phytochemicals their synonym, and description of crude drugs of Artemisinib and Diodgenin

Phytochemicals
1) Artemisinin
2) Diosgenin

1. Phyto -chemicals

2. ARTEMISININ ARTEMISININ Synonyms : worm weed, Qninghao, sweet wormwood, sweet sagewort, sweet annie, annual wormwood. Biological Source : It is obtained from the leaves and the closed , unexpanded flower heads of Artemisia annua Linn., Family : Asteraceae / Compositae This particular herb has used in the Chinese system of medicine exclusively for the treatment of malaria since more than one thousand years.

3. Artemisia annua is indigenous to China and grows in Europe & America . It is also cultivated in China, Iran , Turkey and Australia . In India- Gujarat , Jammu and Kashmir, Uttar Pradesh , Himachal Pradesh & Karnataka. Out of seven species of Artemisia, only A. annua contains 0.1% of artemisinin . Artemisinin is a sesquiterpene lactone with an internal peroxide linkage. The other constituents are artemisinic acid and arteannuin B . It contains two derivatives a rtesunate and artemether. A.annua also contains volatile oil of about 0.3% to 0.4% along with Artemisia alcohol. Artemisia ketone, camphor, caryophyllene and myrecene . Geographical Distribution : Chemical Constituents

4. Chemically it is an epoxide 1, 2,4 – trioxane ring Classification : Sesquiterpene lactone Molecular Formula : C₁₅H₂₂O₅ Molecular weight : 282.336 g/mol Melting point : 156°-157°C Appearance : Whitish Crystalline Powder Solubility : Soluble -Organic Solvent Slightly Soluble - Oil. CHEMISTRY

5. Fresh leaves are dried below 60° c. powder is extracted with methanol by maceration for 1 hr. Methanol extract partitioned with n- hexane . Hydro alcoholic extract partitioned with ethyl acetate until the n- hexane layer becomes Colourless. Concentrated at controlled temperature at 40°C under vacuum. Artemisinin obtained as fine white crystals after recrystallization with cyclohexane. INDUSTRIAL PRODUCTION

6. TLC method HPLC method HPTLC method TLC method Mobile phase - n-Hexane : Ethyl acetate - ( 75:25 v/v) Stationary phase - Silica Gel-G ( 60 F 2 54 ) Detecting Agent - Anisaldehyde- H 2 SO 4 reagent - heated at 110°c colour measured at 560-610 nm. Spot volume - 10 µl for test & std. Spot Colour - pink R f Value. - 0.28 Room temperature - 25±2°c Relative humidity - 45±2% Estimation :

7. Equipment Model - Waters 501 Column - C18. Column Sample preparation - 5 g powder + n hexane – reflux – 6 hrs at 60 o C – filter – collect in 250 ml amber colour volumetric flask – evaporate until dryness on water bath at 70 o C residue diluted with mobile phase to get final sol n 1000 µ g / ml . Standard preparation – 10 mg of artemisinin 10 ml Mobile phase. Mobile Phase - Phosphate buffer: methanol - ( 60:40 v/v) . ( PH - 6.7) Injection volume. - 20 µl Detector - UV 260 nm Flow Rate - 1 ml/min R t Value. - 4.68 min. Calibration curve was plotted with diff conc. HPLC Method -

8. Sample : 100 mg artemisinin in 10 ml methanol- extraction for 6 hrs – evaporate extract –residue obtained redissolved in 5 ml methanol . Standard : 10 mg artemisinin in 10 ml methanol – 5 ml transfer in 25 ml volumetric flask- dilute to the mark with methanol to get- 0.20 mg/ ml conc. Mobile phase : Petroleum ether : ethyl Acetate - (75:25) Stationary phase : 60 F-254 silica gel Detecting reagent : Vanillin -sulphuric acid reagent Spot volume : 10 µl of test and standard sample spots Spot colour : Pink- coloured spots of artemisinin Detection : at 560 nm R f Value. : 0.51 Values are plotted in calibration curve (con Vs spot area)Extrapolating the graph to x- axis give concentration of test sample HPTLC method -

9. Antimalarial Cerebral Malaria. In Gastric Infections Suppress Inflammatory Immune Reactions Anticancer UTILISATION

10. Tablets Capsules Injectables Toothpastes and mouthwash bodywash Marketed preparation :

11. DIOSGENIN It is steroidal sapogenin obtained from saponin glycosides commonly known as Diosgenin. Synonyms : yam, rheumatism root Biological Source : Dried tuber of the plant Dioscorea deltoidea , Composita , D.belophylla or other species of Dioscorea . Family : Dioscoreaceae D. deltoidea D.belophylla

12. USA and Mexico, North western Himalaya , Kashmir , Punjab, Nepal and China at an altitude of 1000-3000m 75 % of starch ( non edible because of bitter taste ) steroidal saponin glycosides : Dioscin Dioscin on hydrolysis produce diosgenin : steroidal sapogenin 4% to 6% Other sapogenins are smilagenine & epismilagenine , beta yamogenin Enzyme : sapogenease Chemical constituents : Geographical Distribution :

13. Is a steroidal sapogenin Molecular formula : C₂₇H₄₂O₃ Molecular weight : 414.62 g/mol Melting point : 204°C – 207°C Appearance : Crystalline Solubility : Soluble in organic solvent and acetic acid CHEMISTRY

14. METHOD -1 Fresh tubers are cut into small pieces & crushed fermentation , 4-10 days process Fermentation mixture, Hydrolysed with mineral acid filter Filtrate is Washed & dried Diosgenin Extraction and isolation –

15. Cut the tubes into small pieces , dried & powered Hydrolysis of Dioscin is done by mineral acid to liberate the diosgenin Filter After filtration the insoluble fraction is neutralized and washed Extract with non - polar solvent petroleum ether / toluene Diosgenin METHOD- 2

16. Tubers are washed ,dried & sliced Extracted with hot water or 95 % ethanol for several hours Alcoholic extract Concentrated under vacuum The glycoside is precipitated with solvent ether or by lead acetate Hydrolysis & extraction with petroleum ether (40-60° C) Diosgenin METHOD- 3

17. Dried powder Hydrolyzed with 2.5N H2SO4 by reflux or autoclave. Marc washed with 10% sod. Bicarbonate to neutralize acid. Washed with water to make neutral Hydrolyzed powder extracted with benzene for 6-8 hrs. Benzene extract is filtered, residue dissolve in chloroform & conc. by recrystallization. Crystals of diosgenin . Industrial Production – Acid Hydrolysis

18. TLC method : Sample : Rhizome extract Sample preparation . : 1 mg of dried extract in 50 ml chloroform. Standard preparation : 1 mg std. Diosgenin in 1 ml chloroform. Stationary phase : Absorbent pre-coated silica gel G 60 F 254 Mobile phase : n- hexane or Benzene : Ethyl acetate ( 85 : 15 ) Directing agent : Anisaldehyde + methanol + acetic acid. ( 1:1) Observation : Standard - Reddish Pink Colour Spot. Sample - Greenish Brown Colour Spot . Rf value : 0.47 Estimation – 1. TLC 2. HPTLC . 3. HPLC 4. Colourimetry Method

19. Standard preparation : 100 mcg std . Diosgenin in 1 ml methanol Sample preparation : 50 mg sample in 1 ml methanol . Stationary phase : Slice gel G F 254 Mobile phase. : Toluene: Ethyl acetate: acetic acid: formic acid . ( 4:3:1:1 v/v/v) Directing agent : Anisaldehyde - H 2 SO 4 Detection : at 450 nm R f Value. : 0.83 HPTLC method -

20. Column. : C 18 Column Standard preparation : 100 mcg std . Diosgenin in 100 ml methanol - sonicated for 10 Min. to prepare a stock sol n of 1000 µ g / ml.various aliquots are taken & diluted with methanol at appropriate vol m to produce different conc. Sample preparation : 10 mg sample in 10 ml methanol . - sonicated for 10 min & filtered through 0.2 µ PTFE filter . Mobile phase. : methanol : water (90:10 V/V) Detection. : UV at 203 nm Flow rate. : 0.4 ml/ min Injection volume : 10 µl R t Value. : 6.5 minutes HPLC method -

21. Diosgenin is mixed with a solution of antimony trichloride in a mixture of nitrobenzene and methanol. Heating is necessary to develops color for about 20 minutes at 60°c. Absorbance of both standard and sample is measured at 400- 600 nm against Maximum absorbance is at 486 nm . Colorimetric method:

22. As a precursor for steroidal synthesis like several corticosteroids, sex –hormones & Oral –Contraceptive. In treatment of rheumatic arthritis and natural hormone replacement therapy. Used in semi synthesis of progesterone. Help in reducing serum cholesterol level. UTILISATION

23. Tablets Capsules Cream Ointment powder Marketed preparation :

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