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 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.

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(auxins) and shield from

environmental toxins

The Most Important Classes of Flavonoids and their Biological

Significance

classnumber 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 350cream-coloured

pigments of flowers

flavonols 350feeding repellents (?)

in leaves

dihydrochalcons 10 some taste bitter

proanthocyanidins 50 astringent substances

catechins 40some have properties

like 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 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.

CORE STRUCTURES AND

NOMENCLATURE

• The nomenclature offlavonoids proper is straight-forward with the aromatic ringA condensed to theheterocyclic ring C and thearomatic ring B most oftenattached at the C2 position.The various substituents arelisted first for the A and C ringand - as primed numbers - forthe B ring (note that thenumbering for the aromaticrings of the open-chainedprecursor chalcones isreversed).

• (Harborne JB, ed. (1988) TheFlavonoids. 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

FLAVONOIDS & THERE EXAMPLE

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 isovitexinG

OH

OH O

O

OH

G

OH

OH O

O

OH

Vitexin Isovitexin

O

O

Chromone

O

O

Flavone

1

2

34

5

6

7

81'

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.

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.

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

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,sililicacid,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 molybdatecomplexes 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 C15H9O2(OH)+ CH3COCl

C15H9O2(OCOCH3)+HCl

When methylated followed by fusion with KOH,flavonol yields phenol and benzoic acid.Boththese products do no possess methoxyl group.Thisshows that the methoxy group must be present at C3which must have been lost in KOH fusion.

C15

H10

O3

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)2SO

4/NaOH

O

O

C6H6

OCH 3

KOH

Fusion

OH HOOC+

Phenol Benzoic acid

O

O

C6H6

OH

KOH

Boiling

OH

O

OH

OH

C6H5COC 6H5

OH

O

OH

OH

COCH 2OH+ C

6H

5COOH

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+ (C

6H

5CO)

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,quercetinyields phloroglucinol and protocatechinic acid.Alsoquercetin when methylated yields pentamethylquercetin.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.KO

HBoiling

OH

O

OOH

OH

OH

O-Rhamnose

• All the foregoing reactions can be

explained on the basis of structure (I) of

quercetin as follows.

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 3OHC

OMe

OMe

+OH-

OMe

MeO

OH

OMe

MeO

HCl O

OOMe

MeO

OMe

MeO

C5H

11ONO

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 in 2.2.15 cells.[22]

Quercitrin and myricetin 3-O-beta-D-galactopyranoside inhibit HIV-1reverse 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]

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]

Inflammation

Several laboratory studies show quercetin may have anti-inflammatoryproperties,[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 D2production.[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

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]

OH

O

OOH

OH

OH

O-Rutinose

Rutin, also called rutoside, quercetin-3-O-rutinoside and sophorin, is the glycosidebetween the flavonol quercetin and the disaccharide rutinose

Occurrences

Rutin is one of the phenolic compounds found in the invasive plant species Carpobrotus edulisand 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.

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 synthaseactivity.

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 hydroxylfunctional group.

Both quercetin and rutin are used in many countries as medications for blood vesselprotection, 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 vitrothe 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.

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 vitromodels.[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.

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.

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]