AN INTRODUCTION TO PHYTOCHEMICAL METFODOLOGY WITH

SPECIAL REFERENCE TO FLAVONOIDS

Nature is an excellent synthetic organic chemist. Using mild reaction conditions
and a few elemental combinations a large variety of complex molecules are made in and
around us. The atoms are put together in precise arrangements to enable to cany out
different tasks with remarkable specificity. The treasure house of natural chemicals
contain delicate perfumes, spectacularly coloured substances, medicines for numerous
ailments including deadliest of poisons.
It is a famlliar fact that the living world which surrounds us and of which we are an
integral part consists of two kinds of organisms each of which has developed a pattern of
living successfully in its own way. One 1s represented by the animal kingdom and the
other developed by plants with a resulting diversity of quarter of a million species.' The
plant kingdom is a virtual goldmine of new chemical compounds waiting to be
discovered. According to rough estimates,'.' among the 7.5 lakh species of plants',b on
earth, 5 lakh being classified as higher plants and 2.5 as lower plants, only 510% have
been exam~nedfor their chemical constituents and

' or

biological activity. With the

extermination of plant species progressing at an alarming rate in certain areas, even

before the plant has been recorded, much less studied chemically,' increased efforts
directed towards the conservation of gene pools of locally used medicinal plants are
urgent and most needed.
India with its vast area from Kashmir to Kanyakurnari and varying soil and climatic
conditions ranging from tropical to temperate, has one of the world's richest vegetations

(Angiosperms, the largest of all the major groups of living plants, include about 300

families with 12,500 genera and 2,50,000 species), to be aptly called "Botanic garden of
the World". India also has a rich heritage of indigenous drugs from the vedic times. The
'Ayurvedic system' of medicine is strictly Indian in origin and development. More than
2400 remedies have been known from Indian medicinal flora. The remarkable properties
and therapeutic uses of about 700 plant drugs have been recorded by ancient Indian
scholars - Sushrutha. Charaka and Vagbhatta probably earlier than 1000 B.C. Sanskrit
literature written by them contains information about the morphological features of many
medicinal plants, their geographical distribution, condition of growth. the best season for
their maximum potency as well as toxicity.
India like all other countries, has made significant progress by a systematic scient~fc
study of these plant drugs from the pharmacological, chemical, pharmacognostical and
clinical points of view during the past 65 years, bringing to the forefront a large number
of herbs used in Indian indigenous system for their approved efficiency and
administration in modem medicine. Recogn~singthe importance of medicinal plants.
collaborative teamwork for a complete study of plant drugs has been encouraged by the
Indian Council of Medical Research (ICMR), Central Council for Research in Ayurveda
and Siddha (CCRAS) and Councll for Scientific and Industrial Research (CSIR). The
results of various studies on Indian medicinal plants are available as research publications
and a number of books and m o n ~ ~ r a ~ h sCentral
. ~ . ' ~ Drug Research Institute (CDRI),
Lucknow initiated a programme from 1979 to investigate Indian plants which either had a
reputation in folklore medicine or whose extracts showed consistent biological activity
when put to a broad biological screen. So far under this programme over 8000 species
have been screened and biological activity has been confirmed in about 650 plants.'0

~hytochemistry,"'~~
evolved from natural products chemistry is confined to the
study of products elaborated by plants and it has developed as a distinct discipline
between natural product organic chemistry and plant biochemistry in recent years. It
deals with the study of chemical structures of plant constituents, their biosynthesis,
metabolism, natural distribution and biological functions. The fact that only less than 6%
of about 6 lakh species of plants on earth has been investigated, indicates the opportunity
provided and challenges thrown open to phytochemists. The task of phytochemlst is
compounded In accomplishing the charactensation of very small quantity of the
compounds isolable from plants. Phytochemistry also enjoys the application of modem
research for the scientific investigation of ancestral empirical knowledge. It has found
wide and varying application in about all fields of life and civilization.

Its direct

involvement in the field of food and nutrition, agriculture, medicine and cosmetics is well
known for years.

Its contribution even in seemingly remote areas such as plant

phys~ology,plant pathology, plant ecology, paleobotany, plant genetics, plant systematics

and plant evolut~onhas been ~ncreasinglyfelt. One of the more encouraging trends as
phytochemlstry continues to grow and develop as a scientific disipline, is the wider and
wider applicat~onsthat are occuring in agriculture, horticulture and f ~ r e s t r y . ~ ~
Among the phytochemicals, the polyphenolics constitute a distinct group.

They

embrace a wide range of substances which possess in common an aromatic ring bearing
one or more hydroxy substituents or their ether or glycoside derivatives.

These

compounds possess great structural diversity and are of widespread occurrence among
the secondary metabolites. A further feature of this particular group of compounds is
their ability to interact with primary metabolites such as polysaccharides and proteins.

Among the several thousands of naturally occurring phenolic compounds, the flavonoids
are the largest and the most widespread. Though the flavonoids have been found along
with alkaloids, steroids etc., not much attention was paid to their medicinal importance
for a long time. Considerable interest has now been shown in plant flavonoids as human
dietary components, therapeutic agents and as having significant activity in a variety of
isolated animal cell system. Given the importance of flavonoids as components of the
human diet (the daily diet consists approximately Ig of mixed flavonoids) the
pharmacology of these compounds has been studied. The resurgence of interest in the
study of traditional medicines together with the availability of better b~oassayshas seen
renewed efforts to understand the interaction of various flavonoids with mammalian
tissues and enzymes, and an extensive review" is available on this topic.
The flavonoids, one the most numerous and widespread group of natural constituents,
are important to man not only because they contribute to flower colour but also because
many members (eg: coumestrol, phloridzin, rotenone) are physiolog~caily active.
Although the anthocyanins have an undisputed function as plant pigments, the raison d'
etre the more widely distributed colourless flavones and flavonols still remain a mystery.
It is perhaps the challenge of discovering these yet undisclosed functions which has

caused the considerable resurgence of interest in flavonoids during the last few decades.
Flavonoids exhibit a wide range of biological activities, and currently are of particular
interest as potential anticancer agents,28 as insect antifeedants and as natural

insecticide^.^^
Over 4000 chemically unique flavonoids have been identified in plant sources and
new structures are being reported at an ever increasing rate. These low molecular weight

substances, found in all vascular plants are phenyl-benzopyrones (phenylchromones) with

an assortment of basic structures. Primarily recognised as the pigments responsible for
the autumnal burst of hues and many shades of yellow, orange and red in flowers and
food, the flavonoids are found in fruits, vegetables, nuts, seeds, stems, flowers as well as
tea and wine. Flavonoids have definite advantage over alkaloids from the point of view
of pharmacological evaluation, that they occur universally making a search for new
active substances simpler and the overall uniformity in their chemlcal structure providing
easier understanding of Structure Activ~tyRelationship (SAR). The ready a v a ~ l a b ~ l of
~ty
the more common flavonoids in pure form is a definite advantage for the study of thelr
biological activity and evaluation of their pharmacological properties. Flavonoids have
attracted the attention of scientists of d~fferent disciplines and the systemat~c
investigation on their occurrence, natural distribution, chemical nature and biological
functions continues ~nabated.""~
The tern "flavonoid" was first applied about 47 years ago by Geissman and
~inreiner".~'to embrace all those compounds whose structure is based on that of flavone
(2-phenylchromone) (I) having a basic Co-C3-C6 skeleton in common.

When the

heterocyclic ring 1s reduced, it becomes flavan (2-phenylchroman) (11). Flavone (I)

consists of hvo benzene rings (A and B)joined together by a y-ring (ring C). The various
classes of flavonoid compounds differ from one another only by the state of oxidation of
this carbon link. There is a limitation to the number of structures commonly found In
nature, which vary in their state of oxidation froth1 flavan-3-01s (catechin) (111) to
flavonols (3-hydroxy flavones) (IV) and anthocyanins (V). Flavanones (VI), flavanonols
or dihydroflavonols (VII) and the flavan-3, 4-diols (proanthocyanidins) (VIII) are also

included in the flavonoids.

It should be noted that there are also five classes of

compounds (dihydrochalcones or 3-phenylpropiophenones (IX) chalcones or phenyl

styryl ketones (X) isoflavones or 3-phenyl chromones (XI) neoflavones or 4-phenyl
coumarins (XII) and the aurones or 2-benzylidine-3-coumaranones (XIII) which do not
actually possess the basic 2-phenyl chromone (I) skeleton, but are closely related both
chemically and biosynthetically to other flavonoid types, that they are always included in
the flavonoid group.
d ' ~ by the number and
The individual compounds in each class are d ~ s t i n ~ u i s h emainly
orientation of hydroxy and methory groups in the two benzene rings. These groups are
usually arranged in certain restricted pattern in the molecule, reflecting the d~fferent
biosynthetic origin of the two aromatic nuclei~.Thus, in the A ring (I) of the majority of
flavono~dcompounds, hydroxy groups are distributed at either C-5 and C-7 or only at C7 (C-5 and C-7 of flavone become C-2' and C-6' in dihydrochalcones and chalcones and
C-4 and C-6 in aurones) and generally are unmethylated. T h ~ spattern of hydroxylation
follows from the acetate or malonate origln of the ring. The B-ring (I) of flavonoids on
the other hand is usually substituted either by one, two or three hydroxy or methoxy
groups. The rarely methylated pos~tionis C-4' with often methylation at C-3' and C-5'.
The hydroxylation pattern of the B-ring thus resembles that found in commonly occurring
cimamic acid (XIV) and coumarins (XV) and reflects their common biosynthetic origin
from prephenic acid and its congeners.

STRUClWRES OF SOME COMMON

KAVONOID TYl'ES

I Flavone

I1 Flavan

111 Flavan 3-01

IV Flavonol

V Anthocyanidin

VI Flavanone

VII Dihydroflavonol

VIII Flavan 3,4-diol

IX Dihydrochalcone

X Chalcone

XI Isoflavone

XI1 Neoflavone

XI11 Aurone

XIV Cinnamic acid

XV Coumarin

A comparison of the nature and position of' various substituents at different

:arbons of the flavonoid skeleton has been made use of in arriving at certain
peneralisati~ns~~
regarding structure and properties.

Most of the flavonoids occur naturally in conjugated form, usually bound to

sugar, by a hemiacetal linkage. But their conjugation with inorganic sulfates or organic
acids is not unusual.

The sugar free compounds are referred to as aglycones and it is

probable that in most cases they are formed as artefacts during the course of extraction,
since most living tissues contain vely active glycosides which can work even in the
presence of high concentration of organic solvents. The presence of sugars in the
molecule confers sap-solubility to the generally somewhat insoluble flavonoid
compounds.

In anthocyanins the sugar imparts stability to the aglycone. Stability

conferred by glycosylation to flavonols is observed in 3-0-glycosides of quercetin and

myricetin which are not susceptible to oxidation catalysed by phenolase unlike the
corresponding aglycones, presumably because of steric reasons."
of new glycosides are encountered in plants.4z"'

More and more range

An increasing number of flavonoid

glycosides canying sugars in B-ring hydroxyls have been

Conjugation of

flavones and flavonols through glucose with organic ac~ds like malonlc acid and
derivatives of cinnarnic acids have also been reported.s'~s" The number of acylated
flavonoid compounds succeeded by terpenold

counterpart^^^^'^ is on the increase. As a

result of electrophoretic studies, a number of zwitter ionic anthocyanins with malonic

acid and succinic acid linked to C-6 of glucose have been isolated and charactensed."
The sugars found in flavonoid glycosides include simple pentoses and hexoses
(monosides) and di- and tri-saccharides (biosides and triosides) mostly combined through
oxygen at C-1 position of sugars, usually by a P-linkage. In many cases more than one
phenolic hydroxyl group in the flavonoid molecule maybe glycosylated giving rise to
diglycosides and so on. The common sugars encountered are D-glucose, D-galactose, L-

rhamnose, D-xylose, L-arabinose and D-glucuronic acid. D-allose and D-galacturonic

acid are rare and D-apiose is an unusual and uncommon one.
The study of the distribution of flavonoids in plants58is a continuing exercise and
known flavonoids are being regularly discovered from new sources. Flavonoids are
universal in vascular plants, but variation according to phyla, order, family and
populational variation within species have been detailed by Harborne and Turner. 59
Only in 1983, the distribution of flavonoids in animals has been reported6' with
the isolation of 4'-methoxy flavone from scent glands of canadian beaver (castar feber)
and in Lepidoptera. No doubt, the presence of flavonoids in butterflies has been earlier
recognised."
A single plant may contain one or more aglycones with several glycosidic

combinations. For this reason, it is better to examine the aglycones of acid hydrolysate of
the plant extract before examining the nature of glycosides.32 Flavonoids being
polyphenolic compounds give characteristic colour changes when treated with alcohol~c
~ e " , ammonia vapour or alkalis. Positive Shinoda test (Mg-HCI) and formation of
coloured complexes with heavy metal salts are charactenstic of flavonoids.
Standard methods of extraction, separation and chemical characterisation of
flavonoid compounds are described by Peach and ~ r a c e yas~ ~well as ~ a r b o m e . ' ~
Systematic procedure for their identification employing chromatographic methods of
analysis and chemical and spectral methods of identificat~on have been detailed by
~ e i s s m a n , ~Mabry
'
et al," ~ a r k h a m , @ ~ et
a yal,"' Haxborne and ~ a b r ~ and
' ' Linskens
and ~ a c k s o n . ~ ~

The conventional chromatographic methods like column, paper and thin layer are
still in use for separation and purification of the flavonoid compounds. Increase in speed
and efficiency in the separation of mixtures by new techniques like centrifugal TLC
(chromat~tron)~'and high pressure liquid chromatography (HPLC)~'."

have been

achieved. Among the modem separation techniques applied to flavonoids, HPLC has the
advantage over other techniques in regard to sensitivity, rapidity and easy quantification.
Hosttetman and ~osttetman" reviewed the relevant literature on HPLC upto 1980. A
few other publications in t h ~ sfield include R, values by Daigle and Conkeston,'" use of
Bondpak Cis with MeOH-HOAc-H20 as developing system with two pumps by Casteele
et al

'I,

analytical problems in HPLC by Bankova et al.-' Tamma et a17' and Barberan et

aL7' Recently the application of HPLC combined with FABMS has helped in structural
elucidation of anthocyanin pigments.'s On line HPLC-UV spectroscopy, centrifugal thin
~ ~ ~ chromatotron
~~'
and flash c h r o m a t ~ g r a p h y ~are
~ ~ 'also
~
layer c h r ~ r n a t o g r a ~ husing
applied in the isolation of flavonoids. For difficult separations requ~ringvery high
resolution semipreparatory HPLC with automatic fraction collector is an ideal method.
Reverse phase chromatographyB0on chemically bonded phases gives better results for the
separation of plant phenolics.

The complication of irreversible adsorption and

decomposit~onof the solute at the liquid-sol~dInterface in all techniques employing a

solid stationary phase and liquid phase is overcome by barious suppon free liquid-liquid
partition techniques. Among these the two most efficient all liquid chromatographic
techniques, DCCC",'~ and RLCC'~have been employed profitably for quantitative and
qualitative separation by Hosttetman and co~laborators,~~
Aritomi et alg5and Gunasegaran

eta^.'^

However the constant need in natural product chemistry to separate large and
small quantities of complex mixtures efficiently and rapidly is unfortunately seldom
satisfied by the use of any one chromatographic technique. The best results have been
obtained by a combination of several techniques which are often complementary. Paper
electrophoresis is a technique of limited application in flavonoid analysis, since to be
mobile, a flavonoid must be in an ionised state at the pH of the electrolyte. Its useful
application lies in the recognition and identification of flavonoid

and in the

distinction of glycuronides from g ~ ~ c o s i d e s .Relative

'~
mobillties of different flavonoid
sulfates are listed by ~osttetman.~'Electrophoresis finds greater application in the field
of anthocyanins and betacyanins.
The flavonoid once isolated as a homogenous compound is characterised by the
specific colour tests, physical constants, elemental analysis, R, values in various solvent
systems, analysis of hydrolysis products, preparation of derivatives and comparison of
these data with related compounds. Further support for confirmation of the structure of
the flavonoid is achieved by the analysis of different spectral data (UV-V~sible,IR,'H
and "C hWR and MS).
The development in the general methodology of natural product chemistryq' is
readily applicable to flavonoid compounds also. Ultra violet spectroscopy03~92
is still one
of the oldest and useful techniques for flavonoid identification. The UV spectrum with
MeOH and with diagnostic shift reagents can give information on the type of flavonoid as
well as its substitution pattern. The use of AICI3 and AICIJ~HCIW spectra in the precise
determination of the structure of flavonoid compounds was demonstrated by ~ o i r i n ~ '
and the limitation of NaOAc spectra in flavonoid analysis has been reported by Rosler et

al.94 IR s p e c t r o ~ c o p yprovides
~~
valuable information regarding the type of flavonoid as
well as the nature of the substituents like methoxy, methylene dioxy and prenyloxy
groups. It is also used as a 'finger-printing' devise for establishing the identity of two
samples.
Over the past thirty years, chiroptical methods like ORD and CD spectral analysis
have been employed for the determination of stereochemistry of chiral f l a v o n o i d ~ . ~ ~ . ~ '
The exciton chirality method9' employing the application of coupled oscillators in
determination of the chirality of natural products

IS receiving

greater attention.

Mass spectrometry of flavonoids serves as a valuable aid in determining the

molecular weight and probable structure even with very small sample size. The electron
impact mass spectrometry (EIMS)~'~'"1s used for the volatile compounds, the nonvolatile compounds are convened to suitable derivatives like trimethyl silyl ether,
permethyl ether, methyl ester or similar derivatives. When hvo MS are linked in tandem,
it has?ow become poss~bleto employ the first as separator and the second as analyser to
perform direct mixture analysis (Tandem-MS). This multiple stage MS has been
rev~ewedby Roush et al."' Field desorption mass spectrometry (FD-MS) employed for
polar and thermolabile compounds like flavonoids has been reviewed by Schulten and
~ames."'

Desorption chemlcal ionisation mass spectrometry (DCI-MS)"'

using

electrically heated tungsten probe and fast atom bombardment mass spectroscopy (FABMS)'"

using the sample solubilised in polar matrix (like glycerol, thioglycerol

etc.,)deposited in a copper target which is bombarded with energised neutral atoms to

induce ionisation and d e s ~ r ~ t i oappears
n ~ ~ ~to be the most advantageous one for the
analysis of flavonol glycosides. In the chemical ionisation mass spectrometry ( C I - M S ) ' ~ ~

the ions are formed in ion molecular collisions which Include abstraction as primary
process using weak gas phases like CHI, NH3, i-CoHlo in positive CI for protonation. In
negative CI, OMe' as reagent for proton abstraction or C1'as an attachment reagent is
employed. The MS analysis of permethyl ether of glycosides is widely employed for
settling structural problems in C-glycosyl flavone~.'~','"~
Pyrolysis chemical ionisation
mass spectrometry of flavonolds under positive and negative ionisation condit~onsis
o b s e ~ e d ' ~to' yield data charactelistic of both aglycone and sugar residues, providing an
alternative for FD and FABMS techniques. The easy MS differentiation of flavanones
and dihydroflavonols by the characteristic fragments has been

The

application of GC-MS"' analysis to perdeureromethylated derivatives of f l a v o n o i d ~ ' ' ~

has rendered the identification of certain methoxylated compounds easier. Online HPLC~ ~ " b w i be
l l readily accepted by flavonoid researchers. Solution phase secondary ion
mass ~pectrometry"~has proved useful in the determination of molecular weight of
complex flavonoid glycosides. Becchi and ~raisse"' have reported mass analysed ion
kinetic energy (MIKE) and collision activated dissociation MIKE spectra of flavonoids
providing characteristic fragment ions which permit differentiation of the 6- and I or 8substituent location and the position of 0-glycosylation.

Electrospray MS114 has

become quite versatile on account of the amanability of the technique to highly polar and
large sized molecules including biomolecules like proteins.
The continuing rapid development in instrumentation In particular those involving
Nuclear Magnetic Resonance (NMR) over the last hvtnty years has given considerable
impetus to structural elucidation in all fields of natural product chemistry. The proton
magnetic resonance spectroscopy is the established non-destructive method of flavonoid

analysis. Use of high field magnets and computer assistance has made the recording of
high resolution 'H NMR spectra of minute quantity of flavonoid~."~~"' Typical
application of 'H NMR includes determination of oxygenation pattern, number of
methoxy group, distinction of isoflavones, flavanones and dihydroflavonols, number and
nature of sugar present (whether a-liked or P-linked) and detection of hydrocarbon side
chain. Shift reagents provide a method of spreading out PMR absorption signals. The
use of lanthanide shift reagents In positioning of methoxyl groups of flavonoids was
reported by Joseph Nathan et al."'
Of late

''c S M R

has become the most useful technique for the

structural determination of flavonoids. "C resonance signals extending over 2OOppm

provide the nature of the carbon skeleton. Carbon relaxation time measurements being
less difficult is very useful in d~fferentiatingotherwise non-discernible carbon atoms like
C-6 and C-8 in flavonoids. Interglycosidic linkages in the case of d i s a ~ c h a r i d e s ' ~ ~
(rutinose, neohespendose etc.,) type of linkage with aglycone and sugars.and conjugation
with sulfates and organic acids can also be determined. Homonuclear and heteronuclear
correlation spectroscopy127(HOMCOR and HETCOR) and the various decoupling
experiments have reduced the difficulty in the interpretation of 'H and "C spectra of
complex molecules. Fourier transform NMR employing popular pulse sequences like
off-resonance, heteronuclear decoupling, gated heteronuclear decoupling, inverse gated
heteronuclear decoupling and selective proton decoupling has eased the task of
assignment of peaks in a "C NMR spectrum. Refocused INEPT with decoupling is an
alternate of off-resonance decoupling for assigning carbon multiplets. I-modulated spin
echo spectroscopy'28 is yet another method of off-resonance decoupling which gives

positive signal for even number of hydrogen (CHI and quaternary) and negative signal
for carbon with odd number of hydrogen (CH and CH,) attached. Usually a set of routine
2D experiments130including COSY (and / or DQF cosy), relayed COSY [(HOHAHA),
2D NOE (NOESYIROESY) and HETCOR (HMQC)] are used in structure elucidation
exploiting a wealth of information inherent in the data. No doubt, considerably decreased
sample size required for 2D NMR studies at present'"~'32make it more acceptable to
natural product chemist.
If the flavonoid compound can be obtained in a fine crystalline form. X-ray
analysis can help in further confirming the structure as reported in the case of
calycopterin.'"
Structures of organic compounds are usually determined by using data generated
by chemical and instrumental methods. A chemist then invariably attempts to establish
the proposed structure by unequivocal total synthesis. The conventional methods of
synthesis of flavonoids from simple precursors by condensation methods have been
proposed by Baker and ~enkataramen,'" Algar and ~ o b i n s o n , ' ~ bAlgar
d and F I ~ ~ . " ~
These methods of synthesis have been modified by Farkas et ali" and Wagner et

as

illustrated by the synthesis of a number of flavonoids and their methyl ethers. Many
flavonoid compounds have been prepared by simple modification of the existing structure
through nuclear oxidation, nuclear reduction, isomerisation, selective alkylation and
dealkylation, selective glycosylation and partial hydrolysis. Farkas et

accomplished

the synthesis of methoxylated flavones from the corresponding brominated methoxy

chalcones.

The synthesis of 5,6,7,3',4'-pcntamehtoxy flavone (sinensetin) by

dehydrogenation of the corresponding flavanone with Se02 has been achieved by Wagner

et al.IJ9 This flavone has been used as the starting material for the synthesis of a number
of related flavones. Bose et all4' have reported cyclisation and simultaneous dehydration
of the hydroxy chalcone to the corresponding flavone by heating with palladium on
charcoal. A short and facile synthetic route to hydroxylated flavones has been reported
by Nagarathnam and ~ u s h m a n . ' ~ ~
The synthesis of

flavonoid glycosides have been achieved using the a-

acetobromo sugars of pentoses, hexoses or disaccharides and the aglycones in the

presence of catalysts.
Zemplen and

arka as."'

The selective glycosyLation of 7-OH has been achieved by

Synthesis of other glycosides have been accomplished by trans

acylation methods by Nogradi et all" and Wagner et al.'"

glycosyl flavones has been reported by Eade et al.I4\
exemplified by the preparation of 5.7,4'-trimethoxy

The total synthesis of Cmore general synthesis,

ite ex in"^ and other complex ones

has been provided by later workers.14' Thus the synthesis of almost all types of mono
and

di-C-glycosyl .flavones

and

flavone

C-glycosyl-0-glycosides

has

been

accomplished.'48 synthesis of some novel flavonoids has been illustrated by Rakosi et

The chiron approach to the total synthesis of natural products'50 might become a
useful guide in the synthesis of chiral flavonoid compounds. Studies of the selective Oalkylation and dealkylation of flavonoids with anhydrous AIBr3 were reported by Hone et
a~.151
Flavonoids may have existed in nature for over one billion years'52 and thus may
have interacted with evolving organisms over the aeons. Clearly the flavonoids possess
some important purposes in nature, having survived in vascular plants throughout
evolution. Our ideas of the evolution of flavonoids is mainly based on our knowledge of

the overall modes of biosynthesis of the different classes of compounds. Tremendous

progress has been made in the biosynthesis of f l a v ~ n o i d s . ' ~ 'Our
~ ' ~ present
~
knowledge
in flavonoid biosynthesis is based on a combination of earlier results from radioactive
tracer studies in vivo and the more recent data obtained at the enzyme level in vitro. In
the past few years the enzymology of flavonoid biosynthesls has made particularly rapid
progress. Flavonoid biosynthesis can be considered in three stages. The first is the
formation of the basic C6-C3*Cb skeleton through acetate-malonate and shikimic acid
pathway to aromatic compounds. The second stage is concerned with the ways by which
the different classes of flavonoids are synthesised.

The final stage embraces the

elaborat~onof individual compounds withln each flavonoid class, involving steps such as
hydroxylation, glycosylation, methylation etc., Chalcone is considered as the common
intermediate in the biosynthesis of all classes of flavonoids. Insight into three aspects of
the problem of

flavonoid biosynthesls .has come In the past from comparative

a n a t ~ m ~ and
' ~ ~chemical
" ~ ~ genetic studies'" and recently from feeding experiments
w ~ t hradioactive tracers. Major work on chemlcal genetic studies have been carried out
by Grisebach and c o - w ~ r k e r s . ' ~Research
~ " ~ ~ has led to the isolation and characterisation
of enzymes of the pathway of flavonoid biosynthesls. The use of young plant tissues and
cell suspension cultures as source materials have also greatly facilitated the study of
flavonoid biosynthesis at the enzyme level. Roux and ~erreira'~%avehighlighted the
special role of a-hydroxy chalcone as the key intermediate in flavonoid biogenesis. A
comprehensive report on the biosynthesis of flavonoids by

ani it to'" and

Heller and

~ o r k r n a n ,a' ~good
~ account of biosynthesis of shikimate derived phenolic compounds by
~arbome,"' biosynthetic studies in vivo with labelled precursors and biochemistry of

flavonoid biosynthesis by ~ e l l e rare

' ~ useful
~
publications. The importance of flavonoids
and other secondary metabolites in plant biochemistry has been detailed in "the
Biochemistry of plants".'69 Different aspects of mammalian metabolism of flavonoids
have been reviewed by De ds,17' ~cheline,'" ~riffithsl" and Middleton Jr and
~andaswami." Flavonoids are the constituents of the mammalian diet derived from
plants. The ingestion of flavonoids by mammals in the diet or for therapeutic use, brings
them in contact with both intestinal micro-organisms and mammalian tissues which are
capable of biotransformation of flavonold compounds.

The available evidences'''

indicate that the hydrolysis of the flavonoid glycosides to thelr corresponding aglycones,
ring fission and oxidative and reductive transformations are medlated by intestinal microorganisms. Though it 1s cenain that the metabolic changes undergone by flavonoids
occur with~nmammalian tissues, the relative contribution of Individual tissues is not fully
understood.
The flavonoids find exceptional use as taxonomic guide in the classification of
plants.

The reason for prefemng flavonols to other secondary metabolites is their

structural diversity, widespread distribution. comparative stability, easy detection and

Identification and the fact that this group of plant products 1s not actl\ely concerned with
cellular metabolic processes. Any particular flavonoid can be relied to be present in more
or less constant amount in the same tlssue of the same species so long as the plants are
grown under normal physiological conditions. The conspicuous exception to this is the
variation in the relative concentration of p-coumaric acid and caffeic acid (mono and
dihydroxy phenyl propanoid precursors) as well as kaempferol and quercetin which are
insignificant from the taxonomic point of view.

Importance of flavonoids in

chernotaxonomy can be illustrated with a few examples. Phloretin or its 3-hydroxy

derivatives occur in all species of Malus ( ~ o s a c e a e ) . ' No
~ ~ other genus in Rosaceae
contain these compounds. Caviunin, an isoflavone is a taxonomic marker in Dalbergia
species.175 Quercetrin, the major constituent in the parasite Dendropthoe falcata
(Loranthaeceae) growing on plants of different families projects its possible use as a
chemotaxonom~cmarker'7b of this specles. The flavonoid profile characterised by Cglycosyl flavones of Mollugo species17' supported its separation from Aizoaceae of the
Centrospermae and placement in Molluginaceae along with Caryophyllaceae in
Caryophyllales. Among the numerous flavonoids of higher plants the rare and unusual
ones find more acceptance in micromolecular taxonomy. Quite recently a survey of 2'oxygenated flavonoids along with their posstble biosynthetic pathways has been
reported.""
Colour in flowers is the ultimate step in the synthesis of large bioorganic
m?lecules, which are quite exclusively to be found among flavonoids, carotenoids and
quinones. In contrast to carotenoids and qutnones, which always absorb light in the
visible range, flavonoids can exist in both coloured and colourless forms. Therefore,
flavonoids may contnbute directly or ind~rectlyto the visual effect produced, not only on
humans, but also on animals, especially insects, birds and herbivores. Thus, flavonoids
are multifunctional and the function varies according to the need and stress placed upon
the plant and depending on its stage of growth and development. The anthoclor pigments
(chalcones and aurones) usually give yellow to orange colours to the tissues in which
they are located. However, bright yellow colours are generally related to the presence of
c a r ~ t e n ~ i d s . Moreover
"~
those flavonoids which are barely detectable to the human eye

may well be clearly visible to insects, especially to those which feed on plants and which
are frequently involved in the pollination process. Naturally occumng chalcones and
aurones have been reviewed from the point of view of their s t r ~ c t u r e . ' ' ~Analytical
chemical methods most useful in the structural determination of the faintly coloured to
colourless flavones, flavonols and their glycosides have been reported.'''

No doubt, the

anthocyanins are the most important flavonoid plant pigment and their expression is
achieved in a unique way when they are present In flowers. Major advances in the past
~ the aptitude of
decade concern the determination of anthocyanin primary s t ~ c t u r e ' 'and
anthocyanins to give molecular complexes with a large variety of natural and artificial
m o ~ e c u l e s . ~ ' Pnmary structures are the forms recognised during anthocyanin
identification ie: the flavyliurn cations are zwitter ionic or not. Anthocyanin-flavone
glycos~decopigment complexes"J are stabilised by chelation with metal ions or by H-

bonding.'" These copigments are found to protect the anthocyanin in vivo from
nucleophilic attacks resulting, in colour loss.

Plant species now known to contain

malonated anthocyanins in their flowers have been listed by ~ a r b 0 r n e . l ' ~ Being

zwltterionic under suitable conditions, malonated anthocyanins are easily recogn~sedby
use of electrophoretic surveys.18- Several speculations about the role of anthocyanins on
the perception or filtration of light and response to stress factors, includ~ngmicrobial
attack, await further thorough s t ~ d i e s . ~ ~ Anthocyanins
'~''~
may also be important factors
with other flavonoidsiw in the resistance of plant in insect attack. Several physiological
functions for anthocyanins in the general metabolism of plants described in the
literature'" are still rather obscure.

The important function of flavonoids in plants are their protective role as light
screen against damaging UV radiation, as feeding deterrents and protection from
herbivores and as allelopathic agents. In biological systems, the stimulation of protein
degradation as well as protein formation by antibiotics resulted in flavonoid accumulation
implicating the importance of flavonoids in protein s y n t h e ~ i s . ' ~ ~Other
~ ' ~ ' important
functions include their role as anti-oxidants, enzyme inhibitors, precursors of toxic
substances and as photosensitising and energy transferring compounds. ~n control of plant
growth and development, in respiration, photosynthesis, morphogenesis and sex
determination in plants

In general, flavonoids show potential as environmentally less

harmful insecticides, especially when applied systemattcally, and knowledge of their

properties in plants to deter insects can be used to breed more resistant crops. Thus, if
flavonoids are used in integrated pest strategies, and as long as agronomists and plant
breeders are aware of the properties of these compounds and other classes of secondary
substances, they have a real potential for crop protection in tht future.
Apart from the physical and morphological means, chemical means are a major
method of plant defence. A significant range of flavonoids have been encountered as
antifungal agents. A number of flavonoids are being tnduced In plants following fungal
invasion. Some of the flavonoids affect the behaviour, growth and development of
insects due to the~rtoxicity, while flavone glycosides are feeding stimulants.'"

The

metabolic challenges of the plant flavonoids including the condensed tannins to the
insects consists mainly of the variety of phenolic groups. This activity 1s destroyed when
the phenolic OH group is methylated in flavonoids. The structure- activity effects have
been studied for a number of flavonoids for anti-growth and anti-bacterial activity and it

has been found that growth inhibiting activity depends on the presence of ortho
dihydroxy group in ring A and B and not the functional group of ring C and the position
of ring B (at C-2 or C-3 as in flavones or isoflavones). The glycosylation of flavono~ds
also show marked variation in growth inhibition; 3-0-glycosylation inhibits growth but
not 7-0-glycosylation.

The enhanced activity of the flavanones compared to the

corresponding flavones showed that the coplanarity of the flavonold rings of flavones
might hinder their biological efficacy. Different tqpes of bioassay involving several
types of organisms conducted with ~soflavonoidphytoalexins by ~mith"' revealed that
they possessed fung~toxicityand limited antibacterial activtty

The introduction of

~soflavonoid phytoalexlns In plants causes p h y t o t o ~ ~ ~ ~ t y such

" ~ h ~as
' v 'inhibition of
resp~ration,reduced growth of suspension cultures, repressed seed germination, retarted
root growth and electrolytic leakage.
The occurrence and concentration of flavonoids in foodstuff have been reviewed
by ~ e r m a n n . ' " The chem~callymodified anthocyanins have found ~ncreasinguse as
approved colours in formulated food and beverages.

Although different groups of

flavonoids are consumed, the diversity is removed by intestinal micro-organisms

hydrolysing the glycosides.

The flavono~ds combine w ~ t h dietary proteins and

e
carbohydrates and w ~ t hdigestive enzymes and cellular components of the d ~ g e s t ~ vtract.
A small fraction of the flavonoid is absorbed into the blood stream and the unabsorbed

compounds affect many aspects of digestion and health.

An ever-increasing number of pharmacological effects of flavonoids have become

known over the years through the discovery of new plant flavonoids and their

derivative^.'^^^^^^

The

spasmolytic,

antianginal,

antiulcer,

antihepatotoxic.

antiinflammatory, antiallergenic, antimicrobial and antiviral effects of flavonoids are

gossypin,
noteworthy. In recent years a number of flavonoids like baicalin, tax~fol~n,
proanthocyanidins, nepetin, diosmin, fisetin, sophoricoside, (I)-catechin, (-pepicathecin
and 5.7-dimethoxy flavone have been reported to have anti-inflammatory e f f e ~ t s . ' ~ ' ~ ' ~ '
Recently the effect of flavonoids on arachidonate metabolism was reported by Ferrandiz
et al.204 Many semisynthetic flavonoid derivatives also show this effect of which (0-Phydroxy ethyl) rutin and various quercetin derivatives are

Stud~esof

compounds with flavone skeleton were stimulated by recognition of antiallergic effects

Thus, orally effective anti-allergic chromone drugs (kellin, hypolaet~n-8-glycos~de
disodium chromoglycosate etc..) are In use.'"' Antiviral activ~tyof a feu flavonoids were
Investigated by Pusztai et al"'

and observed that hydroxylatlon in 3-position appears to

be a prerequlslte for this activity. Antirhinovirus and antipicronovirus activity of certain

flavonoids have also been reported."'

Anti-cancer activity of a few flavonoids (flavones.

~soflavones,.flavanones, and flavonols) are also reported.'"R~'O' Recentl? 7.8-di-Osubstituted flavans, blflavans and flavones showed cytotoxic a c t i ~ i t y "and
~ 11 has been
establ~shedthat the activity is due to methoxy and 1 or hydroxy group In the structure.
Inhibition of human immunodeficiency virus reverse transcripvase 1s currently
considered a useful approach in the prophylaxis and intervent~onof Acqulred Immuno
Deficiency Syndrome (AIDS) and natural products have been extensively explored as
inhibitors of this enzyme, to discover drugs active against AIDS. Recently 150 pure
natural products have been examined2" and polyphenolic compounds were found to be
responsible for the activity; among flavonoids tested quercetln exhibited moderate

The anti-hepatotoxic effect of flavonoids was first demonstrated by Halm et al2I2

with the flavanolignan, siliybin and its isomers. About 13 flavonoids and coumarins have
been demonstrated2" to be anti-hepatotoxic or liver protective agents. The screening of
plants, recorded in Indian folk medicines, for liver injury has established214that the active
principles are flavonoids.

Anti-ulcerogenic properly of 3-0-methlyl (+)-catechin.

apigenin, and luteolin, anti-diabetic effects of hispidulin and nepetin and analgesic effects
of puerarin are also reported.21s.2'6
Several dihydrochalcone derivatives are used as sweeteners. The bitter taste of
flavanones and thelr neohesperidosides, sweet taste of dihydrochalcone neohesperidoses,
reduction of the bltter taste of flavanone glycosides by flavones and the tastelessness of
the flavone glycostdes irrespective of the nature of the sugars etc were elaborately studied
by Horow~tz and ~ e n t i l i . " ' Although flavonoids have been extensively studied In
mediclnal research for dlberse physiological effects, their role in plants from which they
are derived has not been un+mbiguously established.
Work on different aspects o f flavonoids including discovery of new compounds,
' ' ~ the
biological activity, medicinal properlies erc., are reviewed r e g ~ l a r l ~ ' ~ and
knowledge 1s updated through proceedings of regular international symposia on
flavonoids.'lR Considerable work on the c h e m ~ s t yand pharmacolog~calproperties of a
number of flavonoids has been accomplished in the laboratones of our

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218. Future Trends in Phytochemistry - a Young Scientists Symposium - Rolduc,

Netherlands 10 - 13 May 1998.
The Application of Chromatograph~cMethods in Phytochernistry & Biomedical
Analysls

- Meeting

of the Polish Phytochemical Soc~ety25 - 27 Jun 1998.

46IhAnnual Congress of the Soclety for Medicinal Plant Research in Vienna 31Aug

- 3 Sep 1998.
1 9 ' ~International Conference on Polyphenols - Lille, France i

-4

Sept 1998.

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Ph.D Thesis,

Pondicheny University.
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Plants", Ph.D Thesis, Pondicheny University, India.

224. Publications from our laboratory In the field of flavonoids cited in references 29 &
32-34.