2. 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 in nature and 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).
•However, their occurence is not restricted to flowers but include all
parts of the plant.
•They are secondary mettabolite and effective in CNS disorders.
3. 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
4. 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(auxins) 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
chalcons 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
5. 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
concentracted 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.
6. 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.)
OH
O
Chalcone
A
O O
B
O O
OH
O
OH
Flavanone Dihydroflavonol Flavan-3-ol
O O
O O
OH
O
OH
+
Flavone Flavon-3-ol Anthocyanidin
O
O
Isoflavone
O
Neoflavone
A
B
C
Flavonoids Society For Free Radical Biology and Medicine W. Bors
8. 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.
9. O
O
Chromone
1
7
2'
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
HO O
OH
O
OH
HO O
G
OH
O
OH
Vitexin Isovitexin
O
O
Flavone
2
4 3
5
6
8
1'
3'
4'
5'
6'
10. 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.
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.
11. Cl
O
-
+
OH
-
+
Cl
O
O
H
+
+
O
}Cl
OH
-
Different structures of oxonium salts of flavones.
Flavones exhibit two absorption bands: Band I 330-350 nm and Band II,250-270
12. Ground plant material
Digestion with boiling water
diluted and treated with lead acetate
Filteration
Diluted with water,acidified with HCl and boiled for some hours
Precipitate of sugar
free flavonoids
Exctracted with alcohol &
carried out fractional crystallisation
Water extract
Precipitate of
tannins
Flavonoids in
supernatant liquid
Acetate free flavonoids
13. 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.
14. 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)
15. 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.
16. 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.
17. 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.
18. 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 C15H9O2(OH)+ CH3COCl
C15H9O2(OCOCH3)+HCl
When methylated followed by fusion with
KOH,flavonol yields phenol and benzoic acid.Both
these products do no 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
19. 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 C. Hence flavanol must be 3-
3hydroxyflavone(3-hydroxy-O
2-phenyl-ϒ-chromone)
O
C6H6
OH
Flavanol
On the basis of the above structure of
flavanol,the foregoing reactions can be
explained as follows:
20. 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
C6H5
OH
COC 6H5
OH
O
OH
OH
COCH 2OH
+ C6H5COOH
o-hydroxybenzoyl methanol Benzoic acid
a)
b)
c)
21. 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
C6H5
OMe
O
HI
O C6H5
O
OH
Flavanol
22. 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
CHO+ HO HCl
CHO212011 215103
+CH(CHOH)CHO
3
23. 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.
24. KOH
Fusion
Quercetin Phloroglucinol +
Protocatechuic
acid
methyln Ethanol.KO
Quercetin Pentamethyl quercetin
H
Boiling
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.
25. OH
O
HO O
HO
OH
O-Rhamnose
• All the foregoing reactions can be
explained on the basis of structure (I) of
quercetin as follows.
26. 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.
27. MeO
MeO
OH
COCH 3 OHC
OMe
OMe
+
OH-
OMe
MeO
OH
OMe
MeO
HCl O
OMe O
MeO
OMe
MeO
C5H11ONO
HCl
MeO O
OMe O
NOH
OMe
OMe
H2SO4
MeO O
OMe O
OMe
OMe
O
(i)Enolisation
(ii)HI
MeO O
OMe O
OMe
OMe
O
2,4-dimethoxy- 6 hydroxy acetophenone 3,4-dimethoxy benzaldehyde
quercetin
28. USES
Preliminary research
Antiviral
Hyperoside (which is the 3-O-galactoside of quercetin) is a strong
inhibitor of HBsAg and HBeAg secretion in 2.2.15 cells.[22]
Quercitrin and myricetin 3-O-beta-D-galactopyranoside inhibit HIV-1
reverse transcriptase, all with IC50 values of 60 μM.[23]
Quercetin can also inhibit reverse transcriptase, part of the replication
process of retroviruses.[24] The therapeutic relevance of this inhibition
has not been established.
Asthma
Quercetin is an effective bronchodilator and helps reduce the release of
histamine and other allergic or inflammatory chemicals in the body.[25]
Quercetin has demonstrated significant anti-inflammatory activity
because of direct inhibition of several initial processes of
inflammation.[26]
29. Cancer
Laboratory studies have investigated Quercetin's potential for use
in anti-cancer applications.[27] The American Cancer Society says
while quercetin "has been promoted as being effective against a
wide variety of diseases, including cancer," and "some early lab
results appear promising, as of yet there is no reliable clinical
evidence that quercetin can prevent or treat cancer in humans."
In the amounts consumed in a healthy diet, quercetin "is unlikely
to cause any major problems or benefits."[28]
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.[29]
30. Inflammation
Several laboratory studies show quercetin may have anti-inflammatory
properties,[30][31] and it is being investigated for a wide range of potential health
benefits.[31][32]
Quercetin has been reported to be of use in alleviating symptoms of
pollinosis.[33] An enzymatically modified derivative was found to alleviate ocular
but not nasal symptoms of pollinosis.[34][35][36]
Studies done in test tubes have shown quercetin may prevent immune cells from
releasing histamines which might influence symptoms of allergies.[37][38]
A study with rats showed that quercetin effectively reduced immediate-release
niacin (vitamin B3) flush, in part by means of reducing prostaglandin D2
production.[39] A pilot clinical study of four humans gave preliminary data
supporting this.[40]
Quercetin may have properties of a calcineurin inhibitor, similar to cyclosporin A
and tacrolimus, according to one laboratory study.[41]
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
31. Metabolic syndrome
Quercetin has been shown to increase energy expenditure in rats, but only for short periods
(fewer than 8 weeks).[30] Effects of quercetin on exercise tolerance in mice have been associated
with increased mitochondrial biogenesis.[31] In mice, an oral quercetin dose of 12.5 to 25 mg/kg
increased gene expression of mitochondrial biomarkers and improved exercise endurance.[31]
It has also been claimed that quercetin reduces blood pressure in hypertensive[43] and obese
subjects in whom LDL cholesterol levels were also reduced.[44]
In vitro studies showed quercetin and resveratrol combined inhibited production of fat cells[45]
and vascular smooth muscle cell proliferation.[46]
Supplements of quercetin with vitamin C and niacin does not cause any significant difference in
body mass or composition[47] and has no significant effect on inflammatory markers, diagnostic
blood chemistries, blood pressure, and blood lipid profiles.[48]
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.[49]
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.[50]
34. 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[4] and
antioxidant[5] properties of the plant.
Its name comes from the name of Ruta
graveolens, a plant that also contains rutin.
35. In food
Rutin is a citrus flavonoid glycoside found in many plants
including buckwheat,[6] 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).[6] 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.[7]
Rutin is one of the primary flavonols found in 'clingstone'
peaches.[8]
In the fava d'anta tree, the synthesis is done via a rutin synthase
activity.
36. 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.
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.
37. 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[9] in
subtoxic concentrations, so acts as an inhibitor
of angiogenesis. This finding may have potential
relevance for the control of some cancers.
38. Health effects
While a body of evidence for the effects of rutin and quercetin is
available in mice,[10] rats,[11] hamsters,[12] and rabbits,[13] as well as
in vitro studies,[14] no clinical studies directly demonstrate
significant, positive effects of rutin as dietary supplement in
humans.
Rutin inhibits platelet aggregation,[15] as well as decreases
capillary permeability, making the blood thinner and improving
circulation.[citation needed]
Rutin shows anti-inflammatory activity in some animal and in vitro
models.[16][17]
Rutin inhibits aldose reductase activity.[18] Aldose reductase is an
enzyme normally present in the eye and elsewhere in the body. It
helps change glucose into the sugar alcohol sorbitol.
39. Recent studies show rutin could help prevent blood clots, so
could be used to treat patients at risk of heart attacks and
strokes.[19]
Some evidence also shows rutin can be used to treat
hemorrhoids, varicosis, and microangiopathy.[20]
Rutin increases thyroid iodide uptake in rats without raising serum
T3 or T4.[21]
Rutin is also an antioxidant;[22] compared to quercetin, acacetin,
morin, hispidulin, hesperidin, and naringin, it was found to be the
strongest.[23][unreliable source?] However, in other trials, the effects of
rutin were lower or negligible compared to those of
quercetin.[24][25]
Hydroxyethylrutosides, synthetic hydroxyethyl acetylations of
rutin, are used in the treatment of chronic venous insufficiency.
40. In veterinary medicine
Rutin may have a veterinary use in the
management of chylothorax in dogs and
cats.[26]
Metabolism
The enzyme quercitrinase can be found in
Aspergillus flavus.[27] It is an enzyme in the
rutin catabolic pathway.[28]
