Presentation

TECHNIQUES AND METHODS IN ARTEMISININ PRODUCTION

Semester VAmity Institute of Biotechnology

Amity University RajasthanJaipur2010

 

Submitted by:

Deepraj Singh Sisodiya

INTRODUCTIONArtemisinin: -

Artemisinin and its derivatives are a group of drugs that possess the most rapid action of all current drugs against falciparum malaria.

Producing plant: - Artemisia annua.

Mechanism of action: - The artemisinin radical binds to membrane proteins of parasite and

alkylation reactions eventually cause destruction of the parasite.

Tread Names: -

• ARTECOPE

• Larither

• Larimef

• Larinate

• RAPITHER - AB INJECTION (IM Only)

Material and method

• Extraction technique.• Purification technique.• Analysis technique.

Extraction technique: -

Objective : - To Extract plant leaves content from A.Annua.L plant leaves.: -

• Take 25g of grinded leaves for the sample.

Put them in thimble.• Add 200 ml of the hexane in the distilling pot.• Set the temperature of water bath to 70-80’C• Continue the process for 4 hrs.• Make the total volume of the

extracted solution 250ml.• Distillation of the extracted solution in

rotary evaporator . at 750C and 50 rpm.

Result:-

Crude (Extracted material) weight was 420 mg.

Purification Technique: -

Objective: -Separation of Reaction Mass of method for the preparation of artemether from artemesinin.

Materials:- Column, Silica gel, TLC, Chromatogram Immersion Devise, TLC plate heater.

Method: - • Initial set up of column.

• Made slurry with 60 g of silica and N hexane. Packed the column with the slurry.• Added reaction mass 3g , silica and N hexane mixture as slurry on the surface.

• Purification of component.• Charged the column with the ethyl acetate and N hexane in the ratio of (2:98).• Collected the fraction, Change the conical flask for each 100-150ml collection.

• TLC analysis of the fraction of reaction mass.

• (ethyl acetate :hexane) ethyl acetate :hexane• (97:3) (97:4)

(1 to 60) (60 to 90) (95 to 130) 136 fractions 5 th TLC

• Crystallization of the component.• Beaker named (Artemether alpha) fraction 1 to 60.• Beaker named (Aremether beta ) fraction 95 to 130• Beaker named (Impurity) 136 and after.

Set the temperature of water bath 75 degree C. Set the rotation of the flask 50 RPM.• Result:-

Yellow crystal of artemether alpha formed.

Transparent crystal of beta artemether formation on the surface of flask.

RESULT:-Alpha Artemether :- 1.78g

Beta Artemether: - .65g

furnished pure alpha and beta Artemether 2.43 g .

Analysis Technique: -

Objective: - Sulphated ash analysis of Artemisinin.

Material:-

Conc. Sulphuric acid, Muffle furnace.

Method: -• Weight of blank crucible was X1=120g.• Conditioning.• Burn the sample by heating the crucible along with addition of conc. Sulphuric

acid.• put the crucible in the muffle furnace for 3 hr.

Calculation:-

Weight of left content:- X2-X1= 120.012g-120g=.012g=12mg

Weight of left content 12mg Sulphated ash % = ………………………. = …….. × 100 = 1.2%

Weight of sample used 1000mg

Result:-

The sulphated ash content of 1g artemisinin was 1.2%.

Objective : - To find out Bulk density and Tap density.Material:-Tapping instrument, 25ml measuring cylinder,10-15g of artemisinin.Method: -Sample taken for Bulk and Tap density was 5mg.

Calculation and Result: - Grams of sample taken 5

Bulk density=…………………………………= ………..= .47g/ml

Volume occupied by sample 10.5

 

Grams of sample taken 5

Tap density= ………………………………… = …… =.754 g/ml

Volume occupied by sample after tapping 8.7

 

Objective : - To determine the moisture content in artemisinin.

Material:-

Sample 3g Artemisinine , Karl Fischer 701.Method: -

Sample Taken for analysis was .324g

Result:-

The water content was 4.89%

Objective: - LOD analysis technique.

Material: - 3g Artemisinin ,

MATTLER TOLEDO HR73 .

Result:-

Loss of drying was 0.5%.

Objective: - To determine the melting point of artemisinin.

Material:-

Melting point capillaries, Mortar and pestle, 3g of artemisinin , Compaction wire.

Method:-

setpoint 150 degree C.

grading 5 degree C.

max point. 160 degree C.

RESULT:-

The observed result was. Initial malting point 152 degree C and final malting point 157 degree C.

Objective :- Melting point and enthalpy analysis of artemisinin.

Instrument used:-Differential scanning calorimeter.

Method: - INITIAL OPRETION. OPRETION OF UNIVERSAL SEALING PROCESS. OPRATION OF PYRIS 6 DSC SOFTWARE.

Result:-

Onset = 152.020C, Area = 308.826mj, Data H= 75.3235j/g, Peak =154.090C

Objective: - To find out the standard optical rotation of artemisinine.

Sample preparation:-

Take 3.2 g of artemisinine and dissolve it in 100% methanol.

Method: -• Select mercury lamp.• Select the wavelength 599 by pressing key “waveln” and key enter

press.• For “SORT” feed the concentration by pressing “conc” added the

value in which the sample solution prepared 3.20%. Press the “enter” key.

• After setting this, put the sample firstly “blank” (Methanol) in cell quartz cell & then the pressing key “Auto zero”.

• After this put the sample in quartz cell. Press “Enter”.

Result:- The Specific optical rotation of artemsinin was +64°

Objective : - Purity analysis of artemesinin by using IR spectroscopy.

Instrument: - Infrared spectroscope

Sample preparation:-

Mix 0.5mg of artemesinin and mix it with 500mg KBr. Use mortar and pastel to make fine powder. Put it on the sample pan.

Method : -• Put the KBr in pan. Fix pan with slit then insert it into the instrument. • Put the prepare sample in the pan. Click on instrument->SCAN->Give

file name “Artemisinin” and scan range of 450 to 4000 cm-1. And no of scan 1.

Result : -

The comparison of our batch

product with

standard product will give

similarity of 97.23%.

Object:- Plant leaves artemisinin content of sample with HPTLC.

Material:- Artemisia annua samples and artemisinin standard, HPTLC plates silica gel, Soxhlet extraction, Chromatographic equipment, Immersion device, Automatic TLC Sampler 4, TLC Scanner 3, Win CATS 1.4.2 software.

Sample preparation : - Soxhlet extraction.

Standard preparation:-

Take 10mg artemisinin and dissolve in the 10 ml hexane.

Method: -• Software linomat 5 setup.• Linomat auto sampler set up.• HPTLC plates development.• Scanning of TLC plate.

Standard graph Sample graph

The analysis and calculation of samplegraph in win cats give the artemisinin content was .8mg/ml.

• Object:- Impurity analysis of Sulphamethoxy pyrazine by using Gas chromatography.

Material.

Diluents:-

DMSO:Water(90:10) 

Standard stock solution:-

200mg benzene add in 100ml flask containing 50ml diluents. Conc. 2000ppm.

Preparation of standard.

1.0ml standard stock solution. Add in 100ml diluents. Conc. 0.2ppm.

Transfer 1.0ml of standard solution in dry head space vial and seal it. 

Test solution.

100mg of sample add in 1ml diluents.

Method:-• procedure for operation Totalchrom navigator.• Operation of Instrument Option of Totalchrom navigator.• Operation of Sequence set up.• GC Instrument Setup.

• operating of Head space sampler-Turbomatrix -40 .• Click the RUN option of GC total chrome Navigator

Result: -

Calculation And Result:- AT WS 1 1 1

ppm of benzene =…….....×……….×………×…….×….......×106

AS 100 100 100 WT

WS=200mg, WT=100mg, AT=1995.33, AS=2003.25

1995.33 200 1 1 1

ppm of benzene =…….....×……….×………×…….×….......×106

2003.25 100 100 100 100 

ppm of benzene = 1.992