1. THE IDENTIFICATION OF THE CARDIO ACTIVE GLYCOSIDES
Pharmacognosy
3rd Class, 1st Semester
THE IDENTIFICATION OF THE CARDIO ACTIVE GLYCOSIDES
Lab.4

2. General CHEMICAL TESTS
BALJETS TEST
Aim
The identification of the cardio active glycosides in general.
Equipment and reagents
Test tube
Picric acid
Sodium hydroxide solution
Procedure
Take one ml of fraction A.
Add two drops of picric acid.
Make it alkaline with sodium hydroxide solution.
Results
Turbid, yellow to orange in color.
Discussion
The use of picric acid is to hydrolyze the glycoside to glycone and aglycone parts.
The use of sodium hydroxide is to neutralize the acidity in the solution.
The color is product due to the aglycone portion (genin).

3. KELLER-KILLIANS TEST Aim
The identification of the cardio active glycosides in general.
Equipment and reagent
Test tube
Glacial acetic acid
0.1%of ferric chloride solution
Concentrated H2SO4
Procedure
Take two ml of fraction A and add 3ml of glacial acetic acid.
Add two drops of 0.1%of ferric chloride solution.
Take 1ml of concentrated H2SO4 and add it to the above mixture in drops so as to make two layers.
Results
Two layers are formed:
The upper one has light bright green color.
The lower layer has transparent clear color (H­2SO­4 Layer) the junction appears as a reddish-brown ring

4. Discussion
The color of the acetic acid layer is due to the genin part of the glycoside.
The use of glacial acetic acid is to hydrolyze the glycoside into glycone and aglycone parts.
The addition of 0.1 % ferric chloride is an oxidizing agent needed in the reaction.
The H2SO4 has a high density therefore will be in the lower layer causing charring of the sugar and hence giving the reddish-brown color.

5. OTHER CHEMICAL TEST FOR THE IDENTIFICATION OF THE STEROL GLYCOSIDES
Sterol glycosides (SGs) occur naturally in vegetable oils and fats.
RAYMMONDS REACTION
Aim
To identify the sterol nucleus
Equipment and reagents
Test tube
10%sodium hydroxide solution
1% m-dinitrobenzene
Procedure
To an alcoholic solution of glycoside add 1-2 drops of 10%sodium hydroxide.
Add few drops of an alcoholic solution of 1% m- dinitrobenzene .
Note the color.
Results
Pink color appears

6. 2)KEDDS REACTION Violet color Aim To identify the sterol nucleus
Equipment and reagents
Test tube
1%3, 5-dinitrobenzene acid
0.5 N aqueous methanolic KOH (50%)
Procedure
To a solution of glycoside add a solution of 1%3, 5-dinitrobenzene acid in 0.5 N aqueous methanolic KOH (50%).
Report the color.
Results
Violet color

7. 3)LIEBERMANS STEROL REACTION
Aim
To identify the sterol nucleus
Equipment and reagents
Test tube
Porcelain dish
Anhydrous acetic acid
Concentrated H2SO4
Procedure
Take 5ml of the alcoholic extract in a test tube.
Add 5ml of the anhydrous acetic acid and shake well.
Take 4 drops of the above mixture and place in a porcelain dish.
Add 0ne drop of concentrated H2SO4.
Results
Achange of color from rose, through red , violet, and blue to green. The color is slightly different from compound to compound.
Discussion
This reaction is due to the steroidal part of the molecule and it is characteristic of the aglycone of the scillarenin type (unsaturated steroidal part)

8. Cardenolide in pyridine + Na nitroprusside + NaOH
LEGALS REACTION
The glycoside or the purified extract of the crude drug is dissolved in pyridine. When sodium hydroxide and sodium nitroprusside are added alternatively, a transient blood red color develops. This is a test for the unsaturated lactone ring of the genin.
Cardenolide in pyridine + Na nitroprusside + NaOH
Deep red colour

9. THE IDENTIFICATION OF CARDIO ACTIVE GLYCOSIDE BY CHROMATOGRAPHY
By the use of thin layer chromatography (T.L.C)
The stationary phase=silica gel G
The mobile phase
Chloroform: Ethanol: Water (7:3:1)
Or Ethyl acetate: Methanol: Water (75:10:5)
The standard compound =Oleanderin, Digoxin
The spray reagent=Lieberman's reagent.
Other mobile phase as
Butanone: Xylene: Formamide (50:5:4)
Chloroform: Tetrahydrofuran: Formamide(50:50:6)

10. Procedure
Prepare 100ml of the mobile phase; place it in the glass tank.
Cover the tank with glass lid and allow standing for 45 minutes before use.
Apply the sample spots (fraction A and fraction B) and the standard spot, on the silica gel plates, on the base line.
Put the silica gel plate in the glass tank and allow the mobile phase to raise to 90% of the plate.
Remove the plate from the tank.
Allow drying and then detecting the spots by use of the spray reagent and heat the plate at c for 5-10 minutes in the oven.
Note the spots, and calculate the RF value for each spot.
Note:-
The RF value= distance moved by the sample /distance moved by the solvent.
The RF value should be less than 1 because if the RF value=1 this means that there is no separation, and the sample moved with the solvent.

11. Thank you