A presentation

On

Phytochemical assays of phenolic compounds

Presented by – Gaurav

Reg. No. – 16TMM2363

In guidance of Dr. IP Singh

Department of Natural Products

NATIONAL INSTITUTE OF PHARMACEUTICAL EDUCATION AND

RESEARCH

S.A.S. NAGAR

Phytochemical Study of Phenolic Compounds

Phenolic Compounds - The term phenolic compound embraces a wide

range of plant substances which possess in common an aromatic ring

bearing one or more hydroxyl substituents. Phenolic substances tend to be

water-soluble, since they most frequently occur combined with sugar as

glycosides.

Among the natural phenolic compounds, of which over a thousand

structures are known, the flavonoids form the largest group but simple

monocyclic phenols, phenylpropanoids and phenolic Quinone's all exist in

considerable numbers.

The classic procedure for detecting simple phenols is by means of the intense

green, purple, blue or black color many of them give in solution when 1 %

aqueous or alcoholic ferric chloride is added. This procedure, modified by using

a fresh aqueous mixture of 1 % ferric chloride and 1 % potassium ferricyanide, is

still used as a general means of detecting phenolic compounds. Where as these

phenolic compounds which mainly include

1. Phenols and phenolic acids

2. Phenylpropanoids

3. Flavonoid pigments

4. Anthocyanins

5. Flavonols and flavones

6. Minor flavonoids, xanthones and stilbenes

7. Tannins

8. Quinone pigments

Phenols and phenolic acids

Acid hydrolysis of plant tissues releases a number of ether soluble phenolic acids,

some of which are universal in their distribution. These acids are either associated

with lignin combined as ester groups or alcohol-soluble fraction bound as simple

glycosides . phenols are relatively rare in plants. Hydroquinone is probably the most

widely distributed; others such as Gallic acid , catechol, orcinol, phloroglucinol and

pyrogallol, have been reported from only a few sources.

And for detection of phenols and phenolic acid , usually Thin layer chromatography

method follows on silica gel of simple plant phenols. Key: solvent 1, 10% acetic acid

in chloroform; solvent 2, 45% ethyl acetate in benzene .

Exg . phenols and phenolic acids

1. Gallic acid

2. 3,4-dihydroxybenzoic acid

3. Hydroquinone

(FCR) or Folin's phenol reagent or

Folin–Denis reagent,(sodium 1,2-naphthoquinone-4-sulfonate )

1. 2,5-dihydroxybenzoic acid

2. Rhododendrol , orcinol,

3. p-hydroxybenzoic acid

4. Syringic acid

5. Vanillic acid

6. Salicylic acid

(FCR) or Folin's phenol reagent or

Folin–Denis reagent,(sodium 1,2-naphthoquinone-4-sulfonate)

Give

blue

color

Give blue

color after

fuming

with

ammonia

Detection of phenols and phenolic acid -

1. Orcinol Reac

t

vanillin-HCl.

Give pink

color

PHENYLPROPANOIOS

Phenylpropanoids are naturally occurring phenolic compounds which have an

aromatic ring to which a three-carbon side-chain is attached. They are derived

biosynthetically from the aromatic protein amino acid phenylalanine and they

may contain one or more C6-e3 residues.

Thin layer chromatography separation on microcrystalline cellulose of

cinnamic acids. Solvent 1 = benzene-acetic acid-water (6: 7: 3). Solvent 2 = 15%

acetic acid in water.

In phenylpropanoids compounds like

1. Caffeic acid

2. Pcoumaric acid

3. o-coumaric acid

4. Sinapic acid

5. Ferulic acid.

can be detected as different shades of blue under UV light and fuming

with ammonia.

And where these two other

1. Gallic acid

2. Cinnamic acid

can be detected as different shades of blue under UV light and fuming

with ammonia.

Anthocyanins

The anthocyanins are the most important and widespread group of colouring

matters in plants. These intensely coloured water-soluble pigments are

responsible for nearly all the pink, scarlet, red, mauve, violet and blue colours

in the petals, leaves and fruits of higher plants. The anthocyanins are all based

chemically on a single aromatic structure, that of cyanidin, and all are derived

from this pigment by addition or subtraction of hydroxyl groups or by

methylation or by glycosylation . Examples of Anthocyanins

1 2

3

Chemical test for determination of Anthocyanins :

Sodium Hydroxide Test/ Alkaline reagent test :

About 0.2 gm of plant extract was weighed in separate test tube, 1ml of 2N

Sodium hydroxide was added, and heated for 5 minutes at 100 ± 2°C and

observed for the formation of bluish green color which indicates the

presence of anthocyanin.

Other tests for distinguishing anthocyanin

Procedure Anthocyanin response

1. Heat with 2M HCI

for 5min at 100°C

Color stable (can be

extracted into amyl

alcohol)

2. Add 2M NaOH

dropwise

Changes to blue-green

and slowly fades

3. Chromatography in

1% aqueous HCI

Low to intermediate RF

4.Visible spectrum

in Methanol-HCI

Maximum in the range

505-535nm

Test

(extra

ct)

Flavonoids

Flavonoids have the general structure of a 15-carbon skeleton, which consists of

two phenyl rings (A and B) and heterocyclic ring (C). This carbon structure can be

abbreviated C6-C3-C6. According to the IUPAC nomenclature,[1][2] they can be

classified as.

Flavonols

Kaempferol , Quercetin, Myricetin, lsorhamnetin, Azaleatin.

Flavones

Apigenin, Luteolin, Chrysoeriol, Tricin.

Glycosylflavones

Vitexin, Isovitexin, Orientin, Iso-orientin.

Bifiavonyl

Kayaflavone.

Figure 1– General moiety of flavonoid

Chemical test for flavosnoid –

Shinoda test – crude extract was mixed with few fragment of magnesium ribbon

and concentrated HCL was added drop wise. Pink scarlet color appeared after

few minute which indicate presence of flavonoids .

Alkaline reagent test – crude extract was mixed with 2 ml of 2% oh NaOH .

An intense yellow color was formed and which turned colorless on addition of

few Drops of diluted acid .which indicate presence of flavonoids .

Lead acetate test

The extract (50 mg) is dissolved in of distilled water and to this 3 ml of 10%

lead acetate solution is added. A bulky white precipitate indicates the

presence of phenolic compounds /flavonoids

Other test for flavonoids - The ethanol extract (5 ml) was added to a

concentrated sulphuric acid (1 ml) and 0.5g of Mg. A pink or red coloration

that disappear on standing (3 min) indicates the presence of flavonoids.

Tannins

The word tannins was first applied by Seguin –means the substance

present in plant extracts which are able to combine with animal hides,

prevent their purification and convert in to lather.or Tannins are naturally

occurring complex organic compounds possessing nitrogen free

polyphenols of high molecular weight . . They also precipitate proteins and

alkaloids

Classification of tannins

1. Hydrolysable tannins

2. Condensed tannins

3. Pseudo tannins

Chemical tests: for tannins

1. Gelatin test:

To a solution of tannin, aqueous solution of gelatin and sodium chloride are

added. A white buff coloured precipitate is formed.

2. Goldbeater’s skin test: Soak of small piece of goldbeater’s skin in 2% HCL,

rinse with distilled water and place in a solution to be tested for 5 min . Wash with

D.water and transfer to 1% solution of ferrous sulphate .a brown or black color on

the skin denotes the presence of tannins .goldbeater’s skin is a membrane prepared

from the intestine of ox and behave to untanned hide

3. Phenazone test:

A mixture of aqueous extract of a drug and sodium acid phosphate is

heated and cooled and filtered. A solution of phenazone is added to the

filtrate. A bulky coloured precipitate is formed.

of phloroglucinol.

4. Match stick test (Catechin test):

A match stick is dipped in aqueous plant extract, dried near burner

and moistened with concentrated hydrochloric acid. On warming

near flame, the matchstick wood turns pink or red due to formation

of phloroglucinol.

5. Chlorogenic acid test:

An extract of chlorogenic acid containing drug is treated with

aqueous ammonia. A green colour is formed on exposure to air.

6. Vanillin-hydrochloric acid test:

Sample solution and added vanillin- hydrochloric acid reagent

(Vanillin 1 gm, alcohol 10 ml, concentrated hydrochloric acid 10 ml).

A pink or red colour is formed due to formation .

QuinonesQuinones are coloured and contain the same basic chromophore, that of

benzoquinone itself, which consists of two carbonyl groups in conjugation

with two carbon-earbon double bonds. For their identification, quinones can

conveniently be divided into four groups: benzoquinones, naphthaquinones,

anthraquinones and isoprenoid quinones. The first three groups are generally

hydroxylated, with 'phenolic' properties, and may occur in vivo either in

combined form with sugar as glycoside or in a colourless, sometimes

dimeric, quinol form. Some examples quinons are

Hydroxyquinones may appear during paper chromatographic surveys as

yellow or orange pigments which move to the Phenolic compounds

front in butanolic solvents (e.g. BAW) and which are generally immobile

in aqueous solvents.

Characterization quinones were carried out according the reaction

Borntraeger. In a capsule, 2 mL of the plant extract are evaporated to

dryness. The residue was triturated in 5 mL of HCl diluted 1/5 and then

brought the solution to the boiling water bath for 30 min in a test tube.

After cooling under a stream of cold water, the hydrolyzate was

extracted with 20 mL of chloroform in a test tube. The chloroform layer

was then collected in another test tube and then, 0.5 mL of ammonia

diluted twice was added thereto. The appearance of a color ranging

from red to purple characterizes the presence of quinones.

References - J. B. HARBORNE ,Phytochemical Methods “A Guide to Modern

Techniques of Plant Analysis” 3ed Edition 1978 , page No. 33-80

2- W.C EVANS, ”Trace Pharmacognosy” , 15th Edition ,Page No. -214- 235.

3- RNS Yadav* and Munin Agarwala, “Phytochemical Screening and identification of

some compounds” Journal of Phytology 2011, 3(12): 10-14.

4- S.F. Zohra, B.Meriem, S.Samira, “Phytochemical Screening and identification of

some compounds” J. Nat. Prod. Plant Resour., 2012, 2 (4):512-516