Isolation Extraction Estimation of Artemisinin

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1. artemisinin Afsath.b M Pharm 1 st year Pharmacognosy & phytochemistry MALIK DEENAR COLLEGE OF PHARMACY

2. PLANT PROFILE SYNONYM : Sweet worm wood,sweet annie , sweet sagewort , annual mugwort or annual wormwood BIOLOGICAL SOURCE : Leaves and the closed, unexpanded flower heads of Artemisia annua FAMILY : Asteraceae / Compositae ACTIVE CONSTITUENT : Artemisinin , dihydro artemisisnin , artemisin , artemisic acid USES : Effective against malaria & cerebral malaria Hepatitis B Schistosomiasis (caused by schistosomes ) Several blood parasitic protozoans Against a variety of cancer cell lines including breast cancer Human leukemia Colon Small-cell lung carcinomas Drug-resistant cancers

3. Artemisinin semi-synthetic derivatives are a group of drugs used against plasmodium species ( P. Falciparum , P. Malariae , P. Ovale and P. Vivax .) Chemically, artemisinin is a sesquiterpene lactone containing an unusual peroxide bridge. REQUIREMENTS Raw material Solvents ( methanol, n-hexane, n-heptane, benzene, toluene, chloroform, methylene dichloride, ethyl acetate, acetone, acetonitrile …) Magnetic stirrer … Extraction can be done by 2 ways;- Soxhlet extraction Microwave-assisted extraction

4. The raw material used is plant artemisia annua L. The plant was dried and mashed into powder. The chemicals used are methanol, hexane, and ethyl acetate. EXTRACTION 100 grams of powder herb artemisia annua Macerated using methanol solvent (Erlenmeyer with a magnetic stirrer speed of 700 rpm for 1 hour) This process is done many times until the methanol layer is colorless The extract then was evaporated (using a rotavapor vacuum at a temperature of 40 ° C) U ntil the extract volume to 100 ml.

5. PARTITION OF EXTRACT Partition the extract using 50 ml hexane Partitioning done many times until hexane layer become colorless Two layers got from this process 1. hexane extracts (non-polar fraction) 2. methanol extracts the methanol extract obtained was added 10 ml of distilled water partitioned again using 50 ml of ethyl acetate Partitioning done many times until the ethyl acetate layer is colourless Again we get 2 layers 1.ethyl acetate extract ( semipolar fraction) 2.methanol-water extract (polar fraction ) Each extract was concentrated using a rotavapor at a temperature of 40˚c .

6. SOXHLET EXTRACTION AND TEST SAMPLE PREPARATION 100  mg of fine powder was placed into an extraction thimble extracted with 170 ml of solvent via hot soxhlet extraction method for 6 hours over a water bath The extract was evaporated and redissolved in 5 ml methanol 10   μl of these test solutions was used for quantification purpose .

7. MICROWAVE-ASSISTED EXTRACTION AND TEST SAMPLE PREPARATION 100  mg of fine powder was extracted under the influence of microwave energy using solvents Extraction parameters (160 watts, 120 s, 10 ml per extraction cycle, two extraction cycles, and cleanup with 2 ml of corresponding solvent at the end of second cycle of extraction ) For microwave-assisted extraction (MAE) the same parameters should be followed for every solvent The extract thus obtained was evaporated and redissolved in 5 ml methanol 10   μl of these test solutions was used for quantification purpose.

8. ESTIMATIONS By 2 methods:- TLC Densitometer HPTLC method TLC METHOD Mobile phase – ethyl Acetate : hexane (3:97 ) Stationary phase -- 60 F254 silica gel Detecting reagent -- anisaldehyde -sulphuric acid reagent Spot volume -- 10  μL of test and standard sample spots Spot colour -- Pink- colored spots of artemisinin Room temp. -- 25 +- 2˚C Relative humidity -- 45+- 2%

9. A simple TLC- densitometric technique has been developed for the rapid and accurate analysis of artemisinin in a large number of Artemisia annua plantlets at 540 610  nm using tungsten lamp Values are plotted in calibration curve (con Vs spot area) Extrapolating the graph to x axis give concentration of test sample

10. Hplc method EQUIPMENT MODEL -- WATERS 501 COLUMN -- WATERS C18 DETECTOR -- UV 260 nm MOBILE PHASE -- phosphate buffer : methanol (6:4) – PH – 7.9 FLOW RATE -- 1ml/min Calibration curve was plotted with diff conc

11. SPECIFIC SHEET Description -- colour less needles / white crystalline powder, odourless Identification -- TLC/ HPLC with std Molecular formula -- C15H22O5 Molecular wt -- 282.3 Solubility -- partially insoluble in water very soluble in dichloromethane freely soluble methanol, ethanol MP -- 151-154 c LOSS ON DRYING -- NMT 0.5% STORAGE -- Tightly closed container

12. reference Http://onlinelibrary.Wiley.Com/doi/10.1002/pca.976/abstract Https://www.Hindawi.Com/journals/cri/2014/361405/ Wikipidea.Org Herbal Drug formulation, Paridhavi

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