Qualitative tests for phytochemicals Carbohydrates Molish test: All carbohydrates monosaccharides, disaccharides, and polysaccharides should give

ve a positive reaction, and nucleic acids and glycoproteins also give a positive reaction, as all these compounds are eventually hydrolyzed to monosaccharides by strong mineral acids. Pentoses are then dehydrated to furfural, while hexoses are dehydrated to 5-hydroxymethylfurfural. Either of these aldehydes, if present, will condense with two molecules of naphthol to form a purple-colored product, as illustrated below by the example of glucose:

Proteins Biuret test:

The Biuret reagent is made of potassium hydroxide (KOH) and hydrated copper(II) sulfate, together with potassium sodium tartrate.
[4] [5]

Potassium sodium tartrate

2+ +

is added to complex to stabilize the cupric ions. Proteins in the

alkaline environment reduce Cu color change

to Cu , which forms a coordination complex with proteins, leading to a blue to pink

Amino acids: Ninhydrin test Ninhydrin (2,2-Dihydroxyindane-1,3-dione) is a chemical used to detect ammonia or primary and secondary amines. When reacting with these free amines, a deep blue or purple color known as Ruhemann's purple is produced. Ninhydrin is most commonly used to detectfingerprints, as the terminal amines of lysine residues in peptides and proteins sloughed off in fingerprints react with ninhydrin.[2] It is a white solid which is soluble in ethanol and acetone at room temperature.[3] Ninhydrin can be considered as the hydrate of indane-1,2,3-trione.
The carbon atom of a carbonyl bears a partial positive charge enhanced by neighboring electron withdrawing groups like carbonyl itself. So the central carbon of a 1,2,3-tricarbonyl compound is much more electrophilic than one in a simple ketone. Thus indane-1,2,3-trione reacts readily with nucleophiles, including water. Whereas for most carbonyl compounds, a carbonyl form is more stable than a product of water addition (hydrate), ninhydrin forms a stable hydrate of the central carbon because of the destabilizing effect of the adjacent carbonyl groups. Note that to generate the ninhydrin chromophore, the amine is condensed with a molecule of ninhydrin to give a Schiff base. Thus only ammonia and primary amines can proceed past this step. At this step, there must also be an alpha proton (H* in the diagram) for Schiff base transfer, so an amine adjacent to a tertiary carbon cannot be detected by the ninhydrin test. The reaction of ninhydrin with secondary amines gives an iminium salt, which is also coloured, and this is generally yelloworange in color.

Alkaloids Dragendorffs reagent A solution of potassium bismuth iodide. It is used to determine the presence of alkaloids.

Mayer's reagent. (white precipitate with most alkaloids in slightly acid solutions). Dissolve 1.358g of HgCl2 in 60ml of water and pour into a solution of 5g of KI in 10ml of H2O Add sufficient water to make 100 ml. Iodo-potasium iodide (Wagner's reagent for alkaloids). Dissolve 2g of iodine and 6g of KI in 100ml of water. Hagers reagent :Picric acid (Hagers reagent for alkaloids, wool and silk). Dissolve 1 g of picric acid in 100 mL of wat Terpenoids: Detection of terpenoids on thin-layer chromatography (TLC) using anisaldehyde sulfuric acid reagent
Preliminary chemical examination of TRFPE showed an intense pink colour with the Liebermann Burchard test, indicating rich terpenoid content in the plant, which was then detected on a TLC plate by treatment with anisaldehyde sulfuric acid reagent (Fig. 1 ).

The Liebermann-Burchard reaction is a chemical reaction mainly used as the litmus test cholesterol and more generally phenols . It was originally described by Carl Liebermann 1 then applied to the revelation of cholesterol Burchard 2 , 3 . Reagents: solution of the substance to be identified in the chloroform + sulfuric acid + concentrated acetic anhydride

A few drops of acetic anhydride was added to the solution and then a drop of sulfuric acid is introduced. The solution turns deep red, then purple, blue and finally takes a dark green color. This color changing rapidly with time which makes it even more difficult to define specific staining.

Phenolics and flavonoids:

Shinodas test: To 2ml ethanol extract of different plant parts, 0.5ml concentrated hydrochloric acid and few pellets of magnesium turning were added in a test tube. Pink-tomato red color indicates the presence of flavanoids. To 2ml ethanol extract of different plant parts, 1ml of 1% potassium hydroxide solution was added. Dark yellow color indicates the presence of flavanoids. To 2 ml ethanol extract of different plant parts, 1 ml of 1% aluminum chloride in methanol was added in a test tube. Yellow color indicates presence of flavanols, flavanones and/or chalcones. To 2ml ethanol filtrates for different plant parts, 0.5ml concentrated Hydrochloric acid and few drops from amyl alcohol were added in a test tube and shaken. Red color indicates presence of flavanoidal glycosides.

GLYCOSIDES Glycosides are defined as the organic compounds obtained from plant or animal source which on enzymatic or acid hydrolysis gives one or more sugar moieties along with non-sugar moiety. The sugar moiety is called as glycone & non-sugar moiety called as agycone or genin. Chemically these are acetals or sugar ethers formed by interaction of hydroxyl group each of non-sugar & sugar moiety, with a loss of water molecule. The sugars involved in glycosides are beta-D

glucose,galactose,mannose,rhamnose.digitoxose, cymarose etc. The linkage b\w glycone & aglycone is called glycosidic linkage &on the basis of this linkage alpha & beta stereoisomers are assigned. Classification of glycosides: 1). According to their therapeutic activity : a).cardiotonic: digitalis , thevetia, squill, quabain, strophanthus. b). purgative: senna,aloe, rhubarb,cascara. c).galactogogue: shatavari. d).antidiabetic: gymnena. e).antifungal: henna. f).anthelmentic: quassia. g).diuretic: bearberry, gokhru. h).counter irritant & rubefacient: blackmustard, mylabris, cantharides. 2). Based on type of sugar or glycone: glucosides with glucose. rhamnosides with rhamnose. pentosides with pentose like ribose. digitoxose, cymarose (deoxysugars).

3).Based on type of linkage existing b\w glycone & aglycone part: basically all types of glycosidal likages are occurred by interaction of OH group of glycone & hydrogrn coming through any of the radicals like CH,OH,SH &NH present on aglycone part.hence by elimination of one water molecule ,linkage or bridge formed. a). C-glycosides: (aloin type glycosides) glycone-OH + HC-aglycone glycone-C-aglycone +H2O. ex: anthraquninone glycosides like cascarosides from cascara,aloin from aloe. c-glycosides present in liliaceae family ex: aloe. These are not hydrolysed by heating with dilute acids\ alkalies, but by oxidative hydrolysis with ferric chloride. b).O-glycosides: ex:senna,rhubarb. These are hydrolysed by treatment with acids or alkali in to agycone &sugar. Glycone-OH +HO-aglycone glycone-O-aglycone +H2O. c).S-glycosides: ex: Isothiocyanate glycosides like sinigrin from black mustard. These are formed by interaction of sulfhydryl group of agycone & OH group of glycone. Glycone-OH + HS-aglycone glycone-S-aglycone +H2O. d).N-glycosides : ex:nucleosides. Here the amino group of base reacts with OH group of ribose\ deoxyribose.

Glycone-OH+ HN-aglycone glycone N-aglycone+H20. 4).According to chemical nature of aglycone moiety: a).Anthraquinone glycosides : ex: senna.aloe,rhubarb,cascara,cochineal. b). Sterol or cardiac glycosides : ex: digitalis, thevetia,squill,strophanthus,quabain. c). Saponin glycosides: ex:dioscorea,licorice,shatavari,bramhi,ginseng,gokhru. d). Cyanogenetic or cyanophoric glycosides: ex: bitter almond,wildcherry bark. e).Isothiocyanate glycosides: ex: blackmustard. f).Flavonol glycosides: ex: citrus fruits,gingko. g).Coumarin & furanocoumarin glycosides: ex:ammi,mylabris,tonkabean. h).Aldehyde glycosides: ex: vanilla pods. i). phenol glycosides: ex:bearberry. j).Steroidal glyco-alkaloids: ex: solanum. k).Glycosidal bitters &miscellaneous: ex: gentian, chirata,quassia(bitter wood),kalmeg, gymnema,henna.

Anthraquinone
O 8 7 6 5 O 9 10 4 1 2 4H 3 7 6 5 8

Anthrone
O 1 9 10 H H 4 2 3

Anthranol
OH

H O 8 7 6 5 H 9 10 OH 4 1 2 3

2H

Oxanthrone
Properties of glycosides: Sugar moiety of glycosides facilitates absorption of glycosides & helps in transportation of aglycone portion

at the site of action. Physical,chmical,therapeutic properties are depend on the agycone part. Glycosides are crystalline or amorphous substances which are soluble in water & dilute alcohol with an exception of resin glycosides, but insoluble in organic solvent like chloroform or ether. The aglcone moiety is soluble in non-polar solvents like benzene or ether. Glycosides are easily hydrolysed by water,mineral acids, enzymes. These shows optical activity with laevorotatory effects. These are participate in growth regulation & protection of plant. 1).anthracene glycosides: These considt of different agycone moieties like anthraquinone,anthranol,anthrone,dianthranol,oxanthrone &dianthrone. These are identified by following tests Borntragars test: O-glycosides ( senna) are detected by this test.the drug boiled with dil. H2SO4 ,filtered & to the filtrate benzene or ether or chloroform is added &shaken well. The organic layer is separated to which ammonia is added slowly. The ammoniacal layer shows pink or red color due the presence of anthraquinone glycosides, Modified Borntragars test: indicates the presence of C-glycosides (aloe emodin). The aqueous solution of aloes is treated with ferric chloride solution & dil.Hcl to bring out the oxidative hydrolysis of aloe emodin . the hydrolysis sets free anthraquinone which are collected in organic solvent like CCL4 or ether. The organic layer separated & shaken with dil.ammonia. the ammonical layer shows rose- pink to cherry red color. Indicting the presence of C-glycosides \ aloe emodin. 2).Cardiac glycosides: Aglycone part of cardiac glycosides is a steroidal moiety. Five membered lactone ring called as cardenolide (C23). six membered lactone ring called as bufadienolide (C24). The lactone of cardenolide contain only one double bond & attach to steroidal nucleus through C -17 position. The lactone of bufadienolide contain only two double bond & attach to steroidal nucleus through 17 beta position. Sugar part attach through C-3 ,beta linkage.

Keller-kiliani test: Boiling about 1g of digitalis powder wuth 10 ml 70% alcohol for 2-3 min. filter. To the filtratr 5ml of water & 0.5 ml strong solution of lead acetate ,shakewell & separate filterate. Clear filtrate is treat with equal volume of chloroform & evaporated to yield the extractives.

The extractive dissolved in glacial aceticacid & after cooling 2 drops ferric chlorid e solution are added to it, these contents transferred to a test tube containing the 2ml of con.H2SO4. reddish brown layer change to bluish green color after standing due to presence of digitoxose. Legal test & baljet test also performed. Use: treatment of congestive heart failure.

3).Saponin glycosides: aglycone part of these glycosides has soap like action & produce haemolysis of RBC. Saponin have bitter & acrid taste, cause irritation of mucous membrane. Aglycone part called as sapogenin. Depending on aglycones a). steroidal( tetracyclic triterpenoid saponin).

b).pentacyclic triterpenoid saponins. Uses: licorice-expectorant, Gokhru- diuretic, Bramhi-nervine tonic. Tests: foam test & haemolysis test. 4).Isothiocyanate glycosides: (glucosinolate compounds) aglycone part of this glycosides contain isothiocyanate (-NCS) as it contain sulpher & nitrogrn. Uses: black mustard: counter irritant, rubefacient externally, Emetic as internally. 5).flavonol glycosides: aglycone part contain flavone, flavonol etc. uses: silymarin- liver disorders citrus fruits- vit-c.

6). Cyanogenetic glycosides: The name called because presence of hydrocyanic acid in the aglycone moiety. These are derivatives of nitrile of mandelic acid. These in hydrolysis yields hydrocyanic acid & benzaldehyde. Uses: bitter almond- sedative, demulcent. Wildcherry bark- expectorant , flavouring agent. 7). Glycosidal bitters: uses: gentian- bitter stomachic, tonic. Chirata- antipyretic, stomachic. Quassia- bitter stomachic, anthelmentic. Gymnema- antidiabetic. 8). Steroidal glycoalkaloids: uses: solanum- for steroidal synthesis. 9). Aldehyde glycosides: uses: vanilla pods- flavouring agent.

10). Phenol glycosides: uses: bearberry- diuretic.

11). Coumarin glycosides : uses: mylabris- counter irritant ,rubefacient.

Reference: 1). Pharmacognosy by C.K.Kokate p.no. 167-254. 2). Pharmacognosy by Trease & Evans, p.no.214-327.