Flavonoids are polyphenolic compounds found in plants that act as antioxidants. They have 15 carbon atoms arranged in two benzene rings connected by a 3 carbon chain. Over 4,000 flavonoids have been identified, commonly found as flower pigments but also throughout plants. Major classes include flavones, flavonols, flavanones, and anthocyanidins. Key flavonoids like quercetin and kaempferol are studied for their health benefits such as antiviral and anti-inflammatory properties. Their structures can be elucidated through various chemical tests and reactions.
3. FLAVONOIDS
•The flavonoids are polyphenolic compounds
possessing 15 carbon atoms; two benzene rings
joined by a linear three carbon chain having the
carbon skeleton C6 - C3 - C6 and they are the plant
pigments and they are having polar nature and is
solouble in methanol and water.
• Flavonoids constitute one of the most characteristic
classes of compounds in higher plants. Many
flavonoids are easily recognised as flower pigments
in most angiosperm families (flowering plants).
4. • However, their occurence is not restricted to flowers but
include all parts of the plant.
• They are secondary metabolite and effective in CNS
disorders.
5. Flavonoids
Act like antioxidants. How effective they are depends
on their molecular structural characteristics
Some flavonoids in hops and beer have been found
to have better antioxidant effects than tea or red wine;
most flavonoids are found in fruits, vegetables, teas,
and other drinks.
Flavonoids have been known to have antiviral, anti-
allergic, antiplatelet, anti-inflammatory, antitumor
and antioxidant activities
6. Flavonoids
Polyphenolic
compounds with 15 C
atoms, 2 benzene rings
on linear 3 C chain
Over 4,000
flavonoids
Easily recognized as
flower pigments in most
angiosperm plants but
are not always flower
pigments
In plants they repair
damage and shield from
environmental toxins
The Most Important Classes of Flavonoids and their Biological
Significance
Class
number of known
members
biological significance
(so far as known)
anthocyanin(s) 250 red and blue pigments
Chalcones 60 yellow pigments
Aurones 20 yellow pigments
Flavones 350
cream-coloured
pigments of flowers
Flavonols 350
feeding repellents (?)
in leaves
Dihydrochalcons 10 some taste bitter
Proanthocyanidins 50 astringent substances
Catechins 40
some have properties
like those of tannins
Isoflavonoids 15
oestrogen effect, toxic
for fungi
7. TESTS FOR FLAVONOIDS
The extracts were dissolved in ethanol, filtered and
subjected to following tests.
Shinoda test: The dried extracts were dissolved in 95%
ethanol (5ml) and few drops of concentrated
hydrochloric acid (HCL) were added. Then the
magnesium turnings were put into the solution and
observed for appearance of pink color.
Lead acetate solution test: To small quantity of above
residue, lead acetate solution was added and observed for
appearance of formation of yellow colored precipitates.
8. CORE STRUCTURES AND
NOMENCLATURE
• The nomenclature of flavonoids
proper is straight-forward with the
aromatic ring A condensed to the
heterocyclic ring C and the
aromatic ring B most often
attached at the C2 position. The
various substituents are listed first
for the A and C ring and - as
primed numbers - for the B ring
(note that the numbering for the
aromatic rings of the open-chained
precursor chalcones is reversed).
• (Harborne JB, ed. (1988) The Flavonoids.
Advances in Research. Chapman & Hall.)
O O
O O
OH
O
OH
O O
O O
OH
O
OH
+
Flavanone Dihydroflavonol Flavan-3-ol
Flavone Flavon-3-ol Anthocyanidin
O
OH
Chalcone
A
B
O
O
Isoflavone
O
Neoflavone
A
B
C
Flavonoids Society For Free Radical Biology and Medicine W. Bo
10. FLAVONES
These are yellow pigments which occur in plant
kingdom either in the free state or as glycosides
associated with tannins.These are also known as
anthoxanthins.
Chemically they are hydroxylated derivative of
flavone(2-phenyl-4-chromone) which are partially
alkylated.
In most of the flavones, positions 5 and 7 are
hydroxylated and also one or more positions 3,4,5 are
also hydroxylated.Further positions 3’ and 5’ are often
methylated whereas positions 5,7 and 4’ are usually
unmethylated.
11. When a flavone is hydrolysed with mineral acid, it yields
an aglycon and one or more molecules of sugars. The
sugars are generally glucose, rhamnose etc. Flavones may
exist as C-glycosyl derivatives as well as O-glycosides, eg:
vitexin and isovitexin
G
OH
O
H O
O
OH
G
OH
O
H O
O
OH
O
O
Chromone
O
O
Flavone
1
2
3
4
5
6
7
8
1'
2'
3'
4'
5'
6'
12. PROPERTIES OF FLAVONES
Most flavones are yellow solids
Most flavones are soluble in water, ethanol and dilute
acids and alkalis.
Flavones are precipitated by lead salt.
With ferric chloride, flavones give either a dull green or
a red brown colour.
13. OH
O
+
Cl
-
O
O
Cl
-
+
H
+
OH
O
+
}Cl
-
Different structures of oxonium salts of flavones.
Flavones exhibit two absorption bands: Band I 330-350 nm and Band II,250-270
In acidic medium, flavones are usually more highly
coloured than the bases from which they are derived. In
acidic medium flavones form oxonium salts which impart
this colour. However these oxonium salts are very
unstable in presence of water. The flavones differ in this
respect from the anthocyanidins which give strong
oxonium salts and are found as such in plants.
14. Digestion with boiling water
diluted and treated with lead acetate
Filteration
Diluted with water,acidified with HCl and boiled for some hou
Exctracted with alcohol &
carried out fractional crystallis
Water extract
Precipitate of
tannins
Flavonoids in
supernatant liquid
Ground plant material
Acetate free flavonoids
Precipitate of sugar
free flavonoids
15. SEPARATION & PURIFICATION OF
FLAVONES
PAPER CHROMATOGRAPHY
Convenient means of separating and purifying
flavones on milligram scale
Dried plant material is extracted with either 70 % or 80 % methanol.
The aqueous extract is then concentrated to a small volume in
vacuo and refiltered if necessary.
An aliquot of this concentrate should be applied on Whatman No. 3
filter paper.
16. Separation of the flavones present in concentrate is generally
carried out in the solvent mixture BAW(n-butanol-acetic acid-
water),4:1:5
Individual bands are eluted and concentrated .
Further fractioned in water, 5 % acetic acid.
Purified in n-butanol-ethanol-water(4:1:2.2)
17. THIN LAYER CHROMATOGRAPHY
More sensitive method than paper chromatography.
Layers of microcrystalline cellulose is employed.
Solvent system same as that of PC.
Removal of lipid impurities is essential otherwise
considerable streacking may occur.
Visualisation of plates may be done by viewing the plate in
UV light(336 nm) either in the presence or absence of
ammonia vapour.It is often assisted by the use of layers
which contain a UV-Fluoroscent indicator. Flavonoids
appear as dark spots against a fluoroscent green
background.
Another useful method of detection is brief exposure of the
plate to iodine vapours which produce yellow-brown spots
against white background with most flavonoids.
Both methods are non destructive.
18. COLUMN CHROMATOGRAPHY
Used for large scale separations.
Adsorbents used include cellulose,celite,magnesol-
celite,sililic acid,polyamide and sephadex.
Polyamide is the widely used separation of the different
flavone glycosides being achieved by gradient elution with
water-methanol mixture.
Recently separation of flavanol glycosides as their
molybdate complexes on columns of Sephadex G-25 or
LH-20 is employed.Elution with water followed by 1 M
molybdate will separate mixtures of the common flavone
glycosides on G-25.Alternatively simple mixtures of
flavanol glycosides and aglycons can be separated on the
250 mg scale by adsorption on Sephadex I,H-20 and
subsequent elution with methanol.
19. GAS LIQUID CHROMATOGRAPHY
Not used extensively for the analysis & isolation of
flavanoids.
It is an acceptable method provided the flavanoid is
derivatized to increase it volatility. Trimethylsilyl ether
derivatives have been found most effective for this
purpose, although methyl ether and acetate derivatives
have also been used.
The stationary phases,SE-30 and OV-1 are most
commonly used for the separation of flavonoids.
20. GENERAL METHODS FOR THE ELUCIDATION OF
STRUCTURE OF FLAVONOLS
Flavonol shows characteristic bands at 350-390 nm and 150-270 nm in
ultraviolet spectrum.
The molecular formula of flavonol has been found to be C15H10O3
It can be acetylated to give an ester shows the presence of a hydroxyl
group.
C15H9O2(OH)+ CH3COCl
C15H9O2(OCOCH3)+HCl
When methylated followed by fusion with KOH, flavonol yields phenol
and benzoic acid. Both these products do not possess methoxyl group.
This shows that the methoxy group must be present at C3 which must
have been lost in KOH fusion.
C15H10O3
Methylation
and fusion with KOH
OH
+
COOH
flavonol phenol benzoic acid
21. When flavanol is boiled with an ethanolic solution of
potassium hydroxide, it yields a mixture of o-
hydroxybenzoylmethanol and benzoic acid. The formation of
these products reveals that flavonol contains a hydroxy group
at C3. Hence flavanol must be 3-hydroxyflavone(3-hydroxy-
2-phenyl-ϒ-chromone)
On the basis of the above structure of flavanol,the foregoing
reactions can be explained as follows:
O
O
C6H6
OH
Flavanol
22. O
O
C6H6
OH
Flavanol
+ CH3COCl
O
O
C6H6
OCOCH 3
+ HCl
Acetyl derivative of flavonol
O
O
C6H6
OH
methylation
(CH3)2SO4/NaOH
O
O
C6H6
OCH 3
KOH
Fusion
OH HOOC
+
Phenol Benzoic acid
O
O
C6H6
OH
KOH
Boiling
OH
O
OH
OH
C6H5
COC 6H5
OH
O
OH
OH
COCH 2OH
+ C6H5COOH
o-hydroxybenzoyl methanol Benzoic acid
a)
b)
c)
23. Synthesis
The above structure of flavanol has been confirmed by its
various syntheses
a)Robinson’s synthesis.
In this synthesis ω-methoxy-2-hydroxyacetophenone is
condensed with benzoic anhydride in the presence of its
potassium salt
OH
COCH 2OMe
+ (C6H5CO)2O
O
OMe
C6H5
O
HI
O C6H5
O
OH
Flavanol
24. QUERCETIN
Source: Occurs as glycoside quercetin in the bark
of Quercus tinctoria.
When quercetin is treated with acid,it yields one
molecule of quercetin and one molecule of
rhamnose
C21H20O11 + H2O C15H10O3
+CH3(CHOH)CHO
HCl
25. Molecular formula of quercetin has been found to be
C15H17O7.
As quercetin forms penta acetyl and penta methyl
derivatives,it means that it contains five hydroxyl groups.
By usual tests it has been shown that quercetin does not
contain any methoxy groups.
When fused with potassium hydroxide,quercetin yields
phloroglucinol and protocatechinic acid.Also quercetin
when methylated yields pentamethyl quercetin.The latter
compound when boiled with an ethanolic solution of
potassium hydroxide yields a mixture of hydroxy –ω, 2,4-
trimethoxyacetophenone and veratric acid.
26. Quercetin Phloroglucinol +
Protocatechuic acid
Quercetin Pentamethyl quercetin
6-Hydroxy-ω-2,4, trimethoxyacetophenone
+Veratric acid
All the above facts can be explained if structure (I),i.e,
3,3’,4’,5,7-pentahydroxy flavone is accepted as correct
structure of quercetin.
KOH
Fusion
methyln Ethanol.KOH
Boiling
29. Synthesis.
Finally,the structure of quercetin has been confirmed by its
various syntheses.
Kostanecki’s synthesis
In this synthesis,quercetin is obtained by the condensation
of 2,4-dimethoxy-6-hydroxy acetophenone with 3,4-
dimethoxy benzaldehyde in the presence of NaOH as
follows.
30. MeO
MeO
OH
COCH 3 OHC
OMe
OMe
+
OH-
OMe
MeO
OH
OMe
MeO
HCl O
O
OMe
MeO
OMe
MeO
C5H11ONO
HCl
MeO O
O
OMe
NOH
OMe
OMe
H2SO4
MeO O
O
OMe
OMe
OMe
O
(i)Enolisation
(ii)HI
MeO O
O
OMe
OMe
OMe
O
2,4-dimethoxy- 6 hydroxy acetophenone 3,4-dimethoxy benzaldehyde
quercetin
31. USES
Preliminary research
Antiviral
Hyperoside (which is the 3-O-galactoside of quercetin) is
a strong inhibitor of HBsAg and HBeAg secretion
Quercitrin and myricetin 3-O-beta-D-galactopyranoside
inhibit HIV-1 reverse transcriptase, all with IC50 values
of 60 μM.
Quercetin can also inhibit reverse transcriptase, part of
the replication process of retroviruses. The therapeutic
relevance of this inhibition has not been established.
32. Asthma
Quercetin is an effective bronchodilator and helps reduce the
release of histamine and other allergic or inflammatory chemicals
in the body.
Quercetin has demonstrated significant anti-inflammatory activity
because of direct inhibition of several initial processes of
inflammation.
Eczema
Serum IgE levels are highly elevated in eczema patients, and
virtually all eczema patients are positive for allergy testing.
Excessive histamine release can be minimized by the use of
antioxidants. Quercetin has been shown to be effective in reducing
IgE levels in rodent models.
33. Inflammation
Several laboratory studies show quercetin may have anti-
inflammatory properties, and it is being investigated for a wide
range of potential health benefits.
Quercetin has been reported to be of use in alleviating symptoms
of pollinosis. An enzymatically modified derivative was found to
alleviate ocular but not nasal symptoms of pollinosis.
Studies done in test tubes have shown quercetin may prevent
immune cells from releasing histamines which might influence
symptoms of allergies.
A study with rats showed that quercetin effectively reduced
immediate-release niacin (vitamin B3) flush, in part by means of
reducing prostaglandin D2 production. A pilot clinical study of
four humans gave preliminary data supporting this.
Quercetin may have properties of a calcineurin inhibitor, similar to
cyclosporin A and tacrolimus, according to one laboratory study.
34. Fibromyalgia
Quercetin may be effective in the treatment of fibromyalgia
because of its potential anti-inflammatory or mast cell inhibitory
properties shown in laboratory studies
Cancer
Laboratory studies have investigated Quercetin's potential for use
in anti-cancer applications. The American Cancer Society says
quercetin has been promoted as being effective against a wide
variety of diseases, including cancer.
35. Metabolic syndrome
Quercetin has been shown to increase energy expenditure in rats, but
only for short periods (fewer than 8 weeks). Effects of quercetin on
exercise tolerance in mice have been associated with increased
mitochondrial biogenesis.In mice, an oral quercetin dose of 12.5 to
25 mg/kg increased gene expression of mitochondrial biomarkers and
improved exercise endurance.
It has also been claimed that quercetin reduces blood pressure in
hypertensive and obese subjects in whom LDL cholesterol levels were
also reduced.
In vitro studies showed quercetin and resveratrol combined inhibited
production of fat cells and vascular smooth muscle cell proliferation.
Supplements of quercetin with vitamin C and niacin does not cause any
significant difference in body mass or compositionand has no
significant effect on inflammatory markers, diagnostic blood
chemistries, blood pressure, and blood lipid profiles.
36. Monoamine-oxidase inhibitor
Possibly an active component of heather (Calluna vulgaris),
quercetin was suspected from a bioassay test on crude extracts to
selectively inhibit monoamine oxidase, possibly indicating
pharmacological properties.
Prostatitis
Quercetin has been found to provides significant symptomatic
improvement in most men with chronic prostatitis, a condition
also known as male chronic pelvic pain syndrome.
38. Rutin, also called rutoside, quercetin-3-O-rutinoside and
sophorin, is the glycoside between the flavonol quercetin
and the disaccharide rutinose
Occurrences
Rutin is one of the phenolic compounds found in the invasive
plant species Carpobrotus edulis and contributes to the
antibacterial and antioxidant properties of the plant.
Its name comes from the name of Ruta graveolens, a plant
that also contains rutin.
39. In food
Rutin is a citrus flavonoid glycoside found in many plants including
buckwheat, the leaves and petioles of Rheum species, and asparagus.
Tartary buckwheat seeds have been found to contain more rutin (about
0.8-1.7% dry weight) than common buckwheat seeds (0.01% dry
weight). Rutin is also found in the fruit of the fava d'anta tree (from
Brazil), fruits and flowers of the pagoda tree, fruits and fruit rinds
(especially the citrus fruits orange, grapefruit, lemon, and lime) and
apple; berries such as mulberry, ash tree fruits, aronia berries and
cranberries.
Rutin is one of the primary flavonols found in 'clingstone' peaches.
In the fava d'anta tree, the synthesis is done via a rutin synthase activity.
40. Chemical relatives
Rutin (quercetin rutinoside), like quercitrin, is a glycoside of the
flavonoid quercetin. As such, the chemical structures of both are
very similar, with the difference existing in the hydroxyl
functional group.
Metabolism
The enzyme quercitrinase can be found in Aspergillus flavus. It is
an enzyme in the rutin catabolic pathway.
41. USES
Both quercetin and rutin are used in many countries as
medications for blood vessel protection, and are ingredients
of numerous multivitamin preparations and herbal remedies.
Role as ligand
In humans, it attaches to the iron ion Fe2+, preventing it from
binding to hydrogen peroxide, which would otherwise create
a highly reactive free radical that may damage cells. It is also
an antioxidant.
Furthermore, it has been shown to inhibit in vitro the vascular
endothelial growth factor in subtoxic concentrations, so acts
as an inhibitor of angiogenesis. This finding may have
potential relevance for the control of some cancers.
42. Health effects
While a body of evidence for the effects of rutin and quercetin is
available in mice, rats, hamsters, and rabbits, as well as in vitro
studies,no clinical studies directly demonstrate significant,
positive effects of rutin as dietary supplement in humans.
Rutin inhibits platelet aggregation, as well as decreases capillary
permeability, making the blood thinner and improving circulation.
Rutin shows anti-inflammatory activity in some animal and in
vitro models.
Rutin inhibits aldose reductase activity. Aldose reductase is an
enzyme normally present in the eye and elsewhere in the body. It
helps change glucose into the sugar alcohol sorbitol.
43. Recent studies show rutin could help prevent blood clots, so could
be used to treat patients at risk of heart attacks and strokes.
Some evidence also shows rutin can be used to treat hemorrhoids,
varicosis, and microangiopathy.
Rutin increases thyroid iodide uptake in rats without raising serum
T3 or T4.
Rutin is also an antioxidant; compared to quercetin, acacetin,
morin, hispidulin, hesperidin, and naringin, it was found to be the
strongest.However, in other trials, the effects of rutin were lower
or negligible compared to those of quercetin.
44. Hydroxyethylrutosides, synthetic hydroxyethyl
acetylations of rutin, are used in the treatment of
chronic venous insufficiency.
In veterinary medicine
Rutin may have a veterinary use in the management
of chylothorax in dogs and cats.
45. REFERENCES
1)Organic chemistry of natural products; vol 1 ;
O.P. Agarwal ; pg no :350-406
2)Organic chemistry of natural products ; vol 2;
Gurdeep. R. Chatwal ; pg no : 2.1 – 2.40
3)Organic chemistry ; vol 2 ; stereochemistry &
chemistry of natural products ; I. L .Finar ;
5th edition ; pg no : 425
