PHYTOCHEMICAL TESTS FOR SECONDARY METABOLITES OF PLANT

 

Phytochemical Tests for Plant Secondary Metabolite

Nature always stands as a golden mark to exemplify the outstanding phenomenon of symbiosis. The biotic & a-biotic elements of nature are all interdependent.

Depending upon the type of natural drug under examination, the test solution may be an aqueous extract or alcoholic extract in specific menstrum like petroleum ether, chloroform, ethyl acetate etc. for the tests to be performed. For testing as far as possible clear, transparent solution should be used.

Test for Phenolic Compound

Phenols probably constitute the largest group of plant secondary metabolite which is wide spread in nature. Compound having aromatic moieties. They range from simple structure with one aromatic ring to highly complex polymeric structure.

        Phenolic compounds are important constitute of some medicinal plant & they are utilizes and most of the medicinal.

       The Phenolic compound are acidic in nature due to presence of (-OH) group in it.

·        Classification:-

1.     Simple phenolic compounds(e.g.-Eugenol)

2.     Tannins (e.g.-Galic acid)

3.     Coumarin(e.g.-Coumarine)

4.     Anthraquinone(e.g-Di-anthrone)

5.     Napthaquinone(e.g-Juglon)

6.     Flavonoide(e.g.-Flavones)

7.     Anthocyanidine(e.g-Anthocyanidins)

8.     Lignin(e.g-Magnolol)

 

  1. Phytochemical test of Simple phenolic compounds (Eugenol):- On addition of a drop of Ferric chloride solution to a drop of volatile oil. It gives blue colour. So Eugenol is present.

 

2. Phytochemical test of Tannins:-

  i)Goldbeater’s skin test: Add 2% HCl acid to a small piece of Goldbeater’s skin, rinse it with distilled water & place in the solution to be tested for 5 mins.Then give wash of distilled water & transfer to a 1% ferrous sulphate solution. A brown or black colour on the skin indicates presence of Tannins.

  ii) Ferric chloride test: Treat the extract with chloride solution, blue colour appears if hydrolysable tannins are present & green colour appears if condensed tannins are present.

 iii) Phenazone test: Add about 0.5gm of sodium acid phosphate to 5ml of aqueous extract. Warm it & filter. To the filtrate add 2% phenazone solution, bulky ppt. is formed. This is often coloured.

iv) Gelatin test: To the test solution add 1% gelatin solution containing 10% sodium chloride.ppt. is formed.

v) Test for Catechin: Dip a matchstick in the test solution, dry it & lastly moisten with conc. HCl acid. Then warm the stick near flame. The colour of the wood changes to pink due to phloroglucinol. (Phloroglucinol is formed when catechins are treated with acids.)

vi) Test for chlorogenic acid:Treat the test solution with aqueous ammonia & expose to air gradually, green colour is developed.

 

3. Phytochemical test of Coumarine:-In ammonical solution coumarine shows a blue to bluish green or violet flurisence, this is used for qualitative test for coumarine

 

5. Phytochemical test of Napthaquinone:-

1. Juglon test: Treat 2ml of chloroform extract & 2ml of ethyl ether with dil. Ammonia solution. Pink colour is formed.

2. Dam-karrer test:To the chloroform plant extract add 10% pot hydroxide solution, blue colour develops.

 

6. Phytochemical test of Flavonoide:-

    i) Shinoda test: To the test solution add few magnesium turnings & conc. HCl acid dropwise; pink scarlet; crimson red or occasionally green to blue colour, appears after few minutes.

 ii) Alkaline reagent test: To the test solution add few drops of solution hydroxide solution, intense yellow colour is formed which turns to colourless on addition of few drops of dil. Acid indicate presence of Flavonoide.

iii) Zinc hydrochloride test: To the test solution add a mixture of zinc dust & conc. HCl acid. It gives red colour after few minutes.

 

7. Phytochemical test of Lignin: -                                                  

i) treat the section of drug with conc. HCl acid & phloroglucinol solution, pink colour is formed.

ii) Treat the section of drug with saffranine solution, pink colour is formed.

     iii) Treat the section of drug with thionine solution; after 15mins wash     with alcohol, bluish violet colour is formed.

 

Phytochemical test of Phenolic compounds:-

1. Ammonia test:- A filter paper dipped in an alcoholic solution of the flavonoid in exposed to the vopour of ammonia solution. Yellow colour appearance-positive test.

2. Vanillin test:- To the alcoholic solution of the flavonoid vanillin hydrochloride is added. Pink or purple colour appear-positive test.

 

PHYTOCHEMICAL TEST FOR GLYCOSIDE

Glycoside is the natural pdt. Secondary metabolite. They are obtaining from plant & animal sources. It is having two parts-

(i)Sugar or Glycone. (ii) Nonsugar or Aglycone.

Classification:-

1.     According to the glycosidic lincage.

2.     According to the chemical nature of aglycone moiety.

3.     According to the biological activity.

 

Phytochemical test for specific Glycoside:-

·        Cardiac Glycoside:-

i)Kedde’s test.

ii)Raymond’s test.

 i)Kedde’s test: Extract the drug with chloroform;evaporate to dryness.add 1drop of 90%alcohol,2drops of 2%3,5-dinitro benzoic acid in 90%alcohol.make alkaline with 20% NAOH solution, purple colour is produced.the colour reaction with 3,5-dinitro benzoic acid depends on the presence of alfa-bita unsaturated lactones in the aglycone.

ii)Raymond’s test: Treat the test solution with hot metabolic alkali, violet  colour is produced.

·        Saponine Glycoside:-

1. Froth formation test.

2. Haemolysis test.

1. Froth formation test: Place 2ml solution of drug in water in a test-tube, shake well; stable froth (foam) is formed.

2. Haemolysis test: Add 0.2ml of solution of saponine to 0.2ml of blood in normal saline & mix well. Centrifuge & note the red supernatant compare with controltube containing 0.2ml of 10% blood in normal saline dil.with 0.2ml of normal saline.

·    Phytochemical test:-

1. Brontrager’s test.

2. Lugal test.

3. Bulget test.

4. Killer-killani test.

1. Brontrager’s test:- Drug is boiled with dil.Sulfuric acid & filter. To the filtrate benzene or chloroform is added & shaken well. The organic layer is separated & to which ammonia is added slowly. The ammoniacal layer shows pink to red colour due to presence of glycoside.

2. Lugal test:- The extract is dissolve in Pyridine & Sodium nitropruside solution & made alkaline. It will give pink or red colour in presence of glycoside.

3. Bulget test:- Extract is treated with Sodium picrate solution. It will give yellow to orrange colour due to presence of glycoside.

4. Killer-killani test:- 1gm of finely powder of Digitalis leafs are treated with 10ml of 70% alcohol & Kept for 2-3mins & filter. To the filtrate & 0.5ml of water & shaken well & filter. To the clear filtrate add equal vol. of chloroform & evaporated to yield the extractive. The extractive is dissolved in Glacial acetic acid & cool. To it 2drops of ferric chloride solution is added & transfer to a test-tube. To the test-tube 2ml of conc. Sulfuric acid is added. A reddish brown layer appearing bluish green colour after standing it observed due to the presence of Glycoside.

 

PHYTOCHEMICAL TEST FOR ALKALOID

Alkaloids are plant & natural pdt. All alkaloids are insoluble in water. But it salt firm are soluble in water. It is having alkaline in nature. Alkaloids have many therapeutic activities. Sometime they are toxic.

·        Phytochemical test:-                                                               

1. Mayer’s reagent.

2. Dragendroffs reagent.

3. Hayer’s reagent.

4. Bognos reagent.

5. Muroxide test.

 1. Mayer’s reagent:- 1.36gm of Mercuric chloride dissolved in 60ml of distilled water(A).5gm of Potassium chloride dissolved in 20ml of distilled water(B). (A) & (B) are mixed & vol. is adjusted to 100ml of distilled water. It gives ppt. in positive response of Alkaloid.

 2. Dragendroffs reagent:- 14gm of Sodium iodide is boiled with    5.2gm of basic bismuth carbonate in 50ml of Glacial acetic acid & stands for 10mins.Then it is stirred & it is allow to stands for over night & filter of the ppt. of Sodium acetate crystal. To 40ml of reddish brown filtrate, 160ml of Ethyl acetate, 1ml of water are added. Then strong solution is preserve in the amboured colour container. It gives orange brown colour & positive response of alkaloid.

3. Hayer’s reagent:- A saturated solution of picric acid & it gives yellow ppt. in positive response of alkaloid.

4. Bognos reagent:-  1.27gm of iodine & 2gm of potassium iodide are dissolved in 5ml of water & made up the vol. 200ml with distilled water. It gives reddish brown ppt. & positive response of alkaloid.

5. Muroxide test:- The sample is mixed with a very small amt. of potassium chlorate & a drop of HCl.Evaporated to dryness & expose in the residue to ammonia vapour. It gives purple colour & positive response of xanthene’s & purines alkaloid.

 

PHYTOCHEMICAL TEST FOR PROTEIN

Proteins are polymers of a-amino acids bonded by peptide linkages. Their molecular weights range from 5000 to many millions. They occur in all living cells. Without proteins life would not be possible.

·        Determining Protein Structures:-

   • X-ray crystallography is one of the primary means of getting     high-resolution protein structures. It is based on Bragg scattering of x-rays (λ = 0.2 – 2 Å) from electron density surrounding the atoms in a protein. Higher electron density leads to more scattering.

·        Phytochemical test:-

1. Xanthoproteic test.

2. Biuret test.

3. Ninhydrin test.

4. Millon’s test.

5. Hopkins-Cole test.

6. Heat test.

7. Test with trichloroacetic acid.

 

1. Xanthoproteic test: - When a protein is warmed with conc. nitric acid, a yellow colour is produced.

2. Biuret test:- A small amt.of NaOH solution is added to the protein. After mixing the two reagents, copper sulphate solution is added. Violet colour is produced.

Protein+ NaOH+Copper sulphate= Violet colour.

3. Ninhydrin test:- When proteins are boiled with a dil. aqueous solution of ninhydrin, a violet colour is produced.

Protein+ Ninhydrin= violet colour.

4. Millon’s test:- The Millon’s reagent consists of mercury dissolved in nitric acid. When Millon’s reagent is added to a protein, a white ppt. is formed.

        Protein+ Millon’s reagent= White ppt.

5. Hopkins-Cole test:- Conc. Sulfuric acid is added down the side of test-tube contain a solution of protein & glyoxalic acid, to form a layer. A violet ring appears between the two layers.

6. Heat test:-Heat the test solution in a boiling water bath, proteins gets coagu lated.

7. Test with trichloroacetic acid:- To the test solution add trichloroacetic acid, ppt. is formed.

 

PHYTOCHEMICAL TEST FOR STERIODS

The steroids are a family of compounds widely distributed in plants & animals. Common to the structure of all compounds of   this class is a tetracyclic framework composed of the phenanthrene nucleus to which is fused at the 1, 2-positions cyclopentene rings.

·        Classification:-

1. Fungus steroids

·        Ergosterols

2. Plant steroids

·        Phytosterols/ Brassinosteroids

 3. Animal steroids

 i) Insect steroids

·        Ecdysteroids

ii) Vertebrate steroids

a) Corticosteroids

·        Glucocorticoids/ Mineralocorticoids

 b) Sex steroids (Sex hormones)

·        Androgens/ Estrogens

 

Phytochemical test:-

1.Libermann-Burchared test

2.Salkowski test

3.Sulfur powder test 

                                                       

1. Libermann-Burchared test: Treat the extract with few drops of acetic anhydride, boil & cool. Then add Conc. Sulfuric acid from the side of the test-tube, brown ring is formed at the junction two layers & upper layer turns green which shows presence of steroids.

2. Salkowski test: Treat the extract with few drops of Conc. Sulfuric acid; red colour at lower layer indicates presence of steroids.

3. Sulfur powder test: Add small amt. of Sulfur powder to the test solution, it sinks at the bottom.

 

PHYTOCHEMICAL TEST FOR TERPENOIDS

Terpenoids are regarded as derivatives of polymers of isoprene.Terpenoids are abundantly available in volatile oils. They occur widely in the leaves & fruits of higher plants as conifers, citrus & eucalyptus. They consist of complex mixture of either the hydrocarbons having general formula (C5H8) or their oxygen derivatives (terpens, alcohol, aldehydes, ketons, acids & esters.).

·        Classification:-

1. Monoterpenes (e.g.-Geranial)

2. Sesquiterpenes (e.g-Zingiberene)

3. Diterpenes (e.g.-Vitamin-A)

4. Triterpenes (e.g.-Ambergris)

5. Tetraterpenes (E.g-Crocin)

 

·        Phytochemical test:

 

1. Libermann-Burchared test:Treat the extract with few drops of acetic anhydride, boil & cool. Then add Conc. Sulfuric acid from the side of the test-tube, brown ring is formed at the junction two layers & lower layer turns deep red colours which show presence of Triterpenes.

2. Salkowski test: Treat the extract with few drops of Conc. Sulfuric acid; formation of yellow colour at lower layer indicates presence of Triterpenes.

3. Sulfur powder test: Add small amt. of Sulfur powder to the test solution, it sinks at the bottom.

 

PHYTOCHEMICAL TEST FOR FIXED OIL

 

These are the reserve food materials of plant & animals. Those, which are liquid at 15.5 degree to 16.5 degree, are called as fixed oil. Fixed oil derives from plant sourses, occur in seeds. They possess the following properties:

1. Fats & oils are thick, viscous, yellow coloured liquids with characteristic odour.

2. They are non-volatile & cannot be distilled.

3. They do have food value & can be saponified.

4. They turn rancid on storage due to free acidity.

5. Fats & oils are esters of glycerol & various straight chained monocarboxylic acids, known as fatty acids.

 

·        Phytochemical test:-

 

  1. Using sodium hydro: Mix 1ml 1% copper sulphate solution & 5 drops of the fixed oil or fat. Then add 5drops of10%sodium hydroxide solution. A clear blue solution is obtained which shows glycerin is present in the sample. The cupric hydroxide formed in the reaction does not ppt. out as it is soluble in glycerin.

2. Using sodium hydrogen sulphate:Take 5drops of the sample in a test-tube & add a pinch of sodium hydrogen sulphate.Pungent odour emanates from the tube indicating glycerin is present in the sample. The pungent odour is due to the formation of acrolein.

 

PHYTOCHEMICAL TEST FOR TANNINS

Tannins are one of the most widely occurring groups of natural subs. in different families of higher plants. These secondary metabolites are present in solution form in the cell sap & also in distinct vacuols.They are used in medicines for allied purposes, mild antiseptics; in treatment of diarrhoea & to check small haemorrhages.They contain the mixture of complex organic substance in which polyphenols are present, generally with o-dihydroxy or o-tryhydroxy groups on a phenyl ring.normaly they have fairly high molecular weight & unlike alkaloids, are devoid of nitrogen. Tannins form colloidal solution with water & are non crystalline subs. In solution, they show acidic reaction due to phenols. They are soluble in alcohol, dil.alkalies, insoluble in organic solvents except acetone.

·  Classification:-

 

1. Hydrolysable tannins: This tannins are hydrolysed by acids or enzyme quickly & the pdt. are hydrolysed by galic acids or Ellagic acid.

2. Condensed tannins: They are also called as non-hydrolysable tannins.

E.g. - Black catechu, Pale catechu.

·        Phytochemical test of Black catechu:

i) Presence of catechin, Black catechu gives pink or red colour with vanillin & HCl acid.

ii) Lime water when added to aqueous extract of Black catechu gives brown colour, which turns to red ppt.on standing for some time.

iii) Green colour is produced when ferric ammonium sulphate is added to dil solution of Black catechu. By the addition of NAOH, the green colour turns to purple.

·        Phytochemical test of Pale catechu:

i) It gives test for catechin by dipping a match stick in HCl acid & warming it near flame similar to Black catechu.

ii) Small quantity of drug is warmed with chloroform & filtered in porcelain dish & evaporated to dryness. Due to presence of chlorophyll, it shows greenish yellow colour.

iii) With a mixture of vanillin & HCl acid, it shows pink or red colour.

·        Phytochemical test:-

 

1. Ferric chloride test: They show colour reaction with iron salts. Ferric chloride gives bluish black or brownish green colour; potassium ferricyanide with ammonia gives deep red colour.

2. Goldbeater’s skin test: Goldbeater’s skin is a prototype of untanned fresh skin of an animal and is obtained as a membrane from the intestine of ox. This membrane is treated with HCl acid, rinsed with distilled water & then placed in tannin solution for 5mins.it is followed by washing with distilled water & putting in ferrous sulphate solution. A brown or black colour is developed on the skin due to tannin.

Tannins are ppt. by 2% solution of phenazone, the tannin solution being prepared with sodium acid phosphate.

3. Phenazone test: Add about 0.5gm of sodium acid phosphate to 5ml of aqueous extract. Warm it & filter. To the filtrate add 2% phenazone solution, bulky ppt. is formed. This is often coloured.

4. Gelatin test: To the test solution add 1% gelatin solution containing 10% sodium chloride.ppt. is formed.

5. Test for catechin: Dip a matchstick in the test solution, dry it & lastly moisten with conc. HCl acid. Then warm the stick near flame. The colour of the wood changes to pink due to phloroglucinol. (Phloroglucinol is formed when catechins are treated with acids.)

6. Test for chlorogenic acid: Treat the test solution with aqueous ammonia & expose to air gradually, green colour is developed.

 

PHYTOCHEMICAL TEST FOR CARBOHYDRATES

Carbohydrates were defined as a group of compounds composed of carbon, hydrogen & oxygen in which the later two elements are in the same proportion as in water. Carbohydrates can be classified into three main groups:-

i) Naturally occurring organic compound.

ii) It can be represented by formula-Cm.

iii) It is polyfucntional compound.

 

Classification:-

1. Monosaccharide (e.g. =a-D-glucose)

2. Oligosaccharide

i) Disaccharide (e.g.-Sucrose=Glucose+Fructose)

ii) Trisaccharide

(E.g-Raffinose= Glucose+Fructose+Galactose)

3. Polysaccharide (e.g.-Starch, Insulin, cellulose)

·        Phytochemical test

 

1. Molisch’s test: To the test solution add few drops of alcoholic a-naphthol, then add few drops of conc. sulfuric acid through sides of test-tube, purple to violet colour ring appears at the junction.

2. Barfoed’s test: 1ml of test solution is heated with 1ml of Barfoed’s reagent on water bath, if red cupric oxide is formed, Monosaccharide is present. Disaccharide are prolong heating about 10mins.may also cause reduction, owing to partial hydrolysis to Monosaccharide.

3. Salivanoff’s test (test for ketones): To the test solution add crystal of resorcinol & equal vol. of conc. HCl acid & heat on a water bath. rose colour is produced. (E.g-Fructose, honey.)           

4. Test for pentose: To the test solution add equal vol. of. HCl acid containing a small amt. of phloroglucinol & heat, red colour is produced.

5. Osazone formation test: Heat the test solution with solution of phenylhydrazine hydrochloride, sodium acetate & acetic acid. Examine the yellow crystals formed under microscope. These crystals are of characteristic shape for particular sugars.