chapter i a brief general introduction chehistry...
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CHAPTER I
A BRIEF GENERAL INTRODUCTION TO CHEHISTRY AND
BIOLOGICAL ACTIVITY OF FWVONOIDS
The discovery and use of sulpha drugs, antibiotics
such as penicillin, and syn?hetic drugs led to a dramatic
decline in the popularity of medicinal plant use in therapy.
Now, the pendulum has swung to the other extreme. A
resurgence of interest in the study and use of medicinal
plants has been taking place during the last two decades.
Also a considerable growth has occurred in popular, official
and commercial interest in the use of natural products. WHO
has done its share to further awareness of the importance of
traditional medicine to the majority of the world's
population, and to promote increased rational exploitation
for all people of the world of safe and effective practices,
including the use of medicinal plants. The success of any
health system depends on the ready availability and use of
suitable drugs on a sustainable basis. Medicinal plants have
always played a key role in world health.
"Health for all by the year 2000" is both a goal
and a process which engages each nation of the world to
improve the health of its people. This concept calls for 6
level of health that will permit all people to lead a
socially and economically productive life.
Medicinal plants are commonly avail able in
abundance, especially in the tropics. They offer immediate
access to safe and effective products for use in the
treatment of illness through self-medication. Furthermore,
plants are valuable for mode* medicine in four basic ways:
(i) They are used as sources of direct therapeutic
agents;
(ii) They serve as a raw material base for the
elaboration of more complex semi-synthetic chemical
compounds;
(iiil The chemical structures derived from plant
substances can be used as models for new synthetic
compounds;
(iv) Finally plants, applying the concept of
chemotaxonomy, can be used for the discovery of new
compounds.
Plant-derived medicines are used in self-
medication in all cultures. However only a fraction of the
world's plants has been studied. Even so, human kind has
already reaped enormous benefit.
By the application of modern techniques of
isolation and pharmacological evaluation, many new plant
drugs find their way to medicine as purified substances
rather than in the form of older galenical preparations.
Chemical plant taxonomy genetical studies involving
secondary metabolites, tissue culture, the study of effects
of chemicals on plant metabolites and the induction of
abnormal syntheses in plants are new areas pursued by an
inter-disciplinary approach to develop new and more potent
medicinal agents. The term 'medicinal phytochemistry' is
synonymous with the interdisciplinary scientific approach to
the development of drugs from plants.
Plants are considered to be medicinal if they
possess pharmacological activities of possible therapeutic
use. These activities are known generally as a result of
continued methods of trial and error1. With the increasing
awareness of side effects like cytotoxicity, genotoxicity
etc., present day investigation in the discovery of new drug
demands an array of strict systematic evaluation by a team
of scientists drawn from a number of disciplines. There is
no single philosophy that will lead to the discovery of new
2 and more potent plant drug . The contribution of natural products to medicine
3
during the period after 1950 have been outstanding. Today
emphasis of pharmaceutical research and development is on
the creation of therapeutic, prophylactic and diagnostic
substances with specific functions and minimum side effects
in particular applications and here the plant derived
substances have the advanuge of being tool for modern
medicine satisfying these conditions. Hence the latest
trend in the advanced countries indicates preference4 for
natural drugs over synthetic ones.
Another recent aspect of interest in plant drug
research is the concept of a non-specifically increased
resistance (NSIR) of an animal to diseases attributable to
other substances, besides the active principles responsible 5 for specific biological activity . This probably justifies
the use of many of the plant drugs os household remedies by
natives of many countries from ancient times and hence
warrants their evaluation in more detail.
India, with its vast area from Kashmir to
Kanyakumari and varying soil and climatic conditions ranging
from tropical to temperate, has one of the world's richest
vegetations, comprising approximately 130,000 species of
plants belonging to about 120 families, to be aptly called,
'The Botanic Garden of the World'. India 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 ear&r than 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 toxic
properties.
India, like all advanced countries, has also made
significant progress by a systematic scientific study of
these plant drugs from the pharmacognostical, chemical,
pharmacological and clinical points of view during the past
50 years, bringing to the forefront a larger number of herbs
used in Indian indigenous system for their approved
efficiency and administration in modern medicine.
Recognising the importance of medicinal plants,
collaborative team work 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 the Council of Scientific and
Industrial Research (CSIR). The results of various studies
on Indian Medicinal plants are available in as many a s over
10,000 research publications and a number of books and
m~no~raphs~-'~. Central Drug Research Institute (CDRI 1 ,
Lucknow, India, initiated a programme from 1979, to
investigate Indian plants which either had a reputation in
folklore medicine or whqqe extracts showed consistent
biological activity when put to a broad biological screen.
So far under this programme over 3000 species has been
screened; biological activities have been confirmed in about
415 plants18.
A few of the notable contributions made in Indian
plant products during the past twenty five years include the
work on the alkaloids of Piper speciesr9, Ancistrocladus
2 2 heyneanus20, Cocculus laurifolius2', Murraya koenegii , 2 3 Tylophora asthamatica , Alstonia scholaris 24 and
25 Phyllanthus niruri , oxygen heterocycles of Seaeli
~ i b i r i c u m ~ ~ , Garcinia ~ ~ e c i e s ~ ' - ~ ~ , Artocarpus species 29-.31
and Mollugo species 32-33, terpenoids from Aguillaria
34 35 36 agallocha , Zingiber zerumbt , Commiphora mukul , Ailanthus m a l a b a r i ~ a ~ ~ , Cleistanthus ~ o l l i n u s ~ ~ , Thevetia
n e r i f o l ~ a ~ ~ and Sesamum 1 a ~ i n i a t u m ~ ~ , withanolides from
withania4' species, physalins from Physalis species 42-43 and
saponins and sapogenins from a variety of plants 64-48
Triterpenoid saponins isolated during the period 1982 to
1989 together with their occurrence, the newer methodology
used in their isolation and structure elucidation and the
biological activity were reviewed by Mahato
Besides the continuing general reviews on triter-
penoids50-52, a few specific reviews have been published,
e.g. on pentacydic t r ~ t e r ~ e a p i d s ~ ~ , on cycloartane compounds - detected in 43 species belonging to 34 genera and 32
f a m i l ~ e s ~ ~ , and on the constituents of Azadirachte. indicaS5.
An excellent review on bacterial triterpenoids giving an
account on triterpenoid occurrence as well as function in 5 6 bacteria has been published .
Srivastava and Kulshreshtha 57 reviewed the
biologically active polysaccharides isolated from natural
sources during 1977 to 1988. Also latest reports on new
medicinal plants and/or additional information have appeared
recently 58959. Detailed bibliography in respect of number
of medicinal plants covering pharmacognosy, physiology,
pharmacology, phytochemistry and utilization has been
regularly reported in Medicinal and Aromatic Plant Abstracts
(MAPA); so Ear 58 plants have been covered besides
abstracting a broad spectrum of research investigation on
6 0 plants . ~ h ~ t o c h e m i s t r ~ ~ ~ - ~ ~ , evolved from natural products
chemistry 68-71 is confined to the study of products
elaborated by plants and it has developed as a distinct
discipline between natural products organic chemistry and
plant biochemistry 7 2 - 7 4 in recent years. It deals with the
study of chemical structures of plant constituents, their
biosynthesis, metabolism, natural distribution and
biological functions. The fQct that only less than 6 per
cent of about six lakhs species of plants on earth has been
investigated, indicates the opportunity provided and
challenges thrown to phytochemists. The task of the
phytochemist is compounded in accomplishing the
characterization of very small quantity of the compounds
isolable from plants. Phytochemistry also enjoys the
application of modern research for the scientific
investigation of ancestral empirical knowledge. It has found
wide and varying application in almost 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 physiology, plant pathology,
plant ecology, palaeobotany, plant genetics, plant
systematics and plant evolution has been increasingly felt.
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 substitutents 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 w c h 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. 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 chemical structures providing easier understanding of
structure - activity relationship ( S A R ) . The reedy
availability of many of the more common flavonoids in pure
form is a definite advantage for the study of their
biological activity and evaluation of their pharmacological
properties. More than 4500 flavonoids have been
characterized till now and new structures are being reported
at an ever increasing rate. Flavonoids have attracted the
attention of scientists of different disciplines and the
systematic investigation on their occurrence, natural
distribution, chemical nature and biological functions
continues unabated 75-81
The term 'flavonoid' was first applied about forty
years ago by Geissman and ~ i n r e i n e r ~ * ~ ~ ~ to embrace all
those compounds whose structure is based on that of flavone
(2-phenylchromone) (I) having a basic C6-C3-C6 carbon
skeleton in common. When the heterocyclic ring is reduced,
it becomes flavan (2-phenylchroman) (11). Flavone (I)
consists of two benzene rings (ring A and 9) joined together
by a three-carbon link which is formed into a f-pyrone ring
(ring C ) . The various classes of flavonoid compounds differ
from one another only by the state of oxidation of thia
carbon link. There is a limitation to the number of
structures commonly found in nature, which vary in their
state of oxidation from flavan-3-01s (catechinl (111) to
flavonols (3-hydroxyflavones) (IV) and anthocyanins (V).
Flavanones (VI), flavanonols or dihydroflavonols (VII) and fl9
the flavan - 3,4 - dFols (proanthocyanidins) e also
included in the flavonoids. It should be noted that there
are also five classes of compounds (dihydrochalcones or
3-phenyl propiophenones (1x1; chalcones or phenylstyryl
ketones (X); isoflavones or 3-phenylchromones (XI);
I . FLAVONE
l l I. FLAVAN - 3 - OL
11. FLAVAN
IV. FLAVONOL
V. ANTHOCYANIDIN VI. FLAVANONE
VII. DIHYDROFLAVONOL
I X . DIHYDROCHALCONE
VI I I . FLAVAN -3,4-DIOL
X . CHALCONE
X I . ISOFLAVONE XII. NEOFLAVONE
0
XI I I . AURONE
CH = CH - COOH
XIV . ClNNAMlC ACID
neoflavones or 4-phenylcoumarins (XI11 and the aurones or 2-
benzylidine-3-coumaranones (XIrD) which do not actually
possess the basic 2-phenylchromone ( I ) skeleton, but are
closely related both chemically and biosynthetically to
other flavonoid types that &ey are always included in the
flavonoid group.
The individual compounds within each class are
distinguished mainly by the number and orientation of
hydroxyl and methoxyl groups distributed in the two benzene
rings. These groups are usually arranged in restricted
pattern in the molecule, reflecting the different
biosynthetic origins of the aromatic nuclei. Thus, in the
A-ring ( I ) of the majority of flavonoid compounds, hydroxy
groups are distributed at either C-5 and C-7 or only at C-7
(C-5 and C-7 of flavone become C-2' and C-6' in
dihydrochalcones and chalcones and C-4 and C-6 in auronee)
and generally are unmethylated.
This pattern of hydroxylation follows from the
acetate or malonate origin of the ring. The B-ring (I) of
the flavonoids on the other hand is usually substituted by
either one, two or three hydroxyl or methoxyl groups. The
rarely methylated position is 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 cinnamic acid
(XIV) and coumarins (XV) and reflects their common
biosynthetic origin from prephenic acid and its congeners.
Most of the flavonoids occur naturally in
conjugated form, usually bound to sugar, by a hemiacetal .rr
linkage. But their conjugation with inorganic sulphates 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 very active
glycosidases which can work even in the presence of high
concentration of organic solvents. The presence of sugars
in the molecule confers sap-solubibility to the generally
somewhat insoluble flavonoid compounds. In anthocyanins the
sugar imparts stability to the aglycones. 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
84 corresponding aglycones, presumably due to steric reasons . More and more range of new glycosides are encountered in
plants. An increasing number of flavonoid glycosides 85,86
carrying sugars in B ring hydroxyls have been reported . Conjugation of flavones and flavonols through glucose
organic acids like malonic acid and derivatives of cinnamic
acids have also been reported87'88. As a result of
electrophoretic studies, a number of zwitter ionic
anthocyanins with malonic acid and succinic acid linked to
8 9 C-6 of glucose have been isolated and characterized . The sugars found in flavonoid glycosides include
simple pentoses and hexosea .Itnonosides) and di- and
trisaccharides (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 may be glycosylated giving
rise to diglycosides and so on. The common sugars 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
plants 75,76,90 is 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 population variation within species 91 has been detailed by Harborne and Turner .
Only in 1985, the distribution of flavonoids in
animals has been reported92 with isolation of
6'-methoxyflavone from scent glands of Canadian beaver
(Caster feber) and in Lepidoptera; no doubt, the presence of 93
flavonoids in butterflies has been earlier recongnised .,
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 hydrolysis of the
plant extract before examining the nature of the 7 7 gl ycosides . Flavonoids king polyphenolic give
characteristic colour changes when treated with alcoholic
~e~', ammonia vapour or alkalis. Positive Shinoda test
(Mg-HC1) and formation of coloured complexes with heavy
metal salts are characteristic of flavonoids.
Standard method of extraction, separation and
chemical characterization of flavonoid compounds are
65 described by Peach and race^^^ as well as Harborne . Systematic procedure for their identification employing
chromatographic methods of analysis and chemical and
spectral methods of identification have been detailed by
~eissrnan~~, Mabry g ,195, arkh ham^^, Jay g ,197, Harborne
9 8 and ~ a b r ~ " and Linskens and Jackson . The conventional chromatographic methods like
column, paper and thin layer are still in use for the
separation and purification of the flavonoid compounds.
Increase in speed and efficiency in the separation of
mixtures by new techniques like centrifugal TLC"
(Chromatotron, CTLC) and high pressure liquid
chromatography 98-10u (HPLC) has been achieved. Flash
chromatography or in combination with centrifugal TLC,
column chromatography (on polyamide and on Sephadex LH 20)
and low pressure liquid chromatographylO1 are often used t o
separate samples weighing 100 mg to 10 gm in less than half
an hour. For difficult separatfbns requiring very high
resolution semipreparatory HPLC with automatic fraction
collector is an ideal method. Reverse phase
chromatography102 on chemically bonded phases gives best
results for the separation of plant phenolics. The
complication of irreversible absorption and decomposition of
the solute at the liquid-solid interface in all techniques
employing a solid stationary phase and liquid phase, is
overcome by various support free liquid-liquid partition
techniques. Among these, droplet counter-current
(DCCC) and rotation locul ar counter - current chromatography lo5 (RLCCC) have found wide
application in the field of flavonoids and other
polyphenolics. However, the constant need in natural
products chemistry to separate large and small quantities of
compl9x mixtures efficiently and rapidly is unfortunately
seldom satisfied by the use of any one chromatographic
technique. The best results have been obtained by a
cornination of several techniques which are often 106 complementary . Paper electrophoresis107 is a technique
of limited application in flavonoid analysis since, to be
mobile, a flavonoid musc be ill an ionized state at Llie pll of
the electrolyte. Its useful application lies in the
recognition and identification of f l avonoid sul phates108*109
110 and in the distinction of glycuronides from glycosides . Relative mobilities of different flavonoid sulphates ore
listed by ~ostettmannl'l. Electrophoresis find greater
application in the field of anthocyanins and betacyanins.
'The f 1 avolioid , OIICC i sol a trd ns ;I I~<~nit~~:riirnus
compound is characterized by tt~c specific cr>l our tests,
physical constants, clcmc~~tnl arlnl ysls, HE vnlucss 1 1 1 \~;ir.ic~~~s
solvent systems, analysis of hydro1 ysis products,
preparation of derivatives and compariso~i of thcsc. data witl~
related compounds. Further support for confirmation of the
structure of a flavonoid is acl~icved by the analysis of
different spectral data (UV - Visible, IR, 'H and 13c Nb1R
and M S ) .
'The devei opments, in t11c gclicral ~~~cth<~dol ugy o f
natural products chemistry1'' is readily applicable to
flavonoid compounds also. Ultra Violet-Visible spectroscopy
is still one of the useful techniques available for
flavonoid structural elucidation. The UV spectrum in
methanol and with various diagonistic shift reagents give
information about the type of flavonoids, oxygenation
pattern and kstitution. The use of A1Cl3 and A1C13/HCl UV
spectra in the precise determination of the structure of
f lavonoid compounds was demonstrated by ~ o i r i n ~ ~ ~ and the
limitation of NaOAc spectra in flavonoid analysis has been
reported by Rosler ,1113. The infrs-red spectra 114
provide information about therlass of compound and nature
of certain substituents. It is frequently used as finger
print device for establishing the identity of two samples.
Over the past 30 years chiroptical methods like
ORD and CD spectral analysis have been employed for the
determination of stereochemistry oE chirel flavo-
noids '15,'16. The exciton chirality method1" employing the
application of coupled oscillators in determining the
chirality of natural products is receiving greater
attention.
Mass spectrometry of flavonoids serves as e
valuable aid in determining the moleculer weight end
probable structure even with very small sample size. The
electron impact mass spectrometry 1189119 (EIMS) is used for
the volatile compounds; the nonvolatile compounds are
converted to suitable derivatives like TMS ether, permethyl
ether, methyl ester or similar derivatives. When two mass
spectrometers are linked in tandem, it has now become
possible to employ the first as separator and the second as
analyser to perform direct mixture analysis (TANDEM-MS).
This multiple stage mass spectrometry has been reviewed by
Roush ,1120. Field desorption mass spectrometry (FDMSI
employed for polar and thermolabile compounds like
121 flavonoids has been reviewed by Schulten and Gamea . Desorption chemical ionisation m s s spectrometry122 (DCIMS)
using electrically heated tungsten probe and fast atom
bombordment mass spectrometry123 (FABMS) using the sample
solubilized in polar matrix (like glycerol, thioglycerol,
m-nitrobenzyl alcohol, etc.,) deposited in a copper target
which is bombarded with energized neutral atoms to induce
ionization and desorption appears124 to be the most
advantageous one for the analysis of flavonoid glycosides.
In the chemical ionization mass spectrometry125 (CIMS) the
ions are formed in ion-molecular collisions which include
abstraction as primary process using weak gas phase like
CH4, NH3,i-C4H10 in positive CI for protonation. In
negative CI, 0 ~ e - as reagent for proton abstraction or CL-
as an attachment reagent is employed. The MS analysis of
permethyl ether of glycosides is widely employed for
settling structural problems in C-glycosyl flavones 126,127
Pyrolysis chemical ionization mass spectrometry of
flavonoids under positive and negative ionization conditions
is observed12' to yield data characteristic of both sglycone
and sugar residues, providing an alternative for FD and
FABMS techniques. The easy differentiation of flavanones
and dihydroflavonols by the characteristic fragments has
been reported129. The application of CC-MS'~' analysis to
perdeuteromethylated derivatives of flavonoids 13' has
rendered the identification of certain methoxylated
compounds easier. Online WLC-MS'~' will be readily
accepted by flavonoid researchers. Solution phase secondary
ion mass spectrometry132 has been proved useful in the
determination of the 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
chracteristic fragment ,ions which permit differentiation of
the 6-and/or 8-substituent location and the position of
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 flavonoids 134s135, Shift reagents provide a
method of spreading out PMR absorption signals, The use of
lanthanide shift reagents in the determination of position
of methoxyl groups of flavonoids was reported by Joseph- 136 Nathan et a1 .
Of late, NMR spectroscopy 137-143, has become
the most useful technique for the structural determination
of flavonoids. 13c resonance signals extending over 200 ppm
provide the nature of the carbon skeleton. Carbon
relaxation time measurement, e i n g less difficult is very
useful in differentiating otherwise nondiscernable carbon
atoms like C-6 and C-8 in flavonoids. Interglycosidic
1 inkages in the case of disa~charides'~~ ( rutinose,
neohesperidose, etc.), type of linkage with aglycone and
sugars and conjugation with sulphates and acids can also be
determined. Homonuclear and heteronuclear correlation
spectroscopy 14' (HOMCOR and HETCOR) and the various
decoupling experiments have reduced the difficulty in the
interpretation of 'H and NMR spectra of complex
mol ccul es. Fourier transform NMK employing popular pulse
sequences like off-resonance, heteronuclear decoupling,
gated heteronuclear dccoupling, inverse gated heteronuclear
decoupling and selective proton decoupling has eased the
task of assignment of peaks in a NMR spectrum.
J-modulated spin echo spectroscopy146 is yet another method
of off-resonance decoupling which gives positive signal for
carbon with even number of hydrogen (CH2 and quarternary)
and negative signal for carbon with odd number of hydrogen
(CH and CH3) attached. The flavonoid glycoside from violet
blue flowers of Primula polyantha was completely
characterized as quercctin 3-0-(p-D-glucopyranosyl (1->Z)-P-
D-glucopyranosyl (1->6)-p-D-glucopyranoside) by ZD-NMR 147 methods . Also the structures of a few cytotoxic
flavonoids were established unambiguously with NMR
assigr~mc~lLs based of, ' t i - 'H tOSY, 'H-'~c III.1.COK and
selective INEPT experiments1h8.
If the flavonoid compounds can be obtained in a
fine crystalline form, X-ray analysis can help in further
confirming the structure as reported in the case of
~ a l ~ c o p t e r i n ~ ~ ~ . Final confirmation is always desired to be
established by unquivocal total synthesis. The synthesis of
a number of £1 avonoid gl ycosides (0-gl ycosides,
C-glycosides, 0-glycosyl-C-glycosides) has been
r ~ ~ x ~ r t e d ~ ~ , ' ~ , ' ~ ~ . Synthesis of some novcl El nvonoid
derivatives has been illustrated by Rakosi dl5'. The
'Chiron' approach to the total synthesis of natural
products152 might become a useful guide in the synthesis of
chiral flavonoid compounds.
Great progress has been made in the understandine
of bio-synthesis of flavonoids 153-155. our present
knowledge in flavonoid biosynthesis is based on a
combination of earlier results from radioactive tracer
studies in vivo and more recent data obtained at the enzyme
level in vitro. In the past few years, the enzymology of
f l nvo~loitl I~iosy~~Lhcsis lrns made pnrticul or1 y r n p l d progress.
Flavonoid biosynthesis can be considered 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 seqnd stage is concerned with
the ways by which the different classes of flavonoids are
synthesized. The final stage embraces the elaboration of
individual compounds within 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
all three aspects of the problem of flavor~oid biosynthesis
has come in the past from comparative anatomy 156-158 and
chemical genetic studies159 and recently from feeding
cxpcrilnc~~Ls wlL11 ra(lioncLivc tracers. Ma Jor work on
chemical genetic studies have been carried out by Grisebach
and co-workers 160-162. Recent research has led to the
isolation and characterization of enzymes of the pathway of
flavonoid biosynthesis. The use of young plant tissues and
cell suspenion cultures as source materials has also greatly
facilitated the study of flavonoid biosynthesis at the
enzyme level. Roux and ~ e r r e i r a l ~ ~ 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 ani it to'^^, a good account of
biosynthesis of shikimate derived phenolic compounds by
~arborne'~~, biosynthetic studies 5 viva with labelled
precursors and biochemistry of flavonoid biosynthesis by
~ e l l e r l ~ ~ , ' ~ ~ are useful publications. The importance of
flavonoids and other secondhy metabolites in plant
biochemistry has been detailed in "The Biochemistry of
i'lantstt7'. Ilifferent aspects of mammalian metabolism of
flavonoid compounds have been reviewed by De ~ d s l ~ ~ ,
Schel ine lb9 and ~riffiths'~'. Flavonoids are 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 microorganisms
and mammalian tissues which are capable of biotransformation
of f 1 avonoid compounds. The avail able evidences
indicate1709171 that the hydrolysis of flavonoid glycosides
to their corresponding aglycones, ring fission and oxidative
and reductive transformations are mediated by intestinal
microorganisms. Though it is certain that the metabolic
changes undergone by flavonoids occur within mammalian
tissues, the relative contribution of individual tissues is
not fully understood.
The f 1 avonoids find exceptional use as taxonomic
guide in the classification of plants. The reason for
preferring flavonoids to other secondary metabolites is
thcir structural diversity, widespread distribution,
comparative stability, easy detection and identification and
thc fact that this group of plant products is not actively
concerned with cellular metabolic processes. Any particular
flavonoid can be re1 ied to bepresent in more or less
constant amount in the same tissue of the same species so
long as the plants are grown under normal physiological
condition. The conspicuous exception to this is the
variation in the relative concentrations of p-coumaric acid
and caffeic acid (mono - and dihydroxphenyl propanoid
precursors) as well as kaempferol and quercetin which are
insignificant from the taxonomic point of view. Importance
of flavonoids in chemotaxonomy can be illustrated with a few
examples. Phloretin or its 3-hydroxy derivatives occur in
all specics of Malus (~osaccae)'~~. No othcr genus in
Rosaceae contain these compounds. Caviunin, an isoflavone
is a taxonomic marker in Dalbergia species173. Quercitrin,
the major constituent in the parasite Dendropthoe falcate
(Loranthaceae) growing on host plants of different families
projects its possible use as a chemotaxonomic marker174 of
this species. The flavonoid profile characterized by
C-glycosyl flavones of Mollugo species175 supported its
separation from Aizoaceae of the Centrospermae and placement
in Mol luginaceae along with Caryophyll aceae in
Caryophyllales. Among the numerous flavonoids of higher
plants tl~c rare and unusual ones Eind more acceptance in
micromolecular taxonomy. Quite recently a survey of
2'-oxygenated flavonoids along with their possible
biosynthetic pathways has been presented176.
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 most significant function of the sap-soluble flavonoids
is their ability to impart colour to the plants in which
they occur. They are mostly responsible for the orange,
scarlet, crimson, mauve, violet and blue colours as well as
contributing much to yellow, ivory and cream shades. Brown
and black pigments found in nature are either due to the
products of oxidation of El avonoitls ant1 re1 atcd phenolic
compounds or to their chelates with metals. Anthocyanin - £1 avone 81 ycoside copigment complexes177 are stab1 ized by
chelation with metal ions or bondin in^'^^. These copigments
are found to protect the anthocyanins from
nucleophilic attacks resulting in colour loss. Carotenoids
and chlorophylls are the other colouring materials in higher
plants. The anthocyanins serve as anti-microbial agent,
effective growth inhibitors and in pollination ecology and
seed dispersal by attracting insects and animals. Both
fruit and flower colours provide immense aesthetic pleasure;
conscious selection of colour varieties among garden plants
and horticultural crops has been in practice for a long
time. Furt\ier, in vivo studies of anthocyanins show that
acylation with hydroxy cinnamic acids and dibasic acids like
malonic acid and succinic aciddtabilises anthocyanina and
significantly protects them from photooxidative
degradation179. The important functions of flavonoids in
plants are their protective role as light screen against
damaging UV radiation, as feeding and protection from
herbivores and as allelopathic agents. In biological
systems, the stimulation of protein degradation as well as
inhibition of protein formation by antibiotics resulted in
the flavonoid accumulation implicating the importance of
flavonoids in protein synthesis 180,181. Other important
Eunctions 182,183, include their rolr n~ onti-oxidnntr,
enzyme inhibitors, precursors of toxic substances and aa
photosensitizing and energy transferring compounds, in
control of plant growth and development, in respiration,
photosynthesis, morphogenesis and sex determination in
plants.
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 induced
in plants following fungal invasion. Some of the flavonoids
affect the behaviour, growth and development of insects due
to their toxcity, while flavone glycosides are feeding
The metabolic challenges of the f lavonoids
including the condensed tanninbto the insects consista
mainly of the variety of phenolic groups. This activity is
destroyed when the phenolic 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
orthodihydroxy group in ring A and B and not the functional
group of ring C and the position of the ring B (at C-2 nr
C-3 as in flavones or isoflavones). The glycosylation of
flavonoids also show marked variation in growth inhibition;
3-0-glycosylation inhibits growth but not 7-0-glycosylation.
The enhanced activity of flavanones compared to the
corresponding flavones showed that the coplanarity of the
flavonoid rings of flavones may hinder their biological
efficacy. Different types of bio-assay involving several
types of organisms conducted with isoflavonoid phytoalexinr
by revealed that they possessed fungitoxicity and
limited antibacterial activity. The introduction of
isof 1 avonoid phytoalexins in plants caure~
phytotoxicity186~187 such as inhibition of respiration,
reduced growth of suspension cultures, repressed seed
germination, retarded root growth and electrolytic leakage.
The flavonols quercetin and kaempferol and their
glycosides rutin and isoquercitrin exhibited strong
antifungal activity188. Apigen~n, apigenin 7-0-glucoside,
echinacin and echinaticin were assayed against germination
of conidia of Alternaria tenuissima, which incites leaf
blight disease in Cajanus cajan (Pigeon pea). All showed
high efficacy against the pathogen. Echinacin was high1
effective and considered the most promising antifungal 189 agent .
The occurrence and concentration of flavonoids in
food stuff have been reviewed by ~ermannl~'. The chemically
modified anthocyanins have found increasing use as approved
colours I n formulated food and beverages. Although
different groups of flavonoids are consumed, the diversity
is removed by intestinal microorganisms hydrolysing the
glycosides. The flavonoids combine with dietary proteins
and carbohydrates and with digestive enzymes and cellular
components of the digestive tract. A small fraction of the
flavonoid is absorbed into the blood stream and the
unabsorbed compounds affect many aspects of digestion end
health.
An ever-increasing number of phsrmacological
effects of flavonoids have become known during the past
fifty years through the discovery of new plant flavonoids
and their derivatives80a81. The spasmolytic, hypoglycaenic,
antianginel, analgesic, amulcer, antihepatotoxic,
antiinflammatory, antiallergic, antimalarial, antimicrobial
and antiviral effects of flavonoids are noteworthy. In
recent years a number of flavonoids like baicalin,
taxifolin, gossypin, nepetrin, proanthocyanidins, diosmin,
fisetin, sophoricoside, hypolaetin 8-0-glucoside,
naringenin, ( + ) - catechin, ( - 1 - epicatechin and 5,7-
dimethoxyflavone have been reported to have antiinflammatory
effects 191-195. Recently the effect of flavonoids on
196 arachidonate metabolism was reported by Ferrandiz . Many semisynthetic flovonoid derivatives also show t h i ~
effect of which (0-p-hydroxy ethyl) rutin and various 197 quercetin derivatives are important . Studies of
compounds with flavone skeleton were stimulated by
recognition of antiallergic effects. Thus, orally effective
antiallergic chromone drugs (kellin, hypolaetin 8-glycoside,
disodium chromoglycosate, etc) are recently in use197. More
detailed investigation has been done on this aspect by
employing various citrus flavonoids for their effect on
human basophil histamine release stimulated by several
secretagogues, and it was found that apigenin, luteolin,
tangeretin, sinensetin, nobiletin and kaempferol were very
active inhibitors of basophil histamine release and
neutrophil betaglucuronidase relase, and thereby posaesa
vivo antiallergic Many flavonoids were - studied200-202 for antiviral acavity and observed that
hydroxylation in 3-position appears to be a prerequisite for
this activity. Antirhinovirus and anti-picronovirus
203 activity of certain flavonoids have also been reported . Anticancer activity of a few flavonoids (flavonea,
isoflavones, f lavanones and f lavonols are also
reported 204-207. Recently 7,8-di-0-substituted f lavans,
biflavans and flavones showed cytotoxic activity14' and it
has been established that the activity was due to methoxy
and/or hydroxyl groups in the structure.
Inhibition of human immuno deficiency virus
reverse transcriptase is currently considered as useful
approach in the prophylaxis and intervention of Acquired
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 examinedzo8 and polyphenolic
compounds were found to be responsible for the activity;
among flavonoids tested, quercetin exhibited moderate
activity.
The antihepatotoxic effect of flavonoids wan first
demonstrated by Halm a1209 with the flavanolignan,
silybin and its isomers. About thirteen flavonoids and
coumarins have been demonstrated210 to be antihepatotoxic or
liver protective agents. The screenQg of plants, recorded
in Indian folk medicines, for liver injury has
established2" that the active principles are flavonoide.
Antiulcerogenic property of 3-0-methyl ( + I catechin,
apigenin and luteolin, antidiabetic effects of hispidulin
and nepetin and analgesic effects of puerarin are also
reported 2129213. Elavonoids ere known to inhibit a number
of enzymes such as aldose r e d ~ c t a s e ~ ~ ~ ~ ~ ~ ~ xanthine
oxidase 218 219 216y217, phosphodiesterase , ca2+ ATPase , ~ i ~ o o x ~ ~ e n a s e ~ ~ ~ , cycloxygenase221 etc.
Several dihydrochalcone derivatives are used as
sweeteners. The bitter taste of flavanones and their
neohesperidosides, sweet taste of dihydrochalcone
neohesperidosides, reduction of the bitter tastes of
flavanone glycosides by flavones and the tastelessness of
the flavgne glycosides irrespective of the nature of sugars
were elaborately studied by Horowitz and ~ e n t i l i ~ ~ ~ .
Although flavonoids have been extensively studied in
medicinal research for diverse physiological effects, their
role in plants from which they are derived has not been
unambiguously established.
Work on different aspects of flavonoids including
discovery of new compounds, biological activity, medicinal
properties, etc. are regularly reviewed7b-81,191s223~224 and
the knowledge is updated through proceedings of regular
international symposia on f lavon0iS8~~~. Considerable work
on the chemistry and pharmacological properties of a number
of flavonoids has been accomplished in the laboratories of
our department 226-230
33
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