METHODS IN BIOTECHNOLOGY

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Natural
Products
Isolation
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Second Edition
Edited by

Satyajit D. Sarker
Zahid Latif
Alexander I. Gray

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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

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METHODS IN BIOTECHNOLOGY

Wales, United Kingdom

Alexander I. Gray
Phytochemistry Research Lab
Department of Pharmaceutical Sciences
University of Strathclyde
Glasgow, Scotland, United Kingdom

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© 2006 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.

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Cover design by Patricia F. Cleary

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Preface

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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

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Satyajit D. Sarker
Zahid Latif
Alexander I. Gray

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Preface to First Edition

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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


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 scientists 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 experience of natural products work, but where there is great biodiversity and, hence,
great potential for utilizing and sustaining that biodiversity through the discovery of novel, useful natural products.
Richard J. P. Cannell

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In memory of Richard John Painter Cannell—b. 1960; d. 1999

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Contents
Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v
Preface to First Edition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vii
Contributors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xi
1 Natural Product Isolation

Satyajit D. Sarker, Zahid Latif, and Alexander I. Gray . . . . . . . . . . 1


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
ix

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Contents

14 Isolation of Marine Natural Products

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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


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

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1
Natural Product Isolation
An Overview
Satyajit D. Sarker, Zahid Latif, and Alexander I. Gray
Summary

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

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1. Older strategies:

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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 microorganisms (1). However, in most cases the term natural products refers to secondary metabolites, small molecules (mol wt
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Natural Product Isolation

Fig. 1. An example of natural product drug discovery process (bioassayguided approach).

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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 different 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.

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Presented a large number of crude drugs from natural
1550 BC
sources (e.g., castor seeds and gum arabic)
460–377 BC Hippocrates, ‘‘The
Described several plants and animals that could be
Father of Medicine’’ 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
Presented information and pictures of medicinal
(herbals)
plants
BC

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Natural Product Isolation


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 Penicillium 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 (

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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

All secondary metabolites produced by one natural source that are not produced 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 chemical fingerprinting or metabolomics study (see Chap. 9).

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Natural Product Isolation

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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 characterize 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:

3. Choice of extraction method

Maceration.
Boiling.
Soxhlet.
Supercritical fluid extraction.
Sublimation.
Steam distillation.


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,

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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 concentrations 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.

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6. Isolation


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and EtOAc, CHCl3, DCM, or n-hexane, followed by an assay to determine the
distribution of compounds in solvent fractions.
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 property 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.
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).
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 biological activity. If the initial extraction of natural products is carried out at
a high temperature, the test for heat stability becomes irrelevant.

5.

High-performance thin-layer chromatography (HPTLC).
Multiflash chromatography (e.g., BiotageÕ).
Vacuum liquid chromatography (VLC).
Chromatotron.
Solid-phase extraction (e.g., Sep-PakÕ).

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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

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Natural Product Isolation

Fig. 2. Isolation of microbial natural products: spirocardins A and B from
Nocardia sp.

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Fig. 3. Isolation of microbial natural products: cispentacin from B. cereus.