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.
6. 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 μL 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
9. 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
10. • 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
11. • 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.
12. • 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
ArtemisininFront. Plant Sci., 31 January 2018
13. • 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
14. • The chromatographic separation was carried out on
precoated silica gel plate G60 F254 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
16. 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 other species of Artemisia.
Family Asteraceae.
17. Identification test
• 60. • 1 gm powder boil with 10 ml of alcohol
& filtered, NaOH heated- red color
18. 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 μl & eluted using a methanol:
water (9:1). With flow rate 1ml/min. • C18
column used.
19. 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.
