Waterman & Mole. Analysis of Phenolic Plant Metabolites. Blackwell 1994

BLACKWE
METHODS IN ECOLOGY
Analysis of
Phenolic Plant Metabolites
PETER G.WATERMAN
Phy~ochemisrlyResearch Laboratories
D~parlmenlof PharmflceuticaF Sciances
Urtiv~rsilyof Strathclyde, UK
SIMON MOLE
School of Biological Sciences
Uniwrsiry of Nebraska at Lincoln, USA
OXFORD
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Contents
The Methods in Ecology Series, vi

Preface, vii
1 Structure and biosynthesis of phenolic compounds, 1
2 Patterns in the distribution of phenolic secondary metabolites, 36
3 Why are phenolic compounds so important? 44
4 Extraction and chemical quantification, 66
5 Biochemical techniques for tannins, 104
6 Methodsforin vivostudies, 134
7 Qualitative and quantitative separation methods, 143
8 Structure elucidation of phenolics, 168
9 For the future? Current and future utility of chemoecological studies
based on phenolics, 199
References, 210
Subject index, 231
Chemical substance and group of substances index, 235
- --
The Methods in Ecology Series
The explosion of new technologies has created the need for a set of
concise and authoritative books to guide researchers through the wide
range of methods and approaches that ate available to ecologists. The
aim of this series is to help graduate students and estaMished scientists
choose and employ n methodology suited to a particular problem. Each
volume is not simply a recipe book, but takes a critical look at different
approaches to the solution of a problem, whether in the laboratory o r in
the field, and whether involving the collection or the analysis of data.
Rather than reiterate established methods, authors have been encour-
aged to feature new technologies, often borrowed from other disciplines,
that ecologists can apply to their work. Innovative techniques, properly
used, can offer particularly exciting opportunities for the advancement of
ecology.
Each book guides the reader through the range of methods available,
letting ecotogists know what they could, and could not, hope to learn by
using particular methods or approaches. The underlying principles are
discussed, as well as the assumptions made in using the methodology, and
the potential pitfalls that could occur - the type of information usually
passed on by word of mouth or learned by experience. The books also
provide a source of reference to further detailed information in the
literature. There can be no substitute for working in the laboratory of a
real expert on a subject, but we envisage this Methods in EcoPogy Series
as being the 'next best thing'. We hope that, by consulting these books,
ecologists will learn what technologies and techniques are available, what
their main advantages and disadvantages are, when and where not to use
a particular method, and how to interpret the results.
Much is now expected of the science of ecology, as humankind struggles
with a growing environmental crisis. Good methodology alone never
solved any problem, but bad or inappropriate methodology can only
make matters worse. EcoIogists now have a powerful and rapidly growing
set of methods and tools with which to confront fundamental problems of
a theoretical and applied nature. We hope that this series will be a major
contribution towards making these techniques known to a much wider
audience.
John H.Lawton
Gene E. Likens
Preface
Our goal for this volume is to provide information and methods for the
study of phenolic plant metabolites in ecological research programmes.
We have taken a rather broad view and have included not only methods
of quantitative and qualitative analysis but also those for isolation and
identification of individual compounds.
The opening chapter deals with the chemical classification of phenolic
compounds and the biosynthetic routes used to achieve this in living
organisms. This is valuable as background information and should help to
make sense of the bewildering diversity of naturally occurring chemical
structures. The second chapter makes some observations about the
evolution of these metabolites and the distribution patterns that we see
today.
In the third chapter we have attempted to provide an outline as to why
phenolic compounds have attracted such attention among ecologists. This
review has of necessity been very selective as there is enough material in
the literature to warrant a book in its own right. We hope that in the
space available we have been able to identify the achievements and point
out some of the problems.
Chapters 4, 5 and 6 deal with sample preparation, extraction and
chemical, biochemical and in vivo methods for the quantification of
phenolics and tannins. In each case the most widely used methods have
been detailed and, after taking a deep breath, we have made recom-
mendations as to which we would regard as the most appropriate for
ecological studies. We have erred on the side of caution in our selection
and have generally recommended tried and tested procedures that shouId
be of value for a good many years into the future.
The next two chapters deal with the study of individual phenolics rather
than with their collective chemical or biological analysis. In the first
the emphasis is on chromatographic methods that can be employed
to separate, identify and quantify individual components of complex
mixtures. The second deals with the problem of finding the structure of a
previously unknown phenolic compound. Initially this was felt to be
outside the scope of a book this size but as the text evolved the inclusion
of this information seemed to become more warranted. While few
ecologists wiIl perform the types of analyses detailed in this chapter, the
knowledge gained from reading it should be of value if they are faced
with trying to persuade a chemist to carry out a structure elucidation.
i
vlll Preface
Finally, we return to the role of phenolics in ecological studies all"

make some suggestions concerning future areas of research, involving
phenolics, that seem to us to be particularly important. Research on the
ecological impact of phenolics continues to expand rapidly and we are
mindful that there are already a number of additional papers that warrant
inclusion.
The authors extend their thanks to Jeffrey Harbome, Clive Jc)ReS
and John Lawton for reading various drafts and for their many useful
comments. One of us (S.M.) wishes to acknowledge the United States
Department of Argiculture (CSRS grant 89-37153-4467) for finartcial
support while writing this book.
Finally, to those of you who have undertaken or are undertalcing
studies involving phenolic metabolites we hope this work is of inte:rest
and value - and good luck!
P.G.W.
S.M.
CHAPTER 1
Structure and biosynthesis of

phenolic compounds
1.1 Introduction
The plant world ik not colored green; it is colored morphine, cafleine,
tannin, p heno], terpene, canavanine, latex, phytohaem -agglutinin, oxalic
acid, snponin, L-dopa, etc. (Janzen, 1978)
The above statement encapsulates the realization by ecoiogists during

the 1960s and 1970s that plants were not just collections of ceHulose,
lipids, carbohydrates and proteins but also contained a vast range of
other molecules. While the presence of these chemicals has been recog-
nized only relatively recently, humans 'have unknowingly exploited them
throughout history for economic purposes (tannin, dyestuffs, etc.), as
medicines (almost everything we used until this century), to aid in food
gathering (fish and arrow poisons) and in crop protection.
The numbers of these compounds identified to date may now exceed
100000 (Buckingharn, 1993), many of which are characteristic of only one
'
or very few species. As they have appeared to have no intrinsic role in the
physiological processes of the producer they have become known as
secondary metabolites and as the products of secondav metabolh. But
as secondary metabolic processes have their own specific complement of
specialist enzymes, are under strict genetic control, and have appreciable
energetic and metabolic requirements it is difficult to accept the once
widely held belief that they are waste products.
The recognition that secondary metabolites were bound to be en-
countered by other organisms interacting with the producer and that
through their chemical properties and biological activity they could influ-
ence that interaction slowly infiltrated into ecological thinking in the
1960s and 1970s (see Chapter 3). However, once this potential function
was recognized the investigation of these interactions became one of the
fastest growing areas of ecology (Harborne, 1988a). It has even gained its
own name - chemical ecology. This is a truty interdisciplinary subject
which, if to be tackled by an individual rather than by a research team,
calls for expertise in two very separate areas, ecoIogy and analytical
organic chemistry. It is fortunate that some of the pioneer practitioners of
chemical ecology were skilled in both and were able to set standards for
others to aspire to, but the fact remains that ignorance of chemistry has
marred the work of many ecologists. This is not meant as a specific cri-
ticism of ecologists; the same is true to an even greater degree when
2 Chapter 1
chemists have attempted to work in this area without appropriate eco-

logical knowledge or assistance,
The most commonly investigated group of secondary metabolites
are those known collectively as phenolics. Attempts to interpret their
role and ecological impact have given us some of the best examples of
chemical ecology but have also illustrated many of the pitfalls of the
discipline. The purpose of this book is to provide the scientist contem-
plating work in chemical ecology and using phenolic secondary meta-
bolites with a text which will alert them to the various methods available
for their quantification, isolation and identification as well as the advantages
and disadvantages of each of these. To this end there are chapters dealing
with general structure and biosynthesis, distribution, procedures for
sample collection, chemical and biological methods for quantification,
chromatographic and other techniques for separation and spectroscopic
methods for identification. In addition we have attempted to put the
study of phenolic metabolites into an ecological context, hopefully as-
sisting the reader to interpret results to the full without over-interpreting
them.
Why deal only with phenolics and not with the other common groups
of metabolites such as alkaloids and steroidal triterpenes? It is partly
because we are restricted by the length of this book. However, there are
other reasons. Phenolic compounds are ubiquitous in plants, and methods
to quantify them can, to a degree, be approached in a general manner.
That is, we can construct assay procedures that will give us information
about concentrations and activity of phenolic compounds that can be
employed with most organisms: it is far less easy to do this for other
groups of secondary metabolite such as alkaloids (Waterman, 1993a).
Likewise, while the array of biological activity exhibited by phenolic
compounds is very considerable, it is possible to relate some types of
activity to readily quantifiable levels of phenolic compounds in a manner
which is impossible for other types of secondary metabolite. For example,
the statement that a plant extract contains 4% weight by weight of
condensed tannin conveys far more information a bout potential ecological
impact than would the statement that the extract contains 4% weight by
weight of alkaloids. Condensed tannins have certain predictable properties
whatever the source; the alkaloids could be as different as strychnine and
caffeine. It is for these reasons that phenolics have been a major focus of
chemical ecology, and we expect that this will continue to be the case in
the future.
1.1.2 What do we mean by phenolic?

The term 'phenolic' is used to define substances that possess one or more
hydroxyl (OH) substituents bonded onto an aromatic ring. The name
derives from the simple parent substance phenol (1).Compounds that
I" Structure and biosynthcsia 3
@-
have several or many phenolic hydroxyl substituents are often referred to
as polyphenols. However, it must be recognized that wt all hydroxyl-
groups are phenolic; they are equally likely to occur bonded to non-
aromatic cyclic or to non-cyclic structures ( e .g. ethanol 2a, cholesterol 2b)
in which case they do not have the properties of a phenol.
An important property of phenolic hydroxyl groups is their acidity,

which is due to the propensity for the bond between the oxygen and
hydrogen to break (Scheme 1.la) to form the corresponding negatively-
charged phenoxide ion (3). The phenoxide exhibits enhanced water solu-
bility, particularly in the presence of simple metal cations such as sodium
or organic cations like ammonium (NHdf ). While all phenolic compounds
exhibit these properties the degree to which this ionization occurs can be
modified by other substituents on the aromatic nucleus. For example, the
addition of two nitro (NOz) groups to the C-2 and C-4 positions in phenol
makes the compound a stronger acid than acetic acid.
Scheme 1.1 (a) Formation of the phenoxide ion and delocaliza-

tion, (b) derivation of the radical species, and (c) hydrogen bond-
ing between a phenol (gallic acid) and caffeine (after Haslam,
1989).
a)
oOL
0": fJo--oo
u
- + -
3 3a 3b
0°4QL~~;
-
- .wO
b)
U
4 48 4b Continued
.,I !
4 Chapter 1
Scheme 1.1 Conrinued
cl HOOC
COOH
HO
OH
Notable properties of phenols include:

1 the ability of the phenoxide ion to delocalize, that is to move the
negative charge into the aromatic ring system to form hemiquinone anions
(3a, 3b) in which the charge resides on a carbon rather than on the oxy-
gen (Scheme 1. la). Such negatively charged centres can attract psitively
charged (electrophilic) a1kylating groups (see below).
2 the potential for the phenoxide ion to lose a further electron (E) to
form the corresponding radical (4) which can also delocalize (4a, 4b,
Scheme 1 . lb). Two such radicals can undergo a process known as oxida-
tive coupling in which covalent carbon- carbon or carbon-oxygen bonds
are formed. We will also return to this prmss later in the chapter.
3 the capacity of phenols to form hydrogen bonds with other molecules
through interaction between the acidic (positively charged) phenolic
hydrogen and basic (negatively charged) centrcs in other molecules. An
example of this is the interaction between caffeine and three molecules of
gallic acid (Scheme 1.1~;as described by Haslam, 1989).
These properties are all of consequence in the biosynthesis of phenolic
compounds and are also centraI to many of the interactions between the
producer and other organisms in its environment (see Chapters 3 and 9).
The versatility shown by organisms, particularly plants, in the pro-
duction of secondary metabolites is quite staggering, particularly when
the very limited range of buiiding blocks that they employ is taken into
account (we below). Among the many structural classes of secondary
metabolite (e.g. alkaloids, flavonoids, triterpenes, etc.) most include
some products that can be classified as phenolic. However, in some
classes this phenolic nature arises as the result of secondary modifications
of the initial metabolite (e.g. most terpenes), while in other classes a
phenolic nature is preordained because of the mechanism of formarion.
In the remainder of this chapter we first describe the various major
groups of phenolic metabolites in terms of the structural skeleta and we
Structure and biosvnthesis 5
hope to make you familiar with their nomenclature. We will then attempt
to rationalize these structural types by explaining their metabolic origins.
1.2 Major classes of naturdly occurring phenolic compounds
I -2.1 Simpk phenols possessing a single aromatic ring

Such compounds are certainly very widespread (in reatity probably ubiq-
uitous) in higher and lower plants. They can be divided into subgroups
depending on the number of carbons that occur in a side-chain (C,)
attached to a phenolic, aromatic (Ch) nucleus (e.g. C6C,).In practice the
number n is usually between zero and three in higher plants but phenols
with long alkyl chains are also known.
102.1.1 c6c0
The simple C 6 q phenols encountered are most frequently derived from
the trihydroxy compounds pyrogaliol (5) and phloroglucinol (6) and,
less commonly, the dihydroxy substitution patterns of catechol (5a),
resorcinol (6a) and hydroquinone (7). The pyrogallol, phloroglucinol and
catechol hydroxylation types also occur very widely as part of more
complex molemIes, as does the monohydroxylated aromatic ring. The
resorcinol and hydroquinoue patterns are much less common in complex
structures.
1.2.1.2 c,c,
The most important phenol in this class is gallic acid (81, in which the
single carbon side-chain is a carboxylic acid. As well as occurring as
a discrete entity, gallic acid and its dimeric form ellagic acid are the
common phenolic components of hydrolysable tannins (see below). Be-
cause they are acids they have the capacity to combine with hydroxyl-
containing compounds (both phenols and alcohols) to form esters (Scheme
1.2). As a result gallic acid and other phenolic acids can often be found
added to other metabolites which may themselves not be phenolic. Other
important C6C1 compounds include the willow metabolites such as sali-
cylic acid (9) and salicylaldehyde (10). Protocatechuic acid (111, with the
catechol (5a) oxygenation pattern, is also quite widespread.
Scheme 1.2 Formation of ester bond between acid and phenolic or
alcoholic hydroxyl.
.-e, - OP.S
o+~
phew3
r---------7
--.c ------
J
acid
-HI.
ester
1.2.1.3 C6C2
The acetophenones, of which xanthoxylin (12) is a typical example, are
the commonest C6C2derivatives but these are less widely distributed than
either CbClor C6C3c~rnpoumds.The oxygenation of C6C2 compounds
can follow the phloroglucinol (6) pattern, ar in 12, or can be simpler, as
in para-h ydroxyphenylacetic acid (13).
1.2.2.4 c6c3
CoIIectively these compounds are often called phenylpropenes and phen-
ylpropanes, depending upon the presence (in the case of pbenyipropene)
nr absence (in the case of phenylpropane) of a double-bond in the
3°C side-chain. The hydroxy cinnamic acids, para-coumaric acid (14),
caffeic acid (151, ferulic aid (16) and sinapic acid (17), are among the
commonest of all phenolic substances. Numerous compounds uccur with
this C6C3 system modified by changes in substitution patterns on the
aromatic nucleus or by ~nodificationof the oxidation level in the side-
chain, as in wniferyl alcohol (18) and eugenol (19). Cnniferyl alcohol is
central to the formation of lignin, a high molecular weight polymer
which, while undoubtedly phenolic, is not trcated in this volume because
of its insolubiIity and consequent biochemical inertness. That is not to say
that lignin and lignification are not important in studies of food selection
by herbivores. However, lignin is best quantified by means of the stan-
dard proximate fibre anaIysis methods (van Soest , 1977).
Like gallic acid, the cinnamic acids are able to esterify phenols at the
hydroxyl substituents (Scheme 1.2). Cinnamic acid esters occur as 'add
ons' to numerous other classes of metabolites: pnra-wumaric and ferulic
acids in particular are often found esterified to the hydroxyls of sugar
molecules, both in secondary metabolites and in cell walls.
19
16 OCH; H
1.2.2 More complex metabolites based on the CbC3skeleion
1.2.2.1 Lignans (C6C3 dimers)

Not to be confused with lignin, lignans are products of the linking (dimer-
ization) of two phenylpropene or phenylpropane precursors. Complexa-
tion between the C6c3parent monomers (usually coniferyl alcohol, 18)
can be either simple, as in nordihydroguaiaretic acid (NDGA) (201,
or more complex, as in pinoresinol (21). Hordatine-A (22) illustrates
bonding between one aromatic nucleus and thc Cg sidc-chain of the
8 Chapter 1
second participating monomer rather than between the two C3 side-

chains. Hordatine-A is unusual in having further linked with an amine
(the decarboxylated form of the amino acid arglnine) to form an amjde
(the CO-NH hk). Hordatine-A does, of course, remain phenolic, even
though it can also be classed as an alkaloid.
1.2.2.2 Coumarin~
The simple cournarin or benzopyran-2-one system has a cyclized C6C3
skeleton which occurs widely. Somc coumarins such as umbelliferone (23)
and aescuIetin (24) are common and are often found in glywsidic form,
e.g. aesculin (25). Other mumarins that have attracted considerable
attention among chemical ecologists are furocoumarins, but these are
rarely phenolic. For example, one of the most common h~rocoumarinsis
xanthutoxin 126); the corresponding phenolic analogue, xant hotoxol (27),
is much rarer.
1.2.2.3 Chromon~s
The benzopyran ring system is very common in the plant kingdom but
it is more extensively encountered as a benzopyran-&one (28) rather
than the benzopyran-2-one form seen in cuumarins (e.g. 23). In most
cases the benzopyran-4-one nucleus is further substituted at C-2. Alkyl
substituents occur sporadically giving compounds, usually termed
chromones, and exemplified by aloesin (29) and eugenin (30).
1.2.3 Metabolites with a CaCo-2C6 carbon skeletmt

This heterogeneous grouping contains a number of different skeleta1
classes none of which are encountered very frequently in higher plants.
Structure and biosynthtsis g
The aryl-pyrones are bicyclic compounds exemplified by aloenin (31).

Pyrones exist in which the two rings are joined directly by a carbon-
carbon bond, as in 31, or where there are a number of linking carbons
(usually two) between the rings (cf. 32). Many of the known pyrones
are non-phenolic because of methylation of the hydroxyls. Stilbenes and
dsocoumarins are characterized by possessing two aromatic nuclei in a
C6C2Garrangement; most members of this class are phenolic. Pinosylvin
(32)is a simple example of a fully unsaturated phenolic stilbene. Lunularic
acid (33) has Iost the double-bond in the Cz linking chain and has an
additional carboxylic acid group. In hydrangeol (34) the carboxylic acid
has cyclked to give what is known as an isocoumarin nucleus.
1.2.4 Metabolites with a CdC3C6skeleton

The CBC3C6skeleton IS the structural type encountered most often in
the investigation of phenolic compounds. It forms the backbone for all
flnvonoids, l~eoflavonoidsand condensed tannins and, in a modified form,
for isoflavonoids. According to Harborne (1488b) some 4000 flavonoid
structures are known, of whidi the majority wiU be phenolic, There is no
sign that the rate at which new flavonoids are being reported is declining.
1.2.4.2 Flavonoids
Most metabolites with the benzopyran-Cone nucleus (cf. 28) are char-
acterized by having an aromatic substituent at C-2, and these axe known
collectively as flavonoids. Virtually all flavonoids carry oxygenation, often
still including unmodified phenolic groups, on the aromatic ring of the
benzopyran (ring A) and many also on the substituent at C-2 (ring B). Six
of the most common types of flavonoid are illustrated: (i) flavanones-
naringenin (39) and pinocembrin (36) ; (ii) flavanols- taxifohn (37),(iii)
flavones-luteolin (38); (iv) flavonols- kaempferol(39), quercetin (40) and
10 Chapter 1
myricetin (41); (v) flavan-3-01s-catechin (42); and (vi) anthocyanidins-

pelargonidin (43), cyanidin (44) and delphinidin (45). These examples are
all common or relatively common compounds in their various subclasses.
They represent the tip of the iceberg. Modifications that occur commonly
include loss of oxygen substituents, further oxygenation on different
carbons, me thylation, prenylatioo (5-C units of terpene origin) and sly-
cosylation (addition of sugars). Methyk tion, prenylation and glymsylation
can all reduce or even eliminate the phenolic nature of a flavonoid.
Dimeric flavonoids (biflavonoids) are known, arnentoflavone (46) being a
typical example (see also condensed tannins, below).
Some compounds classified as flavonoids do not retain the 2-
phenylbenzopyran-4-onenucleus. In chalcones, such as the yellow flower
Structure and biosynthesis 11
pigment carthamin (47), the oxy gen-containing heterocycljc ring has

upened leaving a double-bond system, referred to as the a and flcarbons.
In dihydrochalcones, such as the anti-fungal compound phloridzin (a),
the double-bond has been reduced. Another ,group of yellow flower
pigments are the aurones, such as aureusidin (49), where the oxygen-
containing ring contains one less carbon than in typical flavonoids. This is
known as the benzofuran-3-onesystem.
I . 2.4.2. Neoflavonoids
In neoflavonoids the benzopyran skeleton has been modified by the
additional aromatic ring being attached at C-4, and there is a carbonyl at
C-2 making the benzopyran system similar to that found in mumarins.
In some compounds which are often classified as neoflavonoids the C-4
substitucnt is a saturated 3- or 5-C alkyl group, in which case they do not
conform to the C6C3C6 skeleton. Two examples are the strangely named
insecticidal cornpounds from Mammaa americana (Guttiferae), marnmea
coumarin AlAA (50) and mammea BlBA (51).
I2 Chapter 1
1.2.4.3 Isofivonoids
ln the isoflavonoids the carbon skeleton has become a 3-phenylbenzopyran-
4-one in which it is no longer possible to detect a CdC3C6 pattern (it
appears as C6C2+C6). Within this modified skeleton modifications anal-
agws to those occurring in the flavonoids are found, e .g. isoflavones such
as genistein (52) and formononetin (53) and isoflavanones such as cajanol
(54). Further cyclizations Iead to additional complexity and give many
important bioactive compounds such as: (i) rotenoids- rotenone (55) is
the most important rotenoid (but note it is non-phenolic); (ii) pterocar-
pans-erycristagalhn (56); and (iii) coumestans-coumestrd (57).
I .2.5 Tannins
Few, if any, classes of secondary metabolite have attracted as much
attention from ecologists as tannins or, as they are often called, poly-
phenols; however it should be recognized that the two terms are not
really synonymous, since not all polyphenols are tannins. In the past 20
years tannins have occupied centre stage in chemical ecology, both in
Structure and biosynthesis T3
the development of apparency and resource allocation hypotheses and

through their presumed feeding deterrent activity (see.Chapters 3-5 and
9), a presumption not always sustained by experiment (see Chapter 3).
It is possible to recognize three chemically distinct types of tannin,
each originating from one of the previously discussed groups of simpler
phenols.
1.2.5.1 Phlarotannins
The least complex and least well known type (they have only been
recognized in the last 10 years) are the phlorotannins. These substances
appear to be made up entirely by polymerization of phloroglucinoI (6) or
phloruglucinol units further substituted by halogens. The phloroglucinol
monomers are Bnked through a mixture of carbon-carbon or carbon-
oxygen bonds (5%) to give large polymers.
1.2.5.2 Hydrolysable tannins

Hy drolysable tannins are, like phlorotannins, reliant on one major phe-
nolic building block, in this case gallic acid (8) and its derivatives. The
other consistent feature of hydrolysable tannins is the presence of a sugar
(hexose) core onto which gallic acid can be linked by esterification. One
glucose unit can accommodate esterification with five gdlic acids if it is
in the cyclic (pyranose) form. The simplest hydrolysable tannin is, there-
fore, pentagalloylgiucose (59). In practice the situation is usually more
complex with gaHic acid groups often bonding to each other to give
dimeric forms (@a) which can, when hydrolysed from the hexose by acid,
form ellagic acid (60b), Casuarictin (61) is st typical example of a hy-
drolysable tannin based on a cyclic pyranose sugar. However, the sugar
can also exist in a linear form, and a number of hydrolysable tannins
involving ellagic acid and Aavan-3-01 substituents have recently been
isolated and identified which show this feature, e.g. stenophyllanin (62).
Recent advances in methods of purification and structure elucidation have
14 Chapter 1
Structure and biosynthesis f5
greatly increased our knowledge of hy drolysabIe tannins. These have

been reviewed by Haslam (1989).
1.2.5.3 Condemed bannim

The most widely distributed group of tannins are the condensed tannins.
Here the structure is made up of the linkage of a series of monomers
based on the ftavan-3-01 (42) nucleus or a derivative thereof. These
normally bond between C-4 of one unit and C-8 (more rarely C-6) of
another. Variations can occur through differences in the number of mon-
omers that become linked, the positions between which linkage occurs,
the oxygenation patterns on rings A and B of the Aavan-3-01 units and,
more subtly, in the three-dimensional (stereochemical) relationship that
exists between the aromatic substituent at C-2, the hydroxyl at C-3 and
the inter-monomer bond on C-4. Consider the two most common mon-
omers, catechin (42) and epicatechin (63). The difference between these
flavan-3-01s is the relative stereochemistry of the hydroxyl on C-3 and the

phenyl on C-2. The C-2 substituent is unchanging but the C-3 substituent
varies. In 42 they are on the opposite sides of the ring (trans) while in 63
they are on the same side (cis) (Fig. 1.1).
Fig. 1.1 Three-dimensional structures of catechin (42, R =H,R1== OH)and epicatechin

(63, R = OH,RI=W).
16 Chapter 1
The C-4 bond is generally formed on the side uf the ring opposite to
the C-3 hydroxyi (i.e. tram). Thus there are four options for formation of
a dimer involving epicatechin and catechin. Epicatcchin can bond with
either catechin (64, procyanidin-B1) or epicat echin (65, procyanidin-B2).
Alternatively atechin can link with either catechin (66, procyanidin-B3)
or epicatechin (67, procyanidin-B4). The next stage of polymerization
(to a trimer) will, obviously, again increase the number of possible com-
binations (to 27. An additional source of variation in condensed tannins
is the potential to add substituents to the ring hydroxyls, notably the C-3
hydroxyl. Among the common additional substituents are galiic acid and
simple cinnamic acids.
1.2.6 Quinones, benzophenones and allied substances

These are a range of metabolites of moderate to wide distribution that
includes mono-, bi-, tri- and polycyclic aromatic compounds, many of
which are quinonoid (with two carbonyls, usually opposite one another in
a ring) as well as phenolic. Typical examples include naphthaquinotzes
(juglone, 68), a~zthraquinunes (emodin , 69) and hypericin (70). The
quinoid compounds are always highly coloured, but can sometimes exist
in reduced forms which are less coloured ur even colourless. For example,
juglone (68) is a highly active phytotoxin that exists in walnut in a
reduced form and as a glycoside (71). Oxidation of the chalcone car-
thamin (47) to the quinoid form carthamone (72) is responsible for the
change of the flower colour ofsafflower from yellow to red during ageing.
The most common forms of benzophenone plant metabolites are the
xantbnes , such as gentisin (73) and mangiferin (74) Depsides, of which
lecanoric acid is an example (75), occur widely in lichens, which are also a
major source of xanthones.
Structure and biosynthesk I?
1.2.7 AIkakoids and terpenes

There are many ways to classify natural products and these axe not all
mutually exclusive. For instance there are currently estimated to be some
7000 alkaloids and probably many more terpenes. Such &mpounds can
also be phenolic, although the frequency of phenols is much reduced,
particularly among the terpenes.
Alkaloids are one of the major groups of natural products and many
of them exhibit considerable bioactivity. The central chemical feature of
18 Chapter 1
alkaloids is that they contain nitrogen and are basic (1.e. alkali-like),
and as such they have solubility properties opposite to those of phenolics.
However, some compounds among those classes of alkaloids which con-
tain aromatic rings in their structure are also phenohc. Some examples
are reticuline (76) and morphine (771, where the phenolic hydroxyls arise
as part of the natural oxidation pattern of the metabolite, and reserpine
(78), where the phenolic nature is due to esterification with gaIIic acid.
The duplicity of phenolic and basic characters gives such alkaloids a
grcater range of solubility than their non-phenolic counterparts.
Among the terpenoids phenols only rarely occur. This stems from the
largely saturated nature of the rnevalunic acid precursor and the law level
of oxygenation arising from the early stages in the biosynthetic processes
leading to terpenoids. Yet despite this, phenolic compounds do occur at
all levels of terpene molecular complexity, from the simple monoterpenes
to compIex diterpenes and triterpenes. Two examples are thymol (79),
which is a monoterptne with mitd antiseptic activity, and the diterpene
picrosalvin (SO), which is a more powerful antibiotic.
Structure and biosrnthesis 18
1.2.8 'Masked' phen olits

When considering the phenolic composition of a material it is necessary
to bear in mind the possibiiities for a hidden 'phenolic potential'. The
presence of a phenol can often be masked by the addition of a sugar unit
to the phenolic hydroxyl to form a glycoside in which sugar and phenolic
compound (the aglycone) are linked by an ether bond (Scheme 1.3). This
'masking' effect can also be achieved by esterification (Scheme 1.2).
Scheme 1.3 Formation of ether link between phenol and sugar (in
this case the P-glucopyranose form of glucose).
___t
phe nu1 alcohol ether
The addition of glucose or a similar sugar (glycosylation) can reduce

phytotoxicity and has the advantage of enhancing water solubility which
Eould assist -with transportation of the metabolite in the plant, or in an
ingesting animal. scopolin (81) is a non-phenolic coumarin glycoside
which has the potential, through the activity of P-glucosidase or acid
hydrolysis (for example in the gut), to convert into the phenolic coumarin
scopoletin (82). The 'masking' effect can involve not only sugars but also
other ether groupings, notably the prenyloxy substituent which is usually
-
very easily removed, or the methyl group, which is generally more stable.
For example, isoimperation (83) is non-phenolic, indeed the prenyloxy
(O-CH2CH=C(CH3)2) substituent makes imperatorin fat-soluble (lipo-
philic). However the prenyloxy group is labile to acid and under appro-
priate conditions can decompose to give xanthotoxot (27), a phenolic
20 Chapter 1
furornumarin. Such a change could occur naturally in, for example, the
gut of a herbivore. The corresponding methyl ether xanthotoxin (26) is
much Iess readily converted into xanthotoxol; only in certain circum-
stances are methyl groups lost as readily from ethers as sugars and
isoprenyl units.
Lass of ether and ester substituents could also occur because of poor
collection or extraction procedures and so give a false impression of the
phenolic content of a plant or plant extract.
1.3 The biosynthetic origins of phenolic compounds

In the remainder of this chapter we will look at the mechanisms by which
the types of structures discussed previously are formed. There are several
comprehensive, well illustrated, texts dealing with this subject of second-
ary metabolite biosynthesis in detail (Geissmann & Crout, 1969; Stumpf
& Conn, 1980-1981; Haslam, 1985; Mann, 1987; Herbert, 1989). We
do not intend to try and prbcis these but rather to highlight the mast
important routes and mechanisms leading to phenolic compounds. The
value in this is that an understanding of the basis of secondary metab-
olism is a prerequisite to making sense of the, apparently bewildering,
range of secondary metabolites with which one is confronted. A word of
warning. Here we are trying to establish in your mind the building blocks,
how they fit together, and the form of the find product. We are not
concerned with the enzymology or the minutiae of processes. The result is
a rather simplistic and mechanistic approach.
An understanding of biosynthesis can also be directly useful to the
chemical ecologist in various situations. For instance, the choice of a
suitaMe radioactive tracer to label a specific allelochemical may depend
on biosynthetic knowledge, and judgements relating to chernosysternatic
relationships usually require an appreciation of biosynthetic routes.
Another particularly topical use of biosynthetic knowledge has been the
calculation of the costs involved in the production of allelochemicals
(Chapin, 1989; Waterman, 1993b).
This survey will demonstrate that the vast majority of phenolic md-
ecules owe their origin to one or more of three buitding blocks, erythrose-
4-phosphate (84), phosphoenol pyruvate (85) and acetyl co-enzyme A
(86). Of these erythrose-4-phosphateand phosphoenol pyruvate are jointly
responsible for what is known as the shikimic acid pathway, while acetyl
co-enzyme A and its activated form, malonyl co-enzyme A, are central to
the potyketide or acetate pathway. Acetyl co-enzyme A can also be
converted into mevalonic acid (87) which is the starting point for the vast
array of terpene compounds.
H2C=C-COOH H3C-C-SCo-A H3C, /O%

I I1
riHOH OP 0 CH,OH
CH20P H~ H
COOH
84 85 86
87
1.3.1 Bu Ming the building blocks

The process of biosynthesis commences, of course, with photosynthesis.
BasicaIIy photosynthesis does two things:
I it captures energy which can be stored in energy-rich molecules that
can later be expended in driving chemical reactions and
2 it fixes carbon by adding one carbon atom, from carbon dioxide, to
a 5-C sugar (ribulose-l,5-diphosphate)and thereby increases the carbon
economy.
carbon Furation occurs as part of the Calvin Cycle which has three
primary functions. Firstly, it produces more of the ribulose-l,5-diphosphate
which is recycled to capture further carbon dioxide. Secondly, it produces
6-C (hexose) sugars which are transported out of the system and stored
as starch (metabolic reserve) or catabolized through the Embden-
Meyerhof pathway leading to the tricarboxylic acid cycle through inter-
mediates such as phosphoenol pyruvate (85) and acetyl co-enzyme A
(86). Thirdly it produces erythrose-4-phosphate (84) which can be taken
from the Calvin Cycle for the purpose of building secondary metabolites.
The major features of the Calvin Cycle and the derivation of 84-87 are
shown in Scheme 1.4.
Scheme 1.4 The Calvin cycle and derivation of erythrose-4-P (841,

phosphoenol pyruvate (85), acetyl co-enzyme A (86) and mevalonic
acid (87).
5C
/
-/ Phnk~synrhe\~s------c Z x 7C
4C +7
. C 7C
4
7C + 3c- 2 x 5C
86
117
22 Chapter 1
1.32 Phenolic me&bokites h i e d sol& on ace@ e o - e n p e A

(the polyketides.
The impact of acetyl co-enzyme A (86) in phenolic secondary metabolism
originates in its ability to polymerize to give chains of carbons (Scheme
1.5) in which every second carbon has a carbonyt (keto) function (hence
poIyketide). This is not a spontaneous reaction. Condensation of two
molecults of acetic acid can be achieved in the laboratory as depicted in
Scheme 1Sa, but this requires a major input of energy. In biosynthesis
the condensation of two molecules of acetyl co-enzyme A is facilitated by
first converting it into nalonyl co-enzyme A (88) by the addition of
carbon dioxide. This changes the acetyl methyl group into a highly active
methylene and so assists the process of polymerization to occur without
the need for high investment of energy. During the condensation the
carbon dioxidc which was added as an activator is Iost (Scheme 1.5b).
This condensation is a repetitive process; the 4-C chain formed can then
go o n to a a with another acetyl co-enzyme A unit to build a 6-C polymer
and so on. Chain lengths can be greater than 20 carbons before modifying
reactions occur.
i Scheme 1.5 Combination of acetate groups to build polyketides. In

(a) where there is no activation much energy is required; in (b) the
malonyl co-enzyme A unit ($8) activates the process.
What folluws the completion of the polymerization process is rela-

tively simple and, in chemical terms, consistent. The pnlyketide chain can
be considered to possess a terminal acid from which the co-enzyme has
been lost (although it is doubtful that this is really the case). Cyclization
of the polyketide chain can occur by elimination of the elements of water
to farm six-membered ring systems. In Scheme 1.6 the simplest example
of this, the cyclization of the polyketide polymer containing three acetate
units ((2-C),, where n = 31, is shown. The initial product of the cycliza-
tion is the triketide (89) but this structure is not favoured and rapidly
undergoes transformation (taut omerisrn) into the thermodynamically
more stable aromatic form (e.g. 6 , phloroglucinoi) in which there are
three phenolic hydroxyl groups. This uxygenation pattern, in which al-
ternate carbons carry oxygen atoms is the unmistakabre trademark of the
acetate-derived secondary metabolite.
Scheme 1.6 Cyclization of a triketide chain to form phloroglucinol

(6)-
0
o*Cr-C*o 6
H/ H'
89
In Scheme 1.7 the results of cyclizations of larger polyketide units are

shown. ,For example, where the polyketide chain is four acetate units long
then it can give an acetophenone (90, cf. 12) or a benzoic acid such as
orsellinic acid (91). Five acetate units can lead to a chromone (92, cf. M),
six a phenylpyrone (93, cf. 31), seven another chromone with a larger C-2
substituent (94, cf. 29) and eight an anthrone (95).
Scheme 1.7 Cyclization of a series of polyketide chains of varying

lengths .
It =4
OH 0
- H:O
HO OH
YO
Continued
, , 24 Chapter 1
Scheme I ,7 Continued
0
COON
- HO OH
91
0vc:;20
n =5
0 0
___L
OH
7-------I
-H20
_3
OH 0
-30
0 U
92
n=6
-2H70
__C
0
- 31
93
OH
I1 s7 r----- --,
CH-, 0 CH3 0
29 -
C'H3 O 94 CHI 0 Cumhued
Scheme 1.7 Continued

rr =8
0 0
-
-3H20
HO
95
Two important points are shown in the examples in Scheme 1.7.

Firstly, it is possible for pdyketide chains of a given length to cyclize in
different ways (i.e. 90 and 91). This is presumed to be due to the
cyclization process actually occurring on a carrier protein, the shape
of which dictates the positions that will participate in ring formation.
Secondly, in some cases (94, 95) the final product has an odd number of
carbon atoms, indicating that one carbon has been lost. This is invariably
the terminal 'carboxylic acid' carbon. While as a resuIt of this 94 and 95
have lost the predictable even number of carbons they do retain the
characteristic residual oxygenation pat tern of acetate metabolites. Ident-
ifying acetate derivatives only becomes more troublesome when, through
secondary reduction reactions, oxygens are lost or gained. This is quite
often seen in anthracene compounds. For example emodin (69) requires
additional oxidation of 95 to give the anthraquinone, while in chrys-
ophanol anthrone (96) thc hydroxyl group has been lost from the C-6
position of 95.
A further way of increasing complexity among polyketide products

is through secondary reactions that link pre-existing metabolites. Two
obvious examples of this are depsides, in which two molecules of orsel-
28 Chapter 1
linic acid (91) are joined through an ester linkage (Scheme 1.2) to give
lecanoric acid (75). A second mechanism, also touched upon earlier, is
through formation of a radical generated from a phenoxide ion (Scheme
l.la,b), Where such species exist the charge can reside on the oxygen or
be taken into the aromatic ring, Two such radicals can subsequently be
removed by formation of a bond between the atoms on which they were
residing. The phlorotannins (e.g. 58) are the result of such a process,
which is known as oxidative coupling. We will note several other ex-
amples of oxidative coupling later in this chapter. It is a common process
in phenolic metabolites.
Scheme 1.8 The electrophilic addition process, in which a posi-

tively charged isoprene (mevalonic acid derived) species is added
to negatively charged centres derived from a phenoxide ion.
a)
j;&.;
-
H3C,
c,C-Z:
H
' -Hz0
- JJy-cH.
$-CH~OP CHrOP -Op- L C H :
q i i H
0
b) OH 0 OH 0
3":&
,
HO HO
O
&
OW
-
0
--0&c~3
OH O
X
-acn \"'
HO
011 0
97
I
A similar process (eIectrophilic addition) also leads to increasing

complexity. The acetophenone metabolite (97) is a good example of
this, showing the addition of two 5-C prenyl units (mevalonic acid derived)
to activated (electron-rich) centres in the acetophenone molecule. These
prenyl units are produced from mevalonic acid as pyrophosphates which
on loss of the phosphate retain a positive charge, ideal for interaction
with the negative chrgcd phenoxide ion (Scheme 1.8a). In the example
shown, one of these prenyl units bonds with an oxygen and the other with
a carbon (Scheme 1.8b).
1.3.3 Phenolic rneaoiites based solely on the shikimic acid path way
The shikimic acid route to secondary metabolites is altogether more
complex and convoluted than the acetate pathway. The building blocks
involved are erythrose-4-phosphate (84) and phosphoenol pyruvate (85).
These are linked in a sequence of reactions to yield shikimic acid (98),
which is characterized by a C6C1skeleton and the oxygenation pattern of
pyrogallol (5). Gallic acid (8) is the fully aromatized form of 98 but as will
be noted later its formation is probably not as straightforward as this
observation might lead one to suppose.
Although we talk of the shikimic acid pathway, the reality is that the
pathway does not start to generate most of its products until further
general structural developments beyond shikimic acid have taken place.
Thus, addition of a second molecule of phosphoenol pyruvate (85) to
shikimic acid leads to the formation of chorisrnic acid (99), and it is at this
point that the route to the amino acid tryptophan (100) separates. From
chorismic acid (99) the main pathway leads to phenylpyruvic acid (101)
and para-hydroxyphenylpyruvic acid (102), where we at last see the
typical 'shikimate' C6C3skeleton. From here, by the addition of nitrogen,
the amino acids phenytalanine (103) and tyrosine (104) are generated
from 101 and 102, respectively. The development of the shikirnic acid
pathway to this p i n t is outlined in Scheme 1.9. The three amino acids
100, 103 and 1Od are perhaps the most important precursors for the
synthesis of alkaloids, at least in terms of the number of alkaloids they
generate (Cordell, 1981).
Proliferation of shikimate-derived non-nitrogenous phenolics is fo-
cused through one of these mino acids, phenylalanine (103) (Scheme
1.10). Through the mediation of the critically important enzyme phenyl-
alanine ammonia lyase (PAL) (Harborne, 1990)phenylalanine is converted
into irans-cinnamic acid (105). This is followed by a sequence of aromatic
hydroxylation and subsequent methylation reactions which lead to the
formation of para-cnumaric acid (14), caffeic acid (15), ferulic acid (16)
and sinapic acid (17). Further diversification can occur through modifi-
cation of the cinnamjc acid side-chain. For example reduction of the
28 Chapter 1
Scheme 1.9 Derivation of aromatic amino acids through the shikimic

acid route.
a COOH
PO - A1*CH, Co(3H
R -
"-;c.@.tH
2
H0
COOH
-
H I1 OH OH
H OH
84
98 , 99
I
COOH * I
- a~fln:"""
H
1
R R
loo
103 R = H 101 R = H
1w R = OH 1W R=OH
double-bond and carboxylic acid of ferulic acid leads to coniferyl alcohol

(18) while further reduction to eliminate the side-chain oxygen com-
pletely and rearrangement of the double-bond gives eugenol (19).
Through oxidation reactions the side-chain can be shortened and it is
probable that para-hydroxyphenylacetic acid (131, protocatechuic acid
(11) and catechol (Sa) arise from 14, 15 and 15, respectively, by dim-
ination of part or all of the side-chain. It also seems. probable that this is
the most common route of formation for gallic acid (a), although some
evidence does exist that it may also be formed, in some plants, directly
from shikimic acid (Haslam, 1986).
The coumarins represent an interesting modification of cinnamic acid
metabolism. In almost all naturally occurring coumarins the precursor
appears to be para-coumaric acid which has to undergo two critical
modifications: the double-bond must change from the trans to the cis
configuration and the aromatic nudeus must undergo oxidation ortho to
the side-chain to give 2,4-dhydroxy-ck-cinnamic acid (106). It is then
possible for elimination of water tts occur between the carboxylic acid and
hydroxyl leading to siglpIe coumarins such as 23-25, It is still debatable
whether in 23-25 substitution occurs at C-6 prior to cyclization (e.g. 15 is
the precursor) or whether the further oxidation 'of the aromatic ring
occurs after coumarin formation (in which case 14 is the precursor).
Scheme 1.10 Proliferation of simple shikimic acid metabolites.
m2 r COOH 0C)H
\. -
1#
" 14 13
-
'I
I*
.:I:/ +.. /5 / 11
23-
no I- 16 18
1M
I
1 h
A further group of simple shikimate-derived secondary metabolites
are the lignans and here we return again to the phenomenon of radical
formatian and oxidative coupling (Schemes 1.1 and 1.8). Using nordihy-
droguaiaretic acid (20), which is probably a derivative of para-coumaric
acid or the corresponding alcohol, as an example (Scheme I. 11) it is easy
to rationalize the biusynthetic events which occur. From para-coumaric
acid a series of radicals can be formed (107-110) culminating in that in
which the B-carbon of the 3-C side-chain is the activated position (e.g.
110). Bringing two such radicals together leads to the required J3-$ bond
and from there it needs only reduction to eliminate the oxygen (this could
have happened earlier in the process) and return to the fully phenolic
form by regaining the hydrogen lost in the initial radical formation to give
nordihydroguaiaretic acid.
In pinoresin01 (21) the mechanism is a little more complex but can
again by readily rationalized. Starting from coniferyl alcohol (18) the
initial formation of the p-p bond needs only to be followed by two
further cyclizations involving, in each case, the alcohol of one monomer
and the a-C of the other.
But note that such oxidative coupling need not initially involve only P-
C radicals. In hordatine-A (22) one p-C radical has interacted with a
second radical equivalent to 108. As in nordihydroguaiaretic acid this
30 Chapter 1
Scheme 1.11 Formation of a simple lignan through radical for-

mation and oxidative coupling of a cinnamic acid precursor.
a)
COOH COOH COOH COOH
I 1 I
cn B
&- 4
1
I %H
CH
HO HO 0 HO 0 Q
107 I08 109 110
b)
OH
_If
0
110
20
has been followed by a secondary modification, this time involving- the

phenolic
- oxygen of one unit and the a-carbon of the other.
coupling-between two cinnamic acid units can be followed by very
extensive secondary modifications. This is seen to greatest effect in com-
pounds called neolignans (e.g. 111) where one of the aromatic nuclei has
been partially reduced.
1.3.4 General comments on acetate and shikimate-den'ved phenolics

From this brief review of acetate and shikimate metabolism it can be
seen that there are two different ways of producing similar phenolic
compounds. The normal pathway of origin can usually be recognized
without great difficulty because of the inherently different patterns of
oxidation, but we have also seen that comparable skeletons can originate
from either route. Irrespective of pathway once the phenol is formed,
mechanisms such as oxidative coupling and substitution with nucleophilic
mevalonate-derived and other units, can then operate for further
modification.
1.3.5 Phenolk compoundF derivedfrom the combination of

shikimate and aceme pathways
Biosynthetic pathways cannot be viewed in isolation. Many types of
secondary metabolite arise by the combination of more than one path-
way, The combination of shikimate and acetate in the form C6C3+ (Qn,
where n is usually three (i. e . C6GC6),gives rise to a number of groups of
phenolic natural products of which the Aavonoids are by far the most
important.
The stilbene Iunularic acid (33) represents a simple example of the
amalgamation of the two pathways. The precursors are para-dihydro-
cournaric acid (112) and a polyketide chain made up of three acetate units
(Scheme 1.12). Linkage between the two participants involves the acid
group of para-cournaric acid and the terminal methyl of the polyketide
and occurs with the typical loss of the elements of water followed by the,
again typical, polyketide cyclization. In lunularic add there is then Ioss of
one aromatic hydroxyl. Pinosylvin (32) is formed in a similar manner
employing cinnamic acid (105) as the shikimate component. In this case
the acetate part retains the full hydroxylation pattern but there is loss of
the carboxylic acid.
Scheme 1.12 Combination of shikimate and acetate pathways to

form stilbenes and related compounds.
.---
---- H
H -
-HP
no HO
112
33 - OH
6
32 Chapter 1
The flavonoids are generated in a comparable manner to the stilbenes

but here the acid of the cinnamic acid participant is not involved in the
cyclization of the acetate moiety (Scheme 1.13). The bicyclic chalcone
skeleton (113) might logically appear to be the first product but in prac-
tice chalcones normally convert spontaneously into flavanones (e .g. 114).
For chalcones to be stable certain substitution requirements are necessary;
in the two examples given earlier (47, 48) glycosyIation provides that
stability. Once formed the flavanone nucleus can undergo modifications
involving the pyran-+one ring. These can involve oxidation (either adding
oxygen or removing hydrogen to form a new double-bond) or reduction
(addition of hydrogen) to give such widespread classes of flavonoids as
flavanols (37), flavones (38) and Ravonols (39-41). Some metabolites,
such as the flavan-3-01s (42) and anthocyanidins (43-45), require more
extensive modification of the pyran system involving the loss of the
carbonyl at C-4. Aurones (115) represent an unusual cyclization of
the chalmne intermediate involving the P-C of the double-bond of the
chalcone-type intermediate (1 13). Oxidation of the cinnarnic acid-derived
B-ring probably occurs after formation of the flavonoid skeleton whereas
two phenolic hydroxyls are formed in the acetatederived A-ring as part of
the initial cyclization process.
The isoflavonoid skeleton (116, and 52-57) originates through a shift
of the aromatic ring from C-2 to C-3 of the pyran and this is again
fallowed by an extensive series of modifications involving the pyran ring
Scheme 1.13 Combination of acetate and shikimate precursors to

form flavonoids and isoflavonoids.
0
- HO
115
\ 37-46 Conrklz~ed
I
b
Scheme 1.13 Continued

HO
\
52-9
116
-
(many changes are analogous to those in the flavonoids) or the formation
of further ring systems which may, as in the rotenoid skeleton (55).
incorporate additional carbon derived from a methoxyt substituent.
Other modifications that can occur to the flavanoid involve dimer-
ization or polymerization. Biflavonoids such as amentoflavone (46) can
once again be explained through oxidative coupling reactions (cf. Scheme
1.11). The condensed tannins seem to be formed from Aavan-3,4-diols
(117), the precursors of the catechins . The condensation between mon-
omers involves the formal lass of the elements of water (Scheme 1.14).
As has already been explained, the products of that condensation will
vary according to the stereochemistry of the monomers (64-67).
Scheme 1.14 Polymerization of flavan-3,4-diol units leading to

condensed tannins. The (WN) bond at C-3 means that the hydroxyl
can be in either possible configuration, so increasing the number of
"
stereochemical possibilities (see Fig. 1.1).
OH
HOw o ~ o *
117 )xL
64-67
OH /OH:
:H '
42
63 OH
OH
The neoflavonoid skeleton offers a further interesting biosynthetic

analogy to the flavonoids. With those neoflavonoids in which C-4 carries
an aromatic substituent, such as mammea coumarin AlAA (SO), cinnamic
acid and acetate components are clearly distinguishable and synthesis can
34 Chapter 1
be presumed to involve linkage between cinnarnic acid and the acetate

chain at the a-C of the former, foilowed by cyciization of the acetate and
then closure of the pyran ring (Scheme 1.15). In mammea coumarin
BlSA (51) we have a compo~indthat is almost certainly all acetate-
derived and which may well involve the comparable series of reactions
but employing two separate chains of three acetates, one the 'normal'
polyketide and the other a fatty acid (CH3(CH2)2CH=CHCOOH).
Scheme 1.15 Alternative acetatelshikimate combination leading to

the 4-phenylneoflavonoids.
--2H I_C
-50
In the xanthones (73,74) the shikimate element is C6C1 rather than

C6C3but otherwise the mechanism of formation is similar to those already
described (Scheme 1.16).
Scheme 1.16 Formation of xanthones from acetate and a C6C1

shikimate-derived precursor.
H~)I1
&;~H;Q 1 0-;
-- H(l OH
HO 0
OH HO OH
1.3.6 Phenolic compounds origimu%g from mevahnic acid

As already noted the very important mevalonate or terpene pathway
plays a relatively minor part in the production of phenolic secondary
metabolites. It is beyond the scope of this volume to discuss the biosyn-

thesis of mevalonate compounds but they are generally simple to re-
cognize because of the central role of the 5-C isoprene unit (118). Of the
two examples of phenolic terpenoids cited earlier thymol (79) is formed
from two such units and picrosalvin (80) from four units. Metabolites with
two, three, four, six, and eight of these 5-C units are a11 commonplace in
nature.
One deployment of mevalonate which we sometimes encounter in

phenolic metabolites is the simple 5-C prenyl (3-methylbutenyl) sub-
stituent which can Occur as either a substituent on aromatic carbon or
phenolic oxygen as in the acetophenone (97) or involved in pyran or
furan ring systems, as in rotenone (55) and the furocoumarin xanthotoxin
(26). In xanthotoxin the mevalonate nature is not readily recognizable as
three of the five carbons have been lost.
In a few cases these prenyl groups can be incorporated into other
molecules to give aromatic rings. Typical examples are the anthraquinone
pseudopurpurin (119) and the carbazole alkaloid mukonidine (120). In
both of these molecules the carbons of rnevalonate origin are highlighted*.
They have become aromatic and carry phenolic hydroxyl groups,
1.4 Concluding comments

If you are a relative newcomer to natural products-chemistry, then this
chapter should have increased your familiarity with the names and struc-
tural pat terns (skeleta) of the commonly encountered groups of phe-
nolics. Beyond this our main biological message has been to shcw the
conservatism of both starting materials and building mechanisms that
organisms employ in the production of phenolics.
Finally we caution the reader that in a book of this size we canhot
be exhaustive in our coverage, and that a specialized background on
particular kinds of phenolics can only be obtained by consulting a similarly
specialized treatise (e.g. Harborne, 1989).
CHAPTER 2
Patterns in the distribution of

phenolic secondary metabolites
2.2 Introduction
It should be obvious from what has been said in Chapter 1 that the for-
mation of secondary metabolites is governed by the presence of enzymes
to catalyse each step in the pathway. Soma of these enzymes seem rather
adaptable and able to catalyse comparable reactions on a number of
substrates, but others are highly specific, These enzymes are direct prod-
ucts of the genetic coding of the producer and so the capacity and ver-
satility of secondary metabolic processes in a species must directly reflect
its genetic history. In this respect it can be argued that the products of
metabolic pathways are more dosely linked to the genome of a species
than are some of the anatomical characters often employed in classifica-
tion, and that the distribution of secondary metabolites will closely follow
phylogenetic relationships. The corollary to this is, of course, that we
should, to some extent, be able to anticipate and predict the secondary
metabolites that a species will be able to synthesize from its taxonomic
position, assuming of course that it has been correctly placed using non-
chemical characters. The importance of being able to adopt a phylogenetic
approach to ecology has recently been discussed (Wanntorp el al., 1990;
Berenbaum & Seigler , 1992).
Unfortunately, this argument is far too simplistic. There are a number
of factors that confound the observation of simple phylogenetic patterns
in the distribution af secondary metabolites. Available evidence begins to
suggest that what we observe in the secondary metabolic profile of a
species reflects only a small part of the potential of that species for
secondary metabolite production. The products of tissue culture are often
not seen in the whole plant. This may indicate that in the whole plant,
enzyme and potential substrata are being kept apart by compartmen-
talization or that some enzymes or enzyme systems that are usually
dormant can be activated under atypical conditions. Wink and Witte
(1983) have detected traces of the alkaloid lupanine in cell suspension
cultures of about 50 out of a selection of 100 species from widely varying
taxonomic positions. Many of the positive species were not expected to
produce this type of alkaloid. They suggest that the genes responsible for
the production of this class of alkaloid may be widely distributed within
the plant kingdom, but are generally 'silent'. Only in some of these
plants, mostly legumes, are these genes activated for alkaloid production.
Patterns in distribution 37
The degree to which a pathway is expressed can be influened by many

extrinsic factors, both biotic and abiotic (Waterman & Mole, 1989).
Yet, taking into account all these caveats, secondary metabolites do,
quite often, exhibit patterns of distribution that are recognizable and
interpretable in terms of phylogenetic relationships. The study of such
relationships is generally referred to as chemotaxonomy or chemical
systematics (Harborne & Turner, 1984; Waterman & Gray, 1987). In this
chapter we will briefly review current ideas on the evolution of secondary
metabolites and the distribution of the groups of phenolic compounds first
outlined in Chapter 1.
Before doing this there is one important point to stress and that is that
while the co-occurrenceof the same or closely allied metabolites in two
species may well reflect a phylogenetic relationship it does not necessarily
do so. fn the course of evolution it is certain that many biosynthetic
mechanisms have been lost (either through deletion of genetic encoding
or 'switching off' the gene) and have later reappeared, either by the
gene being switched back on, or through independent evolution in other
phylogenetic lines. Differentiation between true affinity and between
parallel and convergent biosynthetic pathways is a major problem of
chemical systematics.
2.2 The evolution of phenolic secondary metabolism

We are here in a highly speculative area and what follows in this section
should be treated with circumspection! However, enough data are now
available on the occurrence and distribution of secondary metabolites
for there to be discernible trends and these have been interpreted in
evolutionary terms.
It was Luckner (1972) who observed that the post tyrosine and phenyl-
alanine parts of the shikimic acid biosynthetic pathway (see for example
Schemes 1.10- 1.16) were most highly developed and widely exploited
in land plants. This argument has been taken further by Kubitzki (1987)
who states that:
It is self-evident that the origin and early evolution of land plants
must have been intimately linked to the expansion of the
phenylpropanoid (shikirnate) metabolism. Certainly cutin and
lignin, and probably flavonoids as well, were indispensible
components of the upswing in land plants in the Silurian and
Devonian.
Cutin, which is vital to water retention, contains simple cinnarnic acids as
esterifying groups. Lignins are polymers of cinnamyI alcohols which are
necessary for mechanical functions in a high-gravity environment. The
flavonoids, which may have been critical to early land plants as ultraviolet
98 Chapter 2
(UV) screens (Caldwell, 1981) contain a cinnamic acid unit linked with an
acetate-derived triketide (Scheme 1.13).
If you examine current knowledge of secondary metabolism of some
lower plant phyla then Kubitzki's argument does seem to be supported.
PMorotannins (58) are derived wholly from acetate and their occurrence
appears at present to be restricted to brown algae (Ragan & Glombitza,
1986). However, their distribution is subject to considerable geographical
variability (Steinberg, 1988). By contrast, shikimic acid metabolites are
notable by their absence from algae except for a subset of the green
algae, the Charophycaceae, which produce flavonoids and hydrolysabfe
tannins. It has been suggested by several authorities (see Kubitzki, 1987)
that these green algae, unlike other algae, are closely allied to land plants
and may have actually returned to an aquatic environment from a ter-
restrial or semi-terrestrial life form.
In fungi there appears to again be a bias toward acetate-derived
secondary metabolites but the shikimate pathway also makes an appear-
ance, notably in the use of the aramatic amino acids for the production of
alkaloids (antibiotics) and small peptides. However, as noted by Wat and
Towers (1979), where non-nitrogenous shikimate metabolites are found,
it is normally the case that production originates at the phenylpyruvate
stage (e.g. 101, 102, Scheme 1.9) of the pathway rather than at the
cinnamic acid stage. That is, fungi tend to use C6C3 building blocks
generated before formation of amino acids, and not from the 'post-
phenylalanine' cinnamic acids.
In the Bryophyta the shikimic acid pathway has become more exten-
sivety used in the production of secondary metabolites, notably flavonoids
and cinnamyl alcohols. But, as pointed out by Kubitzki and Gottlieb
(1984), there still seem to be limitations to its exploitation. They suggest
that its involvement is restricted to thnse areas of metabolism where the
cinnarnic acids can be activated by initial combination with co-enzyme A
(notablv by maIony1-S-Co-A), a combination that leads to the formation
of flavomids (Scheme 1.13).
Kubitzki and Gottlieb (1984) argue that the further evolution of
shikimic acid-derived phenolics, first seen widely in the pteridophytes,
depended on the critical step of reduction of cinnamoyl-S-Co-A (vari-
ously substituted in the aromatic nucleus) to the corresponding cinnamy1
alcohols (e.g. 16-18). These reduced dihydrocinnamic acids could then
combine with pdysaccharides to strengthen cell walls and eventually, in
the gymnosperms, polymerize with their congeners to form lignins. It is
also in the gymnosperms that further oxidation and reduction of free
cinnamyl alcohols may have startcd to occur, leading to the formation of
wholly shikimic acid-derived secondary metabolites such as lignans. With
the production of lignin came the advent of condensed tannins, which are
themselves made up of highly rcduccd flavoiioids. T h e woody habit and

condensed tannin production are phenomena that are closely, but not
completely, correlated (Bate-Smith, 1962; Kubitzki, 1987).
These oxidation and reduction processes appear to reach their climax
in what are usually regarded as 'relatively primitive' angiosperms where,
for example, sinapyl alcohol (17) replaces coniferyl alcohol (16) in iignin
production and where the complex neolignans (111) occur. From here on,
however, in the further evolution of angiosperms, both monocotyledons
and dicotyledons, these processes appear to go into reverse. The capac-
ity to oxidize and reduce the cinnamic acid unit lessens, although it is not
completely lost. The significance of other parts of the shikimate pathway
also sccm to regress. In non-phenolic alkaloid metabolism, tyrosine and
phenylalanine lose their importance as precursors, to be replaced by
anthranilic acid and tryptophan, derivatives which arise from chorismic
acid (99, Scheme 1.10). Hydrolysable tannins, formed either by oxidation
of the cinnamic acid side-chain or direct from shikimic acid, also become
important in what are generally regarded as moderately advanccd angio-
sperm families. A useful pictorial scheme describing key elements of this
phytochemical evolution has been presented by Kubitzki and Gottlieb
(1984) and a modified version is reproduced here (Fig. 2.1). Phylogenetic
constraints an the production of secondary metabolites have also recently
been discussed by Beren baum and Seigler (1992).
This scheme should only be regarded as the most broad of templates.
While in supposedly 'modern' angiosperms there is a resurgence of acetate-
derived compounds (and non-phenolic products of mevalonic acid - i.e.
terpenes), flavonoids continue to be produced as do relatively simple
cinnamic acid derivatives. It has been suggested (Molgaard, 1985) that
the continued occurrence of such ubiquitous conipounds as caffeic acid
(15) may indicate their function as a 'carbon sink' for surplus photo-
synthate, an idea that is to some extent convergent with the resource
allocatiori hypothesis that currently holds sway in ecological circles (see
Chapters 3 and 9).
2.3 Distribution of individual classes of secondary metabolites

It is not our purpose here to supply exhaustive information on the dis-
tribution of individual compounds or groups of compounds. To do that
would require a book many times the size of this one. Information on the
occurrence of secondary metabolites and the sources from which they
have bccn obtained is available through Chemical Abstracts and can
be accessed manually or by computer; searching under chemical and
botanical name is possible. This is certainly the most comprehensive
source for establishing the distribution of any individual compound or any
of the classes of compounds that were discussed in Chapter 1. Other
i 40 Chapter 2
Angiospmae .
Filicinae
acetate - flavonoids
Bryophyta
Fungi Tri- and

Simple cinnamic acid
ACETATE tetra-
derivatives
terpenes
Algae
Acetate Shikirnate Mevatonate
FIg. 2.1 Changes in use of secondary metabolite precursors ('building blocks') during the
evolution ofthe Dimtyledonese (modified from Kubifzki & Gottfieb, 1984).
important sources of data include the recently published Dictionary of

Natural Products (Buckingham, 1993, which is also available on CD-
ROM) ; Chcrnotmonornie der gCEanzen (Hegnauer, L 963 - 19Y2), which is
an outstanding series and certainly the best for comparative taxonomic
data; The Flavonoids series (Harborne, Mabry & Mabry, 1975;Harborne
& Mabry, 1982; Harborne, 1988b) for all classes of flavonoid, and the
Royal Chemical Society series, Natural Produciv Repom, which wiU

cover most classes of phenolics at fairly regular intewals.
2.3-1 Simple cinnarnic acid-derivedphenolics

Simple phenols are rather rare, hydroquinone (7) probably being the
mast widely distributed. C6C1compounds are common in some taxa, e.g.
9 and 10 in Salk and Popdus and 11 in the Gentianaceae. Gallic acid (8)
is widely distributed in angiosperms and is also found in some green
algae; its distribution in by drolysable tannins will be discussed later.
Simple C6Cz compounds also seem to be relatively uncommon, but like
other small phenolic compounds might have been overlooked by chemists
interested in terpenes or alkaloids.
Cinnamic acid derivatives are common in higher plants and you
should expect to find them in most plants if you try hard enough. Pre-
dominating substitution patterns do vary be tween different parts of the
plant kingdom and this has been discussed in detail by Molgaard (1985).
These acids will often be found as esters attached to other metabolites or
as simple or quite complex glycasides such as verbascoside (121). Dimeric
cinnamic acid derivatives (lignans) are widespread in gymnosperms and
angiosperms (Ayres & Loike, 1991) and achieve a high level of oxidation
in the neolignans (e.g. 111) which appear to be largely restricted in
distribution to the Lauraceae and some allied woody families.
The coumarin nucleus (23-25) occurs widely but more complex furo-
and pyranocoumarins (26,27) are of restricted distribution with centres of
proliferation in the Rutaceae and Apiaceae, and to a lesser degree the
Moraceae and Fabaceae . Murray (1991) lists almost 1000 compounds
bearing the coumarin nucleus and while some of these are more correctly
considered as isoflavonoids or neoflavonoids and others are aberrant
derivatives of acetate biosynthesis, this is still an extraordinary number
and reflects the potential for biosynthetic diversity based on a common
nucleus.
2.3.2 Structural classes primarily acetde-derived

Depsides are typical lichen metabolites (e.g. 75) and over 200 different
structures are known (Culberson & Elix, 1989). Like all lichen metab-
widely distributed, not only among angiosperms but also in ferns and
fungi, Xanthones are another group of phenolic metabolite that occur
quite mmmonly in lichens.
2.3.4 Tannins
Mole (1994) has updated the early work of Bate-Sznith (1962) and con-
cludes that fewer families are characterized by the typical presence of
tannins than had previously been reported. There is, in fact, considerable
variation in the occurrence of tannins within a family.
As already noted phlorotannins are restricted to brnwn algae while
condensed tannins tend to associate with l i p i n production and there-
fore with the woody habit in higher plants. Exceptions to this corre-
lation indude: (i) condensed tannin-producing herbs in Fabaceae,
Rosaceae, Gerniaceae and (ii) arboreal taxa In Oleaceae. Verbenaceae,
Bignoniaceae, Viataceae and Asteraceae that are free of condensed
tannins. These anomalies probably reflect evolutionary development:
condensed tannin-producing herbs have arisen from arboreal species and
have not yet lost the capacity for tannin production while arboreal species
that lack condensed tannins may reflect a return to the arboreal habit
from an herbaceous ancestry in which the capacity to produce tannins had
been lost. However, it seems very likely that the ca~acityto produce
condensed tannins has also been lost from individual species {or varieties
within a species) in farliilies that are entirely arboreal and have never lost
the arboreal habit.
Hydrolysable tannins occur in some green algae and widely in angio-
sperms, although less widely than condensed tannins. Okuda, Yoshida
and Hatano (1993) have reviewed the distribution of hydrolysable tannins
in the Dicotyledonae and have found that, in contrast to the widespread
occurrence of condensed tannins, hydrolysable tannins are confined
almost entirely to the Choripetalae. Different hy drolysable tannin oli-
gomers occur in taxonomically r elcv ant patterns in the Rosaceae (Okuda
el al., 1992).
CHAPTER 3
Why are phenolic compounds

so important?
3.1 Introduction
The decision to analyse ecological samples chemically can be arrived at in
many ways. At one extreme, the investigator may be working on a system
in which earlier work has demonstrated a pivotal role for some specific
phenolic allelochemical. Alternatively, there may be just a vague aware-
ness that phenolics might be important and be worth investigating. Many
ecologists faU between these extremes and need to gain some information
about exactly what to measure and how the results might be interpreted.
At this level, the aim of this chapter is to introduce to you the kinds of
ecological interactions in which phenolics have been found to play a role
as ailelochemicals semu Whittaker and Feeny (1971). To an extent, this
chapter catalogues the ecological effects of phenolics. The positive value
in this is that it provides easy ecological access to the chemist, or to the
ecologist seeking information on effects other than those of focal interest
in a particular study. Unfortunately, this chapter is also something of an
indictment of our subject. Its organization reflects a current situation in
which we have no useful organizing principles for phenolic structure and
ecological activity relationships!
Given this current state of play, there are good reasons why you
should familiarize yourself with the material in this chapter. We illustrate
the broad diversity of ecoIogica1 interactions mediated by compounds
that can all be classified as phenolics. This is vital background for the
interpretation of chemical information in ecology as it warns us that we
cannot infer the biological role of a compound with certainty from a
simple knowledge of what kind of substance it is. A further point to
stress is that where several allelochemically active and biosynthetically
interrelated metabolites are produced by a plant, one ecological interac-
tion may have the potential to influence another through this biosynthetic
association. These biosynthetic linkages provide constraints to life-history
evolution and they also caution us against assuming linear relationships
between single adaptive needs and the quantities of allelochemicals
produced by plants.
Chemical ecology has advanced very much as an ecological discipline
concerned with the 'how it works' aspects of ecological interactions. This
kind of knowledge may have direct and beneficial economic applications.
For instance, in agriculture a knowledge of plant-animal interactions at
the chemical level has been important in pest and range management.
Importance of phenolic compounds 46
Even simple measurements of total phenolics have become valuable in

the assessment of non-traditional feeds for livestock (Reed, 1986) or as a
tool for wildlife managers in exploring habitat quality (Robbins, 1983).
In addition to this array of mechanistic knowledge, there is also a
considerable body of ecological and evolutionary theory dependent on
studies of allelochemidly mediated interactions. Investigations involving
phenolics have been critical to the development of apparency theory
(Feeay, 1976; Rhoades & Cates, 1976) and resource allocation ideas
(Coley, Bryant & Chapin, 1985). Our appreciation of co-evolution,
induced defences in plants and cyclic plant-herbivore dynamics has also
been dependent on work with phenolics (Schultz & Baldwin, 1982;
Lindroth & Batzli, 1986; Schafer el d., 1989). Indeed, it is becoming the
case that a critical interpretation of these ideas requires chemical as well
as ewlogicd knowledge. This situation is analogous to the need for a
mathematical background in order to fully comprehend other aspects of
ecologicd theory.
3.2 Ecological interactions

In keeping with our emphasis on plant natural products, we have takcn
something of a botanical perspective in organizing our survey of ecolo-
gical interactions. As we shall demonstrate, phendics can play a role in
virtually any interaction a piant can have with its environment, biotic or
abiotic. In terms of the biotic environment, these interactions may be
within the autotrophic level (allelopathy), or with consumers of either
living or dead plant material. With regard to the consumers of living
plants, both pathogens and herbivores can be involved. Sequestration
of phytochemicals by herbivores also leads to roles for plant-derived
phenolics in interactions between primary and secondary consumers.
Thus, at the ecosystem level, phenolics can mediate interactions that
directly or indirectly link autotrophs to each other, to saprotrophs, to
pathogens, or to herbivores and their predators,
3.2.1 Plant- herbivore intermiions

The degree to which herbivores exploit food resources available to them
has been a key topic of ecology ever since the 'why the world is green'
dcbate was reopened by ecologists with a knowIedge of chemistry such as
Ehrlich and Raven (1%4), Levin (1971), Feeny (1975), Janzen (3975) and
Rhoades and Cates (1976). So far as the ecology of plant phenolics is
concerned, plant- herbivore interactions are the most widely studied
interactions which these chemicals mediate. A key factor in the develop-
ment of this topic has been the considerable headway made with two very
general groups of proximate assay techniques, those for 'total phenolics'
and 'tannins'. More detailed studies of particular chemicals have also
48 Chapter 3
been carried out and the outcome of both approaches are examined
below. We begin with a consideration of the pre-ingestive process of
food selection and then consider studies of the post-ingestive effects of
phenolics.
3.2.2 Tot& phenolics, tannins a d food selection

Plant phenolics have been implicated in food selection by many species of
animals as diverse as elephants (Jachmann, 1989) and ants (Seaman,
1984). Herbivores using almost every conceivable nutritional system
have been examined. Thus placental mammals, monogastric, ruminant,
ruminant-like and hind gut fermenters have all been studied. Marsupial
mammals and birds have also been examined, as has ant-fungus sym-
bioses (Nichols-Orians, 1991). Since our last review of this area (Mole &
Waterman, 1987a) many new investigations have been made, although
much remains to be done. Recent work includes that by Cooper and
Owen-Smith (1985); Tahvanainen et al. (1985); Smallwood and Peters
(1986); Foley and Hume (1987); Robbins et al. (1987a); Greig-Smith
(1988); Marks et al. (1988); Marquis and Batzli (1989); Sewello and
Kirkpatrick (1989);Clausen et al. (1990), and Distel and Provema (1991).
Among the insects a wide taxonomic range from the Lepidoptera,
Orthoptera, Memiptera, Hymenoptera, Diptera , and Coeleoptera have
been studied, including grass, forb and tree feeders. Arachnids and snails
have also been examined, as have symbiotic systems such as leaf feeding
ants that utilize fungal gardens to process their food. With respect to
tannins, much of this work has been reviewed by Bernays, Cooper-Driver
and Bilgener (1989) which is, in our opinion, the best available review of
herbivory and plant tannins in general. In contrast to work on vertebrates,
most recent work with insects has concerned specific chemicals and has
been less dependent on aggregate assays such as those for total phenolics.
Although most of the research has been directed towards terrestrial
ecosystems, marine systems are the latest outcome of endeavours to
examine the role of plant phenolics in plant -herbivore interactions
{Steinberg, 1986; Hay & Fenical, 1988).
The typical result from all these food selection studies has been that
most herbivores avoid consuming levels of phenolics that are in excess of
their normal diet; this is particularly true where tannins are involved
(Mole & Waterman, 1987a; Bernays, Cooper-Driver & Bilgener, 1989;
Cork & Foley, 1991). It is unfortunate that we are only able to express
this result as a probability. Part of the uncertainty is that phenolics are
just one part of the equation in food selection. A herbivore may pre-
ferentially feed on a tannin-rich food source such as an acorn because the
benefit of the very energy-rich nut may offset the cost of consuming the
tannins (Smallwood & Peters, 1986). Dietary fibre and nitrogen nutrition
are other important factors and the ratio between plant nitrogen content,
and phenolic plant constituents (including lignin in fibre) may be the best
guide to food selection (Waterman et al., 1988; Cork & Foley, 1991).
Apart from nutrients, other allelochemicals may prevent feeding on pZants
low in phenolics,
One fact to emerge over recent years is the wide range in the level of
phenolics that a herbivore can tolerate. This can range from essentially no
intake of phenolics through to specialization to feed on plants with high
levels of phenolic compounds (Atsatt & Ingram, 1983; Cork & Foley,
1991). Such differences can be seen within particular groups of related
herbivores such as the ruminants, where grazers tend to be less able to
cope with phenolics than browsers (Robbins el aE., 1987a,b). The same
comparison may also be seen among forb and grass feeding grasshoppers
(Bernays, Cooper-Driver & Bilgener, 1989). This variation in what is a
typical intake ofphenolics for a herbivore has played an important role .in
our understanding of how herbivore communities function, especially
when seen in context with phenological changes in the levels of plant
phenolics (Feeny, 1968; and see below).
In summary, the value of proximate assays for plant phenolics in food
selection studies is that they assist in defining the range of suitable plant
foods for a herbivore. There has been considerable empirical success in
using total phenolics and tannin assays to explain feeding preferences
among herbivores, particularly polyphagous ones. This probably works
because phenolics and tannins are so widespread that they are not so
likely to be diluted down in a diverse diet; whereas the effects of other
potential chemical antifeedants, of more restricted distribution, are.
Assays for total phenolics and tannins are a major focus of Chapters 4-6
and much additional information is presented about them there.
3.2.3 Individui phenols and food selection

Feeding behaviour is incredibly varied across the range of organisms
considered above. At one extreme are the highly poIyphagous colobine
monkeys of old world tropical rain forests that may consume material
from over a hundred plant species from many families. At the other
extreme are stenophagous insects that are restricted to single host plant
species. There are only a few dietary specialist mammals (Vaughn, 1982;
Atsatt & Ingram, 1983; Freeland, 1991) and even these have diets which
would be considered oligophagous or even polyphagous for an insect.
Insects are generally much more restricted in host plant choice than
mammals and so much more sophisticated cues are thought to be used in
their food selection behaviour (Harborne, 1987).
Often, in insects, larval feeding is actually determined by the oviposi-
tion behaviour of the adults. Analysis for phenolic compounds has played
48 Chapter 3
an important role in studies of oviposition behaviour (Feeny, 1992) and a

good example of the painstaking approach needed to identify which
particular phenolics attract oviposition is provided by the work of Feeny,
Rosenberry and Carter (1983). A key aspect of oviposition behaviour is
that it is often mixtures of substances that cue oviposition. h the case of
the black swallowtail (Papiliopolyxenes) which oviposits on carrot leaves,
Feeny et aJ. (1988) found tram-chlorogenic acid (127) and luteolin-7-(6-
malonyl-o-glucoside) (128) to be active components in the cue. For a
citrusfeeding swallowtail Nishida et ab. (1987) found that four flavonoids
were active in eliciting oviposition. Honda (1986) reported two flavanone
glycosides as active oviposition stimulants for Papilio protenor on the host
plant (Citrusnatsudaidai), but showed that several co-occurring flavon-
oids were inactive. Phenolics may actually be active as oviposition deter-
rents when they occur in non-host plants (Tabashnik, 1987). In summary,
patterns of an insect's oviposition may depend on both the presence of
deterrents and the absence of stimulants in non-hosts as well as the
presence of stimulants and absence of deterrents in the host.
Apart from oviposition behaviour, particular phenolics have bean

identified in the acceptance or rejection of many plant foods during
feeding behaviour. For insects, Kim, Koh and Fukami (1985), found that
a mix of eight glucosylflavones elicited probing behaviour for several
species of plant hopper feeding on rice. Some other examples (Harborne,
1988a) of flavonoid feeding stimulants include:
1 the use of isoquercitrin (129) and morin (130) by the silkworm (Bombh
mori);
2 6;-methoxyluteolin-7-rhamnoside (131) by the Agasicles beetle;
3 catechin-7-xyloside (132) by the elm bark-beetle;
4 taxifolin (37),pinocembrin (36) by Scolym rneditermnew.
Structure- activity studies (Lane et al., 1985) have shown that isoflav-
onoids which are able to assume a rotenone-like configuration (cf. 55) are
effective feeding deterrents fox root feeding beetles.
While the emphasis has bken on Bavonoids, other types of phenolic
compounds can be involved in such interactions, Phenylpropanoids such
as methylisoeugenol (133) and asarone (134) are attractive to the carrot
root fly (Guerin, Stadler & Buser, 1983). The simple phenolic deriva-
tives salicin (135) and populin (136) act in conjunction with luteolin-7-
glucoside to attract the willow feeding beetle Chrysomela vigintipunct~ta
(Matsuda & Matsuo, 1985). For the grasshopper Bootetix argentam, a
specialist on Larrea hidentata, the surface resin constituent nordihydrox- '
yguaiaretic acid (20) is a feeding stimulant but for more oligophagous and
polyphagous grasshopper species that consume this plant, this lignan is a
feeding deterrent (Chapman, Bernays & Wyatt, 1988). Simple neolignans
such as rnagnolol (137) have proved to present a significant barrier to
host plant use by some of the less polyphagous taxa of Papilio (Nitao et
, 1992).
dal.
Apart from being gustatory cues, phenolics can also nkdiate her-
bivore feeding when they occur as sticky andlor reactive substances on
plant surfaces. Trichomes that prevent feeding by aphids and larvae of
other insects often utilize phenolics and a phenoloxidase-peroxidase
system which is reported to combine with phenolics to immobilize insects
when these glandular hairs are ruptured by insect movements (Duffey,
1986). Plant surfaces or glanduIar hairs may also have contact sensitizers
that deter feeding. Examples (Gerhold, Craig & Mumma, 1984; Harborne,
1987) include the simple flavonoid 5,8-dih ydroxflavone (138) and anac-
ardic acids such as 0-pentadecenylsalicylic acid (139).
In this section we have emphasized insects. With respect to mammals,

while tannins and uncharacterized phenolics may be the classic feeding
deterrents (Feeny, 2976; Rhoades & Cates, 1976;Cork & Foley, 1991;
Chapters 4-6), there is also evidence for the role of particular defined
substances. For instance, simple phenolics such as fraxetin (140) and
allied coumarins are important in bullfinch feeding behaviour (Greig-
Smith, 1988). An important study (Jakubas, Gullion & Ciausen, 1989;
Jakubas & Gullion, 1990) showed that ruffed grouse feeding behaviour
was well correlated to coniferyl benzoate (141) levels in quaking aspen
but not related to levels of tannin or total phenolia. I
As an indication of where further sophistication in chemica1 and

behavioural analyses may take us, Clausen et al. (1990) have demon-
strated different feeding responses of snowshoe hare to different, chem-
ically defined, types of tannin. Provenza et 01. (1990) have studied the
behaviourat subtleties of conditioned flavour aversion in response to
condensed tannins and Sullivan ei ai. (1992) have demonstrated the
ability of pinosylvin (32) to deter snowshoe hare from feeding, an effect
they suggest to be triggered by the olfactory pathway. As Glendining
(1992) has shown, we may gain additional mechanistic insights into
such behaviours through understanding the details of tannin-protein
interactions in the mouth.
As consumers ofplant foods we are ourselves responsive to phenolics
such as vanillin (142) (vanilla), and eugenol (19) (banana). Closely
related compounds can be both attractive and repelkat. Naringin (143) is
bitter tasting while the related dihydrochalcone (144) is 500 times sweeter
than sugar (Harborne, 1988a). Even so, we again emphasize that while
phenolics are clearly important, they are only active in food selection
along with inputs provided by the other nutrients present.
3.2.4 Post-ingestive eflects

The ubiquity of plant phenolics means that all herbivbres must consume
them to some extent. Considerable interest has been focused on the
52 Chapter 3
nutritional and physiotogical consequences of this. Obviously, study of

these interactions is usuafly one step removed from the field because of
the need to use captive animals in controlted feeding trials. Nevertheless,
such work is often deemed necessary to the understanding of particular
food habits or habitat usage. General issues central to the chemical
ecology of secondary metabolite consumption can all be found in studies
involving phenolics. These issues involve the mechanisms of action of
altelochemicals, the costs of xenobiotic metabolism, and the counter
adaptations to particular defence traits. Attention has mostly been focused
on tannins, isoflavonoids and photodynamic substances such as furocou-
marins, although some attention has been given to virtually all classes of
phenolics (Harborne , 1988a).
Since the assertion of Feeny (1976) that tannins acted as quantitative
defences by reducing the digestion of nutrients by herbivores, consider-
able efforts have been made to substantiate this claim. This has led to
work modeling the digestive process in v i m as well as much in vivo
investigation. The role of tannins in plant-herbivore systems has been
extensively reviewed elsewhere (Bernays, 1981; Butler eb al., 1986; Mole
& Waterman, 1987a; Bernays, Cooper-Driver & Bilgener, 1989) and
we explore it in some detail in later chapters when we address the
appropriate measurement techniques for tannins. These studies have
also led to an interest in the potential for synergistic interactions be-
tween tannins with other al~elochemicals,such as saponins or cyanogenic
glywsides, that occur in the same diet (Freeland, Calcott & Anderson,
1985; Goldstein & Spencer, 1985).
While work with tannins has focused on digestibility reduction in the
gut, phenolics may well be absorbed into the.body and disrupt other
physiological processes. A key issue here has been when and whether
absorption into the body is possible. With tannins this has rarely been
considered because of their large size and polar nature, nevertheless, the
realization that hydrolysable tannins may hydrolyse after ingestion has led
to studies of their toxicity (Murdiati, McSweeny & Lowry, 1992). In
contrast with tannins, most other phenolics are much smaller and they
can often be absorbed across lipid membranes due to the presence of
lipophilic sub groupings within the molecule. Few phenolics are acutely
toxic, an exception baing the effects of the rotenoid group of isoflavonoids
on insects (Birch, Crombie & Crombie, 1985; Lane et al., 1985); however
many are thought to be effective toxins to naive consumers.
A group of phenolics receiving current attention is simple phenolic
glycosides such as are found in many plants but notably in birch and
willow (Palo, 1984; Lindroth & Pajutee, 1987; Lindroth, Scriber: &
Hsia, 1988; Kelly & Curry, 1991). While not acutely toxic, the action of
glycosidases is able to liberate the phenolic aglycone and potentiate their
activity as toxins (Woodhead & Cooper-Driver, 1979; Reichardt, Clausen

& Bryant, 1988). Detoxification of these and other phenolics has also
been a focus of investigation (Lindroth & Batzli, 1983; Lindroth, Scriber
& Hsia, 1988; Bernard er al., 1989; Lindroth, 1989). Such investigations
are still in their infancy and we have a very incomplete knowledge about
the metabolic routes or costs of such xenobiotic metabolism.
Ideas are also changing with regard to the kind of substances likely to
act as digestion inhibitors. Evidence now suggests that a few relatively
low molecular weight phenolics can act in this way, as well as the polar,
high mul'ecular weight tannins. Examples are NDGA (20) (Rhoades &
Cates, 1976) and platyphylloside (145) (Sunnerheim et al., 1988). By
contrast, several studies (Manuwoto & Scriber, 1986; Mole, Butler &
lason, 1990; Mole et al., 1990) investigating the effects of tannins in
feeding trials indicate toxic as well as antidigestive activity and it may be
that in future we will have to reconsider the possibility of these effects.
Some phenolics exert subtle and non-lethal influences on herbivores.

For instance the isoflavonoids genistein (52) and formononetin (53),
present in clover, act as oestrogen analogues and reduce the fertility
of sheep grazed on pasture containing this species (Harborne, 1988a).
Another isoflavonoid and oestrogen mimic, coumestrol (57), has also
been isolated from legumes. It is at least a possibility that these substances
are active on natural populations of herbivores as well as in their role
as part of the phytoalexin based defences of plants against pathogens
(Harbo~ne,1988a). It has been suggested (Bhargava, 1989) that Aavanoids
can antagonize the androgenic action of testosterone, in contrast to the
antagonism of oestrogens cited above. Simple phenolics are also ahought
to b active in regulating mammalian reproduction. Examples include
para-mumaric (14) and ferulic (16) acids which inhibit reproduction in
h f i c r ~ 5montlanus and the hydroxyamic acid DIMBOA (146) which
appears to act as a feeding deterrent to Schizaphis graminiurn and is the
precursor of 6-methoxybenzoxazolinone which stimulates reproduction
in Micro- (Harbarne, 1988a). Simple phenolics are also important in
altering other endocrine functions as demonstrated by the aatithyrotropic
activity attributed to 3,Cdihydroxycinnamic acid derivatives (Auf molk el
al., 1985).
54 Chapter 3
Another facet uf this array of developmental protesses controlled

by plant metaboIites concerns the polymorphism found in Nemoria
arizonaria. In this bivoltine species of geometrid moth, catkin-like morphs
are produced in spring while twig-Iike morphs are produced in the sewnd
generation. Both rnarphs are adaptive in camouflaging the insects from
avian predators, catkins only being present in the spring. Artificial diet
studies revealed that seasonal trends in tannin levels in the oak trees
on which the insect feeds were possible cues for morph determination
(Greene, 1989).
Much interest has been fomsed on the light activated toxicity shown
by certain phenolics. Examples include the phenolic quinone hypericin
(70) (Knox, Sammuels & Dodge, 1987) and the furocoumarins (Swain &
Downum, 1990). Among the insects that feed on,furocoumarin-containing
umbellifers, several adaptations are found that mitigate their effect.
These include leaf rolling to avoid light and the presence of oxidative
enzymes to detoxify them (Sandberg & Berenbaum, 1989). Toxicity is
caused by the binding of these coumarin derivatives to DNA and so there
is a general toxicity to all herbivores including both insects and generalist
mammals such as the hyrax (Ashkenazy el al., 1985). Studies on the
degree to which insect specialists can detoxify furocoumarins (Berenbaum,
Zanged & Nirao, 1986) have made a major contribution to our under-
standing of the chemical mediation of co-evolution.
3.2.5 Third trophik level eflects

Beyond the plant-herbivore interaction, plant chemicals can be saques-
tered and further metabolized by insects for their own defence (Jones et
al., 1988). This seems to be the case for the chrysomelid beetle Chrysomela
aenicoh which converts the phenolic glycoside salicin (135) to sali-
caldehyde (10). In a similar system also involving willow beetles, Denno,
Larsson and Olmstead (1990) have provided evidence for the ability of a
beetle (Phratora vitellinae) with comparable capacity for salicaldehyde
sequestration to obtain enemy-free space through these defences. Thus a
third trophic level interaction can be demonstrated. Interestingly, these
beetles deveIoped less rapidly in salici11 rich hosts, but such hosts were
selected for larval development by parental ovipostion preferences. The
clear implication here is that parental oviposition on salicin rich hosts
leads to reduced mortality in their offspring due to predation. In contrast
to Phratora viteflinae, another willow feeding beetle (GolerucetZu lineola),

that is unable to convert salicin to salicafdehyde, fed on a wider range of
willows including nutritionally superior ones low in salicin (Denno,
Larsson & Olmstead, 1990).
These examples are far from the only cases of phenolics being se-
questered by insects. Fung and Herrebout (1987) and Fung (1987) have
demonstrated the sequestration of food plant derived dihydrochalcones
and coumarins by Ypponorneuta mahalebelk and Y. rnalinellus, respec-
tively. Other phenolics have been reported in the defensive secretions of
grasshoppers, cockroaches and mites (Eisner et al., 1977; Maschwitz &
Tho, 1978; Jones et al., 1987). One species of tick has also been reported
to use simple phenolics as pheromones (Wood et al., 1975).
Not all arthropods observed to use phenolics are herbivores (e.g. ticks
and mites) and the presumably synthesize the phenolics de novo and do
not use plant foods as their source in any direct way. This raises the
probIem of to what extent may herbivorous arthropods also synthesize
some of the phenolics used for their defence, e.g. as in the secretions
of grasshoppers. In at least one case, Romalea gufata, there is well
documented variation of defence secretion chemistry that parallels chemical
variation in the diet (Jones et a]., 1987). A clear example of the seques-
tration of phenolics from dietary sources is also provided by Wilson
(1985a, 1986, 1987), who has documented the accumuIation of flavonoid
glycosides in the wings of butterflies. In the case of Lysandru corion the
pattern varies according to food plant use while in Melanargia galathea
the naturally static pattern suggested a less varied diet. Another report of
plant flavonoid derived wing pigmentation is for grasshoppers (Hopkins
& Ahmad, 1991) but in none of these cases is there any clear reason as to
why the Ravonoids are accumulated.
3-2.6PolZinution and seed dispersal

One of the classic plant-animal interactions where there is a well es-
tablished role for phenolics is that of plant pollination. Here flavonoid-
based pigments, together with carotenoids (some of which can also be
phenolic), play a pivotal role in the determination of Bower colour and
thus pollinator preferences. Despite the taxonomic ubiquity of flavonoids
(Chapter 2) there is good evidence that pollinators can exert strong
selective forces in determining the pigmentation of flowers (Harborne &
Smith, 1978; Scogin & Freeman, 1987). Harborne (1988a) gives an
excellent account of the biochemistry of plant pollination in general: here
we provide only a brief outline.
The ffavonoids important in flower colour determination are both
those that absorb in the wavelength range visible to the human observer
and also those which absorb in the ultraviolet (UV) range visible to many
56 Chapter 3
insects such as bees. In the first of these groups we are mostly concerned
with the anthocyanidins (e.g. 43-45) and, occasionally, chalcones (e.g.
47) and aurones (49). UV-absorbing pigments are typically ffavones (e .g.
38) or flavonols (e.g. 39-41). Non-phenolic compounds often influence
flower colour and can be entirely responsible for some colours, notably
yellows. However, phenolics can provide red, orange, yellow, blue,
purple, violet, black, white and UV visua! cues to pollinators.
Probably the most important flowcr colour pigments are the anthocy-
anidins. The three most widespread members of this group of flavonoids
are pelargonidin (43), cyanidin (44) and delphinidin (45) which typically
occur as glycosides (known as anthocyanins). These pigments are re-
sponsible for orange-red to mauve pigmentation. Occasionally the
3-deoxyanthocyanidins apigeninidin [147) and leuteolinidin (148) are found
in yellow and orange-yellow flowers. All five of these substances are very
similar stmcturally, differing only in hydroxylation patterns. Minor
modifications to these structures, such as methylation of one or more of
the A or B ring hydroxyls, can result in marked colour changes. One
more complex way in which pigment colour is modified is by the co-
occurrence of anthocyanins with other colourless flavones or flavonol
glycosides (called co-pigments) which interact with the chromophore to
modify petal colour. A similar modifying effect can be achieved by the
attachment of aromatic compounds such as cinnamic acids to the sugar
groups of anthocyanins. Metal ion co-pigments are yet another group of
substances that produce these effects. One well studied example of co-
pigments influencing flower colour is in Hydranga macrophylla where
aluminium ions and caffeoylquinnic acid interact with delphinidin-3-
glucoside to give a blue sepal colour (Takeda, Kariuda & ltoi, 1985).
Colour intensity is influenced by the concentration of the pigments, an
extreme case being that of apparently black Rowers which have very high
delphinidin concentrations in their petals,
Flavonoids colourless to the human eye and which occur in white

flowers are glycosides based on flavone or flavonol aglycones. Bands of
tissue rich in such flavonoids (called honey guides) point pollinators
to nectar and are often found on petals irrespective of their colour.
Harborne and Nash (1984) have detailed the variety of pigments re-
sponsible for U V patterning in the genus PobentilZa:
While flower odours are normally non-phenolic terpenes, they can be
phenolic, such as vanillin (142) and eugenol (19). These can also be
important in mediating plant-disperser interactions where they occur in
fruit. Once more, flavonoid pigments can be involved, now signalling the
ripeness of fruit through colour change. Just as the synthesis of odours
and pigments may increase during ripening, feeding deterrents need to be
catabofized or otherwise inactivated. Studies of the de-astringency of
tannin-containing persimmons revcal that the latter process may occur
with tannins being deactivated by a reaction with aldehydes produced in
the ripening fruit (Matsua & Itoo, 1982). An alternative viewpoint is
that polysaccharides formed during ripening may disrupt the binding of
tannins and proteins in ripe Fruit (Ozawa, Lilley & Haslam, 1987).
3.2.7 Phnt-puthogen interactions

The interactions between plants and pathogenic micro-organisms have
traditionally been studied within the discipline of plant: pathology, which
has developed quite separately from the ecological mainstream which has
focused on plant - animal interactions. From a phytocentric point of view,
chemical defences that are active against both herbivores and microbial
pathogens are needed. Some secondary metabolites certainly are effective
against both classes of enemies. However, because of the disciplinary
division outlined above it has rarely been the case for plant metabolites to
be tested for activity against both microbes and animals.
Plant pathologists have traditionally recognized two types of anti-
infectiunal defence chemicals in plants: those that are present constitutively
and those where de novo synthesis is induced as a result of an infection.
Thesc two groups are thus pre- and post-infectional defences, respectively.
Below, we give examples of the substances involved.
Phenolics that are constitutively present in plants, and that are be-
lieved to confer disease resistance, include simple compounds such as
hydroxystilbenes, isoflavonoids, fitv ones and hordarines. Examples from
these five groups include catechol (Sa) in onion buIbs, pinasylvin (32) in
the heartwood of pine trecs, luteone (149) in lupin leaves, pinocembrin
(36) in cottonwood and hordatine (221 in barley leaves (Harborne, 1987,
1988a). New groups of fungistatic substances arc: still being found (Tornas-
Lorente ei a/., 1989) and antifungal roles are also being proposed for
familiar substances such as apigeninidin (147) (Snyder & Nicholson, 1990)
and other anthocyanins (Coley & Aide, 1989). Condensed tannins are
also reported to be active against fungi (Azaizeh at ol., 1990), includ-
ing plant pathogens (Brownlee et a / ., 19901, but this is not aIways con-
firmed (Anagnostakis, 1992).
58 Chapter 3
Active antibiotic substances are often not the only phenolics present
in plant tissues and so the measurement of total phenolics may not
correlate well with disease resistance. A key point always to be remem-
bered is that essentially all phenolics are antibiotic, but in a passive sense
that contrasts with the active and aggressive antibiotic activity seen in a
true al~elochemical.The substances referred to above are essentialty the
'first line' of chemical defence. For dead tissues such as the heartwood of
trees they are also the only possible line of chemical defence as further
metabolism is not usually an option (but see Kemp & Burdon, 1986).
In live tissues, there is the possibility of further metabolism after
infection. One option is simply to synthesize more of the defensive
substances present before infection or to release stored but inactive forms
of defence chemicals. Phenolics such as scopolin (81) and chlorogenic
acid (127) may be involved in such responses (Harborne, 1988a). A
problem here is to distinguish whether such changes are always adaptive
or simply a result of the disease deranging the plant's metabolism.
The one system of chemical defence in plants thought to be specific to
microbial infection is where substances not found before infection are
synthesized as a result of infection. The problem in studies of such
compounds has always been to be certain that these metabolites axe of
defensive significance. Where a defensive significance is apparent, such
compounds are called phytoalexins. The speed of their induction and
their toxicity are both vital to their utility to the plant. The substances
reported to be involved are mostly structurally complex and include
isoflavonoids (e.g. 52-57), stilbenes (32) and benzofurans (124), although
the simple phenolic acid, benzoic acid, is produced in apples (Harborne,
1988a).
Recent work by Schaffer et a/. (1989) has verified the phytoalexin
concept for at least one system involving flavonoid production in peas.
Here disease susceptibility on the part of the plant has been related to the
inability to produce the isoflavonoid pisatin (150). Disease resistance is
conferred by the capacity of the plant to produce pisatin, but only where
the pathogen is unable to detoxify this compound. The particular con-
tribution of Schaffer et nl. (1989) has been to show that pathogenicity on
the part of the pathogen is genetically linked to the presence of genes for
a variant of cytochrome P-450 with pisatin demethylase activity, which is
able to catalyse pisatin detoxification. With this genetic link between

pathogenicity and pisatin detoxification so clearly estabtished, the defen-
sive role of pisatin against fungi unable to detoxify it is also established.
3.2.8 Higher phnts: aklelopathy and soil processes

3.2.8.1 Alkelopathy
Higher plants are thought to interact with each other through two main
processes: (i) competition for physical resources such as light and mineral
nutrients; and (ii) allelopathy mediated by toxic substances used to inhibit
or kill competing plants.
To many plant ecologists, allelopathy has traditionally been seen
as an insufficiently well demonstrated phenomenon with a minor or non-
existent role, relative to competition, in the fieId situation. Fuerst and
Putnam (1983) set out criteria for demonstrating the existence of either a
competitive or an allelopathic effect, and they conclude that, in both
cases, the methodologies used in experimentation often lead to incon-
clusive resuIts. The general problem is that the interaction is not cleanly
traced from a substance active in controlled laboratory conditions through
to a demonstration that the same substance is present at the same level
and is responsible for the same response in the field. For instance,
Elakovich and Stevens (1985) extrapolate a natural role for nordihidro-
guairetic acid (20) as an atlelopathic agent from Petri dish type seedling
bioassays. Similar studies concerning phenolics include Schumacher, Thill
and Lee (1983); Paszkowski and Krerner (1988); Singh, Tamma and Nigg
(1989). Where microbes have been present in experimental systems, their
intervention has been shown to change the chemistry and toxicity of the
putative allelopathic agents (Liebl & Worsham, 1983). Field studies
measuring natural levels of putative allelopathic agents often down-play
their importance (Proksch, Weissenbock & Rodriguez, 1985) or suggest
an intervening role for microbes (Liebl & Worsham, 1983, and below).
Many ecologists also criticize field experiments where ecologically irre-
levant species are used as bioassay test organisms (e.g. as in the lettuce
seed germination assay). This is perhaps unjustified as much of the
impetus concerning allelopathy studies has come from agricultural needs
to understand changes in soil fertility , particularly in no-tillage agricultural
practices. One other misgiving about aZZelopathy is that the substances
involved are often relatively common (e.g. ferulic acid, 16) and seem ill-
suited to mediate specific ecological interactions.
Even if allelopathy has not yet been demonstrated with the rigour it
deserves, there is much evidence to lend credence to its being a real
phenomenon. Several of the important examples supporting the idea of
allelopathy concern phenolic met a bolites. The best known implicates the
60 Chapter 3
walnut tree which releases juglone (6%) into the soil as inactive glywside
precursors which are then hydrotysed to yield juglone by soil %microbes.
The Californian chapemal shrubs A denustoma fasicula~mand A rctosfa-
phylos glandulosa are also thought to liberate many water soluble inhibi-
tors which may act as allelopathic agents. These studies are reviewed by
Harborne (1988a).
3.2.8.2 Litter decay and plant mineral nutrition

There is no doubt that plant secondary products enter the soil system
through leaching or litter fall. Plants can be seen to liberate specific
quantities of phenolic materials in the soil in which they grow (Whitehead,
Dibb & Hartley, 1982). Such substances may exert significant autoal-
IeIopathic effects on the species that produces them (Kil & Lea, 1987).
Soil conditions may in turn affect the quantities of phenolics liberated
into the soil by plant roots (Muller , Kalisz & Luken, 1989). Phenolics
may thus have a role in soil formation and fertility with the potential for
feedback between low soil nutrient conditions leading to high inputs of
phenolics and still lower fertility (Rice & Pancholy, 1973; Janzen, 1974).
Such concerns are not new (Bloomfield, 1957). For instance Handley
(1954) related the presence of tannins to the mode of leaf litter decom-
position and thence to 'mull' and 'mar' type formation in European forest
soils. Such concerns are once again topical through work such as that by
Nicolai (1988) and Stout (1989). The effects of plant phenolics on plants
growing in soil are now under investigation in general studies of min-
eralization processes as well as a component of allelopathy studies (Turner
& Rice, f 975; Hall, Blum & Fites, 1983).
Other roles for phenolics in the soil environment involve the media-
tion of luteolin (38) in the nodulation of legumes by Rhizobium (Peters,
Frost & Long, 1986). In this case, the flavonoid-containingroot exudate
controlled the infective response of the bacterium. Root exudates also
appear to be used as signals by less benign organisms which recognise the
presence of the host root by specific phenolics. For instance xenognosin-
A (151) and 2'-hydroxyfomononetin (152) are haustorial inducers for
the parasitic plant Agalinis purpurea which parasitizes Astragaltls spp.
(Grisebach, 1987).
3.3 Abiotic constraints and plant development

For all the attention that has been given to phenolics as allelochemicals,
they are still h t and foremost plant metabolites, and they must be
synthesized within the context of plant metabolism as a whole. Much
recent attention has been directed to the way abiotic factors such as
climate and mineral nutrition constrain plant growth and chemical corn-
position (Waterman & Mole, 1989). The aim of this section is to discuss
the present state of knowledge of this area. This has become an important
subject for study in its own right as weIl as a necessary background for
understanding abiotic limits that may constrain a plant's interaction with
its biotic environment.
One other issue we raise is the question of whether phenolics have
any physiological roles in plants. Such primary, rather than secondary,
metabolic roles seem particularly likely in respect to withstanding drought
and high intensity solar radiation and may also involve adaptations
to soil conditions (Kimura & Wada, 1989). In seasonally changing envir-
onments, seasonal and ontogenetically canstrained changes are hard to
separate, nevertheless, we conclude this section by assembling what is
known a bout developmentaIly controlled changes in the metabolism of
phenolics. In overview, our object here is to introduce the causes of
variation in production of plant phenolics that are likely to be encount-
ered irrespective of the plant's interaction with other organisms. The
following sections deal with 'natural' abiotic factors, however, clear
evidence is also becoming available that pollutants can have an appreci-
able influence on the metabolism and production of phenolics (Jordan et
al., 1991).
3.3 .I Solar irradiance and UV light

There is a well established positive relationship between the intensity of
solar radiation and the quantity of phenolics produced by plants. Usually
this has been observed as a rise in total phenolics in plants grown in sunny
conditions relative to shady ones, but it can also be seen at the intra-
individual level by comparing plant parts exposed to differing amounts of
light (Bryant et lal., 1987; Mole, Ross & Waterman, 1988 - see Fig. 3.1;
Waterman, Ross & McKey, 1984). Similar results have been seen for
particular types of phenolics (Bryant, 1987; Mote, Ross & Waterman,
1988). We know of no exceptions to this positive relationship between the
accumulation of phenolics and light intensity. The explanation usually
advanced to account for this is the carbon-nutrient balance hypothesis
(Bryant, 1987; and see below).
An adaptive interpretation of this response in terms of plant physiolo-
gical needs is that the phenolics are produced as a way of reducing the
photodestruction of exposed tissues. This is seen as being padcularly
82 Chapter 3
1-f I
1
1
I
I
I
I I i
0 10 20 30 40 50 60
Light intensity (as % of maximum incident light)
Flg. 3.1 Relationship between light intensity and levels of phenolics (+)and
condensed tannins (I leaflets of Acaciapennafa (from Mole, Ross & Waterman, 1988).
in)
likely where UV light is being absorbed (del Moral, 1972). There is

some evidence (Caldwell, 1981; Mancinelli, 1981; Wellmann, 1981) that
phenolics with appropriate UV absorptive properties can be induced by
UV light of the wavelength range that a plant could experience under
field conditions. The predominant substances involved in these responses
are flavones and flavonols (Grisebach, 1987) which absorb UV-B radia-
tion (280-320 nm) while leaving photosynthetically active radiation
unaffected. Interestingly, UV light has been used to induce isoflavonoid
phytoalexin synthesis (Ingham, 1982), although as yet there is no role
proposed for these compounds as UV screens. There is evidence that the
external resins on the desert shrubs Elytropappas rhinacerotb, Larrea
triderttata and Eriodictyon californicum have the optical properties nec-
essary to absorb UV radiation (Rhoades, 1977; Johnson, 1983). Where
such compounds are produced internally it seems logical that, for maximum
effectiveness, they should be produced in the upper epidermal layer.
There is now some support for this hypothesis, even though factors
determining the production of phenolics appear to be only partly as-
sociated with UV protection (Lovelock, Clough & Woodrow, 1992).
To summarize, there is evidence to suggest that light induced ac-
cumulations of phenolics are adaptive as UV screening responses but we
caution against blanket acceptance of this hypothesis. Tannins and many
Importance of pknolic compounds 63
simple phenolics have absorption maxima at wavelengths considerably

shorter than UV-I3 and so UV screefiing is unlikely to be a universal
explanation for all the phenofics produced in response to fight. Often, the
overflow hypothesis (see below) is a more appealing explanation for their
accumulation.
3.3.2 Drought
Plants close their stomata and curtail photosynthesis during periods of
water shortage and thus one might expect a negative relationship between
water shortage and the synthesis of phenolics. In reality, there seems to
be no clear relationship between phenolic biosynthesis and water stress
(Gershenzon, 1984). Recent information (Worner , 1988, 1990) suggests
that xylem pressure and tannin synthesis may actually be linked but that
the relationship can be either positive or negative, depending on the
degree of water stress suffered by the plant. With so little known about
this topic, it is clearly an area wide open for further research,
A more definite role for phenolics in relation to plant -water relations
has been proposed for lipophilic resins accumulated on the external
surfaces of plants. Examples include the resins of Erw dictyon, Diplacus
and Larrea species (Rhoades, 1977; Johnson & Brain, 1985) where an
integrated antidesiccant, UV screen and antiherbivore defence role has
been proposed.
3.3.3 Nutrients and the carbon -nutrient bdmce

Of all the nutrients supplied to plants through the sail, only the effects of
nitrogen and phosphorous have been studied to any great extent, although
some information is available for sulphur and a few metal ions, such as
potassium (Gershenzon, 1984; Waterman & Mole, 1989). The general
observation is that lower quantities of phenolics are produced in condi-
tions of abundant nitrogen supply (Bryant, 1987; Price ei al., 1989). For
phosphorous the situation is not so clear-cut (Bryant et al., 1987).
Rather than nutrients alone, it is more usual to consider the produc-
tion of phenolics in relation to the ratio of fixed carbon resources to
nutrient resources in the plant (Larsson et a)., 1986). At the intraspecific
level, the production of C-,H- and 0-but not N-containing aljelo-
chemicals is antipicated to show a positive relationship with the carbon-
nutrient ratio (Bryant, Chapin & Klein , 1983; Gershenzon, 1984; Water-
man, Ross & McKey , 1984). These ideas also reflect earlier proposals, at
the interspecific level, of Janzen (1974) and Gartlan er al. (1980). These
authors correlated low soil nutrient status with high leveIs of phenolics in
tropical vegetation.
64 Chapter 3
The interpretation placed on such results is that the accumulation of

phenolics when the carbon-nutrient ratio is high, is due to a relative
excess of fixed carbon being available to the plant. Thus if extra minerals
are not available in a sunny versus a shady habitat, then the plant is only
able to use the photosynthate for C-, H- and 0-containing metabolites,
such as phenolics. Essentially this is an argument following the law of
mass action and it has been put in a more metabolically detailed form by
Hasiam in his 'overflow' hypothesis (Haslam, 1985; Waterrnan & Mole,
1989).
The carbon -nu trient hypothesis (CoIey, Bryant & Chapin, 1985) and
its accompanying economic world view is today's dominant paradigm in
this branch of ecology. One problem with this concept is that plants of
inherently different growth rates seem to differ in the degree to which
their phenolic metabolism is sensitive to carbon -nutrient balance (Schultz,
Nothnagle & Baldwin, 1982; Bryant et al., 1987). This developmental
aspect of the problem has been a focus of work by Coley (1987) and by
Herms and Mattson (1992). This is an active area of research, and
perhaps one where the current paradigm may soon become outdated.
3.3.4 Seasonal and developmental changes

In the field situation, studies of seasonal effects on the production of
phenolics will invariably be confounded with changes under the control of
developmental processes internally (horrnonaIly) controlled by the plant:
we thus consider both together. Seasonal changes in plant phenolics
have been an important focus for study since the work of Feeny (1968)
and have continued up to the present day (Cooper-Driver et ral., 19'77;
Mauffette & Oechel, 1989). It is frequently found that as the season
progresses andlor the leaves age, then levels of phenolics increase or at
least rise to a plateau level (Parker, 1977; Glyphis & Puttick, 1988).
Usually seasonal changes in climate, i.e. abiotic factors, are used to
account for changes in phenolics. Coley (1987) and Harper (1989) also
consider the changing carbon-nutrient balance of growing leaves, assuming
that carbon resources are more profitably used for growth in young leaves
rather than older ones. There are contradictory reports of young leaves
having higher levels of phenolic secondary metabolites than mature
ones (Waterman & McKey, 1989) as well as reports of non-linear trends
where dilution effects on leaf expansion come into play (Homer, 1988).
Rises and subsequent declines or declines and rises, are reported by
Schultz, Nothnagle and Baldwin (1982) ; Wilson (1984); Lindroth, Batzli
and Seigler (1986) and Lindroth, Hsja and Scriber (1987). In general,
source-sink relations of growing plants can be expected to constrain the
levels of phenolics accumulated by growing plants (Lilov & Angelova,
19871, but their dynamics are still unresolved.
3.4 ConcIuding comments

The overall view that we hope readers have gained.from this chapter is
the probable interdependence of several ecological aspects of a plant's
biochemical relationship to its environment that derive from the biosyn-
thetic constraints on the production of allelochemicals. The ultimate
sources of these constraints appear to be based upon the physical re-
sources supplied by the environment (carbon-nutrient balance) as well as
organismal physiology, as determined by the genome which reflects
phylogenetic factors associated with both recent and ancient history
(Wanntorp et al., 1990). An understanding of these points is a funda-
mental prerequisite to the ecologicai interpretation of data from the
chemical assay techniques described in the remainder of this book.
For example, the NGDA (20) in Larrea resins has been discussed in
the context of antiherbivore defence, allelopathy ,drought resistance and
UV screening! AH these aspects of its potential function need to be
considered when evaluating its role in the current ecology of Larrea. If
ultimate (evolutionary) questions are to be examined, then consideration
of material presented in Chapter 2 will be important for understanding
why Larrea produces NDGA rather than some other phenolics. More
detailed analyses, using the methodologies proposed by Wanntorp et al.
(1990), would also be needed to assess questions about NDGA and its
adaptive value to Larrea.
In closing this chapter, we return our focus to current ecological
effects. Flavonoids and thus flavonoid metabolism have appeared in every
instance of our consideration of how phenolics mediate or are influenced
by plant -environment interactions. The clear message from our current
knowledge is that adaptive functions cannot be safely inferred from
chemical structures alone and that quantitative variation in the produc-
tion of phenolics may not be simply related to selection for any single
adaptive utility.
CHAPTER 4
Extraction and
chemical quantification
4.1 Introduction
The goal of Chapter 4 is to introduce you to the pleasures and perils of
extracting and then quantifying plant phenolics. Initially we will cover
sample preparation and extraction procedures. This is followed by details
on the steps needed to make quantitative assays of phenolics in biological
material. All the most important and commonly used techniques are
described. These include methods for both total phenolics and particular
types of phenolics. These assay procedures have been mainstays of much
ecological work: of all the techniques in this book, these are the least
specific chemically but the most simple technically. This technical sim-
plicity means that they are, inevitably, often the methods of choice where
the number of samples to be processed is large. Once equipped with a
sound grasp of the rationale behind these procedures, investigators can
optimize their own system for the particular material being studied.
4.2 From the living organism to the extract

The general problem we are faced with is that the chemicals we wish to
study originate from within living organisms. Once stressed in any way,
the metabolic state of the organism may change and with it the quantity
and quality of these chemicals. This can be a problem with live material
even before harvesting. After harvesting a plant or plant part, initial
changes may be quantitative and subtle. For aII cells, as they die and sub-
cellular compartmentation breaks down, enzymes come into contact with
potential substrates to which they are not normally exposed. As cell
membranes degrade and water is lost, the concentration of these enzymes
and substrates increases, further facilitating their reaction. The loss of
membrane integrity also increases the potential for oxidation. Oxidation
reactions are a particular problem with phenolics, as the phenol group
can easily be transformed into a reactive quinone on exposure to air (see
Chapter 1). Polymerization reactions are particularly likely subsequent to
quinone formation.
In short, the chemicals in a plant cut and left to dry in the sun are
likely to be different from those in the living uncut plant. The objective in
preparing an extract is generally to transfer the chemicals which represent
the living (physiological) state into solution and avoid those which re-
present the pathological state. For this reason, the process usually begins
with killing the tissues i.e. halting all metdolic activity as quickly as
66
Extraction and quantification 67
possible. In all subsequent procedures the aim is to preserve the chemical

state of freshly killed tissue and to bring its chemical constituents into
solution.
The practical process of preparing an extract can be considered in
three stages. Firstly there is the collection and preservation of samples.
Secondly there is, usually, a need to physically degrade the sample before
the final stage of adding a liquid extractant to bring the phytochemicals
into solution. Sometimes all three processes happen together, as in the
case of putting live material into a blender together with a solvent. More
usually samples are collected in the field and then ground and extracted in
the laboratory. Chemical changes in the material are mostly encountered
in the first and last of these processes.
We are confident that few readers will approach these topics with a
sense of enormous excitement, yet it should be clear that measurements
of plant phenolics are only as good as the extracts on which they are
based and so this topic is an important one.
4.2.1 Collection of material

A vital prelude to sample collection is having thought about what pre-
cisely to collect. For instance, many animals are seed feeders but some
may feed on ripe fallen seeds while others feed on ripe or unripe seeds
still attached to the plant. Many chemical differences may exist between
seeds in these situations, notwithstanding that they are all from the same
species. Likewise, in studies of howling monkeys Glander (1982) noted
feeding preferences among conspecific trees which may possibly be due to
differing secondary chemistry. Thus, the actual plants and plant parts that
a herbivore eats need to be collected; parts from nearby conspecifics or
even other parts of the same plant may not be appropriate. As noted in
Chapter 3, factors generating intraspecific differences in plant chemistry
include age, phenological stage, disease, genotype, edaphic factors and
microclimate. Consideration of any or all of these may be important in
selecting material. Two recent studies documenting the effect of leaf age
and stage of development on levels of total phenolics and tannins are
provided by Pandey and Makkar (1991) and Makkar, Dawra and Singh
(1991).
Hoarding of material, as commonly seen in rodents, adds the addi-
tional dynamics of moulding and the accumulation of fungal metabolites
in the food as a cause for consideration. Food handling may also mean
that only certain anatomical parts of an item are eaten. Often chemically
interesting differences are seen between eaten and discarded parts.
Experience, imagination and common sense are all required to select
samples. Clearly, the above observations show that it can often be helpful
to have some sense for sources of chemical variation in any specific
68 Chapter 4
system, but these are things each individual researcher must sort out for
him or herself.
We now continue from the point at which the investigator has selected
samples, what next? At this point the critical factors are twofold: (i) how
far is the field site from processing facilities? (ii) what is the nature of the
biological material? So much work involves the collection of leafy plant
material that the following two sections deal with this. The methods
discussed here may also be suitable for flowers, buds, small stems and
even whole plant samples. A further section covers variations on the
themes developed below as they apply to materials with a high water
content, such as fleshy fruits or animal samples. Finally, a section deals
with the recovery of material present on the surfaces of plants rather than
inside tissues.
4.2.1.1 Collection close to the laboratory

The two ideal conditions for material to arrive for analysis are fresh
or, failing that, freeze dried or frozen. For fresh material, whole plant
samples or material such as excised leaves need to be treated with care. If
plants are stuffed into plastic bags and allowed to warm up and wilt or
sweat during transport they will not be in the physiological condition
they were in the field by the time they are extracted! A better general
starting point will be fresh material kept moist, in the dark and on ice.
Such material should survive even a couple of hours' transport to the
laboratory and still be in excellent condition. Another collection method,
which is generally recognized as safe, is to cut stems under water to
prevent vascular cavitation and then bring plant material back to the
laboratory fully hydrated (e.g. in a vase). Kleiner (1991) has recently
reported the stability of tannin and total phenolics measures of oak
foliage for up to 48 hours in material collected and maintained in this
way. Even so, these comments are generalization and even the seemingly
appropriate methods can at times be inapplicable. As an illustration of
this Lindroth, Hsia and Scriber (1987) report that phenolic glycoside
composition of quaking aspen can be 'dramatically' altered by freezing
but that transport of material on ice and immediate processing was
acceptable.
For those with access to high-technology, samples can be collected into
liquid nitrogen and then freeze dried in a portable freeze drier at the
earliest opportunity. This involves transporting a Dewar flask of liquid
nitrogen to the field site: such flasks usually retain nitrogen for several
weeks before it boils away. Foliage is placed in labelled paper bags which
are then dropped into the liquid nitrogen. Samples freeze nearly instantly
and are then taken back to the freeze drier. Several days' samples can be
accumulated and stored in a desiccator until transport back to the main
laboratory. Such sample preservation techniques make the study of
actively metabolized substances possible. The obvious constraints are

the cost of the equipment, the liquid nitrogen supply and the need for
electricity at or near the field site. A word of warning concerning freeze
drying: where phenolic compounds are to any extent volatile there are
likely to be appreciable losses during the drying process.
Without going to the extreme of taking liquid nitrogen into the field, it
is quite possible to place fresh plant material directly into a freeze drier
and dry it without being frozen first. The limit here is the proximity of the
freeze drier to the plants in the field. A major problem with freeze dried
material is keeping it as dry as possible after lyophilization, the 'product'
tending to be hygroscopic. As during the freezing process it can be
assumed that all cell membranes have been broken and that on wetting,
enzymes and substrates present in the material will begin to react, there is
clearly scope for major changes to occur in poorly stored material. Thus,
even freeze drying, widely regarded as a 'safe' procedure, can lead to
variation in extractable phenolics, notably condensed tannins (Cork &
Krockenberger, 1991). This danger of reaction is also present when
frozen material thaws. While material can simply be frozen, given a
freezer near the field site, thawing can be a problem when transporting
fresh frozen material back to a main laboratory. Solid carbon dioxide is
usually needed to prevent this during prolonged transit. Express mail
service and well insulated containers can now be used to cover large
distances and have material arrive frozen or, at the very least, cold.
Servello and Kirkpatrick (1989) have used this method to obtain over
1000 crop samples from hunt shot grouse from 11 states in the USA, all
taken in one season.
4.2.1.2 Collection far from the laboratory

Here we are considering expeditions to remote areas of the world or
other instances where the luxuries of freezers and freeze driers may
not be available. Less ideal sample preservation techniques have, of
necessity, to be used and invariably introduce some level of uncertainty
into the analysis because of unknown and uncontrolled post-mortem
changes. For qualitative analyses, the loss of substance may not be disas-
trous as long as some remains to be detected. A more worrisome problem
is that new compounds may be created in pathological reactions. For
quantitative results to hold true with imperfectly preserved material, then
the greater the quantity of substance present and the more metabolically
inactive it is the better. Tannins have long been analysed using material
dried in the sun or in herbarium driers and the general consensus seems
to be that this does not invalidate the data as long as the problem is
recognized and interpretation takes it into account. Gartlan et al. (1980)
carried out a comparative study of fkesh and heat dried leaves of several
species and found that there was a general loss of extractable phenolics
during drying. However, with a few exceptions there was correlation
between the fresh and oven dried data sets, and it was concluded that, for
comparing species using non-specific or specific tannin assay procedures,
drying in this manner still gave useful comparative data. Further and
quantitative information on the effects of various drying conditions has
been provided by Makkar and Singh (1991) who did not find significant
differences in total phenolics and tannin measures due to outdoor and
oven drying conditions for oak foliage.
Practical methods for preserving leaves entail drying out the material
as quickly as possible after collection to minimize chemical changes as
cells die. Even in the humid tropics, the direct midday sun will dry out
leaves quite quickly, especially if the leaves are hung upside down to
expose the stomata on the undersides. Less extreme conditions, such as
well vented herbarium driers set to 6WC, are an excellent means to dry
samples as long as the leaves are put in loosely: leaves in well packed
herbarium presses tend to be well steamed before they dry. We do not
recommend drying at temperatures above 60°C.
4.2.1.3 Material with a high water content

Where they cannot be transported fresh or frozen, some materials are
unsuitable for the above methods because of their high water content.
Even if frozen first, fleshy fruits are usually impossible to freeze dry,
because water takes too long to sublime from the interior. In some
instances this problem can be overcome by cutting sections and drying
these separately. At remote sites fruits, insects, faeces, digesta and other
materials are often 'pickled'. Technical (or potable!) forms of alcohol are
the most suitable pickling solvents. It is important to keep the pickled
sample in a sealed container with enough alcohol so that water in the
sample does not dilute it to the extent that the sample rots. The container
needs to be sealed as substances present in the sample will be extracted
into the pickling fluid so this will probably also need to be analysed. It is
vital that the total volume of pickling fluid added be recorded for use in
any calculations designed to find the concentration of leached substances
in the original fresh sample.
For the investigator planning to take a preservation medium to a field
site, or to use a substance other than alcohol, anything that includes
formaldehyde (formalin) should not be used as this reacts with phenolics
to make them unsuitable for further work.
4.2.1.4 External resins

Most phenolics that ecologists will encounter are produced inside plant
cells, however there are some that figure prominently as components of
resins on external surfaces. These are usually secreted by glands or
glandular hairs. The best known example of a plant with such an external
resin is creosote bush (Larrea m'dentata). Such resins can be highly
complex mixtures, often including terpenoids as well as shikimate-derived
substances. The one common feature they generally possess is solubility
in non-polar solvents. Simply washing the leaf surface with chloroform or
methylene chloride will usually remove surface resins, but if material is
washed for too long epidermal and other cell membranes will rupture,
so material removed may not be just the external resin. As a guide,
plant epidermal cells are not photosynthetic, except for stomata: if the
washings turn green with chlorophyll then internal tissues are being
extracted. The reader is referred to Elakovich and Stevens (1985) for
further information on creosote bush lignans and to Atkinson and
Blakeman (1982) for a report dealing with flavanone exudates. In gen-
eral, solvent evaporation from a sample of external resin washed off a
plant will yield a sample and a weight of resin per unit of plant material
washed (Bryant, 1987).
As a practical approach, it is usually possible to take solvents and
sample bottles for washings directly to the plant. As long as the washings
are kept cool and in the dark they will transport well for further use. One
final point is that flavonoid components of resins are sometimes fully
methylated and may thus not be phenolics in the strict sense.
4.2.1.5 Summary
Table 4.1 summarizes recommendations for collecting samples of leafy
material and transporting them to the laboratory. We have ranked
methods in order of preference. The lower the ranking of the method the
more sensitive we consider it to be to post-mortem changes in the sample.
If the substances you are interested in are not subject to much post-
mortem change, or your analyses are qualitative, then the first few
methods may be more effort than is necessary.
Table 4.1 Sample collection methods for leafy material
1 Live (fresh) material at laboratory

2 Liquid nitrogen frozen (in field)
Subsequent transport to laboratory frozen or lyophilued
3 Fresh material taken frozen or lyophilized near field
Subsequent transport to laboratory frozen or lyophilized
4 Fresh material transported on ice more than 3-4 hours
5 Samples dried in vented (herbarium) oven at c hO0C
Subsequent transport as dry samples
6 Samples dried in direct sun, uncontrolled wnditions
Subsequent transport dry
7 Samples transported warm for several hours with no attempt at preservation
72 Chapter 4
4.2.2 Sampk preparation

In this section we cover the treatment of samples from their arrival in the
laboratory to the addition of solvent. The real focus for our attention
here is the physical degradation of the sample so as to increase the
efficiency of the extraction process. If there is to be any delay before
extraction, wet material should be frozen while dry material needs to be
kept dry and cool.
The general object of the exercise is to crush every cell in the material
and release its contents to the extracting solvent. The traditional way of
doing this for fresh material is to use a pestle and mortar with acid-
wash~dsand. Usually some solvent (extractant) is added in the process
and so both the physical degradation of the sample and its extraction are
simultaneous. Microscopic analysis can be used to ensure that all cells are
ruptured and that extraction is probably complete. Anyone who has
attempted this exercise will know just how difficult it is to achieve! One
improvement on the traditional use of a pestle and mortar is to grind
fresh tissues in liquid nitrogen before adding any extractant (Lindroth,
Hsai & Scriber, 1987). This will freeze the material so cold that it will
shatter to a h e powder when ground and can then be extracted, without
much further grinding, by the addition of solvent.
The real problem with a pestle and mortar is that as one tires the
samples tend to become progressively less well ground. Mechanical
devices may not be quite as effective but they have the virtue of con-
sistency. Perhaps the best homogenizers are of the 'Polytron' type. These
have a circle of counter-rotating 'teeth' which are capable of turning quite
fibrous plant tissues into tiny fragments. We do not recommend Waring-
type blenders for anything but the softest of materials as these have a
tendency to leave vascular tissues relatively unscathed and often wrapped
around the blades.
The standard grinding equipment for dry material is the Wiley Mill.
Careful investigators will report the mesh size of the ground particles and
keep this factor as a constant in all their work. Some typical procedures
for lyophilized material are given below but there is really no one standard
to follow. One important precaution with Wiley Mills is that samples
with a high lipid content must be defatted. For instance, Fung (1987)
defatted insects prior to grinding and analysing them for plant-derived
dihydrochalcones. Besides animal derived 'material, grains and other
seeds often need to be defatted. If they are not, then a paste rather than a
powder is produced, the usual effect being to clog the mill.
In general, fresh material is more difficult to work with than dried
material which can be ground to a dry powder with relative ease. The
biological variation in water content also adds to the variability of results
expressed on a fresh weight basis, and thus our preference is that where
Table 4.2 Storage and grinding of lyophilized material
Storage Meshlmm Reference
-15°C in desiccator 60 Martin & Martin (1983)

Sealed jar, room temperature 60 Mattson & Palmer (1988)
Room temperature 1 mm Jonasson et al. (1986)
-20°C 40 Marquis & Batzli (1989)
-20°C 0.85 mm Lindmth, Batzli & Seigler (1986)
Room temperature 20 Mole (current practice)
possible dry material should be used. The general approach is to dry to a

constant weight with steps being taken to prevent the subsequent uptake
of water from the atmosphere. If dry samples are kept refrigerated they
must not be opened until they equilibrate with room temperature; other-
wise there is an 'open invitation' for water to wndense on them.
Some examples of procedures adopted are given in Table 4.2.
4.2.3 Extraction
The objective here is very simple: to remove all phenolic substances
from the solid residue of plant material by bringing them into solution.
Physically we are dealing with a solvation and a diffusion process. Phe-
nolics will leach out of the sample faster if they are very soluble in the
extractant, if their concentration in the extractant is low relative to that in
the sample and if the temperature is high. It is generally true that
solvation processes take longer from dry material versus fresh material as
all the components need to be rewetted and essentially 'unstuck' before
they enter the extract.
From this analysis it might naively be assumed that we only have to:
(i) choose a suitable solvent; (ii) extract the sample repeatedly with large
volumes of fresh solvent; and (iii) do all this at an elevated temperature.
Unfortunately there are problems with this almost every step of the way.
Some phenolics, like those present in creosote bush resin, are in-
soluble in water but soluble in non-polar solvents. Others, such as some
phenolic glywsides, are most soluble in water. Phenolics, while obviously
to a degree polar are, however, often most soluble in solvents less polar
than water. If a plant is being extracted prior to total phenolics analysis
then clearly one wants to use a solvent in which every phenolic in the
plant will dissolve. This is perhaps too much to expect, but some solvents
can approach this ideal. The extent to which they do so will depend on
exactly which phenolics are in the plant. Different plants with different
phenolics of different polarities may be best extracted with different
solvents optimized to meet their needs. This fact largely explains the
74 Chapter 4
variety of solvents used (see Table 4.3). Some other considerations also
apply to extract choice. For example, acetone-containing extracts should
not be used in the direct proanthocyanidin assay for condensed tannins.
This is unfortunate because acetone is an excellent solvent for these
tannins (Foo & Porter, 1980; Cork & Krockenberger, 1991). For the
vanillin assay for condensed tannins, water needs to be excluded from the
methanolic solvent used. This is a problem as condensed tannins do not
dissolve well in methanol. We return to these issues later but mention
them at this point in order to show that decisions made about an ex-
tractant affect more than just extract production.
Our recommendation for solvent selection is that the reader use a
mixture of water and one of the following: methanol, ethanol or acetone.
Aqueous methanol (50%) is a popular choice (see Table 4.3). This should
be tried first but also be prepared to try several others based on different
solvents and with more or less water. After extracts have been made
with several of these, they should be assayed and the most appropriate
adopted for regular use. There is no real mystery to solvent selection but
it is a matter of trial and error. If a survey of a large number of different
species is to be made it may not be possible to optimize for each of them
so an arbitrary selection may have to be made. In such circumstances we
suggest selection of a commonly used system.
Repeated extraction of a sample with fresh solvent is the best way to
maintain a steep diffusion gradient favouring the loss of phenolics from
the sample to the extract solution. Using large volumes of liquid relative
to the sample also helps. The catch here is that this is labour intensive
and that one can end up with a small amount of phenolic material
dissolved in a huge volume of extract. The alternative approach is simply
to wait longer for extraction to become complete. In Table 4.3 we tabu-
late the volume of extractant used per gram of sample and indicate
whether the sample was fresh or dry. We also tabulate the number of
times each sample was extracted and the time taken for the extraction.
Most investigators also make some attempt to stir the system during
extraction. The beginner may be bewildered by the variety of approaches
taken: clearly there is no single and standard method!
A charitable interpretation of Table 4.3 is that each procedure was
fully optimized to suit the particular situation in which it was used. At
least some investigators indicate such efforts (Seevers & Daly, 1970;
Martin & Martin, 1983; Glyphis & Puttick, 1988). It is also likely that
many such methods are based on intuition and on the time and equip-
ment available.
We recommend working at room temperature or in a cold room.
Place the sample in a flask with at least 100 times its volume of extractant
and use an orbital shaker or similar device to keep things moving at least
overnight. When pressed for time heating will speed things up (Gartlan et
al., 1980), but you must be sure that the substames of interest are stable.
Where this is the case then the Soxhlet apparatus is a tried and tested
extraction procedure. If things must be kept cool then repeated extraction
with fresh solvent has to be resorted to. Sonicating samples with their
extractant can also be useful.
Once liquid has been added to a sample reactions can occur quickly.
Optimizing all the parameters in Table 4.3 for a given system may pay
off if one is anticipating a substantial effort in analysing a particular
kind of sample. Extracting samples for insufficient time or at too low a
temperature may lead to incomplete extraction. Very long extractions at
too high a temperature may lead to the degradation and loss of material.
Lindroth, Hsai and Scriber (1987) indicate having had to work around
this problem.
We conclude by suggesting that each investigator should anticipate
having to make the effort to optimize the yield of phenolics extracted
from his or her samples and not simply trust to whichever method ap-
pears to have worked in the past. The range of values in Table 4.3 is
impressive but also worrisome. Extract volume to sample weight ratio
ranges from 2 to 200. Extraction times vary from 30 seconds to 96 hours,
and extractions are repeated between one and five times at temperatures
between the freezing and boiling points of water.
4.2.4 On being q u a n W v e
The methods presented in the rest of this chapter and much of the rest of
the book are quantitative. To accomplish these the practitioner needs
manual dexterity and attention to detail when using balances, volumetric
glassware, spectrophotometers and other scientific instruments. There is
also a need to be able to make simple biochemical calculations. While
the required level of manual dexterity tends to be learnt through bitter
practical experience, the arithmetical skills can to some extent be obtained
from a book such as this: some tips are given below.
Samples often start as a ground powder of measured weight (Wig)
that is some fraction of the whole sample that was collected (whole
sample weight, W2g). If the fraction of the sample to be analysed (Wt) is
extracted into some volume of solvent (V,ml), then a small volume of
this might then be taken (V2ml) and diluted with some more solvent
(V3ml) before further analysis. Some fraction (V4ml) of the diluted
sample (V,) will actually be used in an assay. If the assay is spectro-
photometric, then an absorbance reading will be generated from the
sample contained in volume V4.
Suppose that the standard curve (for explanation of standard curve
see below) for your assay procedure indicates that the volume of diluted
Table 4.3 Initial extracts and extractants
Solvent mllp dlf Time Number of times Temperature References and notes
70% Acetone 48 h It Dement & Mwney (1974) +0.1% Vit C

70% Acetone 5 min rt Broadhurst & Jones (1978) +0.19/0 Vit C
70% Acetone ? rt Stafford & Lester (1980)
80% Acetone 20 h It Jukunen-Tiitto (1985a)
Acetone 30min tnlii Hansson et al. (1986)
Acetone 2h 50" Zudcer (1982)
Acetone 30 min rt Palo (1984)
50% Ethanol 30 min inid Gartlan et 01. (1980)
70% Ethanol 12 h n W i n (1984)
80%Ethanol 48 h rt Lindroth & Batzli (1986)
95% Ethanol 5min n Johnson & Schaal(1957)
Water ? It Del Amo, Ramirez & Espejo (1986)
50% Methanol 30 rnin boil Martin & Martin (1983)
50% Methanol 1h It Hageman (1988)
50% Methanol l0min 95" Jonasson et al. (1986)
50% Methanol 20 min 90" Tempe1 (1981)
50% Methanol 30 s rt Maufette & Oechel(1989)
50% Methanol 5min boil Becker & Martin (1982)
50% Methanol 10 min 95" Bryant (1987)
50% Methanol 1h 70" Baldwin, Schultz & Ward (1987)
50% Methanol 24 h rt Nascimento & Langenheim (1986)
50% Methanol ? hot Foley & Hume (1987)
70% Methanol 10 s It Walters & Stafford (1984)
76% Methanol 24 h rt Mattson & Palmer (1988)
80% Methanol 5 min O"s0 Lindroth, Hsai & Scriber (1987)
80% Methanol ? rt Woodhead & Cooper-Driver (1979)
80% Methanol %h rt McDougal & Parks (1986)
80% Methanol ? boil Cooper-Driver et al. (1977)
80% Methanol ? rt Coley &Aide (1989) +1% HC1
Methanol ? cold Scalbert, Monties & Favre (1988)
Methanol 1h rt Jakubas, Gullion & Clausen (1989) +1% HCI
Methanol 2 min rt Seevers & Daly (1970)
Methanol lmin rt Price & Butler (1977)
b, bark; d, dry powder; f, fresh macerated leaf; g, ground grain; st, vortex/blender/vigorous stirring; sx, soxhlet; so, sonicate; rt, room temperature,
vit C., ascorbic atid, sometimes used as an antioxidant.
78 Chapter 4
sample V, contains Z mg of some phenolics, you are then faced with

working out how much phenolic material was present in the original
sample of weight W2. This is a very typical situation that students beginn-
ing with these techniques often have trouble facing. This kind of logic
problem is situation dependent, i.e. dependent upon the actual dilution
and mass transfer operations you carried out. In this case the total weight
of phenolics (P) in the whole sample (W2) is given by
We assume that you will be able to work out how this equation was
reached, and we hope that doing this will help you approach your own
dilution problems. For practice with other types of calculations, such as
those needed to make buffer or reagent solutions, the reader is referred
to books on the subject such as that by Segel(1975).
We must stress the essential need for accuracy and keeping track of
sample handling operations: take notes as you go! Every time a volume
or weight is measured variability and scope for error rises. Errors may be
due to either the instruments or to the operator. The former will probably
not be a problem for comparative measurements made with the same
equipment (unless a component is slowly failing), while practice can
improve operator skills.
4.3 Spectrophotometric assays
Spectrophotometry is one of the more easily mastered quantitative tech-
niques, and one of the most valuable. In essence it involves measuring the
amount of light of a particular wavelength (UV or visible 220-850nm)
that is absorbed by a liquid sample. The measurement can be used
to determine'the concentration of a particular substance or group of
substances.
The mathematics behind the quantitative use of spectrophotometry is
governed by the Beer-Lambert Law. This states that the fraction of light
absorbed by a solution is proportional to: (i) the distance the light travels
through the solution (the path length); (ii) the chemistry of the substance
or part of the substance responsible for absorbing the light (the chromo-
phore); and (iii) the concentration of that chromophore. The law states
that the attenuation of light occurs as an exponential rather than as a
linear loss in intensity. Thus the intensity of incident light (Ib) and
emergent light (I,) that has passed through the solution is related to the
concentration (c) and the distance travelled (I). These variables are
related by the following equation, where a is a constant:
The quantity is known as the transmittance and can be expressed as

a decimal fraction or as a percentage. While this can be read directly off

most spectrophotometers, scientific results are generally presented in
units of absorbance (sometimes called optical density), i.e. logfb/Ia.
Absorbance can also be directly read from spectrophotometers. Note that
absorbance is linearly related to concentration, unlike transmittance.
Finally we must consider how absorption data is to be presented. For
a constant optical path length (1, usually 1cm), the absorbance is related
to concentration via the constant (a). This constant is variously known as
the 'absorbancy index', 'absorption co-efficient' or 'extinction coefficient'
(E). If concentrations are specified in molllitre then E is termed the
molar extinction coefficient. If the molecular weight of the substance(s)
under analysis are unknown it is common to calculate E for a 1%solution
(El%). In practice E values may be encountered in two ways: (i) you may
have a solution of known concentration for which you wish to report
E; or (ii) you may wish to determine the concentration of a material
for which E is published. Published E values refer to specified solvent
conditions, path lengths, and wavelengths that must be recreated exactly
in order to use. the data. Similarly, you must specify these conditions if
you wish to report such data.
For visible wavelength spectrophotometry (colorimetry) of phenolics it
is generally necessary to use a chemical reaction to produce a specific colour
in direct and linear proportion to the amount of phenolic material present.
By selecting a wavelength of light that is maximally absorbed by the
absorbing part of the molecule (chromophore) generated in the assay, the
sensitivity of the measurement is maximized. Thus light of 760nm wave-
length is used to detect the blue colour generated in the Prussian Blue
assay for total phenolics (see below). In general calorimeters that employ
coloured filters are perfectly adequate for the assays described below. For
instance, Price and Butler (1977) designed the Prussian Blue assay for use
with a 'Klett' calorimeter employing a red filter. Spectrophotometers
(which use a diffraction grating to produce light of a particular wave-
length) are superior instruments, but the main point is to make all
comparative measurements on the same instrument, no matter what that
is. If results must be compared between laboratories some form of stan-
dardization is usually needed to put all results on an absolute basis.
In most of the assays discussed below, phenolics are quantified by first
making measurements on solutions of known concentration of standard
substances. When absorption measurements are plotted against the
known concentration of phenolics in solution, linear relationships are
generally obtained for the reasons outlined above (these are often re-
ferred to as 'standard curves'!). Concentrations of phenolics in extracts
can then be determined by measuring their absorbance in the same way I
80 Chapter 4
and then comparing these results to those obtained with solutions of

known concentration.
Such measurements are simple in concept. Technical difficulties
usually arise because in ecological and biochemical work in general,
impurities and other contaminants occur in the solutions being measured.
To obtain the absorbance due to the substance being assayed in the
absence of any interference, one needs to set up an otherwise identical
solution, impurities and all, that lacks only the substance being measured.
This control or 'blank' has its absorbance subtracted from the sample
being measured. The concept of the blank is simple, but its construc-
tion can present technical difficulties as outlined for each assay below.
Understanding how best to construct 'blank' reactions is often critical to
interpreting spectrophotometric data. This is especially true where ideal
blanks cannot be constructed and apparently false-positive results are
encountered.
4.4 Total phenolics

Many ecological studies report measurements of total phenolics in bio-
logical materials. The conceptual basis of the measurement is to quantify
the total concentration of phenolic hydroxyl groups present in the extract
being assayed, irrespective of the particular molecules in which they
occur. Such assays in no way provide information as to the particular
phenolic compounds present. It is surprising to find that the assays still
most commonly in use today were originally designed to quantify the
phenolic amino acid tyrosine (Folin & Denis, 1912a).
There are three commonly used methods for total phenolics and
examples of their use in ecological studies are listed in Table 4.4. There
are two techniques due to Folin and w-workers and one due to Price and
Butler. Each has particular merits which we will endeavour to highlight.
However, for most research questions, any one technique will yield
perfectly acceptable and publishable results and we do not espouse a
particular technique to the exclusion of the others. We recommend the
new investigator to read through our comments about each method and,
in the first instance, to choose the method that seems most appropriate
to his or her laboratory circumstances.
One point to note from Table 4.4: the Association of Official
Agricultural Chemists' (AOAC) method for tannins in wine and distilled
spirits (AOAC, 1970) is, in reality, an assay for all phenolics (i.e. total
phenolics) and not just tannins. We will return to such distinctions later.
For the present, we emphasize that the investigator needs to take re-
sponsibility for his or her own research and fully understand the workings
of the techniques employed, i.e. what they actually detect and hob
dependable the results are.
Table 4.4 Exam~lesof methods used for analysis of total phenols
Reference Source reference Standard
Folin-Ciou~l~eu method
Leszczynski (1985) Seevers & Daly (1970)
Glyphis & Puttick (1988) Singleton & Rossi (1%5) Tannic acid
ServeUo & Kirkpatrick (1989) Singleton & Rossi (1965) GaUic acid
Foley & Hume (1987) F o l i & Ciocalteu (1927)
J b e n - T i i t t o (1985a) - Phenol
Seevers & Daly (1970) - Chlorogenic acid
F&-Denis method
Jonasson ef al. (1986) - Tannic acid
Baldwin, Schultz & Ward (1987) Swain & Hillis (1959) Tannic acid
Martin & Martin (1983) Swain & Hillis (1959) Tannic acid
Mauffette & Oechel(1989) Swain & Hillis (1959) OD
Zucker (1982) Swain & W i s (1959)
Hartley & Lawton (1987) Bergelson, Fowler & Hartley (1986)
Nascimento & Langenheim (1986) AOAC (1970) C
Wilson (1984) AOAC (1970) Tannic acid
Kedrowski (1983) Swain & Goldstein (1964) OD
Lindroth, Batzli & Seigler (1986) Swain & Goldstein (1964) OD
Price eta[. (1989) Zucker (1982) OD
Manson & Palmer (1988) Rosenblatt & Peluso (1941) Tannic acid
Johnson & Schaal(1957) - Chlorogenic acid
Mole & Waterman (198%) Gartlan ef al. (1980) Tannic acid
Gartlan et nl. (1981) Folin & Denis (1912a) Tannic acid
Price-Butler method
Del Amo, Rarnirez & Espejo (1986) Price & Butler (1977) Tannic acid
Jakubas, Gullion & Clausen (1989) Price & Butler (1977) Gallic acid
Mole eta!. (1990) Price &Butler (1977) Tannic acid
Standards: OD, optical density; C, constant but non-specific substance.
4.4.1 The Folin-Denis method

In 1912 Folin and Denis published two papers (Folin & Denis, 1912a,b),
one concerning phosphotungstic-phosphomolybdic compounds as colour
reagents and the second concerning the use of such reagents in the
determination of tyrosine in proteins. While the method is quite capable
of detecting tyrosine-containing proteins, it is rarely used for this today.
However, as seen in Table 4.4, it remains the most commonly used
method for measuring total phenolics (Scheme 4.1).
The chemistry behind the Folin-Denis reaction is simple to use
although the structure of the inorganic complex formed remains unre-
solved. The reaction is a reduction-oxidation in which the phenolate ion
is oxidized under alkaline conditions while reducing the phosphotungstic-
phosphomolybdic complex in the reagent to a blue-coloured solution (the
chromophore). The Folin-Denis reagent is not commercially available
and requires'preparation. Full detail;; of the procedure, following the
82 Chapter 4
AOAC (1975), are given in Scheme 4.1. The assay involves mixing the
Folin-Denis reagent and an aliquot of the extract together in a large
volume of water. The addition of sodium carbonate, which acts as an
alkali, then begins the development of the blue colour. Spectrophoto-
metric measurements are made after a measured time has elapsed for
colour formation.
Scheme 4.1 The Folin-Denis method
Folin- De& reagent

To a llitre round bottom flask, add 750ml deionized water,
sodium tungstate (lOOg), phosphomolybdic acid (20g) and or-
thophosphoric acid (50ml). Reflux for 2h, then cool and dilute
to llitre with deionized water. Use of glass beads to prevent
bumping during reflux is advised.
Saturated sodium carbonate solution

Mix anhydrous sodium carbonate and deionized water in the ratio
35g per 100ml. Dissolve solids at 70-80°C and cool overnight.
Seed solution with sodium carbonate decahydrate and after crystal-
lization, filter through glass wool.
Procedure
Add sample ( I d ) to a lOOml volumetric flask containing de-
ionized water (about 60ml). Swirl contents to mix. Add Folin-
Denis reagent (5ml) and mix again. After 1min and before amin,
add the sodium carbonate solution (loml), record the time as time
zero, and mix again. Make up to lOOml with deionized water.
Stopper the flask and mix thoroughly by inverting several times.
(A common error is to fail to mix liquid from the neck of the
flask). After 30min (to within lmin) record the absorbance of the
reactants at 760nm.
See also 4.4.4 General procedures and standards
The assay has been substantially improved since 1912, particularly in

relation to optimizing reagent and sample volumes (Singleton & Rossi,
1965) and in using a standard 30-minute colour development time. One of
the most obvious problems is the occasional development of a white
precipitate that prevents spectrophotometric measurement. The Folin-
Ciocalteu method (see below) was devised to circumvent this problem
Extraction and auantification 83
and to improve the sensitivity of the assay in other ways. However, most
investigators still use the older Folin-Denis propdure, perhaps because
the reagent is somewhat easier to prepare.
We recommend the use of 1mi sample, 5 ml reagent, lOml saturated
sodium carbonate, 100ml total volume: this is, in essence, the AOAC
procedure (AOAC, 1975). The original literature justifying this viewpoint
can be accessed through the review by Singleton and Rossi (1965) al-
though these authors actually favour the Folin-Ciocalteau procedure.
Variations that predate the Singleton and Rossi review (e.g. Rosenblatt
& Peluso, 1941; Johnson & Schaal, 1957; Swain & Hillis, 1959; Swain &
Goldstein, 1964) should no longer be used. The principal disadvantage of
these earlier techniques is that the conditions used are such that the
relationship between concentration of phenolics and colour produced may
become non-linear (i.e. the Beer-Lambert Law is not followed).
This does not mean that there are not limits to the linear range of
the Folin-Denis assay, even following the AOAC (1975). It is possible to
saturate the assay with so much phenolic material that no additional blue
coloration develops. The option of adding more than 5ml of reagent
should not be taken as precipitation problems often arise: instead, dilute
the extract. As noted above precipitates sometimes form anyway, espec-
ially if the sample is high in potassium salts. In this case the reaction
mixture should be centrifuged. Filtering the solution is not recommended
as significant amounts of the blue complex can be adsorbed to the filter
paper. Another source of variation, common to almost all versions of the
Folin-Denis procedure, is the degree of saturation of sodium carbonate
which varies with temperature and alters the pH in the assay and thus the
results. The Folin-Ciocalteu procedure is not prone to this error.
Despite the fact that the Folin-Denis procedure was invented to assay
an amino acid there is little cause for concern that proteins will affect the
results of total phenolics assays. Tyrosine has only a single phenolic
hydroxyl and is but one of the 20 or so different amino acids i~ proteins.
Equal weights of proteins and of phenolics, such as gallic acid, have vastly
different numbers of phenolic hydroxyls. Add to this the general insolu-
bility of proteins in the solvents used to extract phenolics and one can see
that any interference is minimal, even when levels of phenolics are quite
small.
4.4.2 The Folin-Ciocalteu method

Folin and Ciocalteu (1927) improved upon the Folin-Denis reagent by
making it more sensitive to reduction by phenolics and less prone to
precipitate. The first of these improvements was achieved by increasing
;he ratio of molybdenum to tungsten and using liquid bromine to oxidize
any traces of molybdenum-tungsten blue in the freshly prepared reagent.
84 Chapter 4
The precipitation problem can be solved by addition of lithium sulphate.

A description of its preparation and use, as modified by Singleton and
Rossi (1965), is given in Scheme 4.2.
Scheme 4.2 The Folin-Ciocalteu method
The Folin-Cwcalteu reagent

To a llitre round bottom flask, add 700ml deionized water,
sodium tungstate (100g), phosphomolybdic acid (25g), lOOml
concentrated hydrochloric acid and 85% orthophosphoric acid
(501111). Reflux for 10h, cool and add lithium sulphate (150g). Use
of glass beads to prevent bumping during reflux is advised. Add a
few drops of liquid bromine so that the final reagent is yellow in
colour (not green). Boil off excess bromine from open flask (in a
fume hood!). Dilute to 1 litre with deionized water.
Sodium carbonate solution

Mix anhydrous sodium carbonate and deionized water in the
proportion 20 g per 100ml.
Procedure
Add sample (lml) to a 100ml volumetric flask containing
60-75m1 deionized water. Swirl contents to mix. Add Folin-
Ciocalteu reagent (5 ml) and mix again. After 1min and before
8min, add the sodium carbonate solution (15 ml), record the time
as time zero, and mix again. Make volume up to l00ml exactly
with deionized water. Stopper the flask and mix thoroughly by
inverting it several times. After 2h (to within 1-2min) record the
absorbance of the reactants at 760nm.
The Folin-Ciocalteu method gives, on average, 30% more colour

than the older method for a given sample (Singleton & Rossi, 1965). This
has recently been reconfirmed by both Julkunen-Tiitto (1985a) and Price
et al. (1989). In some phenolics (e.g. salicylic acid, 4-methylumbelliferone)
the colour yield is more than doubled relative to that with the Folin-
Denis reagent (Singleton & Rossi, 1965). The Folin-Ciocalteu procedure
also has the reputation of being less prone to interference by non-
phenolics and for all the above reasons it has been recommended over the
Folin-Denis procedure. There is little to be said against this procedure,
but the reagent preparation may put some off as may the 2 hour wait
for wlour development. Most of the literature references for its use
(Table 4.4) are recent, suggesting that this method is gaining acceptance
among ecologists.
4.4.3 The Price and Butler method

The method pioneered by Price and Butler (1977) also exploits an oxida-
tion-reduction reaction. Here also the phenolate ion is oxidized, but this
time ferric ions are reduced to the ferrous state and then detected by the
formation of the Pmssian Blue wmplex (Fe4[Fe(CN)&) with a potassium
ferricyanide-containing reagent. This method is 65 years younger than the
Folin-Denis method and is much less extensively tried and tested. In our
hands, and those of undergraduate technicians, it has worked well for the
routine assay of plant phenolics and is easier to perform than either Folin
technique. Unfortunately, a rigorous comparison of the methods is not
yet available.
The reagents and procedures that we reproduce below (Scheme 4.3)
are those described by Price and Butler (1977), slightly modified to reflect
current use in Butler's laboratory. As with the Folin assays, a small
Scheme 4.3 The Price and Butler method
Ferric chloride reagent

Prepare a 0 . 1 solution
~ of femc chloride (Feel3) in 0 . 1 hy- ~
drochloric acid. Filter the h a 1 solution until it is clear (yellow),
i.e. without turbidity.
Potassium ferricyanide reagent

0 . 0 0 8 ~K3Fe(CN)6 (= 2.632s per litre) in deionized water.
Procedure
Add exactly 251111 deionized water to a 50ml container. Add
sample (250 p1) and mix. Add 3 ml ferric chloride reagent and mix.
After 3min add 3ml potassium ferricyanide reagent and mix.
After a further 15min,* read the absorbance at 720nm.
* i.e. l8min after the beginning of the assay. In the original paper,
Price and Butler (1977) only waited 10min for colour development
as solutions grew turbid after 15-20min. If turbidity occurs use
this shorter time. The drawback here is that colour formation is
not quite maximal.
88 Chaoter 4
volume of sample is added to water, mixed, and subsequently the two

reagents are added in order. The reactants are then mixed again and
allowed to stand while a blue colour develops. Because the reagent
preparation does not require the use of a reflux apparatus, and that 50ml
test tubes or Erlenmeyer flasks can be used in place of 100ml volumetric
flasks, this method is particularly worthy of consideration by those oper-
ating outside a chemistry laboratory or on a limited budget.
4.4.4 General procedures and standards

The obvious must first be stated: always use analytical grade reagents.
The procedures outlined in Schemes 4.1 to 4.3 indicate how to generate
a colour reaction from an extract containing phenolic material. Two
questions must be confronted at this point. Firstly, what is a suitable
blank reaction against which to compare the sample, or to put it another
way, what should be used to zero the spectrophotometer? Secondly, how
can the absorbance reading obtained be converted to an understandable
quantity of phenolics in the plant?
4.4.4.1 The blank

Note that in each assay the sample volume taken from the extract repre-
sents only 1% of the reaction mixture that is finally assayed. Thus any
pigment in the extract will usually be so diluted that it will not interfere
significantly with the reading obtained. Under most conditions an appro-
priate blank will therefore consist of all the reagents including the solvent
in which the sample is dissolved, with the reagents being added at the
proper times and in proper sequence. This solution can then be used to
zero the spectrophotometer after the correct time and before measurement
of the sample. The addition of the appropriate solvent may seem a
frivolous exercise as these are for the most part unreactive and colourless
liquids. On the one hand this should be done just so that you can say,
with your hand on your heart, that the only factor differentiation between
the blank and the assay was the sample. On the other hand it can have
real importance: Price and Butler (1977) note that methanol-containing
solvents have a slight effect on the reaction of ferric chloride and that
they need therefore to be present in the blank for chemical reasons.
4.4.4.2 Standardization
The value of quantitative assay data is inordinately increased if calibrated
according to some reference standard. In addition to the fact that to most
readers the statement that a plant sample contains the equivalent of
40mglg tannic acid imparts more immediate information than stating that
it has an E(,%) of 60, standardization also controls variation in the
calibration of spectrophotometers from laboratory to laboratory. The
negative side to this is that to the uninformed reader 'equivalent of

40mg/g tannic acid' translates solely in terms of tmnic acid, whereas in
truth there may be no tannic acid in the sample at all! Standardization is
clearly vitally important to the value of your results and it is worthwhile
examining in more detail methods of presentation.
Using E(*%). If, in an assay, 5Wmg of plant material is extracted into

50ml of solvent and l m l of that solution is used in the preparation of
lOOml of a reaction mixture, then what is effectively being assayed is the
quantity of phenolics in 500/(50 x 100) mg of plant material (i.e. 0.1 mg
in lWml of solution, or 0.001%). If this gives an absorbance reading of
0.35 then to convert the value to an E(iY6)it is necessary to multiply by
1000 (e.g. E(ioh) = 350). E(146) data can be used to compare relative
concentrations, measured by a particular assay, within a series of samples.
Using smndardr based on a specific substance. To convert absorbance

readings to quantities of phenolics one needs to perform the assay on
a standard substance using a series of samples containing known concen-
trations. Given that the assay obeys the Beer-Lambert law, then a plot
of the absorbance readings against the known concentration will give a
straight line (Fig. 4.1). Such a plot of absorbance against the quantity of
substance assayed is called, somewhat paradoxically, a standard curve. If
you have a standard curve for a phenolic compound x then it becomes
easy to translate an absorbance value for an extract into a value based on
Concentration (mgil)
Fig. 4.1 Standard curve for tannic acid using Folin-Denis reagent (from Mugedo &
Waterman, 1992).
88 Chapter 4
x directly from the standard curve. If we return to the example above,

where a solution of 0.1 mg per 100ml of the sample gives an absorbance
of 0.35, comparison with the plot for the standard (Fig. 4.1) shows that
this is equivalent to 4.5mgA (or 0.45mg1100ml) of tannic acid. Thus
0.1 mg of sample contains the equivalent of 0.45mg of tannic acid and its
phenolic content can be expressed as 45% of tannic acid equivalents.
The problem with standardization occurs because one has to make
a choice of a specific standard for assays which in reality measure a
wide range of substances. The key point to remember here is that the
absorbance produced in all three total phenolics assays increases for each
phenolate ion that becomes oxidized in the assay. Consider the series of
simple phenols illustrated by structures 8-17. Their molecular weights
vary less than twofold from 122 for salicaldehyde (10) to 224 for sinapic
acid (17) yet their capacity to react with the various total phenol reagents
varies fourfold from approximately 57 mass units per phenolic group in
gallic acid (8) to 224 mass units per phenolic group in 17.
This will cause problems when estimating the concentration of phenolics
in uncharacterized samples. For instance, suppose gallic acid (8) was
chosen for construction of the standard curve and it was deduced that for
each mg of gallic acid per ml of sample, the assay yielded a colour
reaction measuring z absorbance units. This factor z can then be used to
work out the weight of phenolics present in extracts (as illustrated above)
as long as all the phenolics present had a molecular weight of 170 and
have three phenolic hydroxyls. If sinapic acid (17) were present in the
samples and not gallic acid then we would be in error by a factor of about
fourfold. Where one has no idea of what phenolics are present in a plant
then this problem is insoluble.
Using standards based on a non-specific substance. The only empirical

solution to this problem is to prepare a large batch of phenolics from the
plant under investigation and use this as a standard. Even if such a
standard is never characteriked it can be used for comparative purposes
and to correct for run to run variation in the assay. If carefully preserved
(desiccated, deep frozen, in the dark and under nitrogen will cover most
problems!) it should be usable for some time. Of course if the study is
comparative across species this option does not hold. The only options
then are to use E&) values or to trust to a general standard.
Reference standardv for comparisons between laboratories. These need to

be widely available and adopted by consensus. Examination of Table 4.4
shows that tannic acid is the substance most widely used as a standard for
total phenolics measurements. When tannic acid or another standard is
used results must be presented to explicitly state that the samples assayed
contained a quantity of phenolics that gave an absorbance in the assay

equivalent to a particular quantity of tannic acid. The usual phraseology
is to say that the sample contained so many mglg tannic acid equivalents,
(often abbreviated to TAE). It is worth reiterating the point that the
sample does not necessarily contain any tannic acid. Today ecologists
seem well aware of this distinction, but the previous literature is littered
with examples where total phenolics assays have mistakenly been assumed
to be specific for a particular type of tannin, when they will actually
detect any substance covered in this book! This problem is seen in the
AOAC assay for tannins in wine and spirits (AOAC, 1965). Wine contains
phenolics that are predominantly tannins and the use of a total phenolics
assay is a reliable guide, but only because of this previous characterizationof
the sample. For a chemically unknown species of plant, positive results
with the AOAC assay offers no proof of the presence of tannins.
These problems of interpretation have led some investigators to
abandon the use of standards so that people do not misinterpret their
work. We think it unfortunate that in doing so they have also prevented
others from using their data for comparative purposes.
One other important point in presenting standardized data is to be
sure methodology is adequately described and properly referenced. Take,
for example, the various citations given for the use of the Folin-Denis
method (Table 4.4). Often a multitude of sins can be hidden by ambiguous
citations. For instance Hartley and Lawton (1987) cite Bergelson, Fowler
and Hartley (1986), who in turn claim to be following Swain and Hillis
(1959) and Martin and Martin (1982). Suspicions are raised when Bergelson,
Fowler and Hartley (1986) also state that the Folin-Ciocalteu reagent
was used in their work. The two cited papers correctly used the Folin-
Denis reagent. However, closer examination reveals that sample, reagent
and sodium carbonate solution volumes differ in all of these studies!
Fortunately total phenolics assays are relatively robust and insensitive to
such variations. Bergelson, Fowler and Hartley (1986) also used a tannic
acid standard to relate their absorbance results to a widely accepted unit
of measurement. We thus have considerable confidence in their data
despite their unique methodology. Nevertheless, we cannot be sure that
they were always working within the limits of the Beer-Lambert Law or
that they would have recognized such problems. Other methods described
in this book do not stand such variation and so require much more
attention to detail.
4.4.5 Zntetfering substances: ascorbic acid

Any substance capable of being oxidized by the reagents of the various
total phenolics assays will yield the reduced (coloured) forms of the
reagents and appear as a phenolic. Ascorbic acid (153, vitamin C) is one
90 Chapter 4
commonly occurring non-phenolic capable of doing this. Indeed, Johnson

and Schaal (1957) report that ascorbic acid produces a slightly higher
absorbance than chlorogenic acid (127). Peng and Jay-Allemand (1991)
report that addition of ascorbic acid or sodium metabisulphate greatly
enhanced the protein-precipitating activity of tannins extracted from
walnut. The likely explanation for this is that the phenol groups of the
tannin remained fully functional because of the presence of the antioxidant.
On a more reassuring note, Johnson and Schaal (1957) show that the
levels of natural ascorbate in extracts tended to be so low as to be of
negligible concern in total phenolics assays. Thus, wounded potato tissues
can accumulate over 200mg/100g phenolics but never more than
3 mgI100g ascorbate. Johnson and Schaal (1957) give details of an inde-
pendent method for the determination of ascorbate which can be used to
test this assumption. One way in which ascorbic acid can become a
problem is its exogenous addition to samples, particularly as an antioxidant.
Some authors recommend its use in extraction buffers (see Table 4.4)
and in these instances the crude extracts cannot then be used for total
phenolics assays but must first have the ascorbate removed.
4.4.6 Totalphenolics methods to avoid

Some older methods for total phenolics which have fallen into disuse and
one recent and novel method are, in our opinion, best disregarded in
favour of the methods discussed above. Given the satisfactory and com-
monly used techniques we have described, we see no need to complicate
the interpretation of published data by the use of other published methods
based on diierent principles. For the sake of completeness, we have
presented brief details of these alternative techniques below.
The one recent addition to total phenolics techniques is due to Reed et
al. (1985) who have introduced a gravimetric technique based on the
precipitation of phenolics with ytterbium. The chemical basis of this
reaction is not completely understood, raising doubts about the pdssibility
of precipitation with non-phenolics. Lowry and Sumpter (1990) have
recently reported a more worrisome problem in that the reaction can fail
to detect phenolics. Specifically, they failed to obtain precipitates of
ytterbium with extracts of leaves containing 40% by dry weight of the
common phenolic glywside rutin.
A permanganate based redox method has been used for phenolics,

as have iron, molybdenum and tungsten dependent techniques. The
permanganate procedure, known as the Nebauer-Loewenthal method
(Singleton & Rossi, 1965), has also been incorporated in several protein
precipitation methods for tannins, such as those in tea (AOAC, 1965).
Simple complexation of phenolics with iron has been used as the basis for
total phenolics assays due to the ability of ferric chloride to form a
coloured complex with phenolics. Bums (1963) utilized this reaction as
did Hagerman and Butler (1978) when detecting protein precipitated
phenolics. We tried to develop this reaction further (Mole & Waterman
(1987b), noting that the colour of the complex with tannins varied according
to their type. We no longer advocate the use of this reaction as other
methodologies appear superior. Swain and Hillis (1959) also note the use
of some coupling reagents, including Gibbs reagent, for phenolics, but we
have seen no recent use of these.
The last approach to the measurement of phenolics that is occasionally
used is direct UV spectrophotometry at 280nm, utilizing the strong
absorbance of many phenolic compounds at this wavelength. Unfortunately
the molar extinction coefficients of phenolics are so varied as to preclude
this as a technique for the quantification of phenolics in general. UV
detection is commonly used for high performance liquid chromatography
(HPLC) analysis (see Chapter 7), but only for separated compounds.
Even here, molar extinction coefficients need to be known before quanti-
fication can be accomplished.
4.5 Condensed tannins

The chemical structure of condensed tannins has been dealt with in some
detail in Chapter 1, while their chemical ecology and protein precipitating
properties are discussed in Chapter 5. Here we are concerned with
spectrophotometric assays for condensed tannins based upon detecting
them by their specific chemical attributes. There are far fewer techniques
to deaf with here than was the case for total phenolics; the only two of
proven value in ecological terms being the vanillin and proanthocyanidin
assays, although the recently described para-dimethylaminocinnamalde-
hyde method (Delcour & de Varebeke, 1985) may, in future, be worthy
of consideration. For the routine quantification of condensed tannins
with a single assay, we strongly recommend the more widely used proan-
thocyanidin assay (Table 4.5). The two assays work according to different
principles and can give widely varying results for the same tannin (Fig.
4.2). This has led some investigators to use both techniques in tandem.
The vanillin assay is also of use in the estimation of condensed tannin
polymer length by spectrophotometric means.
92 Chapter 4
Table 4.5 Examples of methods used for analysis of condensed tannins
Reference Source reference Standard
Proartlhocymidin
Foley & Hume (1987) Dement & Mooney (1974) C
Baldwin, Schultz & Ward (1987) Bate-Smith (1975) C
Glyphis & Pnttick (1988) Swain & H i s (1959) Quebracho
Maufette & Oechel(1989) Swain & Hillis (1959) OD
Nascimento & Langenheim (1986) Swain & HiUis (1959) C
Jakubas, Gullion & Clausen (1989) Watterson & Butler (1983) OD
Jachmann (1989) Martin & Martin (1982) OD
Jonasson et al. (1986) Martin & Martin (1982) Quebracho
Gartlan et a!. (1980) Bate-Smith (1977) Quebracho
Vanillin reaction
Jonasson et al. (1986) Burns (1971) Quebracho
Mosjidis, Peterson & Mosjidis (1990) Sarkar & Howarth (1976) C
Standards. OD, optical density; C, constant but non-specific substance.
Catechin
Fig. 4.2 Comparison of the estimation of condensed tannin by proanthocyanin (CT, mglg)
and vanillin (Vme, mglg) assays in different plant materials (from Mole & Waterman,
1987b).
4.5.1 Proanthocyanidin method

This method involves the hydrochloric acid catalysed depolymerkation of
condensed tannin in butanol to yield a red anthocyanin product that can
be detected spectrophotometrically. The older literature also refers to this
as the leucoanthocyanin method (Swain & Hillis, 1959), reflecting an
earlier term for colourless substances that yield anthocyanins after acid
treatment. In the procedure described it is condensed tannins that are
detected (Watterson & Butler, 1983) and proanthocyanidins is the more
appropriate description.
Swain and Hillis (1959) can be credited with creating a viable assay
procedure using a butanol-hydrochloride reagent. The method has been
continuously modified since then, major developments being concerned
with: (i) optimization of the proportions of acid and butanol and the
reaction time; (ii) introduction of ferrous iron as a catalyst to speed up
and improve the depolymerization activity; and (iii) recognition of the
sensitivity of the reaction to water and other solvents added with the
sample. This last point is perhaps the most critical to ecologists working
with crude extracts of fresh plant material.
The most important contribution to this development is that of Porter,
Hrstich and Chan (1986). They have optimized, and most importantly,
publicized the use of iron as a catalyst in the reaction. Until recently
reagents both with iron (e.g. Gartlan et al., 1980) and without (Swain &
Hillis, 1959) were being employed even though the utility of iron had
been recognized by Govindarajan and Mathew (1965). Reactions have
also been carried out in mixtures of butanol and concentrated hydrochloric
acid ranging from 95 :5 (Swain & Hillis, 1959), 80 :20 (Martin & Martin,
1982), 70:30 (Jakubas, Gullion & Clausen, 1989) to 60:40 (Maufette &
Oechel, 1989). Besides altering the acid concentration in the reaction this
also radically alters the water content (see below). All investigators have
heated these reactions, but for times varying from 2 hours (Foley &
Hume, 1987) to 10 minutes (Jakubas, Gullion & Clausen, 1989). Clearly
all users would benefit from order being brought to this situation. This
reaction would then have the potential to be highly comparable between
laboratories.
We advocate the general use of a procedure similar to that of Porter,
Hrst~ihand Chan (1986). This is detailed in Scheme 4.4. Critical points
include the 40-minute reaction time and the use of screw capped test
tubes to prevent volume changes by solvent evaporation.
The 6% optimum water content for the reaction (Porter, Hrstich &
Chan, 1986) includes that in the reagent as well as any introduced with
the solvent. Concentrated hydrochloric acid (specific gravity 1.18) is 74%
water, thus the reagent contains 3.7% water assuming the butanol to be
anhydrous. Thus, no more than 200pl of water must be added to the
reagent through the addition of the sample. If any excess is present then
the reaction will be highly sensitive to this. The additiqn of the sample in
anhydrous methanol is one solution but unfortunately tannins are not
very soluble in this solvent. Any acetone present will reduce the yield of
anthocyanin colour. Thus, carefully formulated alcohol-water mixtures
94 Chapter 4
Scheme 4.4 Proanthocyanidin method
Butanol reagent
Add ferrous sulphate heptahydrate (0.7g) to 50ml conc. HCl in a
1 litre volumetric flask and swirl until the salt dissolves. Add n-
butanol up to 1 litre volume.
Procedure
Add 7ml reagent and 500pl sample to a screw cap test tube, cap
and mix. Heat at 9S°C (or use boiling water bath) for between
40min and 1h. Cool and read absorbance at 550nm. (Caution:
carry out this procedure behind a screen. During heating pressure
will build up in the tubes and if there is any flaw they could burst.)
Blank reaction (to zero spectrophotometer)

Make a 'blank reagent' by using 50ml water in place of the 50ml
HCI. Otherwise proceed as above.
See also 4.5.4 Standards and false-positive results
usually need to be used as solvent. The precise mixture should be reported

and kept an experimental constant, as should the volume added to the
reagent. The extent of the 'water problem' also depends on whether
dry or fresh plant material was extracted. A plant macerated in 100%
methanol may actually yield an extract in 75% methanol. Our recom-
mendation is for the use of dry samples extracted with 50% methanol
because this is such a popular extraction medium. The addition of 500 p1
of 50% MeOH to 7ml reagent gives a total water content of 6.8%.
Unlike the total phenolics assays, where the sample is diluted 100
times in the assay, here the dilution is much less. Highly coloured extracts
can affect the absorbance of the assay solutions and care is needed in
the use of appropriate blanks. If the colour is too intense for accurate
measurement it should be diluted with reagent. Because there are some
substances present in plants that give red colours with the reagent without
heating (Watterson & Butler, 1983) an unheated reagent-sample mixture
is not recommended as a blank. The use of a 'blank reagent' in which
the acid component of the normal reagent is replaced with water is
appropriate. Problems still arise in this assay as chlorophyll goes brown
on heating with acid relative to heating without acid. Thus tannin-free but
chlorophyll-cdntaining extracts will assay slightly positive relative to their
blank reactions. This is unavoidable but not indicative of the presence
of tannins. Such readings are usually very small in comparison to the
1
0 0.5 1 1.5 2 2.5 3 3.5
Concentration of procyanidin polymer (Oh x 1000)
Fig. 4.3 Typical 'discontinuous' standard curve for the proanthocyanidin assay.
absorbances of samples that really do contain tannin and are best reported as
zero in the absence of any clear red coloration. If in doubt check for
condensed tannins by thin layer chromatography (TLC) (see Chapter 7).
There is one final problem with the assay of which all too few investi-
gators are aware. The 'standard curve' relating absorbance to tannin
concentration (Fig. 4.3) is not a straight line but a biphasic curve. In this
assay a standard curve may have to be interpolated by hand. The reason
for this problem is unknown as is the detail of the mechanism for the
production of anthocyanins from condensed tannins. Porter, Hrstich
and Chan (1986) show support for the involvement of three kinds of
reactions, autocatalysis, autooxidation and direct oxidation but beyond
this we are ignorant.
As a development of the proanthocyanidin assay Watterson and
Butler (1983) demonstrated that the flavones apigenin and luteolin (38)
formed unstable anthocyanins with the butanol reagent without heating.
These products are heat labile and decompose so as not to interfere
with the regular proanthocyanidin assay. Watterson and Butler (1983)
recommend that spectrophotometric measurements are made after an
hour.
4.5.2 Vanillin method

This method-depends on the ability of vanillin to form coloured complexes
with condensed tannin polymers. Unlike the proanthocyanidin reaction it
is critically dependent on timing and temperature and is thus technically
S Chapter 4
more difficult. The enormity of temperature induced errors is perhaps

best illustrated by the finding (Price, van Scoyoc & Butler, 1978) that a
change from 24.6"C to 26OC produces an 11% change in absorbance
readings. This is the main reason for our view that this is not an appro-
priate method for an investigator intending to use only one assay for
condensed tannins. The method is nevertheless tried and tested and has
been used in modified forms (Butler, 1982; Mole & Waterman, 1987b)
for the estimation of condensed tannin polymer lengths (see below).
The original vanillin method is due to Burns (1971) and has been
widely used in assessing tannins in cereal grain. Maxson and Rooney
(1972) quickly realized that between and within laboratory variation was
a problem. The following procedure (Scheme 4.5) is based on the work of
Price, van Scoyoc and Butler (1978) who have provided the most detailed
analysis of this method. The main problem of the assay is that all solutions
must be in 100% methanol as the assay is particularly sensitive to water.
Scheme 4.5 The vanillin method for condensed tannins
Vanillin reagent
Make two solutions, one of 8% conc. HCl in methanol, the other
of 1% vanillin in methanol. Make the vanillin solution on the day
of use, by combining equal volumes of these two solutions to give
a reagent of 4% HCl and 0.5% vanillin in methanol.
Procedure
The sample (in 100% methanol) and reagent solutions must
be at exactly 30°C before starting. We recommend the use of a
thermostat controlled water bath. At time zero add 1ml of sample
to 5 ml of reagent and mix. Maintain assay at 30°C until 20min has
elapsed. Measure the absorbance at 500 nm.
Note: It is suggested that test tubes are wrapped in aluminium foil
to exclude light.

Make a 'blank reagent' of 4% HCI in methanol and use this in
place of the vanillin reagent. Some plant constituents will react to
form a red colour with this (Watterson & Butler, 1983) and the use
of such a blank is crucial to the defection of potential false-positive
reactions.
See also 4.5.4 Standards and false-positive results
This puts a severe strain on the use of the assay because, as already
noted, most tannins do not dissolve well in 100% 'methanol. The tannins
in cereals seem rather unusual in being very soluble in this solvent.
The vanillin reaction is also said to be light sensitive and it has been
recommended (Porter, 1989) that reactions should be performed in test
tubes painted black or wrapped in aluminium foil.
4.5.3 Comparison of vanillin andproanthocyanidin methods

The proanthocyanidin method depends on acid degradation of the con-
densed tannin polymer to yield coloured anthocyanins. Most tannins yield
either cyanidin (44) or delphinidin (45) or a mixture of these. Perhaps the
greatest problem with the method is that these anthocyanins exhibit
different spectra and absorption maxima, with, at 550nm, delphinidin
having the higher molar absorption coefficient. This leads to a relative
over estimation of condensed tannin if it is rich in prodelphinidins relative
to procyanidins. Thin layer chromatography (Chapter 7) or scanning
spectrophotometry (Chapter 8) can be used to resolve the question of
which particular type of tannin one is dealing with while measurements of
absorbance at three wavelengths can be used to quantify their relative
abundance (Bate-Smith, 1977).
The vanillin reaction does not involve a depolymerization, instead
vanillin reacts with the flavonoid A-ring at C-6 forming a complex that is
not dependent on the hydroxylation pattern on the B-ring (Fig. 4.4) and
Coloured product
Fig. 4.4 Reaction between vanillin and the G6 position of the &+vanunit of a condensed
tannin.
98 Chapter 4
is thus not differential in its response to cyanidin or delphinidin yielding

residues. The reaction is limited to flavonoids with meta oriented unsub-
stituted hydroxyls, a saturated C-2-C-3 bond and lacking a C-4 carbonyl
(Sarkar & Howarth, 1976). This makes the assay largely condensed tannin
specific. However dihydrochalwnes and flavan derivatives will also react,
as will phloroglucinol based phlorotannins.
In comparing the two condensed tannin assays, we would expect
variation in results for tannins differing in their cyanidin to delphinidin
yield. It must also be remembered that interllavan bonds in condensed
tannins can be C-4-C-6 as well as C-4-C-8 (see Chapter 1). For every C-
6-C-4 linkage there is one less site for the vanillin reaction and thus
further cause for disparity between the two assays. It is not so much that
one assay is right or wrong; both detect different attributes of tannins.
For comparative purposes we believe investigators are best served by the
adoption of the proanthocyanidin assay as a standard so that methodology is
removed as a factor when different laboratories compare results. This is
not to say that additional biologically interesting information cannot be
obtained from the vanillin assay.
4.5.4 Standara3 and false-positive results

Unlike the total phenolics assays, where tannic acid has become a com-
monly accepted standard, there is no really suitable equivalent for con-
densed tannins. The closest is quebracho tannin, which several laboratories
have used (see Table 4.5). The problem is that this is a crude hot water
extract of the wood from Schinopsis balansae. While used as a commercial
leather tannin, quebracho is not a single defined chemical substance, nor
does it pretend to be. It is likely to be as variable as the biology of the
trees from which it is obtained. Additional variation is likely because the
several companies that market quebracho are all likely to have different
sources of supply.
The marketing of a wnstant source of pure condensed tannin would
solve the problem. Hagerman and Butler (1989) proposed to act as
suppliers of crude quebracho together with instructions for its purification.
However, without any central quality control of the fini~hedproduct it is
doubtful that this would solve the problem. In lieu of this we recommend
the continued use of crude quebracho as the best available solution to the
problem. In the unlikely event that all investigators begin using the same
methodology, simply reporting absorbance values at 550nm might be
better. However, this seems unlikely given the usual state of flux in
spectrophotometric techniques. Catechin has been used as a standard for
the vanillin assay, but as this cannot be used with the proanthocyanidin
assay, it precludes all possibility of comparing results between the two
assays.
With regard to false-positive results, there are two dangers with assays
for condensed tannins. The most frequently encountered is the failure to
construct a proper blank. In both assays sufficient concentrations of
chlorophyll and other plant pigments are present to give some detectable
absorbance or even slight turbidity. Turbidity, if slight, can usually be
corrected by filtration or centrifugation. If pure reagent, without any
sample, is used to zero the spectrophotometer it should come as no
surprise if a positive absorbance is recorded. Ideally a separate blank
should be constructed for every sample. When all samples contain about
the same amount of pigment this often seems tedious. But if you strive
for accuracy, there is no simple short cut. The following puts the problem
in perspective. Suppose the typical blank reaction has an absorbance
(relative to the pure reagent) that is only 1-2% of the assay reactions,
then you may choose to ignore this error and zero the spectrophotometer
on pure reagent. For larger blank values which are relatively constant for
all samples, you may wish to zero the spectrophotometer on one and
again ignore any errors this causes. Such short cuts will always introduce
some error and variability which you usually trade off against your time
and effort.
The second source of false-positives is with some flavans, such as 3-
deoxyanthocyanins, which produce a colour with acid in the absence of
vanillin. Several reports are in the literature where investigators ap-
pear to have omitted the controls which would have compensated for
these.
4.5.5 Condensed tannin polymer length by colorimetry

Condensed tannins are unbranched linear polymers and so if some
method of estimating the ratio of internal to chain terminating units could
be devised, then it would be possible to assess the average polymer length
of a mixture of condensed tannins. In the proanthocyanidin assay, the
longer a condensed tannin is, the more anthocyanin pigment it will
release, while in the vanillin assay the more steric hindrance there will be
as vanillin molecules attempt to react at the free A-ring positions. By
this logic, Goldstein and Swain (1963) pointed out that a ratio of the
absorbance of a sample in the proanthocyanidin assay to that in the
vanillin assay should rise as polymer length rises. Butler, Price and
Brotherton (1982) examined the vanillin reaction in a variety of solvents
and showed particular chain terminating unit sensitivity when the solvent
was glacial acetic acid rather than methanol. The procedure for conducting
the reaction in glacial acetic acid is described below (Scheme 4.6).
The major drawback of this procedure is that tannins are even less
soluble in glacial acetic acid than they are in methanol and so this method
may not be possible for some tannins. Few ecologists have used these
100 Chapter 4
techniques, but comparative data on their performance is available (Mole

& Waterman, 1987b).
For assessment of polymer length there are probably better procedures,
notably gel permeation (Jones, Broadhurst & Lyttleton, 1976; Williams,
Porter & Hemingway, 1983) and nuclear magnetic resonance (see Chapter
8). The virtues of the colorimetric techniques are again that they are
simple, cheap and, perhaps, useful in the exploratory phases of investiga-
tions or where the other techniques are impracticable.
Scheme 4.6 Vanillin method using glacial acetic acid
Vanillin reagent
Make two solutions in glacial acetic acid, one of 8% conc. HCl and
the other of 1% vanillin. Make the vanillin solution on the day of
use by combining equal volumes of the two solutions to give a
reagent of 4% HCl and 0.5% vanillin in glacial acetic acid.
Procedure
The sample (in glacial acetic acid with up to 6% methanol to aid
solubility) and reagent solutions must be at exactly 30°C before
starting: we recommend the use of a thermostat controlled water
bath. At time zero add 1ml of sample to 5ml of reagent and mix.
Maintain assay at 30°C for 5min (to within a few seconds) and
then measure the absorbance at 510nm.
Note: It is suggested that test tubes are wrapped in aluminium foil
to exclude light.

Make a 'blank reagent' of 4% HCl in glacial acetic acid (i.e.
omit the vanillin) and use this in place of the vanillin-containing
reagent. Some plant constituents will react to form a red colour
with this (Watterson & Butler, 1983) and the use of such a blank is
crucial to the detection of potential false-positive reactions.
4.6 Hydrolysable tannins

We have remarked previously that quantitative measurements of hydro-
lysable tannins are conspicuous by their absence in investigations of
ecological samples (Mole & Waterman, 1987b). The reason for this is the
obvious one: the lack of suitable assay techniques. Within the last two
years this position has changed due to work in Hagerman's laboratory.
While we are hopeful that practical methods for hydrolysable tannins can
now become part of the chemical ecologist's standard repertoire, previous
experience with methods for total phenolics and condensed tannins warn
us that even good techniques seldom survive without modification. Below
we outline methods for both gallotannins and ellagitannins.
4.6.1 Gallotannins
Inoue and Hagerman (1988) essentially rediscovered the work of Thies
and Fischer (1972) and developed the latters' assay for gallic acid, with
the addition of a hydrolysis step to yield gallic acid from gallotannins
prior to assay. The basis of the reaction is the intense colour produced by
reaction between gallic acid and the dye rhodanine (Scheme 4.7). The
sensitivity of the assay is O.Olmg gallic acid which is sufficient to avoid
most problems with interfering pigments and other substances potentially
present in extracts. While the assay requires accurate timing it has the
advantage of working well at room temperature.
Scheme 4.7 Rhodanine method for gallotannins
Reagent
0.667% methanolic rhodanine solution.
Sample preparation
Mix 5ml 1~ HzS04 and 1ml sample (in 70% acetone) in an
ampoule or screw cap test tube. Either vacuum seal after freezing
contents or seal after venting under nitrogen to remove oxygen.
Heat seal ampoule or tube for 26h at 100°C. Cool, make up to
50ml with deionized water. (Caution: shield test tubes during
heating.)
Procedure
At time zero mix 1ml prepared sample and 1.5 ml reagent. Exactly
5 min later add 1ml of 0.5 M KOH. After 2.5 min dilute to 25 ml
with water and mix before reading the absorbance at 520 nm after
a further 5- 10min.
There is one other technique available, the iodate technique of Bate-

Smith (1977), which has also been used by Marks et al. (1988). This assay
is sensitive to both timing and temperature and there are problems with
side reactions and turbidity. The assay has never been investigated
102 Chapter 4
in as much detail as that utilizing rhodanine and for this and the above
practical reasons we feel that it is not an appropriate method, especially
for crude plant extracts. For workers dealing with relatively pure tannins
these problems may be insignificant and the method workable.
4.6.2 EUagitmnins
Once again there is an older method (Bate-Smith, l973a) and a newer
one (Wilson & Hagerman, 1990). Both assays depend on the reaction of
ellagitannins or their hydrolysis product, hexahydroxydiphenic acid
(HHDP), with sodium nitrite to yield a nitrosylated chromophore. There
the similarities end as the reaction takes place in different solvents and
yields different coloured products.
In our hands (Mole & Waterman, 1987b) the Bate-Smith method
(Scheme 4.8) has proved unreliable while others have noted interfering
side reactions, particularly with gallic acid (Scalbert, Monties & Favre,
1988). This method requires the use of nitrogen to exclude oxygen but is
otherwise relatively less cumbersome than the newer method of Wilson
and Hagerman (1990). The latter (Scheme 4.9) requires a careful and
time-consuming hydrolysis of the tannin to prepare ellagic acid for analysis.
The actual analysis is carried out in pyridine which necessitates the use of
a fumehood. The major claim of the new assay is that it is less susceptible
to interference, particularly by other phenolics. Its sensitivity is O.lpg,
which is impressive. It is too early to pronounce a method of choice, but
for those with good laboratory facilities, and able to make the effort, we
would recommend the method of Wilson and Hagerman.
Scheme 4.8 Bate-Smith method for ellagitannins
Sample preparation
Crude extract in 50% methanol.
Reagent
6% sodium nitrite in water.
Procedure
Mix 2 ml sample and 160pl of 6% aqueous acetic acid in a cuvette
and then remove oxygen by gently bubbling a stream of nitrogen
through the mixture for 15min. Add 160pl of reagent and flush
with nitrogen for a further 15min. Seal the cuvette with a teflon
stopper and record the absorbance at 600nm after 1h.
Scheme 4.9 Wilson and Hagerman method for ellagitannins
Sample preparation
Mix 5ml 1~ H2S04 and 1ml sample (in 70% acetone) in an
ampoule or screw cap test tube. Either vacuum seal after freezing
contents or seal under nitrogen. Heat the sealed ampoule or tube
for 10h at 100°C. After hydrolysis cool to O°C in an ice bath and
vacuum filter through a membrane filter. Wash residue with ice
cold acidic aqueous acetone (70 :30: 1 acetone :water :HC1). Dry
both filter and residue under nitrogen and then dissolve both in
1Oml pyridine. (Caution: shield tubes during heating.)
Reagent
0.1% sodium nitrite in water.
Procedure
Mix 2.10ml prepared sample (in pyridine) and 0.1 ml conc. HCl
reagent and bring reaction to 30°C. At time zero add 0.1 ml reagent
and measure absorbance at 538nm. Incubate for 36min and
then measure absorbance at the same wavelength. The difference
between the two absorbance measurements is proportional to the
ellagic acid concentration. Glassware must be glass (not plastic)
and be scrupulously clean.
CHAPTER 5
Biochemical techniques for tannins
5.1 Introduction
Second only to measurements of total phenolics, tannins are the most
frequently analysed group of phenolics in ecological studies. Earlier we
gave an overview of the types of studies in which tannins have been
measured (Chapter 3), the chemical nature of tannins (Chapter I), and
their taxonomic distribution (Chapter 2). Chapter 4 focused on assays for
tannins exploiting their chemistry by the use of colorimetric reagents.
So, why another chapter on methods for tannins? One answer is that
many researchers will, with some justification, dispute the idea that tan-
nins can usefully be defined as a specific group of chemicals. Instead,
there has been a long tradition of defining tannins as phenolics which
have the ability to precipitate proteins from aqueous solutions. Using this
operational definition, the only sure way to detect and quantify a tannin is
to measure its potential for protein precipitation.
Furthermore, many workers have studied tannin-protein interactions
with a view to modelling the in vivo effects of tannins in vitro. Rather
than the simple quantification of tannins there are methodologies which
can be used to learn something about the potential physiological and
ecological effects of tannins on herbivores. Methods for this separate line
of enquiry are also presented here.
5.1.2 Defiloiiionsfor tannins

The word 'tannin' has long been used as the name for organic substances
present in the water-soluble extracts of certain plants, which are able to
convert animal hide to leather. The longest association of the word is thus
with tannery and tanning, although today most leather is made without
using traditional vegetable tanning materials. The application to specific
groups of structurally characterized chemicals in defining tannins is rela-
tively recent.
Techniques to quantify tannins that pre-date the methods presented
in Chapter 4 rely on methodologies used by the leather trades. In this
industry it was recognized that tannins were able to precipitate proteins
from aqueous solutions as well as transform hide into leather. Because
precipitation assays take minutes relative to the months taken to trans-
form hide into leather, precipitation assays were adopted for the quality
control of tanning agents. The protein of choice for precipitation assays
Biochemical techniques 105
Table 5.1 Definitions of tannins
Operational
Water-soluble phenolic natural products that can precipitate proteins from aqueous solution
(Mole & Waterman, 1987b,c)
Chemr'cal, by biasynthetic group
e.g. condensed tannins, hydrolysable tannins, phlorotannins (See strnctnres 58,59 and
64-67 and Mole & Waterman, 1987b; Haslam, 1989)
Chemical, by chemical character&tics
'True tannins' have: (i) molecular weights in the 1000-3000 range; (ii) sufficientphenolic
hydroxyl groups to complex with proteins and other macromolecules containing carbonyl
and amino groups; and (i) hydrogen bonds formed with macromolecules that are
susceptible to auto-oxidation to form covalent linkages (Swain, 1979)
became gelatin, which is a preparation of collagen, the most important

protein in skin (Gustavson, 1956).
Tannins have also been recognized as being responsible for hazes in
beer and as astringents in foods, beverages and certrain pharmaceutical
applications. In each case the mechanism of action of the tannin has been
through complexation with proteins. We have thus recognized the opera-
tional definition of tannins as water-soluble phenolic natural products that
are protein precipitants (Mole & waterman; 1987a,b). This contrasts with
chemically based definitions of tannins (see Table 5.1).
It would be simple if all water-soluble phenolic natural products that
precipitated proteins were members of one of the chemically defined
groups of tannins and vice versa. Unfortunately this is not the case. For
instance, not all hydrolysable tannins precipitate proteins equally well and
there has been a long running and parallel debate concerning condensed
tannins (Haslam, 1981). For condensed tannins it seems that both the
longest and the shortest polymers may be less effective than oligomers:
the very longest polymers may not even be water-soluble. For these and
other reasons Swain (1979) used other chemical attributes of tannins in
order to characterize them as a coherent group (see Table 5.1). Deriva-
tive names such as prototannins, p-tannins and oxytannins have been
reserved for molecules that have some but not all of the defining charac-
teristics. Swain's scheme has never been adopted, but his work still
illustrates the complexity of trying to give a chemically based definition of
a tannin.
For our purposes, it is important to understand the basis for the
different definitions of the word tannin in order to properly appreciate
the difficulties of translating results measured in one paradigm into an-
other. For instance, equal quantities of condensed tannins, as determined
by the proanthocyanidin assay, cannot be assumed equal in their protein
106 Chapter 5
precipitating abilities if they come from different sources and are thus
chemically different. This is because they may differ in polymer length or
other structural attributes such that they interact differently with proteins.
For a detailed treatment of this point see Hemingway and Karchesy
(1989) and the various reviews cited therein. In samples of condensed
tannin (e.g. quebracho) which are in fact crude extracts from plants which
contain many different compounds, there may be other types of tannin
present which affect protein precipitation but not the condensed tannin
assay.
5.2 Tannin-protein interactions: an overview
5.2.1 Bonding in tannin-protein complexes

The polyphenolic nature of tannins immediately suggests that they are
likely to form hydrogen bonds in solution (McManus et al., 1981). The
principal evidence that this does occur comes from the disruption of
iannin-protein precipitation by hydrogen bonding solvents such as
, -
formamide and its N-substituted derivatives (Hagerman & Butler, 1978,
1980a). Other supporting evidence comes from the chemical nature of
material with a high specificity for binding tannins such as polyvinyl
pyrrolidone ( P W , 154). The fact that tannins and proteins do not typi-
cally precipitate when the pH of the solution exceeds the pK, of the
phenolic hydroxyl indicates that the phenol groups act as hydrogen
bond donors with the peptide carbonyl oxygens acting as the acceptors
(Hagerman, 1989).
Hydrogen bonding is thought to be augmented by hydrophobic

phenomena in which the aromatic nuclei of the tannin interacts with
hydrophobic regions in proteins (Haslam, 1989). Again evidence for this
comes principally from studies of solvent disruption effects (Oh et al.,
1980; Hagerman & Butler, 1981) and such bonding is particularly evident
at acidic pH (McManus et al., 1985).
The absence of tannin-protein interactions when pH exceeds the pK,
of the phenolic hydroxyls suggests the absence of any ionic bonding
because the phenolic hydroxyls are the only ionizable groups present
in tannins. covalent bonds are not thought to initially be involved in
complex formation because of abundant evidence for the reversibility of
tannin-protein interactions (see below). However, when tannin-protein
+
TANNIN PROTEIN TANNIN + PROTEIN
(Low TIP ratio) (High TIP ratio)
SOLUBLE TIP , INSOLUBLE TIP

COMPLEX Lecithin/cholate COMPLEX
I Disruption of protein
tertiaty structure
DIGESTIBILITY OF
PROTEIN ENHANCED
Fig. 5.1 Processes involved in the tannin-protein (TR)

1
LOSS OF NITROGEN TO
CONSUMERIHERBIVORE
interaction
complexes are allowed to remain associated in the presence of oxygen

it often becomes impossible to completely dissociate them and thus oxida-
tive covalent links are then likely. It is thought that amino acid side-
chains of a nucleophilic nature, such as lysine or cysteine, are predomi-
nantly involved in covalent bonding (Pierpoint, 1969).
The biochemistry underlying the interaction of tannins with proteins
remains somewhat obscure. The complexes formed may be either soluble
or insoluble. Many low molecular weight phenolics will bind to proteins
as ligands but not all will cause their precipitation. At least two stages of
interaction can be envisaged: (i) an initial complexation (bonding) of
tannins to proteins to form soluble complexes (aggregates of tannin and
protein); and (ii) further association of these by cross-linkage to form
larger and ultimately insoluble complexes seen as precipitates (Mole &
Waterman, 1987a; Haslam, 1989). These processes are surhmarized in
Fig. 5.1. (Appel (1993) has drawn attention to the importance of oxida-
tion in the allelochemical activity of tannins. This paper is strongly
recommended.)
If this scenario is correct there are several key points to be recognized.
Perhaps the most important is that there is no fixed stoichiometry to the
reaction: the relative composition of these tannin-protein complexes is
variable and dependent on the particular conditions in which they are
formed. The transition from an optically clear solution to a hazy one and
finally to one where precipitates form is also acutely dependent on the
particular conditions present.
108 Chapter 5
5.2.2 Solution conditions influeming cornplex formation

In biological systems the universal solvent is water but in many assays
organic solvents are used to dissolve tannins and are thus incorporated
into the assay mixture. As solvents can easily disrupt the interaction
between tannin and protein (see above) their quantities should be both
minimized and kept as an experimental constant. The most important
factor to control when measuring protein precipitation is usually pH. In
general, protein precipitation is enhanced by more acidic conditions as
hydrophobic bonding is augmented. However, this is not the most im-
portant aspect of pH. The really critical effect of pH is in determining the
ionization state of amino acids in the proteins. For example, if there is a
single protein structure present (a common assay situation but not often
found in nature) and it carries a net charge in solution, then there must
be a repulsion between the protein molecules and thus tannin-protein
complexes will not aggregate and precipitate. To maximize the pre-
cipitation reaction it should take place at the isoelectric pH (PI) of the
protein, where it carries no net charge (Hagerman & Butler, 1978;
Fig. 5.2).
The need to control pH requires the use of buffers and this brings a
further set of problems. Many modern biochemical buffers are made with
biochemicals which are not found naturally in biological systems and
Fig. 5.2 Precipitation of three different proteins in relation to pH and PI. Pepsin (+,pI
1.0). trvpsin (H, PI 10.1), BSA (A, pI 4.9). AS,, measures concentration in solution. (Data
taken from G e r m a n and Butler, 1978.)
tannins may interact with these in undesirable ways. For instance, con-
densed tannins will precipitate with phthalate and borate based buffer
salts while even some pure amino acids will form precipitates with tannins
(Mole & Waterman, 1987c) and so not even these should be used. We
advise the use of phosphate buffers at moderate ( 1 0 0 ~ concentrations
)
as systems within which to form precipitates. As well as pH, the ionic
strength of solutions (which includes buffer concentration) needs to be
kept constant as precipitation may be sensitive to this factor at low ionic
strengths (Mole, 1986).
In many practical assays of tannin-protein precipitation, it is neces-
sary to redissolve the precipitates, at least in part reversing the tannin-
protein interaction. The objective here is usually to obtain either the
tannin or protein in solution prior to a chemical assay. Agents that
accomplish this reversal also prevent the formation of precipitates in the
first place and so these should be avoided when setting up a system to
precipitate proteins with tannins. There are three very effective treat-
ments to disrupt tannin-protein complexes.
1 Addition of organic solvents.
2 Increasing pH.
3 Addition of detergents.
The first two of these treatments will act on tannin-protein bonding and
increase repulsion between proteins in the complex, so promoting disrup-
tion. In general, a pH two units in excess of the pI of the protein will
substantially, if not entirely, dissolve a precipitate. Detergents (surfactants)
will bind to complexes and aid their dissolution as well as denaturing
proteins and altering their affinity for tannins (Blytt, Guscar & Butler,
1988). Hagerman and Butler (1978) employed all three of these appro-
aches in their assay techniques, which we discuss in detail below. Other
agents used to disrupt tannin-protein complexes include phenol and
caffeine (Hagerman, 1989), but these are not in current use.
All we have said so far applies to 'tannins' and 'proteins' in general.
Problematically, there are differences to be seen between different tan-
nins when their interactions with a particular protein are compared.
Similarly, proteins also differ in their behaviour to a 'standard' tannin.
With respect to proteins, pI (see above) is perhaps their most important
characteristic but there are other structural features that are important
(see below).
5.2.3 The nature of the tannin

Some caution has to be exercised when interpreting the literature relating
to variation in the properties of tannins as the substrates employed vary
in purity. Data from experiments utilizing highly purified tannins (of a
single molecular structure) provide valuable information about structure-
110 Chapter 5
0.6 -I 4
0 2 4 6 8 10 12 14
Degree of polymerization
Fig. 5.3 Relative astringency of condensed tannin polymers of different degrees of

polymerization and composition. +, pure procyanidin; H, pure prodelphinidin; A , mixed
procyanidinlprodelphinidin dimers. (From Porter & Woodmffe, 1984.)
activity relationships. Data using crude tannin-containing extracts (such

as herbivores obtain by chewing leaves) indicate the likely magnitude of
biologically important differences. Thus both kinds of data are useful but
they must be interpreted differently. Mole and Waterman (1987c,d) have
documented differences in the protein precipitating abilities of different
crude tannins as have others (Martin & Martin, 1982; Wisdom, Gonzalez-
Coloma & Rundel, 1989).
Evidence that these differences may be directly attributable to chemi-
cal variation in the structure of the tannin rather than other co-occurring
substances in the crude extracts comes from work with pure tannins.
Beyond the mere possession of phenolic hydroxyl groups, the disposition
in space and the size and flexibility of the rest of the molecule to which
phenolic hydroxyls are attached may be important in determining how
specific tannin molecules interact with proteins. With respect to con-
densed tannins, molecular weight has been considered to be the most
important factor in determining protein precipitation capacity (Fig. 5.3;
Porter & Woodruffe, 1984; Ezaki-Furuichi et al., 1987; Haslam, 1989).
The monomeric condensed tannin precursors catechin (42) and epicatechin
(63) are not protein precipitants, but dimers of these are capable of weak
precipitation reactions. Trimers are more active but little additional
Biochemical techniques 11 1
precipitation capacity is achieved beyond four or five unit oligomers

(Roux, 1972; Bate-Smith, 1973a; Butler, 1982; Asano et al., 1984; Artz et
al., 1987). Similar results have also been reported for the interaction of
tannins with PVP (Tilstra et al., 1989). There is evidence for differences
caused by variation in the disposition of phenolic hydroxyls on the carbon
skeleton of condensed tannins such that polymers based on prodelphinidin
(155), procyanidin (156) and profisetinidin (157) are distinguishable in
their activity (Bate-Smith, 1975; Asano et al., 1984; Asquith & Butler,
1986).
For hydrolysable tannins, Beart, Lilley and Haslam (1985) showed

that increasing numbers of galloyl groups added to protein precipitating
ability but that further modification of structure, as found in many nat-
urally occurring hydrolysable tannins, decreased precipitation capacity.
Conformational flexibility as well as the number of galloyl groups is
important in the protein precipitating capacities of hydrolysable tannins
which may vary by at least an order of magnitude between different
structures (Ozawa, Lilley & Haslam, 1987). Few studies have compared
pure condensed and hydrolysable tannins. Haslam (1974) found dif-
ferences between them, but in general these are unlikely to exceed the
differences within each group (Hagerman & Klucher, 1986; Hagerman,
1989).
5.2.4 The nature of the protein

Since the observation (Hagerman & Butler, 1981) that proteins differed
markedly in their affinity for binding to tannin, irrespective of their PI,
there has been considerable interest in elucidating the characteristics of
proteins that determine this. The amino acid composition of a protein has
been the focus of most attention. Many results indicate that proteins
rich in proline (158) have high affinity for tannins (Hagerman & Butler,
1981). Such proteins include gelatin (collagen), prolamine proteins from
seed coats and salivary proteins of some animals (Austin et al., 1989;
112 Chapter 5
Hagerman, 1989). Synthetic proline-rich analogues such as PVP and

polyproline also have high affinity for tannins. Curiously, polyhydroxy-
proline and poly(pro-gly-pro) are reported not to have high affinities
for tannin-binding and Hagerman (1989) believes interchain hydrogen
bonding reducing availability of carbonyls to phenolic hydroxyls may
explain this. Other exceptions to the rule include some proline-rich
salivary proteins with low binding affinities (Mole, Butler & Iason, 1990).
Proteins with low affinities for tannins tend to be of low molecular weight
andlor globular and compact structurally. Glycoproteins may have low
affinities for tannins although there are again exceptions (Strumeyer &
Malin, 1975; Asquith et al., 1987). Our understanding of how tannins
bind to specific proteins is still unsatisfactory. The important point is that
we can recognize large differences between (see F$. 5.4) and
there are indications that there is biological significance in this (Mehansho,
Butler & Carlson, 1987; Mole, Butler & Iason, 1990).
Tannins will interact with many other biological macro-and micro-
molecules. A technical problem in working with tannins is that they also
Competitor protein added (nM)
Fig. 5.4 Variation in the biding capacity of proteins with a tixed amount of tannin (BSA
used as standard protein). (Modified from Hagerman & Butler, 1981.)
bind strongly, if not irreversibly, to most solid chromatographic supports,

including cellulose. Indeed, Haslam (1974) has long argued that con-
densed tannins have a structural role in plant cell walls due to their
capacity to bind to cellulose and proposed that the loss of astringency
of ripening fruit is due to inactivation of tannins by their binding to
carbohydrates (but see Matsuo & Itoo, 1981). Other molecules to which
tannins can bind include nucleic acids and alkaloids (Hagerman, 1989).
5.2.5 Soluble tannin-protein complexes

Virtually all of our knowledge of tannin-protein interactions has come
through studies of reactions yielding a precipitate. Precipitates are visible
and easily separated and quantified. Soluble complexes have mostly been
ignored by both chemists and biologists, so we know little about them
chemically or whether they are biologically significant. We can infer
the existence of soluble tannin-protein interactions from Scatchard
plots (van Buren & Robinson, 1%9), from spectroscopic measurements
(Zanobini, Vanni & Firenzuoli, 1967; Tilstra et al., 1989) and from the
effects of tannins on biochemical reactions where no precipitation occurs
(Mole & Waterman, 1985). Much more work is needed to understand
these soluble systems. In simply using protein precipitation to quantify
tannins we need to optimize our techniques to minimize the amounts of
tannin and protein that combine in the soluble form because these will
not be detected as precipitates! On the other hand it must never be
forgotten that what is going on in solution may have a greater ecological
impact than what we see in terms of precipitation.
5.3 Quantification of tannins

Unlike the situation with the chemical assay techniques described in
Chapter 4, there are a multitude of techniques for quantifying tannins
through their interaction with proteins. Two classes of technique can be
recognized: (i) those that determine some aspect of a tannin-protein
precipitation reaction; and (ii) those that determine the inhibition of
an enzyme catalysed reaction by a tannin. The first of these is far more
widely used; the second approach is usually seen only in relation to
studies attempting to model the digestive process (Mole & Waterman,
1985; and see below) or to accurately characterize specific tannins (Ozawa,
Lilley & Haslam, 1987). We will first concentrate on the tannin-protein
precipitation approach.
From the myriad of permutations and combinations of techniques to
choose from we return to the basic definition of a tannin as a protein
precipitant and so advocate methods that actually determine the quantity
of a protein precipitated by a tannin, and not the tannin precipitated by a
protein. Below we detail the practical considerations underlying these
114 Chapter 5
methods and then describe what we feel to be the best of the currently
practised methods.
5.3.1 Optimizingprecipitation reactions

In order to trust a measurement of tannin concentration obtained by
protein precipitation some appreciation for whether the reaction is work-
ing properly is essential. Any reader of this book will be capable of
mixing an aqueous solution of tannin with one of protein to instantly
generate a precipitate. Unfortunately that is not the end of the matter. In
order to replicate the precipitation of a particular quantity of protein by
a particular tannin, the reader will already be aware (see the Introduc-
tion to this chapter) that the following conditions must be kept as experi-
mental constants.
I The specific protein and tannin used.
2 The pH of the solution.
3 The buffer concentration (and composition).
In addition, the solution should be as free as possible from contaminants
and so pure protein and tannin should be used where feasible. The
complete absence of surfactants and other interfering small molecules
should also be maintained. If the sample was not dissolved in water, any
organic solvent used to dissolve the tannin should be minimized and kept
as a constant over all experiments. It is essential that not even traces
of acetone contaminate precipitation reactions (Hagerman & Robbins,
1987). What must next be confronted is the additional need to minimize
and control the formation of soluble tannin-protein complexes.
If a tixed quantity of tannin is titrated with steadily increasing amounts
of protein, an increase in the quantity of protein precipitated is seen:
as more protein is added more can combine with tannin to form a
precipitate. Eventually a point is reached where no additional precipita-
tion is observed: the capacity of the tixed quantity of tannin to precipitate
protein has been reached. Sometimes, as more protein is added, the
quantity of precipitate formed stays constant so that in graphical terms a
plateau is obtained. However, it has been found that in some cases as
more protein is added in the titration, then the quantity of precipitate can
diminish (Mole & Waterman, 1987~).In essence, the point at which
maximum precipitation is obtained represents an optimal stoichiometry
for the insoluble complex. When there is an excess of either tannin or
protein then the presence of soluble complexes becomes more favoured.
knother manifestation of this effect is the occurrence of thresholds for
precipitation. Here, tannin can be added to protein (or vice versa) with-
out any precipitation until a certain ratio of tannin to protein is
obtained at which precipitates begin to form. The particular ratio of
tannin to protein found at precipitation maxima and at precipitation
(a)
Phenolics
in solution
BSA in 0.8
precipitate 0.7
\ E!
c
0.6
$0 0.5
2 0.4
4
0.3
0.2
0.1
0 . . . . . . . , . . .
0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5
BSA mglml
Id)
BSA mglml BSA rnglml
Fig. 5.5 Precipitation a w e s for complexation of tannins with BSA. (a) quebracho tannin;
(b) tannic acid; (c) Pinw radiata; and (d) oak tannin. (From Mole & Waterman, 1987c.)
thresholds varies with the absolute concentrations of the components. To

summarize, the quantity of precipitate obtained from a particular pair of
tannin and protein reactants is a function of the absolute and relative
quantities of these two components present in the system (all other
factors being constant!).
Figure 5.5 shows the results (Mole & Waterman, 1987c) when four
crude preparations of tannins from different species were titrated against
a single protein in otherwise constant conditions. We agree with Hager-
116 Chapter 5
man and Robbins (1987) that the most appropriate way to determine the
maximum capacity of a tannin to precipitate proteins would be to conduct
such titrations and record precipitation maxima or points at which the
plateau is reached. However, such determinations are time-consuming.
Mole and Waterman (1987~)give a methodology for these measurements
but at present this represents an ideal not typically achieved in ecological
studies. Measurements are usually made in conditions of excess protein,
but with the assumption that the excess is not so great as to favour soluble
complexes. The reader is advised to check such assumptions for him or
herself, as we describe below.
5.3.2 Protein precipitation methods

Table 5.2 lists the range of protein precipitation techniques used recently,
ordered in terms of the protein used to precipitate with tannin. There are
other procedures in the literature, but these tend to be found in papers
dealing with method development. We have restricted Table 5.2 to studies
actually using methods to answer ecological questions. Clearly, the use of
haemoglobin as the protein to be precipitated has been very popular.
Several variants of the haemoglobin procedure exist, and perhaps even
more variety exists among the second most popular group of techniques,
those using bovine serum albumin (BSA).
Tabk 5.2 Protein precipitation techniques in current use
Protein User(s) Source of method
Haemoglobin Baldwin, Schultz & Ward (1987) Schultz, Baldwin & Nothnagle (1981)
Haemoglobin Bryant (1987) loc. cit
Haemoglobin Horner (1988, 1990) loc. cit.
Haemoglobin Jonasson et al. (1986) loc. cit.
Haemoglobin Schultz, Baldwin & Nothnagle (1981)
Haemoglobin Bate-Smith (1977) Bate-Smith (1973b)
Haemoglobin Glyphis & Puttick (1988) loc. cit.
Haemoglobin Muller, Kalisz & Luken (1989) 1oc. cit.
Haemoglobii Villena & Plister (1990) loc. cit.
Haemoglobin Maufette & Oechel (1989) Mills (1986)
Plant protein Armory & Schnbert (1987)
&Gluwsidase Becker &Martin (1987,) Goldstein & Swain (1965)
&GLucosidase Martin & Martin (1982) loc. cit.
Blue-BSA Hoppe et al. (1990) Asquith & Butler (1985)
Blue-BSA Robbins et al. (1987b) loc. cit.
BSA Martin &Martin (1982) Hagerman & Butler (1978)
BSA Robbins et al. (1987a) Martin & Martin (1982)
BSA Amory & Schubert (1987)
BSA Wilson (1984)
BSA Mole & Waterman (1987~) Hagerman & Butler (1980b)
Gelatin Tem~eI(1981) AOAC (1%)
We first describe what we consider to be the best approach to utilizing

haemoglobin and then describe our current method of choice, which
utilizes BSA. The haemoglobin methods are too widely used to be ignored
but we believe they have significant drawbacks.
5.3.2.1 Haemoglobin precipitation methods

Bate-Smith (1973b) was the first to introduce haemanalysis of tannins and
the concept of relative astringency. Haemanalysis was his term for the
estimation of tannins by measuring their ability to precipitate haemo-
globin. Prior to 1973 various proteins had been explored such as saliva
and milk proteins as well as the technical products casein and collagen.
Methods using tannin to precipitate these proteins had proved either
impractical or extremely laborious. The principal attraction of haemo-
globin to Bate-Smith was its chromophore, which allowed for its direct
spectrophotometric assay in the red region of the visible spectrum, where
few other substances present in plant extracts are likely to interfere. The
basic principle of the assay is very simple: tannin-containing extracts
are added to a solution of haemoglobin, precipitates are subsequently
removed by centrifugation, and finally the loss of absorbance is measured
relative to a suitable unprecipitated control.
Bate-Smith (1973b) and subsequent users of this technique (Schultz,
Baldwin & Nothnagle, 1981) have stated that the critical requirement is
for the haemoglobin to be in its native state (not in any way denatured).
This means it must be generated directly through the haemolysis of red
blood cells, so requiring a reliable source of fresh blood. Bate-Smith used
his own blood. Quite apart from the discomfort and questionable hygiene,
working with a bleeding finger can be quite messy! Schultz, Baldwin
and Nothnagle (1981) opted for the use of blood collected from a slaugh-
terhouse. This was centrifuged to remove cell debris after the haemolysis
step but before tannins were added. This seems to be a significant impro-
vement. Preserved human blood was not found suitable because of added
anti-coagulants and commercial freeze dried blood products are unsuit-
able due to denaturation.
Given a suitable source of blood the technique is relatively strai-
ghtforward. The procedure described in Scheme 5.1 closely follows that
of Schultz, Baldwin and Nothnagle (1981). The one significant variation
we have made is to ensure that the volume of solvent due to the plant
extract remains constant. Schultz, Baldwin and Nothnagle (1981) do not
do this, but in view of the possibility of organic solvent effects on tannin-
protein interactions we think it may be important. In particular acetone-
containing solvents or those containing more than 50% alcohol should be
avoided.
The critical role of blank reactions should be clear from Fig. 5.6,
118 Chapter 5
Scheme 5.1 Haemoglobin precipitation assay for tannins
Stock haemoglobin solution

Collect fresh blood from a steer at slaughter and dilute with sev-
eral volumes of cold water (5°C) to haemolyse cells. Return the
haemoglobin solution to the laboratory on ice, centrifuge free of
cell debris, store at S°C and use within 90h.
Method
Add up to lml of the plant extract to a centrifuge tube, making
the volume up to l m l with the same solvent as is present in the
extract. Then add l m l of water and mix. Finally add 1 ml of
diluted haemoglobin solution, mix for 15s and then centrifuge at
7500g for 30min. Prepare a blank without any plant extract
(replace extract with 1ml solvent).
Measurement
Pour the supernatant solution into a spectrophotometer cuvette
and record the absorbance at 578nm. Do not record results from
turbid (cloudy) reactions. Schultz, Baldwin and Nothnagle (1981)
recommend adjusting the stock haemoglobin solution to such
a concentration that blank reactions have an absorbance of 2.0.
Measurements of haemoglobin precipitation should be standardized
by reference to tannic acid.
which shows the sensitivity of haemoglobin precipitation to the haemo-

globin concentration. At high haemoglobin levels a threshold effect is
evident where substantial amounts of tannin can be added before there is
a sudden, large response in terms of precipitation. At low concentrations
the haemoglobin is rapidly precipitated with little or no response as more
tannin is added. We have already explored the rationale for these effects.
The individual investigator must optimize the system to suit the range
of tannin concentrations encountered in his or her samples. The haemo-
globin solution concentration should be adjusted so that the typical
sample precipitates approximately 50% of the haemoglobin present. This
minimizes errors due to: (i) the threshold effect; and (ii) the precipitation
of all the haemoglobin leaving unprecipitated tannins in solution. Where
exceptional samples are encountered they must be diluted or concen-
trated as necessary after which they may be measured against the same
blank reaction as the other samples, with the dilution and concentration
factors being accounted for in subsequent calculations. Once an appro-
3.5 -
3 --
0 0.2 0.4 0.6 0.8 1

Tannic acid (mglml)
Fig.5.6 Variation in the precipitation profile of four haemoglobin mlutions of different

initial concentration. (From Schultz, Baldwin & Nothnagle, 1991.)
priate haemoglobin concentration has been found for an assay, then a

standard curve may be obtained by replacing the plant extracts with
solutions of known concentrations of tannic acid dissolved in the same
solvent. The standard curve obtained from these data can then be used to
determine the equivalent amount of tannic acid present in the plant
extracts. Results obtained with this technique are usually reported as
tannic acid equivalents (TAE).
Our misgivings with this popular method extend well beyond the
problem of blood supply. One problem is that of soluble, or at least
unprecipitable, tannin-protein complexes in the supernatant solution
used for absorption measurements. These are reported to cause turbidity
problems (Bate-Smith, 1973b) which interfere with spectrometric
measurements. Another unrelated source of interference is caused by the
ability of some extracts to form methaemoglobin which has different
spectral characteristics to normal haemoglobin (Bate-Smith, 1977). Yet
another cause for concern is that haemoglobin will certainly not be the
only protein in the assay solution. Tannins differ in the specificity with
which they bind to haemoglobin in relation to other proteins, which may
lead to them appearing to differ in relative astringency when they may
120 Chapter 5
0.8 s
0.7-
0.6 --
-E
C 0.5--
0
m
9
8 0.4--
0 100 200 300 400 500 600 700

Concentration (pglml)
Fig. 5.7 Variation on the precipitation of BSA with two tannins
only differ in whether they preferentially precipitate with haemoglobin or

with some other blood protein, such as serum albumin.
We are also far more impressed by the linearity of standard curves
published for precipitation assays using BSA than for those using haemo-
globin. Figure 5.6 presents the best available curve for haemoglobin, and
this is positively curvaceous when compared to the curve for BSA (Fig.
5.7). While standard curves are acceptable, linear relationships are much
easier to construct, interpret and analyse statistically. Good 'linear' curves
showing little spread in the data points also suggest that a technique is
robust and likely to be replicable between laboratories.
5.3.2.2 BSA precipitation methods

There are several protein precipitation methods which employ BSA as
the tannin precipitated protein. The differences between these procedures
almost all relate to the methods used to detect the protein precipitated
rather than the actual precipitation reaction itself. This is the case be-
cause most colorimetric methods for quantifying proteins are subject to
interference from phenolics, and circumvention of this problem has re-
quired some effort in terms of method development.
The most popular precipitation reaction, and the one favoured here, is
that due to Hagerman and Butler (1978, 1980b). The BSA is dissolved in
an acetate buffer at the pH of its isoelectric point. Additional ionic

strength is provided in the reaction medium by the presence of salt. The
conditions are optimized for BSA precipitation and there is no potential
for interference by other proteins, unlike in haemanalysis procedures.
The tannin-containing plant extracts are added to the BSA solution and
precipitates are collected by centrifugation. Unlike haemanalysis, there is
no need for the plant extract to be perfectly clear as spectrophotometric
measurements are not made on the supernatant solution. Instead the
precipitates are analysed and their protein content is determined.
Details of the precipitation reaction conditions are given in Scheme
5.2. Two equally suitable techniques for the determination of protein
content in the precipitate are then outlined in Scheme 5.3. Many of the
concerns voiced about the haemoglobin precipitation reaction also apply
here. The reaction is sensitive to the absolute and relative concentrations
of tannin and protein present. We recommend using exactly the same
BSA concentration as given in Scheme 5.2 and then diluting the plant
extract so that typical samples precipitate a significant and easily meas-
urable proportion of protein. At one extreme, a small amount of tannin
relative to a large amount of BSA may lead to soluble complexes rather
than precipitates (Hagerman & Robbins, 1987). On the other hand one
must not come close to precipitating all the protein or the assay will not
be sensitive to samples containing atypically large amounts of tannin.
The assay must be set up correctly with a linear standard curve being
constructed and measurements should fall within the limits of the standard
curve's range of linearity. Samples that do not fall within this range will
need appropriate concentration or dilution.
Once the precipitate has been obtained the next step is to measure the
Scheme 5.2 The BSA precipitation reaction
Protein solution
Prepare a 0 . 2 ~acetate buffer (pH 5.0) containing 0 . 1 7 ~sodium
chloride and l.Omg/ml BSA (Fraction V). If using blue-dye label-
led BSA prepare the above solution with the protein at 2.0mglml.
Precipitation reaction
Add l m l of the tannin-containing extract to 2ml of the protein
solution. The extract may be dissolved in water containing up to
50% methanol but no acetone. Mix and allow to stand at room
temperature for 15min. Centrifuge at the top speed of a clinical
centrifuge (5000g) and discard the supernatant.
amount of protein present. We suggest three, and describe two, methods
for this. Each has its own peculiar advantages and disadvantagv. The
simplest to use in practice is the 'blue-dye labelled BSA' method in which
a chromophore is attached to the BSA so that the presence of the protein
can literally be seen in solution. This method was pioneered by Asquith
and Butler (1985) and involves a relatively simple reaction to produce the
coloured BSA. Blue-dye labelled BSA is made by dissolving 2g BSA and
150mg Remazol brilliant blue R in 40ml of 1% sodium bicarbonate
solution (adjusted to pH 8.2). After this mixture has been stirred for 30
minutes it is dialysed against the same buffer used in the precipitation
reaction outlined in Scheme 5.2 (0.214 acetate buffer (pH 5.0) containing
Scheme 5.3 Measuring precipitated protein
Blue-dye labelled BSA

Prepare an aqueous solution that contains 1% wlv sodium dodecyl
sulphate (SDS), 5% vlv triethanolamine and 2046 vlv isopropanol.
Add 2.0ml of this solution to the precipitate and mix to resuspend
the tannin-protein mixture. When an optically clear solution has
been obtained measure the absorbance of the solution at 590nm.
Make measurements relative to a control containing 1ml of the
protein solution used in the precipitation reaction and 1ml of the
solution used to resuspend the precipitate. This control will have
the absorbance expected for 100% precipitation of the protein by
tannin. Also use a 0% control employing tannin-free solvent in the
precipitation reaction.
Normal BSA
Add 1.2ml of 1 3 . 5 NaOH
~ to the precipitate, loosely cap the test-
tube and mix to resuspend the precipitate. Hydrolyse at 120°C for
30min using either a heating block or an autoclave (Makkar,
Dawra & Singh, 1987). After hydrolysis, cool the reactions and
add 2ml glacial acetic acid, again cool and mix. Add aliquots of
the neutralized hydrolysate to a ninhydrin reagent and assay the
amino acids released colorirnetrically. Our preference is to use the
Sigma Chemical Co. reagent, heating the reagent and aliquot of
hydrolysate at 1 W C for 15min exactly and then diluting and
cooling this reaction with 50% aqueous ethanol prior to measuring
absorbance at 570nm. It is also possible to prepare ninhydrin
reagent 'in house' rather than use a commercial preparation
(Makkar, Dawra & Singh, 1987).
Biochemical techniaues 123
0 . 1 7 ~sodium chloride). It is then diluted with this buffer to give a

2.0mgiml solution of blue-dye labelled BSA which is used in the pre-
cipitation reaction in place of the undyed BSA. This procedure has the
advantages of haemoglobin and haemanalysis. Indeed, it is superior to
haemanalysis in that a pure protein from a convenient source is used.
Excellent linear standard curves have been published (Asquith & Butler,
1985; Fig. 5.7). Its disadvantages, relative to other BSA techniques, are
that the blue dye molecule, which is covalently attached to the BSA,
lowers the affinity of the protein for tannin somewhat, hence its use at
2.0mgJml rather than at l.Omg/ml. This is tantamount to blue-dye
labelled BSA being a different protein and thus the technique is not
exactly a BSA technique.
An earlier variation on this 'tagged-BSA' theme was to attach a
radioactive label to BSA (Hagerman & Butler, 1980b). This is an excel-
lent technique, which uses scintillation counting to detect the protein in
the precipitate after its resuspension. unfortunately, this technique seems
destined for obscurity as the use of radioisotopes becomes more tightly- .
controlled and less convenient as a technique for occasional use. We have
chosen not to describe this technique, but for those already equipped for
radiochemical work, this method (Hagerman & Butler, 1980b) is worth
looking into.
The third technique is the most laborious but uses an unadulterated
form of BSA in the precipitation reaction. After centrifugation, the
tannin-protein complex is subjected to an alkaline hydrolysis to yield
amino acids which are then detected colorirnetrically by reaction with
ninhydrin. The method was first used by Mole and Waterman (1985) and
Marks, Buchsbaum and Swain (1985) and was then further developed by
Makkar, Dawra and Singh (1987). Details of what we currently believe to
be the best procedure are given in Scheme 5.3. As two laboratories
independent of our own have developed the method, we feel confident in
recommending it. The one and only note of caution is that the ninhydrin
reagent is very sensitive to oxidation and must be kept sealed when not in
use, preferably under nitrogen.
There are other methods for measuring proteins, most of which we
feel should not be used under any circumstances! The main problem is
that some, if not most, methods for protein quantification are subject to
interference by phenolics. Commonly, methods for proteins rely on
chromogenic redox reactions, typically involving the oxidation of the
phenolic hydroxyl of tyrosine. Redox based methods for proteins include
the Lowry, Biuret and more recent BCA procedures (Smith et al., 1985)
which utilize a copper reagent. As noted previously, even the Folin
methods were originally developed to detect tyrosine residues. Detection
of proteins by UV is also dependent, to a degree, on the phenolic
tyrosine residue and its absorbance will be swamped by the W absorb-
ance of any phenolics present. The Bradford dye binding method is again
susceptible to interference by phenolics (Compton & Jones, 1985) and so
this method has had to be abandoned for measuring proteins in the
presence of tannins. While Martin and Martin (1982) did attempt to
use Bradford's method after removing phenolics chromatographically,
they later abandoned this in favour of an amido black method (Martin,
Rockholm & Martin, 1985) which appears to be an entirely suitable
method, but one that has not been adopted by others. For more detail on
these points we recommend the work of Amory and Schubert (1987) who
came to similar conclusions as ourselves. We reiterate the need to avoid
the 'standard techniques for proteins' which only work well in the absence
of phenolics. Instead, we strongly suggest the hydrolysis to amino acids
followed by ninhydrin detection, which does not appear to be adversely
affected by any phenolics that may be present.
5.3.3 Other protein precipitation methotis

The only other method of real note is the AOAC method for tea tannins
(AOAC, 1%5) and its variants, which utilize gelatin as the protein to be
precipitated by tannins, In these techniques the protein is simply used to
remove phenolics from solution allowing for their determination by
measuring the total phenolic content of the solution before and after
precipitation (Tempel, 1982). This method makes no direct measurement
of the proteins (or the precipitated phenolics) and thus we do not regard
it as a suitable method for the assay of tannins, even if it is quite
appropriate for detecting them on a qualitative basis. In systematic work
a more primitive gelatin precipitation method has been extensively used
to detect the presence of tannins but only Tempe1 (1981, 1982) appears to
have applied this technique to quantitative ecological work and we do not
recommend its further use in this context. The traditional hide powder
method used in the leather trade (Society of Leather Trades' Chemists,
1965) is an exceedingly laborious method in which the amount of tannin
precipitated with hide powder is assayed through a gravimetric method.
Because of the complexity and time-consuming nature of the method it is
definitely not recommended. Comparisons with more rapid methods of
protein precipitation analysis that we do recommend indicates good
correlation between these techniques (Seigler et al., 1986; Mugedo &
Waterman, 1992).
5.3.4 Other methods

Tannin-protein precipitation reactions are the best way to quantify
tannins in the broadest operational sense and we have reviewed what we
consider the best approaches to doing this above. One other noteworthy
Biochemical techniaues 125
method is the radial diffusion assay developed by Hagerman (1987). This

has the unique advantages of not requiring a centrifuge or a spectro-
photometer, which will be attractive to those who wish to have results in
the field. We also include a discussion of techniques for determining the
quantity of phenolics present in tannin-protein precipitates. While we do
not see these as appropriate for the quantification of tannins, it is some-
times useful to gain an assessment of the proportion of phenolics in an
extract which actually precipitate with protein. Finally, we discuss the
optimization of protein precipitation assays for tannins and the use of
several assay techniques to measure the specific activity of a particular
tannin or tannin mixture.
5.3.4.1 Radial diffusion assay

Hagerman (1987) developed a technique in which a tannin-protein
interaction is allowed to develop in an agar gel. The method involves the
incorporation of BSA (the protein) into an agar medium which is then
poured into Petri dishes and allowed to set. These can be stored at 4°C
for future use. Wells are cut into the gel with a cork borer and aliquots of
plant extracts are placed into these. Over time the tannin diffuses out and
forms a ring of precipitate around the well, the diameter of which can be
related to the concentration of tannin present. The results are far from
instantaneous (96-120 hours development time). This is a method which
may gain in popularity because of its technical simplicity. One other
advantage is that the extracts placed in the wells in the agar can be quite
crude (e.g. unfiltered primary extracts of plant tissues) and they may
contain high levels of organic solvents, even acetone (Hagerman, 1987).
The method is detailed in Scheme 5.4 but readers wishing to attempt this
are also advised to consult the original publication for photographs illus-
trating the nature of the experimental results. Jakubas, Gullion and
Clausen (1989) and Peng and Jay-Allemand (1991) have already made
use of the method in ecological studies. While the method looks promis-
ing, at present it remains very much less tried and tested than the
methods described above. On a cautionary note, it must be remembered
that the method relies on the diffusion of tannin through agar (a polysac-
charide) and protein. We know little about that diffusion mechanism and
the effects that concentration may have on it, and suspect that if it were
understood we would face serious complications.
5.3.4.2 Assay of protein precipitated phenolics

On occasion it is of interest to know how much of the phenolic material
present in a extract can function as a tannin in the sense that it actually
coprecipitates with proteins. Hagerman and Butler (1978) devised a
method for this which is dependent on the formation of wloured com-
126 Chapter 5
Scheme 5.4 Radial diffusion method for tannins
Preparation of agar-BSA plate

Prepare a 1% (wlv) solution of agarose in the following buffer:
5 0 m acetic acid, 6 0 ascorbic
~ acid, adjusted to pH 5.0 with
sodium hydroxide. Bring the solution to the boil to dissolve the
agarose and cool to 45'C and add BSA to 0.1% (wlv). Pour 9.5 ml
aliquots into 8.5cm diameter Petri dishes, store at 4°C. Cut 4mm
diameter wells with a cork borer. Holes should be at least 1.5cm
apart.
Assay
Place 8pl aliquots of crude extract into the wells. Additional
aliquots may be applied to the wells as solvent is absorbed. Standard
curves are prepared using solutions of tannic acid. After the addi-
tion of samples to the wells replace Petri dish lid and seal with
parafilm and incubate at 30°C for 96-120h.
Reading results
Once the precipitin rings have developed, measure two diameters
at right angles to each other and use the square of the average
diameter as a measure of the concentration of tannins present.
This should produce a linear standard curve for tannic acid.
plexes between iron and the phenolics present in resuspended tannin-

protein precipitates. The method is presented in Scheme 5.5 and can also
be converted into a useful Prussian Blue assay for total by
following the ferric chloride reagent with the potassium ferricyanide
reagent used in that assay: this increases the sensitivity of the assay and
makes it directly compatible with total phenolics assays.
5.3.4.3 Assay optimization and specgc activity determination

In Fig. 5.5 we showed examples of tannin-protein interactions where the
quantity of precipitate formed actually falls when an excess of protein is
present. Our interpretation of such data is that they are evidence for
soluble tannin-protein complexes which can form in situations where
there is a relative excess of either tannin or protein. In practice, this
means that when an assayed value for the protein precipitating capacity of
a tannin or extract has been obtained, then it may repay the investigator
to repeat the measurement with both higher and lower concentrations of
protein present in the assays, rather than to accept the data from a single
Biochemical techniques ln
Scheme 5.5 The analysis of phenolics in tannin-protein precipitates
BSA solution
Prepare a 0 . 2 ~
acetate buffer (pH 5.0) containing 0.17111 sodium
chloride and 1.0mglml BSA (Fraction V).
SDS reagent
Prepare an aqueous solution containing 1% wlv sodium dodecyl
sulphate (SDS), 5% vlv triethanolamine and 20% vlv isopropanol.
Ferric chloride reagent

0.01 M FeC13 in 0.01 N HCI.
Assay
Add l m l of the tannin-containing extract to 2ml of the BSA
solution. The tannin extract can be dissolved in water containing
up to 50% methanol, but no acetone. Mix and allow to stand at
room temperature for 15min. Centrifuge at the top speed of a
clinical centrifuge (5000g) and discard the supernatant. Add 4.0ml
of the SDS solution to the precipitate and mix to resuspend the
precipitated material. Add 1ml of the ferric chloride reagent, mix
and measure absorbance at 510nm, 15-30min after mixing.
comparison. Such data can indicate whether the original assays were
made on a plateau, at an optimum or in sub-optimal conditions. Read-
justment of assay conditions may be warranted for particularly critical
measurements.
For measurements made where protein precipitation is close to maximal,
for a given quantity of tannin, then Hagerman and Butler (1980a, 1981)
defined the 'specific activity' of the tannin extract or isolate as the weight
of protein (BSA) precipitated per unit weight of oxidizable (total phenolic)
material present. This is a particularly useful way to characterize a tannin
and we encourage its use. Broadly, it is the ratio obtained by dividing the
weight of protein precipitated by a tannin by the total phenolics content
of the assay. The protein precipitated must be determined with a protein
precipitation assay and a Prussian Blue or Folin reaction should be used
for the total phenolics measurement. Where the tannin is a crude extract
containing non-tannin phenolics, then the determination of phenolics in
the precipitate can be a useful measurement, especially when made in
comparison with results obtained for a pure tannin.
128 Chapter 5
5.3.5 Standards and units of measurement

As any tannin will precipitate a protein, it is possible to standardize one
in terms of another, even if they are chemically very different. The
current position reflects this in that tannic acid, a hydrolysable tannin, is
the almost universally accepted standard for all protein precipitation
techniques. Typically the results of precipitation assays are presented in
terms of the weight of tannic acid which would have given the equivalent
precipitation of protein to that observed in the assay. The acronym
TAE (tannic acid equivalent) has been widely used to designate this.
We recommend the continued use of tannic acid in the construction of
standard curves for protein precipitation based assays for any tannins.
Given the ready availability of this material we cannot support the use of
a crude, investigator-specific preparation of tannin as standard. Where
such are used, data will become more useful to others if converted to
TAE before publication.
5.4 I n viho models of the effects of tannins

There has always been a strong desire on the part of ecologists to in-
terpret the results of protein precipitation assays for tannins as being
somehow more biologically meaningful than results from chemical assays.
We conclude this chapter by exploring the methods used to determine the
biological significance of tannin-protein interactions. This work is en-
tirely separate in its aim from that discussed above. Thus far we have
been concerned with simply answering the quantitative analytical ques-
tion of how much tannin is present. Now we are concerned with the
qualitative aspects of how tannin-protein interactions might be of signi-
ficance in biological systems. Put simply, the objective is to construct in
vitro models of biological systems. As is the case with mathematical
models, those that have been created are usually simplistic and it is
the ways in which they fail to describe reality that has yielded most
biologically important insights.
To contribute useful data, a model must incorporate some new and
unique insight and so there are few standard techniques. Nevertheless,
some basic themes have emerged. Understanding the role of tannins in
enzyme inhibition has been of primary importance, and as a cause of
inhibition could be the precipitation of proteins, two diverging lines of
work have developed from this. One concerns the formation and precipi-
tation of tannin-protein complexes in biologically realistic conditions and
the other concerns the specificity with which tannins bind to particular
proteins when given a choice of different protein substrates.
5.4.1 Inhibition of enzyme activity

Ecological interest in the inhibition of enzyme activity by tannins can be
best said to have begun with the study of the inhibition of tryptic diges-
tion of casein by Feeny (1969). At low pH, inhibition was acute because
casein was precipitated from solution leaving the trypsin with little sub-
strate. At high pH the digestion rate was more substantial as a tannin-
casein precipitate did not form. These results led to the conclusion that
the high midgut pH in gypsy moth larva is adaptive because it prevents
the inhibition of proteases by tannins for analogous reasons, i.e. pre-
cipitates do not form because of this. In this system there was an implicit
assumption that the tannin acted primarily upon the substrate protein. If
this were not the case then at high pH, a direct inhibition of trypsin would
be expected due to its alkaline pI (see Fig. 5.2). Subsequently, it has been
shown that this assumption is reasonable for a similar (trypsin-BSA)
system where the differing affinities for tannin binding shown by these
proteins indicated that the inhibition of proteolysis was due to substrate
binding, rather than direct enzyme inhibition by the tannin (Mole &
Waterman, 1987d).
In Feeny's experiment, attention was primarily directed towards
ensuring the physiologically appropriate pH for the digestive system.
Numerous studies have now been conducted to explore other parameters.
One improvement has been to use substrate proteins isolated from plants
and proteases from the guts of appropriate herbivores. Rarely have in-
vestigators done both! Trypsin has been the usual choice of protease, and
given the ubiquity of serine proteases, this is perhaps reasonable for
mammals. Evidence that this may not be the case for insects comes from
the considerable variety of pH-activity profiles and sensitivities to specific
inhibitors that have been demonstrated for insect proteases (Wolfson &
Murdoch, 1990). Few investigators have chosen substrate proteins that
are or resemble appropriate plant proteins. We fault ourselves here
(Mole & Waterman, 1985) for the use of BSA as the substrate. Little
is known about the interaction of tannins with the important dietary
proteins such as RUBISCO except for the work initiated by Martin and
Martin (1983). These authors (Martin & Martin, 1984; Martin, Martin &
Bernays, 1987) used crude preparations of insect gut fluids to digest
RUBISCO and made the important discovery that in vivo conditions
were such that the inhibition of proteolysis by tannic acid was unlikely
for both Manduca sexta and Schistocerca gregaria. They concluded that
surfactants are an important factor in preventing the tannin-mediated
precipitation of proteins in digestive systems, thus eliminating the main
cause of protease inhibition. De Veau and Schultz (1992) have recently
confirmed that lysophospholipids from the mid-guts of lepidopteran
larvae were able to inhibit the protein-tannin interaction but further
report that at high pH tannins can precipitate these surfactantsqualitatively.
It has been shown that the ratio of tannin to substrate protein in a
proteolytic system is important, as a relatively small amount of tannin
may denature the substrate protein and so speed up the proteolytic
130 Chapter 5
reaction and aid the digestion of protein (Mole & Waterman, 1985).
Similar arguments have been advanced to explain tannin-induced in-
creases in $-lactoglobulin digestion (Oh & Hoff, 1986, 1988) in a system
modelling human digestion. As a model system for exploring mammalian
digestion, Oh and Hoff (1986, 1988) present what is perhaps the state-of-
the-art for monogastric systems, except for their omission of surfactants.
Biologically relevant surfactants present in both insects and mammals can
clearly alter the way in which tannins affect a proteolytic system (Martin
& Martin, 1984; Mole & Waterman, 1985; Martin, Martin & Bernays,
1987; de Veau & Schultz, 1992).
A cynical observer might conclude that, because in vitro models of
digestion can be used to show tannins inhibiting, potentiating or not
affecting proteolysis, the work on modelling digestion has been of little
predictive utility. On the contrary, we feel that the situation is much
advanced since the seminal work of Feeny (1969). Our capacity to com-
prehend the complexity of factors involved is far better, and because
there are examples of animals that are damaged, helped and left unaf-
fected by dietary tannins (Bernays, Cooper-Driver & Bilgener, 1989) it
may even be the case that our various models do indeed reflect reality in
these particular cases. Work modelling conditions specific to particular
organisms will be required to investigate this possibility.
For the investigator wishing to pursue this line of enquiry, the basic
approach is to obtain physiological information as to the pH, buffering
and other solutes present in a particular digestive system and then match
as closely as possible the levels of enzymes, substrates and tannins present
for a digestion reaction. Models explicitly considering the con-
ditions and residence times for digesta in different comvartments of the
digestive system may be the direction future work will ta'ke. It is also the
case that other digestive activities besides proteolysis need to be con-
sidered, such as carbohydrate digestion. Another area of research with
which this work overlaps is the bioassay approaches used to study diges-
tion in ruminants and other herbivores that ferment their food. We deal
with this in the next chapter.
5.4.2 Precipihtion of proteins

One of the critical issues in studying the inhibition of digestive processes
is to decide whether the tannin binds to the substrate protein, or the en-
zyme, or both, and whether a precipitate or a soluble complex is formed,
or both. This has recently been a particular growth area for research.
One direct link between this work and ecological theory relates to the
status of tannins as quantitative defences sensu Feeny (1976). If tannins
bind specifically to proteases then, if each protease catalyses the destruc-
tion of a thousand substrate molecules, three orders of magnitude less

tannin are required to inhibit the enzyme catalysed reaction than if
tannins bind to the substrate molecules only. In essence this marks the
difference between tannins and the specific trypsin inhibitory proteins
produced by some plants. It has been important to find out whether
tannins are specific in which proteins they bind to, and if so, what kinds
of proteins are most likely to bind with tannin.
Work by Hagerman and Butler (1981) first demonstrated that tannins
did exhibit differences in the specificity with which they bound to proteins
and that proteins rich in proline were particularly likely to be bound by
tannin when in competition with other proteins. In general, digestive
enzymes do not exhibit strong affinities for tannins and there is at least
suggestive evidence that salivary proteins may further protect the gut by
strongly binding to tannins as they enter the mouth (Asquith et al., 1987).
This is an active and still controversial area of research. However, the
basic technique for determining the affinity of a tannin for one protein
relative to another is well established and is presented as the competitive
binding assay in Scheme 5.6.
In concept, the technique involves comparing the amount of pre-
cipitate obtained when a fixed amount of tannin is added to a solution
containing a controlled amount of one protein, both in presence of and in
the absence of a second protein. The reaction is set up so that the
precipitation by tannin of the first protein can be measured independently
of any precipitation of the second protein. This is achieved by labelling
the first protein, either by radioactivity or by a dye. If any tannin binds to
the second protein, then less tannin can bind to and precipitate the first
protein. Part of the elegance of the technique is that the results do not
depend on a precipitate forming with the second protein; all that is
required is that the second protein binds the tannin for the precipitation
of the labelled protein to be affected.
In practice BSA is the protein against which others have been com-
pared, and tannic acid has been most widely used as the precipitant.
Experiments are performed to measure the quantity of the competing
protein that must be present to 50% inhibit the precipitation of the BSA.
On a weight for weight basis, between protein variation has been found
to vary over several orders of magnitude. Figure 5.8 shows some typical
results. The technique has been used to assess the tannin-binding affinity
of enzymes, salivary and potential dietary proteins such as those in the
seed coats of grain. Once the relative affinity of tannin binding is known
for a specific pair of proteins and one tannin, it is also possible to
compare tannins while keeping the pair of proteins constant (Asquith &
Butler, 1985).
132 Chapter 5
Scheme 5.6 The competitive binding assay
Standard protein substrate

Prepare a 0 . 2 ~acetate buffer (pH 5.0) containing 0 . 1 7 ~sodium
chloride and 2.0mg/ml blue-dye labelled BSA (Fraction V, Scheme
5.3 for labelling technique).
Tannin-protein interaction
Prepare a solution of tannin in methanol such that 0.41111of tannin
solution will precipitate between 70-80% of the protein when
mixed with a solution of 0.5 ml of the above blue-dye labelled BSA
solution and a further l.lml of 0 . 2 ~acetate buffer (pH 5.0)
containing 0 . 1 7 ~sodium chloride. To prepare precipitates, mix
the above solutions and allow to stand at room temperature for
15min. Centrifuge at the top speed of a clinical centrifuge (5000 g)
and discard the supernatants.
Assay of protein precipitation

Prepare an aqueous solution that is 1% w/v sodium dodecyl sul-
phate (SDS), 5% v/v triethanolamine and 20% v/v isopropanol.
Add 2.0ml of this solution to the precipitate and mix to resuspend
the precipitated material. Once an optically clear solution is
obtained in which all the precipitated material has redissolved,
measure the absorbance of the solution at 590nm. Make measure-
ments relative to a control containing 1d of the protein solution
used in the precipitation reaction and 1d of the solution used to
resuspend the precipitates. This control will have the absorbance
expected for 100% precipitation of the protein by tannin. Also use
a 0% control employing tannin-free solvent in the precipitation
reaction.
Protein comparison
Dissolve the competitor protein in additional 0 . 2 ~acetate buffer
(pH 5.0) containing 0 . 1 7 ~sodium chloride and use 1.1ml aliquots
of this protein at various concentrations to replace the l.lml
buffer in the above reaction. By trial and error, determine the
concentration of competitor protein needed to 50% inhibit the
precipitation of the blue-dye labelled BSA. This can best be
determined by the graphical presentation of results given in Fig.
5.8.
I
0 0.02 0.04 0.6 0.08 0.1 0.12
Gelatin added (mg)
Fig. 5.8 Competative binding experiment; a typical result.
As with the precipitation assay for tannins that used blue-dye labelled
BSA, this method derives from an earlier technique using radio labelled
BSA (Hagerman & Butler, 1981). The original is probably the superior
method because of the greater sensitivity with which radioactivity can be
detected and is recommended to investigators equipped for work with
radioisotopes.
5.5 Concluding comments

In this chapter we have covered representative methods that deal speci-
fically with the interaction between a tannin and a protein or proteins.
We have ignored the fact that a tannin may also complex with other kinds
of substrate such as carbohydrate polymers or small molecular weight
toxin molecules such as alkaloids. If studying the biochemistry of the
tannin-protein interaction you must never forget that you are taking
substrates out of context. The techniques available are complex enough
and sufficiently difficult to interpret when treated in this simplistic way.
This is, we fear, the best that we can do at the moment, particularly when
experiments are confined to the in vitro level.
CHAPTER 6
Methods for in vivo studies
6.1 Introduction
In this chapter we will try to indicate where the application of chemical
expertise can aid in the design and interpretation of experiments involv-
ing live organisms. Such experiments are the logical next step beyond the
in vitro models with which we concluded the previous chapter. We are
concerned with experiments that range from invasive physiological studies
through to feeding studies with defined diets and captive animals. In
essence we are considering any in vivo system in which some aspect of an
organism interacting with a phenolic allelochemical is to be analysed. In
such experiments the plant phenolic is not usually in the physical wndi-
tion encountered in the field and so considerations of how the material is
deployed become a problem.
Our focus here is on plant-animal interactions because least is writ-
ten about the methodology in this area. We focus our attention on three
aspects in particular. Firstly, the construction of defined diets for feed-
ing trials where we believe more attention needs to be given to the
bioavailability of phenolics. Secondly, how the application of standard
assays for phenolics may be employed for materials such as digesta, urine
and faeces. We conclude our coverage of feeding studies with a review of
techniques for examining the process of detoxification, beginning with a
consideration of the chemical consequences of caching behaviour and
then tracing these processes through to the degradation of ingested and
absorbed compounds.
As well as feeding studies, other forms of bioassay are important in
the study of phenolics. These may involve the analysis of behavioural
responses (e.g. oviposition) or toxicity (e.g. to micro-organisms). They
are considered in a final section aimed at providing access to the literature
on bioassay methods for the whole spectrum of interactions in which a
phenolic compound might be tested for its activity.
6.2 Diet construction for feeding trials

The premier requirement for a feeding trial is the proper formulation of a
diet. This can range from live and whole plants to artificial diets that are
precisely formulated and defined in chemical terms. Many points of
relevance to the selection of plant material have already been discussed in
Chapter 4, particularly with regard to the preparation of extracts for total
phenolics. Even where unadulterated 'fresh' plant material is to be fed,
In vivo studies 135
herbivores will often reject wilted or defrosted material in favour of

that which is fully turgid. Where live but excised plant material is fed
it is critical to keep it fresh and monitor for any pathological changes
(Wolfson, 1988). Later in this chapter we will touch on cases where
animals naturally choose to eat material that is far from fresh, such as
rotted and/or cached material.
While many studies have concerned interspecific comparisons of plant
foods in feeding trials (e.g. Atsatt & Ingram, 1983), the use of whole
plant material will leave between species differences in non-phenolic
plant attributes as uncontrolled variables. To circumvent this, one ap-
proach is to use a plant deficient in phenolics as a control diet and then
apply extracts of phenolics to this for experimental treatments. This will,
however, generate a multitude of new problems. First and foremost, the
topical application of phenolics. This leaves the allelochemical on the
plant surface and with the exception of external resins, phenolics are
typically intracellular and not encountered until the first bite has been
taken. Particularly for insects with taste receptors in their feet, this may
give spurious feeding behaviours. Phenolics are prone to oxidation while
on the plant surface and are also more likely to interact there with animal
proteins, whereas in real situations they should first interact with plant
proteins. Some investigators recommend the use of ascorbic acid (vitamin
C) as an antioxidant to help prevent this, but this will add an 'acid taste',
as well as complicating the assay procedure (see Chapter 4).
The solvent in which phenolics are topically applied to foods is typically
volatile and organic which leads to the consideration of three other
factors. One is the importance of applying as much solvent to the control
plants as to the test plants. A second is to ensure that the final amount of
phenolic applied matches that in the plants which the diet is intended to
mimic (Steinly & Berenbaum, 1985). It is rare that actual analyses of
diets are reported to verify that the intended quantity of phenolics has
been applied. A third twist to working with phenolics dissolved in organic
solvents is that these solvents have a tendency to dissolve materials that
are natural on the surface and to run to the edges of the leaf surface and
evaporate leaving more natural and applied material deposited there.
'Edge effects' are also a problem with the application of material to filter
paper disks in bioassays. Insect herbivores, in particular, may naturally
start to feed at the edge, or choose not to do so if all the extract ended up
there. One advantage of filter paper over leaves in preference trials is that
sheets can be soaked in a chemical and uniformly dried so that identical
disks can then be cut from the sheets. After drying, quantitative measure-
ments of added materials can be made. Other substrates that have been
found useful in such preference tests are glass fibre filter disks, which
have the advantage of not turning to a mush when wetted. J. Harbome
136 Chapter 6
Table 6.1 Composition of diets: example protocol
Diet*
Ingredient 1 2 3 4 5 6 7 8
Tannin 1 1.72 1.72

Tannin 2 3.0 3.0
Tannin 3 1.05 1.05
Propranolol 0.05 0.05 0.05 0.05
Cellulose 3.05 1.33 0.05 2.00 3.00 1.28 0.00 1.95
Basal mix All diets %.95%,to complete total diet
* Diet 1 is a control, diet 5 controls for the effect of propranolol, diets 2 , 3 and 4 and diets
6 , 7 and 8 are used to explore the effects of tamins without and with the presence of
propranolol respectively. Basal mix is a nutritious diet supplemented to National Research
Council Requirements with vitamins and minerals. Mulose is added as an inert diluent for
the other ingredients Listed above so that the total nutrient content of all diets is controlled.
(personal communication) has also found communion wafers to be a

palatable substrate in preference trials involving slugs.
For feeding rather than preference trials, the incorporation of ma-
terial into artificial diets can overcome the problems associated with
topical and/or localized deposits. For chemicals active in small amounts,
then material can be simply mixed into a diet. However, for substances
such as tannins which may be fed at as much as 5% or even 10% of a
diet, this is insufficient. Here an inert substance needs to be added to the
control so that nutrients are of equal concentrations in all diets. For an
example see Table 6.1 which shows the construction of diets for an
experiment designed to estimate the interaction of three types of tannin
with the drug propranolol (Mole, Rogler & Butler, 1993). Each tannin
was fed at a different proportion of the diet, but to give the same total
phenolics content for each diet. This example illustrates the use of inert
bulk (e.g. cellulose) to keep all diets isocaloric for the monoga$tric
mammals to which the diet was eventually fed.
Artificial diets may work when fed as dry powders, or may need
pelleting. It is possible to impregnate commercial pelleted diets with
chemicals in some instances (Jung & Batzli, 1981). One technique of
controlling for differences in the physical characteristics of plants is to
feed ground plant material. Such diets may be mixtures of plant material
with other powdered ingredients and there is much to recommend this
procedure as it obviates the need for extract preparation. However, there
are a couple of potential problems. One is that the grinding should be fine
enough so that the animal cannot sort the diet into components. Se-
In vivo studies 137
condly, the formulation of such diets as agar gel suspensions can be

helpful, particularly for insects (Greene, 1989), but this can over-dilute
diets. In general, the water content of the final diet must match that in
the fresh plant material. In artificial diets that contain both phenolics and
water, there is much to recommend the incorporation of realistic amounts
of ascorbic acid as an antioxidant (up to 1%).
Finally some further comments on what is lost when phenolics are
added to a diet after extraction. A principal point of issue is that in the
artificial environment phenolics will no longer first react with the plant
constituents that would surround them under normal conditions. In the
case of tannins the consequences of this will be evident from what has
been said in Chapter 5. Microscopic studies reveal that tannins are often
restricted to particular parts or cells of a plant organ or tissue (Zobel,
1986) and, as a consequence, some cellular constituents are more likely
to be reacted with than others. As an example, in sorghum grain the
tannins are present in the outer seed coat layer, in close proximity to the
proline rich kafirin proteins. These are thus ideally placed to interact with
tannins. When exogenous tannins are simply added to a diet of ground
sorghum meal thejr effects on digestion can be different (Mole, Rogler &
Butler, 1993). As Wolfson (1988) argues, an 'artificial diet can be just
that - artificial!' Berenbaum (1986), has observed that there is a suspicious
association between the use of artificial diets and the finding that tannins
inhibit digestion, while opposite results are more likely with natural diets.
Yet another problem identified by the work of Berenbaum is the
possibility of synergistic effects between allelochemicals (Berenbaum &
Neal, 1985). This is a caution against single factor experiments and overly
simplified artificial diets. Fahey and Jung (1989) make some interesting
observations on this point: relatively innocuous phenolics present in a
natural diet may cause other phenolics to be toxic by competing for mixed
function oxidases and other detoxification systems. It is also possible
that such indirect effects may be found through the capacity of phenolics
to chelate metal ions and interact with vitamins. Understanding the
synergistic interaction of phenolics with each other and other allelo-
chemicals is a subject deserving considerably more attention than has so
far been the case.
6.2.1 Analyses for feeding trials

Once a feeding trial has begun, it is normal to ,collect faeces, urine,
digesta, blood and other kinds of samples. The handling, analysis and
interpretation of such materials and the data obtained from such studies
often needs to differ from that for plant material. Problems arise when
experimenters neglect to consider the physical surroundings of plant
phenolics once they have entered animal bodies. In contrast to live plant
138 Chapter 6
tissues, the conditions are typically warm and wet in the gut, as well as
being potentially extreme in terms of pH and redox potential. These
conditions favour chemical reactions, and yet more reactions are likely
once samples are exposed to air, either naturally or upon dissection.
The practical consequence of this is that phenolics can rapidly become
covalently bound into insoluble complexes with proteins and other
biopolymers in the gut. Condensed tannins may be present in the faeces
but no longer in an extractable form. Thus, it is not reasonable to expect
that tannin assays for food and faeces (weighted for consumption and
egestion) can be used to compute the digestibility of tannins. They may
have been digested, but they may also have simply been rendered in-
soluble in faecal material. We are thus at odds with those such as Cork,
Hume and Dawson (1983) who have interpreted the apparent digestion of
tannins as evidence for their degradation.
Animal tissues are also lipid rich compared to plants, and may need to
be defatted (after lyophilization) in order to aid extraction of phenolics.
Frequent collection of frass, faeces and urine will best preserve phenolics.
Collection of other tissues on dry ice or in liquid nitrogen may be appro-
priate, especially for digesta. Once dried, defatted and ground faecal and
other animal samples can usually be treated as normal for use in assays
such as those for total phenolics. Urine will generally fail to lyophilize
due to its urea content but can be preserved frozen and then used direct
in assays. Besides analysis for phenolics in urine and faeces, there has
also been an interest in monitoring other indicators of detoxification
processes. Chief among these are the detection of glucuronide production
and any changes in nitrogenous waste products. Urea and uric acid can be
measured using- commercially available kits. Lindroth and Batzli (1983)
provide a methodology for investigating glucuronidation in an ecological
study and we discuss the excretion of metabolites arising from detoxifica-
tion processes below.
6.3 Detoxification involving phenolics
6.3.1 Caching behaviour and detox~fiationbefore feeding

Mammals, such as small rodents, will often bury or hoard foodstuffs.
In part, this is almost certainly a response to seasonal abundance but
there is also a potential adaptive role. Phenolics can often be rendered
inextractable and, as a consequence, probably less toxic by wetting and
burial (Smallwood & Peters, 1986). Information about these processes
can be found in the literature on human ecology and food processing
(Mehansho, Butler & Carlson, 1987; Mukuru et al., 1992). Geophagy,
where clays from the soil mix with plant foods may also be a significant
means by which phenolics are detoxified, at least by primates (Johns &
Duquette, 1991).
In vivo studies 139
6.3.2 Xenobwtic metabolism after ingestion

One important non-event in many feeding studies occurs when phenolics
known to be present in the diet fail to emerge in the urine or faeces.
While somewhat peripheral to the topic of this book it is of considerable
importance to try to understand what happens to phenolics once ingested.
This section attempts a brief o v e ~ e wof the metabolism of xenobiotics
and a guide to the methodologies involved. A recent comprehensive
review of the literature on this subject has been published (Fahey & Jung,
1989), although this is limited to phenolics in vertebrate systems.
One of the ecologically interesting points that can be made is that
even in monogastric systems, the metabolism of ingested phenolics is
likely to be begun by microbes. These are responsible for 0-methylation,
dehydroxylation, and decarboxylation reactions in simple phenolics and
the reduction of double-bonds in cinnamic acids. The initial attention
of gut microbes can also lead to the formation of phenols, through
hydrolysis of a glycoside to the corresponding phenol (i.e. 'unmasking' a
phenol- see Chapter 1). Once in the body phenolics can be 'masked'
by glycosylation, often with glucuronic acid (159) or with sulphate ions.
Such products are water-soluble and can be voided in the urine or bile
(Millburn, 1978). Glucuronidation reactions and the production of
ethereal sulphates are typical for higher molecular weight phenolics.
Smaller phenolics may be degraded to produce hippuric acid (160) which
appears as the most abundant aromatic compound in the urine of mam-
malian species. At least some hippuric acid is believed to arise from
dietary sources, such as cinnamic acid. In ruminants, in particular, Fahey
and Jung (1989) consider that it is quite possible that non-aromatic
products may be formed from phenolics and that these need not show any
obvious relation to their dietary origin. In contrast, some phenolics may
emerge in the urine that are not obvious or expected. Such is the case
with the production of enterolactone (161) in humans, which is presumed
to arise from bacterial action on lignin.
For flavonoids consumed by mammals, ring fission leading to hydroxy-
phenylacetic or proprionic acids is widely reported (Scheline, 1978).
140 Chapter 6
Less extensively metabolized products are frequently encountered in a

'masked' form, for example as glucuronides. With regard to tannins,
the hydrolysable type is apparently degraded to some extent so as to
yield methylated derivatives of gallic acid or glucuronides (Murdiati,
McSweeny & Lowry, 1992). Condensed tannins may be partly metab-
olized but the products are as yet unknown. Evidence for the microbial
degradation of tannins is becoming stronger (Cork, Hume & Dawson,
1983; Osawa, 1990; McArthur & Sanson, 1991) but at present we still
remain ignorant as to what to assay in terms of excreted metabolites.
From the above it should be obvious that one cannot expect to feed a
phenolic and recover it quantitatively in the excretory products of the
animal. Even if one is lucky enough to find metabolites that are still
recognizable, extensive work will usually have to go into their recogni-
tion. Particularly in the case of alkylated products, the general utility of
the assay for total phenolics is reduced because it will only be sensitive to
phenol groups that escape alkylation. Instead, chromatography using thin
layer chromatography (TLC), high performance liquid chromatography
(HPLC) gas chromatography (GC), analytical counter current or electro-
phoresis (see Chapter 7) may allow for the detection of new but unidentified
peaks in the excretory products (urine, bile) of test animals fed phenolics
relative to controls. The help of a trained chemist may then be needed if
it becomes important to identify the particular products, one exception
here being hippuric acid, a common metabolite for which several chro-
matographic methods have long been available (Zweig & Sherma, 1970).
Variation in urea and uric acid production in feeding trials is easily
monitored using commercial kits. Radio-tracers can also be used to detect
xenobiotic metabolites, given a suitable source to feed. The work of Bull
et al. (1984) provides an excellent illustration of an entomological study in
which this was done. Again, a considerable chemical expertise and the
methodologies discussed in Chapters 7 and 8 are needed if the ultimate
goal is to identify the breakdown products from phenolics.
The alternative approach to measuring xenobiotics produced from
phenolics is to estimate detoxification metabolism indirectly by monitor-
ing changes in the levels of the enzymes involved. This approach had
been applied to insects with particular success (Brattsten, 1988), although
the interpretations that can be placed on measured levels of enzyme
activity are not always clear (Gould, 1984). Besides glucuronidation
assays (Lindroth & Batzli, 1983), Lindroth has also pioneered the meas-
urement of b-glucosidase, quinone reductase, cytochrome-c reductase, 0-
demethylase, esterase and glutathione transferase activities in ecological
studies (Lindroth, 1988, 1989; see also Bernard et al., 1989). All these
techniques are classical enzymological methods and outside, the scope of
the present text as regards any detailed treatment. Interest in these
In vivo studies 141
procedures and the interaction of plant phenolics with detoxification

systems seems likely to remain topical (Reichardt; Clausen & Bryant,
1988) and we hope this brief review will have drawn the possibilities and
problems of this area to the reader's attention.
6.3.3 Bioassays
Bioassay procedures tend to be investigator specific; indeed it is often
hard to define exactly what constitutes a bioassay. At one extreme they
blend in with field work, at the other with more intensive physiological
and toxicological experiments. It is hard, therefore, to generalize about
techniques for bioassays beyond extolling the virtues of clear thinking and
common sense. For such general advice readers may wish to consult
Wolfson (1988), who provides a useful summary of major pitfalls.
Table 6.2 Examples of bioassay techniques
Typelactivity Target organism References
Allelopafhy
Seed germination Seeds (various) Kil & Lea (1987)
Ipomoea l a m a Liebl & Worsham (1983)
Lettuce Elakovich & Stevens (1985)
Seedling growth Ipomoea lacunosa Liehl& Worsham (1983)
Crop plants Elakovich & Stevens (1985)
Antimicrobial
Fungal growth Soil fungi Paszkowski & Kremer (1988)
Cladosporum herbarum Tomas-hrente et al. (1989)
Bacterial growth Escherischa coli Swain & Downum (1990)
Streptococcw sp. Osawa (1990)
Antiviral Herpes simplex Takechi u al. (1985)
Antifeedantloviposition
m w Cmtelytra zealandica Lane et al. (1985)
Artificial seed Callosobmchur maculatus Birch, Crombie & Crombie (1985)
Probing behaviour Plant hoppers Kim, Koh & Fukami (1985)
Oviposition Papilio polyxenes Feeny et al. (1988)
Pieris rapae Tabashnik (1987)
Animal growth and development
Nutritional index Helwthis zea Reese, Chan & Waiss (1982)
Insect moulting Manduca sexta stamp (1990)
Monogastric growth Lab. rat Mole et at. (1990)
Lwmys salvini Janzen, Fellows & Waterman (1990)
Avian growth Chicken Featherstone & Rogler (1975)
Canada goose Buchsbaum, Valiela & Swain (1984)
Ruminant growth Deer Robbins (1983)
Animal toxicily
F i b , LC, Tilapia Balza et al. (1989)
Insect, LC,, Heliothis zea Neal (1989)
142 Chapter 6
The purpose here is simply to present an array of bioassay procedures

applicable to the interactions of plant phenolics with plants, mammals,
birds, insects, molluscs, fungi, viruses, bacteria, etc. While readers should
be able to construct their own appropriate bioassay procedures for the
organisms with which they work, suitable protocols for other systems
may not be on hand. For instance, the worker interested in oviposition
stimulants may, periodically, wish to test for other potential activities of
particular compounds, such as their antimicrobial effects. Table 6.2 aims
at addressing these needs. In most cases 'off the shelf organisms of
dubious ecological relevance are the targets. These are perhaps the best
bet for the investigator wishing to test whether substances of interest
have any activity at all. After positive results from pilot studies, more
time-consuming experiments with appropriate organisms will need to
be devised before the study becomes truly ecological. We have already
discussed the shortcomings of only usfng such bioassay approaches with
respect to allelopathy in Chapter 3. Similar reservations can be made
against other techniques listed in Table 6.2. However some familiarity
with these techniques is advisable if there is a need for either a bioassay-
directed search to isolate a compound from a crude extract of known
activity, or if some kind of biological quality control is required when
isolating and purifying substances. Thus these techniques may be appro-
priate for use in conjunction with the isolation, purification and identifica-
tion techniques dealt with in the following chapters.
CHAPTER 7
Qualitative and quantitative

separation methods
7.1 Introduction
If you are contemplating or have already decided that you need to quan-
tify or identify specific phenolic compounds in material under analysis, it
is important that you read this introductory section first and then take
some hard-headed decisions about what you want to achieve and whether
you have the necessary wherewithal to do so. If, on reflection, you decide
to proceed then in this chapter we endeavour to point you in the right
direction, and if your concern is with extraction, separation and purifi-
cation, this chapter will guide you to the appropriate techniques. Some of
the simpler aspects of quantitative and qualitative analysis using chrom-
atographic techniques are also covered here. In Chapter 8 we will proceed
to the next logical step, the subsequent chemical characterization of
individual 'unknown' compounds.
The problems outlined in Chapters 7 and 8 can take you deep into the
realms of the organic and analytical chemist. There remains, among a
significant subset of biologists, a fear of crossing this boundary. Such fears
are largely unfounded; no-one should be afraid of chemistry. What you
must be aware of, however, is that there is an enormous variation in the
complexity of the problems you might face. Some you will be able to cope
with but others will clearly require expert assistance as the skills involved
and the equipment needed are highly specialized. Equipment costs will
also vary markedly. You can, for example, sometimes achieve the neces-
sary chromatographic separation with filter paper, a glass tank and a
small quantity of solvent at a trivial cost. At the other end of the scale
you may need a gradient high performance liquid chromatography (HPLC)
system with a photo-diode array detector and a total cost of over &20000!
You may be able to establish the identity of an individual substance by
means of very simple co-chromatography or, if you are dealing with
unknown and perhaps novel compounds, you may need sophisticated
mass spectrometry and nuclear magnetic resonance spectroscopy, in which
case you will require access to equipment costing hundreds of thousands
of pounds, and you will generate results which may well call for the assist-
ance of a specialist before they can be interpreted.
So think carefully before you decide. The following are some of the
questions you need to consider, although perhaps not all at the beginning
of the exercise; for example, question 5 might well not need to be faced
immediately.
143
144 Chapter 7
1 Do I need information on individual phenolic compounds or is simply

knowing that changes in total phenolics occur sufficient? L
2 Does the information required need to be quantitative? Or is it suf-

ficient to be able to identify that different compounds are being produced?
3 If quantitative data are needed then are relative concentrations of
phenolics sufficient or do I need absolute values?
4 Before I start, am I sure I have established what is already known
about the phytochemistry of the species I am interested in? It may be that
much of the groundwork has been done. A survey of what is already in
the literature is an essential prerequisite that can stop you reinventing the
wheel! However, a word of caution. Do not necessarily believe all that
you read: chemists are often less rigorous than they should be when
identifying the sources of their isolates.
5 Must I know what the structures of some or all of the potentially
unknown compounds are? Or do I just need to verify that what is to be
expected is indeed there? If the latter then can I get standards for
compounds others have reported to be present? This could be by either
buying them commercially or by writing to scientists who have reported
them, either from this or from another species. Remember that a com-
pound is a compound irrespective of which Latin binomial it came from.
6 If I get out of my depth is there anyone I can turn to? As a rule of
thumb the further down 1-5 you reached before you stopped saying yes
to the initial question the more likely you are to need help. Try and
identify the potential chemical support available to you, both the people
and their facilities. Discuss what you want to do and ascertain the level of
potential back up. Vague enquiries about problems or about the use of
facilities are often met with incomprehension and lack of interest. If you
have thought your way through the problem, know your aims, and are
aware of the potential difficulties you should find much more interest and
willingness to support and, if necessary, collaborate.
7.1.1 Preparation of samples

The initial problem to be faced is the possibility of decomposition. Selec-
tion and collection of samples is a field exercise while their extraction is
almost certainly delayed until return to the laboratory. When a plant part
is harvested metabolism does not stop: reactions continue until some
essential component is exhausted. Equally importantly other reactions
may be initiated or their role magnified. For example, glucosidases will
continue to operate and may liberate phenols from previously non-
phenolic glycosides. Wounding of tissues (which is inevitable to some
extent during harvesting) will lead to the production of phenol oxidases
which will alter chemistry and may render compounds difficult or impos-
Separation methods 145
sible to extract. There are ways of minimizing these problems and they
have been considered in Chapter 4.
Back in the laboratory the next problem to be confronted is: do you
extract immediately and store extracts for analysis or do you dry and
store samples until the analysis can be performed? Whatever the choice
it must be remembered that, if there is any likelihood of requiring quan-
titative information, the sample weight and the volumes of extractant
used must be recorded, both from any work done in the field and from
subsequent laboratory operations.
There are clear advantages to immediate grinding and maceration (or
refluxing) of material in your chosen solvent followed by immediate
analysis. If immediate analysis is not feasible then samples must be stored
but this always introduces further imponderables. The optimum approach
will often be to extract so that the resulting solutions can then be con-
centrated, freeze dried and stored at -70°C in the dark until further
processing can take place. However, it should be noted that even heeze
drying can lead to the loss of volatile phenolic compounds. If samples
have been collected as dry material, do not grind them until extraction is
to be undertaken. Grinding increases the surface area and so enhances
the likelihood of chemical changes, such as oxidation, taking place.
7.1.1.1 Extraction
There are two problems to confront, the extraction method to use and
the choice of solvent. Methods will generally be based on cold or hot
extraction but there is great variety among approaches, as is indicated by
Table 4.3. For the larger quantities of material that must be extracted to
provide enough substance for structure determinations, the continuous
solvent extraction Soxhlet apparatus is probably the preferred method for
thermostable compounds. It is often worthwhile to spend time at this
stage trying out the various options. But remember that the methods
presented in Table 4.3 are intended to extract phenolics in general and
can only provide a crude extract for further purification. Often these
methods can be improved upon if the target of the phytochemical analysis
is less general than 'phenolics'. For instance, you might experiment with
less polar solvents such as ethyl acetate, particularly if your target(s)
are partially methylated or acetylated or are free aglycones and not
glycosides. Glycosides will normally require alcohol or aqueous alcohol as
the extractant.
7.1.1.2 Establishing the complexity of the extract

When searching for some types of secondary metabolite such as alkaloids
a prerequisite is to extract, clean up the extract, and perform a standard
148 Chapter 7
colour test to see if alkaloids are present. In our view this is unnecessary
if you are looking for phenolics - almost invariably some will ,be present
in the crude extract. The initial phase in an analysis of an extract con-
taining an unknown variety of phenolic compounds will usually be to
resolve the complexity of the problem; to do that the extract must be
somehow split up so that the individual wmpounds are visualized.
The first step in such analysis of a new material will probably involve
either paper chromatography (PC) or thin-layer chromatography (TLC),
quite possibly two-dimensional. These methods will be dealt with below.
What they will do is cause the compounds in the extract to spread over
the plate or paper and those that are phenolic can then be detected by a
number of different methods (Table 7.1). The most generally useful of
these is examination under UV light. Phenols are by definition aromatic
wmpounds and will as a consequence always either fluoresce or quench
W light. The latter effect can be seen when phenolics appear as dark
Table 7.1 General methods for the detection of phenolic wmpounds on chromatograms
Reagent Observation
UV light (254 nm) Various fluorescence wlours, most wmmon are violet, blue
and yellow. Also quenching
UV light + ammoma Enhanced or changed fluorescence when compared with W
vapour light alone
Folin-Ciocalteu or Blue or grey spots, may not be seen until after fuming with
Folin- Denis ammonia (spray with 20% aq. sodium carbonate, dry, spray
again with reagent diluted 1:3 with water)
Ferric chloride (1-5%) Phenols turn blue or green, hydroxamic acids red
in 0.5 M HC1
Nitroso-para-nitroaniline Mix 5m10.5% Cnitroaniline in 2 M HCI with 0 . 5 d 5 % aq.
sodium nitrite. Add 15ml20% aq. sodium acetate solution.
various w l o m
Diazotized sulphanilic Dissolve 4.5 g sulphanilic acid in 4 5 d 1 2 HCI
~ (warm if
acid necessary) and make up to 500ml with water. Cool l0ml (ice
bath) and add l O d 4 . 5 % aq. sodium nitrite solution. Stand at
P C for 15min (stable 3 days). For use mix with equal quantity
10% aq. sodium carbonate solution. Various colours (probably
the best spray reagent)
Fast Blue salt B 0.5% aq. Fast Blue salt B and 0.1 M sodium hydroxide. Mix
before use
10% vanillin in conc. HCI Usually red or pink wlours (variable)
1YO potassium White to yellow spots on a mauve background
permanganate in 0 . 5 ~ (Warning: powerful oxidizing agent; do not use with oxidizable
suiphuric acid media such as cellulose)
Table 7.2 Methods for hydrolysis of glycosides (can be performed on fresh or dried plant
material)
Type of glycoside Method
0-glycosides 1 2~ HCI in methanol (1 : 1) at 100°C for 1 h, preferably under nitrogen

2 2~ NaOH and borax- same conditions as above. Acidify for
extraction of phenol
3 b-glucnronidase ex marine mollusc-20min (also various other
general or specific hydrolases (b-gluwsidase) or esterases (tannase)
C-glycosides Shake with 10 ml5% ferric chloride solution for 5 min, add 5 ml2 M HCI
and heat to 100°C for lOmin
spots on TLC plates impregnated with a fluorescent indicator (often

designated as PZs4plates). The colours and intensity of phenolics that
fluoresce can often be modified by exposing the chromatogram to
ammonia vapour which can be most revealing and lead to the identification
of specific classes of phenolics. In addition to UV light spray reagents,
with varying degrees of specificity, may be useful.
Two particular points should be remembered. Firstly, always use more
than one solvent system for the preliminary analysis. You may eventually
select one as appropriate for your needs but if you don't initially ex-
periment you could well be working with 'spots' that you think are dis-
crete compounds but which are, in reality, mixtures. Secondly, perform
analyses on hydrolysates as well as the original extract. As previously
noted many phenols can be masked by formation of 0- or C-glycosides
and these may be released in the systems you are studying. Various
methods for performing hydrolysis are noted in Table 7.2.
7.1.2 Preliminnry steps in pur@cation of phenolic extracts

A crude alcoholic extract of, for example, a leaf is a mixture of con-
siderable complexity. Before any serious attempt is made to identify or
quantify individual compounds it is often advisable or even necessary to
carry out some general clean-up processes. In doing so you must always
be aware of potential problems such as unwanted hydrolysis and chemical
modification occumng during the work. These are a factor with phenolics
as with most other compounds. Concentrating extracts under excessive
heat is dangerous, as is the often cited ploy of extracting acidic com-
pounds (i.e.-phenols) into aqueous bases such as dilute sod& hydroxide
or sodium carbonate. Precipitation of extracts with lead acetate is another
technique that is best avoiied.
Three relatively simple procedures which can help are defatting
(particularly for seeds and fruits), gel filtration and reverse-phase micro-
columns (for small samples).
Defatting can be done either by extraction with hexane or petroleum
ether prior to the main alcohol extraction, or by washing the concentrated
aqueous alcoholic extract with herane after removal of most of the
al&hol and then using a separating funnel to recover the aqueous phase.
If defatting is undertaken be aware that some phenolics are, to a degree,
lipophilic, as is seen in the methods used for rotenoid isolation by Birch,
Crombie and Crombie (1985). It is also a mistake to assume that in
washing one extract with an immiscible solvent, both solvents do indeed
stay unmixed. For instance, low molecular weight condensed tannins
which are insoluble in pure ethyl acetate can be quite soluble in the water
saturated ethyl acetate present in an ethyl acetate-water partition. Be
prepared to lose some material in such procedures, and check all extracts,
before they are discarded, to find out what is being lost.
Gel filtration is often the method of choice for cleaning extracts.
Sephadex LH-20 gel (Pharmacia) is stable in organic solvents and a short
column will rapidly exclude high molecular weight compounds while
requiring fairly long elution times for those with a lower molecular weight.
This is an ideal way to remove chlorophyll, polysaccharides and proteins.
However, Sephadex LH-20 can, on occasion also act as a useful separating
medium, notably with tannins (see for example, Cai et al., 1991). Where
the gel filtration technique really scores as a clean-up procedure is,
therefore, with low molecular weight phenolics, such as flavonoids.
Other column clean-up methods that might be employed include ion
exchange, polyamide and reverse-phase silica gel. Polyamide is good for
small quantities but it is tiresome and laborious if working with large
sample numbers or amounts of extract. It also tends to irreversibly bind
with some phenols. Ion exchange, in which the phenol is linked to a
weakly acid resin and then removed with alkali, is good for high through-
put but is an inherently dangerous technique likely to lead to breakdown
of compounds; it is not a technique for the beginner. Small (1-2g)
'throw-away' reverse phase columns are now commercially available and
are excellent for the rapid elution of polar phenolic compounds. For
rapid 'cleaning' of large numbers of small extracts for comparative chrom-
atographic analysis they are of great value.
An exceptionally good and complete review of separation techniques
for tannins is that by Karchesy et al. (1989).
Note that the procedures described above are not aimed at isolating
a single compound but primarily at removing unwanted groups of metab-
olites, such as fats, basic compounds and non-phenolic pigments. The aim
here has been to generate an extract in which the major, ideally the only,
components are the phenolics. You will rarely achieve that goal but you
can approach it and so make the separation procedures outlined below

more efficient.
7.2 Separating and quantifying phenolics by

chromatographic methods
7.2.1 Paper chromatography (PC)

This was the original method of chromatographic analysis. Today it is
often discounted because of its limited power of resolution, which is due
to slow rates of running and consequent high levels of diffusion of spots.
However, for phenolic compounds it can still offer good value for money.
It has the virtues of being cheap, requiring minimal skills, and being able
to provide, relatively rapidly, small samples of purified compounds. Do
not be seduced by more expensive and complex techniques through
assuming that they will necessarily produce better results.
PC can be run using ordinary grade Whatman paper and can be
achieved using either an ascending or a descending solvent flow. Solvents
are generally water or alcohol based and a number of the most commonly
used are given in Table 7.3; others have been listed by Harborne (1989).
Two of the most versatile and widely used are the upper layer of an n-
butanol, glacial acetic acid, water (4: 1:5) system and a mixture of glacial
acetic acid, concentrated HCl and water (30:3:10), which is known as
Forestal solution. Detection of compounds is most often accomplished by
Table 7.3 Some examples of PC and TLC systems (data from Harborne, 1989)
Stationary phase Solvent Types of compound

- - - -
Cellulose Glacial acetic acid :conc. Anthocyanidins, flavones, flavonols

(PC or TLC) HCl :water (30: 3 :10)
n-butanol :glacial acetic Anthocyanins, awones, chalcones,
acid :water (4: 1:5 -upper Bavonol and flavone glycosides,
layer) stilbenes
10% aq. acetic acid Coumarins
Isobutanol: acetic acid: water Hydrolysable tannins,
(14:1:5) proanthocyanidins
Silica gel Petrol: ethyl acetate (7 :3) Naphtbaquinones
Hexane:ethyl acetate (17: 3) Benzoquinones
Ethyl acetate :methanol: water Anthraquinones
(100: 17: 13)
Chloroform:acetic acid (9 :1) Phenolic acids
Ethyl acetate: methanol (19: 1) Lignans ,
Toluene :ethyl formate :formic ~ f i v o n o i h sesterified
,
acid (5:4: 1) hydroxycinnamic acids
150 Chapter 7
SOLVENT 1
5,
/-1
k-i
0 0
@' '
,-.
00
'.-, I
0
,'-a
\--
,<-\
4*.; 1 0
'\<' 0
0 - Point of application
*
SOLVENT 2
Fig. 7.1 Two-dimensional paper or thin layer chromatogram.
using UV light or the non-chamng spray reagents described in Table 7.1.

It is, of course, not possible to use oxidizing agents or concentrated acids
as detection reagents as they destroy the paper. Many PC solvents are
acidic and this can be essential if the analysis is to involve anthocyanins as
their colour will fade in the absence of acid.
One particularly useful procedure that can be adopted with PC is two-
dimensional chromatography. Here a sample is run with one solvent; the
paper is then dried and run again, at right angles to the original direction,
using a second solvent (Fig. 7.1). This can lead to the separation of quite
complex mixtures of phenolics.
If small amounts of a compound are required pure for further analysis
(such as UV spectrophotometry) then PC may well be the simplest
method of production. A PC can be run, the spots visualized under UV
light and then removed by cutting out the paper containing them. Each
compound can then be eluted from the paper with ethanol and the W
spectrum obtained. Del Moral (1972) exemplifies the use of this method
in the identification and quantification of chlorogenic acid. A similar
chromatographic isolation and quantification for ferulic acid is described
by Turner and Rice (1975).
7.2.2 Thin-layer chromatography (TLC)

For relatively cheap analysis and separation of small amounts of com-
pounds, TLC has now commonly replaced PC. Originally TLC required
learning the skill of spreading the stationary phase on glass plates and this
is still likely to be the case where compounds are to be isolated or unusual
procedures are to be employed. However, for a small outlay it is now
possible to purchase machine spread analytical layers on either a plastic
or metal foil backing. These commercial plates are certainly much better
than 'home made' alternatives and because they are mass produced the
quality is more even and so results are more reproducible. Another
advantage of commercial plates is cost effectiveness; metal or plastic
backed plates can be cut to the appropriate size with a pair of scissors -you
do not have to use a whole 20 x 20cm plate for one analysis when you
may be able to see a separation of a sample applied to a (large) postage
stamp-sized piece of TLC plate in a fraction of the time and for a fraction
of the cost.
A number of TLC solid phases are available. Microcrystalline cel-
lulose may be used to translate the results of PC directly into a more
rapid and reproducible technique, using the same solvent systems. As an
alternative to cellulose, plates made up with polyamide can sometimes
give better resolution for glycosides. For simple phenolics (i.e. generally
those that are neither glycosides nor polyphenols) silica gel can offer the
best results. However, in many cases silica gel will cause streaking. This
can sometimes be overcome by first running the plates in analytical grade
dilute HCI or H2S04 to remove metal ions which otherwise chelate with
phenolics, adding to the streaking problem.
Reverse phase silica gel, in which the silicic acid has been reacted with
long chain hydrocarbons offers another alternative. Here the surface of
the plate becomes lipophilic which results in the most polar compounds
being eluted first. Reverse phase is most commonly used with HPLC (see
below) -TLC lacks the resolving power to make this rather expensive
technique routinely worthwhile.
TLC is certainly generally to be preferred to PC as a method for
separation of mixtures. Solvents are usually of a less polar nature, based
on organic solvents (Table 7.3), sometimes incorporating small amounts
of acids. Another advantage of silica gel is that it is stable to strong
oxidizing acids which can therefore be used in spray reagents.
7.2.2.1 Standardizing PC and TLC Data - the Rfvalue
In theory if you keep the conditions constant in a chromatographic system
a compound should always run the same distance (measured to the centre
of the compound spot) relative to the distance moved by the solvent
front: this fraction is known as the Rf value. Unfortunately it is not a
perfect world and in practice, however carefully conditions are controlled
there will be small fluctuations in the Rfvalue a compound gives in any
system. This can be compensated for by including a known standard in
the run and standardizing Rfvalues on that compound. Whatever you do
the point to note is that an Rfvalue is a fragile thing! A quoted value of
0.52 in a given system is not written on a tablet of stone and may not be
reproducible.
7.2.3 Strategy for use of TLC and PC in the analysis of

phenolic extracts
1 Make a crude extract of about 5-log plant in aqueous methanol.
Test an aliquot for the presence of tannins by protein precipitation.
2.1 Subject a portion of the extract to hydrolysis for glycosides (prefer-
ably for both 0 and C, but at least for 0 ) using methods outlined in
Table 7.2.
2.2 Take part of the hydrolysate and partition into diethyl ether, separate
and concentrate the ether extract. Do separate two-dimensional TLC of
the original extract and ether soluble hydrolysate on silica gel with P254
indicator (acetic acid :chloroform 1:9 versus ethyl acetate :toluene 9 :11).
Examine under UV light (254 nm) and than spray plate with either Folin
or ferric chloride reagent (see Table 7.1). Count UV absorbing and blue
to green staining spots as phenolics. Phenolics separating with an Rfvalue
between 0.95 and 0.05 include, but are not limited to, simple phenols.
Such simple phenolics as gallic and salicylic acids do not fluoresce under
UV light but quench to give dark absorbing spots as do some other
phenolics. The presence of gallotannins can be verified by the detection
of gallic acid in the hydrolysate (co-chromatograph with authentic gallic
acid).
2.3 Repeat 2.2 using microcrystalline cellulose TLC plates and perform
two-dimensional TLC (water versus n-butanol :glacial acetic acid :water
4: 1:5, upper layer). Substances with Rf between 0.05 and 0.95 and that
fluoresce blue - or mauve in UV light (try with and without ammonia)
and also give a blue reaction with Folin or femc chloride reagents are
likely to be hydroxycinnamic acids or their derivatives. Spots reacting
this way may also have been seen on the previous TLC. If these acids
are suspected, a better yield will be obtained from a mild(er) alkaline
hydrolysis of the plant extract (Harbome, 1984). Cinnamic acid is one
exception to be aware of, as it fluoresces yellow under UV.
2.4 Take another part of the acid hydrolysate and extract with ethyl
acetate, concentrate the ethyl acetate layer and perform one-dimensional
TLC on cellulose using Forestal mixture (glacial acetic acid:concentrated
HC1: water 30 :3 :10) as the solvent. Detect anthocyanins as mauve-
purple-red-orange or occasionally yellow spots in visible light. Chalcones
and aurones (yellow flower pigments) give red colours with ammonia
when present. Under UV light, spots revealed by bright yellow fluoresc-
ence are likely to be flavonols while flavones appear dull brown but
fluoresce yellow-green in UV after fuming with ammonia. Biflavonyls
remain dull brown even after this treatment.
2.5 If the aqueous phase of the hydrolysate remains coloured after ethyl
acetate extraction, reextract with amyl alcohol. Anthocyanidins are ex-
tracted particularly successfully by this procedure and can be chromato-
graphed after evaporating the amyl alcohol. In particular, the production
of cyanidin and/or delphinidin by the hydrolysis of condensed tannins
can be confirmed by their Rfs and colours on Forestal chromatograms:
cyanidin 0.49, magenta; delphinidin 0.32, purple.
3 By performing the two-dimensional chromatograms from 2.1 and 2.2
above and the Forestal chromatograms (2.3, 2.4) and spraying each with
the reagents that you employed for total phenolics determination (Folin
or ferric chloride) you will see which systems give the greatest reactivity
and so gain some sense of whether the total phenolics are likely to be
all one substance, or mostly flavonoids, or phenylpropanoids or simple
phenolics. By performing the same TLC analyses with the unhydrolysed
extract, phenolic aglycones present in the plant will be detected. Thus the
comparison of TLCs of hydrolysed and unhydrolysed extracts can provide
information as to whether the live plant contains free or glycosylated
phenolics. For flavonoids in particular, it is likely that several glycosides
of a single aglycone will be present. A good two-dimensional system to
resolve these is with cellulose and n-butano1:glacial acetic acid:water
(4:1:5, upper layer) as one solvent and 5% acetic acid as the other.
Detection of tannins by protein precipitation will suggest the probable
of gallic acid or ellagic acid (from hydrolysable tannins) and/or anth-
ocyanidins (from condensed tannins). This will be confirmed if these
phenolics are detected only, or in much higher concentrations, in TLCs of
the hydrolysates rather than in TLCs of the crude extracts.
4 If you now wish to definitively identify the substance present in a
particular spot (e.g. the most abundant one), you may still be able to
do this by TLC alone but it will require luck. Because Rfs of similar
substances can be so close, Harborne (1984) recommends that co-
chromatography with an authentic standard in at least four different
chromatographic solvent systems is required for reasonable certainty. We
agree. This means that you will need to find published Rfvalues for four
154 Chapter 7
chromatographic systems for the group of phenolics that you believe your
spot belongs to. Then you should duplicate these systems and determine
the Rfs for your spot. If all four Rfs are in reasonable agreement with
those published for a known compound then you should still try to further
verify the identity by obtaining an authentic sample and show exact co-
chromatography in all four systems. Needless to say, reactions with
several suitable spray reagents should also match for the unknown and
the authentic marker.
You may be lucky, particularly if you are concerned with a common
aglycone. However the level of excitement at discovering the presence of
myricetin or chlorogenic acid in yet another plant may not be so high as
these are so common. If the phenolic is exotic and rare then be prepared
to be very frustrated as this is precisely the situation where Rf data and/or
standards are likely to be unavailable. Further frustration arises when
your Rfs may match a known compound in some systems but not others
and you cannot tell whether the room temperature or brand of TLC plate
(or any one of a number of other variables) is the real cause of the
mismatch. Worse still, you may successfully identify an aglycone knowing
that some unknown glycoside is actually present in the plant. TLC can be
used to identify the sugar but its point of attachment is harder to identdy.
If you really want to identify the substance and have been unsuc-
cessful by chromatography alone, at the very least you now need to find a
preparative TLC system to generate a small pure sample for UV-VIS
spectrophotometry and then proceed to Chapter 8.
7.2.4 Preparative TLC (PTLC)

Whereas for analytical work TLC plates usually have a thickness of
0.1-0.25mm they may be spread to between 0.5 and 2mm thickness for
preparative TLC. For PTLC the technique usually employed is to apply
the extract as a streak about 2cm up from the bottom of the plate (Fig.
7.2) and then after running the plate the bands formed are visualized (by
UV light or by spraying the edges of the plate) and are then scraped from
the rest of the plate and extracted from the absorbant by washing with a
polar solvent, usually methanol. The most difficult point to get right in
PTLC is the polarity of the solvent. It is generally better to employ a
solvent that moves compounds less far than that which is most appropriate
for analytical TLC. One useful strategy is to use a solvent which moves
no important compounds in the extract more than an Rfof 0.3 and to
carry out double or triple elution. That is, the plate is run, dried, run
again, etc.
A variant of PTLC that is initially fairly expensive to acquire but
which is often very rewarding is the centrifugal PTLC system. This con-
sists (Fig. 7.3) of a circular plate, coated with a 1-4mm layer of ab-
SOLVENT
I
Sprayed portion of plate
Fig. 7.2 Preparative TLC plate with extract applied.
sorbant, which is spun in an inert (nitrogen) atmosphere, usually under a

UV transparent quartz screen. The sample to be separated is infiltrated
onto the inner edge of the circle in the least polar solvent possible.
Elution can take place by passing the appropriate solvent (pumped or
gravity fed) over the plate and collecting it in fractions when it is spun off
the outer edge of the plate into the solvent gutter. Resolution is several
times better than TLC, solvent mixtures of increasing polarity can be
used during a single run, and samples of up to 400mg material can be
applied and separated. The passage of phenolic compounds on the ab-
sorbant can be followed using UV light.
7.2.5 Column chromatogtnphy (CC)

The various forms of CC, other than the very short 'clean-up' columns
already mentioned, are primarily preparative techniques and tend to be
more expensive than TLC as they use more media and solvent. Make
sure the amounts of material they can provide are really required before
embarking on what could be a messy business.
The most typical form of CC is a long, thin column of absorbant on
top of which is placed the sample, usually after it has been dried onto a
small amount of the same absorbant. The column is then eluted with a
156 Chapter 7
tral spindle
Application
of solvent
at inner edge of \ Rotation

absorbant
Fig. 7.3 Circular preparative TLC procedure.
range of solvents of increasing polarity. As a rule of thumb a well packed,

efficient column should be able to cope with about 1g of extract for every
50-100g of absorbant. CC is really rather a difficult procedure and needs
practice to perfect. Its resolving power is limited and it will often need to
be followed by PTLC or some other refining procedure to obtain pure
compounds.
Vacuum liquid chromatography (VLC) is a 'quick and dirty' CC
procedure in which the absorbant bed is reduced in depth and increased
in breadth. Its purposed is not to achieve complete separation but to 'cut'
the extract up into a number of bands each containing a few compounds.
This is achieved by using a solvent sequence of increasing polarity and
collecting large fractions of 50-200ml which are sucked through the
absorbant bed under a mild vacuum.
Flash chromatography (FC) is essentially the opposite to VLC. Here a
long column is set up, with a relatively ,small amount of extract placed at
the top. The solvent is then pushed through under pressure. FC is the
most difficult of all the CC techniques and also the slowest; it is, however,
perhaps the most likely to result in a pure product.
For all these CC procedures the most widespread absorbant is again
silica gel and both this and alumina will separate simple phenolic com-
pounds if the solvent is right. These are not to be recommended where
polyphenols are expected as there is poor resolution and excessive streak-
ing. Reverse phase silica gel is available but it is relatively expensive.

Microcrystalline cellulose and polyamide, with partly aqueous solvents,
offer promising alternatives for polyphenols and glycosides. The main
problem with both of these is that they have low porosity and as a result
columns are slow running, even using the FC technique. One potential
answer to slow flow rate is to mix the absorbant with Celite or polyvinyl
pyrrolidone (PVP) to make a more porous filter bed (NB- don't use PVP
for tannins!). An example of the use of several CC procedures in the
separation of phenolic compounds, including glycosides (Scheme 7.1), is
given by Conner et al. (1990).
7.2.6 Gas chromatography (GC)

Compared with what has been discussed above, gas chromatography is
a technique that brings additional dimensions and possibilities for the
quantitative and qualitative analysis of phenolics, particularly where their
structure or structural class is known. It will rarely, if ever, be of use
in isolating 'unknown' compounds, or for their subsequent identification
unless coupled with mass spectroscopy (see Chapter 8). Co-chromato-
graphy against known standards is really the only avenue towards wm-
pound identification with this technique.
The GC procedure consists of partition of components between a
mobile gas phase (helium, hydrogen or nitrogen) and a stationary phase
which is normally a non-volatile liquid, but can be a solid. The operating
temperature of the column can be varied during chromatography so that
compounds of lower volatility can be eluted from the column by raising
the temperature during the analysis (i.e. temperature gradient elution).
Conventionally, separation is achieved using either a glass column about
2m long with a diameter of 4-5 mm or a (fused silica) capillary column of
25-50m length and 0.2-0.4mm internal diameter. The latter is far more
expensive but does hold out the possibilities of far better resolution.
Various methods are available to detect compounds when they are
vented from the column. The most commonly employed is the flame
ionization detector (FID) in which the material is burnt in a hydrogen
flame which induces a small electric current which can be measured. The
FID response is proportional to the number of oxidizable carbons in
the molecule and is, therefore, semi-quantitative. An FID will normally
detect compounds down to the level of about 10-'g. The electron capture
detector (ECD) is similar in concept to the FID but instead of using a
hydrogen flame as an electron source passing between two electrodes it
employs a radioisotope. Although more expensive it has the advantage of
increased sensitivity and is reported to be of value down to concentrations
of 10-~g. Both have been successfully used with phenolics. Greatest
sensitivity in GC analysis is achieved with the linked GC-mass spec-
158 Chapter 7
Scbeme 7.1 Separation of phenolic compounds by dolumn and

thin-layer chromatography
Freeze dried methand extract (2509)

Dissolve in water, partition
with ethyl acetate and with
chloroform-isopropanol(1.1)
water soluble organic sdvent soluble
I
concentrate
supernatant
homonataloln (5.5 g)
Concentrate to solid (28 g).
Separate on acid-washed silica
gel column eluting with chloroform
containing increasing amounts of
methanol
I I
2.5% methanol 5% methanol 7.5% methanol
I
10% methanol
novel chromone
I
rnlxture
I
mixture
Polyamide:PVP (1 :1) Polyamide:PVP (1 :1)
column eluting column eluting with
with water water then PTLC
aloerastn B (150 mg) aloesin (100 mg)
supernatant
Pdyamide:PVP (1 :1)
column e h n g with
water
2'-0-tlgloylalaosln (105mg)
trometry system which also adds the possibility for identification of un-
known constituents (see Chapter 8).
If considering GC as a method to analyse phenolics and assuming that
you have a suitable instrument available then two major decisions have to
be made. Firstly, what type of stationary phase should be used? Secondly,
how is it best to present the samples to the column?
7.2.6.1 The stationary phase

There are some 200 different stationary phases available presenting, to
the uninitiated, a bewildering choice! In reality only a few need to be
considered and most of these are listed in Table 7.4. It is vital to take
particular note of the upper temperature limit of the particular stationary
phase and stay within those limits: once a column is 'cooked' it is useless
and if it was the capillary type then that will be expensive!
7.2.6.2 Sample preparation

These comments assume that the sample has already been subjected to a
preliminary 'clean up' (see above) and is therefore in a suitable condition
for GC analysis. The additional necessity here is that the compounds
need to be volatile and this is naturally the case for only a few phenolics;
those that are volatile will often still 'tail' badly on the column giving
asymmetric and non-quantitative peaks. Almost invariably therefore a
vital procedure to be undertaken is sample derivatization to convert the
non-volatile phenolics into volatile derivatives.
The most common procedure adopted in derivatization is the pro-
duction of trimethylsilyl (TMS) ethers. This can be readily achieved using
a number of commercially available silylating agents which are reacted
with the sample in pyridine. Alternatives to silylation include methylation
(best for acids), acetylation, and trifluoroacetylation (good for sugars).
Some examples are given in Table 7.4.
A very good example of the use of GC in the analysis of phenolics
is the study of the bud exudate of Populur lasiocarpa by Greenaway,
Table 7.4 Some examples of the use of GC in the analysis of phenols
Compound type Column type Derivatives Conditions Reference
Simple phenols Carbowax ZQM tert-butyl ethers 90-200" Doring et 01. (1985)
Phloroglucinols Carbowax ZQM methyl ethers 100-185" Pyysalo & Widen (1979)
Simple glyws~des SE-52 TMS ethers 190-295" Julkunen-Tiitto (1985b)
Phenolic acids CP-Sil5CB TMS ethers 140' Ford & Hartley (1988)
Phenylacetic acids OV-17 methyl esters 40-250' Liebich & Pickert (1985)
Bud exudates OV-1 TMS ethers 85-300" Greenaway, SMysbrook
& Whatley (1988)
160 Chapter 7
Scan
Fig. 7.4 GC separation of TMS ethers of phenolic compounds from Populw laviocarpa
(after Greenaway, Scaysbrook & Whatley, 1988).
Scaysbrook, and Whatley (1988). Separation of some 60 phenolic com-

pounds (Fig. 7.4) was achieved using TMS derivatives prepared by heat-
ing a small amount of the extracted material dissolved in 50 pl of pyridine
and 100N of bis(trimethylsilyl)trifluoroacetamide containing 1% trime-
thylchlorosilane at 100°C for 30minutes. The resulting derivatives were
chromatographed on a SO m X 0.3 mm inner diameter silica column bonded
with OV-1 using helium (20psi) as canier gas. The injector temperature
was set at 310°C and the oven temperature was raised from 85°C to 310°C
at a rate of 3"CImin.
In our view GC is an attractive method for the analysis of relatively
simple phenolic compounds and can be very powerful where quantitation
of known compounds or an analysis of quantitative differences between
samples is required. On the topic of quantitation there are two proce-
dures that can be adopted depending on the type of quantitative infor-
mation required.
1 Relative concentrations of components. This can readily be established
by simply expressing the area under any given peak as a percentage of the
total area for all periks (other than the solvent). This can be achieved by
the use of a simple electronic integrator.
2 Absolute concentration. To obtain an absolute value for concentration
of a component in terms of the weight of plant material used it is neces-
sary to add an internal standard. In such cases all stages of sample pre-
paration must be undertaken in a quantitative manner and the standard
added, usually at the stage of preparation of the sample for GC injection,
so that the concentration injected is known. It is preferable to have the
standard elute from the column in a relatively unpopulated part of the
elution profile and not at either the beginning or the end. Selection of
the correct standard can be tricky problem.
7.2.6.3 Reporting GC data

Literature on GC procedures can often be frustrating for what it leaves
out. The following should be obligatory: (i) length and diameter of
column; (ii) the stationary phase and its concentration; (iii) the gases used
and flow rates; (iv) type of detector; (v) temperatures at injection port, in
the oven and at the detector; and (vi) temperature changes in the oven
during the run. It is in our view preferable to report the elution profile of
compounds in terms of relative retention times (RR,) against a standard
compound. The standard compound can be introduced or a known com-
ponent can be used. Retention times alone are not very reliable. More
sophisticated methods of indicating elution profiles do exist but they are
rarely necessary.
7.2.7 High performance liquid chromatography (HPLC)

The processes of separation involved in HPLC are comparable to those of
TLC and CC but particle size of the absorbant is much reduced (3-104
and solvents need to be pumped through the columns under high pres-
sure. Simple HPLC systems use one pump which pumps a single solvent
or solvent mixture through a column. This is known as isocratic HPLC.
More complex gradient systems use multiple pumps governed by a wm-
puter and pump progressively changing mobile phases which can involve
several solvents. Almost any solvent can be used with HF'LC but care
must be taken with quality; columns are easily ruined by impurities such
as metal ions. Solvents must be degassed, otherwise under high pressure
gases will be forced out of solution and will interfere with uniform
pumping of solvents and with detection. Degassing is easily achieved,
most commonly by passing helium through the solvent for about 20
minutes prior to its use. Column efficiency and reproducibility can be
enhanced by working at a constant temperature.
For analytical HPLC the column can vary from 5-25 cm in length and
4-6- in inner diameter, with a typical flow rate of 1-Zmllmin. For
small scale separation column inner diameter can be increased to 1-2.5 cm
and column length to 50cm with a concomitant increase in.flow rates of
solvents (up to 100ml/rnin). The most common column packing used for
the analysis of phenolics is reverse phase silica, usually employing a CI8
(octadecyl silica, ODs) or more rarely Cs-ODs stationary phase. These
wlumns, contrary to normal silica columns, elute polar compounds more
rapidly than non-polar compounds. There are a plethora of different
makes of column, each of which may have its own advantages for doing a
particular job. On the whole, however, most makers supply a good
162 Chanter 7
Table 7.5 Examples of HPLC systems for the separation of phenolic compounds
Compound type Column type Solvent References
Simple phenols ODS lOpm 5% aq. HCOOH; MeOH Van Casteele, Geiger
& Sumere (1983)
Resorcinols ODs 5 pm Yeung, Nacht & Gans
(1981)
Phenolic acids ODS3pm 5% aq. HCOOH; 75% aq. Wilson (1985b)
MeOH
Gallic acid ODS5pm 0.5% aq. phosphoric acid; Delahaye & Verzele
0.5% phosphoric acid in (1983)
MeOH
Hydroxy- 1.5% aq. phosphoric acid; Meurer et al. (1988)
cinnamoyl amides 1.5% aq. phosphoric acid +
+
20% acetic add 25%
acetonitrile in water
Anthocyanins ODs various Phosphate buffers and Strack & Wray (1989)
acetonitrile (common
solvents)
Tannins ODS 10 pm 1 mu phytic acid in 0.1% Putnam & Butler
acetic acid; (1989)
aceto~trile-water-
acetic acid (800:200: 1) Scalbert, Monties &
water-phosphoric acid Favre (1988)
(990: 1); methanol-
phosphoric acid (990: 1)
Xanthone Acetonitrile-water-acetic Hayashi & Yamagishi
glycosides acid (u):80: 1) (1988)
quality ODS column. Phenyl columns, where the ODs is replaced by a

phenyl ether, are also reported to be useful (Blo, Dondi & Corrado,
1984). One practical point is that columns are generally very expensive
and with fairly crude plant extracts they can deteriorate fairly rapidly.
Protection of the main column by adding, in line, a small guard column
containing the same absorbant is always worthwhile and can increase the
effective life of a column many times over. Sowe examples of useful
HPLC systems and the solvents they employ are given in Table 7.5.
The information given in Table 7.5 is meant only to indicate some
possibilities. Many HPLC systems will deal quite readily with a wide
range of phenolic substances. An excellent example (Fig. 7.5) is the study
of phenolics from Norway spruce needles (Strack et al., 1989) where
HPLC analysis on a 4 p ODS column of 25cm X 4mm dimensions,
eluting with a two step linear gradient (Table 7.6) at 1.5mllmin allowed
.w
m
I I I
0 10 20 30 40 50 0 10 20 30 40 50
Time (min) Time (min)
Rig. 7.5 HPLC separation of phenolic compounds in Noway spruce needles (from Strack
et d., 1989), showing variation in spectrophotometricresponses due to detection at different
wavelengths.
Table 7.6 Gradient solvent system employed by Strack et a[. (1989)
Xme (min) 0 40 70
Solvent A (1.5% aq. phosphoric acid) 100 f50 0

Solvent B (water-acetonitrile-methanol (1 :1 :1) 0 40 100
the separation
. of simple phenols and their glycosides, lignans, flavanones,
flavones and flavone glycosides, stilbenes and cinnamic acids.
Strack et al. (1989) employed a UV detector in their study and this
is by far the most common method to use in HPLC investigations. How-
ever, UV detection does have one drawback in that when used at a fixed
wavelength the results it will give can be very misleading. This is why
Strack et al. used two different wavelengths (Fig. 7.5). Figure 7.6 shows
the UV detection traces for the HPLC separation of a mixture of caffeic
acid (15) and catechol(5a) where the detector was set at: (i) 280 nm; and
(ii) 320 nm. This discrepancy is due to differential UV absorption of the
two compounds at these wavelengths (Fig. 7.7) and obviously has major
implications for any attempt to employ UV detection in the quantitative
164 Chapter 7
.15
280 nm
A n - AU
.15-.05
320 nm '
I
- AU
-
7 -.05
0 10 20 30 40 50 60
Minutes
Fig. 7.6 UV detector responses for equimolar samples of a caffeic acid and catechol run at
280nm and 320nm. AU, absorption units.
250 300 350 400 450 500
- .15
- '1
- .05
-0
...-....
r'
250 300 350 400 450 500
Wavelength 250-502 nm
Fig. 7.7 UV spectra of caffeic acid (broken line) and catechol (solid line).
Time range (minutes)
Fig. 7.8 Photo-diode array generated 'contour plots' for the HPLC analysis of caffeic acid
and catechol.
analysis of mixtures of unknown compounds. This problem can to some

extent be overcome by the use of photodiode array detectors which have
the capacity to measure UV absorption simultaneously at many different
wavelengths. Results from photodiode array detectors can be visualized
in a number of different ways, including contour plots of the type shown
in Fig. 7.8.
While UV detectors are and will certainly remain the most widely
used there are alternatives. Most notable among these are fluorescence
detectors (usually they require preparation of a fluorescent derivative)
and electrochemical detectors. The latter may well prove to be a very
useful method for detection of phenolic compounds.
For analytical use of HPLC the same arguments apply as were dis-
cussed under GC, with of course the added probIem of variable detector
response if the analysis is aimed at the quantification of a range of
structures with variable chromophores. However, despite these problems
it is a very important technique and the preferred procedure for most
investigations that involve the analysis of complex mixtures of phenolics.
It is equally valuable, in the preparative mode, in the final purification of
single compounds, particularly where the need is for small amounts of
highly pure material.
7.2.8 Solvent-solvent partition or countercurrent methods

Partition of organic compounds between two immiscible liquid phases
has always been a valuable method for separating different classes of
166 Chapter 7
Table 7.7 Some examples of the use of countercurrent chromatography in isolation of

phenolic compounds
Compound type Solvent(s) References
Simple phenolics Chloroform-methanol-water Muira, Panetta & Metzger

(37 :37 :26) (1988)
n-Butanol-saturated aq. barium Weisz, Harkey & Ito (1986)
chloride (1 :1)
Flavonoids Chloroform-methanol-water (4 :3:2) Zhang, Cai & Ito (1988)
Condensed n-Butanol-1 M sodium chloride Putnam & Butler (1985)
tannins containing 1 m~ phytic acid (1: 1)
Polyphenols Chloroform-methanol-water Marston & Hostettmaon (1987)
(43:37:20)
Anthocyanins n-Butanol-acetic acid-water (4: 1 :5) Francis & Andersen (1984)
Billavonoids Chloroform-n-butanol-methanol- Markham, Andersen & Viotto
water (10: 1 :10:6) (1988)
Xanthones Chloroform-methanol-n-propanol- Hostettmann, Hostettmann &
water(9:12:1:8) Sticher (1979)
compounds. For example chloroform-soluble phenolic compounds can be

partitioned into a dilute aqueous sodium hydroxide or sodium carbonate
solution by virtue of the fact that, at the interface between the two
phases, they can be converted into the water-soluble phenates. If the
partition rate is as low as 50:50 then by successively washing the chloro-
form with five batches of the alkaline solution, in excess of 95% of the
material (50 + 25 + 12.5 + 6.25 + 3.125) can be transferred into the
aqueous phase.
It is much more difficult to use this technique to purify individual
compounds from complex mixtures but two related procedures, droplet
counter current chromatography (DCCC) and centrifugal counter current
chromatography (CCCC) are now available. Both of these provide the
facilities to permit a very high number of transfers between two immis-
cible liquid phases and, if you get the solvents right, very high levels of
separation are possible. DCCC is the older of the two techniques and in
its normal form consists of a series of some 300 narrow bore glass tubes
filled with a stationary liquid phase. Through this a mobile phase is
passed as discrete droplets. The material to be separated will partition
between the two phases and individual compounds with varying partition
coefficients will be found either retained within the series of tubes or will
be eluted with the mobile phase. Detection of compounds eluted can be
achieved by using a UV detector. CCCC differs in that control of solvent
movement is governed by means of a pump and the use of centrifugal
forces. This technique has the advantages of greater control, use of less
solvent, and greater resolution which allow some scope for analytical,
quantitative use as well as qualitative or preparative studies. CCCC seems
to be the more practical procedure to adopt if there is a choice. Useful
introductions to the technique have been provided by Hostettmann,
Hostettmann and Marston (1986) and Conway (1989).
In our view the value of this procedure is underestimated, particularly
for the separation of mixtures of glycosides. Some examples of the use of
countercurrent chromatography are given in Table 7.7.
7.2.9 Electrophoresis
Paper electrophoresis has been utilized in the analysis of phenolics but
in most cases can be regarded as inferior to paper chromatography.
Exceptions are to be found in charged species such as anthocyanins and
flavonoid salts, such as sulphates. Examples of these specialized uses can
be found in Harborne (1989).
CHAPTER 8
Structure elucidation of phenolics
8.1 Introduction
If you are dealing with a plant extract containing phenolic compounds
then it is intellectually satisfying to know the structural identity of at least
the more important of these compounds. Intellectually satisfying it may
be but before embarking on the quest for a structure be sure that you
need to know it! Reasons why such knowledge could be vital include:
studies of herbivore adaptation involving detoxification mechanisms; the
understanding of induced responses to extrinsic factors; and the identifi-
cation of individual compounds responsible for some observed biological
effect. It is possible (even probable) that the compound(s) that interest(s)
you are already known and to confirm structures can sometimes then be
simple. But, on the other hand, there is the chance that they might be
novel and complex, containing sugars and esterlfying groups in addition
to the aromatic phenolic skeleton.
If the problem you are confronted with does not resolve readily using
one of the short-cuts listed in the next section then it is very likely that
you will have to enlist the help of an organic chemist who knows his or
her way about the problems of identifying natural products. They may, in
turn, have to go for assistance to specialists in one or more of the very
sophisticated (and expensive) spectroscopic procedures. We have tried in
., this chapter, as in the last, to deal with methods in a sequence in which
complexity and difficulty of interpretation increases. The rule is, once
again, start simple!
8.1.1 Short cireuiting the irien&pcation problem

In structure elucidation of 'unknown' natural products one of the most
common events is the reinvention of the wheel! In other words, much
effort goes into the isolation and identification of a compound which is
already known, perhaps even from that very species, or the presence of
which was predictable. A sensible rule to adopt is that prior to any study
that is likely to lead to an attempt at isolation and identification the
literature should have been thoroughly examined for relevant informa-
tion. The most exhaustive source for such information is Chemical Ab-
stracts and this should be searched under both Latin binomial and common
names. Note, this should not just be under the binomial you are using but
also under any others that have been used at any time in the recent past.
A 'feel' for the chemistry of the genus and family can be obtained by
Structure elucidation 169
reference to Chemotaxonomie der Pfanzen (Hegnauer, 1963-1992). This

is, in essence, a reminder of what was said in Chapter 2. There is, often, a
degree of predictability in what will occur where.
There are, however, caveats to be added. Most important is to re-
member to treat what you find in the literature with a healthy scepticism.
Chemists are not, in general, overly worried about taxonomy and no-
menclature and identification of botanical sources is sometimes inaccur-
ate. Reported structures may also be wrong - it happens!
If there are recent relevant publications on your species, or one that is
closely allied and may contain the same compounds, then you should
attempt to obtain small amounts of the reported compound or com-
pounds. Only 1-5 mg are necessary for comparison using chromatographic
and spectrophotometric methods. It is also possible to buy some natural
from commercial sources, although quite often the prices.charged
are excessive. If funds are available it will certainly be worthwhile build-
ing up a library of the simpler phenolics, notably the variously substituted
benzene derivatives (5-ll), the common cinnamic acids (14-17), and
perhaps even a few of the simpler flavonoids such as the flavonols (43-45).
8.1.2 Comparison of an unknown with an authentic sample

As noted above an 'authentic' sample can be purchased commercially or
acquired through a request made to a scientist who has isolated and
identified it previously. While such samples are always sent in good faith
a prudent worker will still treat them with a degree of caution. One of us
spent many unfruitful hours trying to relate a commercial sample of
hypericin (70) to a semi-pure extract painstakingly obtained by repeated
column chromatography. The commercial sample (the supplier shall re-
main nameless) eventually turned out to be more-or-less pure chlorophyll!
It pays, therefore, to confirm identity of an authentic sample by as many
of the methods described below as time and circumstances permit.
The obvious first step in a comparison is co-chromatography. Thin-
layer chromatography (TLC) is the cheapest and quickest but remember
not to rely on a single TLC system. There are, at most, 100 measurable
Rfvalues so that common sense tells you that on a single system there is a
significant chance that Rf values for different compounds will be the
same. Our recommendation is to employ four systems at least. High
performance liquid chromatography andlor gas chromatography are other
valuable comparative techniques and have the advantage that unknown
and authentic samples can be injected together for a direct comparison.
Comparative UV spectra (see below) is another useful test that can be
performed in very small amounts of substance.
If more material is available and it is pure then a mixed melting point
will be useful. If the authentic compound has a sharp melting point then
170 Chapter 8
on mixture with your sample of the same compound there should not
be any change in melting point. Other spectroscopic procedures (all
discussed below) can now come into the reckoning. Infra-red (IR) gives a
great deal of detail. Little of that information is easily understood but this
is not particularly important; two superimposable IR spectra are very
good evidence of co-identity even if most of the bands are not rationalized.
Nuclear magnetic resonance (NMR)may allow the separation of isomeric
forms of the same general structure while measurement of optical activity
in any of several ways will allow the distinction between isomeric forms
and stereoisomers. These last few points may sound like 'ultra nit-picking'
by a chemist but sensory perception and other forms of bioactivity are
known to be radically altered by stereochemical variation.
8.1.3 Isolating compoundsfor identification

The mechanisms and procedures for dealing with the isolation of phe-
nolics have been dealt with in Chapter 7. All of the chromatographic
andlor partition procedures discussed there are, with varying degrees of
difficulty, capable of being scaled up to yield milligram (or sometimes
greater) quantities of pure compounds. It is almost certainly through
these procedures that any isolation and purification will take place.
8.2 Identification by spectrophotometric and

spectroscopic techniques
In the rest of this chapter the assumption is made that you have a 'need'
to identify a specific phenolic compound and have been able to obtain it
in pure form (although quite possibly only as a 'smear' in the bottom of a
test tube). Some of the procedures to be considered are based on direct
measurement of the ability of a compound to absorb energy in the visible,
UV or IR portions of the electromagnetic spectrum. Ultra violet/visible
(UVNis) spectrophotometry is a technique that will be readily available
to almost anyone and, as we will see, can impart a great deal of very
valuable information. It also requires very small (pg) amounts of material.
Infra-red spectrophotometry is analogous to UVNis in many ways but
sample preparation is more of a problem and, unless for comparison with
an authentic sample, the resulting absorption profile (spectrum) is far
more difficult to interpret.
Other techniques (optical rotation (OR), optical rotatory dispersion
(ORD), circular dichroism (CD)) also examine the way in which a solu-
tion of a substance interacts with light in the UVNis range. Here,
however, it is not absorption that is being considered but the capacity
(if any) of the substance to influence the way in which the light behaves,
notably by causing polarized light to 'bend' (so-called chiroptical methods).
Simple OR measurements can be made in any well equipped laboratory

but ORD and CD are somewhat more specialized techniques.
The remaining two procedures are the most powerful in terms of
actually assigning structures to unknown substances. Their operation and
the interpretation of results obtained from them are far more difficult and
certainly beyond the scope of the beginner. Here you need to collaborate!
Mass spectroscopy (MS), in its various forms, examines the effects of
bombarding a molecule with small particles (most commonly electrons)
and analysing the way in which it breaks up (fragments). The procedure
will often give the molecular weight and also information of various
structural elements within the molecule. It is the only one of the pro-
cedures outlined in this section that is actually destructive of material,-but
quantities required are again very small (less than 1mg).
-
Nuclear magnetic resonance (NMRI is the most informative of all the
techniques and through it the intramolecular environments and spatial
relationships between protons ('H isotope) can be investigated. The tech-
nique can also be used to study the carbon skeleton and the relationships
between hydrogen and carbon atoms. Unfortunately, however, for car-
bon NMR only the 13C isotope is suitable and this makes up only 1%of
the natural carbon in the molecule. This makes 13cNMR inherently
insensitive and despite the considerable advance in instrument sensitivity
due to modern techniques this means that samples have to be of several
milligrams.
Note: One structure elucidation technique not discussed here is X-Ray

crystallography. If a structure is intractable and you can grow a good
crystal then this technique represents the ultimate in structure deter-
mination. Your part in the process will almost certainly end when you
hand over the crystal.
8.2.1 Ultra-violet and visible spech.ophotometry

Samples for UV (220-370nm) and/or visible (370-800nm) spectropho-
tometric analysis can be made up in any UV-transparent solvent. This
requirement eliminates the use of substances such as benzene, pyridine
and acetone. Traditionally, the most widely employed solvents are eth-
anol or methanol, and we would advise the use of these. Sample pre-
paration is perfectly straightforward, consisting of: (i) dissolving the test
substance in the solvent and ensuring the absence of particles; and (ii) the
transfer of an aliquot to a suitable silica cuvette (usually 1cm optical path
length). A second 'matched' cuvette is filled with the solvent and acts as a
blank.
The spectrum is preferably measured by a scanning, double-beam,
instrument in which two identical light beams pass through the solvent
172 Chapter 8
(blank) and test cells and the degree of absorption is assayed by com-
paring the light intensities on the detectors after passage through the test
and blank solutions. In the course of the experiment the wavelength of
the light passing through the cuvettes is varied at a constant rate between
pre-ordained minimum and maximum wavelengths. It is quite feasible to
produce such measurements on a single-beam instrument of the type
used in the assay procedures described in Chapters 4 and 5. However,
this requires the instrument to be zeroed at each wavelength and the
absorption then taken - a process that may need to be repeated up to 200
times (or more) to obtain a spectrum measured to 1nm resolution. Three
points in particular should be noted.
1 The need for matching cells as variation between test and blank
caused by imperfections in the cuvettes will lead to spurious differences in
apparent absorption. This is easily checked by initially filling both test
and blank cells with solvent alone and running the experiment with these.
A 'straight-line' is expected but in practice a small deviation (1-2%) is
generally acceptable.
2 The need to use silica cells if analysis involves measurement in the UV
region. Glass cells, which are perfectly acceptable for measurement in the
visible range, will absorb UV light!
3 At the beginning of the experiment you must decide whether or not
you wish the UVNis spectrum to be quantitative. If you are simply
interested in producing a spectrum and observing the wavelengths at
which maxima and minima occur then it is only necessary to prepare a
solution of the test substance and make a dilution (probably by trial and
error) at which all maxima can be measured without being above the
selected absorbance maximum. However, it is often valuable to calculate
molar extinction coefficients for maxima and minima and to be able to do
this it is necessary that the exact concentration of a solution is known.
The molar extinction coefficient ( E ) , at any given wavelength, is com-
puted using the following equation:
where, A = absorbance, 1 = path length, c = concentration in M.

The extinction coefficients (maximum and minimum) are often ex-
pressed to the power of loglo (log E ) . The UVNis maxima of some typical
(common) phenolic compounds are given in Table 8.1. All measurements
were obtained in methanol or ethanol.
Light is absorbed by functional elements within molecules that are
called chromophores. From your viewpoint all that it is necessary to know
is that the smallest UV-absorbing component is the double-bond, as
found in the carbonyl group. Acetic acid, for example, will absorb at
208nm while acetone absorbs at 166 and 189nm. As a chromophore
grows in size it will absorb at higher wavelengths. Thus the benzene ring
Strncture elucidation 173
Table 8.1 UVIVisible maxima of some common types of phenolic mmpounds
Compound W maxima (nm)
Simple phenols
catechol(5a)
phloroglucinol(6)
Simple aldehydes and acids 250-325 (usually two bands)
gaUic acid (8) 270
salicylaldehyde (10) 243,325
protocatechuic acid (11) 258,292
Cinnamic acids 285-330 (two bands)
para-coumaric acid (14) 295,312
sinapic acid (17) 302,327
Stilbenes 280-330 (2-3 bands)
lunularic acid (33) 281,287,310
Flavanones and flavanols 270-295 (main hand) with smaU secondary usually
310-350
289 (326)
Flavones 245-350 (three bands)
lutwlin (38) 253,267,291(sh), 349
Flavonols 250-380 (up to 4 bands)
kaempferol(39) 266,294(sh), 322(sh), 367
quercetin (40) 255,269(sh), 301(sh), 370
myricetin (41) 254,272(sh), 301(sh), 374
Anthocyanins 270-280,500-550 (solvent 0.1% HCI in methanol)
Chalcones 220-270,340-390 (latter of higher intensity)
Aurones Main band usually 380-430
Coumarins 246-350 (2-5 hands)
scopoletin (82) 252,259,297,344
xanthotoxin (26) 247,262,293
Xanthones 230-390 (up to 4 bands of decreasing intensity)
250-450 (3 bands naphthoquinones and 4 bands
anthraquinones)
juglone (68) 249,345,422
emodin (69) 254,267,290,440
sh, shoulder.
system found in simple phenols (e.g. 6-11) absorbs in the 260-280nm

region (see Table 8.1) while phenylpropenoids (e.g. 14-17) and cou-
marins (e.g. 82) have maxima at higher wavelengths (over 300nm). What
is more, as chromophores get bigger and more complicated, distinct
absorption maxima can be seen which are due to just part of the whole
chromophore.
Note that the size of a chromophore is not simply the sum of the
174 Chapter 8
Fig, 8.1 Flavone (A) and flavanone (B) skeletons. The former wnsists of a single
chromophore, the latter is two distinct chromophores separated by the saturated nature of
the 213 bond.
number of double-bonds present. Consider the flavone and flavanone

skeletons (Fig. 8.1) and their UV spectra (Table 8.1). In the flavone the
chromophore extends through the whole molecule because the double-
bonds are conjugated with each other (that is they are linked to each
other as they are separated by only one C-C bond). By contrast in the
flavanone there is discontinuity because of the absence of a 213 double-
bond. This means that to all intents and purposes the flavanol consists
of two relatively small chromophores and this is confirmed by the oc-
currence of its major maximum below 300nm whereas the extended
chromophore of the flavone leads to major bands above 300nm.
The situation is made more complicated by the fact that substituting
groups (known as auxochromes) have the capacity to alter the position at
which maxima occur. In Table 8.1 an example of the effect of different
hydroxylation patterns on the absorption maxima in the flavones (39-41)
can be seen. It is not just hydroxyls that can act as auxochromes: methyl
groups (both C-methyl and 0-methyl), amino groups, sugars and halides
21 have a modifying effect on maxima.
8.2.1.1 Reagents used to modify spectra
Further information can often be gained from a UV spectrum by the use
of modifying reagents. Their application has reached a considerable level
of sophistication in work on flavonoids, where they can often be suc-
cessfully used to predict substitution patterns by locating the positions of
free hydroxyl groups (Mabry, Markham & Thomas, 1970; Harborne,
1984).
A modification in the UV spectra of all phenolic compounds is caused
by the addition of a small amount of sodium hydroxide. This leads to
ionization of the phenol with the production of the corresponding phen-
ate ion (see Scheme 1.la). This is seen in changes in the maxima observed;
most typically movement of the maximum at longest wavelength to an
even greater wavelength. This is known as a bathochromic shift and can
be small (c. 10nm) or very large (60nm or more). Examination of the
1;
i
i
1
Fig. 8.2 Pen relationship between carbonyl and phenolic hydroxyl. This leads to hydrogen
bonding and will show a strong bathochromic shift with aluminium hydroxide.
effect of sodium hydroxide is achieved by running the UV spectrum and

then taking the cuvette and adding to the alcoholic solution 2 drops of a
2~ solution of sodium hydroxide and repeating the measurement. This is
a simple, although not foolproof, method of confirming that a substance
is phenolic. Note that sodium hydroxide will cause some phenolic com-
pounds to decompose, catechol (5a) being a good example.
Other modifying reagents that are in common use are more selective.
A 5% solution of aluminium chloride can cause a large bathochromic
shift where carbonyl and phenolic hydroxyl groups are in a peri relation-
ship (Fig. 8.2). This reagent allows rapid distinction between the ben-
zopyrans (29, no shift) and (30, strong bathochromic shift). Addition of
sodium acetate powder to the methanol or ethanol solution (effectively
forming sodium methoxide or ethoxide) will cause bathochromic shifts
only where there are phenolic groups that are particularly prone to ox-
idation, typically the C-7 hydroxyl (Mabry, Markham & Thomas, 1970).
Further addition of boric acid to the methoxide or ethoxide solution will
cause additional bathochromic shifts where two phenolic hydroxyls occur
adjacent to one another. For example both quercetin (40) and myricetin
(41) would show a borate-induced bathochromic shift, but kaempferol
(39) would not.
8.2.2 Fluorescence spectrophotomeby

Some molecules, including many phenols, have the capacity to emit
energy in the form of light after irradiation with energy at a different
wavelength. This is the phenomenon known as fluorescence. Relatively
little use has been made of fluorescence in the identification of phenolics.
Fluorescence maxima for simple phenolic aldehydes and acids can be
anything from 365 nm for protocatechuic acid to 506 nm for saiicylaldehyde.
Fluorescence detectors are useful and very sensitive in HPLC analysis.
8.2.3 Chiroptical techniques

The importance of optical rotation has already been mentioned. AS
phenolic compounds are inherently aromatic, chirality or asymmetric
176 Chapter 8
centres, are not generally of importance. However, non-aromatic moi-

eties can bring in asymmetry (see for example 21, 22, 34-37). Optical
activity can give important information on the cisitruns composition of
monomeric units of condensed tannins (Porter, 1989). Much can be told
about the stereochemistry of condensed and hydrolysable tannins by
means of circular dichroism measurements, paying particular attention to
the shape and positive or negative values of maxima in the 200-280nm
region (Okuda et al., 1982; Porter, 1988).
8.2.4 Infra-red spectrophotometry

Infra-red instruments are designed along lines comparable to UV spec-
trophotometers. There are several methods of running samples. For solids
the most commonly used is to mix a small amount of the solid sample
with potassium bromide, which is IR transparent, and then convert this to
a disk by placing it in a dye and compressing it. The disk is then placed
in a holder and scanned, the spectrum arising from the differential ab-
sorption of energy by the compound in the disk at different wavelengths.
No blank disk is required. Alternatively, samples may be dispersed in
liquid paraffin (nujol mull) and then placed between two rock salt plates
when running the spectrum. Unfortunately nujol mull has an IR spectrum
of its own, and so blanks out part of the spectrum.
Infra-red spectra of organic compounds are usually quite complex,
producing a far greater number of maxima than UV spectra. Infra-red
spectra are much harder to interpret than UV spectra and in practice
only the parts of the spectra between 3000 and 36001cm and 1800 and
1600icm are usually of any immediate practical value. A strong absorp-
tion band in the former region (often broad) invariably indicates the
presence of a hydroxyl group (not necessarily a phenolic hydroxyl). Be-
ware of two facts: (i) weaker and generally sharper bands can indicate
N-H rather than 0 - H ; and (ii) be sure the potassium bromide is dry
otherwise water from the damp inorganic compound will show up in the
spectrum. Intense, sharp, bands in the 1650-1800icm range are indicative
of the presence of a carbonyl substituent and the exact absorption position
can sometimes be used to identify the structure in which the carbonyl is
involved (i.e. aldehyde, acid, lactone, etc.). Carbonyl absorption can also
occur between 1650 and lMX)lcm, in which case it may be difficult to
distinguish from bands caused by double-bonds and aromatic nuclei.
Having given you this information we would not encourage you
to spend much time or effort on IR spectrophotometry, except to
verify coidentity with an authentic sample. In this context being able to
super-impose spectra of authentic and unknown compounds in the aptly
named 'fingerprint' region (1500-804Ylcm) can offer compelling proof of
coidentity.
Structure elucidation 1i7
8.2.5 Mass spectrometry

Put at its simplest the procedure of mass spectrometry involves the
introduction of a substance to an ion source which bombards it and causes
molecules to be modified by becoming positively or negatively charged,
or causes the breaking bonds to form charged fragments (daughter ions).
These resulting ions are separated in a mass analyser according to their
masslcharge (mlz) ratio. These fragments and their relative intensity can
then be detected. The technique is highly sensitive (below 10-"g in some
cases) and while it does destroy material the quantities used are usually
insignificant. With sophisticated double focusing instruments where a
specimen can be run against a standard fluorocarbon polymer it is pos-
sible to gain accurate molecular weights for individual fragments, and
these fragments can be solved for a unique empirical formula. Where the
material under analysis is stable enough that some will survive charged
without fragmentation then this allows identification of the molecular
weight and empirical formula of the compound. The ion with the largest
masslcharge ratio on the spectrum is, therefore, very often the molecular
ion. This procedure uses far less material than would be used in the
traditional elemental analysis that would otherwise be necessary to find
the empirical formula.
There are two major decisions confronting you in the use of mass
spectrometry: (i) which kind of ion source is most appropriate; and (ii)
how do I interpret the results?
8.2.5.1 Ion sources

The most commonly available mass spectrometers employ the electron
impact ionization procedure. This requires the sample to be vaporized
and transferred into the ion source at very low pressure (c. 10K6Torr).
Here the vapour is bombarded by electrons of a predetermined energy
level (70eV is widely used). This is sufficient to cause fragmentation of
most organic molecules. Temperature of the source and applied eV can
be varied to alter fragmentation patterns. A common problem of using
electron impact with some phenolics is that they simply lack the volatility
required for transfer into the source. In such cases (e.g. biflavonoids,
tannins, glycosides) simple electron impact is a non-starter! This volatility
problem can often be overcome by forming derivatives, such as the
permethylated compound (we generally advise against acetates as MS
derivatives).
A fairly widely available alternative to electron impact is chemical
ionization, in which the ion source contains excess of a gas which readily
forms ions that can react with the sample. The most widely used reactant
gases for this purpose are ammonia or methane. The reactant gas is
bombarded with electrons with an energy of about 300eV at a pressure of
178 Chapter 8
about lTorr, whereupon ions are formed which can transfer tg the
sample under study. Thus, using chemical ionization the molecular ion
[MI+ will be replaced by a pseudomolecular ion which will analyse for
+
[M R]+ (where R is usually H). Ions produced in this way are relatively
stable so that with chemical ionization there is less fragmentation and the
pseudomolecular ion is generally more prominent than the molecular ion
would be in a comparable electron impact experiment. Volatility can still
be a problem.
Field ionization and desorption techniques are much less widely avail-
able but are valuable in studying polyphenolic compounds with low vola-
tility. Field ionization consists of vaporizing the sample followed by its
exposure to a powerful electric field at very low pressure ( ~ O - ~ ~ o r r ) .
This causes loss of an electron to give a charged molecule but there is
very little fragmentation. The procedure is therefore very valuable in
supplying information on molecular weights of compounds. Field desorp- -
tion is a derivative of field ionization in which the material is ionized prior

to vaporization and where vaporization can, as a consequence, often be
achieved at lower temperature. This makes field desorption particularly
valuable when thermal lability is suspected.
Finally, among procedures readily available, there is fast atom bom-
bardmeni mass spectroscopy (FAB-MS). Here the ionization process uses
an inert gas (usually xenon) in which atoms have been accelerated and
these bombard the sample which has previously been dispersed in a liquid
(usually glycerol). The bombardment generates a range of fragments
including pseudomolecular ions as well as smaller fragments. FAB-MS
is exceptionally useful where volatility is low or absent or where the
molecular weight of the sample is large. It has proved very useful in
studies of hydrolysable tannins (Porter, 1989).
8.2.5.2 Interpreting spectra

The value of mass spectroscopy is not that it tells us a great deal about
phenolic groups per se. About all you can expect is the loss of fragments
of 17 mass units, representing OH- ions which have been 'knocked off
the parent aromatic ring. What it does do is give you much information
on the molecule containing the phenolic groups and also, in many cases,
it will give some indication of the location of the phenolic hydroxyls
within the molecule. Let us take as an example the analysis of simple
flavones, a type of compound common to many plant species. The flavone
nucleus is relatively stable to electron impact mass spectroscopy, so a
major fragment is usually the molecular ion. However, fragmentation will
occur and a fragmentation pathway common to all flavones involves
breaking two bonds to give the fragments A and B (Scheme 8.1), as
shown for the simple flavone lapigenin (162, R = R,=R2=H).
Shucture elucidation 179
Scheme 8.1 Electron impact mass spectrum of apigenin and mon-

omethyl ethers. Formation of fragments identifying A-ring (A) and
B-ring (B) substituents.
R?o
OR, O
/ \
wyc,;+.
OR1 O
.+tcHD O R 3
'A' '8'
Let us consider the situation where one of the three hydroxyl groups
of apigenin has been substituted to give the corresponding methoxyl.
There are, obviously, three possibilities (Table 8.2). Electron-impact
mass spectroscopy will permit you to localize that methoxyl in either the
A or B fragment. If it is in the latter then the problem of structure
identification is resolved; if it is in A you still have a problem, one that is
better resolved by use of UV spectrophotometry (see above) or NMR
spectroscopy (see below).
Table 8.2 A and B hagments in apigenin monomethyl ethers
Substitution
Fragment A Fragment B
RI R2 R3 m/z mlz
This sort of argument can often be used to assign substituents. How-

ever, what must be recognized is that the fragment B from Scheme 8.3
would be given by two other flavonoids isomeric with apigenin (e.g. 163
and 164). Even more complex is the A fragment where the two hydroxyls
could theoretically be found in any of six substitution patterns. Further-
more where the substitution is made up of one methoxyl and one hy-
180 Chapter 8
droxyl the number of possible arrangements for the A-ring increases to

12! There are some features of fragmentation patterns which might allow
distinction between some of these isomers but, in most cases, once MS
has shown you that the A-ring has either two hydroxyls or one methoxyl
plus one hydroxyl it is better to proceed to another technique to further
explore the structure.
The problems inherent in electron impact mass spectroscopy are illus-
trated by reference to another flavonoid, kaempferol-3-(2,3-diacetoxy-4-
para-coumaroyl)rhamnoside (165). This strongly antifungal flavonol gly-
wside is induced by herbivory on the leaves of Myrica gale and has been
shown to be a potent fungicide (Carlton et al., 1991). It was isolated
by HPLC. Attempts to obtain an electron impact mass spectrum were
unsuccessful, because of the facile degradation of parts of the molecule in
the electron beam, a problem made worse by the need to use high inlet
temperatures because of low volatility. Thus, the electron impact spec-
trum revealed the presence of acetate (mlz 43), the para-coumarate ester
(6. 14, m/z 147) and kaempferol (39, mlz 286). To establish the full
dimensions of the molecule it was necessary to turn to fast atom bom-
batdment mass spectroscopy (Carlton et al., 1990). With the sample
suspended in 3-nitrobenzylalcohol (rather than glycerol) FAB-MS gave
an [M + H]+ ion at m/z 663 and an [M + Na]+ ion at 685, which permitted
the assignment of the empirical formula as 662 mass units (C34H30014).
This indicated that there was also a hexose sugar. Furthermore a major
fragment at mlz 377 solved for that hexose plus two acetate and one para-
wumarate ester (166), a major step forward in the identification of the
compound.
8.2.6 Nuclear magnetic resonance spectroscopy

The ultimate spectroscopic procedure in the structure elucidation armoury
is NMR spectroscopy. This technique makes use of the phenomenon
of magnetic resonance, whereby some atomic nuclei can take up elec-
tromagnetic energy at characterisdc wavelengths to attain a higher res-
onance state. This is studied by using powerful magnetic fields and applying
radiation in the range 60-600MHz (the wavelength depends on the
magnetic field strength) and measuring the absorption of radiation.
Among the isotopes that exhibit NMR the two that are critical to us are
'H and 13c.The technique depends on being able to measure very small
losses of energy that occur over very narrow wavelength bands so the
sophistication of the equipment is considerable, making an NMR instru-
ment rather expensive.
For our purposes spectra are obtained in the solution state, the sample
being dissolved in a small quantity of an appropriate solvent. For 'H
NMR the minimum amount of sample needed is, with a modern instru-
ment, in the order of 0.5-lmg but the general maxim is 'the more the
better'. 13C NMR needs more material because the natural abundance of
the 13cisotope is only 1%, so that a lOmg sample is, for NMR purposes,
only a 0.1 mg sample as far as carbon NMR is concerned. Samples for 'H
NMR are dissolved in a range of solvents in which 'H has been replaced
by 2~ (deuterium) which does not resonate in overlapping parts of the
spectrum to 'H, e.g. CDC13 (deuteriochloroform) and D 2 0 (deuterium
oxide). Traditionally the sample has contained tetramethylsilane (TMS)
to give a standard resonance but this is now often replaced by reference
to the residual 'H signal arising from the 'H in the deuterated solvent
(the resonance position, 6, of the solvent is still assigned on the basis of
TMS = 6 0).
Modem instruments acquire data from repetitive broad band pulsing
over the appropriate radiation wavelengths with data acquisition by com-
puter, followed by Fourier transformation of the data. Thus, a simple 'H
NMR spectrum of a 1mg sample of an unknown might result from some
2000 scans taken at &second intervals. The final spectrum will then be
transformed and will be seen as a series of signals on a chart that is
measured in 6 (delta) units. The 6 value of TMS is arbitrarily set at 0 and
each 6 unit then represents one millionth of the wavelength employed;
that is for a 60MHz spectrometer each 6 unit represents 60Hz and for a
600MHz instrument it represents 600Hz. This may seem a little confusing
but what must be remembered is that the resonance position or chemical
shift of a proton (that is the wavelength at which it resonates in relation
to TMS) varies proportionally with the applied magnetic field. So, if when
placed in the magnetic field of a 60MHz instrument a proton resonates at
6 2.5 (2.5 x 60 = 150Hz from TMS) then if placed in the magnetic field
182 Chapter 8
Tabk 8.3 Examples of some approximate 6 values for 'H nuclei
'H 'H
C-CH3 c=-cH-C
=C-CH3 CXH-OH
COCH, (acetyl) (XH-0-ester
N-CH3 =CH-C (olefinic)
0-CH3 aromatic ring
C-CH2-C -CHO (aldehyde)
=C-CH-C -COOH
CO-CH2-C C-OH (alcohol)
0-CH,-C C-OH (phenol)
of a 600MHz instrument it will still resonate at 6 2.5 (which is now

1500Hz distant from TMS). What is achieved by moving to these very
powerful 'high-field' instruments is some increase in sensitivity but, more
importantly, considerable enhancement of resolution.
With a 'H NMR spectrum the frequencies at which protons resonate
varies over a total of about 20 6 units, although the vast majority of
signals are between 6 0 and 6 10. Where a signal will occur is largely
dictated by the environment of the proton within the molecule. Some
examples of 6 ranges for different protons are given in Table 8.3. Two
points should be made now. Firstly, in some cases groups of protons are
all essentially identical and therefore will all occur together as one signal.
Examples include the three hydrogen atoms of a methyl group, the six
hydrogen atoms of an unsubstituted benzene ring and the twelve methyl
protons of TMS. Secondly, 'H NMR is essentially a quantitative tech-
nique, the area under a signal will approximate to the number of protons
involved in its formation.
Looked at simply, where a proton will be found is decided primarily
by the electronegativity of its surroundings, the more electronegative then
the further away from TMS it will resonate. This is exemplified by the
increase in 6 values for CH3 resonances as the nucleus to which it is
attached goes from a saturated carbon to an unsaturated carbon, a car-
bonyl, nitrogen and then oxygen (Table 8.3). Secondary effects (rarely
capable of more than 1 6 unit modification) occur through bonds (induc-
tive) and through space (anisotropic) causing further variation while the
solvent used and the temperature at which the experiment is run will also
have an effect. Whatever the problems of signal overlap, it is obvious
from Table 8.3 that a great deal can be told about a proton simply from
the 6 value at which it resonates.
For protons attached to oxygen chemical shift values can be very
varied i d in addition the signals can be lost by the process of exchange.
Ng. 8.3 Deuterium exchange.
An example of exchange is shown in Fig. 8.3 where phenol is being

analysed in the presence of deuterium oxide. The important factor here is
that the 0 - D 'replacement' is invisible to the proton spectrum. The
likelihood of replacement occurring is, to a large degree, dependent on
the solvent. Solvents in which there is a readily replaceable proton (i.e.
4 0 , CD30D) are almost guaranteed to mean loss of OH signals, as are
any solvent systems where there is free water present. In the latter case
what happens is that the 0 - H proton is in a constant exchange with the
population of 0 - H protons from the free water and as this exchange
happens more rapidly than the spectrum can be measured then the
separate OH resonance is not observed but merges with the 'water'
signal.
The exchange process can be deliberately employed to confirm the
presence of 0 - H protons. This is done by running the spectrum in
CDC13 and then adding 2 drops of DzO, shaking, and rerunning the
spectrum. The 'disappearance' of signals indicates that they were 0 - H
(or possibly N-H or S-H).
Chemical shift phenomena are mirrored in 13cNMR. Here, however,
the 6 range over which resonances can occur is far larger: a total of some
230 6 units. Furthennore a carbon will occur, in an unmodified spectrum,
as a singlet, a doublet, a triplet or a quartet (Fig. 8.4). This reflects the
number of hydrogen atoms attached directly to the carbon; if it is none
then the carbon is a singlet, one a doublet, etc. Providing there is no
serious signal overlap it is, therefore, a straightforward operation, to
establish how many CH3, CH2, CH and C atoms there are in an unknown
molecule.
The interaction between a carbon and the attached hydrogen(s) is
known as heteronuclear spin-spin coupling. As such coupling is observed
between nuclei under many circumstances then it is a phenomenon that
expands further than the amount of information that can be gathered
from an NMR spectrum. Coupling between protons ('H-'H coupling =
homonuclear coupling) will occur where protons are on adjacent nuclei
(i.e. where they are three bonds apart), and over a greater number of
bond linkages (up to six bonds) where certain spatial configurations
184 Chapter 8
Singlet Doublet Triplet Quartet

Fig. 8.4 Direct 'H-I3C coupling patterns.
occur. Where protons occur on the same carbon but are not in identical
environments (that is they are non-equivalent) then they can also exhibit
spin-spin coupling. The rule is that each proton will split every proton
with which it can couple into two signals; the distance between those two
signals, measured in Hz, is called the coupling constant (J). Coupling
constants between protons can vary from the lower limit of resolution of
the instrument (0.5%) up to a maximum of just over 20%. The oc-
currence of coupling is particularly valuable in allowing an understanding
of the relationships between protons in a molecule. Coupling occurring
over one bond length will often be referred to as 'J coupling, over two
bonds, '3, etc.
As an example let us look at 'H-'H coupling patterns in aromatic or
olefinic systems. Within an aromatic nucleus there are three possible
relationships between two protons (Fig. 8.5). These can readily be dis-
tinguished by 'H NMR spectroscopy as is shown in Fig. 8.5. On an iso-
lated olefinic bond there are two possible relationships between protons
on the adjacent carbons, cis or @am;these again are readily distinguished
by coupling constants. If we turn to the kaempferol derivative (165) we
can observe in the aromatic part of the spectrum (6 6.0-8.0) three series
of proton couplings (Fig. 8.6) that tell us, unambiguously, that we have
one pair of aromatic protons that are meta to each other (a, b; 4~ = 2Hz),
four protons (as two pairs) showing ortho coupling (cld and elf; 3~ =
Stactwe elucidation 185
orrho mem P"
Fig. 8.5 Simple olefinic/aromatic relationships between protons and their coupling
constants.
I I I I
7.5 7.0 6.5 6.0
Delta
Fig. 8.6 Aromatic portion of the 'H NMR spectrum of kaempferol-3-(2,3-diacetoxy-4-pm-

coumaroyl)rhamnoside(165).
186 Chapter 8
8 Hz) that indicate the presence of two para substituted aromatic nuclei,
and a pair of signals (g, h; 3J = 15Hz) indicating a trans double-bond.
When discussing mass spectroscopy the problem of differentiating
between possible patterns of A- and B-ring hydroxylation patterns in
apigenin (162) was noted (six possibilities and three possibilities respec-
tively). 'H NMR coupling will immediately solve the B-ring problem as
each of the three possibilities will give a unique series of splitting patterns
among the four protons. It will reduce the number of options for the A-
ring to three; where the two aromatic protons are adjacent (i.e. show
ortho coupling - three possibilities), where the two protons are separated
by a carbon bearing a hydroxyl (i.e. show meta coupling - two possibilities)
or where the two protons are opposite one another (i.e. show para
coupling - one possibility only). As a unique chemical shift value for a 5-
OH (6 12-14 due to H-bonding to the carbonyl) or H-5(c. 6 8.0) can be
recognized it is further possible to distinguish between the various meta
and ortho options.
13C-'H coupling (heteronuclear coupling) can also occur over two
and three bonds. Above (Fig. 8.4) we considered only direct (one-bond)
C-H coupling, which is normally in the range of 150-200Hz. C-H
coupling, of much smaller dimensions (in the order or 7-lo&), can
be observed over two or three bond lengths and exploitation of this
represents another powerful tool for gathering information on a mol-
ecule. It will be discussed further below.
Coupled spectra are often very complex and it can be difficult to
resolve just what is coupling with what. This can be overcome by decoup-
ling experiments in which by irradiating a specific proton in the spectrum
at its resonating wavelength it is possible to nullify its coupling capacity,
and so simplify the signals of those protons with which it was coupling.
This is called spin-spin dewupling and has great practical value in struc-
ture elucidation. While in the past decoupling was normally done as a
series of single irradiation experiments in which one proton at a time was
decoupled it is now generally done universally, using a two-dimensional
procedure (see below).
When running carbon spectra, it is also usual to perform decoupling of
protons by irradiating all the protons at the same time so that each carbon
resonance occurs as a singlet, as well as to run various forms of coupled
spectra. 13C NMR spectra are now usually obtained by procedures with
acronyms such as JMOD, INEPT, DEPT, which depict direct C-H
bonding by giving positive or negative signals rather than simple multi-
plicities which can overlap and then become difficult to interpret.
Yet another valuable procedure of NMR spectroscopy is the study of
the nuclear Overhauser effect (nOe). This is a phenomenon that occurs
through space and not through bonds. If a nucleus is irradiated at its
resonance frequency (exactly as in spin-spin decoupling) energy can be

transferred through space to other nuclei with the result that the size of
those signals is enhanced. If, using a computer, the original spectrum is
subtracted from the spectrum produced with irradiation then differences
in intensity will remain as a residual spectrum, the residual signals indi-
cating groups close (spatially) to the irradiated signal. If we return to the
problem of differentiating between apigenin-5-methyl ether and apigenin-
7-methyl ether, which proved impossible by routine electron impact mass
spectrometry (see Table 8.2) it is easy to see that nOe would readily
resolve the problem. In the 5-methyl ether (R,= CH3) irradiation of the
methoxyl resonance would enhance only one proton (H-6), whereas if the
methoxyl was at C-7 (R2 = CH3) then both H-6 and H-8 would be
enhanced.
With modem sophisticated NMR instruments it is possible to separate
the collection of chemical shift data from other effects to produce two-
dimensional spectra in which the classical one-dimensional spectrum oc-
cupies the diagonal. Where the information relates to a homonuclear
study the additional data can be observed as crosspeaks at the inter-
section of the horizontal and vertical lines from the two resonances
involved. Where it is heteronuclear then the interaction is observed as a
cross peak at the intersection of the 'H and 13C chemical shift values on
the two-dimensional spectrum. These procedures are all based on dif-
ferent sequences of pulses of energy used to manipulate resonances of
the nuclei; each different sequence is known by a different acronym
(invention of new and more bizarre acronyms is a major pastime of NMR
spectroscopists).
Two-dimensional procedures can be used to investigate spin-spin
coupling through techniques such as COSY, COSY-45, TOCSY
(HOHAHA) and long-range COSY, while nOe can be examined using
NOESY. There is a procedure for examining C-C direct coupling (the
INADEQUATE technique) but because of the low abundance of the 13C
isotope, which makes the chances of adjacent 13cnuclei fairly remote,
this procedure needs prohibitively large amounts of material.
The simplest heteronuclear two-dimensional procedures are those that
examine direct C-H interactions. Among the most popular of these are
HETCORR, XH-CORR, H-C-COBIDEC and HMQC. The last of
these is the most interesting because it is a so-called proton-detected
experiment. That is, the effect of the pulse sequence applied is examined
by studying the proton frequency. The other three procedures are carbon-
detected. Because of the greater abundance of 'H, proton-detected pro-
cedures are more sensitive and can be used with smaller samples. For
example a spectrum obtained in 2 hours by means of HMQC might take 6
or 7 hours by H-C-COBIDEC.
168 Chapter 8
Long-range C-H coupling over two or three bonds can be studied

using COLOC or, in the inverse (proton-detected) mode, HMW.
Let us finish this brief review of NMR by considering the results of a
series of experiments on the relatively simple compound 3'-methoxy-4'-
hydroxy-trans-cinnamaldehyde (167). For this compound the techniques
described are the equivalent of using a very large steamroller to crack a
very small nut. However, it does illustrate the value of the various
procedures. The following spectra are shown: (i) one-dimensional 'H
spectrum (Fig. 8.7); (ii) two-dimensional H-H COSY (Fig. 8.8); (iu)
one-dimensional decoupled I3C NMR spectrum (Fig. 8.9); (iv) one-
dimensional JMOD 13C NMR spectrum (Fig. 8.10); (v) two-dimensional
H-C COBIDEC (Fig. 8.11); (vi) two-dimensional H-C HMBC (Fig.
8.12); and (vii) two-dimensional H-H NOESY (Fig. 8.14). The reson-
ance positions of all protons and carbons and their major interactions are
given in Table 8.4.
The 'H NMR spectrum (Fig. 8.7) shows signals for an aldehyde
proton (a) as a doublet, five aromatic/olefinic protons as a doublet (b), a
double doublet (c), a doublet (d), a doublet (e) and a double doublet ( f ) ,
Tabk 8.4 'H and "C chemical shift values for 167
c/H 6H mdt. I(&) 3JH-~
mult, multiplicity (coupling pattern); s, singlet; d, doublet; dd, double doublet; br.s., broad
singlet. ?Ic_, coupling from HMBC spectrum.
Fig. 8.7 'H NMR spectrum of 167 run in CDCI, at 400MHz.
a broad phenolic 0 - H proton (g) and a 3-proton singlet (h) which is at

the right 6 value for a methoxyl. Of the various couplings, that of (b) and
one of those of ( f ) are about 16Hz, while (a), one of (c), (e) and the
second of ( f ) are all about 8Hz, and the second in (c) and that of (d) are
about 2Hz. From the information provided in Fig. 8.5 it is obvious that
these relate to trans, cislortho, and rneta, couplings respectively.
190 Chapter 8
Fig. 8.8 H-H COSY spectrum of 167.
The spin systems observed in the one-dimensional spectrum can be

resolved by the two-dimensional H-H COSY spectrum (Fig. 8.8). Only
that part of the spectrum involving protons (a) to ( f ) are shown as neither
(g) nor (h) have any visible couplings. The diagonal one-dimensional
spectrum running from bottom left to top right is clearly visible; cross-
peaks which are not on that diagonal represent H-H spin-spin inter-
actions. The most immediately obvious cross peak is that associated with
(a) and ( f ) which tells us immediately that H(f) is coupling with and
therefore adjacent to H(a). The aldehyde proton (Ha) cannot be adjacent
to any further protons (it has only one coupling) but H(f) shows a second
coupling, which can be traced to H(b). Thus, we now know that in this
molecule there are couplings of H(a) to H(f) which must be about 8 Hz,
and H(f) to H(b) which must be about 16Hz. This means that H(b) and
H(f) are tram-coupled across a double-bond and that the aldehyde must
be linked to the end of the double-bond with H(f), as H(a) and H(f)
couple. This yields a partial structure (168).
Using the same arguments H(e) is obviously coupled to H(c), the
coupling constant being SHz, and, although close together, the cross-
peak for an H(c)/H(d) interaction (of 2Hz) can also be seen. This spin
system indicates one ortho-coupled (He), one meta-coupled (Hd) and one
ortho and meta-coupled (Hc) proton. This gives another partial structure
(169) to which 168, a methoxyi and a hydroxyl must be linked to the three
Rs.
Now, let us turn to the 13cspectrum. The simple H-decoupled spec-

trum (Fig. 8.9) shows 10 carbon signals of which that at 6 193.8 must be
the aldehyde carbon and that at 6 56.21 is the methoxyl carbon. The
remaining eight must be associated with the aromatic ring (six required)
and the double-bond (two required). The JMOD spectmm (Fig. 8.10)
permits more resolution of the carbons. Negative signals for CH and CH3
carbons allow us to distinguish which among the aromatic and olefinic
carbons cany a hydrogen. To an experienced interpreter of NMR spectra
the fact that there are two carbons without hydrogen at 6 149.19 and
147.19 is a sure sign that these are two adjacent oxygen-bearing carbons
and allows further refinement of 169 in which we now require the meth-
oxyl and hydroxyl to be next to one another. This leaves two possible
structures, 167 and 170.
The assignment of the aromatic and olefinic carbon resonances can

be unequivocally established by the two-dimensional H-C COBIDEC
I94 Chapter 8
ppm 180 160 140 120 1M) 80 60
Fig. 8.11 Identification of direct H-C coupling in 167 by the H-C COBIDEC technique.
experiment (Fig. 8.11). Here there is no diagonal; a proton spectrum

occupies one side of the spectrum and a carbon spectrum the other.
Cross-peaks, where the horizontal from the proton spectrum bisects the
vertical from the carbon spectrum denotes a direct C-H coupling.
By this stage we have the structure resolved to either 167 or 170,
and all protons and carbons unambiguously assigned except for the
three carbons not bonded directly to hydrogen. Their assignment can be
achieved by studying long-range C-H coupling, which has been done
here using the HMBC technique (Fig. 8.12). In this example all 3J
couplings for the molecule are visualized and have been depicted in Fig.
8.13 and listed in Table 8.4. There are three critical observations.
1 Coupling of H(e) and H(f) to the 6 126.85 resonance.
2 Coupling of the methoxyl and H(e) to the 6 147.19 resonance.
Fig. 8.12 Long-range H-C-C-C-coupling in 167, shown through the HMBC spectrum.
The 'noise' on the methoxyl proton channel is normal; true interactions are readily
distinguishable from the noise.
3 Coupling of H(c) and H(d) to the 6 149.18 resonance.

All carbon resonances are now unambiguously assigned and the fact that
H(e) and not H(c) and H(d) couple with the same carbon as the methoxyl
protons also fixes the position of this substituent, so confirming structure
167.
The placement of the methoxyl can be further confirmed by appli-
cation of the NOESY (two-dimensional nuclear Overhauser) technique
(Fig. 8.14). Superficially this looks like the COSY, the difference being
196 Chapter 8
Fig. 8.13 'I H-C couplings visible in the HMBC spectrum of 167.
..
P P ~ 9 8 7 6 5 4
Fig. 8.14 H-H NOESY spectrum of 167 showing interaction of the methoxyl protons.
that what is measured is effects through space rather than through bonds
(see above). The important cross-peak observed here associates the meth-
oxyl with the me&-coupled aromatic proton H(d), so requiring that they
be adjacent.
8.2.6.1 Studying tannins by NMR

There has long been interest in the use of NMR spectroscopy to assist in
the analysis of structure, polymer composition and stereochemistry of
tannins (for reviews see Haslarn, 1989; Okuda, Yoshida & Hatano, 1989).
In recent years the technique has been widely used for the study of hy-
drolysable tannins but its value for gathering information on the structure
of condensed tannin polymers has not been so thoroughly exploited.
However, a recent paper by Cai et al. (1991) has illustrated the potential
application of NMR for the analysis of these polyphenols.
Cai et al. (1991), working with the exudate of Croton lechleri man-
aged, by HPLC, to isolate a number of condensed tannin polymers the
molecular weights of which were estimated (up to 2130 daltons) by FAB-
MS. The composition of each polymer was examined by a combination
Scheme 8.2 Thiolysis of condensed tannin polymers.
a:
F+
HO H
*' OH
Y
H
H
+
H
198 Chapter 8
Fig. 8.15 'H NMR spectrum of the 'H-2' region of the spectra of a mixture of flavan-3-01
and flavan-3~14benzylthioethe~sobtained from the condensed tannin of Croton lechleri
(after Cai et al., 1991). a , epigalloylcatechin-4-benzylthioether; h, epigallocatechin;
c, epimtechin-4-benzylthioether; d, catechin; e, gallocatechin; f, gallocatechin-4-
benzylthioether; g, catechin-4-benzylthioether;h, epicatechin (not visible).
of thiolysis using toluene-a-thiol followed by 'H NMR spectroscopy of

the resulting flavan-3-01s and their benzylthioethers. The hydrolytic pro-
cedure (Shen et al., 1986) generates the 4-benzylthioethers for internal
units of the polymer chain while the terminal units are not thioalkylated
(Scheme 8.2). The H-2 signal (Fig. 8.15) for each of the potential hydro-
lysis products can be distinguished when spectra are run in deuterioacetone;
catechin (& 4.55, doublet, J = 6.7Hz), epicatechin (SH 4.88, singlet),
gallocatechin ( 6 4.51,
~ doublet, J = 7.3Hz), epigallocatechin (SH 4.82,
singlet), catechin-4-benzylthioether (&, 4.91, doublet, J = 9.7Hz),
epicatechin-4-benzylthioether ( 6 ~ 5.30, singlet), gallocatechin-4-
benzylthioether (&, 4.89, doublet, J = 9.6Hz), epigallocatechin-4-
benzylthioether (6H 5.21, singlet). From a careful quantitative analysis of
the H-2 resonances in the 'H NMR spectrum of the mixture of products
of the thiohydrolysis of each purified polymer it is possible to determine,
with a high degree of certainty, both the size and the composition of the
polymer, although where more than one flavan-3-01 structure occurs it
does not tell you the internal sequence.
CHAPTER 9
For the future? Current and

future utility of chemoecological
studies based on phenolics
9.1 Introduction
In Chapter 3 we surveyed current understanding of the role of phenolics
in mediating ecological interactions, from a 'how it works' viewpoint. To
date the majority of practitioners of chemical ecology have focused more-
or-less entirely on understanding how chemical signals are transmitted
and received between individual organisms. Consequently the major suc-
cesses of 'chemical ecology' have been in providing mechanistic explana-
tions for ecological phenomena at the population level. This focus has
turned chemical ecology into a discipline of peripheral interest for many
ecologists, because such studies do not contribute directly to the questions
that require resolution at the ecosystem or community levels. Nor does
the mechanistic focus of chemical ecology directly address questions of
evolutionary importance which provide the driving motivation for much
of mainstream ecology.
It can be argued that, if chemical ecology is to prosper as a valid
approach to ecology in general, then it needs to become more than a set
of tools. These chemical techniques must become fundamentally in-
tegrated into ecological studies investigating questions of wide interest
that could not otherwise be answered. If this happens then students who
invest their energies in understanding this interdisciplinary area will be
repaid by being able to contribute work of theoretical interest rather than
work of narrow technical interest. The situation is very much analogous
to that dealing with mathematical approaches to ecology. Chemistry and
mathematics are both areas that not everyone feels comfortable with.
Among those who do not deal well with these subjects there is often the
feeling that advocates of the mathematical or chemical approaches get so
caught up in their specialization that they may become side-tracked from
doing work of real ecological relevance. It is in this context that we now
wish to further review the contribution of studies involving phenolic
secondary metabolites to ecology. Our goal is to illuminate areas where
chemical studies are currently well integrated into modern ecology and to
see where future developments might lie.
With this goal in mind, we have divided this chapter into two parts.
In the first of these we cover some current foci of ecological work at
population, community and ecosystem levels, where studies involving
phenolics are of integral importance. In the second we deal with devel-
199
200 Chapter 9
opments in plant defence theory, studies that are motivated by con-

siderations of a more evolutionary nature.
9.2 Phenolics and current ecological problems

In Chapter 3 we largely considere; constitutive chemical factors, such as
those that acted as antifeedants or oviposition stimulants to herbivores.
Our one departure from this was with respect to pathogens, where we
considered phytoalexins, which are induced defences. In addition to
pathogens abiotic factors and herbivores are widely reported to be able
to influence the types and quantities of phenolics produced by a plant.
In particular these factors may 'induce' the formation of additional
quantities of phenolics.
Over recent years the phenomenon of phytochemical induction of
phenolic metabolites due to attack by herbivores has been given con-
siderable attention (see reviews by Karban & Myers, 1989 and Raupp &
Tallamy, 1990 and papers by Tempel, 1981; Walters & Stafford, 1984;
Leszczynski, 1985; Mattson & Palmer, 1988; Hartley, 1988 and Hartley &
F i n , 1989). In the field, such herbivore induced effects will be observed
together with genetic, developmental and abiotically induced changes,
acting with or against one another and it is not surprising to find that the
evidence for one or other of these effects is often confounded by the
other components (Johnson & Brain, 1985; Hansson et al., 1986; Feibert
& Langenheim, 1988). In essence, the current view is that plant phenolic
metabolism must be considered to be under some form of integrated
metabolic control.
While the regulatory controls remain a 'black box' mechanism, a
widely employed general hypothesis is that external inputs that can be
superimposed on the genotypic chemistry of the individual include a
series of biotic and abiotic factors in the environment. With this starting
point, we now explore the ecological ideas that have been developed with
some significant input from studies considering phenolic allelochernicals.
9.2.1 laoprrldon interactions and induced &fence

T k suggestion that a plant can respond to herbivory by an elevation in
the levels of defensive compounds has led to much speculation that this
could be an adaptive response designed to deter further herbivory (see
for example, Schultz & Baldwin, 1982; Rhoades, 1985; Karban & Myers,
1989). Unfarfunately, the robustness of results purporting to show this
effect can often be questioned (Fowler & Lawton, 1985) and it is clear
that much remaim to be done to substantiate the hypothesis (Karban &
Myers, 1%9). Particular probtems centre on the questions: (i) do the
substances (phenolics) that are induced actually deter herbivory or signifi-
cantly alter feeding behaviour? (Hartley, 1988; Raupp & Sadof, 1989);
For the future? 201
and (ii) does the spatial distribution of damage induced phenolics have
the effect of directing the further attention of herbivores to less valuable
plant parts or to areas where they will be more prone to predation?
(Edwards & Wratten, 1989). Another subject in need of investigation is
the effectiveness of induced phenolics against secondary pathogen infec-
tions (Del Amo, Ramirez & Espejo, 1986). While there is some evidence
that they could be active against both herbivore and pathogen (Russell et
al., 1978; Sutherland et al., 1980) the case is far from proven.
Whatever the adaptive impact on target organism(s) arising from the
biochemical changes that occur, it does seem to be established that
herbivores can impact plant chemistry to an appreciable extent. This must
be considered as a variable along with abiotic and genetic factors influenc-
ing plant chemistry. It is this suite of factors leading to variable plant
chemistry that provides the baseline against which to consider the poten-
tial for reciprocal changes. These may be brought about by ecological
interactions of primary consumers with producers and secondary con-
sumers and, consequently, the impact may be over several trophic levels.
If we accept that, to some extent, herbivore success is dependent on
the levels of plant phenolics, then one important line of work must be to
establish whether such biotically mediated changes in plant quality can be
observed at the level of population dynamics. The conceptual basis to
extend this to three trophic levels (plant-herbivore-predator) is now
well established (Barbosa & Letourneau, 1988) as is the role of phenolics
in systems where plants may mediate apparent competition or mutualistic
interactions between consumers (e.g. plant-herbivore, pathogen), see
Karban, Adamchak and Schnathorst (1987).
Investigations of these topics have been linked closely with those on
cyclic plant-herbivore interactions, or more generally to predator-prey
interactions. For example, the induction of chemical defences in food
plants has been central to the formulation of hypotheses relating cyclic
variation in food plant quality to herbivore population cyclicity in micro-
tine voles (Lindroth & Batzli, 1986). The attraction of induced chemical
change here is as a physical basis for introducing time lags into cyclic
dynamics: results may differ for plant species that induce and/or return to
the uninduced condition at different rates. Substantive evidence for cyclic
relationships has yet to be obtained, and it is unlikely that it will ap-
pear as long as the induced defence hypothesis remains tentative. The
fact that phenolics such as tannins do not usually mediate tightly coupled
relationships between plants and stenophagous herbivores is also a key
consideration.
Alternative reasons for periodic changes in plant phenolics, such as
climatic and edaphic factors, are also being explored (Jonasson et al.,
1986; Laine, 1988). Such ideas have been considered to play an important
202 Chapter 9
role in insect population dynamics (Khoades, 1985) and there is much

current work that focuses on the ways in which plants may mediate
climatic and edaphic effects on herbivore populations via chemical
changes altering their quality as nutrients.
Currently this area of investigation remains in its infancy. The lesson
it has given us so far is for a need to increase sophistication in the way we
analyse and understand the chemical quality of foodstuffs and the likely
dynamic changes in this quality. For the chemist, important aspects of
food quality are likely to include basic nutrients such as proteins, carbo-
hydrates, limiting vitamins or essential amino acids, as well as the more
exotic secondary metabolites, some of which are covered in this text.
Recognition that both primary and secondary metabolites and fibre,
water and other relatively inert plant constituents contribute to indices
of plant quality will be important in the future. Field workers can no
longer look to the chemist to supply 'the answer' in terms of single
allelochemicals measured on a per unit weight of leaf basis either. In
seasonal environments, herbivores may be sensitive to changes in the
abundance and age structure of populations of leaves within a single
species, which may have different chemical qualities, necessitating more
sophisticated sampling procedures in advance of chemical analyses; i.e.
sampling what the animal actually eats over the time period when it eats.
It has been suggested that variation in the levels of alIefochemicals
over various spatial and temporal scales may constitute an important part
of plant defence. This idea seems to be tacitly accepted in much of the
work reviewed for this volume yet testing these ideas rarely gets beyond
the anecdotal presentation of the hypothesis (Mole & Waterman, 1988).
In spatial terms, intraplant variation is thought to be a mechanism for
steering herbivores away from growing parts of the plant or of forcing
them to gather nutrients in a position where they are exposed to pre-
dators. The capacity of pests or pollinators to locate plants may also
depend on the spatial distribution of individuals and their juxtaposition
with other species giving different chemical signals. Chemical variation
within host plant communities is the ultimate manifestation of these
effects and a subject we know little about. Larger scale topographic,
edaphic and microclimatic variation may also influence the functioning of
allelochemicals at the population and community levels (Waterman &
McKey, 1989),controlling local variation in herbivore population densities.
9.3 Community and ecosystem patterns of secondary metabolites

At an organizational level beyond the consideration of particular popula-
tions of interacting species, there are ecosystem and further community
level effects to be evaluated. In these circumstances phenolics may be
non-specific as to their target organism. This is thought to be the case
For the future? 203
with tannins, in respect of their role in deterring herbivores and in

allelopathy, where superabundant production of this class of allelo-
chemical causes dramatic effects on other aspects of the community which
may be clear to see.
Plant phenolics enter the soil via decaying vegetation and are thought
to affect mineralization and nutrient cycling and thus all of the plant
community in the vicinity of the plants generating those phenolics (Janzen,
1974; Waterman & McKey, 1989). Blackwater rivers and white sand soils
in tropical systems are viewed as a classic and well known example of
these effects, impacting both the plant and animal communities in an
ecosystem (Janzen, 1974). Recent work on tannin deposition in con-
iferous forests is just beginning to quantify the nature of these inputs
(Tiarks ef al., 1989). The importance of phenolic deposition in run-off
and stream communities is also an active and important area for research
(Stout, 1989).
There is an important contrast between: (i) population level studies
where the precise chemistry is critical in determining which substances a
herbivore may detoxify or use for its own defence; and (ii) the relatively
gross analysis of how all phenolics may generally alter soil or stream
processes. This begs the question of the relative importance of these two
extreme types of ecological effect and the extent to which molecular
(structural) diversity is of general adaptive value in natural products.
For instance, alkaloids (Cordell, 1981) and procyanidin oligomers and
polymers (Hemingway & Karchesy, 1989) are both incredibly diverse in
their precise molecular structures. We can at present conceive of far more
adaptive roles for chemical diversity in the former than we can for the
latter group of natural products.
At this point we can take a divergent line of enquiry. So far in this
discussion we have considered the functional level and the mechanics
of present day ecosystems. Another aspect for consideration is at the
historical or evolutiona~level where we must be concerned about the
prior assembly processes that have led to present day ecosystems, and
the degree to which component organisms are co-adapted to each others'
presence.
9.4 Evolutionary ecology and plant defence theory

The prerequisite for considering questions concerning evolution is to have
some appreciation for genetics and the inheritance of phenolics as plant
traits. The following review of these prerequisites is brief, largely because
little is known about this topic, both with regard to quantitative and
qualitative variation. We will then examine current theoretical ideas
about plant chemical defence with particular regard for their evolutionary
components. Finally, we conclude with an overview of where new ad-
204 Chapter 9
vances seem likely in this field and where systems involving the study of
plant metabolites seem likely to continue to play an important role.
9.4.1 Genetic variation in plant phenolics

To an ecologist working with natural plant populations it usually becomes
clear that any one individual, or any one part of an individual, is not
chemically identical with comparable units in the population. Once one is
aware of this problem, genetic variation that is not in itself of interest
can often be accounted for by a suitable sampling programme and
experimental design.
Where the focus is evolutionary, genetic variation may be the raison
d'ttre for the study, as for example the situation where the investigator is
interested in material for selection experiments. In crop plants, tradi-
tional breeding has identified genes for tannins in sorghum and for flav-
onoids in beans and corn (Haskins & Gon, 1988). Genetic analysis has
also been used to establish the effect of selection as a constraint on the
variability of phenolic levels expressed in the phenotypes (chemotypes)
of various cultivated lines. As expected, there is a loss of chemical
variation as lines are selected for specific agronomic traits (Sanlaville, Jay
& Guiard, 1988).
In natural populations genetic variation is ubiquitous and is usually to
be seen where specific compounds are identified and multiple sample
surveys performed. For example, flavonoid variation has been found in a
wide ranging polyploid complex of Arnica cordifolia (Wolf & Denford,
1983) and Semple and Averett (1975) report considerable flavonoid
variability in Xanthisma texanum. In Piiyrogamma triangularis variation
in secondary metabolite profiles were attributed to diploid and tetraploid
chemotypes (Star et al., 1975). However, genetic variation is not always
so clear cut. In separating genetic and environmental components in
flavonoid patterns in Quercur rubra it was shown that the former was the
less important (McDougal & Parks, 1986). This has also been demon-
strated for the production of tannins in Berula allegheniensis and Acer
saccharurn (Baldwin, Schultz & Ward, 1987). Most studies trying to
separate the input of these two components into chemical variation are
recent and it is hoped that identification of the genetic element can be
exploited in the future, especially in studies where fitness costs of either
-
producing or not producing phenolics are to be studied (see, for example,

Coley, 1986).
In contrast to the relatively plastic variations that are of importance in
microevolutionary studies, the genome also codes for those relatively
invariant traits that determine an organism's fundamental nature. Such
morphological and developmental constraints are innate factors that
need to be taken into account as they set patterns seen at the macroevolu-
For the future? 205
tionary level. For example, the influence of morphological parameters on

the internal organization of plant vascular tissues have been found to be
important in resource allocation (Watson & Casper, 1984; Haukioja et
al., 1990). The ecological effects of this are seen where hypotheses of
plant resource allocation and plant responses to multiple, environhental
factors shape our ideas about the causes of variation in phenolic produc-
tion (Bazzaz et al., 1987; Chapin et al., 1987; Waterman & Mole, 1989).
9.4.2 Plant phenolics and plant defence theory

Until recently, it has been assumed that rates of microevo~utionary
change were rapid and that communities of organisms were sufficiently
responsive to change that they were near some kind of equilibrium in
their interactions with their environment. With regard to the biotic en-
vironment, 'arms race' style co-evolution as envisaged by Ehrlich add
Raven (1964), was assumed to be the main type of evolutionary process
underlying changes in allelochemically mediated interactions.
Two well explored factors thought to influence the distribution and
abundance of phenolic compoudds in plant communities are described by
the apparency (Feeny, 1976; Rh~i-ides& Cates, 1976) and resource
allocation hypotheses (Bryant, Chapiiti & Klein, 1983; Coley, Bryant &
Chapin, 1985). These ideas bridge the gap between population and com-
munity levels and also attempt to link functional explanations of how
communities work and evolutionary explanations for diversity. Appar-
ency theory was developed as an explicitly co-evolutionary hypothesis
while the more recent resource allocation and growth rate arguments are
based on the assumption that plants are physiologically adapted to their
present day environments. These ideas are briefly reviewed below.
9.4.2.1 Apparency
The idea that plants vary in the degree to which they are available to, or
likely to be discovered by, other organisms such as herbivores, has had
a strong influence on the development of ecology and chemieal ecology
in particular. These ideas were coalesced in the Apparendy Theory
(Feeny, 1976; Rhoades & Cates, 1976) which enables predidtions to be
made concerning the likely defence chemistry that would be adopted by
abundant, long-lived, perennial (apparent) plants relative to shod-lived,
rare and herbaceous plants. The mechanistic underpinnings of this con-
cept, with its reliance on the dose-response properties of allelochemicals
and the anti-digestive mechanism of action of tannins, are now in a state
of disrepute because of considerable research into the mechaaism of
action of these substances (Chapter 5). However, the idea of apparency is
still widely cited and it clearly did, and perhaps still does, have some
value as a predictive tool in identifying which allelochemicals can be
206 Chapter 9
anticipated in a community of plants (Grubb, 1993). Less charitably, the

difficulties in unambiguously quantifying apparency seem to defy both the
acceptance and refutation of hypotheses depending on this concept.
9.4.2.2 Resource allocation

As an alternative to the co-evolutionary explanations for allelochemical
distribution offered by apparency theory, others have suggested that the
nutrient resources available to plants largely determine the quantity and
types of allelochemicals that they produce (Bryant, Chapin & Wein,
1983; Coley, Bryant & Chapin, 1985). Some differences of opinion
appear to exist regarding whether the primary constraint is in factors
which are external to the plant and related to nutrient supply or whether
the focus should be on innate factors that control growth rate and nutri-
ent composition.
Writers who espouse the resource allocation ideas are essentially
concerned with the relationships of plants to environments in which they
are native and to which they are, in large part, adapted by natural
selection. The major advantage of this concept has been the focus on
plant physiological factors to explain the distribution of ailelochemicals
rather than the mechanism of action of the allelochemicals or the hard-to-
quantify apparency of the producer. Like apparency, both the extrinsic
factors and the plant physiology approaches to resource allocation make
use of microevolutionary explanations with regard to the current, in situ,
adaptive value of secondary plant metabolites. In essence, all these ideas
-
attempt to address the question of how the present day inter- and in-
traspecitic distributions of different secondary metabolites have come to
be. The key plant attribute analysed is its present day life history.
9.4.3 Historical approaches to chemical ecology

We consider that both of the preceding approaches have been valuable
but that this work is now at an impasse. We can predict, with reasonable
accuracy, that long-lived, large, evergreen plants will produce tannins
more often than not by apparency arguments. We can be more sure of
our predictions if we limit ourselves to such plants as they occur in sunny
locations in nutrient-stressed and climatically extreme environments by
using resource allocation ideas. Unfortunately, exceptions will still be
encountered and this obstacle lies across the path to further improvement
in our predictions using these ideas.
At the macro-evolutionary level, phylogenetic explanations have been
advanced to explain patterns of secondary metabolite distribution. These
were discussed in some detail in Chapter 2. If there were real faith in the
predictive power of either apparency or resource allocation then the
question that ecologists often address to phytochemists -'does my plant
For the fuhlre? 207
contain compound X?'-would not so often be necessary. Not all herbs

are tannin-free and not all trees are full of tannins but don't contain
toxins. The particular type(s) of phenolics produced by a plant, as well as
those it cannot produce, is a genetically determined trait. Groups of
phenolics produced sometimes find practical use as taxonomic markers.
Take for example the phenolic flavanone glycoside hesperidin. It occurs
commonly in the Rutaceae, in herbaceous species from xerophytic envi-
ronments and from forest canopy trees of tropical rain forests. It is a
marker for the family, not for the species growing in any particular
environment or with any particular level of apparency (Hegnauer,
1963-1992).
Recent work on cladistic methods (Brooks & McLennan, 1991) has
provided new approaches to the question of whether community level
allelochemistry is determined by the present day interactions ar by the
history of component species prior to community assembly. This remains
a little explored question (Waterman et al., 1988; Waterman & McKey,
1989) although the notion of w-evolution (Thompson, 1988) is now
substantially less appealing than it once was due to the rigour of the
modem historical approach. Thus, Ehrlich and Raven (1964) conceived
of co-evolution as a particular process whereas Brooks and McLennan
(1991) have analysed it in terms of the separate components of co-specia-
tion and co-adaptation. The conceptual framework is now available to
compare clades of putatively co-evolved species and determine the extent
to which speciation events in one lineage parallel those in another.
Should such parallelism prove to be the case, then a further and equally
important question can be asked. Do changes in mutually adaptive traits
in one species match reciprocal changes in the other? New standards have
thus been set before the original use of the term 'co-evolved' can be
appropriately applied to organisms that today appear mutually adapted to
each other's presence. While such studies may be 'flavour of the month',
they also force a sober reassessment of the extent to which chemical
adaptations are linked to speciation and cladogenic versus anagenic
change in co-adapted species.
Present ecological theory has been dominated by the idea that plant-
herbivore relations are relatively labile and that co-evolved changes
in plant chemistry can occur relatively rapidly. We need to face the
paradox that chemotaxonomy and its use of phenolics and other classes of
&lelochemicals, such as alkaloids, reminds us that apparently ancient
chemical characteristics persist as useful takonomic markers. These are
either selectively neutral or they constrain present day ecological interac-
tions by not being selectively neutral! A vast amount of work has been
aimed at finding 'co-evolutionary' explanations for secondary metabolites.
Determination of the extent to which they are neutral or selective will be
208 Chapter 9
an interesting goal for future research and one that can be expected to
build on current information about plant phenolics and use the methods
provided in this book.
9.4.4 The future of phnt defence theory and sftuIies with phenolics
Plant defence theory aims to describe the ways in which plants are able to
survive in the presence of herbivores. As we have seen, the theory has
been elaborated by Feeny (1976); Rhoades and Cates (1976); Bryant,
Chapin and Klein (1983) and Coley, Bryant and Chapin (1985) who all
used arguments in which phenolics were extremely important. A recent
review and extension of this work has been provided by Herms and
Mattson (1992) who continue the theme of resource allocation based
ideas while attempting to integrate information about developmental and
other organismal constraints that reflect important historical factors.
Explicit recognition of historical effects has also been voiced by Edwards
(1989) who has put forward the notion of neutral defence, which he
believes is exemplified by tannins.
Grubb (1993) has synthesized many of these ideas as they apply to
understanding the present day defensive characteristics of plants in cur-
rent environments. The appeal of his work is that he accepts that the
theory may have to be complex and that he is explicit in not addressing
other levels of organization beyond his interest. Thus he does not make
predictions at the level of plant physiology as the resource allocation
theory does.
Like Grubb (1993), we do not see previous theoretical work as having
generated mutually incompatible theories, rather the reverse is true. If
this is the case then we need to take a catholic approach in the kinds of
work we regard as making legitimate contributions to our knowledge. In
any general theory of plant defence, the approach of Grubb (1993) will
need to be extended to allow the formulation of theoretical ideas appro-
priate to other organizational levels, such as the physiological one.
As an umbrella structure, we think there are useful parallels between
the theoretical framework surrounding studies of plant defence and what
has become known as life history theory (Steams, 1992). F i t l y , chemical
ecology research crosses levels of biological organization, leading to an
attempt to integrate physiological and genetic insights with ecological
studies of organisms made in the field (Herms & Mattson, 1992). Secondly,
optimality arguments and the economic metaphor underlie parts of this
theory (Bloom, Chapin & Mooney, 1985; Gulmon & Mooney, 1986) as
well as more mechanistic and physiologically conceived studies where
concerns about currency concepts, resource allocation issues and trade-
offs permeate the debate (Chapin, 1989; Bauaz et al., 1987). Thirdly,
there has been a considerable growth in studies which relate the pheno-
For the future? 209
typic expression of defensive or life history traits to their underlying

genetic basis (Fritz & Simms, 1992). Fourthly, phytogenetic systematic
studies have led to substantial efforts to understand lineage specific con-
straints to plant-herbivore interactions (Farrall & Mitter, 1990; Futuyma
& McCafferty, 1990).
Our conclusion from this is that we expect plant defence theory to
become less prescriptive and to emulate life histo6 theory (Stearns, 1992)
by accepting a wide variety of experimental and methodological appro-
aches. We hope that this will be the norm in future contributions to plant
defence theory and we see no reason why any one approach should be
more valid than another. In the course of this book, we have encountered
studies involving plant phenolics at every level of biological interest,
whether it was physiological, genetic, ecological or phylogenetic. With
this and the broad umbrella of current theory in mind, we are confident
that all the information and techniques presented in this book can be
used, with imagination, to make further advances in this field.
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Subject index
Page numbers in italic indicate figures Calvin cycle 21

carbon-nutrient balance 63-4
abiotic constraints and plant development CCCC systems 166
61-4 chemoecological studies 44,199-209
drought 63 communitylecosystem patterns of
nutrients and carbon-nutrient balance secondary metabolites 202-3
63-4 ecological interactions 45
seasonal and developmental changes 64 ecological problems 200-2
solar irfadiance and UV light 61,62-3 evolutionary ecology and plant defence
acetate pathway 22-6,27 theory 203-9
metabolites 41,42 future studies 208-9
see also shikimatelacetatecombimed genetic variation in plant phenolics
pathway 204-5
Adenostoma fasiculatum 60 historical approaches 206-8
Agalririrpurpurea 60 plant phenolics and plant defence theory
Agasicles beetle 48 211
- .5.-
-13
algae 43 population interactions and induced
see also tannins defence 200-2
allelopathy 59-60,141 see also importance of phenolic
Aloe 42 compounds
animal growth and development, bioassays chiroptical techniques 175-6
141 chromatography 149-67
animal toxicity, bioassays 141 column 155-7,158
antifeedantlovipositionassays 141 gas 157,159,160-1
antimicrobial assays 141 reporting of data 161
apparency 205-6 sample preparation 159-61
Arctostaphylos glandulosa 60 stationary phase 159
Asteraceae 42 high performanceliquid 161,162,163-5
paper 149,150-1
Bate-Smith method 102 standardization 152
bioassays 141-2 strategy for use 152-4
biosynthesis 20-35 solvent-solvent partitionlcountercnrrent
acetate pathway 22-6,27 165,166
building blocks 21 thin-layer 149,150,151,158
combined shikimic acidlacetate pathways preparative 154,155,1>6,
31-4 standardization 152
mevalonic acid pathway 34,35 strategy for use 152-4
shikimic acid pathway 27,28-30 Chrysomela aenicolir,sequestration of plant
birch, phenolic glycosides 52 chemicals by 54
Bombirmori 48 Chrysomela vigintipunctata 49
Boo~etixargentam 49 Citrus ~ I r ~ d n i d a48
i
bovine serum albumin precipitation assay for Cladosporumherbarum, bioassay 141
tannins 120-4 classes of phenolics 5-20
Bryopbyta 38 acetophenones 6
alkaloids and terpenes 17,18
caching behaviow and detoxification before aryl-pyrones 9
feeding 138 chromones 8
Callosobruchur maculatur, bioassay 141 cinnamic acids 6
232 Subject index
coumarins 8 standards and false-positive results

flavonoids 9,lO-11 98-9
isocOumarins 9 vanillin method 95-7
isoflavonoids I2 hydrolysable tannins 101-3
lignans 7-8 ellagitannins 102-3
'masked' phenolics 19-20 gallotannins 101-2
neoffavonoids 11 living organism to extract 66-78
phenylpropanes/phenylpropanoids 6,7, close to labotatory 68-9
48 couection of material 67-70
phenylpropenes 6,7 external resins 70-1
quinones and benzophenones 16,17 extraction 73-5
single aromatic ring 5-7 far from laboratory 69-70
stilbenes 9 leafy material 71
tannins 12,13-I6 material with high water content 70
colorimetry 99-100 quaatitation 75,76-7,78
co-pigments 56 sample preparation 72, 73
competitive biding assay for tannins 132, spectrophotometric assays 78-80
133 total phenolics 80, 81-91
Cosleiytra zealandim, bioassay 141 ascorbic acid interference 89-90
creosote bush, see Larrea iridentata blank 86
Folin-Ciocalteu method 83-5
DCCC systems 166 Folin-Denis method 81-3
de-astringency 57 methods to avoid 90-1
detoxificaiion 138-42 Price and Butler method 85-6
bioassays 141-2 procedures and standards 86-9
caching behaviour 138 standardization 86,87-9
of pisatin 59 see also separation methods
xenobiotic metabolism after ingestion
139-41 Fabaceae 42
diet construction for feeding trials 134-5, feeding trials, diet construction 134-5,
136-8 136-8
distribution patterns 36-43 flower wlour 55-57
evolution of phenolic secondary meta- Folin-Ciocalteu method 83-5
bolism 37-9 Folin-Denis method 81-3
individual classes 39, 40- 1 food selection
acetate-derived 41, 42 individual phenols 47,M-51
acetatelshikimate-derived 42,43 total phenolics and tannins 46-7
cinnamic atid-derived 41 fungi 38
tannins 43
drought 63 Galemella lineola, sequestration of plant
chemicals by 55
GC systems 159
electrophoresis 167 genetic variation 204-5
elm-bark beetle 48 glycosides, hydrolysis 147
Elytropappw rhinocerotis 62
Eriodictyon californicum 62 haemoglobm precipitation assay for tannins
Escherischia coli, bioassay 141 117-18,119,120
evolution of phenolic metabolites 37-39 Heliothis zea, bioassay 141
extraction and quantification 66-103 Herpes simplex, bioassay 141
condensed tannins 91,92-100 HPLC systems 162
colorimetry 99-100 Hydrangea macrophylla 56
comparison with proanthocyanidii
method 97-8 importance of phenolic compounds 44-65
comparison with vanillin method abiotic constraints and plant development
97- 8 61-4
proanthocyanidin method 92-4,95 drought 63
Subject index 233
nutrients and carbon-nutrient balance deuterium exchange I83

63-4 tannins 197-8
seasonal and developmental changes nutrients 63-4
64
solar irradiance and UV light 61,62- Papilw polyxenes 48
3-
' bioassay 141
allelopathy 59-60 Papilio prorenor 48
individual phenols and food selection 4, phenolics analysis in tannin-protein
48-51 precipitates 125-6,127
litter decay and plant mineral nutrition pheromones 55
60 Phratora vifellinae, sequestration of plant
plant-herbivore interactions 45-6 chemicals by 54
plan-pathogen interactions 57-9 phytoalexin 53
pollination and seed dispersal 55,56-7 Pieris rapae, bioassay 141
post-ingestive effects 51-2,53-4 pigments 56
third trophic level effects 54-5 plant defence theory 203-9
total phenolics, tannins and food future studies 208-9
selections 46-7 genetic variation in plant phenolics
see alro cbemoecological studies 204-5
ionization of phenolics 4 historical approaches 206-8
insects, oviposition behaviour 47,48-51 plant phenolics and UH-6
in vivo studies 134-42 plant-herbivore interactions 45-6
analysis for feeding trials 137-8 plant-pathogen interactions 57,58-9
detoxification involving phenolics 138- pollination and seed dispersal 55,56-7
42 Populup 41
bioassays 141-2 post-ingestive effects 51-2,53-4
caching behaviour and detoxification Potenlilla 57
before feeding 138 precipitation assays for tannins
xenobiotic metabolism after ingestion BSA 120-4
139-41 haemoglobin 117-18,119,120
diet construction for feeding trials 134- optimization 114,115-16
5.135-8 Price and Butter method 85-6
Ipomoea lacunosa, bioassay 141 proanthocyanidin method 92-4,95,97-8
proline in tannin-protein interactions 111
Larrea tridentata 49,62 properties of phenolic compounds 2.3,
external resins 62,65,70-1 4-5
lichens 41-2 Pyonomeufamahalebellup, sequestration of
Liomys salvini, bioassay 141 plant chemicals by 55
litter decay and plant nutrition 60 Pyonomeuta dinellus, sequestration of
Lysandra corion, sequestration of plant plant chemicals by 55
chemicals by 55 radial diffusion assay for tannins 125,126
reactions, acid-base, ionization, radicals 4
Mammea arnericana 11 ether link to sugar 19
Manduca sexra 129 resins 49,62
bioassay 141 resource allocation 206
'masked' phenolics 19-20 Rhizobium, nodulation of legumes 60
mass spectrometry 177-80 R o d e o gutata, sequestration of plant
interpreting spectra 178,179,I80 chemicals by 55
ionsources 177-8
Melanargia galathen, sequestration of plant Salir 41
chemicals by 55 Schirrocerca gregaria 129
Microtus montanus 53 Schizaphis graminium 53
scolytus mediterrmeus 48
Nemoria arizonaria 54 seasonalldevelopmentalchanges 64
nuclear magnetic resonance spectroscopy secondary metabolites 1-2
181,182,183-5,186-7,188,189-98 separation methods 143-67
234 Subject index
column chromatography 155-7,158 chiroptical techniques 175-6

electrophoresis 167 comparison of unknown and authentic
gas chromatography 157,159,160-1 samples 169-70
reporting of data 161 fluorescence spectrophotometry 175
sample preparation 159-62 infra-red spectrophotometry 176
stationary phase 159 isolation of compounds 170
high perfoGance liquid chromatography mass spectrometry 177-80
162,163-5 interpretation of spectra 178,179,180
paper chromatography 149,150-1 ion sources 177-8
standardization 152 nuclear magnetic resonance spectroscopy
strategy for use 152-4 181,182,183-5,186-7,188,189-98
preliminary purification 147-9 tannins 197-8
sample preparation 144-7 W and visible spectrophotometry 171-2,
complexity of extract 145,146,147 173,174,175
extraction 145
solvent-solvent partitionlcountefcu~~ent tannic acid equivalents 119
methods 165,166-7 tannins see chemical substance index
thin-layer chmmatograpby 151,158 third level trophic effects 54-5
preparative 154,155,156 Tilnpia, bioassay 141
standardization 152 total phenolics, extraction and quantifi-
strategy for use 152-4 cation 80.81-91
see also extraction and quantification ascorbic acid interference 89, 90
shikimatelacetate combined pathway 31-4 Folin-Ciocalteu method 83-5
metabolites 42,43 Folin-Denis method 81-3
shikimate pathway 27.28-30.37 methods to avoid 90-1
see also shikimatelacetate combined Price and Butler method 85-6
pathway procedures and standards 86-9
solar irradiance and UV light 61,62-3 blank 86
spectmphotometricassays 78-80 standardization 86,87-9
spectmphotometry trichomes 51
fluorescence 175
infra-red 176 vanillin method 95-8
ultra-violet and visible 171-2, 173,
174-5 willow, phenolic glycosides 52
Streptococcus spp., bioassay 141 Wilson and Hagerman method 103
structure of phenolics 168-98
Chemical substance and group of
substances index
acetophenone 23,35 trans-cinnamic acid 27,29

acetophenones 6,23,35,42 cinnamic acids 29,31,139
acetyl co-enzyme A 20,21 derivatives 41
metabolites 41,42 Wmaxima 173
see aho acetate pathway cinnamyl alcohols 38
aesculetin 8 condensed tannins 15,43
aesculin 8 wniferyl alcohol 6,7,39
alkaloids 2,17,18 wniferyl benzoate 50
aloenin 9 para-coumaric acid 6,7,27,29
aloesin 8 post-ingestive effects 53
anacardic acids 50 radicals 29
amentoflavone 10,33 W maxima 173
anthocyanidins 10,32,56 wumarins 8,28,41,50,54
Wmaxima 173 W maxima 173
anthraquinones 16,25,42 mumestans 12
UVmaxima 173 wumestrol 12,53
anthrone 23,25 post-ingestive effects 53
apigenin 179 cutin 37
apigeninidin 56, 57 cyanidin 10, 56, 97
aryl-pyrones 9
asarone 49,50 delphinidm 10,56,97
ascorbic acid 89-90 delphinidin-3-glycoside 56
aureusidin I1 depsides 16,17,41
aurones 32,42,56 dihydrochalwne 51,54
UV maxima 173 para-dihydrocoumaric acid 31
3,4-dihydroxycinnamic acid derivatives 53
benzofurans 42,58 5,8-dihydroxyflavone 50
benzofuran-3-one system 11 DIMBOA 53
benzophenones 16,17
benzopyrans 42,175 ellagic acid 13.14
benzopyran ring system 8 ellastannins, extraction and quantification
benzoquinones 42 102-3
e m d m 16,17,25
caffeic acid 6, 7,27 UVmaxima 173
cajanol I 2 enterolactone 139
catthamin I 1 ppicatechin 15, 16
carthamone 16,17 , erycristagallin 12
casuarictin 13,14 erythrose-4-phosphate U),21, 27
catechin-7-xyloside 48,49 eugenin 8
catechin 15 eugenol 6,7,51,57
catechol 5,28,57, 175
UV maxima 173 ferulic acid 6, 7,27
chalwnes 32,56 allelopathy 59
UVmaxima 173 post-ingestive effects 53
t ~ a t I S - ~ h l ~ r ~acid
g e n i 48,58,90
~ flavan-3,4-diols 33
chorismic acid 27,28,39 flavan-3-01s 10, 15,32
chromones 8,23,24,42 flavanols 9,1O, 32
chrysopanol anthrone 24 W maxima 173
236 Chemical substance and group of substances index
flavanones 9,10 leuteolinidin 56

glycosides 48 lignans 7-8,49
structure 174 synthesis 30
UV maxima 173 lignins 6,37
flavones 32,56 lunularic acid 9.31
structure I74 Wmaxima 173
W maxima 173 luteolin 9,10,60
hvonoids 9,10-11,37-8,48,56 Wmaxima 173
5-deoxyAavonoids 42 luteolin-7-(6-malonylgluwside) 48
glycosides 55 luteone 57,58
pigments 55
flavonols 9,10,32,56 magnolol 49, SO
Wmaxima 173 malonyl co-enzyme A 22
formononetin 12,53 Mammea americana I1
post-ingestive effects 53 mammeaB/BA 11
fraxetin 50 mammea mumarin AlAA 11,33
furocoumarins 54 mangiferin 16,17,42
'masked' phenolics 19-20
gallic acid 5,6,13,27,28,41,88 6-methoxyluteolin-7-rhamnoside 48,49
W maxima 173 4-metboxyparawtoin 42, 43
gallotannins, extraction and quantification methyliweugenol 49.50
101-2 mevalonic acid 20,21
genistein 12,53 morin 48,49
post-ingestive effects 53 morphine 18
gentisin 16.17 mukonidine 35
glucosyMavones 48 myricetin 10, 175
glywsides, hydrolysis 147 W maxima 173
hippuric acid 139 naphthoquinones 16,42

hordatine-A 7,29,57 UVmaxima 173
hydrolysable tannins 13,43 naringenin 9,ZO
ellagitannins 13, 102 W maxima 173
gallotannins 13,101 naringin 51
hydrangeol 9 neoflavonoids I1
hydroquinone 5,41 neolignans 30,39,41,49
para-hydroxyphenylacetic acid 6,28 nordihydroguaiaretic acid 7,29,49,53
para-hydroxyphenylpyruvic acid 27,28 allelopathy 59
2'-hydroxyfonnonetin 60
hypericin 16,17,54 orsellinic acid 23,24,25-6
isocoumarins 9 pelargonidin 10,56

isoflavonoids I2,58 0-pentadecenylsalicylicacid 50
isoquercitrin 48,49 pentagalloylglucose 13,14
phenol 3
juglone 16,17,60 phenylalanine 27,28
glycoside 16,17 phenylpropanes 6 , 7
W maxima 173 phenylpropenes 6, 7
phenylpyrone 23,24,42
kaempferol 9,10, 175 phenylpyruvic acid 27,28
mass spectrometry 180 phloridzin I1
UV maxima 173 phloroglucinol 5,6,13
kaempferol-3-(2,3-diacetoxy-4-para- W maxima 173
coumaryl) rhamnoside 180 phlorotannins 13,26,38
kawain 42,43 phosphoenol pyruvate 20,21,27
phytoalexin 53
lecanoric acid 16,17,26 picrosalvin 18, 35
Chemical substance and group of substances index 237
pinocembrin 9,1O, 48,57 assay of protein precipitated phenolics

pinoresinol 7,29 125-6
pinosylvia 9,31,51,57 competitive K i n g assay 132,133
pisatin 58 optimidng precipitation reactions 114,
platyphylloside 53 115-16
polyketides 22 protein precipitation methods 116-24
polyvinylpyrrolidone(PVP) 106, 112 BSA 120-4
populin 49,50 haemoglobin 117-18,119,120
Populur 41 radial diffusion assay 125, 126
procyanidin I11 standards and units of measurement
procyanidin-B1 16 128
procyanidin-B2 16 condensed 15-16
procyanidin-B3 16 extraction and quantification 91,92-
prodelphinidin I11 100
profisetinidin 111 colonmetry 99-100
proline 111 proanthocyanidin method 92-4, 95,
protocatechuic acid 5,6,28,175 97-8
W maxima 173 standards and fake-positive results
pseudopurpurin 35 98-9
pteroearpans 12 vanillin method 95-8,100
pyrawcoumarins 41 definitions 104,105-6
pyrogailol 5,27 ecobgical interactions 46-7
extraction and quantification
quercetin 10,175 condensed 91,92-100
UV maxima 173 hydrolysabk 100-3
quinones 16,17 hydrolysable 13,14-15
extraction and auantification 100-3
reserpine 18 ellagitamins 102-3
resins 49,62 gallotannins 101-2
resorcinol 5 in vitro effects models 128-33
reticuline 18 inhibition of enzyme activity 218-30
rotenoids 12, 33 precipitation of proteins 130-2,133
rotenone 12,35,49 nuclear magnetic resonance spectroscopy
197-8
salicin 49, SO, 54,552 phlorotannins 13,26,38,43
salicylaldehyde 5,6,54, 88, 175 post-ingestive effects 52
protein interactions 106-13
UVmaxima 173
salicylic acid 5 , 6 bonding 106,107
scopoletin 19 nature of protein 111,112-13
W maxima I73 nature of tannin 109,110,111
soluble tannin-protein complexes
mpolin 19,58
shikimic acid Z l solution conditions predisposing to
metabolites 29 108-9
taxifolin 9, 10, 48
sinapic acid 6, 7,27,88
terpenes 17,18
UVmaxima 173
thamin 16,17
sinapyl alcohol 7,39
thymol 18, 35
stenophyllanin 13,14
triketide 23
stilbenes 9,58
tryptophan 27,28
UVmaxima 173
tyrosine 27.28
tannic acid equivalents 119
tannins 12-16,43,60 umbelliferone 8
biochemical techniques 104-33
assay optimization and specific activity vanillin 51,57
determination 126-7 verbascoside 41
238 Chemical substance and group of substances index
xanthones 16,17,34,43 xanthotoxol 8,19

UV maxima 173 xanthoxylin 6
xanthotoxin 8,20,35 xenognosin-A 60
UVmaxima 173
Analysis of Phenolic Plant Metabolites
Phenolic plant metabolites (both tannins and simple phenolics) have been the
focal point for many investigations in the fast developing discipline of chemical
ecology and they have been reported to have a major impact in many areas of
plant-animal, plant-pathogen and plant-plant interactions.
This book outlines the various classes of phenolic compounds likely to be
encountered by biologists, dealing with their structural diversity, distribution,
and examples of their ecological significance. The major part of the book is
concerned with methods for quantitation, isolation and identification, including
detailed methods for both chemical and biochemical assays. As with all books in
the Methods in Ecology series, the background to the methodology is explained,
the use of methods in varying ecological sitwations is discussed, and the
problems of interpreting results are considered.
The book is written primarily for biologists, both as a relatively non-
technical introduction to the chemistry of phenolic compounds and as a practical
aid to their analysis by the non-specialist.
The Methods in Ecology Series

The aim of this series is to provide ecologists with concise and authoritative
books that will guide them in choosing and applying an appropriate
methodology to their problem. New technologies are a feature of the series.
GLIM for Ecologists Geographical Population Analysis
M.J. Crawley B. Maurer
,1993. 336 pages, 65 illustrations 1994. 144 pages, 35 illustrations
Stable Isotopes in Ecology

K. Lajtha & R. Micbener
1994. 352 pages, 64 illustrations
BLACKWELL SCIENTIFIC I S B N 0-b32-029b9-2

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The Methods in Ecology Series, vi

The Methods in Ecology Series

Finally, we return to the role of phenolics in ecological studies all"

Structure and biosynthesis of

The above statement encapsulates the realization by ecoiogists during

chemists have attempted to work in this area without appropriate eco-

1.1.2 What do we mean by phenolic?

An important property of phenolic hydroxyl groups is their acidity,

Scheme 1.1 (a) Formation of the phenoxide ion and delocaliza-

Scheme 1.1 Conrinued

Notable properties of phenols include:

1.2 Major classes of naturdly occurring phenolic compounds

I -2.1 Simpk phenols possessing a single aromatic ring

1.2.2 More complex metabolites based on the CbC3skeleion

1.2.2.1 Lignans (C6C3 dimers)

second participating monomer rather than between the two C3 side-

1.2.3 Metabolites with a CaCo-2C6 carbon skeletmt

The aryl-pyrones are bicyclic compounds exemplified by aloenin (31).

1.2.4 Metabolites with a CdC3C6skeleton

myricetin (41); (v) flavan-3-01s-catechin (42); and (vi) anthocyanidins-

pigment carthamin (47), the oxy gen-containing heterocycljc ring has

the development of apparency and resource allocation hypotheses and

1.2.5.2 Hydrolysable tannins

greatly increased our knowledge of hy drolysabIe tannins. These have

1.2.5.3 Condemed bannim

flavan-3-01s is the relative stereochemistry of the hydroxyl on C-3 and the

Fig. 1.1 Three-dimensional structures of catechin (42, R =H,R1== OH)and epicatechin

1.2.6 Quinones, benzophenones and allied substances

1.2.7 AIkakoids and terpenes

1.2.8 'Masked' phen olits

phe nu1 alcohol ether

The addition of glucose or a similar sugar (glycosylation) can reduce

1.3 The biosynthetic origins of phenolic compounds

H2C=C-COOH H3C-C-SCo-A H3C, /O%

1.3.1 Bu Ming the building blocks

Scheme 1.4 The Calvin cycle and derivation of erythrose-4-P (841,

1.32 Phenolic me&bokites h i e d sol& on ace@ e o - e n p e A

i Scheme 1.5 Combination of acetate groups to build polyketides. In

What folluws the completion of the polymerization process is rela-

Scheme 1.6 Cyclization of a triketide chain to form phloroglucinol

In Scheme 1.7 the results of cyclizations of larger polyketide units are

Scheme 1.7 Cyclization of a series of polyketide chains of varying

Scheme 1.7 Continued

Two important points are shown in the examples in Scheme 1.7.

A further way of increasing complexity among polyketide products

Scheme 1.8 The electrophilic addition process, in which a posi-

A similar process (eIectrophilic addition) also leads to increasing

Scheme 1.9 Derivation of aromatic amino acids through the shikimic

double-bond and carboxylic acid of ferulic acid leads to coniferyl alcohol

Scheme 1.10 Proliferation of simple shikimic acid metabolites.

Scheme 1.11 Formation of a simple lignan through radical for-

107 I08 109 110

has been followed by a secondary modification, this time involving- the

1.3.4 General comments on acetate and shikimate-den'ved phenolics

1.3.5 Phenolk compoundF derivedfrom the combination of

Scheme 1.12 Combination of shikimate and acetate pathways to

The flavonoids are generated in a comparable manner to the stilbenes

Scheme 1.13 Combination of acetate and shikimate precursors to

Scheme 1.13 Continued

Scheme 1.14 Polymerization of flavan-3,4-diol units leading to

Waterman & Mole. Analysis of Phenolic Plant Metabolites. Blackwell 1994

Waterman & Mole. Analysis of Phenolic Plant Metabolites. Blackwell 1994