Podophyllotoxin

Isolation Podophyllotoxin Method I

1. The powdered rhizome or roots of P. emodi are extracted with methanol.

2. The extract obtained is reduced under vacuum.

3. The semi-solid mass obtained is put into acid (10ml HCl in 100ml water).

4. The precipitate formed is allowed to settle and filtered.

5. The filtrate is decanted and washed with cold water.

6. The resin obtained is dried, which gives a dark brown amorphous powder of podophyllin.

7. This powder is extracted with chloroform and purified by repeated recrystallisation from benzene (either alone or a mixture of alcohol and benzene).

8. The resultant is washed with petroleum ether or hexane to yield podophyllotoxin.

Isolation Podophyllotoxin Method II

1. The chloroform-soluble fraction is dissolved in alcohol.

2. The resultant is refluxed with neutral aluminium oxide.

3. This makes the solution light yellow in colour.

4. To this alcoholic solution, benzene is added to obtain podophyllotoxin with a yield of 95-98%.

Isolation Podophyllotoxin Method III

1. The roots or rhizomes of P. emodi are extracted over a bed of neutral alumina with solvents like benzene, toluene, xylene, etc., for 1.5-4 hours.

2. The resultant is re-crystallised from organic solvents such as hot benzene, toluene, and xylene to yield pure podophyllotoxin (95-97%).

3. Podophyllotoxin is a tetrahydronaphthalene derivative having hydroxy and lactone groups.

4. The attachment at cis-position is responsible for its purgative property and the attachment at trans-position is responsible for its anti-cancer property

Isolation Podophyllotoxin Method IV

1. Powder the root and rhizome and extract with methanol or ethanol and concentrate the extract under vacuum.

2. Treat the semisolid mass with acid (10 ml HCl in 1000 ml water) and cool to 10°C slowly.

3. Decanted the settled precipitate and wash it with cold water.

4. On drying this precipitate we get amorphous dark brown powder known as podophyllin.

5. Extract podophyllin with chloroform and purify by recrystallisation with benzene or benzene and ethyl alcohol mixture and then wash with hexane/ petroleum ether which gives the commercial podophyllotoxin.

Podophyllotoxin Identification Test:

1. 0.5gm of the drug is macerated with 10ml of alcohol and filtered.

2. The filtrate is added with 0.5ml of strong copper acetate solution.

3. The Formation of a brown precipitate confirms the presence of podophyllotoxin.

4. Podophyllotoxin shows violet-blue colour when treated with 50 % sulphuric acid solution

Thin layer chromatography of Podophyllotoxin:

• Dissolve podophyllotoxin in methanol and spot the solution on Silica gel G TLC plates.

• Elute the TLC plates in a solvent system containing toluene: ethyl acetate (5:7).

• Sulphuric acid should be used as a detecting reagent.

• Compare with the standard by scanning 280nm densitometric scan.

• Violet colour spot will be seen with approx Rf value 0.39.