Artemisinin.pptx

Pharmacognosy

1. ARTIMISININ

2. Artemisinin Source: Leaves &unexpanded flower heads of Artemisia annua (Sweet worm wood) FAMILY : Asteraceae Chemistry: a  sesquiterpene lactone Internal peroxide linkage located in the 7-membered ring responsible for antimalarial activity Epoxide 1,2,4-trioxane ring Whitish crystalline powder(156-157 C) Soluble in methanol, dichloro methane, organic solvents. Partially soluble in water.

3. Isolation Plant material, dried coarsely powdered and extracted with pet. ether Filter & dried extract Re-dissolved in CHCl3 + Acetonitrile (precipitate waxes, sugars) Filter & the filtrate is concentrated Subjected to silica gel CC Elute with CHCl3-ethyl acetate Crystallized from the fraction containing artimisinin , using ethanol.

4. Production of artemisinic acid by yeast fermentation A. annua  genes encoding artemisinic acid biosynthetic enzymes , was identified Those  enzymes were expressed in yeast for production of amorphadiene , & its oxidation to artemisinic acid. Artemisinic acid is isolated from the fermentation medium Further , it is chemically converted to artemisinin .

5. ESTIMATIONS HPTLC method Sample: dissolved in CHCl3 Std : Dissolved in CHCl3 Mobile phase –Petroleum ether : ethyl Acetate (1:2) Stationary phase -- 60 F-254 silica gel Detecting reagent – Vanillin - sulphuric acid reagent Spot volume -- 10 μ L of test and standard sample spots Spot colour -- Pink-colored spots of artemisinin Detection at 560 nm Values are plotted in calibration curve (con Vs spot area) Extrapolating the graph to x- axis give concentration of test sample

6. HPLC METHOD Column: C-18 Detector: UV 260 nm Mob phase: Phosphate buffer: methanol(6:4) Flow rate : 1 ml/min Calibration curve was plotted with different conc.

7. Utilization Antimalarial effect by its rapid blood schizonticidal activity. Artemisinin and its semisynthetic derivatives are a group of potent drugs for treating cerebral malaria . Act against both chloroquine sensitive and resistant strains Plasmodium falciparum and P. vivax malarial parasite

8. PLANT PROFILESYNONYM : 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

9. 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. 

10. Ref;   https://doi.org/10.3389/fpls.2018.00087 Stephanie H. Kung , Sean Lund,  Abhishek Murarka ,  Derek McPhee  and  Chris J. Paddon * Approaches and Recent Developments for the Commercial Production of Semi-synthetic Artemisinin Front . Plant Sci., 31 January 2018

11. 2.4 TLC of AA, AM and AB The linear ascending development was carried out on RP18 F254 S TLC plate, 20610 cm in a Camag twin-trough chamber (20620 cm) which was presaturated with mobile phase 0.2% TFA in water/ACN (35: 65, v/v). Samples and standards were applied on the plate as 6-mmwide bands with the automatic TLC applicator, ATS4 under N2 gas flow, 10 mm from the lower edge of the plate. The application parameters were identical for all the analyses performed. The chromatogram was allowed to develop for 20 min to a height of 8 cm at room temperature (25 € 28C) and 50 € 5% relative humidity. After the development, TLC plates were dried in a current of air with the help of an air drier in a wooden chamber with appropriate ventilation. The dried plate was dipped into freshly prepared reagent consisting of glacial acetic acid/concentrated H2SO4 / anisaldehyde (50:1:0.5, v/v/v) followed by heating (TLC plate heater, Camag ) at 1108C for 5 min to visualize the bands of AA, AM and AB. Sesquiterpenes AA, AM and AB were quantified by means of five-point calibration curve considering peak areas with Camag TLC scanner 3, equipped with winCATS software version 1.2.3, slit width 660.4 mm, wavelength 426 nm in absorption-reflectance mode, scanning speed 20 mm/s and data resolution 100 lL /step

12. The chromatographic separation was carried out on precoated silica gel plate G60 F 254  using  n -hexane-ethyl acetate-acetic acid (2:1:0.1) as the mobile phase, and densitometric analysis was carried out in absorbance mode at 540 nm after derivatization with anisaldehyde spraying reagent. The visualized the bands of AM was quantified by means of five-point calibration curve considering peak areas with Camag TLC scanner 3, equipped with winCATS software version Ref : https://doi.org/10.1556/1006.2016.29.5.3

13. Artemisin  

14. Artemisin Artemisin is a sesquiterpene lactone obtained from leaves and unexpanded flowed buds of Artemisia annua (sweet warmwood ) Artemisia cina , A. brevifolia , A. maritima and o ther species of Artemisia. Family Asteraceae .

15. Identification test 60.  • 1 gm powder boil with 10 ml of alcohol & filtered, NaOH heated- red color

16. Analysis: 1) IR: 2mg drug mix with 98 mg of KBr which dried for 24 hr. at temp. 105 °C. • Analysed at : 4000 cm-1 to 400 cm-1. • 2) UV : 1mg drug mix with 10 ml methanol & analysed λ 200-400 nm. • 3) TLC : 1 mg dissolved with 5 ml of ethyl acetate. • Stationary phase: Silica gel 60 F254 • Mobile Phase: Ethyl acetate: hexane (3:97 or 7:93) • 4) LC – MS : 1 mg drug mixed with 1 ml methanol & injected as 20 μ l & eluted using a methanol: water (9:1). With flow rate 1ml/min. • C18 column used.

17. Isolation • Take powder macerate with solvent methanol. • Performed in magnatic stirrer with speed of 700 rpm for 1 hr. • This process repeted till methanol layer became colorless. • Ext. then evaporated using rotavapour vaccum at temp. 40 °C. untill volume to 100 ml. • The ext. solu . Was partitioned using 50 ml haxene . Untill a colorless haxane layer was obtained. • 1: hexane ext. & 2: methanol ext. • methanol ext. add 10 ml water & 50 ml ethyl acetate. Partitioned untill ethyl acetate layer become colorless. • By this process, ethyl acetate & methanol-water ext. obtained. • evaporated using rotavapour vaccum at temp. 40 °C.