Natural Products Isolation Techniques
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Transcripts - Natural Products Isolation Techniques
M E T H O D S I N B I O T E C H N O L O G Y ᮀ 2 0TM
Satyajit D. Sarker
Alexander I. Gray
Satyajit D. Sarker
Alexander I. Gray
Natural Products Isolation
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,
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
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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
13. Supercritical Fluid Methods and Protocols, edited by John R. Williams and Anthony A.
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
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
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M E T H O D S I N B I O T E C H N O L O G Y ™
M E T H O D S I N B I O T E C H N O L O G Y
Satyajit D. Sarker
Pharmaceutical Biotechnology Research Group
School of Biomedical Sciences
University of Ulster at Coleraine
Coleraine, Northern Ireland
Molecular Nature Limited
Plas Gogerddan, Aberystwyth
Wales, United Kingdom
Alexander I. Gray
Phytochemistry Research Lab
Department of Pharmaceutical Sciences
Glasgow, Scotland, United Kingdom
University of Strathclyde
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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.
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
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
reference guide to all of the available techniques for the more
experienced among us.
Satyajit D. Sarker
Alexander I. Gray
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
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
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
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
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
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
RUSSELL A. BARROW • Microbial Natural Product Research Laboratory,
Department of Chemistry, The Australian National University,
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,
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,
STEVEN M. MARTIN • Cubist Pharmaceuticals (UK) Ltd, Slough,
JAMES B. MCALPINE • Ecopia BioSciences Inc., Frederick Banting,
Saint Laurent, Quebec, Canada
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,
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,
Natural Product Isolation
Satyajit D. Sarker, Zahid Latif, and Alexander I. Gray
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;
Products of natural origins can be called ‘‘natural products.’’ Natural
products include: (1) an entire organism (e.g., a plant, an animal, or a
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
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
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
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
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.
Fig. 1. An example of natural product drug discovery process (bioassay-
Natural Product Isolation 3
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
History of Natural Product Medicine
Period Type Description
(knowledge of life)
Introduced medicinal properties of plants and other
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
Presented information and pictures of medicinal
4 Sarker et al.
century, a number of top selling drugs have been developed from natural
products (vincristine from Vinca rosea, morphine from Papaver somni-
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
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
3. by indicating new modes of pharmacological action that allow complete
synthesis of novel analogs (e.g., synthetic analogs of penicillin from Penicil-
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
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).
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.
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
d. Supercritical ﬂuid extraction.
f. Steam distillation.
The fundamentals of various initial and bulk extraction techniques for
natural products are detailed in Chapters 2 and 3.
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
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.
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.
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
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).
5. Solid-phase extraction (e.g., Sep-PakÕ).
Natural Product Isolation 9
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
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.
Fig. 2. Isolation of microbial natural products: spirocardins A and B from
Natural Product Isolation 11
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.
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
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
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
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).
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
14 Sarker et al.
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
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
Fig. 6. Isolation of plant natural products: saponins from S. salzmanniana.
16 Sarker et al.
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
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
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.
—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
C J mod., nOe-diff., and so on.
b. Two-dimensional techniques: 1
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.
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.
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
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
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
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-
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
, 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.
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
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
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
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.
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.
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.
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.
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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-
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Rev. 13, 77–98.
Natural Product Isolation 25
Initial and Bulk Extraction
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.
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.
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
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.
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
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
22.214.171.124. 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.
126.96.36.199. 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
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
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
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.
188.8.131.52. 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).
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.
184.108.40.206. 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
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
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.
220.127.116.11. 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.
18.104.22.168. 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
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
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
Physicochemical Properties of Some Common Solvents Used in Natural
Solubility in water
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
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
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
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
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
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
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.
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