Phytochemical Screening of plants

Dr. SINDHU K.

M. V. Sc. SCHOLAR,

DEPT. OF VPT,

COVAS,

Pookode.

• Phytochemicals have two categories:

Primary & Secondary constituents.

• The phytochemical analysis Commercially

value.

• Great interest in pharmaceutical companies for the

production of the new drugs for curing of various

diseases.

Qualitative Quantitative• Steroids,

• Reducing sugars,

• Triterpenoids,

• Sugars,

• Alkaloids,

• Phenolic compounds,

• Flavonoids,

• Saponins,

• Tannins,

• Anthroquinones,

• Amino acids.

• Determination of total

alkaloids,

• Total flavonoids,

• Total phenolics,

• Total saponins,

• Total tannins,

• Total glycosides.

Standard procedures

• Sofowara (1993).

• Trease and Evans (1989).

• Harborne (1973).

Qualitative analysis methods

Detection of alkaloids

• The individual extract is dissolved in dilute hydrochloric acid

and filter.

• The filtrate was further tested with following reagents for the

presence of alkaloids.

• Filtrate was treated with potassium bismuth iodide solution (Dragendroff’s reagent).

• Formation of orange red precipitate indicated the presence of alkaloids.

Dragendroff’s Test:

• Filtrate was treated with saturated aqueous solution of picric acid (Hager’s reagent).

• Presence of alkaloids were confirmed by the formation of yellow coloured precipitate.

Hager’s Test:

• Filtrate was treated with potassium mercuric iodide solution (Mayer’s reagent).

• Formation of a whitish yellow or cream coloured precipitate indicated the presence of alkaloids.

Mayer’s Test:

Detection of carbohydrates

Dissolve 2g extract in 5 ml distilled

water & filter it.

The filtrates were used to test for the presence of

carbohydrates.

Molisch’s Test:

• Filtrate was treated with 2 drops of alcoholic α-naphthol solution in a test tube, shaken

• Add conc. sulphuric acid from the side of the test tube.

• Development of a violet ring @ the junction of two liquid confirmed the presence of carbohydrates

Detection of reducing sugars

Benedict’s test:

• Filtrate was treated with Benedict’s reagent & boil in a thermostatic

• water bath for 5 minutes.

• Formation of an orange red precipitate indicated the presence of reducing sugars.

Fehling’s Test

• Filtrate was acidified with dil. Hydrochloric acid, neutralized with alkali & heated with Fehling’s A & B solutions.

• Formation of red precipitate indicated the presence of reducing sugars.

Detection of saponins

Froth Test:

• Extract was diluted with distilled water to 20 ml & shaken in a graduated test tube for 15 minutes.

• Formation of 1 cm layer of foam indicated the presence of saponins.

Foam Test:

• Small quantity of the extract was shaken with 2 ml of water.

• Persistence of foam produced for ten minutes indicated the presence of saponins.

Detection of phytosterols

Small quantity of extract dissolved

in 5 ml of chloroform

Salkowski’s Test:

On adding a few drops of conc. Sulphuric acid.

Allow the solution to stand

Formation of brown ring indicated the presence of

phytosterols

LibermannBurchard’s test:

The chloroform extracted solution was treated with

few drops of acetic anhydride.

Boil & cool.

Add conc. sulphuric acid.

Formation of a bluish

green colour solution confirmed the presence of

phytosterols.

Detection of phenolic compounds:

• Treat the extract with 3-4 drops of ferric chloride solution.

• Formation of bluish black colour indicated the presence of phenols.

Ferric Chloride Test:

• Treat the extract with 3ml of 10% lead acetate solution.

• A bulky white precipitate indicated the presence of phenolic compounds.

Lead Acetate Test:

Detection of tannins:

Take 0.5 g of the dried powdered

plant

Boil 0.5g sample in 20 ml of

water in a test tube.

Filter the above

mixture

Add few drops of 0.1% ferric

chloride.

Development of a brownish green or a

blue-black

colouration indicated the presence of tannins

Detection of flavonoids:• Treat the extract with few drops of sodium

hydroxide solution.

• Formation of intense yellow colour, which becomes colourless on further addition of dilute acid, indicated the presence of flavonoids.

Alkaline Reagent Test

• Treat the extract with few drops of lead acetate solution.

• Formation of yellow precipitate indicated the presence of flavonoids.

Lead acetate Test:

• Add a few drops of ferric chloride solution to the extract solution.

• Development of intense green colour indicates the presence of flavonoids.

Ferric chloride Test:

Detection of proteins and amino acids:

• Treat the test solution with few drops of Millon’s reagents.

• when warmed , a white precipitate is formed which changes to a brick red or disappears: indicates the presence of proteins & A.A.

Millon’s Test:

• Treat the test solution with few drops of 2% of copper sulphate solution

• Add 1ml of ethanol followed by excess of potassium hydroxide pellets

• formation of pink colour in the extract layer indicates the presence of Pr.

Biuret Test:

• Add Ninhydrin reagent to the test solution & boiled for few minutes.

• Formation of blue colour indicated the presence of amino acids.

Ninhydrin Test:

Detection of terpenoids:

Salkowski test:

Mix 2 ml of chloroform to extract solution

carefully added conc. Sulphuric acid (3 ml) to

form a layer.

A reddish brown colouration of the interface indicated

the presence of terpenoids.

Detection of cardiac glycosides

Keller-Killani test

Add 1ml of conc. sulphuric acid,

Appearance of brown ring @

the interface indicate the deoxysugar characteristic of

cardenolides

Appearance of a violet ring below the brown ring & a

greenish ring in the acetic acid layer confirmed the results.

Treat the extract with 2 ml of glacial acetic acid containing

one drop of ferric chloride solution.

Test for fixed oils and fats:

Spot Test:

• Place small quantity of the extract in between two filter papers.

• Oil stain produced with any extract showed the presence of fixed oils and fats in the extracts.

Saponification test:

• Add few drops of 0.5N alcoholic potassium hydroxide extract with few drops of phenolphthalein solution.

• Heat on a water bath for 1-2 hours.

• Formation of soap indicated the presence of fixed oils and fats in the extracts.

Test for gums and mucilages

Dilute small quantity of the

ethanolic extract with water

Add ruthenium red solution.

A pink colour production showed the

presence of gums and mucilages.

Quantitative determination of phytochemicals

• Total phenols determination: Hagerman A., Muller I., Makkar

H. (2000).

• Total alkaloid determination: Harborne.J. (1973).

• Total flavonoids determination: Kumaran A, Karunakaran R.

(2006).

• Total tannins determination: Van-Burden T, Robinson W.

(1981).

• Total saponins determination: Obdoni B, Ochuko P. (2001).

Determination of total phenolic compounds(Hagerman A,

Muller I, Makkar H, 2000)

• Weigh accurately 100 mg of the extract of the sample &

dissolved in 100 ml of triple distilled water (TDW).

• Transfer 1 ml of this solution to a test tube & add 0.5 ml 2N of

the Folin-Ciocalteu reagent.

• Add 1.5 ml 20% of Na2CO3 solution & make volume up to 8 ml

with TDW followed by vigorous shaking.

• Finally allow to stand for 2 hours.

• Take the absorbance at 765 nm. (Spectroscopic determination).

• Data use: To estimate the total phenolic content using a standard

calibration curve obtained from various diluted concentrations of

gallic acid.

Determination of total alkaloids (Harborne J, 1973)

• Weigh 5 g of the sample & add 5g sample into a 250 ml beaker.

• Add 200 ml of 10% acetic acid in ethanol & cover the beaker with aluminum

foil

• Allow to stand for 4 hour.

• Filter the extract & concentrated on a water bath to one-quarter of the original

volume.

• Add concentrated ammonium hydroxide drop wise to the extract until the

precipitation was complete.

• The whole solution was allowed to settle

• Collect the precipitate & wash with dilute ammonium hydroxide and then filter.

• The residue is the alkaloid, which was dried and weighed

Determination of total flavonoids

(Kumaran A, Karunakaran R. 2006)• The method is based on the formation of the flavonoids - aluminium

complex which has an absorptivity maximum at 415nm.

• Mix 100μl of the plant extracts in methanol (10 mg/ml) with 100 μl of

20 % aluminum trichloride in methanol

• Add a drop of acetic acid, and then diluted with methanol to 5ml.

• After 40 minutes read the absorption @ 415 nm.

• Blank samples were prepared from 100 ml of plant extracts and a

drop of acetic acid, and then diluted to 5ml with methanol.

• The absorption of standard rutin solution (0.5 mg/ml) in methanol was

measured under the same conditions.

• All determinations were carried out in triplicates.

Determination of total tannins

(Van-Burden T, Robinson W. 1981).

• Weigh 500 mg of the sample & transfer to a 50 ml plastic bottle.

• Add 50 ml of distilled water & shaken for 1 hour in a mechanical

shaker.

• Filter the above mixture into a 50 ml volumetric flask & make up to

the mark.

• Pipette out 5 ml of the filtrate into a test tube & mix with 2 ml of 0.1

M FeCl3 in 0.I N HCl & 0.008 M potassium ferrocyanide.

• Measure the absorbance @ 120 nm within 10 min.

Determination of total saponins

(Obdoni B, Ochuko P. 2001)

• Ground the samples & 20 g of each were put into a conical flask

• Add 100 cm3 of 20% aqueous ethanol.

• Heat the samples over a hot water bath for 4 hour with continuous

stirring @ about 55°C.

• Filter the mixture.

• Re-extract the residue with another 200 ml 20% ethanol.

• The combined extracts were reduced to 40 ml over water bath @

about 90°C.

• The concentrate was transferred into a 250 ml separatory funnel &

add 20 ml of diethyl ether; shake vigorously.

• The aqueous layer was recovered while the ether layer was discarded.

• Repeat the purification process.

• Add 60 ml of n-butanol & wash twice with 10 ml of 5% aqueous sodium

chloride.

• Heat the remaining solution in a water bath.

• After evaporation the samples were dried in the oven to a constant weight.

• The saponins content was calculated using standard formulae.

Qualitative Quantitative• steroids,

• reducing sugars,

• triterpenoids,

• sugars,

• alkaloids,

• phenolic compounds,

• flavonoids,

• saponins,

• tannins,

• Anthroquinones,

• amino acids.

• Determination of total

alkaloids,

• Total flavonoids,

• Total phenolics,

• Total saponins,

• Total tannins,

• Total glycosides.

Steps Involved in the Extraction of Medicinal Plants

1. Size reduction2. Extraction3. Filtration4. Concentration5. Drying6. Packing

7. Storage

Size reduction

Different extraction methodsmaceration,infusion, percolation, digestion, decoction, hot continuous extraction (Soxhlet), aqueous-alcoholic extraction by fermentation, counter-current extraction, microwave-assisted extraction, ultrasound extraction (sonication), supercritical fluid extraction, phytonic extraction (with hydrofluorocarbon solvents).

Different methods of drying

I. Natural drying Sun drying Shade drying

II. Artificial drying Tray drying Vacuum dryers Spray dryers

GARBLING / DRESSING

• Process of removal of sand & foreign organic part from same plant.

Eg: removal of excessive stem.removal of excessive stalk in case of cloves.careful removal of rhizomes from roots & rootlets.removal of iron piece using magnet in caster beans.bark removal from gum acacia.

Packing

• Pressed & balded leaf drugs like senna, vinca.• Amber color bottle cod liver oil

(protect from direct sunlight)• Closed container asfoetida (prevent loss of volatile oil)• Kerosine tins colophony, balsam of tolu.• Goat skin aloe preparations.• Packing in big masses colophony (control auto oxidation).• Packing one side the other cinnamon bark /quill (prevent

volatilization of oils).• Simple packing crude drugs from root & seeds.

Storage & preservation of crude drugs

Sterile, Closed containers, Moisture free, air tight.

• Physical & chemical damages• Insect & mould attacks

References

I. HERIN SHEEBA D. GRACELIN A. JOHN DE BRITTO & P. BENJAMIN JEYA RATHNA KUMAR.2013. QUALITATIVE AND QUANTITATIVE ANALYSIS OF PHYTOCHEMICALS IN FIVE PTERIS SPECIES. Int J Pharm Pharm Sci, Vol 5(1): 105-107 .

II. Adarsh Krishna T.P., Ajeesh Krishna T.P., Sanyo Raj V.N., Juliet S., Nair S.N., Ravindran R. and Sujith S. 2013.Evaluation of phytochemical constituents and proximate contents of the ethanolic leaf extract of Tetrastigma leucostaphylum (Dennst.) Alstone (Vitaceae) found in Western Ghats of Kerala, India Res. J. Pharmaceutical Sci. 2(10): 1-6.

III. Prashant Tiwari, Bimlesh Kumar, Mandeep Kaur, Gurpreet Kaur, Harleen Kaur. 2011. Phytochemical screening and Extraction: A Review Internationale Pharmaceutica Sciencia 1(1): 98-106.

IV. Google images.