Chemical Tests For Glycosides: General and specific
CHEMICAL TESTS FOR GLYCOSIDES
1. Introduction: Glycosides are natural occurring molecule which carry a sugar group which is bounded by its anomeric carbon to other group by a glycosidic bond.
Glycosidic bond have the property of bind a sugar molecule to another molecule. Basically, a substachnce which carry a glycosidic bond is known as glycoside.In the structure of glycoside, sugar group is known as glycone and the nonsugar group is known as aglycone.
2. Classification:
a. On the basis of glycone:
• Glucoside: If glucose group is present in glycoside, then it is known as glucoside.
• Fructoside : If fructose group is present in glycoside, then it called fructoside.
• Glucuronide: If glucunoric acid is present , then it is known as glucunronide.
b. On the basis of aglycone: on the basis of presence of nature of aglycone group classes are as follows:
• Sterol and triterpenoid glycosides
• Anthraquinone glycosides
• Cyanogenetic and cyanophoric glycosides
• Saponin glycosides
• Flavone glycosides
• Coumarin glycosides
• Steroidal or cardiac glycoside
3. Chemical tests according to classes:
a. Sterol and triterpenoid glycoside:
• General tests :
(1) Antimony trichloride test: An alcoholic extract of drug →evaporate → dry → make extract with chloroform + saturated solution of antimony trichloride in chloroform containing 20% acetic anhydride → appearance of pink colour on heating → presence of steroids and triterpinoids.
(2) Tetranitro methane test : Alcoholic extract of drug + tetranitro methane solution →formation of yellow colour → presence of .sterol and triterpenoid.
• Specific tests for sterol :
(1) Libermann burchard test : Alcoholic extract of drug → evaporated → dry →extracted with CHCl3 + few drops of acetic anhydride + conc.salphuric acid ( from the side wall of test tube) → appearance of violet ring → blue colour → presence of sterol group in drug.
(2) Salkowaski test : Alcoholic extract of drug→evaporated →dry → extracted with CHCl3 + conc. H2SO4( from side wall of test tube) →appearance of yellow colored ring (at the junction of two liquids) → turn to red → presence of sterol group in drug
• Specific test for triterpenes :
Trichloro acetic acid test: Drug + saturated trichloro acetic acid → coloured precipitate → presence of triterpenes .
2. Anthraquinone glycosides :
Specific tests: For c- types of anthraquinone glycosides
(i) Modified brontrager’s test : 1 gm of drug sample + 5ml of dilute HCl + 5 ml of ferric chloride →Boil for 10 min. on waterbath → cool (on room temperature) →filter → extract of filterate with carbon tetrachloride or benzene + equal amount of ammonia solution →appearance of pink to red colour → presence of anthraquinone moiety.
(ii) Borntrager’s test :
1gm of drug sample + 5-10 ml of dilute HCl + 10 min. boil on waterbath and filter + extract of filterate with CCl4 or benzene + equal amount of ammonia solution to filterate + shake → appearance of pink to red colour → indicate presence of anthraquinone moiety.
3. Cyanogenetic and cyanophoric glycoside :
• Specific tests for cyanogenetic glycoside:
(i) Cuprocyanate test : Saturate the filter paper in freshly prepared solution of guaic resin + dissolved in ethanol → dry . → make contact of that filter paper with dilute solution of CuSO4 →place it with contact of drug sample → Generation of HCN gas with the appearance of stain → the presence of cynogenetic glycoside.
(ii) Feeriferrocyanide test: 1 gm of drug sample + 5 ml of alcoholic KOH → transfer it to aqueous solution of FeSO4 and FeCl3 → keep it on room temperature for10 minutes. → transfer whole solution to 20 % HCl →appearance of Prussian blue colour → the presence of cynogenetic glycosides .
(iii) Precipitation of mercury from HgNO3 :Generation of HCN gas by→reduction of aqueous mercurous nitrite solution → metallic Hg → the presence of cyanogenetic glycosides.
• Specific tests for cynophoric glycosides:
(i)Sodium picrate test : small amount of drug sample → humidification with water in a conical flask + few drops of conc. H2SO4 → appearance of brick colour → presence of cynophoric glycoside.
4. Saponin glycosides:
(i) Haemolysis test : A drop of blood on slide + few drops of aq. saponin solution → appearance of ruptured red blood cells →the presence of sapnonin glycoside.
(ii) Foam test : 1 gm of sample drug + 10 to 20 ml of water →well shaked →generation of froths → the presence of saponins.
5. Flavonoid glycosides :
General test:
(i) Shinoda test : alcoholic extract of 1 gm of drug + magnesium turning+ dilute HCl → appearance of red colour → the presence of flavonoids.
• alcoholic extract of 1 gm of drug sample + zinc turning + dil. HCl →appearance of deep red colour → turns to magenta colour → the presence of dihydro flavonoids( other type of flavonoid glycoside).
(ii) Ammonia test : To the alcoholic solution of 1 gm of drug sample , when filter paper dipped and after that exposed to ammonia vapor, appearance of yellow spot on the filter paper indicates the presence of flavonoid.
(iii) Vanilin HCl test :
Alcoholic solution of drug sample + vanillin HCl → appearance of pink colour→ presence of flavonoid
6. Coumarin glycoside :
specific tests:
(i) Fluorescence test :
Alcoholic extract of drug sample + NaOH solution (1N) → generation of blue – green fluorescence → indicates presence of coumarins.
(ii) Ferric chloride test :
Concentrate alcoholic extract of drug sample + few drops of alcoholic FeCl3 solution →appearance of dark green colour → turned to yellow after some time on addition of conc. HNO3 → indicates the presence of coumarins.
7. Cardiac glycosides :
Specific test:
(i)3,5-dinitro benzoic acid test :
Alcoholic solution of drug sample + few drops of NaOH + 2 % solution of 3,5- dinitro benzoic acid →appearance of pink colour → indicates the presence of cardiac glycosides.
General tests :
(i) Legal test :
Alcoholic extract of drug + equal volume of water + 0.5 ml of strong lead acetate solution → well shake →filter → filtrate extracted with same volume of CHCl3→CHCl3 extract evaporate → dryness → remainder dissolve with 2 ml of pyridine and sodium nitropruside + NaOH solution to make it alkaline → appearance of pink colour → indicates the presence of aglycon or glycoside moiety.
(ii) Baljet test :
Section of drug containing cardiac glycoside ( thick section of leaf of digitalis) →dipped into sodium picrate solution → appearance of yellow to orange colour → indicate the presence of aglycon moiety.
(iii)Keller – kiliani test :
Alcoholic extract of drug + equal volume of water + 0.5 ml of strong lead acetate solution → shake → filter→ filtrate extracted with same amount of CHCl3 → CHCl3 extract evaporated to dry → remainder dissolved in 3 ml of glacial acetic acid + few drops of FeCl3 solution + 2ml of conc. H2SO4→ reddish brown layer → turns to bluish green → indicate the presence of aglycon ( digitoxose).
MULTIPLE CHOICE QUESTIONS:
1. General tests for the identification of sterol and triterpenoid glycosides in a drug sample
(a) Borntrager’s test
(b) Antimony trichloride test
(c) Tetranitro methane test
(d) Both b and c
2. Salkowaski test used for the identification of which glycoside moiety in a drug sample ?
(a) Sterol glycoside
(b) Anthraquinone glycoside
(c) Cynophoric glycoside
(d) Flavonoid glycoside
3. Legal test used for the identification of which glycoside:
(a) Anthraquinone glycoside
(b) Coumarine glycoside
(c) Cardiac glycoside
(d) all
4. Vanillin HCl test is used in the identification of :
(a) Cardiac glycoside
(b) Anthraquinone glycoside
(c) Flavonoid glycoside
(d) Cynophoric glycoside
5. Specific test for the identification of cardiac glycoside:
(a) Baljet’s test
(b) Legal test
(c) Keller – kiliani test
(d) 3,5- dinitro benzoic acid test
6. Reagent used in the fluorescence test of coumarin glycoside:
(a) KOH
(b) NaOH
(c) FeCl3
(d) CuSO4
7. Modified borntrager’s test used in the identification of which glycoside?
(a) Cardiac glycoside
(b) Flavonoid glycoside
(c) Saponin glycoside
(d) Anthraquinone glycoside
8. Haemolysis test used in the identification :
(a) Sterol glycoside
(b) Triterpinoid glycoside
(c) Saponin glycoside
(d) All
9. Sodium picrate test used in the identification of which glycoside?
(a) Cynophoric glycoside
(b) Cynogenic glycoside
(c) Saponin glycoside
(d) Anthraquinone glycoside
10. In salkowaski test which colour ring appear at the junction of two liquid mixed?
(a) Yellow
(b) Red
(c) Pink
(d) Both a and b
ANSWERS:
1. Both b and c
2. Steroid glycoside
3. Cardiac glycoside
4. Flavonoid extract
5. 3,5- dinitro benzoic acid test
6. NaOH
7. Anthraquinone glycoside
8. Saponin glycoside
9. Cynophoric glycoside
10. Both a and b
REFERENCES:
(1)Evans W. C, Editors. Trease and Evans Pharmacognosy. New York, Saunders Elsevier; 2009. p.304,347.
(2) Biren shah, A.K. Seth,Textbook of Pharmacognosy & phytochemistry. New Delhi. Elsevier :2010 p. 233-234.