M E T H O D S I N B I O T E C H N O L O G Y ᮀ 2 0TM
Natural
Products
Isolation
Second Edition
Edited by
Satyajit D. Sarker...
Natural Products Isolation
John M. Walker, SERIES EDITOR
21. Food-Borne Pathogens, Methods and Protocols, edited by Catherine Adley, 2006
20. Natural...
M E T H O D S I N B I O T E C H N O L O G Y
Natural Products
Isolation
SECOND EDITION
Edited by
Satyajit D. Sarker
Pharmac...
© Humana Press Inc.
999 Riverview Drive, Suite 208
Totowa, New Jersey 07512
www.humanapress.com
All rights reserved. No pa...
v
Preface
The term “natural products” spans an extremely large and diverse
range of chemical compounds derived and isolate...
vi Preface
reference guide to all of the available techniques for the more
experienced among us.
Satyajit D. Sarker
Zahid ...
vii
Preface to First Edition
Biodiversity is a term commonly used to denote the variety of species and
the multiplicity of...
viii Preface
they wish to isolate a small molecule from a biological mixture. However, there
may also be something of inte...
ix
Contents
Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v
Prefac...
x Contents
14 Isolation of Marine Natural Products
Wael E. Houssen and Marcel Jaspars . . . . . . . . . . . . . . . . . . ...
xi
Contributors
RUSSELL A. BARROW • Microbial Natural Product Research Laboratory,
Department of Chemistry, The Australian...
xii Contributors
PATRICK MORRIS • Ecopia BioSciences Inc., Frederick Banting,
Saint Laurent, Quebec, Canada
LUTFUN NAHAR •...
1
Natural Product Isolation
An Overview
Satyajit D. Sarker, Zahid Latif, and Alexander I. Gray
Summary
There has been a re...
microorganism) that has not been subjected to any kind of processing or
treatment other than a simple process of preservat...
Fig. 1. An example of natural product drug discovery process (bioassay-
guided approach).
Natural Product Isolation 3
2. Natural Products: Historical Perspective
The use of natural products, especially plants, for healing is as ancient
and ...
century, a number of top selling drugs have been developed from natural
products (vincristine from Vinca rosea, morphine f...
Only a small fraction of the world’s biodiversity has been explored for
bioactivity to date. For example, there are at lea...
It is also necessary to seek answers to the questions related to the expected
outcome of the extraction. These include:
1....
13–15). For initial fractionation of any crude extract, it is advisable not
to generate too many fractions, because it may...
and EtOAc, CHCl3, DCM, or n-hexane, followed by an assay to determine the
distribution of compounds in solvent fractions.
...
6. Droplet countercurrent chromatography (DCCC).
7. High-performance liquid chromatography (HPLC).
8. Hyphenated technique...
Fig. 2. Isolation of microbial natural products: spirocardins A and B from
Nocardia sp.
Natural Product Isolation 11
20-hydroxyecdysone and ponasterone A, using a combination of solvent
extraction, SPE, and preparative RP-HPLC, was outline...
by ecdysteroid bioassay/RIA revealed the presence of ecdysteroids in the
60% MeOH–H2O fraction, which was then subjected t...
Similar purification of fraction 3 yielded ponasterone A (purity 99%, Rt
5.2 min) and limnantheoside B (purity 99%, Rt 10.8...
3. The extract solution may not have been prepared in a solvent that is
compatible with the mobile phase, so that a large ...
4. Most of the active component(s) spread across a wide range of fractions,
causing undetectable amounts of component(s) p...
8. ‘‘Poor-Yield’’ Problem
Poor yield or poor recovery is one of the major problems in natural
product isolation. For examp...
or microbe under investigation could sometimes provide additional hints
regarding the possible chemical class of the unkno...
The following basic points should be borne in mind when carrying out
assays of natural products (2):
1. Samples dissolved ...
of cell growth (18,19), etc. Most of the modern bioassays are microplate-
based and require a small amount of extract, fra...
10.2. Antibacterial Serial Dilution Assay Using Resazurin as
an Indicator of Cell Growth
Antibacterial activity of extract...
10.2.3. Preparation of 96-Well Plates and Assay
The top of the 96-well plates is labeled appropriately. For evaluating the...
without isolation and purification (21). Because of the phenomenal
progress made in the area of MS and NMR in the last few ...
4. Cragg, G. M. and Newman, D. J. (2001) Natural product drug discovery in
the next millennium. Pharm. Biol. 39, 8–17.
5. ...
19. Sarker S. D., Eynon E., Fok K., et al. (2003) Screening the extracts of the
seeds of Achillea millefolium, Angelica sy...
2
Initial and Bulk Extraction
Ve´ronique Seidel
Summary
Currently, there is a growing interest in the study of natural pro...
1. Introduction
The natural products of interest here are small organic molecules
(mol wt 2000 amu approx.), which are als...
2. Method
2.1. Extraction of Plant Natural Products
Plants are complex matrices, producing a range of secondary metabolite...
2.1.1.2. COLLECTION AND IDENTIFICATION
The whole plant or a particular plant part can be collected depending on
where the ...
direct sunlight is advised to minimize chemical reactions (and the formation
of artifacts) induced by ultraviolet rays. To...
2.1.2.1. MACERATION
This simple, but still widely used, procedure involves leaving the pulver-
ized plant to soak in a sui...
2.1.2.3. PERCOLATION
In percolation, the powdered plant material is soaked initially in a sol-
vent in a percolator (a cyl...
maceration or percolation. However, the main disadvantage of Soxhlet
extraction is that the extract is constantly heated a...
water) condense and the distillate (separated into two immiscible layers)
is collected in a graduated tube connected to th...
Extractions can be either ‘‘selective’’ or ‘‘total.’’ The initial choice of the
most appropriate solvent is based on its s...
low-polarity constituents. The ‘‘total’’ extract is evaporated to dryness,
redissolved in water, and the metabolites re-ex...
2.2.1. Isolation and Fermentation
Because of the enormous diversity of the microbial world, it is not a sim-
ple task to s...
fermenters as opposed to flasks (e.g., actinomycetes and filamentous fungi
can grow as two different morphologies, hyphae or...
metabolites will be retained if the medium (aqueous solution) is passed
through a column packed with a hydrophobic adsorbe...
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Natural Products Isolation Techniques

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  • 1. M E T H O D S I N B I O T E C H N O L O G Y ᮀ 2 0TM Natural Products Isolation Second Edition Edited by Satyajit D. Sarker Zahid Latif Alexander I. Gray Natural Products Isolation Second Edition Edited by Satyajit D. Sarker Zahid Latif Alexander I. Gray
  • 2. Natural Products Isolation
  • 3. John M. Walker, SERIES EDITOR 21. Food-Borne Pathogens, Methods and Protocols, edited by Catherine Adley, 2006 20. Natural Products Isolation, Second Edition, edited by Satyajit D. Sarker, Zahid Latif, and Alexander I. Gray, 2005 19. Pesticide Protocols, edited by José L. Martínez Vidal and Antonia Garrido Frenich, 2005 18. Microbial Processes and Products, edited by Jose Luis Barredo, 2005 17. Microbial Enzymes and Biotransformations, edited by Jose Luis Barredo, 2005 16. Environmental Microbiology: Methods and Protocols, edited by John F. T. Spencer and Alicia L. Ragout de Spencer, 2004 15. Enzymes in Nonaqueous Solvents: Methods and Protocols, edited by Evgeny N. Vulfson, Peter J. Halling, and Herbert L. Holland, 2001 14. Food Microbiology Protocols, edited by J. F. T. Spencer and Alicia Leonor Ragout de Spencer, 2000 13. Supercritical Fluid Methods and Protocols, edited by John R. Williams and Anthony A. Clifford, 2000 12. Environmental Monitoring of Bacteria, edited by Clive Edwards, 1999 11. Aqueous Two-Phase Systems, edited by Rajni Hatti-Kaul, 2000 10. Carbohydrate Biotechnology Protocols, edited by Christopher Bucke, 1999 9. Downstream Processing Methods, edited by Mohamed A. Desai, 2000 8. Animal Cell Biotechnology, edited by Nigel Jenkins, 1999 7. Affinity Biosensors: Techniques and Protocols, edited by Kim R. Rogers and Ashok Mulchandani, 1998 6. Enzyme and Microbial Biosensors: Techniques and Protocols, edited by Ashok Mulchandani and Kim R. Rogers, 1998 5. Biopesticides: Use and Delivery, edited by Franklin R. Hall and Julius J. Menn, 1999 4. Natural Products Isolation, edited by Richard J. P. Cannell, 1998 3. Recombinant Proteins from Plants: Production and Isolation of Clinically Useful Compounds, edited by Charles Cunningham and Andrew J. R. Porter, 1998 2. Bioremediation Protocols, edited by David Sheehan, 1997 1. Immobilization of Enzymes and Cells, edited by Gordon F. Bickerstaff, 1997 M E T H O D S I N B I O T E C H N O L O G Y ™
  • 4. M E T H O D S I N B I O T E C H N O L O G Y Natural Products Isolation SECOND EDITION Edited by Satyajit D. Sarker Pharmaceutical Biotechnology Research Group School of Biomedical Sciences University of Ulster at Coleraine Coleraine, Northern Ireland United Kingdom Zahid Latif Molecular Nature Limited Plas Gogerddan, Aberystwyth Wales, United Kingdom Alexander I. Gray Phytochemistry Research Lab Department of Pharmaceutical Sciences TM Glasgow, Scotland, United Kingdom University of Strathclyde
  • 5. © Humana Press Inc. 999 Riverview Drive, Suite 208 Totowa, New Jersey 07512 www.humanapress.com All rights reserved. No part of this book may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, microfilming, recording, or otherwise without written permission from the Publisher. Methods in Biotechnology™ is a trademark of The Humana Press Inc. All papers, comments, opinions, conclusions, or recommendations are those of the author(s), and do not necessarily reflect the views of the publisher. This publication is printed on acid-free paper. ∞ ANSI Z39.48-1984 (American Standards Institute) Permanence of Paper for Printed Library Materials. Cover design by Patricia F. Cleary For additional copies, pricing for bulk purchases, and/or information about other Humana titles, contact Humana at the above address or at any of the following numbers: Tel.: 973-256-1699; Fax: 973-256-8341; E-mail: orders@humanapr.com; or visit our Website: www.humanapress.com Photocopy Authorization Policy: Authorization to photocopy items for internal or personal use, or the internal or personal use of specific clients, is granted by Humana Press Inc., provided that the base fee of US $30.00 per copy is paid directly to the Copyright Clearance Center at 222 Rosewood Drive, Danvers, MA 01923. For those organizations that have been granted a photocopy license from the CCC, a separate system of payment has been arranged and is acceptable to Humana Press Inc. The fee code for users of the Transactional Reporting Service is: [1-58829-447-1/0 $30.00]. Printed in the United States of America. 10 9 8 7 6 5 4 3 2 1 eISBN 1-59259-955-9 Library of Congress Cataloging-in-Publication Data Natural products isolation. – 2nd ed. / edited by Satyajit D. Sarker, Zahid Latif, Alexander I. Gray. p. cm. – (Methods in biotechnology; 20) Includes bibliographical references and index. ISBN 1-58829-447-1 (acid-free paper) – ISBN 1-59259-955-9 (eISBN) 1. Natural products. 2. Extraction (Chemistry) I. Sarker, Satyajit D. II. Latif, Zahid. III. Gray, Alexander I. IV. Series. QD415.N355 2005 547Ј.7–dc22 2005017869 2006 6
  • 6. v Preface The term “natural products” spans an extremely large and diverse range of chemical compounds derived and isolated from biological sources. Our interest in natural products can be traced back thousands of years for their usefulness to humankind, and this continues to the present day. Compounds and extracts derived from the biosphere have found uses in medicine, agriculture, cosmetics, and food in ancient and modern societies around the world. Therefore, the ability to access natural products, understand their usefulness, and derive applications has been a major driving force in the field of natural product research. The first edition of Natural Products Isolation provided readers for the first time with some practical guidance in the process of extraction and isolation of natural products and was the result of Richard Cannell’s unique vision and tireless efforts. Unfortunately, Richard Cannell died in 1999 soon after completing the first edition. We are indebted to him and hope this new edition pays adequate tribute to his excellent work. The first edition laid down the “ground rules” and established the techniques available at the time. Since its publication in 1998, there have been significant developments in some areas in natural product isolation. To capture these developments, publication of a second edition is long overdue, and we believe it brings the work up to date while still covering many basic techniques known to save time and effort, and capable of results equivalent to those from more recent and expensive techniques. The purpose of compiling Natural Products Isolation, 2nd Edition is to give a practical overview of just how natural products can be extracted, prepared, and isolated from the source material. Methodology and know- how tend to be passed down through word of mouth and practical experience as much as through the scientific literature. The frustration involved in mastering techniques can dissuade even the most dogged of researchers from adopting a new method or persisting in an unfamiliar field of research. Though we have tried to retain the main theme and philosophy of the first edition, we have also incorporated newer developments in this field of research. The second edition contains a total of 18 chapters, three of which are entirely new. Our intention is to provide substantial background information for aspiring natural product researchers as well as a useful
  • 7. vi Preface reference guide to all of the available techniques for the more experienced among us. Satyajit D. Sarker Zahid Latif Alexander I. Gray
  • 8. vii Preface to First Edition Biodiversity is a term commonly used to denote the variety of species and the multiplicity of forms of life. But this variety is deeper than is generally imagined. In addition to the processes of primary metabolism that involve essentially the same chemistry across great swathes of life, there are a myriad of secondary metabolites—natural products—usually confined to a particular group of organisms, or to a single species, or even to a single strain growing under certain conditions. In most cases we do not really know what biological role these compounds play, except that they represent a treasure trove of chem- istry that can be of both interest and benefit to us. Tens of thousands of natural products have been described, but in a world where we are not even close to documenting all the extant species, there are almost certainly many more thou- sands of compounds waiting to be discovered. The purpose of Natural Products Isolation is to give some practical guidance in the process of extraction and isolation of natural products. Literature reports tend to focus on natural products once they have been isolated—on their struc- tural elucidation, or their biological or chemical properties. Extraction details are usually minimal and sometimes nonexistent, except for a mention of the general techniques used. Even when particular conditions of a separation are reported, they assume knowledge of the practical methodology required to carry out the experiment, and of the reasoning behind the conditions used. Natural Products Isolation aims to provide the foundation of this knowledge. Following an introduction to the isolation process, there are a series of chapters dealing with the major techniques used, followed by chapters on other aspects of isolation, such as those related to particular sample types, taking short cuts, or making the most of the isolation process. The emphasis is not so much on the isolation of a known natural product for which there may already be reported methods, but on the isolation of compounds of unknown identity. Every natural product isolation is different and so the process is not really suited to a practical manual that gives detailed recipe-style methods. However, the aim has been to give as much practical direction and advice as possible, together with examples, so that the potential extractor can at least make a rea- sonable attempt at an isolation. Natural Products Isolation is aimed mainly at scientists with little experi- ence of natural products extraction, such as research students undertaking natural products-based research, or scientists from other disciplines who find
  • 9. viii Preface they wish to isolate a small molecule from a biological mixture. However, there may also be something of interest for more experienced natural products scien- tists who wish to explore other methods of extraction, or use the book as a general reference. In particular, it is hoped that the book will be of value to scientists in less scientifically developed countries, where there is little experi- ence of natural products work, but where there is great biodiversity and, hence, great potential for utilizing and sustaining that biodiversity through the discov- ery of novel, useful natural products. Richard J. P. Cannell In memory of Richard John Painter Cannell—b. 1960; d. 1999
  • 10. ix Contents Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v Preface to First Edition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vii Contributors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xi 1 Natural Product Isolation Satyajit D. Sarker, Zahid Latif, and Alexander I. Gray . . . . . . . . . . 1 2 Initial and Bulk Extraction Véronique Seidel . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27 3 Supercritical Fluid Extraction Lutfun Nahar and Satyajit D. Sarker . . . . . . . . . . . . . . . . . . . . . . 47 4 An Introduction to Planar Chromatography Simon Gibbons . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77 5 Isolation of Natural Products by Low-Pressure Column Chromatography Raymond G. Reid and Satyajit D. Sarker . . . . . . . . . . . . . . . . . 117 6 Isolation by Ion-Exchange Methods David G. Durham . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 159 7 Separation by High-Speed Countercurrent Chromatography James B. McAlpine and Patrick Morris . . . . . . . . . . . . . . . . . . 185 8 Isolation by Preparative High-Performance Liquid Chromatography Zahid Latif . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 213 9 Hyphenated Techniques Satyajit D. Sarker and Lutfun Nahar . . . . . . . . . . . . . . . . . . . . . 233 10 Purification by Solvent Extraction Using Partition Coefficient Hideaki Otsuka . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 269 11 Crystallization in Final Stages of Purification Alastair J. Florence, Norman Shankland, and Andrea Johnston . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 275 12 Dereplication and Partial Identification of Compounds Laurence Dinan . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 297 13 Extraction of Plant Secondary Metabolites William P. Jones and A. Douglas Kinghorn . . . . . . . . . . . . . . . . 323
  • 11. x Contents 14 Isolation of Marine Natural Products Wael E. Houssen and Marcel Jaspars . . . . . . . . . . . . . . . . . . . . 353 15 Isolation of Microbial Natural Products Russell A. Barrow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 391 16 Purification of Water-Soluble Natural Products Yuzuru Shimizu and Bo Li . . . . . . . . . . . . . . . . . . . . . . . . . . . . 415 17 Scale-Up of Natural Product Isolation Steven M. Martin, David A. Kau, and Stephen K. Wrigley . . . . . 439 18 Follow-Up of Natural Product Isolation Richard J. P. Cannell . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 463 Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 507
  • 12. xi Contributors RUSSELL A. BARROW • Microbial Natural Product Research Laboratory, Department of Chemistry, The Australian National University, Canberra, Australia RICHARD J. P. CANNELL • Formerly, Glaxo Wellcome Research and Development, Stevenage, Herts, UK LAURENCE DINAN • Inse Biochemistry Group, Hatherly Laboratories, University of Exeter, Exeter, Devan, UK DAVID G. DURHAM • School of Pharmacy, The Robert Gordon University, Aberdeen, Scotland, UK ALASTAIR J. FLORENCE • Department of Pharmaceutical Sciences, University of Strathclyde, Glasgow, Scotland, UK SIMON GIBBONS • Centre for Pharmacognosy and Phytotherapy, The School of Pharmacy, University of London, London, UK ALEXANDER I. GRAY • Phytochemistry Research Laboratories, Department of Pharmaceutical Sciences, University of Strathclyde, Glasgow, Scotland, UK WAEL E. HOUSSEN • Marine Natural Products Laboratory, Chemistry Department, Aberdeen University, Aberdeen, Scotland, UK MARCEL JASPARS • Marine Natural Products Laboratory, Chemistry Department, Aberdeen University, Aberdeen, Scotland, UK ANDREA JOHNSTON • Department of Pharmaceutical Sciences, University of Strathclyde, Glasgow, Scotland, UK WILLIAM P. JONES • College of Pharmacy, Medicinal Chemistry and Pharmacognosy, University of Illinois at Chicago, Chicago, IL DAVID A. KAU • Cubist Pharmaceuticals (UK) Ltd, Berkshire, UK ZAHID LATIF • Molecular Nature Limited, Plas Gogerddan, Aberystwyth, Wales, UK A. DOUGLAS KINGHORN • College of Pharmacy, Medicinal Chemistry and Pharmacognosy, Ohio State University, Columbus, OH BO LI • Kunming Institute of Botany, Chinese Academy of Science, Kunming, China STEVEN M. MARTIN • Cubist Pharmaceuticals (UK) Ltd, Slough, Berkshire, UK JAMES B. MCALPINE • Ecopia BioSciences Inc., Frederick Banting, Saint Laurent, Quebec, Canada
  • 13. xii Contributors PATRICK MORRIS • Ecopia BioSciences Inc., Frederick Banting, Saint Laurent, Quebec, Canada LUTFUN NAHAR • School of Life Sciences, The Robert Gordon University, Aberdeen, Scotland, UK HIDEAKI OTSUKA • Department of Pharmacognosy, Graduate School of Biomedical Sciences, Hiroshima University, Minami-ku, Hiroshima, Japan RAYMOND G. REID • Phytopharmaceutical Research Laboratory, School of Pharmacy, The Robert Gordon University, Aberdeen, Scotland, UK SATYAJIT D. SARKER • Pharmaceutical Biotechnology Research Group, School of Biomedical Sciences, University of Ulster at Coleraine, Coleraine, Northern Ireland, UK VERONIQUE SEIDEL • Phytochemistry Research Laboratories, Department of Pharmaceutical Sciences, University of Strathclyde, Glasgow, Scotland, UK NORMAN SHANKLAND • Department of Pharmaceutical Sciences, University of Strathclyde, Glasgow, Scotland, UK YUZURU SHIMIZU • Department of Biomedical and Pharmaceutical Sciences, University of Rhode Island, Kingston, RI STEPHEN K. WRIGLEY • Cubist Pharmaceuticals (UK) Ltd, Slough, Berkshire, UK
  • 14. 1 Natural Product Isolation An Overview Satyajit D. Sarker, Zahid Latif, and Alexander I. Gray Summary There has been a remarkable resurgence of interest in natural product research over the last decade or so. With the outstanding developments in the areas of separation science, spectroscopic techni- ques, and microplate-based ultrasensitive in vitro assays, natural product research is enjoying renewed attention for providing novel and interesting chemical scaffolds. The various available hyphenated techniques, e.g., GC-MS, LC-PDA, LC-MS, LC-FTIR, LC-NMR, LC-NMR-MS, CE-MS, have made possible the preisolation analyses of crude extracts or fractions from different natural sources, isolation and on-line detection of natural products, chemotaxonomic studies, chemical finger printing, quality control of herbal products, derepli- cation of natural products, and metabolomic studies. While different chapters in this book are devoted to a number of specific aspects of nat- ural product isolation protocols, this chapter presents, with practical examples, a general overview of the processes involved in natural product research, starting from extraction to determination of the structures of purified products and their biological activity. Key Words: Natural products; secondary metabolite; extraction; isolation; bioassay. 1. Introduction Products of natural origins can be called ‘‘natural products.’’ Natural products include: (1) an entire organism (e.g., a plant, an animal, or a 1 From: Methods in Biotechnology, Vol. 20, Natural Products Isolation, 2nd ed. Edited by: S. D. Sarker, Z. Latif, and A. I. Gray ß Humana Press Inc., Totowa, NJ
  • 15. microorganism) that has not been subjected to any kind of processing or treatment other than a simple process of preservation (e.g., drying), (2) part of an organism (e.g., leaves or flowers of a plant, an isolated animal organ), (3) an extract of an organism or part of an organism, and exudates, and (4) pure compounds (e.g., alkaloids, coumarins, flavonoids, glycosides, lignans, steroids, sugars, terpenoids, etc.) isolated from plants, animals, or microor- ganisms (1). However, in most cases the term natural products refers to sec- ondary metabolites, small molecules (mol wt <2000 amu) produced by an organism that are not strictly necessary for the survival of the organism. Con- cepts of secondary metabolism include products of overflow metabolism as a result of nutrient limitation, shunt metabolism produced during idiophase, defense mechanism regulator molecules, etc. (2). Natural products can be from any terrestrial or marine source: plants (e.g., paclitaxel [TaxolÕ ] from Taxus brevifolia), animals (e.g., vitamins A and D from cod liver oil), or microorganisms (e.g., doxorubicin from Streptomyces peucetius). Strategies for research in the area of natural products have evolved quite significantly over the last few decades. These can be broadly divided into two categories: 1. Older strategies: a. Focus on chemistry of compounds from natural sources, but not on activity. b. Straightforward isolation and identification of compounds from natural sources followed by biological activity testing (mainly in vivo). c. Chemotaxonomic investigation. d. Selection of organisms primarily based on ethnopharmacological informa- tion, folkloric reputations, or traditional uses. 2. Modern strategies: a. Bioassay-guided (mainly in vitro) isolation and identification of active ‘‘lead’’ compounds from natural sources. b. Production of natural products libraries. c. Production of active compounds in cell or tissue culture, genetic manipula- tion, natural combinatorial chemistry, and so on. d. More focused on bioactivity. e. Introduction of the concepts of dereplication, chemical fingerprinting, and metabolomics. f. Selection of organisms based on ethnopharmacological information, folk- loric reputations, or traditional uses, and also those randomly selected. A generic protocol for the drug discovery from natural products using a bioassay-guided approach is presented in Fig. 1. 2 Sarker et al.
  • 16. Fig. 1. An example of natural product drug discovery process (bioassay- guided approach). Natural Product Isolation 3
  • 17. 2. Natural Products: Historical Perspective The use of natural products, especially plants, for healing is as ancient and universal as medicine itself. The therapeutic use of plants certainly goes back to the Sumerian civilization, and 400 years before the Common Era, it has been recorded that Hippocrates used approximately 400 dif- ferent plant species for medicinal purposes. Natural products played a prominent role in ancient traditional medicine systems, such as Chinese, Ayurveda, and Egyptian, which are still in common use today. According to the World Health Organization (WHO), 75% of people still rely on plant-based traditional medicines for primary health care globally. A brief summary of the history of natural product medicine is presented in Table 1. 3. Natural Products: Present and Future Nature has been a source of therapeutic agents for thousands of years, and an impressive number of modern drugs have been derived from natural sources, many based on their use in traditional medicine. Over the last Table 1 History of Natural Product Medicine Period Type Description Before 3000 BC Ayurveda (knowledge of life) Chinese traditional medicine Introduced medicinal properties of plants and other natural products 1550 BC Ebers Papyrus Presented a large number of crude drugs from natural sources (e.g., castor seeds and gum arabic) 460–377 BC Hippocrates, ‘‘The Father of Medicine’’ Described several plants and animals that could be sources of medicine 370–287 BC Theophrastus Described several plants and animals that could be sources of medicine 23–79 AD Pliny the Elder Described several plants and animals that could be sources of medicine 60–80 AD Dioscorides Wrote De Materia Medica, which described more than 600 medicinal plants 131–200 AD Galen Practiced botanical medicines (Galenicals) and made them popular in the West 15th century Kra¨uterbuch (herbals) Presented information and pictures of medicinal plants 4 Sarker et al.
  • 18. century, a number of top selling drugs have been developed from natural products (vincristine from Vinca rosea, morphine from Papaver somni- ferum, TaxolÕ from T. brevifolia, etc.). In recent years, a significant revival of interest in natural products as a potential source for new medicines has been observed among academia as well as pharmaceutical companies. Several modern drugs (~40% of the modern drugs in use) have been devel- oped from natural products. More precisely, according to Cragg et al. (3), 39% of the 520 new approved drugs between 1983 and 1994 were natural products or their derivatives, and 60–80% of antibacterial and anticancer drugs were from natural origins. In 2000, approximately 60% of all drugs in clinical trials for the multiplicity of cancers had natural origins. In 2001, eight (simvastatin, pravastatin, amoxycillin, clavulanic acid, azithro- mycin, ceftriaxone, cyclosporin, and paclitaxel) of the 30 top-selling medi- cines were natural products or their derivatives, and these eight drugs together totaled US $16 billion in sales. Apart from natural product-derived modern medicine, natural products are also used directly in the ‘‘natural’’ pharmaceutical industry, which is growing rapidly in Europe and North America, as well as in traditional medicine programs being incorporated into the primary health care sys- tems of Mexico, the People’s Republic of China, Nigeria, and other devel- oping countries. The use of herbal drugs is once again becoming more popular in the form of food supplements, nutraceuticals, and complemen- tary and alternative medicine. Natural products can contribute to the search for new drugs in three different ways: 1. by acting as new drugs that can be used in an unmodified state (e.g., vincris- tine from Catharanthus roseus). 2. by providing chemical ‘‘building blocks’’ used to synthesize more complex molecules (e.g., diosgenin from Dioscorea floribunda for the synthesis of oral contraceptives). 3. by indicating new modes of pharmacological action that allow complete synthesis of novel analogs (e.g., synthetic analogs of penicillin from Penicil- lium notatum). Natural products will certainly continue to be considered as one of the major sources of new drugs in the years to come because 1. they offer incomparable structural diversity. 2. many of them are relatively small (<2000 Da). 3. they have ‘‘drug-like’’ properties (i.e., they can be absorbed and metabolized). Natural Product Isolation 5
  • 19. Only a small fraction of the world’s biodiversity has been explored for bioactivity to date. For example, there are at least 250,000 species of higher plants that exist on this planet, but merely 5–10% of these have been investigated so far. In addition, reinvestigation of previously studied plants has continued to produce new bioactive compounds that have drug poten- tial. Much less is known about marine organisms than other sources of natural products. However, research up to now has shown that they represent a valuable source for novel bioactive compounds. With the development of new molecular targets, there is an increasing demand for novel molecular diversity for screening. Natural products certainly play a crucial role in meeting this demand through the continued investigation of the world’s biodiversity, much of which remains unexplored (4). With less than 1% of the microbial world currently known, advances in technol- ogies for microbial cultivation and the extraction of nucleic acids from environmental samples from soil and marine habitats will offer access to an untapped reservoir of genetic and metabolic diversity (5). This is also true for nucleic acids isolated from symbiotic and endophytic microbes associated with terrestrial and marine macroorganisms. Advent, introduction, and development of several new and highly spe- cific in vitro bioassay techniques, chromatographic methods, and spectro- scopic techniques, especially nuclear magnetic resonance (NMR), have made it much easier to screen, isolate, and identify potential drug lead compounds quickly and precisely. Automation of these methods now makes natural products viable for high-throughput screening (HTS). 4. Extraction The choice of extraction procedure depends on the nature of the source material and the compounds to be isolated. Prior to choosing a method, it is necessary to establish the target of the extraction. There can be a number of targets; some of these are mentioned here. 1. An unknown bioactive compound. 2. A known compound present in an organism. 3. A group of compounds within an organism that are structurally related. 4. All secondary metabolites produced by one natural source that are not pro- duced by a different ‘‘control’’ source, e.g., two species of the same genus or the same species grown under different conditions. 5. Identification of all secondary metabolites present in an organism for chemi- cal fingerprinting or metabolomics study (see Chap. 9). 6 Sarker et al.
  • 20. It is also necessary to seek answers to the questions related to the expected outcome of the extraction. These include: 1. Is this extraction for purifying a sufficient amount of a compound to charac- terize it partially or fully? What is the required level of purity (see Note 1)? 2. Is this to provide enough material for confirmation or denial of a proposed structure of a previously isolated compound (see Note 2)? 3. Is this to produce as much material as possible so that it can be used for further studies, e.g., clinical trial? The typical extraction process, especially for plant materials (see Chap. 13), incorporates the following steps: 1. Drying and grinding of plant material or homogenizing fresh plant parts (leaves, flowers, etc.) or maceration of total plant parts with a solvent. 2. Choice of solvents a. Polar extraction: water, ethanol, methanol (MeOH), and so on. b. Medium polarity extraction: ethyl acetate (EtOAc), dichloromethane (DCM), and so on. c. Nonpolar: n-hexane, pet-ether, chloroform (CHCl3), and so on. 3. Choice of extraction method a. Maceration. b. Boiling. c. Soxhlet. d. Supercritical fluid extraction. e. Sublimation. f. Steam distillation. The fundamentals of various initial and bulk extraction techniques for natural products are detailed in Chapters 2 and 3. 5. Fractionation A crude natural product extract is literally a cocktail of compounds. It is difficult to apply a single separation technique to isolate individual com- pounds from this crude mixture. Hence, the crude extract is initially separated into various discrete fractions containing compounds of similar polarities or molecular sizes. These fractions may be obvious, physically discrete divisions, such as the two phases of a liquid–liquid extraction (see Chap. 10) or they may be the contiguous eluate from a chromatography column, e.g., vacuum liquid chromatography (VLC), column chromatography (CC), size-exclusion chromatography (SEC), solid-phase extraction (SPE), etc. (see Chaps. 5, Natural Product Isolation 7
  • 21. 13–15). For initial fractionation of any crude extract, it is advisable not to generate too many fractions, because it may spread the target compound over so many fractions that those containing this compound in low concen- trations might evade detection. It is more sensible to collect only a few large, relatively crude ones and quickly home in on those containing the target compound. For finer fractionation, often guided by an on-line detection technique, e.g., ultraviolet (UV), modern preparative, or semipreparative high-performance liquid chromatography (HPLC) can be used. 6. Isolation The most important factor that has to be considered before designing an isolation protocol is the nature of the target compound present in the crude extracts or fractions. The general features of the molecule that are helpful to ascertain the isolation process include solubility (hydrophobicity or hydrophilicity), acid–base properties, charge, stability, and molecular size. If isolating a known compound from the same or a new source, it is easy to obtain literature information on the chromatographic behavior of the target compound, and one can choose the most appropriate method for isolation without any major difficulty. However, it is more difficult to design an isolation protocol for a crude extract where the types of com- pounds present are totally unknown. In this situation, it is advisable to carry out qualitative tests for the presence of various types of compounds, e.g., phenolics, steroids, alkaloids, flavonoids, etc., as well as analytical thin-layer chromatography (TLC), (see Chap. 4) or HPLC profiling (see Chaps. 5, 8, and 9). The nature of the extract can also be helpful for choos- ing the right isolation protocol. For example, a MeOH extract or fractions from this extract containing polar compounds are better dealt with using reversed-phase HPLC (RP-HPLC). Various physical properties of the extracts can also be determined with a small portion of the crude extract in a series of small batch-wise experiments. Some of these experiments are summarized below. 1. Hydrophobicity or hydrophilicity: An indication of the polarity of the extract as well as the compounds present in the extract can be determined by drying an aliquot of the mixture and trying to redissolve it in various solvents cover- ing the range of polarities, e.g., water, MeOH, acetonitrile (ACN), EtOAc, DCM, CHCl3, petroleum ether, n-hexane, etc. The same information can be obtained by carrying out a range of solvent partitioning, usually between water 8 Sarker et al.
  • 22. and EtOAc, CHCl3, DCM, or n-hexane, followed by an assay to determine the distribution of compounds in solvent fractions. 2. Acid–base properties: Carrying out partitioning in aqueous solvents at a range of pH values, typically 3, 7, and 10, can help determine the acid–base prop- erty of the compounds in an extract. It is necessary to adjust the aqueous solution or suspension with a drop or two of mineral acid or alkali (a buffer can also be used), followed by the addition of organic solvent and solvent extraction. Organic and aqueous phases are assessed, preferably by TLC, for the presence of compounds. This experiment can also provide information on the stability of compounds at various pH values. 3. Charge: Information on the charge properties of the compound can be obtained by testing under batch conditions, the effect of adding various ion exchangers to the mixture. This information is particularly useful for designing any isolation protocol involving ion exchange chromatography (see Chap. 6). 4. Heat stability: A typical heat stability test involves incubation of the sample at ~90 C for 10 min in a water bath followed by an assay for unaffected compounds. It is particularly important for bioassay-guided isolation, where breakdown of active compounds often leads to the loss or reduction of bio- logical activity. If the initial extraction of natural products is carried out at a high temperature, the test for heat stability becomes irrelevant. 5. Size: Dialysis tubing can be used to test whether there are any macromole- cules, e.g., proteins, present in the extract. Macromolecules are retained within the tubing, allowing small (2000 amu) secondary metabolites to pass through it. The necessity of the use of any SEC in the isolation protocol can be ascertained in this way. The chromatographic techniques used in the isolation of various types of natural products can be broadly classified into two categories: classical or older, and modern. Classical or older chromatographic techniques include: 1. Thin-layer chromatography (TLC). 2. Preparative thin-layer chromatography (PTLC). 3. Open-column chromatography (CC). 4. Flash chromatography (FC). Modern chromatographic techniques are: 1. High-performance thin-layer chromatography (HPTLC). 2. Multiflash chromatography (e.g., BiotageÕ). 3. Vacuum liquid chromatography (VLC). 4. Chromatotron. 5. Solid-phase extraction (e.g., Sep-PakÕ). Natural Product Isolation 9
  • 23. 6. Droplet countercurrent chromatography (DCCC). 7. High-performance liquid chromatography (HPLC). 8. Hyphenated techniques (e.g., HPLC-PDA, LC-MS, LC-NMR, LC-MS-NMR). Details about most of these techniques and their applications in the isolation of natural products can be found in Chapters 4–9 and 13–16. A number of isolation protocols are presented in Figs. 2–6. 6.1. Isolation of Spirocardins A and B From Nocardia sp An outline of the general protocol described by Nakajima et al. (6) for the isolation of diterpene antibiotics, spirocardins A and B, from a fer- mentation broth of Nocardia sp., is presented in Fig. 2. The compounds were present in the broth filtrate, which was extracted twice with EtOAc (half-volume of supernatant). The pooled EtOAc fraction was concen- trated by evaporation under vacuum, washed with an equal volume of water saturated with sodium chloride (NaCl), and further reduced to obtain an oil. This crude oil was redissolved in a minimal volume of EtOAc and subjected to silica gel CC eluting with n-hexane containing increasing amounts of acetone. It resulted in two fractions containing spirocardin A and spirocardin B, respectively, as the main components. Further purification was achieved by silica gel CC and RP-HPLC. For silica gel CC at this stage, an eluent of benzene–EtOAc mixture was used. Nowadays, benzene is no longer in use as a chromatographic solvent because of its carcinogenicity. 6.2. Isolation of Cispentacin From Bacillus cereus Konishi et al. (7) presented an isolation protocol (Fig. 3) for an antifun- gal antibiotic, cispentacin, from a fermentation broth of B. cereus. This is an excellent example of the application of ion-exchange chromatography in natural product isolation. The broth supernatant was applied directly onto the ion-exchange column without any prior treatment. The final step of the isolation process employed CC on activated charcoal to yield cispen- tacin of 96% purity, which was further purified by recrystallization from acetone–ethanol–water. 6.3. Isolation of Phytoecdysteroids From Limnanthes douglasii A convenient method (Fig. 4) for the isolation of two phytoecdyste- roid glycosides, limnantheosides A and B, and two phytoecdysteroids, 10 Sarker et al.
  • 24. Fig. 2. Isolation of microbial natural products: spirocardins A and B from Nocardia sp. Natural Product Isolation 11
  • 25. 20-hydroxyecdysone and ponasterone A, using a combination of solvent extraction, SPE, and preparative RP-HPLC, was outlined by Sarker et al. (8). Ground seeds (50 g) were extracted (4Â24 h) with 4Â200 mL MeOH at 50 C with constant stirring using a magnetic stirrer. Extracts were pooled and H2O added to give a 70% aqueous methanolic solution. After being defatted with n-hexane, the extract was concentrated using a rotary evaporator. SPE (Sep-Pak fractionation) of the concentrated extract (redissolved in 10% aq MeOH) using MeOH–H2O step gradient, followed Fig. 3. Isolation of microbial natural products: cispentacin from B. cereus. 12 Sarker et al.
  • 26. by ecdysteroid bioassay/RIA revealed the presence of ecdysteroids in the 60% MeOH–H2O fraction, which was then subjected to HPLC using a preparative RP-column (isocratic elution with 55% MeOH–H2O, 5 mL/ min) to yield five fractions. Fractions 2 (Rt 18–20 min) and 3 (Rt 33– 36 min) were found to be bioassay/RIA positive. Further NP-HPLC ana- lyses of fraction 2 on NP-semiprep diol column (isocratic elution with 6% MeOH in DCM, 2 mL/min) produced 20-hydroxyecdysone (purity 99%, Rt 13.1 min) and limnantheoside A (purity 99%, Rt 19.2 min). Fig. 4. Isolation of plant natural products: phytoecdysteroids from L. douglasii. Natural Product Isolation 13
  • 27. Similar purification of fraction 3 yielded ponasterone A (purity 99%, Rt 5.2 min) and limnantheoside B (purity 99%, Rt 10.8 min). 6.4. Isolation of Moschatine, a Steroidal Glycoside, From Centaurea moschata Moschatine, a steroidal glycoside, was isolated from the seeds of C. moschata (9). The isolation protocol (Fig. 5) involved successive Soxhlet extraction of the ground seeds with n-hexane, CHCl3, and MeOH, followed by preparative RP-HPLC (C18 preparative column, isocratic elu- tion with 55% MeOH in water, 5 mL/min). Final purification was carried out by RP-HPLC using a semipreparative C6 column, eluted isocratically with 45% MeOH in water, 2 mL/min, to yield moschatine with a purity of 98%. 6.5. Isolation of Saponins From Serjania salzmanniana The isolation of antifungal and molluscicidal saponins (Fig. 6) from S. salzmanniana involved the use of silica gel CC followed by counter- current chromatography (10). An unconventional feature of the final preparative TLC stage was the use of water as a nondestructive visualiza- tion ‘‘stain.’’ The TLC plate turned dark (wet) when sprayed with water, except those regions represented by the sapogenins, which because of their hydrophobicity, remained white (dry). 7. Quantification The yield of compounds at the end of the isolation and purification pro- cess is important in natural product research. An estimate of the recovery at the isolation stage can be obtained using various routine analytical techniques that may involve the use of a standard. In bioassay-guided isolation, the compound is monitored by bioassay at each stage, and a quantitative assessment of bioactivity of the compound is usually carried out by serial dilution method (see Note 3). Quantitative bioactivity assess- ment provides a clear idea about the recovery of the active compound(s) and also indicates whether the activity results from a single or multiple components. During the isolation process, if the activity is lost or reduced to a significant level, the possible reasons could be as follows: 1. The active compound has been retained in the column. 2. The active compound is unstable in the conditions used in the isolation process. 14 Sarker et al.
  • 28. 3. The extract solution may not have been prepared in a solvent that is compatible with the mobile phase, so that a large proportion of the active components precipitated out when loading on to the column. Fig. 5. Isolation of plant natural products: moschatine, a steroidal glycoside from C. moschata. Natural Product Isolation 15
  • 29. 4. Most of the active component(s) spread across a wide range of fractions, causing undetectable amounts of component(s) present in the fractions. 5. The activity of the extract is probably because of the presence of synergy among a number of compounds, which, when separated, are not active individually. Fig. 6. Isolation of plant natural products: saponins from S. salzmanniana. 16 Sarker et al.
  • 30. 8. ‘‘Poor-Yield’’ Problem Poor yield or poor recovery is one of the major problems in natural product isolation. For example, only 30 g of vincristine was obtained from 15 t of dried leaves of V. rosea (or C. roseus) (11). Similarly, to obtain 1900 g of TaxolÕ , the felling of 6000 extremely slow-growing trees, Taxus brevifolia, was necessary to produce 27,300 kg of the bark. To tackle this poor-yield problem, especially in the case of TaxolÕ , a meeting was orga- nized by the National Cancer Institute in Washington, D.C., in June 1990, where four suggestions were made: 1. Finding a better source for the supply of TaxolÕ , such as a different species or a cultivar of Taxus, or a different plant part or cultivation conditions. 2. Semisynthesis of TaxolÕ from a more abundant precursor. 3. Total synthesis of TaxolÕ . 4. Tissue culture production of TaxolÕ or a close relative. Out of these four ways, the most successful one was semisynthesis. While three successful total syntheses of TaxolÕ have been achieved, they have not been proven to be economically better than the semisynthetic approach. 9. Structure Elucidation In most cases of extraction and isolation of natural products, the end point is the identification of the compound or the conclusive structure elucidation of the isolated compound. However, structure elucidation of compounds isolated from plants, fungi, bacteria, or other organisms is generally time consuming, and sometimes can be the ‘‘bottleneck’’ in nat- ural product research. There are many useful spectroscopic methods of getting information about chemical structures, but the interpretation of these spectra normally requires specialists with detailed spectroscopic knowledge and wide experience in natural product chemistry. With the remarkable advances made in the area of artificial intelligence and com- puting, there are a number of excellent automated structure elucidation programs available that could be extremely useful (12,13). If the target compound is known, it is often easy to compare preliminary spectroscopic data with literature data or to make direct comparison with the standard sample. However, if the target compound is an unknown and complex natural product, a comprehensive and systematic approach invol- ving a variety of physical, chemical, and spectroscopic techniques is required. Information on the chemistry of the genus or the family of plant Natural Product Isolation 17
  • 31. or microbe under investigation could sometimes provide additional hints regarding the possible chemical class of the unknown compound. The fol- lowing spectroscopic techniques are generally used for the structure deter- mination of natural products: 1. Ultraviolet-visible spectroscopy (UV-vis): Provides information on chromo- phores present in the molecule. Some natural products, e.g., flavonoids, isoquinoline alkaloids, and coumarins, to name a few, can be primarily char- acterized (chemical class) from characteristic absorption peaks. 2. Infraredspectroscopy(IR):Determinesdifferentfunctionalgroups,e.g.,—C¼O, —OH, —NH2, aromaticity, and so on, present in a molecule. 3. Mass spectrometry (MS): Gives information about the molecular mass, mole- cular formula, and fragmentation pattern. Most commonly used techniques are: electron impact mass spectrometry (EIMS), chemical ionization mass spectrometry (CIMS), electrospray ionization mass spectrometry (ESIMS), and fast atom bombardment mass spectrometry (FABMS). 4. NMR: Reveals information on the number and types of protons and carbons (and other elements like nitrogen, fluorine, etc.) present in the molecule, and the relationships among these atoms (14). The NMR experiments used today can be classified into two major categories: a. One-dimensional techniques: 1 HNMR,13 CNMR,13 CDEPT, 13 CPENDANT,13 C J mod., nOe-diff., and so on. b. Two-dimensional techniques: 1 H-1 H COSY,1 H-1 H DQF-COSY, 1 H-1 H COSY-lr,1 H-1 H NOESY,1 H-1 H ROESY,1 H-1 H TOCSY (or HOHAHA),1 H-13 C HMBC,1 H-13 C HMQC,1 H-13 C HSQC, HSQC- TOCSY, and the like. In addition to the above-mentioned spectroscopic techniques, X-ray crystallographic techniques provide information on the crystal structure of the molecule, and polarimetry offers information on the optical activity of chiral compounds. 10. Assays Chemical, biological, or physical assays are necessary to pinpoint the target compound(s) from a complex natural product extract. At present, natural product research is more focused on isolating target compounds (assay-guided isolation) rather than trying to isolate all compounds present in any extract. The target compounds may be of certain chemical classes, have certain physical properties, or possess certain biological activities. Therefore, appropriate assays should be incorporated in the extraction and isolation protocol. 18 Sarker et al.
  • 32. The following basic points should be borne in mind when carrying out assays of natural products (2): 1. Samples dissolved or suspended in a solvent different from the original extraction solvent must be filtered or centrifuged to get rid of any insoluble matter. 2. Acidified or basified samples should be readjusted to their original pH to prevent them from interfering with the assay. 3. Positive and negative controls should be incorporated in any assay. 4. Ideally, the assay should be at least semiquantitative, and/or samples should be assayed in a series of dilutions to determine where the majority of the target compounds resides. 5. The assay must be sensitive enough to detect active components in low concentration. Physical assays may involve the comparison of various chromato- graphic and spectroscopic behaviors, e.g., HPLC, TLC, LC-MS, CE-MS LC-NMR, and so on, of the target compound with a known standard. Chemical assays involve various chemical tests for identifying the chemical nature of the compounds, e.g., FeCl3 can be used to detect phenolics, Dragendorff’s reagent for alkaloids, 2,2-diphenyl-1-picrylhydrazyl (DPPH) for antioxidant compounds (15,16), and so on. Bioassays can be defined as the use of a biological system to detect pro- perties (e.g., antibacterial, antifungal, anticancer, anti-HIV, antidiabetic, etc.) of a crude extract, chromatographic fraction, mixture, or a pure com- pound. Bioassays could involve the use of in vivo systems (clinical trials, whole animal experiments), ex vivo systems (isolated tissues and organs), or in vitro systems (e.g., cultured cells). In vivo studies are more relevant to clinical conditions and can also provide toxicity data at the same time. Disadvantages of these studies are costs, need for large amount of test compounds/fractions, complex design, patient requirement, and difficulty in mode of action determination. In vitro bioassays are faster (ideal for HTS), and small amounts of test compounds are needed, but might not be relevant to clinical conditions. The trend has now moved from in vivo to in vitro. Bioassays available today are robust, specific, and more sensi- tive to even as low as picogram amounts of test compounds. Most of them can be carried out in full or semiautomation (e.g., using 96- or 384-well plates). There are a number of biological assays available to assess various activities, e.g., Drosophila melanogaster BII cell line assay for the assess- ment of compounds with ecdysteroid (see Note 4) agonist or antagonist activity (17), antibacterial serial dilution assay using resazurin as indicator Natural Product Isolation 19
  • 33. of cell growth (18,19), etc. Most of the modern bioassays are microplate- based and require a small amount of extract, fraction, or compound for the assessment of activity. While it is not the intention of this chapter to dis- cuss at great length various assays presently available, a summary of two typical assays used in natural product screening, the DPPH assay and anti- bacterial serial dilution assay using resazurin as indicator of cell growth, is presented here as an example. Details on various types of bioassays used in the screening of natural products are available in the literature (20). 10.1. DPPH Assay for Antioxidant Activity DPPH (molecular formula C18H12N5O6) is used in this assay to assess the free radical scavenging (antioxidant) property of natural products (15,16). Quercetin, a well-known natural antioxidant, is generally used as a positive control. DPPH (4 mg) is dissolved in MeOH (50 mL) to obtain a concentration of 80 mg/mL. This assay can be carried out both qualitatively and quantitatively using UV-Vis spectrometer. 10.1.1. Qualitative Assay Test extracts, fractions, or compounds are applied on a TLC plate and sprayed with DPPH solution using an atomizer. It is allowed to develop for 30 min. The white spots against a pink background indicate the anti- oxidant activity. 10.1.2. Quantitative Assay For the quantitative assay, the stock solution of crude extracts or frac- tions is prepared using MeOH to achieve a concentration of 10 mg/mL, whereas that for the test compounds and positive standard is prepared at a concentration of 0.5 mg/mL. Dilutions are made to obtain concentra- tions of 5Â10À2 , 5 Â 10À3 , 5 Â 10 À 4 , 5 Â 10À5 , 5 Â 10À6 , 5 Â 10À7 , 5 Â 10À8 , 5Â10À9 , 5 Â 10À10 mg/mL. Diluted solutions (1.00 mL each) are mixed with DPPH (1.00 mL) and allowed to stand for 30 min for any reaction to take place. The UV absorbance of these solutions is recorded at 517 nm. The experiment is usually performed in triplicate and the average absorption is noted for each concentration. The same procedure is fol- lowed for the standard (quercetin). 20 Sarker et al.
  • 34. 10.2. Antibacterial Serial Dilution Assay Using Resazurin as an Indicator of Cell Growth Antibacterial activity of extracts, fractions, or purified compounds can be assessed and the minimal inhibitory concentration (MIC) value deter- mined by this assay (18,19). Sufficient amounts of dried crude extracts are dissolved in dimethyl sulfoxide (DMSO) to obtain stock solutions of 5 mg/mL concentration. For purified compounds, the concentration is normally 1 mg/mL. Ciprofloxacin or any other broad-spectrum antibiotic could be used as a positive control. Normal saline, resazurin solution, and DMSO were used as negative controls. The antibacterial test is performed using the 96-well microplate-based broth dilution method, which utilized resazurin solution as an indicator of bacterial growth. All tests are gener- ally performed in triplicate. 10.2.1. Preparation of Bacterial Species The bacterial cultures are prepared by incubating a single colony over- night in nutrient agar at 37 C. For each of the bacterial species, 35 g of the bacterial culture is weighed into two plastic centrifuge tubes using aseptic techniques. The containers are covered with laboratory parafilm. The bac- terial suspension is then spun down using a centrifuge at 4000 rpm for 10 min. The pellets are resuspended in normal saline (20 mL). The bacterial culture is then centrifuged again at 4000 rpm for another 5 min. This step is repeated twice to obtain a ‘‘clean’’ bacterial culture for the purpose of the bioassay. The supernatant is discarded and the pellets in each of the centrifuge tubes are resuspended in 5 mL of normal saline. The two bacter- ial suspensions of the same bacteria are added aseptically to a sterile universal bottle, thereby achieving a total volume of 10 mL. The optical density is measured at a wavelength of 500 nm using a CE 272 Linear Readout Ultraviolet Spectrophotometer, and serial dilutions are carried out to obtain an optical density in the range of 0.5–1.0. The actual values are noted and the cell-forming units are calculated using equations from previously provided viability graphs for the particular bacterial species (19). The bacterial solution is diluted accordingly to obtain a concentra- tion of 5Â105 CFU/mL. 10.2.2. Preparation of Resazurin Solution One tablet of resazurin is dissolved in 40 mL sterile distilled water to obtain standard resazurin solution. Natural Product Isolation 21
  • 35. 10.2.3. Preparation of 96-Well Plates and Assay The top of the 96-well plates is labeled appropriately. For evaluating the activity of two different extracts, 100 mL of the extracts in DMSO, cipro- floxacin, normal saline, and resazurin solution is pipetted into the first row. The extract is added to two columns each, while the controls to one column each. Normal saline (50 mL) is added to rows 2–11. Using fresh sterile pipet tips, 50 mL of the contents of the first row is transferred to the second row. Serial dilutions are carried out until all the wells contain 50 mL of either extracts or controls in descending concentrations. Resazurin solu- tion (10 mL) is added, which is followed by the addition of 30 mL of triple- strength broth (or triple-strength glucose in the case of Enterococcus faeca- lis) to each of the wells. Finally, 10 mL of bacterial solution of 5Â105 CFU/ mL concentration is added to all the wells starting with row 12. The plates are wrapped with clingfilm to prevent bacterial dehydration, and then incubated overnight for 18 h at 37 C. The presence of bacterial growth is indicated by color change from purple to pink. 11. Conclusion Currently, there are a number of well-established methods available for extraction and isolation of natural products from various sources. An appropriate protocol for extraction and isolation can be designed only when the target compound(s) and the overall aim have been decided. It is also helpful to obtain as much information as possible on the chemical and physical nature of the compound(s) to be isolated. For unknown natural products, sometimes it may be necessary to try out pilot extraction and isolation methods to find out the best possible method. At the time of choosing a method, one should be open-minded enough to appreciate and weigh the advantages and disadvantages of all available methods, particularly focusing on their efficiency and, obviously, the total cost involved. Continuous progress in the area of separation technology has increased the variety and variability of the extraction and isolation meth- ods that can be successfully utilized in the extraction and isolation of natural products. For any natural product researcher, it is therefore essen- tial to become familiar with the newer approaches. In most cases, ex- traction and isolation of natural products are followed by structure determination or confirmation of the purified components. With the intro- duction of various hyphenated techniques (see Chap. 9), it is now possible to determine the structure of the compound as separation is carried out, 22 Sarker et al.
  • 36. without isolation and purification (21). Because of the phenomenal progress made in the area of MS and NMR in the last few decades, it has now become possible to deduce the structure of a compound in micro- gram amounts (22–24), thereby further blurring the boundaries between analytical and preparative methods. 12. Notes 1. The conclusive structure determination of an unknown complex natural product using high-field modern 1D and 2D NMR techniques requires the compound to be pure, 90%. The known structure of a compound can be deduced from a less pure one. In X-ray crystallographic studies, materials are required in an extremely pure state, 99.9% pure. For bioassays, it is also important to know the degree of purity of the test compound. The most reliable assay result can be obtained with a compound of $100% purity, because it excludes any possibilities of having activities resulting from minor impurities. 2. If the extraction is designed just to provide enough material for confirmation or denial of a proposed structure of a previously isolated compound, it may require less material or even partially pure material, because in many cases this does not require mapping out a complete structure from scratch, but per- haps simply a comparison with a standard of known structure. 3. Approximate quantification can be performed by assaying a set of serial dilu- tions of every fraction at each stage of the separation process. To detect the peaks of activity, it is often necessary to assay the fractions at a range of dilu- tions, which approximately indicate the relative amounts of activity (propor- tional to the amount of compound present) in each fraction. Thus, the fraction(s) containing the bulk of the active compounds can be identified, and an approximate estimation of the total amount of activity recovered, relative to starting material, can be obtained. 4. Ecdysteroids, invertebrate steroidal compounds, are insect-molting hor- mones, and have also been found in various plant species. References 1. Samuelsson, G. (1999) Drugs of Natural Origin: A Textbook of Pharmacog- nosy. 4th revised ed. Swedish Pharmaceutical Press, Stockholm, Sweden. 2. Cannell, R. J. P. (1998) How to approach the isolation of a natural product, in Natural Products Isolation. 1st ed. (Cannell, R. J. P., ed.), Humana Press, New Jersey, pp. 1–51. 3. Cragg, G. M., Newmann, D. J., and Snader, K. M. (1997) Natural products in drug discovery and development. J. Nat. Prod. 60, 52–60. Natural Product Isolation 23
  • 37. 4. Cragg, G. M. and Newman, D. J. (2001) Natural product drug discovery in the next millennium. Pharm. Biol. 39, 8–17. 5. Cragg, G. M. and Newman, D. J. (2001) Medicinals for the millennia—the historical record. Ann. N. Y. Acad. Sci. 953, 3–25. 6. Nakajima, M., Okazaki, T., Iwado, S., Kinoshita, T., and Haneishi, T. (1989) New diterpenoid antibiotics, spirocardins A and B. J. Antibiot. 42, 1741–1748. 7. Konishi, M., Nishio, M., Saitoh, K., Miyaki, T., Oki, T., and Kawaguchi, H. (1989) Cispentacin, a new antifungal antibiotic I. Production, isolation, phy- sicochemical properties and structure. J. Antibiot. 42, 1749–1755. 8. Sarker, S. D., Girault, J. P., Lafont, R., and Dinan, L. (1997) Ecdysteroid xylosides from Limnanthes douglasii. Phytochemistry 44, 513–521. 9. Sarker, S. D., Sik, V., Dinan, L., and Rees, H. H. (1998) Moschatine: an unusual steroidal glycoside from Centaurea moschata. Phytochemistry 48, 1039–1043. 10. Ekabo, O. A., Farnsworth, N. R., Henderson, T. O., Mao, G., and Mukherjee, R. (1996) Antifungal and molluscicidal saponins from Serjania salzmanniana. J. Nat. Prod. 59, 431–435. 11. Farnsworth, N. R. (1990) The role of ethnopharmacology in drug develop- ment, in Bioactive Compounds from Plants (Chadwick, D. J. and Marsh, J., eds.), John Wiley and Sons, New York, pp. 2–21. 12. Blinov K. A., Carlson D., Elyashberg M. E., et al. (2003) Computer assisted structure elucidation of natural products with limited 2D NMR data: appli- cation of the StrucEluc system. Magn. Reson. Chem. 41, 359–372. 13. Steinbeck, C. (2004) Recent developments in automated structure elucida- tion of natural products. Nat. Prod. Rep. 21, 512–518. 14. van de Ven, F. J. M. (1995). Multidimensional NMR in Liquids: Basic Principles and Experimental Methods, Wiley-VCH, New York, USA. 15. Takao, T., Watanabe, N., Yagi, I., and Sakata, K. (1994) A simple screening method for antioxidants and isolation of several antioxidants produced by marine bacteria from fish and shellfish. Biosci. Biotechnol Biochem. 58, 1780–1783. 16. Kumarasamy, Y., Fergusson, M., Nahar, L., and Sarker, S. D. (2002) Biological activity of moschamindole from Centaurea moschata. Pharm. Bio. 40, 307–310. 17. Dinan, L., Savchenko, T., Whiting, P., and Sarker, S. D. (1999) Plant nat- ural products as insect steroid receptor agonists and antagonists. Pestic. Sci. 55, 331–335. 18. Drummond, A. J. and Waigh, R. D. (2000) Recent Research Developments in Phytochemistry. vol. 4 (Pandalai, S. G., ed.) Research Signpost, India, pp. 143–152. 24 Sarker et al.
  • 38. 19. Sarker S. D., Eynon E., Fok K., et al. (2003) Screening the extracts of the seeds of Achillea millefolium, Angelica sylvestris and Phleum pratense for antibacterial, antioxidant activities and general toxicity. Orient. Phar. Exp. Med. 33, 157–162. 20. Hostettmann, K. and Wolfender, J.-L. (2001) Application of liquid chroma- tography and liquid chromatography/NMR for the on-line identification of plant metabolites, in Bioactive Compounds from Natural Sources (Tringali, C., ed.), Taylor and Francis, New York, USA, pp. 31–68. 21. Viletinck, A. J. and Apers, S. (2001) Biological screening methods in the search for pharmacologically active natural products, in Bioactive Com- pounds from Natural Sources (Tringali, C., ed.), Taylor and Francis, New York, USA, pp. 1–30. 22. Neri, P. and Tringali, C. (2001) Applications of modern NMR techniques in the structure elucidation of bioactive natural products, in Bioactive Com- pounds from Natural Sources (Tringali, C, ed.), Taylor and Francis, New York, USA, pp. 69–128. 23. Peter-Katalinic, J. (2004) Potential of modern mass spectrometry in structure elucidation of natural products. International Conference on Natural Pro- ducts and Physiologically Active Substances (ICNOAS-2004), Novosibirsk, Russia. 24. Peter-Katalinic, J. (1994) Analysis of glycoconjugates by fast-atom-bomb ardment mass-spectrometry and related ms techniques. Mass Spectrom. Rev. 13, 77–98. Natural Product Isolation 25
  • 39. 2 Initial and Bulk Extraction Ve´ronique Seidel Summary Currently, there is a growing interest in the study of natural products, especially as part of drug discovery programs. Secondary metabolites can be extracted from a variety of natural sources, including plants, microbes, marine animals, insects, and amphibia. This chapter focuses principally on laboratory-scale processes of initial and bulk extraction of natural products from plant and microbial sources. With regard to plant natural products, the steps required for the preparation of the material prior to extraction, including aspects concerning plant se- lection, collection, identification, drying, and grinding, are detailed. The various methods available for solvent extraction (maceration, per- colation, Soxhlet extraction, pressurized solvent extraction, ultra- sound-assisted solvent extraction, extraction under reflux, and steam distillation) are reviewed. Further focus is given on the factors that can influence the selection of a method and suitable solvent. Specific extraction protocols for certain classes of compounds are alsodiscussed. Regardingmicrobialnaturalproducts,thischaptercoversissuesrelating to the isolation of microorganisms and presents the extraction methods available for the recovery of metabolites from fermentation broths. Methods of minimizing compound degradation, artifact formation, extract contamination with external impurities, and enrichment of extracts with desired metabolites are also examined. Key Words: Solid–liquid extraction; extraction methods; initial extrac- tion; bulk extraction; maceration; percolation; Soxhlet extraction; ultra- sonification; pressurized solvent extraction; extraction under reflux; steam distillation; infusion; decoction; broth fermentation. 27 From: Methods in Biotechnology, Vol. 20, Natural Products Isolation, 2nd ed. Edited by: S. D. Sarker, Z. Latif, and A. I. Gray ß Humana Press Inc., Totowa, NJ
  • 40. 1. Introduction The natural products of interest here are small organic molecules (mol wt 2000 amu approx.), which are also frequently called secondary metabolites and are produced by various living organisms. The natural material (or biomass) originates from several sources including plants, microbes (e.g., fungi and filamentous bacteria), marine organisms (e.g., sponges, snails), insects, and amphibia. Unlike the ubiquitous macromole- cules of primary metabolism (which are nutrients and factors fundamental for survival, (e.g., polysaccharides, proteins, nucleic acids, lipids), second- ary metabolites comprise a range of chemically diverse compounds often specific to a particular species, which are not strictly essential for survival. Nevertheless, there is a growing interest in their study (particularly as part of drug discovery programs) as they represent a formidable reservoir of potentially useful leads for new medicines. Prior to any isolation and purification work, natural products have to be extracted (or released) from the biomass. This could be with a view to iso- late a known metabolite or to isolate and characterize as many compounds as possible (some of unknown structure) in the context of a systematic phy- tochemical investigation. An initial extraction is performed typically on a small amount of material to obtain a primary extract. This can be as part of a pharmacological study or to gain preliminary knowledge on the exact nature and amount of metabolites present in the material. Once specific metabolites have been identified in the initial extract, it may then become desirable to isolate them in larger quantities. This will involve either recol- lecting a larger amount of plant material or increasing the scale of the fer- mentation. In both cases, a bulk or large-scale extraction should follow. Since natural products are so diverse and present distinct physicochem- ical properties (e.g., solubility), the question to address is how can these metabolites be extracted efficiently from the material under investigation. Solvent-extraction methods available for the initial and bulk laboratory- scale extraction of natural products from plant and microbial sources (solid–liquid extraction mainly) are presented in this chapter. Focus is also made on particular procedures, useful for removing unwanted interfering contaminants and enriching the extract with desired metabolites. Various other available natural product extraction methods are discussed in Chapters 3, 10, and 13–16. 28 Seidel
  • 41. 2. Method 2.1. Extraction of Plant Natural Products Plants are complex matrices, producing a range of secondary metabolites with different functional groups and polarities. Categories of natural pro- ducts commonly encountered include waxes and fatty acids, polyacetylenes, terpenoids (e.g., monoterpenoids, iridoids, sesquiterpenoids, diterpenoids, triterpenoids), steroids, essential oils (lower terpenoids and phenylpropa- noids), phenolics (simple phenolics, phenylpropanoids, flavonoids, tannins, anthocyanins, quinones, coumarins, lignans), alkaloids, and glycosidic deri- vatives (e.g., saponins, cardiac glycosides, flavonoid glycosides). Several approaches can be employed to extract the plant material. Although water is used as an extractant in many traditional protocols, organic solvents of varying polarities are generally selected in modern methods of extraction to exploit the various solubilities of plant constituents. Solvent- extraction procedures applied to plant natural products include maceration, percolation, Soxhlet extraction, pressurized solvent extraction, ultrasound- assisted solvent extraction, extraction under reflux, and steam distillation. 2.1.1. Preparation of Plant Material 2.1.1.1. SELECTION Any plant species and plant parts, collected randomly, can be investi- gated using available phytochemical methods. However, a more targeted approach is often preferred to a random selection. The plant material to be investigated can be selected on the basis of some specific traditional ethnomedical uses (see Note 1). Extracts prepared from plants and used as traditional remedies to treat certain diseases are more likely to contain biologically active components of medicinal interest. Alternatively, the plant can be selected based on chemotaxonomical data. This means that if species/genera related to the plant under investigation are known to con- tain specific compounds, then the plant itself can be expected to contain similar compounds. Another approach is to select the plant with a view to investigate a specific pharmacological activity. Additionally, work can be carried out on a particular group of natural products, a plant family, or on plants from a specific country or local area. Some plants can be selected following a combination of approaches. The use of literature databases (see Chap. 12) early in the selection process can provide some preliminary information on the type of natural products already isolated from the plant and the extraction methods employed to isolate them. Initial and Bulk Extraction 29
  • 42. 2.1.1.2. COLLECTION AND IDENTIFICATION The whole plant or a particular plant part can be collected depending on where the metabolites of interest (if they are known) accumulate. Hence, aerial (e.g., leaves, stems, flowering tops, fruits, seeds, bark) and under- ground (e.g., bulbs, tubers, roots) parts can be collected separately. Only healthy specimens should be obtained, as signs of contamination (fungal, bacterial, or viral) may be linked to a change in the profile of metabolites present. Collection of plant material can also be influenced by other fac- tors such as the age of the plant and environmental conditions (e.g., tem- perature, rainfall, amount of daylight, soil characteristics, and altitude). In some cases, it can be challenging, if not hazardous. This is particularly true if the targeted plant is a species of liana indigenous to the canopy (60 m above ground level!) of a remotely accessible area of the rain forests. It is important to take these issues into account for recollection purposes to ensure a reproducible profile (nature and amount) of metabolites. It should be stressed that the plant must also be identified correctly. A specialized taxonomist should be involved in the detailed authentication of the plant (i.e., classification into its species, genus, family, order, and class). Any features relating to the collection, such as the name of the plant, the identity of the part(s) collected, the place and date of collection, should be recorded as part of a voucher (a dried specimen pressed between sheets of paper) deposited in a herbarium for future reference. More details on this particular aspect can be found in Chapter 13. 2.1.1.3. DRYING AND GRINDING If the plant is known to contain volatile or thermolabile compounds, it may be advisable to snap–freeze the material as soon as possible after col- lection. Once in the laboratory, the collected plants are washed or gently brushed to remove soil and other debris. Frozen samples can be stored in a freezer (at À20 C) or freeze-dried (lyophilized) (see Note 2). It is usual to grind them subsequently in a mortar with liquid nitrogen. Extracting the pulverized residue immediately or storing it in a freezer to prevent any changes in the profile of metabolites (1,2) is advisable. It is, however, a more common practice to leave the sample to dry on trays at ambient temperature and in a room with adequate ventilation. Dry conditions are essential to prevent microbial fermentation and subsequent degradation of metabolites. Plant material should be sliced into small pieces and distributed evenly to facilitate homogenous drying. Protection from 30 Seidel
  • 43. direct sunlight is advised to minimize chemical reactions (and the formation of artifacts) induced by ultraviolet rays. To accelerate the drying process (especially in countries with high relative humidity), the material can be dried in an oven (see Note 3). This can also minimize enzymatic reactions (e.g., hydrolysis of glycosides) that can occur as long as there is some residual moisture present in the plant material. The dried plant material should be stored in sealed containers in a dry and cool place. Storage for prolonged periods should be avoided, as some constituents may decompose. The aim of grinding (i.e., fragmentation of the plant into smaller parti- cles) is to improve the subsequent extraction by rendering the sample more homogenous, increasing the surface area, and facilitating the penetration of solvent into the cells. Mechanical grinders (e.g., hammer and cutting mills) are employed conveniently to shred the plant tissues to various par- ticle sizes. Potential problems of grinding include the fact that some mate- rial (e.g., seeds and fruits rich in fats and volatile oils) may clog up the sieves and that the heat generated may degrade thermolabile metabolites. 2.1.2. Range of Extraction Methods A number of methods using organic and/or aqueous solvents are employed in the extraction of natural products. Supercritical fluid extrac- tion (which uses carbon dioxide in a supercritical state as the extractant), a solvent-free and environment-friendly method of extraction, is discussed in Chapter 3. Solvent extraction relies on the principle of either ‘‘liquid–liquid’’ or ‘‘solid–liquid’’ extraction. Only the latter is described here, and theoretical and practical aspects related to liquid–liquid extraction are covered in Chapter 10. In solid–liquid extraction, the plant material is placed in con- tact with a solvent. While the whole process is dynamic, it can be simplified by dividing it into different steps. In the first instance, the solvent has to diffuse into cells, in the following step it has to solubilize the metabolites, and finally it has to diffuse out of the cells enriched in the extracted meta- bolites. In general, extractions can be facilitated by grinding (as the cells are largely destroyed, the extraction relies primarily on the solubilization of metabolites) and by increasing the temperature (to favor solubilization). Evaporation of the organic solvents or freeze-drying (of aqueous solu- tions) yields dried crude extracts (see Note 4). Initial and Bulk Extraction 31
  • 44. 2.1.2.1. MACERATION This simple, but still widely used, procedure involves leaving the pulver- ized plant to soak in a suitable solvent in a closed container at room temperature. The method is suitable for both initial and bulk extraction. Occasional or constant stirring of the preparation (using mechanical shakers or mixers to guarantee homogenous mixing) can increase the speed of the extraction. The extraction ultimately stops when an equilibrium is attained between the concentration of metabolites in the extract and that in the plant material. After extraction, the residual plant material (marc) has to be separated from the solvent. This involves a rough clarification by decanting, which is usually followed by a filtration step. Centrifugation may be necessary if the powder is too fine to be filtered. To ensure exhaus- tive extraction, it is common to carry out an initial maceration, followed by clarification, and an addition of fresh solvent to the marc. This can be performed periodically with all filtrates pooled together. The main disadvantage of maceration is that the process can be quite time-consuming, taking from a few hours up to several weeks (3). Exhaus- tive maceration can also consume large volumes of solvent and can lead to the potential loss of metabolites and/or plant material (see Note 5). Furthermore, some compounds may not be extracted efficiently if they are poorly soluble at room temperature. On the other hand, as the extrac- tion is performed at room temperature, maceration is less likely to lead to the degradation of thermolabile metabolites. 2.1.2.2. ULTRASOUND-ASSISTED SOLVENT EXTRACTION This is a modified maceration method where the extraction is facilitated by the use of ultrasound (high-frequency pulses, 20 kHz). The plant pow- der is placed in a vial. The vial is placed in an ultrasonic bath, and ultra- sound is used to induce a mechanical stress on the cells through the production of cavitations in the sample. The cellular breakdown increases the solubilization of metabolites in the solvent and improves extraction yields. The efficiency of the extraction depends on the instrument fre- quency, and length and temperature of sonication. Ultrasonification is rarely applied to large-scale extraction; it is mostly used for the initial extraction of a small amount of material. It is commonly applied to facil- itate the extraction of intracellular metabolites from plant cell cultures (4). 32 Seidel
  • 45. 2.1.2.3. PERCOLATION In percolation, the powdered plant material is soaked initially in a sol- vent in a percolator (a cylindrical or conical container with a tap at the bottom) (see Note 6). Additional solvent is then poured on top of the plant material and allowed to percolate slowly (dropwise) out of the bottom of the percolator. Additional filtration of the extract is not required because there is a filter at the outlet of the percolator. Percolation is adequate for both initial and large-scale extraction. As for maceration, successive perco- lations can be performed to extract the plant material exhaustively by refilling the percolator with fresh solvent and pooling all extracts together. To ensure that percolation is complete, the percolate can be tested for the presence of metabolites with specific reagents (see Chap. 4). There are several issues to consider when carrying out a percolation. The extent to which the material is ground can influence extracts’ yields. Hence, fine powders and materials such as resins and plants that swell excessively (e.g., those containing mucilages) can clog the percolator. Furthermore, if the material is not distributed homogenously in the con- tainer (e.g., if it is packed too densely), the solvent may not reach all areas and the extraction will be incomplete. Both the contact time between the solvent and the plant (i.e., the percolation rate) and the temperature of the solvent can also influence extraction yields. A higher temperature will improve extraction but may lead to decomposition of labile metabolites. The other disadvantages of percolation are that large volumes of solvents are required and the process can be time-consuming. 2.1.2.4. SOXHLET EXTRACTION Soxhlet extraction is used widely in the extraction of plant metabolites because of its convenience. This method is adequate for both initial and bulk extraction (see Note 7). The plant powder is placed in a cellulose thimble in an extraction chamber, which is placed on top of a collecting flask beneath a reflux condenser. A suitable solvent is added to the flask, and the set up is heated under reflux. When a certain level of condensed solvent has accumulated in the thimble, it is siphoned into the flask beneath. The main advantage of Soxhlet extraction is that it is a continuous process. As the solvent (saturated in solubilized metabolites) empties into the flask, fresh solvent is recondensed and extracts the material in the thimble continu- ously. This makes Soxhlet extraction less time- and solvent-consuming than Initial and Bulk Extraction 33
  • 46. maceration or percolation. However, the main disadvantage of Soxhlet extraction is that the extract is constantly heated at the boiling point of the solvent used, and this can damage thermolabile compounds and/or initiate the formation of artifacts. 2.1.2.5. PRESSURIZED SOLVENT EXTRACTION Pressurized solvent extraction, also called ‘‘accelerated solvent extrac- tion,’’ employs temperatures that are higher than those used in other meth- ods of extraction, and requires high pressures to maintain the solvent in a liquid state at high temperatures. It is best suited for the rapid and repro- ducible initial extraction of a number of samples (see Note 8). The pow- dered plant material is loaded into an extraction cell, which is placed in an oven. The solvent is then pumped from a reservoir to fill the cell, which is heated and pressurized at programmed levels for a set period of time. The cell is flushed with nitrogen gas, and the extract, which is automati- cally filtered, is collected in a flask. Fresh solvent is used to rinse the cell and to solubilize the remaining components. A final purge with nitrogen gas is performed to dry the material. High temperatures and pressures increase the penetration of solvent into the material and improve metabo- lite solubilization, enhancing extraction speed and yield (5). Moreover, with low solvent requirements, pressurized solvent extraction offers a more economical and environment-friendly alternative to conventional ap- proaches (6). As the material is dried thoroughly after extraction, it is pos- sible to perform repeated extractions with the same solvent or successive extractions with solvents of increasing polarity. An additional advantage is that the technique can be programmable, which will offer increased reproducibility. However, variable factors, e.g., the optimal extraction temperature, extraction time, and most suitable solvent, have to be deter- mined for each sample. 2.1.2.6. EXTRACTION UNDER REFLUX AND STEAM DISTILLATION In extraction under reflux, plant material is immersed in a solvent in a round-bottomed flask, which is connected to a condenser. The solvent is heated until it reaches its boiling point. As the vapor is condensed, the sol- vent is recycled to the flask. Steam distillation is a similar process and is commonly applied to the extraction of plant essential oils (a complex mixture of volatile constitu- ents). The plant (dried or fresh) is covered with water in a flask connected to a condenser. Upon heating, the vapors (a mixture of essential oil and 34 Seidel
  • 47. water) condense and the distillate (separated into two immiscible layers) is collected in a graduated tube connected to the condenser. The aqueous phase is recirculated into the flask, while the volatile oil is collected sepa- rately. Optimum extraction conditions (e.g., distillation rate) have to be determined depending on the nature of the material being extracted (see Note 9). The main disadvantage of extraction under reflux and steam dis- tillation is that thermolabile components risk being degraded. 2.1.3. Selection of an Extraction Method and Solvent The ideal extraction procedure should be exhaustive (i.e., extract as much of the desired metabolites or as many compounds as possible). It should be fast, simple, and reproducible if it is to be performed repeatedly. The selec- tion of a suitable extraction method depends mainly on the work to be carried out, and whether or not the metabolites of interest are known. If the plant material has been selected from an ethnobotanical point of view, it may be worthwhile reproducing the extraction methods employed traditionally (if they are reported) to enhance the chances of isolating poten- tial bioactive metabolites. Traditional methods rely principally on the use of cold/hot water, alcoholic, and/or aqueous alcoholic mixtures to obtain pre- parations that are used externally or administered internally as teas (e.g., infusions, decoctions). Boiling solvent can be poured on the plant material (infusion) or the plant can be immersed in boiling solvent (decoction). If a plant has already been investigated chemically, a literature search can indi- cate the extraction methods employed previously. However, this does not exclude the possibility of choosing an alternative method that may yield dif- ferent metabolites. If a plant is being investigated for the first time, the lack of information on suitable extraction methods leaves the choice to the inves- tigator. The selection will be governed by the nature and amount of material to be extracted. If large amounts are to be extracted, the ease of transfer from initial to bulk scale must also be considered. Extraction processes can employ water-miscible or water-immiscible solvents. The solvent selected should have a low potential for artifact formation, a low toxicity, a low flammability, and a low risk of explosion. Additionally, it should be economical and easily recycled by evaporation. These issues are particularly important in the case of bulk extraction where large volumes of solvents are employed. The main solvents used for extraction include aliphatic and chlorinated hydrocarbons, esters, and lower alcohols (Table 1) (see Note 10). Initial and Bulk Extraction 35
  • 48. Extractions can be either ‘‘selective’’ or ‘‘total.’’ The initial choice of the most appropriate solvent is based on its selectivity for the substances to be extracted. In a selective extraction, the plant material is extracted using a solvent of an appropriate polarity following the principle of ‘‘like dissolves like.’’ Thus, nonpolar solvents are used to solubilize mostly lipophilic compounds (e.g., alkanes, fatty acids, pigments, waxes, sterols, some terpenoids, alkaloids, and coumarins). Medium-polarity solvents are used to extract compounds of intermediate polarity (e.g., some alkaloids, flavo- noids), while more polar ones are used for more polar compounds (e.g., fla- vonoid glycosides, tannins, some alkaloids). Water is not used often as an initial extractant, even if the aim is to extract water-soluble plant consti- tuents (e.g., glycosides, quaternary alkaloids, tannins) (see Chap. 16). A selective extraction can also be performed sequentially with solvents of increasing polarity. This has the advantage of allowing a preliminary separation of the metabolites present in the material within distinct extracts and simplifies further isolation (7). In an extraction referred to as ‘‘total,’’ a polar organic solvent (e.g., ethanol, methanol, or an aqueous alcoholic mixture) is employed in an attempt to extract as many compounds as possible. This is based on the ability of alcoholic solvents to increase cell wall permeability, facilitating the efficient extraction of large amounts of polar and medium- to Table 1 Physicochemical Properties of Some Common Solvents Used in Natural Products Extraction Solvent Polarity index Boiling point ( C) Viscosity (cPoise) Solubility in water (% w/w) n-Hexane 0.0 69 0.33 0.001 Dichloromethane 3.1 41 0.44 1.6 n-Butanol 3.9 118 2.98 7.81 iso-propanol 3.9 82 2.30 100 n-Propanol 4.0 92 2.27 100 Chloroform 4.1 61 0.57 0.815 Ethyl acetate 4.4 77 0.45 8.7 Acetone 5.1 56 0.32 100 Methanol 5.1 65 0.60 100 Ethanol 5.2 78 1.20 100 Water 9.0 100 1.00 100 36 Seidel
  • 49. low-polarity constituents. The ‘‘total’’ extract is evaporated to dryness, redissolved in water, and the metabolites re-extracted based on their partition coefficient (i.e., relative affinity for either phase) by successive partitioning between water and immiscible organic solvents of varying polarity (see Chap. 10) (8,9). Specific protocols during which the pH of the extracting aqueous phase is altered to solubilize selectively groups of metabolites (such as acids or bases) can also be used. For instance, these are applied to the extraction of alkaloids (which occur mostly as water-soluble salts in plants). On treat- ing the plant material with an alkaline solution, the alkaloids are released as free bases that are recovered following partition into a water-immiscible organic solvent (10). Subsequent liquid–liquid extractions and pH modifi- cations can be performed to separate the alkaloids from other nonalkaloi- dal metabolites (see Chap. 10). Alternatively, alkaloids can be extracted from the plant material in their salt form under acidic conditions (11). Acidic extraction is also applied to the extraction of anthocyanins (12). However, one drawback of the acid–base treatment is that it can produce some artifacts and/or lead to the degradation of compounds (13–15). Finally, single solvents or solvent mixtures can be used in extraction protocols. When a solvent mixture is necessary, a binary mixture (two mis- cible solvents) is usually employed. In a Soxhlet extraction, it is preferable to use a single solvent simply because one of the solvents in the mixture may distill more rapidly than another. This may lead to a change in the solvent proportions in the extracting chamber. 2.2. Extraction of Microbial Natural Products Microorganisms are also a valuable source of chemically diverse and potentially useful metabolites. To date, mostly filamentous bacterial spe- cies of the genus Streptomyces (Actinomycetes) and fungal species of the genera Penicillium and Aspergillus have been used for the extraction and isolation of their metabolites, which have important medical applications (e.g., antibiotics, immunosuppressants, hypocholesterolemic and antican- cer agents). The search for novel microbial metabolites has been driven by the need for new antibiotics to combat the ever-increasing number of pathogenic microbes that are resistant to current antimicrobial agents. Aspects related to the selection, culture, and extraction of the microbial biomass are presented below. Further details on the purification and char- acterization of microbial metabolites are provided in Chapter 15. Initial and Bulk Extraction 37
  • 50. 2.2.1. Isolation and Fermentation Because of the enormous diversity of the microbial world, it is not a sim- ple task to select, identify, and culture pure strains that produce potentially bioactive metabolites. As many microorganisms are found in the soil, the investigation of microbial metabolites usually starts with the collection of soil samples. A wide variety of environments (e.g., soils of unusual compo- sition or those from different climatic areas) can be explored to search for novel strains. The sample collected is typically prepared as a suspension in water, and appropriate dilutions of the supernatant are plated on a solid (agar) medium. Streptomyces species are widely found in the soil and will grow well on normal nutrient agar. The isolation of other species usually requires the use of selective media (e.g., MacConkey’s medium for Gram- negative bacteria), the use of antibacterial/antifungal agents (e.g., nystatin to inhibit the growth of molds and fungi), and/or particular incubation conditions (e.g., thermophilic strains require incubation at 50 C) (see Note 11). Once individual colonies are obtained, they are subcultured sev- eral times on different media until they display purity (morphologically and microscopically). Pure strains are commonly stored in liquid nitrogen or freeze-dried in the presence of a cryoprotective agent (see Note 12). To enable cell growth and metabolite production, the isolated strains are transferred from stock to liquid broth (see Note 13). The culture (or fermentation) is carried out initially in flasks containing a liquid medium before the strain is transferred to small fermenters (stain- less steel closed vessels) and the whole process is scaled up. In flask fermen- tation, the culture is grown in a nutrient broth dispensed in flasks that are sealed and placed on a rotary shaker at a defined temperature (see Note 14). This allows a relatively good set-up to monitor both the growth rate of the biomass and the production of metabolites. It also provides a means of carrying out initial studies to optimize culture conditions and increase metabolite production (see Note 15). When performing studies in a small fermenter, the culture is grown under controlled conditions (see Note 16). The process can be scaled up once the effects of other important parameters for metabolite production have been optimized (e.g., aeration, stirring speed, temperature, pH, oxygen, and carbonic acid concentration), and the absence of external contamination has been ascer- tained. It is important that the growth of the producing strain be consis- tent to ensure reproducible productivity. This may not be true in cases where the morphology of the culture is different while growing in 38 Seidel
  • 51. fermenters as opposed to flasks (e.g., actinomycetes and filamentous fungi can grow as two different morphologies, hyphae or pellets). 2.2.2. Selection of Extraction Methods When selecting an extraction procedure for microbial metabolites, the following considerations should be borne in mind. Microbial metabolites are often produced in low yields, and one strain can yield a complex mixture of compounds. The metabolites may be completely or partially excreted by the cells into the (extracellular) medium or they may be present within the cells (intracellular). If metabolites of a certain type are expected, it is possi- ble to refer to previously published protocols. The situation is more difficult when the strain is new, the metabolites are hitherto unknown, or the aim is to extract as many metabolites as possible. As for plant material, water- miscible and immiscible organic solvents, e.g., ethyl acetate (EtOAc), dichloromethane (DCM), n-butanol, methanol (MeOH), and so on, are used for the extraction of microbial metabolites (Table 1). A variety of approaches can be employed in the recovery of microbial metabolites from fermentation broths. If the metabolites of interest are not only associated with the cells but are also present in the medium, a whole-broth solvent extraction is usually required to solubilize both intra- and extracellular compounds. In some cases, the fermentation broth may be freeze-dried prior to the extraction (16,17). Alternatively, it can be clarified first by separating the microbial cells from the liquid medium prior to extraction. Clarification is achieved by filtration or centrifugation depending on the broth’s physical properties (e.g., consistency) and the morphology and size of cells (see Note 17). Extraction is simpler if the metabolites are either entirely in the liquid medium, or adsorbed onto or located within the cells. For metabolites associated with the cells, it is advi- sable to perform the clarification (removal of media constituents and other contaminants) prior to the extraction (18). The preliminary removal of physical ‘‘impurities’’ (e.g., cells, cell debris, insoluble medium compo- nents) is also advantageous if the metabolites of interest are principally extracellular. The extraction of compounds is then performed by partition- ing the medium (aqueous phase) between a water-immiscible organic solvent (19,20). Changing the pH of the aqueous phase and selecting a sol- vent into which the desired metabolites partition efficiently can extract metabolites selectively depending on their pKa and partition coefficients. Adsorption procedures can also be employed in the extraction of metabolites from the medium. These exploit the fact that most secondary Initial and Bulk Extraction 39
  • 52. metabolites will be retained if the medium (aqueous solution) is passed through a column packed with a hydrophobic adsorbent. Following washes with water to elute inorganic salts and highly polar material (desalting step), elution with organic solvents (e.g., MeOH, acetone) or aqueous mixtures of organic solvents yields an extract enriched in the metabolites (see Note 18) (21,22). The adsorbent can sometimes be added directly to the fermentation broth to ‘‘trap’’ metabolites as they are produced (23). In most cases, it will be necessary to obtain larger amounts of the micro- bial metabolites identified in the initial extract. This may be to carry out biological tests and/or to design structural analogs to investigate structure activity relationships. In such cases, not only is a larger volume of fermen- tation required but also good overall yield of metabolites is necessary for subsequent bulk extraction. 3. Conclusion Plants and microorganisms produce complex mixtures of natural pro- ducts, and the selection of the best protocol for an efficient extraction of these substances is not a simple task. ‘‘Classic’’ solvent-based procedures (e.g., maceration, percolation, Soxhlet extraction, extraction under reflux, steam distillation) are still applied widely in phytochemistry despite the fact that they lack reproducibility and are both time- and solvent- consuming. This is principally because they only require basic glassware and are convenient to use for both initial and bulk extraction. Accelerated solvent extraction is a newer instrumental technique. While it offers some advantages over conventional methods (mainly efficiency and reproduci- bility), it is best suited for initial rather than bulk extraction. It has found a wider application in industry (where large numbers of extracts have to be produced in an efficient and reproducible way) rather than in academia. To date, mainly plant and microbial sources have been investigated for their metabolites. However, it is important to remember that researchers are only beginning to explore other biotopes (e.g., the marine environ- ment, insects) and that many plants and microorganisms have not yet been characterized. Moreover, several species among the bacteria known are yet to be cultured under laboratory conditions (24). This leaves much scope for the potential discovery of novel and/or useful natural products in the future. 40 Seidel

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