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FLAVONOIDS

Prepared by, ROSHNI ANN BABY, M.PHARM PART I.

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).

• However, their occurence is not restricted to flowers but include all parts of the plant.

• They are secondary metabolite and effective in CNS disorders.

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

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

Classnumber of known

membersbiological significance

(so far as known)

anthocyanin(s) 250 red and blue pigments

Chalcones 60 yellow pigments

Aurones 20 yellow pigments

Flavones 350cream-coloured

pigments of flowers

Flavonols 350feeding repellents (?)

in leaves

Dihydrochalcons 10 some taste bitter

Proanthocyanidins 50 astringent substances

Catechins 40some have propertieslike those of tannins

Isoflavonoids 15oestrogen effect, toxic

for fungi

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.

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. Bors et al. .5

FLAVONOIDS & THEIR EXAMPLES

Flavone:- Luteolin, Apigenin, Tangeritin Flavonol:- Quercetin, Kaempferol, Myricetin,

Fisetin, Isorhamnetin,

Pachypodol, Rhamnazin Flavanone:- Hesperetin, Naringenin,

Eriodictyol, Homoeriodictyol Flavanonol- Taxifolin, Dihydrokaempferol

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.

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

OH O

O

OH

G

OH

OH O

O

OH

Vitexin Isovitexin

O

O

Chromone

O

O

Flavone

1

2

345

6

78

1'

2'

3'4'

5'

6'

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.

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.

ISOLATION OF FLAVONOIDS

Digestion with boiling water

diluted and treated with lead acetate

Filteration

Diluted with water,acidified with HCl and boiled for some hours

Exctracted with alcohol & carried out fractional crystallisation

Water extract

Precipitate of tannins

Flavonoids in supernatant liquid

Ground plant material

Acetate free flavonoids

Precipitate of sugar free flavonoids

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.

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)

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.

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.

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.

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

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

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

OCH3

KOH

Fusion

OH HOOC+

Phenol Benzoic acid

O

O

C6H6

OH

KOH

Boiling

OH

OOH

OH

C6H5COC6H5

OH

OOH

OH

COCH 2OH+ C6H5COOH

o-hydroxybenzoyl methanol Benzoic acid

a)

b)

c)

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

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

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.

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

OH

O

OOH

OH

OH

O-Rhamnose

• All the foregoing reactions can be explained on the basis of structure (I) of quercetin as follows.

KOH Fusion

OH

OHOH

OH

OH

COOH

+

(I)

Phloroglucinol Protocatechuic acid

O

O

OH

OH

OH

OH

OH

OMe

O

OMe

MeO

O

OMe

OMe

(CH3)2SO4 /methylation ethanolic KOH

MeO

OMe

HOOC

OMe

MeO OH

COCH2OMe

+

6-Hydroxy-w-2,4, trimethoxyacetophenone Veratric acid

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.

MeO

MeO

OH

COCH 3 OHC

OMe

OMe

+OH-

OMe

MeO

OH

OMe

MeO

HCl O

OOMe

MeO

OMe

MeO

C5H11ONO

HCl

MeO O

OOMe

NOH

OMe

OMe

H2SO4

MeO O

OOMe

OMe

OMe

O

(i)Enolisation

(ii)HI

MeO O

OOMe

OMe

OMe

O

2,4-dimethoxy- 6 hydroxy acetophenone 3,4-dimethoxy benzaldehyde

quercetin

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.

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.

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.

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.

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 composition and has no significant effect on inflammatory markers, diagnostic blood chemistries, blood pressure, and blood lipid profiles.

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.

RUTINOH

O

OOH

OH

OH

O-Rutinose

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.

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.

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.

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.

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.

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.

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.

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

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