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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