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

EXTRACTION Equipment and reagents Aim: - to isolate the cardio active glycoside Equipment and reagents Large beaker &two medium size beakers Two conical flask Sintered glass (Sintered glass is a glass mesh used for filtration. It can be used instead of filter paper). Centrifuge &centrifuge tube Separatory funnel Water bath or rotary evaporator 70% ethanol Lead sub acetate (Lead sub acetate is added to precipitate tannins and other unwanted material) 10% sodium phosphate solution (10%sodium phosphate is added to take the excess of lead sub acetate)

Cont… Chloroform-ethanol (3:1 v/v) (Use of chloroform-ethanol in partition is due to the fact that the chloroform will take the genin part while the ethanol will take the glycoside there will be no loss in the glycoside) Anhydrous sodium sulphate (Anhydrous sodium sulphate is added in during mixture since the anhydrous form will act as an adsorbent) 4 N HCL (4N HCL is used to hydrolyze the glycoside to glycone and aglycone parts) Chloroform (Use of chloroform alone is to extract the genin part So fraction A, will contain the whole glycoside, while fraction B will contain only the genin part)

Procedure Maceration 10 gm of the powdered leaf in 100ml of the 70%ethanol for 24hr Filtrate the macerate through a sintered glass to yield a clear filtrate Place 60 ml of the alcoholic extract in conical flask Add 25 ml of 0.1% of lead sub acetate Mix thoroughly and standing for two minutes

Placing the mixture in the centrifuge tubes and centrifuging for several minutes Decant and take the supernatant into a flask and add 35 ml of 10% sodium phosphate solution Mix thoroughly and centrifuge then divide the mixture you had into two divisions FRACTION A FRACTION B

FRACTION A Take one of the two divisions and put in a separatory funnel Shake it with three quantities each of 50 ml of chloroform –ethanol (3:1 v/v) Dry by the addition of a small quantity of anhydrous sodium sulphate and allow standing for a few minutes Decant the chloroform-ethanol extract and reduce the volume to about 1ml on rotary evaporator or (water bath) Results Fraction A contains the whole glycoside

FRACTION B Place the other division of the extract in a conical flask, and then add 6ml of 4N HCL Place in a boiling water bath for 20 minutes cool the hydrolysate and transfer to a separatory funnel (Boiling is to accelerate the process of hydrolysis). Partition with three quantities each of 25ml of chloroform Dry with a small quantity of anhydrous sodium sulphate Decant the chloroform and reduce the volume to about 1ml on a rotary evaporator (or water bath) Results Fraction B:- contains the genin part only

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