Advances in
FOOD AND NUTRITION RESEARCH VOLUME
55
ADVISORY BOARDS KEN BUCKLE University of New South Wales, Australia
MARY ELLEN CAMIRE University of Maine, USA
ROGER CLEMENS University of Southern California, USA
HILDEGARDE HEYMANN University of California, Davis, USA
ROBERT HUTKINS University of Nebraska, USA
RONALD JACKSON Quebec, Canada
HUUB LELIEVELD Global Harmonization Initiative, The Netherlands
DARYL B. LUND University of Wisconsin, USA
CONNIE WEAVER Purdue University, USA
RONALD WROLSTAD Oregon State University, USA
SERIES EDITORS GEORGE F. STEWART
(1948–1982)
EMIL M. MRAK
(1948–1987)
C. O. CHICHESTER
(1959–1988)
BERNARD S. SCHWEIGERT (1984–1988) JOHN E. KINSELLA
(1989–1993)
STEVE L. TAYLOR
(1995–
)
Advances in
FOOD AND NUTRITION RESEARCH VOLUME
55 Edited by
STEVE L. TAYLOR University of Nebraska, Lincoln
AMSTERDAM • BOSTON • HEIDELBERG • LONDON NEW YORK • OXFORD • PARIS • SAN DIEGO SAN FRANCISCO • SINGAPORE • SYDNEY • TOKYO Academic Press is an imprint of Elsevier
Academic Press is an imprint of Elsevier 84 Theobald’s Road, London WC1X 8RR, UK Radarweg 29, PO Box 211, 1000 AE Amsterdam, The Netherlands Linacre House, Jordan Hill, Oxford OX2 8DP, UK 30 Corporate Drive, Suite 400, Burlington, MA 01803, USA 525 B Street, Suite 1900, San Diego, CA 92101-4495, USA First edition 2009 Copyright # 2009 Elsevier Inc. 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. ISBN: 978-0-12-374120-2 ISSN: 1043-4526 For information on all Academic Press publications visit our website at elsevierdirect.com Printed and bound in USA 09 10 11 12 10 9 8 7 6 5 4 3 2 1
CONTENTS
Contributors
vii
1. Ginsenosides: Chemistry, Biosynthesis, Analysis, and Potential Health Effects
1
Lars P. Christensen Introduction Chemistry Biosynthesis Analysis Potential Health Effects of Ginsenosides Conclusion References I. II. III. IV. V. VI.
2. Adherence, Anti-Adherence, and Oligosaccharides: Preventing Pathogens from Sticking to the Host
2 23 37 45 64 79 80
101
Kari D. Shoaf-Sweeney and Robert W. Hutkins Introduction Route of Infection Adherence Basics Specific Pathogen–Host Interactions Intestinal Target Tissues Bacterial Adhesins Common Bacterial Adherence Mechanisms Anti-Adhesives Conclusions and Future Prospects References I. II. III. IV. V. VI. VII. VIII. IX.
3. Lung Disease in Flavoring and Food Production: Learning from Butter Flavoring
102 103 105 108 111 114 117 128 139 140
163
Nancy Sahakian and Kathleen Kreiss I. II. III. IV.
Introduction Respiratory Tract Anatomy and Defense Mechanisms Medical Tests Used to Diagnose Lung Disease Types of Occupational Respiratory Disease
164 165 165 169
v
vi
Contents
V. Flavoring-Related BO VI. Recognition of Emerging Occupational Respiratory Disease
in the Food Industry
179 186
VII. Prevention of Known Occupational Respiratory Diseases
in the Food Industry References
4. Beneficial Health Properties of Psyllium and Approaches to Improve Its Functionalities
187 188
193
Liangli (Lucy) Yu, Herman Lutterodt, and Zhihong Cheng I. Introduction II. Beneficial Health Effects of Psyllium III. Approaches to Improve the Functionality, Safety,
and Biological Activity of Psyllium References
5. Starch Gelatinization
194 195 204 215
221
Wajira S. Ratnayake and David S. Jackson Introduction Starch: Importance and Sources Starch Structure and Crystallinity Starch Gelatinization Theories and Models Starch Annealing and Its Relationship to Gelatinization Glass Transition and Gelatinization Contradicting Theories: What Is Gelatinization? References I. II. III. IV. V. VI. VII.
Index
222 223 224 230 253 255 258 260 269
CONTRIBUTORS
Numbers in parentheses indicate the pages on which the authors' contributions begin.
Zhihong Cheng
Department of Nutrition and Food Science, University of Maryland, College Park, Maryland 20742, USA (193) Lars P. Christensen
Research Center Aarslev, Department of Food Science, Faculty of Agricultural Sciences, University of Aarhus, Kirstinebjergvej 10, DK-5792 Aarslev, Denmark (1) Robert W. Hutkins
Department of Food Science and Technology, University of NebraskaLincoln, Lincoln, Nebraska 68583, USA (101) David S. Jackson
Department of Food Science and Technology, University of NebraskaLincoln, Lincoln, Nebraska, 68583-0919, USA (221) Kathleen Kreiss
Centers for Disease Control and Prevention, National Institute for Occupational Safety and Health, Division of Respiratory Disease Studies, Morgantown, West Virginia 26505, USA (163) Herman Lutterodt
Department of Nutrition and Food Science, University of Maryland, College Park, Maryland 20742, USA (193) Wajira S. Ratnayake
Department of Food Science and Technology, University of NebraskaLincoln, Lincoln, Nebraska, 68583-0919, USA (221) Nancy Sahakian
Centers for Disease Control and Prevention, National Institute for Occupational Safety and Health, Division of Respiratory Disease Studies, Morgantown, West Virginia 26505, USA (163)
vii
viii
Contributors
Kari D. Shoaf-Sweeney
School of Molecular Biosciences, Washington State University, Pullman, Washington 99164, USA (101) Liangli (Lucy) Yu
Department of Nutrition and Food Science, University of Maryland, College Park, Maryland 20742, USA (193)
CHAPTER
1 Ginsenosides: Chemistry, Biosynthesis, Analysis, and Potential Health Effects Lars P. Christensen*
Contents
I. Introduction II. Chemistry A. Chemical structure of ginsenosides and distribution in plants B. Ginsenosides produced during steaming and drying of ginseng C. Pharmacokinetics and metabolism of ginsenosides III. Biosynthesis IV. Analysis A. Sample extraction B. Thin-layer chromatography C. Gas chromatography D. High-performance liquid chromatography E. Nuclear magnetic resonance spectroscopy F. Capillary electrophoresis G. Enzyme immunoassay H. Near infrared spectroscopy V. Potential Health Effects of Ginsenosides A. Anticarcinogenic effects B. Immunomodulatory effects C. Anti-inflammatory and antiallergic effects D. Antiatherosclerotic and antihypertensive effect E. Antistress activities
2 23 23 32 33 37 45 45 46 48 49 61 62 62 63 64 64 69 71 72 73
*Research Center Aarslev, Department of Food Science, Faculty of Agricultural Sciences, University of Aarhus,
Kirstinebjergvej 10, DK-5792 Aarslev, Denmark Advances in Food and Nutrition Research, Volume 55 ISSN 1043-4526, DOI: 10.1016/S1043-4526(08)00401-4
#
2009 Elsevier Inc. All rights reserved.
1
2
Lars P. Christensen
F. Effects on the CNS G. Effect on metabolic processes H. Antidiabetic effects VI. Conclusion References
Abstract
74 76 77 79 80
Ginsenosides are a special group of triterpenoid saponins that can be classified into two groups by the skeleton of their aglycones, namely dammarane- and oleanane-type. Ginsenosides are found nearly exclusively in Panax species (ginseng) and up to now more than 150 naturally occurring ginsenosides have been isolated from roots, leaves/stems, fruits, and/or flower heads of ginseng. Ginsenosides have been the target of a lot of research as they are believed to be the main active principles behind the claims of ginsengs efficacy. The potential health effects of ginsenosides that are discussed in this chapter include anticarcinogenic, immunomodulatory, anti-inflammatory, antiallergic, antiatherosclerotic, antihypertensive, and antidiabetic effects as well as antistress activity and effects on the central nervous system. Ginsensoides can be metabolized in the stomach (acid hydrolysis) and in the gastrointestinal tract (bacterial hydrolysis) or transformed to other ginsenosides by drying and steaming of ginseng to more bioavailable and bioactive ginsenosides. The metabolization and transformation of intact ginsenosides, which seems to play an important role for their potential health effects, are discussed. Qualitative and quantitative analytical techniques for the analysis of ginsenosides are important in relation to quality control of ginseng products and plant material and for the determination of the effects of processing of plant material as well as for the determination of the metabolism and bioavailability of ginsenosides. Analytical techniques for the analysis of ginsenosides that are described in this chapter are thin-layer chromatography (TLC), high-performance liquid chromatography (HPLC) combined with various detectors, gas chromatography (GC), colorimetry, enzyme immunoassays (EIA), capillary electrophoresis (CE), nuclear magnetic resonance (NMR) spectroscopy, and spectrophotometric methods.
I. INTRODUCTION Ginsenosides are triterpenoid saponins found nearly exclusively in ginseng and they have been the target of a lot of research as they are believed to be the main active principles behind the claims of ginsengs efficacy. Ginseng refers to species within the genus Panax (Araliaceae
Ginsenosides
3
family) that comprise approximately 14 species of slow-growing perennial plants with fleshy roots (Choi and Wen, 2000). They grow naturally in the Northern Hemisphere in eastern Asia and North America and are cultivated in minor scale in the Northern part of Europe. The most widely used Panax species is P. ginseng (Korean or Asian ginseng). It was first cultivated around 11 BC and has a medical history (as wild herb) stretching back more than 5000 years (Yun, 2001a,b), and is considered as one of the most valuable medicinal herbs in traditional Asian medicine. Another member of the genus is P. quinquefolium (American ginseng), which was valued by the American Indians long before the arrival of Europeans in the New World and since the eighteenth century American ginseng has been cultivated in North America for medicinal purposes. Other commonly used Panax species in herbal medicine are P. japonicus (Japanese ginseng), P. notoginseng (Sanchi ginseng), and P. vietnamensis (Vietnamese ginseng). Ginseng is also one of the most commonly used herbal medicinal remedies by American consumers. In Europe, ginseng is also counted among the top retail products, but unlike the United States, where ginseng preparations are classified as dietary supplements, ginseng products in Europe and particularly in Germany are treated as drugs. To day, most of the research has been focused on P. ginseng that has been used in traditional Asian medicine as a tonic and a panacea that can promote longevity. Nowadays, ginseng is used mainly to increase resistance to physical, chemical, and biological stress and boost general vitality. This activity of ginseng has been described as ‘‘adaptogenic’’ in most of the alternative medicine literature. However, immune system modulation, antistress activities, and antihyperglycemic activities are among the most notable features of ginseng in in vitro studies and in clinical trials. Extensive recent in vitro, in vivo, and epidemiological research also suggests that ginseng may have a cancer preventive effect. The roots of ginseng is the plant part of ginseng that has mainly been used for medicinal purposes and consequently most investigations have been performed on ginseng roots, although medicinal effects of other plant parts of ginseng has been demonstrated. Up to now more than 150 naturally occurring ginsenosides have been isolated from roots, leaves/stems, fruits, and/or flower heads of Panax species, of which most of them can be classified into two groups by the skeleton of their aglycones, namely dammarane-type and oleanane-type (Fig. 1.1; Table 1.1). Furthermore, studies of changes in ginsenoside composition due to different traditional processing of ginseng roots such as white and red ginseng have been undertaken, and this has led to the identification of a series of ginsenosides in processed ginseng products with interesting bioactivities of which many are considered as being degradation products of naturally occurring ginsenosides.
4
Lars P. Christensen
FIGURE 1.1
Protopanaxadiol-type ginsenosides HO
R2O 20 S
12
3
R1O
6
20(S)-protopanaxadiol (20(S)-PPD, R1 = R2 = H)
No.
Ginsenosides
R1
R2
–Glc –Glc2–1Glc –Glc2–1Glc –Glc2–1Glc –Glc2–1Glc –Glc2–1Glc6– Malonoyl –Glc2–1Glc –Glc2–1Glc6– Malonoyl –Glc2–1Glc –Glc2–1Glc –Glc2–1Glc6– Malonoyl –Glc2–1Glc –Glc2–1Glc6– Malonoyl –Glc2–1Glc
–Glc –Glc6–1Ara(p)4–1Xyl –Glc6–1Ara(f)2–1Xyl –Glc6–1Glc3–1Xyl –Glc6–1Glc –Glc6–1Glc
15 16
Ginsenoside F2 Ginsenoside Ra1 Ginsenoside Ra2 Ginsenoside Ra3 Ginsenoside Rb1 Ginsenoside malonyl-Rb1 Ginsenoside Rb2 Ginsenoside malonyl-Rb2 Ginsenoside Rb3 Ginsenoside Rc Ginsenoside malonyl-Rc Ginsenoside Rd Ginsenoside malonyl-Rd Ginsenoside 20(S)–Rg3 Ginsenoside Rh2 Ginsenoside Rs1
–H –Glc6–1Ara(p)
17
Ginsenoside Rs2
18
Ginsenoside Rs3
19
Chikusetsusaponin la
–Glc –Glc2–1Glc6– Ac –Glc2–1Glc6– Ac –Glc2–1Glc6– Ac –Glc6–1Xyl
1 2 3 4 5 6 7 8 9 10 11 12 13 14
–Glc6–1Ara(p) –Glc6–1Ara(p) –Glc6–1Xyl –Glc6–1Ara(f) –Glc6–1Ara(f) –Glc –Glc –H
–Glc6–1Ara(f) –H –H (continued)
Ginsenosides
FIGURE 1.1
Protopanaxadiol-type ginsenosides (Continued ) HO
R2O 20 S
12
3
R1O
6
20(S)-protopanaxadiol (20(S)-PPD, R1=R2=H)
No.
Ginsenosides
R1
R2
20
Chikusetsusaponin III
–H
21
Chikusesusaponin VI
22 23 24 25
Gypenoside IX (¼ notoginsenoside Fd) Gypenoside XV Gypenoside XVII Notoginsenoside D
–Glc2–1Glc 6| 1 Xyl –Glc2–1Glc 6| 1 Xyl –Glc
26
Notoginsenoside Fa
27
Notoginsenoside Fc
28 29 30 31 32 33
Notoginsenoside Fe Notoginsenoside K Notoginsenoside L* Notoginsenoside L* Notoninsenoside R4 Pseudo-dinsenoside F8
34
Pseudo-ginsenoside RC1 Quinquenoside R1
35
–Xyl2–1Glc –Glc –Glc2–1Glc2– 1 Xyl –Glc2–1Glc2– 1 Xyl –Glc2–1Glc2– 1 Xyl –Glc –Glc6–1Glc –Glc –Glc2–1Glc –Glc2–1Glc –Glc2–1Glc 6| Ac –Glc2–1Glc6–Ac –Glc2–1Glc6–Ac or –Glc2–1Glc
–Glc6–1Glc
–Glc6–1Xyl
–Glc6–1Xyl –Glc6–1Glc –Glc6–1Glc6–1Xyl –Glc6–1Glc –Glc6–1Xyl –Glc6–1Ara(f) –Glc –Glc2–1Ara(p) –Glc6–1Glc –Glc6–1Glc6–1Xyl –Glc6–1Ara(p)
–Glc –Glc2–1Glc6–Ac or –Glc2–1Glc
5
6
Lars P. Christensen
FIGURE 1.1
Protopanaxadiol-type ginsenosides (Continued ) HO
R2O 20 S
12
3
6
R1O
20(S)-protopanaxadiol (20(S)-PPD, R1 = R2 = H)
No.
Ginsenosides
R1
R2
36
Quinquenoside I
–Glc
37
Quinquenoside II
38
Quinquenoside III
39 40
Quinquenoside V Yesanchinoside J
–Glc2–1Glc6– (E)-2-Butenoyl –Glc2–1Glc6– (E)-2-Octenoyl –Glc2–1Glc 6| Ac –Glc2–1Glc –Glc2–1Glc 6| Ac
–Glc6–1Glc –Glc –Glc6–1Glc4–1aGlc –Glc6–1Glc6–1Xyl
*Ginsenoside 30 and 31 has been given the name notoginsenoside L by different authors (Yoshikawa et al., 2001; Ma et al., 1999).
OR2 HO
20 R
12
3
R1O
6
No.
Ginsenosides
R1
R2
41 42 43
Ginsenoside 20(R)–Rd Ginsenoside 20(R)–Rg3 Ginsenoside 20(R)–Rs3
–Glc2–1Glc –Glc2–1Glc –Glc2–1Glc6–Ac
–Glc –H –H
Ginsenosides
FIGURE 1.1
Protopanaxadiol-type ginsenosides (Continued ) OH 25
HO 20 R
OH
12
3
6
HO
20(R)-dammarane-3 β, 12 β, 20, 25-tetrol (25-OH PPD, 44)
HO
GlcO
25 20 S
12
3
CH2OH
6
Glc1–2GlcO
Quinquenoside L2(45)
HO
R2O
25 20 S
OH
12
3
R1O
6
No.
Ginsenosides
R1
R2
46 47 48 49 50
Majoroside F4 Notoginsenoside A Quinquenoside L3 Vina-ginsenoside R8 Yesanchinoside H
–Glc –Glc2–1Glc –Glc –Glc2–1Glc –Glc2–1Glc
–Glc –Glc6–1Glc –Glc6–1Glc –Glc –Glc6–1Xyl
7
8
Lars P. Christensen
FIGURE 1.1
Protopanaxadiol-type ginsenosides (Continued ) HO R2O
25 20 S
OOH
12
3
6
R1O
No.
51 52 53 54 55 56
Ginsenosides
R1
R2 2 1
Floralginsenoside E Floralginsenoside F Floralginsenoside G Floralginsenoside O Notoginsenoside E Notoginsenoside K*
–Glc – Glc –Glc –Glc2–1Glc6–Ac –Glc2–1Glc –Glc2–1Glc –Glc2–1Glc
–H –Glc –Glc –Glc6–1Ara(f) –Glc –Glc6–1Glc
*The name notoginsenoside K has been used for ginsenoside 56 (Yoshikawa et al., 1997a, 1998, 2001) and for ginsenoside 29 (Ma et al., 1999; Sun et al., 2005b).
OH HO GlcO
24 20 S
25
12
3
RO
6
No.
Ginsenosides
R
57
Ginsenoside 24(R)–Rg7 Ginsenoside 24(S)–Rg7 (¼ Majoroside F2) 24(S)–Vina-ginsenoside R9 24(R)-Vina-ginsenoside R9 (¼ Majoroside F1)
–Glc
58 59 60
–Glc –Glc2–1Glc –Glc2–1Glc
Ginsenosides
FIGURE 1.1
Protopanaxadiol-type ginsenosides (Continued ) OOH HO R2O
24 20 S
25
12
3
6
R1O
No.
Ginsenosides
R1
61 62 63 64 65
Ginsenoside I (epimer of 60) Ginsenoside II (epimer of 59) Floralginsenoside H Floralquinquenoside D Notoginsenoside C
R2 2 1
–Glc – Glc –Glc2–1Glc –Glc2–1Glc6–Ac –Glc –Glc2–1Glc
–Glc –Glc –Glc –Glc –Glc6–1Glc
O HO R2O
24 20 S
25
12
3
6
R1O
No.
66 67
Ginsenosides
R1
R2 2 1
Ginsenoside III Notoginsenoside B
–Glc – Glc –Glc2–1Glc HO GlcO 20 S 12
3
Glc1–2GlcO
6
Quinquenoside L1 (68)
–Glc –Glc6–1Glc
24 25
9
10
Lars P. Christensen
FIGURE 1.1
Protopanaxadiol-type ginsenosides (Continued ) OH 20 12
3
6
RO
No.
Ginsenosides
R
69 70 71
Ginsenoside Rk1 Ginsenoside Rk2 Ginsenoside Rs5
–Glc2–1Glc –Glc –Glc2–1Glc6–Ac
OH 20 12
3
RO
6
No.
Ginsenosides
R
72 73 74
Ginsenoside Rg5 Ginsenoside Rh3 Ginsenoside Rs4
–Glc2–1Glc –Glc –Glc2–1Glc6–Ac
Ginsenosides
FIGURE 1.1
Protopanaxadiol-type ginsenosides (Continued ) OH HO
GlcO 20 S
24
25
OH
12
3
6
Glc1–2GlcO
Vina-ginsenoside R13 (75) Glc6–1Glc HO
O
25 20 S
OH
12
3
6
Glc1–2GlcO
Koryoginsenoside R2 (76) O R2O 20 S 12
3
R1O
No.
77 78 79
6
Ginsenosides
Chikusetsusaponin LN4 Chikusetsusaponin LT5 Chikusetsusaponin LT8
R1
R2 6 1
–Glc – Xyl –Glc –Glc
–Glc6–1Ara(p) –Glc6–1Glc –Glc
11
12
Lars P. Christensen
FIGURE 1.1
Protopanaxatriol-type ginsenosides (Continued ) R3O
R4 O 20 S
12
3
6
R1O
OR2 20(S)-protopanaxatriol (20(S)-PPT, R1 = R2 = R3 = R4 = H)
No. Ginsenosides
R1
R2
R3
R4
80 81 82 83 84 85
–H –H –H –Glc –H –H
–H –H –H –H –H –H
–Glc –Glc6–1Ara(p) –Glc6–1Ara(f) –Glc –Glc –Glc
–H –H
–H –H –H –H –Glc2–1Rha –Glc2–1RhaMalonoyl –Glc2–1Glc –Glc2–1Glc
–H –H
–H –Glc
–H –H
–Glc –Glc6–Ac
–H –H
–Glc –Glc
–H
–H
–Glc
–H
–Glc6– Malonoyl –Glc2–1Rha
–H
–H
–H
–Glc
–H
–H
–H –H
–Glc –H
–H –H
–H
–H
–CH3 –Glc6–1Ara(p)4– 1 Xyl –Glc –H
–H
–Glc2–1Rha
–H
86 87 88 89 90 91
92 93 94 95 96
Ginsenoside F1 Ginsenoside F3 Ginsenoside F5 Ginsenoside la Ginsenoside Re Ginsenoside malonyl-Re Ginsenoside Rf Ginsenoside 20gluco-Rf Ginsenoside Rg1 Ginsenoside acetyl-Rg1 Ginsenoside malonyl-Rg1 Ginsenoside 20(S)–Rg2 (¼ Chikusetsusaponin I) Ginsenoside 20(S)–Rh1 Ginsenoside Rh5* Chikusetsusaponin L5 Chikusetsusaponin L10 Floralginsenoside M
–Glc6–1Ara(f) (continued)
Ginsenosides
FIGURE 1.1
13
Protopanaxatriol-type ginsenosides (Continued)
No. Ginsenosides
R1
R2
R3
R4
97 Floralginsenoside N 98 Floralginsenoside P
–H –Glc2– 1 Glc
–Glc2–1Rha –H
–H –H
–Glc6–1Ara(p) –Glc6–1Ara(p)
R3O
R4O 20S
12
3
R1O
6
OR2 20(S)-protopanaxatriol (20(S)-PPT, R1 = R2 = R3 = R4 = H)
No.
99 100 101 102 103 104 105 106 107
Ginsenosides
Floralquinquenoside E Notoginsenoside M ** Notoginsenoside N Notoginsenoside R1 Notoginsenoside R2 Notoginsenoside R3 Notoginsenoside R6 Notoginsenoside U Koryoginsenoside R1
R1
R4
–Glc – Rha
–H
–Glc6–1Xyl
–H
–Glc6–1Glc
–H
–Glc
–H –H –H –H –H –H –H
–Glc4–1Glc –Glc2–1Xyl –Glc2–1Xyl –Glc –Glc –H –Glc6–(E)-2Butenoyl –Glc2–1Rha 6| Ac –Xyl
–H –H –H –H –H –H –H
–Glc –Glc –H –Glc6–1Glc –Glc6–1aGlc –Glc6–1Glc –Glc
–H
–Glc
–H
–H
–H
–H
–Glc
Pseudo-ginsenoside RS1
–H
109
Pseudo-ginsenoside RT3 Vina-ginsenoside R4
–H –Glc2– 1 Glc
2 1
R3
–H
108
110
R2
*Ginsenoside 93 isolated from the roots of Panax vietnamensis and named ginsenoside Rh5 by Tran et al. (2001) should not be confused with ginsenoside Rh5 (141) isolated from the leaves of P. ginseng (De-Quiang et al., 2001). **The name notoginsenoside M has been used for both ginsenoside 100 (Yoshikawa et al., 2001) and for ginsenoside 116 (Ma et al., 1999).
14
Lars P. Christensen
FIGURE 1.1
Protopanaxatriol-type ginsenosides (Continued ) OH HO 20 R 12
3
HO
6
OR
No.
Ginsenosides
R
111 112
Ginsenoside 20(R)–Rg2 Ginsenoside 20(R)–Rh1
–Glc2–1Rha –Glc
R3O
R4O
25 20 S
OH
12
3
R1O
6
OR2
No.
Ginsenosides
R1
R2
R3
R4
113
Chikusetsusaponin L9a Majoroside F6 Notoginsenoside H Notoginsenoside M
–H
–H
–Glc
–H
–Glc –H –H
–Glc2–1Rha –Glc2–1Xyl –Glc
–H –H –H
–Glc –Glc –Glc
114 115 116
Ginsenosides
FIGURE 1.1
Protopanaxatriol-type ginsenosides (Continued ) HO
R3O
25 20 S
OOH
12
3
6
R1O OR2
No.
Ginsenosides
R1
R2
R3
117 118 119 120 121 122 123
Ginsenoside Rh6 Floralginsenoside B Floralginsenoside D Floralginsenoside I Floralginsenoside K Floralquinquenoside A Floralquinquenoside C
–H –H –Glc –H –Glc2–1Glc –H –H
–H –Glc –H –Glc2–1Rha –H –Glc –Glc2–1Rha
–Glc –Glc –Glc6–1Ara(f) –Glc –Glc –H –H
OH 25
HO 20R
OH
12
3
6
HO OH 20(R)-dammarane-3 b, 6 a,12 b, 20, 25-pentol (25-OH PPT, 124)
15
16
Lars P. Christensen
FIGURE 1.1
Protopanaxatriol-type ginsenosides (Continued ) R4 R2O R3O
24 20 S
25
12
3
HO
6
OR1
No. Ginsenosides
R1
R2
125 Chikusetsusaponin L9bc 126 Floralginsenoside A 127 Floralginsenoside C 128 Floralginsenoside J
–H
–Glc –H
129 130 131 132 133
–Glc –H –Glc2– 1 Rha Floralginsenoside La –Glc2– 1 Rha Floralginsenoside Lb –Glc2– 1 Rha Floralquinquenoside B –Glc2– 1 Rha –H Ginsenoside M7cd Notopanaxoside A –Glc
R3
R4
–OH
–H –H –H
–Glc –OOH –Glc 6–1Ara(p) –OOH –Glc –OOH
–H
–Glc
–H
–Glc
–H
–H
–a-OH or –b-OH –a-OH or –b-OH –OOH
–H –H
–Glc –H
–OH –OH
O HO
GlcO 20 S
24
12
3
HO
6
OGlc Vina-ginsenoside R25 (134)
25
FIGURE 1.1
Protopanaxatriol-type ginsenosides (Continued ) OH R2O
HO
24
20S
25
OH
12
3
6
HO
OR1
No.
Ginsenosides
R
135 136 137
Ginsenoside Rg6 Ginsenoside Rk3 Ginsenoside Rs7
–Glc2–1Rha –Glc –Glc6–Ac
OH 20 12
3
6
HO
OR
No.
Ginsenosides
R
138 139 140
Ginsenoside Rh4 Ginsenoside Rs6 Ginsenoside F4
–Glc –Glc6–Ac –Glc2–1Rha 25 24
OH OH 20 12
3
HO
6
OGlc Ginsenoside Rh5 (141)
FIGURE 1.1
Protopanaxatriol-type ginsenosides (Continued ) OH HO
R2O 24
20S
25
OH
12
3
6
HO
OR1
No.
Ginsenosides
R1
R2
142 143 144
Notoginsenoside J Quinquenoside L9 Vina-ginsenoside R12
–Glc –Glc2–1Rha –Glc
–Glc –H –H
GlcO O 20 12
3
6
HO
OH Ginsenoside Rh8 (145)
23
O
OGlc
H
20
12
3
HO
6
OH Ginsenoside Rh9 (146)
19
Ginsenosides
FIGURE 1.1
Ocotillol-type ginsenosides HO 25
CH2−R2 24 S
O OH 20 S 12
3
HO
6
OR1 24(S)-Ocotillol (R1 = R2 = H)
No.
Ginsenosides
147 148 149 150 151 152
24(S)–Majonoside R1 24(S)–Majonoside R2 Protopanaxatriol oxide II 24(S)–Pseudo-ginsenoside F11 24(S)–Pseudo-ginsenoside RT4 Vina-ginsenoside R1
153
Vina-ginsenoside R2
154 155
Vina-ginsenoside R5 Vina-ginsenoside R6
156
Vina-ginsenoside R14
R1
R2 2 1
–Glc – Glc –Glc2–1Xyl –H –Glc2–1Rha –Glc –Glc2–1Rha 6| Ac –Glc2–1Xyl 6| Ac –Glc2–1Xyl4–aGlc –Glc2–1Xyl 6| 1 aGlc –Glc2–1Xyl
–H –H –H –H –H –H –H –H –H –OH
20
Lars P. Christensen
FIGURE 1.1
Ocotillol-type ginsenosides (Continued ) HO 25
24 R
O OH
20 S
12
3
6
HO OR 24(R)-Ocotillol (R = H)
No.
Ginsenosides
R
157 158
24(R)–Majonoside R1 24(R)–Majonoside R2 (¼ 24(R)–Pseudoginsenoside RT2) 24(R)–Pseudo-ginsenoside F11 24(R)–Pseudo-ginsenoside RT5 24(R)–Vinaginsenoside R1
–Glc2–1Glc –Glc2–1Xyl
159 160 161
–Glc2–1Rha –Glc –Glc2–1Rha 6| Ac
Oleanolic acid type ginsenosides 12 17
R4O
O O O
O OR5
3
6
OR2 R3 O OR1 Oleanolic acid 3-O-β-glucuronide (R1 = R2 = R3 = R4 = R5 = H)
21
Ginsenosides
No.
Ginsenosides
R1
R2
R3
R4
R5
162
Ginsenoside Ro (¼ Chikusetsusaponin V) Ginsenoside Ro methyl ester Ginsenoside ROA Chikusetsusaponin lb Chikusetsusaponin IV Chikusetsusaponin IVa Hemsloside-Ma3 Pseudo-ginsenoside RP1 Pseudo-ginsenoside RT1 Zingibroside R1
–Glc
–H
–H
–H
–Glc
–Glc
–H
–H
–CH3
–Glc
–Glc –H –H –H
–H –H –H –H
–H –Ara(f) –Ara(f) –H
–H –Glc –H –H
–Glc6–1Glc –H –Glc –Glc
–Glc –Xyl
–Ara(p) –H –H –H
–H –H
–Glc –H
–Xyl
–H
–H
–H
–Glc
–Glc
–H
–H
–H
–H
163 164 165 166 167 168 169 170 171
OH 12
OH 17
O OGlc
O
O
3
O
6
O OH Polyacetyleneginsenoside Ro (172) HO O
HOH2C O OH HO
OH
Panaxatriol-type ginsenosides R2 24
O HO 20 S 12
3
6
HO
OR1 20(S)-panaxatriol (20(S)-PT, R1 = R2 = H)
No.
Ginsenosides
R1
R2
173 174
Vina-ginsenoside R10 Vina-ginsenoside R11
–Glc –Glc2–1Xyl
–OH –OH
Dammarenediol-type ginsenosides R2O 20S 12
3
6
R1O
20(S)-dammarenediol (R1 = R2 = H)
No.
Ginsenosides
R1
R2
175 176 177
Notoginsenoside I Vina-ginsenoside R3 Yesanchinoside I
–Glc2–1Glc –Glc –Glc2–1Glc
–Glc6–1Glc –Glc2–1Glc –Glc6–1Glc6–1Xyl
Miscellaneous type ginsenosides
25 24
HO
23
OH H
20
12
3
HO
H 6
O−Glc6−Rha Ginsenoside Rg8 (178)
O
Ginsenosides
OH
23
R2O 20
12
3
7
R1 O
OH
No.
Ginsenosides
R1
R2
179 180 181 182
Ginsenoside Rh7 Notoginsenoside G Quinquenoside IV Yesanchinoside G
–H –Glc2–1Glc –Glc2–1Glc –Glc2–1Glc
–Glc –Glc –Glc6–1Glc –Glc6–1Xyl
FIGURE 1.1 Ginsenosides isolated from fresh and/or processed ginseng material (roots, leaves, fruits, flower buds), including medicinal preparations based on plant material from ginseng species. Ara(p) ¼ a-L-arabinopyranosyl; Ara(f) ¼ a-L-arabinofuranosyl; Glc ¼ b-Dglucopyranosyl; Rha ¼ a-L-rhamnopyranosyl; Xyl ¼ b-D-xylopyranosyl; Ac ¼ acetyl.
The fact that ginseng is a very popular phytomedicine used all around the world, a huge quantity of work has been carried out during the last 30–40 years in order to develop analytical methods for the identification, quantification, and quality control of ginsenosides in raw materials, extracts, and marketed products as well as for the determination of their metabolization in humans and their pharmacokinetics. Among all the classical techniques usually employed for phytochemical analysis highperformance liquid chromatography (HPLC) combined with ultraviolet (UV) and/or mass spectrometry (MS) detection has been the method of choice for the analysis of ginsenosides in the last 20 years. The aim of this chapter is to highlight the present state of knowledge on the chemistry, biosynthesis, analysis, and pharmacological effects of ginsenosides. The latter clearly demonstrate the potential health effects of this interesting group of compounds.
II. CHEMISTRY A. Chemical structure of ginsenosides and distribution in plants Ginsenosides with a few exceptions share a similar basic structure, consisting of a saturated 1,2-cyclopentanoperhydrophenanthrene (sterane or gonane) steroid nucleus. They are classified into two groups by the skeleton of aglycones, namely dammarane-type and oleanane-type. Ginsenosides
TABLE 1.1 Distribution of ginsenosides in different plant parts (fresh, dried, and/or processed) of Panax species (Araliaceae) and Gynostemma pentaphyllum (Cucurbitaceae)
24 Family/species
Araliaceae Panax bipinnatifidus Seem. (¼ P. japonicus C. A. Meyer var. bipinnatifidus (Seem.) C. Y. Wu et Feng; P. pseudoginseng Wall. var. bipinnatifidus (Seem.) H. L. Li) P. ginseng C. A. Meyer
Common name
Ginsenosides Roots/rhizomes
Leaves/stems
Fruits/flower buds
References
Featherleaved bamboo ginseng
5, 12, 84, 101, 158, 162, 166, 167
—
—
Zhu et al., 2004
Korean ginseng, Chinese ginseng, Asian ginseng
2–14, 15a–18a, 32, 34, 35, 42a, 43a, 69a– 72a, 74a, 76, 84, 86–88, 91, 92, 102, 103, 107, 111, 112, 135a– 140a, 159b, 162–164, 172
1, 5, 7, 10, 12, 57, 58, 80, 81, 84, 88, 92, 112, 117, 132, 141, 145, 146, 179, 20(S)-PPT, 20(S)-PT
5, 7, 10, 12, 15, 24, 34, 44, 51– 55, 61–63, 66, 73, 80–82, 84, 88, 91, 92, 96– 98, 108, 110, 112, 114, 118– 121, 124–130, 20(S)-PPD
Besso et al., 1982; Dou et al., 2001; Fuzzati et al., 1999, 2000; Haijiang et al., 2003; Kim et al., 1995; 1996; Kitagawa et al., 1983; Kite et al., 2003; Lui and Staba, 1980; Liu et al., 2005; Ma et al., 2005; Morita et al., 1983; Nakamura et al., 2007a; Park et al., 2002a,b,c; Qui et al., 1998, 2001;
P. japonicus C. A. Meyer (¼ P. pseudoginseng Wall. subsp. japonicus (C. A. Meyer) H. Hara)
Japanese ginseng
5, 9, 10, 12, 19, 20, 26–28, 32, 40, 50, 84, 88, 91, 103, 159, 162, 163, 165–167, 177, 179, 182
78–81, 95, 96, 114, 125
—
Ryu et al., 1997; Samukawa et al., 1995; Sanada et al., 1974a,b; Sanada and Shoji, 1978; Shibata, 2001; Uvarova et al., 2000; Wan et al., 2007; Wang et al., 2004, 2007; Yahara et al., 1976a,b, 1979; Yoshikawa et al., 2007a,b; Yu et al., 2005; Zhang et al., 2002; Zhu et al., 2004 Kondo et al., 1971; Lee et al., 1977; Lin et al., 1976; Lui and Staba, 1980; Morita et al., 1983; Tanaka et al., 1985; Yahara et al., 1977, 1978; Zhu et al., 2004; Zou et al., 2002 (continued)
25
26 TABLE 1.1
(continued)
Family/species
P. japonicus C. A. Meyer var. angustifolius (Burk.) Cheng et Chu P. japonicus C. A. Meyer var. major C. Y. Wu et K. M. Feng P. notoginseng (Burk.) F. H. Chen ( ¼ P. pseudoginseng Wall. var. notoginseng (Burk.) G. Hoo and C. J. Tseng)
Common name
Ginsenosides Roots/rhizomes
Leaves/stems
Fruits/flower buds
References
Narrowleaved Japanese ginseng
5, 12, 88, 162, 166, 167
—
—
Zhu et al., 2004
Pear ginseng
5, 12, 87, 91, 103, 147, 148, 156, 158, 162, 166, 167 1–7, 9, 10, 12, 14, 22, 24– 32, 35–39, 42, 47, 55, 56, 65, 67, 69, 72, 80, 84, 87, 88, 91, 92, 100– 106, 115,
—
—
Morita et al., 1982, 1983; Tanaka et al., 1985; Tohda et al., 2002; Zhu et al., 2004
—
—
Kite et al., 2003; Komakine et al., 2006; Lai et al., 2006; Li et al., 2005; Lui and Staba, 1980; Ma et al., 1999; Matsuura et al., 1983; Sun et al., 2005b, 2006; Wan et al., 2007;
Sanchi ginseng,
P. pseudoginseng Wall. var. angustatus Hara P. pseudoginseng Wall. subsp. himalaicus Hara
P. quinquefolium L.
116, 132, 135, 136, 138, 140, 142, 159b, 162b, 175, 180 20, 21, 86, 103, 162, 166, 167 Himalayan ginseng
American ginseng
1, 5, 9, 12, 24, 84, 88, 91, 102, 103, 109, 148, 150, 151, 158–160, 162, 166, 167, 169, 170 1, 5–15, 24, 34– 39, 47, 56, 65, 80, 84, 88–92, 102, 103, 112, 140, 159,
Yoshikawa et al., 1997a,b, 2001; Zhang and Cheng, 2006; Zhou et al., 1981; Zhu et al., 2004 —
—
Kohda et al., 1991
9, 12, 33, 84, 159
—
Morita et al., 1983; Tanaka et al., 1985, 2000; Tanaka and Yahara, 1978
5, 7, 9, 10, 12, 14, 45, 48, 68, 84, 91, 92, 143, 159b, 162b, 176
5, 7, 9, 10, 12, 14, 16, 24, 33, 34, 38, 55, 61, 62, 64, 81, 83, 84, 88, 91, 92, 99, 111, 112, 122,
Christensen et al., 2006; Corbit et al., 2005; Dou et al., 2006; Kite et al., 2003; Ligor et al., 2005; Lui and Staba, 1980; Ma et al., 2006; Nakamura (continued)
27
28 TABLE 1.1
(continued)
Family/species
Common name
Ginsenosides Roots/rhizomes
Leaves/stems
162, 167, 178, 181
P. stipuleanatus H. T. Tsai et Feng P. trifolius L.
Pingpien ginseng Dwarf ginseng
P. vietnamensis Ha et Grushv.
Vietnamese ginseng
Fruits/flower buds
123, 131, 143, 150, 159–161
166, 167
—
—
5, 9, 10, 12, 28, 162
5, 9, 10, 12, 28, 162
—
5, 7, 9, 10, 12, 24, 26, 32, 35, 40, 49, 59, 60, 75, 84, 87, 88,
—
—
References
et al., 2007b; Popovich and Kitts, 2004a; Wan et al., 2007; Wang et al., 1998, 2001b,c, 2006a; Wills and Stuart, 2001; Wood et al., 2006; Yoshikawa et al., 1998, 2001; Zhu et al., 2004 Zhu et al., 2004 Lee and Marderosian, 1981, 1988; Lui and Staba, 1980 Duc et al., 1993, 1994a, b; Tohda et al., 2002; Tran et al., 2001; Zhu et al., 2004
P. vietnamensis Ha et Grushv. var. fuscidiscus K. Komatsu, S. Zhu et S. Q. Cai P. zingiberensis C. Y. Wu et K. M. Feng Cucurbitaceae Gynostemma pentaphyllum Makino a
Vietnamese ginseng
92, 93, 102, 105, 108, 110, 112, 134, 138, 144, 147– 156, 162, 167, 168, 173, 174, 176 5, 10, 12, 84, 88, 103, 158, 162
—
—
Zhu et al., 2004
Ginger ginseng
5, 12, 88, 92, 103, 162, 166, 167, 171
—
—
Tanaka et al., 1985; Tran et al., 2003; Zhu et al., 2004
Jiaogulan, five-leaf ginseng
—
1, 5, 6, 9, 10, 13, 14, 22–24
—
Cui et al., 1999; RazovskiNaumovski et al., 2005
This ginsenoside is known to be unique or characteristic for processed ginseng (red ginseng). This ginsenoside has only been detected in trace or minute amounts by TLC from this ginseng plant part and hence the presence is questionable (Lui and Staba, 1980).
b
29
30
Lars P. Christensen
within the dammarane-type consist mainly of three types classified according to their genuine aglycone moieties: protopanaxadiol (PPD), protopanaxatriol (PPT), and ocotillol, whereas ginsenosides of the oleanane-type are classified according to their aglycone oleanolic acid (Fig. 1.1). Other types of ginsenosides isolated from ginseng species include panaxatrioltype and dammarenediol-type ginsenosides (Fig. 1.1). The major ginsenosides in the roots of ginseng include the 20(S)-PPDs Rb1 (5), Rb2 (7), Rc (10), and Rd (12) and the 20(S)-PPTs Re (84) and Rg1 (88) as well as the malonyl derivatives of the ginsenosides Rb1, Rb2, Rc, and Rd (6, 8, 11, and 13; Fig. 1.1), which normally account for over 90% of the total ginsenoside content in ginseng roots (Christensen et al., 2006). Consequently, these ginsenosides are used as markers of ginseng quality. The four malonyl derivatives of the ginsenosides Rb1, Rb2, Rc, and Rd together with ginsenoside Ro (162) and similar esterified ginsenosides are termed ‘‘acidic ginsenosides,’’ while the others are termed ‘‘neutral’’ ginsenosides. The most used ginseng roots in herbal medicine and dietary supplements are those from P. quinquefolium (American ginseng) and P. ginseng (Asian or Korean ginseng). The ginsenoside profiles of these species can be distinguished by ginsenoside Rf (86), which is detectable in P. ginseng but not in P. quinquefolium and several other ginseng species (Table 1.1). Ginsenoside ratios are also indicative of the different types of ginseng even though they may differ among species. The ocotillol-type triterpene 24(R)-pseudo-ginsenoside F11 (159) is present in P. quinquefolium whereas it only seems to be present in very minute amounts in P. ginseng, and hence a high ginsenoside Rf/24(R)-pseudoginsenoside F11 ratio (>700) clearly distinguishes Asian ginseng from the American species and may be used to determine whether P. ginseng is contaminated with P. quinquefolium and vice versa (Chan et al., 2000; Li et al., 2000). In addition, a high Rb1/Rg1 ratio with values around 10 or greater is usually indicative of P. quinquefolium, while low Rb1/Rg1 values usually between 1 and 3 are characteristic for P. ginseng (Fuzzati, 2004; Li and Fitzloff, 2002b). The PPD group of ginsenosides dominates quantitatively in P. quinquefolium in contrast to P. ginseng where the PPT group ginsenosides occur in highest amounts (Fuzzati, 2004; Wan et al., 2007; Wang et al., 1999). Finally, it appears that the profiles of malonyl-ginsenosides can be used to distinguish the species P. ginseng, P. quinquefolium, and P. notoginseng. In P. quinquefolium, the levels of malonyl-Rc and malonyl-Rb2 relative to malonylRb1 are lower than in P. ginseng. In P. notoginseng, the most abundant malonyl-ginsenosides is malonyl-Rb1 and some nonidentified isomeric forms of this ginsenoside (Kite et al., 2003). P. notogingseng (Sanchi ginseng) is an example of another ginseng species used frequently in herbal medicine, although mostly in Chinese medicine (Lai et al., 2006; Sun et al., 2006). The roots of P. notoginseng and/or products based on this species have been intensively investigated for ginsenosides (Lai et al., 2006; Li et al., 2005; Sun et al., 2006; Zhang and Cheng, 2006; Table 1.1).
Ginsenosides
31
This species seems to be rich both quantitatively and qualitatively in ginsenosides of the PPD- and PPT-type (Sun et al., 2006; Wan et al., 2007), and the amounts of dammarane-type ginsenosides are clearly higher in P. notoginseng compared to the morphologically related species P. ginseng and P. quinquefolium (Wan et al., 2007). However, ginsenosides of the ocotilloltype and oleanolic acid type seem to be absent in P. notoginseng (Table 1.1), although the presence of minute amounts of ginsenoside Ro (162) (oleanolic acid type) and 24(R)-pseudo-ginsenoside F11 (159) (ocotillol-type) has been indicated in a single investigation of dried roots of this species (Lui and Staba, 1980). The absence or presence of very minute amounts of ginsenosides of the ocotillol-type and oleanolic acid type in P. notoginseng clearly distinguishes this species from other ginseng species investigated (Table 1.1). Oleanolic acid type ginsenosides (162–172) seem to be typical constituents of ginseng species and are in particular characteristic for P. ginseng, P. japonicus (Japanese ginseng), P. pseudoginseng subsp. himalaicus (Himamayan ginseng), P. vietnamensis (Vietnamese ginseng), and P. zingiberensis (ginger ginseng) (Table 1.1). Ocotillol-type ginsenosides (147–161) also seem to be typical constituents of ginseng species, although they only seem to be characteristic for P. japonicus var. major (pear ginseng), P. pseudoginseng subsp. himalaicus, P. vietnamensis, and P. quinquefolium (Table 1.1). The root/rhizome is the plant part most used in herbal remedies of ginseng and consequently most investigation on ginsenosides has been performed on this plant part. However, ginsenosides are also present in the aerial parts of ginseng species as demonstrated for P. ginseng, P. japonicus, P. pseudoginseng subsp. himalaicus, P. quinquefolium, and P. trifolius (Table 1.1). In particular, the aerial parts of P. ginseng and P. quinquefolium have been intensively investigated for ginsenosides that have in addition to typical root ginsenosides resulted in the isolation of several new and characteristic ginsenosides such as the floralginsenosides A–P (51–54, 63, 96–98, 118–121, and 126–128), La (129), and Lb (130) from flower buds of P. ginseng (Nakamura et al., 2007a; Yoshikawa et al., 2007a,b) and the floralquinquenosides A–E (64, 99, 122, 123, and 131) from the flower buds of P. quinquefolium (Nakamura et al., 2007b). The species Gynostemma pentaphyllum ( Jiaogulan or five-leaf ginseng) of the Cucurbitaceae is especially rich in gypenosides (gynosaponins), which mainly exists as dammarane-type triterpene glycosides closely related to the ginsenosides (Razmovski-Naumovski et al., 2005). Indeed, a few of the over 90 gypenosides isolated from G. pentaphyllum have been shown to be identical with ginsenosides Rb1 (gypenoside III), Rc, Rb3 (gypenoside IV), Rd (gypenoside VIII), F2, Rg3, malonyl-Rb1, malonylRd, Rf, gypenoisde XVII, gypenoside IX, and gypenoside XV (Fig. 1.1) (Cui et al., 1999; Kuwahara et al., 1989; Razmovski-Naumovski et al., 2005). These ginsenosides make up to 25% of the total gynosaponin content in the plant and is the first example of ginsenosides found outside of the
32
Lars P. Christensen
Araliaceae family (Liu et al., 2004). Because of its phytochemical similarity with the more expensive ginseng root, G. pentaphyllum has attracted much interest as a potential new plant drug. Pharmacological studies of G. pentaphyllum have shown that the plant exhibits similar biological activities as the ginseng root probably because of its relatively high ginsenoside content (Razmovski-Naumovski et al., 2005). The content of total and individual ginsenosides does not only vary between plant organs and species. In particular, the content of ginsenosides in ginseng roots also depends on growing conditions and age of the roots, and internal root size (root hairs, lateral roots, and main roots) (Christensen et al., 2006; Court et al., 1996b; Soldati and Tanaka, 1984; Wills and Stuart, 2001).
B. Ginsenosides produced during steaming and drying of ginseng The roots/rhizomes of P. ginseng are normally prepared before they are used as herbal medicine by drying the fresh roots/rhizomes after peeling off (white ginseng) or prepared by steaming the whole roots/rhizomes followed by drying (red ginseng). Ginsenosides Rb1 (5), Rb2 (7), Rc (10), Rd (12), Re (84), Rg1 (88), and Rg2 (91) are major constituents of white and red ginsengs, while ginsenosides such as quinquenoside R1 (35), Rh1 (91), Rh2 (15), 20(S)-Rg3 (14), 20(R)-Rg3 (42), Rg5 (72), Rg6 (135), Rs1–Rs7 (16–18, 71, 74, 137, 139), and Rk1–Rk3 (69, 70, 136) seem to be characteristics for red ginseng as well as the R-epimers of Rg2 (111) and Rh1 (112) (Kim et al., 1996; Kitagawa et al., 1983; Park et al., 2002a,b; Ryu et al., 1997; Shibata et al., 1985). Ginsenoside Rg3 is one of the main artifact components of red ginseng and heat-processed ginseng (Kitagawa et al., 1983; Kwon et al., 2001; Park et al., 2002a,b,c). In addition, white ginseng also contains the malonyl ester of ginsenosides Rb1, Rb2, Rc, and Rd (6, 8, 11, and 13; Fig. 1.1). In red ginseng, the malonyl group, which is originally attached at the 600 -position of the glucosyl moiety of the above-mentioned ginsenosides, is released and the glycosyl moiety at C20-OH is partly loss of to yield, for example, the deglycosylated ginsenosides Rh1, Rh2, and Rg3 as the artifacts. The ginsenosides Rk1–Rk3, Rs4–Rs7, Rg5, and Rg6 are examples of dehydrated ginsenosides generated by the loss of water from the corresponding ginsenosides with a free hydroxyl group at C-20. For example, ginsenoside Rg5 and Rg6 are dehydrated ginsenosides of Rg3 and Rg2, respectively (Fig. 1.1). The acetyl group remains at the 600 -position of the glycosyl moiety of some ginsenosides in red ginseng, such as quinquenoside R1 and ginsenosides Rs1–Rs7, because steaming seems to inactivate the deacetylating enzymes (Shibata, 2001). Finally, it has been shown that storage of dried ginseng roots and its powder at low temperatures (5 C) can last for at least 3 months without
Ginsenosides
33
any significant loss of ginsenosides (Davidson et al., 2004; Wills and Stuart, 2001). However, it is recommended that such herbal products be stored in packing protecting the products from light and atmospheric moisture (<10%) in order to reduce oxidative and enzymatic degradation of the ginsenosides (Wills and Stuart, 2001).
C. Pharmacokinetics and metabolism of ginsenosides Medicinal herb products or dietary supplements are normally taken orally, including those based on ginseng. When most medicinal herbs/dietary supplements are taken orally, their constituents are brought into contact with gastric fluids (stomach acids) and by the microflora and enzymes in the large intestine before being absorbed from the gastrointestinal tract. Consequently, a part of the compounds originally present in the medicinal herb products/dietary supplements are metabolized to other components that may be more or less active than the undecomposed (intact) metabolites. The metabolic fate of the components of herbal medicines/dietary supplements therefore could be the key to a better understanding of their biological activity and the pharmacological actions of individual components. Furthermore, it has been suggested that the microfloral metabolic activity is affected by diet change and physiological factors, rather than by variations in the bacteria flora, and could be important in relation to the pharmacological actions and effects of bioactive constituents (Kim et al., 2006). The metabolism and absorption of ginsenosides have been studied intensively in recent years in order to explain the pharmacological actions of ginsenosides and their contribution to the clinical efficacy of ginseng products. From the many investigations that have been undertaken to elucidate the fate of ginsenosides through the gastrointestinal tract using acids, enzymes, and human intestinal bacteria, it is clear that a large part of the intact ginsenosides is metabolized/transformed to ginsenosides with more enhancing biological effects compared with the intact ginsenosides, and that the degradation products of ginsenosides identified in plasma and urine most likely are a result from the metabolism of ginsenosides in the gastrointestinal tract. For ginsenosides of the PPD-type, such as Rb1 (5), Rb2 (7), and Rc (10), it has been demonstrated in both in vitro and in vivo studies that intestinal human bacteria transform PPD ginsenosides to 20-O-b-glucopyranosyl-20 (S)-PPD (compound K, which is also sometimes named M1 or IH-901) and then finally to 20(S)-PPD via a stepwise cleavage of the sugar moieties as illustrated in Fig. 1.2 (Akao et al., 1997, 1998; Hasegawa et al., 1996, 1997, 2000; Shibata, 2001; Tawab et al., 2003; Wakabayashi et al., 1997). The observed intestinal degradation product compound K after oral administration of PPD ginsenosides clearly suggests the presence of bacterial b-glucosidase enzymes that are capable of hydrolyzing the glycosidic linkage. Human intestinal bacteria having b-glucosidase activity that
34
Lars P. Christensen
Glc6−1Glc HO O
Ginsenoside Rb1
Glc1−2GlcO
Stomach
−Glc HO
HO
20(S)-Ginsenoside Rg3 + hydrated ginsenoside Rg3
Glc1−2GlcO
−Glc −Glc
Ginsenoside Rh2
Glc
Glc
HO O
HO O
Intestine
−Glc
Ginsenoside F2
GlcO
Ginsenoside Rd
Glc1−2GlcO
−Glc Glc HO HO
HO O −Glc
HO
Compound K
HO
20(S)-Protopanaxadiol
FIGURE 1.2 Possible metabolism (degradation pathway) of protopanaxadiol-type ginsenosides such as ginsenoside Rb1 by stomach acidity and by human intestinal bacteria.
metabolizes PPDs to compound K includes Eubacterium sp. (Akao et al., 1997; Bae et al., 2000, 2004), Prevotella oris (Hasegawa et al., 1997), Streptococcus sp., and Bifidobacterium sp. (Bae et al., 2000, 2004). However, the transformation of the ginsenosides and production of compound K have shown to be very different between human intestinal bacteria (Bae et al., 2000, 2004; Tawab
Ginsenosides
35
et al., 2003), and hence the efficiency of conversion and the transformation pathways of ginsenosides may differ greatly due to the diversity of the resident microflora between individuals (Lee et al., 2002a). Compound K has been shown to induce an antimetastatic or anticarcinogenic effect by blocking tumor invasion or preventing chromosomal aberration and tumorgenesis and hence could be one of the key bioactive metabolites after oral intake of ginseng products explaining their potential anticancer activity as described in Section V.A (Lee et al., 1999; Wakabayashi et al., 1997). On the other hand, it has also been shown that the ginsenosides Rb1, Rb2, and Rc can be decomposed to ginsenoside Rg3 (14) by mild acid treatment, such as stomach acids (Bae et al., 2004; Han et al., 1982), although a few investigations have indicated that PPD ginsenosides are hardly decomposed in the stomach but reach the intestine in intact forms (Tawab et al., 2003). However, if ginsenoside Rg3 is produced in the stomach, it can be metabolized to ginsenoside Rh2 (15) and/or 20(S)-PPD by human intestinal bacteria as demonstrated in some in vitro studies (Bae et al., 2002, 2004). This is also in accordance with pharmacokinetic studies in rats that after intragastric administration of 10 mg/kg ginsenoside Rg3 resulted in plasma concentrations of approximately 40 and 100 ng/ml of ginsenoside Rh2 and 20(S)-PPD, respectively, 4 h after administration (Xie et al., 2005b). Ginsenoside Rh2 and 20(S)-PPD also seem to be much more physiological active than the intact ginsenosides Rb1, Rb2 and Rc. Ginsenoside Rh2 and 20(S)-PPD have, for example, been shown to be much more cytotoxic against tumor cell lines compared with the intact ginsenosides (Bae et al., 2004). For ginsenosides of the PPT-type, such as Rg1 (88) and Re (84), several studies have demonstrated that these compounds are metabolized to ginsenoside Rh1 (92) and ginsenoside F1 (80) and then finally to 20(S)-PPT under different conditions via a stepwise cleavage of the sugar moieties as illustrated in Fig. 1.3 (Hasegawa et al., 1996, 1997; Tawab et al., 2003). After oral administration of ginsenoside Rg1, the compound may be transformed in the stomach to ginsenoside Rh1 and hydrated derivatives of Rh1 (Han et al., 1982; Sun et al., 2005a; Tawab et al., 2003). However, not all ginsenoside Rg1 is hydrolyzed in the stomach and intact ginsenoside Rg1 may reach the large intestine, where it is metabolized by intestinal bacteria to ginsenoside F1 and 20(S)-PPT (Hasegawa et al., 1996; Tawab et al., 2003). Ginsenoside Re, on the other hand, may be hydrolyzed by gastric fluids to ginsenoside Rg2 (91) that is then converted in the intestine to ginsenoside Rh1 by the elimination of rhamnose through intestinal bacteria (Fig. 1.3). Intact ginsenoside Re may also reach the large intestine where it can be metabolized by intestinal bacteria to ginsenoside F1 and 20(S)-PPT via ginsenoside Rg1. Besides intestinal bacteria, several food microorganisms have shown to be able to produce specific forms of ginsenosides, including those produced by intestinal human bacteria (Chi et al., 2005; Kim et al., 2006). This clearly shows that it
Glc
Glc HO O
HO O
Ginsenoside Rg1
HO
Ginsenoside Re
HO
OGlc–Rha
OGlc
Stomach
−Glc HO HO
HO OGlc −Glc
−Glc HO HO
20(S)-Ginsenoside Rh1 HO + hydrated ginsenoside Rh1
20(S)-Ginsenoside Rg2 +
OGlc−Rha hydrated ginsenoside Rg2 −Rha
−Glc
Intestine
Glc Ginsenoside Rh1
HO O
Ginsenoside Rg1 −Glc
Ginsenoside F1
HO
Ginsenoside F1
OH
FIGURE 1.3 Possible metabolism (degradation pathway) of protopanaxatriol-type ginsenosides such as ginsenoside Rg1 and Re by stomach acidity and by human intestinal bacteria.
Ginsenosides
37
would be feasible to develop a specific bioconversion process to obtain specifically designed functional products by the appropriate combination of ginsenoside substrate and specific microbial enzymes from food microorganisms (Chi et al., 2005). Pharmacokinetic studies in rats and/or humans have demonstrated that ginsenosides when taken orally can be detected in plasma and urine samples as intact ginsenosides or deglycosylated degradation products (Hasegawa et al., 1996, 1997, 2000; Li et al., 2004a,b, 2007a,b). The main degradation products detected in urine and plasma samples after oral intake of PPD and PPT ginsenosides are the monoglycosylated ginsenosides compound K, ginsenoside Rh1 and ginsenoside F1 and hydrated products of these, which clearly indicates that ginsenosides are transformed in the gastrointestinal tract as illustrated in Figs. 1.2 and 1.3, and further that the metabolized products are absorbed from the human gastrointestinal tract. Deglycosylated ginsenosides are usually more readily absorbed into the bloodstream acting as active compounds than the corresponding undecomposed ginsenosides (Tawab et al., 2003). The concentrations of the deglycosylated ginsenosides in plasma after oral administration of ginseng products have been shown to be in the range where a significant physiological effect can be expected, whereas the bioavailability of intact ginsenosides is very poor compared with the deglycosylated ginsenosides. Consequently, the direct physiological effect of intact ginsenosides in vivo can therefore be discussed and clearly need further investigations. In humans, compound K and ginsenoside F1 are usually detected in plasma from 7 h after the intake of ginseng and in urine from 12 h after the intake, whereas ginsenoside Rh1 is detectable from 1 h in plasma and 3 h in urine after oral intake (Tawab et al., 2003). Investigations on the pharmacodynamics of compound K after intravenous administration to mice have shown that compound K is mostly excreted as bile; however, some compound K are esterified with fatty acids at C-3 of the aglycone moiety or C0 -6 of the glucose moiety in the liver (Hasegawa et al., 2000). Consequently, the esterified forms of compound K are accumulated longer in the liver than compound K itself. Further, it has been shown that esterified compound K inhibits tumor growth more than compound K in vivo (Hasegawa et al., 2000). These results suggest that liver enzymes also play a role in the metabolism of ginsenosides and in the formation of active principles of ginsenosides in the body.
III. BIOSYNTHESIS Ginsenosides are biosynthesized via the isoprenoid pathway in the cytosol with mevalonic acid as the precursor for isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), which are the two C5 starting units in the biosynthesis of ginsenosides and other terpenoids
38
Lars P. Christensen
(Dewick, 2004; Haralampidis et al., 2001; Linsefors et al., 1989). By using expressed sequence tag analysis, it has been possible to identify several candidate genes encoding for the enzymes geranyl diphosphate synthase (GPS), farnesyl diphosphate synthase (FPS), and squalene synthase (SS), involved in the various biosynthetic steps from IPP and DMAPP to squalene (Fig. 1.4A) (Choi et al., 2005; Jung et al., 2003; Lee et al., 2004a; Tansakul et al., 2006). Further candidate genes that appear to play a critical and important role in the biosynthesis of ginsenosides have been identified. However, further elucidation of the structure and physiological function of these genes is necessary before their role in the ginsenoside biosynthesis can be determined (Choi et al., 2005; Luo et al., 2005). As illustrated in Fig. 1.4A, triterpenes are not formed by an extension of adding an IPP C5 unit to a growing chain of isoprenoid units such as farnesyl diphosphate. Instead, two molecules of farnesyl diphosphate (C15) are joined tail to tail to yield the hydrocarbon presqualene diphosphate (Fig. 1.4A). Loss of the diphosphate group from presqualene diphosphate results in the formation of an unfavorable primary cation, which, however, via a Wagner-Meerwein 1,3-alkyl shift will lead to the formation of a more favorable tertiary carbocation and achieve the required C-1–C-10 bond. Breaking the original C-1–C-20 bond will then give an allylic cation that via supply of hydride from an NADPH cofactor will result in the formation of squalene (Blagg et al., 2002; Dewick, 2004; Haralampidis et al., 2001; Torssell, 1983). The next step in the biosynthesis of ginsenosides is the formation of squalene-2,3-oxide (squalene oxide or oxidosqualene), a reaction that is catalyzed by squalene epoxidase requiring O2 and NADPH cofactors. If squalene oxide is suitablly positioned and folded on the enzyme surface, the resulting polycyclic triterpene structures formed can be rationalized in terms of a series of cyclizations, followed by a sequence of concerted Wagner-Meerwein migrations of methyls and hydrides. Thus, if squalene oxide is folded on the cyclized enzyme, cycloartenol synthase, it will approximate a chair-boat-chair-boat conformation, and the transient tetracyclic protosteryl cation will then be produced with these conformational characteristics (Dewick, 2004). This cation may then undergo a series of Wagner-Meerwein 1,2 shifts, thus, for example, creating cycloartenol (Fig. 1.4B), the precursor of a series of plant sterols such as b-sitosterol (Lee et al., 2004a; Tansakul et al., 2006). Should squalene oxide be folded on to another type of cyclase enzyme, in a chairchair-chair-boat conformation, then the transient dammarenyl cation will be formed with different stereochemical features to the prostosteryl cation (Dewick, 2004). Direct quenching of the dammarenyl cation with water leads to epimeric C-20 dammarenediols (Fig. 1.4B), which are precursors of the dammarane-type triterpene saponins (Dewick, 2004; Haralampidis et al., 2001; Tansakul et al., 2006; Torssell, 1983). Most ginsenosides have S configuration at C-20 and it has been demonstrated that water addition
C5 A
Mevalonic acid
OPP
C10
IPP
C15 DMAPP
GPS
Isomerase
OPP
OPP
FPS Farnesyl PP (FPP)
Geranyl PP (GPP) OPP
FPP
Squalene synthase (SS)
DMAPP C30
1⬘
H 1⬘ OPP 1 3
PPO
2⬘
2⬘ 3⬘
3
2
H Presqualene PP
3⬘
+ 2
1
Allylic cation (from FPP)
1. −OPP 2. Wagner-Meerwein 1,3-alkyl shift 3. NADPH 1 3
2
2⬘
Squalene epoxidase (SE) 3⬘
O2, NADPH
1⬘
Squalene
O Squalene oxide
FIGURE 1.4
(Continued)
B
Squalene oxide
12
Cycloartenol synthase H+
Plant sterols 3
O HO
Cycloartenol
Chair−boat−chair−boat
HO 20 S 12
Dammarenediol synthase
H+ O
Dammarane-type triterpene saponins
3
HO Chair−chair−chair−boat
Dammarenediols
β-Amyrin synthase 12
H 3
Oleanane-type triterpene saponins
H
HO β-Amyrin
FIGURE 1.4 Proposed biosynthetic route for the biosynthesis of (A) squalene oxide (squalene-2,3-oxide) via the isoprenoid pathway and (B) triterpene saponins of the dammarane-type and oleanane-type from squalene oxide. PP, diphosphate group; GPS, geranyl phosphate synthase; FPS, farnesyl phosphate synthase; NADPH, nicotinamide adenine dinucleotide phosphate.
Ginsenosides
41
to the dammarenyl cation is stereospecific and that quenching of the carbocation by water addition, which terminates the cyclization reaction, is strictly enzymatic controlled (Kushiro et al., 1997; Tansakul et al., 2006). A few ginsenosides of the dammarenediol type have been isolated from the roots of ginseng species, including P. notoginseng (175), P. vietnamensis (176), and P. japonicus (177) (Table 1.1, Fig. 1.1), and appear to be directly biosynthesized from dammarenediol followed by glycosylation (Fig. 1.5). Hydroxylation of dammarenediol at C-12 leads to PPD and further hydroxylation at C-6 then to PPT, oxidation processes that most likely proceed via cytochrome P450 monoxygenase enzymes (Choi et al., 2005; Haralampidis et al., 2001; Jung et al., 2003; Tansakul et al., 2006). Biosynthesis of ginsenosides from PPD and PPT triterpene aglycones involves glycosylation primarily at the C-3 and/or C-20 positions on the skeleton for PPD-type ginsenosides (1–79) and at C-6 and C-20 positions for PPTtype ginsenosides (80–82, 84–97, 99–109, 111, 112, 115–120, 122–146), although a few PPT-type ginsenosides such as ginsenoside Ia (83); chikusetsusaponin L10 (95) and L9a (113); floralginsenoside P (98), D (119), and K (121); vina-ginsenoside R4 (110); and majoroside F6 (114) have different glycosylation patterns with glycosylation at C-3 and/or C-12 positions. The glycosylation of triterpene aglycones proceeds via triterpeneglucosyltransferase enzymes, of which several candidates have been identified (Wang et al., 2005, 2006b; Yue and Zhong, 2005). The biosynthesis of ocotillol-type ginsenosides has to the best of my knowledge not been investigated. However, they may be biosynthesized via epoxidation of the double bond at C-24–C-25 followed by an intramolecular nucleophilic attack of the hydroxyl group at C-20 as illustrated in Fig. 1.6 (route a). On the other hand, a nucleophilic attack of the hydroxyl group in position C-20 at C-25 of the epoxide group will then lead to the rare panaxatriol-type ginsenosides, such as vina-ginsenoside R11 (174) (Fig. 1.6, route b) isolated from the roots of P. vietnamensis (Table 1.1). Addition of water to the epoxide group will finally lead to hydrated PPDtype ginsenosides (Fig. 1.6, route c), such as notoginsenoside J (142). Alternatively the dammarenyl cation may undergo a Wagner-Meerwein 1,2-alkyl shift to give the baccharenyl cation. A pentacyclic ring system can now be formed by cyclization on the double bond, giving a new five-membered ring and the tertiary lupenyl cation (Fig. 1.7). Ring expansion in the lupenyl cation by bond migration gives the oleanyl system that by Wagner-Meerwein 1,2-hydride shifts leads to b-amyrin, which is the precursor for the oleanane-type triterpene saponins including those found in ginseng (Dewick, 2004; Haralampidis et al., 2001; Tansakul et al., 2006; Torssell, 1983), as illustrated in Fig. 1.7. As described above, the cyclization of squalene oxide is a biosynthetic branching point not only for phytosterols and triterpenes but also for dammarane- and oleanane-type ginsenosides. In ginseng, the enzyme
42 +
HO 20 S
Dammarenediol synthase (DS)
12
12
H2O DS
3
O
Glycosylation
3
HO
HO Dammarenediol
Dammarenyl cation
Squalene oxide
Dammarenediol-type
Oxidation Glc HO O 12
3
HO
6
HO HO
HO HO
20 S
20 S 12
Glycosylation 3
3
6
HO
HO
OH 20(S)-protopanaxatriol
OGlc Ginsenoside Rg1
20 S 12
Oxidation
20(S)-protopanaxadiol Glycosylation
Protopanaxatriol-type Glc6−1Glc HO
O 20 S
12
Protopanaxadiol-type
3
Glc1−2GlcO Ginsenoside Rb1
FIGURE 1.5 Possible biosynthetic route for ginsenosides of the dammarenediol-, protopanaxadiol-, and protopanaxatriol-type via dammarenediol.
b a
HO HO
24
HO HÖ
25
20 S
12
Epoxidation 3
HO O
O
c
H2Ö
6
3
6
HO
OH 20(S)-protopanaxatriol
Squalene oxide
20 S
12
OH a
Glycosylation
c
Glycosylation
b
Glycosylation Glc HO
HO
OH
O
24 S
25 20 S
24
OH
OH
12
O HO 20 S
24
O
12
HO 3
HO
20 S
6
12 3
OGlc Notoginsenoside J Protopanaxatriol-type
HO 3
HO
6
OGlc2−1Xyl Vina-ginsenoside R11
6
OGlc2−1Glc 24(S)-majonoside R1 Ocotillol-type
Panaxatriol-type
FIGURE 1.6
Possible biosynthetic route for ginsenosides of the ocotillol-, panaxatriol-, and protopanaxatriol-type via 20(S)-protopanaxatriol.
43
+ H
H
3
3
HO
O
H +
Wagner-Meerwein 1, 2-alkyl shift
b-amyrin synthase
H
HO Dammarenyl cation
Squalene oxide
Baccharenyl cation
+
H 3
H
Wagner-Meerwein 1, 2-hydride shifts
H
+ H H
H
H
H
3
HO
H
3
H
HO
HO b-amyrin
Lupenyl cation
Oleanyl cation
Oxidation
O
Glycosylation H
H 3
OH
H
3
H
O
Oleanolic acid type OGlc
Glc1-2GlcAO
HO Oleanolic acid
FIGURE 1.7
Ginsenoside Ro
Possible biosynthetic route for ginsenosides of the oleanolic acid type from squalene oxide.
Ginsenosides
45
cycloartenol synthase catalyzes the production of cycloartenol, and b-amyrin synthase is involved in the biosynthesis of oleanane-type triterpenes (Fig. 1.2). The cyclization of squalene oxide into the dammarane skeleton/dammarenediol is catalyzed by squalene oxide (oxidosqualene) cyclase (Abe et al., 1993; Haralampidis et al., 2001; Kushiro et al., 1997), which has recently been identified as dammarenediol-II synthase (Tansakul et al., 2006). The formation of the various types of ginsenosides from dammarenediol and b-amyrin proceeds via various hydroxylation, oxidation, and glycosylation reactions that are catalyzed by various enzymes, of which only a few have been characterized (Choi et al., 2005; Haralampidis et al., 2001; Jung et al., 2003; Yue and Zhong, 2005). A special type of polyacetyleneginsenoside named polyacetyleneginsenoside Ro (172) (Fig. 1.1) and isolated from the roots of P. ginseng (Zhang et al., 2002) is clearly formed from the oleanane-type ginsenoside Ro (162) and the polyacetylene panaxytriol by a simple esterification reaction. This ginsenoside is an example of a compound biosynthesized from two independently biosynthetic pathways, the mevalonate pathway and the acetate pathway. Polyacetylenes are biosynthesized from oleic acid by b-oxidation and various dehydrogenation and oxidation steps (Christensen and Brandt, 2006), and hence originate from the acetate pathway.
IV. ANALYSIS Several qualitative and quantitative analytical techniques have been developed for the analysis of ginsenosides for the evaluation of quality of ginseng products and to determine the effect of processing of ginseng roots on the content of ginsensoides as well as for the determination of the metabolism and bioavailability of ginsenosides in vitro and in vivo. These analytical techniques include thin-layer chromatography (TLC), highperformance liquid chromatography (HPLC) combined with various detectors, gas chromatography (GC), colorimetry, enzyme immunoassays (EIA), capillary electrophoresis (CE), nuclear magnetic resonance (NMR) spectroscopy, and spectrophotometric methods.
A. Sample extraction Many types of extraction procedures have been employed for the extraction of ginsenosides from fresh or dry ginseng plant material as well as from ginseng preparations. Characteristic for most of the extraction methods is the use of methanol or ethanol or different aqueous mixtures of these two solvents, which also clearly enhance the extraction performances of these compounds compared with pure methanol or ethanol at room temperature (Anderson and Burney, 1998; Christensen et al., 2006; Fuzzati, 2004; Lou et al., 2006a). In order to enhance the recovery of
46
Lars P. Christensen
ginsenosides, various extraction experiments have been conducted at room temperature or using heat (Du et al., 2004; Christensen et al., 2006; Chuang and Sheu, 1994; Corbit et al., 2005; Fuzzati, 2004; Lou et al., 2006a; Popovich and Kitts, 2004a) or sonification (Corbit et al., 2005; Court et al., 1996a; Fuzzati, 2004; Li and Fitzloff, 2002a,b; Lou et al., 2005) as well as supercritical fluid extraction (Wang et al., 2001a; Wood et al., 2006), microwave-assisted extraction (Chen et al., 2007; Kwon et al., 2003; Shu et al., 2003), and enzyme-assisted extraction (Chen et al., 2007). The use of heat for the extraction of ginsenosides may improve the total yield of ginsenosides but has also proved to degrade the thermally unstable malonyl-ginsenosides into the corresponding neutral ginsenosides. For example, Court et al. (1996a) showed that ginsenosides were partial degraded (50%) after 5 h of soxhlet extraction with pure methanol and after minimum 20 h, total conversion of the malonyl-ginsenosides to their corresponding neutral ginsenosides was achieved (Court et al., 1996a). Based on the huge amount of literature on extraction of ginsenosides from fresh or dried plant material, it appears that simple extraction with 80% of methanol, at room temperature, under stirring or sonification is optimal for the extraction of both neutral and acidic ginsenosides (Christensen et al., 2006; Lou et al., 2006a), whereas extraction with 100% methanol by refluxing at 60–65 C for approximately 1 h seems to be an optimal extraction procedure of neutral ginsenosides (Corbit et al., 2005).
B. Thin-layer chromatography In the 1960 and 1970s when the first ginsenosides were isolated from ginseng, the ginsenosides were named Rx according to their Rf values on TLC (x ¼ o, a1, a2, b1, b2, b3, c, d, e, f, g1, g2, h1, etc, Fig. 1.1) where x corresponds to the sequence of Rf value of the spots on the TLC plate from the bottom to the top (Shibata et al., 1966). As the ginsenosides often separate chromatographically in groups on TLC containing the same genuine aglycone, this nomenclature seemed to be reasonable at the time with relative few known ginsenosides. Today, this nomenclature system is not practiced anymore. TLC is still commonly used in the analysis of plant material/extracts for ginsenosides due to its easiness of use, low cost, and versatility (Corthout et al., 1999; Dallenbach-Toelke et al., 1987; Fuzzati, 2004; Glensk et al., 2001; Lee and Marderosian, 1981; Ludwiczuk et al., 2002; Lui and Staba, 1980; Schulten and Soldati, 1981; Tani et al., 1981; Vanhaelen-Fastre´ et al., 2000). Detection of ginsenosides by TLC is usually achieved by using sulfuric acid alone or its mixtures with aromatic aldehydes like vanillin or anisaldehyde. TLC has been shown to be useful to discriminate ginseng species and products based on their content of ginsenosides (Corthout et al., 1999; Ludwiczuk et al., 2002), and in the United States Pharmacopoeia and the European
47
Ginsenosides
0.0 Start •
Standard mixture
Ro
Rb2, Rc
Rb1
Rg2 Rg1 Rf Rd Re
Solvent system I
•
American ginseng
1.0 Front
Pharmacopoeia, TLC is still employed for the identification test of root plant material. P. quinquefolium and P. ginseng can, for example, be discriminated for their ginsenoside composition by two-dimensional (2D) TLC using a mixture of chloroform, methanol, and water (13:7:2) as the first developing system and a mixture of water, n-butanol, and ethyl acetate (5:4:1) as the second solvent developing system (Fig. 1.8). Using this 2D-TLC system, it is possible to separate the ginsenosides Rb1 (5), Rb2 (7), Rc (10), Rd (12), Re (84), Rf (86), Rg1 (88), Rg2 (91), and Ro (162) (Fig. 1.8; Lui and Staba, 1980), where in particular, ginsenoside Rf is characteristic for P. ginseng and therefore commonly used to discriminate this ginseng species from P. quinquefolium as described in Section II.A. The
Front 1.0
Rg1, Rg2 Rf
Rg2
Solvent system II
Re Rd
Start 0.0
Rg1 Re Rd Rc
Rc Rb2
Rb1, Rb2 Ro
• Standard mixture
Rb1 Ro
• American ginseng
• American ginseng
FIGURE 1.8 One-dimensional TLC separation of a standard mixture containing the ginsenosides Ro, Rb1, Rb2, Rc, Rd, Re, Rf, Rg1, and Rg2 and one- and two-dimensional TLC separation of ginsenosides of Panax quinquefolium (American ginseng) root extract. Solvent system I ¼ first developing system containing chloroform–methanol–water (13:7:2); Solvent system II ¼ second developing system containing water–n-butanol– ethyl acetate (5:4:1).
48
Lars P. Christensen
reproducibility and accuracy of this form for TLC is, however, limited because of the unstable coloration of the developing reagents, and is therefore not suitable for quantification. In contrast, densiometric determination of ginsenosides by high-performance TLC (HPTLC) offers the advantage of being reproducible, accurate, and selective and having relatively low detection limits (Corthout et al., 1999; Dallenbach-Toelke et al., 1987; Vanhaelen-Fastre´ et al., 2000). Ginsenosides of P. ginseng roots and preparations have been quantified using HPTLC on silica gel F254 using chloroform, ethyl acetate, methanol, and water in the ratio 15:40:22:9 as developing system. The ginsenosides were revealed by anisaldehyde reagent and quantification of the ginsenosides Ra1 (2), Rb1, Rb2, Rc, Rd, Re, Rf, Rg1, and Rg2 were performed by scanning the plates at 535 nm in emission mode. The method was successfully validated with regard to linearity, precision, and accuracy and with a detection limit for the ginsenosides of approximately 10 ng/spot (Corthout et al., 1999). Vanhaelen-Fastre´ et al. (2000) have also successfully used HPTLC for the densitometric determination of major ginsenosides (Rb1, Rb2, Rc, Rd, Re, and Rg1) in P. ginseng by employing vapors thionyl chloride for detection, allowing the quantification of ginsenosides both in absorbance mode (l ¼ 275 nm) and in fluorescence mode (lexcitation ¼ 366 nm, lemission ¼ 400 nm) with a practical lower quantification limit of 100 ng. The relative low detection limits of ginsenosides on TLC have also made it possible to study the pharmacokinetics of these compounds in vivo. Odani and colleagues (Odani et al., 1983a,b; Takino et al., 1982) developed a TLC-dual-wavelength method that could be used to investigate the absorption, distribution, metabolism, and excretion of ginsenoside Rb1 and Rg1 in rats. They showed that degradation and metabolism of intact ginsenosides occurs in the stomach and large intestine of rats in accordance with other pharmacokinetic studies of ginsenosides in animal and humans (see Section II.C). The developed TLC-densiometry method was also found to be useful for quantiative analysis of ginsenoside Rb1 and Rg1 in the different tissue and biofluid samples (Odani et al., 1983a,b).
C. Gas chromatography In the beginning of 1980 when the analysis of ginsenosides by GC was developed, the analysis of ginsenosides by GC was usually performed directly on the trimethylsillyl derivatives of ginsenosides. However, efficiency of the separation and the number of ginsenosides detected and quantified at that time was only 7 with ginsenoside Rf (86) and Rg1 (88) being superimposed in the GC analysis (Bombardelli et al., 1980). Today GC analysis is performed on highly efficient capillary GC columns and the strategy today for GC analysis of ginsenosides is first to hydrolyze the ginsenosides followed by trimethylsillyl-derivatization of their genuine
Ginsenosides
49
aglycones in order to simplify and improve the sensitivity of the GC analysis (Cui, 1995; Cui et al., 1993, 1996, 1997, 1998; Fuzzati, 2004). Oxidative alkaline cleavage of the glycosidic chains is preferable compared with acid hydrolysis as the former method does not cause epimerization, hydroxylation, and cyclization (Cui et al., 1993, 1998). The GCmethodology, however, does not allow evaluating complex pattern of the different types of ginsenosides in ginseng and ginseng products. Consequently, the GC methodology is rarely used for the analysis of ginsenosides. However, GC-MS has shown to be sensitive enough to allow the detection and quantification of the genuine aglycones of ginsenosides 20(S)-PPD and 20(S)-PPT in human urine samples after oral administration of ginseng preparations using panaxatriol as internal standard. Among 65 samples collected from athletes stating the consumption of P. ginseng preparations, Cui et al. (1996) were able to detect and quantify 20 (S)-PPT and 20(S)-PPD in 60 samples with a concentration between 2 and 35 ng/ml urine for 20(S)-PPT and a concentration of lower than 7 ng/ml urine for 20(S)-PPD in all samples. In a similar study on human urinary excretion of ginsenosides after ingestion of ginseng preparations, the same levels of ginsenosides in urine samples was found (Cui et al., 1997). 20(S)-PPT occurred in at least sixfold higher concentration than 20(S)-PPD even though the daily intake of 20(S)-PPD-type ginsenosides was at least twofold higher than the intake of 20(S)-PPT-type ginsenosides. These findings clearly suggest that the uptake, metabolism, and excretion of 20(S)-PPD-type ginsenosides differ from those of 20(S)-PPT-type ginsenosides (Cui et al., 1997).
D. High-performance liquid chromatography HPLC has been the method of choice for the analysis of ginsenosides in most studies on ginseng plant material and preparations (Asafu-Adjaye and Wong, 2003; Chuang and Sheu, 1994; Li et al., 2001b; Ma et al., 1995; Pietta et al., 1986a; Samukawa et al., 1995; Soldati and Sticher, 1980; van Breemen et al., 1995; Wang et al., 1999, 2006a), gastric fluids (Pietta et al., 1986b), plasma/serum ( Jeoung et al., 2005; Li et al., 2004a; Qian et al., 2006; Sun et al., 2005a), urine samples (Li et al., 2004b; Qian et al., 2006), feces (Qian et al., 2006), and tissues (Li et al., 2006), because of its speed, sensitivity, and ability to analyze polar compounds. Different techniques have been used for the detection of ginsenosides such as UV, evaporate light scattering detection (ELSD), fluorescence, and MS.
1. UV detection Among the different techniques for detection of ginsenosides, UV is the most employed technique because it is by far the most widespread detector type either as a simple UV-detector or in the form of the more
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Lars P. Christensen
advanced diode array detectors. However, as ginsenosides in general are poor chromophores, their detection is limited to short wavelengths at 200–205 nm. The level of sensitivity for HPLC-UV detection of ginsenosides is not ideal, and because many compounds absorb light at short detection wavelength, many compounds may interfere with the analysis (Fuzzati, 2004; Yap et al., 2005). This feature also limits the choice of solvents and mobile phase modifiers to improve separation. Separation of ginsenosides is commonly achieved by using a reversed-phase C18 column with water or phosphate or ammonium acetate buffers and acetonitrile mixtures as mobile phases in isocratic or in gradient elution mode (Fuzzati, 2004; Lou et al., 2006b). The use of phosphate buffer and its concentration has been shown to be very important in order to obtain an optimal resolution of ginsenosides, in particular malonylated and other esterified ginsenosides (Christensen et al., 2006; Fuzzati, 2004). Effective separations of neutral and acidic ginsenosides have been obtained using 10 mM KH2PO4 buffer (pH5.8–5.9) in combination with aqueous acetonitrile as mobile phases in gradient elution. Such a mobile phase system has, for example, been used in a comparative study on 37 commercial samples of ginseng radix (Chuang et al., 1995). Content of the major ginsenosides Rb1 (5), Rb2 (7), Rc (10), Rd (12), Re (84), Rf (86), Rg1 (88), Rg2 (91), and Ro (162) and the malonylated derivatives of Rb1 (6), Rb2 (8), and Rc (11) (Fig. 1.1) was determined in white and red P. ginseng (Korean ginseng) as well as in P. quinquefolium (American ginseng) and P. notoginseng (Sanchi ginseng) roots. According to this study, P. notoginseng possesses the highest total content of ginsenosides followed by P. quinquefolium and white P. ginseng. Red ginseng was characterized by the absence of malonyl-ginsenosides due to the heating procedure connected with the production of this type of ginseng as described in Section II.B. The use of phosphate buffer has also been used to determine the distribution of acidic and neutral ginsenosides in fresh or processed roots of Panax species and the ratio between acidic and neutral ginsenosides in the roots (Christensen et al., 2006; Chuang et al., 1995; Wills and Stuart, 2001). For example, in a recent study by Christensen et al. (2006), it was found that the total ginsenoside content and most individual ginsenosides except for ginsenoside Rg1 varied significantly between different root sections of different diameters of P. quinquefolium, that is root hairs, lateral roots, and main roots of ginseng (Table 1.2; Figs. 1.9 and 1.10). Further, it was shown that the concentration of malonyl-ginsenosides constitutes approximately one-third of the total ginsenoside content in American ginseng roots, independent of size of root sections (Table 1.2). These results are in accordance with similar investigations on P. quinquefolium and other ginseng species (Chuang et al., 1995; Wills and Stuart, 2001) and clearly show that malonyl-ginsenosides contribute significantly to the total content of ginsenosides in ginseng roots. The fact that ginseng
TABLE 1.2 The content of ginsenosides (mg/kg fresh weight) in different root sections [diameter 0.5–2.5 mm (root hairs); 5.0–10.0 mm (lateral); 15.0–20.0 and >20.0–38.0 mm (main roots)] of fresh roots from 6-year-old Panax quinquefolium (American ginseng) plants grown in Denmark (Christensen et al., 2006) Ginsenosides (mg/kg fresh weight) Root size class according to root diameter
Rg1
Re
Rb1
Rc
Rb2
Rd
Ro
0.5–2.5 mm 5.0–10.0 mm 15.0–20.0 mm >20.0–38.0 mm
800a 500a 510a 650a
7420a 4450b 4290b 4390b
8070a 6260b 5910b 6230b
3170a 900b 530c 380c
910a 280b 180c 160c
2650a 1250b 740c 550c
310c 470bc 540b 780a
Means within a column followed by different letters are significantly different (p 0.05).
Total malonylMalonyl- Malonyl- Malonyl- Total Rc Rd ginsenosides ginsenosides Rb1
4850a 4420ab 3820b 3780b
520a 260b 170b 200b
2290a 1280b 920c 790c
31000a 20100b 17600b 17900b
7660a 5960b 4910bc 4770c
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Lars P. Christensen
Root hairs
Main root
Lateral roots
FIGURE 1.9 Ginseng roots from 6-year-old American ginseng plants (Panax quinquefolium) grown in Denmark with root hairs, lateral roots, and main roots. Ginseng roots within the same species may not only differ in content of ginsenosides but also in root size.
roots vary in ginsenoside content between root types/diameters may be used to make differentiated ginseng herbal remedies and/or to select and improve the genotypes of ginseng with respect to a higher yield and content of ginsenosides (Christensen et al., 2006). However, if phosphate buffers are not used in the mobile phases, the resolution of acidic ginsenosides such as malonyl-ginsenosides are very poor and in most cases, these ginsenosides will not be visible in the HPLC chromatograms using UV-detection as illustrated in Figs. 1.10 and 1.11. The importance of the malonyl-ginsenosides (6, 8, 11, and 13) must be recognized because of their potential to undergo demalonylation by heat and hydrolysis. This clearly affects the concentration of total ginsenosides because malonyl-ginsenosides are often present in significant amounts in ginseng roots as mentioned previously. Hence, it is important that malonyl-ginsenosides are quantified when evaluating the quality of ginseng preparations and products. The use of phosphate or other types of buffers such as ammonium acetate is therefore recommended in the HPLC analysis of ginsenosides. Malonyl-ginsenosides may also be determined indirectly even though they cannot be observed in the HPLC chromatograms (Court et al., 1996a; Du et al., 2004; Wills and
53
Ginsenosides
A FaOH
Absorbance at 203 nm (mAU)
300
200
g PaOH b
100
h j
d f a
c
i
e
k
0 0
20
40
60
80
100
B 200
Absorbance at 203 nm (mAU)
g 150 b PaOH 100
FaOH
d
50 a
c
f h j i k
e
0 0
20
60 40 Retention time (min)
80
100
FIGURE 1.10 HPLC chromatograms of 80% aqueous methanolic ginseng extracts. (A) Root hairs (root diameter: 0.5–2.5 mm) and (B) main roots (root diameter: 15.0–20.0 mm) from a fresh root of a 6-year-old American ginseng (Panax quinquefolium) plant. Ginsenosides: a ¼ Rg1 (88), b ¼ Re (84), c ¼ Ro (162), d ¼ malonyl-Rb1 (6), e ¼ malonyl-Rc (11), f ¼ malonylRd (13), g ¼ Rb1 (5), h ¼ Rc (10), i ¼ Rb2 (7), j ¼ Rd (12), k ¼ gypenoside XVII (24) (Figure. 1.1). Polyacetylenes: PaOH ¼ panaxydol; FaOH ¼ falcarinol. Separations performed on a PurospherÒ STAR reversed-phase (RP)-18 end-capped column (5 mm; 2504 mm id) with mobile phases consisting of solvent A [10% MeCN–90% 10 mM KH2PO4, pH ¼ 5.82 (v/v)] and solvent B [75% MeCN–25% H2O (v/v)]. Solvent gradient: 0 min 0% B, 5–15 min 15% B, 26 min 20% B, 36 min 22% B, 45 min 33% B, 50 min 35% B, 55 min 40% B, 75 min 80% B, 90–105 min 100% B, and 115 min 0% B.
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Lars P. Christensen
Absorbance at 203 nm (mAU)
600
g
400
b
200 j k a
h i
0 0
20
40 60 Retention time (min)
80
FIGURE 1.11 HPLC chromatogram of 80% aqueous methanolic extract from a fresh root of a 6-year-old American ginseng (Panax quinquefolium) plant. Polyacetylenes removed by extracting the roots with ethyl acetate before extracting with aqueous methanol. Ginsenosides: a ¼ Rg1 (88), b ¼ Re (84), g ¼ Rb1 (5), h ¼ Rc (10), i ¼ Rb2 (7), j ¼ Rd (12), and k ¼ gypenoside XVII (24) (Figure 1.1). Separations performed on a PurospherÒ STAR reversed-phase (RP)-18 end-capped column (5 mm; 2504 mm id) with mobile phases consisting of solvent A [10% MeCN–90% H2O (v/v)] and solvent B [75% MeCN–25% H2O (v/v)]. Separations performed by the following solvent gradient: 0 min 0% B, 5–15 min 15% B, 26–36 min 25% B, 45 min 35% B, 50 min 40% B, 60–74 min 100% B, and 84–89 min 0% B.
Stuart, 2001). In a study by Wills and colleagues, malonyl-ginsenosides were determined by an indirect HPLC method in which samples of ginseng were analyzed twice. In the first analysis, the amount of neutral ginsenosides was quantified in the extract, after which the extract was hydrolyzed and then analyzed second time for ginsenosides (Du et al., 2004; Wills and Stuart, 2001). The purpose of the hydrolysis process was to convert the malonyl-ginsenosides to their respective neutral ginsenosides. The HPLC chromatograms of both the original and hydrolyzed extracts were compared, and the concentration of malonyl-ginsenosides in the original extract was calculated. The indirect method may also be useful to identify esterified ginsenosides (Court et al., 1996a) as commercial standards are not available for these compounds.
Ginsenosides
55
HPLC-UV has also been shown to be useful for the detection and quantification of ginsenosides in biological fluid and tissues. The pharmacokinentics of ginsenoside Rb1, Rd, Rg1, and notoginsenoside R1 (102), which are the four main ginsenosides in P. notoginseng, have been determined by HPLCUV in rat serum and various tissues after oral and intravenous administration of ginsenoside extract from P. notoginseng (Li et al., 2004a, 2006). The serum and tissue samples were pretreated with solid phase microextraction prior to analysis by HPLC-UV in order to remove excessive interferences and to improve selectivity and sensitivity of the four ginsenosides.
2. Evaporate light scattering detection The main problems encountered in performing HPLC-UV analyses of ginseng for ginsenosides are the high level of baseline noise and relatively poor sensitivity due the weak UV absorption and the limit choice of solvents and mobile-phase modifiers for improved separation (see Section IV.D.1 and Fuzzati, 2004). ELSD is a fast and relatively cheap and straightforward analytical method based on mass detection in which the chromatographic elute is nebulized by a gas stream (nitrogen) and the vapor enters a heated tunnel, where the solvent evaporates. The resulting analyte particles pass through a narrow light beam, and the scattered light is collected by a photomultiplier. The ELSD signal depends on the number of particles and size of analytes. Because ELSD only responds to nonvolatile analytes, it generates a stable baseline even by gradient elution and hence the use of volatile modifiers in the eluents, in order to obtain better selectivity, can be used by ELSD (Fuzzati, 2004). Consequently, HPLC-ELSD is normally more sensitive than, for example, HPLC-UV, although Li and Fitzloff (2002a) concluded in a direct comparison of these two method when used to quantify the ginsenosides Rb1 (5), Rb2 (7), Rc (10), Rd (12), Re (84), and Rg1 (88) in P. ginseng and P. quiquefolium that HPLC-ELSD and HPLC-UV are comparable with regard to sensitivity and reproducibility. However, in another comparative study by Li and Fitzloff (2001), a fast HPLC-ELSD method was developed for the determination of 24(R)-pseudo-ginsensoide F11 (159), a minor ocotillol-type ginsenoside in P. quinquefolium that has been reported to improve memory performance (see Section V.F). 24(R)pseudo-ginsensoide F11 was separated with a Sperisorb ODS-2 C18 column using a gradient of acetonitrile and water. Comparison between UV and ELSD detection showed very poor UV absorption due to no double bond in the structure of this ginsenoside. The detection limit of ELSD for 24(R)-pseudo-ginsenoside F11 was approximately 50 ng whereas the detection limit by UV detection was approximately 1050 ng (Li and Fitzloff, 2001). HPLC-ELSD have been used to isolate, detect, and quantify all types of ginsenosides from various types of fresh and processed plant material of
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Lars P. Christensen
ginseng species and preparations (Cao et al., 2003; Fuzzati, 2004; Fuzzati et al., 2000; Kim et al., 2000; Park et al., 1996c; Wan et al., 2006a,b, 2007). White and red ginseng have been analyzed by HPLC-ELSD for their content of the ginsenosides Rb1, Rb2, Rc, Rd, Re, Rf (86), Rg1, Rg2 (91), Rg3 (14), and Rh1 (92) (Park et al., 1996c). Complete separation of these ginsenosides was achieved within 35 min with a LiChrosorb NH2 column using an acetonitrile-water-2-propanol gradient system. The detection limits (S/N ¼ 3) of the ginsenosides ranged from 35 to 150 ng, clearly illustrating its higher sensitivity. This method, for example, has been used to show that steaming of P. ginseng roots at high temperatures enhances the yield of ginsenosides such as Rg5 (72) and 20(S)-Rg3 (14) and 20(R)-Rg3 (42) in red ginseng, which appear to have great impact on the health effects of ginseng (see Section V). Ginsenosides Rg3 and Rg5, which were absent in raw ginseng, were detected after steaming, whereas ginsenoside Rg3 and Rg5 were the most abundant in the material steamed at 120 C, accounting for 39% and 19% of total content of ginsenosides, respectively (Kim et al., 2000). Processing of ginseng roots normally also produces a wide variety of other less polar ginsenosides as described in Section II.B, and a HPLC-ELSD method that simultaneously separates and detects both polar ginsenosides and the less polar ginsenosides such as ginsenoside F4 (140), Rg3, Rg5, Rg6 (135), Rk1 (69), Rk3 (136), Rs3 (18), Rs4 (74), Rs5 (71) together with the 20(R) epimers of Rg2 (111), Rh1 (112), Rg3, and Rs3 (43) has been developed (Kwon et al., 2001). Separations were achieved within 45 min with an RP-C18 column using an acetonitrile-water-acetic acid gradient system. Also acidic ginsenosides such as malonyl-Rb1 (6), malonyl Rb2 (8), malonyl-Rc (11), and malonylRd (12) have been successfully determined by HPLC-ELSD together with 13 common neutral ginsenosides in P. ginseng roots. The compounds were separated on a Hypersil BDS C18 column with 8 mM ammonium acetate (pH 7 with ammonium hydroxide) and acetonitrile as mobile phase in a linear programmed gradient system (Fuzzati et al., 2000). Furthermore, HPLC-ELSD methods for the simultaneous determination of ginsenosides obtained by pressurized liquid extraction of different parts of P. notoginseng have recently been described (Wan et al., 2006a,b). Eleven major ginsenosides namely notoginsenoside R1 (102), ginsensoides Rb1, Rb2, Rb3 (9), Rc, Rd, Re, Rf, Rg1, 20(S)-Rg2, and 20(S)-Rg3 were determined by HPLC-ELSD with detection limits between 18 and 98 ng. Separations were achieved in 60 min using a Zorbax ODS C18 column eluting with a gradient consisting of acetonitrile and water.
3. Fluorescence detection Fluorescence is one of the most sensitive detection methods in HPLC analyses. However, as ginsenosides do not contain a suitable fluorescence chromophore they have to be derivatized before detection. Shangguan
Ginsenosides
57
et al. (2001) described a novel precolumn derivatization method for the quantitative determination of ginsenosides by HPLC with fluorescence detection. The double bond at the C24–C25 position was converted into an aldehyde group by means of ozonolysis. Reaction of the aldehyde group with 9-fluorenylmethoxycarbonyl (FMOC) by hydrazine formed the ginsenosides FMOC-hydrazone following separation by RP-HPLC with gradient elution using methanol-water-0.1% TFA as eluent. Detection was performed with fluorescence (excitation 270 nm and emission at 310 nm) and the detection limits for ginsenosides such as Rb1 (5) and Rg1 (88) were approximately 1 and 2 ng, respectively. However, the limitation of this method is the requirement for a double bond at C24–C25 and hence it cannot be used for the detection of all types of ginsenosides (Fig. 1.1). Still as the most common ginsenosides possesses a double bond at C24–C25, this method can still be considered useful for the detection of ginsenosides. Another HPLC method using photoreduction fluorescence detection has been described for the analysis of the ginsenosides Rb1, Rb2 (7), Rd (12), Re (84), and Rg1 (Park et al., 1995). Ginsenosides were separated on a LiChrosorb NH2 column using acetonitrile and aqueous 2-tert-butylanthraquinone (t-BAQ) solution. The column effluent was passed through a 45 cm PTFE capillary tube coiled around a 10 W UV lamp to reduce t-BAQ in combination with the analyte (ginsenosides) to a highly fluorescent dihydroxy anthracene derivative that was detected by fluorescence detection (excitation 400 nm and emission 525 nm). The method showed reasonable detection limits between 100 and 1000 ng for ginsenosides, which is comparable with those obtained by UV detection (Park et al., 1995).
4. Mass spectrometry With the development of sophisticated ionization techniques including electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI), HPLC-MS techniques have been successfully applied to the online analysis of ginsenosides in extracts and biological fluids (Fuzzati, 2004). In terms of sensitivity and specificity, an MS detector is better than UV or ELSD. Among the various MS methods, the HPLC-MS-MS (or just LC-MS-MS) technique is to date the most sensitive method for detection and quantification of ginsenosides. LC-APCI-MS has been shown to very useful for the characterization of both neutral ginsenosides as well as thermolabile malonyl-ginsenosides in ginseng extracts (Ma et al., 2005). However, LC-MS with ESI interface is a highly sensitive and soft ionization technique for the LC-MS analysis of thermolabile compounds and is considered to be the best method for the analysis of ginsenosides as it can overcome most problems associated with the thermolabile malonyl-ginsenosides and low molecular ion abundance levels. LC-ESI-MS is characterized by abundant adduct formation
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Lars P. Christensen
and combined with collision-induced dissociation (CID) methodology, the LC-ESI-MS technique has been shown to be very useful for structure characterization of ginsenosides (Ackloo et al., 2000; Cai et al., 2002; Chan et al., 2000; Cui et al., 2000, 2001; Fuzzati et al., 1999; Kite et al., 2003; Li et al., 2000; Song et al., 2005; van Breemen et al., 1995; Wang et al., 1999). Both positive and negative ionizations of ginsenosides have been studied. The glycosidic linkages, the aglycone, and the attached sugar(s) can be determined by CID MS-MS analyses of [MþH]þ and [MH] ions. Moreover, some alkali and transition metal cations may form strongly bonded attachment ions with the ginsenosides, and positive mode quasi-molecular [MþH]þ ions are therefore often observed together with [MþLi]þ, [MþNa]þ, and/or [MþK]þ. As a result, their CID spectra of the metal attachment ions show a variety of structurally characteristic fragmentation patterns that can give important information about the structure of ginsenosides. Ackloo et al. (2000) conducted CID experiments on metal-attachment ions for the characterization of ginsenosides. Positive ESI-MS experiments with alkali metal ions such as Liþ and Naþ and transition metal cations such as Co2þ, Ni2þ, and Zn2þ were found to be useful in determining the molecular masses of the ginsenosides, and their CID spectra showed a variety of structure-related fragmentation patterns that could be used to determine the identity of the aglycone, the type of attachment positions of sugars to the aglycone, and the nature of the O-glycosidic linkages in the appended disaccharides. LC-ESI-MS and LC-ESI-MS-MS analysis have been used to detect 25 ginsenosides in P. ginseng roots (Fuzzati et al., 1999). The ginsenosides were separated on a reversed-phase Hypersil BDS C18 column using a binary eluent (aqueous 8 mM NH4OAc, buffered to pH 7 with NH4OH and acetonitrile) under gradient conditions. The investigation revealed the presence of several minor ginsenosides not described previously, including two isomers of ginsenoside a1 (2) and a2 (3) and several malonyl-ginsenosides (Fuzzati et al., 1999). In this study, the neutral ginsenosides exhibited the quasi-molecular ion [MH], together with adduct ions [MþOAc] and [MCH2OþAcO] and doubled-charged adduct species such as [MHþOAc]2 and [Mþ2 OAc]2. Malonyl-ginsenosides exhibited the quasi-molecular ion [MH] together with the quasi-molecular ion [MCO2H] and adduct ions such as [MCO2þOAc], [MCO2þ2 OAc]2, and [MCO2HþOAc]2. The presence of double-charged adduct species is due to the thermal instability of malonyl-ginsenosides. The MS-MS spectra of the ginsenosides exhibited fragmentation pattern corresponding to the successive loss of the glycosidic units including the [Aglycone –H] ions. However, the technique did not allow a complete structural identification of the isomers and some of the malonyl-ginsenosides (Fuzzati et al., 1999). LC-MS-MS in negative ionization mode has also been applied to investigate the
Ginsenosides
59
in vivo metabolism of ginsenoside Rb1 (5) in rats (Qian et al., 2006). Oxygenation and deglycosylation were found to be the major metabolic pathways of Rb1 in rat. A total of nine metabolites were detected in urine and feces samples collected after intravenous and oral administration of Rb1. Deglycosylated metabolism of Rb1 generated other ginsenosides as major metabolites such as ginsenoside Rd (12), Rg3 (14), F2 (1), Rh2 (15), or compound K (Fig. 1.2), which clearly indicates that ginsenoside Rb1 may have many pharmacological activities and may be used as a prodrug (Qian et al., 2006). A prodrug is a pharmacological substance that is administered in an inactive or significantly less active form. Once administered, the prodrug is metabolized in vivo into the active compound and therefore intact ginsenosides, such as ginsenoside Rb1, may therefore be used as prodrugs toward diseases where they are less effective compared to their in vivo degradation products. LC-ESI-MS in negative ion mode using selected ions monitoring has been used to develop a method for rapid quantification of ginsenoside Rg1 (88) and its secondary glycoside Rh2 and the aglycone 20(S)-PPT in rat plasma in order to study the pharmacokinetics of ginsenoside Rg1 (Sun et al., 2005a). The mass spectra of ginsenoside Rg1, Rh2, and 20(S)-PPT revealed beside quasi-molecular ion [MH] also negative adduct ions [MþCl] at m/z 835.50, 673.75, and 511.35, respectively. Sensitivity of the method was further improved by addition of NH4Cl to the mobile phase. The detection limits for ginsenoside Rg1 in deprotonated ion mode [MH] was 100 pg and 12.5 pg in adduct ion mode [MþCl]. Consequently, the latter method was used for pharmacokinetic studies of ginsenoside Rg1 in rat plasma. A similar LCESI-MS method was used to detect and quantify ginsenoside Rg3 (14) and its metabolites in rat plasma (Xie et al., 2005b) for the study of the pharmacokinetics of this pharmacological active ginsenoside (see Section V). Finally, LC-ESI-MS-MS in negative ionization mode has also been employed for the quantification of ginsenosides Rb1, Rb2 (7), Rc (10), Rd, Re (84), Rf (86), and Rg1 in commercial samples of P. ginseng and P. quiquefolium. Separations were performed on a narrow bore Zorbax C18 column with water and acetonitrile as mobile phases. Although ginsenoside Rg1 and Re coeluted under these conditions, they could be quantified separately using differences in molecular ions and product ions (Ji et al., 2001). Concentrations of the other ginsenosides were determined by peak area of the most abundant product ions. LC-ESI-MS-MS in positive ion mode has been employed for the determination of ginsenosides such as Rb1, Rb2, Rc, Rd, Re, Rf, and Rg1 in root extracts of P. ginseng and P. quinquefolium (Chan et al., 2000; Wang et al., 1999). In the study of Wang et al. (1999), the quantification was performed by selected reaction monitoring choosing [MþH]þ as the precursor ion and monitoring the most abundant fragment ion that was a disaccharide ion for ginsenoside Rb1, Rb2, Rc, Rd, and Re and the [aglyconeþH3H2O]þ ion for Rf and Rg1.
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Detection limits were 2 pg on column. Another LC-ESI-MS-MS method by Li et al. (2000) illustrates the use of a triple-quadrupole mass spectrometer for the analysis of ginseng extracts and for the differentiation of isobaric ginsenosides. Li et al. (2000) investigated the presence and the concentration ratio of ginsenoside Rf and 24(R)-pseudo-ginsenoside F11 (159) in P. ginseng and P. quinquefolium based on their baseline chromatographic separation and unambiguous identification using MS-MS. The two ginsenosides were separated with a narrow-bore Waters Spherisorb C18 column eluting with 0.1% acetic acid and 5 mM sodium acetate and acetonitrile containing 0.1% acetic acid under gradient conditions and monitored using the multiple reaction monitoring precursor/product ion pairs m/z 823 [MþNa]þ!365 and 801 [MþH]þ!143 during LC-MS-MS analysis with detection limits of 120 pg on-column. Li et al. (2000) found that 24(R)-pseudo-ginsenoside F11 was abundantly present in P. quinquefolium and only present in minor amounts in P. ginseng and that ginsenoside Rf appears to be absent or under the detection limit in P. quinquefolium, clearly showing that these ginsenosides can be used to distinguish P. ginseng and P. quinquefolium, as described in Section II.A. While LC coupled with quadrupole MS has been extensively used for especially the quantification of ginsenosides, ion trap MS and quadrupole-time of flight (Q-TOF) MS provide several advantages in the structural analysis of ginsenosides. Ion trap MS with positive and negative ionization modes with its ability to perform ESI combined with multistage MS (MSn) have been used to analyze ginsenosides rapidly in plant extracts and to provide their structural information (Cui et al., 2000, 2001). In particular, the fragmentation pathways of the quasimolecular [MH] ion resulted in several significant signals corresponding to the cleavage of the glycosidic bonds and sugars, allowing a relatively straightforward interpretation of the MSn spectra for structure elucidation of ginsenosides (Cui et al., 2000). The effect of metal cationization (Liþ, Naþ, Kþ, Agþ) on CID of ginsenosides has been investigated by ESI-MSn (Cui et al., 2001). Metal-cationized ginsenosides were found to have characteristic fragmentation patterns that were found to be useful for convenient screening and identification of ginsenosides in mixtures. Although a lot of structural information can be obtained through CID in the course of ESI-MS-MS or ESI-MSn analysis, high-resolution mass spectrometry (HRMS) analysis provides more detailed and more accurate structural information that can be used to identify, especially unknown degradation products or metabolites of ginsenosides. HRMS analysis of ginsenosides has been performed by Q-TOF MS in combination with MSMS and have shown to be useful not only for confirming the molecular composition but also for studying the structures of various isomers of ginsenosides by applying CID technique (Cai et al., 2002; Song et al., 2005).
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Accurate mass measurement for both parent and fragment ions from the HRMS analysis provides information of elemental composition of the analytes. Fragmentation pathways obtained from the interpretation of MS-MS spectrum and assignment of the accurate mass of each fragment ion can not only be used to verify the fragmentation pattern but also be used for differentiating ginsenoside isomers (Cai et al., 2002; Song et al., 2005). HRMS analysis by Q-TOF MS seems to be a very useful technique especially for pharmacokinetic and metabolic studies of ginsenosides due to its sensitivity and selectivity. Recently, Q-TOF MS in combination with ultra-performance liquid chromatography (UPLC) had also been demonstrated to be a powerful tool for herbal metabolomics to discriminate differentially processed herbs such as raw and steamed P. notoginseng (Chan et al., 2007). As demonstrated, the UPLC-TOF-MS-based metabolomics approach is promising for the quality control of ginseng and the holistic standardization of ginseng herbal extracts for clinical studies. Finally, UPLC-TOF-MS may also find use for metabonomic studies of endogenous metabolites as well as metabolized ginsenosides in biofluids and tissue samples in order to provide further information on the potential health effects of ginsenosides.
E. Nuclear magnetic resonance spectroscopy Herbal remedies contain different constituents in which characteristic metabolomic NMR fingerprints can be assigned. Therefore, NMR spectroscopy has been shown to be useful in the quality control of different herbal products such as ginseng (Qin and Zhao, 1999; Yang et al., 2006), St. Johns wort (Bilia et al., 2001), arnica (Bilia et al., 2002), and many other herbs. Yang et al. (2006) recently demonstrated the application of 2D NMR spectroscopy for quality control of ginseng products. By combining 2D J-resolved NMR spectroscopic methods with principal component analysis, they were able to distinguish different ginseng preparations as well as white and red ginseng roots from each other based on their metabolic profiling. The most important constituents of the metabolic profiling of ginseng products and roots were ginsenosides, polysaccharides, mono- and disaccharides, amino acids, fumaric acid, and inositol. Another application of NMR spectroscopy was demonstrated by Kang et al. (2005) who demonstrated by using a combination of NMR spectroscopy and molecular dynamics simulations that ginsenoside 20(S)-Rg3 inhibited Naþ channel activity but not 20(R)-Rg3. The different effect on Naþ channel activity observed for 20(S)- and 20 (R)-Rg3 may explain the different effects observed for these enantiomers in relation to tumor cell invasion and metastasis (Azuma and Mochizuki, 1994; Section V.A).
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F. Capillary electrophoresis Very few studies have described the use of CE for the analysis of ginsenosides (Glo¨ckl et al., 2002; Iwagami et al., 1992). Glo¨ckl et al. (2002) described a very fast and reliable method for the analysis of ginsenosides Rb1 (5), Rb2 (7), Rc (10), Rd (12), Re (84), Rf (86), and Rg1 (88) in ginseng extracts and preparations using micellar electrokinetic chromatography. Capillary zone electrophoresis was not applicable due to the absence of charge in ginsenosides. The analysis was performed using 100 mM borate and 80 mM cholate (pH 10) as mobile phases and a capillary length of 75 cm. Chloramphenicol was used as internal standard. The ginsenosides were separated within 20 min and detected using UV at 200 nm. The analyses of the ginseng extract showed good separation of all ginsenosides with except of ginsenoside Rf, which coeluted with other components in the extract. Validation of the method was performed for the quantification of the major ginsenoside Rb1 evaluating linearity, precision, and accuracy.
G. Enzyme immunoassay Enzyme immunoassay (EIA) and enzyme-linked immunosorbant assay (ELISA) are almost synonymous and both methods are based on the principle of immunoassay, that is, it is a biochemical test that measures the concentration of a compound using the reaction of an antibody or antibodies to its antigen. The immunoassay takes advantages of the specific binding of an antibody to its antigen and hence monoclonal antibodies (MAbs) are often used as they usually only bind to one site of a particular molecule. This often provides a more specific, accurate, rapid, and sensitive test for specific compounds, although cross-reactivity occurs. EIA or ELISA techniques has been developed for the qualitative and quantitative determination of ginsenosides in plant extracts and biological fluids using both polyclonal antibodies and MAbs (Fukuda et al., 2000a,b, 2001; Kanaoka et al., 1992; Morinaga et al., 2006; Shoyama et al., 1999; Tanaka et al., 2006; Yoon et al., 1998). The first step in the development of EIA methods is the synthesis of a hapten–carrier protein conjugate. Bovine serum albumin (BSA) in combination with ginsenosides particularly have been used for the preparation of specific MAb in mouse against ginsenoside Rb1 (5), F1 (80), Rf (86), Rg1 (90), and Rg2 (91) (Fukuda et al., 2000a; Morinaga et al., 2001; Shoyama et al., 1999; Tanaka et al., 1999) and for the establishment of ELISA assays for the determination of immunoaffinity concentration for ginsenosides (Fukuda et al., 1999, 2000b, 2001). Recently, a simple procedure using periodate oxidation has been developed for coupling ginsenosides with BSA (Morinaga et al., 2006; Yoon et al., 1998). Both the synthetic hapten–carrier protein conjugates and the MAb produced were characterized by matrix-assisted
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laser desorption mass spectrometry (MALDI-TOF-MS) (Morinaga et al., 2001, 2006) and have, for example, been used to develop an ELISA assay for the determination of total ginsenoside content in ginseng (Morinaga et al., 2006). The total content of ginsenosides determined by the ELISA was in good agreement with HPLC-UV determinations. The effective measuring range of this ELISA assay was between 20 and 400 ng/ml for total ginsenosides when using ginsenoside Re (84) as standard. Furthermore, a highly sensitive ELISA method has been developed for the determination of 20(S)-PPT ginsenosides (Jung et al., 2002). Polyclonal antibodies raised against ginsenoside F1-BSA showed high reactivity to 20(S)-PPT ginsenosides and minor reactivities to other ginsenosides. Using ELISA, the detection and quantification range was from 50 pg/ml to 20 ng/ml, and the method was proven to be useful for the determination of 20(S)-PPT ginsenosides in biological fluids. An immunochromatographic assay for detection of ginsenosides Rb1 and Rg1 has been developed that uses anti-ginsenoside Rb1 and antiginsenoside Rg1 MAbs and a detection reagent that contains colloidal gold particles coated with anti-ginsenoside Rb1 and anti-ginsenoside Rg1 MAbs (Putalun et al., 2004). This qualitative assay system was found to be useful for a rapid screening method for the detection of ginsenoside Rb1 and Rg1 in plants and plant preparations in concentrations down to 2 mg/ml. Finally, Western and Eastern blotting methodologies have been used to detect and quantify ginsenosides in ginseng in picomole concentrations (Fukuda et al., 1999, 2000b, 2001; Tanaka et al., 2006). The use of this methodology has allowed the direct immunocrystolocalization of ginsenosides and/or individual ginsenosides directly in fresh ginseng roots, thus showing that the highest content of ginsenosides is found primarily in the endodermis cells, followed by the exodermis tissue and the radial vascular bundle (Shoyama et al., 1999; Tanaka et al., 2006).
H. Near infrared spectroscopy Near infrared spectroscopy (NIRS), a technique based on absorption and reflectance of monochromatographic radiation by samples over a wavelength range of 400–2500 nm, has been successfully applied for food composition analysis, for food quality assessment, and in pharmaceutical production control. NIRS can be used to differentiate various samples via pattern recognitions. The technique is fast and nondestructive method that does not require sample preparation and is very simple to use compared too many other analytical methods such as HPLC. The drawback of NIRS, however, is that the instrument has to be calibrated using a set of samples typically 20–50 with known analyte concentrations obtained by suitable reference methods such as HPLC in order to be used for quantitative analyses. Simultaneous quantification of the
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ginsenosides Rb1 (5), Rb2 (7), Rc (10), Rd (12), Re (84), Rg1 (88), Ro (162), and the malonylated-ginsenosides 6, 8, 11, and 13 (Fig. 1.1) in P. quinquefolium roots has been performed by NIRS (Ren and Chen, 1999). In this study, the NIRS was calibrated by analyzing 26 samples of P. quinquefolium roots for their content of ginsenoisdes by HPLC-UV, and for each sample, NIR spectra were collected over 400–2500 nm. The HPLC and spectral data obtained were used to calibrate and cross-validate the NIR instrument for measuring the individual ginsenosides. A similar investigation using NIRS have been used to quantify the ginsenoside Rb1, Rb2, Rd, Re, Rf (86), and Rg1 in P. notoginseng (Chen and Sorensen, 2000). These investigations showed that the NIRS methods for the quantitative determination of ginsenosides are comparable with those obtained by HPLC-UV.
V. POTENTIAL HEALTH EFFECTS OF GINSENOSIDES There is extensive literature on the beneficial effects of ginseng. Pharmacological effects of ginseng have been demonstrated in the central nervous system (CNS), the cardiovascular system, and the immune system. Furthermore, extensive preclinical and epidemiological studies have demonstrated that ginseng and ginseng products have potential cancerpreventive effects as well as effects on hyperglycemia (Gillis, 1997; Shibata, 2001; Sticher, 1998; Yun, 2001a, 2003). The active components in ginseng consist mainly of polysaccharides, polyacetylenes, and ginsenosides, of which the ginsenosides are considered to be the major active principles of ginseng (Sticher, 1998). The ginsenosides have demonstrated an ability to target different types of tissues, producing an array of pharmacological responses. Since ginsenosides may produce effects that are different from one another, and single ginsenosides and/or their metabolized products may initiate multiple actions in the same tissue, the overall pharmacology of ginseng and ginseng products is very complex. In the following, some of the most interesting pharmacological effect of ginsenosides, and hence their potential health promoting effects are discussed.
A. Anticarcinogenic effects Ginsenosides have been shown to exert anticarcinogenic effects in vitro and in vivo through different mechanisms. Several ginsenosides show direct cytotoxic and growth inhibitory effects against tumor cells, whereas others have been shown to inhibit metastasis and tumor growth. Results from epidemiological and cohort studies with white and red ginseng have clearly demonstrated that they have nonorganic specific preventive effect against cancer and that this effect is likely to be due to their content of
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ginsenosides, in particular ginsenoside 20(S)- and 20(R)-Rg3 (14, 42), Rg5 (72), and Rh2 (15) (Yun, 2001a, 2003).
1. Cytotoxic and antitumor activity The cytotoxic and antiproliferative effects of ginsenosides toward human and animal cancer cell lines have been demonstrated in numerous investigations. In a study, Wang et al. (2007) tested the cytotoxicity of 10 ginsenosides (20(S)-PPD, 5, 12, 14, 15, 44, 84, 88, 91, and 124), isolated from the fruits of P. ginseng, toward several human cancer cell lines, including breast cancer cell lines (e.g., MCF-7 cells), lung cancer cell lines (e.g., H838 cells), and prostate cancer cell lines (e.g., LNCaP and PC3 cells). Among the ginsenosides tested, ginsenoside 20(S)-PPD, Rh2 (15), and ginsenoside 20(R)-25-OH PPD (44) showed substantial activity in all cell lines and were clearly the most effective inhibitors of cancer cell growth and proliferation (Wang et al., 2007). For 20(R)-25-OH PPD, the IC50 values for most cell lines were in the range of 10–60 mM, which was at least twofold lower than for any of the other ginsenosides tested. Both 20(S)-PPD and 20(R)-25-OH PPD increased programmed cell death (apoptosis) and cell cycle progression in a dose-dependent manner, whereas these effects were less pronounced for ginsenoside Rh2. It is notably that 20(R)-25-OH PPD had stronger effect than ginsenoside 20(S)-Rg3 (14) on cell growth inhibition with IC50 values being 5- to 15-fold lower than for ginsenoside Rg3 (Wang et al., 2007), a compound already being marketed for cancer therapy (Liu and Ye, 2004; Shibata, 2001). Furthermore, ginsenoside Rb1 (5), Rd (12), and Rg3 had little or no effect on cell growth and proliferation. The results from the study of Wang et al. (2007) clearly suggest that the structural type of dammarane saponin, the number of sugar moieties, and differences in the substituent groups in the side chain of the aglycone affect the anticancer activity of ginsenosides. This is also in accordance with a study of Popovich and Kitts (2002) who found that both ginsenoside 20(S)-Rh1 (92) and Rh2 with a single sugar moiety had antiproliferative effects on human leukemia cells (THP-1), while 20(S)-Rg3 with two sugar moieties did not have a substantial antiproliferative effect on the cells. The effect on cell proliferation of ginsenoside Rh2 was furthermore found to be of the same magnitude as the aglycones 20(S)-PPD and 20(S)-PPT, whereas the inhibitory effect of ginsenoside 20(S)-Rh1 was tenfold less. Furthermore, the presence of sugars in PPD and PPT aglycone structures seems to reduce the potency to induce apoptosis as PPD and PPT was found to induce apoptosis to a higher extent than ginsenoside Rh2, whereas Rh1 did not induce apoptosis (Wang et al., 2007). This indicates that the position of sugar moieties at C-3 or C-6 also play a role in the anticancer effect of ginsenosides (Odashima et al., 1985; Popovich and Kitts, 2002; Wang et al., 2007). It has been suggested that the antiproliferative effects of ginsenosides and other bioactivities are dependent
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on the ability of ginsenosides to interact with cell membrane functions, and hence their hydrophobic character (Attele et al., 1999; Popovich and Kitts, 2002, 2004b; Wang et al., 2007). This is also in accordance with the structure–activity studies on the antiproliferative effects of ginsenosides and the enhanced activity observed for fatty acid conjugate ginsenosides (Hasegawa et al., 2000, 2002). Ginsenosides of the 20(S)-PPD family is the best-studied group of ginsenosides with regard to anticancer effect of which ginsenoside Rh2 is one of the best studied ginsenosides. Ginsenoside Rh2 has been shown to suppress proliferation in a number of human cancer cells, including breast, colorectal, prostate, hepatic, intestinal, melanoma, and animal cell lines (Bae et al., 2004; Kikuchi et al., 1991; Kim et al., 1999c; Lee et al., 1996; Odashima et al., 1985; Oh et al., 1999; Ota et al., 1991; Park et al., 1997; Popowich and Kitts, 2004b; Wang et al., 2007). The antiproliferative effect of Rh2 appears to be linked to its ability to induce apoptosis and/or by arresting cell cycle progression. For example, Rh2 has been reported to activate caspase-3 protease, a major proenzyme involved in apoptosis, and to arrest cell cycle progression at the G1 stage of MCF-7 human breast cancer cells, SK-HEP-1 hepatoma cells, and B16-BL6 melanoma cells ( Jin et al., 2000; Lee et al., 1996; Oh et al., 1999; Park et al., 1997) and to inhibit tumor growth in vivo of nude mice bearing human ovarian cancer cells (Kikuchi et al., 1991; Nagata et al., 1998; Tode et al., 1993). The antiproliferative effects toward cancer cells of other PPD ginsenosides such as Rg3, Rg5 (72), Rs3 (18), and Rs4 (74) also seem to be due to their ability to induce apoptosis and to perturb normal cell cycle events (Liu et al., 2000; Kim et al., 1999a,b, Min et al., 2006), although the antiproliferative effects of ginsenosides, including PPD and PPT, toward renal proximal tubule cells may be due to a decrease of c-fos and c-jun gene expression (Han et al., 2002). Compound K, the metabolized ginsenoside of some of the major ginseng PPD ginsenosides, such as ginsenoside Rb1, Rb2, Rc, and Rd, as described in Section II.C, has shown to be cytotoxic and to inhibit proliferation of a number of cancer cells such as B16-BL6 mouse melanoma cells and activated rat hepatic stellate cells in a dose-dependent manner, and to induce morphological changes and apoptotic cell death at concentrations between 24 and 40 mM (Lee et al., 2000; Park et al., 2006; Wakabayashi et al., 1998). So despite that some PPD ginsenosides do not show any significant cytotoxic and antiproliferative effects toward cancer cells even in high concentrations, they are to be considered as important prodrugs due to their metabolization into compound K in vivo (see Section II.C). Much less is known about the cytotoxic and antiproliferative effects of the 20(S)-PPT family of ginsenosides. Ginsenoside Rh1 has been reported to inhibit proliferation of the NIH 3T3 mouse fibroblast cell line but did
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not influence growth of B16 melanoma cells. Further, it was shown in the study of Wang et al. (2007) that ginsenoside Rh1 and related PPTs such as Re, Rg1, and Rg2 had no or only a minor antiproliferative effect when tested against human cancer cell lines. However, one of the main metabolized ginsenosides after oral administration of PPT-type ginsenosides is 20(S)-PPT as described in Section II.C. The antiproliferative and antitumor effect of 20(S)-PPT has been clearly demonstrated (Hasegawa et al., 2002) and as in the case of PPD-type ginsenosides, many PPT-type ginsenosides may be considered as important prodrugs due to their metabolization into, for example, 20(S)-PPT in vivo (see Section II.C).
2. Inhibition of tumor cell invasion and metastasis The prevention of cancer metastasis is important in order to improve the prognosis of cancer patients. The most characteristic step of cancer metastasis is tumor cell invasion of surrounding tissues and vasculature. Kitagawa and colleagues developed an invasion model for estimating tumor cell invasion ability in vitro (Kitagawa et al., 1995; Shinkai et al., 1996). In this model, tumor cells are seeded on a primary cultured monolayer of host cells, such as mesothelial or endothelial cells. The tumor cells penetrate the monolayer and grow and form tumor cell colonies underneath the monolayer. The capacity of penetration of tumor cells in vitro corresponds well with that of in vivo implantation into test animals. Thus, the in vitro model allows studying the effects of substances on tumor cell invasion. By using this in vitro model, more than 10 ginsenosides have been tested for the inhibition of tumor cell invasion and metastasis (Kitagawa et al., 1995; Shinkai et al., 1996). Ginsenoside 20(R)-Rg3 (42) has been found to be a potent inhibitor of invasion of several tumor cells including heaptanoma (MM1), melanoma (B16FE7), human small lung carcinoma (OC10), and human pancreatic adenocarcinoma (PSN-1) cells whereas ginsenoside Rb2 (7), 20(R)-Rg2 (111), and 20(S)-Rg3 (14) have only shown little inhibitory activity on tumor cell invasion. Neither ginsenoside Rc (10), Re (84), Rh1 (91), Rh2 (15), nor 20(R)-Rh1 (112) was found to have any effect in the model. As demonstrated by Azuma and Mochizuki (1994) and Mochizuki et al. (1995), the enantiomers 20(S)and 20(R)-Rg3 appear to have significant inhibitory effect on tumor metastasis growth as demonstrated in vitro on two highly metastatic tumor cells, B16-BL6 melanoma and colon 26-M3.1 carcinoma, and in vivo by tumor inoculation of B16-BL6 melanoma in mice. However, the effects of 20(S)- and 20(R)-Rg3 against pulmonary metastasis in vitro and in vivo appear to be different, with 20(S)-Rg3 showing the weakest effect in vivo and the strongest effect in vitro compared with 20(R)-Rg3 (Azuma and Mochizuki, 1994). Furthermore, as ginsenoside Rh2 with no inhibitory effect on metastasis but with clear antiproliferative effects toward a wide range of cancer cells, as described in Section V.A.1, indicates that
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ginsenosides exert stereo- and structure-specific biological actions suggesting that the mechanisms of their actions on cell growth and invasive locomotion are not necessarily the same. The mode of action of ginsenoside Rg3 on cell invasion and metastasis has been suggested to be related to its ability to inhibit intracellular Ca2þ increase without affecting the protein phosphorylation (Shinkai et al., 1996).
3. Inhibition of tumor angiogenesis Angiogenesis is a physiological process involving the growth of new blood vessels from preexisting vessels and is considered as a normal process in growth and development, as well as in wound healing (Fan et al., 2006; Folkman, 1995). However, this is also a fundamental step in the transition of tumors from a dormant state to a state where the tumor cells grow rapidly (malignant state). Inhibition of angiogenesis therefore prevents tumor growth, proliferation, and secondary metastasis and is essential in the prevention and treatment of cancer (Fan et al., 2006; Folkman, 1995). Only a few studies on the angiosupressive effects of ginsenosides have been performed and mainly concern the ginsenosides Rb2 (7) and 20(R)-Rg3 (42). Sato et al. (1994) studied the effect of ginsenoside Rb2 on angiogenesis and metastasis produced by B16-BL6 melanoma cells in syngeneic mice. Intravenous administration of ginsenoside Rb2 on day 1, 3, or 7 after tumor inoculation resulted in a remarkable reduction in the number of vessels oriented toward the tumor mass, but did not cause a significant inhibition of tumor growth. The angiosuppressive effect was dose-dependent in the range 10–500 mg/mouse. In contrast, intratumoral or oral administration of ginsenoside Rb2 caused a marked inhibition of both neovascularization and tumor growth. Ginsenoside Rb2 did not affect the growth of rat lung endothelial cells but inhibited in a dosedependent fashion the invasion of rat lung endothelial cells into the reconstituted basement membrane (Matrigel), which is considered to be an essential event in tumor neovascularization. Multiple administrations of ginsenoside Rb2 after the intravenous inoculation of B16-BL6 melanoma cells resulted in a significant inhibition of lung metastasis as compared with the untreated control. The results suggest that the inhibition of tumor-associated angiogenesis by ginsenoside Rb2 may partly contribute to the inhibition of lung tumor metastasis. Yue et al. (2006) examined the ability of ginsenoside 20(R)-Rg3 to interfere with the various steps of tumor angiogenesis. Ginsenoside 20(R)-Rg3 was, for example, found to inhibit the proliferation of human umbilical Vein endothelial cells (HUVEC) with an IC50 of 10 nM. Ginsenoside 20(R)-Rg3 also dose dependently suppressed the capillary tube formation of HUVEC on the Matrigel from 1 to 1000 nM in the presence or absence of 20 ng/ml vascular endothelial growth factor (VEGF). The tumor angiosupressive effects and inhibiting effect of metastasis of ginsenoside Rb2 and 20(R)-Rg3 are probably related to their inhibitive effect on the release of VEGF from tumor cells.
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B. Immunomodulatory effects The immunomodulatory (immunosuppressive and/or imunostimulatory) activities of ginsenosides are closely related to their anticarcinogenic, antiinflammatory, and antiallergic activities. The immune responses are controlled by T helper (Th) cells and can broadly be categorized into cellularmediated responses (cell-mediated immunity) mediated by Th1 cells and macrophages and antibody (antibody-mediated immunity) responses directed by Th2 cells. Th cells are involved in activating and directing other immune cells such as cytotoxic T cells and natural killer (NK) cells, and hence are particularly important in the immune system. The development and differentiation of Th cells are strictly regulated by antigenpresenting dendritic cells (DCs). DCs that generate Th1 responses may be used to prevent or treat pathological conditions that are caused by infections and malignant disorders via secretion of type 1 cytokines such as interferon-g (IFN-g) and interleukin-2 (IL-2) to facilitate T-cell-mediated cytotoxicity (Takei et al., 2004). In contrast, DCs that generate Th2 responses may be used to prevent or treat conditions in which Th1 responses are disturbed, for example, contact allergy and autoimmune disorders, by secretion of type 2 cytokines, such as IL-4 and IL-10, to help B cells to secrete protective antibodies (Takei et al., 2004). Therefore, any compound capable of modulating especially macrophage activation and/ or function, and hence the production of small and large lymphocytes (e.g., NK, T, and B cells), is important in the prevention and treatment of tumors, infectious agents, and chronic inflammatory diseases (e.g., rheumatoid arthritis, asthma, and atherosclerosis) (Rhule et al., 2006). It is well known that various ginseng species have different immunomodulatory activities and that the main active components are ginsenosides. Yu et al. (2005) investigated various PPT-type ginsenosides isolated from P. ginseng leaves (20(S)-PPT, panaxatriol (20(S)-PT), F1 (80), Re (84), Rg1 (88), Rh1 (92), and 20(R)-Rh1 (112)) for their ability to differentially modulate type 1 and type 2 cytokines production from murine splenocytes. Ginsenosides F1 and Rg1 were found to influence type 2 cytokines production through regulation of the expression of, for example, IL-4 while ginsenosides Rh1 and 20(R)-Rh1 influenced type 1 cytokines production by regulation of the production of IL-12 and the expression of IFN-g and T-bet, the latter being a specific Th1 transcription factor that is thought to initiate development of Th1 while inhibiting Th2 differentiation (Yu et al., 2005). The results clearly showed that PPT-type ginsenosides have different immunomodulatory effects including both immunostimulatory and immunosuppressive effects. This is also in accordance with a study of Cho et al. (2002) who found that the ginsenosides Rb1 (5), Rb2 (7), Re (84), and Rg1 (88) differently modulated lymphocyte proliferation induced by T lymphocyte mitogens [e.g., concanavalin A (Con A))] and the B lymphocyte mitogen, lipopolysaccharide
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(LPS), as well as cytokine IL-2, a potent trigger of lymphocyte proliferation. Ginsenoside Rb1 and Re significantly enhanced Con A-induced lymphocyte proliferation whereas Rg1 did not affect the proliferation. On the other hand, Rb2 strongly blocked the mitogen-induced lymphocyte proliferation with IC50 values between 21.8 and 29 mM and moreover Rb2 inhibited Con A-stimulated IL-2 production with an IC50 of 13.3 mM. This clearly shows that ginsenoside Rb2 is a very potent immunosuppressive agent. Ginsenosides Rb2 and Rb1 had no suppressive effects on the proliferation of IL-2stimulated CD8þ T cells whereas Re and Rg1 showed strong suppressive effects with IC50 values of 57.5 and 64.7 mM, respectively. These results clearly indicate that ginsenosides may modulate lymphocyte proliferation and that the immunosuppressive effects of ginsenosides toward tumor necrosis factor (TNF)-a cytokine production and T cell proliferation are different. Ginsenosides of P. notoginseng and P. ginseng, such as Rb1, Rb2, and Rg1, have also shown to strongly suppress the production of TNF-a in macrophages treated with LPS (Cho et al., 2001; Rhule et al., 2006). Furthermore, these ginsenosides also seem to suppress the production of other inflammatory cytokines, such as IL-6 and IL-1b (Rhule et al., 2006), and hence demonstrate that widely distributed ginsenosides possesses antiinflammatory and immunosuppressive properties in vitro. The activation of macrophages and hence the production of various types of lymphocytes has been shown to be important for the prevention and treatment of tumors and infectious diseases. Ginsenoside Rg1 has been reported to have mainly immunomodulatory effects that increases both humoral and cell-mediated immunities by enhancing activity of Th cells and NK cells responsive to given antigens (Kenarova et al., 1990; Lee et al., 2004b; Lee and Han, 2006). Furthermore, it has been reported that ginsenoside Re activates microphage function to kill tumor cells (Plohmann et al., 1997) and that ginsenosides from red ginseng in combination with other constituents in red ginseng such as melanoidins (Maillard reaction products) have immunomodulatory effects (Lee et al., 2002b). The immunomodulatory effects of red ginseng that may be effective in defending against infections and tumors seem to be closely related to the ability of the constituents of red ginseng to stimulate the production of the multifunctional cytokine TNF-a by macrophages (Lee et al., 2002b). Finally, it has been shown that maturation of DCs is promoted by metabolized ginsenosides such as compound K. Takei et al. (2004) showed that mature DCs differentiated with compound K enhance the differentiation of naive T cells toward the Th1 type depending on IL-12 secretion, which clearly suggests that compound K has immunostimulatoty effects and that this compound is involved in the cancer preventive effects of ginseng and that compound K may be used on DC-based vaccines for cancer immunotherapy (Takei et al., 2004).
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C. Anti-inflammatory and antiallergic effects The anti-inflammatory and antiallergic properties of ginsenosides are more or less directly linked to their immunostimulatory and anticarcinogenic effects as well as in diseases where inflammatory conditions play a significant role such as in atherosclerosis and neurodegenerative diseases. Allergic diseases of type 1, such as asthma, allergic rhinitis, atopic dermatitis, and food allergy afflict up to 20% of the human population in many countries (Park et al., 2003). Allergen reactivity in these allergic diseases is based on immunoglobin E (IgE)-mediated pharmacological processes in a variety of cell populations, in particular basophils and mast cells. Degranulation of basophils and mast cells with antigen cross-linked IgE releases histamine, prostaglandins, leukotrienes, and cytokines affecting macrophages, lymphocytes, eosinophils, and neutrophils, causing tissue injuries and inflammatory diseases. Cytokines and/or bacterial LPS induce nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) expression in, for example, macrophages and hence a production of nitric oxide (NO) and prostaglandins (PGs), respectively. A sustained production of NO and PGs has been implicated in the pathogenesis of inflammatory diseases and cancer (Gillis, 1997). Several ginsenosides have shown to reduce the expression of iNOS and COX-2 and to inhibit the production of NO and PGs in macrophages as well as the inhibition of nuclear factor (NF)-kB transcription factor, which regulates iNOS and COX-2 gene expression. Ginsenoside Rh1 (92) and Rh2 (15) and ginsenoside 20(S)-PPT, a metabolite of, for example, Rh1 or Rg1 (88), and compound K, a metabolite of, for example, ginsenoside Rb1 (5), have been reported to inhibit the production of NO and PGE2 and to inhibit the activation of NF-kB, in LPS-stimulated murine macrophages (RAW 264.7 cells) (Oh et al., 2004; Park et al., 1996a, 2003, 2004, 2005). The inhibition of NF-kB and COX-2 expression has also been demonstrated for compound K in mouse ear edema induced by the prototype tumor promoter 12-O-tetradecanylphorbol-13-acetate (Lee et al., 2005). The results suggest that these ginsenosides can inhibit NO and PGs production by regulation of the signal transduction related to the activation of NF-kB. The anti-inflammatory effects of ginsenosides have also been demonstrated in microglial cells, which are resident macrophages of the CNS (Bae et al., 2006; Wu et al., 2007). Wu et al. (2007) found that the PPDs ginsenoside Rb2 (7) and Rd (12) and the PPTs ginsenosides Rg1 and Re (84) were able to inhibit LPS-induced NO formation and TNF-a production due to the inhibition of NF-kB in N9 microglial cells. Correspondingly, Bae et al. (2006) demonstrated that ginsenosides Rg3 (14) and Rh2 were able to inhibit the production of NO and the expression of COX-2, TNF-a, and IL-1b in BV-2 microglial cells induced by LPS and IFN-g, while they increased the expression of the anti-inflammatory
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cytokine IL-10. Thus, these ginsenosides may be used in the prevention or treatment of inflammatory diseases, such as allergic inflammation and neurological diseases (e.g., Alzheimer’s and Parkinson’s diseases) as well as cancer (Bharti and Aggarwal, 2002; Oh et al., 2004; Wu et al., 2007). The antiallergic effect of ginsenosides has been studied in vitro and in vivo on rodent peritoneal mast cells and on IgE-induced passive cutaneous anaphylaxis (PCA), the latter being a model for study of type 1 sensitivity reactions. Ginsenosides Rb1, Rc (10), Rd, F2 (1), and Rh1 have been shown to inhibit histamine and/or leukotriene release from peritoneal mast cells (Choo et al., 2003; Park et al., 2004; Ro et al., 1998), whereas ginsenoside Rh1, Rh2, and compound K have been shown to be potent inhibitors of the PCA reaction in rodents (Choo et al., 2003; Park et al., 2003, 2004). The inhibitory activity of Rh1, Rh2, and compound K on the PCA reaction was found to be more potent than the commercial antiallergic drug disodium cromoglycate (Choo et al., 2003; Park et al., 2003, 2004). These ginsenosides furthermore showed a membrane stabilizing effect and it has been suggested that this membrane stabilizing effect, which may prevent membrane perturbations, is the main cause for their antiallergic activity (Choo et al., 2003; Park et al., 2003, 2004).
D. Antiatherosclerotic and antihypertensive effect Many studies have shown that ginseng has a protective effect on the development of atherosclerosis that may lead to myocardial infarction and other cardiovascular diseases. The preventive effects on cardiovascular diseases of ginseng include its potential antihypertensive and antiatherosclerotic effects. Ginsenosides are likely to be responsible for some of these effects as they have been shown to have inhibitory effects on platelet aggregation and to suppress thrombin formation as well as an effect on blood vessel contraction. One of the major effects of ginsenosides on the cardiovascular system is due to their ability to reduce sympathetic nerve activity and with increase vascular relaxation resulting in lowered blood pressure. This relaxing effect of ginsenosides on the cardiovascular system is partially due to the release of endothelial NO or a labile nitroso compound that liberates NO. NO relaxes blood vessels, in part, by stimulating the production of cyclic GMP in the smooth muscle (Kim et al., 1994). Ginsenosides have depressant action on cardiomyocete contraction that may be mediated, in part, through increased NO production. This is also in accordance with several animal studies in rats that have demonstrated that ginsenosides such as ginsenoside Rb1 (5), Re (84), and Rg1 (88) cause endothelium-dependent vascular relaxation and an increase in the tissue content of cyclic GMP in rat aorta, and hence an increased NO production (Chen, 1996; Kim et al., 1994; Kang et al., 1995a,b; Scott et al., 2001). The
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improved effect of red ginseng on the vascular endothelial dysfunction in patients with hypertension is possibly due to an increased production of NO (Sung et al., 2000), although it has been shown that ginsenosideinduced vasorelaxation may also involve Ca2þ activated Kþ channels in vascular smooth muscle cells (Li et al., 2001a). It has also been shown that ginsenosides enhance cerebral blood flow in rats (Kim et al., 2002b) and reduces plasma cholesterol levels and prevent the formation of atheroma in the aorta of rabbits fed on high cholesterol diet (Kang et al., 1995b). This antiatherosclerotic effect of ginsenosides such as ginsenoside Rg2 (91), 20(S)-Rg3 (14), and 20(R)-Rg3 (42) may be due to their strong inhibitory activity on platelet aggregation (Kimura et al., 1988; Matsuda et al., 1986), regulation of cyclic GMP and cyclic AMP levels, and their inhibitory effect on the conversion of fibrinogen to fibrin (Matsuda et al., 1986; Park et al., 1996b). Ginsenosides have been furthermore shown to be relatively potent platelet-activating factor antagonists ( Jung et al., 1998) as well as potential regulators of total cholesterol, triglycerides, and high-density lipoprotein (HDL) cholesterol (see Section V.G) and anti-inflammatory compounds (see Section V.C), which also may play a role in the prevention of the development of atherosclerosis and other cardiovascular diseases (Liu and Xiao, 1992).
E. Antistress activities Cortisol (corticosterone) is a vital hormone produced by the adrenal cortex and is often referred to as the ‘‘stress hormone’’ as it is involved in the response to stress. IL-6 is a multifunctional cytokine produced by a variety of cells, including immune cells (macrophages, T, and B cells), fibroblasts, neurons, and glial cells, in response to infection, trauma, and stress (Gadient and Otten, 1997). It is well known that stress increases cortisol and plasma IL-6 level, and hence these are good antistress markers. For example, the catecholamines, norepinephrine (noradrenaline), and epinephrine (adrenaline) are involved in the increase of plasma IL-6 level induced by CNS stimuli such as stress, whereas cortisol can suppress plasma IL-6 levels (Reichlin, 1993; Takaki et al., 1994). Kim et al. demonstrated that several common ginsenosides [e.g., Rc (10) and 20(S)-Rg3 (14)] as well as compound K (Fig. 1.2), the major intestinal metabolite of PPD ginsenosides, are able to inhibit stress-induced cortisol levels in mice, clearly indicating the potential antistress activity of ginsenosides (Kim et al., 1998a, 2003a). Furthermore, Kim et al. (2003b) investigated the effect of ginseng saponins on plasma IL-6 in nonstressed and immobilization-stressed mice. Ginseng total saponins, ginsenosides Rb2 (7), Rd (12), and Rg1 (88) administered intraperitoneally attenuated the stress-induced increase in plasma IL-6 level. Intracerebroventricular injection of each ginsenoside did not affect plasma IL-6 level induced by immobilization stress. Ginsenosides Rb2 and
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Rd were shown to significantly decrease IL-6 level in basal state macrophage cells (RAW 264.7) and to decrease the catecholamine (norepinephrine and epinephrine)-induced IL-6 release as well. Ginsenoside Rg1 effectively blocked epinephrine- but not norepinephrine-induced IL-6 release. It was suggested that the inhibitory action of ginseng saponins against the immobilization stress-induced increase of IL-6 level is partly periphery, mediated by blocking catecholamine-induced increase of IL-6 level in macrophages rather than in CNS (Kim et al., 2003b). The antistress effect of ginseng total saponin and ginsenoside Rg3 and Rb1 toward immobilization stress has also been demonstrated by investigating the brain level of endogenous polyamines (Lee et al., 2006b), which are essential for cellular growth, proliferation, regeneration, and differentiation of the brain, and are also well-known stress stimuli markers. In this study, it was found that ginsenoside Rg3 and Rb1 blocked the activity of the enzyme ornithine decarboxylase, involved in the metabolism and catabolism of polyamines, and attenuating the levels of the polyamine putrescine. Thus, ginsenoside Rg3 and Rb1 may play a neuroprotective role in the immobilization-stressed brain (Lee et al., 2006b).
F. Effects on the CNS Various ginseng species have been shown to have both stimulatory and inhibitory effects on the CNS, and may modulate neurotransmission. Ginsenosides, and in particular ginsenoside Rb1 (5), Rg1 (84), and Re (88), seem to play a major role in these effects (Attele et al., 1999; Rausch et al., 2006).
1. Memory, learning, and neuroprotection Central cholinergic systems have been implicated in mediation learning and memory processes (Perry, 1986). Because scopolamine is a cholinergic receptor antagonist, the performance impaired by scopolamine may result in a dysfunction of central cholinergic mechanisms, and hence may result in memory deficits (Yamaguchi et al., 1995, 1997). Results from animal studies have shown that Rb1, Rg1, and Re prevent scopolamine-induced memory deficits (Benishin et al., 1991; Yamaguchi et al., 1995, 1996b). Yamaguchi et al. (1996a) showed that sugar moieties at C-6 and C-20 as in ginsenoside Rg1 and Re are important for the ameliorating effect of ginsenosides on the performance impaired by scopolamine in rats. These ameliorative effects of Rg1 and Re have been shown to be closely related to an increase of choline acetyltransferase activity in the medial septum of young and aged rats (Yamaguchi et al., 1997). Ginsenoside Rb1 and Rg1 have also shown to be capable of partially reversing scopolamine-induced amnesia by improving cholinergic activity and having partial neurotrophic and neuroprotective effects (Radad et al., 2004b).
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Furthermore, it has been demonstrated that ginsenoside Rb1 increases the uptake of choline in central cholinergic nerve endings (Benishin, 1992), and facilitates the release of acetylcholine from hippocampal slices (Benishin et al., 1991; Lee et al., 2001). These results clearly suggest that ginsenosides may facilitate learning and improve the basic synaptic transmission as well as nerve growth. Ginsenosides also seem to have a neuroprotective effect where nerve growth also plays an important role. Ginsenosides have in several in vitro studies shown to increase survival of cultured neuronal cells and to enhance the outgrowth of their neuritis. For example, ginsenoside Rb1 has shown to increase the neurite outgrowth of cultured cerebral cortex neurons (Kim et al., 1998b; Sugaya et al., 1988) and to stimulate neurite outgrowth of PC12 cells in the absence of nerve growth factor (Rudakewich et al., 2001). The ability of ginsenosides to regenerate neuronal networks has also been demonstrated in SK-N-SH cells for PPD-type saponins such as ginsenoside Rb1 and Rb3 (9) and notoginsenoside R4 (32) and Fa (26), while PPT- (84, 87, 88, 91, 102, 103), ocotillol- (148, 150–153, 155), and oleanolic acid type (162, 167) saponins had no effect on neurite outgrowth (Tohda et al., 2002). This clearly indicates that some ginsenosides are able to extend axons and dendrites in neurons that may compensate for and repair damaged networks in, for example, the dementia brain. Furthermore, it has been shown that ginsenosides Rb1 and Rg1 protect neurons from excitotoxicity induced by, for example, glutamate and oxidative stress caused by hydrogen peroxide and promote neurite lengths and neurite numbers of dopaminergic cells after exposure to 1-methyl-4-phenylpyridinium (Radad et al., 2004a,b), which is an active metabolite selectively toxic to dopaminergic neurons in vitro. Interestingly, ginsenosides Rb1 and Rg1 also seem to be able to reverse the cell death caused by 1-methyl-4-phenylpyridinium (Rudakewich et al., 2001). These beneficial effects of ginsenosides, and in particular ginsenoside Rb1 and Rg1, are primarily mediated through scavenging of free radicals and improving energy metabolism (Radad et al., 2004b) as well as their ability to block Ca2þ over-influx into neuronal cells and inhibit Naþ channel activity (Kim et al., 1998b, 2002a, 2005b; Lee et al., 2006d; Radad et al., 2004b). In particular, Ca2þ loading exceeding the capacity of Ca2þ regulating mechanisms could activate several cell death-related genes and pathways leading to apoptosis and cell death (Radad et al., 2006; Rausch et al., 2006; Said et al., 2000). Glutamate that is a major neurotransmitter in the mammalian nervous system not only plays a role in the development of the brain and learning but is also a potent neurotoxin when present in excess at synapses (Plaitakis and Shashidharan, 2000; Rausch et al., 2006). Glutamate excitotoxicity has been shown to contribute to neuronal degeneration in acute conditions such as stroke, epilepsy, hypoglycemia, and chronic
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neurodegerative diseases such as Alzheimer’s, Parkinson’s, and Huntington’s diseases (Radad et al., 2006; Rausch et al., 2006). Although the pathogenesis of glutamate toxicity is not fully understood, it appears that ginsenosides and ginseng plants may have beneficial effects toward the above-mentioned diseases due to their neurotrophic and neuroprotective effects (van Kampen et al., 2003). Also the anti-inflammatory effects of ginsenosides seem to play an important role in neurodegenerative diseases such as Alzheimer’s disease ( Joo et al., 2005), which is primarily caused by cell death due to chronic inflammation and cell stress.
2. Neurotransmitter modulation
Ginsenosides have in in vitro studies shown that they may modulate nerve transmission by decreasing or even increasing the availability of neurotransmitters (Kimura et al., 1994; Tsang et al., 1985; Xue et al., 2006). Xue et al. (2006) demonstrated that both ginsenoside Rb1 (5) and Rg1 (88) increased neurotransmitter release in undifferentiated and differentiated PC12 cells. The promoted neurotransmitter release of ginsenoside Rb1 was found to be due to an increasing phosphorylation of synapsin phosphoproteins through the cyclic AMP-dependent protein kinase pathway, whereas the similar effects observed for ginsenoside Rg1 were independent of the phosphorylation of the synapsins. On the other hand, Tsang et al. (1985) demonstrated that ginseng extracts, and hence also ginsenosides, concentration dependently inhibits the uptake of g-aminobutyric acid (GABA), glutamate, dopamine, norandrenaline, and serotonin in rat brain synaptosomes. GABA is an inhibitory neurotransmitter in mammalian CNS and it has been shown that ginsenosides compete with agonists for binding to GABAA and GABAB receptors and hence modulate neurotransmission (Kimura et al., 1994). The regulation of GABAergic neurotransmission may be important in the action of ginsenosides.
G. Effect on metabolic processes The effects of ginseng and ginsenosides on metabolic processes are in particular due to their ability to activate peroxiome proliferator-activated receptors (PPARs) that are transcription factors and part of a large family consisting of steroid/thyroid hormone receptors. Ginsenosides have been shown to change the expression of PPAR a and g. PPAR a is expressed in the kidney, the liver, the muscles, as well as in adipose tissues and activation results in upregulation of genes involved in the triglyceridelowering effects through transcriptional activation of apolipoprotein (apo) C-III and lipoprotein lipase (Auwerx et al., 1996; Hertz et al., 1995) as well as fatty acid b-oxidation (Desvergne and Wahli, 1999). PPAR a has also been demonstrated to increase the concentration of HDL levels
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through induction of apo A-I and -II gene expression in humans (Staels and Auwerx, 1998). Thus, PPAR a is an important regulator of lipid metabolism and hence is an important target for the prevention and treatment of lipid disorders, cardiovascular diseases, obesity, and diabetes. PPAR g is primarily expressed in adipose tissue and is also involved in lipid metabolism and plays an important role in insulin resistance and hence is an important molecular target for particular obesity and diabetes as discussed in more detail in Section V.H. It has been shown that ginseng and a mixture of ginsenosides obtained by extraction are able to regulate total cholesterol, triglycerides, and HDL cholesterol in vitro and in vivo and hence are able to regulate lipid metabolism through activation of PPAR a (Yoon et al., 2003). This is also in accordance with another study where ginsenoside Rf (86) was identified as one of the major active components that regulate lipoprotein metabolism by interacting with PPAR a acting as a PPAR a antagonist (Lee et al., 2006a). Ginsenoside Rf may therefore have therapeutic applications in relation to the prevention and treatment of various diseases, including lipid disorders, cardiovascular diseases, obesity, and diabetes.
H. Antidiabetic effects According to the World Health Organization (WHO), more than 180 million people suffer from diabetes and more than 90% of these have type 2 diabetes (T2D) and this number is likely to be doubled by 2030 due to the increasing prevalence of obesity (Wild et al., 2004). T2D is characterized by insulin resistance, low fasting glucose levels (hyperglycemia), and high concentrations of triglycerides in the blood and hence also the risk for especially cardiovascular diseases. In recent years, clinical trials and animal experiments have demonstrated that ginseng and ginsenosides are able to lower blood glucose (Attele et al., 2002; Cho et al., 2006; Chung et al., 2001; Lee et al., 2006c; Sotaniemi et al., 1995; Vuksan et al., 2000; 2001; Xie et al., 2002a,b, 2004, 2005a; Yun et al., 2004), to increase insulin sensitivity (Attele et al., 2002; Han et al., 2006; Shang et al., 2007; Yun et al., 2004), and to regulate lipid metabolism (Han et al., 2006; Yoon et al., 2003; Yun et al., 2004), and even to reduce body weight (Attele et al., 2002; Han et al., 2005; Kim et al., 2005a; Sotaniemi et al., 1995; Xie et al., 2002a,b, 2005a; Yun et al., 2004). Hence, ginsenosides may be used for the prevention and treatment of T2D. However, antidiabetic effects have only been demonstrated for a few specific ginsenosides including Rb1 (5), Re (84), Rh2 (15), and the aglycone 20(S)-PPT. Antidiabetic effect has been demonstrated for ginsenoside Re in ob/ob diabetic mice (Attele et al., 2002) and ginsenoside Rh2 has been shown to increase insulin secretion and to lower plasma glucose in Wistar rats (Lee et al., 2006c). Furthermore, it has been shown that the antidiabetic
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effects of ginsenoside Rb1 (Shang et al., 2007) and 20(S)-PPT (Han et al., 2006) are probably related to their ability to activate peroxisome PPAR g. PPAR g is a member of the nuclear receptor of ligand-activated transcription factors that regulate the expression of key genes involved in lipid and glucose metabolism and adipocyte differentiation (Desvergne and Wahli, 1999; Tobin and Freeman, 2006). PPAR g is primarily expressed in adipose tissue, and activation of PPAR g improves the ability of adipocytes to store lipids, thereby reducing lipotoxicity in muscle and liver (Bays et al., 2004). The genes expressed by activation of PPAR g depend largely on the type of activating ligand present as these recruit a different set of cofactors (Semple et al., 2006). Hence, the transcriptional response of the PPAR g can result in either cofactors that lead to increased lipid storage and decreased energy expenditure (e.g., transcriptional factor-2 (TIF-2)) or recruitment of cofactors that lead to increased insulin-stimulated glucose uptake and positive regulation of glucose metabolism and energy expenditure (e.g., the steroid receptor coactivator-1) (Burgermeister et al., 2006; Schupp et al., 2005). In general, the activation of PPAR g causes body-wide lipid repartitioning by increasing the triglyceride content in adipose tissue and lowering free fatty acids triglycerides in circulation, liver, and muscle, thereby improving insulin sensitivity (Han et al., 2006). The thiazolidinediones (TZDs) are full PPAR g agonist often prescribed in the clinical treatment of T2D, as insulin sensitizing drugs. However, severe side effects such as edema development, weight gain, heart enlargements, and hepatoxicity are seen in relation to the use of TZDs (Larsen et al., 2003; Pan et al., 2006). These undesirable side effects are believed to be caused by the fact that TZDs are full PPAR g agonists (Barroso et al., 1999). Partial PPAR g agonists are ligands that activate PPAR g in a more selective way than full agonists and furthermore appear to promote more beneficial recruitment of cofactors, and hence are not believed to have the same side effects as full agonists. The potential antidiabetic effects of ginsenoside Rb1 and 20(S)-PPT have been shown to be due to promotion of adipocyte differentiation via PPAR g activation and increased activation of glucose transporter 4 (GLUT4) associated with insulin sensitivity in adipocyte tissue (Han et al., 2006; Shang et al., 2007). Therefore, Rb1 and 20(S)-PPT seem to improve insulin resistance by reducing lipotoxicity in muscle and liver through increasing ability to store lipids in adipocytes and enhancing insulin sensitivity through increase of GLUT4 expression in adipocyte. However, further investigations of the antidiabetic effects of Rb1 and 20(S)-PPT are needed to classify these ginsenosides as potential antidiabetic agents with no side effects. Some investigations have indicated that the hypoglycemic effect of ginseng products depends both on the ginsenoside content and on the
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profile, clearly indicating that the antidiabetic effect of ginseng depends on the concentration of single ginsenosides and that not all ginsenosides possesses antidiabetic effects (Sievenpiper et al., 2003a,b, 2004; Vuksan et al., 2000, 2001).
VI. CONCLUSION Ginsenosides are a unique group of compounds that have demonstrated a multiple of pharmacological effects including anticarcinogenic, immunostimulatory, antiatherosclerotic, antihypertensive, and antidiabetic effects as well as effects on the CNS and stress. The various pharmacological effects of ginsenosides are probably due to their resemblance in chemical structure/nature with triterpenoid steroid hormones and their amphiphilic nature being able to intercalate into plasma membranes (Attele et al., 1999). Their amphiphilic properties of ginsenosides can lead to changes in the membrane fluidity, and thus affect membrane function, elicitating a cellular response. Moreover, like steroid hormones, they are lipid-soluble signaling molecules that can traverse the plasma membrane and interact with membrane anchored receptors, and ion channels as well as nuclear receptors initiating genomic effects (Attele et al., 1999). Pharmacokinetic studies have also confirmed that ginsenosides are bioavailable and that to some extent they are metabolized in the gastrointestinal tract to deglycosylated ginsenosides whose ability to traverse membranes are even better than intact ginsenosides, which further add to the multifunctional pharmacological activities of this group of compounds. Finally, the diversity in ginsenoside structures, including structural isomerism (Fig. 1.1), furthermore contributes to the multifunctional pharmacological effects of ginsenosides. Certainly, the argument can be raised that evidence for most pharmacological effects of ginsenosides has been obtained from in vitro studies, many of which have not been confirmed in vivo and certainly not in humans with the exception of ginsenoside 20(S)-Rg3 (14) and 20(R)-Rg3 (42), which is used in anticancer treatment (Shibata, 2001). Nevertheless, the demonstration of the health effects of ginseng and ginseng preparations in epidemiological and cohort studies and the fact that ginsensoides are bioavailable and can initiate effects at the plasma membrane by interacting with multireceptor systems and that ginsenosides are able to traverse the membrane and produce genomic effects clearly indicates that ginsenosides have potential health effects in humans. The pharmacological effects of ginseng and ginseng preparations and hence their quality are clearly dependent on their content of ginsenosides.
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Consequently, many types of analytical methods have been developed for the identification and quantification of ginsenosides in raw materials and preparations. In particular, analytical HPLC combined with UV, evaporate light scattering, and/or MS detection have shown to be useful for the analysis of nearly all types of ginsenosides. So far, focus have been primarily on the quantification of major ginsenosides in roots from P. ginseng, P. quinquefolium, and P. notoginseng such as ginsenoside Rb1 (5), Rb2 (7), Rc (10), Rd (12), Re (84), Rf (86), Rg1 (88), and Rg2 (91) as these compounds are the most common ginsenosides and have shown a multifunctional pharmacological effects. However, the pharmacological effects of many ginsenosides and their potential metabolized products have so far not been investigated that also include many of the new types of ginsenosides isolated in recent years from the aerial parts of several ginseng species (Table 1.1). Therefore, many interesting pharmacological effects of ginsenosides are yet to be discovered and perhaps this may reveal further and new insight in the pharmacological effects of ginsenosides and their potential health effects that in the end may result in new ginseng herbal remedies or medicinal products.
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Yu, J.-L., Dou, D.-Q., Chen, X.-H., Yang, H.-Z., Guo, N., and Cheng, G.-F. (2005). Protopanaxatriol-type ginsenosides differentially modulate type 1 and type 2 cytokines production from murine splenocytes. Planta Med. 71, 202–207. Yue, C.-J., and Zhong, J.-J. (2005). Purification and characterisation of UDPG: Ginsenoside Rd glucosyltransferase from suspended cells of Panax notoginseng. Process Biochem. 40, 3742–3748. Yue, P. Y. K., Wong, D. Y. L., Wu, P. K., Leung, P. Y., Mak, N. K., Yeung, H. W., Liu, L., Cai, Z., Jiang, Z.-H., Fan, T. P. D., and Wong, R. N. S. (2006). The angiosuppressive effects of 20(R)-ginsenoside Rg3. Biochem. Pharmacol. 72, 437–445. Yun, T. K. (2001a). Panax ginseng—a non-organ-specific cancer preventive? Lancet Oncol. 2, 49–55. Yun, T. K. (2001b). Brief introduction of Panax ginseng C.A. Meyer. J. Korean Med. Sci. 16 (Suppl.), S3–S5. Yun, T. K. (2003). Experimental and epidemiological evidence on non-organ specific cancer preventive effect of Korean ginseng and identification of active compounds. Mutat. Res. 523/524, 63–74. Yun, S. N., Moon, S. J., Ko, S. K., Im, B. O., and Chung, S. H. (2004). Wild ginseng prevents the onset of high-fat diet induced hyperglycemia and obesity in ICR mice. Arch. Pharm. Res. 27, 790–796. Zhang, H., and Cheng, Y. (2006). Solid-phase extraction and liquid chromatographyelectrospray mass spectrometric analysis of saponins in Chinese patent medicine of formulated Salvia miltiorrhizae and Panax notoginseng. J. Pharm. Biomed. Anal. 40, 429–432. Zhang, H., Lu, Z., Tan, G. T., Qiu, S., Farnsworth, N. R., Pezzuto, J. M., and Fong, H. H. S. (2002). Polyacetyleneginsenoside-Ro, a novel triterpene saponin from Panax ginseng. Tetrahedron Lett. 43, 973–977. Zhou, J., Wu, M., Taniyasu, S., Besso, H., Tanaka, O., Saruwatari, Y., and Fuwa, T. (1981). Dammarane-saponins of Sanchi-ginseng, roots of Panax notoginseng (Burk.) F. H. Chen (Araliaceae): Structures of new saponins, notoginsenosides-R1 and -R2, and identification of ginsenosides-Rg2 and -Rh1. Chem. Pharm. Bull. 29, 2844–2850. Zhu, S., Zou, K., Fushimi, H., Cai, S., and Komatsu, K. (2004). Comparative study on triterpene saponins of ginseng drugs. Planta Med. 70, 666–677. Zou, K., Zhu, S., Meselhy, M. R., Tohda, C., Cai, S., and Komatsu, K. (2002). Dammarane-type saponins from Panax japonicus and their neurite outhgrowth activity in SK-N-SH cells. J. Nat. Prod. 65, 1288–1292.
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CHAPTER
2 Adherence, Anti-Adherence, and Oligosaccharides: Preventing Pathogens from Sticking to the Host Kari D. Shoaf-Sweeney* and Robert W. Hutkins†
Contents
I. Introduction II. Route of Infection III. Adherence Basics A. Adherence kinetics B. Adherence specificity C. Adherence affinity IV. Specific Pathogen–host Interactions A. Lectin–carbohydrate interactions B. Protein–protein interactions C. Hydrophobin–protein interactions V. Intestinal Target Tissues A. Cell surface structures B. ECM components C. Host cell adhesive components VI. Bacterial Adhesins A. Fimbrial adhesins B. Afimbrial adhesins VII. Common Bacterial Adherence Mechanisms A. Salmonella B. Helicobacter pylori C. Enteropathogenic Escherichia coli (EPEC) D. Enterohemorrhagic Escherichia coli (EHEC) E. Uropathogenic Escherichia coli (UPEC)
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*School of Molecular Biosciences, Washington State University, Pullman, Washington 99164 Department of Food Science and Technology, University of Nebraska-Lincoln, Lincoln, Nebraska 68583
{
Advances in Food and Nutrition Research, Volume 55 ISSN 1043-4526, DOI: 10.1016/S1043-4526(08)00402-6
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2009 Elsevier Inc. All rights reserved.
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VIII. Anti-adhesives A. Adhesin-based vaccines B. Host-derived anti-adhesins C. Probiotics as anti-adhesives D. Adhesin analogs E. Receptor analogs IX. Conclusions and Future Prospects References
Abstract
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For many pathogenic bacteria, infections are initiated only after the organism has first adhered to the host cell surface. If adherence can be inhibited, then the subsequent infection can also be inhibited. This approach forms the basis of anti-adherence strategies, which have been devised to prevent a variety of bacterial infections. In this chapter, the molecular basis by which respiratory, urinary, and gastrointestinal tract pathogens adhere to host cells will be described. The five general types of anti-adherence agents will also be reviewed. The most well-studied are the receptor analogs, which include oligosaccharides produced synthetically or derived from natural sources, including milk, berries, and other plants. Their ability to inhibit pathogen adherence may lead to development of novel, food-grade anti-infective agents that are inexpensive and safe.
I. INTRODUCTION For most pathogenic bacteria, infection or colonization of host tissues depends, in large part, on the ability of the organism to somehow withstand the normal flux or flow within the particular tissue and to ‘‘stick’’ to the surfaces of the intended target. In fact, adherence of pathogens to host cell surfaces can be considered an essential first step in the infection process (Savage, 1977, 1984). Adherence, however, is not random or indiscriminant, but rather is cell- and tissue-dependent. That is, a particular organism recognizes specific receptors located on the host cell surface and then attaches itself to those receptors using specific adhesin molecules. Thus, an enteric pathogen, such as Salmonella enterica, expresses adhesins that bind to targets found in the gastrointestinal tract (GIT), but not in the urinary tract. Conversely, uropathogenic Escherichia coli (UPEC) can adhere to cells that line the urinary tract, but is poorly equipped to stick to other tissues. Importantly, adhesins are rightfully considered virulence factors; their absence renders the organism incapable of causing an infection. Likewise, if the receptor is absent or adherence is otherwise blocked, infection is similarly impeded.
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For the majority of cases, carbohydrate- or oligosaccharide-bearing moieties are the most common host cell receptors recognized by pathogenic bacteria, although protein-type receptors also exist that are associated with adherence to connective tissues (collagens) or injured tissues that are on the mend (fibronectin, laminin). The identification and characterization of these specific bacteria–host interactions have not only revealed important insights into the molecular mechanisms of pathogenesis, but have also provided a basis for development of anti-infection agents. If the receptor targets are known, then it is possible to identify molecules that mimic those receptors and to use those molecules as decoys. Averting adherence by physically blocking bacterial contact with intended receptors has provided the rationale for development of several anti-adhesion strategies (Kahane and Ofek, 1996; Karlsson, 1998; Kelly and Younson, 2000; Ofek et al., 2003b; Sharon and Ofek, 2000; Zorf and Roth, 1996). Such approaches are attractive for several reasons. First, anti-adherence approaches could provide an effective alternative to antibiotics, whose use has led to the emergence of antibiotic-resistant pathogens. Second, some of the proposed anti-adherence substrates occur naturally in milk, foods, and plants. Recently, for example, food-grade oligosaccharides that are used as prebiotics were also found to have antiadhesive activity. Finally, the efficacy of many novel anti-adhesive agents has already been reported. These include adhesin-based vaccines, host-derived anti-adhesives, probiotics, adhesin analogs, and receptor analogs. This chapter will provide an overview of the research on anti-adhesion agents. Particular attention will be devoted to the anti-adherence agents derived from or found naturally in foods. In addition, the pathogen infection process, the architecture of host epithelial cell surfaces, and the chemistry and mechanisms involved in bacterial interactions with host cell surfaces will also be reviewed.
II. ROUTE OF INFECTION Enteric bacterial pathogens must maneuver through a lengthy stretch of hazardous terrain before they reach their intended target or infection site within a host. Initially, they must tolerate salivary enzymes having various hydrolytic activities in the mouth, followed by exposure to shedded epithelial cells in the esophagus that may prevent local bacterial adherence (Pearson and Brownlee, 2005). In the stomach, bacteria must endure another severe environment created by the secretion of digestive enzymes and hydrochloric acid (up to 0.1 M concentration and a pH as low as 1.0). Once bacteria reach the intestines, they then encounter mechanical,
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chemical, and physical barriers in the form of luminal flow, a mucus layer, commensal bacteria, secreted anti-microbial proteins and peptides, and a host immune response (Pearson and Brownlee, 2005). The severity of these barriers against pathogens within the intestines is dependent upon the physiological dynamics and the prepared mucosal defenses of the target tissues. In the small intestine, the rapid luminal flow, the low pH, and the presence of digestive enzymes ward off many invaders. The apical surface of the small intestine contains an abundance of intestinal villi ranging from 10 to 40 per mm2 of mucosal surface area (Laux et al., 2005), that, through ciliary action, also help avert bacterial adherence. These villi contain varying types of epithelial cells, including columnar absorptive enterocytes, goblet cells that secret mucus, and cells that secrete anti-microbial proteins (Falk et al., 1998; Ouellette and Selsted, 1996). Additionally, there is a high rate of epithelial cell turnover that assists in preventing adherence (Laux et al., 2005; Pearson and Brownlee, 2005). The conditions within the large intestine are much less strenuous on invading pathogens. The luminal flow is less vigorous, there is a neutral pH, and the epithelial cell turnover is around tenfold slower than that of the small intestine (Falk et al., 1998; Xu and Gordon, 2003). Moreover, the surface of the large intestine does not have villi and is relatively smooth, but still contains absorptive enterocytes, goblet cells, and anti-microbial secreting cells (Falk et al., 1998). Goblet cells are present in many of the gastrointestinal tissues of humans. They secrete a protective gel-like mucus layer that covers the stomach, small intestine, and large intestine (Allen et al., 1984; Forstner et al., 1995). This mucus is chiefly composed of large filamentous gelforming glycoproteins called mucins (Forstner et al., 1995), that provide both a loosely adherent surface layer and a firmly adherent underlying layer (Atuma et al., 2001). Mucus is thought to act as a medium for protection, lubrication, and transport between the lumen and the epithelial cell surface (Forstner et al., 1995). With respect to bacterial adherence in the GIT, however, the most important role of mucus is as a protective barrier against unwanted bacterial invaders. The mucus layer not only acts as a physical barrier by blocking binding sties at the epithelial cell surface, but it also contains proteins, lipids, and nucleic acids that may deter pathogen adherence, including defensins, lysozyme, anti-adherence molecules, secreted IgA and IgM, and other resident microorganisms (Pearson and Brownlee, 2005). Although the mucus layer acts as a barrier to some invading pathogens, it also supports the growth and maintenance of a number of commensal bacteria in the GIT. It acts as an initial binding site, a source of nutrients for growth, and is a niche where these bacteria can replicate and potentially compete with other newly introduced bacteria (Laux et al., 2005). Therefore, tissues that produce mucus have the potential to provide the host
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with a well established microflora that is thought to be essential for GIT balance (Tannock, 1999). The thickness of the mucus layer differs in varying portions of the GIT. It is thinner and discontinuous in the small intestine and significantly thicker and continuous in the stomach and large intestine (Atuma et al., 2001). The mucus layer in the stomach acts as a protectant against the digestive enzymes and high acid content that would otherwise destroy the tissue (Allen et al., 1984), rather than for the growth and maintenance of microflora. The mucus layer in the large intestine fosters immensely diverse, highly competitive bacterial populations (Tannock, 1997, 1999). Consequently, pathogenic microorganisms that attempt to infect tissues that support large populations of indigenous microflora must vigorously compete with these bacteria to become established.
III. ADHERENCE BASICS Many of the general molecular mechanisms involved in pathogen binding, including specificity, overall binding kinetics, and affinity, are now established (Ofek and Doyle, 2000). First, the ability of pathogens to survive and initiate infection within a host is reliant on their ability to adhere to host cell tissues. Secondly, adherence involves the interaction of complementary molecules on the surface of the bacteria and the surface of the host epithelium, respectively. These principles emphasize the importance of understanding the molecular mechanisms behind adherence. This is especially true when designing new approaches to prevent pathogen infection by interrupting the adhesion process.
A. Adherence kinetics Adherence is very intricate and requires a strict series of events that eventually leads to stable bacterial–host interactions. As a bacterium approaches a host cell, it must overcome repulsive forces generated by the negative charges found on both the host tissues and the bacterial surface (Ofek et al., 2003a). Varying attractions and interactions account for the ability of bacteria to prevail over these forces, including van der Waals’ attractions, Coulombic forces, hydrophobic interactions, and eventually complementary interactions. The van der Waals’ attractions and Coulombic forces allow a bacterium to move to within 5 nm of the host cell surface (Busscher and Weerkamp, 1987). Once this occurs, bacterial binding follows a two-step kinetic model (Hasty et al., 1992). The first step occurs when the bacteria overcomes repulsive forces and becomes loosely and reversibly bound to the host cell surface through hydrophobic interactions. These interactions are mediated by
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hydrophobins on the bacterial surface that interact with hydrophobic moieties (e.g., fatty acids) on the host cell surface (Rosenberg and Doyle, 1990; Rosenberg and Kjelleberg, 1986; Rosenberg et al., 1996). The second kinetic step occurs when a strong irreversible complex is established between a specific bacterial adherence molecule and a complementary host receptor (Fig. 2.1) (Ofek and Doyle, 2000). The precise chemical interactions between an adhesin and its receptor are also important. For example, direct- and water-mediated hydrogen bonds are the most important interactions within the carbohydraterecognition domain in carbohydrate-binding adhesins on the host cell surface (Weis and Drickamer, 1996). Nonpolar van der Waals’ interactions and hydrophobic ‘‘stacking’’ of the receptor oligosaccharide rings with aromatic amino acid side chains of the bacterial adhesin protein also contribute to oligosaccharide–protein interactions. X-ray structural
Hydrophobic interactions Electrostatic interactions
+ −
Complementary interactions
50 nm Bacterium 10–20 nm Bacterium
<5 nm Bacterium
FIGURE 2.1 Description of bacterial adherence. Three distinct interaction regions are illustrated. When the bacterium is greater than 50 nm from the host cell, van der Waal’s interactions occur. Upon moving to within 10–20 nm, both van der Waal’s interactions and Coulombic forces attempt to overcome an intense repulsion generated by the net negative charges on opposing surfaces. Fimbriae and other polymers that have a small diameter can effectively overcome this repulsion, as repulsive forces decrease in proportion to the diameter of the particles approaching each other. When the bacterium reaches to within 5 nm, complementary binding (lectin–carbohydrate interactions) is required, which may also involve stabilizing hydrophobin–hydrophobin and charge–charge (electrostatic) interactions. Adapted from Busscher and Weerkamp (1987) and Ofek et al. (2003a).
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analysis indicates that the hexose units of some oligosaccharide receptors interact with adhesins through hydrogen bonding, while nonpolar interactions form between other portions of the oligosaccharide and protein (Weis and Drickamer, 1996). Additionally, hydrophobic interactions and hydrogen bonds surrounding the adhesin–oligosaccharide complex promote more stable adherence (Duncan-Hewitt, 1990).
B. Adherence specificity Bacterial adherence molecules are referred to as adhesins (Finlay and Falkow, 1989). The most well-studied and abundant group of adhesins (at least among bacterial pathogens) are the proteinaceous bacterial lectins that recognize complementary oligosaccharides on the host cell surface. Structurally, these lectin adhesins have a small globular carbohydraterecognition domain that represents a relatively shallow indention on the surface of the protein (Weis and Drickamer, 1996). Subtle chemical diversity within this indentation allows for the selectivity of each adhesin to its target or cognate oligosaccharide receptor. Consequently, this results in the exquisite ability of bacteria to differentiate between slightly dissimilar oligosaccharide structures on the host cell surface. Thus, the overall specificity of a bacterium for a particular host is contingent on the presence of definitive oligosaccharide receptors (Ofek et al., 1978; Sharon and Ofek, 1986). For example, E. coli strains that express the K99 adhesin bind specifically to N-glycolylneuraminyllactosyl ceramide. Animals, such as newborn piglets, that have these particular oligosaccharide sequences on their intestinal cell surface, are susceptible to infection by this pathogen. Humans do not possess these structures and are accordingly resistant to infection by K99-expressing E. coli (Ono et al., 1989). This binding specificity also explains the attraction a pathogen has for a particular host tissue. Streptococcus pneumoniae targets oligosaccharide structures present on human respiratory tract tissues (Andersson et al., 1983; Smit et al., 1984), while most diarrheagenic E. coli pursue the oligosaccharides on intestinal tissues (Ofek et al., 1977). However, specificity is not entirely a function of the presence or absence of particular oligosaccharide structures. For example, E. coli that possess mannose-specific adhesins do not colonize all mannose-containing tissues (Ofek and Doyle, 2000). Therefore, adhesion is apparently due to a combination of factors including oligosaccharide presentation and orientation. Additionally, specificity may be context-dependent in that the structures surrounding the bacterial adhesin and complementary host cell receptor must be in suitable positions and have agreeable charges for any host–pathogen interactions to occur (Ofek and Doyle, 2000; Weis and Drickamer, 1996).
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C. Adherence affinity The affinity of an adhesin for an oligosaccharide is a significant feature of bacterial adherence. Generally, a single oligosaccharide molecule would have a low affinity for its corresponding protein, within, for example, a range of micro- to millimolar (Weis and Drickamer, 1996). However, increasing the valency of the protein–oligosaccharide interaction significantly enhances the affinity of the protein for its intended target. Bacteria create these multivalent oligosaccharide-binding proteins by assembling individual protein subunits that contain numerous individual oligosaccharide-binding sites into a filamentous structure (Vijayan and Chandra, 1999; Weis, 1997; Weis and Drickamer, 1996). These multi-unit structures can concurrently bind to numerous individual oligosaccharides on the host cell via a ‘‘Velcro-like’’ mechanism, increasing avidity for the target (Mulvey et al., 2001).
IV. SPECIFIC PATHOGEN–HOST INTERACTIONS Identifying and characterizing the direct molecular contact points between bacterial adhesins and host receptors are central to developing novel strategies to prevent infection via adhesin–receptor interference. Three main types of adhesin–receptor interactions have been described, lectin– carbohydrate, protein–protein, and hydrophobin–protein (Courtney et al., 1990; Cywes et al., 2000; Hanski et al., 1992; Hasty et al., 1992; Sylvester et al., 1996; Szymanski and Armstrong, 1996; Wu et al., 1996). Lectin–carbohydrate interactions are often found along the surface of the host cell, and involve the glycolipids, glycoproteins, and proteoglycans found in the glycocalyx layer. Protein–protein interactions usually involve the extracellular matrix (ECM) components of the host cell (discussed in Section IV.B.). Hydrophobin–protein interactions are thought to take place during the early stages of bacteria–host contact before specific lectin–carbohydrate or protein–protein interactions occur. Each of these will be discussed below in more detail.
A. Lectin–carbohydrate interactions The lectins involved in lectin–carbohydrate interactions are either located on the bacterial cell surface or in the host epithelial cell surface. An example of the latter is the host cell lectin CD44 that binds to hyaluronic acid moieties in the capsule of the Gram-positive pathogen Streptococcus pyogenes (Cywes et al., 2000). Host lectins have only recently been found to be complementary to Gram-negative pathogen oligosaccharide structures. Lipopolysaccharides (LPS) or lipooligosaccharides anchored to the outer membranes of Vibrio mimicus (Alam et al., 1996) and Pseudomonas aeruginosa (Zaidi et al., 1996) have been shown to serve as adhesins to mucosal cells and mucus components, most probably by recognizing lectins on the host cell surface (Jacques, 1996).
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The most well-studied bacteria–host interactions are those involving bacterial protein lectins and complementary oligosaccharide ligands on the host cell surface. In Gram-negative bacteria, lectins usually exist in the form of polymorphic fimbriae or pili. They are often made up of hundreds of protein subunits that bind host oligosaccharides (Nuccio and Baumler, 2007; Ofek and Doyle, 1994; Sharon and Ofek, 1986). Lectins in Grampositive bacteria are often positioned within the peptidoglycan layer or are anchored in the cytoplasmic membrane so that they traverse the peptidoglycan layer and extend beyond the cell wall (Ofek et al., 2003a). Determining the specificity of bacterial lectins to their cognate oligosaccharide ligands has been the subject of considerable interest, but has also proven to be experimentally challenging. Several approaches have been described, including direct-binding assays, measurement of in vitro cell adherence to tissue culture cells in the presence of oligosaccharides, and determining virulence in vivo when exogenous oligosaccharide receptors are present (Ascencio et al., 1993; Barthelson et al., 1998; Brennan et al., 1991; Firon et al., 1987; Giannasca et al., 1996; Hanisch et al., 1993; Jagannatha et al., 1991; Krivan et al., 1988; Ofek et al., 1977; Rajan et al., 1999; Stromberg et al., 1990; Teneberg et al., 2004). The interactions between UPEC FimH adhesin and human host glycoproteins are among the best described, and are discussed, in detail, later in this chapter. Table 2.1 lists some of the oligosaccharide structures found on host cell surfaces that are complementary to pathogen lectins.
B. Protein–protein interactions Protein–protein interactions are usually associated with the exposed ECM proteins and proteoglycans that are normally found at the basolateral surface of the mucosa. They become available for binding when the host cell surface has been compromised. There are a number of bacterial proteins that have been found to bind one or more of the ECM components including fibronectin, laminin, collagen, and elastin. These proteins are commonly referred to as MSCRAMMs (microbial surface components recognizing adhesive matrix molecules) (Patti et al., 1994). The bestillustrated protein–protein interactions are those involving fibronectinbinding bacterial proteins that, accordingly, adhere to fibronectin in the ECM of host enterocytes. Several bacteria have been found to express fibronectin-binding proteins (FnBPs) (Hasty et al., 1994). However, only a few studies have shown a definitive interaction between an adhesin and fibronectin as it pertains to adherence. As a result, the frequency of this adherence mechanism in nature remains to be determined (Ofek et al., 2003a). Over six different FnBPs have been identified in S. pyogenes (Finlay and Caparon, 2000); some of these will be discussed in more detail below (Section VI.B.).
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TABLE 2.1 Examples of pathogen oligosaccharide adherence sites on host mucosal surfacesa Organism
Escherichia coli Type 1 pili P-fimbriae S-fimbriae CFA/1 K1 F5 (K99) Bordetella pertussis Haemophilus influenza Helicobacter pylori Klebsiella pneumoniae Mycococcus pneumoniae Neisseria gonorrhoea Neisseria meningitidis Pseudomonas aeruginosa Salmonella typhimurium Streptococcus pneumoniae Streptococcus suis a
Target molecule
Target tissue
Man(a1–3)[Man(a1–6)]Man Gal(a1–4)Gal NeuAc(a2–3)Gal(b1–3)GalNAc NeuAc(a2–8)GlcNAc(b1–4)GlcNAc NeuGc(a2–3)Gal(b1–4)Glc Gal(b1–3)GalNAc(b1–4)Gal(b1–4)Glc [NeuAc(a2–3)]0,1 Gal(b1–4)GlcNAc(b1–3)Gal(b1–4)GlcNAc NeuGc(a2–3)Gal(b1–4)Glc(NAc) Fuc(a1–2)Gal(b1–3)[Fuc(a1–4)]Gal Man
Urinary Urinary Neural Intestinal Endothelial Intestinal Respiratory
Stomach Stomach Respiratory
NeuGc(a2–3)Gal(b1–4)Glc(NAc)
Respiratory
Gal(b1–4)Glc(NAc)
Genital
[NeuAc(a2–3)]0,1 Gal(b1–4)GlcNAc(b1–3)Gal(b1–4)GlcNAc Gal(b1–3)Glc(NAc)(b1–3)Gal(b1–4)Glc Man
Respiratory
Gal(b1–4)GalNAc [NeuAc(a2–3)]0,1 Gal(b1–4)GlcNAc(b1–3)Gal(b1–4)GlcNAc Gal(a1–4)Gal(b1–4)Glc
Respiratory
Respiratory Intestinal Intestinal Respiratory Respiratory
Adapted from Karlsson (1989) and Ofek and Doyle (1994).
C. Hydrophobin–protein interactions Hydrophobin–protein interactions include those bacterial surface components that promote adhesion to host cell surfaces via hydrophobic moieties that are often thought to be nonspecific (Rosenberg and Doyle, 1990; Rosenberg and Kjelleberg, 1986; Rosenberg et al., 1996).
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Hydrophobin–protein interactions enable the bacterial cell to overcome any repulsive forces at the host cell surface. However, there may also be some degree of specificity involved, as in the case of lipoteichoic acid (LTA) found on the surface of S. pyogenes that exhibits reversible adherence (Courtney et al., 1990; Hasty et al., 1992).
V. INTESTINAL TARGET TISSUES Epithelial cells of the mucosal surface are the first to come into contact with invading pathogens, and consequently are the first to be colonized during infection (Ofek et al., 2003a). However, when epithelial cells are disrupted by injury or by tissue or organ insults, the exposed underlying structural components or ECM become prime targets for bacterial adherence. Therefore, understanding the biology of host cells and tissues is critical in identifying mechanisms of bacterial adherence, especially when developing novel methods to prevent selective pathogen adherence. The intestinal epithelia is generally characterized as structurally simple, in that it is made up of a single layer where the apical side of each cell borders the lumen and each basal side sits on the basal lamina or basement membrane. The basal lamina is primarily composed by twodimensional sheets of type IV collagen, but also includes laminins, heparan sulfate proteoglycans, fibronectin, and other ECM components (Erickson and Couchman, 2000). The epithelial cell layer itself, is made up of absorptive enterocytes (Trier and Madara, 1981), mucus secreting goblet cells (Moe, 1955), undifferentiated crypt cells (Trier and Madara, 1981), Paneth cells with large secretary granules (Elms, 1976; Erlandsen and Chase, 1972), enteroendocrine cells, and gut-associated lymphoid tissue (GALT) (Roy et al., 1987) containing Peyer’s patches (Trier and Madara, 1981), which are predominately composed of antigen-sampling M cells (Owen and Jones, 1974). Individual cells are bound to the ECM by transmembrane integrins, and are bound to each other via several multiprotein complexes including tight junctions (occludins and claudins), adherens junctions (cadherins), and gap junctions (Ofek et al., 2003a). Beneath the basement membrane is a network of connective tissues including fibril-forming collagen (type I, II, and III), fibronectin, elastin, various cell types like mast cells and macrophages, and neural and vascular elements (Trier and Madara, 1981). Depending on the location within the GIT, the apical surface of the epithelium may be organized into microvilli, and is covered by a carbohydrate-rich glycocalyx layer and a mucus layer composed of high-molecular weight glycoproteins that collectively protect the epithelium from pathogens when structurally intact (Neutra and Forstner, 1987). However, when the integrity of the apical cell surface is compromised, or when the host is confronted by pathogens that
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are capable of invading enterocytes from the basal side, the structural components associated with the basal membrane and ECM become key receptors in bacterial adherence.
A. Cell surface structures Under normal circumstances, bacteria first encounter the carbohydraterich mucus and glycocalyx layers on the apical surface of the gastrointestinal epithelium. The mucus layer is mainly composed of large filamentous gel-forming glycoproteins called mucins that may or may not be membrane-bound, are naturally highly sialylated, and are continuously in motion down the GIT (Forstner et al., 1995). Pathogens must either penetrate through or bind to the mucus layer to initiate infection (Ofek et al., 2003a). Consequently, bacterial motility and chemotaxis through the mucus layer may play very important roles in adherence for many pathogens (Eaton et al., 1992; Freter et al., 1981; Takata et al., 1992; Young et al., 2000). On the other hand, some nonmotile pathogenic bacteria are still capable of complete colonization and full virulence. For example, nonmotile, flagellated S. enterica serovar Typhimurium and E. coli F-18 remain virulent in animal models (McCormick et al., 1988, 1990). However, it should be noted that the involvement of motility and chemotaxis in mucus penetration is not well understood (Laux et al., 2005). Additionally, many human and animal enteric pathogens have been shown to bind to mucus components in vitro, including enteropathogenic E. coli (EPEC) (Mack and Sherman, 1991; Smith et al., 1995), pathogenic E. coli strains with K88 (Cohen et al., 1983), K99 (Laux et al., 1984; Lindahl and Carlstedt, 1990), RDEC-1 (Mack and Sherman, 1991), and 987P adhesins (Dean, 1990), Clostridium difficile (Karjalainen et al., 1994, 2001; Tasteyre et al., 2001; Waligora et al., 2001), Campylobacter spp. (Sylvester et al., 1996), Salmonella serovars (Vimal et al., 2000), Yersinia spp. (Mantle and Husar, 1994), Shigella dysenteriae 1 (Sudha et al., 2001), and Helicobacter pylori (Van de Bovenkamp et al., 2003). It is unclear how binding of mucus components facilitates in vivo adhesion to the epithelial cell surface. However, it is likely that adherence to mucus components in vitro reflects events associated with the early stages of infection rather than later stages of colonization (Laux et al., 2005), and that in some cases, binding of mucus components appears to be positively correlated to enhanced colonization (Cohen et al., 1985; Vimal et al., 2000). The glycocalyx is a general term that refers to the dense mat of variable, highly glycosylated integral membrane glycoproteins, glycolipids, and proteoglycans that are presented on the epithelial cell surface (Ito, 1969). They are thought to play a recognition role in cell growth, differentiation, and cell–cell interactions (Brandley and Schnaar, 1986; Roseman, 1985), as well as in malignancy (Hakomori, 1984; Prokazova
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et al., 1988; Yogeeswaran, 1983) and modulation of receptor-mediated membrane processes (Hanai et al., 1988; Usuki et al., 1988). The diversity and density of saccharides in the glycocalyx layer make it an exceptionally appealing surface for lectin-bearing bacteria (Mirelman and Ofek, 1986). Several studies have shown that bacteria are capable of binding internal or terminal saccharide sequences of glycolipids (Bock et al., 1985; Karlsson, 1989; Krivan et al., 1988). For example, a number of bacteria have been shown to bind specifically to lactosylceramide, including Bacteroides spp., Clostridium spp., Shigella spp., S. enterica serovar Typhimurium, and E. coli spp. (Karlsson, 1989). Interestingly, lactosylceramide is not present in the human small intestinal epithelium (Bjork et al., 1987), but is found in abundance in the colonic epithelium (Holgersson et al., 1988), which appears to coincide with the target tissue for most of the aforementioned pathogens. Thus, receptor specificity to particular saccharide structures in the glycocalyx is of utmost importance to many enteric pathogens.
B. ECM components The components of the ECM are also important bacterial receptors when they become accessible to invading pathogens. Studies have shown that several pathogenic bacteria bind to ECM components, including collagens (Holderbaum et al., 1986; Visai et al., 1990; Wagner et al., 2007), laminins (Moran et al., 2005; Plotkowski et al., 1996; Speziale et al., 1982; Switalski et al., 1987; Valkonen et al., 1994), fibronectin (Dorsey et al., 2005; Dramsi et al., 2004; Froman et al., 1984, 1987; Monteville and Konkel, 2002), vitronectin (Liang et al., 1997; Valentin-Weigand et al., 1988), hyaluronan (Cywes et al., 2000), elastin (Downer et al., 2002), and proteoglycans (Alvarez-Dominguez et al., 1997; Ascencio et al., 1993; Bergey and Stinson, 1988; Fleckenstein et al., 2002; Guo et al., 1998; Isaacs, 1994). In general, these proteins and proteoglycans function together, when necessary, to facilitate wound healing and inflammation, resulting in a vulnerable provisional ECM that is susceptible to bacterial colonization and host cell entry (Preissner and Singh Chhatwal, 2005). Besides the recuperative aspect of ECM components, they are essential contributors to the cellular shape, orientation, differentiation, and metabolism of a variety of cellular systems (Ruoslahti and Obrink, 1996; Timpl and Brown, 1996).
C. Host cell adhesive components Other cellular components that mediate cell–cell or cell–ECM interactions have also been shown to have bacterial specificity, including integrins (Coburn et al., 1998; Leong et al., 1990; Rezcallah et al., 2005; Wang et al., 2006; Watarai et al., 1996), cadherins (Mengaud et al., 1996), and selectins
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(Ho et al., 1998; Sandros et al., 1994). Integrins are expressed ubiquitously and have multiple functions depending on their location in the body. In the small intestine, integrins have been shown to not only mediate cell interactions with the ECM and basement membrane, but also activate various signaling pathways that leads to the modulation of gene expression (Lussier et al., 2000). For example, Shigella flexneri (Watarai et al., 1996), Staphylococcus aureus (Fowler et al., 2000), and Yersinia spp. (Isberg et al., 2000) use integrin, cadherin, and selectin receptors, either directly or indirectly, to make cellular contact and then initiate invasion pathways into epithelial cells. With every new bacterial receptor identified, an exciting opportunity emerges for receptor decoy discovery that could prevent infection by these pathogens. Table 2.2 lists some of the specific interactions of bacterial species with host ECM and adhesive components.
VI. BACTERIAL ADHESINS The initial adherence of pathogens to host cell surfaces is considered an essential step in colonization and infection (Savage, 1977, 1984). Therefore, identifying the bacterial molecules that mediate adherence has been a major area of research, especially since these molecules may serve as targets for anti-adherence strategies. As discussed previously (Section VI), the detailed interactions between a pathogen and a host cell are often mediated by proteinaceous surface structures on the bacterial surface. These bacterial proteins are referred to as adhesins (Finlay and Falkow, 1989), and are most often found on the tips of bacterial fimbriae or pili (fimbrial adhesins), but may also be anchored in the bacterial membrane so that it can be presented on the bacterial outer membrane (afimbrial adhesins) (Sharon and Ofek, 1986). Models of fimbrial and afimbrial adhesins of some human pathogens are discussed here.
A. Fimbrial adhesins Often UPEC strains carry many different adhesins, two of these are type 1 fimbriae ( fim) and pyelonephritis-associated pili (pap) (Berglund and Knight, 2003; Mulvey, 2002; Schilling et al., 2001). These adhesins allow the organism to take advantage of its local environment by regulating cross-talk between the fim and pap operons (Xia et al., 2000), ultimately resulting in a genetic on/off switch. In the lower urinary tract, type 1 fimbriae binds a high-mannose glycoprotein, uroplakin Ia (Firon et al., 1987; Wu et al., 1996; Zhou et al., 2001), resulting in cystitis (Ronald et al., 2001). The P-pili are used by UPEC in escalating urinary tract infections (UTI) to bind galabiose-containing glycolipid receptors in the kidney that
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TABLE 2.2 Important extracellular matrix and adhesive components use as receptors by pathogensa ECM/adhesive components
Fibronectin
Collagen
Vitronectin Laminin
Integrins Heparan sulfate E-cadherin Uroplakin CD48 a
Microorganism
Adhesin
Staphylococcus aureus Group A streptococci
FnbA, FnbB PrtF1/Sfb1, PrtF2, LTA SOF/SfbII, M3 protein FnBA, FNnBB FnB, GfbA FbpA CadF ShdA, MisL
Group C streptococci Group G streptococci Listeria monocytogenes Campylobacter jejuni Salmonella enterica serotype Typhimurium Staphylococcus aureus Group A streptococci Streptococcuc parasanguis Legionella pneumophila Staphylococcus aureus Neisseria meningitidis Staphylococcus aureus Group A streptococci Shigella Yersinia Neisseria gonorrhoeae Listeria monocytogenes E. coli E. coli
Can M proteins, FNB54 FimA Mip 60-kDa protein NhhA
IpaB, IpaC Invasin Opa proteins Internalin A Type 1 fimbriae Type 1 fimbriae
Adapted from Finlay and Caparon (2005) and Ofek et al. (2003a).
initiates pyelonephritis (Dodson et al., 2001; Lund et al., 1987). The role of type 1 fimbriae in the GIT has not been elucidated (Bloch et al., 1992), and the receptors are undefined in this milieu (Bouckaert et al., 2005). However, the type 1 fimbriae are by far the most prevalent adhesin in UPEC strains (Brinton, 1959; Buchanan et al., 1985). Many proteins are necessary to construct these fimbriae including FimA that forms a rigid helical rod, FimF and FimG subunits that makes up the short tip fibrillum, and FimH presented at the end of the tip fibrillum that is responsible for adherence through a carbohydrate-recognition domain for mannose (Jones et al., 1995; Krogfelt et al., 1995). Although the primary FimH receptor is
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uroplakin Ia, FimH also recognizes glycoproteins with one or more N-linked high-mannose structures and is able to agglutinate yeast cells (Firon et al., 1984; Krogfelt et al., 1995; Ofek et al., 1977). More recently, the FimH lectin has been shown to bind with a high affinity to butyl a-Dmannosides particularly those with longer alkyl tails, aryl mannosides, and fructose (Bouckaert et al., 2005). Accordingly, inhibiting FimH– receptor interactions have been shown to prevent bacterial adherence to the bladder epithelium and as a result may prevent infection (Langermann and Ballou, 2003; Langermann et al., 1997, 2000; Thankavel et al., 1997). Both natural and synthetic mannose terminal structures have been shown to interrupt FimH-mediated UPEC adherence (Aronson et al., 1979; Firon et al., 1987; Nagahori et al., 2002; Old, 1972), and with the recent discovery of high affinity mannoside and fructose receptors, more anti-adherence candidates are sure to follow, increasing the likelihood of developing potential vaccines against these pathogens.
B. Afimbrial adhesins Many afimbrial adhesins have been identified in pathogenic bacteria including E. coli, H. pylori, Bordetella pertussis, Neisseria species, Yersinia species, Haemephilus influenzae, Campylobacter jejuni, S. aureus, and Streptococcus species (Finlay and Caparon, 2005). These adhesins have been shown to bind ECM components in an attempt to initiate infection. Some of the most well-studied afimbrial adhesins are FnBPs belonging to the MSCRAMM family of adhesins that are expressed by S. aureus and S. pyogenes (Patti et al., 1994). Fibronectin is a large dimeric glycoprotein found in plasma, the ECM, and on eukaryotic cell surfaces. It is responsible for host cellular processes like adhesion, migration, and differentiation (Hynes, 1990), but is also a common substrate for bacterial attachment that ultimately results in host cell internalization (Fowler et al., 2000). The FnBPs of S. aureus and S. pyogenes have similar structural organization and Fn-recognition mechanisms (Joh et al., 1999). In general, FnBPs are surface proteins anchored in the cell wall that contain an LPXTG motif found in most surface-associated proteins of Gram-positive bacteria (Fischetti et al., 1990), and have a short positively charged C-terminal tail where Fn recognition occurs within sequence repeats of 35–40 amino acid residues (called FnBRs) (Patti et al., 1994). In some instances, two Fn-binding domains have been identified, as in the case of UR, which is upstream of sfbI in S. pyogenes. Currently, SfbI is the model FnBP for Fn recognition. It contains two high-affinity and several lower-affinity binding sites for Fn (SchwarzLinek et al., 2003, 2004, 2006). Each FnBR in the C-terminus can potentially bind to one dimer of Fn, which in turn, contains two binding domains for integrins (Schwarz-Linek et al., 2006). Therefore, SfbI is thought to serve as a molecular bridge between the bacteria and host cell integrins in a
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FnBP-mediated internalization of S. pyogenes (Ozeri et al., 1998). Additionally, SfbI has been shown to recruit other ECM components like collagen I and IV that aid in escaping the host immune system (Dinkla et al., 2003). Around 50% of the S. pyogenes clinical isolates express the sfbI gene (Natanson et al., 1995). However, only five different FnBPs contain FnBRs that can potentially assist bacteria in adherence and invasion (Schwarz-Linek et al., 2006). The presence of multiple FnBPs could possibly explain how S. pyogenes is able to colonize different host tissue and confer various tissue tropisms. The identification of the SfbI adhesin has contributed to the recent development of vaccines composed of SfbI-derived peptides conjugated to either the diphtheria toxoid or used with the Lipid Core Peptide (LCP) delivery system. These vaccines have been shown to confer protective immunity to BALC/c mice when challenged intranasally with lethal doses of S. pyogenes (Olive et al., 2007; Schulze et al., 2006).
VII. COMMON BACTERIAL ADHERENCE MECHANISMS Human microbial pathogens that possess the ability to adhere to host tissues have a distinct advantage over those that do not, in that they are better equipped to evade and resist the defense systems of their host. There are numerous adherence mechanisms that have been described to date. However, those that are most commonly studied originate from pathogens that colonize the GIT and genital-urinary tract, including Salmonella, H. pylori, and pathogenic serotypes of E. coli.
A. Salmonella The adherence mechanisms involved in Salmonella infection have been studied in great deal. Disease associated with S. enterica serovars is initiated by attachment to and invasion of host cells, followed by subsequent inflammation of the lamina propria and lymph nodes (Darwin and Miller, 1999). Several genetically defined fimbrial or piliar adhesins contribute to the initial attachment and the overall infection process of Salmonella. Some of these include type 1 fimbriae (Fim), plasmid-encoded (PE) fimbriae, long polar (LP) fimbriae, and thin aggregative fimbriae (curli). However, many other putative fimbrial operons have been identified within various S. enterica serovar genomes, but the expression of these proteins is currently undefined. The type 1 fimbriae in S. enterica serovars are encoded by the fimAICDHF operon (Collinson et al., 1996b) and are morphologically similar to, but antigenicly different from the type 1 fimbriae of E. coli (Korhonen et al., 1980). These fimbriae are composed primarily of FimA
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protein subunits, but binding specificity is determined by the FimH subunit on the tip of the fimbrial shaft, which has an affinity to mannose residues, as described previously (Clegg and Swenson, 1994; Klemm and Krogfelt, 1994). Recent studies have shown that the S. enterica serovar Typhimurium FimH adhesin mediates adherence to HeLa, HEp-2, and mouse intestinal epithelial cells (Boddicker et al., 2002; Hancox et al., 1997; Thankavel et al., 1997). Additionally, both S. enterica serovar Tyhimurium and S. enterica serovar Enteritidis have been shown to bind human colon carcinoma cell line HT-29 and human bladder cancer cell line Hu1703He via their type 1 fimbriae FimH adhesin (Kisiela et al., 2006). However, there appears to be significant heterogeneity in receptor specificities for particular mannosylated compounds among type 1 fimbriae, not only from different bacterial genera, but also from within the same species. These differences have been attributed to allelic variants in the FimH adhesin, where a disparity in one or two amino acid residues leads to differing receptor affinities and specificities (Boddicker et al., 2002; Kisiela et al., 2006; Sokurenko et al., 1994, 1995). These allelic differences likely mediate host specificity and target tissue specificity via the type I fimbriae adhesion. The Salmonella LP fimbriae, encoded by the lpfABCDE fimbrial operon, were first identified in S. enterica serovar Typhimurium and thought to have been acquired by horizontal transfer during evolution, as the flanking sequences of this operon are homologous to those in E. coli K-12 (Baumler, 1997). Although the expression of the lpf operon in a nonfimbriated E. coli strain results in the appearance of polar filaments, there is no conclusive evidence that LP fimbriae are polar on Salmonella. The lfpABCDE operon has been implicated in the colonization of murine Peyer’s patches and reported to be important in the early stages of oral infection (Baumler et al., 1996c; Norris et al., 1998). Using the mouse small intestine model of infection, a mutation in an outer membrane protein (OMP) thought to be an usher for fimbrial assembly (lpfC), resulted in reduced colonization of the Peyer’s patches, but not villous enterocytes (Baumler et al., 1996c). In vivo, this mutation alone had a minimal effect on virulence in BALC/c mice, as did a single mutation in invA, a type III secretion system (TTSS) gene required for invasion. However, these mutations in conjunction, led to a 150-fold increase in oral LD50 compared to the wild type or either single mutant, leading to the conjecture that Salmonella must be intimately adhered to target cells to invade a murine host via its TTSS and that LP fimbriae may be the means by which Salmonella accomplishes this feat (Jones et al., 1994; Norris et al., 1998). The PE fimbriae, encoded by the pefBACD operon contained on the virulence plasmid pSLT, has been found in only four Salmonella serotypes, S. typhimurium, S. choleraesuis, S. paratyphi, and S. enteritidis (Baumler et al., 1997). The PE fimbria has been demonstrated to mediate adherence to the
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mouse small intestine and also appears to be involved in the initiation of fluid accumulation (Baumler et al., 1996a,b). Nicholson and Low (2000) reported that the expression of the pefBACD operon is under methylationdependent transcriptional regulation, similar to the pap operon in E. coli, and that expression only occurs in conditions of low pH and O2 rich medium. Additionally, the expression of SrgA, a disulfide oxidoreductase, is also required for the production of PE fimbriae (Bouwman et al., 2003). Specifically, the disulfide bond within the major structural subunit of the PE fimbriae, PefA, must be oxidized by SrgA in order for the fimbriae to be assembled and for the maintenance of PefA stability. Another fimbrial adhesin that mediates the adherence of Salmonella to host cells is the thin aggregative fimbriae, often referred to as Tafi. Collinson et al. (1991) identified this adhesin in Salmonella enteritidis, and the operon was termed agf. However, because the Tafi homolog in E. coli was first termed curli and the operon termed csg (Arnqvist et al., 1992; Collinson et al., 1992), Tafi was renamed with the csg nomenclature (Romling et al., 1998). The curli fimbriae have been found to be essential for numerous Salmonella virulence mechanisms including accelerating amyloidosis in mice, binding fibronectin, and enhancing adherence and invasion of eukaryotic cells (Arnqvist et al., 1992; Dibb-Fuller et al., 1999; Kim and Kim, 2004; La Ragione et al., 2000; Lundmark et al., 2005; Sukupolvi et al., 1997). Curli-producing bacteria tend to auto-aggregate, which has been suggested to enhance the survival of Salmonella spp., in hostile environments like stomach acid or other detrimental milieus (Collinson et al., 1993). Interestingly, the genes involved in curli production are organized into two adjacent divergently transcribed operons, agfBAC and agfDEFG (Collinson et al., 1996a), which are both required for curli biosynthesis and assembly (Collinson et al., 1993). Additionally, curli are the only fimbriae dependent on the extracellular nucleation– precipitation assembly pathway, which deviates from other assembly pathways as fiber growth occurs extracellularly (Hammar et al., 1996). Recently, a non-fimbrial adhesin, SiiE, has been identified in S. enterica serovar Typhimurim. Although little is known about SiiE, it has been found to mediate contact-dependent adhesion to HeLa cell surfaces (Gerlach et al., 2007). SiiE is a type 1 secretion system (T1SS) secreted protein encoded in the Salmonella pathogenicity island 4 and might functionally resemble the type 1 fimbrial adhesins. More work is needed to elucidate the true role of SiiE in adherence in vivo.
B. Helicobacter pylori H. pylori is one of the main causes of human chronic gastritis, resulting in various diseases including peptic ulcers, gastric adenocarcinomas, and mucosa-associated lymphoid tissue (MALT) lymphomas (Williams
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and Pounder, 1999). At least 50% of the world population is infected with H. pylori (Hocker and Hohenberger, 2003), but less than 30% of those are symptomatic (Das and Paul, 2007). The variability in disease frequency and severity of clinical outcome has been attributed, in part, to variable expression of at least two virulence genes, the cytotoxin associated gene (cagA), which is encoded within the cag pathogenicity island (PAI) and the vacuolating toxin A (vacA). Importantly, virulence is also associated with expression of an outer membrane-bound adhesin encoded by babA2 (Atherton et al., 1995; Blaser, 1996; Gerhard et al., 1999). Due to the very low pH in the human stomach (pH 1–2), adherence and colonization are a significant challenge. Thus, rather than colonizing the intensely acidic stomach lumen, H. pylori colonizes the mucin layer that covers the gastric mucosa. This latter microenvironment has a near neutral pH and is much more suitable for survival (Salyers and Whitt, 2002). Therefore, H. pylori must not only be highly motile in an effort to penetrate the mucus barrier, but must be able to maintain their presence there. Not surprisingly, adhesins associated with the carbohydrate-containing moieties within the mucus layer have been identified, including H. pylori– neutrophil-activating protein (HP–NAP). In addition, adhesins have been described that allow H. pylori to adhere directly to the epithelial cell surface. These include BabA, AlpA and AlpB, SabA, HopH, HopZ, and HorB, all of which belong to the H. pylori OMP family 1 (Alm et al., 2000). Originally, HP–NAP was found to induce neutrophil adhesion to endothelial cells in vitro and in vivo (Kurose et al., 1994; Yoshida et al., 1993). This is supported by the observation that HP–NAP has a high affinity for glycosphingolipids ending in a linear NeuAca3Galb4GlcNAc b3Galb4GlcNAcb sequence (Teneberg et al., 1997), which is found in the glycosphingolipid fraction of human neutrophils (Karlsson et al., 2001). However, more recently, HP–NAP has been isolated from the OMP fraction of H. pylori and shown to have an entirely different function in the bacterial cell membrane (Namavar et al., 1998). Namavar et al. (1998) found that HP–NAP may be responsible for the adhesion of H. pylori to sulfated mucins. Consistent with these findings, HP–NAP has a high affinity for sulfatide (SO3Gal1b-Cer) and gangliotetraosyl ceramide (SO3Galb3GalNAcb4Galb4Glcb1-Cer), which are not found on human neutrophils (Teneberg et al., 1997). Additionally, H. pylori and purified HP–NAP bind to SO3–3-Gl, SO3–NAcGlc, the blood group antigen SO3–3Lewis a, and sulfo-Lewis x and Lewis x antigens (Namavar et al., 1998). Moreover, bovine milk and fucoidan, components of dietary seaweed that have been shown to have an anti-ulcer effect (Shibata et al., 1998), block sulfatide-mediated and Lewisb-mediated adherence of H. pylori to gastric cells (Hata et al., 1999; Shibata et al., 1999, 2003). As noted above, BabA is the best-characterized H. pylori adhesin. BabA binds Lewisb moieties on gastric epithelial cell surfaces. However, not all
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H. pylori isolates express the BabA protein. In fact, only about half of the isolates studied by Hennig et al. (2004) expressed a detectible level of BabA protein and of those, there was considerable variation with regard to binding Lewisb in vitro. Additionally, Ilver et al. (1998) reported that 63 (66%) out of 95 H. pylori isolates were able to bind Lewisb moieties. On the other hand, Gerhard et al. (1999) reported that the presence of babA2 genotype is a good indicator for the ability of strains to express the Lewisb-binding adhesin, and that there is a strong correlation among the expression of babA2, Lewisb adherence, and gastric cancer. H. pylori also expresses a sialic acid-binding adhesin (SabA), which binds inflamed gastric mucosa (Mahdavi et al., 2002). The expression of SabA receptors, sialyl-Lewis x (sLex) and sialyl-Lewis a (aLea) glycans, is activated by an inflammatory response, in which they help recruit white blood cells to tissue in peril (Alper, 2001). SabA has also been shown to be a prerequisite for the nonopsonic activation of human neutrophils, an inducer of oxidative metabolism, and to be essential for phagocytosis induction via binding sialylated neutrophil receptors (Petersson et al., 2006; Unemo et al., 2005). The minimal binding epitope described for SabA is a NeuAca2–3Gal disaccharide, but longer gangliosides and glycoconjugates allow better binding, as do sialylated structures lacking fucose constituents (Aspholm et al., 2006). Both AlpA and AlpB have been shown to be involved in H. pylori adherence to gastric epithelial cells and gastric tissue sections, in vitro (Obenbreit et al., 1999, 2000). Additionally, recent studies have shown that AlpB and, to a lesser extent, AlpA are required for colonization of the guinea pig stomach (de Jonge et al., 2004). Additionally, the alp genes may play an important role in the early stages of infection, as the transcription of alpA is tenfold higher 1 h after infection versus 1 week after infection. However, alpA is also transcribed throughout the first 3 months of infection in vivo, suggesting an active role in maintaining infection (Rokbi et al., 2001). More work is needed to elucidate the complete function of alpA and alpB in H. pylori infection in humans. The role of other putative OMP adhesins, including HopZ, HopH, and HorB is still relatively poorly understood. Peck et al. (1999) showed that a hopZ isogenic mutant greatly reduced adherence to AGS cells (human gastric adenocarcinoma epithelial cells). The HopH protein once designated the ‘‘outer inflammatory protein’’ (OipA), because it was associated with an increase in interleukin-8 secretion from epithelial cells in vitro and heightened gastric inflammation in vivo, has also been implicated in bacterial adherence. However, de Jonge et al. (2004) showed that both hopZ and hopH isogenic mutants were able to colonize guinea pigs and the wild-type strain, confirming previous observations in a mouse model (Yamaoka et al., 2002). The HorB protein has also been found to be involved in the adherence of H. pylori to AGS cells and in the production
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of LPS O-polysaccharide chains (Snelling et al., 2007), which has been suggested to have a role in adherence to gastric sections (Edwards et al., 2000), albeit later studies have discredited H. pylori LPS as an adhesin (Mahdavi et al., 2003). The loss of HorB production reduces the ability of H. pylori to colonize Helicobacter-free BALB/c mice and the expression of horB has been detected in gastric biopsies of two culture-positive human subjects (Snelling et al., 2007). More research is needed to completely elucidate the role of these putative adhesins in H. pylori infection. Undoubtedly, more OMP adhesins will be identified. However, these adhesins mentioned above and other more recently discovered OMPs must be confirmed as true adhesins and not simply auxiliary proteins that are necessary for the presentation of a functional adhesin.
C. Enteropathogenic Escherichia coli (EPEC) Infections by EPEC strains are one of the major causes of infant diarrhea in developing countries (Cravioto et al., 1991; Levine et al., 1988), and recently have been recognized as a contributing factor in childhood diarrhea in the United States as well (Cohen et al., 2005). Generally, infection causes acute and chronic diarrhea, vomiting, and low-grade fever with a higher mortality rate in developing countries where treatment may be inadequate (Clausen and Christie, 1982; Levine and Edelman, 1984). EPEC is characterized according to the unique genetic attributes that encode its distinctive multi-step infection process, and, in particular, the formation of ‘‘attaching and effacing’’ (A/E) lesions on the brush border surface of the small intestine (Moon et al., 1983). The organism is also known for its ability to form three-dimensional microcolonies on the surface of host gastrointestinal epithelium cells (Kaper, 1996). Scaletsky et al. (1984) described this microcolony formation as ‘‘localized adherence’’ (LA). A/E lesion formation is manifested by a degeneration of the intestinal brush border surface at the site of attachment, followed by microvilli effacement, and the assemblage of highly organized pedestal structures mediated by individual bacteria (Frankel et al., 1998). The genes necessary for A/E lesion formation are found in the locus of enterocyte effacement (LEE) PAI and include structural components of the TTSS apparatus and secreted translocator and effector proteins (Frankel et al., 1998). Before LA and A/E lesion formation can occur, however, EPEC must first attach to the host cell surface via adhesins. Several adhesins have been implicated in the initial adherence of EPEC to small intestine enterocytes including type IV bundle forming pili (BFP), TTSS EspA filaments, intimin, and flagella. However, in a recent study, Cleary et al. (2004) found no evidence that adhesive factors other than BFP and EspA are able to support initial EPEC adherence. Recently, LifA, whose gene sequence has significant homology to the efa-1 and toxB genes in enterohemorrhagic
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Escherichia coli, EHEC O111 and EHEC O157:H7, respectively, has also been suggested to play a role in adherence in the absence of BFP (Badea et al., 2003). The BFP of EPEC has been shown to be very important in the pathogenicity of this organism (Bieber et al., 1998). It serves as a contact for bacteria–bacteria interactions and microcolony formation (Giron et al., 1991), auto-aggregation (Vuopio-Varkila and Schoolnik, 1991), dispersal of bacteria from microcolonies (Bieber et al., 1998; Knutton et al., 1999), and, as noted above, has been implicated in the initial adherence of EPEC to host epithelial cells (Donnenberg et al., 1992; Giron et al., 1991; Tobe and Saskawa, 2002). Although strains that lack BFP are still capable of causing ‘‘diffuse’’ binding and promoting pedestal formation in vitro (Cleary et al., 2004; Rosenshine et al., 1996), they are about 200-fold less virulent than the wild-type parent strain in vivo (Bieber et al., 1998). Thus, BFP appears to be a convincing candidate as the primary adherence factor. Among the BFP receptor molecules that have been proposed, most are oligosaccharides. Exogenous molecules that resemble BFP receptors interfere with LA, thus competitive-binding inhibition assays are very useful when investigating these candidate receptors. For example, N-acetylgalactosamine completely inhibits LA to HeLa cells, thus, it may act as a receptor for BFP on the host cell surface (Scaletsky et al., 1988). Another group found that locally adhering EPEC bound to asialo-GM1, asialoGM2, globoside, and lacto-N-neotetraose, which all share the sequence GalNAc(b1–4)Gal (Jagannatha et al., 1991). In addition, LA was inhibited by fucosylated tetra- and pentasaccharides in several strains of BFPexpressing EPEC (Cravioto et al., 1991), suggesting a role of these sugars as binding sites. Additionally, phosphatidylethanolamine (PE) has also been implicated as a potential receptor for BFP (Foster et al., 1999), and recently, Hyland et al. (2008) reported that N-acetyllactosamine (LacNAc) was a possible receptor for EPEC with BFP composed of a-bundlin. Because EPEC are still able to bind to host cells, albeit less efficiently, without BFP, other factors may also be involved in adherence. Recent studies have demonstrated that EspA filaments promote an attenuated adherence of BFP deficient EPEC strains to the brush borders of Caco-2 cells (Knutton et al., 1998). EspA is the major component of the long hollow filamentous needle complex of the TTSS that directly contacts the host cell and that facilitates injection of bacterial effector proteins, EspB and EspD, into the host cytoplasm for A/E lesion formation (Daniell et al., 2001). EspA-mediated adherence is less efficient than BFP binding, presumably due to the small number of filaments (12 EspA filaments) produced per bacterium (Daniell et al., 2001). The nature of the interaction is currently unknown (Cleary et al., 2004), and host cell receptors have not been thoroughly investigated. However, recent data suggests that cholesterol may play an important role in adherence and type III secretion
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in the absence of BFP. Allen-Vercoe et al. (2006) found that bacterial adherence and delivery of effector proteins were abolished after treatment of HeLa cells with the cholesterol-depleting agent, methylb-cyclodextrin. Additionally, lipid rafts, where cholesterol is localized, were necessary for pedestal formation by EPEC. Therefore, cholesterol may serve as an adherence site for EPEC when BFP is absent. Flagella are also thought to play a role in the initial attachment of EPEC to host cells (Giron et al., 2002). Giron et al. (2002) discovered that flagella extended outward from microcolonies while BFP were tightly associated with the microcolony, suggesting that flagella may tether the bacteria to the host and BFP may simply mediate microcolony formation. However, other workers suggested that flagella were produced by adherent EPEC, but found no evidence that implicated flagella in adherence (Cleary et al., 2004). Consequently, more work is needed in this area. Several other proteins have been suggested to act as putative adhesins, but additional work is needed to determine their precise role in adherence. Intimin, for example, is an OMP that directly binds Tir, a translocated intimin receptor inserted in the host cell membrane by the bacteria through the TTSS after initial contact has occurred. This interaction ultimately prompts A/E lesion formation (Frankel et al., 1998; Kenny et al., 1997). Cleary et al. (2004) suggested that intimin cannot support initial bacterial adhesion as EPEC strains that lacked BFP and EspA could not adhere to two types of epithelial cells in the absence of Tir. However, intimin has been shown to bind other host cell receptors, such as b1 integrins (Frankel et al., 1996) and nucleolin (Sinclair and O’Brien, 2002, 2004). These receptors have been shown to be necessary for EPEC to modulate host cell tight junctions (Dean and Kenny, 2004), an event that may precede the onset of diarrhea (Guttman et al., 2006). In a recent study, Hernandes et al. (2008) found that atypical EPEC strain 1551–2 that lacks BFP was able to form loose microcolonies after 6 h of infection via intimin omicron, suggesting that intimin omicron is responsible for the LA phenotype observed and that this strain may express an additional novel adhesive structure. The protein LifA, which has been characterized as the EPEC toxin lymphostatin (Klapproth et al., 1996, 2000), has also been implicated as a potential EPEC adherence factor. The lifA gene has high homology to the efa-1 and toxB genes in EHEC strains (Badea et al., 2003), which have been proposed to influence adherence to epithelial cells (Stevens et al., 2002; Tatsuno et al., 2001). However, the true role of these genes in EHEC adherence is still unclear (Torres et al., 2005). An initial EPEC adherence study using a lifA mutant found no difference in adherence to cultured epithelial cells (Klapproth et al., 2000). However, a subsequent study found that LifA may play a role in adherence in the absence of BFP (Badea et al., 2003). Therefore, the function of lifA in adherence remains to be established.
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D. Enterohemorrhagic Escherichia coli (EHEC) EPEC and EHEC strains share many of the same virulence and adherence factors. In particular, EHEC strains also contain the LEE, PAI and they form characteristic A/E lesions on cultured mammalian cells and in animals (Knutton et al., 1989; Tzipori et al., 1986). In fact, intimin, which is essential in EPEC pathogenesis as discussed above, is also found in EHEC O157 and other EHEC serotypes (Huppertz et al., 1996; Yu and Kaper, 1992). Although intimin is one of the only adhesins in EHEC demonstrated to play a role in colonization in vivo (Donnenberg et al., 1993), many other putative fimbrial and afimbrial adhesins have been identified in EHEC strains including Efa1 (Nicholls et al., 2000), LP fimbriae (Torres et al., 2002), curli (Kim and Kim, 2004), F9 (type I pilus homolog) (Low et al., 2006), E. coli common pilus (ECP) (Rendon et al., 2007), and type IV pilus (TFP) (Srimanote et al., 2002). However, their role in in vivo adherence and host colonization remains unclear. The most recent and well-studied EHEC adhesins are discussed below. The Efa1 adhesin was identified in 2000 by Nicholls et al. (2000) through transposon mutagenesis of a clinical non-O157:H7 EHEC isolate of serotype O111:H-. They found that Efa1 promotes adherence to Chinese hamster ovary (CHO) cells, human red blood cells agglutination, and autoaggregation. Additionally, the efa1 gene was present in 116 strains of attaching–effacing EPEC and EHEC, but not in 91 nonattaching–effacing E. coli strains. E. coli O157:H7 strains lack efa1, but do encode toxB, a truncated version of the efa1 gene. A toxB mutant exhibits reduced adherence to cultured epithelial cells (Stevens et al., 2004). However, toxB had an indirect effect on adherence to epithelial cells by modulating the production and secretion of proteins that play a role for A/E formation in EHEC. The receptors for Efa1 and ToxB have not been identified and their true role in vivo is unknown. The lpfABCC’DE chromosomal fimbrial operon identified in EHEC O157:H7 has high similarity to the LP fimbriae (lfp) operon of S. enterica serovar Typhimurium (Perna et al., 2001). In one recent study (Torres et al., 2002), LP fimbriae were proposed to participate in the interactions of EHEC with eukaryotic cells by assisting in microcolony formation, as isogenic E. coli O157:H7 lpf mutants showed slight reductions in adherence to tissue culture cells and formed fewer microcolonies compared to the wild-type strain. A second locus within the EHEC O157:H7 genome has homology to LP fimbriae in Salmonella and shares an overall 31% identity to proteins in the previously mentioned lpf operon in EHEC O157:H7. Additionally, a similar region has been identified in Shiga toxin-producing E. coli O113:H21 (Doughty et al., 2002). A mutation in the O113:H21 LP fimbriae results in decreased adherence to epithelial cells. Together, these data suggest that LP fimbriae may play a role in
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EHEC adherence in vitro. However, the role of these LP fimbriae in vivo remains undefined. The TFP in EPEC, termed BFP, have been shown to play a very important role in infection in animal models as discussed above. However, until recently, no TFP has been identified in EHEC strains. Xicohtencatl-Cortes et al. (2007) identified hemorrhagic coli pilus (HCP), a TFP, in EHEC O157:H7. It is composed of a 19-kDa pilin subunit (HcpA) and is encoded by the hcpA gene. The HCP was found to be composed of bundles of fibers that formed physical bridges between bacteria adhering to human and bovine host cells. Although the expression of HCP was only induced under strict growth and environmental conditions in vitro, the authors showed that hcpA was expressed in vivo by testing the sera of HUS patients and healthy individuals for antibodies against HcpA. Only HUS patient sera had antibodies that recognized HcpA. Additionally, the disruption of hcpA gene reduces EHEC adherence to cultured epithelial cells and bovine and porcine explants. Consequently, these data suggests that EHEC O157:H7 possesses TFP that are important intestinal colonization factors that contribute to the pathogenesis of this microorganism. Recently, other putative EHEC fimbrial adhesins have been identified, including the ECP and the F9 fimbriae. The ECP is composed of a 21-kDa pilin subunit whose amino acid sequence corresponds to the ecpA gene present in all E. coli genomes. Isogenic ecpA mutants of EHEC O157:H7 or fecal commensal E. coli demonstrated significant reduction in adherence to cultured epithelial cells (Rendon et al., 2007). The F9 fimbriae in EHEC O157:H7 were identified by Low et al. (2006) and found to promote colonization in 1–2-week-old calves. Mutation of the major F9 subunit gene in EHEC O157:H7 resulted in reduced levels of shedding in weaned calves, but did not reduce the level of colonization at the terminal rectum, indicating that the adhesin is not responsible for rectal colonization, but may contribute to colonization at other intestinal sites.
E. Uropathogenic Escherichia coli (UPEC) UPEC is the primary cause of UTI in the developed world. These infections are ascending infections in that they usually start in the bladder and move up the urinary tract toward the kidneys, and then possibly entering the bloodstream. If the bacteria do enter the bloodstream, any organ is susceptible to infection and other more life threatening conditions may develop, including pneumonia and meningitis. As with other pathogens, the ability of UPEC to colonize the bladder and kidney in animal models is dependent on its ability to adhere to uroepithelial cells (Hagberg et al., 1983). The most common UPEC adhesins are type I, P, F1C, S, and Auf fimbriae and the Afa/Dr afimbrial adhesins (Oelschlaegar et al., 2002). Recently, the sequencing of three UPEC genomes have revealed the
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presence of several additional gene clusters with homology to some of the existing UPEC fimbrial adhesins already identified (Brzuszkiewicz et al., 2006; Chen et al., 2006; Welch et al., 2002). The type I fimbriae will not be discussed here as it has been described above for E. coli and Salmonella. The pyelonephritis-associated pili (P fimbriae), like the type I fimbriae, is a chaperone-usher class of fimbriae and is the most extensively studied adhesin. These fimbriae are used by UPEC to bind galabiose-containing glycolipid receptors in the kidney that initiates pyelonephritis (Dodson et al., 2001; Lund et al., 1987). Acute pyelonephritis is the most serious UTI in that it may lead to scarring of the kidneys, resulting in kidney damage, kidney failure, and even sepsis. The P fimbriae are encoded by the pap genes. They are structurally comprised of around 1000 copies of PapA, the major subunit protein, which polymerize to form a rigid structure that connects to PapE and PapF (minor subunit structures), and PapG, the receptor-binding adhesin, at the distal end (Kuehn et al., 1992; Lindberg et al., 1987). There are three different PapG isoreceptor-binding variants (PapGI, -II, and -III) (Stromberg et al., 1990). Each PapG variant binds different isoreceptors that contain a common Gal(a1–4)Gal moiety linked to a ceramide group that anchors the receptor in the lipid bilayer of the host cell (Hakomori, 1990). Differences in the PapG receptor type and distribution on different host cell surfaces have been shown to dictate variations in host tropism of P-fimbriated E. coli (Stromberg et al., 1990). For example, the class II papG allele has been shown to be primarily associated with pyelonephritis and bacterimia, while the class III papG allele is associated with human cystitis and with genitourinary infections in dogs and cats (Johnson, 1998; Johnson et al., 2000; Otto et al., 1993). However, little is known about the clinical association of the class I papG allele, as this allele is found less frequently. Recently, an extensive review has been published by Lane and Mobley (2007) detailing the role of P fimbriae in adherence and persistence in UPEC. Several other fimbrial adhesins have been identified in UPEC strains; however, much less is known about these adhesins. The F1C fimbriae (Foc) resembles type I fimbriae in genetic organization and organelle structure (Klemm et al., 1994; van Die et al., 1991). Backhed et al. (2002) identified two F1C receptors, galactosylceramide and globotriaosylceramide, both with phytosphingosine and hydroxy fatty acids. However, the ceramide portion of the glycosphingolipid receptor was found to confer binding specificity. The authors also reported that human renal epithelial cells produce proinflammatory chemokine interleukin-8 in response to F1C-mediated attachment, suggesting a role in F1C-mediated attachment in mucosal defense against bacterial infection. The S-fimbriae, which resembles the F1C fimbriae at the amino acid level (van Die et al., 1991), mediates adherence to sialic acid-containing glycolipids or glycoproteins, and are associated with sepsis and meningitis in newborn infants
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(Korhonen et al., 1985; Parkkinen et al., 1986). The Auf fimbriae, encoded by the aufABCDEFG gene cluster, were found to be significantly associated with UPEC (Buckles et al., 2004). Although the deletion of the entire auf gene cluster had no effect on the ability of UPEC to colonize the kidney, bladder, or urine in a murine model, aufA was detected in the urine from infected mice by RT-PCR. Therefore, the true role of the Auf fimbriae in UPEC pathogenesis is still unclear. The Afa/Dr family of UPEC afimbrial adhesins has also been shown to promote initial attachment to host cells. Interestingly, the Afa/Dr family consists of both fimbrial and afimbrial members as determined by electron microscopy (Bilge et al., 1989; Garcia et al., 1996). However, recent high-resolution structural studies have shown that the previously characterized afimbrial adhesin, Afa-3, may comprise fine filaments that are not detectible after preparation for electron microscopy (Anderson et al., 2004). The Afa/Dr adhesins are encoded by genes designated A through E, with E typically encoding the structural adhesin (Le Bouguenec et al., 2001). Most of the Afa/Dr adhesins bind to the Dra blood group antigen present on CD55 (also known as decay-accelerating factor, DAF). DAF is an eukaryotic cell membrane protein that regulates complement cascade and protects cells for autologous complement-mediated damage (Lublin and Atkinson, 1989). In fact, studies have shown that the level of host cell colonization by Afa/Dr-expressing E. coli is directly proportional to the extent of host cell CD55 expression (Selvarangan et al., 2000). Other Afa/ Dr adhesins, including F1845, Dr, and Afa-3 have also been shown to bind to three members of the carcinoembryonic antigen-related adhesin molecules (CEACAM) family: CEA, CEACAM1, and CEACAM6 in diffusely adhering E. coli (Berger et al., 2004). Additionally, many studies have indicated that both Dr and Afa-3 adhesins recognize a5b1 integrin (Guignot et al., 2001; Plancon et al., 2003). While other fimbrial and afimbrial adhesins such as F9 (Ulett et al., 2007a) and Antigen 43 (Ulett et al., 2007b) have been shown to promote persistence in the UTI, their proposed function relates to aggregation and biofilm formation, rather than initial attachment and thus are not discussed here.
VIII. ANTI-ADHESIVES The ability to adhere to host tissues is an essential step for infection by many pathogenic microorganisms (Finlay and Falkow, 1989). Adherence not only provides the pathogen with the means to initiate colonization of the host cell surface, but it also enhances resistance against host cleansing or clearing mechanisms, such as flow of lumen contents in the intestinal tract, airflow through the lungs, and urine flow through the urinary tract. In addition, once bacteria have adhered, they are privy to nutrients that
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support colonization and survival (Ofek and Doyle, 1994). Adhered bacteria are also positioned to facilitate toxin delivery or to invade host tissues. It is now well accepted that the initial phase of most bacterial infections involves attachment of the pathogen to host cell receptors via bacterial adhesins, as discussed above in Section VI (Fig. 2.2A). Therefore, by disrupting adhesin–receptor interactions, it may be possible to prevent initial adherence and subsequent infection. The disruption or inhibition of pathogen attachment to host cells via anti-adherence agents has attracted considerable attention for several reasons. First, this approach is considered more gentle and ecologically sound compared to alternative approaches, such as using chemotherapy or antibiotic treatments (Karlsson, 1998). Some of the candidate antiadhesive agents are even found naturally in foods. In addition, although some resistance to anti-adhesive agents may possibly occur, dissemination of bacterial strains that are resistant to anti-adhesives will likely occur at a significantly lower rate compared to antibiotic-resistant strains (Ofek et al., 2003a). There are some potential limitations of anti-adhesive strategies that must also be recognized. Pathogenic bacteria often encode genes for more than one type of adhesin, and, via a process known as phase variation (Henderson et al., 1999), express adhesins on either a random or perhaps ‘‘as-needed’’ basis. Therefore, a cocktail of different anti-adhesive agents D
C B
A
Receptor analog
Adhesin analog
Bacterium
Adhesin
Cellular receptor
Infection
Probiotics
Adhesin analogs
Receptor analogs
FIGURE 2.2 Schematic illustration of adherence (A) and anti-adhesive agents: probiotics (B), adhesin analogs (C), and receptor analogs (D).
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that target several adhesins or a single agent that has a broad spectrum of anti-adhesion activity may be necessary (Ofek et al., 2003a). Many bacterial adhesins and host receptors have been identified and studied in great depth, as described in Section VII. Therefore, with every new adhesin or receptor discovered, a new opportunity arises to develop an anti-adherence mechanism that may inhibit or block the adhesin– receptor interaction, which is the ultimate aim of anti-adhesion therapy (Kahane and Ofek, 1996; Moricout et al., 1990; Ofek and Doyle, 1994). In actual practice, there are several types of anti-adhesive mechanisms including adhesin-based vaccines, innate host-derived anti-adhesives, probiotics, adhesin analogs, and receptor analogs.
A. Adhesin-based vaccines Averting infection by blocking adhesion with adhesin vaccines can be conferred both passively and actively (Ofek et al., 2003a). Passive immunity was shown in a study that demonstrated that suckling piglets acquired immunity from their mother who were previously vaccinated with K88 fimbriae and other related adhesins from enterotoxigenic E. coli (ETEC) while pregnant (Moon and Bunn, 1993). It was assumed that milksecreted antibodies prevented infection by blocking bacterial adherence (Ofek et al., 2003a). In another study, anti-Streptococcus mutans monoclonal antibodies against SA I/II adhesins were applied to the tooth surfaces of human volunteers that had been chemically cleared of oral S. mutans microflora (Ma et al., 1998). The control subjects were positive for S. mutans within 2–3 months, while the passively immunized group was free of S. mutans for at least a year. However, this passive immunity may have been the result of competitive exclusion, as SA I/II antibodies were not detectable one day after infection (Kelly and Younson, 2000). Nonetheless, adhesin-based vaccines appear to be a promising area of study, and may some day, greatly contribute to reducing infections in some populations. Active immunity appears to be functional in animal models through the stimulation of both IgG and secretory IgA antibodies (Ofek and Doyle, 1994). It has been suggested that mucosal IgG-mediated immunity, as opposed to systemic immunity, can improve the protective effect of adhesin-based vaccines (Mestecky et al., 1997; Wisemann et al., 1999). Active immunity against UTI has been extensively investigated. The type 1 fimbriae, FimH adhesin complex and its periplasmic chaperone, FimC, provided immunity against E. coli in the urinary tract of both mice and nonhuman primates. The potential effectiveness of adhesin vaccines in animals has gained considerable attention, and development of multiple adhesin vaccines in humans is likely to follow.
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B. Host-derived anti-adhesins The innate immunity of a host plays a significant role in preventing infection. Body fluids have an abundance of potential anti-adhesive molecules. However, only a few of them have been found to be effective against pathogens (Ofek et al., 2003b). The anti-adhesive agents that have the most potential to inhibit bacterial attachment are those associated with the host mucosal surface including secreted mucins (Ofek et al., 2003a). Mucins, as discussed previously in Section II, protect the mucosal surface from invading pathogens by a permissive protective barrier. However, other components of the mucus layer may provide some innate immunity, as recent studies have shown that some mucins and other mucosal surface components can inhibit bacterial adherence in vitro (Mack and Sherman, 1991; Wu et al., 1996). This is promising data, but may only be effective in luminal areas where the mucus flow is relatively fast so that the bacteria can be cleared with the mucus. In luminal areas where the mucus layer is thick and moving slowly, bacterial binding to these potential anti-adhesins may accomplish the opposite effect by facilitating bacterial penetration of the mucus so infection can continue. Much more research is needed in this area to determine efficacy of host-derived anti-adhesins.
C. Probiotics as anti-adhesives Probiotic bacteria are by definition, ‘‘nonpathogenic, live microbial, monoor mixed-culture preparations, which, when administered to humans or animals in adequate amounts, confer a health benefit on a host by improving intestinal microbial balance’’ (Fuller, 1989; Havenaar and Huis in’t Veld, 1992; Havenaar et al., 1992; Salminen et al., 1998). The most widely used probiotics are lactobacilli and bifidobacteria, but other microorganisms, including E. coli, enterococci, bacilli, and yeasts have also been used (Holzapfel and Schillinger, 2002). Both lactobacilli and bifidobacteria have been shown to have an inhibitory effect on many enteric pathogens (Saavedra, 1995). A number of mechanisms have been attributed to these antagonist effects, such as decreasing the luminal pH by the production of short chain fatty acids, competition with pathogens for nutrients, production of inhibitory compounds like bacteriocins, and competing for adhesin receptors on the host cell surface thereby inhibiting pathogen adherence (Bernet et al., 1994; Sanders, 1993). All of these mechanisms are important and potentially play a role in probiotic functionality. However, the competitive binding of probiotics to host tissues at the expense of pathogens is clearly an anti-adhesive effect (Fig. 2.2B). Several probiotic organisms have been shown to adhere to the intestinal mucosa ( Jacobsen et al., 1999). For example, Lactobacillus acidophilus
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(Bernet et al., 1994; Coconnier et al., 1992) and L. casei (Hudault et al., 1997) were shown to bind to Caco-2 cells at relatively high numbers, and this adherence may reduce or displace adherence of enteric pathogens, including Samonella enterica serovar Typhimurium, EPEC, and Yersinia enterolitica. In some cases, the probiotics do not have to be viable or even bind directly to the pathogen target receptor to affect adherence of pathogens. In one study, heat-killed L. acidophilus LB was shown to inhibit ETEC attachment to polarized Caco-2 cells (Chauviere et al., 1992). However, elevated concentrations of heat-killed L. acidophilus were required. Because ETEC utilize CFA/I, CFA/II, and CFA/III receptors to bind host cells and L. acidophilus does not express adhesins for these receptors, the authors suggested steric hindrance as the explanation for adherence inhibition. Recently, surface-layer proteins (Slps) extracted from Lactobacillus helveticus were shown to act as anti-adhesives against E. coli O157:H7 (Johnson-Henry et al., 2007). The Slps in many Lactobacillus species have been found to, among other things, assist in bacterial adherence to host tissues (Frese et al., 2005). Extracted Slps from L. helveticus reduced E. coli O157:H7 adherence and A/E lesion formation on both HEp-2 and T84 cells, suggesting that probiotic binding may interrupt the infectious process of some intestinal pathogens (Johnson-Henry et al., 2007). The role of probiotics as anti-adhesives is somewhat unclear in that, although in vitro studies are promising, there have been no carefully controlled clinical human studies to test the effect of probiotics as anti-adhesives (Ofek et al., 2003a).
D. Adhesin analogs The rationale for adhesin analogs is based on the assumption that soluble, exogenous bacterial adhesins will bind to their intended receptor, thereby competitively blocking pathogen adherence to those same receptors (Fig. 2.2C). This type of anti-adhesive has been mostly impractical to use because they are almost always macromolecules, are not readily available, must be used in high concentrations, and due to their innate nature may be toxic and/or immunogenic (Ofek et al., 2003a). Despite these drawbacks, however, new technologies have allowed the development of some potential adhesin analogs. Both proteinaceous and non-proteinaceous analogs have been studied. Examples include a synthetic 20 amino acid adhesin peptide sequence copied from S. mutans and LTA of groups A and B streptococci. The synthetic peptide mimics a S. mutans adhesin that binds a salivary protein on dental surfaces and was shown to inhibit bacterial adherence to immobilized salivary receptors in vitro. In vivo, this peptide hindered the recolonization by S. mutans on teeth that had been cleared of the
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normal microflora as compared to the control groups that had been treated with saline or placebo peptides (Kelly et al., 1999). Although these results would appear promising, S. mutans is able to utilize other adhesins that bind to cell surfaces, especially in the presence of sucrose. Thus, the application of multiple analogs may be required for this approach to be effective (Ofek et al., 2003a). In another study, non-proteinaceous LTA was used as an antiadhesive against group A streptococci (Dale et al., 1994). The nasal cavities of mice were treated with the adhesin analog and a group A streptococcal suspension was then administered. Colonization and death were significantly reduced in the LTA-treated mice compared to the control mice. Although the potential of adhesin analogs was demonstrated in this study, LTA may not be a practical anti-adhesive because of its potential toxicity. Group A streptococci are also thought to bind the CD44 receptor on epithelial cells via the presence of a hyaluronic acid capsule (Cywes et al., 2000). Thus, the anti-adhesive properties of hyaluronic acid have also been examined. Mice that had been orally treated with hyaluronic acid and then challenged by group A streptococci have fewer adhered cells and resisted colonization (Cywes et al., 2000). However, the exact mechanisms accounting for this inhibition are not known.
E. Receptor analogs Inhibition of bacterial adherence via receptor analogs is the most wellstudied of the anti-adhesive mechanisms. This strategy is based on the observation that bacterial adherence is often mediated by interactions between bacterial surface proteins and complimentary oligosaccharide receptors located at the surface of host cells. Soluble oligosaccharides that resemble or mimic host oligosaccharide receptors interrupt the adherence process by acting as receptor analogs or decoys. More precisely, rather than binding to host cells, pathogens bind to the soluble oligosaccharide decoys and are displaced from the intestinal tract preventing infection initiation and subsequent host tropism (Fig. 2.2D). Although many of the receptor analogs that have been studied are derived synthetically, there are numerous reports describing antiadherence activities from natural sources, such as milk, berries, and other foods (Table 2.3). Moreover, there is now considerable evidence demonstrating that soluble oligosaccharides specific for an adhesin can competitively inhibit binding to target cells not only in the GI tract, but also in a variety of other tissues (Aronson et al., 1979; Barthelson et al., 1998; Bouckaert et al., 2005; Firon et al., 1987; Hyland et al., 2006; Nagahori et al., 2002; Zorf and Roth, 1996).
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TABLE 2.3 Synthetic and naturally occurring anti-adhesive receptor analogs Receptor analogs
Synthetic N-acetyl-galactosamine Methyl a-mannoside Sialylated NeuAca2–3(or 6) Galb1 Globotriose Commercial galactooligosaccharides Natural Human milk oligosaccharides
Egg-yolk-derived sialyloligosaccharides Cranberry extracts
Green tea extracts
Carrot extracts Mannooligosaccharides
Pathogen
Tissue
Enteropathogenic E. coli Uropathogenic E. coli Streptococcus pneumoniae E. coli shiga toxins Enteropathogenic E. coli
Intestinal
Shigella Campylobacter jejuni Pathogenic E. coli varotypes Salmonella enteritidis
Intestinal Intestinal Intestinal
Helicobacter pylori Uropathogenic E. coli Streptococcus mutans Helicobacter pylori Proprionibacterium acnes Staphylococcus aureus Enteropathogenic E. coli Salmonella Klebsiella
Intestinal Urinary
Uropathogenic E. coli
Urinary Respiratory Intestinal Intestinal
Intestinal
Oral Intestinal Epithelial Epithelial Intestinal Intestinal Respiratory/ Urinary Urinary
1. Studies with synthetically derived analogs As described earlier in Section IV.A., several of the carbohydrate sequences that serve as receptors for enteric pathogens have been identified. For EPEC, these receptors are located on the surface of host epithelial cells and are often comprised of galactose, N-acetyl-galactosamine, lactosyl
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glycans, and fucosylated and sialylated oligosaccharides (Vanmaele et al., 1999). These residues, if derived synthetically and added exogenously, would, therefore, have the potential to serve as effective anti-adhesives. Several studies support this hypothesis (Alvarez-Dominguez et al., 1997; Ascencio et al., 1993; Hyland et al., 2006; Klapproth et al., 2000). In several of these studies, carbohydrate receptors were conjugated to bovine serum albumin (BSA), forming lactosyl-BSA, N-acetyl-galactosamine-BSA, or other BSA-glycoconjugates, and then used in adherence assays (Hyland et al., 2006). Adherence inhibition of nearly 99% was achieved for N-acetylgalactosamine-BSA. Galactosyl-, fucosyl-, and other mixed glycoconjugates gave intermediate levels of inhibition (about 50%). The ability of synthetically derived carbohydrate analogs to protect against infection in vivo (in animals) was first reported by Aronson et al. (1979). They found that methyl a-mannoside inhibited UTI in mice that were administered E. coli expressing type 1 fimbriae. Subsequently, polyvalent sialylated oligosaccharides that terminate in NeuAca2–3(or 6) Galb1 were shown to prevent nine strains of S. pneumoniae from binding to human cells derived from the upper respiratory tract (Barthelson et al., 1998). Finally, it also appears that oligosaccharide receptor analogs may not only prevent the adhesion of bacterial cells to host tissues, they also neutralize toxins produced by some bacteria. Shiga toxins (Stx), Stx1 and Stx2c, produced by Stx-producing E. coli (STEC) human strains were neutralized by globotriose-expressing recombinant E. coli, revealing a potential treatment for these infections (Paton et al., 2001).
2. Studies with naturally occurring analogs The possibility that pathogens can be inhibited by naturally occurring anti-adhesive substances is especially attractive and has captured significant attention. The initial evidence that such substances might exist was based on the long-standing observation that breast-fed infants appeared to suffer from fewer diarrheal diseases than formula-fed infants (Dewey et al., 1995; Grulee et al., 1934; Hagberg et al., 1983; Huffman and Combest, 1990; Kramer et al., 2001; Kunz and Rudloff, 1993; Newburg et al., 2005). This apparent reduction in infection by diarrheal pathogens has been attributed to several components in human breast milk, including lactoferrin, casein peptides, and human milk oligosaccharides (HMOs) (Coppa et al., 2006; de Araujo and Giugliano, 1999; Rhoades et al., 2005). The concentration of free oligosaccharides, in particular, may reach levels as high as 10 g/L in mature milk (Chaturvedi et al., 2001; Newburg et al., 2004), which would make these oligosaccharides the third largest solid constituent in human milk (Newburg, 2000). These oligosaccharides can be found in nonconjugated (free) or conjugated form (glycolipids, glycoproteins). Some of the oligosaccharides appear to function as prebiotics (Coppa et al., 2004), which stimulate the growth of lactobacilli and
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bifidobacteria in the infant gut (Gibson and Roberfroid, 1995), and, by virtue of short chain, volatile acid production, inhibit enteric pathogens. While the prebiotic effect of milk oligosaccharides likely accounts for a substantial positive effect on gut and overall health of nursed infants, it is now evident that HMOs also act as adhesin analogs against invading pathogens (Brand-Miller et al., 1994; Crane et al., 1994; Kobata, 2003; Morrow et al., 2004; Newburg et al., 2004; Ruiz-Palacios et al., 2003). In recent years, human milk-derived oligosaccharides have been reported to have anti-adhesive activity against several pathogenic bacteria. For example, HMOs were reported to inhibit Shigella and Campylobacter and various pathotypes of E. coli via an anti-adherence mechanism (Kunz and Rudloff, 1993; Kunz et al., 2000; Newburg, 1997; Sharon, 2006). The oligosaccharide fraction of human colostrums also inhibited adherence of EPEC to HEp-2 cells (Cravioto et al., 1991). In addition, the fucosylated fraction of HMOs inhibited binding of C. jejuni to HEp-2 tissue culture cells (Ruiz-Palacios et al., 2003) and protected infants from diarrheal diseases (de Araujo and Giugliano, 1999; Morrow et al., 2005). Similarly, the fucosylated fraction of HMOs that contain H-2 blood group epitope was found to inhibit the binding of C. jejuni to monolayers of HEp-2 cells in vitro (Ruiz-Palacios et al., 2003). This fraction also inhibited Campylobacter colonization of mice in vivo and inhibited binding of invasive pathogenic campylobacter to human intestinal mucosa ex vivo. Another study showed that the sialylated oligosaccharide fraction prevented enterotoxigenic and uropathogenic strains of E. coli from binding and agglutinating calf and human erythrocytes (Martin-Sosa et al., 2002). In fact, in one of the first reports demonstrating the protective effects of milk oligosaccharides, the authors suggested that sialic acid-linked oligosaccharides were involved (Gyorgy et al., 1974). Conversely, others have suggested that neutral, rather than sialylated breast milk oligosacccharides were responsible for adherence inhibition (Asakuma et al., 2007; Coppa et al., 1990; Newburg, 1997). Collectively, the data strongly suggest that HMOs are exceptional anti-adhesives. Several other dietary saccharides have also been found to inhibit bacterial adherence in both animals and humans. In one report, eggyolk-derived sialyloligosaccharides and their derivatives were found to inhibit S. enteritidis infection and lethality in BALC/c mice when administered orally (Sugita-Konishi et al., 2002). This study also established that an immune response was not stimulated in cultured macrophages by these oligosaccharides, ruling out an immunological response as the cause for reducing infection and death. Similar anti-adherence activity against S. enteritidis, S. enterica serovar Typhimurium, and E. coli O157:H7 was also reported using non-immunized egg yolk powder (Kassaify et al., 2005). In this study, a high density lipoprotein fraction reduced adherence
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of Salmonella and, to a lesser extent, E. coli O157:H7, although the exact mechanism responsible for this inhibition could not be established. Numerous studies have shown that cranberry extract has extensive anti-infection, and, in particular, anti-adhesive properties (Burger et al., 2002; Howell, 2007; Howell et al., 2001; Puupponen-Pimia et al., 2005; Sharon and Ofek, 2002). Initially, cranberry juice became an area of interest due to its well-known beneficial effects in UTI (Moen, 1962; Papas et al., 1968; Sternlieb, 1963). Several clinical trials have substantiated these initial observations. In one study, elderly women drank 300 ml of cranberry cocktail or a placebo every day for 6 months (Avorn et al., 1994). In another study, young women drank 50 ml of cranberry–lingonberry juice concentrate diluted in 200 ml of water every day for 6 months (Kontiokari et al., 2001). Both studies showed a significant reduction in the incidence of bacteria in urine samples as compared to the placebo. In another recent study, cranberry juice was administered to patients with H. pylori infections and who were receiving antibiotic therapy (Shmuely et al., 2007). Although there were no overall differences in eradication of H. pylori among the treatments and control groups, eradication in female subjects fed the cranberry juice was significantly higher. Direct evidence showing that cranberry juice components have antiadhesive activity have also been described (Howell, 2007; Shmuely et al., 2007). The high concentration of fructose that is found in cranberry juice has an affect on adherence, in that it has been found to inhibit, in vitro, type 1 fimbriae-mediated E. coli adhesion (Zafriri et al., 1989). However, fructose would not be expected nor as evidence been shown, to indicate that it has anti-adherence activity in vivo (Howell, 2007). Rather, at least two other components of cranberries are now thought to be responsible for the anti-adherence activity. In particular, several studies have shown that proanthocyanidins (a flavonoid, also referred to as a condensed tannin) and other high-molecular weight compounds inhibit adherence of UPEC (Howell et al., 1998, 2001; Shmuely et al., 2004). Despite these reports, however, the precise mechanism for the observed inhibition in adherence is not clear. Howell (2007) has suggested that the cranberry components act as receptor analogs and inhibit adherence of E. coli fimbriae to host cell surfaces. In addition, alteration in cell surface properties or electric potential, cell morphology, or fimbrial length may also contribute to reduced adherence (reviewed in Howell, 2007). In addition to reducing urinary and stomach infections, cranberry juice has also been shown to have anti-adhesive activity against oral bacteria, such as S. mutans (Guo et al., 1998; Weiss et al., 2002). In the latter study, the ability of saliva-coated S. mutans to adhere to saliva- or glucancoated hydroxyapatite in the presence of 25% cranberry juice was greatly reduced by 40–85% as compared to the control, indicating that cranberry
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juice effectively blocked bacterial adherence to binding sites in salivary pellicle and in glucans (Koo et al., 2006). Pectic-type and other water soluble oligosaccharides have also been suggested to have anti-adherence activity (Guggenbichler et al., 1997; Kastner et al., 2002; Lee et al., 2006). In one recent report (Lee et al., 2006), a high-molecular weight (80,000 Da) extract from green tea reduced adherence of H. pylori by up to 40% to a human gastric epithelial cell line. In addition, similar levels of inhibition of Propionibacterium acnes (a skin pathogen) and S. aureus to a fibroblast epithelial cell line were also observed. In another study, an aqueous extract from carrots blocked EPEC to HEp-2 cells and to human mucosal cells (Kastner et al., 2002). The active material was found to be an acidic oligosaccharide containing trigalacturonic acid. Finally, another group of oligosaccharides that have attracted attention for their potential anti-adhesive activity are the mannooligosaccharides (MOS). The MOS can be extracted from natural sources, produced synthetically, or can also be derived inexpensively from food-grade yeast cell walls, which are rich in mannan. These MOS products are sometimes included in feed rations for beef cattle, swine, and poultry, although their use in humans has not yet been considered (Castillo et al., 2008; Franklin et al., 2005; Hooge, 2004). Importantly, mannan contains a-linked mannose residues that are known to inhibit the adhesion of many enterobacterial species including Salmonella, Klebsiella, and E. coli (Bouckaert et al., 2006; Sharon, 2006). In the latter study, UPEC, and other E. coli pathotypes, were found to vary as much as 100-fold in their affinity to various oligomannosides, suggesting that receptor analog activity depends on structure and bond type, and that some analogs may be better than others as anti-adhesive agents.
3. Commercial prebiotics as anti-adhesives More recently, commercial prebiotic oligosaccharides have been reported to also have adherence–inhibition activity. Ordinarily, prebiotics are thought to exert beneficial effects to the host primarily by selectively influencing the growth of desirable lactobacilli and bifidobacteria in the colon (Gibson and Roberfroid, 1995). However, the similarity of some of these prebiotics to those found in nature, particularly those found in human breast milk (discussed above in Section VIII.E.2.), would suggest that they may also function as anti-adherence agents. Although commercially available galactooligosaccharides (GOS) clearly have a different composition from that of the natural GOS present in human milk, they do share a general structural similarity. Therefore, commercial GOS
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would be expected to have many of the same attributes that naturally occurring, milk-derived GOS possess (Boehm and Stahl, 2007). Indeed, commercial GOS, like human milk GOS, is bifidogenic, both in vitro and when fed to infants. Recently, the Hutkins’ lab evaluated several such prebiotics for antiadherence activity (Fig. 2.1), and showed that GOS, obtained as a foodgrade material, significantly inhibited adherence of EPEC to HEp-2 and Caco-2 cells (Shoaf et al., 2006). In addition, GOS also reduced the number of adhered microcolonies by 50% and the microcolony size (number of cells per microcolony) by 70%. This suggests that GOS may specifically be targeted to an adherence factor that is also responsible for microcolony formation. Specifically, BFP have been shown to mediate both microcolony formation (Giron et al., 1991) and LA. Thus, the ability of GOS to interfere with BFP formation is especially important, given the role of BFP as an initial adherence factor in EPEC pathogenesis. The application of commercial oligosaccharides as anti-adhesive agents is not restricted to the GIT, but may also extend to the urogenital tract, where mannosides and yeast mannan have been shown to have anti-adherence activity (Aronson et al., 1979; Ofek et al., 1977). These and other findings provide convincing evidence that commercial, food-grade oligosaccharides may serve as anti-infective agents against pathogenic microorganisms. This approach offers tremendous advantages as these agents are food-grade, safe, inexpensive, and importantly, could reduce reliance on antibiotics. Over-administration of antibiotics, both clinically and in animal agriculture, has led to bans in the European Union and a search for alternative treatments to reduce bacterial infections (Mountzouris et al., 2006).
IX. CONCLUSIONS AND FUTURE PROSPECTS Research aimed at understanding bacterial pathogenesis has established the importance of bacterial adherence in disease. This research has led to the identification of a number of both bacterial adhesins and potential host cell receptors. By understanding the detailed interactions between a bacterial adhesin and host receptor, it is possible to develop new mechanisms to prevent bacterial adhesion, thereby averting disease. Many promising anti-adhesion mechanisms have been developed and studied, but much more work is needed, both in vitro and in vivo, to establish the feasibility of these mechanisms.
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CHAPTER
3 Lung Disease in Flavoring and Food Production: Learning from Butter Flavoring Nancy Sahakian* and Kathleen Kreiss*
Contents
I. Introduction II. Respiratory Tract Anatomy and Defense Mechanisms III. Medical Tests Used to Diagnose Lung Disease IV. Types of Occupational Respiratory Disease A. Anaphylaxis, allergic conjunctivitis/rhinitis, and allergic asthma B. Irritant-induced asthma, work-aggravated asthma, and chronic bronchitis C. Hypersensitivity pneumonitis D. Emphysema E. Bronchiolitis obliterans V. Flavoring-related BO A. NIOSH investigation of flavoring-related BO B. Recommended interventions C. Timeline of the emergence of flavoring-related BO and the industry and regulatory agency response D. Liability VI. Recognition of Emerging Occupational Respiratory Disease in the Food Industry VII. Prevention of Known Occupational Respiratory Diseases in the Food Industry References
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*Centers for Disease Control and Prevention, National Institute for Occupational Safety and Health, Division of Respiratory Disease Studies, Morgantown, West Virginia 26505 Advances in Food and Nutrition Research, Volume 55 ISSN 1043-4526, DOI: 10.1016/S1043-4526(08)00403-8
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2009 Elsevier Inc. All rights reserved.
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Abstract
Nancy Sahakian and Kathleen Kreiss
Workers in the food industry are exposed to multiple respiratory hazards that include irritants, allergens, and substances capable of causing destruction and scarring of the lungs. Cases of constrictive bronchiolitis obliterans, a severe potentially disabling lung disease, have been identified in workers exposed to flavorings. Workplace engineering controls, work practices, and respiratory protection can minimize potential exposures. Medical surveillance of workers exposed to known respiratory hazards will help to identify disease early, facilitate the prompt removal of workers from the causative exposure(s), and prevent further worsening and/or permanence of disease. When companies or employees suspect occupational respiratory disease, they can involve public health agencies to investigate any excess risk of lung disease, risk factors among processes and exposures, and effectiveness of interventions, if needed.
I. INTRODUCTION In 2000, the National Institute for Occupational Safety and Health (NIOSH) of the Centers for Disease Control and Prevention (CDC) became aware of eight former workers of a microwave popcorn facility who had been diagnosed with a rare lung disease, bronchiolitis obliterans (BO). Further investigation determined that exposure to artificial butter flavoring was the causative agent. Additional cases of flavoring-related BO were subsequently identified in workers in the microwave popcorn-, flavor-, and diacetyl-1 manufacturing industries. Worksite interventions helped to prevent BO in other workers exposed to flavoring chemicals. In this chapter, we describe the anatomy and defense mechanisms of the respiratory tract, medical tests used to diagnose respiratory diseases, occupational respiratory diseases, specific respiratory hazards in the food industry, how exposures can be reduced, and how workers can be monitored through medical surveillance to allow early identification and protection of affected workers. Finally, we outline the discovery that occurred as a result of the public health response to cases of BO in workers in the microwave popcorn and flavoring industries. This information can assist in future identification of other occupationally related respiratory diseases in the food industry.
1
Diacetyl is a major component of butter flavoring.
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II. RESPIRATORY TRACT ANATOMY AND DEFENSE MECHANISMS The upper respiratory tract includes the nasal passages, sinuses, pharynx (mouth and throat), and larynx (voice box). The lower respiratory tract includes the trachea, bronchial tree (bronchi, bronchioles, terminal bronchioles, and respiratory bronchioles), air sacs (alveoli), and supporting tissue (interstitium). The internal caliber of the airways decreases with sequential branching of the bronchial tree. Bronchioles have a diameter of about 1 mm. Terminal bronchioles have a diameter of about 0.3–0.5 mm. Compared to the larger airways, smaller airways are composed of proportionately less cartilage and more smooth muscle; at the level of the terminal bronchioles, smooth muscle totally circumscribes the airways. Rapid exchange of gases between the air in the air sacs and the blood in the capillaries is facilitated by the thin air sac wall that is only several cells thick and by the proximity of capillaries (the smallest blood vessels). Large inhaled particles are entrapped by hairs in the nasal passages. Many of the smaller inhaled particles are entrapped in the mucus layer that coats the respiratory tract. Small particles that reach the air sacs are engulfed and ingested by immune cells called macrophage cells. Particles that contain irritating chemical substances can damage the site in the respiratory tract where they impact; however, even inert particles can damage the respiratory tract at high enough concentrations. The mucus layer also absorbs highly water-soluble chemicals, such as ammonia and formaldehyde, and prevents these chemicals from penetrating deeper into the lung. Mucous glands within the respiratory tract, as well as goblet cells on the internal surface of the respiratory tract, create the mucus. Additionally, cells with hair-like projections, called cilia located alongside goblet cells, propel the mucus layer with its entrapped particles and chemical contaminants upward where the mucus is either swallowed or coughed out. The lungs are able to repair minor injuries by replacing damaged cells with normal lung cells. Repetitive or excessive injury may result in permanent loss of lung tissue or repair with scar tissue. Scarring can occur within the air sacs, the airways, or the supporting tissue of the lung.
III. MEDICAL TESTS USED TO DIAGNOSE LUNG DISEASE A spirometry test is a breathing test in which a person takes as deep a breath as possible and blows out quickly and completely into a tube connected to a spirometry machine (Table 3.1). Lung measurements obtained from this test include forced expiratory volume in one second (FEV1), the amount of air blown out in one second; forced vital capacity (FVC), the total amount of air blown out; the FEV1/FVC ratio; and the
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TABLE 3.1 Pulmonary tests Pulmonary test
Description
Results
Spirometry
After inhaling as much air as possible, the patient blows out as quickly and completely as possible into a tube connected to a spirometry machine When a spirometry test shows airways obstruction, an inhalable medication that relaxes the muscles in the small airways is administered and the spirometry test is repeated Spirometry tests or PEFR measurements are repeated five or more times each day during the waking hours over a 3- to 4-week period
Airways obstruction (asthma, emphysema, BO). Restrictive pattern (HP)
Bronchodilator trial
Serial spirometry or serial peak expiratory flow rate (PEFR) measurements
Bronchoprovocation test
Methacholine challenge test
The baseline FEV1 is compared to FEV1 following a simulated occupational exposure or a controlled exposure to an occupational agenta The baseline FEV1 is compared to FEV1 following administration of
Reversible airways obstruction suggests the presence of asthma
Lower FEV1 or lower PEFR measurements on workdays compared to non-workdays are suggestive of work-related asthma A drop in FEV1 suggests workrelated asthma
A drop in FEV1 following a low dose of methacholine (continued)
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TABLE 3.1
(continued)
Pulmonary test
Description
Antibody test
methacholine, an inhalable drug that can cause the muscles in the small airways to contract Blood tests that detect antibodies to an allergen; reactions to skin pricking with antigen imply allergic sensitization
Diffusing capacity of the lung for carbon monoxide (DLCO)
High-resolutioncomputed tomography (HRCT) of the chest
a
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A test dose of carbon monoxide is inhaled and measurements are obtained to determine how much of this is absorbed. This measurement is used to estimate how well other gases are exchanged by the lungs Detailed radiological images that provide a three-dimensional picture of the lungs
Results
suggests the presence of asthma
A positive test indicates antibody sensitization, which is associated with allergic rhinitis, allergic conjunctivitis, and allergic asthma A low DLCO may be caused by: (1) scarring of the lung tissue in HP; (2) obliteration of the small airways in severe BO; and (3) destruction of the air sacs in emphysema HRCT can detect: (1) scarring of the lung tissue in HP; (2) air trapping in the air sacs during expiration in BO; and (3) destruction of the air sacs in emphysema
Bronchoprovocation tests using an occupational agent are infrequently performed because only a few medical facilities in the United States are equipped to perform this test.
peak expiratory flow rate (PEFR), the fastest rate at which air can be exhaled. FEV1, FVC, and PEFR are compared to average values for a person of the same height, age, gender, and race to yield percent predicted values; for example, a test result that is 80% of the average value
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would be 80% of predicted value. Additionally, there are normal ranges with lower limit of normal values for lung measurements of a person of a specific height, age, gender, and race. A pattern of airways obstruction is identified if the FEV1 and FEV1/FVC are below the lower limit of normal and the FVC is normal. In this case, the individual has an adequate lung capacity but is unable to blow the inhaled air out quickly due to resistance in the airways. Symptoms associated with this type of lung abnormality are wheezing and chest tightness. A restrictive pattern is identified if the FEV1/FVC ratio is normal and the FVC is below the lower limit of normal. An individual with a restrictive pattern is unable to take in a full breath; however, he or she is able to quickly exhale the inhaled air. Symptoms associated with this type of lung abnormality include shortness of breath on exertion. An inhalable medication that relaxes the muscles in the airways (bronchodilator) is frequently administered when airways obstruction is identified. In this bronchodilator trial test, the spirometry test is subsequently repeated and compared to the results from the initial spirometry test. If there is substantial improvement in lung function with the administration of the bronchodilator, the airways obstruction is reversible. An example of a lung disease with reversible airways obstruction is asthma, in which symptoms occur episodically when airways obstruction occurs. If there is little or no improvement after the administration of the bronchodilator, the airways obstruction is fixed. An example of a lung disease with fixed airways obstruction is BO, where there is scarring of the airways. In the methacholine challenge test, subjects inhale a drug that can cause the muscles in the small airways to contract. People with asthma will have a reduction in their FEV1 after inhaling a low dose of this drug, reflecting increased ‘‘irritability’’ or bronchial hyperreactivity. To make an association between occupational exposures and asthma, serial spirometry, serial measurements of PEFR, provocation with a simulated occupational exposure or an occupational agent, and antibody tests are frequently conducted. In work-related asthma, repeat spirometry and/or PEFR measurements five or more times daily during the waking hours over a 3- to 4-week period may show lower FEV1 or PEFR measurements on workdays compared to non-workdays. A drop in FEV1 with a simulated occupational exposure and/or with exposure to an occupational agent is also suggestive of work-related asthma. If there is exposure to a known occupational allergen, then antibody sensitization may be tested with a blood test (radioallergosorbent test) that detects antibodies specific to the allergen or with a skin prick test. The ability of the lungs to exchange gases is measured by the diffusing capacity of the lung for carbon monoxide (DLCO). In this test, ability of the lungs to absorb a test dose of carbon monoxide is measured and is used to approximate their ability to exchange oxygen and carbon dioxide.
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Inflamed lung tissue (hypersensitivity pneumonitis, HP), complete obliteration of a large proportion of the airways (severe BO), and damage to the air sacs (emphysema) will reduce the DLCO measurement. High-resolution computed tomography (HRCT) of the chest is able to detect inflammation and scarring of the supporting lung tissue, air trapping in the air sacs during expiration, and extensive destruction of air sacs; findings that are present in HP, BO, and emphysema, respectively.
IV. TYPES OF OCCUPATIONAL RESPIRATORY DISEASE Respiratory disease may occur as a result of one of three mechanisms: immunological sensitization and response, irritation, and injury followed by destruction and/or scarring. In most immunological respiratory diseases, a period of time is required for a person’s immune cells to become sensitized; subsequent exposure results in respiratory symptoms. The period of time from first exposure to onset of symptoms is referred to as the latency period. Immunological diseases that are antibody mediated consistently have latency periods. These include anaphylaxis, allergic conjunctivitis/rhinitis, and allergic asthma. HP is a cell-mediated immunological disease that may result in symptoms in sensitized individuals within hours after a high-intensity exposure or after months of lowintensity exposures. In diseases due to irritation, there is generally injury to the lining of the respiratory tract, resulting in mucus production and possibly permanent damage of the respiratory tract. Irritating chemicals and particles may also cause irritant-induced asthma, especially in the case of an overwhelming exposure. BO is an example of a respiratory disease due to injury followed by scarring. Emphysema is a respiratory disease characterized by injury followed by permanent destruction of the air sacs. Shortness of breath, cough, and wheeze are common symptoms of respiratory diseases (Table 3.2). These shared symptoms make misdiagnosis by physicians common if the diagnosis is based solely on reported symptoms. Medical tests help to distinguish the respiratory diseases from each other (Table 3.3). Other pitfalls in medical diagnosis include diagnosing common diseases (such as asthma, emphysema, and chronic bronchitis) instead of the actual rarer diseases (such as BO); and not considering occupational exposures as the cause.
A. Anaphylaxis, allergic conjunctivitis/rhinitis, and allergic asthma An allergen is a chemical or substance that results in an immunoglobulin E-mediated (antibody) allergic response. Allergens are usually high molecular weight organic compounds. The production of antibodies
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TABLE 3.2 Symptoms of occupational lung diseases Respiratory disease
Predominant symptoms
Allergic asthma Irritant-induced asthma Work-aggravated asthma Chronic bronchitis
Episodic wheeze, chest tightness, shortness of breath, and cough
Hypersensitivity pneumonitis
Emphysema Constrictive BO
Daily cough that produces phlegm for three or more consecutive months out of a year Progressive shortness of breath, cough, and weight loss (low-intensity exposures); shortness of breath, cough, muscle achiness, chills, sweating, and fatigue on workdays (high-intensity exposures) Progressive shortness of breath and cough Progressive shortness of breath and cough
(sensitization) may occur following inhalation, ingestion, or contact with the eyes or skin. Subsequent exposure results in symptoms of hives, anaphylaxis (see the following paragraph), conjunctivitis/rhinitis, and/ or asthma. The route of sensitization may differ from the route of exposure responsible for symptoms: for example, an individual sensitized by airborne exposures at work may develop allergic respiratory symptoms following ingestion of the same allergen in a food (Acero et al., 1998). Sensitization can be detected by a blood test (such as the radioallergosorbent test) or a skin prick test for specific allergens. Because of the risk of provoking a life-threatening event, a skin prick test is not performed on individuals with a history of anaphylaxis. Anaphylaxis is a severe systemic allergic reaction, which can be fatal. Frequently the first symptom is itchy hives (welts) within minutes of exposure. Swelling of the larynx, with constriction of the air passage and a rapid drop in blood pressure quickly follow. Treatment includes immediate removal from exposure, administration of epinephrine, and strict avoidance of reexposure. Symptoms of allergic conjunctivitis and rhinitis include red, itchy, watery eyes, watery nasal discharge, nasal congestion, and sneezing. Asthma is characterized by episodic airways obstruction and symptoms of wheeze, chest tightness, shortness of breath, and cough. Asthma symptoms are due to contraction of the smooth muscles, swelling of the lining, and mucus in large and small airways (Table 3.4). Symptoms generally appear or worsen within several hours after exposure. Once an exposure association is made, early removal from exposures may result in cure with
TABLE 3.3
Medical test results for occupational lung diseases
Test
Lung examination
Spirometry
Bronchodilator trial
Methacholine challenge Diffusing lung capacity for carbon monoxide (DLCO) High-resolutioncomputed tomography (HRCT) of the chest
Occupational asthma or workaggravated asthma
Wheeze during an asthma attack May show airways obstruction Airways obstruction improves
Hypersensitivity pneumonitis
Emphysema
Constrictive BO
Crackles (rales), occasionally wheeze
Decreased breath sounds Airways obstruction
Crackles (rales), wheeze Airways obstruction
Airways obstruction does not improve Normal reactivity Decreased
Airways obstruction does not improve Normal reactivity Normal or decreased
Large holes in the lungs (bullae)
Air trapping during exhalation
May be normal, or may show airways obstruction or a restrictive pattern Airways obstruction may minimally improve
Increased reactivity Normal
May show increased reactivity
Normal
Inflammation of the interstitium, scarring of the lung tissue, and/or small nodules
Decreased
172 TABLE 3.4 Description and causes of occupational lung diseases in the food industry Respiratory disease
Site of disease
Description
Known causes in the food industry
Allergic asthma
Bronchi, bronchioles, terminal bronchioles
Antibody-mediated response to inhaled allergens with contraction of the smooth muscles in the walls of the airways, swelling of the airways, and mucus secretion in the airways
Irritant-induced asthma
Bronchi, bronchioles, terminal bronchioles
Workaggravated asthma
Bronchi, bronchioles, terminal bronchioles
Contraction of the smooth muscles in the walls of the airways, swelling of the airways, and mucus secretion in the airways due to inhaled irritants Contraction of the smooth muscles in the walls of the airways, swelling of the airways, and mucus secretion in the airways due to nonspecific occupational exposures or occupational allergens to which the worker was previously sensitized
Cereal flour, buckwheat flour, soy flour, seafood allergens, pork, sesame seeds, sunflower seeds, lupin, spinach, sarsaparilla root dust, cocoa, coffee dusts, green tea, egg protein, lactalbumin, milk powder, casein, honey, a-amylase, glucoamylase, pectinase, gluconase, pepsin, pectin, spices, carmine, flavorings A single high-intensity or multiple low-intensity exposures to chlorine gas, bleaching agents, cleaning agents, and fumigants Cold air, dusts, aerosol sprays, smoke, fumes, occupational allergens to which the worker was previously sensitized
Chronic bronchitis
Bronchi, bronchioles
Constrictive BO
Terminal and respiratory bronchioles
Emphysema
Alveoli, respiratory bronchioles Interstitium
Hypersensitivity pneumonitis
Enlarged mucous glands and an increased number of goblet cells produce an excessive amount of mucus Injury of the airways results in scarring and in some cases complete obliteration of the air passages Destruction of the walls of the air sacs and scarring of and weakening of the walls of the small airways Cell-mediated response to substances results in the formation of scar tissue in the supporting tissue of the lung
Bakery and food-processing exposures
Diacetyl, butter flavoring, overheated cooking oil
–
Penicillium in cheese or cheese casings, green coffee dust, Botrytis cinerea on grapes, Aspergillus oryzae in soy sauce, grain weevils, carmine
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the disappearance of episodic asthma symptoms. Continued exposure may result in an increased likelihood of: (1) persistent asthma with asthma symptoms even when no longer exposed, with symptoms at nighttime, after exercise, with respiratory infections, and with exposure to cold air or irritants; and (2) scarring of the airways resulting in fixed airways obstruction. Smokers and individuals with a history of asthma, hay fever, or eczema are at greater risk for developing occupational allergic asthma. Occupational allergens in the food industry include mammalian and avian proteins, and allergens from plants, mold, bacteria, spices, and seafood (Bernstein et al., 1999). Enzymes, which have multiple applications within the food industry, are frequently allergens. a-Amylase derived from Aspergillus oryzae is used to stimulate the growth of yeast and facilitate the rising of bread. Glucoamylase derived from A. niger is used in the production of high-fructose corn syrup. Pectinases are used to assist in the removal of the pith from fruit and to clarify fruit juices. Rennet, proteases, and lipases are used in cheese production. Spirometry and bronchodilator trial tests that indicate reversible airways obstruction and/or airways reactivity when challenged with methacholine are used to diagnose asthma. Occupational allergic asthma is subsequently diagnosed when respiratory symptoms have begun after hire and after a latency period of several weeks to several years, and there is an indication that asthma symptoms or the status of asthma are worse on workdays. Changes in asthma status suggestive of occupational asthma include serial spirometry tests or PEFR measurements that show lower FEV1 or PEFR measurements on workdays compared to nonworkdays; and improvement in the methacholine challenge test after the worker has been removed from work exposures for several months. Although largely unavailable, provocation tests with allergens cause a decline in FEV1. Allergic rhinitis and asthma commonly occur in bakers and fish and seafood processing workers (Bernstein et al., 1999). Occupational asthma has additionally been reported in egg-processing workers (Smith et al., 1987); coffee workers (Osterman et al., 1985; Zuskin et al., 1981); green tea factory workers (Shirai et al., 2003), spice factory workers and workers who handle spices (An˜´ıbarro et al., 1997; Falleroni et al., 1981; Fraj et al., 1996; Lemie`re et al., 1996; Sastre et al., 1996; Seuri et al., 1993; Zuskin et al., 1988); cocoa-processing workers and workers who handle cocoa (Perfetti et al., 1997; Zuskin et al., 1998); and natural food dye (carmine) production workers (Lizaso et al., 2000; Rodriguez et al., 1990; Tabar-Purroy et al., 2003). Case reports of occupational asthma include a candy worker exposed to lactalbumin (Bernaola et al., 1994) and a bakery worker exposed to milk powder (Toskala et al., 2004), a candy worker and a jam-manufacturing worker exposed to pectin (Cohen et al., 1993; Kraut et al., 1992), a worker in
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a meat tenderizer manufacturing plant exposed to papain (Novey et al., 1979), a meat kneader exposed to casein (Rossi et al., 1994), a meat processor exposed to pork meat (Labrecque et al., 2004), bakery workers exposed to sesame and sunflower seeds (Alday et al., 1996; Keskinen et al., 1991; Vandenplas et al., 1998), a worker who handled lupin powder (Campbell et al., 2007), a pasta factory worker exposed to spinach powder (Schuller et al., 2005), a herbal tea worker exposed to sarsaparilla root dust (Vandenplas et al., 1996), a cereal maker exposed to honey (Johnson et al., 1999), fruit-processing workers exposed to pectinase and gluconase (Sen et al., 1998), and a cheese maker exposed to pepsin (An˜´ıbarro and Fontela, 1996). Asthma associated with occupational exposure to flavorings has also been reported (CDC, 2007a). The impact of occupational asthma in food industry workers was quantified in a random population survey of workers in New Zealand that found food processors (other than bakers) to be about 2.5 times more likely to report wheeze than office workers (Fishwick et al., 1997a). Bakers’ asthma and allergic rhinitis are the most common forms of occupational respiratory disease in the food industry. Among bakery workers, the prevalence of occupational asthma and allergic rhinitis ranges from 5% to 7% and 15% to 20%, respectively (Houba et al., 1996). Bakers’ asthma is most often due to cereal flours (such as wheat, rye, and barley flour) and less frequently due to buckwheat flour, soy flour, fungal a-amylase, fungal glucoamylase, proteases, cellulases, xylanase, molds, and storage mites (Bauer et al., 1986; Merget et al., 2001; Quirce et al., 1992, 2002; Tarvainen et al., 1991). Antibody sensitization rates for wheat flour and a-amylase among bakers range from 5% to 25% and 2% to 15%, respectively. Estimated wheat flour-related incidence rates among bakers are 10 cases of antibody sensitization per 1000 workers per year, and 3–4 cases of respiratory allergy per 1000 workers per year (Heederik and Newman Taylor, 1999). The diagnosis of respiratory allergy in bakery workers is made by the presence of nasal and/or airways symptoms after an initial symptom-free period (latency period) of months to several years. Rhinitis often occurs first, followed by the development of asthma; however, asthma in the absence of rhinitis is not uncommon. Asthma symptoms develop within minutes to hours of exposure at work and may persist for 24 h or more. A temporal relationship with work may only be discovered during 1or 2-week vacations. Supporting medical tests include: (1) positive radioallergosorbent blood test or skin prick test specific to flour proteins and/or a-amylase allergen; (2) reversibility of airways obstruction when a bronchodilator is administered and/or airways reactivity when challenged with methacholine; and (3) serial lung function tests that improve away from work. Once sensitized, workers will become symptomatic with exposure to very low concentrations of airborne allergen. Because of this, the only
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effective solution is a change in their work. As an interim measure until a change in work is feasible, the worker may wear a respirator with a particulate filter to minimize exposures. The amounts of flour allergen and a-amylase per gram of dust vary considerably, making airborne dust concentration a poor measure of allergen exposure in bakeries. Use of environmental allergen levels to assess health risk is challenging due to the lack of standardization of immunoassays among analytical laboratories. As yet, there are no recognized occupational exposure limits for flour dust, flour allergens, or a-amylase. The absence of regulation, however, does not imply safety for this classic example of occupational asthma. Prevention measures include eliminating activities that generate high levels of dust, such as emptying bags, compressing empty paper bags that previously contained flour or dough improvers, dusting dough, dry sweeping, and use of pressurized air. Potential solutions include use of divider oil to prevent dough adhesion to surfaces instead of dusting with flour, automated forming instead of dough-braking, closed transfer of flour, exhaust ventilation that circumscribes the dough mixer, use of flow table exhaust systems if dusting dough is unavoidable, covering dough mixers during the dough-making process, and the use of encapsulated or dissolved enzyme formulations (Burstyn et al., 1997; Heederik and Newman Taylor, 1999). Medical surveillance of food industry workers is suggested for workers exposed to allergenic materials. A typical program includes: (1) baseline and periodic skin prick or radioallergosorbent tests for specific antibodies (antibody sensitization); and (2) instruction of workers to promptly report new upper or lower respiratory symptoms (or worsening of prehire symptoms) to the facility director of safety. Workers who develop antibody sensitization or symptoms are medically evaluated by a pulmonologist or allergist. Workers subsequently diagnosed with occupational rhinitis or asthma are relocated away from further exposure. There is no standard recommended interval for antibody sensitization testing. In the detergent-manufacturing industry where workers are exposed to proteases, amylases, lipases, and cellulases, it has been suggested to test workers every 6 months for the first 2 years, and thereafter every 2 years. This decision was based on studies that have shown that the risk of antibody sensitization is highest during the first 2 years of exposure (Nicholson et al., 2001).
B. Irritant-induced asthma, work-aggravated asthma, and chronic bronchitis Irritant-induced asthma usually occurs following a single or multiple accidental high-intensity exposures. Repetitive lower intensity exposures to irritants may also cause asthma (Balmes, 2002). Once asthma develops,
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its symptoms may occur with exposure to nonspecific stimuli, such as cold air, dusts, aerosol sprays, smoke, and fumes. Chlorine gas, bleaching agents, cleaning agents, anhydrous ammonia, and fumigants are exposures in the food industry known to cause irritant-induced asthma. Exposure to endotoxin, a cell wall component of gram-negative bacteria, has been associated with respiratory symptoms and a reversible workrelated change in lung function among potato processors (Zock et al., 1998). In work-aggravated asthma, a worker with preexisting asthma has worsening of asthma symptoms due to exposures in the workplace. These exposures may be irritant chemicals, cold air, or allergens to which the worker was sensitized prior to hire. The diagnosis is made if the asthma was not active within 2 years before the hire date and there is: (1) worsening of asthma symptoms on workdays; and (2) serial spirometry tests and/or repeat PEFR measurements suggesting a work-related pattern. Chronic bronchitis is defined as a daily cough that produces phlegm for three consecutive months out of a year. It is due to enlarged mucous glands and an increased number of goblet cells that result in an excessive amount of mucus. Involvement of goblet cells is particularly important due to their location in smaller airways where the mucus can interrupt airflow. Chronic bronchitis is most commonly caused from cigarette smoking. However, a random population-based study of 20- to 44-yearold workers demonstrated that current and former smokers in the food industry were two to three times more likely to report phlegm production compared to office workers who also were current or former smokers (Zock et al., 2001). Another study found chronic bronchitis to be 3 times more likely in food processors and chronic bronchitis with airways obstruction to be 26 times more likely in bakers, compared to office workers (Fishwick et al., 1997b).
C. Hypersensitivity pneumonitis HP is an uncommon lung disease caused by cell-mediated sensitization to organic dusts or chemicals. With high-intensity exposures, symptoms can include shortness of breath, cough, muscle achiness, chills, sweating, and fatigue that occur on workdays. Complete normalization of symptoms and lung function can occur with early removal of workers from exposure. If exposure continues, permanent lung damage from scarring can occur. Symptoms associated with low-intensity exposure include progressive shortness of breath and cough, as well as weight loss. The slow progression of symptoms and the persistence of symptoms away from work that occur with low-intensity exposure may result in delayed recognition of the disease by both workers and physicians. Medical evaluation often includes a restrictive spirometry pattern and may reveal crackles
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(rales) on chest examination, decreased ability of the lungs to exchange gases, and immunoglobulin G antibodies specific to the causative agent. Workers in the food industry at risk for developing HP include cheese workers exposed to Penicillium in cheese or cheese casings, coffee workers exposed to green coffee dust, wine growers exposed to Botrytis cinerea on grapes, soy sauce brewers exposed to A. oryzae, millers exposed to grain weevils in wheat flour, and natural dye production workers exposed to carmine (Christiansen et al., 1981; Schuyler, 1998).
D. Emphysema In emphysema there is destruction of the walls of the air sacs. Lung function tests demonstrate fixed airways obstruction due to collapse of airways during exhalation and a decreased ability of the lungs to exchange gases. Physical examination of the lungs usually reveals decreased breath sounds. Approximately 20% of cases of emphysema are due to occupational causes and the remaining 80% are mostly due to smoking. Smokers with emphysema should not be assumed to have smoking-related emphysema, particularly young smokers. This is because only about 15% of smokers will ever develop emphysema and most individuals with smoking-related emphysema will become symptomatic in their fifties after having smoked 20 or more pack-years (ATS, 1995). Risk of emphysema is increased among food industry workers: a large US health survey found airways obstruction to be 2.1 times more prevalent in food products manufacturing workers than in office workers after adjusting for smoking and other factors (Hnizdo et al., 2002). The causes have not been investigated.
E. Bronchiolitis obliterans BO is a rare lung disease. In constrictive BO, there is inflammation and injury of the small airways that lead to the air sacs (terminal and respiratory bronchioles). Subsequent repair mechanisms result in scarring and narrowing or complete obliteration of the affected airways. The disease is patchy in distribution so that all sections of the lung are not equally affected. Symptoms consist of cough and shortness of breath with exertion, which typically do not improve away from the causative exposure. With continued exposure, the scarring involves increasingly greater amounts of the lung and workers become increasingly short of breath. Scarring and breathlessness are permanent, resulting in the consideration for lung transplants in some workers. Spirometry tests reveal fixed airways obstruction. HRCT of the chest with inspiratory and expiratory images may show air trapping in the expiratory images. Unlike emphysema, the lungs ability to exchange gases is unaffected until late in the
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disease. Lung biopsy typically shows inflammation and scarring of the small airways. However in individuals with BO, lung biopsies frequently fail to make the diagnosis. Characteristics of flavoring-related constrictive BO are described in the literature (Akpinar-Elci et al., 2004); a lung biopsy is usually not required to make this diagnosis. In addition to diacetyl and butter flavorings, overheated cooking oil has also been reported as a cause of constrictive BO (Simpson et al., 1985).
V. FLAVORING-RELATED BO A. NIOSH investigation of flavoring-related BO In 2000, eight cases of BO among former workers of a microwave popcorn plant resulted in an investigation that identified a new occupational hazard in the food industry. BO is a rare lung disease, so the occurrence of eight cases in a small rural community was unusual. The fact that they had all worked at the same plant gave rise to suspicion of a common cause. The persistence of symptoms away from work (typical in this disease) in these eight workers delayed recognition of a work-related illness by physicians. One pulmonologist caring for these workers called the US Occupational Safety and Health Administration (OSHA); however, the compliance officer who visited the plant could not find any known pulmonary hazard. Ultimately, an occupational medicine physician who reviewed medical records on the eight workers for a workers’ compensation attorney reported the cases to the Missouri Department of Health and Social Services, which, in turn, requested assistance from NIOSH. All eight workers had become ill while employed at the microwave popcorn plant with the earliest case starting in 1993. Four of the eight workers had worked in the flavor-mixing room and four had worked in the microwave popcorn packaging area. Six of the eight workers had FEV1 measurements less than 40% of predicted (criterion of the Social Security Administration for total disability from respiratory disease); four were on lung transplant waiting lists. NIOSH conducted a medical survey of current workers and an environmental survey of the facility in November 2000 (Kreiss et al., 2002a). Workers added salt, heated butter flavoring, and coloring agents to heated soybean oil (130 F) in large mixing tanks. The flavoring mixture was pumped into heated holding tanks on an open mezzanine level and then added to popcorn in microwavable bags in the microwave popcorn packaging lines below (Fig. 3.1). There was no physical barrier between the flavor-mixing tanks, holding tanks, and microwave popcorn packaging lines. Heating of the flavors and of the flavored soybean oil resulted in volatilization of the flavoring ingredients. Chemical vapors from the
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FIGURE 3.1 Sentinel microwave popcorn plant with holding tanks in the mezzanine above microwave popcorn packaging line.
mixing tanks on the main floor and holding tanks on the mezzanine level exposed microwave popcorn packaging workers on the main floor of the plant. Quality control workers popped the microwave popcorn and opened popped bags to assess the quality of the popcorn. Workers did not use respiratory protection. A thermal desorption tube air sample from the mixing room demonstrated a complex spectra of over 100 volatile organic compounds, with ketones predominating. Using diacetyl as a marker of exposure, NIOSH measured diacetyl air concentrations based on 8-h time-weighted averages (8-h TWA). The mean diacetyl air concentrations based on work area air samples collected over multiple days were 37.8 parts per million (ppm) for the mixing tank room, 2.0 ppm for the microwave popcorn packaging line, and 0.5 ppm for the quality control room. The prevalence of airways obstruction in this workforce was 3.3 times greater than expected when compared to national data. Nineteen of the 21 workers identified with airways obstruction had fixed airways obstruction. Cumulative worker exposures were calculated in parts per million diacetyl-years and workers were placed in equally sized worker categories of least, minimally, moderately, and most exposed. When successive categories of least to greatest exposed workers were compared, it was found that the proportion of workers with airways obstruction increased and average-percent-predicted FEV1 decreased, demonstrating an exposure–response relationship with diacetyl. Five of
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six quality control workers had airways obstruction despite relatively low diacetyl air concentrations in this work area (Kreiss et al., 2002a). Potential hazards in these quality control workers may have been caused by peak exposures to a combination of volatile organic compounds unique to this work area. Quality control workers are exposed to momentary bursts of volatile organic compounds when they open freshly popped bags of popcorn. The high temperatures associated with popping microwave popcorn generated different proportions of volatile organic chemicals than present in the mixing and microwave popcorn packaging areas. NIOSH subsequently evaluated five other microwave popcorn plants and analyzed aggregated data from all six plants. In five of the six plants, mixers and/or microwave popcorn packaging workers had medical findings consistent with BO. Mixers had a lower mean-percent-predicted FEV1 compared to other workers. Microwave popcorn packaging workers in plants where mixing tanks were located in proximity to the packaging lines had a higher prevalence of airways obstruction compared to microwave popcorn packaging workers in plants where the tanks were isolated in a separate room with the door closed. The mean work area 8-h TWA diacetyl air concentrations in plants where cases of BO occurred were 0.2– 37.8 ppm in mixing rooms and 0.3–1.9 ppm in microwave popcorn packaging areas (Kanwal et al., 2006). (The NIOSH sampling method #2557 for diacetyl was subsequently found to underestimate diacetyl air concentrations in samples collected in high-humidity conditions; development of an alternate sampling method is currently in progress.) In 2006 and 2007, NIOSH investigated two flavor-manufacturing plants. Medical testing of 29 production workers identified 7 workers with fixed airways obstruction who had worked in plated-powder and/or liquid flavoring work areas; 4 of these workers had severe fixed airways obstruction (CDC, 2007b,c). Mean work area diacetyl air concentrations (using NIOSH method #2557) for plated-powder production and liquid production in one of these plants were 0.25 and 0.02 ppm, respectively (CDC, 2007b). Animal inhalation studies were conducted at NIOSH in which rats were exposed to vapors from a butter flavoring used at the sentinel microwave popcorn plant. Rats were exposed for 6 h to vapors with diacetyl concentrations of 203, 285, 352, or 371 ppm. Exposure to butterflavoring vapors of 285 or more ppm diacetyl resulted in damage to the bronchi; nasal damage was seen in all the exposed rats (Hubbs et al., 2002). Studies using pure diacetyl vapors demonstrated nasal damage in rats exposed for 6 h to 198 or more ppm diacetyl (Hubbs et al., 2008). The degree of inflammation and damage to the nose, larynx, trachea, and bronchi in exposed rats is concentration dependent (Hubbs et al., 2008). Due to the absence of mouth breathing, the larger surface area of the nose in rodents compared to humans, and the high water solubility of diacetyl,
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much of the observed injury in rodent studies is in the upper airways. In order to demonstrate animal findings more comparable to health effects experienced by humans, a study conducted at the National Institute of Environmental Health Sciences placed liquid diacetyl in the oropharynx of mice. The liquid that was subsequently inhaled resulted in scarring at the level of the smallest airways. Diacetyl inhalation studies demonstrated inflammation of the nose, larynx, and bronchi when mice inhaled 100 or more ppm of diacetyl for 1 h/day, 5 days/week for 4 weeks (Morgan et al., 2006). Butter flavoring is implicated in BO among microwave popcorn workers. Diacetyl, a major component of butter flavoring, causes BO based on cases of BO among chemical manufacturing workers who made diacetyl (van Rooy et al., 2007) and animal studies that showed respiratory effects from diacetyl alone. Other chemicals in flavorings are also likely to cause or contribute to BO based on: (1) reports of BO in flavor-manufacturing workers thought to be due to acetaldehyde (Lockey et al., 2002); (2) lung disease in flavored popcorn-manufacturing workers for whom diacetyl was not detected in the air (CDC, 2007a); and (3) rat butter-flavoring experiments demonstrating respiratory damage in excess of that attributable to diacetyl alone (Kreiss et al., 2002b).
B. Recommended interventions Over 2000 different chemicals are used to formulate artificial flavors (FEMA, 2004). Flavoring chemicals are generally volatile. Flavored powders produced by flavor-plating or spray dried processes may be inhaled if these powders become airborne. Implementation of engineering controls in flavor-manufacturing plants and food production plants can minimize workers’ exposure to flavoring chemicals. Of special concern are processes that involve heat (as heat will increase the volatilization of flavoring chemicals) and processes that agitate powders. High-exposure processes are best enclosed in a separate room under negative pressure and venting of the exhaust air to the outdoors distant from the air intake vents for the facility. Effective use of local exhaust ventilation will reduce exposures in mixing, blending, and spray-drying operations, as well as exposures produced during the pouring and measuring of ingredients; respective applications include use within mixing tanks, around the perimeter of blenders and spray dryers, and at the point of operation of pouring and measuring activities. Mixing vessels should remain lidded during mixing processes. The process of adding ingredients to vessels should be converted to an automated closed process. Respirators with particulate filters and organic vapor cartridges should be used as an interim measure until engineering controls successfully eliminate worker exposures.
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Safe exposure levels to diacetyl and other flavor ingredients are not known. It is likely that both peak and average exposures of diacetyl and other chemicals are important. Exposure monitoring for diacetyl is recommended with the intention of keeping diacetyl peak and 8-h TWA concentrations as low as possible. Eight-hour TWA and peak diacetyl exposures for workers diagnosed with flavoring-related BO illustrate how greatly these measurements may differ. The mean 8-h TWA for the mixing room of the sentinel microwave popcorn plant where workers with flavoring-related BO had worked was 37.8 ppm diacetyl (range: 2.3–97.9); months later, using a direct-reading instrument (Fourier transform infrared spectroscopy, FTIR), the diacetyl level inside the headspace of the holding tank was measured as high as 1230 ppm (CDC, 2006). The mean 8-h TWA for the powder production work area in a flavor-manufacturing plant where other workers with flavoring-related BO had worked was 0.2 ppm diacetyl (range: 0.002–0.790) (using NIOSH method #2557), and the peak diacetyl level using FTIR was 210 ppm (CDC, 2007b). Some exposed workers in the flavor-manufacturing and food production industries who have developed severe respiratory disease experienced symptoms within months of exposure to flavoring chemicals. Other exposed workers with fixed airways obstruction did not have any obvious symptoms. Many workers with flavoring-related BO were initially misdiagnosed by their physicians as having asthma, emphysema, or chronic bronchitis. Because of the severity, permanency, short latency, and the possibility of misdiagnosis of this disease, exposed workers should be part of a medical surveillance program that includes spirometry testing. If airways obstruction is identified, a bronchodilator should be administered and spirometry repeated to establish the presence of fixed airways obstruction. Spirometry should be initially conducted at time of hire (baseline test) and should be repeated every 6 months. Newly hired workers with preexisting lung disease should be medically evaluated to determine whether work exposures place them at increased risk for progression of their lung disease. Workers with posthire onset of fixed airways obstruction or a 15% or greater drop in FEV1 from the baseline value should be: (1) referred to a pulmonologist for medical evaluation; (2) relocated to prevent further exposures; and (3) allowed to return to their previous job only if their physician does not find fixed airways obstruction or 15% or greater decline in FEV1, or engineering controls are implemented at the worksite to control exposures and there is physician oversight. In workplaces with a case of work-related fixed airways obstruction, coworkers should have spirometry testing more frequently, such as every 3 months. Quality spirometry is required both to minimize the number of workers who are inappropriately referred to pulmonologists for evaluation and to identify workers who may have decreased lung function compared to their baseline spirometry test. Poor coaching by the spirometry technician may
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result in a poor effort by the worker being tested, which may be reflected by inaccurately low lung volume results. If there is poor effort for an interval spirometry test, the worker may be needlessly referred to a pulmonologist for a medical evaluation; if there is poor effort for a baseline test, a subsequent interval spirometry test may not detect a large drop in FEV1 when it exists. If a large decline in FEV1 is detected, prompt removal of affected workers may help maintain their lung function tests within the normal range and prevent disability. Spirometry tests from on-site and off-site testing locations frequently are of poor quality and do not meet quality criteria of the American Thoracic Society (Miller et al., 2005). Initial and refresher training of spirometry technicians, periodic quality assurance evaluation of spirometry tests completed by individual spirometry technicians, and quality spirometry testing equipment help to insure good quality tests.
C. Timeline of the emergence of flavoring-related BO and the industry and regulatory agency response BO in microwave popcorn workers established a newly identified cause of occupational lung disease. However, the problem had been present several decades earlier when two workers in a company that made flavored cornstarch and flour mixes were reported to have BO (CDC, 1986). The workers were nonsmokers in their twenties who developed progressive shortness of breath and severe fixed airways obstruction within 5–8 months of hire. Two former mixers in the same company aged 36 and 38 who were exsmokers (22 and 10–15 pack-years, respectively) and who did not have symptoms were identified to have moderate airways obstruction, likely due to exposure to flavorings, given their age and smoking histories. At the time, an association between butterflavoring exposures and BO was not made, although one of the workers had attributed the symptoms to Cinna Butter, and diacetyl was one of the common ingredients used in the plant. Other workers with flavoring-related BO were not appropriately diagnosed, even after reports of flavoring-related BO were published in the scientific literature (Kreiss et al., 2002a; Lockey et al., 2002; Parmet and von Essen, 2002), public health communications (CDC, 2002), and the press. Frequent misdiagnoses by physicians included asthma, bronchitis, and emphysema due to a presumptive diagnosis, an incomplete medical evaluation, and/or failure to make a connection with occupational exposures. In 2002, 14 cases of flavoring-related BO among microwave popcorn workers and flavor-manufacturing workers were reported (CDC, 2002; Kreiss et al., 2002a; Lockey et al., 2002; Parmet and von Essen, 2002). The NIOSH Alert on flavoring-related lung disease was disseminated in 2004 to flavor and food manufacturers and regional OSHA offices (CDC, 2004).
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In that same year, the Flavor and Extract Manufacturers Association (FEMA) disseminated to its member companies a list of 34 high-priority chemicals and 49 low-priority chemicals used in the flavoring industry that were suspected to be respiratory hazards (FEMA, 2004). Exposure control and medical surveillance of workers with spirometry testing were recommended in both the NIOSH and FEMA communications. From 2006 to 2007 additional cases of flavoring-related lung disease were reported in microwave popcorn-manufacturing workers (Kanwal et al., 2006), flavormanufacturing workers (CDC, 2007d), diacetyl-manufacturing workers (van Rooy et al., 2007), and workers who popped corn and coated the hot popcorn with powdered cheese and jalapeno flavorings (CDC, 2007a). In July 2006, labor unions petitioned OSHA for an emergency temporary standard for diacetyl. In July 2007, OSHA began a National Emphasis Program to address the hazards and control measures associated with working in the microwave popcorn industry. Under this program, OSHA prioritized inspections to this industry. In September 2007, due to congressional input and renewed pressure by labor unions, OSHA began formal rulemaking for occupational exposure to diacetyl and food flavorings containing diacetyl. Formal rulemaking usually requires several years to allow for a thorough review of the scientific literature, assessment of the economic impact of a regulation, and stakeholder input. Frequently, OSHA decides that no regulation should be established. Consumer concerns about food product safety increased in September 2007 when information of a possible case of flavoring-related BO in a patient who had daily consumed two or more bags of extra-butterflavored microwave popcorn for 10 years was released to the press (Harris, 2007). A single case of disease is insufficient to make a causal association between disease and exposure if the association cannot be tested by other means. Because no other cases of BO among microwave popcorn consumers have been reported in the medical literature or to government agencies, the risk to consumers is unclear. However, the perceived risk by consumers motivated flavor-manufacturing companies to reformulate artificial butter flavoring used in microwave popcorn. Two possible substitutes for diacetyl are starter distillate and diacetyl trimer. Starter distillate is a diacetyl-containing product of a fermentation process. Diacetyl trimer is a molecule that contains three diacetyl molecules. The inclusion of these alternative substances neither eliminates diacetyl nor assures safety for workers.
D. Liability Workers’ compensation benefits pay workers for medical expenses and lost wages due to occupational injury or illness. In exchange for carrying workers’ compensation insurance, companies are protected against legal
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suits by their employees. Workers with flavoring-related BO have pursued workers’ compensation from their employers and have been involved in third-party litigation against flavor-manufacturing companies (frequently in the form of class action suits).
VI. RECOGNITION OF EMERGING OCCUPATIONAL RESPIRATORY DISEASE IN THE FOOD INDUSTRY There are a number of barriers to the recognition of currently unknown causes of occupational respiratory disease in the food industry. Food additives, including diacetyl, are classified by the US Food and Drug Administration as ‘‘generally recognized as safe’’ (GRAS). This classification is based on published studies of safety by consumption or on a substantial history of consumption by a significant number of consumers. The GRAS classification does not address safety of inhalation exposures to workers in the food industry and may give employers and workers a false assurance of safety. Physicians frequently do not explore potential causative agents when evaluating individual patients suffering from a common lung disease, such as asthma. Rarer lung diseases, such as BO, may be misdiagnosed as a common disease, such as asthma. The lack of work-related symptoms in some occupational respiratory diseases (such as HP due to low-intensity exposures, emphysema, and BO) can delay recognition of an occupational cause. Additionally, physicians may attribute lung disease in smokers to smoking even at ages younger than middle age when smoking-related obstruction is improbable. If a new cause of occupational respiratory disease is suspected, the most appropriate action for employers, employees, or physicians is to contact public health agencies, such as local or state health departments or NIOSH. Public health agencies can utilize teams of medical staff, industrial hygienists, and epidemiologists to investigate the potential problem. Such multidisciplinary investigations can assess exposure, test for health effects, describe process-related risk factors, define exposure–response relationships, and make recommendations to control exposures. Referral to OSHA is generally not productive as this is a regulatory agency, and compliance officers address exposure levels of known regulated chemicals and substances. Biological plausibility of potential causative agents can be established through animal models in industry-funded investigations, research institutions, or federal agencies. Effectiveness of control interventions can be evaluated through longitudinal follow-up of exposed worker populations.
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The extent of flavoring-related lung disease in food production is an emerging endeavor outside of microwave popcorn- and flavoringmanufacture industries. Butter flavorings are used in snack foods, baked goods, candies, and dairy products. In December 2007, the media raised concern about diacetyl risks in food service workers using butterflavored fats on grills (Schneider, 2007). Much more work needs to be done to characterize the exposures and risks associated with working in these food production and food service industries.
VII. PREVENTION OF KNOWN OCCUPATIONAL RESPIRATORY DISEASES IN THE FOOD INDUSTRY Exposure to hazardous agents can be minimized through enclosure of work processes, use of lids on containers of mixing vessels, use of local exhaust ventilation, mandatory use of respirators, and eyes and skin protection. Emphasis should be placed on engineering controls to reduce exposure rather than reliance on respiratory protection. Multiple problems are inherent with respirator use. These include inadequate fit, respirator malfunction, inappropriate use, and failure of workers to consistently use respirators whenever exposed. For volatile exposures, a NIOSH-certified full-facepiece, negative-pressure respirator with organic vapor cartridges is the minimum level of respiratory protection recommended. If there are also particulate exposures, as is the case in powder flavor manufacturing, then particulate filters should be used in tandem with the organic vapor cartridges. A comprehensive respiratory protection program (OSHA, 2007) includes a written program, a program director, initial medical clearance and annual fit-testing, filter and cartridge change-out schedules, training, and seal checks whenever the respirator is used. Prehire and periodic spirometry testing with bronchodilator trial may help to identify new-onset reversible airways obstruction (asthma), as well as fixed airways obstruction and excessive fixed FEV1 decrements (emphysema, BO). Many workers in the food industry are exposed to occupational allergens. For these workers, prehire and periodic testing for antibody sensitization will identify workers who may go on to develop allergic respiratory diseases, including asthma. Periodic symptom questionnaires and reporting of respiratory symptoms to the director of safety will target individuals who would benefit from further medical evaluation. For workers with occupational allergic rhinitis, decreased exposures and frequent medical follow-up may have utility in preventing progression to allergic asthma. Finally, if an occupational respiratory disease is diagnosed, avoidance of further exposure is prudent.
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Rodriguez, A., de la Cuesta, C. G., Olaguibel, M. J., Tabar, A., and Santos, F. (1990). Occupational asthma due to inhaled carminic acid dye: Case report. Clin. Exp. Allergy 20(Suppl. 1), 43. Rossi, G. L., Corsico, A., and Moscato, G. (1994). Occupational asthma cause by milk proteins: Report on a case. J. Allergy Clin. Immunol. 93, 799–801. Sastre, J., Olmo, M., Novalvos, A., Iban˜ez, D., and Lahoz, C. (1996). Occupational asthma due to different spices. Allergy 51, 117–120. Schneider, A. (2007). Flavoring additive puts professional cooks at risk. Seattle PostIntelligencer. December 21, 2007. Schuller, A., Morisset, M., Maadi, F., Kolopp Sarda, M. N., Fremont, S., Parisot, L., Kanny, G., and Moneret-Vautrin, D. A. (2005). Occupational asthma due to allergy to spinach powder in a pasta factory. Allergy 60, 408–409. Schuyler, M. (1998). Hypersensitivity pneumonitis. In ‘‘Fishman’s Pulmonary Diseases and Disorders’’ (A. P. Fishman, J. A. Elias, J. A. Fishman, M. A. Grippi, L. R. Kaiser, and R. M. Senior, eds), 3rd ed., pp. 1085–1097. McGraw-Hill, New York. Sen, D., Wiley, K., and Williams, J. G. (1998). Occupational asthma in fruit salad processing. Clin. Exp. Allergy 28, 363–367. Seuri, M., Taivanen, A., Ruoppi, P., and Tukiainen, H. (1993). Three cases of occupational asthma and rhinitis caused by garlic. Clin. Exp. Allergy 23, 1011–1014. Shirai, T., Reshad, K., Yoshitomi, A., Chida, K., Nakamura, H., and Taniguchi, M. (2003). Green tea-induced asthma: Relationship between immunological reactivity, specific and non-specific bronchial responsiveness. Clin. Exp. Allergy 33, 1252–1255. Simpson, E. G., Belfield, P. W., and Cooke, N. J. (1985). Chronic airflow limitation after inhalation of overheated cooking oil fumes. Postgrad. Med. J. 61, 1001–1002. Smith, A. B., Bernstein, D. I., Tar-Ching, A. W., Gallagher, J. S., London, M., Koff, S., and Carson, G. A. (1987). Occupational asthma from inhaled egg protein. Am. J. Ind. Med. 12, 205–218. Tabar-Purroy, A. I., Alcarez-Puebla, M. J., Acero-Sainz, S., Garcia-Figueroa, B. E., EchechipiaMadoz, S., Olaguibel-Rivera, J. M., and Quirce-Gancedo, S. (2003). Carmine (E-120)induced occupational asthma revisited. J. Allergy Clin. Immunol. 111, 415–419. Tarvainen, K., Kanerva, L., Tupasela, O., Grenquist-Norde´n, B., Jolanki, R., Estlander, T., and Keskinen, H. (1991). Allergy from cellulose and xylanase enzymes. Clin. Exp. Allergy 21, 609–615. Toskala, E., Piipari, R., Aalto-Korte, K., Tuppurainen, M., Kuuliala, O., and Keskinen, H. (2004). Occupational asthma and rhinitis cause by milk proteins. J. Occup. Environ. Med. 46, 1100–1101. van Rooy, F. G. B. J., Rooyackers, J. M., Prokop, M., Houba, R., Smit, L. A. M., and Heederik, D. J. J. (2007). Bronchiolitis obliterans syndrome in chemical workers producing diacetyl for food flavorings. Am. J. Respir. Crit. Care Med. 176, 498–504. Vandenplas, O., Depelchin, S., Toussaint, G., Delwiche, J. P., Vande Weyer, R., and SaintRemy, J. M. (1996). Occupational asthma caused by sarsaparilla root dust. J. Allergy Clin. Immunol. 97, 1416–1418. Vandenplas, O., Vander Borght, T., and Delwiche, J. P. (1998). Occupational asthma caused by sunflower-seed dust. Allergy 53, 907–908. Zock, J. P., Hollander, A., Heederik, D., and Douwes, J. (1998). Acute lung function changes and low endotoxin exposures in the potato processing industry. Am. J. Ind. Med. 33, 384–391. Zock, J. P., Sunyer, J., Kogevinas, M., Kromhout, H., Burney, P., Anto´, J. M., and E.C.R.H.S. study group (2001). Occupation, chronic bronchitis, and lung function in young adults. Am. J. Respir. Crit. Care Med. 163, 1572–1577. Zuskin, E., Valic, F., and Kanceljak, B. (1981). Immunological and respiratory changes in coffee workers. Thorax 36, 9–13.
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CHAPTER
4 Beneficial Health Properties of Psyllium and Approaches to Improve Its Functionalities Liangli (Lucy) Yu,* Herman Lutterodt,* and Zhihong Cheng*
Contents
Abstract
I. Introduction II. Beneficial Health Effects of Psyllium A. Hypolipidemic effects B. Reducing hyperglycemia C. Cancer prevention D. Laxative effect E. Possible effects on gastrointestinal system F. Possible adverse effects III. Approaches to Improve the Functionality, Safety, and Biological Activity of Psyllium A. Physical and mechanical approaches B. Conventional enzymatic approaches C. Solid-state enzymatic procedures D. Chemical modification of psyllium References
194 195 195 201 202 203 203 204 204 204 206 207 214 215
Psyllium is an excellent dietary source for both soluble and insoluble fibers and has been used in supplemental and food products for its beneficial health effects. The strong water-absorbing and gelling capacities have made it a great challenge to incorporate psyllium in foods at the level needed to claim health benefits on the label. This review is focused on the approaches to improve the functionality,
* Department of Nutrition and Food Science, University of Maryland, College Park, Maryland 20742
Advances in Food and Nutrition Research, Volume 55 ISSN 1043-4526, DOI: 10.1016/S1043-4526(08)00404-X
#
2009 Elsevier Inc. All rights reserved.
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sensory property, and bioactivity of psyllium. Also included is a brief summary of the health beneficial effects of psyllium, along with its possible adverse effects. The information may be useful for those in psyllium research and functional food development. Key Words: Psyllium, Cholesterol lowering, Solid-state enzymatic reaction, Functionality. ß 2009 Elsevier Inc.
I. INTRODUCTION Psyllium is a mucilaginous material prepared from the seed husk of the plants of the Plantago genus including but not limited to P. ovata, P. psyllium, and P. indica, which grow in certain subtropical regions. Psyllium is a highly branched acidic arabinoxylan (Kennedy et al., 1979). The xylan backbone has both (b1!4) and (b1!3) linkages. Other monosaccharides present in psyllium are D-galactose, D-rhamnose, D-galacturonic acid, 4-O-methyl-D-glucuronic acid, and 2-O-(2-D-galactopyranosyluronic acid)-L-rhamnose (Chan and Wypyszyk, 1988). Psyllium has been investigated for its potential health benefits and its applications in food and other consumer products such as hair-setting lotions and drug delivery systems (Chan and Wypyszyk, 1988; Singh, 2007). Psyllium is well recognized for its laxative activity, cholesterollowering capacity, potential in reducing the risk of colon cancer and hyperglycemia, and possible application in the treatment of irritable bowel syndrome and in body weight control (Anderson et al., 1990; Arjmandi et al., 1992; Bijkerk et al., 2004; Ganji and Kies, 1994; Hannan et al., 2006; Hara et al., 1996; Kang et al., 2007; Marlett et al., 2000; Park et al., 1997; Pittler and Ernst, 2004). In addition to its beneficial health effects, psyllium also has functional contributions in foods or other consumer products. For instance, psyllium may be used as a deflocculant in paper and textile manufacture, an emulsifying agent, a binder or lubricant in meat products, and a carbohydrate-based fat replacer to be used in low fat/low total calorie foods (Haque and Morris, 1994). However, the strong hydrophilic and gelling properties of psyllium make it a real challenge to incorporate psyllium in food/beverage formula at the level required to have a health claim on the label. A substantial amount of time is required for complete dispersal of psyllium in an aqueous system containing other ingredients including sugar even with vigorous agitation due to its water-absorbing and gelling capacities (Rudin, 1985). An unpleasant slimy mouth feeling is also related to these properties. Beverages are a preferred carrier of nutraceuticals. Adding a sufficient amount of psyllium into a beverage formula is impossible
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due to its strong gel-forming capacity. Many previous studies have been conducted to improve its functional/biological/sensory properties to enhance its food and nonfood applications (Kumar and Verma, 2007; Yu and Perret, 2003b). This review summarizes the beneficial health properties of psyllium, and the approaches to improve its physicochemical and biological properties.
II. BENEFICIAL HEALTH EFFECTS OF PSYLLIUM A. Hypolipidemic effects The Division for Heart Disease and Stroke Prevention (DHDSP) of the Centers for Disease Control (CDC) classifies coronary heart disease (CHD) as the leading cause of death in the United States, and a major cause of disability. It costs billions of dollars to deal with this public health concern (Anderson et al., 1990). Several factors influence the incidence of CHD. High blood cholesterol, or hypercholesterolemia, which is categorized as serum cholesterol levels above 240 mg/dl, has been conclusively linked to increased risk of CHD (Anderson et al., 1988; Gupta et al., 1994). Cholesterol causes plaque formation which narrows the blood vessels, and increases the risk of heart attacks. Serum concentrations of lowdensity lipoprotein (LDL), high-density lipoprotein (HDL), and triacylglycerols (TG) are associated with the risk of CHD. Psyllium has been found to lower blood lipid levels and the risk of CHD in a number of previous studies (Anderson et al., 1988, 2000a,b; Everson et al., 1992; Ganji and Kies, 1996; Gupta et al., 1994; Olson et al., 1997; Pastors et al., 1991; Roe et al., 1988; Sola et al., 2007; Sprecher et al., 1993; Turley et al., 1994). A large number of in vivo studies using rats demonstrate the efficacy of the lipid-lowering activity of psyllium compared against different dietary fibers. Anderson et al. (1994) observed that feeding rats with a diet rich in soluble fiber significantly reduced their serum and liver cholesterol concentrations, compared to an insoluble fiber diet. They fed the rats with 10 different fibers, including psyllium, pectin, and guar gum. Cellulose, which has no significant hypocholesterolemic effect, was used as the control fiber. Test diets contained 60 g dietary fiber/kg diet, with 10 g cholesterol and 2 g cholic acid/kg diet. At this level of intake, over a period of three weeks, psyllium had the most impact on serum lipid levels, lowering serum and liver cholesterol concentrations by 34% and 53%, respectively, compared to the cellulose control. In another study, psyllium significantly reduced plasma and liver cholesterol concentrations in female rats fed a cholesterol-enriched diet (Terpstra et al., 2000a). This reduced cholesterol level was accounted for by an observed 26% increase in fecal excretion of bile acids, primarily b-muricholic acid.
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The results were based on a 3 g psyllium/100 g diet. Rats fed 10% psyllium diet (with 0.5% cholesterol) were found to have lower serum cholesterol levels, as well as higher HDL-cholesterol levels when compared with those fed with an equal amount of cellulose (Kritchevsky et al., 1995). The mechanisms involved in the cholesterol-modulating effect of psyllium fiber have been explored by various studies. Cholesterol 7a-hydroxylase is the key regulatory enzyme in the synthesis of bile acids, converting cholesterol to bile acids. The activity of this important rate-determining enzyme has been observed to increase in a dose-dependent manner when a psyllium-supplemented diet is fed to rats (Buhman et al., 2000; Matheson et al., 1995). The liver makes bile acids from cholesterol. Increased bile acid synthesis therefore implies increased removal of cholesterol from circulation, resulting in decreased liver and serum cholesterol levels. In Matheson et al. (1995), after 28 days of feeding the rats with a 5% psyllium supplemented diet, the activity of cholesterol 7a-hydroxylase increased twofold compared to the cellulose control at the same level of supplementation. Psyllium also reduced liver total cholesterol concentrations when cholesterol was added to the diet. Buhman et al. (2000) went further to explain from their results that the increase in the bile acid pool was achieved by the regulation of not only cholesterol 7a-hydroxylase, but also involved the regulation of ileal apical sodium-dependent bile acid transporter (ASBT) and 3-hydroxyl-3-methylglutaryl (HMG) CoA reductase mRNA levels. The antinutritional properties of dietary fiber are a concern, and some fibers have actually demonstrated this undesirable effect, both in human and animal studies. Results from the study by Perez-Olleros et al. (1999) showed no effect on macronutrient utilization by a 100 g psyllium fiber/kg diet in rats. They however observed a reduction in protein utilization compared to the cellulose control. Several hamster feeding studies indicate that psyllium reduces plasma cholesterol concentrations by primarily increasing fecal bile acid excretion (Terpstra et al., 2000b; Trautwein et al., 1998, 1999). Both Terpstra et al. (2000b) and Trautwein et al. (1998) attributed the observed increase in fecal bile acid excretion to an increase in the ratio of the secondary bile acid deoxycholic acid to lithocholic acid. Trautwein et al. (1999) observed that in addition to lowering plasma concentrations of cholesterol, psyllium also reduced plasma levels of TG to an extent similar to that induced by cholestyramine, an effective pharmacological intervention in the regulation of cholesterol levels. Psyllium has been observed to enhance the cholesterol-lowering activity of cholestyramine in hamsters (Daggy et al., 1997; Turley et al.,1994). Combining psyllium and cholestyramine in a diet (5% psyllium and 0.5% cholestyramine) increased total bile acid excretion by as much as 79% (Daggy et al., 1997). Turley et al. (1994) observed a dose-dependent increase in sterol loss in a combination of psyllium with a low dose of cholestyramine. It appears from the data reviewed that
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psyllium, in hamsters, exerts a greater hypocholesterolemic effect on liver cholesterol concentrations than serum cholesterol levels (Table 4.1). The hypocholesterolemic effects of psyllium in guinea pigs are similar to the other animals already discussed. An increase in fecal bile acids of as much as threefold was observed when guinea pigs were fed with a diet containing at most 10 g psyllium/100 g diet, compared to the cellulose control (Romero et al., 2002). Plasma triglycerides and LDL cholesterol were also 34% and 23% lower in the same study. Lecithin cholesterol acyltransferase (LCAT) and cholesterol ester transfer protein (CETP) activities were significantly affected. Fernandez et al. (1995) suggested from their results that psyllium lowered plasma cholesterol levels by inducing cholesterol 7a-hydroxylase and HMG CoA reductase, and suppressing acyl CoA cholesterol acyltransferase (ACAT) activities. Upregulation of apolipoproteins B and E receptors was also observed to be influenced by psyllium intake, contributing to the lower plasma cholesterol levels. Increased secretion of apolipoprotein B and upregulation of LDL receptors might also contribute to the reduced plasma cholesterol concentrations induced by psyllium (Fernandez et al., 1997). Some studies suggest that the metabolic response to the cholesterol-lowering effect of psyllium in guinea pigs may be gender dependent (Fernandez et al., 1995; Roy et al., 2000). Under low cholesterol intake conditions, both male and female guinea pigs exhibited similar responses in their plasma cholesterol concentrations to dietary psyllium supplementation (Fernandez et al., 1995). However, under stressed hormonal conditions (as induced in this study by ovariectomization to mimic menopause in the female subjects), the psyllium diet exhibited a diminished hypocholesterolemic effect (Roy et al., 2000). In addition to lowering cholesterol and other blood lipid concentrations, psyllium has been shown to exhibit a potential antioxidant effect (Vergara-Jimenez et al., 1999). Psyllium has been evaluated in human subjects for its effectiveness in the control of mild to moderate hyperlipidemia. In pilot human studies, it lowered serum cholesterol levels by 5–20% with daily psyllium doses from 3.5 to 24 g (Gupta et al., 1994). The study conducted by Gupta et al. (1994) showed that two daily doses of 3.5 g psyllium significantly reduced total serum cholesterol (19.7%), LDL-cholesterol (23.7%), and triglycerides (27.2%) in patients with non-insulin-dependent diabetes mellitus (NIDDM). Everson et al. (1992) found psyllium to lower LDL-cholesterol in 50% of hypercholesterolemic men. Their study also concluded that the cholesterol-lowering effect of psyllium was due primarily to its stimulation of bile acid synthesis. This might be achieved by promoting the regulatory enzyme that catalyzes the conversion of cholesterol to bile acids. Some studies have examined the hypocholesterolemic effect of psyllium when it was used as an adjunct to diet therapy in controlling hyperlipidemia (Anderson et al., 2000a; Bell et al., 1990; Gupta et al., 1994;
TABLE 4.1
Effect of dietary psyllium on plasma and liver cholesterol concentrations in hamstersa
Number Cholesterol per Days in diet Psyllium Fat in group on diet (%) in diet (%) diet (%)
Plasma cholesterol (mmol/l)
Liver cholesterol (mmol/g)
Decrease (%)
Decrease (%) References
8
21
0.2
5
10
Cellulose Psyllium 8.84 6.52 26
Cellulose Psyllium 42.9 15.8 63
10
28
2
20
NR
6.81
2.83
58
ND
ND
–
12
35
0.4
5
5
9.72
6.69
31
107.3
114.0
–6
10
35
0.12
8
20
5.54
2.96
47
92.8
10.5
89
16
28
0.1
7.5
10
10.30
3.15
69
42.9
6.7
84
8/10
17
0
7.5
4.6
3.20
2.11
34
6.5
5.2
19
8
18
0.1
7.5
10
8.61
4.11
52
20.0
6.2
69
14
56
0.1
3
10
5.72
4.21
26
25.6
15.0
41
ND, not determined; NR, not reported. Table adapted from Terpstra et al. (2000b). a Statistical significance was P<0.05 in all studies except Trautwein et al. (1993).
Daggy et al. (1997) LengPeschlow (1993) Trautwein et al. (1993) Trautwein et al. (1998) Turley et al. (1991) Turley et al. (1994) Turley and Dietschy (1995) Terpstra et al. (1998)
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Olson et al., 1997; Wolever et al., 1994). Wolever et al. (1994) concluded that psyllium mixed with foods had a greater influence on serum cholesterol levels than when it was taken alone. Considering that one of the proposed mechanisms of psyllium action might be interfering with lipid absorption (Anderson et al., 1990; Wolever et al., 1994), it makes sense that it has an increased effect on total cholesterol levels when taken with food. When a psyllium-enriched cereal was administered together with a low-fat diet in hypercholesterolemic adults, the combination resulted in an improved blood lipid profile (Olson et al., 1997). Stoy et al. (1993) recorded modest increases in the total cholesterol and LDL-cholesterol-lowering effect of a standard low-fat, low-cholesterol diet, when the diet was administered together with a ready-to-eat psyllium cereal. In another study, a psylliumenriched cereal, as part of a step-1 diet, reduced total cholesterol and LDLcholesterol by an additional 5.9% and 5.7%, respectively, compared to the 3.8% reduction by the step-1 diet alone in patients with mild to moderate hypercholesterolemia (Bell et al., 1990). The step-1 diet is the introductory diet recommended by the National Heart, Lung, and Blood Institute’s National Cholesterol Education Program (NCEP) for patients with high cholesterol. The step-1 diet contains not more than 30% fat (as a percentage of total calories), with no more than 10% as saturated fat, and less than 300 mg/day cholesterol (American Heart Association, http://www. americanheart.org/presenter.jhtml?identifier¼4764). A study in hypercholesterolemic children revealed a 7% reduction in LDL-cholesterol concentrations when subjects on a low-fat diet were fed a psyllium-enriched cereal (Davidson et al., 1996). The hypocholesterolemic effect of psyllium has been found to persist in long-term use. In a 26-week, placebocontrolled study, subjects with hypercholesterolemia were treated with 5.1 g psyllium two times a day, and showed 4.7% and 6.7% reductions in serum total cholesterol and LDL-cholesterol concentrations, respectively (Anderson et al., 2000b). In another long-term study, a 5.3% reduction in LDL-cholesterol, effected by treating hypercholesterolemic patients with 10.2 g psyllium seed husk, persisted throughout the 24-week treatment period (Davidson et al., 1998). In summary, all these data suggest, conclusively, that psyllium does lower serum and liver cholesterol concentrations, and may increase HDLcholesterol levels. Psyllium appears to have a greater influence on LDL-cholesterol levels than on total serum cholesterol and triglyceride concentrations (Table 4.2). The significance of this hypocholesterolemic behavior in reducing the risk of CHD is still being debated. A metaanalysis conducted by Brown et al. (1999) concludes that, within the practical range of intake (3 g), dietary fiber, including psyllium, lowers total and LDL-cholesterol by 0.13 mmol/l, which they considered only a small contribution in the dietary intervention against hypercholesterolemia. The precise mechanisms involved are still under investigation.
TABLE 4.2
Hypocholesterolemic effect of psyllium in humans
Number of subjects
26 24 37
Status of subjects
Type of diet
Mild to moderate hypercholesterolemia NIDDMa with hyperlipidemia Primary hypercholesterolemia
Normal (<300 mg cholesterol)
Psyllium (g/day)
Decrease (%)
References
10.2
TC 14.8
LDL-C 20.2
TG 12.7
7
19.7
23.7
27.2
High fat
10.2
5.8
7.2
NR
81 28
CVDb
Low fat Low saturated fat
10.2 10.5
4.2 3.76
6.4 6.9
2.79
248
Hypercholesterolemia
Step 1 diet
10.2
4.7
6.7
NR
NR, not reported; TC, total cholesterol; LDL-C, LDL cholesterol; TG, triglycerides. a Non-insulin-dependent diabetes mellitus. b Cardiovascular diseases.
Anderson et al. (1988) Gupta et al. (1994) Sprecher et al. (1993) Sola et al. (2007) Anderson et al. (2000b)
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Some researches suggest that psyllium binds to and reduces the reabsorption of bile acids in the small intestine in a fashion similar to bile acidbinding resins like cholestyramine, increasing its excretion (Everson et al., 1992; Turley et al., 1994; Wolever et al., 1994). Another mechanism proposed is that psyllium fiber reduces fat absorption by interfering with digestive enzyme activity and/or altering the absorptive surface of the small intestine (Anderson et al., 1990). Reduced rate of carbohydrate absorption (Wolever et al., 1994), interference in hepatic cholesterol metabolism by short chain fatty acids (SCFAs) produced by bacterial fermentation of psyllium in the colon (Wolever et al., 1994), and influencing hormonal (insulin and glucagon) control of lipid metabolism (Anderson et al., 1990) are some other mechanisms that explain the CHD risk-reducing effect of psyllium. These mechanisms are however not necessarily mutually exclusive (Wolever et al., 1994).
B. Reducing hyperglycemia Water-soluble fibers are well known for their potential in moderating postprandial glucose and insulin concentrations in non-insulin-dependent diabetic patients if taken with meals (Hannan et al., 2006; Pastors et al., 1991). Psyllium is traditionally used in India for treatment of diabetes and has been evaluated in animal models and human pilot studies for its effectiveness in reducing hyperglycemia and the possible mechanisms involved in this beneficial activity (Anderson et al., 1999; Hannan et al., 2006; Song et al., 2000). A recent rat feeding study (Hannan et al., 2006) evaluated the hot-water extractable components of P. ovata for their potential in reducing hyperglycemia at a daily dose level of 0.5 g/kg body weight/day. It was found out that the psyllium preparation was able to significantly inhibit the rise of blood glucose level induced by the oral intake of glucose or sucrose, with no alteration in fasting levels of blood glucose and insulin status. The administration of psyllium also reduced sucrose absorption in the gastrointestinal tract, decreased intestinal glucose absorption during the 30 min of perfusion, and increased gastrointestinal motility, while it had no effect on disaccharidase activity. It was concluded that the psyllium preparation may reduce hyperglycemia by suppressing intestinal glucose absorption. In an earlier study, psyllium was able to enhance blood glucose disposal or improve insulin sensitivity by significantly increasing the skeletal muscle plasma membrane GLUT-4 protein expression without phosphatidylinositol 3 (PI3)kinase activation in stroke-prone spontaneously hypertensive rats (Song et al., 2000). Insulin enhances glucose uptake into skeletal muscle by transporting GLUT-4, a glucose transporter, from the intracellular membrane to the plasma membrane through PI3-kinase activation.
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A number of human pilot studies indicated the potential for application of psyllium in improving postprandial glycemic index and insulin sensitivity (Anderson et al., 1995 , 1999; Sierra et al., 2002; Ziai et al., 2005). For instance, the addition of psyllium in the diet was shown to reduce postprandial serum glucose and insulin concentrations in hypercholesterolemic men (Anderson et al., 1995), and in type-2 diabetic human subjects (Pastors et al., 1991; Sierra et al., 2002). In 1999, Anderson and others reported that psyllium at a level of 5.1 g/day and twice a week for 8 weeks resulted in 11% and 19% reduction of all-day and postprandial glucose concentrations after lunch in men with type-2 diabetes (Anderson et al., 1999). This observation was supported by a later study in which psyllium intake significantly decreased fasting plasma glucose (Ziai et al., 2005). It needs to be pointed out that conflicting results were obtained for the possible residual effect of psyllium intake after the second meal in type-2 diabetic human subjects (Anderson et al., 1999; Clark et al., 2006; Pastors et al., 1991). In addition, these previous studies suggest that addition of psyllium to a conventional diet is a safe approach for improving the glycemic index (Anderson et al., 1999; Ziai et al., 2005), and may not adversely affect the bioavailability of dietary minerals and vitamins (Sierra et al., 2002).
C. Cancer prevention Psyllium has been implicated in the prevention of cancer, particularly colon and breast cancers (Morita et al., 1999; Nakamura et al., 2004, 2005). The mechanism of this observation is not clearly understood, but various theories have been put forward. Psyllium intervention in colon cancer is thought to be due to its fermentation in the distal colon by the in vivo bacterial flora, converting this soluble fiber into SCFAs (Morita et al., 1999). These SCFAs include acetate, propionate, and n-butyrate. Research by Morita et al. (1999) suggested that psyllium might slow down the fermentation of high amylase corn starch, making it possible for fermentation of the starch to take place in the distal colon, which is the common site of colon cancer. The presence of SCFA, specifically n-butyrate, has been associated with slowing the reduction of proliferation of cancer cells and induction of differentiation of mucosa cells. Five different combinations of psyllium and wheat bran, with comparative levels of wheat bran to psyllium of 12% and 0%, 8% and 2%, 6% and 3%, 4% and 4%, and 0% and 6%, were evaluated and compared for their effects on chemically induced mammary tumorigenesis in F344 rats (Cohen et al., 1996). After 19 weeks on the treatment diets, rats on 1:1 (wheat bran/psyllium) had the lowest rate of mammary tumorigenesis, and rats on the other combined fiber diets or on the psyllium-alone diet had an intermediate rate of tumorigenesis. Rats on a diet with higher level
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of psyllium had lower fecal estrogen excretion, although rats on different diets had no differences in their circulating estrogens or urinary estrogen excretions. In addition, suppression of bacterial b-D glucuronidase activity was observed with psyllium intake (Cohen et al., 1996). The relationship between bacterial b-D glucuronidase activity and mammary tumor development was not clear, and other phytochemicals such as phytates, isoflavonoids, and protease inhibitors may contribute to the overall anticarcinogenesis activity.
D. Laxative effect Psyllium fiber has long been used as a laxative. It absorbs water in the large intestine and swells, increasing fecal bulk (http://www.hort. purdue.edu/newcrop/afcm/psyllium.html). It also increases bowel movement by stimulating contraction of the colon walls. In a study involving 15 healthy adults, it was found out that a component of psyllium, unlike other viscous fibers, was not fermented in the colon (Marlett et al., 2000). It formed a gel that acted as a lubricant, facilitating propulsion of colon contents. This resulted in bulkier stools with higher moisture content. The results of this study were based on 15 g/day of psyllium husk. Another study examined the laxative effect of psyllium in subjects suffering from chronic idiopathic constipation (McRorie et al., 1998). The subjects were treated with psyllium and docusate for 2 weeks following a 2-week placebo control phase. At the end of the study, psyllium was found to have a more efficacious laxative effect than the docusate, increasing stool water content significantly over the period of the study. The stool softening effect was observed to increase as the study progressed, suggesting that continued use of psyllium may increase its laxative effect. The study showed that psyllium was a better stool softener than docusate in patients suffering from idiopathic constipation.
E. Possible effects on gastrointestinal system Psyllium has been investigated for its possible influence on the gastrointestinal system (Bijkerk et al., 2004; Cavaliere et al., 2001; Satchithanandam et al., 1996). In 2004, Bijkerk and others reviewed the available information and concluded that psyllium might play a role in treatment of irritable bowel syndrome and irritable bowel syndrome-related constipation (Bijkerk et al., 2004). They also suggested performing additional clinical studies for investigating the effect and tolerability of psyllium use in primary care. It was believed that the beneficial effects of psyllium in treatment of irritable bowel syndrome were associated with its anticonstipation activity. Interestingly, psyllium has also been shown to slow down the gastric emptying time and colon transit, which may benefit the
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consumers with diarrhea and pain (Washington et al., 1998) and those using pancreatic lipase inhibitor for its antiobesity effect (Cavaliere et al., 2001). Diarrhea and abdominal cramping were reported side effects for orlistat, a pancreatic lipase inhibitor for treatment of obesity.
F. Possible adverse effects Many previous studies detected the possible adverse effects of psyllium intake, although others demonstrated that psyllium is generally safe for human consumption. Psyllium may alter nutrient and drug absorption, reduce food intake or appetite suppression, decrease caloric availability, increase bloating and flatulence, cause abdominal pain, and elicit anaphylactic symptoms (Lantner et al., 1990; Roe et al., 1988; Stevens et al., 1987). The allergic symptoms of psyllium exposure including oral intake has been reviewed and discussed briefly by Lantner et al. (1990). The effects of psyllium intake on mineral absorption have been evaluated in a number of previous studies (Asvarujanon et al., 2004; Heaney and Weaver, 1995; Luccia and Kunkel, 2002a,b). In 2002, psyllium was shown to reduce calcium bioavailability and induce undesirable changes in bone composition in weanling Wistar rats (Luccia and Kunkel, 2002b), though an in vitro study showed that psyllium had no binding of exogenous calcium (Luccia and Kunkel, 2002a). This observation was in contrast to that observed in an earlier human study, which concluded that a commercial form of psyllium preparation at typical therapeutic levels had little practical effect on the availability of co-ingested calcium (Heaney and Weaver, 1995). It was also reported that the viscosity and fermentability of psyllium might be associated with its capacity in suppressing mineral absorption. A reduction in viscosity and fermentability may decrease its inhibitory effects on calcium, magnesium, and zinc absorption. In addition, psyllium may slow down intestinal gas transition and increase gas production, which is associated with the pronounced gaseous symptoms such as bloating and uncomfortable abdominal distension (Gonlachanvit et al., 2004).
III. APPROACHES TO IMPROVE THE FUNCTIONALITY, SAFETY, AND BIOLOGICAL ACTIVITY OF PSYLLIUM A. Physical and mechanical approaches It has been a continuous effort to improve the physicochemical, functional, sensory, and biological properties of psyllium for promoting its food utilization and enhancing its safety. It is a great challenge to disperse psyllium in water or aqueous solutions even with vigorous agitation
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because of its extremely strong water-absorbing capacity. Several approaches have been investigated for their potential in improving the water dispersibility of psyllium powder. The first approach was to use psyllium with a wide range of particle sizes (Rudin, 1985). However, these psyllium preparations were not sufficiently more dispersible than the commonly used forms with uniform particle sizes. Changes of particle size distribution also could not improve the adverse sensory impact of psyllium in food formulations. Powell and Patel (1982) disclosed that coating psyllium granules with polyvinylpyrrolidone, polyethylene glycol, or their combination may substantially improve its water dispersibility. Addition of polyvinylpyrrolidone may reduce the granule friability. Coating was carried out in an anhydrous solvent system containing ethanol and methanol (Powell and Patel, 1982). Later in 1985, Rudin disclosed that agglomeration of psyllium in water could be prevented or reduced by coating psyllium particles with a food-grade emulsifier (Rudin, 1985). The coating might be accomplished by blending the emulsifier, psyllium, and cereal bran in pure ethanol, followed by evaporation of the solvent in air at ambient temperature. It was proposed that the food-grade emulsifier might consist of a mixture of monoglycerides, diglycerides, sodium stearyl lactylate, hydrophilic ethoxylated sorbitan monoesters, maltodextrin, lecithin, and a combination of them (Rudin, 1985). In 1993, a mechanical procedure was developed to prepare a novel psyllium preparation, psyllium nuggets (Wullschleger et al., 1993). Psyllium was first blended with selected combinations of wheat bran, corn bran, oat bran, different flours, sugar, high fructose corn syrup, gums, salts, and food-grade acids. The resulting blends were subjected to extrusion under certain conditions. The extruded nuggets can be used to make ready-to-eat cereal products with an improved flavor and texture. The nuggets and the ready-to-eat cereal products were reported to retain their cholesterol-lowering activities (Wullschleger et al., 1993). However, the resultant psyllium preparation was not evaluated for its water-absorbing and gelling properties. The resultant psyllium preparations also may not be safe for consumers who have the restricted intake of sugar and salt. Changing the pH or adding one or more food-grade acids with or without a coating could delay the gelling rate of psyllium and improve its dispersibility in liquids (Barbera, 1993, 1995; Barbera and Burns, 1993). It was disclosed that several food-grade acids such as citric, ascorbic, malic, succinic, tartaric, and phosphoric acids, as well as monopotassium phosphate and the mixture of these acids could reduce the gelling capacity of psyllium and improve the sensory properties of the final food formulation containing the psyllium preparation (Barbera, 1993). It was also reported that maltodextrin coating combined with food-grade acid (s) with or without sugar was able to improve the mixability and
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dispersibility of psyllium in liquids (Barbera, 1993, 1995). The food-grade acids could be citric, malic, succinic, ascorbic, tartaric, or phosphoric acids, or monopotassium phosphate and mixture of them at a level not less than 0.5% in a final psyllium preparation (Barbera, 1995). Both maltodextrin and sugar may further alter the behavior of psyllium during food formulation and processing. A physical approach was also applied to reduce the allergenicity of psyllium and psyllium-containing food products (Wullschleger, 1993). Psyllium was subjected to a thermal treatment under certain pressure and moisture content for a predetermined time period to destroy the allergenic proteins. The preferred treatment conditions included a temperature of 245–265 F, pressure range of 14–20 psi, and a time period between 55 and 75 min. This procedure may eliminate up to 100% of the allergenicity of psyllium. In summary, there have been several physical/mechanical means developed to improve the functionality, safety, and sensory properties of psyllium. These previous investigations have indicated the possibility to improve the physicochemical, sensory, biological properties of psyllium for its optimal applications in foods. However, none of them could sufficiently solve the strong gelling and extreme water-uptake problems of psyllium.
B. Conventional enzymatic approaches Enzymatic approaches have been developed to improve the functionality and safety of psyllium (Allen et al., 2004; Nielsen, 1993; Yu, 2003a,b; Yu and Perret, 2003a,b; Yu et al., 2001). Psyllium exposure could lead to asthma, allergic rhinitis, and anaphylaxis (Lantner et al., 1990; Nielsen, 1993). It was noted that the allergenic proteins in psyllium husks were water extractable. An enzymatic procedure was developed to treat psyllium husks with selected proteases in aqueous slurry to eliminate the allergenic protein fractions mainly by hydrolytic reactions (Nielsen, 1993). This procedure did not generate any toxic decomposition products or cause any undesirable changes in functionalities of psyllium. The procedure was easily performed, involving the preparation of the psyllium slurry, addition of the protease, enzymatic reaction at a pH close to the optimal pH of the proteolytic enzyme for a selected time period, and followed by inactivation of the enzyme (Nielsen, 1993). Suitable proteases include but are not limited to trypsin, chymostrypsin, and pronase E at a final concentration ranging from 100 to 2000 IU per liter of slurry at about 37 C. The possible effects of the protease treatment on the functionality of psyllium were not reported in this invention disclosure. Enzymatic procedures were also developed to improve the functionality of psyllium (Yu, 2003a,b; Yu and Perret, 2003a,b; Yu et al., 2001). In 2001,
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it was disclosed that carbohydrase treatment of a psyllium slurry might be able to reduce its water-absorbing and gelling capacity, thereby improving its functionality during food formulation and processing (Yu et al., 2001). The potential effective carbohydrases included xylanases, cellulases, hemicellulases, arabanases, pentosansases, b-glucanases, and the various combinations of these enzymes. The enzymatic reaction might be conducted in water or a buffer with a pH value close to the optimal pH of the selected carbohydrase(s). The modified psyllium preparations were shown to have reduced gelling capacity measured as gel hardness and adhesiveness. The enzyme-treated psyllium was able to reduce serum total and LDL cholesterol levels, and serum triglycerides in male Golden Syrian hamsters (Yu et al., 2001). The disadvantage of this enzymatic procedure was the requirement of a freeze-dry procedure to remove moisture from the enzyme-treated psyllium. The involvement of the freeze-dry step limited the possibility to scale this procedure up for commercial production of the psyllium-derived food ingredients with improved functionality. However, the results from this invention disclosure supported the hypothesis that (a) change of chemical/molecular structures of psyllium may alter its functional characteristics and its biological activity; (b) breaking of the xylan backbone may result in a reduced gelling capacity and improvement of the gel properties, associated with an improved dispersive effect and decreased water-absorbing capacity; and (c) psyllium preparations with reduced gelling and waterabsorbing capacities may retain their health beneficial properties. In other words, the biological activity is not completely determined by the same structural factor(s) responsible for its water-absorbing/gelling properties.
C. Solid-state enzymatic procedures Later in 2003, a solid-state enzymatic procedure was developed to reduce the water-absorbing and gelling capacities of psyllium (Yu, 2003a,b; Yu and Perret, 2003a,b; Yu et al., 2003). In a typical solid-state enzymatic reaction, original psyllium was mixed with the enzyme and the solid reaction mixture is kept at ambient temperature until the inactivation of the enzyme, which terminates the reaction (Fig. 4.1 comparing solid and liquid reactions). The resulting solid mixture is the modified psyllium product with about 100% total yield. No chemical was added to the reaction mixture and no chemical waste is generated from the reaction. This ‘‘solid-state’’ procedure involves a limited amount of water in the enzymatic reaction and requires no additional step after enzyme inactivation for removing moisture content in the modified psyllium preparations, and thus can be easily scaled up for commercial production, whereas the product from the conventional liquid phase enzymatic reaction was a rubbery gel and required a freeze-drying step to remove
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FIGURE 4.1 Psyllium products from solid (left) and liquid (right) enzymatic reactions. (Adapted from Yu, 2003b.)
moisture (Fig. 4.1 comparing solid and liquid reactions) (Yu, 2003a,b; Yu and Perret, 2003a,b; Yu et al., 2003). A number of food-grade enzymes have been evaluated for their potential to improve psyllium functionality under the solid-state reaction conditions including Shearzyme 500L with xylanases activity, Pentopan Mono BG with pentosannase activity, and Viscozyme L with a combined activity of cellulase, hemicellulases, xylanases, arabanases, and b-glucanase. All three food-grade enzyme preparations are commercially available and may be obtained from Novo Nordisk Biochem North American, Inc. (Franklinton, NC). The modified psyllium samples were evaluated and compared to original psyllium for their water-absorbing capacity and gelling properties, as well as their fiber contents. The water absorption capacity was determined following a protocol described previously (Elizalde et al., 1996). In brief, all samples were equilibrated in a low relative humidity (RH) chamber for 48 h. Then, samples were transferred into a high RH chamber and exposed to moisture for a preselected time period. The dry matter and the absolute amount of absorbed water were determined. All three tested food-grade enzymes were able to dose-dependently reduce the water-absorbing capacity of psyllium, although their effectiveness on a per enzyme concentration basis differed (Yu and Perret, 2003a,b). Viscozyme L at levels of 19.2 and 36 units/g of psyllium significantly decreased the water-absorbing capacity of psyllium, and a 49% reduction
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Water absorbing rate (mg/g psyllium/min)
in the rate of water absorption was detected in the psyllium sample treated with Viscozyme L at a level of 36 units/g of psyllium under the experimental conditions (Fig. 4.2). The enzyme-treated psyllium samples were evaluated and compared with the original raw psyllium for their surface structures using a scanning electron microscope (SEM) technique. SEM analysis showed that solid-state enzyme treatment resulted in a smoother surface on the psyllium particles as compared to original raw psyllium and the control which went through the solid-state reaction without enzyme added (Fig. 4.3). It was suggested that solid-state enzymatic treatment reduced the total surface area which may partially explain the reduced water absorption rate of these modified psyllium preparations (Yu and Perret, 2003a,b; Yu et al., 2003). The gelling properties of the modified psyllium under the solid-state enzymatic reaction conditions were evaluated using a TA-XT2 texture analyzer (Texture Technologies Corp., Scarsdale, NY) (Boune and Comstock, 1981; Paraskevopoulou and Kiosseoglou, 1997; Pons and Fiszman, 1996). A known amount of psyllium was mixed into water with agitation. After incubation at room temperature for 3 h, gel samples were subjected to a double compression test (Yu and Perret, 2003a,b; Yu et al., 2003). Shearzyme 500L and Viscozyme L were more effective than Pentopan Mono BG in reducing the gelling capacity of psyllium on a per same enzyme concentration basis (Yu and Perret, 2003a,b; Yu et al., 2003).
1.2 1.0 0.8 0.6 0.4 0.2 0.0
0
2.4 4.8 9.6 19.2 Viscozyme L amount (U/g psyllium)
36
FIGURE 4.2 Effects of Viscozyme L on water uptaking capacity of psyllium. 0, 2.4, 4.8, 9.6, 19.2, and 36 represent the final Viscozyme L concentrations of 0, 2.4, 4.8, 9.6, 19.2, and 36 units/g of psyllium in the solid-state reactions, respectively. Means are reported and the vertical bars represent the standard deviation of each data point (n ¼ 3). (Adapted from Yu et al., 2003).
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FIGURE 4.3 Surface structures of psyllium preparations determined by SEM. (A) Psyllium treated with 0 enzyme, (B) psyllium treated with 120 units of Shearzyme 500 L, (C) raw psyllium, and (D) psyllium treated with Viscozyme L at a level of 30 units/g psyllium, under the experimental conditions (A and B are adapted from Yu, 2003b, while C and D are adapted from Yu et al., 2003).
Shearzyme 500 L and Viscozyme L were able to dose-dependently reduce the gelling capacity of psyllium. Figure 4.4 presents the effects of Viscozyme L on gelling properties of modified psyllium. Compared to the original psyllium, Viscozyme L treatment dose-dependently reduced the heights of all four peaks and altered the shape of the two negative peaks, indicating that the modified psyllium formed a weaker gel and might have reduced sliminess and less coating effects on sensory receptors in the mouth. Taking into account the water-absorbing and gelling properties of the modified psyllium, the solid-state enzymatic procedure may serve as an effective approach for improving the functionality and sensory properties of psyllium for promoting its food applications. It is well accepted that the soluble fiber is the primary contributor for the beneficial health effects of psyllium especially the cholesterollowering activity, while the insoluble fiber may also have a contribution. The effects of solid-state enzymatic treatment on fiber contents were investigated for the modified psyllium. Shearzyme 500 L, Viscozyme L, and Pentopan Mono BG treatments all caused loss of soluble fiber under
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19.2 36
40.0 20.0 0.0 0.0 -20.0 -40.0
5.0
10.0
15.0
20.0
Psy Time (s)
FIGURE 4.4 Effects of Viscozyme L treatment on gelling properties of psyllium. 4.8, 19.2, and 36 represent the final Viscozyme concentrations of 4.8, 19.2, and 36 units/g psyllium in the solid-state reaction mixtures, respectively, and Psy represents the original psyllium sample. The setting time was 3 h for all gel samples (redrawn from Yu et al., 2003).
the solid-state enzymatic reaction conditions, but had no influence on insoluble fiber contents (Fig. 4.5) (Yu and Perret, 2003a,b; Yu et al., 2003). A higher enzyme concentration was associated to a lower soluble fiber content in the modified psyllium products, although doubling the enzyme level did not result in doubled reduction in soluble fiber (Yu and Perret, 2003a,b; Yu et al., 2003). It was also noted in these previous studies that the loss of soluble fiber under the solid-state reaction conditions was less than that under the liquid state enzymatic reaction conditions (Yu and Perret, 2003b). It was proposed that other chemical reactions such as isomerization or acceptor reaction may occur under the solid-state reaction conditions with the limited amounts of free water molecules available (Yu and Perret, 2003b). The potential synergistic effect between enzymes on psyllium functionality was tested using Pentopan Mono BG and Shearzyme 500 L under the solid-state reaction conditions (Yu and Perret, 2003a). Pentopan Mono BG and Shearzyme 500 L exhibited synergistic effect in reducing the water-absorbing capacity of psyllium, but not in altering the gelling properties of psyllium. Addition of Shearzyme 500 L in the Pentopan Mono BG reaction resulted in further loss of soluble fiber from the psyllium preparation. These data warrant further investigation to develop enzyme combinations for improving functionality, sensory properties, and health benefits of psyllium.
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A 90
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15 S
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15 S
10 0P &1 2S
40 0P
20 0P
10 0P
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Ps y
0
Enzyme levels
FIGURE 4.5 Effects of solid-state enzyme treatment on (A) soluble fiber and (B) insoluble fiber contents in psyllium. P, the Pentopan Mono BG; S, the Shearzyme 500 L from Novo Nordisk Ferment Ltd. (Switzerland); 100P, 100 units of P; 12S, 12 units of S; and Psy stands for the commercial psyllium husks, the starting material for the solid-state enzymatic reaction (re-drawn from Yu and Perret, 2003a).
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In addition, modified psyllium preparations under the solid-state reaction conditions were evaluated for their hypolipidemic effects using hamsters (Allen et al., 2004). Original psyllium was included as the positive control, and cellulose was used as a fiber control. Hamsters were fed 0.2 wt.% cholesterol diets with 12% cellulose or 5% cellulose plus 7% original or enzymatically modified psyllium preparations. During 5 weeks of feeding period, psyllium addition did not reduce total food intake. Both modified psyllium preparations were as effective as the original psyllium in reduction of the total plasma, LDL, and HDL cholesterol, but cellulose did not cause similar reductions under the same experimental conditions (Fig. 4.6). These psyllium preparations also exhibited similar effects in enhancing the bile acid extraction. Interestingly, one of the modified psyllium preparation was able to significantly reduce the total body weight gain over the 35 days of feeding (Fig. 4.7), suggesting its potential utilization in body weight control (Allen et al., 2004). These previous studies indicate that solid-state enzymatic treatments may be an effective approach to improve not only the functionality but also the biological activity of psyllium, and additional research a Total cholesterol HDL cholesterol LDL cholesterol Triglyceride
300
250 Plasma lipid mg dL−1
a 200
100
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b
b
b
a
b
b
b
150
a
a
b
a
0 Cellulose
Raw psyllium
Modified psy-1
Modified psy-2
Diet
FIGURE 4.6 Effect of solid-state enzyme treatment on hypolipidemic activities of psyllium. Plasma lipid concentrations in hamsters were measured at day 35. Values are mean SEM (vertical bars) for nine animals per group. Within each response parameter, values not sharing common letters are significantly different, P<0.05. Modified Psy-1 and modified Psy-2 represent the two modified psyllium preparations using the Viscozyme L and the Shearzyme 500 L, respectively, under the solid-state reaction conditions (re-drawn from Allen et al., 2004).
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33 a
a
28 a Weight gain (g)
23
18
b
13
8
3 −2
Cellulose
Modified psy-1
Raw psyllium
Modified psy-2
Diet
FIGURE 4.7 Comparison of enzymatically modified psyllium with cellulose and original raw psyllium for their effects on body weight gain in hamsters. Total body weights gained over the 35 days of feeding are reported. Values are mean SEM (vertical bars) for nine animals per group. Values not sharing common letters are significantly different, P<0.05. Modified Psy-1 and modified Psy-2 represent the two modified psyllium preparations using the Viscozyme L and the Shearzyme 500 L, respectively, under the solidstate reaction conditions (re-drawn from Allen et al., 2004).
involving more food-grade enzymes is requested to further explore the opportunity. Additional research is also required to further investigate the relationships between the chemical/molecular structures and the physicochemical/health-benefit properties of psyllium.
D. Chemical modification of psyllium Grafting and networking may modify the mechanical, chemical, and functional properties of polymers and enhance their utilization for some purposes, such as for water treatment (Kumar and Verma, 2007; Mishra et al., 2003). Psyllium derivatives were prepared by grafting acrylonitrile onto psyllium molecules using a ceric ammonium nitrate and nitric acid system (Mishra et al., 2003). The resulted grafted psyllium samples were not soluble in commonly used solvents or their combinations. In 2007, methacrylic acid derivatives of psyllium were prepared using ammonium persulfate as initiator and cross-linked using N,N-methylenebisacrylamide as the crosslinker (Kumar and Verma, 2007). The modified psyllium
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showed different swelling and thermal degradation behaviors with the possible application in water treatment. These research activities suggest the potential of improving psyllium functionality and biological activity through chemical modifications, although these two grafted psyllium preparations were mainly for nonfood uses. Chemical methods have also been developed to eliminate the allergenicity of psyllium (Ndife, 1993). The alkaline treatment, including aqueous sodium hydroxide or potassium hydroxide, at a ratio of 2–20 g of psyllium weight per 100 ml alkali with a concentration of 0.1–0.5 N for 10–60 min, might significantly decrease the allergenicity of psyllium.
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Bijkerk, C. J., Muris, J. W. M., Knottnerus, J. A., Hoes, A. W., and de Wit, N. J. (2004). Systematic review: The role of different types of fibre in the treatment of irritable bowel syndrome. Aliment. Pharmacol. Ther. 19, 245–251. Boune, M. C., and Comstock, S. H. (1981). Effect of degree of compression on texture profile parameters. J. Texture Stud. 12, 201–216. Brown, L., Rosner, B., Willett, W. W., and Sack, F. M. (1999). Cholesterol-lowering effects of dietary fiber: A meta-analysis. Am. J. Clin. Nutr. 69, 30–42. Buhman, K. K., Furumoto, E. J., Donkin, S. S., and Story, J. A. (2000). Dietary psyllium increased expression of ileal apical sodium-dependent bile acid transporter mRNA coordinately with dose-responsive changes in bile acid metabolism in rats. J. Nutr. 130, 2137–2142. Cavaliere, H., Floriano, I., and Medeiros-Neto, G. (2001). Gastrointestinal side effects of orlistat may be prevented by concomitant prescription of natural fibers (Psyllium mucilloid). Int. J. Obes. 25, 1095–1099. Chan, J. K. C., and Wypyszyk, V. (1988). A forgotten natural dietary fiber: Psyllium mucilloid. Cereal Foods World 33, 919–922. Clark, C. A., Gardiner, J., McBurney, M. I., Anderson, S., Weatherspoon, L. J., Henry, D. N., and Hord, N. G. (2006). Effects of breakfast meal composition on second meal metabolic responses in adults with type 2 diabetes mellitus. Eur. J. Clin. Nutr. 60, 1122–1129. Cohen, L. A., Zhao, Z., Zang, E. A., Wynn, T. T., Simi, B., and Rivenson, A. (1996). Wheat bran and psyllium diets: Effects on N-methylnitrosourea-induced mammary tumorigenesis in F344 rats. J. Natl. Cancer Inst. 88, 899–907. Daggy, B. P., O’Connell, N. C., Jerdack, G. R., Stinson, B. A., and Setchell, K. D. R. (1997). Additive hypocholesterolemic effect of psyllium and cholestyramine in the hamster: Influence on fecal sterol and bile acid profiles. J. Lipid Res. 38, 491–502. Davidson, M. H., Dugan, L. D., Burns, J. H., Sugimoto, D., Story, K., and Drennan, K. (1996). A psyllium-enriched cereal for the treatment of hypercholesterolemia in children: A controlled, double-blind, crossover study. Am. J. Clin. Nutr. 63, 96–102. Davidson, M. H., Maki, K. C., Kong, J. C., Dugan, L. D., Torri, S. A., Hall, H. A., Drennan, K. B., Anderson, S. M., Fulgoni, V. L., Saldanha, L. G., and Olson, B. H. (1998). Long-term effects of consuming foods containing psyllium seed husk on serum lipids in subjects with hypercholesterolemia. Am. J. Clin. Nutr. 67, 367–376. Elizalde, B. E., Pilosof, A. M. R., and Bartholomai, G. B. (1996). Empirical model for water uptake and hydration rate of food powders by sorption and Baumann methods. J. Food Sci. 61, 407–409. Everson, G. T., Daggy, B. P., McKinley, C., and Story, J. A. (1992). Effects of psyllium hydrophilic mucilloid on LDL-cholesterol and bile acid synthesis in hypercholesterolemic men. J. Lipid Res. 33, 1183–1192. Fernandez, M. L., Ruiz, L. R., Conde, A. K., Sun, D. M., Erickson, S. K., and McNamara, D. J. (1995). Psyllium reduces plasma LDL in guinea pigs by altering hepatic cholesterol homeostasis. J. Lipid Res. 36, 1128–1138. Fernandez, M. L., Vergara-Jimenez, M., Conde, K., Behr, T., and Abdel-Fattah, G. (1997). Regulation of apolipoprotein B-containing lipoproteins by dietary soluble fiber in guinea pigs. Am. J. Clin. Nutr. 65, 814–822. Ganji, V., and Kies, C. V. (1994). Psyllium husk fiber supplementation to soybean and coconut oil diets of humans: Effect on fat digestibility and faecal fatty acid excretion. Eur. J. Clin. Nutr. 48, 595–597. Gonlachanvit, S., Coleski, R., Owyang, C., and Hasler, W. L. (2004). Inhibitory actions of a high fibre diet on intestinal gas transit in healthy volunteers. Gut. 53, 1577–1582. Gupta, R. R., Agrawal, C. G., Singh, G. P., and Ghatak, A. (1994). Lipid-lowering efficacy of psyllium hydrophilic mucilloid in non insulin dependent diabetes mellitus with hyperlipidaemia. India J. Med. Res. 100, 237–241.
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psyllium in reducing serum cholesterol levels in hypercholesterolemic patients on high- or low-fat diet. Ann. Intern. Med. 119, 545–554. Stevens, J., Levitsky, D. A., VanSoest, P. J., Robertson, J. B., Kalkwarf, H. J., and Roe, D. A. (1987). Effect of psyllium gum and wheat bran on spontaneous energy intake. Am. J. Clin. Nutr. 46, 812–817. Stoy, D. B., LaRosa, J. C., Brewer, B. K., Mackey, M., and Meusing, R. A. (1993). Cholesterollowering effects of ready-to-eat cereal containing psyllium. J. Am. Diet. Assoc. 93, 910–912. Terpstra, A. H. M., Lapre, J. A., de Vries, H. T., and Beynen, A. C. (1998). Dietary pectin with high viscosity lowers plasma and liver cholesterol concentration and plasma cholesteryl ester transfer protein activity in hamsters. J. Nutr. 128, 1944–1949. Terpstra, A. H. M., Lapre, J. A., de Vries, H. T., and Beynen, A. C. (2000a). Hypocholesterolemic effect of dietary psyllium in female rats. Ann. Nutr. Metab. 44, 223–238. Terpstra, A. H. M., Lapre, J. A., de Vries, H. T., and Beynen, A. C. (2000b). Transiency of the different cholesterolaemic responses to dietary cellulose and psyllium in pigs and two strains of hamsters. J. Anim. Physiol. Anim. Nutr. 84, 178–191. Trautwein, E. A., Siddiqui, A., and Hayes, K. C. (1993). Modeling plasma lipoprotein-bile lipid relationships: Differential impact of psyllium and cholestyramine in hamsters fed a lithogenic diet. Metabolism 42, 1531–1540. Trautwein, E. A., Rieckhoff, D., Kunath-Rau, A., and Erbersdobler, H. F. (1998). Psyllium, not pectin or guar gum, alters lipoprotein and biliary bile acid composition and fecal sterol excretion in the hamster. Lipids 33, 573–582. Trautwein, E. A., Kunath-Rau, A., and Erbersdobler, H. F. (1999). Increased fecal bile acid excretion and changes in the circulating bile acid pool are involved in the hypocholesterolemic and gallstone-preventive actions of psyllium in hamsters. J. Nutr. 129, 896–902. Turley, S. D., and Dietschy, J. M. (1995). Mechanisms of LDL-cholesterol lowering action of psyllium hydrophillic mucilloid in the hamster. Biochim. Biophys. Acta 1255, 177–184. Turley, S. D., Daggy, B. P., and Dietschy, J. M. (1991). Cholesterol-lowering action of psyllium mucilloid in the hamster: Sites and possible mechanisms of action. Metabolism 40, 1063–1073. Turley, S. D., Daggy, B. P., and Dietschy, J. M. (1994). Psyllium augments the cholesterollowering action of cholestyramine in hamsters by enhancing sterol loss from the liver. Gastroenterology 107, 444–452. Vergara-Jimenez, M., Furr, H., and Fernandez, M. L. (1999). Pectin and psyllium decrease the susceptibility of LDL to oxidation in guinea pigs. J. Nutr. Biochem. 10, 118–124. Washington, N., Harris, M., Mussellwhite, A., and Spiller, R. C. (1998). Moderation of lactulose-induced diarrhea by psyllium: Effects on motility and fermentation. Am. J. Clin. Nutr. 67, 317–321. Wolever, T. M. S., Jenkins, D. J. A., Mueller, S., Boctor, D. L., Ransom, T. P. P., Patten, R., Chao, E. S. M., McMillan, K., and Fulgoni, V., III (1994). Method of administration influences the serum cholesterol-lowering effect of psyllium. Am. J. Clin. Nutr. 59, 1055–1059. Wullschleger, R. D. (1993). Heat treatment for decreasing the allergenicity of psyllium seed husk products. US Patent 5,271,936. Wullschleger, R. D., Chen, S. C., Bowman, F. A., and Hawblitz, L. V. (1993). Ready-to-eat cereal containing psyllium. US Patent 5,227,248. Yu, L. (2003a). Method for improving psyllium functionality by solid-state reaction(s). US Patent 20030211179. Yu, L. (2003b). Structural modification to improve psyllium functionality. Food Factors in Health Promotion and Diseases Prevention. ACS Symp. Ser. 851, 392–399. Yu, L., and Perret, J. (2003a). Effects of solid-state enzyme treatments on the water-absorbing and gelling properties of psyllium. LWT-Food Sci. Technol. 36, 203–208.
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CHAPTER
5 Starch Gelatinization Wajira S. Ratnayake* and David S. Jackson*
Contents
Abstract
I. Introduction II. Starch: Importance and Sources III. Starch Structure and Crystallinity A. Birefringence and starch crystallinity B. Granular structure IV. Starch Gelatinization Theories and Models A. Early studies B. Semi-cooperative theory C. Water availability theory D. Crystallite stability theory E. Sequential phase transitions (first amorphous and then crystalline) theory F. Three-stage (partial melting, recrystallization, and total melting) phase transition theory G. Application of Flory’s theory to explain starch gelatinization V. Starch Annealing and Its Relationship to Gelatinization VI. Glass Transition and Gelatinization VII. Contradicting Theories: What Is Gelatinization? References
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Starch occurs as highly organized structures, known as starch granules. Starch has unique thermal properties and functionality that have permitted its wide use in food products and industrial applications. When heated in water, starch undergoes a transition process, during which the granules break down into a mixture of
*Department of Food Science and Technology, University of Nebraska-Lincoln, Lincoln, Nebraska, 68583-0919 Advances in Food and Nutrition Research, Volume 55 ISSN 1043-4526, DOI: 10.1016/S1043-4526(08)00405-1
#
2009 Elsevier Inc. All rights reserved.
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polymers-in-solution, known as gelatinization. The sequence of structural transformations that the starch granule undergoes during this order-to-disorder transition has been extensively researched. None of the published starch gelatinization theories can fully and adequately explain the exact mechanism of sequential structural changes that starch granules undergo during gelatinization. This chapter analyzes several published theories and summarizes our current understanding of the starch gelatinization process. Key Words: Starch, gelatinization.
ß 2009 Elsevier Inc.
ABBREVIATIONS DSC NMR RS Tg To Tp Tc Tm TMA XRD
Differential scanning calorimetry Nuclear magnetic resonance Resistant starch Glass transition temperature DSC onset temperature DSC peak temperature DSC conclusion (or end) temperature Melting temperature Thermomechanical analysis X-ray diffraction
I. INTRODUCTION Starch is the major component of human diet and is also used for many food and nonfood/industrial applications (Gaillard, 1987; FAO, 2006a). A significant quantity of starch is further processed by subjecting it to various forms of chemical and physical modifications, resulting in starches with even greater functionality attributes (Zobel, 1992). Both unmodified (native) and modified starches are used as bulking agents, thickeners, stabilizers, viscosity builders, and gel formers. Starch is also used to produce various hydrolysis products, such as maltose, maltodextrins, and cyclodextrins, by acid and/or enzyme conversion methods. Starch is obtained from a variety of plant sources. Corn, cassava, sweet potato, wheat, and potato are the major sources of food starch while sorghum, barley, rice, sago, arrowroot, etc. serve as minor sources of starch in different localized regions of the world (Gaillard, 1987; Ratnayake and Jackson, 2003). Raw starch granules do not disperse in cold water. This limits the use of raw native starches for food as well as industrial applications, and therefore starch is often cooked during product-manufacturing
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processes. Cooking (or heat treatment) causes structural and often molecular changes in granular and polymeric structures of starch. Functional properties of starch are directly influenced by hydrothermal (heat and moisture) treatment or processing conditions. When raw starch granules are heated in water, the semicrystalline nature of their structure is reduced or eliminated and the granules break down, forming a viscous solution; solution viscosity depends on starch source and concentration. Heat-induced starch granule breakdown in water is known as gelatinization. The unique starch gelatinization processes, and the subsequent formation of viscous solutions when starch granules are heated in water, have fascinated researchers since the early 1800s. It is known that granules undergo a sequence of structural changes during gelatinization. These structural changes, however, are poorly understood and are the subject of frequent research studies. Gelatinization is described as a transition of starch granules from an ordered state to a disordered state. This chapter critically evaluates and summarizes the ‘‘evolution’’ of currently accepted theoretical descriptions of starch gelatinization process.
II. STARCH: IMPORTANCE AND SOURCES Starch is one of the most abundant plant polysaccharides and is a major source of carbohydrates and energy in the human diet (Zobel and Stephen, 1995). Starch is the most widely used hydrocolloid in the food industry (Wanous, 2004), and is also a widely used industrial substrate polymer. Total annual world production of starch is approximately 60 million MT and it is predicted to increase by additional approximately 10 million MT by 2010 (FAO, 2006b; LMC International, 2002; S. K. Patil and Associates, 2007). Corn/maize (Zea mays L.), cassava (also known as tapioca—Manihot esculenta Crantn.), sweet potato (Ipomoea batatas L.), wheat (Triticum aestivum L.), and potato (Solanum tuberosum L.) are the major sources of starch, while rice (Oryza sativa L.), barley (Hordeum vulgare L.), sago (Cycas spp.), arrowroot (Tacca leontopetaloides (L.) Kuntze), buckwheat (Fagopyrum esculentum Moench), etc. contribute in lesser amounts to total global production. Starch is isolated from plant sources by various methods (Ratnayake and Jackson, 2003; Zobel, 1992). The method of starch isolation depends on the nature and composition of the raw material source. Most food starches are isolated and purified on a commercial scale and then used as ingredients by food manufacturers. Commercial food starches are generally classified based on both botanical origin and functionality. With the increasing availability of modified starches prepared for specific food applications, starch manufacturers tend to emphasize and market starch with a secondary focus on botanical source. In fact, for food
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manufacturers, ingredient functionality is the most important consideration when choosing starch for food products. Starch functional properties depend on many factors that include both granular and molecular (polymeric) characteristics. Starch physicochemical properties depend on both its botanical source and any form of physical or chemical modification applied to starch during its preparation into an ingredient. Extensive research on starch has focused on its botanical sources, functionality, chemistry, and nutritional/health effects.
III. STARCH STRUCTURE AND CRYSTALLINITY Native or raw starch occurs as small semispherical (microscopic) structures called granules. The size, shape, and molecular arrangement inside the granules depend on the species, cultivar, variety of the source plant, and the genetic–environment interactions (Ratnayake and Jackson, 2003; Trubell, 1944; Zobel, 1988a). Regardless of the plant source, all starches are made up of d-glucopyranose-based polymeric structures. In starch polymers, individual glucose units are linked together by either a(1-4) or a(1-6) glycosidic bonds. The starch biosynthetic pathway generally results in two different kinds of polymers being formed, namely, amylose and amylopectin. Amylose is essentially a linear polymer with a(1-4) linkages [and 2–5% a(1-6) bonds] and has a molecular weight less than 1105. Amylopectin is a branched polymer, which has both a(1-4) and a(1-6) bonds with a molecular weight ranging from 50 to 500 million (Banks and Greenwood, 1975; Thomas and Atwell, 1999). The molecular weights and degrees of branching in a given starch polymer depend on its source (Blanshard, 1987; Zobel, 1992; Zobel and Stephen, 1995). The linear branch chains of the amylopectin molecule contain an average of 20–25 a(1-4)linked anhydroglucose residues. These linear branches are linked by a(1-6) glycosidic bonds to form a highly branched structure (Hizukuri, 1986; Manners, 1989; Fig. 5.1). For detailed information on how starch polymers are synthesized in plants, readers may refer to reviews and articles by Denyer et al. (1997), Smith et al., (1997), Erlander (1998), Kossmann and Lloyd (2000), Ball and Morell (2003), James et al., (2003), and Preiss (2004). The polymer composition of starch is genetically controlled; mutations are found naturally or plant breeding techniques can be used to obtain high-amylose or high-amylopectin (waxy) starches (Blennow, 2004; Bogracheva et al., 1999; Tetlow, 2006). The basic structure of the starch granule has been recognized and documented in the literature since the late 1800s (Czaja, 1978; Donald, 2004; French, 1944; FreyWyssling, 1969; Jane, 2006; Kraemer, 1902; Meyer and Gibbons, 1951; Oates, 1997; Trubell, 1944; Vermeylen et al., 2004; Wang et al., 1998). Early reports were mainly based on optical microscopy results, which
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B2
A
A ∅
B3
A
B1 B1
B2
B1 A B1 c.l. = 12−16
c.l. = 27−28
Crystalline region
Intercrystalline amorphous region
FIGURE 5.1 Cluster model of amylopectin. A and B denote nomenclature of branch chains, F¼reducing end, c.l. ¼ chain length in degree of polymerization. Reprinted from Carbohydrate Research, Vol. 147, Hizukuri (1986), Polymodal distribution of the chain lengths of amylopectin, and its significance, Pages 342–347, with permission from Elsevier.
revealed granule shapes, sizes, and the presence of growth rings (Fig. 5.2) inside granules from different sources (Leach, 1914). Although the existence of growth rings is obvious in starch granules, the biochemical origins and physiological importance of these rings are poorly understood. Concentric growth rings or striations inside the starch granule were initially thought to be a result of ‘‘periods of relative inactivity of deposition of starchy material’’ during granule synthesis (Trubell, 1944). Later, it was proposed that these growth rings were formed as a result of a ‘‘photosynthetically controlled supply of starch precursor’’ in wheat starch, but not in potato starch (Buttrose, 1962). Subsequent research during the last few decades has not completely explained the origin of growth rings, although the enzyme activity (starch synthases) involved in the process are well documented (Fig. 5.3). More recent reports (Pilling and Smith, 2003) indicate that a variety of factors, including circadian rhythms, physical mechanisms, and diurnal rhythms, control growth ring formation in starch granules.
A. Birefringence and starch crystallinity Starch granules, when observed under polarized light, exhibit an optical birefringence pattern known as a ‘‘Maltese cross’’ (Fig. 5.4), which implies a high degree of molecular order within the granule (Greenwood, 1979).
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A
B
C
D
E
F
G
FIGURE 5.2 Growth rings in potato starch granules developed under different environmental conditions. Scale bars on A and G represent 10 mm; all other scale bars represent 5 mm. (A and B) Plant grown in 16 h of light at 18 C and 8 h of dark at 15 C. (C and D) Plant grown in constant light and constant temperature (18 C). (E and F) Microtuber grown in continuous darkness at 25 C for 12–16 weeks. (G) Plant grown in 20 h of light at 18 C and 20 h of dark at 15 C (Pilling and Smith, 2003; reproduced with permission from American Society of Plant Biologists).
Birefringence (or double refraction) is the decomposition of a light ray into two rays when it passes through certain types of crystalline material. This occurs only when the material is anisotropic, that is, the material has different characteristics in different directions. Amylose and amylopectin polymers are organized into a radially anisotropic, semicrystalline unit in the starch granule. This radial anisotropy is responsible for the distinctive
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Sucrose a Amylose
Starch Amylopectin
UDP glucose e ADP glucose
Fructose b
g
f ADP glucose
PPi c Glucose 1-P
h
Glucose 1-P
d Fructose 6-P
Glucose 6-P Cytosol
h
Glucose 6-P
Plastid
FIGURE 5.3 Biochemical pathway of starch biosynthesis from sucrose. The major metabolites and enzymes involved in the conversion of sucrose to starch in storage organs. Carbon enters the plastid either as a hexose phosphate or as ADPglucose. Enzymes are: (a) sucrose synthase; (b) UDPglucose pyrophosphorylase; (c) ADPglucose pyrophosphorylase; (d) phosphoglucomutase; (e) starch synthase (GBSSI); (f) starch synthase and starch-branching enzyme; (g) ADPglucose transporter; (h) hexose phosphate transporter. PPi: inorganic pyrophosphate (Smith et al., 1997; Reprinted with permission from the Annual Review of Plant Physiology and Plant Molecular Biology, Volume 48, #1997 by Annual Reviews. www.annualreviews.org.)
FIGURE 5.4 Potato starch granules viewed under polarized light (magnification, 400). Reprinted from Encyclopedia of Food Sciences and Nutrition (Second Edition), Jackson (2003b), Starch – Structure, properties and determination, Pages 5561–5567, with permission from Elsevier.
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Maltese cross (Blanshard, 1979). The crystallinity of starch is caused essentially by amylopectin polymer interactions (Banks and Greenwood, 1975; Biliaderis, 1998; Donald, 2004; Hizukuri, 1996). An illustration of currently accepted starch granule structure is given in Fig. 5.5. It is believed that the outer branches of amylopectin molecules interact to arrange themselves into ‘‘crystallites’’ forming crystalline lamellae within the granule (Fig. 5.5; Tester et al., 2004). A small number of amylose polymers may also interact with amylopectin crystallites. This hypothetical structure has been derived based on the cluster model of amylopectin (Hizukuri, 1986; Robin et al., 1974; Fig. 5.1). The crystallinity exhibited by starch granules can be measured, both qualitatively and quantitatively, by X-ray powder diffraction (Eliasson et al., 1987; Hizukuri, 1961; Jauncey and Pennell, 1933; Katz, 1928; Mizuno et al., 1998; Nara et al., 1978; Ziegler et al., 2005; Zobel, 1988b). Degree of crystallinity and the nature of crystalline and noncrystalline structures and their relationship within granules are major factors that determine starch properties. Starch crystallinity, determined by X-ray diffraction (XRD), is influenced by starch moisture content; positions and intensities of radiation maxima are affected by the degree of hydration (Cleven et al., 1978; Waigh et al., 1997). Trubell (1944) suggested that changes in X-ray patterns were due to water entering into the crystalline structure and anisotropy, and therefore was due to ‘‘crystal-like’’ structures within granules. Qualitative measurement is usually the determination of XRD patterns (Gernat et al., 1990; Zobel, 1988b; Zobel et al., 1988; Fig. 5.6). Quantitative measurements include polymorphic composition and degree of relative crystallinity compared to a given reference crystalline material (Bogracheva et al., 1997; Cairns et al., 1997; Nara et al., 1978; see Box 1 for more details on starch XRD).
B. Granular structure Although the origin of granular assembly is being further refined (Pilling and Smith, 2003; Smith, 2001; Smith et al., 1997), it is generally accepted that granules contain amorphous and crystalline domains arranged in alternating concentric rings that create a semicrystalline environment within the granule. It is also understood that the crystalline domains are mainly composed of amylopectin while bulk amorphous domains are made up of amylose traversed by noncrystalline regions of amylopectin. The crystalline patterns of starch are classified into three distinct groups, A, B, and C based on their polymorphism. During late 1970s, Wu and Sarko published a series of articles (Sarko and Wu, 1978; Wu and Sarko, 1978a,b) elucidating the nature of these A, B, and C polymorphs in starch crystalline structure. The A polymorph crystallizes in an orthogonal unit cell with slightly distorted hexagonal packing and 8 water molecules per unit cell, whereas the B polymorph crystallizes in a hexagonal
Intercrystalline amorphous lamella
Crystalline lamella
Starch granule
Amylopectin polymer arrangement
Bulk amorphous region
FIGURE 5.5 Schematic diagram of starch granule structure. Adapted from Donald et al. (1997), Starch – Structure and functionality, ISBN 0854047425, with permission from Woodhead Publishing Ltd., Cambridge, UK.
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A B
C
V 10
20 Reflection angle 2q
30
FIGURE 5.6 Classes of X-ray diffraction patterns exhibited by different starches. In general, the A pattern is exhibited by cereal starches. The B pattern is exhibited by tuber (potato) starches, and the C pattern is exhibited by root (cassava) and legume (pea) starches. The Verkleisterungspektrum or V pattern is exhibited by amylose–lipid complexes. Zobel (1988b), Starch crystal transformations and their industrial importance. Starch/Sta¨rke, 40, 1–7. Copyright Wiley-VCH Verlag GmbH & Co. KGaA. Reproduced with permission.
unit cell with a more open hexagonal packing and 36 water molecules per unit cell (Fig. 5.8). The C polymorphic structure is a mixture of A and B unit cells and, therefore, is considered to be intermediate between A and B forms in packing density and structure. These different polymorphisms display distinctly different XRD patterns (Fig. 5.6).
IV. STARCH GELATINIZATION THEORIES AND MODELS The properties exhibited by starch during gelatinization are governed by several factors, including granule size and shape. When raw starch is heated in excess water, granules swell and lose their birefringence. After the granules are swollen to a maximum volume, they burst dispersing ‘‘starch substance’’ forming a colloidal dispersion in water (Alsberg, 1928).
A. Early studies In the 1940s, researchers identified that when starch is heated in water there were three distinct stages in the granule disruption process. During the first phase, water is slowly and reversibly absorbed by granules,
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BOX 1 X-RAY DIFFRACTION
In early 1900s, it was discovered that starch gave an X-ray spectrum of a crystalline, not amorphous, substance (Katz, 1928). Researchers suspected that starch exhibited both amorphous and crystalline characteristics in XRD experiments, elucidated mainly from a theoretical point-of-view (Sponsler, 1923). An article published by Katz and van Itallie (1930) was paid great interest by researchers and caused XRD to be used as a ‘‘popular’’ analytical tool in the mid- to late twentieth century. In this article, Katz and van Itallie (1930) explained how starches can be classified into three different groups: A, B, and C based on the XRD patterns. XRD can estimate, qualitatively and quantitatively, both crystalline and amorphous phases of starch granules. Therefore, theoretically speaking, the loss of granular structural order and changes in both amorphous and crystalline domains during gelatinization can be monitored and estimated by XRD (Fig. 5.7). Developments in knowledge, techniques, and instrumentation, since Katz and van Itallie’s findings, have permitted better, reliable, and highly reproducible procedures for X-ray analyses of starch. Both small-angle and wide-angle XRD have been extensively used during last two decades to characterize starch properties. Comprehensive reviews (Chandrasekaran, 1998; Zobel, 1988b) and numerous theories (Cairns et al., 1997; Eliasson et al., 1987; Garnat et al., 1990; Hizukuri, 1961; Jenkins and Donald, 1997; Nara et al., 1978; Mizuno et al., 1998; Schreiner and Jenkins, 1983) on how to determine starch relative crystallinity and polymorphic composition using XRD have been published. When starch granules are completely gelatinized, they exhibit an amorphous XRD pattern (with no peaks) as opposed to native or partially gelatinized starch that displays XRD profiles with peaks (Fig. 5.7). The peaks in XRD profiles are known to represent the crystallinity, and the large ‘‘background’’ area of the profile represents the amorphous phase of starch. During gelatinization, starch crystallinity is lost at a rate and to an extent depending on the intensity of hydrothermal treatment applied. Although XRD has traditionally been used as a tool to characterize starch (at static temperatures), it can also be used to determine changes during gelatinization of starch. However, there are some drawbacks to using XRD in starch gelatinization experiments. Gelatinization, as we understand now, is not only associated with crystalline order, but is also influenced by structural changes in the amorphous region. XRD does not detect or account for the structural changes that occur in the amorphous regions of the starch granules. continued
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(continued )
Intensity (counts per sec) - diffractograms offset for display purposes
BOX 1
Completely gelatinized starch
Partially gelatinized starch
Native starch
3
9
15
21 Angle (q/2 q)
27
33
FIGURE 5.7 X-ray diffraction profiles of native (ungelatinized), partially gelatinized, and completely gelatinized (amorphous) tapioca starch. Reprinted from Carbohydrate Polymers, Vol. 67, Ratnayake and Jackson (2007), A new insight into the gelatinization process of native starches, Pages 511–529, 2007, with permission from Elsevier.
(A) X-Ray powder diffraction requires relatively dry samples. Therefore, to measure the degree of gelatinization by XRD, some postprocessing of gelatinized samples is required. Consequently, the results of X-ray analysis may depend on the nature and extent of sample changes that occur during the postgelatinization treatments such as moisture removal and preservation. (B) X-Ray intensities (or peak intensities) depend on sample moisture content. Therefore, to obtain reproducible data, the moisture contents of all the samples analyzed in the experiment should be constant (usually between 10–18% w/w) depending on the analytical requirements.
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(C) A standard, universally acceptable method is not used to estimate the % relative crystallinity of starch by researchers. The differing procedures of estimating % relative crystallinity have produced conflicting data interpretations (Cheetham and Tao, 1998; Mizuno et al., 1998; Nara et al., 1978). (D) We have found that the method of Wakelin et al. (1959), as used by Nara et al. (1978) to calculate the % relative crystallinity, is a reliable and reproducible procedure to determine the degree of gelatinization of starch by XRD powder diffractometry. This procedure, however, is not straightforward and needs suitable reference samples for 100% crystalline and 100% amorphous material, and appropriate computer software to analyze data. A
B
FIGURE 5.8 Unit cells (outlined in each diagram) and helix packing in A and B polymorphs of starch. Reprinted from Carbohydrate Research, Vol. 61, Wu and Sarko (1978b), The double helical molecular structure of crystalline A-amylose, Pages 27–40, with permission from Elsevier.
swelling them to a limited extent. The viscosity of such a suspension does not increase noticeably during this stage. These granules, if subsequently dehydrated, retain their birefringence and return to their original appearance (French, 1944, 1950). The second phase of swelling starts within a small temperature range, characteristic to the particular starch, in which granules increase their volume to a larger extent by absorbing considerable amounts of water while rapidly losing birefringence. During this second phase, which is marked by a sudden rise in viscosity of the starch–water suspension, a small amount of starch becomes solubilized. After traversing the critical temperature at which the second phase
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changes occur, granules do not return to their original shape when cooled and/or dehydrated. During the third phase of swelling, at further increasing temperatures, the granules become formless sacs and the more soluble portion of starch leaches out into the suspension. Ultimately, this suspension is filled with swollen granule sacs, and contains very few intact granules. On cooling, this mixture has the capacity to form gels (French, 1944, 1950). Early studies failed to reveal the exact mechanism of starch gelatinization. Before the structure of the starch granule was characterized in detail, some researchers discovered that granules, which had been solubilized by the action of acids, were no longer capable of swelling. However, these granules could be disintegrated and dispersed in hot water (French, 1944, 1950; Trubell, 1944). With an increasing understanding of starch granular structure, crystallinity, and polymorphic forms (Zaslow, 1965), it was established that gelatinization begins in the more water-accessible amorphous regions of the granule where intermolecular bonding is weak. The use of a Kofler hot stage microscope with polarized light became the method of choice to measure gelatinization, as the loss of birefringence could be easily detected (Leach, 1965). By the 1960s, it was discovered that the initial change in granular structure during gelatinization was swelling without physical disintegration. Researchers suggested that the degree and kind of association within the granules controlled the swelling behavior of a particular starch (Leach, 1965). It also was observed that some cereal starches, such as regular cornstarch, sorghum, and rice, exhibited a two-stage swelling pattern. This phenomenon was linked to the possible presence of two distinct groups of internal bonding forces inside granules, that is, amorphous and crystalline regions (Leach, 1965). With the wide use of polarized light hot stage microscopy to determine starch gelatinization, the gelatinization temperature was defined as the temperature at which 98% of the granules lose birefringence when viewed under a polarized light microscope (Collison, 1968; Watson, 1964). Goering et al. (1974) investigated uncertainties associated with using birefringence loss to define starch gelatinization temperature by comparing the technique to a glucoamylase enzyme treatment procedure (Shetty et al., 1974; Table 5.1). The discrepancies in measurements were found to be caused by the limitations of measuring birefringence and the nonhomogenous structure of different starch granules within a given sample. See Box 2 for more information on estimation of starch gelatinization by enzymatic methods (Table 5.2).
B. Semi-cooperative theory Marchant and Blanshard (1978) studied starch gelatinization using a light scattering system to measure birefringence. This small-angle light scattering device (Marchant et al., 1977) was used to measure the intensity of
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TABLE 5.1 Comparison of birefringence loss and degree of gelatinization in different starchesa
Starch
Corn
Wheat
Rice
Potato
a
Temperature ( C)
Loss of birefringence (%)
Degree of gelatinization (%) by glucoamylase method
65 70 75 55 60 65 70 75 80 85 60 65 70 75 80
2 89.6 100 46.8 77.3 100 – 88.7 99.3 100 0 0 45 100 –
13 51 66 9 27 70 81 61 72 77 3 36 44 55 64
Adapted from Goering et al. (1974).
total scattered light which is the summed average of the polarization crosses from birefringent granules. They suggested that three processes (1) water diffusion into the starch granule, (2) loss of birefringence or ‘‘hydration facilitated melting,’’ and (3) granular swelling predominantly after loss of birefringence occur during gelatinization. They proposed that the loss of birefringence was a result of ‘‘solvation-assisted helix to coil transition.’’ Marchant and Blanshard (1978) also proposed that helices, which cause birefringence, are aggregated together by hydrogen bonding and are radially oriented within the starch granule. They disputed theories that described starch gelatinization as a highly cooperative or ‘‘allor-none’’ process. According to Marchant and Blanshard (1978), starch granules are not coherent crystal structures. Whole granules do not act as the primary thermodynamic unit, but the crystallites within the granule are basic to their thermal behavior, while amorphous regions also play a distinctive role. They further indicated that previously published theories did not account for the granular swelling that occurs before phase transition. Based on previously published reports, Marchant and Blanshard (1978) concluded that the starch granule was a highly concentrated and condensed gel system in which junction zones between polymers make crystallites. The stability of those crystallites and the polymer chain conformations within the amorphous regions were described as mutually
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BOX 2 GELATINIZATION MEASUREMENT BY ENZYMATIC METHODS
An estimation of starch gelatinization using glucoamylase was reported by Toyama et al. in 1966. The degree of starch gelatinization was estimated by selective digestion of gelatinized starch using glucoamylase [E.C. 3.2.1.3] enzyme followed by determination of D-glucose concentration (Shetty et al. 1974). The principle behind this method is that glucoamylase does not attack intact/raw starch granules. Shetty et al. (1974) argued that the enzymatic gelatinization measurement method they proposed was more accurate and sensitive compared to other methods because: (1) enzymatic methods are more substrate (glucose) specific and, therefore, prevent unnecessary carbohydrate degradations during analyses; and (2) glucoamylase converts gelatinized starch into glucose as opposed to other enzymes, such as aamylase or b-amylase which convert starch into larger molecules (maltose or dextrins). According to the method of Shetty et al. (1974), the degree of starch gelatinization can be estimated as % Starch gelatinization ¼ ½ðX BÞ 100=T where X¼percentage of starch removed by glucoamylase digestion, B¼correction factor (from digestion of intact starch by glucoamylase), and T¼total starch percentage (estimated by dispersion in 90% DMSO followed by glucoamylase digestion). Goering et al. (1974) used the enzymatic method of Shetty et al. (1974) to determine degree of starch gelatinization and discovered that the enzymatic method indicated significantly higher degrees of gelatinization compared to optical birefringence techniques. Most commonly available commercial glucoamylase preparations are contaminated with small amounts of a-amylase; this might cause ‘‘raw starch digestion’’ resulting in an overestimation of percent gelatinization. independent, with the crystallites being essentially ‘‘isolated’’ units in terms of their energy relationship with each other. They also suggested that starch polymer chains in the amorphous regions were susceptible to rearrangement under appropriate conditions; these rearrangements would impact the granule’s gelatinization characteristics. Considering the nonuniform nature of granular structure and distinctive roles of amylose and amylopectin, Marchant and Blanshard (1978) described the gelatinization as a ‘‘semi-cooperative’’ process. In its first phase, the semicooperative process describes a situation where a significant portion of granule crystallites are melted and the resultant changes induce the remaining crystallites, along with the entire granular structure, to disintegrate in a second phase. According to Marchant and Blanshard (1978),
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TABLE 5.2 Starch gelatinization (in excess water) parameters reported by various researchersa
Starch type
Regular corn High-amylose corn (70% amylose) Waxy corn Wheat Rice (15% amylose) Waxy rice Oat Potato Sweet potato Yam (Dioscorea alata) Cassava/Tapioca Black bean Chickpea Lentil Navy bean Smooth pea Pinto bean Proso millet Amaranth (Amaranthus hypochondriacus)
Onset ( C)
Peak ( C)
End ( C)
Enthalpy (J/g)
64.0 68.9
69.0 80.5
75.5 106.1
13.0 11.5
66.0 57.1 61.5 76.1 60.0 61.6 67.3 70.2 63.9 66.9 59.4 60.7 66.0 60.8 72.0 69.0 63.0
70.7 61.6 70.0 81.1 63.5 65.9 72.7 74.4 70.5 76.5 64.7 66.1 75.1 66.9 75.0 73.9 70.0
78.4 66.2 78.6 87.0 70.5 79.4 79.6 80.9 82.7 83.0 71.1 76.1 85.0 73.4 81.0 81.8 78.0
15.5 10.7 7.1 19.2 13.5 17.0 13.6 20.9 8.5 12.4 9.7 12.6 13.2 13.8 15.4 14.9 10.5
Adapted from Donovan (1979), Jackson (2003a), Jyothi et al. (2005), Cruz-Orea et al. (2002), Hoover et al. (2003), Hoover and Ratnayake (2002), Varavinit et al. (2003), Atichokudomchai et al. (2002), Liu et al. (2002), Valetudie et al. (1995), Shi and Seib (1992), Yanez et al. (1991), Tomita et al. (1981), Liu et al. (2005), Jane et al. (1999). a The values given in this table represent randomly selected data from many studies or a particular cultivar/ variety used in the study. Starch phase transition parameters, measured by DSC, may change depending on many factors, including variety, cultivar, hybrid, starch:water ratio in the sample, and scanning/experimental conditions.
energy characteristics of the crystallites in any given starch granule lie within a narrow range resulting in an approximately 1.5 C gelatinization range for a single granule. However, in a population of starch granules, the gelatinization range is much wider due to differences between granules. The temperature range required to gelatinize a starch sample, therefore, reflects the differences in energy characteristics of its constituent granules. According to Marchant and Blanshard (1978), during synthesis, it is possible for an individual starch granule to have a more consistent physiological environment to acquire a relatively uniform set of internal crystallites, which would be different in character from other individual granules. Although this argument does not directly affect their
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semi-cooperative gelatinization theory, it also should be noted that at any given time not one but many starch granules are being synthesized in the same physiological environment. Therefore, it is possible to have granules containing crystallites with similar characteristics in a given starch sample. Such an argument is also consistent with starch gelatinization as expressed by a differential scanning calorimetry (DSC) endotherm peak, which often is the result of many individual granules melting simultaneously. With the application of differential thermal analysis (DTA) and DSC to starch characterization and analysis (Stevens and Elton, 1977; Wootton and Bamunuarachchi, 1979), researchers had new tools to study the gelatinization process and associated granular structural changes. In the classical method to measure starch gelatinization using DSC, a small sample is heated with excess water in a hermetically sealed pan against a reference pan (see Box 3 for details). DSC allowed researchers to use a wide range of moisture levels to study gelatinization, which was impossible with birefringence studies. Moreover, with DSC, it was possible to express the heat energy required for granular structure phase transition during gelatinization.
C. Water availability theory Donovan (1979) studied starch gelatinization at both low and high moisture levels using DSC. He observed that as moisture levels were decreased (water:starch volume fractions less than 0.45) DSC endotherms become biphasic. The first (typical) endotherm decreased in area, and the second (high temperature) endotherm shifted to even higher temperatures as water content was decreased (Fig. 5.9). Donovan (1979) suggested that these two endotherms represented two distinct mechanisms by which ordered regions of starch granule undergo hydration facilitated phase transition. This explanation assumes that the crystalline regions are the same throughout the starch granule and that the phase transition mechanism depends only on moisture availability. Donovan (1979) argued that the single endotherm at excess water content occurs due to an order–disorder phase transition of starch granules. He further explained that granule swelling occurred due to water imbibition into amorphous regions first, which facilitated the ‘‘stripping’’ of starch chains from crystallite surfaces.1 ‘‘Stripping starch chains form the surfaces of 1
Starch crystallinity is caused by parallely arranged starch polymers which act as crystals. No evidence has been presented in published literature to describe the actual physical nature of ‘‘starch crystallites.’’ It should not be considered that starch granules contain ‘‘crystals’’ or ‘‘crystallite particles’’ formed by starch polymers. Starch crystallinity represents the relative arrangement of starch polymers in granules, not the presence of physical ‘‘crystals.’’
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BOX 3 DIFFERENTIAL THERMAL ANALYSIS AND DIFFERENTIAL SCANNING CALORIMETRY
DTA is a technique that involves heating or cooling a sample, compared with a reference, under identical conditions, and recording the differential temperature against time or temperature. Absorption or evolution of heat, caused by changes in the sample during heating/ cooling can be observed relative to the reference, which is normally an inert material. Although the DTA technique is widely used to investigate thermal transitions of different compounds, there were strong discrepancies and confusion related to establishing reaction kinetics based on DTA results (Kissinger, 1957; Reed et al., 1966; Thompson, 1966). DTA experiments on thermal behaviors of starch and other carbohydrates were first reported in 1950s (Morita, 1956a,b; Morita, 1957; Morita and Rice, 1955). With the development of analytical instruments, DSC operating based on heat flux (TA Instruments, New Castle, DE) and power compensation (Perkin-Elmer, Waltham, MA) mechanisms controlled by computers became more commonly available. DSC permitted precise control and measurement of energy changes during thermal transitions compared to older DTA instruments (Androsch et al., 2000; Danley, 2003; Dong and Hunt, 2001). The differences between DTA and DSC techniques and instrumentation have been well explained (Brown, 2002; Riga and Collins, 2000). Stevens and Elton (1971) published an early study using DSC to investigate the starch gelatinization process. They characterized a variety of starches and discovered that the DSC profiles reveal both qualitative (nature of water absorption into the granules, effects of damaged starch on thermal behavior, etc.) and quantitative (energy required to gelatinize) data. The DSC instruments that were available during the early 1970s used relatively large samples (500 mg) (Stevens and Elton, 1971), and manually controlled heating and cooling mechanisms required considerable time to complete several scans. Different types of thermal references, such as fine glass beads with silicone oil (Stevens and Elton, 1971); water or buffer (Donovan, 1979); empty sealed pan, that is atmospheric air (Eliasson, 1980; Wada et al., 1979; Wootton and Bamunuarachchi, 1979); and liquid paraffin (Evans and Haisman, 1982), were used in early DSC starch gelatinization experiments. The rationale for selecting a particular thermal reference type was usually unreported, and there was not an agreement on when and why a particular thermal reference should be selected. In order to obtain reliable starch thermal property data, Yu and Christie (2001) and Randzio et al. (2002) indicated that careful consideration of sample continued
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BOX 3
(continued )
preparation (weight or volume ratios of water and starch), pan type, and scanning conditions are extremely important. Numerous studies have been conducted on starch phase transitions using DSC as the primary or only experimental tool. Some theories and mechanisms suggested for starch phase transition have been proposed solely based on DSC experiments (Donovan, 1979; Evans and Haisman, 1982; Nakazawa et al., 1984; Pravisani et al., 1985; Randzio et al., 2002; Zhong and Sun, 2005). Not having a general consensus on a standard practice for conducting DSC experiments has created confusion and discrepancies in data interpretation; there is still an ongoing debate on what happens to starch structures and the molecular associations during specific stages of DSC enthalpic transitions.
crystallites’’ was merely a hypothesis for which there is no substantial direct experimental evidence. Donovan (1979) argues that when sufficient water is present at low temperatures, water is nonuniformly distributed within the starch granules and that there are high concentrations of water present near some crystallites. As these localized high concentrations of water are heated, Donovan suggested that the crystallites in nearby areas undergo polymer stripping from their surfaces, causing a single DSC endotherm. It could be possible that at low moisture levels, some crystallites melt at low temperatures by water-facilitated plasticization that allows optimum hydrogen bonding transfer from starch polymer clusters to water. Some crystallites would also melt at high temperature without the plasticizing effect of water; this may be a realignment of crystallites by hydrogen bond transfer among starch polymers. This theory does not explain, however, why low moisture levels facilitate melting of some crystallites as a group (representing a distinctly separate high-temperature endotherm) rather than just resulting in a broadened endotherm. In the presence of sufficient (i.e., excess) water, all the crystallites are pulled apart by swelling and therefore none remains to be melted at high temperatures, eliminating the high-temperature endotherms. Donovan used Flory’s crystallite melting theory (Flory, 1953) to explain these observations. Flory’s theory, discussed in Section IV.G, applies to perfect crystals at equilibrium. Starch, on the other hand, is semicrystalline, and the distribution of amorphous regions within the granule is not uniform. Donovan’s theory of crystallite melting (1979) by two different mechanisms at low moisture levels is appealing, and it is reasonable to assume that Donovan’s explanation refers more to removal of starch
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0.81
Endothermic heat flow
0.64
0.55
0.51
0.45 0.38
0.36
0.30
300 mcal/⬚C
0.28 50
60
70
80
90 100 110 120 130 140 Temperature (⬚C)
FIGURE 5.9 DSC profiles of potato starch at different water contents (volume fraction of water indicated next to each profile). Heating rate¼10 C/min. Donovan (1979), Phase transitions of starch-water system. Biopolymers, 18, 263–275. Copyright Wiley-VCH Verlag GmbH & Co. KGaA. Reproduced with permission.
polymers from crystallite arrangements due to increased temperature in the presence of water. Consequently, it cannot be argued that crystalline melting in starch granules during gelatinization is an absolute result of water availability (i.e., diluent), although water absorption by amorphous regions likely impacts subsequent structural changes during heating. Donovan’s results (1979) confirm the importance of water availability as a fundamental property in the phase transition process of starch granules.
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D. Crystallite stability theory Evans and Haisman (1982) investigated the gelatinization behavior of potato starch by studying starch granule equilibrium states with glycerol and sodium hydroxide solutions, using DSC and colorimetric (blue dextran exclusion) methods. They argued that starch gelatinization was related essentially to crystallite melting within starch granules and explained the biphasic endotherm (Section IV.C) as a result of melting least stable crystallites first, thereby removing some of the constraints that restrict the granule from absorbing additional external water. According to Evans and Haisman (1982), there are two phases of water in starch granules: (1) the ‘‘tightly’’ bound phase, approximately 20% of the amount, absorbed by granules; and (2) a ‘‘loosely’’ bound phase that is available for gelatinization. They found that the DSC measured onset temperature (To) increased rapidly below 0.6 g water per 1 g starch (29% water volume fraction) and stayed essentially constant between 0.6 and 2.0 g water per 1 g starch (29–57% water volume fraction) (Fig. 5.10). Changes observed in To and Tp, and the peak separation pattern
Temperature (⬚K)
400
380
Single peak
360
340
320
Shoulder developing Single peak into separate peak
1.0 2.0 Water content (g/g dry starch)
3.0
FIGURE 5.10 Change of gelatinization temperatures (initial¼, final¼o) of potato starch based on water content. Evans and Haisman (1982), The effect of solutes on the gelatinization temperature range of potato starch. Starch/Sta¨rke, 34, 224–231. Copyright Wiley-VCH Verlag GmbH & Co. KGaA. Reproduced with permission.
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that depended upon water content, were depicted as evidence for existence of more-stable (perfect) and less-stable (less-perfect) crystallites in starch granules. Evans and Haisman (1982) used Flory’s theory (1953) of polymer melting in the presence of a diluent to explain starch gelatinization. They assumed that Flory’s theory can be applied to the melting behavior of less-perfect crystals (starch). They also assumed that volume fractions in Flory’s equation should be based on granules rather than the entire starch–water system because once the starch granules are swollen to their maximum capacities, changes in the external medium (water) would not affect granule composition. Flory’s theory, however, applies to hypothetical perfect crystals at equilibrium (see Section IV.G for details). According to Evans and Haisman (1982), the gelatinization process starts with melting of the least stable crystallites (corresponding to the To in DSC endotherms) with subsequent progressive melting into more-stable crystallite domains. They also assumed that the melting of less-stable crystallites at lower temperatures releases constraints on morestable crystallites, and makes those crystallites and remaining crystallites melt faster at lower temperatures than they might ordinarily melt. This argument is essentially a combination of Marchant and Blanshard’s semicooperative theory (1978) and Donovan’s water availability theory (1979). Evans and Haisman (1982) also argued that the gelatinization temperature range (the difference between the onset and end temperatures) is determined by the distribution of least stable crystallites in the granules. They explained that the biphasic endotherm at low moisture levels was a result of cooperative melting of some crystallites in the presence of sufficient water followed by ‘‘true’’ melting of remaining crystallites in the absence of free water. They claimed that Donovan’s theory (1979) was invalid because the ‘‘stress’’ created by swelling of amorphous regions could not occur within a very narrow temperature range, and such a stress could not rapidly rise as crystals melt. According to more recent research (Ratnayake and Jackson, 2007; Sahai and Jackson, 1999; Vermeylen et al., 2006), it is quite obvious that an assumption that DSC endotherms represent crystallite melting during starch gelatinization process is not valid. If DSC endotherms of starch– water systems represent only crystallite melting, water absorption into starch granules should occur without any energy utilization. In reality, however, this is not the case. Despite the limited and reversible water absorption that occurs initially, much of the water absorption-driven starch granule swelling occurs only when starch–water mixtures are heated. This means that starch granules need energy to absorb water and swell, that is, it is an endothermic process. Evans and Haisman (1982), however, assumed not only that Flory’s theory can be applied to starch, which is semicrystalline and not in equilibrium during gelatinization, but also that the DSC endotherm is an absolute result of crystallite
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melting. There is sufficient recent research evidence (Ratnayake and Jackson, 2007; Zobel et al., 1988) to suggest that starch ‘‘crystallinity’’ measured by XRD and DSC do not measure the same or similar phenomena, but are assessing two different phenomena that take place during the gelatinization process. By the 1980s, it had been established that starch gelatinization involved starch granule hydration and swelling, birefringence loss, crystallite melting or loss of crystallinity, polymer leaching, and irreversible loss of granular structure. It had been found that structural and thermodynamical changes in the starch granules during gelatinization could be monitored by various analytical techniques such as the birefringence end point method, viscosity measurements, amylose-iodine binding methods, enzymatic digestibility, nuclear magnetic resonance, light scattering/ extinction, solubility and sedimentation methods, colorimetric methods (Congo red and blue dextran), XRD, and DSC (Ghiasi et al., 1982; Lund, 1984; Shiotsubo, 1984; Shiotsubo and Takahashi, 1984; Zobel, 1984).
E. Sequential phase transitions (first amorphous and then crystalline) theory Nakazawa et al. (1984) conducted low-temperature treatment-in-water2 studies on regular rice starch using DSC and detected that at temperatures below To, the amorphous phase became mobile but the crystalline region did not. Some amorphous portions were restructured into morestable forms. Tp shifted to higher temperatures as holding times increased when the sample was treated a below its To (Fig. 5.11). They also observed that the transitions associated with granular restructuring were detected as a sharp peak at high Tp (Fig. 5.11) but they were not detectable by XRD (Fig. 5.12) and concluded that the structural features detected by DSC and XRD were independent of each other. In regular rice starch, they observed a biphasic endotherm in DSC when starch–water mixtures are held for certain periods at constant temperatures. These endotherms merged into a single endotherm with a higher Tp, when held for longer times (Fig. 5.11). Nakazawa et al. (1984) suggested that starch granules are not at equilibrium with water during gelatinization, especially when DSC is used to determine gelatinization properties. To obtain an equilibrium condition they annealed rice starch granules in water for different time and temperature combinations. DSC gelatinization temperatures (To and Tp) were observed shifting to higher temperatures with an increasing degree of annealing (Fig. 5.13). According to Nakazawa et al. (1984) the energy 2
Some of these low-temperature treatments meet the hydro-thermal conditions of classic starch annealing procedures. Annealing is normally conducted below gelatinization temperatures in the presence of excess water (Jacobs and Delcour, 1998; Tester and Debon, 2000; Ozcan and Jackson, 2003).
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50%
5m Endothermic heat flow
0
30 m
0.0145 mcals/sec 18 h
45 h 50
60
70 80 Temperature (⬚C)
90
FIGURE 5.11 DSC endotherms of rice starch annealed at 70 C (times are indicated next to each profile) [adapted from Nakazawa et al. (1984); reproduced with permission from Japan Science and Technology Agency].
provided during lower temperature treatments rearranged the starch polymers, and at high temperatures (75 C) the crystallite structures became completely amorphous (Fig. 5.12). Their results also showed that approximately 80% amylopectin-containing (20% amylose) native starch required more energy to become amorphous during hightemperature treatments compared to 100% amylopectin-containing starch. They also concluded that increased water facilitates the mobility of starch molecules so that the crystalline to amorphous transition takes place at lower temperatures. Nakazawa et al. (1984) argued that when starch–water mixtures (30–50% starch) are held at a certain temperature (55–80 C), for a certain period (0–45 h), and depending on the time–temperature combination, starch granules increase their amorphous portion and decrease their crystalline portion. These amorphous and crystalline phases melted sequentially during DSC phase transition experiments. Their experiments
Wajira S. Ratnayake and David S. Jackson
Relative intensity (counts per sec)
246
Native
40 ⬚C, 100 hr
55 ⬚C, 64 hr
75 ⬚C, 22 hr
Diffraction angle (q/2q)
FIGURE 5.12 X-ray diffraction patterns of annealed rice starch (treatment conditions are indicated above each profile) [adapted from Nakazawa et al. (1984); with permission from Japan Science and Technology Agency].
showed that when starch–water mixtures are held above gelatinization temperatures (75 C), the second endotherm disappeared (Fig. 5.13). Nakazawa et al. ’s argument (1984), however, does not confirm their own sequential phase transition theory. If longer times and high (above gelatinization) temperatures decreased the crystalline:amorphous ratio within starch granules, the resulting granules should show single DSC endotherms at correspondingly lower transition temperatures (because supposedly amorphous regions undergo ‘‘gelatinization’’ or phase transition at lower temperatures). Their DSC results (Fig. 5.14), however, indicated that the peak temperature (Tp) increased and the enthalpy changed without a particular pattern with increasing subgelatinization temperatures. This observation could be better explained by the crystallite stability theory (Evans and Haisman, 1982). Colonna and Mercier (1985) suggested a theory, similar to sequential phase transition theory of Nakazawa et al. (1984), after studying a variety of starches using DSC and amylography (viscosity measurements during heating/cooling). They argued that the low-temperature endotherms of
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30% 0
Endothermic heat flow
5m
15 m
0.0145 mcal/s
1.5 h
45 h 50
60
70
80
90
Temperature, ⬚C
FIGURE 5.13 Effect of annealing (degree of annealing increases along X axis) on DSC transition temperatures of rice starch held at 55 C for specific times (indicated next to each profile) (Nakazawa et al., 1984; reproduced with permission from Japan Science and Technology Agency).
multipeak DSC profiles represented a ‘‘normal gelatinization transition’’ (although it was not fully outlined in the published report, it could be assumed that this transition occurred in the presence of sufficient water to act as the diluent for some crystallites), and endotherms at high temperatures correspond to ‘‘true melting’’ of starch crystallites. Colonna and Mercier (1985) attributed the first transition to disorientations of the amorphous region without crystallite melting (Fig. 5.15). However, in their illustration (Fig. 5.15), the bidirectional arrows associated with the second transition seem surprising since crystallite melting during gelatinization is irreversible unless it undergoes retrogradation (a recrystallization phenomena that is not thought to include the same crystallization sites as those found in native granules). Colonna and Mercier (1985) indicated that at sufficient water contents, starch polymers in amorphous regions increased their mobility, causing irreversible swelling in areas
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A
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90 Peak temperature, ⬚C
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75 80 85
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FIGURE 5.14 (A) Change of Tp based on annealing temperature and time. (B) Change of enthalpy based on annealing temperature and holding time (Nakazawa et al., 1984; reproduced with permission from Japan Science and Technology Agency).
partially restricted by crystallites. Upon a further increase in temperature, crystallite melting, considered independent of swelling, allowed more polymer chain movement to destabilize the entire granular structure. A similar theory was suggested by Pravisani et al. (1985), who investigated potato starch gelatinization using DSC and microscopy. Nakazawa et al. (1984) did not consider granular structural changes that take place
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Intact structure
Mobile amorphous phase and intact crystalline phase
Complete disintegration of structure
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1st transition
2nd transition
3rd transition
FIGURE 5.15 Phase transitions of starch structures during gelatinization. Reprinted from Phytochemistry, Vol.24, Colonna and Mercier (1985), Gelatinization and melting of maize and pea starches with normal and high-amylose genotypes, Pages 1667–1674, with permission from Elsevier.
during annealing (holding starch–water mixtures for long durations at specific time–temperature combinations)3 to explain their observations. There is sufficient evidence in the literature to support the observed starch structural changes, especially in the intermolecular bonds, during annealing (Kohyama and Sasaki, 2006; Qi et al., 2004; Tester et al., 2001). Although Nakazawa et al.’s results (1984) do not adequately explain the nature of sequential phase transition of amorphous and crystalline portions in starch granules during gelatinization, their observations highlight 3 The term ‘‘annealing’’ is not completely applicable to all the treatments they used because some of the holding temperatures are above the gelatinization temperature of rice starch.
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the limitations of using DSC alone to determine gelatinization properties of starch. DSC explains the ‘‘net result’’ of what takes place during gelatinization in terms of energy dynamics, but does not provide a complete picture of the process.
F. Three-stage (partial melting, recrystallization, and total melting) phase transition theory Biliaderis et al. (1986) argued that the previously mentioned gelatinization theories on solvent (water) facilitated starch crystallite melting were imperfect because they did not consider the semicrystalline nature of starch. According to Biliaderis et al. (1986), starch, being a semicrystalline material, undergoes reorganization during heating in DSC experiments. During slow DSC scans (between 1 and 5 C/min) and after the onset (To) of the first peak, mobile polymers rearrange in the crystalline region. They also postulated that such a process could take place during annealing. They found that the biphasic endotherm observed by others could be forced to merge into a single endotherm at high heating rates (20– 30 C/min). Biliaderis et al. (1986) argued that the reorganization of starch polymers during gelatinization was similar to annealing, and it was the reason for biphasic endotherms at low moisture levels. Therefore, according to these workers, the starch gelatinization process is composed of three phases: partial melting, recrystallization, and final melting. They argued that this three-phase theory better corresponded to nonequilibrium crystallite melting during gelatinization. They also reported that both annealing and recrystallization processes depended on moisture content and these processes became less prominent at high moisture levels. Biliaderis et al. (1986) also reported that DSC transitions did not truly represent initial semicrystalline structure phase transitions of starch, but represented a composite result of melting and reorganization that occurred during thermal analysis. Polymer mobility in the bulk amorphous region was restricted by its close association with some parts of crystalline domains. In addition, they suggested that the intercrystalline amorphous region (Fig. 5.5) does not carry ‘‘normal’’ amorphous characteristics due to strains imposed by crystalline domains. They argued that these structural differences associated with starch’s semicrystalline nature, compared to a completely crystalline material, explained the multiple melting profiles observed at low moisture levels. If this is the sequence of changes that takes place during starch gelatinization, applicability of the ‘‘fringed micelle’’ model (Billmeyer, 1984; Flory, 1953; Slade and Levine, 1987, 1995; Wunderlich, 1973) becomes invalid because that model assumes, for gelatinization purposes, that starch structures are two-phase systems in which crystallites are dispersed in a homogenous amorphous matrix and that the two phases behave independently.
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FIGURE 5.16 Spontaneous ordering of linear polymers in a semicrystalline material. Bundles of dark lines represent crystalline regions. Reprinted from Flory (1953): Paul J. Flory, Principles of Polymer Chemistry, Copyright # 1953 Cornell University Press and Copyright # 1981 Paul J. Flory. Used by permission of the publisher, Cornell University Press.
G. Application of Flory’s theory to explain starch gelatinization Numerous attempts (Cruz-Orea et al., 2002; Donovan, 1979; Evans and Haisman, 1982; Lelievre, 1973) have been made to explain starch gelatinization and phase transition using Flory’s theory of the melting point depression (Flory, 1953) in semicrystalline systems. According to Flory (1953), polymers having regular chain structures undergo spontaneous ordering referred to as crystallization (Fig. 5.16). This takes place when chain axes are aligned parallel to one another forming bundle-like structures (Fig. 5.16). The individual polymer chains may occur in a fully extended or a less-extended (spiral) configuration. Ordering of polymers to form crystals, sometimes, may not be accepted as genuine crystallization. In semicrystalline material, submicroscopic crystallites occur (imbedded) in a residual amorphous matrix. The proportion (%) of crystalline material depends on crystallization conditions (Flory, 1953). The ‘‘degree of perfection’’ of crystallites is variable and depends on annealing conditions. According to Flory (1953), annealing is keeping the polymer at a temperature slightly below its melting temperature. When a polymer is annealed, depending on the time, temperature, and pressure combination, it is possible to obtain a range of polymer interactions from amorphous to crystalline and intermediate-level interactions between the two ‘‘extremes.’’ These intermediate states are called ‘‘mesomorphic
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states,’’ which are characteristic to semicrystalline material and are metastable (i.e., not at an equilibrium state) in nature. Annealing is known to both quantitatively and qualitatively improve crystalline order (Flory, 1953). According to Flory (1949, 1953), the crystallite perfection is a measure of the amount of intermediate material located at the crystalline to amorphous transition boundary. Boundary regions or ‘‘intermediate-order’’ material presumably melts at lower temperatures than relatively more highly ordered internal regions (Flory, 1953). More perfect crystals have negligible amounts of intermediate-order material at the crystalline–amorphous transition region, resulting in a sharp melting point. Apart from the intermediate material at transition regions, crystallite size also plays an important role in melting point values. Small crystallites result in depressed melting points due to surface free energy (Flory, 1953). According to Flory (1953), melting point sharpness and reproducibility are indicators of the perfect crystalline state. The melting temperature (Tm) was described as the equilibrium melting temperature for a hypothetical perfect macroscopic crystal. When Flory’s theory (1953) of melting point depression is applied to starch gelatinization (or phase transition) in the presence of water, the situation can be described as follows. At equilibrium state, the chemical potentials between amorphous (mu) and crystalline repeating units (mu) of two phases are equal: ð5:1Þ mu ¼ mou The temperature at which this condition is satisfied is known as melting temperature or melting point (Tm). Melting temperature depends on the composition of the amorphous phase. In a starch–water system, water acts as a diluent and then Tm is regarded as the temperature at which the specific composition is similar to that of a standard solution. If the amorphous phase of starch is pure, the chemical potential (mu) is the same as its chemical potential at the standard state (mu ) at the same temperature and pressure: mu mou ð5:2Þ The chemical potential of the polymer is affected by ‘‘impurities’’ such as solvents or copolymerized units. For an equilibrium condition in the presence of water as the diluent, the melting temperature of starch (Tm) would be lower because mu in the presence of diluent is less than mu. For the starch–water system at equilibrium, the difference between the chemical potentials of the crystalline phase and the phase in the standard state (pure polymer at the same temperature and pressure) must be equal to the decrease in chemical potential of the polymer unit in solution relative to the same standard state (Flory, 1953). By considering the free energy of fusion per repeating unit and volume fraction of water (diluent), the
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melting point depression due to the presence of diluent can be expressed as follows (Flory, 1953): 1 1 R Vu o ¼ ðV1 wV12 Þ Tm Tm DHu V1
ð5:3Þ
¼ equilibrium melting where Tm ¼ actual polymer melting point, Tm temperature, R ¼ gas constant, DHu ¼ enthalpy of fusion per repeating unit, Vu¼molar volume of polymer repeating unit, V1 ¼ molar volume of diluent, and x ¼ Flory-Huggins interaction parameter. It is believed by most researchers, for the purpose of theoretical explanations, that the amorphous and crystalline domains of starch granules present in relatively mutually exclusive domains and the amorphous domains are not in an equilibrium state. Moreover, it cannot be assumed that water enters amorphous regions of the granules ‘‘effortlessly’’ (if it is the case, starch granules should, at least partially, disperse in cold water). Flory’s theory is valid for systems under constant pressure. When DSC is used to evaluate starch–water systems, increasing heat also increases the pressure due to the increase in water temperature within the hermetically sealed pans. When starch is gelatinized in excess water, the water first acts as a diluent in the amorphous phase. When excess water is available, the fluid (or amorphous) phase never becomes a saturated phase; saturation is also a requirement of Flory’s theory of melting point depression in semicrystalline material in the presence of a diluent. Polymers within the crystalline and amorphous regions are not homogenous (in terms of both polymer type and polymer chain length). The presence of homogenous polymer composition is a requirement to apply the theory to explain melting point depression. Flory’s theory is applicable to a pure crystal of infinite size. Starch ‘‘crystals’’ are made up of much smaller size amylopectin branch interactions, which may be described as ‘‘crystallite blocks’’ (Takahashi and Yamada, 1998). The size of these ‘‘crystals’’ may depend on the amylopectin branch chain lengths (Jane et al., 1999). These concerns raise questions on the applicability of Flory’s theory to explain starch gelatinization.
V. STARCH ANNEALING AND ITS RELATIONSHIP TO GELATINIZATION Starch annealing involves heating starches with sufficient hydration below their To to facilitate molecular mobility (Tester et al., 2001). Annealing is defined as ‘‘a physical treatment that involves incubation of starch granules in excess (>60% w/w) or at intermediate (40–55% w/w) water content during a certain period of time at a temperature above the glass
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transition temperature, but below the gelatinization temperature (Gough and Pybus, 1971; Jacobs and Delcour, 1998; Zeleznak and Hoseney, 1987). During annealing, granule composition remains essentially unchanged, but Tp increases significantly (Gomes et al., 2004; Kohyama and Sasaki, 2006; Tester and Debon, 2000; Tester et al., 2001; Fig. 5.13). Qi et al. (2004) studied the effect of annealing on gelatinization properties in an A-type cereal starch (waxy barley) using a-amylase and 2 M hydrochloric acid hydrolyses to remove amorphous domains, thus isolating crystalline regions. Annealing (48 C for 7 days in excess water) increased the DSC melting enthalpy and narrowed the phase transition temperature range. Annealing starch in the presence of a-amylase did not significantly change the DSC melting enthalpy, but narrowed the transition temperature range. Onset, peak and end temperatures also increased in enzymetreated samples compared to their native counterparts. Qi et al. (2004) suggested that annealing caused ordered crystalline domains and they entered into ‘‘more optimally registered’’ structures within the granules. This optimization of crystalline order, they concluded, controls the gelatinization properties of annealed starch by restricting hydration. However, it is not clear what structural changes or modifications take place within granules during so-called optimization or ‘‘optimal registration’’ processes. Other recent reports (Kohyama and Sasaki, 2006) also indicate that changes occur within crystalline regions during annealing. Marchant and Blanshard (1978) reported that starch polymer chains in the amorphous regions were susceptible to rearrangement under appropriate conditions, and that such rearrangements significantly affected the thermal behavior of starch granules. The nature of structural changes taking place inside starch granules during annealing is still under debate. The results from DSC studies on starch annealing could be better explained by assuming an existence of nonmutually exclusive domains of crystalline and amorphous phases in starch. Most of the current theories on starch structure, gelatinization, and annealing assume discrete crystalline and amorphous domains in starch granules. Jenkins and Donald (1995) suggested that amylose, which is presumed to be mainly in the amorphous regions of the starch granules, could cocrystallize with amylopectin in the crystalline domains. If it is assumed that the amorphous and crystalline phases ‘‘blend’’ into each other gradually within an ‘‘intermediate’’ phase that has a semicrystalline structure (i.e., a crystalline structure ‘‘disturbed’’ by amorphous phase), the starch annealing phenomena could be explained differently. In the intermediate phase between the crystalline and amorphous domains, portions of amylopectin molecules are kept from making crystallites by interfering amylose molecules (or amorphous phase). When starch in excess water is heated below gelatinization temperatures (annealing treatment conditions), water first enters the amorphous regions creating increased polymer (amylose)
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Crystalline phase Annealing Intermediate phase Amorphous phase
Increased crystalline and reduced intermediate phase volumes Additional annealing
Intermediate phase substantially reduced or eliminated
FIGURE 5.17 Proposed theory for the existence of an intermediate phase between amorphous and crystalline domains to explain starch annealing.
mobility. With increased annealing (i.e., treatment intensity), the polymers in the intermediate phase become more mobile. This increased polymer mobility reduces the interruptions to the crystalline phase and, with a potential decrease in the intermediate phase, the crystalline phase increases in size/volume yielding ‘‘more perfect crystallites’’ (the term used by many researchers to describe this phenomenon) (Fig. 5.17). This increased ‘‘perfection’’ and increased volume of the crystalline phase results in the observed larger, and sharper DSC enthalpies at higher peak temperatures in annealed starches (Fig. 5.13), compared to their native counterparts. In other words, ‘‘imperfect’’ molecular organizations at this hypothesized intermediate phase result in the wider and smaller DSC enthalpies seen at low peak temperatures (Tp) in native (nonannealed starches).
VI. GLASS TRANSITION AND GELATINIZATION When an amorphous material exists in a glassy state, it is hard and brittle. In a rubbery state, the material is soft and pliable. An amorphous material, at solid state (also referred to as glass), does not flow, but the molecules are randomly distributed as if they were in liquid state. When this ‘‘glass’’ is heated, it softens and eventually becomes a fluid. However, this is not a first-order transition and therefore occurs over a range of temperatures called the glass transition temperature (Tg). The state
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Temperature
Tg
Rubbery
Glassy
% solids
FIGURE 5.18
Glass transition diagram for an amorphous material.
(glassy or rubbery) of an amorphous material depends on its moisture content. When the corresponding plasticizing moisture content (Wg) increases (or the proportion of solids decreases), the glass transition temperature (Tg) decreases. Fig. 5.18 is commonly used to illustrate glass transition of an amorphous material. Slade and Levine (1991) proposed ‘‘an idealized state diagram’’ for an aqueous solution of a hypothetical, glass forming, small carbohydrate (glass forming, small carbohydrates are amorphous) to illustrate the critical locations of solute-specific subzero Tg (Tg0 ) and the maximum practical amount of plasticizing moisture (Wg0 , which is calculated as [mass of soluteunfrozen water]/[weight % of water]) (Fig. 5.19). According to this theory, the kinetics of all constrained relaxation processes (such as translational and rotational diffusion) governed by the mobility of a waterplasticized polymer matrix in a glass forming system vary between distinct temperature/structure domains. These domains are divided by the glass transition. The material (water, solute mixture) below Tg is in the glassy solid state of very low mobility and very slow diffusion. At above Tg, but below Tm, the material is at a viscoelastic, rubbery liquid state of increased mobility and diffusion. It has been suggested that glass transition is an important physicochemical event that controls the phase transition process of starch (Biliaderis, 1998). According to Biliaderis (1998), the ‘‘fringe-micelle’’ model (Fig. 5.16) does not permit assignment of a definite Tg for most starches. This is because the change in heat capacity during phase
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Temperature
Tgsolute
TmH2O
Tg⬘
Tg⬘ H2O
Wg⬘ Water
FIGURE 5.19 Schematic state diagram of temperature vs % weight of water for an aqueous solution of a hypothetical, glass-forming, small carbohydrate. Reprinted from Critical Reviews in Food Science and Nutrition, Vol.30, Slade and Levine (1991), Beyond water activity: Recent advances based on an alternative approach to the assessment of food quality and safety, Pages 115–359, with permission from Taylor and Francis (http:// www.informaworld.com).
transition sometimes does not follow the change in crystallinity, although there are reports indicating that Tg increases with increasing crystallinity (Jin et al., 1984; Kalichevsky et al., 1992; Lim et al., 2000; Zeleznak and Hoseney, 1987). Tg also depends on the moisture (water) content of the starch–water system (Mizuno et al., 1998; Tananuwong and Reid, 2004; Zeleznak and Hoseney, 1987); increasing water contents decreases Tg (Fig. 5.19) in a high-moisture system. In a low-moisture system (8–30% moisture), Tg decreases with the increasing moisture content (Chung et al., 2002). It is generally known that the glass transition is not always affected by changes in water content (starch:water ratio) because the plasticizing effect of water reaches a plateau after approximately 50% water (Huang et al., 1994; Lim et al., 2000; Tananuwong and Reid, 2004). Zeleznak and Hoseney (1987), contradicting the report of Maurice et al. (1985) and Biliaderis et al. (1986), suggested that the glass transition occurs at considerably lower temperatures than To. The argument was that the Tg
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observed by previous authors was the sharp volume increase due to crystalline melting detected by TMA. It is obvious that several factors, mainly the moisture content and degree of relative crystallinity, influence the Tg of a given starch. Matveev et al. (1998) studied glass transition in eight different starches and suggested that the observed positive changes in heat capacity during starch ‘‘melting’’are a sum of both the heat capacities of hydration and glass transition. They also hypothesized that the arrangement of amylose within the crystalline domains of starch granules, that is, the nature of amyloseamylopectin cocrystallization, is also responsible for the observed differences in Tg among different starches. It could be argued that the relative amounts of amylose and amylopectin in a given starch also influence the glass transition process. Relationships between the proportions of amylose/amylopectin and glass transition phenomenon have not been studied in detail. In food processing and other operations that use starch as a raw material, gelatinization takes place under conditions that make glass transition and related effects insignificant. Glass transition of high-moisture starch–water systems takes place below freezing (0 C) temperatures and in low-moisture systems well below room temperature (<25 C).
VII. CONTRADICTING THEORIES: WHAT IS GELATINIZATION? The ordered state of the ‘‘regular’’ starch granule is adequately explained and well documented in the literature. Current theories, however, do not adequately explain changes in granular structure during gelatinization, especially in high amylose and waxy starches. Microscopic studies have revealed the presence of granular ‘‘sacs’’ or ‘‘ghosts’’ in starch–water dispersions after gelatinization (Debet and Gidley, 2007; Derek et al., 1992; Gotilieb and Capelle, 2005). These ‘‘ghosts’’ or granule sacs undoubtedly influence the rheological and functional properties of gelatinized starch solutions. There is very limited information available on the transitions that take place from native granule to ‘‘ghosts’’; their structural and chemical composition is poorly characterized. Sahai and Jackson (1999) studied the gelatinization behavior of regular cornstarch granules with differing sizes/densities using DSC. They found that DSC transition temperatures did not change significantly among different granule size/density fractions of cornstarch. However, melting enthalpies were different among fractions, indicating differences in granular structures. They also observed a decrease in the onset (To) and peak (Tp) with decreasing moisture levels (from 80% to 50%), and suggested that the ‘‘least stable crystallites melt first’’ theory provided in previous reports (Donovan and Mapes, 1980; Evans and Haisman, 1982) did not
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adequately explain starch gelatinization behaviors. In addition, studies on annealed starch fractions showed that the disappearance of secondary transitions in DSC were not clearly explained by the three-phase transition theory outlined by Nakazawa et al. (1984) and Biliaderis et al. (1986). Sahai and Jackson (1999) concluded that enthalpic transitions at various moisture levels did not represent a single phenomenon, but numerous changes occurring simultaneously. They further reported that one or more of these changes are predominantly responsible for DSC patterns at a given moisture level, and therefore any single prevailing theory is inadequate to explain starch gelatinization process. There are numerous other reports published on starch phase transitions during gelatinization (Karlsson and Eliasson, 2003; Liu et al., 2002; Randzio et al., 2002; Tolstoguzov, 2003; Varavinit et al. 2003, Zhong and Sun, 2005). These studies reemphasize various aspects of previously published starch gelatinization theories. The explanations given to account for various thermal behaviors of starches, however, are inconsistent. This highlights the need for new approaches to explain starch gelatinization. Different starches undergo phase transitions differently. The nature, extent, and sequence of structural changes during phase transition depend on the original (native) structure, amylose to amylopectin ratio, and other polymer characteristics that can be attributed to the botanical source of a given starch and its growth environment. A universally acceptable model must be sufficiently comprehensive and flexible to explain the differing gelatinization processes of various starches. The mechanism of starch phase transition partially depends on the amount of available water. It appears that the nature of structural changes that occur within starch granules at ‘‘low’’ (less than 65% v/v) water is different from what takes place at high or excess (above 65% v/v, generally around 80% v/v water is considered as ‘‘excess water’’ in DSC experiments) water levels. As a result, the same theory or model may not be suitable to describe what takes place at different water levels. Alternatively, it could be argued that the nature of the changes that take place in the starch granule structure during gelatinization are highly influenced by the amount of diluent (water) available. Depending on the amount of water present, the rates and extents of some mechanisms involved may change leading to contrasting observations at low- and high-water gelatinization experiments. Some reports, such as by Donovan (1979), suggest that there is a continuum of change in the gelatinization mechanism rather than a sudden change between low- and high-water systems (Fig. 5.9). Starch gelatinization cannot be fully explained using DSC as the only experimental tool. As discussed in previous sections, DSC measures the net endothermic transitions that take place during starch gelatinization. It is known that the DSC parameters obtained for the same sample depend on heating rate (Fig. 5.20; Shiotsubo and Takahashi, 1984); transition
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Calorimeter power/mV mg−1 K−1
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Heating rate 2.3 K min−1
10
1.3 K min−1
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1 K min−1 0.1 K min−1 55
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FIGURE 5.20 Change in DSC enthalpy depending on heating rate (Shiotsubo and Takahashi, 1984; reproduced with permission from Japan Science and Technology Agency).
temperatures (To, Tp, and Tc) increase with increasing heating rates. It is not possible to determine an appropriate or standard heating rate in DSC because very slow heating rates might impose an annealing-like effect on granules before gelatinization, and a very fast heating rate would increase the lag between the measured and the actual sample temperature. The use of different heating rates, starch:moisture ratios, and instrumentation limitations have added more confusion in interpreting starch gelatinization using DSC data. Starch phase transitions occur in a wide temperature range. The phase transition process starts at temperatures as low as 35–40 C, depending on the type of starch. In contrast to what was previously believed, it is now understood that amylose and/or amorphous phases also play significant roles in the phase transition process (Ratnayake and Jackson, 2007; Vermeylen et al., 2006). Theories that describe gelatinization and phase transition in terms of crystallite melting, therefore, are unlikely to adequately explain the phenomena. In summary, it is evident that starch gelatinization is not an absolute result of crystallite melting. Hence, it should not be considered a simple order-to-disorder phase transition of starch structures.
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Atichokudomchai, N., Varavinit, S., and Chinachoti, P. (2002). Gelatinization transitions of acid-modified tapioca starches by differential scanning calorimetry (DSC). Starch/Sta¨rke. 54, 296–302. Ball, S. G. and Morell, M. K. (2003). From bacterial glycogen to starch: Understanding the biogenesis of the plant starch granule. Ann. Rev. Plant Biol. 54, 207–223. Banks, W. and Greenwood, C. T. (1975). In ‘‘Starch and Its Components.’’ Edinburgh University Press, Edinburgh. Biliaderis, C. G. (1998). Structures and phase transitions of starch polymers. In ‘‘Polysaccharide Association Structures in Food’’ (R. H. Walter, ed.), pp. 57–168. Marcel Dekker Inc., New York, NY. Biliaderis, C. G., Page, C. M., Maurice, T. J., and Juliano, B. O. (1986). Thermal characterization of rice starches: A polymeric approach to phase transition of granular starch. J. Agric. Food Chem. 34, 6–14. Billmeyer, F. W. (1984). In ‘‘Textbook of Polymer Science,’’ 3rd Wiley Interscience, New York, NY. Blanshard, J. M. V. (1979). Physicochemical aspects of starch gelatinization. In ‘‘Polysaccharides in Foods’’ (J. M. V. Blanshard and J. R. Mitchell, eds.), pp. 139–152. Butterworths & Co. (Publishers) Ltd., London. Blanshard, J. M. V. (1987). Starch granule structure and function: A physicochemical approach. In ‘‘Starch: Properties and Potential’’ (T. Galliard, ed.), pp. 16–54. John Wiley and Sons, Chichester. Blennow, A. (2004). Starch bioengineering. In ‘‘Starch in Food’’ (A.-C. Eliasson, ed.), pp. 97–127. CRC Press, Boca Raton, FL. Bogracheva, T. Y., Ring, S., Morris, V., Lloyd, J. R., Wang, T. L., and Hedley, C. L. (1997). The use of mutants to study the structural and functional properties of pea starch. In ‘‘Starch: Structure and Functionality’’ (P. J. Frazier, A. M. Donald, and P. Richmond, eds.), pp. 230–237. Royal Society of Chemistry, Cambridge. Bogracheva, T. Y., Cairns, P., Noel, T. R., Hulleman, S., Wang, T. L., Morris, V. J., Ring, S. G., and Hedley, C. L. (1999). The effect of mutant genes at the r, rb, rug 3, rug 4, rug 5, and lam loci on the granular structure and physico-chemical properties of pea seed starch. Carbohydr. Polym. 39, 303–314. Brown (2002). Introduction to thermal analysis: Techniques and applications. Second edition. Springer-Verlag, New York. Buttrose, M. S. (1962). The influence of environment on the shell structure of starch granules. The Journal of Cell Biology. 14, 159–167. Cairns, P., Bogracheva, T. Y., Ring, S. G., Hedley, C. L., and Morris, V. J. (1997). Determination of the polymorphic composition of smooth pea starch. Carbohydr. Polym. 32, 275–282. Chandrasekaran, R. (1998). X-ray diffraction of food polysaccharides. In ‘‘Advances in food and nutrition research’’. Vol. 42. (S. Taylor, ed.), pp. 131–210. Academic Press, San Diego, CA. Cheetham, N. W.H. and Tao, L. (1998). Variation in crystalline type with amylose content in maize starch granules: X-ray powder diffraction study. Carbohydrate Polymers. 36, 277–284. Chung, H.-J., Lee, E.-J., and Lim, S.-T. (2002). Comparison in glass transition and enthalpy relaxation between native and gelatinized rice starches. Carbohydr. Polym. 48, 287–298. Cleven, R., van den Berg, C., and van den Plas, L. (1978). Crystal structure of hydrated potato starch. Starch/Starke 30, 223–228. Collison, R. (1968). Swelling and gelatinization of starch. In ‘‘Starch and Its Derivatives’’ ( J. A. Radley, ed.), pp. 168–193. Chapman and Hall Ltd., London. Colonna, P. and Mercier, C. (1985). Gelatinization and melting of maize and pea starches with normal and high-amylose genotypes. Phytochemistry 24, 1667–1674.
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INDEX A N-Acetyl-galactosamine, 134, 135 Acidic ginsenosides in roots, UV detection, 50–54 Acrylonitrile–psyllium grafted copolymer, 214 Adherence (pathogenic bacteria), 101–161 common mechanisms of, 117–128 inhibitors, 103, 128–139 kinetics, 105–106 specificity, 107 Adhesins, 114–117 adhesion mechanisms, 117–128 analogs as anti-adhesins, 129, 132–133 Adipocytes and ginsenosides, 78 Afa adhesins, 128 Afimbrial adhesins, 116–117 Airway see Respiratory system Alkaline treatment of psyllium, 214 Allergy ginsenoside effects, 71–72 occupational causes, 169–176 psyllium-induced, 204 reducing, 206, 215 AlpA and Alp B, 121 Alveolitis, extrinsic allergic see Hypersensitivity pneumonitis a-Amylase glucoamylase preparations contaminated with, 236 respiratory allergy, 175–176 Amylopectin polymers in starch, 224 high-amylopectin starches, 224 structure, 226–228 Amylose polymers in starch, 224 high-amylose starches, 224 structure, 226–227 Anaphylaxis occupational causes, 170 passive cutaneous, ginsenosides and, 72 Angiogenesis, tumor, ginsenoside effects, 68
Anisotropy, starch granules, 226–227 Anti-adhesive agents, 103, 128–139 Antibodies (use) allergen sensitization tests, 167, 168 anti-adhesin, 130 anti-ginsenoside, 62–63 see also Vaccines Anticancer effects see Cancer Antihypertensive effects, ginsenosides, 72–73 Anti-inflammatory effects of ginsenosides, 71–72 Apoptosis and ginsenosides, 66 Asthma (occupation-related), 169–177 allergic, 169–176 irritant-induced, 172, 176–177 work-aggravated, 176–177 Atherosclerosis and ginsenosides, 72–73 Atmospheric pressure chemical ionization (APCI), ginsenosides, 57 Auf fimbriae, 128 B BabA, 120, 120–121 Bacteria intestinal (commensal) see Microflora pathogenic, adherence see Adherence Baking industry, respiratory allergy, 175–176 BFP (bundle-forming pili) enterohemorrhagic E. coli, 126 enteropathogenic E. coli, 122–123 Bifidobacteria, anti-adhesin activity, 131, 136, 138 Bile acids and psyllium, 201 Birefringence of starch granules, 225–228 loss in gelatinization, 233–235 Bowel see Intestine Breast cancer ginsenoside effects, 65, 66 psyllium effects, 202–203 Bronchiolitis obliterans/BO (occupationrelated), 178–186
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270
Index
Bronchiolitis obliterans/BO (occupationrelated) (cont.) flavoring-related, 164, 179–186 medical test results, 171 site/description/causes, 173 Bronchitis (occupation-related chronic), 170, 177 medical test results, 173 site/description/causes, 173 Bronchodilator trial, 166, 168 in occupational lung disease, 171 Bronchoprovocation test, 166, 168 Bundle-forming pili see BFP Butter flavorings (in microwave popcorn), 164, 179, 181, 182, 184, 185, 187 2-tert-Butylanthraquinone, 57 C Cadherins and pathogen adherence, 113, 114 Calcium and psyllium, 204 Calorimetry, differential scanning (DSC), starch gelatinization studies, 238–247, 249, 250, 253, 254, 256, 258–260 Cancer (prevention and management) ginsenoside effects, 35, 64–68, 70 psyllium effects, 202–203 Capillary electrophoresis, ginsenosides, 61 Carbohydrase treatment of psyllium, 207 Carbohydrates (incl. oligosaccharides), bacterial receptor, 103, 106–108 analogs, anti-adhesive activity, 133–139 in specific pathogen–host interactions, 108–117 Carbon monoxide diffusing capacity (DLCO), 167, 168–169 in occupational lung disease, 171 Carcinogenesis see Cancer Cardiovascular effects, ginsenosides, 72–73 see also Coronary heart disease Carrot extracts, 134, 138 Central nervous system and ginsenosides, 74–76 Chemical modification of psyllium, 214–215 Chest, high-resolution CT see High-resolution CT Chikusetsusaponin, 4, 11, 12, 14, 16, 20 Cholesterol-lowering activity of psyllium, 195–201, 213 Cholinergic system and ginsenosides, 74–75 Chromatography (of ginsenosides) gas, 48–49
immunostaining after (immunochromatography), 63 liquid see Liquid chromatography thin-layer, 46–48 Coating of psyllium granules, 205 Collagen-binding adhesins, 113, 115, 117 Collision-induced dissociation (CID) methodology, ginsenosides, 58 Colon/colorectal cancer ginsenoside effects, 66, 67 psyllium effects, 202 Compensation, flavoring-related bronchiolitis obliterans, 185–186 Compound K (M1; IH-901), 37 anticancer activity, 66 intestinal synthesis, 33–35 Computed tomography, high-resolution see High-resolution CT Conjunctivitis, occupational allergic, 169–170 Constipation, psyllium in, 203 Corn starch gelatinization, birefringence loss, 235 Coronary heart disease, hypolipidemic effects of psyllium in prevention of, 195–201, 213 Coulombic forces and pathogen adherence, 105 Cranberry extracts, 134, 137–138 Crystalline structure/crystallites of starch, 225–228 crystallite melting theory (of Flory), 240, 242–243, 250–253 crystallite stability theory, 241–244 polymorphic groups, 228–230 water availability theory and, 238–241 Csg operon, 119 Cycloartenol, biosynthesis, 45 Cytokines and ginsenosides, 69, 71, 73 Cytotoxicity, ginsenosides, 65–67 D Dammarane-type triterpene saponins, 3, 23, 40 biosynthesis, 38, 40, 41, 45 P. notoginseng, 30 20(R)-Dammarane-3b, 6a, 20, 25-pentol, 44 20(R)-Dammarane-3b, 12b, 20, 25-tetrol, 7 Dammaranediol-type ginsenosides, 41 20(S)-dammaranediol, 22 Dammarenyl cation, 38–41, 41
Index
Defences immune, ginsenoside effects, 69–70 respiratory tract, 165 Dendritic cells, ginsenoside effects, 69, 70 Diabetes ginsenosides and, 77–79 psyllium and, 201–202 Diacetyl (in microwave popcorn), 179 Differential scanning calorimetry (DSC), starch gelatinization studies, 238–247, 249, 250, 253, 254, 256, 258–260 Differential thermal analysis (DTA), starch gelatinization studies, 238, 239–240 Dimethylallyl diphosphate (DMAPP), 37, 38, 39 Donovan’s water availability theory (for starch gelatinization), 238–241, 243 Dr adhesins, 128 E Eastern blots, ginsenosides, 63 ECP, 126 Efa-1 adhesin, 122, 124, 125 Egg yolk-derived sialyloligosaccharides, 134, 136–137 Electrophoresis, capillary, ginsenosides, 61 Electrospray ionization (ESI), ginsenosides, 57–60 ELISA (enzyme-linked immunosorbant assay), ginsenosides, 62 Emphysema, occupation-related, 178 medical tests results, 171 site/description/causes, 173 Emulsifier-coated psyllium granules, 205 Enterohemorrhagic E. coli adhesion, 125–126 Enteropathogenic E. coli adhesion, 122–124 Enzymatic methods of psyllium treatment, 206–214 conventional, 206–207 solid-state, 207–214 of starch gelatinization measurement, 234, 236 Enzyme immunoassays, ginsenosides, 61–62 Epithelial cells, intestinal, as pathogen target, 111 Escherichia coli adhesion, 107, 122–128 enterohemorrhagic, 125–126 enteropathogenic, 122–124 target molecules, 110 uropathogenic (UPEC), 102, 114, 126–128
271
EspA, 122, 123 Ethanol extraction of ginsenosides, 45 Evaporate light scattering detection of ginsenosides, 55–56 Excitotoxicity and ginsenosides, 75–76 Extracellular matrix (ECM) and pathogen adherence, 111, 113–114 F F1C fimbriae, 127 F9 fimbriae, 125 Fecal bulk, psyllium effects, 203 Fibronectins, 115 proteins (adhesins) binding to (FnBPs), 109, 113, 115, 116–117 Fimbrial (pilar) adhesins, 114–116 E. coli enterohemorrhagic, 125–126 uropathogenic, 126–128 Salmonella, 117–118 Flagella, enteropathogenic E. coli, 124 Flavoring-related bronchiolitis obliterans, 164 Floralginsenoside, 8, 9, 12, 13, 15, 16 Floralquinquenoside, 9, 15, 16 Flory’s theory (of starch gelatinization), 240, 242–243, 250–253 Flours, respiratory allergy, 175–176 Flower buds, ginsenosides, 24–29 Fluorescence detection of ginsenosides, 56–57 Fruits, ginsenosides, 24–29 G GABA and ginsenosides, 76 Galacto-oligosaccharides, 134, 138–139 Gas chromatography, ginsenosides, 48–49 Gastric acids and ginsenosides, 33, 34, 35, 36 Gastrointestinal effects of psyllium, 203–204 Gelatinization of starch see Starch Ginseng see Panax Ginsenosides, 1–99 analysis, 45–64 biosynthesis, 37–45 chemistry, 23–45 classification, 2, 3, 23 health benefits, 64–79 metabolism, 33–37 pharmacokinetics, 33–37, 48, 55, 79 preparation from ginseng, 32–33 sample extraction, 45–46 Glass transition, starch, 255–258
272
Index
Globotriose, 134, 135 Glucoamylase, starch gelatinization estimation using, 234, 236 Glucose blood ginsenoside effects, 77, 78–79 psyllium effects, 201 in starch, units of, 224 Glucose transporter 4 (GLUT-4) ginsenosides and, 78 psyllium and, 201 Glutamate and ginsenosides, 75–76 Glycocalyx and pathogen adherence, 111, 112–113 Glycosidic bonds, starch, 224 Goblet cells airway, 165 intestinal, 104 Granules, starch disruption see Starch, gelatinization structure, 224–229 GRAS (‘‘generally recognized as safe’’) classification, 186 Green tea extracts, 134, 138 Gynostemma pentaphyllum, distribution of ginsenosides, 29, 31 Gypenoside, 5 H HcpA, 126 HDL-cholesterol, psyllium effects, 196, 199 Heart disease, coronary, hypolipidemic effects of psyllium in prevention of, 195–201, 213 Helicobacter pylori adhesins, 119–120 Hemsloside-Ma3, 30 Hepatic cancer cells, ginsenoside effects, 66, 67 High-density lipoprotein (HDL)-cholesterol, psyllium effects, 196, 199 High-performance liquid chromatography of ginsenosides, 49–61 High-performance thin-layer chromatography of ginsenosides, 48 High-resolution CT in lung disease, 167, 169 occupational lung disease, 171 High-resolution MS, ginsenosides, 60–61 HopH, 121 HopZ, 121 HorB, 121–122 Hot stage microscopy, starch gelatinization measurement, 234
Human milk oligosaccharides, 134, 136 Hydrogen bonds and pathogen adherence, 105–106 Hydrophobic interactions (and hydrophobins) and pathogen adherence, 105, 108, 110–111 Hyperglycemia reduction see Hypoglycemic effects Hyperlipidemia, psyllium effects, 195–201, 213 Hypersensitivity pneumonitis (extrinsic allergic alveolitis), occupation-related medical test results, 171, 177–178 site/description/causes, 173 symptoms, 170 Hypertension and ginsenosides, 72–73 Hypoglycemic effects ginsenosides, 77, 78–79 psyllium, 201–202 Hypolipidemic effects of psyllium, 195–201, 213 I IH-901 see Compound K Immunization, adhesin active (¼vaccination), 117, 130 passive, 130 Immunoassays, ginsenosides, 61–62 Immunomodulatory effects of ginsenosides, 69–70 Infection, bacterial adherence in see Adherence routes, 103–105 Inflammation, ginsenoside effects, 71–72 Insulin sensitivity ginsenoside effects, 78 psyllium effects, 201–202 Integrins and pathogen adherence, 113, 114 Interleukin-6 and ginsenosides, 72–73 Intestine (bowel) cancer, ginsenoside effects, 66 microflora see Microflora pathogens in routes of infection, 103–105 target tissues, 111–114 pharmacokinetics and metabolism of ginsenosides, 33–35 psyllium effects on movement, 203 Intimin, 124 Invasion, tumor, ginsenoside effects, 35, 67–68 Irritable bowel syndrome, psyllium, 203
Index
Irritant-induced asthma, 172, 176–177 Isopentenyl diphosphate (IPP), 37, 38, 39 K Kofler stage microscopy, starch gelatinization measurement, 234 Koryoginsenoside, 11, 13 L Lactobacillus, anti-adhesin activity, 131–132 Lactosylceramide and pathogen adherence, 113 Laxative effects of psyllium, 203 LDL-cholesterol, psyllium effects, 197, 199, 213 Learning and ginsenosides, 74–75 Leaves, ginsenosides, 24–29 Lectin–carbohydrate interactions in bacterial adhesion, 108, 108–109, 113 Legal liability, flavoring-related bronchiolitis obliterans, 185–186 Leukemia cells, ginsenoside effects, 65 Liability, flavoring-related bronchiolitis obliterans, 185–186 LifA, 124 Lipid-lowering activity of psyllium, 195–201, 213 Lipoteichoic acid (LTA), 132–133 Liquid chromatography of ginsenosides high-performance, 49–61 and mass spectrometry (LC-MS), 57–60 ultra-performance, 61 Liver (hepatic) cancer cells, ginsenoside effects, 66, 67 Long polar (LP) fimbriae, Salmonella, 117, 118 Low-density lipoprotein (LDL)-cholesterol, psyllium effects, 197, 199, 213 LpfABCC’DE fimbrial operon, enterohemorrhagic E. coli, 125 LpfABCDE fimbrial operon, Salmonella, 118 Lung see Respiratory system Lymphocytes, ginsenoside effects, 69–70 Lymphostatin, 124 M M1 see Compound K Macrophages, ginsenoside effects, 70 Majonoside, 19, 20 Majoroside, 7, 8, 14
273
Malignancy see Cancer Malonyl-ginsenosides, 23–32 HPLC-UV detection, 50–54 mass spectroscopy, 59 Manno-oligosaccharides, 134, 138 Mass spectrometry (MS), ginsenosides, 57–61 Medical surveillance for food industry allergy, 176 Melanoma cells, ginsenoside effects, 66, 67, 68 Melting of polymer in starch gelatinization Flory’s theory, 240, 242–243, 250–253 in three-stage phase transition theory, 250 Memory and ginsenosides, 74–75 Metabolism ginsenoside effects on, 76–77 of ginsenosides, 33–37 Metastasis, tumor, ginsenoside effects, 35, 67–68, 68 Methacholine provocation test, 166–167, 168 in occupational lung disease, 171 Methacrylic acid derivatives of psyllium, 214 Methanol extraction of ginsenosides, 45 Methyl-a-mannoside, 134, 135 Mevalonic acid, 37 Microbial surface components recognizing adhesive matrix molecules (MSCRAMMs), 109, 116 Microflora, intestinal, 105 ginsenoside metabolism by, 33–35, 36 Milk (human) oligosaccharides, 134, 136 Monoclonal antibodies to ginsenosides, 62–63 to S. mutans, 130 MSCRAMMs (microbial surface components recognizing adhesive matrix molecules), 109, 116 Mucus (and mucins) intestinal, and pathogen adherence, 104–105, 112, 131 respiratory tract, defence role, 165 N National Institute for Occupational Safety and Health (NIOSH) and microwave popcorn flavoring, 164, 179–182 Near infrared spectroscopy, ginsenosides, 63–64 Neovascularization, tumor, ginsenoside effects, 68
274
Index
Neurological effects, ginsenosides, 74–76 Neurotransmitters and ginsenosides, 76 Neutrophil-activating protein, H. pylori, 120 Nitric oxide and ginsenosides, 71–72 Notoginsenoside, 5, 7, 8, 9, 13, 14, 16, 18, 22, 23 Nuclear magnetic resonance spectroscopy, ginsenosides, 61 O Obesity, orlistat, 204 Occupational respiratory disease, 164, 169–176 new/emerging, recognition, 186–187 prevention, 187 types, 169–179 Occupational Safety and Health Administration (OSHA) and flavoringrelated bronchiolitis obliterans, 179, 185 Ocotillol (and ocotillol-type ginsenosides), 19, 23, 30, 31 biosynthesis, 41, 43 Oleanane-type triterpene saponins, 3, 23, 40 biosynthesis, 41, 45 Oleanolic acid-type ginsenosides, 20, 23, 30, 31 biosynthesis, 44 Oligosaccharides as bacterial receptors see Carbohydrates Orlistat, 204 Outer membrane proteins (OMPs) as adhesins, 120–122 Ovarian cancer cells, ginsenoside effects, 66 P P-fimbriae/pili (pyelonephritis-associated pill), 114–115, 127 Panax (ginseng), 44–45 bipinnatifidus (feather-leafed bamboo ginseng), 24 distribution of ginsenosides in, 24–29 ginseng (Korean/Asian/Chinese ginseng), 3, 24–25, 30 japonicus (Japanese ginseng), 3, 25–26 notoginseng (Sanchi ginseng), 3, 26, 30–31 pseudoginseng, 26, 27 quality markers, 30
quinquefolium (American ginseng), 3, 27–28, 30 steaming and drying, 32–33 stipuleanatus (Pingpien ginseng), 28 trifolius (Dwarf Ginseng), 28 vietnamensis (Vietnamese ginseng), 3, 28–29 zingiberensis (Ginger ginseng), 29 20(S)-Panaxatriol, 21 Pancreatic cancer cells, ginsenoside effects, 67 Pap (pyelonephritis-associated pili), 114–115, 127 Passive cutaneous anaphylaxis and ginsenosides, 72 Pathogenic, bacteria, adherence see Adherence Peak expiratory flow (PEF), 166, 168 PefBACD fimbrial operon, Salmonella, 118–119 Pentopan Mono BG treatment of psyllium, 208, 209, 210–211, 211 Peroxiome proliferator-activated receptors (PPARs) and ginsenosides, 76–77 and diabetes, 78 Pharmacokinetics, ginsenosides, 33–37, 48, 55, 79 Phase transition theory (starch gelatinization), 244–250, 258–260 sequential, 244–249 three-stage, 250, 258 Phosphate buffer, ginsenoside detection, 50–54 Pili see Fimbrial adhesins Plant(s) ginsenosides in, 2–3 distribution in, 23–32 starches from, 222–223, 223 Plantago, 194 Plasmid-encoded (PE) fimbriae, Salmonella, 117, 118–119 Pneumonitis, hypersensitivity see Hypersensitivity pneumonitis Polarized light, starch gelatinization measurement, 234 Polyacetyleneginsenoside Ro, 21, 45 Polymers melting (in starch gelatinization) see Melting psyllium effects on functional properties, 214–215
Index
Polyvinylpyrrolidone-coated psyllium granules, 205 Popcorn (microwave) flavoring, 164, 179–182, 183, 184, 185, 186 Potato starch gelatinization, 241–242, 247 birefringence loss, 235 Prebiotics as anti-adhesins, 103, 135–136, 138–139 Probiotics as anti-adhesins, 129, 131–132 Prodrug, ginsenoside Rb1 as, 59 Prostaglandins and ginsenosides, 71 Prostate cancer cells, ginsenoside effects, 65, 66 Protein(s), as adhesin analogs, 132–133 Protein–protein interactions in bacterial adhesion, 108, 109 Proteolysis, psyllium, 206 Protopanaxadiol (PPD)-type ginsenosides, 4–23 anticancer effects, 65, 66 biosynthesis, 41, 42 gas chromatography, 49 pharmacokinetics and metabolism, 33–37 Protopanaxatriol (PPT)-type ginsenosides, 12, 13, 19, 23 anticancer effects, 65, 66–67 biosynthesis, 42, 43 ELISA, 63 gas chromatography, 49 metabolism, 36 Pseudo-ginsenoside, 5, 13, 19, 20 Psyllium, 193–219 adverse effects, 204 reducing, 204–214 health benefits, 194, 195–204 improving functionality and biological activity, 194–195, 204–214 Pyelonephritis-associated pili (pap), 114–115, 127 Q Quadrupole-MS (LC-MS), ginsenosides, 57–60, 61 Quinquenoside, 5, 6, 7, 9, 18, 22 R Receptors for bacteria, 102–103, 106–107 analogs as anti-adhesins, 129, 133–139 carbohydrates as see Carbohydrates
275
in specific pathogen–host interactions, 108–117 Respiratory system (inc. lung), 163–198 anatomy and defences, 165 cancer cells, ginsenoside effects, 65, 67, 68 disease, 163–192 diagnosis, 165–167 occupational see Occupational respiratory disease Rhinitis, occupational allergic, 169–170, 174, 175, 176, 187 Rhizomes see Roots and rhizomes Rice starch gelatinization, birefringence loss, 235 Roots and rhizomes containing ginsenosides, 24, 31 preparation by steaming and drying, 32–33 UV detection of ginsenosides, 50–54 S S-fimbriae, 127–128 SabA, 121 Salmonella adherence, 117–118 Saponins, triterpinoid see Triterpinoid saponins Selectins and pathogen adherence, 113, 114 Semi-cooperative theory of starch gelatinization, 234–238 Sequential phase transitions theory (starch gelatinization), 244–249 SfbI, 116 Shearzyme 500L treatment of psyllium, 208, 209–210, 210–211, 211 Sialic acid-binding adhesin, 121 Sialylated oligosaccharides, anti-adhesive, 134, 135 egg yolk-derived, 134, 136–137 SiiE adhesin, 119 SLPs (surface-layer proteins) of Lactobacillus, 132 Spirometry, 165–168 in occupational lung disease, 171 popcorn workers, 183–184 Squalene, biosynthesis, 38, 39 Squalene oxide, 38–45 cyclization, 41–45 Starch, 221–267 functional properties, 223 gelatinization, 221–222, 223, 230–260 annealing and its relationship to, 244, 245, 246, 253–255
276
Index
Starch (cont.) glass transition and, 255–258 theories and models, 230–253 importance, 222 sources and isolation, 222–223, 223 structure, 224–230 Stems, ginsenosides, 24–29 Stomach acids and ginsenosides, 33, 34, 35, 36 Streptococcus S. mutans adhesin analog activity against, 132–133 passive immunization, 130 S. pyogenes/group A adhesins, 116 analogs, 133 Stress, ginsenoside effects, 72–73 Surface-layer proteins of Lactobacillus, 132 Sympathetic nerve activity and ginsenosides, 72 T Tafi operon, 119 2-Tert-butylanthraquinone, 57 TFP (type IV pilus), enterohemorrhagic E. coli, 126 Thiazolidinediones, 78 Thin-layer chromatography of ginsenosides, 46–48 Three-stage phase transition theory (starch gelatinization), 250, 258 Time-of-flight (TOF) MS of ginsenosides quadrupole, 60 ultra-performance LC and, 61 ToxB, 122, 124, 125 Transition metal cations and ginsenosides, 58 Triterpenes, biosynthesis, 38 Triterpinoid saponins, 2 dammarane-type see Dammarane-type triterpene saponins oleanane-type see Oleanane-type triterpene saponins TTSS (type III secretion system), 118, 122, 123, 124 Tumors, malignant see Cancer Type III, 122, 123
U Ultra-performance liquid chromatography of ginsenosides, 61 Ultraviolet detection, ginsenosides on HPLC, 49–55 Uropathogenic E. coli (UPEC), 102, 114, 126–128 US Occupational Safety and Health Administration (OSHA) and flavoringrelated bronchiolitis obliterans, 179, 185 UV detection, ginsenosides on HPLC, 49–55 V Vaccines, anti-adhesin, 117, 130 Van der Waals’ forces and pathogen adherence, 105, 106 Vascular effects, ginsenosides, 72–73 Vina-ginsenoside, 7, 8, 11, 13, 16, 18, 19, 20, 22 Viscozyme L treatment of psyllium, 208–209, 209–210, 210–211 W Water (in starch gelatinization), 230–253 sequential phase transitions theory, 244–249 two phases, 242 water availability theory, 238–241, 243 Western blots, ginsenosides, 63 Wheat starch gelatinization, birefringence loss, 235 Work-related respiratory disease see Occupational respiratory disease X X-ray diffraction of starch granules, 228, 231–233 gelatinization studies, 244 Y Yesanchinoside, 5, 7, 22, 23 Z Zingibroside, 20