Abstract:
A compound is extracted from vegetable material, wherein the vegetable material is reduced and treated with a solvent. According to the invention, the vegetable material is frozen using a liquid nitrogen and in frozen condition reduced in size. According to two important embodiments, the vegetable material comes from the ginkgo tree, in particular fresh leaves, and water is used as a solvent. The invention also relates to a pharmaceutical preparation and a cosmetic preparation comprising as active component a compound obtained by the method according to the invention.

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
     This application is a 371 of PCT/NL99/00379, filed Jun. 18, 1999, which claims priority from Dutch patent application serial number 1009437, filed Jun. 18, 1998. 
    
    
     FIELD OF THE INVENTION 
     The present invention relates to a method of extracting a compound from vegetable material, wherein the vegetable material is reduced in size, subjected to an extraction treatment using a solvent, after which the undissolved parts are separated from the solvent comprising the compound. 
     BACKGROUND OF THE INVENTION 
     This type of method is quite old. Examples are the infusion of tea and the preparation of tinctures. In both cases the undissolved parts are usually separated from the solvent comprising the compound. This may be accomplished by, for example, filtering or centrifugation. 
     The disadvantage with the known methods is that to a lesser or greater degree the active compound(s) stay behind in the separated insoluble parts. 
     The object of the present invention is to provide a method as described in the preamble, by which the yield of active compound can be increased. 
     To this end the method is characterized in that the vegetable material is frozen using a liquified gas and in frozen condition reduced in size prior to being extracted with the solvent. 
     In this invention the vegetable material is effectively opened up and a higher percentage of yield is assured. Liquid nitrogen is very suitable for this purpose because of its inert (non-oxidative) nature and low price. 
     European patent 0 477 968 describes a method of preparing an extract of Ginkgo biloba by treating the leaves with aqueous acetone, an aqueous alkanol with 1 to 3 C atoms, or anhydrous methanol at a temperature of 40-100° C. After the removal of solid parts, this solution is treated with ammonium sulphate, methyl ethyl ketone, and the resulting extract is extracted with a higher alkanol. After several additional steps an extract is obtained comprising 20 to % by weight of flavoglycosides, 2.5 to 4.4% by weight of ginkgolides A, B, C and J, and 2.0 to 4.0% by weight of bilobalide. The described method of extraction is very laborious. 
     European patent 0 431 535 describes a method of preparing an extract from Ginkgo biloba leaves that essentially corresponds with the method described above. In addition, lead salt is used for further purification. For this reason this extraction method is also very laborious while moreover using a lead compound that is bad for both the environment and human health. 
    
    
     DETAILED DESCRIPTION 
     According to an important embodiment, the vegetable material is chosen from a group comprising plant parts of the ginkgo tree (Ginkgo biloba) and Huperzia serrata. 
     Such vegetable material proves to extract excellently by the method outlined in the present invention at hand, yielding pharmaceutically high-grade solutions. Ginkgolides and flavonoids can be extracted from plant parts of the ginkgo tree. Huperzine can be isolated from plant parts of the Huperzia serrata. All these compounds possess interesting pharmaceutical properties. 
     The most preferable plant parts of the ginkgo tree to be used are fresh leaves which have been dried shortly before treatment, with water being used as the solvent. 
     Surprisingly, this method has proven that with fewer processing steps a purer product can be obtained. In the present application the term water is understood to mean a liquid comprised of mostly water that may contain up to 10% water-soluble alcohol or ketone, such as acetone. The water can also comprise salt, which prevents undesirable compounds from dissolving and/or assists the precipitation of such compounds. The water may also comprise enzymes that weaken the cell structure of the plant material. Though the pH can vary within a wide range, it will generally be lower than 7. In order to increase the level of flavonoids, the extracted leaves may be washed again with water to which a completely water-miscible organic solvent has been added, such as 40% acetone. The preparation of an extract from ginkgo leaves is known in the art. With this preparation the ginkgo leaves are dried after which they are extracted using alcohol or ketone, which may contain some water. In order to obtain a product with a sufficiently high content of active compound, further laborious refinement steps are necessary, increasing the cost of the product. 
     In order to obtain a high-grade product, the plant parts of the ginkgo tree should be obtained from a 1 to 15-year-old ginkgo tree, preferably from a 3 to 10-year-old tree and most preferably from a 5-year-old tree. 
     To obtain a preparation with a suitable ginkgolide/flavonoid ratio (such as 1:4 or higher), the pH of the water preferably lies between 4 and 7. 
     To further increase the yield, it is advantageous to add an enzyme such as cellulase to break down the cell walls after reduction. 
     Preferably extraction with water is then preformed for a maximum of 3 hours. 
     In order to ensure a high level of purity, the extraction treatment is suitably carried out at a temperature from 10 to 60° C., preferably 20 to 40° C. 
     One favourable embodiment for obtaining a composition containing the compound and having a long shelf life involves drying the solution containing the compound, which may conveniently be achieved by spray-drying or freeze-drying. 
     Suitably the solvent is eliminated from the solvent containing the compound, and the residue is extracted with a further solvent. 
     In this manner it is possible to obtain a purer preparation comprising the compound. For the extraction of ginkgo methylisobutylketone (MIK) or tert.-butylether (TBE) may conveniently be used. 
     In accordance with the above the invention also related to two preparations. 
     The invention relates in particular to a pharmaceutical preparation comprising an active component containing a compound obtained according to a method outlined by the invention, together with a pharmaceutically acceptable excipient or carrier. 
     Such a pharmaceutical preparations may be used, for example, to treat patients with Alzheimer&#39;s disease, patients with symptoms caused by prolonged contact with organic solvents, etc. The pharmaceutical preparations according to the invention can be administered in any form, particularly oral or parenteral administration will be preferred. 
     The invention also relates to a cosmetic preparation containing an active component comprised of a compound obtained according to a method outlined by the invention, together with a cosmetically acceptable topical carrier. 
     Such a cosmetic preparation can help to keep skin young and supple. 
     The invention will now elucidated with the aid of the following exemplary embodiment. 
     EXAMPLE 1 
     Within 72 hours of being picked, 10 g of ginkgo leaves are contacted with liquid nitrogen (3 ml). The ginkgo leaves are crushed using a porcelain mortar. Then 100 ml of water is added as well as 1:100% w/v of ULTRA-ZYME AFP-L and CELLUBRIX L. (Novo Nordisk Ferment Ltd., Switzerland); this is incubated for 2 hours at 50° C. and a pH or 5.5 while being mixed and subsequently extracted at a pH of 4-5. The liquid, containing as active components ginkgolides and flavonoids, is separated from the solid parts by means of filtering over a large-mesh filter and then subjected to microfiltration over, for example, a 0.2 μm hydrophilic filter and subsequently freeze-dried (extract A). 
     EXAMPLE II 
     The method described in Example I is repeated. In the same manner described above, the residue resulting from the extraction is extracted for 1 hour with 40% acetone solution and subsequently freeze-dried (extract B). 
     Water is added to both freeze-dried extracts A and B (0.1 g extract per ml water). The obtained solutions are extracted with (1:1 v/v) methylisobutylketone (MIK) or tert.-butylether (TBE). These extractions are repeated twice and the fractions are combined for each solvent. The solvents are evaporated by means of a film evaporator. 
     As it is difficult to adequately eliminate MIK, the residue is after evaporation dissolved in a 3:2 mixture of acetone and water (first acetone is added until the residue is dissolved, then gradually water is added under heating to 50° C. and sonication). Subsequently the acetone is evaporated and the aqueous solution is freeze-dried. 
     The resulting residues are analyzed with the aid of HPLC. A 150 * 3 mm ODS-3 Inertsil 5 column (Chrompack) was used. Isocratic elution, with as mobile phase methanol/water (for ginkgolides) or methanol/buffer 55/45 v/v (for flavonoids) was performed with 1 ml/min. Buffer composition: 1:2 g/l ammonium dihydrogen phosphate with phosphoric acid at pH=2.5. 
     Retention times for Bilobalide, Ginkgolide A and B: 4.9, 9.0 and 10.1 min. Detection by means of a refractive index detector. 
     Retention times for the flavonoids Quercetin, Kaempferol and Isorametine: 8.9, 15.5 and 16.9 min. Detection at 360 nm. 
     The date a summarized in Table I. 
     
       
         
               
               
               
               
             
               
               
               
               
               
               
               
             
           
               
                   
                 TABLE I 
               
               
                   
                   
               
               
                   
                 Treatment 
                 Ginkgolides *) 
                 Flavonoids *) 
               
               
                   
                   
               
             
             
               
                   
               
             
          
           
               
                   
                 I 
                 water 
                   
                 (1, 57) 
                   
                 (0, 44) 
               
               
                   
                 II 
                 water/acetone 
                   
                 (1, 20) 
                   
                 (1, 04) 
               
               
                   
                 III 
                 I + MIK 
                 115 
                 (35, 8) 
                 50, 8 
                 (15, 8) 
               
               
                   
                 IV 
                 II + MIK 
                 19, 9 
                 (30, 8) 
                 24, 5 
                 (38) 
               
               
                   
                   
                 total 
                 134, 9 
                   
                 75, 3 
               
               
                   
                 V 
                 I + TBE 
                 79, 4 
                 (51, 1) 
                 15, 4 
                 (9, 9) 
               
               
                   
                 VI 
                 II + TBE 
                 14, 0 
                 (41, 3) 
                 5, 6 
                 (16, 5) 
               
               
                   
                   
                 total 
                 93, 4 
                   
                 21 
               
               
                   
                   
               
               
                   
                 *) in mg. The percentage of dry matter is indicated in brackets.  
               
             
          
         
       
     
     The above table shows that extraction with MIK and TBE improves the purity of the product. Extraction with MIK results in a higher yield, while extraction with TBE is useful if a higher yield of ginkgolides in relation to flavonoids is desired.