flavonoids such as quercetin, naringenin, and kaempferol, and coumarins. Thus, it can be said that natural compounds are AOX, antifungal, and molting inhibitors, which can play a role as a model for search of new botanical pesticides, and nutraceutical and bioactive compounds. The percentage of larvae that reached pupation decreased in all tested compounds in comparison to the control (Table 1). The most important effect was observed with stilbene 14, gedunin 19, toosendanin 21, b-dihydroagarofurans 22, and 23, clerodane 28, Argentatin A 30, sargachromenol 32, sargahydroquinoic acid 35, 6,7-dihydroxycoumarin 40, and pinoresinol 47, photogedunin epimeric mixture, photogedunin acetate mixture, which reduced successful pupation into a range between 0% and 25% and caused a significant delay in pupation time. The effects of these substances on reducing insect growth, decreasing the percentage of emergence, and increasing mortality of S. frugiperda are similar to those of other natural products (Arnason et al., 1987, 1993).
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Inspection of the structure of the most active compounds isolated from these plants suggests that the presence of oxygenated function was necessary for the activity displayed by the gedunin-type and phenolic molecules against S. frugiperda. Almost all tested compounds except cedrelanolide and methoxylated flavones showed comparable activity to the commercial insecticide toosendanin, which suggests potential for further development of these materials. However, none of these substances has been found with the outstanding activity of azadirachtin (Govindachari et al., 1995; Kraus, 1995).
Concluding remarks In summary, the insecticidal activity of extracts may be due to synergistic effects shown by the phenolic components of the mixtures in the test system used in this investigation. These facts are indicative of the potency of the methanol extracts. Thus, the effect of compounds on reducing insect growth, increasing development time, and increasing mortality of S. frugiperda is similar to that of gedunin and more potent than the MeOH extract from C. salvadorensis (Ce´spedes et al., 2000; Caldero´n et al., 2001). The sites and mode of action of these compounds and extracts are being investigated and probably correspond to a combination of antifeedant action as midgut phenol oxidase, proteinase, AChE, tyrosinase, or other PPOs and cuticle synthesis inhibition, as well as molting, sclerotization, toxicity, and nerval system inhibition, as has been found for other phenolics and terpenoids (Miyazawa et al., 1997; Kubo and Kinst-Hori, 1999a, 1999b; Kubo, 2000; Kubo et al., 2000; Simmonds, 2003) and extracts (Feng et al., 1995). In addition, the presence of an orcinol or catechol group seems to be important for these activities as shown for the most potent compounds in this chapter. Furthermore, a great percentage of larvae that reached pupation decreased with the application of phenolics in comparison to control, which might be due to the inhibition by tyrosinase as well or to the accumulation of proteinase inhibitors (Tamayo et al., 2000). The activity of these plants, their metabolites, and MeOH extracts is comparable to the insect growth regulators gedunin and toosendanin, which suggests potential for further development of these materials.
Future perspectives The plant kingdom offers a rich source of a wide range of structural biodiversity of natural secondary metabolites. One of the most recent trends in fungal and insect pest control is to reduce heavy reliance on synthetic pesticides and to move towards biodegradable substances. Synthetic pesticides of broad spectrum have been widely used as the main tools for controlling weeds, and fungal and insect pest, which are highly toxic to many living organisms as well as to the environment. Hence, new biorational and specific trends to pest control should be developed. In this chapter, we presented an update of our findings in this field, those approaches resulting from our studies on inhibition of growth (including larval growth, pupation, and emergence) and of the enzymes involved in key processes of insect life, specially modifying the apolysis during molting, sclerotization, pupation, and
Natural compounds as antioxidant and molting inhibitors
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emergence. Since these approaches refer to control of insect pests, many of them can be extrapolated and also considered suitable for medicinal chemistry studies, because the mechanism of action of these inhibitors is similar to that from human and other animals (Guerrero and Rosell, 2004, 2005). The study of AChE and other phenol oxidases inhibitors, as well as IGR substances, can help in the search of new natural compounds for the treatment of Alzheimer’s disease (Cummings, 2000) and Parkinson’s disease (Weinstock et al., 2003). Therefore, there is an increasing expectation about the research on enzymes inhibition by those compounds of botanical origin that could serve as lead compounds for the development of important substances with agrochemical and pharmacological properties. Thus, by studying the plant organisms that protect themselves against the pest attack, we can learn to control this attack in an ecological way and in addition can get pharmacologically active substances.
Acknowledgments We are very grateful to Prof. Isao Kubo (Department of ESPM, U. C., at Berkeley), Prof. Murray B. Isman (Faculty of Agricultural Sciences, U. British Columbia, Vancouver), and Prof. David S. Seigler (Plant Biology Dept. University of Illinois, at Urbana-Champaign), for the great and valuable help given to authors. The bioassays involving insecticidal to FAW larvae were skillfully conducted by Mrs. Laura Lina (Biological Control Lab., CEIB, UAEM, Cuernavaca, Morelos State) and in similar form the antifungal activity by Mrs. Ana Ma. Garcı´ a (Phytochemistry Lab, FESIztacala, UNAM) whose technical support is greatly appreciated. The works were partially supported by grants: IN243802 and IN211105 from DGAPA-UNAM.
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Schultz TP, Hubbard TF, Jin L-H, Fischer TH, Nicholas DD. (1990) Role of stilbenes in the natural durability of wood: fungicidal structure-activity relationships. Phytochemistry 29:1501–1507. Seigler DS. (1997) Plant secondary metabolism. Boston: Kluwer Academic Publishers, p. 790. Shimizu K, Kondo R, Sakai K. (2000) Inhibition of tyrosinase by flavonoids, stilbenes and related 4-substituted resorcinols: structure-activity investigations. Planta Medica 66:11–15. Shirota O, Morita H, Takeya K, Itokawa H, Iitaka Y. (1994) Cytotoxic aromatic triterpenes from Maytenus ilicifolia and Maytenus chuchuhuasca. J Nat Prod 57(12):1675–1681 (17 ref.). Simmonds MSJ. (2003) Flavonoid-insect interactions: recent advances in our knowledge. Phytochemistry 64:21–30. Singh M, Khokhar S, Malik S, Singh R. (1997) Evaluation of neem (Azadirachta indica A. Juss) extracts against American bollworm, Helicoverpa armigera (Hubner). J Agric Food Chem 45:3262–3268. Stivala LA, Savio M, Carafoli F, Perucca P, Bianchi L, Maga G, Forti L, Pagnoni UM, Albini A, Prosperi E, Vannini V. (2001) Specific structural determinants are responsible for the antioxidant activity and the cell effects of resveratrol. J Biol Chem 276(25):22586–22594. Sumner LlW, Mendes P, Dixon R. (2003) Plant metabolomics: large-scale phytochemistry in the functional genomics era. Phytochemistry 62:817–836. Swain T. (1979) Tannins and lignins. In: Rosenthal GA, Janzen DH, editors. Herbivores: their interactions with secondary plant metabolites. New York: Academic Press, pp. 657–682. Tamayo MC, Rufat M, Bravo JM, San Segundo B. (2000) Accumulation of a maize proteinase inhibitor in response to wounding and insect feeding and characterization of its activity toward digestive proteinases of Spodoptera littoralis larvae. Planta 211:62–71. Torres P, Avila JG, Romo de Vivar A, Garcı´ a AM, Marı´ n JC, Aranda E, Ce´spedes CL. (2003) Antioxidant and insect growth regulatory activities of stilbenes and extracts from Yucca periculosa. Phytochemistry 64:463–473. Valladares G, Defago MT, Palacios S, Carpinella MC. (1997) Laboratory evaluation of Melia azedarach (Meliaceae) extracts against the Elm leaf beetle (Coleoptera: Chrysomelidae). J Econ Entomol 90:747–750. Weinstock M, Gorodetsky E, Poltyrev T, Gross A, Sagi Y, Youdim M. (2003) A novel cholinesterase and brain-selective monoamine oxidase inhibitor for the treatment of dementia camorbid with depression and Parkinson’s disease. Prog Neuro-Psychopharmacol Biol Psych 27:555–561. Wollenweber E, Do¨rr M, Fritz H, Papendieck S, Yatskievych G, Roitman JN. (1997) Exudate flavonoids in Asteraceae from Arizona, California and Mexico. Z Naturforsch 52c:301–307. Yu Q-Sh, Zhu X, Holloway HW, Whittaker NF, Brossi A, Greig NH. (2002) Anticholinesterase activity of compounds related to geneserine tautomers. N-Oxides and 1,2-oxazines. J Med Chem 45:3684–3691. Zdero Ch, Bohlmann F, Niemeyer H. (1992) Furolabdanes and linear diterpenes from Gutierrezia resinosa. Phytochemistry 31:1723–1726. Zdero Ch, Bohlmann F, Solomon JC, King RM, Robinson H. (1989) ent-Clerodanes and other constituents from Bolivian Baccharis species. Phytochemistry 28:531–542. Zhang M, Chaudhuri SK, Kubo I. (1993) Quantification of insect growth and its use in screening of naturally occurring insect control agents. J Chem Ecol 19:1109–1118.
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Rai and Carpinella (eds.) Naturally Occurring Bioactive Compounds r 2006 Elsevier B.V. All rights reserved.
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CHAPTER 2
Pesticides based on plant essential oils: from traditional practice to commercialization MURRAY B ISMAN, CRISTINA M MACHIAL
Introduction With very few exceptions, botanical insecticides in commercial use have been used in traditional practices dating back at least 150 years, and often much longer (Jacobson and Crosby, 1971; Philoge`ne et al., 2005). Not unlike pyrethrum, rotenone and neem, plant essential oils or the plants from which they are obtained have been used for centuries to protect stored commodities or to repel pests from human habitations. Unlike other botanicals however, plant essential oils have a long history of human uses apart from pest control, notably as fragrances, flavourings, condiments or spices, as well as medicinal uses. It is ironic then, that development of plant essential oils as commercial insecticides has only occurred within the past decade. In noting the disparity between the vast number of plants shown to poison, deter or repel insects and the actual number of botanical insecticides in commercial use, I previously suggested that there are three barriers to commercialization of botanicals (Isman, 1997). These are: (1) availability of the plant material on a sustainable basis; (2) standardization of extracts based on quantification of active ingredients; and (3) governmental approval, normally requiring costly toxicological evaluation of the candidate product. Given the worldwide use of essential oils in the perfume, food and beverage industries, oils from many species are available in abundance yearround, and their major constituents are either well characterized or are themselves available in a relatively high degree of purity. Thus the major barrier to their use as pesticides may be government approval, but in the United States, several plants and their derivatives are exempt from registration owing to their widespread use in foods and generally acknowledged safety to humans. In the present chapter, the chemistry of plant essential oils, their biological activities in arthropods and fungi, and their potential as botanical pesticides in commercial practice is discussed.
Naturally occurring bioactive compounds
30
Sources and chemistry of essential oils Plant essential oils are obtained through steam distillation of the foliage or other plant parts of terrestrial plants in a limited number of taxa. Among the better-known families from which essential oils are frequently obtained are the mint (Lamiaceae), myrtle (Myrtaceae), citrus (Rutaceae) and carrot (Apiaceae) families. Most essential oils are highly complex mixtures of mono- (C10) and sesquiterpenes (C15), including biogenetically related phenols (phenylpropenes and cinnamates). These compounds are usually responsible for the characteristic odours and/or flavours of the plants from which they are obtained. The predominant group, the monoterpenes, are often cyclic, with a saturated (e.g. pulegone, Figure 1) or unsaturated hexacycle (e.g. limonene) or an aromatic system (e.g. thymol). Bicyclic (e.g. 1,8-cineole) and acyclic examples (e.g. citronellal) are not uncommon. CHO
O O
(+)-Limonene
(+)-Pulegone
1,8-Cineole
Citronellal
OH OCH3
OH
OH CHO Eugenol
Cinnamaldehyde
Thymol
Carvacrol
OCH3
O
Menthone
trans-Anethole
Fig. 1. Structures of some essential oil constituents with documented insecticidal and/or antifungal activities.
Pesticides based on plant essential oils
31
Functional groups include alcohols (e.g. thymol), aldehydes (e.g. citronellal) and ketones (e.g. menthone), but simple hydrocarbons also occur (e.g. limonene). Owing to their generally low boiling points (140–180 1C for monoterpenes, >200 1C for sesquiterpenes), all are relatively volatile, accounting for their use as fragrances and as insect repellents. While many of the monoterpenoids are chemically stable at ambient temperature, their volatility usually renders them nonpersistent in the environment. Among the best known essential oils with bioactivity against insects and other pests are clove oil (eugenol), thyme oil (thymol, carvacrol), mint oil (menthol, pulegone), lemongrass oil (citronellal, citral), cinnamon oil (cinnamaldehyde), rosemary oil (1,8-cineole) and oil of oregano (carvacrol). It is important to emphasize the intraspecific variability in chemical composition of plant essential oils. Sources of compositional variability can include the plant part extracted, phenological state of the plant, and time of year, as well as climatic and soil variations. Numerous studies have also confirmed that some species have distinct chemotypic races or populations, often separated geographically (Salgueiro et al., 1997; Pascual-Villalobos and Ballesta-Acosta, 2003; Angioni et al., 2004). In an investigation of the essential oils isolated from two rosemary ecotypes from Italy, the major constituent, 1,8-cineole, ranged from as low as 7.3% by weight to a high of 55.3% and a-pinene ranged from 11.5% to 30.3% (as isolated from apical, intermediate and lower foliage) (Flamini et al., 2002).
Biological activity Mode of action in arthropods The rapid onset of toxicity from essential oils or their constituents in insects and other arthropods points to a neurotoxic mode of action. In the American cockroach this is manifest as hyperactivity followed by hyperextension of the legs and abdomen culminating in rapid knockdown or immobilization, often in less than 30 s (Enan, 2001). Several essential oil monoterpenes have been shown to be weak inhibitors of acetylcholinesterase (AchE) in insect and mammalian preparations (Ryan and Byrne, 1988; Miyazawa et al., 1997), but toxicity in vivo did not correlate with AchE inhibition in vitro. More recently Enan (2001, 2005a) has provided evidence that many essential oil constituents poison insects by blocking octopamine receptors. Octopamine, synthesized from tyramine, is a neurotransmitter and neuromodulator in arthropods and may have neurohoromonal influences as well. Several essential oil constituents were demonstrated to be mild antagonists of octopamine binding activity in the cockroach nervous system, however, other such compounds were found to be agonists and yet others were without effect. Notable among the last group was thymol, one of the most toxic essential oil compounds to insects (Table 2). Enan (2005b) has also provided evidence that at least some essential oil constituents may act via the tyramine receptor cascade in the fruit fly Drosophila melanogaster. Physical effects, viz. dissolution and disruption of cell membranes, or blockage of the tracheal system in insects cannot be ruled out as alternative mechanisms of toxicity.
Naturally occurring bioactive compounds
32
Efficacy of essential oils to insects Table 1 lists a number of studies in which plant essential oils have been screened for bioactivity against insects. It should be readily apparent that the majority of studies to date have utilized stored product pests, particularly beetles such as Sitophilus, Tribolium and Acanthoscelides species, in which case fumigant toxicity has been investigated (Shaaya et al., 1991; Regnault-Roger et al., 1995; Obeng-Ofori and
Table 1 Screening studies with plant essential oils against insects Essential oil
Insect tested
Reference
53 species 31 species 28 species
Choi et al. (2003) Singh et al. (1989) Shaaya et al. (1991)
11 species
Trialeurodes vaporariorum Sitophilus oryzae Sitophilus oryzae, Rhyzopertha dominica, Oryzaephilius surinamensis, Tribolium castaneum Drosophila auraria
22 species
Acanthoscelides obtectus
4 species
Sitophilus oryzae, Tribolium confusum, Ephestia kuehniella Pediculus humanus Cydia pomonella Musca domestica Acanthoscelides obtectus
5 species 27 species 31 species 22 species 13 species
23 species Vetiver, Vetiveria zizanioides Patchouli, Pogostemon cablin Chamaecyparis obtuse Minthostachys andina, Hedomea mandonianum Tansy, Tanacetum vulgare Garlic, Allium sativum Nutmeg, Myristica fragrans Tetradenia riparia Holy basil, Ocimum sauve
Oryzaephilus surinamensis, Rhyzopertha dominica, Sitophilus oryzae, Tribolium castaneum Lipaphis pseudobrassicae Coptotermes formosanus Coptotermes formosanus Callosobruchus chinensis, Sitophilus oryzae Rhodnius neglectus, Triatoma infestans Choristoneura rosaceana Tribolium castaneum, Sitophilus zeamais Tribolium castaneum, Sitophilus zeamais Zabrotes subfasciatus Sitophilus zeamais, Rhyzopertha dominica, Sitotroga cerealella
Konstantopoulou et al. (1992) Regnault-Roger and Hamraoui (1994) Sarac and Tunc (1995) Mumcuoglu et al. (1996) Landolt et al. (1999) Singh and Singh (1991) Regnault-Roger et al. (1993) Shaaya and Kostjukovsky (1998) Sampson et al. (2005) Maistrello et al. (2001) Zhu et al. (2003) Park et al. (2003) Fournet et al. (1996) Larocque et al. (1999) Ho et al. (1996) Huang et al. (1997) Weaver et al. (1994) Bekele et al. (1996)
Pesticides based on plant essential oils
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Table 2 Toxicity of some essential oil constituents to insects Compound
Insect tested
Effect/dose
Reference
Eugenol
Spodoptera litura
LD50 ¼ 157.6 mg/larva
Sitophilus granaries
LD50~2.5 mg/beetle
Coptotermes formosanus Musca domestica Diabrotica virgifera Drosophila melanogaster Aedes aegypti
LD50~40 ppm (sand)
Hummelbrunner and Isman (2001) Obeng-Ofori and Reichmuth (1997) Cornelius et al. (1997)
Pulegone
Periplaneta americana Musca domestica Diabrotica virgifera Peridroma saucia Spodoptera litura Ostrinia nubilalis
Thymol
Citronellal
d-Limonene
Menthone
Drosophila melanogaster Musca domestica Drosophila melanogaster Drosophila melanogaster Spodoptera litura Culex pipiens molestus Spodoptera litura Callosobruchus maculatus Musca domestica Drosophila melanogaster Musca domestica Diabrotica virgifera Spodoptera litura Callosobruchus maculates Sitophilus oryzae Blattella germanica Musca domestica Sitophilus oryzae Drosophila melanogaster Peridroma saucia
LD50 ¼ 77 mg/fly LD50 ¼ 12 mg/beetle LD50 ¼ 4 mg/cm2 LD50~0.5 mg/43 cm2 paper LC50 ¼ 148 mg/cm2 paper LD50 ¼ 39 mg/fly LD50 ¼ 38 mg/beetle LD50 ¼ 753.9 mg/larva LD50 ¼ 51.6 mg/larva LC50 ¼ 26.3 ppm (in diet) LD50 ¼ 170 mg/4 mm dia paper LD50 ¼ 29 mg/fly LD50 ¼ 2600 mg/4 mm dia paper LD50 ¼ 5 mg/cm2 LD50 ¼ 25.4 mg/larva LC50 ¼ 36 mg/l LD50 ¼ 111.2 mg/larva
Lee et al. (1997) Isman (unpubl. Data) Bhatnagar et al. (1993) Ngoh et al. (1998) Lee et al. (1997) Harwood et al. (1990) Hummelbrunner and Isman (2001) Lee et al. (1999) Franzios et al. (1997) Lee et al. (1997) Franzios et al. (1997) Isman (unpubl. Data) Hummelbrunner and Isman (2001) Traboulsi et al. (2002)
LC50 ¼ 5.5 ml/l
Hummelbrunner and Isman (2001) Don-Pedro (1996)
LD50 ¼ 66 mg/fly LD50 ¼ 27 mg/cm2
Lee et al. (1997) Isman (unpubl. Data)
LD50 ¼ 50 mg/fly LD50 ¼ 88 mg/beetle LD50 ¼ 273.7 mg/larva
Lee et al. (1997)
LC50 ¼ 8.3 ml/l LC50 ¼ 61.5 ml/l LC50 ¼ 23 ml/l LD50 ¼ 98 mg/fly LC50 ¼ 12.7 ml/l LD50 ¼ 1290 mg/4 mm diameter paper LD50 ¼ 1735 mg/larva
Hummelbrunner and Isman (2001) Don-Pedro (1996) Lee et al. (2001) Coats et al. (1991) Lee et al. (1997) Lee et al. (2001) Franzios et al. (1997) Harwood et al. (1990)
(Continued)
Naturally occurring bioactive compounds
34 Table 2 (continued ) Compound
Insect tested
Effect/dose
Reference
Trans-anethole
Tribolium castaneum Musca domestica
LC50 ¼ 48.1 ml/l LD50 ¼ 75 mg/fly
Drosophila melanogaster Spodoptera litura
LD50 ¼ 7 mg/cm2
Ho et al. (1997) Marcus and Lichtenstein (1979) Isman (unpubl. Data)
Blattella germanica Cinnamaldehyde
Tribolium castaneum Coptotermes formosanus Trichoplusia ni
LD50 ¼ 65.5 mg/larva 80% mortality at 159 mg/cm2 LC50 ¼ 0.70 mg/cm2 96% mortality at 1 mg/g paper LD50 ¼ 176.3 mg/larva
Hummelbrunner and Isman (2001) Chang and Ahn, 2001 Huang and Ho (1998) Chang and Cheng (2002) Isman (unpublished)
Reichmuth, 1997; Shaaya and Kostjukovsky, 1998; Lee et al., 2001). Blood-feeding insects (Pediculus, Triatoma) have also been screened (Muncuoglu et al., 1996). Surprisingly, fewer crop pests have been examined in this regard. When all the data are reviewed collectively, certain plant species emerge as being consistently active against insects, although their rankings by potency vary greatly between pest species. Among the more active essential oils are those from thyme, oregano, basil, rosemary and mint, but much more empirical testing using less common plant species and a wider array of pest species will undoubtedly reveal particularly valuable biological activities (Shaaya and Kostjukovsky, 1998; Isman, 2005). Efficacy of pure constituents to insects Eight of the more potent essential oil constituents (monoterpenes and phenylpropenes) are listed in Table 2, along with their toxicity to various insect species if reported. Structures of these compounds are shown in Figure 1. Comparisons of toxicity among insect species for any particular compound are seldom relevant because of the differences in route of administration or exposure method between investigations. On the other hand, some studies have made direct comparisons of different compounds in one or more pest species (Lee et al., 1997; Hummelbrunner and Isman, 2001; Sampson et al., 2005). Accordingly, thymol and pulegone emerge as the most potent individual compounds, at least based on bioassays against the house fly Musca domestica, the tobacco cutworm Spodoptera litura and the turnip aphid Lipaphis pseudobrassicae. However, relative toxicity of individual compounds among pests can also be idiosyncratic. For example, in comparing the phenols eugenol and carvacrol and the monoterpenes a-terpineol and terpinen-4-ol for toxicity against four insect pest species and the twospotted spider mite, eugenol was the most toxic to two species (fruit fly and corn rootworm), terpinen-4-ol to two others (rice weevil and the spider mite), and carvacrol to the fifth (tobacco cutworm) (Isman, 2000). Comprehensive investigation into structure–activity relations of the toxicity of monoterpenes to insects has yielded few insights (Rice and Coats, 1994a), and efforts to enhance potency of
Pesticides based on plant essential oils
35
selected terpenes and phenols through derivitization produced very few analogues more potent than the parent natural products (Rice and Coats, 1994b; Tsao et al., 1995). In general though, phenols (e.g. thymol, eugenol) tend to be more active, both as insecticides and as fungicides, than the related monoterpenes. Essential oils as insect repellents The major use of insect repellents is for personal protection against biting arthropods, particularly mosquitoes. For almost 50 years the most heavily used and reliable insect repellent has been DEET (N,N-diethyl-m-touamide) (Peterson and Coats, 2001). One of the few viable alternatives to DEET for application to human skin has been preparations containing oil of citronella ( ¼ lemongrass oil). A number of such products containing citronella, often mixed with mint oil, are marketed in the United States, in spite of research indicating that they are far less efficacious than DEET (Fradin and Day, 2002). Other oils used in personal repellents include cinnamon oil, clove oil and soybean oil. Presumably these products, with modification, could be useful for protection of domestic animals (e.g. equestrian applications). Another use of repellents is for the removal of cockroaches and the prevention of reinfestation in human habitations and other buildings such as hospitals and restaurants. Certain essential oil constituents clearly repel cockroaches in a laboratory setting (Ngoh et al., 1998) and it is likely that at least part of the efficacy of commercial pest-control products based on essential oils is a consequence of ‘‘flushing’’ cockroaches from their harbourages and discouraging their return. Impregnating repellents into packaging to prevent insect infestation represents another potential use of essential oils that has yet to be realized. It is important to note that repellents could also prove useful in integrated pest management of agricultural crops, particularly in the context of a stimulodeterrent diversionary strategy (SDDS) or ‘‘pushpull’’ strategy (Miller and Cowles, 1990). Finally, it should be mentioned that certain essential oil constituents are effective attractants for some insects. For example, geraniol and eugenol are used as lures in traps for the Japanese beetle Popillia japonica and methyl eugenol has been used to trap Oriental fruit fly Dacus dorsalis (Vargas et al., 2000). Cinnamyl alcohol, 4-methoxy-cinnamaldehyde, cinnamaldehyde, geranylacetone and a-terpineol are attractive to adult corn rootworm beetles (Diabrotica spp.) (Hammack, 1996; Petroski and Hammack, 1998). Efficacy of oils and pure constituents to mites Some studies of the effects of essential oils or pure constituents against both parasitic and free-living mites are listed in Table 3. The tracheal mite Acarapis woodi and the varroa mite Varroa jacobsoni are economically important parasites of the honeybee Apis mellifera and there are few viable treatments for infested bee colonies. Menthol, thymol and some other compounds have been shown to have moderate efficacy as mite fumigants in beehives, although the difference in toxicity between the mites and their host bees (as little as 2–3 fold) requires that these substances be used precisely in practice. Essential oils rich in 1,8-cineole (tea tree oil, eucalyptus oil) have been shown to be effective against house dust mites. Several oils and their constituents are
Naturally occurring bioactive compounds
36
Table 3 Acaricidal activity of some essential oils and constituents Effective oil or compounds
Species tested
Reference
Menthol, thymol Blend of thymol, camphor, menthol and eucalyptus oil Citronellal, pulegone, terpin4-ol, piperitone Lavender oil, menthol, linalool Thymol, eugenol, menthol Wormwood oil, tansy oil Eucalyptus oil, Thuja oil, Melaleuca oil
Acarapis woodi Varroa jacobsoni
Ellis and Baxendale (1997) Calderone and Spivak (1995)
Ornithonyssus sylviarum
Carroll (1994)
Tyrophagus longior
Perrucci (1995)
Psoroptes cuniculi Tetranycus urticae Dermatophagoides pteronyssinus, D. farinae Tetranychus urticae
Perrucci et al. (1995) Chiasson et al. (2001) Yatagai (1997)
Tetranychus urticae, Phytoseilus persimilis Tetranychus urticae
Choi et al. (2004)
Carvomenthenol, terpinen-4ol, eugenol 53 plant essential oils Rosemary oil
Lee et al. (1997)
Miresmailli et al. (2006)
effective against the phytophagous spider mites, as commercial miticides based on rosemary or cinnamon oils attest (Isman, 2000; Miresmailli et al., 2006). Efficacy of oils and pure constituents to fungi Many plant essential oils and their major constituents have demonstrated antifungal activity against a range of plant pathogenic fungi including those responsible for both pre- and postharvest diseases. Some examples are listed in Table 4. Unlike insects, different fungal species yield more consistent results – thyme oil and its major phenolic constituents thymol and carvacrol are clearly active against most fungal species tested. The mechanism of action of these compounds against fungi is unknown but may be related to their general ability to dissolve or otherwise disrupt the integrity of cell walls and membranes. As a cautionary note, many of these oils and pure compounds show considerable phytotoxicity to plants at concentrations only slightly above those required for control of plant pathogenic fungi, presumably because plant cells are affected by a similar mechanism. Toxicity to mammals and other nontarget organisms Given the widespread use of plant essential oils in fragrances, as flavouring agents or condiments in food and in aromatherapy, we might expect these materials to be without appreciable toxicity in mammals. For most of the essential oil constituents toxic to insects and fungi this is the case (Table 5), although some may cause irritation of the skin, eyes and mucous membranes in concentrated form. But there are some notable exceptions. Pulegone is considerably toxic to rats (i.p. LD50 ¼ 150 mg/kg), although data on oral, acute toxicity has not been published. Pennyroyal oil, the source of pulegone, has been implicated in both human and animal poisonings
Pesticides based on plant essential oils
37
Table 4 Antifungal activity of some essential oils and constituents Effective oil or compounds
Species tested
Reference
Palmarosa, red thyme, cinnamon, clove bud oils Thymol, carvacrol
Botrytis cinerea
Wilson et al. (1997)
Botrytis cinerea, Monilinia fructicola Rhizoctonia solani, Fusarium moniliforme, Sclerotinia sclerotiorum Fusarium oxysporum 22 species of fungi Aspergillus niger Aspergillus spp. Aspergillus flavus
Tsao and Zhou (2000)
Thymol, carvacrol
Clove oil Cymbopogon spp. oils Cymbopogon nardus oil Thyme, oregano oils Thyme, oregano, cinnamon, basil, peppermint oils Thyme, oregano, marjoram, dictamus oils Thymol, eugenol, citronellol, cinnamaldehyde Thyme, mint, lavender oils
Muller-Riebau et al. (1995)
Penicillium digitatum
Bowers and Locke (2000) Singh et al. (1980) De Billerbeck et al. (2001) Paster et al. (1995) Montes-Belmont and Carvajal (1998) Daferera et al. (2000)
Seven species of fungi
Kurita et al. (1981)
Fusarium solani, Rhizoctonia solani, Pythium ultimum, Colletotrichum lindemuthianum
Zambonelli et al. (1996)
Table 5 Toxicity of some essential oil constituents to rats Compound
LD50 (mg/kg)a
1,8-Cineole (eucalyptol) Cinnamaldehyde Citral (from citronella oil) Eugenol d-Limonene Menthol Pulegone Terpinen-4-ol Thymol trans-Anethole
2480 1160 (guinea pig) 4960 2680 4600 (lowest published lethal dose) 3180 150 (mouse, intraperitoneal) 4300 980 (mouse ¼ 1800) 2090
a
Oral, acute unless otherwise specified. Source: Food and Agriculture Organization of the United Nations, 1999.
(Anderson et al., 1996). a-Thujone, the toxic constituent of the liqueur absinthe (made from wormwood oil), is also very toxic to rats (i.p. LD5045 mg/kg) (Hold et al., 2000). On the other hand, an essential-oil-based insecticide produced no mortality in rats when fed at 2 g/kg, and the LD50 exceeded 5 g/kg, the normal upper limit for testing required by regulatory agencies (E. Enan, unpubl. data). Eugenol is approximately
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1500 times less toxic than pyrethrum and 15,000 times less toxic than the organophosphate insecticide azinphosmethyl to juvenile rainbow trout Oncorhynchus mykiss, based on 96-h static water tests (Stroh et al., 1998). Effects on other nontarget organisms, namely, pollinators and natural enemies, have yet to be evaluated under field conditions. In the laboratory, a rosemaryoil-based insecticide/acaricide was significantly less toxic to predatory mites (Phytoseilus persimilis) than to their spider mite prey (Tetranychus urtica), although the whitefly parasitoid Encarsia formosa was more susceptible than its whitefly hosts (Miresmailli and Isman, unpublished data). However, most essential-oil constituents degrade quickly in the environment or are rapidly lost from plant foliage through volatilization, minimizing residual contact and favouring immigration or reintroduction of biocontrol agents and the foraging activities of honeybees and other pollinators.
Commercial products and uses In spite of considerable research effort in many laboratories throughout the world and an ever-increasing volume of scientific literature on the pesticidal properties of essential oils and their constituents, surprisingly few pest control products based on plant essential oils have appeared in the marketplace. This may be a consequence of regulatory barriers to commercialization (i.e. cost of toxicological and environmental evaluations) or the fact that efficacy of essential oils toward pests and diseases is not as apparent or obvious as that seen with currently available products. Nonetheless, some U.S. companies have introduced essential-oil-based pesticides in recent years. Mycotech Corporation produced an aphidicide/miticide/fungicide for greenhouse and horticultural crops and for bush and tree fruits based on cinnamon oil with cinnamaldehyde (30% in the EC formulation) as the active ingredient. However, this product is no longer being sold. EcoSMART Technologies has introduced insecticides containing eugenol and 2-phenethyl propionate aimed at controlling crawling and flying insects, under the brand name EcoPCOR for pest control professionals. An insecticide/miticide containing rosemary oil as the active ingredient has recently been introduced for use on horticultural crops under the name EcoTrolTM. Another product based on rosemary oil is a fungicide sold under the name SporanTM, while a formulation of clove oil (major constituent: eugenol), sold as MatranTM, is used for weed control. All of these products have been approved for use in organic food production. Several smaller companies in the U.S. and the U.K. have developed garlicoil-based pest control products and in the U.S. there are consumer insecticides for home and garden use containing mint oil as the active ingredient. Menthol has been approved for use in North America for control of tracheal mites in beehives, and a product produced in Italy (Apilife VARTM) containing thymol and lesser amounts of cineole, menthol and camphor is used to control Varroa mites in honeybees.
Conclusion and future prospects Pesticides based on plant essential oils or their constituents have demonstrated efficacy against a range of stored product pests, domestic pests, blood-feeding pests and certain soft-bodied agricultural pests, as well as against some plant pathogenic fungi
Pesticides based on plant essential oils
39
responsible for pre- and post harvest diseases. They may be applied as fumigants, granular formulations or direct sprays with a range of effects from lethal toxicity to repellence and/or oviposition deterrence in insects. These features indicate that pesticides based on plant essential oils could be used in a variety of ways to control a large number of pests. In general, the efficacy of these materials falls short when compared to synthetic pesticides although there are specific pest contexts where control equivalent to that with conventional products has been observed. Essential oils also require somewhat greater application rates (as high as 1% active ingredient) and may require frequent reapplication when used out-of-doors. Additional challenges to the commercial application of plant essential-oil-based pesticides include availability of sufficient quantities of plant material, standardization and refinement of pesticide products, protection of technology (patents) and regulatory approval (McChesney, 1994; Isman, 2005). Although many essential oils may be abundant and available year round due to their use in the perfume, food and beverage industries, large-scale commercial application of essential-oil-based pesticides could require greater production of certain oils. Pesticides derived from essential oils of relatively rare plants would not be easily produced due to a lack of sufficient quantities of plant material, limiting the essential oils of use to those which are readily available or from those plants which can be easily cultivated (e.g. in plantations) (McChesney, 1994). In addition, as the chemical profile of plant species can vary naturally depending on geographic, genetic, climatic, annual or seasonal factors, pesticide manufacturers must take additional steps to ensure that their products will perform consistently. All of this requires substantial cost and smaller companies may not be willing to invest the required funds unless there is a high probability of recovering the costs through some form of market exclusivity (e.g. patent protection). Finally, once all of these issues are addressed, regulatory approval is required. Although several plant essential oils are exempt from registration in the United States, many more oils are not, and few countries currently have such exemption lists. Accordingly, regulatory approval continues to be a barrier to commercialization and will likely continue to be a barrier until regulatory systems are adjusted to better accommodate these products. Nonetheless, pesticides derived from plant essential oils do have several important benefits. Due to the rapid volatilization of these products, there is a much lower level of risk to the environment than with current synthetic pesticides. Predator, parasitoid and pollinator insect populations will be less impacted as a result of the minimal residual activity, making essential-oil-based pesticides compatible with integrated pest management programs. Further, while resistance development continues to be an issue for many synthetic pesticides, it is likely that resistance will develop more slowly to essential-oil-based pesticides owing to the complex mixtures of constituents that characterize many of these oils. Ultimately, it is in developing countries where the source plants are endemic that these pesticides may ultimately have their greatest impact. However, while commercial development of plant essential-oil-based pesticides may face a variety of problems, it is evident that where human and animal health are at a premium and minimal residues are desirable, it is worth the effort to surmount these challenges. It is therefore expected that these pesticides will find their greatest commercial application in urban pest control, public health, veterinary health, mosquito abatement
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and in stored product protection. In agriculture, these pesticides will be most useful for protected crops (e.g. greenhouse crops), high-value row crops and within organic food production systems where few alternative pesticides are available. As conventional, hazardous insecticides are removed from the marketplace and the cost of developing new products continues to rise, it is likely that pesticides based on plant essential oils will gain further acceptance.
Acknowledgment We thank Dr. E. Enan (Vanderbilt University, Nashville, TN, USA) and Mr. S. Miresmailli (University of British Columbia) for allowing us to include their unpublished data. Supported by grants from NSERC and EcoSMART Technologies Inc. to MBI.
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CHAPTER 3
Natural substrates and inhibitors of multidrug resistant pumps (MDRs) redefine the plant antimicrobials GEORGE P TEGOS
Introduction Efflux mechanisms have become broadly recognized as major components of resistance to many classes of antibiotics. Some efflux pumps selectively extrude specific antibiotics, while others referred to as multidrug resistance pumps (MDRs) expel a variety of structurally diverse compounds with differing modes of action. Based on sequence similarity, multidrug transporter systems are classified into six super-families (Paulsen et al., 2002) (i) ATP binding cassette transporters (ABC), (ii) major facilitators (MF), (iii) resistance nodulation division (RND), (iv) small multidrug resistance (SMR), (v) multidrug and toxic compound extrusion (MATE), and (vi) multidrug endosomal transporter (MET) family. The first five families of multidrug systems are found mostly in microorganisms (the MET family appears restricted to higher eukaryotes) but representatives of all these families are also present in mammalian cells. Multidrug resistant human pathogenic microorganisms are directly associated with serious recalcitrant infections such as cystic fibrosis, nosocomial infections, and infections in immunocompromised patients undergoing anticancer chemotherapy or infected with HIV. A novel and promising approach to deal with multidrug resistance is improving the clinical performance of various antibiotics by the inhibition of efflux pumps. Many companies and research laboratories have undertaken programs to discover and develop efflux pump inhibitors and in many cases their attempts were successful. In addition, it was clearly demonstrated that an efflux pump inhibitor (reserpine) inhibits effectively not only the bacterial but the mammalian MDR ABC-transporter P-glycoprotein (P-gp) as well (Beck et al., 1988; Wang et al., 2001), thus implying that the development of broad range MDR inhibitors is not a chimera. Plants produce an enormous array of secondary metabolites and it is commonly accepted that a significant part of this chemical diversity serves to protect plants against microbial pathogens (Dixon, 2001). These plant substances are classified as either phytoanticipins, compounds present constitutively, or phytoalexins, whose
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levels increase strongly in response to microbial invasion (Morrissey and Osbourn, 1999). Plant compounds are routinely classified as ‘‘antimicrobials’’ on the basis of susceptibility tests that produce minimal inhibitory concentrations (MICs) in the range of 100–1000 mg/ml, orders of magnitude weaker than typical antibiotics produced by bacteria and fungi (MICs of 0.01–1 mg/ml) (Cowan, 1999). Simply because a compound is synthesized in response to pathogen invasion, is required for protecting the plant from a pathogen but shows little activity in an in vitro susceptibility test it is not necessarily an antimicrobial. Such a substance might have a regulatory function, which indirectly increases plant resistance. This analysis suggests that we currently lack a solid rationale for making a functional assignment for the vast majority of plant compounds that have been classified as antimicrobials. Helpful evidence regarding the possible function of plant secondary metabolites is that these compounds often show considerable activity against Gram-positive bacteria, but not against Gram-negative species or yeast (Lewis et al., 2001). Both yeast and Gram-negative bacteria have evolved significant permeability barriers. Gram-negative species have an outer membrane that is a fairly effective barrier for amphipathic compounds and a set of MDRs that extrude amphipathic toxins across the outer membrane. The single membrane of Gram-positive bacteria is considerably more accessible to permeation by amphipathic toxins in regions where MDRs provide limited protection. In yeast, the presence of ergosterol, which decreases permeability, combined with a set of broad-specificity MDRs also provides an effective barrier (Lomovskaya and Lewis, 1992; Nikaido, 1999; Lewis, 2001). Several Gram-positive bacteria invade plants, but the majority of plant pathogens are Gramnegative bacteria, or yeast and related fungi (Rogers et al., 2001). In this chapter we shall emphasize the rationale for characterization of natural substrates of MDR pumps, reporting the identification of the first MDR substrates and inhibitors from plants against bacteria and yeasts. We shall discuss the methodology for the isolation of these compounds as well as the specificity of their activity. Finally we shall analyze the current and future perspectives to natural drug discovery based on this classification of plant antimicrobials.
The quest for natural substrates – rationale Even though MDR pumps extrude structurally unrelated compounds, a general theme emerges if one considers the preferred artificial substrates of most MDRs. These substances are mostly amphipathic cations. It has been suggested that amphipathic cations represent the existing natural substrates of MDR pumps (Lewis, 1999). The simplest MDRs of the SMR family have amphipathic cations as their exclusive substrates, at least in Gram-positive bacteria (Chung and Saier, 2001). Most of the MDR members of the MF super-family extrude exclusively amphipathic cations. The QacA pump only extrudes cations (Mitchell et al., 1998); the NorA pump of the human pathogen Staphylococcus aureus extrudes cations and some quinolones (Kaatz et al., 2000); the BMR pump of Bacillus subtilis extrudes primarily cations and neutral chloramphenicol (Van Bambeke et al., 2000; VazquezLaslop et al., 2000). RND super-family MDRs have a broad substrate spectrum, including amphipathic cations (Elkins and Nikaido, 2002; Mao et al., 2002; Murata
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47
et al., 2002) that are preferential substrates in the case of ABC transporters (Lage, 2003). Simple MDRs like SMRs only export amphipathic cations (Krupka, 1999); MF and MATE MDRs export mainly cations; RND and ABC have broad spectra of specificity that include amphipathic cations as preferred substrates. It appears that a similar need to protect the cell from amphipathic cations evolved in different groups of MDRs (and in different organisms) in spite of a lack of overall homology or similarity in the action mechanism. Quite surprisingly, one does not find amphipathic cations in a general list of natural antimicrobials. (The known cationic antibiotics of the aminoglycoside group such as streptomycin and kanamycin are hydrophilic substances that get smuggled into the cell via specific translocases and are not general substrates of MDRs).
MDR inhibitors – proof of principle Research efforts in a series of academic and industrial laboratories led to the identification of numerous inhibitors of the NorA pump of S. aureus (Aeschlimann et al., 1999; Markham et al., 1999; Gibbons et al., 2003; Kaatz et al., 2003) and MexABOprM, MexCD-OprJ, MexEF-OprN MDRs of Pseudomonas aeruginosa (Renau et al., 1999; Lomovskaya et al., 2001). The inhibitors INF271 (NorA-Influx Co., Chicago, Illinois) and MC207110 (MexAB-OprM, MexCD-OprJ, MexEF-OprN P. aeruginosa Essential Therapautics-Mountain View, CA) have been extremely valuable tools for drug discovery. INF271 identified by screening a synthetic chemical library using ethidium bromide (EtBr) as a substrate in a sub-inhibitory concentration. On the contrary, in the case of MC207110 an array of synthetic compounds and natural product extracts was assayed using strains of P. aeruginosa over-expressing each of the three pumps in the presence of levofloxacin. To qualify as MDR inhibitors hit compounds had to meet several criteria: (i) must enhance the activities of levofloxacin and other antibiotics that are effluxes in strains containing functioning pumps, (ii) must not significantly potentiate the activities of antibiotics in a strain that lacks efflux pumps, (iii) must not potentiate the activities of antibiotics that are not effluxes, (iv) must increase the level of accumulation and decrease the level of extrusion of efflux pump substrates, and (v) must not affect the proton gradient across the inner membrane (Table 1).
MDR mutants and inhibitors valuable tools for natural drug discovery It seems plausible that MDRs evolved in response to natural antimicrobial amphipathic cations and this fact makes these substances difficult to discover in standard screens that employ cells carrying MDR pumps. The development of MDR mutants for the NorA of S. aureus gives an answer to this apparent paradox (Hsieh et al., 1998) and serves as a sensitive tool for drug discovery. The plant isoquinoline alkaloids, berberine, and palmatine resemble artificial MDR substrates such as EtBr and are widely spread among the plant world especially in species of Ranunculales (Schmeller et al., 1997). A susceptibility test to identify MICs for these compounds against the wild type and the NorA mutant of S. aureus was performed. Palmatine
Naturally occurring bioactive compounds
48 Table 1 Natural MDR inhibitors H N
O HN
H2 N
OMe O
O
H N
N H
O
HO
O
NH
OH OMe
O
OH
MeO
N H
INF271
NH2
OH O
MC207110
OMe
5-MHC Berberis sp., Mahonia sp. OH
O
MeO MeO OH
Pheophorbide a
R4O
OR5
2'
3' rha 1' 4'
R3O
OR OMe
O
Chrysoplenol D /Chrysoplenetin Artemisia annua
HO 3 2
O O
OR2 glu 4 1
O
5'
CH3
5
O 6
OR1
6'
1: R1 = R2 = R4 = R5 = H; R3 = CO(CH2)14 CH3 2: R1= -CO(CH2)14CH3; R2 = R4 = R5 = H; R3 = -COPr 3: R1 = R2 = R3 = -COiPr; R4 = -COPr 4: R1 = R2 = R3 = -COiPr; R4 = OAc; R5 = COPr
Neohesperidosides Geranium caespitosum
Dalea versicolor
Dalea versicolor
has very low (>200 mg/ml MIC) and berberine poor (250 mg/ml) activity against wild type S. aureus. The antimicrobial activity of the alkaloids increased sharply in a norA mutant, with an MIC of 50 mg/ml for palmatine and 7.5 mg/ml for berberine. Berberine resembles EtBr and binds to DNA (Jennings and Ridler, 1983). The DNA binding apparently contributes to the antimicrobial activity of berberine. Similar to EtBr, DNA-bound berberine has increased fluorescence and is used as a model plant antimicrobial for uptake studies. Efflux of berberine was more rapid than EtBr. Similarly, the differential rate of berberine accumulation in both strains indicated that these alkaloids represent the first identified natural plant substrates of the MFS MDRs of S. aureus (Stermitz et al., 2000a). The outer membrane of Gram-negative bacteria is a barrier to a broad range of amphipathic compounds, and to all large molecules, which allows for evolving of broadly specific MDRs that pump their various substrates across this barrier.
Natural substrates and inhibitors of MDRs
49
Similarly, Gram-positive species that lack an outer membrane have MDRs with a reported substrate spectrum largely limited to amphipathic cations. Apart from the usefulness of protecting the cell from this class of compounds that actively accumulate in the cell, the relatively slow permeation of amphipathic cations across the cytoplasmic membrane made the evolution of ‘‘cation-pumping’’ MDRs possible, as compared to neutral compounds of similar polarity.
A new series of promising plant antimicrobials Similar studies aimed at finding the basis of Gram-negative bacterial resistance to plant antimicrobials. In Rhizobium etli, there is an operon activated by a number of plant phytoalexins. The operon appeared to code for an RmrAB MDR pump (Gonzalez-Pasayo and Martinez-Romero, 2000). Mutant rmrAB had a diminished ability for root colonization, and increased susceptibility for phytoalexins naringenin and coumaric acid. However, the difference in susceptibility between wild type and mutant is very small, around 30%. In Agrobacterium tumefaciens, coumestrol, an antifungal phytoalexin of soybeans induces expression of an LfeAB MDR (Palumbo et al., 1998). Mutation in the pump increases accumulation of coumestrol in the pathogen, and the mutant was out competed by the wild type in colonizing the plant. Neither the wild type nor the mutant, however, is sensitive to coumestrol. In a slightly different approach a panel of plant antimicrobials was tested by using a set of bacteria representing the main groups of plant and human pathogens and applying a combination of MDR mutants and MDR inhibitors (Tegos et al., 2002). One of the main observations was the strong potentiation of antimicrobial action against strains with disabled MDRs. The activities of the majority of plant antimicrobials were considerably greater against the Gram-positive bacteria S. aureus and B. megaterium and disabling of the MDRs in Gram-negative species led to a striking increase in antimicrobial activity. Thus, the activity of rhein, the principal antimicrobial from rhubarb (Agarwal et al., 2000), was potentiated 100–2000-fold (depending on the bacterial species) by disabling the MDRs. A similar effect was observed with plumbagin (Didry et al., 1994), resveratrol, gossypol, coumestrol, and berberine. Direct measurement of the uptake of berberine confirmed that disabling of the MDRs strongly increases the level of penetration of berberine into the cells of Gram-negative bacteria (Figure 1). Recently the outer-membrane protein-encoding gene tolC in the bacterial plant pathogen Erwinia chrysanthemi EC16 was identified and characterized (Barabote et al., 2003). The gene encodes a 51-kDa protein with 70% identity to its Escherichia coli homolog. The E. chrysanthemi gene functionally complements the E. coli tolC gene in MDR efflux pumps. A tolC mutant of E. chrysanthemi is extremely sensitive to an array of plant-derived chemicals including berberine, rhein, plumbagin, pyrithione, genistein (40 ,5,7-trihydroxyisoflavone), p-coumaric acid and t-cinnamic acid (phenolic acids), (2-mercaptopyridine-1-oxide) and esculetin (6,7-dihydroxycoumarin). This mutant is unable to grow in planta and its ability to cause plant tissue maceration is severely compromised and is defective in the efflux of berberine. The E. chrysanthemi tolC plays an important role in the survival and colonization of the pathogen in plant tissue conferring resistance to plant antimicrobials.
Naturally occurring bioactive compounds
50 200
A
150
RFU
RFU
150
100
50
0
B
200
100
50
0
5
10
15
20
25
0
30
0
5
Time (min)
C
20
25
30
D
200
150
150
RFU
RFU
15
Time (min)
200
100
50
0
10
100
50
0
5
10
15
20
25
30
Time (min)
0
0
5
10
15
20
25
30
Time (min)
200
E
RFU
150
100
50
0
0
5
10
15
20
25
30
Time (min)
Fig. 1. Accumulation of berberine by Gram-negative bacteria. Uptake of berberine without (o) or with MC207110 (i), INF271 (D), MC207110/INF271 (}) by cells of Salmonella typhimurium (A), Pseudomonas syringae (B), Xanthomonas campestris (C), Erwinia carotovora (D), and Sinorhizobium meliloti (E) was measured by fluorescence increase following binding to DNA and expressed as RFU (relative fluorescence units). Berberine was present at a concentration of 30 mg/ml; MC207110 and INF271 were added at the same final concentrations as for the MIC determinations.
Natural MDR inhibitors from plants – an increasing body of evidence, 50 methoxyhydnocarpin and synergy in Berberis plants The rationale behind detecting MDR inhibitory activity is to test the combined action of a plant extract (the nonalkaloid fraction) with a weak plant antimicrobial coming from the same plant added at a sub-inhibitory concentration (Table 2). Extracts that inhibit cell growth in the presence of the antimicrobial and have no
Natural substrates and inhibitors of MDRs
51
Table 2 Identified natural substrates or MDRs O
OCH H3 OCH H3
O
O OH
N +
N +
CH3O OCH3
OMe Berberine
OH O
O
Palmatine O
HO
OH
Plumbagin
O N
O
COOH
SH
O
O
OH
Rhein
Coumestrol
Zink-Pytothione OH
OHC
OH
OH
HO
HO CHO OH
HO
HO
O
O
Me
OH
Me
Resveratrol
Gossypol
Esculetin
O O
O
O
O O
Asarinin
activity when added alone are likely to contain an MDR inhibitor. A bioassay-driven purification is the most common technique used to detect MDR inhibitors. Several Berberis medicinal plants (Berberis repens, B. aquifolia, and B. fremontii) producing berberine were found also to synthesize an inhibitor of the NorA MDR pump S. aureus. The inhibitor 50 -methoxyhydnocarpin (50 -MHC), a flavonolignan, was previously identified in Hydnocarpus wightiana (Flacourtaceae) (Ranganathan and Seshadri, 1974) and reported as a minor component of chaulmoogra oil, a traditional therapy for leprosy (Norton, 1994). 50 -MHC-D is an amphipathic weak acid and is
52
Naturally occurring bioactive compounds
distinctly different from the cationic substrates of NorA. 50 -MHC-D had no antimicrobial activity alone but strongly potentiated the action of berberine and other NorA substrates against S. aureus. MDR-dependent efflux of EtBr and berberine from S. aureus cells was completely inhibited by 50 -MHC-D. The level of accumulation of berberine in the cells was increased strongly in the presence of 50 -MHC-D, indicating that this plant compound effectively disables the bacterial resistance mechanism against berberine (Stermitz et al., 2000a, 2000b). This first identified natural MDR inhibitor is abundant in other Berberis sp. (Musumeci et al., 2003).
Inhibitors against MFS MDRs Identification of 50 -MHC-D intensified the search for natural MDR inhibitors of plant origin and made the idea of synergy in plant antimicrobials more plausible. Bioassay-driven purification and structural determination from various plant sources yielded a number of new MDR inhibitors acting against Gram-positive bacteria with activity similar to 5-MHC pheophorbide a from Berberis sp., and Mahonia (Stermitz et al., 2000a, 2000b, 2001), crysoplenol and crysoplenetin from Artemisia annua (Stermitz et al., 2002), dicaffeoylquinic acids from A. absinthium (Fiamegos et al., 2003), polyacylated neohesperidosides from Geranium caespitosum (Stermitz et al., 2003), diterpenoids from Pinus edulis, (F.R. Stermitz personal communication), and chalcones and a stilbene from Dalea versicolor (Belofsky et al., 2003). Additionally, isoflavones such as genistein, orobol, and biochanin exhibit MDR inhibitory activity against the NorA pump of Gram-positive bacteria (Morel et al., 2003) and aminoquinolines in the AcrAB-TolC pump of E. coli (Mallea et al., 2003).
Inhibitors against ABC transporters P-gp the mammalian ABC transporter is mainly responsible for the unsuccessful chemotherapy of cancers through occurrence of MDR (Leonard et al., 2003). P-gp shares high structural homology with yeast ABC transporters (Saier and Paulsen, 2001). The development of screening methodologies has significantly enhanced the number and the efficiency of available inhibitors (Hiraga et al., 2001; Varma et al., 2003). The inhibitory activity of numerous plant compounds against P-gp such as alkaloids, polymethoxyflavones, diterpenoids, camptothecins (Ulukan and Swaan, 2002), polyphenols (Jodoin et al., 2002), ascorbic acid, vitamins, and others has been extensively studied (Chen and Waxman, 1995; Ikegawa et al., 2000; Foster et al., 2001; Motohashi et al., 2001; Perloff et al., 2001; Deferme et al., 2002; Appendino et al., 2003; Corea et al., 2003; El-Masry and Abou-Donia, 2003). Likewise, many plant sources and products seem quite promising for the identification of new P-gp specific inhibitors: sweet pepper ‘‘Anastasia green’’ (Motohashi et al., 2001), St. John’s Wort (Hypericum perforatum) (Perloff et al., 2001), caper spurge (Euphorbia lathyris) (Appendino et al., 2003), and apricots (Deferme et al., 2003). A novel and interesting inhibitor from G. caespitosum (Figure 2) shows interesting properties. It potentiates berberine in S. aureus, erythromycin in P. syringae pv. maculicola and fluconazole against Saccharomyces cerevisae and Candida albicans.
Natural substrates and inhibitors of MDRs
53
Fig. 2. Geranium caespitosum a paradigm of synergy between plant antimicrobials.
In addition, a structurally similar compound from the same source has no antimicrobial action alone, but combined with the inhibitor is active against P. syringae (a designated pathogen of Geranium sp.) and S. cerevisae (Tegos et al., 2006 (unpublished results)).
Conclusions and future perspectives The experimental data available demonstrate and reassure that plants produce MDR inhibitors against Gram-positive bacteria and yeasts. An effective plant MDR inhibitor of Gram-negative bacteria is still a missing part of the puzzle. Currently, there is only preliminary evidence for this, but the identification of such a compound is more than plausible. A comprehensive library of extracts from the National Cancer Institute (NCI-USA), which consists of over 50,000 extracts from approximately 35,000 plants from all over the world is currently being screened (Tegos et al., 2006 (unpublished results)). The collection represents most genera of vascular plants found in the tropics. The extracts are tested against a panel of representative microorganisms (S. aureus, E. coli, S. cerevisiae) for direct antimicrobial activity. Furthermore, they are tested for MDR inhibitory activity with a sub-inhibitory amount of antibiotic. A pilot screen produced extremely promising hit rates for direct and MDR antimicrobial activity (Figure 3). It is significant to find MDR inhibitory activity against Gram-negative bacteria of which no known natural inhibitors have
Naturally occurring bioactive compounds
54 0.19 0.57 0.76
0.57 0.38 0.19
1.32 2.65 6.25 7.95 10.41
68.75
Inactive
S. cerevisae[MDR]
S. aureus[D], E. coli[D]
S. aureus [D]
S. aureus[D], S. cerevisae[D][D]
S. aureus[D], E.coli[MDR]
S.aureus[MDR]
S. aureus[D], S. cerevisae[MDR]]
S. aureus[MDR], E. coli[MDR]]
S. serevisae [D]
S. aureus[MDR], S. cerevisae[MDR]]
S. aureus[D], E. coli[D], S.cerevisae [MDR]
Fig. 3. Hit rates of extracts from the Natural Products Repository Program (National Cancer Institute-USA, http://dtp.nci.nih.gov/branches/npb/repository.html). A pilot screen of 524 extracts (352 organic/176 aqueous) produced very promising hit rates for the identification of new MDR inhibitors and plant antimicrobials (see text). The majority of hits came from organic extracts. [D] – Direct, [MDR] – MDR-inhibitory activity.
been identified. Extracts with MDR inhibitory activity and without direct detectable activity suggest that antimicrobials from these plants exist. Interestingly, a number of instances exist where a specific part of the same plant produced direct activity against S. aureus; while an extract from another part exhibits MDR inhibitory activity. Closer examination may demonstrate both activities operating at different levels in the entire plant. Similar examples of synergistic activities coming from the same plant are observed in the case of E. coli and S. cerevisiae. The potential activity of plant antimicrobials when they gain access to the microbial cell is only part of the intriguing puzzle. Even if a plant uses a synergistic combination of an antimicrobial and an MDR inhibitor, this does not in itself explain why plants do not come up with self-sufficient antibiotics like aminoglycosides or tetracycline. Plants are stationary, slowly evolving organisms that confront mobile, rapidly evolving pathogens. Resistance to antibiotics such as streptomycin or tetracycline can be gained by a single mutation leading to target modification, or to over-expression of an MDR pump (Lewis, 2001). It is possible that plants employ an antimicrobial strategy aimed at limiting the probability of resistance development. This is apparently the case when plants produce MDR inhibitors, directly targeting the microbial resistance mechanism. Importantly, disabling MDRs also results in a dramatic decrease in acquisition of resistance based on target modification (Zheleznova et al., 1999; Lomovskaya and Watkins, 2001; Markham and Neyfakh, 2001; Lomovskaya et al., 2003). There is another intriguing possibility that plants may employ to limit resistance development. Resistance will not be easily achieved against a substance that hits
Natural substrates and inhibitors of MDRs
55
more than one target. Apparent low activity of an antimicrobial could then be a result of a trade-off – penetration capability is sacrificed in favor of additional functionality, an ability to hit two different targets, while access to the pathogen cell is ensured by a separate molecule, an MDR inhibitor. Do plant antimicrobials hit more than one target? Answering this question might be highly significant for combating the rapid rise of multidrug resistance. Antimicrobials able to act against more than one target would be extremely valuable as therapeutics. Interestingly, a large-scale ‘‘experiment’’ on resistance development to this type of compounds has been performed unwittingly. Synthetic fluoroquinolones act against two different targets – DNA gyrase and topoisomerase (Hooper, 2001, 2002). Resistance by target modification occurs in a series of steps and is a low-probability event. It was shown that blocking MDR pumps of pathogens by an inhibitor decreases resistant mutation development to fluoroquinolones below the limit of detection (o1011). Blocking MDRs increases the effective concentration of fluoroquinolone in the cell and makes the first step in the target modification process ineffective. In retrospect, this means that were fluoroquinolones applied originally in combination with an MDR inhibitor, we might have had a case of largely resistance-proof therapy. It is too late for fluoroquinolones, the resistance to this class of compounds has already developed. But we might still have a chance, or many chances, with potentially dual/multiple target acting plant antimicrobials combined with an MDR inhibitor. There is not sufficient information concerning the plant antimicrobial action mechanisms. Some useful indirect evidence, however, does point to more than one target for at least some compounds. Rhein is effective at mg/ml levels against yeast and Gram-positive bacteria (Cyong et al., 1987; Hatano et al., 1999) and was found specifically to inhibit NADH dehydrogenase in mitochondria (Kean et al., 1970). It is assumed that microbial NADH dehydrogenase is the target of action. At the same time, rhein was reported to be very effective against Gram-positive oral pathogens under anaerobic conditions (Didry et al., 1994) and is very effective against E. coli tolC (defective in major MDRs) under both aerobic and anaerobic conditions. Since NADH dehydrogenase in not functional under anaerobiosis, there should be a second target. Rhein is currently used as a systemic drug against arthritis-administered in the form of a prodrug diacerein (Spencer and Wilde, 1997).
Acknowledgments The author thanks Mahendra Rai for his invitation to participate in this text as well as Kim Lewis (Northeastern University) for continuous support and critical supervision and Frank R. Stermitz (Colorado State University) as a colleague and a mentor in the organic chemistry of natural products. Most of the author’s work was carried out in Kim Lewis Molecular Microbiology Laboratory, Department of Biology at Northeastern University and supported by the NIH grant RO1GM59903 (In search of Natural Substrates and Inhibitors of MDR pumps). Gil Belofsky (University of Tulsa) and John Fiamegos (University of Ioannina) offered valuable feedback information. Susan Harvey and Jennifer Freedman provided significant help revising this manuscript.
56
Naturally occurring bioactive compounds
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Mao W, Warren M, Black DS, Satou T, Murata T, Nishino T, Gotoh N, Lomovskaya O. (2002) On the mechanism of substrate specificity by resistance nodulation division (RND)type multidrug resistance pumps: the large periplasmic loops of MexD from Pseudomonas aeruginosa are involved in substrate recognition. Mol Microbiol 46:889–901. Markham PN, Neyfakh AA. (2001) Efflux-mediated drug resistance in Gram-positive bacteria. Curr Opin Microbiol 4:509–514. Markham PN, Westhaus E, Klyachko K, Johnson ME, Neyfakh AA. (1999) Multiple novel inhibitors of the NorA multidrug transporter of Staphylococcus aureus. Antimicrob Agents Chemother 43:2404–2408. Mitchell BA, Brown M, Skurray RA. (1998) QacA multidrug efflux pump from Staphylococcus aureus: comparative analysis of resistance to diamidines, biguanidines, and guanylhydrazones. Antimicrob Agents Chemother 42:475–477. Morel C, Stermitz F, Tegos G, Lewis K. (2003) Isoflavones as potentiators of antibacterial activity. J Agric Food Chem 51:5677–5679. Morrissey JP, Osbourn A. (1999) Fungal resistance to plant antibiotics as a mechanism of pathogenesis. Microbiol Mol Biol Rev 63:708–724. Motohashi N, Kurihara T, Wakabayashi H, Yaji M, Mucsi I, Molnar J, Maruyama S, Sakagami H, Nakashima H, Tani S, Shirataki Y, Kawase M. (2001) Biological activity of a fruit vegetable, ‘‘Anastasia green,’’ a species of sweet pepper. In Vivo 15:437–442. Murata T, Kuwagaki M, Shin T, Gotoh N, Nishino T. (2002) The substrate specificity of tripartite efflux systems of Pseudomonas aeruginosa is determined by the RND component. Biochem Biophys Res Commun 299:247–251. Musumeci R, Speciale A, Costanzo R, Annino A, Ragusa S, Rapisarda A, Pappalardo MS, Lauk L. (2003) Berberis aetnensis C, Presl. extracts: antimicrobial properties and interaction with ciprofloxacin. Int J Antimicrob Agents 22:48–53. Nikaido H. (1999) Microdermatology: cell surface in the interaction of microbes with the external world. J Bacteriol 181:4–8. Norton S. (1994) Useful plants of dermatology. I. Hydnocarpus and chaulmoogra. J Am Acad Dermatol 31:683–686. Palumbo JD, Kado CI, Phillips DA. (1998) An isoflavonoid-inducible efflux pump in Agrobacterium tumefaciens is involved in competitive colonization of roots. J Bacteriol 180:3107–3113. Paulsen IT, Chen J, Nelson KE, Saier Jr. MH. (2002) Comparative genomics of microbial drug efflux systems. In: ITPaK Lewis editor. Microbial Multidrug Efflux. Norfolk: Horizon Press, pp. 5–21. Perloff MD, von Moltke L, Stormer E, Shader RI, Greenblatt DJ. (2001) Saint John’s wort: an in vitro analysis of P-glycoprotein induction due to extended exposure. Br J Pharmacol 134:1601–1608. Ranganathan KR, Seshadri T. (1974) Minor phenolic components of seed hulls of HydnocarpusWightiana – Constitution of methoxyhydnocarpin. Indian J Chem 12:993–996. Renau TE, Leger R, Flamme EM, Sangalang J, She MW, Yen R, Gannon CL, Griffith D, Chamberland S, Lomovskaya O, Hecker SJ, Lee VJ, Ohta T, Nakayama K. (1999) Inhibitors of efflux pumps in Pseudomonas aeruginosa potentiate the activity of the fluoroquinolone antibacterial levofloxacin. J Med Chem 42:4928–4931. Rogers B, Decottignies A, Kolaczkowski M, Carvajal E, Balzi E, Goffeau A. (2001) The pleitropic drug ABC transporters from Saccharomyces cerevisiae. J Mol Microbiol Biotechnol 3:207–214. Saier Jr. MH, Paulsen IT. (2001) Phylogeny of multidrug transporters. Semin Cell Dev Biol 12:205–213. Schmeller T, Latz-Bruning B, Wink M. (1997) Biochemical activities of berberine, palmatine and sanguinarine mediating chemical defence against microorganisms and herbivores. Phytochemistry 44:257–266. Spencer CM, Wilde MI. (1997) Diacerein. Drugs 53:98–106; discussion 107–108. Stermitz FR, Beeson T, Mueller PJ, Hsiang J, Lewis K. (2001) Staphylococcus aureus MDR efflux pump inhibitors from a Berberis and a Mahonia (sensu strictu) species. Biochem Syst Ecol 29:793–798.
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Stermitz FR, Cashman K, Halligan KM, Morel C, Tegos GP, Lewis K. (2003) Polyacylated neohesperidosides from Geranium caespitosum: bacterial multidrug resistance pump inhibitors. Bioorg Med Chem Lett 13:1915–1918. Stermitz FR, Lorenz P, Tawara JN, Zenewicz LA, Lewis K. (2000a) Synergy in a medicinal plant: antimicrobial action of berberine potentiated by 50 -methoxyhydnocarpin, a multidrug pump inhibitor. Proc Natl Acad Sci USA 97:1433–1437. Stermitz FR, Scriven LN, Tegos G, Lewis K. (2002) Two flavonols from Artemisa annua which potentiate the activity of berberine and norfloxacin against a resistant strain of Staphylococcus aureus. Planta Med 68:1140–1141. Stermitz FR, Tawara-Matsuda J, Lorenz P, Mueller P, Zenewicz L, Lewis K. (2000b) 50 -Methoxyhydnocarpin-D and pheophorbide A: Berberis species components that potentiate berberine growth inhibition of resistant Staphylococcus aureus. J Nat Prod 63:1146–1149. Tegos G, Higginbotham A, Ball A, Stermitz FR, Lewis K. (2006) Neohesperidosides from Geranium caespitosum; versatile multidrug pump inhibitors Chemistry & Biology, submitted. Tegos G, Stermitz FR, Lomovskaya O, Lewis K. (2002) Multidrug pump inhibitors uncover remarkable activity of plant antimicrobials. Antimicrob Agents Chemother 46:3133–3141. Ulukan H, Swaan PS. (2002) Camptothecins: a review of their chemotherapeutic potential. Drugs 62:2039–2057. Van Bambeke F, Balzi E, Tulkens PM. (2000) Antibiotic efflux pumps. Biochem Pharmacol 60:457–470. Varma MV, Ashokraj Y, Dey CS, Panchagnula R. (2003) P-glycoprotein inhibitors and their screening: a perspective from bioavailability enhancement. Pharmacol Res 48:347–359. Vazquez-Laslop N, Zheleznova EE, Markham PN, Brennan RG, Neyfakh AA. (2000) Recognition of multiple drugs by a single protein: a trivial solution of an old paradox. Biochem Soc Trans 28:517–520. Wang EJ, Casciano C, Clement RP, Johnson WW. (2001) Active transport of fluorescent P-glycoprotein substrates: evaluation as markers and interaction with inhibitors. Biochem Biophys Res Commun 289:580–585. Zheleznova EE, Markham PN, Neyfakh AA, Brennan RG. (1999) Structural basis of multidrug recognition by BmrR, a transcription activator of a multidrug transporter. Cell 96:353–362.
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CHAPTER 4
New concept to search for alternate insect control agents from plants ISAO KUBO
Introduction There is no doubt that many plant secondary metabolites affect insect behavior, development and reproduction. Identifying these substances is an important first step in understanding the effect of plants on insect life at the molecular level. Besides its benefits to basic science, accumulation of this kind of knowledge may provide us with a more rational and scientific approach to insect pest control. Continuing search for alternate insect control agents from plants resulted in characterization of many active phytochemicals. Despite their excellent and interesting activities, the use of these botanical insect control agents is still limited. The reason for this limitation is caused mainly by the lack of availability of materials and/or complex molecules for synthetic optimization. In addition, plant defenses to insect attack usually involve multifunctional multichemicals (Kubo and Hanke, 1985). Preliminary artificial diet-feeding assay has indicated that the methanol extract of a medicinal plant Fuchsia tetradactyla (Onagraceae) contains two hydrolyzable tannins in varying quantities. Both tannins inhibited Pectinophora gossypiella larvae when ingested in small amounts. P. gossypiella larvae became much smaller in size from the control due to the ingestion of the tannins, and this activity is likely due to the prooxidant action. Tannins are estimated to be the fourth most abundant biochemical produced by vascular plant tissue after cellulose, hemicellulose and lignin. Tannins are known to play a role in a number of ecological processes in addition to herbivore defense. Because tannins are complex and energetically costly molecules to synthesize, their widespread occurrence and abundance suggests that application of tannins as insect control agents needs to be investigated from practical points of view rather than as leading compounds for synthetic optimization. In addition, both mosquito larvicidal triterpene saponins isolated from the root bark of Pachyelasma tessmannii (Leguminosae) (Nihei et al., 2005) and insect antifeedant limonoids characterized from Croton jatrophoides (Euphorbiaceae) (Kubo et al., 1990; Nihei et al., 2002; 2004) are other examples. The saponins isolated from
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P. tessmannii were found to exhibit potent mosquito larvicidal activity. However, these triterpene saponins have not been further studied as practical mosquito control agents because of the lack of availability. The limonoids isolated from C. jatrophoides showed potent antifeedant activity against two lepidopteran larvae: pink bollworm, P. gossypiella; and fall armyworm, Spodoptera frugiperda. However, these limonoids are complex and energetically costly molecules to synthesize, and are available in minute amounts. Hence, the application of natural products as alternate insect control agents needs to be investigated from a different aspect. The data have been reported as the result of sporadic research, so it is timely to review and synthesize in one paper all that is currently known.
Tannins Gallae Rhois is a unique natural medicine that is used in the manufacture of tannin, tannic acid and gallic acid. It is a kind of gall produced by the gall-forming aphid Melaphis chinensis on the leaves of Rhus javanica. Eggs of M. chinensis are artificially deposited on the fresh leaves of R. javanica to make commercial Gallae Rhois. After the aphid is colonized, the plant tissue undergoes rapid cell division and enlargement. The aphid, during the wingless stage of its complex life cycle, forms galls along the midrib of the leaves from which the aphid parthenogenetically produces winged offspring. The galls are highly irregular and lobed shape, grayish-brown in color, and have a thin but hard wall and large hollow interior (Figure 1). The wall is the source of gallic acid, tannin and tannic acid, and used as the commercial Gallae Rhois. This gall formation may also be a plant defense mechanism to minimize its damage by aphid invasion. The cavity contains numerous dead winged aphid bodies in white
Fig. 1. Gallae Rhois.
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powder that was removed prior to commercial extraction of tannins. It should be noted that the dead aphid bodies are a result of the heating process of Gallae Rhois before their escape. There has been little study to verify the biological activities and chemical analysis of this white powder. In a preliminary thin-layer chromatography (TLC) analysis, this white powder was found to contain mainly two types of chemicals: both nonpolar and polar compounds without anything in between. Non-polar compounds were found to consist mainly of fatty acids and di- and tri-glycerides by gas chromatography–mass spectroscopy (GC–MS) analysis. Interestingly, the ethanol extract of the white powder was found to exhibit growth inhibitory activity against the pink bollworm P. gossypiella in an artificial diet-feeding assay (Chan et al., 1978; Kubo, 1993). The ethanol extract of the commercial Gallae Rhois showed much more potent activity. Plants can defend themselves against insect attack by means of their secondary metabolites, which interfere with many biological activities. Since there is no way to anticipate a priori what effects specifically involve in the growth inhibitory activity against P. gossypiella larvae, the artificial diet-feeding assay (Kubo, 1991) was performed to detect as much of these activities as possible. Importantly, careful observation of the insects throughout the assay is essential. Any difference from the control should not be overlooked (Kubo, 1993). In the current experiment, P. gossypiella larvae became much smaller in size from the control caused by the ingestion of the ethanol extract, but any additional noticeable difference was not observed. Fractionation of the ethanol extract of the white powder of Gallae Rhois was guided by the artificial diet-feeding assay against the pink bollworm. Successive partitioning of the crude extract with n-hexane, CH2Cl2, EtOAc and H2O revealed the biological activity to be retained in the EtOAc layer. Further fractionation of the EtOAc fraction, using droplet counter-current chromatography (DCCC), gave three major compounds. Further purification with Sephadex LH-20 column chromatography gave three purified phenolic compounds (1–3) (see Figure 2 for structures). The 1H-NMR (nuclear magnetic resonance) spectrum of 1 is rather unusual in that it shows only a single peak, corresponding to the aromatic protons near 6.9 ppm, and the other two peaks present are due to the solvent (DMSO and H2O). The simplicity of the spectra reveals the highly symmetrical nature of the compound. The 13 C-NMR data confirm that compound (1) corresponds to gallic acid. Confirmation was also obtained by comparing 1 with an authentic sample. The 1H-NMR spectra of the two remaining compounds, 2 and 3, show the presence of a methyl and ethyl
1, R=H 2, R=CH 3 3, R=CH 2CH3
Fig. 2. Structures of gallic acid and its methyl and ethyl esters.
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group, respectively. In conjunction with their 13C-NMR and EI-MS data, they are characterized respectively as methyl gallate (2) and ethyl gallate (3). Since ethanol was used in the initial extraction, the latter compound is probably an artifact. This was confirmed by the fact that ethyl gallate was not detected when methanol was used for extraction. Similarly, methyl gallate may also be an artifact since methanol was used as a solvent during purification procedure, although this was not confirmed. As a result, the polar part of the ethanol-extractable fraction of the white powder probably consists almost exclusively of gallic acid (Kubo et al., 2003). Interestingly, gallic acid characterized in the white powder could not be detected in the fresh leaves of the original plant R. javanica by high-performance liquid chromatography (HPLC), prior to attack by the gall-forming aphid M. chinensis. In contrast, hydrolyzable tannin is abundant in the fresh leaves. More importantly, this tannin rapidly gets accumulated in the wall after an attack by M. chinensis and produces Gallae Rhois. This process can be termed as a proteinase inhibitor inducing factor (PIIF) (Ryan, 1979), since the aqueous extract of this Gallae Rhois much more strongly inhibited two digestive proteinases tested, chymotrypsin and leucineaminopeptidase, than that of the fresh leaves of R. javanica. It may be logical to assume that gallic acid was derived from hydrolyzable tannin during the gall-forming process in R. javanica by the insect, or by accompanying microorganisms within the parasites’ body, or the plant itself. It appears, therefore, that the releasing mechanism of gallic acid may be one of the key processes to understanding plant defense and/or gall formation. It should be pointed out that the ethanol extract of the white powder did not significantly inhibit the aforementioned two digestive enzymes. The chemicals involved in gall formation are undoubtedly numerous. However, as an initial step to understanding this process, we focused the current study on the biological activity of gallic acid, especially its effects on insects. To facilitate it, tannic acid isolated from Gallae Rhois was also studied for comparison. It should be noted that the structure of tannic acid has not yet been established, although it was reported as the mixture of at least seven related galloylglucoses by HPLC analysis. The basic structure is 1,2,3,4,6-penta-O-galloyl-b-D-glucose to which the depside galloyl group is randomly distributed among C-2, C-3 and C-4 positions (Nishioka, 1983). In the artificial diet-feeding assay against P. gossypiella larvae, both gallic acid and tannic acid showed growth inhibitory activity and the ED50 of gallic acid was found to be 2000 ppm while that of tannic acid was 50 ppm. In other words, tannic acid showed 40-fold more potent growth inhibitory activity in this feeding assay, indicating that P. gossypiella larvae are sensitive to ingested tannic acid but somewhat tolerate gallic acid. It appears that the gallic acid characterized is responsible for the original insect growth inhibitory activity of the ethanol extract of the white powder observed in the preliminary screening. Similarly, tannic acid is responsible for that of the Gallae Rhois ethanol extract. It should be borne in mind that plant secondary metabolites can act in general by a variety of different mechanisms in insects. The sensitivity of P. gossypiella to ingested tannic acid may be a consequence of its extensive chemical modification in the midgut and oxidation is the first thinkable chemical modification. If so, quinones should be the first oxidized structure. Needless to say, quinones are usually highly toxic to insects as well as to many other organisms. For example, quinones may readily react with biologically important
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nucleophilic groups such as sulfhydryl, amino or hydroxyl. Sulfhydryl groups in proteins and lower molecular weight compounds such as glutathione are known to play an important role. The quinones may cause depletion of cytoplasmic and mitochondrial glutathione which functions in eliminating reactive oxygen species (ROS). This depletion can be caused by a direct interaction between the enone moiety and the sulfhydryl group of glutathione by a Michael-type addition. Neither benzoic acid nor 2,4-dihydroxybenzoic acid (b-rosorcylic acid) inhibited insect growth. The result obtained is consistent with the previous work (Elliger et al., 1980). This observation may support that the oxidized products are responsible for the activity. In addition, oxidation also produces ROS in the gut lumen, and this ROS damages biological systems (Pardini, 1995). In fact, alkyl gallates are known to induce ROS production thereby triggering apoptosis pathway in the human leukemia HL60RG cells (Serrano et al., 1998; Sakaguchi, 1999). It seems that the detrimental effects of tannic acid on the non-adapted pink bollworm are the result of ineffective defenses in a consequence of chemical modification in the midgut and/or against ROS generated in the gut lumen (Barbehenn and Martin, 1994; Barbehenn et al., 1996). The possibility that their adverse effects are a consequence of their potential to act as a prooxidant may need to be considered. In fact, gallic acid is known to produce superoxide anion (Serrano et al., 1998). Furthermore, tannic acid may somewhat irreversibly inactivate enzymes (proteins) in the midgut by cross-links, a process known as tanning, prior to being oxidized. This process also needs to be taken into consideration since tannic acid may bind with proteins in the gut and, as a result, inhibit digestive enzymes as well as protein digestion (Feeny, 1976). This can be supported by the observation that tannic acid showed significant inhibitory activity against fungal protease. At the concentration of 3 mg/mL, tannic acid inhibited the enzyme activity 68%, while gallic acid did not show any inhibitory activity up to 6 mg/mL. In addition, tannic acid inhibited the two digestive enzymes, chymotrypsin and leucine-aminopeptidase. Gallic acid also inhibited the same enzymes but much weaker compared to tannic acid. In connection with this, tannic acid is also known to form complexes with heavy metal ions (Scalbert, 1991). For example, gallic acid is known to inhibit iron absorption to the same extent as tannic acid, per mol galloyl groups (Brune et al., 1989) (Table 1). The possibility of involving tanning effect is unlikely but cannot be entirely ruled out since tannic acid is relatively stable compared to other hydrolyzable tannins such as agrimoniin and davidiin (Kubo et al., 1995). Despite numerous investigations, the precise mechanisms of tannin–herbivore interactions are still largely unknown.
Table 1 Scavenging activity of gallic acid and tannic acid Compounds tested Gallic acid Methyl gallate Ethyl gallate Tannic acid
DPPH consumption 6.5170.30 6.3470.12 6.1870.18 78.874.8
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On the other hand, tannic acid isolated from Gallae Rhois was found to inhibit tyrosinase activity with an IC50 of 19 mM that is about 230-fold more potent than that of gallic acid (Kubo et al., 2003). Tyrosinase (EC 1.14.18.1), also known as phenol oxidase (PO), is a copper-containing enzyme widely distributed in microorganisms, animals and plants. In our continuing search for alternative insect control agents from plants (Kubo, 1993), tyrosinase inhibitors have recently been targeted (Kubo, 1997) because tyrosinase is one of the key enzymes in the insect-molting process (Andersen, 1979, 1990). Hence, tyrosinase inhibitors might ultimately provide clues to control insect pests by inhibiting tyrosinase, resulting in incomplete cuticle hardening and darkening (Kramer and Hopkins, 1987). For example, this enzyme is previously reported to highly correlate with aphid resistance of the Solanum plants (Ryan et al., 1982). Finding alternate insect control agents by inhibiting tyrosinase is one of our goals in our continuing search for tyrosinase inhibitors from plants. However, the in vitro results using fungal tyrosinase described is still far from our goal. For example, mushroom tyrosinase used for the initial screening differs somewhat from insects (Andersen, 1990). More importantly, it has not been intentionally overlooked but, as a result, the dynamic function of tyrosinase in insect cuticle has not been thoughtfully taken into account. Thus, tyrosinase does not always exist as the active form in insects (Gupta et al., 2005) and also its inhibitors cannot always reach to tyrosinase in cuticle in sufficient concentration. Tannic acid is a rather bulky polar compound and hence, it unlikely penetrates insect cuticle. Moreover, the reaction time and the amount of available oxygen need to be considered from a practical point of view since insect tyrosinase is an aerobic oxidase. As far as the artificial diet-feeding assay using P. gossypiella larvae is concerned, some tyrosinase inhibitors characterized inhibit insect growth but some do not. The fact that plant secondary metabolites affect by a variety of different mechanisms in insects needs to be kept in mind. The relevance of the results of in vitro experiments in simplified systems to the in vivo situation should be carefully considered. The further study using more appropriate bioassay methods (Londershausen et al., 1996) is currently under investigation (Li and Kubo, 2004). The gall-forming process in which the aphid M. chinensis makes the white powder still cannot be clearly explained on a molecular level, but it may not be illogical to assume that gallic acid is an end product resulting from the defense of the gallforming aphid against the induced defense mechanisms of R. javanica. This may be supported by the previous report that gallic acid is known to be the metabolite of tannic acid when the latter acid was administered to insects (Bernays and Chamberlain, 1980) and sheep (Zhu et al., 1995) orally. Although the effects of tannic acid against M. chinensis could not be directly tested, it should be reasonable to conclude that this aphid can detoxify, at least in part, ingested toxic tannic acid to relatively non-toxic gallic acid. This ability to tolerate ingested tannic acid may rely on its rapid and extensive hydrolysis, similar to being reported to the tolerance of polyphagous grasshoppers (Bernays and Chamberlain, 1980). This can be supported by the finding of gallic acid in large quantities in the white powder, indicating that hydrolysis is the predominant of possible chemical fates of ingested tannic acid. It seems that tannic acid plays an important role in the defense mechanisms of R. javanica as an antimetabolite, resulting from PIIF. Nevertheless, understanding the
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gall-forming mechanism or the defense mechanisms of R. javanica needs to be investigated not only from one aspect but also from a whole and dynamic perspective. In our earlier studies, gallic acid and its methyl ester were often characterized in large quantities from several plants such as a Guatemalan medicinal plant F. tetradactyla (Onagraceae) locally known as ‘‘patayuc’’ and Bersama abyssinica (Melianthaceae) (Kubo and Matsumoto, 1985). However, their weak inhibitory activity against insect’s growth was usually overlooked in the presence of more potent active compounds (Kubo and Matsumoto, 1984). On the other hand, tannins have seen limited success as speciality chemicals. Previous attempts to develop tanninbased chemicals have suffered because of our failure to appreciate fully economic requirements, purity and stability factors and application needs of industrial process. In addition, tannins may not be good leading compounds to develop more effective insect control agents through synthetic optimization because of their complicate structures. Thus, chemical structures of tannins were started to clarify not long ago but the mechanisms of tannin–herbivore interactions has not yet been much studied, especially on a molecular basis. However, tannins are still available as pest control agents for individual farmers, and need to be investigated from more practical points of view. F. tetradactyla contains two hydrolzsable tannins as well as gallic acid in quantities. Both the tannins also inhibited P. gossypiella larvae when being ingested. Tannins are known to play a role in a number of ecological processes in addition to herbivore defense (Kraus et al., 2003). Because tannins are complex and energetically costly molecules to synthesize, their widespread occurrence and abundance suggests that application of tannins as pest control agents needs to be investigated from practical points of view rather than as leading compounds. Extraction and isolation The white powder (350 g) used for chemical analysis was obtained from the galls and extracted with 95% aqueous EtOH for a week (3 ) at an ambient temperature. The solvent was concentrated in vacuo at 40 1C to give a slightly yellowish residue that weighed 41.8 g. The crude extract was successively partitioned between n-hexane CH2Cl2, EtOAc and H2O in this order and subsequent bioassay revealed the biological activity to be retained in the EtOAc fraction. The solvent was evaporated from the bioactive EtOAc fraction in vacuo to give a residue (11.3 g). Then, 2.02 g of this residue was dissolved in 5 mL of the mobile phase and were injected onto DCCC. The ascending method was used with one fraction equivalent to 300 drops. The solvent system consisted of CHCl3–MeOH–H2O–AcOH–MeCoEt (23:42:26:3:6, v/v/v/v/v). Fractions were collected and checked with SiO2 TLC plates using the stationary phase as the developing solvent. Three active compounds were obtained and the further purification by LH-20 column chromatography yielded, gallic acid (1) (83 mg), methyl gallate (2) (17 mg) and ethyl gallate (3) (134 mg). They were identical in all respects including spectroscopic data to authentic samples. Insect feeding assay The artificial diet-feeding assay was carried out as previously described (Chan et al., 1978) with some modifications (Kubo, 1991). The homogeneous incorporation of
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plant extracts or purified chemicals into the diet with the addition of as little heat as possible is an important concern in the artificial-feeding assay. Since there is no way to anticipate a priori what effects will appear, careful observation of the insects throughout the assay is important. Any difference from the control should not be overlooked. For example, P. gossypiella larvae failed their molting cycle caused by the ingestion of the crude extracts of some plants such as Ajuga (Kubo et al., 1981), Podocarpus (Kubo et al., 1984), and Vitex (Kubo et al., 1985; Zhang et al., 1992) species; the insect underwent normal apolysis, but failed to complete ecdysis (Kubo et al., 1983). If materials permit, plant extracts should be fractionated, at least between organic solvent-soluble and -insoluble fractions. In general, water-soluble substances, especially primary metabolites such as sugars and amino acids, are usually feeding stimulants, so they often mask interesting biological activity.
Saponins Despite significant advances in the techniques used to control mosquitoes during recent decades, they still pose a serious public health problem. In addition to the persistent irritation they cause to humans and animals simply by virtue of their bloodsucking behavior and the itching it causes, mosquitoes are also the principal vector of a variety of serious diseases, including malaria, yellow fever, dengue and encephalitis. Worldwide, approximately 2.7 million human deaths occur each year solely as a result of malaria transmitted by mosquitoes (Butler, 1997). By bioassay-guided fractionation using Aedes aegypti larvae, four novel saponins, pachyelaside A, B, C and D, were isolated as potent mosquito larvicidal principles from the root bark of a west African medicinal plant P. tessmannii (Leguminosae) (Nihei et al., 2005) by using recycling HPLC (Kubo and Nakatsu, 1990). Despite their excellent mosquito larvicidal activity, these triterpene saponins are difficult to obtain in quantities and energetically costly complex molecules to synthesize (Figure 3). To cross these hurdles, a series of aliphatic primary alkanols were tested as a model for their mosquito larvicidal activity, since saponins are usually considered as surface-active agents (surfactants). It should be noted that alkanols are among the most versatile of all organic compounds; free and esterified alkanols occur widely in nature. The use of a series of aliphatic primary alkanols applied to water as soluble solutions for the control of mosquitoes in their larval stage. The species used for this study is Culex tarsalis, the principal vector of the Western equine and St. Louis encephalitis viruses common throughout the western US, but preliminary assays against Culiseta incidens suggest that the alkanols tested show similar activity against members of other mosquito genera too (Hammond and Kubo, 1999). In an effort to understand the molecular basis of alkanol toxicity, a variety of naturally occurring unsaturated long-chain alcohols were also tested for comparison. Of these, farnesol was previously reported as acting in some insects as juvenile hormone mimics (Schmialek, 1961). The toxicity of the homologous series of primary alkanols against C. tarsalis larvae was recorded as mortality after 24 h and the results show that larvicidal activity at dodecanol, with an LD50 of 30 mM against the 1st instar larvae and 28 mM against mixed 3rd and 4th instar larvae. Methanol and ethanol were very
New concept to search for alternate insect control agents
Pt 72A C 54 H 88 O 23 Mol. Wt.: 1105.26
H COOH Glc I O
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OH
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H
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Glc I HO O O O HO OH O HO O Glc III HO HO O O OH HO Glc II HO O HO O HO HO OH Glc IV Pt 751 C 67 H 100 O 28 Mol. Wt.: 1353.49 Xyl
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H
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H
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Fig. 3. Structure of potent mosquito larvicidal saponins characterized from P. tessmannii.
weak agents and mortality did not reach 50% at the highest dose tested, 500 mM. Activity increases through the series as chain length is lengthened from propanol to dodecanol, tapers off slightly from dodecanol to pentadecanol, and then cuts off at hexadecanol. Alkanols with greater than 16 carbons were ineffective as mosquito larvicides because they did not display biochemical toxicity and did not consistently
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prevent larvae from surfacing; consequently, they did not produce 50% mortality up to the maximum practical dosage, even when assays were extended to 4 days. The values cited in Table 2 for tridecanol through eicosanol exceed literature values for their maximum solubility in water (Bell, 1973) but experiments at high doses were included because these compounds are far more soluble in cell membranes – the putative active site – than in water. By creating a supersaturated suspension in the aqueous medium, even temporarily, we were able to deliver a high dose to the mosquitoes themselves. Addition of the surfactant Tween-80 to improve solubility increased the toxicity of tridecanol and tetradecanol solubility, and of pentadecanol by nearly a full order of magnitude (Table 2). Activity of alkanols with less than 13 or more than 15 carbons was unaffected by the use of surfactant or sonication. Tween itself had no noticeable toxicity to larvae up to 400 mg/mL, four times the concentration used here. Sublethal doses provoked symptoms of anaesthesia in mosquitoes (e.g. unresponsiveness to outside stimuli such as tapping on the container and cessation of characteristic diving and feeding behaviors).
Table 2 LD50 values of alkanols against C. tarsalis larvae Alkanols tested
LD50 (mM) 1st instar
Methanol Ethanol Propanol Butanol Pentanol Hexanol Heptanol Octanol Nonanol Decanol Undecanol Dodecanol Tridecanol Tetradecanol Pentadecanol Hexadecanol Heptadecanol through Eicosanol Tridecanol+Tweena Tetradecanol+Tween Pentadecanol+Tween Hexadecanol through Eicosanol +Tween 1,10-Decanediol a
>500 >500 171 (727.8) 74.6 (73.66) 20.0 (74.10) 7.55 (71.18) 2.64 (70.322) 1.12 (70.081) 0.260 (70.061) 0.057 (70.015) 0.038 (70.005) 0.030 (70.004) 0.034 (70.003) 0.049 (70.010) 0.302 (70.058) Inactive up to 0.6 Inactive up to 0.6 0.029 (70.003) 0.034 (70.005) 0.034 (70.007) Inactive up to 0.6 Inactive up to 4.6
In combination with Tween-80 added at 100 mg/mL.
3rd and 4th instar
1.02 (70.139) 0.278 (70.026) 0.081 (70.007) 0.046 (70.004) 0.028 (70.002) 0.042 (70.006) 0.039 (70.005) 0.088 (70.028) 0.222 (70.047)
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Whereas there was no appreciable difference between the patterns of physiological toxicity of alkanols to larvae of different instars, the lethal effects of reduced surface tension were most evident against 3rd and 4th instar larvae, which were less able than 1st instars to tolerate prolonged period without air – note the potency of pentadecanol and hexadecanol against late instars as compared to 1st instar. In short, the effectiveness of hexadecanol against late instars only is apparently the result of its physical properties for temporarily lowering surface tension, and not of inherent biochemical toxicity. Statistical analysis of the data showed that the difference in potency of alkanols differing in length by one carbon was highly significant (Po0.01) for C3–C10 and for C14–C16. The difference between pentadecanol alone and pentadecanol+Tween was also highly significant (Po0.01). However, the differences among the most potent homologues (C10–C14) and the effects of combining each of these with Tween were not significant for this data set (n ¼ 5). In order to better understand the significance of the cutoff in activity after pentadecanol, several other alcohols of relevant structure and chain length were tested as well, all in combination with Tween to ensure maximum solubility. It was interesting to note that, whereas hexadecanol was not toxic to 1st instar mosquitoes at the highest concentrations tested, cis-11-hexadecanol was nearly as potent as the strongest of the saturated alkanols. Similarly, the introduction of two (in the case of linoleyl alcohol) or three (linoenyl alcohol) double bonds to octadecanol converted the compound from completely inactive into being among the most potent. The isoprenoid sesquiterpenoid farnesol, which has an overall length of 12 carbons, also showed toxicity comparable to that of dodecanol, leading us to infer the hydrophobic and hydrophilic moieties. The head—tail-type structure is apparently essential for larvicidal activity because decanol was toxic to mosquitoes in the range of 60 mM, while decane-1,10-diol was not toxic even at 4.6 mM. Although the differences in relative toxicity were consistent among the four unsaturated alkenols tested, they were not statistically significant (Po0.01 and n ¼ 5). The objective of the surface tension experiments was to test the hypothesis that the lethal concentration of a given alkanol can be positively correlated to the concentration required to lower surface tension enough to prevent larvae from surfacing for air. Dilute solutions of Ivory soap produce low, yet stable surface tension values, and were used to confirm the results of earlier researchers that some mosquito larvae have difficulty surfacing for air when surface tension (g) – normally about 72 dynes/cm – is decreased to 27–36 dyne/cm. For C. tarsalis we found variability among individuals, but most 1st- and 2nd instar larvae were unable to attach to the surface g when decreased to the range of 28–32 dyne/cm; 4th instar larvae were able to tolerate lower surface tensions, but none were able to surface when g decreased to about 26 dyne/cm. In contrast, the concentration of C3–C10 alkanol that caused 50% mortality in 1st instar larvae produced surface tensions of 62–47 dyne/cm, which allowed even the smallest larvae to surface without difficulty. The concentration of each C3–C10 alkanol necessary to lower g to 30 dyne/cm was on an average eight times than its respective LD50 concentration. Furthermore, for chain lengths up to 10 carbons, many of the dead larvae were found with their respiratory siphons still firmly suspended from the water’s surface, unequivocally eliminating surface phenomena as the cause of death.
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Alkanols with 11–16 carbons, on the other hand, were found to be superior surface tension-reducing agents and even at low doses, larvae attempting to surface for air simply bounce off the air–water interface and sink back downward. Nevertheless, the swiftness of death hinted that even these larvae were succumbing to a biochemical phenomenon long before they could be expected to display the effects of suffocation. For these long-chain alkanols, the role of a biochemical mode of action was demonstrated by using a method that prevented contact with the surface for the course of the assay. Mosquito larvae, especially early instars, can survive for extended periods without access to air, apparently by cutaneous respiration of dissolved oxygen in the water. A glass ceiling such as a Petri dish can be used to trap larvae without air. By creating a test system free of air and its associated interfaces, the container ceiling is effectively made identical to its walls and bottom and, most importantly, the larvae are specifically isolated from any surface-related phenomenon that might wet their siphon opening, reduce its hydrofuge properties or allow leaks into their respiratory siphons during attempts to surface. When we trapped 20 1st instar C. tarsalis larvae under glass without air as a control, 100% were alive and active 16 h later. Fourth instar populations survived only about 3–4 h, apparently due to their higher oxygen requirements and lower surface area-to-volume ratio. Survival time of 1st instar larvae under glass was approximately doubled to 32–48 h by lowering the temperature of water to 8–10 1C. In contrast, 1st instar larvae trapped without air in jars of water treated with decanol or undecanol were immobilized on the bottom and made no further efforts to surface after 5 min; the same result was observed with dodecanol after about 12 min, with tridecanol after an hour, with tetradecanol after 3 h, and with pentadecanol after approximately 9 h. Extrapolation of this time-dependency curve predicts activity for hexadecanol after about 18 h, but no mortality due to hexadecanol nor heptadecanol treatments occurred before control larvae themselves began to die of anoxia, after about 24–30 h. Nevertheless, we could not rule out the possibility that hexadecanol may act by a mechanism similar to – but weaker than – shorter chain homologues, for example as a partial anesthetic. In an effort to extend the survival of larvae trapped without air and thereby allow sufficient time for the possibility that hexadecanol might still show activity at longer exposures, the same air-free experiments were also carried out under refrigeration, at 6 1C, 120 1C and 14 1C. Control larvae were thus able to survive up to 48 h, but we were surprised to find that 260 mM pentadecanol+Tween, which had caused 100% mortality at 20 1C, caused just 20% mortality at 14 1C, and toxicity disappeared entirely at 10 1C or less. Comparatively, the toxicity and time dependency of tetradecanol and the shorter-chain alkanols were unaffected by these lower temperatures, suggesting that the loss of activity is at least partially a function of declining solubility. Subsequent experiments, in which larvae treated with lethal doses of C10–C15 alkanols were transferred to clean water well after the onset of anesthesia had become evident, confirmed that the effects were completely reversible. Within minutes or hours, again depending on chain length, larvae returned to behavior indistinguishable from that of untreated larvae.
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The significance of our findings are twofold: First, they challenge the previously accepted belief that alkanols act as mosquitocides only via suffocation provoked by surface phenomena; second, for studies measuring biological activity of the alkanol series, this is the first-documented instance of cutoff occurring in an animal at a chain length beyond C12. The speed with which alkanols pC15 act as mosquito larvicides is in sharp contrast to the conclusions of previous researchers using insoluble monolayers of dodecanol (McMullen and Hill, 1971), hexadecanol (McMullen and Hill, 1971), and lecithins (McMullen et al., 1977), which were specifically described as producing larval death only after the dissolved oxygen content of the water was depleted, usually overnight. The increasingly time-dependent nature of toxicity as chain length is increased offers further evidence of the biochemical mode of action for alkanols, independent of any suffocation mechanism resulting from reduced surface tension at the air–water interface. Action of farnesol was also rapid, as far as mosquito larvicidal activity is concerned, the rapid lethality may eliminate the possibility of previous conclusion that farnesol is a juvenile hormone mimics (Schmialek, 1961) since hormonal disturbances take time. The toxicity of farnesol against mosquito larvae is apparently via the same mechanism as for unbranched, saturated alcohols. When, for comparison purposes, mosquitoes were treated with Golden Oil, a commercial product sold as a surface-active mosquitocide, 4th instar larvae died after a period of several hours and 1st instar larvae were weakened, but still alive, 24 h later – both results consistent with an explanation of death via suffocation. Alkanols, in contrast, were generally observed to act more quickly on the 1st instar than on the 4th instar larvae. Also, whereas pupae have been shown previously to be more susceptible to monolayers than larvae (McMullen and Hill, 1971; Levy et al., 1982), our preliminary findings indicate that solutions of long-chain alkanols are more toxic to Culex larvae than to the pupae, possibly owing to the pupae’s thicker cuticle and concomitant capacity to resist penetration by foreign substances. Tests with animals in vivo are somewhat limited, but long-chain alkanols are known to produce anesthesia in fathead minnows (Veith et al., 1983), tadpoles (Alifimoff et al., 1989; Miller et al., 1989) and brine shrimp (Chiou et al., 1990), and to cause growth impairment in the ciliate protozoan Tetrahymena pyriformis (Schultz et al., 1990). In our own earlier studies, they showed activity against a variety of Gram-positive bacteria and fungi, but not against Gram-negative bacteria (Kubo et al., 1995). A persistent quandary facing researchers has been the ‘‘cutoff’’ phenomenon in the homologous series of alkanols, whereby potency increases with chain length until reaching a maximum, and the alkanol containing a single additional carbon shows no potency at all, even when duration of exposure lasts several days (Miller et al., 1989). Although attention has been focused on instances where cutoff occurs immediately after dodecanol, the exact length of carbon chain where cutoff occurs clearly varies among genera (Figure 4). Our review of the literature found no other animal (only microorganisms and a protozoan) for which cutoff has been reported at a chain length beyond dodecanol, which may be the result of differences in the membrane composition of test organisms. By comparison, tadpoles were fully anesthetized by nonanol after 30 min, by
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Fig. 4. Comparison of biological activity of alkanols against various organisms. Cutoff in activity against fish and tadpoles occurs after C12, but in mosquitoes not until after C15. Although the endpoints measured are not identical in all test systems, sublethal doses provoked symptoms of anaesthesia in mosquitoes and growth inhibition in microorganisms. &, Tadpole (Rana pipiens), loss of righting reflex, EC50 (Alifimoff et al., 1989); J, Minnow (Pimephales promelas), 96 h LC50 (Veith et al., 1983); r, Protozoan (T. pyriformis), inhibitory growth concentration, EC50 (Schultz et al., 1990); D, S. aureus, minimum bactericidal concentration (MBC) (Kubo et al., 1995); , Mosquito (C. tarsalis), 24 h LD50, Tween used to maximize solubility of alkanols ZC13; B, P. acnes, MBC (Kubo et al., 1995); +, Clostridium botulinum (Huhtanen, 1980), minimum inhibitory concentration.
decanol and undecanol after 60 min, by dodecanol after 120 min, but not by tridecanol, even when exposed for 96 h (Alifimoff et al., 1989). Mosquitoes are also unusual in that the loss of activity is gradual, tapering off from the strongest compounds before disappearing, whereas for most organisms, maximum activity occurs at a given chain length of n, and absolutely no activity is present at the alkanol of chain length n+1. For example, the minimum bactericidal concentration against Propionibacterium acnes was 1.56 mg/mL for hexadecanol, but heptadecanol was completely inactive, even when tested at 800 mg/mL (Kubo et al., 1995). The precise anesthetic mechanism is still not well understood, and attempts to explain it have covered a wide array of biological functions, mostly related to structure and function of lipid membranes and/or proteins (Franks and Lieb, 1994). The increase in activity with chain lengthening has been widely correlated to each compound’s octanol– or lipid–water partition coefficient, and thereby its relative tendency to accumulate in lipid regions of the cell membrane at concentrations
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sufficient to interfere with basic nutrient or iron transport processes (Treistman and Wilson, 1987; Elliott and Elliott, 1994; Weingart and Bukauskas, 1998). Nevertheless, it has been difficult for lipid-based theories to account for the cutoff effect and with this deficiency in mind, other researchers used alkanol inhibition of the lipid-free luciferase enzyme to build a case for anesthetics acting by building directly to a protein pocket of circumscribed dimension (Franks and Lieb, 1994; Abraham et al., 1991). More recently, the effects of alkanols on ligand-gated ion channels in wildtype and mutated neurotransmitter receptors have also led some to conclude that alkanols act directly on membrane proteins, and do not depend on lipid–protein interactions (Mascia et al., 1996; Mihic et al., 1997; Wick et al., 1998). Still, doubt remains as to whether these systems adequately model the site of general anesthesia in animals (Alifimoff et al., 1989; Moss et al., 1991), or for that matter whether anesthetic activity of alkanols is limited to a single mechanism. The fact that sonication, temperature and combination with a surfactant could be used to manipulate mosquitocidal activity nearly 10-fold around the cutoff, supports the notion that the solubility of alkanols in biological membranes and their capacity to perturb membrane lipids at the lipid–protein interface have some bearing on explanation of the cutoff phenomenon. Indeed, there are several recent studies to support this hypothesis. Anesthetic chain lengths were shown to cause perturbation of membrane lipids whereas non-anesthetic alkanols dissolved in membrane lipids without perturbing them (Miller et al., 1989). Fourier transform infrared spectroscopy (FTIR) studies of lipid membrane vesicles in D2O showed that hydrogen bonding of the alkanol hydroxyl to the phosphate moiety in reversed micelles increases up to decanol, and then declines sharply at tetradecanol (Chiou et al., 1990). If alkanols bond to the phosphate moiety in biological membranes the way they do in membrane vesicle, then certainly they would affect the conformation and function of proteins normally held in place by hydrogen bonds at the lipid–protein interface. The surfactant concept can be extended to answer many other questions related to membrane-bound proteins. For example, there has been a long-standing debate on whether alkanols produce their effects in the central nervous system by acting on lipids or on proteins. Alkanols are known to act as general anesthetics, with increasing potency correlated to increasing chain length until a point of cutoff is reached, usually at dodecanol, after which activity disappears entirely. Since anesthesia involves many membrane-bound proteins such as synaptosomal ATPases and acetylcholine receptor, alkanols may alter these membrane-bound proteins in similar fashion, by disrupting and disorganizing the hydrogen bonds at the lipid bilayer–protein interface nonspecifically. Knowledge that introduction of a double bond shifts the cutoff to a longer-chain length in tadpoles (Pringle et al., 1981) – and now in mosquitoes – is further evidence in support of explanations which consider the structural role of lipids rather than isolated action on a protein-binding site, because if cutoff were due to a compound’s hydrophobic moiety exceeding the limited size of a protein pocket, then since the addition of a cis double bond makes the compound even bulkier, it should theoretically provoke cutoff at shorter, rather than longer, alkyl chain lengths. We speculate that hydrogen bonding plays an important role in determining the molecular basis of alkanol activity in mosquitoes and other organisms.
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Alkanols are stable, colorless, inexpensive, biodegradable (Swisher, 1970), and essentially nontoxic to humans (Opdyke, 1973). The fact that they are already approved for use in food products at concentrations comparable to the doses used here may facilitate their approval as insecticides. If further experience bears out the conclusions of earlier workers who reported a cutoff in activity after dodecanol for fish and amphibians, then the narrower spectrum activity of tridecanol, tetradecanol, and pentadecanol against mosquitoes makes these promising candidates for environmentally sensitive pest management programs. The cutoff in anesthetic potency occurs at different chain lengths in different organisms, lending new perspective to the molecular basis of anesthesia. In brief, the mosquito larvicidal activity of alkanols against C. tarsalis larvae depends on the hydrophobic alkyl chain length from the hydrophilic hydroxyl group. The hydrophilic hydroxyl group can be replaced by any hydrophilic groups as long as the ‘‘head and tail’’ structure is balanced. On the basis of this surfactant concept, various phytochemicals previously isolated were tested for their mosquito larvicidal activity against C. tarsalis larvae. As expected, some phytochemicals showed even more potent and selective larvicidal activity compared to aliphatic alkanols. For example, phytochemicals possessing the phenolic moiety as the hydrophilic portions can be expected to exhibit mosquito larvicidal activity. Anacardic acids and cardols isolated from the cashew nut shell liquid (CNSL) are an excellent example for the surfactant concept (Kubo et al., 1986). Although CNSL is available in far greater tonnage, it is neglected in commercial terms, and there is considerable potential for its exploitation. Extraction and isolation of saponins The air-dried root bark (500 g) of P. tessmannii (Leguminosae) was cut into small pieces and extracted with MeOH (500 mL 3) at ambient temperature for 2 weeks. The MeOH extract (70 g) was evaporated to dryness and suspended in 500 mL of H2O, and then it was extracted successively with n-hexane, ethyl ether, CHCl3, EtOAc and n-BuOH to yield four fractions, n-hexane and ethyl ether (3 g), CHCl3 (14 g), EtOAc (10 g), and n-BuOH (30 g). A portion (4 g) of the n-BuOH fraction was subjected by a recycling HPLC (Kubo and Nakatsu, 1990). Column: 50 cm 2.5 cm Asahipack GS-320, 5 mm; mobile phase: MeOH; flow rate: 3 mL/min; detection: refractive index; amount of sample loaded: 500 mg (Nihei et al., 2005). Bioassays Bioassays were conducted at room temperature (2072 1C) using distilled water (20 mL) in 1 oz clear plastic containers made by Plastics Inc. (St. Paul, MN). Alcohol was added by solubilizing them first in acetone, then diluting in water to the appropriate concentration and briefly shaking to ensure mixing. Ten larvae were then pipetted into each 20 mL volume and observed for a minimum of 24 h, when mortality was recorded. Controls were treated with the maximum amount of acetone applied in each alcohol assay, usually 0.1 mL. Acetone itself was found to have no effect on the larvae up to a concentration of 1%.
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All compounds were assayed against 1st instar larvae and octan-1-ol through hexadecane-1-ol were also tested against mixed 3rd and 4th instar larvae. Assays were repeated at least five times for each compound. Compounds were tested at a minimum of five concentrations following range finding tests. Owing to their excessive clumping and general insolubility in water, alkanols with 15 or more carbons were tested only up to 0.6 mM. Although this dose exceeds the measured solubility of long-chain alkanols in water (Bell, 1973), the application of excess material was used as a way to supersaturate the water and thereby deliver a higher dose to the mosquito itself, in whose cell membranes these compounds are much more soluble. For alkanols greater than 10 carbons, bioassays were repeated with the addition of the commercial surfactant Tween-80 (100 mg/mL) to see if improved solubility might increase activity. For assays employing surfactant, shaking was impractical, so solutions were added while sonicating the test water, which helped to maximize solubility and mixing. Larvae were considered dead or moribund if they stopped moving for a prolonged period, were unable to resurface after sinking to the bottom of the container, and were still unable to respond after gentle probing with a small spatula. Mortality among controls was zero for more than 95% of the assays, and in no instance did it exceed 10%. In assays where larvae were trapped without air and prevented from any contact with the surface, each test solution was prepared as described above, then used to fill a 250 mL glass jar above its brim so as to form a meniscus; a Petri dish was then slid horizontally across the mouth of the jar so that no air bubbles remained. The edge was sealed with petroleum jelly to prevent drying and leakage. Time required to manifest toxicity is expressed as the time at which the last bodily movement could be detected from the last survivor among 10 larvae, and values cited are the mean of at least three experiments (Hammond and Kubo, 1999).
Acknowledgments The work was partly reported at the 15th International Plant Protection Congress in Beijing, China. I am indebted to Dr. D. G. Hammond, Dr. J. A. Klocke and Dr. S. Asano for performing mosquito larvicidal and artificial diet-feeding assays, Mr. S. Kawamichi for supplying Gallae Rhois; Dr. M. Takasaki for chemical analysis of Gallae Rhois at an earlier stage of the work, Dr. N. Masuoka for obtaining scavenging antioxidant data and Dr. C. Ce´spedes for assisting the final manuscript preparation.
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Ryan JD. (1979) Proteinase inhibitors. In: Rosenthal GA, Janzen DH, editors. Herbivores: their interaction with secondary plant metabolites. New York: Academic Press, pp. 599–618. Ryan JD, Gregory P, Tingey WM. (1982) Phenolic oxidase activity in glandular trichomes of Solanum berthaultii. Phytochemistry 21:1885–1887. Sakaguchi N, Inoue M, Isuzugawa K, Ogihara Y, Hosaka K. (1999) Cell death-inducing activity by gallic acid derivatives. Biol Pharm Bull 22:471–475. Scalbert A. (1991) Antimicrobial properties of tannins. Phytochemistry 30:3875–3883. Schmialek P. (1961) Double identification in Tenebrio faces and in yeast of substances with juvenile hormone activity. Z Naturforschg 16b:461–463. Schultz TW, Arnold LM, Wilke TS, Moulton MP. (1990) Relationships of quantitative structure-activity for normal aliphatic alcohols. Ecotox Environ Safe 19:247–253. Serrano A, Palacios C, Roy G, Cespo´n C, Villar ML, Nocito M, Gonza´lez-Porque´ P. (1998) Derivatives of gallic acid induce apoptosis in tumoral cell lines and inhibit lymphocyte proliferation. Arch Biochem Biophys 350:49–54. Swisher RD. (1970) In: Swisher RD, editor. Surfactant biodegradation. New York: Marcel Dekker. Treistman SN, Wilson A. (1987) Alkanol effects on early potassium currents in Aplysia neurons depend on chain length. Proc Natl Acad Sci USA 84:9299–9303. Veith GD, Call DJ, Brooke LT. (1983) Structure-toxicity relationships for the fathead minnow, Pimephales promelas: narcotic industrial chemicals. Can J Fish Aquat Sci 40:743–748. Weingart R, Bukauskas FF. (1998) Long-chain n-alkanols and arachidonic acid interfere with the Vm-sensitive gating mechanism of gap junction channels. Eur. J. Physiol. 435:310–319. Wick MJ, Mihic SJ, Ueno S, Mascia MP, Trudell JR, Brozowski SJ, Ye Q, Harrison NL, Harris RA. (1998) Mutations of gamma-aminobutyric acid and glycine receptors change alcohol cutoff: evidence for an alcohol receptor? Proc Natl Acad Sci USA 95:6504–6509. Zhang M, Stout MJ, Kubo I. (1992) Isolation of ecdysteroids from Vitex stricheri using RLCC and recycling HPLC. Phytochemistry 31:247–250. Zhu J, Filippich LJ, Ng J. (1995) Rumen involvement in sheep tannic acid metabolism. Vet Hum Toxicol 37:436–440.
Rai and Carpinella (eds.) Naturally Occurring Bioactive Compounds r 2006 Elsevier B.V. All rights reserved.
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CHAPTER 5
Role of Melia azedarach L. (Meliaceae) for the control of insects and acari: present status and future prospects MARI´A C CARPINELLA, MARI´A T DEFAGO´, GRACIELA VALLADARES, SARA M PALACIOS
Introduction One of the biggest challenges that humanity confronts is providing enough food to the fast growing population of the planet, which is calculated to reach approximately 8 billions of people in the year 2020 (Knight et al., 1997). In order to materialize this, it is necessary to increase food production, which mainly depends on plants. These are often affected by many pests and diseases among which insects play a preponderant role. Commercially available synthetic pesticides are currently the most effective means of pest control. However, the continuous and indiscriminate use of these substances have not only caused adverse effects on mammals health, but have also affected other benefic members of the ecosystem (Theiling and Croft, 1988; C - elik et al., 2005; Chauhan and Gupta, 2005) and the environment in which they are immersed. They are also responsible for the development of resistance in pathogens (Thompson et al., 1993; Bass et al., 2004). Under these circumstances, new ways of pest control must be studied and established. There are many options, but the most promising is the one that involves the secondary metabolites produced by plants, many of which are highly toxic to a wide spectrum of insects and microorganisms. There is a great variety of families of plants that possess potent anti-insect compounds. From the Meliaceae family, strong insecticide molecules have been isolated, the limonoid azadirachtin obtained from Azadirachta indica or Melia azadirachta (neem) being the most potent and studied (Govindachari, 1992; Schmutterer, 1995). Another tree belonging to the Meliaceae family, far less studied than the previously mentioned, is Melia azedarach L., commonly named Paraı´ so, Chinaberry or Persian Lilac. Our research group has been studying the activity of extracts obtained from different structures of M. azedarach on a wide variety of pest insects, either in laboratory or in field experiments. A limonoid (compound 1) has been isolated from this tree as the most potent substance for controlling insects of agronomic importance. In
82
Naturally occurring bioactive compounds
the present chapter, information about the active principles isolated by various investigators from M. azedarach is provided together with their respective bioactivities. Moreover, a description of the effects on feeding, growth, larval and pupal development, fecundity, fertility, toxic effects, as well as alterations in the insect morphology of different extracts and some pure compounds from the tree is reported either in laboratory or in field trials.
Phytochemistry Many compounds have been isolated from different vegetal structures of M. azedarach collected in different places of the world. Most of them have been assayed as antiinsect and cytotoxic compounds showing excellent results. In relation to the first activity, most assays have been carried out on insects belonging to Spodoptera genus and in less measure on Epilachna. Other activities such as antifungal, antibacterial and anti-inflammatory have been less studied (Table 1). Phytochemical analysis of Argentinian M. azedarach ripe fruit extract revealed that alkaloids are absent (Carpinella et al., 1999a); however, Khan et al. (2001), reported the presence of this family of compounds in extracts from leaves, root and stem bark of M. azedarach from Papua New Guinea. Khan et al. (2001) also found tannins and flavonoids in the three mentioned tree structures. These last compounds were also found in the fruit extract from Argentinian M. azedarach (Carpinella et al., 1999a). Riba et al. (1996) followed a bioguided assay purification from a methanolic extract from M. azedarach seeds collected in Spain. After carrying out an HPLC purification, three main fractions were obtained. The first fraction (Fr. 1) obtained at the first 10 min with methanol/water 50:50 did not show any effect on Sesamia nonagrioides (Lepidoptera: Noctuidae) survival while the one obtained between 10 and 20 min of running (Fr. 2) exhibited the highest percentage of mortality till it reached pupation. The fraction obtained with methanol 100% (Fr. 3) showed a 46% of accumulated mortality till pupation on S. nonagrioides. From this fraction four majority products were obtained but only one showed effectiveness (Riba et al., 1996). From Fr. 2, a subfraction which exhibited the major activity was obtained. This subfraction contained two products in a relation 3:1. These products could not be separated; although successive purifications were performed similar chromatograms were obtained (Figure 1; Riba et al., 1996). The authors concluded that it was a decomposition of the majority compound. Carpinella et al. (2002) afforded the same mixture of compounds (compound 1) after carrying out a bioassay-guided isolation from the ethanolic ripe seed kernel extract from M. azedarach from Argentina. The fact that was interpreted as decomposition by Riba et al. (1996) was elucidated by Carpinella et al. (2002) as a mixture of two interchangeable isomeric limonoids, which occur/exist as an equilibrium between isomers A and B. The last one was named ‘meliartenin’ (Carpinella et al., 2002). Compound A was favoured in CH3CN (A/B ¼ 80–70:20–30) while the molar fraction of B increased in CHCl3 (A/B ¼ 53:47) (Figure 2a and b). Note the similarity between chromatograms of Figures 1 and 2a.
Table 1 Pure compounds isolated from extracts obtained from different structures of M. azedarach and their respective bioactivities Origin
Activity
Reference
1-cinnamoyl-3-hydroxy11methoxymeliacarpinin
EtOH ext. (CH2Cl2 fr.) of root bark of Chinese M. azedarach L.
Cytotoxic activity against P388 lymphocytic leukaemia cells, IC50 ¼ 1.5 mg/ml
Takeya et al. (1996b)
1-(2-methylpropanoyl)-3acetyl-11methoxymeliacarpinin
MeOH ext. of root of M. azedarach L. var japonica
No lethal activity on Artemia salina at 100 mg/ml
Fukuyama et al. (2000)
1,12-di-O-acetyltrichilin B
Et2O ext. of root bark of Chinese M. azedarach L.
Antifeedant against S. exigua Hu¨bner (Boisduval) and S. eridania (Boisduval) at 8 mg/cm2
Nakatani et al. (1994b); Huang et al. (1994); Nakatani et al. (1995b); Huang et al. (1995b)
EtOH ext. (CH2Cl2 fr.) of root bark of Chinese M. azedarach L.
Cytotoxic activity against P388 lymphocytic leukaemia cells, IC50 ¼ 0.46 mg/ml
Takeya et al. (1996a)
1,3-dicinnamoyl-11hydroxymeliacarpin
Me2CO and MeOH ext. (CH2Cl2 fr.) of leaves of Indian M. azedarach L.
Insecticide on S. littoralis, LC50 ¼ 2.36 ppm; EC50 ¼ 0.57 ppm
Bohnenstengel et al. (1999)
11-methoxy-20acetylmeliatinin
MeOH ext. of fruit of M. azedarach from Greece
12 a-acetoxyfraxinellone
Et2O ext. of stem bark of Japanese M. azedarach
Kraus et al. (1985) Antifeedant against S. littoralis (Boisduval) at 10 mg/cm2
Nakatani et al. (1998)
Ichthyotoxic on Oryzias latipes at 50 ppm 12-acetoxyamoorastatin ¼ toosendanin ¼ 29deacetylsendanin
Cytotoxic against human tumour cells, ED50 ¼ 0.0007–0.04 mg/ml
Ahn et al. (1994)
Bark of Chinese M. azedarach
Antifeedant against S. litura
Chiu (1989)
Et2O ext. of root bark of Chinese and Japanese M. azedarach
Antifeedant against S. eridania (Boisduval), MIC ¼ 6 mg/cm2
Nakatani et al. (1998); Huang et al. (1995b)
EtOH ext. (CH2Cl2 fr.) of root bark of Chinese M. azedarach L.
Cytotoxic activity against P388 lymphocytic leukaemia cells, IC50 ¼ 0.026 mg/ml
Itokawa et al. (1995)
83
MeOH ext. (EtOAc fr.) of stem bark of M. azedarach var. japonica
Role of Melia azedarach L. (Meliaceae) for the control of insects and acari
Compound
84
Table 1 (continued ) Origin
Activity
Reference
1-O-deacetylochinolide A
MeOH ext. of fruits of Brazilian M. azedarach L.
Cytotoxic activity against HeLa S3 human epithelial cancer cells, IC50 ¼ 1449 mg/ml
Zhou et al. (2004)
1-O-deacetylochinolide B
MeOH ext. of fruits of Brazilian M. azedarach L.
Cytotoxic activity against HeLa S3 human epithelial cancer cells, IC50 ¼ 58 mg/ml
Zhou et al. (2004)
1-O-deacetyl-1-Obenzoylochinolide B
MeOH ext. of fruits of Brazilian M. azedarach L.
Cytotoxic activity against HeLa S3 human epithelial cancer cells, IC50 ¼ 22,638 mg/ml
Zhou et al. (2004)
1-O-deacetyl-1-Otigloylochinolide A
MeOH ext. of fruits of Brazilian M. azedarach L.
Cytotoxic activity against HeLa S3 human epithelial cancer cells, IC50 ¼ 20,374 mg/ml
Zhou et al. (2004)
1-O-deacetyl-1-Otigloylochinolide B
MeOH ext. of fruits of Brazilian M. azedarach L.
Cytotoxic activity against HeLa S3 human epithelial cancer cells, IC50 ¼ 22,443 mg/ml
Zhou et al. (2004)
12-deacetyltrichilin I
EtOH ext. (CH2Cl2 fr.) of root bark of Chinese M. azedarach L.
Cytotoxic activity against P388 lymphocytic leukaemia cells, IC50 ¼ 0.011 mg/ml
Takeya et al. (1996a)
12-hydroxyamoorastatin ¼ 12-deacetyltoosendanin
MeOH ext. (EtOAc fr.) of stem bark of M. azedarach var. japonica
Cytotoxic against human tumour cells, ED50 ¼ 0.01–0.25 mg/ml
Ahn et al. (1994)
EtOH ext. (CH2Cl2 fr.) of root bark of Chinese M. azedarach L.
Cytotoxic activity against P388 lymphocytic leukaemia cells, IC50 ¼ 0.090 mg/ml
Itokawa et al. (1995)
Et2O ext. of root bark of Chinese M. azedarach
Antifeedant S. eridania (Boisduval) 3 mg/cm2
Nakatani et al. (1998); Huang et al. (1995b)
EtOH ext. (CH2Cl2 fr.) of seed kernel of M. azedarach from Argentina
Antifeedant and insecticide against E. paenulata at 4 mg/cm2 and antifeedant against S. eridania at 1 mg/cm2
Carpinella et al. (2002)
Isomer A of compound 1
Naturally occurring bioactive compounds
Compound
MeOH ext. (EtOAc fr.) of stem bark of M. azedarach var. japonica
Cytotoxic against human tumour cells, ED50 ¼ 0.38–13.7 mg/ml
Et2O ext. of root bark of Chinese M. azedarach
Ahn et al. (1994) Nakatani et al. (1998)
Et2O ext. of root bark of Chinese M. azedarach L.
Antifeedant against S. exigua Hu¨bner (Boisduval) and S. eridania (Boisduval) at 8 mg/cm2
Huang et al. (1994); Nakatani et al. (1995b); Huang et al. (1995b)
12-O-acetylazedarachin B
Et2O ext. of root bark of Chinese M. azedarach L.
Antifeedant against S. exigua Hu¨bner (Boisduval) and S. eridania (Boisduval) at 8 mg/cm2
Huang et al. (1994); Nakatani et al. (1995b); Huang et al. (1995b)
12-O-acetyltrichilin B
Et2O ext. of root bark of Chinese M. azedarach L.
Antifeedant against S. exigua Hu¨bner (Boisduval) and S. eridania (Boisduval) at 8 mg/cm2
Nakatani et al. (1994a); Huang et al. (1994); Nakatani et al. (1995b); Huang et al. (1995b)
1-acetyl-2-deacetyltrichilin H
EtOH ext. (CH2Cl2 fr.) of root bark of Chinese M. azedarach L.
Cytotoxic activity against P388 lymphocytic leukaemia cells, IC50 ¼ 0.66 mg/ml
Takeya et al. (1996a)
1-acetyl-3-deacetyltrichilin H
EtOH ext. (CH2Cl2 fr.) of root bark of Chinese M. azedarach L.
Cytotoxic activity against P388 lymphocytic leukaemia cells, IC50 ¼ 0.40 mg/ml
Takeya et al. (1996a)
1-acetyl-3-tigloyl-11methoxymeliacarpinin
EtOH ext. (CH2Cl2 fr.) of root bark of Chinese M. azedarach L.
Cytotoxic activity against P388 lymphocytic leukaemia cells, IC50 ¼ 3.3 mg/ml
Itokawa et al. (1995)
1-acetyltrichilin H
EtOH ext. (CH2Cl2 fr.) of root bark of Chinese M. azedarach L.
Cytotoxic activity against P388 lymphocytic leukaemia cells, IC50 ¼ 0.47 mg/ml
Takeya et al. (1996a)
1-cinnamoilmelianolona
MeOH ext. (CH2Cl2 fr.) of fruit of M. azedarach from USA
Insecticide against Heliothis virescens, EC50 ¼ 0.12 ppm; LC50 ¼ 1.50 ppm and S. frugiperda, EC50 ¼ 0.04 ppm; LC50 ¼ 1.30 ppm
Lee et al. (1987); Kraus et al. (1989); Lee et al. (1991) 85
12-O-acetylazedarachin A
Role of Melia azedarach L. (Meliaceae) for the control of insects and acari
12-hydroxyamoorastatone
86
Table 1 (continued ) Compound
Origin
Activity
Reference
1-cinnamoyl-3-11 dihydroxymeliacarpin
MeOH ext. (CH2Cl2 fr.) of fruit of M. azedarach from USA
Insecticide against H. virescens, EC50 ¼ 0.18 ppm; LC50 ¼ 3.50 ppm and S. frugiperda, EC50 ¼ 0.04 ppm; LC50 ¼ 1.60 ppm
Lee et al. (1991)
1-cinnamoyl-3-acetyl-11hydroxymeliacarpin
Me2CO and MeOH ext. (CH2Cl2 fr.) of leaves of Indian M. azedarach L.
Insecticide against S. littoralis, EC50 ¼ 0.27 ppm; LC50 ¼ 0.48 ppm
Bohnenstengel et al. (1999)
1-cinnamoyl-3-acetyl-11methoxymeliacarpinin
Root bark of Japanese M. azedarach L.
Antifeedant against S. eridania (Boisduval), MIC ¼ 1 mg/cm2
Nakatani et al. (1994a); Huang et al. (1995b)
Cytotoxic activity against P388 lymphocytic leukaemia cells, IC50 ¼ 10.5 mg/ml
Takeya et al. (1996b)
MeOH ext. of fruit of M. azedarach from Greece
Growth regulator on E. varivestis
Growth regulator on E. varivestis No antifeedant against E. varivestis
Kraus et al. (1985, 1987)
Effect on moulting on Tessaratoma papillosa at 50 mg/ml
Chiu (1989)
Stem bark of Okinawan M. azedarach L. EtOH ext. (CH2Cl2 fr.) of root bark of Chinese M. azedarach L. 23bmethoxymeliacarpin Kraus et al. (1985, 1987) MeOH ext. of fruit of M. azedarach from Greece
1-cinnamoyl-3methacrylyl-11hydroxy-meliacarpin
Me2CO and MeOH ext. (CH2Cl2 fr.) of leaves of Indian M. azedarach L.
Insecticide against S. littoralis, EC50 ¼ 0.57 ppm; LC50 ¼ 1.19 ppm
Bohnenstengel et al. (1999)
1-deoxy-3-methacrylyl-11methoxymeliacarpinin
EtOH ext. (CH2Cl2 fr.) of root bark of Chinese M. azedarach L.
Cytotoxic activity against P388 lymphocytic leukaemia cells, IC50 ¼ 47.0 mg/ml
Takeya et al. (1996b)
Naturally occurring bioactive compounds
1-cinnamoyl-3-feruloyl11-hydroxy-22,23dihydroNo antifeedant against E. varivestis 1-cinnamoyl-3-feruloyl11-hydroxymeliacarpin
Antifeedant against S. exigua (Boisduval) at 3 mg/cm2 and S. eridania (Boisduval), MIC ¼ 1 mg/ cm2
Nakatani et al. (1993); Huang et al. (1995b)
1-desacetylnimbolinin B
PE and Et2O ext. of fruit of M. azedarach var. japonica
Antifeedant against E. varivestis
Kraus and Bokel (1981)
1-O-detigloyl-1-Obenzoylohchinolal
MeOH ext. of fruits of Brazilian M. azedarach L.
No cytotoxic activity against HeLa S3 human epithelial cancer cells
Zhou et al. (2004)
1-O-detigloyl-1-Ocinnamoylohchinolal
MeOH ext. of fruits of Brazilian M. azedarach L.
No cytotoxic activity against HeLa S3 human epithelial cancer cells
Zhou et al. (2004)
1-methacrylyl-3-acetyl-11methoxymeliacarpinin
MeOH ext. root of M. azedarach L. var japonica
Lethal on A. salina, LC50 ¼ 19 mg/ml
Fukuyama et al. (2000)
1-tigloil-11-methoxy-20acetylmeliacarpin
Fruit
1-tigloyl-3,20-diacetyl-11methoxylmeliacarpinin
EtOH ext. (CH2Cl2 fr.) of root bark of Chinese M. azedarach L.
Cytotoxic activity against P388 lymphocytic leukaemia cells, IC50 ¼ 100 mg/ml
Takeya et al. (1996b)
1-tigloyl-3-acetyl-11methoxymeliacarpinin
EtOH ext. (CH2Cl2 fr.) of root bark of Chinese M. azedarach L.
Cytotoxic activity against P388 lymphocytic leukaemia cells, IC50 ¼ 3.2 mg/ml
Itokawa et al. (1995)
1,5-dihydroxy-8-methoxy2-methylanthraquinone3-O-a-Lrhamnopyranoside
Stem bark
Srivastava and Mishra (1985)
1,8-dihydroxy-2methylanthraquinone-3O-b-Dgalactopyranoside
Stem bark
Srivastava and Mishra (1985)
Kraus et al. (1987)
87
Et2O ext. of root bark of Chinese M. azedarach
Role of Melia azedarach L. (Meliaceae) for the control of insects and acari
1-deoxy-3-tigloyl-11methoxymeliacarpinin
Table 1 (continued ) Origin
Activity
Reference
29-isobutylsendanin
EtOH ext. (CH2Cl2 fr.) of root bark of Chinese M. azedarach L.
Cytotoxic activity against P388 lymphocytic leukaemia cells, IC50 ¼ 0.034 mg/ml
Itokawa et al. (1995)
2b,3b-dihydroxy-5apregn-17(20)-(2)-en-16one
MeOH ext. of root of M. azedarach L. var japonica
Lethal on A. salina, LC50 ¼ 2.1 mg/ml
Fukuyama et al. (2000)
30-hydroxy-fraxinellone
AcOEt ext. of roots of Italian M. azedarach
3-deacetyltrichilin H
EtOH ext. (CH2Cl2 fr.) of root bark of Chinese M. azedarach L.
Cytotoxic activity against P388 lymphocytic leukaemia cells, IC50 ¼ 0.045 mg/ml
Takeya et al. (1996a)
3-tigloyl-1,20-diacetyl-11methoxylmeliacarpinin
EtOH ext. (CH2Cl2 fr.) of root bark of Chinese M. azedarach L.
Cytotoxic activity against P388 lymphocytic leukaemia cells, IC50 ¼ 48.0 mg/ml
Takeya et al. (1996b)
4,8-dimethoxy-1-vinyl-bcarboline
MeOH ext. (EtOAc fr.) of cortex of M. azedarach L. var japonica
Anti-inflammatory activity (suppression inducible nitric oxide synthase and cyclooxygenase-2 induction) 1.5–10 mM
Lee et al. (2000)
EtOH ext. (CH2Cl2 fr.) of seed kernels of Argentinian M. azedarach L.
Antifungal against Fusarium verticillioides, MIC ¼ 0.4 mg/ml
Anti-inflammatory activity (suppression inducible nitric oxide synthase and cyclooxygenase-2 induction) 2–10 mM
Lee et al. (2000)
Carpinella et al. (2003b) 4-methoxy-1-vinyl-bcarboline 6-acetoxy-3b-hydroxy-7oxo-14b, 15bepoxymeliac-1,5-diene3-O-b-Dglucuronopyranoside
methoxycinnamaldehyde
Cortex of M. azedarach L. var japo´nica
Roots of M. azedarach L. from India
D’Ambrosio and Guerreiro (2002)
Srivastava and Gupta (1985)
Naturally occurring bioactive compounds
4-hydroxy-3-
88
Compound
6-acetoxy-11-a hydroxy 7oxo 14 b, 15 b epoxymeliacin 1, 5 diene 3-O-a-L rhamnopyranoside
EtOH ext. (C6H6 fr.) of seeds of M. azedarach L. from India
6-hydroxy-7methoxycoumarin
EtOH ext. of leaves
Khalil et al. (1979)
6b-hydroxy-4-campesten3-one
PE extract of bark
Nair and Chang (1973)
6b-hydroxy-4stigmasten3-one
PE extract of bark
Nair and Chang (1973)
7a-acetoxy-14b, 15bepoxy-gedunan-1-ene-3O-b-D-glucopyranoside
Stem bark
Saxena and Srivastava (1986)
7-deacetyl-7-oxogedunin
Light PE ext. of stem wood
Okogun et al. (1975)
9a-hydroxyfraxinellone
AcOEt ext. of roots of Italian M. azedarach
D’Ambrosio and Guerreiro (2002)
9b-hydroxyfraxinellone
AcOEt ext. roots of Italian M. azedarach
D’Ambrosio and Guerreiro (2002)
Aesculetin
EtOH ext. of leaves
Khalil et al. (1979)
Amoorastatone
Et2O ext. of root bark of Chinese M. azedarach
Nakatani et al. (1998)
Aphanastatin
Et2O ext. of root bark of Chinese M. azedarach L.
Antifeedant against S. exigua Hu¨bner (Boisduval) and S. eridania (Boisduval), MIC ¼ 4 mg/cm2
Nakatani et al. (1995b); Huang et al. (1995b)
Azedarachin A
Et2O ext. of root bark of Chinese M. azedarach L.
Antifeedant against S. exigua Hu¨bner (Boisduval) and S. eridania (Boisduval), MIC ¼ 4 mg/cm2
Huang et al. (1994); Nakatani et al. (1995b); Huang et al. (1995b)
Srivastava and Gupta (1985) Antibacterial activity
Srivastava (1986)
89
Roots of M. azedarach L. from India
Role of Melia azedarach L. (Meliaceae) for the control of insects and acari
6-acetoxy-7a-hydroxy-3oxo-14b, 15bepoxymeliac-1,5-diene
Table 1 (continued ) Origin
Activity
Reference
Azedarachin C
Et2O ext. of root bark of Chinese M. azedarach L.
Antifeedant against S. exigua Hu¨bner (Boisduval) and S. eridania (Boisduval), MIC ¼ 8 mg/cm2
Nakatani et al. (1995b); Huang et al. (1995a, 1995b)
Azedarachol
Et2O ext. of root bark of M. azedarach var. japonica
Antifeedant against Ajrotis sejetum Denis at 10 mg/cm2
Nakatani et al. (1985)
Azedaralide
Et2O ext. of root bark of Chinese M. azedarach
Antifeedant against S. littoralis (Boisduval) at 10 mg/cm2 Ictiotoxic O. latipes at 50 ppm
Nakatani et al. (1998)
Azedaric acid
Light PE ext. stem wood
Okogun et al. (1975)
Campesterol
Fruit
Hanifa Moursi and AlKhabitib (1984)
EtOH ext. of fruit of M. azedarach
Khalil et al. (1979)
Cinamodiol
MeOH ext. (EtOAc fr.) of seeds of Brazilian M. azedarach
Cinnamic acid
EtOH ext. of leaves
Khalil et al. (1979)
Cycloeucalenone
Light PE ext. stem wood
Okogun et al. (1975)
D-Glucuronic
EtOH ext. (CHCl3 fr.) of root of M. azedarach from India
Keshri et al. (2003)
acid
Kelecom et al. (1996)
Deacetylsalannin
Et2O ext. of root bark of Chinese M. azedarach
Antifeedant against S. eridania (Boisduval) at 20 mg/cm2
Huang et al. (1996a, 1996b)
Fraxinellone
Et2O ext. of stem bark of Japanese M. azedarach
Antifeedant against S. littoralis (Boisduval) at 10 mg/cm2
Nakatani et al. (1998)
Light PE ext. stem wood
Ictiotoxic on O. latipes at 10 ppm
Okogun et al. (1975)
PE ext. of trunk wood
Ekong et al. (1969)
AcOEt ext. of roots of Italian M. azedarach
D’Ambrosio and Guerreiro (2002)
Naturally occurring bioactive compounds
Little antimoulting activity on R. prolixus at 10 mg/ml
90
Compound
Et2O ext. of stem bark of Japanese M. azedarach
No antifeedant against S. littoralis (Boisduval)
Gedunin
Light PE ext. of stem wood
Okogun et al. (1975)
PE ext. of trunk wood
Ekong et al. (1969)
Nakatani et al. (1998)
Ictiotoxic O. latipes at 50 ppm.
EtOH ext. (CHCl3 fr.) of root of M. azedarach from India
Prevents the pregnancy and the number of implants at 50 mg/kg of rat/day
Keshri et al. (2003)
PE ext. of bark M. azedarach from Taiwan
Chiang and Chang (1973); Chang and Chiang (1969)
Kulinone
PE ext. of bark M. azedarach from Taiwan
Chiang and Chang (1973)
Kulolactone
PE ext. of bark M. azedarach from Taiwan
Chiang and Chang (1973); Chang and Chiang (1969)
Melain
Buffer ext. of fruit M. azedarach var. japonica
Protease
Kaneda et al. (1994)
Melain G
Buffer ext. green fruit M. azedarach var. japonica
Protease
Uchikoba et al. (1999)
Melazolide A
AcOEt ext. of roots of Italian M. azedarach
D’Ambrosio and Guerreiro (2002)
Meldenin
EtOH ext. (C6H6 fr.) seeds M. azedarach L. from India
Srivastava (1986)
Meliacarpinin A
Et2O ext. of root bark of Chinese M. azedarach L.
Antifeedant against S. exigua Hu¨bner (Boisduval) and S. eridania (Boisduval) at 1 mg/cm2
Nakatani et al. (1995b)
Meliacarpinin B
Et2O ext. of root bark of Chinese M. azedarach L.
Antifeedant against S. exigua Hu¨bner (Boisduval) and S. eridania (Boisduval) at 1 mg/cm2
Nakatani et al. (1995b) 91
Kulactone
Role of Melia azedarach L. (Meliaceae) for the control of insects and acari
Fraxinellonone
92
Table 1 (continued ) Origin
Activity
Reference
Meliacarpinin C
Et2O ext. of root bark of Chinese M. azedarach L.
Antifeedant against S. exigua Hu¨bner (Boisduval) and S. eridania (Boisduval) at 1 mg/cm2
Nakatani et al. (1995b)
Meliacarpinin D
Et2O ext. of root bark of Chinese M. azedarach L.
Antifeedant against S. exigua Hu¨bner (Boisduval) and S. eridania (Boisduval) at 1 mg/cm2
Nakatani et al. (1995b)
MeOH ext. root of M. azedarach L. var japonica
No lethal activity on A. salina at 100 mg/ ml
Fukuyama et al. (2000)
Meliacarpinin E
Et2O ext. of root bark of Chinese M. azedarach
Antifeedant against S. eridania (Boisduval) at 1 mg/cm2
Huang et al. (1996b)
Meliandiol
Fruit
Han et al. (1991)
Melianin A
Light PE ext. of stem wood
Okogun et al. (1975)
Melianin B
Light PE ext. of stem wood
Okogun et al. (1975)
Melianin B
MeOH ext. root of M. azedarach L. var japonica
Fukuyama et al. (2000)
Melianol
CHCl3 ext. of fruits
Lavie et al. (1967a); Han et al. (1991)
Melianolide
Root bark of Chinese M. azedarach L.
Huang et al. (1996a)
Melianone
CHCl3 ext. of fruits
Lavie et al. (1967a); Han et al. (1991)
Melianoninol
Fruit
Antifeedant against Pieris rapae L.
Han et al. (1991)
Meliantriol
CHCl3 ext. of fruits
Repellent against S. gregaria at 8 mg/ cm2
Lavie et al. (1967b); Ascher (1986)
Naturally occurring bioactive compounds
Compound
EtOH ext. (CH2Cl2 fr.) of seed kernel of M. azedarach from Argentina
Meliatoxin A1 ¼ trichilin D
EtOH ext. (Et2O fr.) of fruit of M. azedarach var. australasica
Meliatoxin A2
Meliatoxin B1
Antifeedant and insecticide against E. paenulata at 4 mg/cm2 and antifeedant against S. eridania at 1 mg/cm2
Carpinella et al. (2002, 2003a) Oelrichs et al. (1983); MacLeod et al. (1990)
Et2O ext. of root bark of Chinese M. azedarach L.
Antifeedant against S. exigua Hu¨bner (Boisduval) and S. eridania (Boisduval) at 8 mg/cm2 (400 ppm)
Nakatani et al. (1994b, 1995b); Huang et al. (1994, 1995b)
EtOH ext. (CH2Cl2 fr.) of root bark of Chinese M. azedarach L.
Cytotoxic activity against P388 lymphocytic leukaemia cells, IC50 ¼ 0.055 mg/ml
Takeya et al. (1996a)
EtOH ext. (Et2O fr.) of fruit of M. azedarach var. australasica
Antifeedant against S. litura at 400 ppm (480 mg/cm2)
Oelrichs et al. (1983); MacLeod et al. (1990)
Et2O ext. of root bark of Chinese M. azedarach L.
Antifeedant against S. exigua Hu¨bner (Boisduval) and S. eridania (Boisduval) at 8 mg/cm2
Huang et al. (1994); Nakatani et al. (1994b, 1995b); Huang et al. (1995b)
EtOH ext. (Et2O fr.) of fruit of M. azedarach var. australasica
Growth inhibitory effect against S. litura at 400 ppm
Oelrichs et al. (1983); MacLeod et al. (1990)
EtOH ext. (CH2Cl2 fr.) of root bark of Chinese M. azedarach L.
Cytotoxic activity against P388 lymphocytic leukaemia cells, IC50 ¼ 5.4 mg/ml
Takeya et al. (1996a)
EtOH ext. (Et2O fr.) of fruit of M. azedarach var. australasica
Oelrichs et al. (1983); MacLeod et al. (1990)
Methyl kulonate
PE ext. of bark of M. azedarach from Taiwan
Chiang and Chang (1973)
Methylene cycloartanone
Fruit and roots of M. azedarach L.
Schulte et al. (1979)
Myristic acid
EtOH ext. (CHCl3 fr.) of root of M. azedarach from India
Prevents the pregnancy and the number of implants at 50 mg/kg of rat/day
Keshri et al. (2003)
Nimbolidin A
PE and Et2O ext. of fruit of M. azedarach var. japonica and from Yugoslavia and Greece
Antifeedant against E. varivestis
Kraus et al. (1980); Kraus and Bokel (1981)
93
Meliatoxin B2
Role of Melia azedarach L. (Meliaceae) for the control of insects and acari
Meliartenin (isomer B of compound 1)
94
Table 1 (continued ) Compound
Origin
Activity
Reference
Nimbolidin B
PE and Et2O ext. of fruit of M. azedarach var. japonica from Yugoslavia and Greece
Antifeedant against E. varivestis
Kraus et al. (1980); Kraus and Bokel (1981)
Et2O ext. of root bark of Chinese M. azedrach L.
Antifeedant against E. varivestis at 0.005%
Bokel (1980)
Antifeedant against S. eridania (Boisduval) at 10 mg/cm2
Huang et al. (1996a, 1996b)
Nimbolin A
Okogun et al. (1975)
PE ext. of trunk wood
Ekong et al. (1969)
Nimbolin B
PE ext. of trunk wood
Ekong et al. (1969)
Nimbolinin B
PE and Et2O ext. of fruit of M. azedarach var. japonica and from Yugoslavia and Greece
Antifeedant against E. varivestis
Kraus et al. (1980); Kraus and Bokel (1981)
Et2O ext. of root bark of Chinese M. azedrach L.
Antifeedant against S. exigua Hu¨bner (Boisduval) and S. eridania (Boisduval) at 20 mg/cm2
Nakatani et al. (1995a, 1995b) Huang et al. (1996a, 1996b)
PE and Et2O ext. of fruit of M. azedarach var. japonica and from Yugoslavia and Greece
Antifeedant against E. varivestis
Kraus et al. (1980); Kraus and Bokel (1981)
MeOH ext. of fruits of Brazilian M. azedarach L.
No cytotoxic activity against HeLa S3 human epithelial cancer cells
Zhou et al. (2004)
PE and Et2O ext. of fruit of M. azedarach var. japonica and from Yugoslavia and Greece
Antifeedant against E. varivestis
Kraus et al. (1980); Kraus and Bokel (1981)
MeOH ext. of fruits of Brazilian M. azedarach L.
Cytotoxic activity against HeLa S3 human epithelial cancer cells, IC50 ¼ mg/ml
Zhou et al. (2004)
Ochinolid A
Ochinolid B
Naturally occurring bioactive compounds
Light PE ext. stem wood
Fruit of M. azedarach L. var. japonica Makino
Fukuyama et al. (1983)
Ohchinolal
Fruit of M. azedarach L. var. japonica Makino
Fukuyama et al. (1983)
Pinoresinol
MeOH ext. of fruits of Brazilian M. azedarach L.
No cytotoxic activity against HeLa S3 human epithelial cancer cells
Zhou et al. (2004)
MeOH ext. (EtOAc fr.) seeds of Brazilian M. azedarach
Inhibited moulting R. prolixus, ED50 ¼ 84 mg/ml
Cabral et al. (1995); Cabral et al. (1999)
EtOH ext. of seed kernels (CH2Cl2 fr.) of Argentinian M. azedarach L.
Antifungal against F. verticillioides, MIC ¼ 1.0 mg/ml
Carpinella et al. (2003b)
Pyroangolensolide
AcOEt ext. of roots of Italian M. azedarach
D’Ambrosio and Guerreiro (2002)
Rutin
EtOH ext. (CHCl3 fr.) of root of M. azedarach from India
Keshri et al. (2003)
Salannal
Et2O ext. of root bark of Chinese M. azedarach
Nakatani et al. (1995a); Huang et al. (1996a, 1996b)
Salannin
EtOH ext. (Et2O fr.) of seeds of M. azedarach L. from India
Srivastava (1986)
Et2O ext. of root bark of Chinese M. azedarach L.
Antifeedant against S. exigua Hu¨bner (Boisduval) and S. eridania (Boisduval) at 20 mg/cm2
Nakatani et al. (1995a, 1995b); Huang et al. (1995b, 1996a, 1996b)
Scopoletin
EtOH ext. of seed kernels (CH2Cl2 fr.) of Argentinian M. azedarach L.
Antifungal against F. verticillioides, MIC ¼ 1.5 mg/ml
Carpinella et al. (2003b)
Sendanin
MeOH ext. of bark and fruit of M. azedarach L. var. japonica
Sitosterol
Fruit
Ochi and Kotsuki (1976); Ochi et al. (1978) Reduce the incidence of induced gastric ulcers in rats
Hanifa Moursi and AlKhabitib (1984) Khalil et al. (1979)
95
EtOH ext. of fruit of M. azedarach
Role of Melia azedarach L. (Meliaceae) for the control of insects and acari
Ohchinin
96
Table 1 (continued ) Origin
Activity
Reference
Teracrylmelazolide A
AcOEt ext. of roots of Italian M. azedarach
D’Ambrosio and Guerreiro (2002)
Teracrylmelazolide B
AcOEt ext. of roots of Italian M. azedarach
D’Ambrosio and Guerreiro (2002)
Trichilin B
Et2O ext. of root bark of Chinese M. azedarach L.
Antifeedant against S. exigua Hu¨bner (Boisduval) and S. eridania (Boisduval) at 4 mg/cm2
Huang et al. (1994); Nakatani (1994b, 1995b); Huang et al. (1995b)
Trichilin H
Et2O ext. of root bark of Chinese M. azedarach L.
Antifeedant against S. exigua Hu¨bner (Boisduval) and S. eridania (Boisduval) at 8 mg/cm2
Nakatani et al. (1994b); Huang et al. (1994); Nakatani et al. (1995b); Huang et al. (1995b)
EtOH ext. (CH2Cl2 fr.) of root bark of Chinese M. azedarach L.
Cytotoxic activity against P388 lymphocytic leukaemia cells, IC50 ¼ 0.16 mg/ml
Takeya et al. (1996a)
Vanillic acid
Fruit
Han et al. (1991)
Vanillin
Fruit
Han et al. (1991)
EtOH ext. of seed kernels (CH2Cl2 fr.) of Argentinian M. azedarach L.
Antifungal against F. verticillioides, MIC ¼ 0.6 mg/ml
Carpinella et al. (2003b)
EC50: effective concentration 50. LC50: lethal concentration 50. MIC: minimum inhibitory concentration. IC50: inhibitory concentration 50. ED50: effective dosage 50. Ext.: extract. Fr.: fraction. AcOEt: ethyl acetate. Et2O: diethyl ether. CH2Cl2: dichloromethane. EtOH: ethanol. MeOH: methanol. PE: petroleum ether.
Naturally occurring bioactive compounds
Compound
Role of Melia azedarach L. (Meliaceae) for the control of insects and acari
97
0.030
C
0.025
AU
0.020 0.015 0.010 0.005 0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 Minutes
Fig. 1. HPLC chromatogram of a subfraction from Fr. 2. Reversed-phase C18 column; mobile phase: 0–20 min 50% MeOH in water; 20 min 100% MeOH. UV detection at 220 nm.(From Riba et al., 1996, with permission.) 23
22
O
OH 18 20
O
12 11 30
OH
9
1
O 3
14
5
H
AcO
HO OH
21
17 16
O
O
12
O
H
AcO
OH 29
11
7
OH H
H HO
O
O
HO
28
A
B 1
It must be noticed that no other compound with similar activity than 1 was found in the seed kernel extract. According to this fact, toosendanin was ruled out as the most active anti-insect compound. Toosendanin which exhibits a similar structure than isomer A was previously reported in bark, root and stem bark extracts of M. azedarach (Chiu, 1989; Huang et al., 1995b; Nakatani et al., 1998). Azadirachtin (aza), whose presence was previously described in paraı´ so tree by Morgan and Thornton (1973) and Luo et al. (1995), was not found (Palacios et al., 1993). Its occurrence was also denied by many other authors (Lee et al., 1991; Ascher et al., 1995; Wandscheer et al., 2004). Reports about aza in M. azedarach extracts could be due to the confusion between M. azedarach and A. indica trees (Schmutterer, 1995; Ho¨rdegen et al., 2003).
Laboratory assays Antifeedant activity on insects of different orders For measuring the antifeedant activity on insects belonging to different orders, extracts obtained from different structures of Argentinian M. azedarach were prepared
98
Naturally occurring bioactive compounds
Fig. 2. HPLC chromatograms of compound 1 (a) dissolved in CH3CN and (b) dissolved in CH3Cl. Each peak corresponded to each isomer A and B. Reversed-phase C18 column; mobile phase: 32% acetonitrile in water. UV detection at 210 nm.
(Valladares et al., 1997, 1999; Carpinella et al., 2002; Banchio et al., 2003; Carpinella et al., 2003a; Valladares et al., 2003). The antifeedant activity of the ethanolic fruit extract measured through choice test was very high showing an antifeedant index [AI (%)] higher than 75% in most studied insects such as adults of the Coleoptera Pantomorus leucoloma Boheman,
Role of Melia azedarach L. (Meliaceae) for the control of insects and acari
99
Priocyphus bosqui (Hustache), Tribolium confusum Duval, Diabrotica speciosa (Germar), Chrysodina sp., Epitrix argentiniensis Bryan, Eumolpinae sp., Plagioneda erythroptera (Blanchard), Xanthogalleruca luteola (Mu¨ller) and adults and larvae of Epilachna paenulata (Germ). The same results were observed on larvae of the Lepidoptera Spilosoma virginica (Fabricius), Anticarsia gemmatalis (Hubner) and Colias lesbias (Fabricius) (Carpinella et al., 2003a). Only Rachiplusia nu Guene´e did not respond to M. azedarach fruit extract. Coleoptera species appeared particularly sensitive to this extract, in contrast with the observations made after treatments with A. indica extracts in which case Lepidoptera was the most affected order (Mordue and Blackwell, 1993). Studies on the above-mentioned insects using senescent leaf extract from M. azedarach showed a potent antifeedant effect on Coleoptera and Lepidoptera (Valladares et al., 2003), being a little more effective on the former ones. Although there is previous information about the activity of extracts or isolated terpenoids from paraı´ so on some species of Spodoptera (Chiu, 1983; Khadr et al., 1986; MacLeod et al., 1990; Schmutterer, 1995; Carpinella et al., 2003a), S. ornithogalli (Guen) (Lepidtera: Noctuidae) was not affected by the leaf extract. In the case of Sitophilus oryzae (L.) (Coleoptera: Curculionidae), the strong rejection towards the leaf extract of M. azedarach observed here disaffirmed the lack of activity of extracts from paraı´ so reported by Imti and Zudir (1997), but corresponded with the positive effects registered on the control of other curculionide (Oroumchi and Lorra, 1993; Fernandes et al., 1996). Many studies on different pest insects have been carried out with M. azedarach extracts obtained from different vegetal structures of the tree, mainly from fruits. Aqueous extracts from fruits showed antifeedant activity against Authonomus grandis Boh. (Coleoptera: Curculionidae) (Fernandes et al., 1996). Adults of Dicladispa armigera and Callosobruchus chinensis, both Coleoptera, were greatly deterred from feeding on rice plants and mung beans; they were respectively treated with leaves and seed extract from M. azedarach collected in Bangladesh as reported by Islam (1986). The effect on D. armigera is in concordance with the activity found in other Chrysomelidae confronted to ethanolic fruit and leaf extracts from Argentinian M. azedarach (Valladares et al., 1997; Carpinella et al., 2003a). Schmutterer (1989) found that M. azedarach bark extracts were inactive up to a concentration of 800 ppm in the E. varivestis larvae leaf-disc feeding test. However, leaf extracts were considerably active as antifeedant at 200 ppm (70% activity) and 400 ppm (100% activity), more than analogous extracts from A. indica. Tewary and Moorthy (1985) tested a petroleum ether extract of drupes of M. azedarach against larvae and adults of Epilachna vigintioctopunctata (Coleoptera: Coccinellidae). The extracts at 0.1% gave 100% protection of leaves. Freshly treated larvae were less parasitized by Pediobius foveolatus (Hymenoptera: Eulophidae) than the control, but on exposure to parasitation 24 h after treatment, larvae were normally parasitized. The parasites that emerged from treated hosts were normal. A choice test with the leaf-disc method on the poliphagous larvae V of Spodoptera litura (Fabricius) (Lepidoptera: Noctuidae) using methanolic extract of seed kernel of Chinese M. azedarach showed an antifeedant concentration 50 (AFC50, concentration that gives 50% of AI), 100 times lower than the one found in no choice test (Chiu et al., 1984). According to the authors, the difference could be attributed to the fact that the full grown larvae were highly sensitive to the extract.
Naturally occurring bioactive compounds
100
Antifeedant activity measured in an artificial diet bioassay on larvae III of Spodoptera littoralis (Lepidoptera: Noctuidae) was related to extract concentration, varying from 11.2% to 56.9% at 10 to 100 ppm respectively. In Agrotis ipsilon (Lepidoptera: Noctuidae) same concentrations produced 7.7–51.1% of deterrency (Schmidt et al., 1997). A 1% methanolic extract from seed kernels of M. azedarach from China showed more than 80% of feeding inhibition on 1st and 2nd instar larvae from the Oriental armyworm Mythimna separata (Walker) (Lepidoptera: Noctuidae) (Chiu et al., 1987). In choice tests performed with Plutella xylostella L. (Lepidoptera: Yponomeutidae) where cabbage leaf discs were dipped in different solutions of methanolic kernel extracts from M. azedarach, the larvae consumed less or no food compared to untreated leaf discs at 24 h from the beginning (Dilawari et al., 1994). Methanolic extracts from fruits and seeds of M. azedarach collected in Spain showed a strong antifeedant activity against larvae II of S. nonagrioides Lefe`bre (Lepidoptera: Noctuidae) (Joan Serra et al., 1998; Juan et al., 2000). Extracts obtained from seed kernels of M. azedarach with petroleum ether showed antifeedant effect against 2nd and 3rd instar nymphs of Nilapavarta lugens Stal. (Hemiptera: Delphacidae). The AC50 (concentration at which 50% of insect were deterred from the plant) corresponded to 1.16% (Chiu, 1983). The activity of meliartenin (as compound 1) was studied under a choice test in comparison with aza and toosendanin (Carpinella et al., 2002). Both compound 1 and aza exhibited similar activity on E. paenulata (Coleoptera: Coccinellidae) larvae showing an AI (%) higher than 90% at 4 mg/cm2, while toosendanin showed same level of effectiveness at 10 mg/cm2. On larvae III of the southern armyworm Spodoptera eridania (Lepidoptera: Noctuidae), same rate of activity was observed at 1 mg/cm2 for compound 1 and aza and at 14 mg/cm2 for toosendanin. MacLeod et al. (1990) reported that the antifeedant activity of meliatoxin A2 measured in choice tests carried out with S. litura is in concordance with the activity of trichilin D reported by Nakatani et al. (1981) on S. eridania showing in both cases high activity at 400 ppm. It is important to say that, even when the concentrations are the same, in accordance with the methodology used by each author, the real quantity of compounds applied on the leaves were different (480 mg/cm2 calculated according to MacLeod et al., 1990 data, against 8 mg/cm2 according to data reported by Nakatani et al., 1995b and Huang et al., 1995a, 1995b). Meliatoxin B1 reduced feeding to a half with respect to control at 400–500 ppm (480–600 mg/cm2, MacLeod et al., 1990). 22
22
O
O
21
21 12
OH O
11 9
19
1
AcO
(CH 3)2CHOCO
H
29
28
H
Meliatoxin A2
OHO
17 30
19 15
O
H
O AcO
18
OH
1
AcO
CH3CH2CHCH3OCO
12 11 9
H
O AcO
H
29
18
28
H
Meliatoxin B1
17 30
15
H OH
O
Role of Melia azedarach L. (Meliaceae) for the control of insects and acari
101
Bentley et al. (1988) showed that the epoxide and furan groups are essential for high antifeedant activity in citrus limonoids. Besides, these substituents in ring D and independent of substitution patterns in ring A and C-28 ester moieties, the 12-OH translate in most potent activity, followed by 12a-acetoxy (Huang et al., 1994; Nakatani et al., 1994b). Acylation of C-29 also reduced the activity (Zhou et al., 1998). According to our results, it seems that 11-OH also ensures a high feeding inhibition. Compounds without the 14-15 epoxide group exhibited weak antifeedant action (Nakatani et al., 1999). Effects on deterrence, survival and development of insects from different orders Order Coleoptera Epilachna paenulata Germ. E. paenulata was used as reference insect for the isolation of the most effective anti-insect compound from the fruits of Argentinian M. azedarach L. Differences in the antifeedant rate were observed when larvae III of E. paenulata were fed with different concentrations of compound 1 in choice or no choice tests (Carpinella et al., 2003a). Results were measured at 24 h from beginning, being the larval stage the same for both tests. The ED50 (effective dosage 50, dosage in which an AI of 50% was obtained) in choice tests corresponded to 0.80 mg/cm2, while in no choice test the ED50 was lower: 0.25 mg/cm2. In no choice test, at 24 h, larvae of E. paenulata fed with cotyledons treated with 1 ate approximately four times less than larvae confronted to aza at all dosages, or to the control. Aza showed an abrupt reduction in food consumption 24 h later. After 6 days of treatment, larvae fed with both compounds exhibited almost null values of food consumption. Compound 1 would act as a primary antifeedant, demonstrated by an immediate rejection of treated food, probably via the gustatory pathway regulated by sensory organs of the mouthparts. Body weight was also affected reaching, depending on concentration, half of weight than those larvae receiving untreated food. Larval mortality showed at 24 h after treatment, 20% of dead larvae fed with 1 while the larvae confronted to aza, to untreated food or the starved ones were all alive. The fact that the larvae confronted to 1 died earlier, even compared to the starved group, suggests a toxic effect. Mortality was complete at 192 h with 0.5 mg/cm2 of 1, while at the same concentration of aza this level of mortality was reached at 240 h. This effect was probably more related to the strong feeding inhibiting effect of the compounds, mortality being due to the reduced food consumption previously mentioned. Epilachna varivestis Muls. A leaf extract from M. azedarach inhibited growth and disrupted metamorphosis of larvae IV of the Mexican bean beetle, E. varivestis (Coleoptera: Coccinellidae). A compound, whose structure was not elucidated, was isolated. A complete mortality was observed with the pure compound at a concentration below 2 ppm (Zhu and Ermel, 1991). Strong growth-disrupting effects were demonstrated by Steets (1975) on 4th larvae E. varivestis fed for 2 days with bean leaves sprayed with 5% and 2.5% extract. None of the larvae pupated and they died within 11 days after the start of the experiment. Similar results were obtained with the first stage of the larvae: only 5%
102
Naturally occurring bioactive compounds
and 20% of larvae reached the second stage when feeding with 2% and 4% extract respectively. € Xanthogalleruca luteola ðMullerÞ. The elm beetle X. luteola (Coleoptera: Chrysomelidae) is the most important pest of the elm tree (Ulmus spp.) in America. Complete larval and adult mortality was observed in no choice test after feeding the insects with 2%, 5% and 10% fruit extract concentration. Development in surviving insects was delayed (Valladares et al., 1997). When the ripe fruit extract was applied as a spray twice with 24 h intervals, no apparent effect on survival of adults was observed (Valladares et al., 1997), whereas in larvae III treated with 5% extract concentration, mortality values were higher than 80%. It was also observed that only a 40% of the treated larvae reached the pupal stage with a delay of 2–6 days compared to control ones (Valladares et al., 1997). Considering the above-mentioned facts, the anti-insect effect was greater when the extract was ingested than when it was topically applied; this indicates that the skin is a strong barrier to be trespassed. This fact must be taken into account in the preparation of formulations. The lack of effect by contact and effectiveness by ingestion was also detected after treatments with neem extracts (Isman, 1994). Hypera postica Gyllenh. An investigation of the activity of aqueous extracts of leaves from M. azedarach collected in Iran was carried out on the cosmopolitan pest of the alfalfa weevil Hypera postica (Coleoptera: Curculionidae) in laboratory assays (Oroumchi and Lorra, 1993). In studies on larvae II–IV high larval-pupal mortality was obtained with leaf extracts at 2.5–10 g of powdered vegetal/100 ml.The feeding activity was reduced and the affected larvae were reduced in size, became darker, refused feeding even on the untreated leaves and gradually lost movement, which resulted in death. In some cases black spots appeared on the body surface of the insects (Oroumchi and Lorra, 1993). A similar reaction also occurs in larvae IV of E. varivestis after treatment with A. indica, which would be attributed to a massive degeneration of the tissue with the resulting liberation of high quantities of cellular debris which stimulates the formation of nodules (Schlu¨ter, 1995). In larval IV period high quantities of phenoloxidase (this enzyme catalyses the cuticular tanning and melanin formation) are available to react with the resulting cellular debris producing, together with the following haemocytic reactions, blackish nodules (Schlu¨ter, 1995). Some survival larvae developed to pupae and adults with normal or abnormal appearance, showing the latter, deformation in wings, legs and parts of the head (Oroumchi and Lorra, 1993). They often did not eliminate the pupal skin, being unable to move and therefore dying. Five percent of parasitism was observed, showing that the active principles did not affect the natural enemies of the weevil (Oroumchi and Lorra, 1993). Diabrotica undecimpunctata howardi Barber. In the search for new active substances that protect seedling corn from injury produced by southern corn rootworm Diabrotica undecimpunctata howardi (Coleoptera: Chrysomelidae), 88 crude extracts obtained by pressure from plants were studied under no choice tests (Landis and Gould, 1988). The only extract that significantly reduced the feeding of the rootworm was the leaf extract from M. azedarach collected in North Carolina.
Role of Melia azedarach L. (Meliaceae) for the control of insects and acari
103
Diabrotica speciosa ðGerm:Þ. Aqueous extracts of leaves, flowers, stems and fruits of M. azedarach collected in Brazil were evaluated against D. speciosa (Coleoptera: Chrysomelidae) (Ventura and Ito, 2000). The most efficient extracts in decreasing food consumption were the ones obtained from flowers and fruits followed by stems and leaf extracts which exhibited an intermediate activity. In a greenhouse experiment in which the flower extract was applied on plants of the common beans Phaseolus vulgaris L. protected from light, the leaf consumed by the beetle was null but when plants were exposed to light, the percentage of consumption increased to about 40%, probably due to the adverse effect of UV rays on the extract (Ventura and Ito, 2000). Tribolium confusum Duv. A population of T. confusum (Coleoptera: Tenebrionidae) was fed with wheat flour to which crushed fruits of M. azedarach collected in Spain were added. Larvae fed with 10–25% of crushed fruit showed an important delay in reaching pupal and larval stage (Del Tı´ o et al., 1996). This delay could be due to the strong antifeedant effect [AI (%) ¼ 88.4] observed by Carpinella et al. (2003) after feeding the weevil with 10% fruit extract. At higher concentrations, a demographic reduction of the insect was observed (Del Tı´ o et al., 1996). Dicladispa armigera Olivier and Callosobruchus chinensis Lucas. Fresh leaves and seeds of ripe fruits of paraı´ so were Soxhlet extracted with various solvents and studied against adults of rice hispa D. armigera (Coleoptera: Chrysomelidae) and the pulse beetle C. chinensis (Coleoptera: Bruchidae) allowed to feed on treated rice plants or mung beans respectively (Islam, 1986). Ovipositional and adult emergence of both insects were respectively deterred and reduced by 1% extracts obtained with methanol, methyl-tert-butyl-ether, methyltert-butyl-ether and methanol, ethanol, n-butanol, n-hexane and acetone being the oviposition most affected in the case of D. armigera. The newly emerged pulse beetle adults were small in size, did not survive more than 6 days and were less active in feeding (Islam, 1986). Order Diptera Liriomyza huidobrensis ðBlanchardÞ. With the aim of measuring the translaminar effect that the ethanolic fruit extract of M. azedarach possesses, larvae I of the leafminer Liriomyza huidobrensis (Diptera: Agromyzidae) were placed on Cucurbita ma´xima seedlings (Banchio et al., 2003). Results showed no significant mortality when the extracts were applied on leaves containing larvae I and III of the leafminer, but an increase in mortality rate of about 60% was observed in pupal stage (Banchio et al., 2003) even at 10% concentration. These results demonstrated the translaminar effect of the extracts even without the addition of surfactant, in accordance with that found by Brunherotto and Vendramim (2001) who suggest that the active principles present in extracts of M. azedarach exhibited translaminar action at producing mortality and delay in larval and pupal phase of the tomato pinworm Tuta absoluta, when the extracts were applied on the outer surface. If the plants were treated with the same extracts at 10% before being infected with females of the miner, the leaves received up to 90% fewer punctures than the control. On the other hand, these plants exhibited a lower number of pupae, while no larval or pupal mortality was observed. The fact that a high pupal mortality was absent
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when the seedlings were treated before exposing them to the adults suggests a shorter residual activity of the extract. In field assays plants of Vicia faba were treated four times with weekly intervals with ripe fruit extract of paraı´ so at 10% at neutral pH and with the addition of 0.5% of Tween 20 (Banchio et al., 2003). A 50% fewer pupae than in plants treated with water (control) and those treated with the synthetic insecticide Tamaron was observed. Pupal survival was also affected (30% of survival), this value being lower than in treatments with Tamaron (80% of survival). Parasitism rates were slightly affected, the difference with the control being much lower than with the use of Tamaron. This result could be due to the delayed action of the extracts, which allows the pupation of the larvae killing them slowly (Banchio et al., 2003). In this way the population of parasitoids is conserved as was observed by Oroumchi and Lorra (1993) and Breuer and Devkota (1990). Aqueous extracts of fruits and leaves of paraı´ so were tested for their efficacy against L. huidobrensis (Abou-Fakhr Hammad et al., 2000b) on naturally infested swiss chard, Beta vulgaris var. cicla L. and in greenhouse experiments on artificially infested cucumber, Cucumis sativus L. Results indicated that Melia fruit extract significantly lowered the number of larvae per swiss chard plant causing deformed larvae, partially brown, rooting and oozing, indicating a growth regulating activity. Leaf extract did not exhibit any effect. In greenhouse experiments, the fruit extract significantly decreased the number of live larvae of the leafminer per cucumber leaf throughout the period of the experiment and with comparable effect to that of synthetic insecticides such as abamectin, cyromazine, imidacloprid and pyrazophos. On its own, leaf extract kept the number of larvae at a significant lower density with respect to control at 20 days after a second application (Abou-Fakhr Hammad et al., 2000b). Anastrepha fraterculus ðWied:Þ. The South American fruit fly, Anastrepha fraterculus (Diptera: Tephritidae) is one of the most important pests in Brazil where the insect has developed resistance to synthetic insecticides (Salles and Rech, 1999). The use of a formulation obtained by aqueous maceration of dry dust of fruits of M. azedarach was studied for the control of the pest. Treatments with doses of the dust at 25–150 g/l produced a decrease in the number of eggs, larvae, pupae or adults. At 150 g/l, malformed pupae and adults were obtained (Salles and Rech, 1999). Orseolia oryzae ðWood-MasonÞ. Orseolia oryzae (Diptera: Cecidomyiidae) is a rice pest that causes important losses in the production of the crop. Studies on the rice gall midge using methanolic extracts of seed kernels of M. azedarach from China showed an oviposition repellence of 83.1% at 1% extract concentration when a choice test was carried out and a 70.7% of repellence with 2% concentration in no choice test (Chiu et al., 1984). Aedes aegypti Linnaeus. Studies of ethanolic extracts from endocarps of M. azedarach collected in Brazil on late larvae III and earlier larvae IV of the mosquito vector of dengue fever, Aedes aegypti 1762 (Diptera: Culicidae) showed an LC50 (lethal concentration 50) of 0.034 g% in an artificial diet assay. This value was lower than that observed when the larvae were not fed at all (Wandscheer et al., 2004).
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Order Lepidoptera Spodoptera litura ðFabriciusÞ. Through a no choice test, MacLeod et al. (1990) studied the effect of meliatoxins A2 and B1, isolated from the ethanolic fruit extract from M. azedarach var. australasica on the 2nd instar of S. litura. Meliatoxin A2 significantly reduced the ingestion of food at 400 ppm (480 mg/cm2), while meliatoxin B1 inhibited the feeding in only 50% at the same concentration. However, this last compound significantly reduced the weight gain at 400 ppm, showing an inhibitory growth effect rather than an antifeedant action. Meliatoxin A2 also reduced the growth but it is apparently due to the inhibition of food consumption. According to the authors, this difference in the activity between both compounds could be attributed to the structure of ring D, showing that the epoxi function confers higher antifeedant activity, and the C-15 keto group would be responsible for the growth inhibition. Spodoptera frugiperda ðJ:E: SmithÞ. The effects of extracts obtained with methanol from fruits of M. azedarach L. collected in Greece were studied in S. frugiperda (Lepidoptera: Noctuidae) (Breuer and Schmidt, 1995, 1996). The larvae fed with 1% and 10% solutions of extract sprayed on lettuce leaves formed smaller pupae and the emerged adults showed deformations and morphogenetic defects. A decrease in feeding was observed and a retarded larval growth and development was detected. When increasing the concentrations of Melia, the ingesta decreased and the larvae digested and/or metabolized the food with less effectiveness. The reduction in growth was not only due to starvation but also due to ingestion of toxic substances (Breuer and Schmidt, 1995). When the experiment was done by topic application no significant differences were observed compared to control (Breuer and Schmidt, 1995). The number of eggs laid by adult females treated from the larval stage was reduced. Adult emergence was not affected. The larval and pupal mortality was increased while increasing the dosages, mainly due to alterations in moulting which could be caused by defects in the neuroendocrine system (Breuer and Schmidt, 1995, 1996). Breuer and De Loof (2000a) obtained different fractions from a methanolic extract of green fruits of M. azedarach collected in Greece. For the assays, the fractions were incorporated into an artificial diet. The growth of S. frugiperda was completely retarded and the larval mortality reached 100% with the ethyl acetate fraction at 1000 ppm, while the petroleum ether and water ones did not show differences with the control. A significant delay in larval development was observed at 10 ppm and upwards and body weights were highly reduced after treatment with 50 ppm of the ethyl acetate fraction (Breuer and De Loof, 2000a). Artificial diet containing 0.01% of an ethyl acetate fraction obtained from unripe fruit of M. azedarach collected in Greece and extracted with methanol resulted in an inhibition of the cholinesterase activity of the larvae of S. frugiperda (Breuer et al., 2003). This effect together with the lower food intake was responsible for the diminished locomotory activity of the larvae. The activity of the NADPH cytochrome c reductase was significantly higher (34%) to that of the control. In the cockroach Leucophaea maderae the effect of 1% extract on the activation of this enzyme was 43% (Breuer et al., 2003). It is known that this detoxification system becomes more
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activated as larvae develop (Breuer et al., 2003), which would explain the lower sensitivity to treatments of the bigger larvae (Breuer and Schmidt, 1996). Rodriguez Hernandez and Vendramim (1998) compared the effect of aqueous fruit extracts obtained from M. azedarach from Brazil with extracts equally obtained from other Meliaceae species on 1st instar larvae of S. frugiperda. Larvae fed with M. azedarach extract, suffered from a longer larval phase with respect to control, being this extract more effective than the extracts obtained from the other Meliaceae. Melia extract also resulted in lower food consumption and body weight compared to other extracts. Less efficient food conversion into biomass was also observed (Rodriguez Hernandez and Vendramim, 1998). Since S. frugiperda consume less food and this was metabolized with lower efficacy after treatments with extract from fruits of Melia (Breuer and Schmidt, 1996; Rodriguez Hernandez and Vendramim, 1998), the content of haemolymph protein was decreased (Schmidt et al., 1998). The reduction in the reserve of this material (necessary for the development of eggs in the adult females) would be the reason for a lower number of eggs laid by S. frugiperda (Breuer and Schmidt, 1996). On the other hand, protein biosynthesis could be affected by M. azedarach compounds. This alteration and the subsequent alteration in neuropeptide production have also been reported for aza (Fritzsche and Cleffmann, 1987; Meurant et al., 1994). Leaves from M. azedarach L. were extracted with hexane, chloroform, methanol and water. Resulting extracts were incorporated to a base of agar in order to feed S. frugiperda (McMillian et al., 1969). Hexanolic and aqueous extracts did not exhibit any effect on larval growth; however, the chloroformic extract at 3 mg equivalents of extract based on dry leaf weight per gram of diet showed smaller larvae. Methanolic extract was less active. With the chloroformic one a decrease in weight of about 100 times compared to control was observed at 30 mg equivalents/g of diet. All larvae died when they were fed with this mentioned concentration. At 10 mg equivalents/g of diet, the mortality of S. frugiperda was 90% and although the remaining 10% pupated, they did not develop into adults. A delay in time till pupation and adult emergence was also observed showing some deformities in the adults at intermediate concentrations (McMillian et al., 1969). When S. frugiperda larvae were fed with corn seedlings treated with the chloroformic extract, a reduction in weight was observed (McMillian et al., 1969). As is observed in this study, the most active extracts were not those obtained with solvents of extreme polarity. This is in accord with the data exposed in Table 1 and by Breuer and De Loof (2000a) where most active principles were those extracted with medium polarity solvents. Spodoptera littoralis ðBoisd:Þ and Agrotis ipsilon ðHufn:Þ. The effectiveness of methanolic extracts enriched by fractionation with petroleum ether and ethyl acetate obtained from fruits of M. azedarach collected in Greece (Schmidt et al., 1997) was studied on 3rd instar larvae of the cotton leafworm S. littoralis (Boisd.), and the black cutworm, A. ipsilon (Hufn.) (Schmidt et al., 1997). After the addition of the methanolic extract at 50 ppm to an artificial diet, consumption was decreased. Weight gain was significantly reduced with the use of the extract at 10 ppm.
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Complete mortality of S. littoralis and A. ipsilon at 50 and 100 ppm respectively was observed (Schmidt et al., 1997). At lower concentrations, pupal weight and development were affected. In both, insect malformations and moulting defects were detected. Adults emerged from larvae previously treated with extract showed a reduction in fecundity and fertility (Schmidt et al., 1997). S. littoralis was slightly more sensitive than A. ipsilon in terms of pupation and adult emergency. Decreasing of body weight may be due to the reduction in food consumption and this is in agreement with the histological findings where destroyed epithelial cells were observed; thus digestion and absorption of the food may be inhibited (Schmidt et al., 1997). This effect was also observed on Schistocerca gregaria and Locusta migratoria after treatments with aza (Mordue and Blackwell, 1993). The fact that larval and pupal intermediates were found, after treatments with methanolic green fruit extract of M. azedarach was due to an increase of juvenile hormone II, S. littoralis being more affected than A. ipsilon (Schmidt et al., 1998). In both insects, the content of haemolymph protein was diminished after treatments with 50 ppm of extract. Heliothis zea ðBoddieÞ. Extracts obtained by McMillian et al. (1969) were incorporated to a base of agar in order to feed larvae I of Heliothis zea (Lepidoptera: Noctuidae) The chloroformic extract at 3 mg equivalents/g of diet was responsible for smaller larvae. All larvae died when they were fed with 10 mg equivalents/g of diet (McMillian et al., 1969). Mythimna separata ðWalkerÞ. Most of the larvae I and II of M. separata did not develop into larvae V and some of them lost weight after treatment with 1% methanolic extract from seed kernels of M. azedarach collected in China. The larvae which reached the last instar were more resistant since the same extract at 2% exhibited only a 60% of feeding inhibition to the 5th and 6th instar although some of them lost weight and died (Chiu et al., 1984) suggesting the presence of toxic substances in M. azedarach extracts. Busseola fusca ðFullerÞ. The level of infection of the maize stalk borer Busseola fusca (Lepidoptero: Noctuidae) in field tests was significantly reduced after two applications of solutions obtained from a macerate in water of dried fruits of M. azedarach L. collected in Ethiopia (Gebre-Amlak and Azeferegne, 1999). An 8.6% of decrease in the number of plants with leaf infestation resulted in a 47.9% of increase in the crop yield. With the same application schedule, solutions of fresh and dried leaves and fruits produce significant reduction in the number of live larvae in each plant after 72 h of the first treatment. After 72 h from the second treatment, results were similar to those obtained with the use of Lamdacyhalotrin (positive control). However, the number of larvae recorded at harvest was not significantly different from the number of larvae obtained from the control, indicating that the protection of the Melia extract did not last for the entire period of growing (Gebre-Amlak and Azeferegne, 1999) in contrast with Lamdacyhalotrin which showed lower degradability. Tuta absoluta ðMeyrickÞ. Aqueous extracts from leaves, branches and unripe and ripe fruit of M. azedarach collected in Brazil were used for controlling the tomato
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pinworm T. absoluta (Lepidoptera: Gelechiidae) (Brunherotto and Vendramim, 2001). Newly emerged larvae of the insect were fed with tomato leaves (Lycopersicon esculentum) treated with extracts from different tree structures until adult emergence. Larval and pupal survival was affected with 0.1% leaf extract. Surviving larvae and pupae showed extended larval development. Male and female pupae weight was lower than the control group even at 0.1%. When the activity of leaf extract was compared to extracts from different structures of the tree, it was observed that the former was the most effective in delaying larval phase. All extracts produce similar larval and pupal mortality, being ripe fruit extract the less effective (Brunherotto and Vendramim, 2001). This similarity in the action is not frequent since, in accordance with what was previously stated by Rodriguez Hernandez and Vendramim (1996), effect on the plants is more drastic in larvae than in pupae because the former are the ones that ingest the treated food. The authors suggested that larvae treated with M. azedarach extracts were not capable of degradating, at least in part, the active principles present in the extracts also affecting the pupal development. This is not in accordance with the findings of Banchio et al. (2003) where larvae of L. huidobrensis treated with complete ripe fruit extract of M. azedarach showed highest mortality percentages in pupal stage. Plutella xylostella L. With the aim of finding an alternative for controlling the diamondback moth, P. xylostella, which has exhibited resistance to different synthetic insecticides (Mehrota, 1991) and even to Bacillus thuringiensis (Tabashnik et al., 1990), Dilawari et al. (1994) studied the effect of a methanolic extract from kernels of M. azedarach. The number of eggs measured when the inner surface of a recipient was sprayed with the crude extract was significantly reduced. The same tendency was also observed for emergency. When the larvae hatching from eggs treated with 10% solution were fed with fresh food, they suffered a higher larval and pupal mortality than controls. Up to 17.7% of the emerged adults suffered malformations (Dilawari et al., 1994). In another assay, chloroform and hexane fractions from kernel extracts of Melia in acetone were topically applied on larvae of P. xylostella at 0.1 mg/3.7 mg of larval weight resulting in 30% and 60% of mortality respectively (reference in Dilawari et al., 1994). In a similar way, the extract was applied on cauliflower cotyledons before placing moth adults on them. There was no effect on the number of laid eggs but hatching was 90% lower (reference in Dilawari et al., 1994). These results suggest that the extracts would also act by contact on P. xylostella eggs, in opposition to what was found by Breuer and Schmidt (1995) on S. frugiperda. With the aim of studying the effect on oviposition, extracts of fruits of M. azedarach were obtained by extraction with diethyl ether (Chen et al., 1996a). Choice and no choice tests were carried out with newly emerged and mated female P. xylostella placed together with rape seedlings (Brassica campestris L.) dipped in the extract solutions. In choice tests, oviposition in treated seedlings was reduced in 49.6–93.5% compared to control at 0.5–4%, while the reduction was lower in no choice test. The number of moths found on the treated seedlings was drastically reduced (Chen et al., 1996a). The effectiveness of the extract was maintained throughout the experiment, indicating that its activity lasts by at least 48 h under laboratory conditions. Besides, there were no signs of phytotoxicity (Chen et al., 1996a).
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When the above-mentioned extracts were applied by dipping plants of Brassica olereaceae L. (Chinese Kale), mortality values over 90% were obtained even when the larvae were treated in 1st, 2nd, 3rd or 4th instar. Some of them died before moulting, while the majority did it for the inability to eliminate the old skin. The diary consumption was significantly reduced at 2% and 4% extract concentration in treatments of all instars. Pupal weight, adult emergence and adult longevity decreased when the larvae were continuously reared with rape leaves treated with 0.05% solution or above. Egg hatchability was significantly reduced when the eggs were directly dipped in 1% extract concentration (Chen et al., 1996b). Studies carried out on two species of parasitoids attacking P. xylostella, Cotesia plutellae (Hymenoptera: Braconidae) and Diadromus collaris (Hymenoptera: Ichneumonidae) showed that aqueous leaf extracts from M. azedarach did not negatively affect the longevity of either parasitoids or the percentage of parasitism (Charleston et al., 2005). Thaumatopoea pityocampa ðDen: & Schiff:Þ. Larvae of the needle devouring caterpillar Thaumatopoea pityocampa (Lepidoptera: Thaumatepoeidae) were put on pine branches of Pinus mugo Turra treated with 1% and 10% of an extract obtained by grounding in methanol ripe fruits of M. azedarach collected in Greece (Breuer and Devkota, 1990). Larvae fed on treated twigs consumed less food than controls and produced very low quantity of faeces. Mortality increased up to 100% depending on the concentration and stage of the larvae. The early death of the caterpillar might be due to the intake of toxic substances, deduced by typical symptoms of poisoning and the lethargic behaviour, or due to the diminished food ingestion (Breuer and Devkota, 1990). The insects on treated branches neither gathered nor built their nest as the control animals did, which could be associated to the repellent effect. As expected, young stage larvae are more sensitive to M. azedarach extract. This monophagous insect seems to be quite sensitive to paraı´ so extract as compared with other poliphagous Lepidoptera such as S. frugiperda or A. ipsilon, perhaps because the later possess a broad spectrum of detoxification mechanism due to its larger nutritional spectrum (Breuer and Devkota, 1990). Field trials on T. pityocampa were undertaken in a pine forest (Breuer and Devkota, 1990) in Greece. In the night of spraying the trees with M. azedarach methanolic ripe fruit extract, the insects rejected feeding on the pine needles. In the case of partially sprayed trees, only the untreated needles were consumed. In the following nights, the caterpillars did not come out of the nests sprayed with 10% Melia extract. A number of T. pityocampa could be seen lethargic or dead hanging on twigs. Inside the nests the larvae were found either dead or lethargic (Breuer and Devkota, 1990). Natural enemies such as the wasp Erigorgus femorata Aubert (Hymenoptera: Ichneumonidae) and the flies Phryxe caudata Rondani and Phorocera grandis Rondani (both Diptera: Tachinidae) were alive and trying to parasitize the lethargic caterpillars (Breuer and Devkota, 1990). These results indicate that M. azedarach extract is highly specific on pest insects. Thaumatopoea processionea L. Thaumatopoea processionea is a Lepidoptera of the Thaumetopoeidae family, whose larvae completely defoliate the oak trees (Breuer
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and De Loof, 1998). Unripe fruits of paraı´ so collected in Greece were extracted with methanol defatted with petroleum ether, and partitioned in water/ethyl acetate. The organic fraction was applied on oak twigs (Quercus robur L.) where larvae of 2nd and 4th instar of T. processionea were placed. In 2nd instar, the faecal production was decreased indicating a strong antifeedant effect of the botanical extracts on the insect. Surviving treated larvae weighed the same throughout the experience (Breuer and De Loof, 1998). Those larvae which have ingested treated twigs were inactive and from day 8 after beginning of experience, the mortality was complete in the concentration of 0.1% or above. Larvae IV fed with twigs sprayed with Melia at 1% stopped its feeding and excretion, and body weight suffered a decrease. Contrary to the effect observed with larvae II, in larvae IV there would be a more important antifeedant effect which masks a possible toxic effect. While control larvae aggregated in one place, the treated young insects separated from each other. The treated larvae were inactive, finally dying at 1% concentration (Breuer and De Loof, 1998). Death was related to problems in moulting probably by the influence of the active principles in the hormonal system and due to the reduced ingestion. The insects could not remove the old skin and were often ‘caged’ in their exuviae. Younger larvae reacted more sensitively to the extracts than the older ones (Breuer and De Loof, 1998). The same organic fraction was tested against T. processionea on Q. robur plantations sprayed with 1% extract (Breuer and De Loof, 2000b). One week after the treatment, the consumed areas in the trees sprayed with the extract were small or almost in traces. In the control trees the caterpillars gathered in groups, while in the treated trees, the insects were often found distributed all over the oak and the larvae did not construct their nest. The insects in the treated trees appeared inactive, and only moved if touched, in coincidence with those previously found in the laboratory assays (Breuer and De Loof, 1998). Two weeks after the beginning, only 10–40% of the caterpillars survived and at 4 weeks, 100% of mortality was registered. The mortality was mainly due to problems during ecdysis. It was also observed that spiders, carabids, scorpionflies and tachinids were fed on weak or death larvae in the treated trees showing that these natural predators were not affected by the extracts (Breuer and De Loof, 2000b). Phthorimaea operculla Zeller. Kroschell and Koch (1996) studied the effect on Phthorimaea operculla (Lepidoptera: Gelechiidae) of aqueous seed extract of M. azedarach at 20% applied on potatoes. The repeated treatment with extract resulted in a lower larval development towards the adult stage when the potatoes were treated before infecting with the eggs of the moths. It was observed that in experiments made with potatoes placed in storage during 3 months and treated before releasing the adults, extract of M. azedarach reduced the frequency of infection (number of infected tubers) in 35.2% compared to controls. The number of points of infection per tuber (intensity of infection) was decreased in treatments with Melia extract (Kroschell and Koch, 1996). Sesamia nonagrioides Lef e bre. Seeds of M. azedarach L. collected in Spain were first extracted with methanol and then defatted with hexane. The resulting extract and fractions obtained from it through the use of preparative HPLC were studied against one of the most important pests of the corn in the Mediterranean area, the
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Mediterranean corn borer S. nonagrioides (Riba et al., 1996). The assays were made with 2nd instar larvae fed either on corn leaves dipped in aqueous solution of the extract at 10 ppb and then transferred to untreated food or with an artificial diet mixed with extract at 450 ppm (prepared with 0.16 g of extract). The isolated fractions were also added to an artificial diet in a concentration equivalent to process 0.16 g of extract. Azadirachtin was used for comparison purposes. While control larvae increase their body weight, those larvae confronted to the extract or to aza maintained the same weight throughout the study (Riba et al., 1996). At the end of the experience, some of the treated larvae increased their weight and then pupating, contrary to those confronted to aza which died at 42 days. Mortality produced by the Melia extract until they reached the pupal stage was 77% while in aza treatments it was 100%, taking into account the very low concentration of M. azedarach extract. While the larvae were fed with the Melia extract, a mortality percentage of 65% was observed whereas the mortality percentage with aza corresponded to 5%; however, this last compound showed a staggering mortality even during or after the treatment until reaching a 100% (Riba et al., 1996). These results are in concordance with those found by Carpinella et al. (2003a) between compound 1 and aza where the first compound acted earlier than the second one. In the artificial diet assay the mortality of the corn borer fed with the M. azedarach extract till pupation was 86% and a delay in development was observed. Larval development was prolonged after treatments with Fr. 2 and Fr. 3 obtained from methanolic seed extract from M. azedarach by HPLC (Riba et al., 1996). The growth speed for larvae confronted to diet with extract or Fr. 2 was significantly different than in the controls, while treatment with Fr. 3 exhibited an intermediate behaviour (Riba et al., 1996). These results showed that the compound present in Fr. 2 was more potent than the one found in Fr. 3. The activity of methanolic extracts from fruits and seeds of M. azedarach collected in Spain at 1000 and 2000 ppm was compared with aza and a commercial product of neem called Mubel, which contained 1.5 ppm of aza using an artificial diet. At 30 days after the ending of the study, only aza, Mubel and M. azedarach seed extracts showed significant differences in weight. Surviving larvae that had recovered at this time pupated with similar weights than the control but with longer development time (Joan Serra et al., 1998; Juan et al., 2000). The difference in the results between both Melia extracts indicates that the active principles of the tree are concentrated in the seed. These differences were also observed in S. littoralis and A. ipsilon (Schmidt et al., 1997). Mubel and seed extract at 2000 ppm showed values of ingest 10 times lower than the ones of the control. The seed extract, Mubel and aza showed high levels of deterrency, being the first two the most effective. This suggests that in Mubel there are other compounds besides aza that increase the activity of the product (Juan et al., 2000). This fact is one of the reasons why the use of complete extracts is recommended over the use of pure compounds. Mortality was complete only in case of Mubel at 75 ppm, while in other assays it corresponded to 6–27%. Order Hemiptera Triatoma infestans Klug. Experiments oriented to study the repellence of ethanolic ripe and unripe fruit and green leaf extracts of M. azedarach collected in Argentina
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were carried out with the hematophagous insect Triatoma infestans (Hemiptera: Reduviidae) or ‘vinchuca’, the vector of Chagas disease (Valladares et al., 1999). For this purpose, treated filter paper refuges were used and the distribution of the insects on them was assessed. The unripe fruit extract was the most effective exhibiting 93% and 80% repellency index on 1st and 4th instar nymphs respectively at environ 11% extract concentration. These results coincide with those reported by Arias and Schmeda Hirschmann (1988) who found high repellency from unripe fruit extract and oil from M. azedarach of Paraguay at 0.5% in contact with 4th instar nymphs of T. infestans. Ovicidal effects measured on eggs sprayed with unripe fruit extract were not observed, nor were alterations in mortality or moulting when placing 1st instar nymphs in contact with treated refuges (Valladares et al., 1999). However, nymphs were smaller and lighter when they were reared on the extract treated refuges for one intermoulting period suggesting that a stronger effect could be observed if the study was carried out along the insect life cycle. Arias and Schmeda Hirschmann (1988) did not find insecticide or moulting activity by contact, but a slight increase in mortality was observed when 4th instar nymphs were topically treated with unripe fruit oil. The lack of effectiveness of the extract by contact could be due to the extract not being ingested, since when feeding 4th instar nymph of Rhodnius prolixus with the lignan pinoresinol, isolated from seeds of M. azedarach collected in Brazil (Cabral et al., 1995) at 100 mg/ ml, a 58% inhibition in the ecdysis of the nymph was observed (Cabral et al., 2000). Pinoresinol was also obtained from ripe fruit of Argentinian M. azedarach (Carpinella et al., 2003b). This compound also produced a high antifeedant effect on R. prolixus instar IV when it was ingested at 500 mg/ml of feeding blood (Cabral et al., 1999). When the compound was applied on the surface of a Petri dish, a low mortality was detected at the highest studied concentration: 100 mg/cm2, with no significant effects on ecdysis (Cabral et al., 1999), but a prolonged intermoulting period was observed in 4th instar larvae of the insect (Cabral et al., 2000). The highest negative effects on pest insects were observed again when extracts or pure compounds were ingested instead of applying them topically. Diaphorina citri Kuwayama. Assays made by spraying the Asiatic citrus psyllid, Diaphorina citri (Hemiptera: Psyllidae), with seed oil from M. azedarach at 2% even after a rain, showed a repellency of 69.7% at 5 days after treatments. Preliminary experiments carried out in large cages showed that the incidence of the yellow shoot disease of citrus of South China, a disease transmitted by the psyllid, was significantly reduced by nearly 70% in young trees sprayed with the previously mentioned emulsion (Chiu et al., 1984). Nilapavarta lugens Stal; Sogotella furcifera ðHorvathÞ; Nephotettix virescens ðDistantÞ. The quantity of food ingested by newly emerged females of N. lugens (Hemiptera: Delphacidae), Sogotella furcifera (Hemiptera: Delphacidae) and Nephotettix virescens (Hemiptera: Cicadellidae) was significantly reduced when they were fed with seed oils of neem and methanolic extract of M. azedarach L., being Melia oil relatively more effective than neem oil (Saxena et al., 1983). When neem oil and Melia extracts were topically applied on S. furcifera, an almost complete mortality was reached at 50 mg/female. Neem oil was more effective on N. lugens than Melia extract. Neither neem oil nor Melia extract was active against
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N. virescens up to 50 mg/female. Even at this last concentration the extracts were slightly toxic on the predator Cyrtorhinus lividipennis (Hemiptera: Miridae) and on the predatory mirid bug Lycosa pseudoannulata (Araneae: Lycosidae). These results indicate that the active principles present in these extracts are rather pest specific (Saxena et al., 1983). Schizaphis graminum ðRondaniÞ. In order to study the systemic effect of methanolic extract obtained from seed kernel of M. azedarach collected in USA or in China, the potting soil of sorghum plants was drenched with emulsions of the extract (Hu et al., 1998). The plants were infected with adults of Schizaphis graminum (Hemiptera: Aphididae). With the addition of 150 mg of Melia extract, the population was slightly reduced (Hu et al., 1998). According to this experience it could not be determined if the poor effect of the M. azedarach extract was due to the lack of systemic translocation or if this extract was not toxic to the insect (Hu et al., 1998). Order Orthoptera Locusta migratoria migratorioides ðReiche & FairmaireÞ. Aqueous, methanolic and petroleum ether extracts of fruits and leaves of M. azedarach collected in Dominican Republic and Germany respectively were assayed in the laboratory against L. migratoria migratorioides (Orthoptera: Acrididae) (Wen and Schmutterer, 1991). Treatments of 1st instar nymphs with aqueous fruit extract at 3% showed 93% of cumulative mortality and when most nymphs were still in instar II, control insects had reached the third stage. Aqueous extract of leaves at 3% applied on 1st instar nymphs and food resulted in a total mortality, while less than 60% mortality was observed when the same concentration was applied only to nymphs. The effect produced by the ingestion was stronger than by contact, this is expected for any substance due to the barrier imposed by the cuticle. Nymphs fed on treated food ate only once and then they rejected the food, even the untreated one. When 1st instar nymphs were treated, lower weights and a delay in reaching the following instar were observed. Treatment with aqueous extract of leaves on nymph III exhibited 93.2% mortality at 3% concentration and a reduction of feeding activity. When nymph IV was treated with methanolic fruit extract at 5% only 23.5% of mortality was observed, increasing to 64.7% when the same concentration was applied to nymphs and food. The weight of treated nymphs was reduced. With the petroleum ether phase, there were no different results to control, suggesting that the activity is due to the presence of polar substances (Wen and Schmutterer, 1991). Schistocerca gregaria ðForsk:Þ. Cabbage leaves were sprayed with different concentrations of an ethyl acetate extract obtained from a methanolic extract from unripe fruits of paraı´ so collected in Greece and confronted with control leaves to 2nd instar nymphs of S. gregaria (Orthoptera: Acrididae) (Breuer and De Loof, 2000a). The development of the larvae was only possible in treatments with extract concentrations below 0.001%. At higher concentrations a strong antifeedant effect was observed. A complete mortality was obtained even at 0.01%. The weight of surviving nymphs treated with 0.01% was threefold lower than the control group (Breuer and De Loof, 2000b).
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The effect of a methanolic extract from fruits of paraı´ so was studied by applying them at 20 and 50 mg/ml on wheat seedlings (Schmidt and Assembe-Tsoungui, 2002). Oral ingestion of the extract reduced the feeding in 3rd instar as in adults of the locust. The larvae showed a decrease in the length of hind femora and elytra and width of head as well as in weight gain. The development of the larvae was prolonged and the moulting process was altered. The oviposition was strongly reduced and the maturation of oocytes was retarded only reaching the stage of previtellogenesis. The normal yellow colour of the males was affected as a step to gregarization (Schmidt and Assembe-Tsoungui, 2002). Order Homoptera Bemisia tabaci ðGenn:Þ. Tests under screen-house conditions were carried out with aqueous extracts of equal parts of leaves and ripe fruits of M. azedarach from Brazil on the vector of the bean golden mosaic virus, Bemisia tabaci (Homoptera: Aleyrodidae) (Nardo et al., 1997). With the aim of measuring toxic effects, adults of the insect were placed together with two plants of P. vulgaris L. cv. carioca sprayed with the extract. A percentage of mortality of 70%, 90% and 100% at 48, 72 and 96 h, respectively, was observed with no significant differences with respect to starved adults (Nardo et al., 1997). These results suggest an antifeedant action of the extract instead of a toxic effect (Nardo et al., 1997). To evaluate the effect on oviposition, adults fed with soybean were confronted to treated or untreated bean plants. The number of pupae found in sprayed plants was half than in water treated plants. However, the extract did not affect the development. On this basis the authors suggest that the extract would affect the number of eggs laid probably due to the antifeedant action and this would be explained because the insect laid the egg during feeding (Gamell, 1974). Aqueous extracts of fresh fruits and leaves of M. azedarach from Beirut were applied at 40% concentration on a single bean leaf. Whole fruits were also extracted with water and methanol and diluted to 20% (higher concentrations of the extracts caused phytotoxicity on tomato leaf). Other fruit extracts were obtained extracting the vegetal material with acetone or methanol after defatting with petroleum ether. The dried extracts dissolved in distilled water were applied on tomato plants (AbouFakhr Hammad et al., 2000a). The fruit and leaf aqueous, methanolic and acetone extracts showed a lower number of B. tabaci adults on treated plants (Abou-Fakhr Hammad et al., 2000a). Mortality of about 23–49% was observed on 1st and 2nd instar of the whiteflies with the use of 20% extracts, the ether and water extracts being the less effective. The highest concentration of methanol extract caused the highest pupal mortality (24%) comparable to Azatin (25%) and water plus Triton 0.2% (23%). These studies indicated that earlier instar was more susceptible to M. azedarch extracts than pupal stage (Abou-Fakhr Hammad et al., 2000a). This could be attributed to the fact that late stages are highly protected by fat bodies (Schlu¨ter, 1995) that may either detoxify or sequester toxins. Extracts of fruit and leaves of M. azedarach collected in Beirut (AbouFakhr Hammad et al., 2001) and from callus obtained from leaf branches of the mentioned tree, even fresh or frozen, were extracted with methanol or water. The resulting extracts (at 20%) were applied to tomato plants (L. esculentum)
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under glasshouse compartment together with adult whiteflies of B. tabaci in a no choice test. Either methanolic or aqueous extracts of leaves, fruits and callus, fresh or frozen, developed significant repellent activity (58–68%) (Abou-Fakhr Hammad et al., 2001). In accordance with the author there would not be differences in using methanol or water as extractive solvents of the fresh or frozen vegetal material. It was found that the mortality of adults confronted to sprayed plants was not significantly different from that of unfed adults, but in both cases it was higher than in the controls. This fact suggests that a possible antifeedant effect would be responsible for death. At the end of the assay, surviving adults in treatments with methanolic and aqueous extracts were transferred to untreated plants. In 1 week the number of eggs laid in treatments was 19.1–22.1 eggs/adult, a value significantly different from the one in controls: 41 eggs/adult. The percentage of emergence was not affected (AbouFakhr Hammad et al., 2001). Order Thysanoptera Thrips palmi Karny. The effectiveness of an extract from seeds of M. azedarach was compared with a sunflower oil and an agricultural oil used at 2% against Thrips palmi (Thysanoptera: Thripidae), a pest that affected the eggplant (Solanum melongena) (Pin˜o´n et al., 1999). With non-conventional products the population of the insects was reduced with higher plant yields registered with M. azedarach extract and sunflower oil. On the other hand, in these plots a high presence of natural enemies of the pest was observed. Order Acari Boophilus microplus ðCanestriniÞ. The efficacy of ripe fruit of M. azedarach L. obtained in Brazil was evaluated against the thick, Boophilus microplus (Acari: Ixodidae), a parasite with economic importance in cattle, dogs and horses (Borges et al., 2003). The fruit extracts were extracted with hexane, chloroform and ethanol and applied on filter papers, which remain in contact with the larvae for 10 min.All the studied extracts were toxic against the larvae, the chloroform extract being the most potent exhibiting 100% of mortality, followed by the hexanic (97%) and the ethanolic one (50%) at a concentration of 0.125%. Hexanic and chloroformic extracts showed more acaricide effectiveness than the ethanolic extract against engorged females of the thick immersed in the solutions of extracts. Egg production and eclosion were inhibited with the hexanic and chloroformic extract and in a lower degree with the ethanolic one (Borges et al., 2003). The same tendency of activity was observed by McMillian et al. (1969) on S. frugiperda. Panonychus citri ðMc GregorÞ. On spraying fields in China with an emulsion of seed oil of M. azedarach at 0.25% a reduction of 85.6% was observed in the population of the citrus red mite Panonychus citri (Acari: Tetranychidae), 1 day posttreatment (Chiu et al., 1984). The effectiveness was comparable to the treatment with Amitraz. A second application on the same trees produced a 98.8% reduction either with the vegetal extract or with Amitraz (Chiu et al., 1984). A spray of seeds oil at 0.5% on citrus trees proved to be comparatively safe to the predator thick Amblysius newsami (Evans), which controlled the citrus red mite, P. citri (Chiu et al., 1984).
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Conclusion and future prospects Today, there is an urgent need of finding new ‘weapons’ for controlling insect attack on plants which are the basis of our food in a direct or indirect way. Extracts obtained with solvents of different polarities from different structures of M. azedarach tree produced a decrease in insects and acari food consumption. This fall in feeding resulted in a diminished weight of the insect and posterior death, even when in some cases this last effect was due to the consumption of toxic compounds. Longer larval and pupal phases were observed after feeding insects with Melia extracts. Alterations in moulting, fecundity and fertility were often observed. These effects resulted in a decrease in the number of pest individuals and therefore a decrease in the damage of crops. According to the above-mentioned results, the extracts and pure compounds isolated from the paraı´ so tree, M. azedarach, could be used for controlling insects of agronomic importance showing effectiveness, availability and seemingly no adverse effects on human and animal health (Carpinella et al., 1999b) or on the environment. The high availability of the renewable resources of the tree such as leaves and fruits as well as their high specificity and potency as antifeedants make it possible to use this natural product for the control of insects which negatively affect plants. In this way, high yields in crops could be obtained guaranteeing a sustainable agriculture.
Acknowledgments This work was supported by FONCyT. MCC gratefully acknowledges receipt of a fellowship from CONICET.
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CHAPTER 6
Bioactivity of fabaceous plants against foodborne and plant pathogens: potentials and limitations DEEPAK ACHARYA, ANIKET GADE, MAHENDRA RAI
Introduction There are various plant diseases caused by pathogenic microbes, which are responsible for reduction in production. The continuous and irrational use of chemicals has resulted into several problems like residues in edible plant parts, resistant strains, and environmental pollution, etc. In the present day agriculture, a lot of emphasis is being given on using eco-friendly means for controlling diseases and pest problems affecting various crops (Thind et al., 2005). There is general growing trend among consumers for more natural rather than synthetic products in a whole range of industries, including food and drink, cosmetic, agricultural, and pharmaceuticals (Bauer et al., 1997; Svoboda and Deans, 1998; Glaser, 1999; Traffic International, 1999; Walton and Brown, 1999; Bansal and Gupta, 2000; Singh et al., 2000; Kamanzi Atindehou, 2002). The use of botanicals for the management of the phytopathogens is gaining ground. Plant extracts and volatile oils may have an important role to play in the preservation of foodstuffs against fungi, in fungicidal application against plant diseases, and in the fight against various human fungal infections. Recent literature has shown the biological activities of plant-extracts, essential oils and their individual pure components, and has documented the inhibitory activity of these substances against the growth of various fungi (Maruzzella and Balter, 1959; Maruzzella and Logeuri, 1959; Maruzzella et al., 1959; Maruzzella et al., 1960; Maruzzella, 1963; Sarvamangala et al., 1993; Awuah, 1994; Bhat et al., 1994; Mwosu and Okafor, 1995; Arellanes et al., 1996; Ezer and Abbasoglu, 1996; Pattnaik et al., 1996; Arras et al., 1997; Singh and Handique, 1997; Dwivedi and Singh, 1999; Abraham and Prakasam, 2000; Jantova et al., 2000; Rao et al., 2000; Rawal and Thakore, 2000; Tsao and Zhou, 2000; Wang and Ng, 2000; Agarwal et al., 2001; Agnese et al., 2001; Alvarez-Castellans et al., 2001; Ye et al., 2001; Abou-Jawdah et al., 2002; Locke, 2002; Pistelli et al., 2002; Muhammad et al., 2003; Rai and Mares, 2003; Ezra et al., 2004; Fukai et al., 2004; Kelemu et al., 2004; Kim and Fung, 2004; Kim et al., 2004; Kone et al., 2004; Randhir et al., 2004; Sato et al., 2004; Usui et al., 2004; Xie et al., 2004; Lee et al., 2005; Romagnoli et al., 2005; Talas-Ogras et al., 2005).
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The family Fabaceae is the third largest family of flowering plants, with approximately 650 genera and nearly 20,000 species (Doyle, 1994). Leguminous plant natural products have been important sources and models of forage, gums, insecticides, fungicides, phytochemicals, and other industrial, medicinal, and agricultural raw materials. Since most of these legume species have not been screened for chemical or biologically active components, it is logical to expect that new sources of valuable substances remain to be discovered (Morris, 1999). The present chapter focuses mainly on antimicrobial potential of plants of family Fabaceae, their bioactive compounds and role in sustainable plant disease management. In fact, a little work has been done in the field of antimicrobial potential of plants of Fabaceae against phytopathogens.
Antimicrobial activities of some important fabaceous plants A review of literature reveals that a significant contribution has been made regarding the antimicrobial potential of family Fabaceae (Mahajan, 1971; Umalkar et al., 1976; Allen and Allen, 1981; Pandey et al., 1982; Lal et al., 1998; Samy, 2000). Antimicrobial activities of some plants are as follows: Abrus precatorius L: A woody twinning plant of the Leguminosae family with characteristic red and black seeds. The leaves are pinnate and glabrous, with many leaflets arranged in pairs. The plant bears orange-pink flowers. The plant produces short and stout brownish pods, which curl back on opening to reveal pendulous red and black seeds, four to six peas in a pod. It grows wild in thickets, farms and secondary clearings, and sometime in hedges. It is most common in rather dry areas at low elevation throughout the tropics and subtropics. The chemical constituents of the plant include: arachidic acid, docosadienoic acid, docosatetraenoic acid, docosatrienoic acid, docosenoic acid, eicosadienoic acid, eicosanoic acid, eicosatrienoic acid, hexadecenoic acid, lauric acid, lignoceric acid, linoleic acid, linolenic acid, myristic acid, octadecadienoic acid, octadecatrienoic acid, oleic acid, palmitic acid, pentadecanoic acid. Antimicrobial activity Dhawan et al. (1977) reported that ethanol/water (1:1) extract of the aerial parts at the concentration of 25.0 mg/ml on agar plate was inactive on Bacillus subtilis, Escherichia coli, Salmonella typhosa, Staphylococcus aureus, Agrobacterium tumefaciens, Microsporum canis, Trichophyton mentagrophytes, and Aspergillus niger. Ether extract of seeds on agar plate demonstrated activity against Staphylococcus aureus (Desai and Sirsi, 1966). The ethanol (95%) extract was active on Escherichia coli and Staphylococcus aureus. Cassia alata L: Ornamental shrub with large pinnate leaves. Flowers are bright yellow in spikes. These are called as ‘‘Roman candle tree’’. The fruit is a legume with two prominent,
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wavy wings extending along its full length. It grows in wet habitats from sea level to 250 m but also cultivated in gardens. The main chemical constituent of this plant is tannin. Antimicrobial activity Dhawan et al. (1977) reported that ethanol/water (1:1) extract of the aerial parts, at a concentration of 25.0 mcg/ml on agar plate was inactive on Bacillus subtilis, Escherichia coli, Salmonella typhosa, Staphylococcus aureus, Agrobacterium tumefaciens, Microsporum canis, Trichophyton mentagrophytes, and Aspergillus niger. Cassia alata is known for antibacterial and antifungal activities (Fuzellier, 1982; Ibrahim, 1995; Selvamani and Latha, 2004). Ogunti et al. (1991) evaluated chloroform extract of dried leaves at a concentration of 5.0 mcg/ml on agar plate and found that it was active on Pseudomonas aeruginosa, Bacillus subtilis, Escherichia coli, Micrococcus luteus, and Staphylococcus aureus. But the extract showed weak activities on Aspergillus fumigatus, Botrydiplodia theobromae, Penicillium italicum, and Trichophyton mentagrophytes. Palacinchamy et al. (1991) reported that ethanol (85%) extract of dried leaves at a concentration of 10.0% on agar plate was active on Escherichia coli, Proteus vulgaris, Pseudomonas aeruginosa, and Staphylococcus aureus. Ibrahim and Osman (1995) found that ethanol (95%) extracts of dried leaves at a concentration of 500 mg/ml on agar plate was active on Microsporum canis, M. gypseum, Trichophyton mentagrophytes, and T. rubrum; weakly active on Aspergillus niger, Cladosporium werneckii, Fusarium solani, and Penicillium species; inactive on Candida albicans, Rhodotorula rubra, and Saccharomyces cerevisiae. Cassia occidentalis L: It is found in many tropical areas of South America, including the Amazon. Indigenous to Brazil, it is also found in warmer climates and tropical areas of South, Central, and North America. The oil of C. occidentalis contains lignoceric acid, linoleic acid, linolenic acid, and oleic acid. Antimicrobial activity Samy (2000) reported that extract of C. occidentalis leaves showed activity against the Gram-negative and Gram-positive bacteria. C. occidentalis leaves and seeds extract varies in activity against pathogenic fungi (Caceres et al., 1991). Cassia tora L: In Sanskrita, it is called as ‘‘Chakramard’’ meaning there by killer of ringworm. It is known as one of the well known fungal agents in Ayurveda. C. tora is an annual indigenous herb. It has yellow flowers. The leaves are applied to ulcers and itching eruptions. The powdered seeds are recommended to keep in water overnight and the ointment formed is suggested to apply on scabies and ringworm infections. The root is also used topically in ringworm (Chakraberty, 1983). Extracts of C. tora was also found to be active against plant pathogenic fungi (Damayanti et al., 1996; Kim et al., 2004).
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Lopez Abraham et al. (1981) reported that acetone and water extracts of dried stem at a concentration of 50% were inactive and ethanol (95%) extract at a concentration of 50% on agar plate demonstrated activity on Neurospora crassa. Ray and Majumdar (1976) reported that ethanol (95%) extract of fruits on agar plate was active on Trichophyton mentagrophytes and T. rubrum. Caceres et al. (1991) found that hot water extract of dried fruit pulp at a concentration of 1.0 ml in broth culture was active on Microsporum canis, Epidermophyton floccosum, and Trichophyton mentagrophytes var. algodonosa. Singh and Pathak (1984) reported that water extract of fresh leaves on agar plate produced strong activity on Ustilago nuda. Kim et al. (2004) reported fungicidal activities of Cassia tora extracts and their active principles against Botrytis cineria, Erysiphe graminis, Phytophthora infestans, Puccinia recondita, Pyricularia grisea, and Rhizoctonia solani using a whole plant method in vivo. At 1 g/L, the chloroform fraction of C. tora showed a strong fungicidal activity against B. cinerea, E. graminis, P. infestans, and R. solani. Emodin, physcion, and rhein were isolated from the chloroform fraction using chromatographic techniques which showed strong and moderate fungicidal activities against B. cinerea, E. graminis, P. infestans, and R. solani. Moreover, aloe-emodin showed strong and moderate fungicidal activities against B. cinerea and R. solani, respectively, but did not inhibit the growth of E. graminis, P. infestans, P. recondita, and Py. grisea. Mucuna pruriens L: A vine of the Papilionaceae family. The seeds of Mucuna pruriens are black in color with pale-brown specks, uniform in shape. The seed coat is hard, thick, and glossy. It originated in India, and is now commonly found throughout the tropics. The chemical constituents include threo-12-octadec-trans-9-enoic acid, threo-12-1, octadec-cis-9-enoic acid, vernolic acid 4.0%, in seed oil. Antimicrobial activity Dhar (1968) evaluated some Indian plants for biological activity and reported that Mucuna pruriens showed the maximum inhibition against pathogenic fungi and bacteria. Pongamia pinnata ðL:Þ Pierre The tree is smooth throughout, reaches a height of 8–25 m. Leaves imparipinnate, shiny; young leaves pinkish-red, mature leaves glossy, deep green; leaflets 5–-9, the terminal leaflet larger than the others. Flowers fragrant, white to pinkish, paired along rachis in axillary, pendent, long racemes or panicles. Calyx campanulate or cup-shaped. Pod short stalked, oblique-oblong, flat, smooth, thickly leathery to sub woody, indehiscent, 1-seeded; seed thick, reniform (Allen and Allen, 1981). P. pinnata is commonly found throughout the Philippines along the seashore. In some localities it extends inland near the borders of the lakes. It also occurs in the Mascarene Islands, and in tropical Asia, across Malaya to Australia. The chemical constituents include – palmitic 3.7%–7.9%, stearic 2.4%–8.9%, arachidic 2.2%–4.7%, behenic 4.2%–5.3%, lignoceric 1.1%–3.5%, oleic 44.5%–71.3%, linoleic 10.8%–18.3%, and eicosenoic 9.5%–12.4%.
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Antimicrobial activity The seed-oil of P. pinnata is generally used in eczema (Sebastian and Bhandari, 1984). Chaurasia and Jain (1978) reported that Pongam oil, a bitter, red brown, thick, nondrying, non-edible oil, 27%–36% by weight, which is used for lubrication and indigenous medicine. Pongam oil showed inhibitory effect on broad-range of microbes, viz., Bacillus anthracis, B. mycoides, B. pulilus, Escherichia coli, Pseudomonas mangiferae, Salmonella typhi, Sarcina lutea, Staphylococcus albus, S. aureus, and Xanthomonas compestris, but did not inhibit Shigella sp. Flavonoids present in the extracts of Pongamia glabra were found responsible for the inhibition of spore germination and mycelial growth of Alternaria solani, Macrophomina phaseolina, and Fusarium udum (Pan et al., 1985). Simin et al. (2002) found that pongarotene, a new rotenoid and karanjin, a known flavonol were isolated from the methanolic extracts of the seeds of P. pinnata demonstrated antifungal activity. Tamarindus indica L: A large tree up to 30 m high, having spreading branches; bark brownish grey, flaked. Leaves are even-pinnate consisting 10–18 pairs of small leaflets, rather closed together, seeds are glossy, dark-brown, embedded in a thick, sticky, and acid-brown envelope. It is cultivated for the edible fruits and as ornamental and shed tree. It contains tannin 7%, arachidic acid, behenic acid, lauric acid, lignoceric acid, linoleic acid, 2.7%–3.4%, linolenic acid, stearic acid (SD OL), geranial, geraniol (EO). Antimicrobial activity Ross et al. (1980) reported that the ethanol (70%) extract was active against several fungi and bacteria, viz., Bacillus cereus, Escherichia coli, Pseudomonas aeruginosa, Staphylococcus albus, and S. aureus. Ethanol 95% extract of fruit on agar plate was active on Escherichia coli and inactive on Staphylococcus aureas. Laurens et al. (1985) found that methanol extract of dried stem-bark at a concentration of 10.0 mg/ ml was active on several Gram-negative organisms and inactive on several Grampositive organisms. A concentration 15.0 mg/ml was active on Sarcina lutea. Trigonella foenum-graecum L: Fenugreek is an erect annual herb, growing about 2 feet high, similar in habit to Lucerne. The seeds are brownish, about 1/8 inch long, oblong, rhomboidal, with a deep furrow dividing them into two unequal lobes, ten to twenty together, in long, narrow, sickle-like pods. Indigenous to the countries on the eastern shores of the Mediterranean. Cultivated in India, Africa, Egypt, Morocco, and occasionally in England. Abdo and Al-Kafawi (1969) reported that oil contains 3-hydroxy-4, 5-dimethyl-2furanene, dihydrobenzofuran, dihydroactinidiolide, muurolene, elemene, selinene. Saponins, based mainly on the sapogenins diosgenin and its isomer yamogenin, gitogenin, and tigogenin. Antimicrobial activity Khanna et al. (1971) demonstrated that Trigonella foenum-graecum can inhibit Candida albicans. Omolosa and Vagi (2001) reported that T. foenum-graecum was
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screened against 26-pathogens and all exhibited broad-spectrum antibacterial activity. Methanol extracts of seeds showed activity against Staphylococcus aureus, Bacillus anthracis, B. mycoides, B. pulilus, Escherichia coli, Pseudomonas mangiferae, Salmonella typhi, Sarcina lutea, and Staphylococcus albus. Bhatti et al. (1996) noted that hexane, chloroform, ethanol, and aqueous extracts of the seeds of T. foenum-graecum showed significant activity. The aqueous and ethanol extracts showed significant antibacterial activity against nine pathogenic bacteria at 50 and 100 mg/ml concentration.
Antimicrobials for management of phytopathogens There are many reports which vouch the antimicrobial activities of fabaceous plants. Maruzzella and Liquroi (1958) and Maruzzella et al. (1960) reported the toxicity of the oil of Cassia sp. against Lentinus lepideus, Lenzites trabea, and Polyporus versicolor. Growth of Aspergillus flavus and Trichoderma viride was inhibited by embryos of green peanut seeds (Lindsey and Turner, 1975). Antimicrobial activity against Agrobacterium tumefaciens and Aspergillus niger was shown by the ethanolic extract of Crotalaria verrucosa (Dhawan et al., 1977). The toxicity of the oil of Caesalpinia sappan was effective against Aspergillus nidulans and methicillin-resistant Staphylococcus aureus (Yadav et al., 1978; Kim et al., 2004b). Jain et al. (1979) reported antimicrobial activity of leaf oil of Dalbergia sissoo against Alternaria solani, Aspergillus flavus, A. fumigatus, A. nidulans, A. niger, Cladosporium herbarum, Cunninghmella sp., Fusarium monoliforme, Helminthosporium sp., Penicillium digitatum, P. italicum, Rhizopus nigricans, and Trichothecium roseum. Grover and Rao (1979) and Sharma and Singh (1979) noted toxicity of the oil of Psoralea corylifolia against Alternaria sp., Aspergillus candidus, A. flavus, A. fumigatus, A. nidulans, A. niger, Cladosporium herbarum, Cunninghmella echinulata, Fusarium monoliforme, F. oxysporum, Helminthosporium sacchari, Microsporum cocci, M. gypseum, Mucor mucedo, Penicillium digitatum, Rhizopus stolonifer, Trichophyton mentagrophytes, T. rubrum, and Trichothecium roseum. Acetone, benzene, and ethanol extracts of the groundnut were active against Alternaria alternata and Curvularia spp. (Kalaichelvan and Mahadevan, 1988). Porwal et al. (1988) reported that alcoholic extract of Butea monosperma demonstrated antifungal activity against plant pathogens, viz., Geotrichum candidum and Penicillium notatum. Methanolic and water extracts of the plant parts and cotyledons of peanuts showed efficacy against Aspergillus parasiticus (Azaizeh et al., 1990). The antifungal activity of the bean chitinase on R. solani was observed by Benhamou et al. (1993). Cajanas cajan was found to be effective against Fusarium oxysporum (Singh et al., 1994). Jurzysta and Waller (1996) reported the antifungal effect of alfalfa (Medicago sativa). Gao et al. (2000) demonstrated that the alfalfa antifungal peptide (alfAFP) defensin isolated from seeds of M. sativa displayed strong activity against the agronomically important fungal pathogen Verticillium dahliae. An antifungal protein designated sativin was isolated from the legumes of the sugar snap (also known as honey pea) Pisum sativum var. macrocarpon. Sativin exerted antifungal activity against Fusarium oxysporum, Coprinus comatus, and Pleurotus ostreatus (Ye et al., 2000). Saxena and Mathela (1996) isolated iridodial beta-monoenol acetate
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from the essential oil of Nepeta leucophylla, and actinidine from N. clarkei and screened for antifungal activities against Aspergillus flavus, A. ochraceus, Penicillium citrinum, and P. viridicatum, all known mycotoxin-producing taxa, and Sclerotium rolfsii and Macrophomina phaseolina, potential soybean pathogens. It was found that iridodial beta-monoenol acetate was most effective against S. rolfsii, while actinidine was highly active against M. phaseolina. Extracts of Gliricidia inhibited the germination of Drechslera oryzae by only 6%. However, in another study, 50 mg of stem chloroform extracts inhibited the growth of Cladosporium cucumerinum and slightly inhibited the growth of Candida albicans (Herath et al., 1998). Esimone et al. (1999) found antibacterial and antifungal activities of leaves of Indigofera dendroides. Fakhoury and Woloshuk (2001) isolated a 36-kDa alphaamylase inhibitor from Lablab purpureus (AILP). AILP inhibited the alpha-amylases from several fungi. The protein inhibited conidial germination and hyphal growth of Aspergillus flavus, which is a fungal pathogen of maize causing an important ear rot disease when plants are exposed to drought and heat stress. The antimicrobial and antifungal properties of several crude extracts and pure saponins, astraverrucins I–VI, from the aerial parts of Astragalus verrucosus were investigated by Pistelli et al. (2002). Simin et al. (2002) reported the antifungal and antibacterial activities of a new rotenoid from Pongamia pinnata. Similarly, a new prenylisoflavone from Ulex jussiaei showed antifungal activity against Cladosporium cucumerinum (Maximo et al., 2002). Chloroform extract of Erythrina vogelii inhibited the growth of Cladosporium cucumerinum (Atindehou et al., 2002). Rao and Joseph (1971) reported that the oil from seed husk of Cicer arietinum was toxic to Cephalosporium sacchari, Ceratocystis paradoxa, Curvularia lunata, Fusarium moniliforme, Helminthosporium sacchari, Physalospora tucumanensis, Rhizopus arrhizus, and Sclerotium rolfsii. Chu et al. (2003) isolated a peptide cicerarin from Cicer arietinum which exerted antifungal activity against Botrytis cinerea, Mycosphaerella arachidicola, and Physalospora piricola. The fungicidal activities of Cassia tora extracts and its active principles were determined against Botrytis cinerea, Erysiphe graminis, Phytophthora infestans, Puccinia recondita, Pyricularia grisea, and Rhizoctonia solani by Kim et al. (2004). The chloroform fraction of C. tora showed a strong fungicidal activity against B. cinerea, E. graminis, P. infestans, and R. solani. Emodin, physcion, and rhein were isolated from the chloroform fraction which showed fungicidal activities against B. cinerea, E. graminis, P. infestans, and R. solani. Furthermore, aloe-emodin demonstrated fungicidal activities against B. cinerea and R. solani, respectively. This study suggests the fungicidal actions of emodin, physcion, and rhein present in C. tora. According to Kelemu et al. (2004), the tropical forage legume Clitoria ternatea is resistant to a number of pathogens and pests. A highly basic small protein was purified from seeds of the plant. The protein, designated ‘finotin’, has broad and potent inhibitory effect on the growth of various important fungal pathogens of plants, namely Rhizoctonia solani, Fusarium solani, Colletotrichum lindemuthianum, Lasiodiplodia theobromae, Pyricularia grisea, Bipolaris oryzae, and Colletotrichum gloeosporioides. It also inhibits the common bean bacterial blight pathogen Xanthomonas axonopodis pv. phaseoli. Flavonol glycoside isolated from the stems of Teramnus labialis showed antibacterial and antifungal activities against plant
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pathogens (Yadava and Jain, 2004). Deepa et al. (2004) reported that the extracts of leaf, root, stem, and the callus obtained from Pseudarthria viscida showed significant inhibitory activity against some fungal pathogens causing major diseases in crop plants and stored food grains. Ng (2004) reported a profile of antifungal proteins and peptides of leguminous plants. The crude methanolic extract of Zuccagnia punctata was active against the fungal pathogens of soybean Phomopsis longicolla and Colletotrichum truncatum (Svetaz et al., 2004). Mandal et al. (2005) reported antibacterial and antifungal activities of saponins from Acacia auriculiformis. They found complete inhibition of conidial germination of Aspergillus ochraceous and Curvularia lunata at 300 mg/ml or less, whereas Bacillus megaterium, Salmonella typhimurium, and Pseudomonas aeruginosa were inhibited at 700 mg/ml or higher concentrations of the mixture. Abbas and Zayed (2005) isolated seven saponins from the aerial parts of Astragalus suberi. These saponins exhibited antibacterial activities against Gram-positive and Gram-negative bacteria and also showed antifungal activity. The heartwood extracts from Acacia mangium and A. auriculiformis was examined on the growth of wood rotting fungi with in vitro assays. A. auriculiformis heartwood extracts had higher antifungal activity than A. mangium (Mihara et al., 2005). Study carried out by Wong and Ng (2005) showed that seeds of Phaseolus vulgaris gave rise to a peptide vulgarinin that manifested an antifungal activity toward Fusarium oxysporum, Mycosphaerella arachidicola, Physalospora piricola, and Botrytis cinerea. It also exhibited the antibacterial action on Mycobacterium phlei, Bacillus megaterium, B. subtilis, and Proteus vulgaris. Peraza-Sanchez et al. (2005) isolated a new Pimarene from Acacia pennatula, which showed in vitro inhibition of growth, sporulation, and germination of Colletotrichum gloeosporioides. A chitinase with antifungal activity was isolated by Wang et al. (2005) from mung bean (Phaseolus mungo) seeds. It exerted antifungal action toward Fusarium solani, Fusarium oxysporum, Mycosphaerella arachidicola, Pythium aphanidermatum, and Sclerotium rolfsii. D’Souza et al. (2005) evaluated 15 native Western Australian legumes for their potential to biologically control Phytophthora cinnamom. Acacia extensa, A. stenoptera, and A. alata along with A. pulchella, were identified with the highest potential for biological control of P. cinnamomi. Acacia urophylla and Viminaria juncea exhibited the least potential for biological control of P. cinnamom.
Future directions and conclusions The search for antimicrobial agents from higher plants, microbes, insects, and vertebrates is continued. Family Fabaceae is known for its antimicrobial potential since time immemorial. Plants like Pongamia pinnata, Cassia tora, Psoralea corylifolia, Caesalpinia sapan, Mucuna pruriens are described in ancient literature for their antimicrobial uses. There is a pressing need to search for new antimicrobial agents for control of pathogenic enemies of crops for the sustainable future. Although, a significant contribution has been made in the field of search for fabaceous-derived natural drugs against human pathogenic fungi, phytopathogens are woefully neglected. Therefore, the pathogens causing diseases in economic plants should be given priority. The peptides play an important role in inhibition of the microbial
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growth. The structural, biochemical, and functional diversity of the proteins found in nature provides an exceptional opportunity for future research (O’Keefe, 2001). The future research should be directed towards search for antimicrobial molecules from the marine microbes and the lower plants like bryophytes and pteridophytes. Unfortunately, microbial resistance is increasing with fast pace. To fight with emerging microbial infections, a strategic ‘natural drug discovery programme’ should be developed, which may open up new vistas in antimicrobial drug research by giving a new generation of broad spectrum, natural drugs/botanicals.
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CHAPTER 7
Screening of plants against fungi affecting crops and stored foods OLI´VIA C MATOS, CAˆNDIDO P RICARDO
Introduction Nature is a rich source of products with interesting and useful biological activities. The use of plants for non-edible purposes is a practice dating back to prehistory. Besides the primary metabolites plants contain a diversity of secondary metabolites related to mechanisms of adaptation and interaction with environment. Collectively, more than 10,000 low-molecular mass compounds are known as secondary metabolites of plants (Cowan, 1999; Dixon, 2001). Simple phenols, phenylpropanoid derivatives, coumarins, flavonoids, tannins, quinones, ethereal oils, isoprene derivatives (iridoids, sesquiterpene lactones, diterpenes, triterpene saponins, steroid saponins), cardiotonic heterosides, alkaloids (pirolidone, pirolysine and piperidine alkaloids, kinolisidine alkaloids, kinoline alkaloids, isokinoline, indole, purine, diterpene and steroide alkaloids), and cyanogene glycosides are examples of such compounds (Mann, 1970; Marston and Hosttetman, 1987; Colegate and Molyneux, 1993; Cowan, 1999). This diversity results in part from an evolutionary process driven by selection for acquisition of improved defence against pests and diseases (Dixon, 2001). Plant secondary metabolites evidencing therapeutic, flavouring, odoriferous, stimulant, poisoning, hallucinogenic or colouring properties still play an important role in human activities. As estimated, there are 250,000–500,000 plant species, but only a small percentage (less than 10%) have ethnobotanical use (Borris, 1996; Moermann, 1996; Cowan, 1999). Several reports on the history of plant applications in folk medicine are known. In the secondhalf of the 20th century, after the development and generalised utilization of synthetic antibiotics, bioactive products derived from plants for antimicrobial application have lost importance. Crop protection from competing weeds, insects and diseases has been necessary for the higher yields in agriculture. Whereas in the early days manual labour was used to solve the weed problem, the manual control of insect infestations was in most cases an impossible task and plant diseases were even more difficult to control.
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Plant diseases, pests and weeds are responsible for crop losses in all continents in spite of the wide search of science and industry for better controlling agents and improved methods (Knight et al., 1997). The first generation of crop protection products consisted of inorganic arsenic, sulphur, copper and mercury compounds. Conventional pesticides, obtained from organic synthesis, which were considered a key for pest and disease control risks to lose importance due to problems of public health, environmental pollution, preservation of ecosystems and wild life habitats (Farrel, 1990). The generalized use of this type of pesticides conduced to serious problems of toxicity, water contamination, persistence in the ecosystems, residues in foods, as well as accumulation in fat tissues of man and animals, and increased pest resistance to them (Lyon and Zalon, 1990). The levels of pollution caused by atrazine and diazinon, an herbicide and an insecticide respectively, measured by Morley (1977), conduced to the definition of rules and restrictions to the use of pesticides, and to the development of strategies to reverse the pollution process (Kuc, 1990; Matos, 2000). Socio-economic and environmental factors, such as, those caused by resistance of pests and microorganisms to synthetic pesticides emphasize the importance of new ways and new strategies for the control of pathogenic agents. The present concept is based on the idea that agro-ecosystem should be managed in ways to hold pest to tolerable levels, making use of natural products, integrated pest management, and the use of pest-resistant crop varieties or even transgenic plants. Natural chemical substances can be useful for crop and food protection. Extracts from Pyrethrum flowers, containing piretrine, made their de´but as household insecticides in the first half of the 19th century and were the first pesticide commercialized under the designation of ‘‘Caucasian insect powder’’ (Banergi et al., 1985), while nicotine, extracted from tobacco, was the first natural insecticide used in crop protection (Casida, 1983; Pumarola-Batlle and Xirau-Vayreda, 1983; Matos, 2000). From the 1970s an increased interest on the study of the plants autoprotective capacity against pathogenic agents or adverse environmental conditions was noted (Bailey et al., 1992). Studies on the discovery of ways of manipulating plants in order to increase or to produce novel chemical molecules have been developed by different research teams. The isolation of several substances synthesized by several plants as a response to biotic or abiotic stresses evidencing insecticidal, fungicidal, bactericidal or herbicidal action have been reported (Lamb and Lawton, 1989; Bouguerra, 1990; Keen, 1990; Matos et al., 1998). In Table 1 some examples of plants analysed for compounds with fungicidal activity are provided. Table 2 illustrates antifungal compounds detected in plant cell cultures and in the respective plant (Constabel and Vasil, 1987; Kutney, 1997).
Fungi as contaminants of crops and foods Phytopathogenic fungi can cause local or general symptoms on their hosts. According to Agrios (1997) the most common necrotic symptoms are leaf-spots (localized lesions on leaves consisting of death and collapsed cells), blight (general and extremely rapid browning death of leaves, branches, twigs, and floral organs), canker (localized necrotic lesion), die-back (extensive necrosis of twigs beginning on their tips and advancing toward their bases), root-rot (disintegration or decay of part of
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Table 1 Plant substances with activity against phytopathogenic fungi Botanical name
Compound
Fungi
References
Ammi visnaga
Khelin Visnagin
Averbeck (1989), Borges et al. (1998)
Arabidopsis thaliana
Ferrulic acid Vanillic acid trans-Cinnamic acid o-Coumaric acid
Brassica napus
Cylobrassin Methoxybrassinin
Carum carvi
Hydroxiacetophenone
Cassia tora
Miscellaneous
Fusarium culmorum Cladosporium cucumerinum Fusarium oxysporum Phytophthora dreshleri Rhizoctonia solani R. solani Alternaria brassicae Botrytis cinerea C. Cucumerinum Fusarium nivale Sclerotinia sclerotium Leptosphaeria maculans Pythium ultimum Rhizoctonia solani F. oxysporum f.sp.dianthi Aspergillus niger
Chelidonium majus
Alkaloids
Aspergillus fumigatus, Fusarium sp.
Chenopodium ambrosioides Coriandrum sativum
Essential oils
Aspergillus flavus
Essential oils
C. cucumerinum Fusarium sp.
Essential oils Dihydroresveratrol
Aspergillus sp. Botrytis cinerea C. cladosporioides
Essential oil
Rhizoctonia solani
Pulegone
C. cucumerinum
Essential oil
Sclerotinia sclerotium
Cuminum cyminum Dioscorea bulbı´fera, D. dumentorum Satureja thymbra Thevetia peruviana Thyma spicata
Walker et al. (2003)
Dahiya and Rimmer (1988)
Curir et al. (1996) Mukherjee et al. (1996) Hejtmankova et al. (1984), Matos et al. (1999) Varma and Dubey (1999) Delgado (1993), Matos and Ricardo (2003) Matos (2001), Matos and Ricardo (2003) Adesanya et al. (1989) Mu¨ller-Riebau et al. (1995) Gata-Gonc- alves et al. (2003) Mu¨ller-Riebau et al. (1995)
all root system of a plant), damping-off (rapid death and collapse of very young seedlings), basal stem rot (disintegration of the lower part of the stem), soft rots and dry rots (maceration and disintegration of fruits, roots, bulbs, tubers, and fleshy leaves), anthracnose (necrotic and sunken ulcerlike lesions of the stem, leaf, fruit, or flower), scab (localized lesions with a scabby appearance on fruit, leaves, etc.), and decline (plants growing poorly, leaves small and yellowish or red, and some
Naturally occurring bioactive compounds
142
Table 2 Efficacy of production of secondary metabolites by plant cell culture Plant species
Metabolite
Cell culture media
Cassia tora Catharanthus roseus
Anthraquinones Ajmalicine
Agar gel Liquid
6.0 1.0
Catharantine Caffeine Berberine Berberine Rosmarinic acid Diosgenine
Liquid Agar gel Liquid Agar gel Liquid Liquid
Chiconine
Coffea arabica Coptis japonica Coleus blumei Dioscorea deltoidea Lithospermum erythrorhizon Macleaya cordata Morindia citrofolia Nicotiana tabacum Panax ginseng Papaver somniferum
Content in the cell culture (% dry weight)
Content in the plant (% dry weight)
Content ratio (cell cultures/ plant)
0.6 0.3
10 3
0.24 1.6 13.0 7.4 15.0 2.0
0.002 1.6 7.0 3.0 2.0
77 1 1.1 5 1
Agar gel
12.0
1.5
8
Chiconine Protopine
Liquid Agar gel
14.0 0.4
1.5 0.32
9.3 1.25
Anthraquinones
Liquid
18.0
2.5
7.2
Ubiquinone-10
Liquid
Ginsengoside Sanguinarine
Agar gel Liquid
0.18 27.0 2.9
0.003 4.5 –
60 6.0 –
Source: Constabel and Vasil (1987) and Kutney (1997).
defoliation can occur). Fungi can also cause excessive enlargement or induced growth and distortion of plant parts, such as: clubroot (enlarged roots appearing like spindles or clubs), galls (enlargement portions of stems, leaves, blossoms or roots), warts (wartlike protuberances on tubers and stems), witches’-brooms (profuse, upward branching of twigs), leaf curls (distortion, thickening, and curling of leaves). Additional symptoms can also occur, like wilt (generalized loss of turgidity and drooping of leaves or shoots), rust (many small lesions on leaves or stems, usually of rusty colour), smut (seed or gall filled with the mycelium or black spores of the smut fungi), mildew (areas on leaves, stems, blossoms, and fruits, covered with whitish mycelium and the fructifications of the fungus). In Table 3, examples of fungi responsible for important crop diseases are presented. The genera Aspergillus and Fusarium are examples of known phytopathogenic fungi reported to be widespread and harmful toxin-producers (Harris and Mantle, 2001; Harvey et al., 2001; Hussein and Brasel, 2001). Aspergillus flavus is a common mold that grows on many substances, especially on grains and nuts, producing the dangerous aflatoxins (Vargas et al., 2001). A. flavus is also referred as being responsible for paranasal invasive aspergillosis, a rare but chronic diseases affecting man (Alrajhi et al., 2001).
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Table 3 Fungi responsible for important crop disease Pathogen
Diseases
Crops
References
Aspergillus flavus A. niger
Synthesis of aflatoxin Grey mold rot or botrytis blight Scab
Peanut
Vargas et al. (2001)
Vegetable and fruit crops Cucumber Pecan Peach
Sherf and MacNab (1986) Agrios (1997)
Ripe rot Antracnose
Blueberry Cucumber
Bailey et al. (1992)
Botrytis cinerea Cladosporium cucumerinum C. effusum C. carpophylum Colletotrichum acutatum Colletotrichum magna Fusarium culmorum F. graminearum F. oxysporum f.sp. conglutinans F. oxysporum f.sp. cubense F. oxysporum f.sp. cumini F. oxysporum f.sp. dianti F. oxysporum f.sp. gladioli F. oxysporum f.sp .lentis F. oxysporum f.sp. lycopersici Leptospheria maculans
Crown rot Corn damping-off Wilt or Fusarium yellows Fusarium wilt Fusarium wilt Fusarium wilt Fusarium yellows, corm rot Fusarium wilt Fusarium wilt
Barley Maize Crucifers Banana Cumin Carnation Gladiolus Lentil Tomato
Mes et al. (1994), Lodha (1995), Singh and Tripathi (1995), Mao et al. (1997), Davanlou et al. (1999), Vargas et al. (2001)
Blackleg
Phoma lingam
Blackleg
Pseudoscorporella herpotrichoides Pythium violae P. Ultimum Phytophthora cinnamomi, P. infestans
Eyespot
Crucifers, cabbages Crucifers, cabbages Winter cereals
Root rot
Carrot
Shafer and Wostemeyer (1994) Shafer and Wostemeyer (1994) Dewey and Priestley (1994) Campion et al. (1998)
Root rot Late blight
Rhizoctonia solani
Soil rot Sheath blight Brown patch Stem canker Southern blight
Ornamental trees, shrubs, wheat Potato Tomato Rice Turfgrass Potato
Sclerotium rolfsii Spongospora subterranea Verticillium dahliae, V. albo-atrum
Powdery scab Vascular wilt
Tomato, pepper, apple Potato Dicotyledoneous crops, potato
Agrios (1997), Drenth and Govers (1994) Thornton et al. (1994)
Jones et al. (1991) Harrison et al. (1994) Carder et al. (1994), Robb et al. (1994)
Infections by Fusarium are responsible for destroying crops or dramatically reducing production yields. The economic importance of Fusarium species is enhanced by their ability to synthesize harmful mycotoxins, such as zearalenone, fusarins, fumonisins or trichothecenes (Koopmann et al., 1994; Blat et al., 1997; Desjardins
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Naturally occurring bioactive compounds
et al., 2000). Despite intensive research, efforts to control Fusarium infections and prevent or eliminate the presence of its mycotoxins in foods have not met with a great deal of success. Fusarium mycotoxins found in foods are generated primarily in the field although some toxin synthesis may occur during storage. Temperature and moisture conditions during the growing season and insect infestations are critical factors affecting fungal infection and toxin synthesis. Toxins like deoxynivalenol (DON) were found in grains of wheat and barley evidencing symptoms of the Fusarium blight (scab). Wet and cool weather during flowering of wheat is conducive to infection with F. graminearum, which produces DON, while late season rainfall increases infection of corn silk with F. verticillioides (F. moniliforme), the main producer of fumonisins (Koopmann et al., 1994; Blat et al., 1997; Desjardins et al., 2000). Even normal-looking wheat kernels may contain DON although concentrations are generally quite low (Munkvold and Desjardins, 1997; Sinha and Savard, 1997). Surveys have demonstrated the worldwide occurrence of fumonisins in cereal grains like corn, barley, oats or rice (Ghebremeskel and Langseth, 2001; Vargas et al., 2001).
History of the use of antifungal compounds Fungi are the major cause of plant diseases and so, most of the phytopharmacological products are aimed at fungal pathogens. Even before the identification of fungi as causal organisms of plant disease, dusting plants with sulphur was a generalized practice. The mixture of copper sulphate and calcium hydroxide (Bordeaux mixture) in 1885 can be considered the beginning of the development of chemical disease control. Fungicides similar to those of sulphur and copper, considered the first generation of fungicides, are still in wide use (Koller, 1999). The synthesis of carbamates and dithiocarbamates, two groups of broad-spectrum fungicides dates back to 1940s. The use of the second generation of fungicides was restricted in several countries due to the possibility of conversion of the most dithiocarbamates into potential carcinogens. Trichloronitrobenzene, quinines, chlorothalonil or pentachloronitrobenzene are other examples of the second generation of fungicides. Despite the restrictions in their use most fungicides of the second generation are still widely used, due to their effectiveness, convenience of handling, low harmful effects on plants and low resistance problems (Borges, 2002). Systemic compounds that can be absorbed by plants and translocated in it to reach the growing pathogen and control the infection constitute a third generation of fungicides. Implemented in the late 1960s, the most representative are the carboxamides and hydroxypyrimidines, highly specific to Basidiomycetes and powdery mildews (Ruberson, 1999). A major focus on the development of systemic fungicides still remains, and families of compounds such as sterol demethylation inhibitors (DMIs) and the morpholines are still of much interest. DMIs current importance lays in the control of cereal diseases in Europe and morpholines are mostly used for controlling cereal powdery mildews. Phenylamide fungicides that include benalaxyl, of urace and oxadiocyl are both used as soil and foliar fungicides for the control of potato late blight and downy mildews. These compounds are fungicides specific for the Oomycete fungi family (Ruberson, 1999; Borges, 2002).
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At the present, studies are focused on the development of a fourth generation of fungicides consisting of chemicals that interfere with specific steps in host–pathogen interactions, either by preventing fungal penetration into the plant or by enhancing the defence system of the pathogens. They are considered as disease control agents with indirect modes of action. The side-effects of these compounds on non-target organisms will be less severe than those caused by the conventional fungicides. Thus far only a few commercial products with such indirect modes of action have been introduced. Examples of fungicides to inhibit host penetration are thalide and carpropamid, developed for the control of rice blast. Probenazole and acibenzolarS-methyl are two examples of compounds that act on the reduction of plant defence reaction.
Interaction of plant products with fungi The term natural fungitoxins has been sometimes used to refer substances of plant origin that cause a significant toxic or inhibitory action on the fungus growth at relatively low concentrations. Flavonoids, isoprenoids and alkaloids with these properties are essential for plant adaptation, participating in a broad range of physiological processes (Harborne, 1987; Mayer, 1989). They can be present in healthy and infected tissues and are considered to be important in the protection of plants even if they do not prevent the infection (Harborne, 1987; Mayer, 1989). Postinhibitins are a group of fungitoxic vascular substances co-existing with the cytoplasmic specific hydrolytic enzymes. Seven classes of these toxins are known, most of them being involved in the plant resistance to one or more phytopathogenic fungi. Some of these compounds are saponins, of sulphurated aminoacids derivatives and several types of glycosides (Harborne, 1987). Examples of antifungal compounds secreted by plant leaves as a response to the attack of a fungus are diterpenoides isolated from Nicotiana glutinosa, isoterpenoides extracted from several species of Lupinus, and a sesquiterpenic lactone produced by Chrysanthemum parthenium (Harborne et al., 1976; Thara et al., 1984; Matos, 2000). Studies on plant organs other than leaves are also common. Roots are, in general, resistant to infection, which means that fungitoxic substances, present or exuded by the epidermis of the roots may play a protective role. In fact, more than twenty substances belonging to the group of isoflavones have been isolated from the roots of Lupinus albus. Borbonol-2, a hydrocarbonated lactone with antifungal activity, was also isolated from the roots of Persea borbonica but was not found in P. indica, which is sensitive to the disease (Harborne et al., 1976; Thara et al., 1984). The area of soil surrounding a plant root, the rhizosphere, is a unique physical, biochemical, and ecological interface between the plant and the environment known. Walker et al. (2003) analysed root exudates of Arabidopsis thaliana elicited by salicylic acid, jasmonic acid and chitosan as well as by two fungal cell-wall elicitors and profiled the secondary metabolites subsequently secreted. Among the several compounds detected 10 were structurally characterized and successfully used on the inhibition of Fusarium oxysporum, Phytophthora drechsleri and Rhizoctonia solani. Studies of Whips (2001) on the relation between plant growth and rhizosphere competence in biocontrol refer the extreme complexity of interactions that can occur
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Naturally occurring bioactive compounds
at the rhizosphere level and provide an extensive list of vascular phytopatogenic fungi (such as Fusarium sp., Rhizoctonia sp. or Pythium sp.) that are controlled through those interactions. One of the most important defence systems of the plant consists in the synthesis de novo of substances generally called phytoalexins, that are not present in healthy plant tissues but are synthesized when plant cells are exposed to microorganisms or other stressing stimulus (Darvill and Albersheim, 1984; Ebel, 1986; Fagboun et al., 1987; Adesanya et al., 1989; Osbourn, 1996). Phytoalexins are compounds of low molecular weight of a heterogeneous group of compounds like isoflavonoids, sesquiternes, diterpenes, polyacetilens and coumarins. Beier (1990) reports the presence of several phytoalexins in the following food plants: alfalfa (vesitol, sativan, medicarpin), pea (pisatin, cinnamylphenols and a methylchalcone), bean (phaseolin), broadbean (wyerone), lima bean (5-deoxikievitol, 8,20 -dihydroxygenistein), soybean (glyceollin), grapevine (viniferin, resveratrol), cotton (gossypol, cadalenes, lacinilenes, hemigossypol), peanut (resveratrol), celery, parsley and parsnip (furanocoumarins), rice (momilactones, oryzalexins), castor bean (casbene), potato (rishitin, hydroxilubimin, phytuberin, a-solanine, a-chalcone, lubimin, solavevitone, phytuberol), pepper (capsidiol), sweet potato (ipomeamarone), carrot (6-methoxymellein, falcarinol), tomato (a-tomatine, rishitin, falcarindiol, falcarinol), tobacco (rishitin, lubimin, phytuberin, phytuberol, solavetivone, capsidiol, glutinosone), eggplant (lubimin). Phytoalexin have long been inferred to be important in the defence of plants against fungal infection (Brooks et al., 1986, 1987). For instance, stilbene phytoalexins are formed in several unrelated plant species, such as peanut (Arachis hypogaea), grapevine (Vitis vinifera) and pine (Pinus sylvestris). Hain et al. (1993) investigated the potential of stilbene biosynthetic genes in a strategy of engineering pathogen resistance. They isolated stilbene synthase genes from grapevine and observed an increased disease resistance (based on the production of a foreign phytoalexin) against Botrytis cinerea in transgenic tobacco expressing the gene at a high level. In Table 4, several phytoalexins of crops elicited by fungi or bacteria are indicated. Since the in vitro plant tissue cultures can synthesize substances different from those of the plant grown in natural conditions, the development of in vitro studies are also important. DiCosmo and Misawa (1985) referred several examples of phytoalexins produced in tissues of different plant species, elicited in vitro by some chemical compounds or extracts of fungi (Table 5).
Plant synthesis of light-activated compounds It has been shown that several compounds when irradiated (photoactive compounds or photosensitizers) can cause toxic reactions in living cells (Matos and Ricardo, 2003). This can be especially important when the photoactive compounds react with arthropods, bacteria or fungi affecting human, animal health or crops. The main classes of plant photosensitizers are polyetilenes, thiophenes and related compounds, coumarins and furanocoumarins, furanocromones, b-carbolines and other alkaloids and complex quinines (Heitz and Downum, 1987; Arnason et al., 1992). By absorbing light photosensitizers trigger chemical modifications of a substrate or
Family
Species
Plant organ
Pathogenic agent
Phytoalexin
Compositae Cruciferae
Artium lappa L. Brassica napus L.
Roots Leaves and stems
Polyacetylenes Methoxibrassinine, cyclobrassinine
Dioscoriaceae
Dioscorea bulbifera L. D. rotundata Poir. Oryza sativa L.
Bulbs and tubers Whole plant Whole plant
Bacteria Leptosphaeria maculans (Desm.) Ces de Nat. Botryodiplodia theobromae (Pat.) Griff. and Maubl. Pyricularia oryzae Carv.
Lupinus albus L. Pisum sativum L. Glycine sp. Glycine max (L.) Merr. Capsicum annum L. Nicotiana tabacum L. Solanum tuberosum L. S. tuberosum L.
Leaves and roots Whole plant Roots Cotyledons Whole plant Leaves Tubers Whole plant
Daucus carota L.
Roots
Gramineae Leguminosae
Solanaceae Umbelliferae
Nectaria haematococca Berk. Phytophthora megasperma Drechsler f.sp. glycine Mucor ramosissimus Samutsevitsch Giberella pulicaris (Fr.) Sacc. Phytophthora infestans (Mont.), Caspary Chaetomium globosum Kunze
Dimethyl batatasine IV Dihydrostilben Carboxylic acid, 2,2-dichloro-3,3-dimethyl cyclopropane Isoflavenoids Pterocarpen Glyceolin Glyceolin Sesquiterpenoids, capsidiol Sesquiterpenoids, diterpenoids
Lubimin Araquidonic and icosapentaenoic acids
Screening of plants against fungi affecting crops and stored foods
Table 4 Induction of phytoalexins in crop plants
6-Methoximeleine
Robeson et al. (1980), Darvill and Albersheim (1984), Amin et al. (1986), Brooks et al. (1986, 1987), Ebel (1986), Zeringue and Hampden (1987), Dahiya and Rimmer (1988), Therelfall and Whitehead (1988), Whitehead et al. (1988), Desjardins et al. (1989), Laks and Pruner (1989), Felicice et al. (2000) and Matos (2000).
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Naturally occurring bioactive compounds
148 Table 5 Production of phytoalexins by cultures of plant cells Class of compounds
Phytoalexins
Plant species
Elicitor
Alkaloids
Acridon Codein Sanguinarine Morphine Ajmalicine Catharantine
Ruta graveolens L. Papaver somniferum L. Catharanthus roseus G. Don.
Benzoquinones
Equinofuran
Coumarins
Psoralen Xanthotoxin Graveolone Diosgenin
Lithospermum srythrorhizon Sieb. and Zucc. Petroselinum crispum (Miller). A.W. Hill
Quitosan, polylysine Products from the fungus cell wall Ribonuclease (denatured) Fungal glycoprotein Fungal polysaccharides Activated charcoal
Steroids Phenylpropanoids
Glyceolinol Kievitone Phaseolin
Naftoquinones
Chiconine
Polyacetylenes
Unspecific Phenylheptatrine
Terpenoids
Phytuberine Sesquiterpenoids
Dioscorea deltoide Wall. Glycine maxima, (L.) Merr. Phaseolus vulgaris L.
Lithospermum erythrorhizon, Sieb. and Zucc. Carthamus tinctorius L. Bidens spinosa L. Solanum tuberosum L. Nicotiana tabacum L.
Fungal polysaccharides Mycelium of fungus Quitosan, polylysine Products from the fungus cell wall Ribonuclease (denatured) Fungal glycoprotein Fungal polysaccharides Agaropectine Fungal polysaccharides Filtered fungus extract Fungus suspension Mycelium of the fungus
Source: DiCosmo and Misawa (1985).
target. Absorption of light generates highly reactive electronic excited states, which can either interact with biomolecules or with oxygen, generating the highly toxic reactive oxygen singlet excited state. UV radiation elicits the synthesis of endogenous photosensitizers by the plant defence mechanisms (Heitz and Downum, 1987; Heitz, 1995). It is considered most relevant the development of new pesticides environmentally safe and harmless to non-target microorganisms for crop protection. The first utilization of light effects was for the control of insects. Presently photosensitizers are used to control weeds, algae, insects, nematodes, viruses, bacteria, yeast and fungi or even tumour cells (Berenbaum, 1987). Plant photosensitizers emerged as relevant compounds for agricultural applications. In the last decades, there has been an increased interest in the plant screening for
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photopesticides. Several plant extracts and a diversity of natural compounds were analysed for their light-dependent antifungal properties (Towers and Champaghe, 1986; Matos and Ricardo, 2003). Compounds known as bioactive in the ground state, such as most of the flavones, can become photoactive by structural modifications (Becker et al., 1993; Borges et al., 1995, 2002).
Factors affecting plant synthesis of bioactive compounds The use of plants for therapeutic purposes has a long history, but the utilization of crude preparations can have some drawbacks, like variation in the amount of the active component with geographic, climate and ecological conditions or with the plant organs and morphology. Interconnection of living organisms, interrelations between biotic and abiotic factors and environmental conditions can induce changes in the synthesis of biologically active metabolites by plants. The diversity of plant compounds also suggests a high probability that synergistic, antagonistic or other undesirable effects can also occur. A synergistic interaction occurs when the combined activity of two or more compounds is greater than that of their individual activities. On the other hand, antagonist interactions are present when a combined activity of two or more chemicals is less than that of their individual activities (Mckey, 1979; Berenbaum, 1985). Although the increased attention on synergistic interactions in the last decade, in bibliography only few studies are reported (Down, 1989; Hay et al., 1994), and one case of antagonistic interactions is known (Terras et al., 1993). Synergy is an adaptive strategy in plant defence that can provide insight into diversity and function of secondary metabolites. Synergic effects can be due to the inhibition of a toxin detoxification, to the modification of an inactive compound rendering it toxic, to an enhanced penetration, transport or accessibility of a toxin to a certain target, or when two independent steps of biosynthetic pathways were affected (Vaara, 1992; Nelson and Kursar, 1999). Variability in collection, treatment and storage of raw material can also cause losses in bioactivity. Although there are several reports of the use of crude extracts in therapy and crop protection the isolation of the bioactive substances can be advantageous. Pure bioactive compounds are easy to formulate and to dose (Colegate and Molyneux, 1993). In Table 3, representative examples of compounds extracted from plants exhibiting activity against phytopathogenic fungi are presented.
Experimental Plant secondary metabolites are synthesized in specialized cells at distinct developmental stages and the complexity of their structures makes their extraction and purification difficult. The plant screening for bioactive compounds require appropriated assays all along the extraction procedure and along the subsequent purification steps. The development of efficient methods for the detection and characterization of natural products of crude plants extracts should consider their preliminary identification at an early stage of separation as a strategic element for guiding selective isolation procedures. Plant screening process can be shorted
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Naturally occurring bioactive compounds
by the development of bioguided plant fractionation towards a certain group of pathogens. Crude extracts were obtained with the adequate solvent that solubilizes a certain group of compounds present in the sample of the plant material to be analysed. The extracts were then analysed by common chromatographic techniques, immediately or after being fractionated with a range of solvents of increased polarity and the active principles were labelled and visualized under UV light (254 and 366 nm). TLC analysis is a rapid and efficient method that combines sensitivity and simplicity and allows the bioguided fractionation analysis. This procedure enables the differentiation between inactive and active compounds directly in the crude extracts or organic fractions. Isolation of no active compounds can be avoided and constituents with novel activity can be isolated. Preparative chromatography was helpful for high throughout purification of the compounds. Separation of the extracts was performed on reversed phase (CC/TLC RP18) with broad gradient mixtures of two or three solvents. Compounds exhibiting interesting antifungal activity were identified by chemical analysis using NMR, UV, GC–MS or by comparison with the correspondent standards.
Bioassays Biological targets The 12 phytopathogenic fungi, viz., Aspergillus flavus, A. niger, Botrytis cinerea, Cladosporium cucumerinum, Fusarium culmorum, F. oxysporum f.sp. cubense, F. oxysporum f.sp. melonis (F.o.m.), F.o.m. race 1,2, F.o.m. race 0, Phytophthora cambivora, Rhizoctonia sp. and Roselinea necatrix were analysed for their sensitivity to plant extracts, and to plant cell culture or to pure compounds, detected or isolated from those extracts. Fungi were supplied by the Centro de Estudos da Ferrugem do Cafeeiro (Center of Coffee Rust Studies) and Departament of Phytopathology of Estac- a˜o Agrono´mica Nacional (E.A.N.), Oeiras, Portugal.
Plant analysis Portuguese native plants were studied for the activity of their constituents against fungi affecting crops or foods. The species Biscutella lusitanica Jordan (Cruciferae), Luzula lactea Link (Juncaceae), Picris spinifera Franco (Asteraceae) and Silene foetida Sprengel (Caryophilaceae) as well as Chelidonium majus L. (Papaveraceae), were obtained by seed germination and grown in the Estac- a˜o Agrono´mica NacionalE.A.N’s green-house. The leaves of Sambucus nigra L. (Caprifoliceae) were collected in the Northeast of Portugal. Thevetia peruviana Schum (Apocynaceae), collected in Macau, was also included in the present studies. Extracts were prepared with organic solvents and tested on several phytopathogenic fungi. Extracts (whole plant) of B. lusitanica, L. lactea, P. spinifera and S. foetida prepared in methanol (MeOH) (1:5 weight/volume) were fractionated in n-hexane (n-hex), chloroform (CHCl3), ethyl
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acetate (EtOAc) and MeOH. Extraction procedures of C. majus (roots and shoots) in MeOH and of T. peruviana (leaves) in EtOAc, MeOH or H2O were performed as described in previous work (Matos et al., 1999; Gata-Gonc- alves, 2003). The leaves of S. nigra were fully dried (in the dark at 35 1C, for 10 days). The extracts were prepared (300 g dry weight/1500 ml of solvent) in water (boiling for 30 min), or in EtOAc, or in EtOH (throughout contact and stirring at 200 rev./min, for 3 days at 12 1C). Aqueous extracts were frozen at 80 1C and lyophilized; those obtained in the organic solvents were dried, by evaporation under vacuum at 40 1C, and resuspended in adequate volume of the solvent used for the extraction to a final concentration of 10–20 mg/ml. All the extracts were submitted to bioautographic fractionation techniques (Matos, 2001). Bioactive fractions were recovered from preparative thin-layer chromatography and concentrations of 1:5 (p/v) were used in the biological tests. Extracts of S. nigra and T. peruviana were tested against B. cinerea, C. cucumerinum and F. culmorum, while the extracts of C. majus were tested on F. oxysporum f.sp. cubense, C. cucumerinum, P. cambivora, Rhizoctonia sp. and Roselinia necatrix. The extracts in n-hexane, CHCl3, EtOAc and MeOH of B. lusitanica, L. lactea, P. spinifera and S. foetida were used for testing C. cucumerinum and F. culmorum.
Cell culture analysis Cell systems can produce different secondary metabolites or in different concentration of those produced by the complete plant. The cell culture of the Portuguese native species B. lusitanica, L. lactea, P. spinifera and S. foetida were included in these studies with phytopathogenic fungi. For the cell cultures, plantlets obtained from seeds aseptically germinated were used. Cells were developed in solid media MS or GB5 (Murashigue and Skoog, 1962; Gamborg et al., 1968) with 1 or 2 mg/l of dichlorophenoxiacetic acid (2,4-D), and incubated at 25 1C. After 3–5 weeks of growth cells were removed to fresh media and the initial media were transferred to Erlenmeyers and MeOH was added to a ratio of 1:2 (weight/volume), stirred on an orbital shaker at 180–200 rev./min, for 24 h, at room temperature. The mass extracted was centrifuged at 3000 g, for 15 min, the supernatant was recovered and fully dried in a rotary evaporator under vacuum. These crude extracts were fractionated by successive direct washing with n-hex, CHCl3, EtOAc and MeOH. Fractions were evaporated to dryness, weighed and re-suspended in the corresponding solvent to a final concentration of 10 mg/ml, and tested on A. flavus, B. cinerea, C. cucumerinum, F. culmorum and F. oxysporum f.sp. melonis. The cells of C. majus were grown in MS liquid culture medium, supplemented with five different combinations of hormones: 20 mg/l of 2,4-dichlorophenoxiacetic acid (2,4-D), 0–0.1 mg/l of kinetin (KIN) and 2.5–5.0 mg/l of gibberelic acid (GA3), for 6 weeks with agitation on an orbital shaker (100 rev./min). The media, after being frozen and lyophilized, were extracted by contact in methanol (1:5 weight/volume). Extracts were filtered and sterilized using a Millipore filter and used for the evaluation of their effectiveness on the control of F. oxysporum f.sp. cubense, C. cucumerinum, P. cambivora, Rhizoctonia sp. and Roselinia necatrix.
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Naturally occurring bioactive compounds
Isolated compounds Active compounds were preliminarily characterized by their reaction on the TLC plates with dyeing reagents (vanillin, MFA, KOH/EtOH, H2SO4/EtOH and Dragendorf reagent). Analysis by capillary gas chromatography followed by mass spectrometry (GC–MS) was performed for T. peruviana, using an Autosystem XL gas chromatograph with a vaporization injector in split mode (1:50) interfaced to a Turbomass Perkin–Elmer mass spectrometer. High-purity helium was used as carrier gas (25 cm/s), with a oven temperature program of 40 1C for 3 min, increased at 5 1C/min to 180 1C, followed by 15 1C/min to 240 1C and finally to 300 1C at 10 1C/min (isothermal 15 min), using a fused-silica capillary column, 30 m 25 mm i.d. 0.32 mm (DB-1; 100% dimethylpolysiloxane) (Gata-Gonc- alves et al., 2003). The characterization of bioactive compounds was also achieved by subjecting chemical standards to similar TLC tests. After elution with the same solvent mixtures and testing against the same fungi, the Rf of the standards and those of the active compounds were compared. Compounds that are frequently present in the plants studied were included in our assays. They were tested together with some of the extracts contributing to an approach to the chemical characterization of the extracts’ components. Stock solutions (102 M) of benzoic acid, caprilic acid, palmitic acid, linoleic acid, oleic acid, (+)-pulegone, isopulegol, ergosterol, citronellol, carvacrol, thymol, flavone (6-hydroxyflavone), coumarin (7-hydroxy-4-methylcoumarin), psoralen, chelerithrine and sanguinarine were prepared. The solvents used for the preparation of these solutions were selected from the solvents used for the extraction of plant material considering their ability to dissolve each of the compounds. For the bioassay the solutions were diluted to concentrations of 1045 104 M. The fungi A. flavus, A. niger, B. cinerea, C. cucumerinum, F. culmorum, F. oxysporum f.sp. melonis (F.o.m.), F.o.m. Y race 1,2, F.o.m. 26 race 1 and F.o.m. MC2 race 0 were the biological targets used in the assays here included. Standards were supplied by Dr. Maria Joa˜o Marcelo Curto from Instituto Nacional de Engenharia e Tecnologia Industrial of Lisbon, by Dr. Anto´nio Mac- anita from Instituto Superior Te´cnico of Lisbon, or were purchased in the market from Aldrich and Sigma.
Assay procedure Fungi were grown and maintained in culture on PDA (Potato Dextrose Agar, Difco Laboratories) at 25 1C in the dark. For the present antifungal tests, 5-, 7- or 10-dayold fungus cultures were used for harvesting the mycelium. Fungi suspensions were prepared by disintegrating the mycelium in a specific liquid medium (Allen and Kuc, 1969). Each fungus suspension was filtered under vacuum through nylon net (100 mm pores) on a Buckner funnel, adding the liquid media until a spore count of about 57.5 104 per ml was achieved for each fungus. Assays in chromatographic plates For the antifungal tests performed on TLC plates, 30 ml of each plant crude extract or fraction, 50 ml of each culture media extract or 20 ml of the solution of the natural
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compounds (104, 2 104 and 5 104 M) were applied. The extracts, the organic fractions and the standard compounds were applied directly into spots of 5–7 mm. The fractions were also chromatographed. After the elution in CHCl3:MeOH (97.5:2.5), the plates were visualized at 254 and 366 nm to visualize the compounds (Matos, 2001; Matos and Ricardo, 2003). Control spots with the solvents were also applied. Fungi were inoculated by spraying the TLC plates with the fungal suspension and these plates were incubated in humid chamber at 23 7 2 1C under continuous dark for 4–5 days. Inhibition areas were revealed as white spots, and qualitatively evaluated by the diameter of the area free of fungus growth. Replicates of each TLC were performed in order to be treated with the dying reagents [H2SO4/EtOH (10%), KOH/EtOH, molybdophosphoric acid (MFA), vanillin, Dragendorff reagent. The standards of benzoic acid, octanoic acid, palmitic acid, linoleic acid, oleic acid, (+)-pulegone, isopulegol, ergosterol, citronellol, carvacrol, thymol, were submitted to similar tests. Assays in liquid media Stock solutions of the standard compounds 3,6-dihydroxyflavone and 7-hydroxy-4methylcoumarin, chelerithrine and sanguinarine and 8-methoxypsoralen of 102– 103 M were previously prepared. From these stock solutions 100–200 ml were taken and added to 20 ml of the liquid fungus suspension. Compounds were solubilized in MeOH (20 ml/ml) to guarantee their solubility in the aqueous media. The samples were firstly incubated for 24 h at 25 1C, in dark, with agitation, in order to allow the fungus adaptation to these conditions. Subsequent to this initial growth, the compounds were added to the samples, with a micropipette directly into the suspension. In the photobiological tests the concentrations of each compound were established by estimating the overlap integral between the absorption spectrum of the compound and the lamp emission spectrum, to assure that, in the final fungus suspension, about the same concentration of species in the excited state was present. Samples containing the 20 ml of spore suspension and 100–200 ml of each of the compounds were placed on an orbital shaker (100 rev./min). This procedure was essential to oxygenate the fungus suspension, to obtain a good contact between the fungus and the compounds that are dissolved in the suspension and, in the case of assays performed under light irradiation, to achieve the best irradiation conditions to activate the compounds to be tested. Six replicates of each sample were prepared. Blank samples, either without any compound or with methanol or each solvent–methanol mixture were also tested. The samples without compound were used to evaluate whether the fungi were growing properly. Results were evaluated 3–5 days after inoculation at 25 1C as the difference between dry weights (Balance Mettler AB204-S, d ¼ 0.1 mg) observed in the samples having the compounds and the samples only with the solvent. Photobiological assays When phototoxic activity was tested the plates or liquid samples were prepared in duplicate, one for testing in the dark and the other for testing under light exposure.
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Naturally occurring bioactive compounds
Incubation was performed also at 2372 1C and the plates, after an initial period (12–24 h) of continuous dark, were exposed to light (8 h/day) for 2–3 days and kept in dark till the end of the assay. A set of five fluorescence lamps (Osram L18w/10 Daylight, emission range 350–730 nm, lmax ¼ 485 nm with 0.62 W/m2 fluence) was used as light source. The intensity per unit area was measured as previously described (Matos, 2001; Borges et al., 2002). The distance between the fluorescent lamps and the samples to be analysed was 30–40 cm. Photoactivity was determined by comparing the results achieved in the dark to those of the correspondent sample submitted to irradiation.
Results and discussion Promising results were obtained with most of the several plant extracts and fractions. The presence of antifungal compounds in the different culture media synthesized by the plant cells was also confirmed. In these experiments the higher efficacy with the lowest quantity of active substance was attempted, in order to avoid or to reduce to a minimum the problems of toxicity to plants, to beneficial microorganisms or insects or even to the environment. In the biological tests we take in consideration that for a natural compound to be considered a fungicide it is necessary that an inhibition of 50% (ED50) of the mycelium’s growth was obtained, with concentrations between 5 and 50 mg/ml. For extracts the concentrations corresponding to the ED50 were between 5 103 and 15 103 mg/ml. In an attempt to characterize the plant constituents responsible for the antifungal activities, the extracts or the extract fractions were chromatographed by liquid chromatography and/or by preparative thin-layer chromatography. Antifungal activity was detected on the TLC plates by the diameter of the halos corresponding to fungus growth inhibition. Antifungal photoactivity was detected by comparing the plates kept in the dark with those submitted to light irradiation (Matos and Ricardo, 2003). Results of assays in liquid media were evaluated by the difference between the mycelium dry weights produced by the fungus in the presence of the active compounds or only the solvent.
Plant extracts Extracts prepared from leaves of S. nigra and T. peruviana with ethyl acetate, water and ethanol or either methanol were tested against B. cinerea, C. cucumerinum and F. culmorum using the liquid media method. As illustrated in Figure 1, for S. nigra, and Figure 2, for T. peruviana, fungi were inhibited by the extracts of both plants. Aqueous extracts were less active then those in EtOAc and in EtOH. With the extracts in EtOAc the level of fungus inhibition reached about 60% for both plants. In the case of S. nigra the extract in EtOH induced inhibitions of about 70–80%, while for T. peruviana the extract in MeOH reduced the fungi growth around 60–70%. As a whole F. culmorum was the fungus that showed higher resistance to all the extracts of both plants.
Screening of plants against fungi affecting crops and stored foods Sambucus nigra
155
B. cinerea C. cucumerinum F. culmorum
Inhibition (%)
100 80 60 40 20 0 EtOAc
EtOH
H2O
Fig. 1. Effect of the leaf extracts of Sambucus nigra obtained in EtOAc, EtOH or H2O on the growth of Botrytis cinerea, Cladosporium cucumerinum, and Fusarium culmorum. Growth inhibition (affected by standard deviation) observed 5 days after inoculation is expressed as a percentage of the growth inhibition observed for the control with the correspondent solvent.
Thevetia peruviana
B. cinerea C. cucumerinum F. culmorum
100
Inhibition (%)
80 60 40 20 0 EtOAc
MeOH
H2O
Fig. 2. Effect of the leaf extracts of Thevetia peruviana obtained in EtOAc, MeOH or H2O on the growth of Botrytis cinerea, Cladosporium cucumerinum and Fusarium culmorum. Growth inhibition (affected by standard deviation) observed 5 days after inoculation is expressed as a percentage of the growth observed for the control with the correspondent solvent.
Results achieved in previous work on the antifungal properties of C. majus (Matos et al., 1999) led to perform with this plant antifungal assays using different genus of fungi as biological targets. Figure 3 presents the sensitivity of the fungi C. cucumerinum, P. cambivora, R. necatrix, Rhizoctonia sp. and Fusarium oxysporum f.sp. cubense (used as control) to the shoot and root methanolic extracts. For the five fungi the mycelium growth reached in the presence of the shoot extracts was quite similar, the inhibitions ranging from 53.5% (Rhizoctonia sp.) to 35.9% (R. necatrix). P. cambivora (26.7%) was the most resistant fungus to shoot extracts while R. necatrix and Rhizoctonia sp. exhibited a reduction in mycelium growth of 48.1%
Naturally occurring bioactive compounds
156
Root
Chelidonium majus L
Leaf Fungus growth (%)
100 80 60 40 20 0 F1
F2
F3
F4
F5
Fig. 3. Effect on the growth of Fusarium oxysporum f.sp. cubense (F1), Cladosporium cucumerinum (F2), Phytophthora cambivora (F3), Roselinea necatrix (F4) and Rhizoctonia sp. (F5) of root and shoot extracts of C. majus obtained in MeOH. Growth inhibition (affected by standard deviation) observed 5 days after inoculation is expressed as a percentage of the growth observed in the presence of the control.
and 53.5%, respectively. F. oxysporum f.sp. cubense and C. cucumerinum were the most sensitive fungi to C. majus root extracts, showing a growth reduction corresponding to 92.6% and 94.3%, respectively. In another experiment, the whole plant material from B. lusitanica, L. lactea, P. spinifera and S. foetida, were extracted in n-hex, CHCl3, EtOAc and MeOH and analysed for their antifungal properties against C. cucumerinum and F. culmorum. The extracts of these plants were less active than those obtained from the cell cultures of same plants (see Table 6). In fact, when assayed without light exposure, the extracts in CHCl3 EtOAc and MeOH were active or slightly active, except in the case of L. lactea, which EtOAc extract was very active against C. cucumerinum (see results included in Table 7).
Cell cultures We also report here assays performed to evaluate the potentiality of different plant cell cultures for the synthesis of antifungal substances. Because bioactive compounds are excreted into the media by the undifferentiated cells, the antifungal properties of each plant cell culture was evaluated based on the concentration of antifungal compounds extracted from the corresponding media. Results of the antifungal assays performed with cells of C. majus, grown in five different liquid media, extracted in methanol are presented in Figure 4. In these assays, a similar pattern of sensitivity of the five fungi to the extracts from the different media was observed. The fungi were insensitive or only slightly affected by the components present in the culture media M4 and M5. A growth reduction of less than 50% was observed with the media M1 and M2. All the fungi were severely affected by M3, which conduced to a growth reduction over 60% to C. cucumerinum, R. necatrix and Rhizoctonia sp., over 65% to F. oxysporum f.sp. melonis and over 80% to P. cambivora. The results achieved in these assays are much relevant since
Plant cells
Media
Extracts
Aspergillus flavus Botrytis cinerea
Cladosporium cucumerinum
Fusarium culmorum
F. oxysporum f.sp. melonis
Biscutella lusitanica
GB5
Luzula lactea
GB5
n-Hex CHCl3 EtOAc MeOH n-Hex CHCl3 EtOAc MeOH n-Hex CHCl3 EtOAc MeOH n-Hex CHCl3 EtOAc MeOH n-Hex CHCl3 EtOAc MeOH n-Hex CHCl3 EtOAc MeOH n-Hex CHCl3 EtOAc MeOH
+ ++ + ++ ++ + + T + + + +++ ++ ++ T + ++ +++ + ++ +
++ +++ +++ +++ +++ + ++ + + ++ +++ +++ +++ +++ ++ + ++ +++ + ++ +
+ + ++ ++ ++ + + + + ++ +++ ++ ++ T + ++ +++ + + +
+ ++ ++ ++ +++ ++ + + + ++ +++ +++ ++ +++ + + ++ +++ + ++ +
MS
Picris spinifera
GB5
MS
Silene foetida
GB5
MS
+ ++ +++ ++ +++ ++ + + + ++ +++ +++ +++ +++ + + ++ +++ + ++ +
157
Bioautographic assays performed in chromatographic plates. Fungus inhibition observed 3 days after incubation at 2372 1C: , inactive; t, traces; +, slight active; ++, active; +++, very active.
Screening of plants against fungi affecting crops and stored foods
Table 6 Sensitivity of Aspergillus flavus, Botrytis cinerea, Cladosporium cucumerinum, Fusarium culmorum and F. oxysporum f.sp. melonis to plant cell media
Naturally occurring bioactive compounds
158
Table 7 Sensitivity of Cladosporium cucumerinum and Fusarium culmorum to plant extracts Plant
Extracts
Biscutella
n-Hex CHCl3 EtOAc MeOH n-Hex CHCl3 EtOAc MeOH n-Hex CHCl3 EtOAc MeOH n-Hex CHCl3 EtOAc MeOH
lusitanica Luzula lactea
Picris spinifera
Silene foetida
Cladosporium cucumerinum
Fusarium culmorum
Dark
Light
Dark
Light
t ++ + + ++ + +++ + + + ++ + + ++ + ++
+ +++ ++ + ++ + +++ + + +++ +++ ++ + ++ t ++
+ + + + ++ + + + + + + + + + +
+ ++ ++ + ++ + + + ++ +++ ++ t + t
Bioautographic assays performed in chromatographic plates. Fungus inhibition observed 3 days after incubation in continuous dark or irradiated with VIS light (8 h/day) at 2372 1C: , inactive; t, traces; +, slight active; ++, active; +++, very active.
M1 M2 M3 M4 M5
Fungus Growth (%)
120 100 80 60 40 20 0 F1
F2
F3
F4
F5
Fig. 4. Effect of the substances produced by cells of C. majus grown in liquid media MS with 2,4-D (20 mg/l) and 2.5 mg/l of GA3 (M1), 5.0 mg/l of GA3 (M2), 0.1 mg/l of KIN (M3), 0.1 mg/l of KIN and 2.5 mg/l of GA3 (M4), 0.1 mg/l of KIN and 5.0 mg/l of GA3 (M5), on the growth of Fusarium oxysporum f.sp. cubense (F1), Cladosporium cucumerinum (F2), Phytophthora cambivora (F3), Roselinea necatrix (F4) and Rhizoctonia sp. (F5). Growth affected by standard deviation, observed 5 days after incubation at 25 1C, is expressed as a percentage of the growth observed with the solvent control.
they indicate that the presence of kinetin promotes the synthesis of most active antifungal compounds. Conversely, the presence of GA3 conduced to the decrease in the synthesis of those compounds. It would be much informative to analyse the several compounds released to those different cell cultures.
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In another experiment, a set of Portuguese endemic wild plant species cultured in vitro in MS or GB5, were also analysed for their ability to synthesize compounds useful for antifungal purposes. The effects of the extracts obtained in n-hex, CHCl3, EtOAc and MeOH of the cell culture of Biscutella lusitanica, Luzula lactea, Picris spinifera and Silene foetida on the growth of A. flavus, B. cinerea, C. cucumerinum, F. culmorum and F. oxysporum f.sp. melonis were evaluated. Results, based on a non parametric evaluation, showed significant differences on the antifungal properties of the cells of the four plants analysed against the five fungi (Table 6). Antifungal effects were highly dependent on the composition of the cell culture media and on the solvent used for the preparation of the extracts. ‘‘As expected, as a whole, the less affected fungi were F. culmorum and A. flavus and C. cucumerinum were the most affected fungi.’’ In the case of B. lusitanica, cells survived only in GB5 medium, and no significant differences were detected on the reduction of fungus growth in the presence of extracts in CHCl3, EtOAc and MeOH. The extracts obtained in n-hexane were quite inactive for most of the fungi, probably due to the low hydro solubility of the substances dissolved in this solvent, L. lactea GB5 cell growth media was an exception since n-hex extracts were active or very active. The components extracted by EtOAc from the MS medium of the L. lactea cells were also very active but only against B. cinerea, C. cucumerinum and F. oxysporum f.sp. melonis. In the case of P. spinifera no significant differences were detected for the CHCl3 extracts of both media, while the EtOAc extracts of the GB5 medium were much more active then those of MS. For this set of plants the methanolic extracts were usually less active, but the GB5 medium of B. lusitanica and S. foetida were active or very active. The assays just described reinforce the idea that in vitro cultures can be useful for the production of antimicrobial compounds as a substitute of the plant. Despite being costly this procedure can avoid the destruction of genetic resources mainly when studies are performed with plants existing in restricted areas or risking extinction.
Photoactivity The extracts obtained from the whole plants of B. lusitanica, L. lactea, P. spinifera and S. foetida, prepared with n-hex, CHCl3, EtOAc and MeOH, were also analysed for their antifungal photoactivity against C. cucumerinum and F. culmorum (Table 7). Only the extracts in CHCl3 and EtOAc of B. lusitanica and those of P. spinifera in CHCl3, EtOAc and MeOH showed increased antifungal activity against both fungi when exposed to light, relatively to the control tested in the dark. The cell culture media of these same plants were also analysed for their photoactivity, and as observed with the plant extracts only the cell culture media of B. lusitanica and P. spinifera showed the presence of antifungal photoactive principles. Results of the antifungal photoactivity against C. cucumerinum of the cell culture media of B. lusitanica extracted in n-hex, CHCl3, EtOAc and MeOH are shown in Figure 5. The extract in CHCl3 showed a good efficacy and photoactivity against C. cucumerinum and the MeOH extract was active mainly in the dark. As in the case of the extracts from the whole plant, the extracts obtained from the cell culture media of P. spinifera in CHCl3 and EtOAc (Figure 6) evidenced the
Naturally occurring bioactive compounds
160
Biscutella lusitanica Inhibition spot (mm in diameter)
12 10 8 6 4 2 0 n-Hexane
CHCl3
EtOAc
MEOH
Fig. 5. Antifungal activity of Biscutella lusitanica cell culture medium (GB5) treated with n-Hex, CHCl3, EtOH and MeOH. Results of the assays performed against Cladosporium cucumerinum observed 4 days after incubation at 2372 1C, in continuous dark ’ or irradiated with VIS light (8 h/day) &. Picris spinifera
Inhibition spots (mm)
15
GB5 (Dark) GB5 (Light) MS (Dark) MS (Light)
10
5
0 CHCl3
EtOAc
Fig. 6. Antifungal activity of Picris spinifera cell culture media treated with CHCl3 and EtOAc. Results of the assays performed against Cladosporium cucumerinum observed 4 days after incubation at 23 1C, in continuous dark ’ or irradiated with VIS light (8 h/day) &.
highest levels of fungus photoinhibition. No significant differences of photoactivity were detected related to the composition of the media.
Isolated compounds T. peruviana, C. majus and P. spinifera were studied in order to identify their bioactive compounds. Capillary GC–MS analysis of the main antifungal fraction of T. peruviana showed to be a complex mixture of several classes of components. More than 25 compounds were identified, representing the terpenes 25.2% and the fatty
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161
acids and derivatives 24.8%. In a previous work, it was found that antifungal activity T. peruviana against C. cucumerinum was mainly due to pulegone (14.3%) the major compound identified (Gata-Gonc- alves et al., 2003). Additional components of T. peruviana, like benzoic, octanoic, palmitic and linoleic acids, isopulegol, ergosterol, citronellol, carvacrol and thymol, were also studied for their antifungal properties. Results of tests performed with those substances against Aspergillus flavus, A. niger, B. cinerea, C. cucumerinum, F. culmorum, F. oxysporum f.sp. melonis, F. oxysporum f.sp. melonis Y race 1,2 and F. oxysporum f.sp. melonis MC2 race 2 are summarized in Table 8. Palmitic, linoleic and oleic acid as well as ergosterol were inactive or slightly active to the tested fungi. Isopulegol and thymol also exhibited a scarce activity. Pulegone and citronellol were active against this set of fungi. Very good inhibitions were achieved with benzoic and octanoic acids as well as with carvacrol, mainly when tested against B. cinerea. In fact, Mu¨ller-Riebau et al. (1995) in studies performed with Satureja thymbra and Thymbra spicata reported that thymol and carvacrol were the main active constituents of those plants. The importance of octanoic acid was also demonstrated in inhibiting the growth of B. cinerea and Penicyllium expansum on stored pears (Matos and Barreiro, 2004). Chelerithrine and sanguinarine, that were detected as responsible for the antifungal activity of the C. majus root and shoot extracts (Matos et al., 1999). Flavone and coumarin were referred as being synthesized by P. spinifera (Matos and Ricardo, 2003), so 6-hydroxyflavone and 7-hydroxy-4-methylcoumarin were analysed for their action against C. cucumerinum, F. culmorum, F. oxysporum f.sp. cubense, F. oxysporum f.sp. melonis 26 race 1 and F. solani. In order to evaluate the potentialities of these compounds as photopesticides they were tested either in the dark or after irradiation (UVA or visible spectrum) (Table 9). Flavone was active against the tested fungi mainly in dark conditions, while coumarin was active or slightly active when irradiated, evidencing higher inhibition than psoralen (Averbeck, 1989) under visible light irradiation (Borges et al., 1995). Chelerithrine and sanguinarine were active against F. oxysporum f.sp. cubense, F. oxysporum f.sp. melonis 26 race 1 and F. solani, mainly when irradiated with UV light.
Conclusions and future perspectives There is a relatively recent knowledge on opportunistic infections in man and animals caused by Fusarium strains (Rebell, 1981; Desjardins et al., 2000) and several reports suggest that diseases caused by Fusarium are responsible for important economic losses in crops (Matos et al., 1999). F. culmorum is a serious pathogen of cereals that infects seedlings, roots and stems (Koopmann et al., 1994). F. solani is responsible for potato wilt causing fusariosis in other vegetables. F. oxysporum is responsible for several vascular wilts (Cook, 1968, 1978; Nelson et al., 1981; Jaspal and Tripa´thi, 1995). Aspergillus infections besides promoting commercial losses in crop yields also produce fungitoxins which can be very dangerous to man and animals. Botrytis cinerea is responsible for fungal attack of foods which in addition to causing rots can make them unfit for consumption by producing mycotoxins (Tripathi and Dubey, 2004).
162 Table 8 Sensitivity of Aspergillus flavus, A. niger, B. cinerea, Cladosporium cucumerinum, Fusarium culmorum, F. oxysporum f.sp. melonis, F. oxysporum f.sp. melonis Y race 1,2 and F. oxysporum f.sp. melonis MC race 0 to isolated compounds Aspergillus flavus
Benzoic acid Octanoic acid Palmitic acid Linileic acid Oleic acid (+)-Pulegone Isopulegol Ergosterol citronellol Carvacrol Thymol
++ ++ ++ + ++ ++
Aspergillus niger
++ + ++ ++
Botrytis cinerea
Cladosporium Fusarium cucumerinum culmorum
+++ +++ t t ++ + + ++ +++ +
++ ++ + + + + t ++ + +
+ ++ ++ + ++ t
F. oxysporum F.o.m. Y race f.sp. melonis 1,2
++ + + ++ ++
+ ++ ++ + ++ ++
F.o.m. MC2 race 0 + ++ ++ + ++ ++
Autobiographic assays performed in chromatographic plants. Fungus inhibition observed after incubation for 3 days at 2372 1C:, inactive; t, traces; +, slight active; ++, active; +++, very active.
Naturally occurring bioactive compounds
Compounds
Fungi
Irradiation condition
Flavone
Coumarin
Psoralen
Chelerithrine
Sanguinarine
C. cucumerinum
Dark UV light VIS light Dark UV light VIS light Dark VIS light Dark VIS light Dark VIS light
++ ++
+ ++ ++ + ++ ++
+ +++ t + +++
+ ++ + + ++ t ++
+ ++ + ++ ++
++ + ++ +
+ ++ + ++
+ + + +
++
++
++
++
F. culmorum F. oxysporum f.sp. cubense F. oxysporum f.sp.melonis 26 race 1 F. solani
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Table 9 Sensitivity of Cladosporium cucumerinum, Fusarium culmorum, F. oxysporum f.sp. melonis Y race 1,2 and MC race 0 to isolated compounds
Assays performed in liquid media. Fungus inhibition observed 3 days after incubation in continuous dark or irradiated with UV or VIS light (8 h/ day): , inactive; t, traces; +, slight active; ++, active; +++, very active.
163
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Naturally occurring bioactive compounds
Fungicides are essential for plant diseases control. The efficacy of the present available fungicides is satisfactory, but a continuous effort to respond to the acquisition of resistance is required. Enhancement of crop quality is an advantage brought on by fungicides to the agricultural output. However, these advantages imply serious economic and environmental costs. As an alternative, the extracts of plants or of plant cell culture or the compounds they contain could be quite effective in the control of fusariosis, aspergillosis and crop infections caused by B. cinerea and C. cucumerinum. In the future many more new natural products useful for crop protection will certainly be identified from diverse natural sources. It is foreseeable, that biotechnology will expand its influence on crop protection. Important research areas for the development of new strategies for the protection of crops and foods will focus in the future on the utilization of natural products, including those that trigger defence mechanisms in plants (Knight et al., 1997; Cowan, 1999), the synergistic effects involving those compounds and synthetic ones as well as diseases control by means of biological interaction (Wink, 1993; Mari and Guizardi, 1998; Carpinella et al., 2003; Qin et al., 2004). Random gene shuffling in plants can be the source of new ‘‘natural’’ products with interesting biological activity and the transgenic technology will also contribute with new tools for crop protection. Even if, at present, synthetic chemicals remain as strong pillars of crop protection the search for natural compounds and new classes of fungicides with novel modes of action will be of increasing importance.
Acknowledgments We are grateful to Professor Anto´nio Mac- anita and Dr. Maria Joa˜o Marcelo Curto for providing facilities and laboratory support. A gift of T. peruviana plant material, from Dr. L. Gata-Gonc- alves and Dr. S. Baptista, and support on the identification of bioactive compounds from Dr. J. M. Nogueira and Dr. C. Moiteiro are also thankfully acknowledged.
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CHAPTER 8
Opportunities and potentials of botanical extracts and products for management of insect pests in cruciferous vegetables TONG-XIAN LIU, HAN-HONG XU, WAN-CHUN LUO
Introduction Cruciferous vegetables include cabbage, Chinese cabbage, broccoli, brussels sprouts, cauliflower, kale, collards, and kohlrabi, and are economically important to vegetable production and consumers around the world. Insect pests that attack these crops include diamondback moth (Plutella xylostella L.), cabbage looper (Trichoplusia ni Hu¨bner), beet armyworm (Spodoptera exigua Hu¨bner) and other armyworm species, (imported) cabbageworm (Pieris rapae L.), corn earworm [Helicoverpa zea (Bodddie)], cross-striped cabbageworm [Evergestis rimosalis (Guenee)], and cutworms in Lepidoptera; cotton aphid (Aphis gossypii Glover), turnip aphid [Lipaphis erysimi (Kaltenbach)], green peach aphid [Myzus persicae (Sulzer)], cabbage aphid [Brevicoryne brassicae (L.)], and whiteflies (Bemisia spp.) in Homoptera; vegetable leaf miners (Liriomyza sativae Blanchard) in Diptera, and others (Capinera, 2001). Due to the importance of crucifer vegetables, synthetic insecticides have been intensively used to control those pests. Although many novel insecticides have been registered to control crucifer pests around the world in recent years, their management still relies heavily on broad-spectrum synthetic insecticides (Talekar and Shelton, 1993). Many insect pests of crucifers have developed resistance to synthetic insecticides in many countries; those insects include P. xylostella, including Btinsecticides (Tabashnik et al., 2003), spinosad (Zhao et al., 2002) and others. In addition, the economic, health, and environmental costs of synthetic insecticides, negative effects on beneficial arthropods, and increasingly stringent government regulation of pesticides has resulted in more interest in the development and use of botanical pest-management products (Ascher, 1993). The role of botanical insecticides in pest management in most industrialized countries is still minimal (Isman, 1999). In recent years, there have been considerable interests in natural products derived from plants that have been registered to control numerous agricultural insect pests (Schmutterer, 1997; Haseeb et al., 2004; Xu et al., 2004).
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For thousands of years, people have recognized that plants are efficient producers of chemical compounds that are used in defense against herbivore attack. Botanical compounds have been used in several parts of the world long before the arrival of synthetic pesticides. For example, farmers in India and China have been using botanical extracts to battle major agricultural pests (Chiu, 1993, 1995; Singh and Singh, 1996), and those botanical insecticides include pyrethrum obtained from the flower of Chrysanthemum cinerariifolium [(Trevir.)Vis.], nicotine from tobacco (Nicotiana tabacum L.), and rotenone from Lonchocarpus nicou (Aublet), Derris indica (Lam.) Bennet, Amorpha fruticosa L., and Tephrosia purpurea L. Whereas plant-derived pesticides or extracts containing active compounds can be used directly, they can also form the basis for synthesizing products with similar or even better insecticidal properties. For example, pyrethrum has been used as the basis for the synthesis of the synthetic pyrethroids, which are important in modern chemical crop protection. The synthetic carbamates are also derived from physostigmine, a natural carbamate from the legume Physostigma venenosum Balf. (Fabaceae). In addition, nontoxic allelochemicals with co-occurring chemicals have been used to control insect pests. Most widespread among these synergists are inhibitors of different insect enzymes, including mixed-function oxidases and esterases (Feyereisen, 1999). In this chapter, we focus on the effects of some crude extracts from plants and some commercially available botanical insecticides on important insect pests of cruciferous vegetables (Table 1). The botanical materials include crude extracts and isolated or purified compounds from various plants species and commercial products. The insect pests tested include Trichoplusia ni, Spodoptera exigua, S. litura, S. spp., Pieris rapae, Helicoverpa spp., Evergestis rimosalis, and Mythimna separate Walker in Lepidoptera, Aphis gossypii, Lipaphis erysimi, Myzus persicae, Brevicoryne brassicae, and Bemisia spp. in Hompotera, Phyllotreta vittata in Coleoptera, and L. sativae in Diptera. The advantages and disadvantages of using those materials, and the potential of these botanical materials in the integrated pest management of cruciferous vegetables are discussed.
Azadirachtin and related products from Azadirachta indica A. Juss Azadirachtin or neem-based insecticides containing azadirachtin derived from extracts of neem tree (Azadirachta indica A. Juss, Meliaceae) have played important roles in crop protection. Azadirachtin, a complex tetranortriterpenoid, has been effectively used against >400 species of insects, including many key crop pests, and has proved to be one of the most promising plant ingredients for integrated pest management at the present time (Schmutterer, 1990, 1992, 1995; Isman, 1999; Walter, 1999). Neem-based compounds display an array of effects on insects, acting inter alia as a phago- and oviposition deterrent, repellent, antifeedant, growth retardant, molting inhibitor, sterilant, and preventing insect larvae from developing into adults (Schmutterer, 1990, 1995). Liang et al. (2003) evaluated three commercial neem-based insecticides in the laboratory for oviposition deterrence, antifeedant effects on larvae, and toxicity to eggs of P. xylostella. The three commercial neem-based insecticides were: AgroneemTM (0.15% azadirachtin, Agro Logistic Systems, Diamond Bay, CA) at
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Table 1 Botanical materials (extracts or commercial products) and their active ingredients that have been tested against major insect pests of crucifer vegetables Plants
Extracts or formulation
Active ingredients
References
Azadirachta indica A. Juss
Neemixs, Agroneems, Ecozins, Neepels
Azadirachtins and many others
Chrysanthemum cinerariaefolium Vis.
Extracts, isolates
Spiro enol ether, pyrethrin, pyrethrum
Daphne tangutica Maxim
Benzene extract, methanol extract, crude extracts
Lignans
Derris trifoliate Lour., D. elliptica (Wallich), D. chinensis (L.) Melia azedarach L. and M. toosendan Sieb. et Zucc.
Extracts, isolates
Rotenone
Wu (1997), Isman (1999), Schmutterer (1990, 1992, 1995), Walter (1999) Xu et al. (2000b, 2000c), Zhang et al. (2001a, 2001b, 2004) Perez-Izequierdo et al. (1992), PerezIzequierdo and Ocete (1994), Chen et al. (2000a, 2000b), Xu (2000a) Chiu (1993), Visetson and Milne (2001), Zeng et al. (2002)
Methanol extracts
Toosendanin
Parthenium argentatum Gray, P. hysterophorus L., P. schottii Greenman, and P. tomentosum DC Rhododendron molle G. Don
Methanol extracts, cold alcohol extract, isolates, petroleum ether extract
Parthenin, argentitatins
Isolates, ethanol extracts
Rhodojaponin III, many others compounds
Sophora alopecuroids L.
Purified alkaloids
Stellera chamaejasme L.
Ethanol extract
Aloperine, matrine, oymatrin, sophoramine, sophocarpine, cytisine, sophoradine 7-Hydro-coumarin, daphnoritin and chamaechromone
Zhang and Chiu (1991, 1992), Wang et al. (1992, 1994, 1999), Chiu (1993, 1995), Ce´spedes et al. (2000) Gajendran and Gopalan (1981), Isman and Rodriguez (1984), Patil et al. (1993), Ce´spedes (2001), Sohal et al. (2002), Sundarajan (2002) Hu et al. (1993, 2000b), Zhong et al. (2000b, 2001a, 2001b, 2001c), Cheng et al. (2002), Zhong (2002) Luo (1995), Luo and Li (1996), Luo and Zhang (2003), Luo et al. (1996, 1997, 1999) Ji et al. (2000), Zhang et al. (2000a, 2000b, 2000c, 2000d, 2000e), Wang et al. (2002), Su (2003)
(Continued)
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174 Table 1 (continued ) Plants
Extracts or formulation
Active ingredients
References
Strophanthus divaricatus (Lour.)
Water extract, 0.05% strophanthin
Huang and Renwick (1994), Hu et al. (2000a)
Tripterygium wilfordii Hook. f. and T. hypoglaucum (level) Hutch.
Acetone extracts, chloroform extracts Ethanol extract
Caudoside, Fcaudostroside, divaricoside, sinlside, isosinotrodise, sarmutodise and Dstrophanthin Rotenoids (rotenone, rotenolone, and 5-,or 6-hydroxylrotenone) wilforine, alkaloids, non-alkaloids, wilfordine, wilforine, wilfortrine
Xanthium sibiricum Patrin ex Widder
Methanol and ethanol extracts
Xanthostrumarin, carboxyatractyloside, xanthatin, atractyloside, xanthin, xanthunin, xanthanodiene, strumaroside
Chiu (1987), Tong and Chiu (1988), Yang et al. (2000), Zhang et al. (2000e, 2000f), Xu et al. (2001, 2002), Zhang et al. (2001c), Luo et al. (2004) Peng (1984), Wang et al. (1999), Zhou et al. (2002)
4.8 mg azadirachtin/l, manufactured by Ajay Bio-Tech (India) Ltd., Erandwane, Pune 411004, Maharastra, India), EcozinTM (3% azadirachtin, AMVAC Chemical Corporation, Los Angeles, CA) at 20 mg azadirachtin/l, and NeemixTM (0.25% azadirachtin, Certis, Columbia, MD) at 20 mg azadirachtin/l. All three neem-based insecticides did not cause significant oviposition deterrence on P. xylostella. Numbers of eggs oviposited by P. xylostella adults on the cabbage leaves treated with the three neem-based insecticides were not significantly different from those treated with water. However, when aluminium foil sheets coated with cabbage juice residue were treated with the three insecticides and were used as egg-laying substrates, significantly fewer eggs were found on the aluminium foil sheets compared with those treated with water. When eggs were treated with Agroneem, Ecozin, and Neemix, 61.6, 66.2, and 75.2% of P. xylostella eggs developed to neonates, respectively, although the larval hatching rates in the treatment of Neemix were not significantly different from that in water control (81.2%). All larvae of P. xylostella fed on the leaves treated with the three neem-insecticides died on or before day 7 compared with 70–74% larvae in the water control surviving to adults. All three neem insecticides exhibited a significant antifeedant effect, and the larvae quickly stopped feeding and dropped off treated leaves, resulting in no or minimal foliar damage. Plutella xylostella larvae that fed on neem-based insecticide-treated leaves were significantly smaller (0.012–0.016 mg per larva, 13.5–14.8 mm in length, and 2.0–2.5 mm in diameter) compared with those fed on water-treated leaves (0.058 mg per larva, 30.2 mm in length, and 4.8 mm in diameter).
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Greenberg et al. (2005) evaluated the same three commercial neem-based insecticides, Agroneem, Ecozin, and Neemix, and a noncommercial neem leaf powder for oviposition deterrence, antifeedant effect on larvae, and toxicity to eggs and larvae of S. exigua in the laboratory. They found that in a no-choice assay, the proportion of eggs laid on the leaves was 2.5- to 9.3-fold higher in the control compared with other treatments. In a two-choice assay, significantly more eggs were deposited on the control leaves than on neem-treated leaves. Significant differences were also detected between pairs of neem treatments, and the treatments could be ranked in the following order of oviposition deterrence: neem leaf powder>Agroneem>Ecozin>Neemix. In a five-choice assay, oviposition preference was significantly lower on neem-treated leaves than on the control. No differences were observed between the neem treatments. Significantly more leaf area was consumed by each beet armyworm instar in the control than any of the neem-based treatments, but differences among the neembased treatments were not detected. Agroneem, Ecozin, and Neemix caused S. exigua egg 78, 77, and 72% of mortality compared with the nontreated control, but neem leaf powder caused 55% mortality, significantly lower than the commercial neem-based insecticides. Three days after treated leaves were offered to the beet armyworm larvae, mortality was not significantly different between neem treatments and the nontreated control. After 5 days of feeding, larval survival in the Agroneem- and Neemix-treatments was 1.5-fold greater than that in the Ecozin treatment and 1.3-fold greater than in the neem leaf powder treatments. After 7 days, larval survival was 61, 60, 33, and 27% in the Agroneem, Neemix, Ecozin, and neem leaf powder treatments, respectively, compared with 93% in the control. Neem-based insecticides have been tested and used for management of P. xylostella and other pest insects on cabbage (Leskovar and Boales, 1996; Perera et al., 2000). The neem-based insecticide, AlignTM (3% azadirachtin, formerly AgriDyne, Salt Lake City, UT), was tested in the field against lepidopterous pests, mainly P. xylostella and T. ni, on cabbage in Texas by Leskovar and Boales (1996). They found that Align significantly reduced larval densities of P. xylostella and T. ni on cabbage plants and foliage damage, and significantly increased marketable cabbage head weights. Neempel 0.3% EC (0.3% azadirachtin) is commercially produced using a new extraction technology developed by the Laboratory of Insect Toxicology, South China Agricultural University in China, and now commercially manufactured by Neemtech (Haikou, Hainan, China). The azadirachtin extraction and isolation process was modified by using a supercritical-fluid chromatography and microwave extraction method as described by Wu (1997). In the new azadirachtin extraction and isolation process, stabilizing agents and some specific epoxidized vegetable oil compounds have been incorporated into the final product, Neempel 0.3% EC, which restrains decompounding of azadirachtin in conditions of sunlight or heat. Neempel 0.3% EC has been tested against P. xylostella and turnip flea beetle, Phyllotreta vittata Fab. (Coleoptera: Chrysomelidae) under field conditions. Xu (unpublished data) found that Neempel was effective against P. xylostella and P. vittata larvae. At the dilutions of 800, 1000, and 1200 , P. xylostella populations were reduced by 81–91% 7 days after application and 81–86% 15 days after application. Percentage of control was 90–92% 7 days after application, and 86–89% 15 days after application. These results were similar to abamectin (1.8% EC, dilution 2000 ) (81 and 91% population reduction and control, respectively). At the dilutions of 1600,
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1800, and 2000 , P. vittata populations declined by 67–84% 7 days after application, and percentage of control was 69–86% 7 days after application, compared with cypermethrin (10% EC) with 52% population reduction and 56% control. In addition to controlling pest insects, many azadirachtin-based insecticides have negligible effects on natural beneficial insects and low environmental impact (Schmutterer, 1990, 1995). Because the neem-based insecticides are not toxic to humans and many beneficial arthropods, and the target pests are unlikely to become resistant, these neem-based insecticides become more amenable for use in pest management programs (Feng and Isman, 1995; Immaraju, 1998; Walter, 1999). However, we need to realize that neem-based formulations also contain numerous bio-active compounds other than azadirachtin, and azadirachtin itself might not always be the most important, or only compound causing the insect responses.
Spiro enol ether analogues and extracts from Chrysanthemum coronarium L. Spiro enol ether isolated from crown-daisy, Chrysanthemum coronarium L. (Asteraceae), has distinct bioactivity against many species of insect pests (Bohlmann and Fritz, 1979; Wu and Wang, 1994). Because of their bioactivity, a series of analogues of spiro enol ethers were synthesized by Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, China (Figure 1). The two of the most promising compounds of spiro enol ether, named after the sequence as Nos. 12 and 20, have been extensively studied in China (Xu et al., 2000b, 2000c; Zhang et al., 2001a, 2004). Xu et al. (2000b) found that the LC50 values of compounds No.12 and No. 20 to 3rd instar S. litura were 945.25 and 1295.76 mg/ml, respectively. Xu et al. (2000c) also found that the LC50s of the compounds No. 12 and No. 20 against the 2nd instar P. xylostella were 498.7 (95% FL: 489.4–558.0) mg/ ml and 299.8 (273.8–325.7) mg/ml, respectively, 5 days after treatment, and that of compound No. 20 is more toxic to the larvae than No. 12. Zhang et al. (2004) found that compound No. 20 prolonged the hatching time of eggs of P. rapae, and the egg hatching rates 3 days after treatment were 49, 36, and 25% at concentrations of 200, 400, and 800 mg/ml, respectively. Zhang et al. (2001b) showed that both analogues of spiro enol ethers had repellent activity against 4th instar larvae of P. rapae, with AFC50 (the concentration causing 50% antifeedant effect) values at 398.88 mg/ml for No. 12 and 280.54 mg/ml for No. 20 24 h after application. The analogues also reduced the weight of larvae and pupae O2N
O O
O O
Compound No. 12
N
Compound No. 20
Fig. 1. Chemical structure of compounds No. 12 and No. 20 isolated from Chrysanthemum coronarium L. (Wu and Wang, 1994).
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of P. rapae and decreased the emergence of adults. Zhang et al. (2004) screened 19 spiro enol ether analogues against the larvae of P. rapae for antifeedant activity. They found that the antifeedant activities of compounds No. 20 and No.12 were higher compared with the other 17 analogues. In a no-choice test, AFC50 values of No. 20 and No.12 against the 3rd instar P. rapae were 226.93 and 370.00 mg/ml, whereas in a choice test against the 4th instars, the AFC50 values were 280.54 and 398.88 mg/ml, respectively, 24 h after the start of the treatment. Compound No. 20 protected cabbage leaves and controlled the larvae of P. rapae effectively. Xu et al. (2000b) determined the antifeedant effects of spiro enol ether’s 19 analogues against the larvae of S. litura. Among the 19 analogues, the highest antifeedant rate of 75.3% was recorded in compound No. 12, followed by 70.2% in compound No. 20. The AFC50 of No. 12 against 3rd instar larvae in preference tests and 4th instar larvae in nonpreference tests were 340.39 and 403.83 mg/ml, respectively. The 4th instar larvae injected with compounds No.12 and No. 20 at 10 mg/ larva, and 66.7 and 87.5% of these larvae died in 24 h, respectively. Meanwhile, 4th instars injected with the two compounds had significantly higher mortality than that of larvae injected with acetone+water (1:1) as controls. Xu et al. (2000b) also found that larvae at 7 days after treatment with compounds No. 12 and No. 20 were significantly smaller than those of the controls, especially in those treated with compound No. 12. Xu et al. (2000c) also found that compounds No. 12 and No. 20 exhibited significant oviposition deterrent activities and larval hatching of P. xylostella, and females oviposited 25.6 and 47.2% fewer eggs on the plants treated with compounds No. 12 and No. 20 at 1000 mg/ml, respectively.
Extracts from Daphne tangutica Maxim. Daphne tangutica Maxim. (Thymelaeaceae) is a local endemic plant, widely distributed in the Qinghai-Tibet plateau of China. Chen et al. (2000a, 2000b) and Xu (2000a) studied the insecticidal activities of the methanol, acetone, ethyl acetate, chloroform, benzene, and petroleum ether extracts from the air-dried, powdered plant tissues. These crude extracts exhibited strong antifeedant effects when applied to 3rd instar S. litura. The AFC50s of the 3rd instar S. litura were 294.77 (130.2–667.6), 111.69 (60.48–206.25), 108.27 (60.10196.65), 102.14 (52.97–195.97), 71.75 (31.65–162.63), and 65.6 (28.4–151.3) g dried powder/l, respectively. Of the extracts, the benzene extract possessed the highest insecticidal activity against the 5th instar P. rapae, followed by the chloroform extract. The petroleum ether extract at a concentration of 500 mg dried powder/ml exhibited 62.3% antifeedant effects. Further bioassay revealed strong stomach toxicity along with antifeedant activity of the extracts against the 5th instar P. rapae at the concentration of 100 mg dried powder/ ml. Moreover, the methanol extract of D. tangutica exhibited high antifeedant and stomach poison effects. Fraction 9 of silica-gel column chromatography proved to be the most active fraction among those partitioned from the methanol extract. Larvae fed on leaf discs treated with fraction 9 grew slowly, and their bodies were significantly smaller than those of the control. Histological study indicated that the midgut tissue of the treated larvae was destroyed and fat bodies atrophied. Preliminary
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physiological reaction studies showed that fraction 9 reduced the content of protein in haemolymph and inhibited the activity of esterase in the midgut (Xu et al., 2000a). Chen et al. (2000a, 2000b) also reported that among root bark, root xylem, stem bark, xylem in stem and leaf had strong antifeedant and stomach poison properties. Residues of different plant parts extracted with benzene were re-extracted with methanol; the leaf extracts showed the most insecticidal activity, implying that the content of active ingredients in the leaf is the highest. Wang et al. (2000) found that the alcohol extract from D. genkwa is a strong antifeedant to P. rapae. Perez-Izequierdo et al. (1992) and Perez-Izquierdo and Ocete (1994) conducted choice tests of ethanol extracts of dried leaves of D. gnidium and Anagyris foetida L. for their antifeedant activity against 5th instar S. littoralis. They found satisfactory antifeedant activity with doses of 30 and 40 mg/cm2 of both extracts. They also found that the ethanol extract of dried D. gnidium leaves exhibited significant antifeedant activity against other insects.
Rotenoids and extracts from Derris spp. The most common bioactive chemicals extracted from genus Derris (Fabaceae) and related genera are rotenoids, which exhibit strong insecticidal activities, including strong contact, fumigant, and stomach poison, and growth inhibition to numerous insect pests, including lepidopteran larvae and aphids (Chiu, 1993; Zeng et al., 2002). Rotenoids include rotenone and other chemicals, such as tephrosin, deguelin, and toxicarol. Besides Derris spp. [Derris elliptica (Wallich) Benth, D. chinesis (L.), and a few others], other plants that possess rotenone include Milletia pachycarpa Baker, Milletia reticulate L., Tephrosia vogelii Hook, and Lonchocarpus spp. (Chiu, 1993). It was found that rotenone inhibits the conversion of nutrients into energy at the cellular level (cellular respiration). After exposure to rotenone, insects quickly stop feeding, and death occurs several hours to a few days. It was believed that rotenone is acting between NADH dehydrogenase and coenzyme Q (Ware, 1994). When the enzymes were inhibited, the ATP level of the insect decreases, resulting in the deficiency of energy and consequently, the insect shows paralysis and slowly dies. Rotenone also inhibits the assembly of spindle body canaliculus and the formation of canaliculus of insects in vitro by reversible conjugation with canaliculus protein. It may change the components of protein in the integument of the lepidopteran larvae, and decrease the content of whole protein. It could also affect succinic acid, mannitol, and some other compounds. It could inhibit the epithelial–mesenchymal transition (EMT) of mitochondrial membranes, thus indirectly affecting the activity of Nicotinamide adenine dinucleotide (NADH) dehydrogenase (Bienen et al., 1991). Rotenone could inhibit 8090% of the reducing enzyme between NADH and CytC, and between NADH and coenzyme Q (Obungu et al., 1999). Elliptone, isolated from the roots of D. elliptica, is an active insecticide in the same chemical family as rotenone. Differences in toxicity, antifeedant properties, inhibition of growth and development, and oviposition deterrence between elliptone and rotenone were evaluated in P. rapae, P. xylostella, Brevicoryne brassicae L., and P. vittata (Zeng et al., 2002). Elliptone exhibited strong toxicity against P. xylostella larvae and P. vittata adults, but significantly lower toxicity against aphids and
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P. rapae larvae compared to rotenone (based on comparison of LC50). Both elliptone and rotenone displayed antifeedant activity. Elliptone, and to a lesser extent, rotenone, had some inhibitory effect on growth, development, and oviposition. Elliptone had a lesser inhibitory effect on NADH–ubiquinone oxidoreductase activity (50% inhibition, IC50, at 5.27 nmol/ml) than rotenone (IC50 at 2.58 nmol/ml), suggesting that the former is a less effective respiratory inhibitor than the latter. It is possible that the higher toxicity of elliptone in leaf-disc immersion trials reflects its lower antifeedant activity and higher ingestion rate. Xie et al. (2004) compared the antifeeding, contact poisoning, and growth-inhibiting activities of the crude extract of the calluses from young leaves and the crude extract of roots of D. elliptica against the 5th instar larvae P. rapae. They found that the AFC50 value at 1 and 2 days after treatment was 2.207 and 1.482 mg/ml, respectively, compared with 2.375 and 1.690 mg/ml from the crude extract of roots. The LD50s of the crude extract of the calluses by topical application was 0.283 mg/larva after 1 day while the crude extract of roots and the standard rotenone showed little toxicity to the larvae. The LC50s of the crude extract of the calluses were 2.050 mg/ml after 2 days, respectively, whereas those of the crude extract of roots and the standard rotenone were 1.239 and 1.190 mg/ml, respectively. They also found that 87.5% of pupae were manifestly inhibited by the crude extract of the calluses, compared with 82.5% by the standard rotenone and 72.5% by the crude extract of roots. Visetson and Milne (2001) used two types of ethanol extraction methods, Soxhlet and stirring soaking from the D. elliptica roots to determine their effects on 3rd instar larvae of P. xylostella. They determined the LD50 values of 24.25 and 89.07 mg/ ml for the Soxhlet and stirring methods, respectively, a 3.7-fold difference.
Rotenoids and extracts from Tephrosia vogelii (Hook f.) Tephrosia vogelii (Hook f.), a perennial shrub, is traditionally used for its ichthyotoxic and insecticidal properties. It originated from tropical Africa and is now widely distributed in Western and Eastern Africa, the Philippine, Cuba, Java, Puerto Rico, Malaysia, and the Southeast US. The purple and white varieties of T. vogelii have been introduced to China from the Philippine and Tanzania where the fast-growing plants with flourish branches and leaves produce high levels of legume. The chloroform extracts were isolated and purified by quick column chromatography, thinlayer chromatography (TLC), and liquid chromatography (LC), and the structures were identified by IR (infrared), MS, GC–MS, and nuclear magnetic resonance spectroscopy (NMR). The results reveal that the main active components of T. vogelii are rotenoids such as rotenone, rotenolone, and 5-, or 6-hydroxylrotenone (Zhang et al., 2000f). The crude extracts and the isolated bioactive ingredients of T. vogelii had strong antifeedant, growth-inhibition, and ovicidal activities against several lepidopteran species. Zhang et al. (2000e) evaluated the antifeedant properties of the acetone extracts of T. vogelii in both choice and no-choice bioassays involving four lepidopteran species, Piers rapae, Spodoptera litura, Mythimna separata Walker, and Plutella xylostella. In choice bioassays, the extract at 5 dilution reduced the feeding of 5th instar P. rapae larvae by 87.3%, which is similar to the antifeeding effect
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caused by 1000 mg toosendanin (12 alpha-acetoxyamoorastatin)/l (88.9%). The same extract dilution reduced feeding by 65.8 and 100.0% for the 5th instar S. litura and M. separata, respectively, and still caused 60.5% feeding reduction at the 200 dilution. In no-choice bioassays, the 100 dilution reduced feeding up to 94.2% for the 5th instars of P. rapae within 48 h, while the 10 dilution reduced feeding of 4th instar P. xylostella by 100%. In another study by Zhang et al. (2001c), the diluted acetone extracts of T. vogelii were applied topically on the dorsal shields of intermediate thoraxes of P. rapae larvae and by feeding P. rapae larvae with cabbage leaves treated with diluted acetone extracts. The results show that in the topical treatments of 100–250 mg/head, the 3–4 instar larvae had a mortality of 100%. Prepupae of P. rapae treated at 200 mg/head at early and late stages had a mortality of 96.1 and 52.4%, respectively, but there were no significant residual effects on their pupae. They also found that topical treatment at 20 mg/head resulted in a mortality of 7.9% to the larvae of S. litura and 8.3% to the larvae of M. separata. In feeding treatments, the growth and development of P. xylostella were inhibited significantly. In a study conducted to determine the effect of extracts from T. vogelii leaves and rotenone on the cuticle of P. rapae larvae and pupae, Xu et al. (2001, 2002) found that the larvae topically treated with acetone extracts from the leaves or rotenone caused malformations on the larvae and pupae. The 5th instars and pupae of P. rapae become malformed pupae exhibiting six different symptoms after treatment with topical application of acetone extracts of T. vogelii (Figure 2). When acetone extracts were diluted 50 to treat the earlier stage of the 5th instar larvae (1–8 h after ecdysis), 74.4% of larvae died, and the others turned into malformed pupae. It was found that the deformation caused by rotenone and its analogues mainly resulted from the influences on decomposition of the old cuticle and synthesis of new cuticle, and significant differences in content of the chitin, total fat, and total protein between the treatment and the check occurred.
Fig. 2. Variations of the malformed pupae of 5th-instar larvae of Pieris rapae after being treated by topical application with acetone extracts from leaves of Tephrosia vogelii (a–f) as compared with untreated control (ck).
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The acetone extract of T. vogelii also caused 65, 93, and 100% antifeedant effects to 4th instar P. xylostella at 0.01, 0.05, and 0.1 mg extract/l (0.114, 0.57, and 1.14 ml/ cm2), respectively, measured as cabbage leaf area eaten, compared with 71, 84, and 85% antifeedant to a 20% EC AZT-VR-K at the same concentrations, respectively (Xu unpublished data).
Toosendanin and extracts from Melia azedarach and M. toosendan Melia azedarach L. and M. taosendan Sieb. et Zucc. (Meliaceae) are widely distributed in China and elsewhere, and the former has been naturalized in the southeastern U.S. Both species contain toosendanin, the most important active ingredient of M. azedarach and M. toosendan, M. azedarach also contains a compound of two interchangeable isomers, the new isomer was called meliartenin. It possesses similar activity to that of azadirachtin and greater than the one showed by toosendanin (Carpinella et al., 2002), which was first isolated and identified in China and was mainly used as anthelmintic against ascariasis (Chiu, 1995; Wang et al., 1999). It possesses strong antifeedant activity at high concentrations against the larvae of Tryporyza incertulas (Walker), Spodoptera venalba, P. xylostella, and Ostrinia furnacalis (Guenee), and acts as a stomach poison at low concentration against larvae of T. incertulas and P. rapae, and it can also inhibit growth (Wang et al., 1992). Methanol extracts of the seed kernels of M. azedarach and M. toosendan, as well as toosendanin were very effective against S. litura as an antifeedant (Chiu, 1993). Fifth instar S. litura larvae were so sensitive to these materials that the AFC50 was found to be as low as 0.0027% in the leaf disc choice test. The 2nd instar larvae reduced feeding by 99.6% in choice tests when the leaves were treated with 1% methanol extract of M. azedarach. The acetone extracts of the leaves, bark, and stems of the two species of chinaberry also were effective as feeding deterrents at a concentration of 1–2% (weight of plant tissue/volume of the solvent). Both the methanol and ethanol extracts showed promising antifeedant activity against the larvae of P. rapae in both choice and no-choice tests. However, it was interesting to find that the larvae almost stopped eating either the treated or untreated leaves after feeding on the bioactive materials for several hours. The larvae were killed when treated with high concentrations of the plant extracts. Growth inhibition, oviposition deterrence, and egg-hatching disruption from treatments with lower concentrations were observed. Mehta et al. (2002) also found that petroleum ether extracts of M. azedarach had significant antifeedant activity on 3rd instar larvae of Pieris brassicae L. Experiments with radioactive isotopes indicated that 3H-toosendanin eaten by P. rapae was found in the blood and then transported to various organs. The phenomenon of antifeeding occurred only after 3H-toosendanin reached the blood. There seemed to be a correlation between the fluctuations of 3H-toosendanin in blood and the response of the insects (Zhang and Chiu, 1991, 1992; Ce´spedes et al., 2000). Zhang and Chiu (1991, 1992) found that by feeding or injecting 5th instars at the dosage of E1–3 mg toosendanin per P. rapae larva, pathological changes in histology could be observed, indicating that the midgut tissues were destroyed. The peritrophic membrane disappeared, so that the food in the gut contacted and damaged the ventriculus tissue.
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The results of field plot trials demonstrated that the vegetables were effectively protected from the injury of P. rapae larvae by spraying these plant extracts. Toosendanin at a concentration of 0.1% gave an effecticeness approximate to that of 0.01%. Therefore, a commercial formulation of toosendanin, 0.5% Toosedarin EC has been recently developed recently by The Botanical Pesticide Research and Development Center, Northwest A&F University at Yangling, Shaaxi, China. Wang et al. (1994) reported isolation of melianoninol, melianone, melianol, meliandiol, meliantriol, vanillic acid, vanillin, and toosendanin from the fruits of M. azedarach and their biological activities toward P. rapae. They found that the triterpenes melianone, melianol, meliandiol, meliantriol, and toosendanin possessed antifeedant activities against P. rapae larvae and only toosendanin showed considerable toxicity to the insect on consumption. The bio-activities of melianoninol, vanillic acid, and vanillin were much lower than the other compounds.
Parthenin, argentatins, and extracts from Parthenium spp. The potentials of extracts in the genus Parthenium (Asteraceae or Compositae), including P. argentatum Gray, P. hysterophorus L., P. schottii Greenman, and P. tomentosum DC. for pest management have been causing considerable attention in recent years. The extracts and chemical compounds from these plants that exhibit insecticidal activities include parthenin, argentatins, and many others. Parthenium argentatum Parthenium argentatum, or guayule, is a shrub containing high amounts of natural rubber. It grows wild in northern Mexico and southwestern US. The plant’s trichomes and pollen contain toxins called sesquiterpene lactones. The major component of these toxins are parthenin and other phenolic acids such as caffeic acid, vanillic acid, anisic acid, chlorogenic acid, parahydroxy benzoic acid, and p-anisic acid, which are lethal to humans and animals (Bauer, 1998). The de-rubberized resin from P. argentatum has been tested for potential as an ovicidal and feeding deterrent against S. exigua (Allan Showler, USDA-ARS, personal communication). Ce´spedes et al. (2001) evaluated argentatin A and B from P. argentatum for their effect on armyworm S. frugiperda (J.E. Smith) larvae. In nochoice diet bioassays with neonate larvae, argentatins A and B and methanol extract of P. argentatum significantly inhibited growth and increased development time. Also, a significant delay in time of pupation, adult emergence, and deformities was observed in most treated groups. Acute toxicity to adults of S. frugiperda was also found, with the methanol extract having an LD50 of 3.10 ppm. Methanol extract and argentatin A inhibited acetylcholinesterase by 93.7 and 90.0% at 5.0 and 50.0 ppm, respectively. Parthenium hysterophorus Although P. hysterophorus is one of the most undesirable weeds in the world (Aneja et al., 1991), its extracts have been studied for feasible botanical insecticides against cruciferous and other vegetable pests, including A. gossypii and L. erysimi, S. litura, and H. armigera.
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Patil et al. (1993) tested the cold alcohol extracts of P. hysterophorus against A. gossypii in the laboratory. Extracts of P. hysterophorus were more toxic against the aphids than the neem extracts. Sohal et al. (2002) tested the petroleum ether extracts of leaves, stems and inflorescence of P. hysterophorus at 500, 1000, 2000, and 5000 mg/ml concentrations in the laboratory for their toxic effects on the mean life span and progeny production of adults of the mustard aphid, L. erysimi. They found a significant decrease in life span and progeny production with all treatments. Among the three plant parts tested, the leaf extract showed the most significant effect in decreasing life span and progeny production in a dose-dependent manner. Gajendran and Gopalan (1981) evaluated the ovicidal activity of various extracts of P. hysterophorus against eggs of S. litura, and they found that the extracts of leaves of young plants, old leaves, inflorescence, stem, and roots applied topically all inhibited egg hatch; the leaf and inflorescence extracts completely prevented hatching when applied to eggs 0–24-h old, while the root and stem extracts inhibited 62.5 and 40.0% hatch, respectively, when applied at a rate of 50 mg/10 eggs. Eggs (48–72-h old) were least affected by low doses (2, 5, and 10 mg) of the extracts. In another laboratory study, Gajendran and Gopalan (1982) found the antifeedant activity of 3% extract from young leaves of P. hysterophorus against the 3rd instar larvae of S. litura could be as high as 55.58%. They also found that the extract from young leaves was superior to the other extracts (inflorescence, leaves, root, and stem from mature plants). Datta and Saxena (2001) studied 11 sesquiterpene lactone derivatives of parthenin from P. hysterophorus, including a pyrazoline adduct of parthenin, its cyclopropyl and propenyl derivatives, anhydroparthenin, a dihydro-deoxygenated product, a formate and its corresponding alcohol and acetate derivatives, a rearranged product, lactone, and hemiacetal. They found that all these derivatives, along with parthenin, had antifeedant action against 6th instar S. litura. They also found that pyrazoline adduct to be the most effective as an insecticide, with LC50 values after 24, 48, and 72 h of 96, 43, and 32 mg/l, respectively. Sundararajan (2002) tested the methanol extract of P. hysterophorus for its insecticidal activity against the fourth instar H. armigera by applying dipping method of the leaf extracts at various concentrations (0.25, 0.5, 1.0, 1.5, and 2.0%). The highest concentration (2%) caused >50% larval mortality 48 h after treatment. Sundararajan (2003) further evaluated the aqueous leaf extracts of P. hysterophorus and two other plant species for their biological activities against the larvae of H. armigera using a dipping method of the leaf extracts at 2, 4, 6, 8, and 10% concentrations. A larval mortality of more than 50% at higher concentrations (8 and 10%) was observed in all the extracts. A reduction in the rate of food consumption and growth was observed in the fourth instar larvae of H. armigera 48 h after treatment in all the extracts at 10% level. Parthenium schottii and P: tomentosum Isman and Rodriguez (1984) used artificial diets incorporating powdered foliage or foliar extracts of P. tomentosum or P. schottii and observed inhibited feeding, growth and dietary use by 3rd-instar larvae of S. exigua relative to larvae feeding on diets incorporating foliage extracts from P. argentatum or an F1 hybrid with P. tomentosum. A mixture of sesquiterpene lactones isolated from P. schottii added to an artificial diet
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inhibited feeding, but did not deter growth or dietary use by larvae. Foliage or foliar extracts of P. schottii added to diets also significantly inhibited growth and dietary use by 3rd-instar larvae of Heliothis zea (Boddie), although there was no reduction in consumption rate. In contrast, powdered inflorescences of P. schottii or extracts thereof, both rich in sesquiterpene lactones, deterred neither feeding nor growth of H. zea when added to artificial diets. Confertin, the principal sesquiterpene lactone of P. schottii foliage and inflorescences, had no effect on feeding or growth of 3rd-instar larvae, even at a concentration which strongly inhibits growth of neonate H. zea larvae. Chemical factors in P. schottii and P. tomentosum foliage appear to inhibit growth of both S. exigua and H. zea, although a contribution to that inhibition by sesquiterpene lactones is uncertain.
Rhodojaponin and extracts from Rhododendron molle G. Don Rhododendron molle G. Don (Ericaceae) is commonly known as yellow azalea, and is well known for its medical and insecticidal uses in China. Several isolates from R. molle have been identified and tested for insecticidal activities against insect pests (Hu et al., 1993; Zhong et al., 2000a, 2001a, 2001b, 2001c; Cheng et al., 2002). Cheng et al. (2000) found that rhodojaponin-III (R-III) was the main toxic component of R. molle and the content of R-III in flower, leaf, base, and stem was about 0.20, 0.14, 0.11, and 0.08%, respectively. Its ethanol extract (1%) of the flower, leaf, and base had antifeedant effects against the larvae of P. rapae and S. litura. The antifeedant rates of flower, leaf, and base to 5th instar P. rapae larvae were 92.8, 86.8, and 74.9%, respectively, and to 3rd instar S. litura were 89.9, 80.1, and 73.5%, respectively. However, the extract of the stems had antifeedant effects. R-III exhibited feeding deterrence against larvae and adults of L. sativae (Hu et al., 2000b), and 500 mg/ml of R-III provided 77.3 and 67.7% of feeding inhibition on 2nd and 3rd instars, respectively. The LC50 values were 208.65 and 300.62 mg/ml against the 2nd and the 3rd instars of L. sativae, respectively. The LC50 value of RIII against L. sativae adults was 159.07 mg/ml 24 h after treatment. Zhong et al. (2000b) determined the activities of R-III, ethyl acetate (EtOAc) extract of R. molle flower extracts as oviposition deterrents and ovicides against P. xylostella. Application of R-III at 0.5 g/l reduced oviposition by 77.5%, and egg eclosion rate by 80.1%, respectively, compared with 5 g EtOAc extract/l which resulted in 55.9% reduction of oviposition and 66.0% of egg mortality 72 h after treatment. Zhong et al. (2001a) determined the insecticidal activities of R-III against S. litura with different extractions. Rhododendron molle flowers were first extracted by methanol and the extract was partitioned with petroleum ether, dichloromethane, and EtOAc with two different extraction methods. The results showed that the EtOAc partitions had the highest activity among the four partitions. R-III isolated from EtOAc extract was the most active constituent compared with other fractions. The AFC50 of R-III against the 4th instar S. litura was 16.48 mg/ml, the LC50 was 16.42 mg/ larva, and the IC50 was 41.70 mg/ml. R-III also had significant antifeeding activity on S. litura (Cheng et al., 2002). The AFC50 values of R-III against the 5th instar S. litura were 16.93, 19.31, and 16.96 mg/ml 24, 48, and 72 h after treatment, respectively. Cheng et al. (2002) studied that antifeeding and toxicity of R-III to S. litura larvae. They found that the AFC50s of R-III against the 5th instar S. litura were 16.93,
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19.31, and 16.96 mg/ml 24, 48, and 72 h after treatment, respectively. After application of R-III at 100 mg/ml, the development of S. litura larvae was as long as 22.4 days, the larval mortality was as high as 39.7%, and the pupating rate was only 34.0%. The weight of the female pupa was reduced to 0.296 g/pupa. They also found that after application of R-III at 500 mg/ml on 3rd instar P. xylostella, the mortality was 53.3% at 4 days after treatment and the pupating rate was only 16.7%. Zhong et al. (2001b) determined the insect growth regulating properties of R-III and EtOAc extracts from R. molle flowers against P. rapae. The IC50 of R-III and EtOAc extract were 6.78 and 70.29 mg/ml against 3rd instars and 13.72 and 346.00 mg/ml against 5th instars, respectively. R-III and EtOAc extract also reduced pupating rate, pupal weight, emergence rate, and extended the duration of development. Thus, development of insects was inhibited significantly and development index decreased. These results also showed that the R-III was superior to toosendanin as an insect growth regulator. Hu et al. (2002) found that ingestion of R-III (1, 3, and 5 mg/dish) by the 5th instar larvae of P. rapae reduced the total haemolymph volume by 40–50%, protein content by 40–50%, carbohydrate content by 14–30%, and glyceride content by an average of 77%, compared with the controls. These results indicated that application of R-III inhibited larval growth and development. Besides R-III, 13 other compounds (numbered from 1 to 13) were isolated from R. molle (Zhong, 2002). Testing with P. rapae and P. xylostella, Zhong (2002) found that the 13 compounds had different insecticidal activities on the two pests: compounds 7, 8, and 10 possessed strong oviposition deterrent activities with 53–75% reduction of P. rapae eggs and 58 to 77% reduction of P. xylostella eggs; compounds 8 and 10 had high antifeeding activities of both P. rapae and P. xylostella; compounds 8, 10, and 12 had insect growth regulating properties (inhibiting larvae’s weight increase and growth, reducing the generation potency and affecting growth rate) on P. rapae; compounds 5 and 10 reduced pupating rate and emergence rate and sublethal effects on oviposition; compounds 4, 8, and 10 were strongly toxic to P. rapae larvae; compounds 5, 6, 7, 8, 9, 10 had strong stomach toxicity to 5th instar P. rapae; and compounds 7, 8, 10, and 11 significantly reduced egg-hatching rates of P. xylostella by 40–58%. To understand the mode of action and optimize rhodojaponin structure, Zhong et al. (2004) also determined the effects of 14 active compounds, previously isolated from R. molle on the cuticle components of S. litura larvae and their structure–activity relationship. The structures of three new compounds, rhodomolin A (R-A), B (R-B), and C (R-C), were elucidated based on IR, UV, NMR, and MS spectroscopic data. When the 4th instar larvae of S. litura were treated with 50 mg/l of R-I, R-III, R-A, R-B, R-C, R-XVIII, grayantoxin III, and azadirachtin, the weight of larvae and pupae and the emergence rate were significantly less than the control, exhibiting greater insect growth inhibition activities than the tested compounds from R. molle. When treated with R-I, R-III, R-A, R-B, R-C, R-XVIII, and azadirachtin, the contents of total larval cuticle protein significantly increased and those of total fats decreased than those of the control, respectively. Furthermore, the contents of water-soluble protein, hydrogen bond combined protein and covalent bond combined protein in the cuticle decreased significantly, while the content of weak bond combined protein increased significantly. However, the content of electrovalence bond
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combined protein was not significantly different from that of the control when larvae were treated with R-III and azadirachtin. They concluded that the mode of action of insect growth inhibition in rhodojaponins is not to inhibit chitin syntheses, but to disturb internal secretion, and significantly decreasing the content of water-soluble protein in cuticle. Their preliminary analysis of the structure–activity relationship showed that substitute group structures of C-2,3-epoxy, C-6, C-10, and C-14 were most important to these grayanoid diterpenids with insect growth-inhibition activity.
Alkaloids and extracts from Sophora alopecuroids L. Sophora alopecuroids L. (Papilionaceae) is known as kudouzi (means ‘‘bitter (ku) bean (dou) seed (zi)’’ in Chinese). It is a perennial leguminous plant with a wide distribution in northwest China. Sophora alpecuroids is also well known as having an abundance of alkaloids with a wide range of useful biological activities including insecticidal activity against some of vegetable pests (Luo, 1995; Luo and Li, 1996; Luo et al., 1996, 1997, 1999; Luo and Zhang, 2003). In the past few years, seven alkaloids (sophoramin, cytisin, matrine, oxymatrine, sophoradine, sloperine, and sophocarpine) were found in methanol and chloroform extracts from S. alopecuroids (Luo and Li, 1996). The major alkaloids in the methanol extracts include 4.29% of matrine, 6.32% of oxymatrine, 1.40% of sophocarpine, 4.29% of sophoramin, and 0.70% of cytisin. The chloroform extract had a slightly different amount of alkaloids, 4.50% of matrine, 8.56% of oxymatrine, 0.68% of sophocarpine, 2.70% of sophoramin, and 0.63% of cytisin. Luo et al. (1996) conducted bioassays of the two extracts from S. alopecuroids against L. erysimi. In the dipping bioassays, the LC50 values for the apterous adults of L. erysimi were 0.8 to 1.2 g/l for both extracts, and no significant difference was detected. In the spray bioassays, the LC50 values for L. erysimi nymphs were 0.7 to 1.1 g/l for both extracts, and, again, there were no significant differences between the two extracts. In another experiment, Luo et al. (1997, 1999) found that the LC50 values against L. erysimi for the three alkaloids, cytosine, anabasine, and nicotine (the latter two as controls), were 432.59, 648.70, and 1090.65 mg/ml, respectively, 48 h after treatment using the cage-dip method, and the results again indicate that cytisine is the most toxic alkaloid against the aphid. Luo et al. (1997) also tested the efficacies of the eight alkaloids from the two extracts of S. alopecuroids on apterous L. erysimi. Results indicated that cytisine was the most effective alkaloid with 96.7 and 100% mortality at 1000 and 2000 P(A)(mg/ml) 48 h after treatment, and the second most effective alkaloid is aloperine which gave much lower mortality, 43.3 and 45.0% at the two concentrations, respectively. All others had little activity on the aphids with 5.0–36.7% mortality. In a field trial on cabbage, Luo and Li (1996) found that the 50% methanol extract effectively reduced the population of L. erysimi when applied on cabbage plants at field rates. The controls were 64.8, 81.1, and 88.7% at 1.0, 5.0, and 10 g/l field rates, respectively, 3 days after application, compared with the untreated control. Similarly, the controls were 78.8, 83.3, and 89.1% at the three field rates, respectively, 5 days after application, compared with the untreated control. At 7 days after application,
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percentages of control were similar to those 6 days after application, ranging from 36.4 to 84.1%. In another field study on cabbage plants, Luo and Li (1996) also found that the methanol extract at 1.0, 5.0, and 10 g/l concentrations exhibited significant repellent activity. Percentages of population reduction were 53.8, 78.1 and 74.2 at 1.0, 5.0 and 10 g/l concentrations, respectively, 3 days after application compared with the untreated control. Similarly, the percentages of reduction were 73.5, 64.5, and 74.3 at 0.05, 0.10, and 0.20% 6 days after application, compared with untreated control. At 9 days after application, percentages of population reductions were similar to those at 6 days after application, ranging from 64.5 to 74.2%. The alkaloids from the extracts from S. alopecuroids exhibited significant synergistic activities when mixed with three commonly used insecticides, profenofos, abamectin, and methomyl (Zhang and Luo, 2002). Abemectin exhibited greatest synergistic effects when it was mixed with cytosine, sophoradine, aloperine, and oxymatrin, followed by matrine, and no synergistic effects when it was mixed with sophocarpine or sophoramine. Methomyl displayed synergistic effects when it was mixed with all alkaloids, although the synergism was not strong when it was mixed with matrine. Profenofos showed distinct synergistic effects when it was mixed with sophoradine, oxymatrine, sophoramine, or cytisine, and lower synergism when it was mixed with aloperine, matrine, or sophocarpine. Luo and Zhang (2003) studied the effects of the seven alkaloids from S. alopecuroids on metabolic esterases of the larvae of P. xylostella. Their results indicate that cytisine and aloperine could inhibit carboxylesterase activity through noncompetitive inhibition. Sophoramine, sophoradine, matrine, oxymatrine, and cytisine could inhibit the activity of acid phosphoresterase, and cytisine could also weakly inhibit the activity of alkaline phosphoresterase. In addition, three alkaloids, cytisine, sophoramine, and sophocarpine could inhibit the activity of glutathione-Stransferase [glutathione transferase] in P. xylostella larvae.
Extracts from Stellera chamaejasme L. Stellera chamaejasme L. (Chinese Starwort or Chinese stellera) is a poisonous plant and is reputed to have medicinal and insecticidal value. It is also a repellant and contact-poison, possibly acting as a stomach poison (Ji et al., 2000; Zhang et al., 2000a, 2000b, 2000c, 2000d, 2002). The antifeedant rate to 5th instar P. rapae was 56.07% by topical application 24 h after at the dosage of 25 mg/larva (Zhang et al., 2000a). Furthermore, the root extract of S. chamaejasme with ethanol also exhibited oviposition deterrent activity and ovicidal activity against P. rapae (Zhang et al., 2000c). Zhang et al. (2000d, 2002) determined the insecticidal activities of the ethanol extract of root of S. chameajasme against P. rapae and Myzus persicae, and they found that the LD50 for the 5th instar P. rapae by stomach poison using the leafdisc sandwich method was 12.32 mg/larva at 2 days after treatment. Leaf dipping in 5 mg/ml extract, the corrected mortality for the 5th instar P. rapae was 100% at 7 days after treatment, and the value of AFC50 for the same larvae was 0.3813 mg/ml. Numerous chemicals have been isolated from the plant (Su et al., 2003), and of these, 7-hydro-coumarin, daphnoritin, and chamaechromone have strong antifeedant
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and growth inhibition activities to 5th instar P. rapae (Wang et al., 2002). The AFC50 of daphnoritin and chamaechromone to the 5th instar P. rapae were 160.57 mg/ml and 229.49 mg/ml 24 h after the treatment and 76.94 mg/ml and 131.30 mg/ml 48 h after the treatment, respectively (Zhang et al., 2000b). Physio-biochemical studies revealed that daphnoritin increased the content of sugar, while it decreased the content of protein in haemolymph. In contrast, chamaechromone decreased the content of sugar and increased the esterase activity in midgut significantly (Zhang et al., 2000d). Wang et al. (2002) studied the biological activity of an ethanol extract from the root of S. chamaejasme collected from Qinghai, China against, P. rapae, P. xylostella, O. furnacalis, S. litura, and M. persicae. Applied at 2.5, 5.0, and 50.0 mg/ml of the ethanol extract, the mortality of the 5th instar P. rapae, the 3rd instar P. xylostella, and the 3rd instar S. litura was 100, 31.0, and 16.7% 7 days after treatment, respectively. The LC50 of the ethanol extract against M. persicae was 0.599 2 mg/ml 2 days after treatment using a leafdipped method. With the ethanol extract at 10 mg/ml for the 3rd instar O. furnacalis, the mortality was 65.5 and 85.7% 7 and 14 days after treatments, respectively. These results showed that the ethanol extract had strong biological activity against P. rapae, O. furnacalis, and M. persicae, whereas it had weak biological activities against S. litura and P. xylostella.
Extracts from Strophanthus divaricatus (Lour.) Hooker & Arnnott Strophanthus divaricatus (Lour.) Hooker & Arnnott (Apocynaceae) [(called ‘‘yang jiao niu’’ in Chinese (goat horn)], is widely distributed in south China. The water extract of S. divaricatus showed contact poison to insects including Coptosoma cribraria ( ¼ cribrarium) (Fabricius) and Tryporyza incertulas (Walker). The extracts of fruit, leaves, and stems of S. divaricatus have strong antifeedant and insecticidal properties on the 4th instar P. rapae. The main secondary metabolites in this plant are caudoside, F-caudostroside, divaricoside, sinlside, isosinotrodise, sarmutodise, and D-strophanthin. Hu et al. (2000a) found that the LC50 and LC95 values of D-strophanthin isolated from this plant were 32.0469 and 379.7181 mg/ml, respectively, against the 4th instar P. rapae. Results from filed trials showed that the commercial product, 0.05% strophanthin was useful for controlling P. rapae. Huang and Renwick (1994) studied the oviposition responses of P. rapae and P. napi oleracea to 18 cardenolides. They found that most of the compounds were deterrent to oviposition by both insects, but to significantly different degrees. P. rapae was strongly deterred by K-strophanthoside, K-strophanthin- beta, cymarin, convallatoxin, oleandrin, erysimoside, erychroside, and gitoxigenin. The most deterrent compounds for P. napi oleracea were erychroside, cymarin, erysimoside, convallatoxin, and K-strophanthoside. They also found that strophanthidinbased glycosides were more of a deterrent than digitoxigenin-based ones, and the number and type of sugar substitutions had profound effects on activity. Cymarin was equally acting as a feeding deterrent to both pest species at all concentrations tested. However, when compared with P. rapae, P. napi oleracea was less sensitive to most cardenolides. P. napi oleracea was insensitive to K-strophanthin-beta and oleandrin at 0.5 104 M, which were an excellent feeding deterrent to P. rapae.
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Extracts from Tripterygium wilfordii Hook. F. and T. hypoglaucum (level) Hutch. Tripterygium wilfordii Hook. F. and T. hypoglaucum (level) Hutch. (Celastraceae) are widely distributed in China and have been used as traditional botanical insecticides and medicinal plants because they contain numerous bioactive chemicals (Yang et al., 2000). Tong and Chiu (1988) determined the toxicity of the extracts of T. wilfordii and T. hypoglaucum against P. rapae larvae in the laboratory and under field conditions. They found that the extracts from the root bark of the two plant species have strong antifeedant, stomach poisons, and growth inhibition properties. Larvae fed on the extracts were paralyzed, and the mid-gut epithelium of their alimentary canal was affected. The larvae finally died of starvation. Tong and Chiu (1988) also found that both alkaloids and nonalkaloids from T. hypoglaucum had high antifeedant activities against the 5th instar P. rapae, and the activities are in the following order: wilforine>total alkaloids>nonalkaloids. The activities of alkaloids from T. wilfordii are in the order: wilfordine>wilforine>wilfortrine. After feeding with extracts from T. wilfordii or wilforine, black spots appeared on the body of larvae (Tong and Chiu, 1988). Microscopic examination of the black area of the larval cuticle shows that the cuticle became thickened and tumors appeared between the epidermis and endocuticle. The epidermis was destroyed, the endocuticle separated from the epidermis and vacuoles formed. Wilforine affected the respiration of the larvae of P. rapae and their body weight was decreased. After eating cabbage leaves treated with wilforine, the nervous system of the larvae was poisoned and showed paralysis; their respiratory rhythm-waves became lower and the CO2 output decreased. The larvae treated with wilforine and those fully starved showed the same change trends in body weight and respiratory rates. It was found that the alimentary canal and the fat bodies were shriveled, and the epithelium of mid-gut and the peritrophic membrane were destroyed. Luo et al. (2004) studied the ethanol extract of the root bark of T. wilfordii and found it had strong insecticidal activities against larvae of M. separata. Three active compounds were isolated by bioassay-guided fractionation of the extract and characterized as triptolide, triptonide, and euonine by IR, 1 H, and 13C NMR and mass spectral analysis. They found that triptolide and triptonide had strong contact activities against 3rd or 5th instar M. separata (LD50 values: 1.6 and 2.9 mg/larva for triptolide and triptonide, respectively), but euonine had no contact activity against the larvae. Antifeedant activity against the 3rd instar M. separata 24 h after treatment was demonstrated; triptolide, triptonide, and euonine gave EC50 (the concentration that produces a level of effect that equals to 50% of the maximum effect) values of 0.25, 0.35, and 0.02 mM, respectively. Activities of triptolide and triptonide were inferior to the positive control represented by toosendanin, whereas euonine was superior to toosendanin. In ingestion bioassays against M. separata, triptolide had the more potent activity with a KD50 (the time taken to knockdown 50% of insects) value of 13.5 mg/g (insect body weight) than toosendanin. Field trials demonstrated that cabbage was well protected from the damage of the larvae of P. rapae after application of 3% ethanol extract from the root bark of T. wilfordii, and the natural enemies of the larvae were not affected (Chiu, 1987; Tong and Chiu, 1988).
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Extracts from Xanthium sibiricum Patrin ex Widder Xanthium sibiricum Patrin ex Widder (Asteraceae) have been used to control some insects for over a thousand years, and its extract was recorded as an effective pesticide against P. rapae in ‘‘Chinese Indigenous Pesticides’’ in 1959 (Peng, 1984). Recent studies showed that it contained many chemicals, such as xanthostrumarin, carboxyatractyloside, xanthatin, atractyloside, xanthin, xanthunin, xanthanodiene, strumaroside, etc. Bioassays indicated that xanthatin and atractyloside exhibited potent stomach-poison and growth inhibition activities as well as antifeedant activity against many insects. He et al. (unpublished data) found that the LD50 of xanthatin against the 5th instar P. rapae was 2.07 mg/larva 24 h after treatment. When treated at the dosage of 50 mg xanthatin/ml, larval pupation density of P. rapae was reduced by 73.3% 4 days after application treatment compared with untreated control. They also studied the mode of action of xanthatin against insects, and found that the contents of sugar and protein in haemolymph of P. rapae larvae decreased significantly, and the activity of esterase in the mid-gut was significantly inhibited. In addition, Zhou et al. (2002) found that the semiochemicals extracted from X. sibiricum deterred feeding and reduced reproduction of M. persicae and L. erysimi. The deterrence rates were 48.1 and 55.7% to M. persicae and L. erysimi, respectively. They also found that the reproduction of the females was reduced by 39.0 and 63.5% for to M. persicae and L. erysimi, respectively. Wang et al. (1999) studied 12 plant species collected from Jiangsu Province, China and tested their extracts for their insecticidal properties against P. rapae. They found that the extracts of X. sibiricum inhibited larval growth and was a stomach poison, but did not act as an antifeedant.
Others Besides the insecticidal plants mentioned above, there is a large number of Chinese medicinal plants that seem to have insecticidal properties. Preliminary studies showed that the following 23 species of plants possessed high antifeedant rates against either P. xylostella or S. litura (Table 2) (Chen and Xu unpublished data). All 23 plant species were extracted with a mixture of methanol, acetone and chloroform (23:30:47) except Osmunda japonica Thunb., Aconitum hemsleyanum Pritz, Picrasma quassioides (D. Don) Benn.(root), Sapium sebiferum (L.) Roxb (stem), Davidia involucrate Baill. var. vilmoriniana (Dode) Hemsl., Pharbitis purpurea (L.) Voigt and Emmenopterys henryi Oliv. (stem), which were extracted with petroleum ether. The results indicate that most of tested plant extracts have strong antifeedant activities on P. xylostella and S. litura with a few exceptions. These results also suggested that more studies are needed to test those with promising insecticidal activities.
Final remark: the pros and cons of botanical insecticides Botanically-derived insecticides
