Flavonoids classification, isolation and identification

Definition:
Group of polyphenolic compounds which
are found in fruits, flowers, seeds &
vegetable.
They are more common in higher plants being
abundant in families, Polygonaceae,
Rutaceae, Leguminosae, Umbelliferae &
Compositae.
Flavonoids (named from the Latin word
flavus meaning yellow, their colour in
nature) are a class of plant secondary
metabolites.

Physiological role:
1- Role in plant defense mechanism:-
They have a very limited role in this respect
due to their low toxicity when compared with
other plant secondary metabolites such as
alkaloids.
2- They are the pigments of flowers and
attract pollinating insects.
3- They play a role in plant growth control
by inhibiting & activating enzymes.

Structure of flavonoids
The flavonoids are possessing 15 carbon
atoms; two benzene rings joined by a
linear three carbon chain the skeleton
can be represented as the C6 - C3 - C6
system.

The three-carbon (-C3-) may be included through an
oxygen bond between the two phenyl rings into:
1- A five-membered heterocyclic ring (furan)
as in aurones.
2- A six-membered heterocyclic ring (pyran) to
give flavonoids which constitute the largest
group.

Flavonoids occur as aglycones, glycosides
and methylated derivatives.
The flavonoid aglycone consists of a benzene ring
(A) condensed with a six membered ring (C) pyran
ring , which in the 2-position carries a phenyl ring
(B) as a substituent.

The flavonoid glycosides:
Glycosides → aglycone (non sugar part)+glycone (sugar
part)
When glycosides are formed, the glycosidic linkage can be
located in positions 3 or 7 and may be L-rhamnose, D-
glucose, galactose or arabinose .

Classification:
They are classified accorrding to chemical structure into:
1-
Flavones:
2-phenylchromen-4-one examples:-
1- Apigenin.
2- Luteolin.
3- Tangeritin.
4- Diosmetin.

4`3`75Flavones
OHHOHOH1- Apigenin
OHOHOHOH2- Luteolin
O-MeHO-MeO-Me3- Tangeritin
O-MeOHOHOH4- Diosmetin

2-Flavonol:
3-hydroxy-2-phenylchromen-4-one.
Examples:
1- Kaempferol.
2- Rutin.
3-Myricetin.
4-Quercetin.
5- Quercetrin.
6- Fisetin.

Ŕ5Ŕ3R7R3Flavonol
HHOHOH1- Kaempferol
OHOHOHOH2-Myricetin
HOHOHOH3-Quercetin
HOHOHO-Rh4- Quercetrin
HOHO-MeOH5- Fisetin

3-Flavanone:
2,3-dihydro-2-phenylchromen-4-
one
examples:-
1-
Hesperetin.
2-
Hespereidin.
3-Naringenin.

4`3`75Flavanone
O-MeOHOHOH1- Hesperetin
O-MeOHO-RutinoseOH2-Hespereidin
OHHOHOH3- Naringenin

4-Flavanonol:
3-hydroxy-2,3-dihydro-2-phenylchromen-4-one
examples:-
1-Taxifolin.
2- Silymarin.
Taxifolin →

5- Flavan:
1- Flavan-3-ol
2- Flavan-4-ol
3- Flavan-3,4-diols

Flavan-3-ol known as flavanol
Examples:-
1- Catechin (β-OH)
2- Epicatechin (α-OH)

6- Isoflavones
3-Phenylchromen-4-one
skeleton
4`753Isoflavone
OHOHOH-1- Genistein
OHO-GluH-2- Daidzin

7-Neoflavonoides
ring B in position 4 (4-phenyl-coumarins)
The Isoflavonoids and the Neoflavonoids can be
regarded as abnormal
flavonoids.

8- Anthocyanidins:
Flavylium (2-Phenylchromenylium) ion skeleton of
anthocyanidins.
e.g
1-Cyanidin. 2-
Delphenidin.

Actions of flavonoids
1-Flavonoids as antioxidants:
The flavones and catechins seem to be the most powerful flavonoids for
protecting the body against reactive oxygen species (ROS).
Flavonoids are oxidized by radicals, resulting in a more stable,
less-reactive radical.
Because of the high reactivity of the hydroxyl group of the
flavonoids, radicals are made inactive. Epicatechin and
rutin are also powerful radical scavengers.
2- Anti ulcer effect:
Majority of peptic ulcers are associated with helicobacter
pylori, a spiral-shaped bacterium that lives in the acidic
environment of the stomach. Quercetin seems to play a
very important role in the prevention and treatment of
peptic ulcer. It acts by promoting mucus secretion, thereby
serves as gastroprotective agent, also quercetin has been
shown to inhibit the growth of helicobacter pylori
bacterium in-vitro studies.

3- Anti atherosclerotic effects:
Atherosclerosis is a condition that results from the gradual
build-up of fatty substances, including cholesterol, on the
walls of the arteries. This build-up, called plaque, reduces
the blood flow to the heart, brain and other tissues and can
progress to cause a heart attack or stroke. This process is
commonly referred to as hardening of the arteries. An
elevated plasma low density lipoprotein (LDL)
concentration is a primary risk factor for the development
of atherosclerosis and coronary artery disease. Flavonoids
seems to suppress LDL oxidation and inflammatory
progression in the artery wall. A Japanese study reported
an inverse correlation between flavonoid intake and total
plasma cholesterol concentrations, other clinical studies, as
mentioned earlier, stated that flavonoid intakes protect
against coronary heart disease.

4- Anti-inflammatory effect:
Cyclooxygenase (COX) is an enzyme that plays an
important role as inflammatory mediator and is involved
in the release of arachidonic acid, which is a precursor for
biosynthesis of eicosanoids like prostaglandins and
prostacyclin. The release of arachidonic acid can be
considered starting point for a general inflammatory
response. Select flavonoids like quercetin are shown to
inhibit the cyclooxygenase pathway. This inhibition
reduces the release of arachidonic acid.

5-Hepatoprotective activity:
Many flavonoids have also been found to possess
hepato-protective activity e.g silymarin, apigenin,
quercetin and naringenin.
The results of several clinical investigations showed
the efficacy and safety of flavonoids in the
treatment of hepato-biliary dysfunction and
digestive complaints, such as sensation of
fullness, loss of appetite, nausea and abdominal
pain.

Isolation of flavonoids
*Glycosides are polar due to sugar moiety and
extractable with alcohol, water and
hydroalcoholic solution.
*Aglycones are extracted with less polar
solvents e.g ether, chloroform and ethyl acetate.

1-Plant preparation:
Flavonoids (particularly glycosides) can be degraded by
enzyme action when collected plant material is fresh or
non dried. It is advisable to use dry, lyophilized, or
frozen samples. dry plant material is used, it is generally
ground into a powder.
2-Solvent choosing:
For extraction, the solvent is chosen as a function of the type
of flavonoid required. Less polar flavonoids (e.g.,
isoflavones, flavanones, methylated flavones, and
flavonols) are extracted with chloroform,
dichloromethane, diethyl ether, or ethyl acetate.
while flavonoid glycosides are more polar and are extracted
with alcohols or alcohol–water mixtures.

The extractions of flavonoid-containing material are still
performed by simple direct solvent extraction, can also be
extracted in a Soxhlet apparatus, first with hexane,to
remove lipids and then with ethyl acetate or ethanol to
obtain phenolics. This approach is not suitable for heat-
sensitive compounds.
A convenient and frequently used procedure is sequential
solvent extraction. A first step,with dichloromethane, for
example, will extract flavonoid aglycones and less polar
material. A subsequent step with an alcohol will extract
flavonoid glycosides and polar constituents.

B- Separation
Preliminary Purification Preparative Methods
HPLC
High-Performance Liquid
Chromatography
MPLC
Medium-Pressure Liquid
Chromatography
Centrifugal Partition
Chromatography

The crude flavonoid extract could be purified by one of the
following methods:-
1- Shaking with Na2CO3 or NaHCO3 in case of
compounds contaning strong acidic OH groups at C-7
and or C-4`followed by acidification.
B-Preliminary
Purification

2- Precipitation with Pb-acetate:
The lead acetate procedure is often
unsatisfactory since some phenolics do not
precipitate; other compounds may co-precipitate
and it is not always easy to remove the lead salt.
3- Chromatographic techniques e.g PC, TLC or
CC.
A short polyamide column, a Sephadex LH-20
column, or an ion exchange resin can be used.

Preparative Methods
The major problems with the preparative
separation of flavonoids:-
sparing solubility in solvents employed in
chromatography.
Poor solubility in the mobile phase used for a
chromatographic separation can induce
precipitation at the head of the column, leading
to poor resolution, decrease in solvent flow, or
even blockage of the column.

In the separation of anthocyanins and
anthocyanin-rich fractions, it is advisable to
avoid acetonitrile and formic acid
Acetonitrile is difficult to evaporate and there is
a risk of ester formation with formic acid.
The choice of method depends on the polarity of
the compounds and the quantity of sample
available.

Conventional open-column chromatography is still
widely used because of its simplicity and its value
as an initial separation step.
Support materials include polyamide, cellulose,
silica gel, Sephadex LH-20, and Sephadex G-10, G-25,
and G-50.
Sephadex LH-20 is recommended for the separation
of proanthocyanidins.
Methanol and ethanol can be used as eluents for
proanthocyanidins, acetone is better for displacing
the high molecular weight polyphenols.

Several preparative pressure liquid chromatographic
methods are available. These can be classified according
to the pressure employed for the separation:
1- High-Performance Liquid Chromatography:-
HPLC is becoming by far the most popular technique for
the separation of flavonoids, both on preparative and
analytical scales.
The difference between the analytical and preparative
methodologies is that analytical HPLC does not rely on
the recovery of a sample, while preparative HPLC is a
purification process and aims at the isolation of a pure
substance from a mixture.

2- Medium-Pressure Liquid Chromatography:-
MPLC is a closed column (generally glass) connected to
a compressed air source or a reciprocating pump.
MPLC columns have a high loading capacity — up to a
1:25 sample-to-packing-material ratio — and are
ideal for the separation of flavonoids.
3- Centrifugal Partition Chromatography:-
Countercurrent chromatography is a separation
technique that relies on the partition of a sample
between two immiscible solvents.
The relative proportions of solute passing into each of
the two phases determined by the partition
coefficients of the components of the solute.

Absence of a solid support has the following
advantages over other chromatographic
techniques:-
1-No irreversible adsorption of the sample.
2-Quantitative recovery of the introduced sample.
3- Reduced risk of sample denaturation.
4-Low solvent consumption.
5-Favorable economics.

Identification and
characterization
A- Test for identification:-
The different types of flavonoides can be characterized by their
colored response to different tests (visible light ) or colored
fluorescence in UV light.
1- Reaction with aluminum chloride:
The different classes of flavonoids give yellow color with AlCl3
but fluoresce differently in UV as shown in the following table .
FlavanonesFlavonolsFlavoneslight
YellowYellowYellowVisible
Pale-brownishYellowish to
Yellowish-green
GreenUV

2- Lead subacetate test:-
all flavonoids give yellowish precipitate with Pb
subacetate.
3-Shinoda´s test for flavanones and
flavonols:-
alcoholic solution + Mg metal ⁄ Hcl , an orange , red or
violet color is produed.
4- Antimony pentachloride test for
chalcones:
alcoholic solution + SbCl5⁄ CCl4 , red or violet color is
produced.

B- Chromatographic characterization by
PC and ⁄ or TLC:-
One or more of the following procedures can be
used for location of flavonoid spots:
1- Under UV before and after spraying with AlCl3
and/or exposure to ammonia vapors.
2- In visible light: e.g. chalcones and aurones
appear bright yellow and turns to orange red on
exposure to ammonia vapor.
3-After spraying with FeCl3, p-anisaldehyde ,
diazotized sulphanilic acid.

Flavonoids classification, isolation and identification

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Flavonoids classification, isolation and identification