Abstract:
Disclosed is a process for manufacturing crude Artemisinin comprising the extraction of  Artemisia annua  from plant material with carbon dioxide or water in a critical physical state such that after extraction, the solvent evaporates completely from the resulting extract.

Description:
FIELD OF INVENTION 
     The present invention relates to a process of extraction of Artemisinin from  Artemisia annua  and to the extract obtainable by said process. 
     BACKGROUND OF THE INVENTION 
       Artemisia annua  has been used in China for centuries to treat malaria. Researchers from all over the world have demonstrated that preparations based on  Artemisia annua  are effective against malaria, even in its most acute forms, caused by infection by the parasite  Plasmodium falciparum.  The main chemical constituent of  Artemisia  responsible for this pharmacological activity has been identified as Artemisinin, a sesquiterpene structure containing an unusual peroxide bridge. The structure of Artemisinin is shown below: 
     
       
                 
         
             
             
         
      
     
     Clinical trials conducted with Artemisinin and semi-synthetic derivatives thereof such as Artesunate have confirmed its efficacy in the treatment of malaria. Artemisinin was recently synthesised, but the complexity of the molecule prevents its large-scale synthesis. This is confirmed by numerous research projects designed to synthesise Artemisinin analogues with a simpler structure. More recent studies have preferred to focus on  artemisia  extracts produced with non-polar organic solvents such as diethyl ether or, more commonly, petroleum ether. 
     However, the known processes do not enable a satisfactory Artemisinin content to be obtained. Moreover, it is very difficult to remove the traces of solvents to obtain a pharmaceutically acceptable product. The solvents are also used in large amounts in the known methods, and have therefore to be discarded or redistilled. 
     In recent years, carbon dioxide in the supercritical state has been used to isolate active pharmaceutical substances from plant materials. This technique has been used on an industrial scale for over 20 years to extract flavourings from herbs and spices. The carbon dioxide is removed rapidly and completely by depressurising the extraction apparatus, thus eliminating the problems associated with disposal of solvents and contamination of the finished product with traces of solvent. Moreover, the selectivity of supercritical carbon dioxide is generally greater than organic solvents of a polarity comparable to ethyl acetate or petroleum ether. 
     Similar procedures can also be successfully employed to extract active components from plant matrices using water in the subcritical state, either alone or mixed with other co-solvents. 
     DESCRIPTION OF THE INVENTION 
     It has now been found that crude Artemisinin can be advantageously obtained by extraction from the green parts of the  Artemisia annua  plant with carbon dioxide or water in the critical state, and their subsequent evaporation from the extract. 
     Another aspect of the invention provides an extract of  Artemisia annua  containing approx. 3% by weight of Artemisinin substantially free from traces of residual organic solvents. 
     Co-eluents, namely small amounts of solvents which can modify the polarity of the mixture, can also be used at the extraction step. For example, methyl alcohol, ethyl alcohol, acetonitrile, ethyl acetate, petroleum ether, n-hexane, and preferably ethanol can be used for that purpose, in order to obtain pharmaceutical grade Artemisinin. Said co-eluents can be used in amounts ranging between 0.5 and 30%, usually 5% by weight. 
     The process according to the invention provides an extract with a high Artemisinin content and no trace of organic solvents. 
     The process according to the invention is preferably performed at an extraction pressure between 0.5 and 100 MPa, usually under a pressure of 50 MPa, and a temperature range between 20 and 150° C. (usually 70-80° C.), with the use of carbon dioxide and/or water in the critical state, with or without the use of co-eluents. 
     The invention is disclosed in greater detail in the example below. 
    
    
     EXAMPLE 
     Dried parts of leaves of the plant  Artemisia annua  were packed into a vessel resistant to pressure. A volume of liquid carbon dioxide, in the ratio of approx. 4:1 of anhydride in the critical state to 100 g of plant material, was forced through the crude material at the speed of approx. 20 rpm. The liquid carbon dioxide was thus collected, and the pressure reduced and balanced with atmospheric pressure to allow the anhydride to be dispersed in the atmosphere. The residue extracted and collected from the extraction chamber presented as an oil or semi-solid, depending on the exact extraction conditions. 
     With carbon dioxide, the following extraction conditions were used: 
     
       
         
               
               
               
               
               
             
           
               
                   
               
               
                 Extraction 
                 Extraction 
                   
                   
                   
               
               
                 pressure 
                 temperature 
                 Speed 
                 Sample 
                 Artemisinin 
               
               
                   
               
             
             
               
                 10 MPa 
                 80° C. 
                 20 g min −1   
                 100 g 
                 0.07%  
               
               
                 20 MPa 
                 80° C. 
                 20 g min −1   
                 100 g 
                 0.9% 
               
               
                 30 MPa 
                 80° C. 
                 20 g min −1   
                 100 g 
                 1.8% 
               
               
                 50 MPa 
                 80° C. 
                 20 g min −1   
                 100 g 
                 3.1% 
               
               
                 70 MPa 
                 80° C. 
                 20 g min −1   
                 100 g 
                 2.9% 
               
               
                   
               
             
          
         
       
     
     The Artemisinin content was determined by liquid chromatography coupled with mass spectrometry (LC/MS). 
     The use of carbon dioxide without the addition of co-elution solvents gives good results: the Artemisinin content obtained from the extract is approx. 3%. 
     Other extraction conditions yield a lower Artemisinin content. 
     As an alternative to the use of carbon dioxide, water in the subcritical state can be employed, and already provides good results without the addition of co-elution solvents. 
     Dried parts of leaves of the plant  Artemisia annua  were packed into a vessel resistant to pressure. A volume of steam, in the ratio of approx. 5:1 of water in the critical state to 100 g of plant material, was forced through the crude material at the rate of approx. 15 ml/10 min. The steam was collected, and the pressure was reduced and balanced with atmospheric pressure to allow the steam to disperse in the atmosphere. The residue extracted and collected from the extraction chamber always presented as an oil or semi-solid, depending on the exact extraction conditions. 
     With water, the following extraction conditions were used: 
     
       
         
               
               
               
               
               
             
           
               
                   
               
               
                 Extraction 
                 Extraction 
                   
                   
                   
               
               
                 pressure 
                 temperature 
                 Speed 
                 Sample 
                 Artemisinin 
               
               
                   
               
             
             
               
                 1 MPa 
                 125° C. 
                 15 ml min −1   
                 100 g 
                 1.6% 
               
               
                 2 MPa 
                 125° C. 
                 15 ml min −1   
                 100 g 
                 2.3% 
               
               
                 5 MPa 
                 125° C. 
                 15 ml min −1   
                 100 g 
                 2.8% 
               
               
                 10 MPa  
                 125° C. 
                 15 ml min −1   
                 100 g 
                 0.7% 
               
               
                 20 MPa  
                 125° C. 
                 15 ml min −1   
                 100 g 
                 0.09%  
               
               
                   
               
             
          
         
       
     
     The Artemisinin content was determined by liquid chromatography coupled with mass spectrometry (LC/MS). 
     For the purpose of direct comparison, the Artemisinin content extracted from the same plant material with conventional solvents such as n-hexane and ethyl acetate only amounted to 0.6%, determined in the same way as for LC/MS. 
     The Artemisinin content in the crude material used, extracted by the conventional method for organic solvent, agrees with the findings published in the literature, namely an interval ranging between 0.01 and 0.6% w/w for  Artemisia annua  extracted with conventional solvents.