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AN INTRODUCTION TO PHYTOCHEMICAL METHODOLOGY
WITH SPECIAL REFERENCE TO FLAVONOIDS
Earth is a planet dominated by plants. 'Ihe green plant is fundamental to all other
life. The oxygen we bnsthe, the nutrients we consume, the fuels we bum and many of
the most important materials we use wen produced by plants.
Plants represent the first stage in the evolution of living things. In the process of
the growth of nature, plants multiplied in number, variety and types. Humanity has
identified as many as 7.5 lakhs species of plants"' on earth, of which 5 lakhs are
classified as "higher plants" and 2.5 lakhs as "lower plants".
The association between plant and man is an age-old process starting from
human civilization. There has always been a race between nature and human
knowledge. The plants sustain nature and nature sustains them. The interdependence of
man and nature increases day by day. If human race makes sensible use of nature,
posterity is bound to be prosperous.
Since their evolution, plants became primarily useful for mankind. Realization
of the importance of plants in the welfare of humanity prompted their systematic study
from different angles. Thus many plant species 6.7 have been subjected to detailed
scientific analysis over the years. Yet not more than 10% of all the plants could be
analyzed and a vast majority still remains uninvestigated. The study continues in
various fields and provides challenges to several groups of plant scientists.
India with its vast area from Kashmii to Kanyakumari and varying soil and
climatic conditions ranging born tropical to temperate has one of the world's richest
vegetations, comprising of about 1,30.000 species of plants belonging to I20 families to
be aptly called "the Botanic Garden of the World." India has a rich heritage of
indigenous dmgs from the Vedic times. The Ayurvedic system of medicine is purely of
Indian origin and development. More than 2400 medics haw been known from Indian
medicinal flora. The remarkable properties and therapeutic uses of about 700 plant
drugs have been recorded by ancient Indian scholan, m. Sushrutha, charaka and
vagbhatta before 1000 B.C. Sanskrit literature written by them contains information
about morphological features of many medicinal plants, their geographical distribution
condition of growth, the best season for their maximum potency as well as their toxic
properties.
Another ancient system of medicine flourished in south India is the siddha
system 8.25. This system was introduced by the siddhars. They were those who attained
or achieved perfection or heavenly bliss. They were the greatest scientists of ancient
times and men of culture, intellectual and spiritual faculties combined with super natural
powers.
Among siddhars, there were eighteen Maha siddhars, viz, Nandhi, Agasthiar,
Mular, Punnakesar, Pulasthiar, Punaikannar. Idaikkadar, Bohar, Pulikaiesar, k a m ,
konganavar, kalangi, Azukannar, Agappiar, Pampani, Theraiar, Kudambaiyar,
Sattainathar. They impaned in different periods of history, not only their knowledge of
medicinal plants but also their philosophy to their students and successors.
According to siddhars school, the human body is composed of 96 tatwas, 72,000
blood vessels, 13,000 nerves, 10 main arteries and 10 vital aim (prana) in the form of a
network. It is owing to the derangement of the three humours, the body becomes liable
to 4448 diseases. There are medicinal plants in India to cure all the above diseases of
mankind (sage ~gasthyu)".
These siddhars did not only cure the dreadful diseases like cancer, lepmsy etc.
by simple herbal powders, but also attained the immo~tality by a plant medicine
popularly called by them as "Muppu".
According to them "Muppu" is composed '' of three salts, separated from a
medicinal plant and reunited, which rejuvenate the human bcdy. Rejuvenation does not
necessarily mean restoring youthfulness to the old, but it simply means the maintenance
of youth without reaching old age, if youth is maintained perpetually it becomes
immortality.
India like all other countries has made significant prognss by a systematic
scientific study of these plant dmgs from the pharmacological, chemical,
pharmacognostical and clinical points of view during the past 65 years. This brought to
the forefront a large number of herbs used in Indian indigenous system for their
approved efficiency and administration in modern medicines.
Rewgnizing the importance of medicinal plants collaborative team work for a
complete study of plant dmgs has teen encowaged by the Indian Council of Medical
Research (ICMR) Central Council for Research in Ayurveda and Siddha (CCRAS) and
council for scientific and Industrial Research (CSIR). The results of various studies on
Indian medicinal plants are availble"". Central Drug Research Institute (CDRI)
Lucknow initiated a programme from 1979 to investigate Indian plants which either had
a reputation in folk medicine or whose extracts showed consistent biological activity
when put to a broad biological screen. So far under this programme over 8000
species were screened and biological activity confirmed in about 650 plants38.
Thorough investigation of siddhar's literatm reveals that each and every plant
contains both poisonous chemicals as well as medicinally useful chemicals. The
medicinal property of the plant alone was retained, by treating this plant with other
simple chemicals or some chemical methods to neutralize the poisonous effect of the
plant. Moreover siddhars have used a mixture of herbal powders in some fixed ratio
than a single herbal powder.
The above studies provide further scope for undertaking research work in several
directions. The phytochemicals both pharmacologically active and poisonous and their
shuctuml changes can be discovered. Besides, there are different results regarding the
s t~chl ra l modifications of pharmawlogically active phytochemicals obtainable by
combining different herbal powders at various proportions. If the modem methods of
investigation of medicinal plants in India with the newly developed scientific approach
are applied, this type of research will provide for the discovery of new effective plant
drugs.
Phytochemistry 3w4, 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 pmduct organic chemistry and plant biochemistry in reffint years. It
deals with the study of chemical stmctms of plant constituents, their biosynthesis,
metabolism, natuml distribution and biological functions. The fact that only less than
10% of about 7.5 lakhs species of plants on earth has been investigated indicates the
oppommity provided and challenges thrown open to phytochemists. The task of the
phytochemist is compounded in accomplishing the characterization of very small
quantity of the compounds isolable from plants. Phytochemisby 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 & i t involvement in the field of food and nutrition, agriculture
medicine and cosmetics, is well known for years. Its contribution even in actmingly
remote areas such as plant physiology, plant pathology, plant ecology, palaeobotany,
plant genetics, plant systematics and plant evolution has been increasingly felt. One of
the more encouraging trends as phytochemisw continues to grow and develop as a
scientific discipline is the wider and wider applications that arc occuning in agriculture,
horticulture and fo~esby'~.
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 primaty metabolites such as plysaccharides and proteins.
Among the several thousands of naturally occurring p l y phenolic compounds,
the flavonoids an the largest and the most widespread. The term "flavonoid" was first
applied about 48 years ago by Geissman and ~inreiner'~" 70 embrace all those
compounds whose structure is based on that of flavone (2-phenyl chromone) (I) having
basic Cg - C) - C 6 skeleton in common when the heterocyclic ring is r e d u d , it becomes
flavan (2-phenyl chroman) (U), Flavone (I) consists of two 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. T h m is a limitation
to the number of muctures commonly found in nature, which vary in their state of
oxidation from flavan 3-01s (catechin) (m) to flsvonols (3- hydroxy flavones) (IV) and
anthocyanins (V). Flavanones (VT), flsvanonols or diiydro flavonols (VQ and the
flavan 3-4-diols (proanthocyanidins) (Vm) are also included in the flavonoids. It should
be noted that there are also five classes of compounds (diiydro chalcones or 3-phenyl
propiophenones (IX) chalcones or phenyl styyl ketones (X) isoflavones or 3-phenyl
chromones (XI) neoflavones or 4-phenyl wumarins (XII) and the aurones or 2-
benzylidine-3-coumatanones (Xm) 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.
The individual compounds in each class are di~tinguished'~ mainly by the
number and orientation of hydroxy and methoxy groups in the two benzene rings. These
groups are usually armnged, reflecting the different biosynthetic origin of the two
aromatic nuclei. Thus, in the A ring (I) of the majority of flavonoid compounds,
hydroxy groups sre distributed at either C-5 or C-7 or only at C-7 (C-5 and C-7 of
flavone become C-2' and C-6' in diiydrochalwnes and chalcones and C-4 and C-6 in
aurones) and generally are unmethylated. This pattern of hydroxylation follows from the
acetate or malonate origin 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 position is C-4' with often methylation at C-3' and C-5'. The hydroxylation
panem of the 9-ring thus resembles that found m commonly occurring c i ~ a m i c acid
(XN) and coumarins (XV) and reflects their common biosynthetic origin from
prephenic acid and its congeners.
STRUCTURES 01; S O M E C O M M O N
FLAVONOID TYPES
I Flavonc
mO OH
I11 Flavan 3-01
I1 Flavan
Q,$3 \ 0 H
0
IV Flavonol
XI Isoflavone XI1 Neoflavone
XI11 Aut-one XIV Cinnarnic acid
A comparison of the nature and position of various substituents at different
carbons of the flavonoid skeleton has ban made use of in arriving at cettnin
genera~izations'~ regarding structw and properties. Most of the f lavonoid~"~~ occur
naturally in conjugated form, usually bound to sugar, by a hemi acetal linkage. But their
conjugation with inorganic sulfates or organic acids is not unusual. The sugar h e
compounds an 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
very active glywsides which can work even in the presence of high concentration of
organic solvents. The presence of sugars in the molecule confers sap-solubililty to the
generally some what insoluble flavonoid compounds. In anthocyanins the sugar imparts
stability to the aglycone. Stability confemd by glycosyiation 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 ". More and more range of new glycosides are encountered in plants 58.63.
An increasing number of flavonoid glywsides carping sugars in 9-ring hydroxyls have
been npo1ted6c~~. Conjugation of flavones and flavonols through glucose with organic
acids like malonic acid and derivatives of cinnamic acids have also been rspo~ted~'~'~.
The number of acylated flavonoid wmpounds succeeded by terpmoid countc~par t s~~~ l2
is on the increase. As a result of electrophoretic studies, a number of miner ionic
anthofyanins with malonic acid and succinic acid linked to C-6 of glucose have been
isolated and characterized".
The sugars found in flavonoid glywsides include simple pentoses and hexoses
(monosides) and di-and tri-saccharides (biosides and triosides) mostly combined
through oxygen at C-l position of sugars, usually by a P- linkage. In many cases, more
than one phenolic hydroxyl group in the flavonoid molecule may be glywsylated giving
rise to diglycosides and so on. The common sugars arc D-glucose, D-galactose,
L r h m o s e , D-xylose, Larabiiose and D-glucuronic acid. Dallose and Dgalacturonic
acid are ran and D-apiose is an unusual and uncommon one.
The study of the distribution of flavonoids in plants",'J is 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 with in species have been detailed by Harbome and ~ u m e r ' ~ . The
presence of flavonoids in butterflies has been rewgnised7' in 1983. In 1985, the
distribution of flavonoids in animals has been reported with the isolation of 4'- methoxy
flavone from scent glands of Canadian beaver (castar feber) and in ~ c ~ i d o ~ t e t a ' ~ .
Standard methods of extraction, separation and chemical characterization of
flavonoid compounds are described by ~ r a c e ~ ' ~ a s well as ~arbome".
Systematic procedure for the flavonoid identification employing chromatographic
methods of analysis and chemical and spectral methods of identification have been
explained by ~eissman'~, ~arbome~ ' , Mabry et alW, Jay et ale', arkh ham' and
Linskens and ~ackson'~.
The conventional chromatographic methods like oolumn, 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 had been achieved by high pressure
liquid chromatography (HPLC). Among the separation techniques applied to flavonoids
HPLC has the advantage over to other techniquesM in regard to sensitivity, rapidity and
easy quantification.
Hosttemnan and ~osnetman" reviewed the relevant literature on HPLC upto
1980. A few other publications in this field include Rt values by Daigle and
Conkcston", use of Bondpak Cis with MeOH-HOAC-H20 as developing system with
two pumps by casteele et a]", analytical problems in HPLC by Bankova et ala8, Tamma
et alB9 and Barberan et alw. The application of HPLC combined with FABMS in the
structural elucidation of anthocyanin pigments was explained by J.B.Hatborne and
~ . ~ . G r a ~ e r ~ ' . On line HPLC-W, flash c h r o m a t ~ ~ r a ~ h ~ ~ ' ~ ~ ~ , centrifugal using
chromatotron are also applied in the isolation of flavonoids. For difficult separations
requiring very high resolution semi preparatory HPLC with automatic fraction collector
IS an ideal method. Reverse phase chromatography97"w, HPLC-DAD and HPLC-
on chemically bonded phases gives better results of the separation of plant
phenolics
The complication of irreversible adsorption and decomposition of the solute at
the liquid-solid interface in all techniques employing a solid stationary phase and liquid
mobile phase is overcome by various support free liquid-liquid partition techniques,
D C C C ' O ' . ~ ~ ~ and R L C C ' ~ have been employed profitably for quantitative and
qualitative separation by Hosttetman and collaborat~rs'~'. Aritani et allM and
Gunasegaran et allo7. However, the constant need in natural product chemistry to
separate large and small quantities of complex mixtures efficiently and rapidly is
unfoltunately seldom satisfied by the w of any one chromatographic technique. The
best results have been obtained by a combination of several techniques, which an often
complementary.
Paper electrophoresis'w is a technique of limited appliation in flavonoid
analysis, since to be mobile, a flavonoid must be in an ionized state at the pH of the
electrolyte. Its useful application lies in the recognition and identification of flavonoid
sulfate^^^^"^ and in the distinction of glycuronides from glycosides"'. Relative
mobilities of different flavonoid sulfates are listed by ~os t te tman~~ ' . Electrophoresis
finds greater application in the field of anthocyanins and bctncyanins.
The flavonoid once isolated as a homogenous compound is characterized by the
specific colow testss2, physical constantss', elemental analysisM, Rf valuesJJ in various
solvent systems, analysis of hydrolysis products, preparation of derivatives and
comparison of these data with related compounds7s. Further support for
confirmation of the structure of the flavonoid is achieved by the analysis of
different spectral data (W-VIS, IR, MS, 'H and "C NMR). '
Ultraviolet spectroscopy is still one of the oldest and useful techniques for
flavonoid identification. The W 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 AlCl' and AlCl3 I HCI UV spectra in the pmise determination of the
structure of flavonoid compound was demonstrated by v o i ~ i n " ~ and the limitation of
NaOAc spectra in flavonoid analysis has been reported by Rosler et all1'.
IR ~pect rosco~y"~ provides valuable information regarding the type of flavonoid
as well as the nature of the substituents like methoxy, mcthylene dioxy and prenyl oxy
groups. It is also used as a "finger - printing" devise for establishing the identity of two
samples.
Mass spectromehy 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)"'~~ used for the volatile compounds, the nonvolatile
compounds are converted to suitable derivatives like trimethyl silyl ether, permethyl
ether, methyl ester or similar derivatives. Field desorption mass spectrometry (FD -
MS) employed for polar and thermolabile compounds like flavonoids has been reviewed
by Schulten and ~ a m e s " ~ . Desorption chemical ionization 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 energized
neutral atoms to induce ionization and d e s ~ r ~ t i o n " ~ appears to be the most
advantageous one for the analysis of flavonol glycosides, when two MS are linked in
tandem, it has now become possible to employ the first as separator and the second as
analyzer to perform direct mixture analysis (Tandem-MS). This multiple stage MS has
been reviewed by Roush et all". In the chemical ionization mass spectrometry (C1 -
M S ) ' ~ ' the ions are formed in ion molecular collisions which include abstraction as
primary process using weak gas phases like C b , NHI, i- C4 HI0 in positive CI for
protonation. In negative CI, OMe- as reagent for proton abstraction or CI- as an
attachment reagent is employed. The MS analysis of permethyl ether of glycosides is
widely employed for senling structural problems in C - glycosyl flavones n2. 12'.
Pyrolysis chemical lonlzatlon mass spectrometry of flavonoids under positwe and
negative ionization cond~t~ons IS obsewedl2' to yield data characteristic of both
aglycone and sugar residues, providing an alternative for FD and FABMS techniques.
The easy MS differentiation of flavanones and dihydro flavonols by characteristic
fragments has been ~ e ~ o n e d ' ~ * . The application of G C - M S ' ~ ~ analysis to
perdeuteromethylated derivatives of flavonoids12' has rendered the identification of
certain methoxylated compounds easier. On line HPLC-MS"' will be readily accepted
by flavonoid researchers. Becchi and ~raisse"~have reported mass analysed ion kinetic
energy (MLKE) and collision act~vated dissociation. MIKE spectra of flavonoids
providing characteristic fragmant ions, which permit differentiation of the 6 -and / o r 8
- substituent location and the position of &glyosylation. Solution phases secondary ion
mass spectrometry''0 has proved useful in the determination of molecular weight of
complex flavonoid glycosides. Electrospray MSI 14 has become quite versatile on
account of the amanability of the technique to highly polar and large sized molecules
~ncluding biomoecules like proteins Recently ESI and MALDl - TOF mass spectromety
in the study of flavonoids and selected components of b~ological interest have been
used131.132
Nuclear Magnetic Resonance (NMR) over the last twenty years has given
considerable encouragement 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 f l a v ~ n o i d s l ~ " ' ~ ~ . Typical application of 'H NMR includes determination of
oxygenation panem. number of methoxy group, distinction of isoflavones, flavonones
and dilydroflavonols, number and nature of sugar present (whether oc - linked or P - linked) and detection of hydrocarbon side chain. Shift reagent provide a method of
spreading out PMR absorption signals. The use of lanthanide shift reagents in
positioning of methoxy groups of flavonoids was reported by Joseph Nathan and et
Of late "C NMR spectroscopy'"~'42 has become the most useful technique for
the structural determination of flavonoids. "C resonance signals extending over 200
ppm provide the nature of the carbon skeleton. Carbon relaxation time measurement
being less difficult is very useful in differentiating otherwise non discemable carbon
atoms like C-6 and C-8 in flavonoids, lnter glycosidic linkages in the case of
disaccharides"' (rutinose, neohesperidose etc.,) type of linkage with aglycone and
sugars and conjugation with sulfates and organic acids can also be determined.
Homonuclear and heteronuclear correlation spectroscopy"4 (HOMCOR and HETCOR)
and the various decoupling experiments have reduced the difficulty in the interpretation
of 'H and " C NMR 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.
Refocussed INEPT with decoupling is an alternate of off-resonance decoupling for
assigning carbon multiplets. I-modulated spin echo'4sspectroscopy is yet another
method of off-resonance decoupling which gives positive signal for carbon with even
nurnber of hydrogen (CHI and quartenary) and negative signal for carbon with odd
number of hydrogen (CH and CH,) attached. At present 2D NMR"~."' methods make it
more acceptable to natural product chemistry with the requirement of decreased sample
size usually a set of routine 2D including COSY (and I or DQF COSY).
relayed COSY [(HOHAHA), 2D NOE (NOESY 1 ROESY) and HETCOR (HMQC)] an
used in the stnrctwal elucidation of flavonoids. Recently NMR techniques have been
extended to clarify the anti oxidative molecular mechanism of ~atechins"~.
Over the past 40 years, chiroptical methods like ORD and C D spectral analysis
have been employed for the determination of stereochemistry of chiral f l a ~ o n o i d s ~ ~ ~ ~ " ' .
The exciton chirality method"' employing the application of coupled oscillators in
determining the chirality of natural products is receiving greater attention.
If the flavonoid compounds can be ubtained in a fine crystalline form, X-ray
analysis can help in further confirming the structure as reported in the case of
c a ~ ~ c o ~ t e r i n ~ ~ ~ .
Final confirmation is always deslred to be established by unequivocal total
synthesis. The conventional methods of synthesis of flavonoids from simple precursors
by condensation methods have been proposed by Algar and ~ o b i n s o n ' ~ ~ , Algar and
FlynnlS5 and Baker and ~enkamaman"~. These methods of synthesis have been
modified by Farkas el a ~ ' ~ ' and Wagner et all" 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. Wagner et all5' accomplished the synthesis of
methoxylated flavones from the corresponding brominated methoxy chalcones. The
synthesis of 5,6,7,3',4'-penta methoxy flavone (sinensetin) by dehydrogenation of the
corresponding flavonone with SeOl has been achieved by Wagner et allJq. This flavone
has been used as the starting material for the synthesis of a number of related flavones.
Bose et allM have reported cyclisation and simultaneous dehydration of the hydmxy
chalcone to the comspondiig flavone by heating with palladium on c h w l . 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-
acetobromosugars of pentoses, hexoses or dissccharides and the aglycones in the
presence of catalysts. The selective glycosylationof 7-OH has been achieved by
Zemplen and arka as'^^. Synthesis of other glycosides have been accomplished by trans
acylation methods by Nogradi et all6' and Wagner et allb4. The total synthesis
of C-glycosyl flavones has been reported by Eade et all" and other complex ones has
been provided by later w o ~ k e r s ~ ~ . ~ ~ ' . The chiron approach to the total synthesis of
natural products168 might become a useful guide in the synthesis of Chiral flavonoid
compounds. Thus the synthesis of almost all types of mono and di-C-glycosyl flavones
and flavone C-glycosyl-0-glycosides has been accomplished'69, synthesis of some
novel flavonoids has been illustrated by Rakosi et al Studies of the selective
0-alkylation and dealkylation of flavonoids with anhydrous AIBr, were reported by
Horie et all".
Our present knowledge In flavonoid biosynthesis 1s based on a combination of
:arl~er results from radioactive tracer stud~es in vivo and the more recent data obtained
at the enzyme level in vitro. In the past few years the enzymology of flavonoid
biosynthesis has made particularly rapid progress. Flavonoid biosynthesis can be
-onsidered in three stages. The first stage is the formation of the basic C6-C3-C6
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 a n synthesized. The final stage embraces the elaboration of individual
compounds with in each flavonoid class, involving steps such as hydroxylation,
glycosylation, methylation etc. Chalcone considered as common intermediate in the
biosynthesis of all classes of flavonoids. Insight into three aspects of the problem of
flavonoid biosynthesis has come in the past from comparative chemical
genet~c"~'"~ studies and recently from feeding experiments with radioactive tracers.
Research has led to the isolation and characterization of enzymes of the pathway of
biosynthesis. 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 ~er re i ra '~ ' have highlighted the special role of a -hydroxy chalcone as
the key intermediate in flavonoid biogenesis. A comprehensive report on the
biosynthesis of flavonoids by Hahlbrock et EI '~ ' , ~ o n g ' ~ ' , Birch et alig3, a good account
of biosynthesis of shikimate derived phenolic compounds by ~ a r b o r n e ' ~ ~ , ~ s n i n o ' ~ ' ,
biosynthetic studies in vivo with labeled precursors and biochemistty of flavonoids
biosynthesis by ~e l le r ' " and Heller and Fork manig7 are useful publications. Recent
trends in the biosynthesis of flavonoids have been discussed by Akashi et Bemeti
et all8'. Kennedy et allw and Fedoreyev et a ~ ~ ~ ' .
The importance of flavonoids and other ~oondary metabolites in plant
biochemistry has been detailed in ''the biochemistry of plants"'92. Diffmnt aspects of
mammalian metabolism of flavonoids have h reviewed by De ~ d s ' ~ ' , ~ c h e l i n e ' ~ ,
~riff i thsl~' , Middleton Ir and ~andaswami'~ and ohm'^. Flavonoids an the
constituents of the mammalian diet derived h m plants. The ingestion of flavonoids by
mammals in the diet or for therapeutic use, brings them in contact with both intestinal
microorganisms and mammalian tissues which arc capable of bio transformation of
flavonoid compounds. The available e v i d e n ~ ' ~ ' indicate that the hydrolysis of the
flavonoid glycosides to their corresponding aglycones, ring fission and oxidative and
reductive transformations arc mediated by intestinal microorganisms. Though it is
cerlain that the metabolic changes undergone by flavonoids occur with in mammalian
tissues, the relative contribution of individual tissues is not fully understood.
The flavonoids find exceptional use as chemotaxonomic guide in the
classification of plants. The reason for preferring flavonol 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 is not
actively concerned with cellular metabolic proccsws. Any particular flavonoid can be
relied to be present in more or less constant amount in the same tissue of the same
species so long as the plants are grown under normal physiological conditions. The
conspicuous exception to this is the vaiation in the relative concentration of pcoumaric
acid md caffeic acid (mono and dihydroxy phenyl pmpanoid precursors) as well as
Laenipferol and quercetln which are ~nslgnificant from the chemotaxonomic point of
view. Importance of flavonoids in chemotaxonomy can be illustrated with a few
examples. A chemosystematic study on ~ u ~ h o r b i a c e o u s l ~ ~ plants was performed using
polyphenolic constituents. The phenolic chracteristics of sub families genera and species
were well distinguished from one another. Hydrolyrable tannins as constituents were
considered to be a valuble chemotaxonomic chancter in elucidating aflematic
relationships among the related taxa. Zizlphus jujuba and Ziziphus vulgarls of
Rhamnaceae have been differentiated chemotaxonomically based on juzaic acid and
betulinic acidIw. Recently 3.4 4imethoxy c i ~ a r n i c acid levels as a tool for the
chemotaxonomic differentiation of 13 species of Cogea canephora var robusla and 7
species Coffea aorb~ca of Rubiaceae family have been studied2w. Further the
occurrence of flavonols and m a n n i t ~ l ~ ~ ' in the species of Rubiaceae can also be used as
a chemotaxonomic marker of the family. Among the numerous flavonoids of higher
plants the rare and unusual ones find more acceptance in micromolecular taxonomy.
Recently chemotaxonomic markers in Calophyllum reysmannri20' of Guttiferae, Bursera
specres20'of Bureseraceae and other have been extensively studied.
The most significant function 20"210 of the sapsoluble flavonoids is their ability
to impart colow to the plants in which they c a w . They are responsible for most orsnge,
scarlet, crimson. mauve, violet and blue colours, as well as contributing much to yellow,
ivory and cream flowers. The only other considetable groups of colouring matters in
higher plants are the lipid-soluble chlorophylls and carotenoids. Chlorophylls-a and b
provide the prevailing g m n of plant leaves, and the carutmoids are the most important
sources of yellow and orange wiours in flowers and fruits.
The importance of anthocyanin2"~217wlow in fruits such as the strawbeny,
cherry, black currant and so on as an aid to seed dispersal by animals is self-evident.
Both fruit and flower colow give immense aesthetic pleasure to man and conscious
selection for wlow varieties among garden plants and horticu)tural cmps has bean
practiced for a very long time. Several physiological functions for anthocyanin in the
general metabolism of plants descritcd in the literature2" 'I9 an still rather obscure.
Anthocyanins may also be important factors with other flavonoids2I9 in the resistance of
plant in insect attack. Several speculations about the role of anthocyanins on the
perception or filteration of light and response to stress factors, including microbial
attack await M e r thorough studies'20, '" Recently anthocyanins as food colours has
been discussed by onn net^'^.
The important function of flavonoids in plants are their protective role as light
screen against damaging W radiation, as feeding deterrents and protection from
herbivores and as allelopathic agents. In biological systems, the stimulation of protein
degradation as well as protein fomlation by antibiotics reaulted in flavonoid
accumulation implicating the importance of flavonoids in protein synthesis 223, 224. Other
important functions include their role as anti - oxidants, enzyme inhibitors, precursors
of toxic substances and as photosensitizing and energy transferring compounds, in
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 systematically, 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 b d e r s an aware of the properties of these compounds and
other classes of secondary substances, they have a real potential for crop protection in
the future.
Apatt from the physical and morphological m a w , chemical means an a major
method of plant defence. The introduction of isoflavonoid phytoalexins in plant causes
phyto toxic it^'. U6 such as inhibition of respiration, reduced growth of suspension
cultures, repressed seed germination, retarded root growth and electrolytic leakage.
Different types of bioassay involving several types of organisms conducted with
isoflavonoids, phytoalexins by ~mith'" revealed that they possessed fungi toxicity and
limited antibacterial activity. A significant range of flavonoids have been encountered
as anti fungal agents2". A number of flavonoids are being induced in plants following
fungal invasion. Some of the flavonoids affect the behaviour, growth and development
of insects due to their toxicity, while flavone glycosides are feeding stimu~ants~'~. The
metabolic challenges of thc plant flavonoids including the condensed tannins to the
insects consists mainly of the variety of phenolic groups. This activity is 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 depmds 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 glywsylation of
flavonoids also show marked variation in growth inhibition; 34-glycosylation inhibits
growth but not 7-0-giywsylation. The enhanced activity of the flavonones wmparcd to
the corresponding flavones showed that the co planarity of the flavonoid rings of
flavones might hinder their biological eficacy.
An ever - increasing number of pharmacological effects of flavonoids have
become known over the years through the discovery of new plant flavonoid and their
derivativcssb.ut~3~ . he anticanceru2, anti anti oxidant23', anti microbial23s, and
anti inflammat~$'~ effects of flavonoids are noteworthy. In ncent years a number o f
flavonoids like baicalin, taxifolin, gossypin, proanthocyanidins, nepctin, diosmin,
fisetinsophoricoside, (t)- catechin, (-)- epicatechin and 5.7-dimethoxyflavone have been
reported to have anti-inflammatory e f f e c ~ ~ ~ ' ~ " ~ .
The effects of a few flavonoids on different types of viruses were investigated by
Pusztai et alZ3' and observed that hydroxylation in 3-position appears to be a pre
requisite for this activity. Anti cancer activity of a few flavanoids (flavones, isoflavones,
flavonones and flavonols) are reported240,i". Anti rhinovirus and anti picronovirus
activity of certain flavonoids have also been reported. The effect of many semi synthetic
flavonoid derivatives on arachidonate metabolism was established of which (0-P-
hydroxy ethyl) rutin and various quercetin derivatives are important.'42. The effect of
naturally occurring flavonoids on this metabolism was also reported by Ferrandiz et
a]"'. Studies of compounds with Flavone skeleton were stimulated by recognition of
anti allergic effects. Thus, orally effective anti-allergic chromone drugs (Kellin,
hplaetin-8-glycoside disodium chromoglycoside etc) are in uselM. Recently 7,s-di-0-
substituted flavans, biflavans and flavones showed cytotoxic activiq" and it has been
established that the activity is due to methoxy and/ or hydroxy group in the stmcture.
This screening of plants, recorded in Indian folk medicines, for liver injury has
e~tablished'~' that the active principles are flavanoids. The anti-hepatotoxic effect of
flavonoids was first demonstrated by Halm et alZdb with the flavanolignan, siliybin and
its isomers. About 13 flavonoids and coumarins have been demonstrated"' to be anti-
hepatotoxic or liver protective agents.
Anti-ulcerogenic property o f 3-0-methyl (+)-catechin, apigenin and
luteolin, anti -diabetic effects of hispidulin and nepetin and analgesic effects o f
puerarin are also reported248."9. Recently herbal remedies for skin disease^^^"^^
obtained from plants used in Unani system of medicine are useful publications.
Inhibition of human imrnunodeticiency virus reverse transcriptase is cumntly
considered a useful approach in the prophylaxis and intervention of Acquired lmmuno
Deficiency syedrome (ALDS) and natural products have been extensively explored as
inhibitors of this enzyme, to discover drugs active against AIDS. One hundnd and fifty
pure natural products have been examinedzs3 and polyphenolic compounds were found
to be responsible for the activity, among flavonoids tested quemetin exhibited moderate
activity.
Work on different aspects of flavonoids including discovery of new compounds,
biological activity, medicinal properties etc., are reviewed regular19's6 and the
knowledge is updated through proceedings of regular international symposia on
flavonoidsZs4. Considerable work on the chemistry and pharmacological properties of a
number of flavonoids has been accomplished in the laboratories of our Institution and
~niversit$'~ '"'.
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Recommended