Isolation and detection of active principles, diosgenin from dioscorea

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BACKGROUND

Diosgenin is obtained from dried tubers of Dioscorea deltoidea (Family- Diascoreaceae). it is mainly found growing in North Western Himalayas from Kashmir and Punjab to Nepal and China. Also found in U.A.S. and Mexico. Synonym is Yam, Rheumatism root. Generally it is a climber containing alternate root. Chemically it contains 75% of starch including diosgenin, sapogenase an enzyme. Itis used for the treatment of rheumatic arthritis and also used as oral contraceptives.1

Aim: Aim of the experiment is to isolate and detect the active constituent, diosgenin present in dioscoria.

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REQUIREMENTS

Apparatus:     Test tube,

Extraction unit,

Refluxtion unit,

Measuring cylinder,

Filter paper,

Funnel,

TLC plate,

Mesh

Chemicals:     H2SO4,

HCl,

Alcohol,

Ethanol,

Hydrocarbon solvent,

Acetone,

Heptanes,

Toluene,

Ethyl acetate,

Silica,

Anisaldehyde.

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PROCEDURE

Isolation of diosgenin

  • Alcoholic extraction method:

The tubers are cut in to small pieces and dried under sun light. Then the dried tuber is subjected to make a powder form and extracted with ethyl alcohol or methyl alcohol two times for six-eight hours. This solution is filtered. The filtrate is concentrated to make a syrupy liquid form and hydrolysed using HCl and H2SO4 for approximately 2-12 hours. About 85% of the crude diosgenin is precipitated. Then precipitates are subjected to filtration following water washing and purified by alcohol.2

  • Acid hydrolysis method:

The powder form of sample was meshed to get 100-200 meshes following refluxtion and heated with 2-4 N mineral acid in autoclave for 2-6 hours. The solution was filtered and the obtained hydrolate is washed with water till it become neutral. By using hydrocarbon solvent, again it is dried and extracted for six hours. The solution is concentrated in to about 25 ml. In refrigerator, it is kept for some time (1 hour). The crystals of diosgenin are filtered out and then it is washed with acetone.2

  • Fermentation cum acid hydrolysis method:

Fresh green leaves is collected and smashed by using a hammer mill. In the fermentation bin, the mesh is placed and allowed for fermentation for 2 days. To reduce the moisture content up to 7-8%, the fermented mesh should be dried. At reduced temperature, by using mineral acid it is subjected to hydrolysis. Finally by using heptanes, the resulting solution is extracted to obtain diosgenin.2

  • Incubation cum acid hydrolysis method:

At 37 degree centigrade, the fresh sample plant material is incubated in water for few days. Acid hydrolysis of the incubated sample was carried out and the obtained hrdrolysed liquid is concentrated. Finally to obtain diosgenin, the solution is extracted by using hydrocarbon solvent. (M.P.=204-207 ºc).2

Detection of diosgenin

TLC method

The obtained purified sample is dissolved in methyl alcohol is spotted in the plate of silica gel and developed in toluene: ethyl acetate (7:3). When the dried plates of silica gel is sprayed with anisaldehyde and sulphuric acid reagent, a dark green spot of diosgenin will appear (Rf=0.34-0.37).2

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CONCLUSION

The active constituent diosgenin was isolated and detected from the root of dioscorea. It is used as cancer chemopreventive, natural alternative to estrogens, rheumatoid arthritis, menstrual disorders.

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REFERENCES

  1. Kokate CK, Purohit AP, Gokhale SB. Druds containing alkaloid (belladonna), Text Book of Pharmacognosy, 51 edition, Nirali Prakashan, pune. 2015: 9-48.
  2. Shah B, Seth AK. Isolation of phytopharmaceuticals, Text book of Pharmacognosy and Phytochemistry, 2nd edition. CBS Publishers & Distributors pvt ltd. 2014: 451.