Abstract:
A method for selectively separating desirable steroidal glycosides from undesirable components present in plant material of the Asclepiadaceae family containing the same. The method comprises contacting the plant material or material derived therefrom (herein: “the material”) with liquid or supercritical carbon dioxide under conditions whereby the desirable steroidal glycosides dissolve in the liquid or supercritical carbon dioxide in preference to the undesirable components, and subsequently recovering the desirable steroidal glycosides from the carbon dioxide solution.

Description:
FIELD OF THE INVENTION 
       [0001]    The present invention relates to the selective separation or extraction of steroidal glycosides from materials containing more than one of these compounds and optionally other components. Most particularly, such materials may be plant matter in which the steroidal glycosides occur naturally. 
       BACKGROUND OF THE INVENTION 
       [0002]    Extracts obtainable from plants of the Asclepiadaceae family, particularly the  Hoodia  genus (formerly the  Hoodia  and  Trichocaulon  genera), have been shown to contain the steroid glycoside 1 which is an appetite suppressing compound (PCT application WO 98/46243). The contents of this prior application are incorporated herein by reference. An extraction process is outlined in the prior application, involving treating plant material with a solvent to extract a fraction having appetite suppressant activity, separating the extraction solution from the rest of the plant material, removing the solvent from the extraction solution and recovering the extract. The solvents specifically mentioned to perform the extraction are one or more of methylene chloride (dichloromethane), water, methanol, hexane, ethyl acetate or mixtures thereof. 
         [0000]    
       
                 
         
             
             
         
       
     
         [0003]    The prior art solvent extraction process tends, however, to result in a mixture of extracted compounds, not all of which have desirable properties. Typically, some may have an unpleasant taste. 
         [0004]    The need for a subsequent fractionation or separation procedure to remove undesirable extracted compounds leads to increased manufacturing costs and complicated procedures, which it is preferable to avoid. 
         [0005]    US Patent Application No. US-A-2003/0152648, the disclosure of which is incorporated herein by reference, describes extraction of pregnane glycosides, particularly stavarosides, from the plant  Orbea variegata,  a member of the Asclepiadaceae family. The extraction process involves initial solvent extraction from dried and ground plant material, using a suitable solvent. It is stated generally that “suitable solvents include water, dilute acids, organic solvents, critical, supercritical or near critical fluid solvents, e.g. carbon dioxide, nitrous oxide, propane, ethane, ethylene and fluorohydrocarbons, and mixtures of any of these”. The preferred solvent is stated to be an alcohol based solvent, the most preferred being ethanol. Ethanol extraction is the only technique shown in any working example. The solvent extraction technique is stated to be usable to extract stavarosides from the plant material, and there is no teaching or suggestion to use it to selectively separate desirable steroidal glycosides from undesirable steroidal glycosides. 
         [0006]    Moore and Taylor ( J. Nat. Prod  1996, 59, 690-693), the disclosure of which is incorporated herein by reference, describe the extraction of the cardiac glycosides, digoxin and acetyldigoxin, from  Digitalis lanata  using near supercritical methanol-modified carbon dioxide. The authors indicate that to effect efficient extraction the extraction solvent was methanol and carbon dioxide where the methanol was in the range of 10-25%, with the optimised conditions employing 20% methanol modified CO 2    
         [0007]    We have now found, surprisingly, that, by using a particular extraction technique described below, specifically the use of liquid or supercritical carbon dioxide, a relatively efficient selective extraction of desirable steroidal glycosides from Asclepiadaceae plant material can be achieved, which involves the selective separation of desirable steroidal glycosides from undesirable steroidal glycosides, reducing or eliminating the need for subsequent processing to remove undesirable components. 
       BRIEF DESCRIPTION OF THE INVENTION 
       [0008]    The present invention provides in the first aspect a method for selectively separating desirable steroidal glycosides from undesirable steroidal glycosides or other compounds present in plant material of the Asclepiadaceae family containing the same, the method comprising contacting the plant material or material derived therefrom (herein: “the material”) with liquid or supercritical carbon dioxide under conditions whereby the desirable steroidal glycosides dissolve in the liquid or supercritical carbon dioxide in preference to the undesirable steroidal glycosides or other compounds, and subsequently recovering the desirable steroidal glycosides from the carbon dioxide solution. 
         [0009]    The use of liquid or supercritical carbon dioxide as an extraction solvent is known, and apparatus for performing the technique are readily available. For a general review of the technology, please refer to Kaiser et al, Pharmazie 56, 907-926 (2001) and Lang et al, Talanta 52, 771-782 (2001), the contents of which are incorporated herein by reference. The conditions of the extraction which may be varied to control the selectivity are primarily the temperature and the pressure of the sealed extraction apparatus. However, the prior art has generally restricted the use of the technique to extraction of active compounds from inactive cell-wall and other material and the use of liquid or supercritical carbon dioxide as a means for selectively separating desirable steroidal glycosides from relatively chemically similar undesirable steroidal glycosides in plant material of the Asclepiadaceae family was not previously known or suggested. 
         [0010]    After an initial extraction from the material, further extraction cycles can be performed on the remaining material if desired, using fresh carbon dioxide, in order to maximise the yield of extracted compounds. 
         [0011]    An initial extraction can be performed using liquid carbon dioxide, and a subsequent extraction performed on the remaining material using supercritical carbon dioxide. Alternatively, both an initial and a subsequent extraction can use liquid carbon dioxide. Still further, both an initial and a subsequent extraction can use supercritical carbon dioxide. The conditions of these sequential extractions can be chosen to preferentially remove particular components from the material. 
         [0012]    The method of the present invention may, if desired, be performed using a flow of carbon dioxide with downstream recovery of the desirable steroidal glycosides. 
         [0013]    The method of the present invention can be used with other extraction procedures for removing other, e.g. non-steroidal, components from the material to be treated. Thus, for example, an initial extraction using liquid carbon dioxide can be performed under conditions selected to preferentially remove undesirable lipophilic compounds such as fatty acids from the material. A subsequent extraction performed on the remaining material according to the present invention can thereby result in an improved yield of the desirable steroidal glycoside. 
         [0014]    An extract produced by the method of the invention can, if desired, be subjected to one or more subsequent extractions, e.g. one or more subsequent solvent extractions using one or more organic solvent to remove any residual unwanted compounds, for example non-steroidal glycosides. The choice of organic solvent(s) will be well within the capacity of a person of ordinary skill in this art, considering the materials being treated. The said one or more subsequent solvent extractions may suitably be performed using organic solvents selected from acetone, ethyl acetate, heptane (e.g. n-heptane), ethanol, hexane (e.g. n-hexane) and any mixture or combination thereof, and at any convenient temperature. 
         [0015]    The material on which the method of the present invention may be performed may be plant material or material derived therefrom. The term “plant material” in this context includes all forms of plant material, including freshly cut plant material, dried or preserved plant material, which may optionally be comminuted, e.g. powdered or crushed. The term “material derived therefrom” in this context includes extracts and decoctions obtained from the plant material. Such extracts may, for example, be obtainable by a non-selective extraction method, such as the prior art solvent extraction method. 
         [0016]    In one embodiment, the desirable steroidal glycoside may comprise a steroidal glycoside having a desirable bioactivity, for example having bioactivity to suppress appetite (particularly through actions at centres in the brain controlling of feelings of hunger and fullness), to treat excessive body weight (particularly above-average body weight for an individual&#39;s age and height), to treat obesity, to reduce total calorific intake of an individual (particularly, total calorific intake over an extended period of at least about two weeks in a lifestyle or environment where calorific foodstuffs and beverages are available substantially ad libitum), or any combination thereof. More particularly, the steroidal glycoside may be a compound of formula 1 as set out above, and the plant may be a plant of the  Hoodia  genus. The plant of the  Hoodia  genus may suitably be selected from  Hoodia gordonii, Hoodia currorii  subsp.  currorrii  and  Hoodia currorii  subsp.  lugardii.  The plant of the  Hoodia  genus is preferably  Hoodia gordonii.    
         [0017]    The undesirable steroidal glycosides or other compounds, from which the desirable steroidal glycoside is to be separated according to the method of the present invention, may, for example, comprise an unpleasant tasting component. 
         [0018]    A multi-step extraction process may use conditions of differing temperature and/or pressure at different stages. Most particularly, conditions of differing pressure may be used. The present invention therefore includes within its scope the use of a multi-pressure extraction of dried plants of the  Hoodia  genus wherein the plant material is initially extracted with carbon dioxide under liquid conditions to extract unwanted lipophilic material such as fatty acids, and then subsequently extracted with carbon dioxide under supercritical conditions to afford an extract containing elevated levels of compound 1, whilst leaving unwanted steroidal glycosides or other compounds with the plant matrix. 
         [0019]    If desired, one or more aqueous or organic co-solvent may be used in the method of the present invention, in conjunction with the carbon dioxide solvent and under the same conditions of temperature and pressure as applied to the carbon dioxide. Any such co-solvent that is present will be used in generally small amounts relative to the carbon dioxide, for example less than about 10% by weight (e.g. about 5% by weight) relative to the amount of carbon dioxide used. The co-solvent may suitably be selected from water, methanol, ethanol, hexane (e.g. n-hexane), heptane (e.g. n-heptane) and any mixture or combination thereof. 
         [0020]    The present invention includes within its scope steroidal glycosides which have been separated from the material by means of the method of the present invention. 
       DETAILED DESCRIPTION OF THE INVENTION 
       [0021]    The taxonomy of plants of the Asclepiadaceae family has been revised in recent years. For example, in 1992 Bruyns reclassified a number of plants to the genus  Hoodia,  which were previously in the genus  Trichocaulon  (Bruyns; Bot. Jahrb. Syst. 115 (2) 145-270 (1993)). One such reclassification related to  Hoodia pilifera,  previously  Trichocaulon piliferum.  The genus  Trichocaulon  now does not exist. 
         [0022]    Work undertaken on identifying molecules present in plants belonging to the genus  Hoodia  has shown that compound 1 is present in addition to a set of steroid glycosides covered by the generic structure 2. 
         [0000]    
       
                 
         
             
             
         
       
     
         [0023]    For example, from  Hoodia gordonii  eleven compounds have been isolated and characterised (Compounds 3-13). 
         [0000]    
       
         
               
             
               
               
               
             
               
               
               
             
           
               
                   
               
               
                 
                   
                             
                     
                         
                         
                     
                   
                 
               
               
                   
               
             
          
           
               
                 Compound 
                 R 1   
                 R 2   
               
               
                   
               
             
          
           
               
                 3 
                 glu-glu-glu- 
                 tig-the-ole- 
               
               
                 4 
                 glu-glu- 
                 tig-mda-cym- 
               
               
                 5 
                 glu-glu-glu- 
                 tig-cym-cym 
               
               
                 6 
                 glu-glu-glu- 
                 tig-ole-cym-cym- 
               
               
                   
                   
                 cym- 
               
               
                 7 
                 glu-glu-glu- 
                 tig-ole-cym- 
               
               
                 8 
                 glu-glu-glu- 
                 ang-mda-cym- 
               
               
                 9 
                 glu-glu-glu- 
                 tig-the-cym- 
               
               
                 10 
                 glu-glu-glu- 
                 tig- 
               
               
                 11 
                 glu-glu-glu- 
                 tig-cym- 
               
               
                 12 
                 glu-glu-glu- 
                 tig-the-ole-cym- 
               
               
                 13 
                 glu-glu-glu- 
                 tig-the-cym-cym- 
               
               
                   
               
               
                 ole = oleandrose, cym = cymarose, glu = glucose, mda = 3-O-methyl-6-deoxyallose, tig = tigloyl, ang = angeloyl, the = thevetose. 
               
             
          
         
       
     
         [0024]    Methanol extraction of dried  Hoodia gordonii  plant material affords extracts containing compound 1 and compounds 3-13. The use of other solvents such as dichloromethane or ethyl acetate similarly produces extracts containing all the steroidal glycosides. 
         [0025]    Surprisingly, it has been found that liquid or supercritical carbon dioxide extraction (for example at pressures of about 300 to about 500 bar and at a temperature of about 55 to about 80° C.) affords selective extraction of compound 1 whilst retaining compounds 3-13 in the plant matrix. The pressure and temperature can be varied outside these limits. 
         [0026]    Certain members of the genus  Hoodia  are known to contain bitter components making the palatability of the plant or extracts thereof poor. Indeed, the native names for  Hoodia gordonii  and  Hoodia currorii  subsp.  lugardii  is ‘bitterghaap’ which may be translated as bitter food. The use of  Hoodia gordonii  as an appetite suppressing agent could be hindered by this bitterness. Surprisingly, it has been found that extraction of plants of the genus  Hoodia  using carbon dioxide extraction affords an extract rich in compound 1 but lacking any unpleasant taste. 
         [0027]    A second aspect of the invention is the use of a multi-pressure extraction of dried plants of the  Hoodia  genus wherein the plant material is initially extracted with carbon dioxide under liquid conditions to extract unwanted lipophilic material such as fatty acids, and then subsequently extracted with carbon dioxide under supercritical conditions to afford an extract containing elevated levels of compound 1, whilst leaving unwanted steroid glycosides, for example compounds 3-13 with the plant matrix. 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWING 
         [0028]    The accompanying drawing shows, purely by way of illustration and without limitation, a suitable carbon dioxide extraction system for use in performing the present invention. 
       
    
    
     DETAILED DESCRIPTION OF THE DRAWING 
       [0029]    A suitable extraction unit (see drawing) consists of extractor and separator vessels as well as of different heat exchangers, pumps, regulation valves and devices. The extractor vessel is filled with the raw material which is to be treated. The required pressure is achieved through a solvent which flows in from a tank. For increasing the pressure, a pump is necessary, which also takes over the transport of the solvent after the required extraction pressure has been achieved. The pressure is maintained by an overflow valve which opens when the required pressure is exceeded and transports the enriched solvent to the separator step. Usually, this separation step is connected with the storage tank of the solvent. The pressure in the separator step and in the storage tank will consequently be the same (with the exception of flow losses) and will correspond to the saturation (evaporation) pressure of the solvent at the respective temperature. 
       EXAMPLES 
       [0030]    The following Examples illustrate, without limitation, the selective extraction of steroidal glycosides from plants of the Asclepiadaceae family using carbon dioxide as the solvent. 
       Example 1  
       [0031]    Dried  Hoodia gordonii  plant material was milled to a fine powder (&lt;1 mm). 700 g of milled material was packed into a single extractor column and extracted with CO 2  at 300 bar and 55° C. Table 1 indicates the quantity of extract obtained during the extraction, as well as the results of the HPLC analysis of the extracts. 
         [0000]    
       
         
               
             
               
               
               
               
               
             
               
               
               
               
               
             
           
               
                 TABLE 1 
               
             
             
               
                   
               
               
                 Yield and composition during the extraction of dried  Hoodia gordonii   
               
               
                 plant material with carbon dioxide at 300 bar and 55° C. 
               
             
          
           
               
                 Extraction 
                   
                   
                   
                 Content of 
               
               
                 Time 
                   
                   
                   
                 compound 
               
               
                 (hours) 
                 Yield (g) 
                 Total Yield (g) 
                 Total Yield % 
                 1 (%) 
               
               
                   
               
             
          
           
               
                 0.5 
                 18.53 
                 18.53 
                 2.65 
                 0.6 
               
               
                 1.0 
                 7.70 
                 26.23 
                 3.75 
                 1.3 
               
               
                 2.0 
                 6.88 
                 33.11 
                 4.73 
                 1.1 
               
               
                 3.0 
                 5.28 
                 38.39 
                 5.48 
                 1.4 
               
               
                 4.0 
                 1.21 
                 39.60 
                 5.66 
                 3.0 
               
               
                 5.0 
                 2.26 
                 41.86 
                 5.98 
                 2.3 
               
               
                 6.0 
                 0.53 
                 42.39 
                 6.06 
                 Not determined 
               
               
                   
               
             
          
         
       
     
       Example 2  
       [0032]    Dried  Hoodia gordonii  plant material was milled to a fine powder. Particle size analysis of this material showed that 85% passed a 600 μm mesh. 700 g of milled material was packed into a single extractor column and extracted sequentially with CO 2  at increasing pressure and temperature. Table 2 indicates the quantity of extract obtained at the four experimental conditions, as well as the results of the HPLC and GC analysis of the extracts. 
         [0000]    
       
         
               
             
               
               
               
               
               
               
             
               
               
               
               
               
               
             
           
               
                 TABLE 2 
               
             
             
               
                   
               
               
                 Yield and composition under increasing pressure and 
               
               
                 temperature conditions of carbon dioxide 
               
             
          
           
               
                   
                   
                 % of nine 
                   
                   
                   
               
               
                   
                   
                 identified 
                 % 
               
               
                   
                 Extracted 
                 steroid 
                 of com- 
                 % tri- 
                 % hydro- 
               
               
                   
                 weight (g) 
                 glycosides 
                 pound 1 
                 glycerides 
                 carbons 
               
               
                   
               
             
          
           
               
                  60 bar 10° C. 
                 5.000 
                 10.9 
                 1.2 
                 39.3 
                 1.4 
               
               
                 100 bar 
                 13.510 
                 43 
                 0.7 
                 17.2 
                 1.3 
               
               
                 25° C.-35° C. 
               
               
                 200 bar 35° C. 
                 1.600 
                 36.5 
                 8.8 
                 11.0 
                 14.8 
               
               
                 300 bar 55° C. 
                 13.290 
                 19.2 
                 4.6 
                 17.9 
                 7.9 
               
               
                   
               
             
          
         
       
     
       Example 3  
       [0033]    A series of extractions were performed using standardised conditions of extraction of milled  Hoodia gordonii  plant material with carbon dioxide at 100 bar at 25° C.-35° C. (LCO 2 ) for 7 hours, followed by extraction at 300 bar at 55° C. (SCO 2 ) for 7 hours. The two extracts obtained were analysed by HPLC to determine that levels of steroid glycosides. Table 3 shows the quantity of input material per batch and the percentage of compound 1 in the two extracts obtained for each of the extraction experiments. 
         [0034]    The input batches of dried  Hoodia gordonii  contain 0.06-0.2% of compound 1. Extraction with liquid carbon dioxide (LCO2) affords an extract in a yield of 0.71-1.94% that contains 0.6-2.7% of compound 1. Compounds 3-13 were not detected in the product. Sequential extraction with supercritical carbon dioxide (SCO2) affords a yellow-green powder which has no bitter taste in a yield of 0.67-2.34% that contains 2.2-5.7% of compound 1. Again compounds 3-13 were not detected in the product. 
         [0000]    
       
         
               
             
               
               
             
               
               
               
               
               
               
               
               
               
             
               
               
             
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 3 
               
               
                   
               
             
             
               
                 The yield and content of compound 1 obtained in the extraction of 
               
               
                 dried  Hoodia gordonii  plant material with sequential liquid and 
               
               
                 supercritical carbon dioxide 
               
             
          
           
               
                   
                 Experiment number 
               
             
          
           
               
                   
                   
                 47/091 
                 47/093 
                 47/094 
                 47/095 
                 47/096 
                 47/098 
                 47/099 
               
               
                   
               
               
                 Plant 
                 Input weight 
                 693 
                 530.2 
                 731.5 
                 743.3 
                 710.1 
                 731.4 
                 742.3 
               
               
                 material 
                 (g) 
               
               
                   
                 % compound 
                 0.20 
                 0.20 
                 0.20 
                 0.14 
                 0.14 
                 0.20 
                 0.20 
               
               
                   
                 1 in input 
               
               
                 LCO2 
                 Mass 
                 8.71 
                 4.76 
                 7.67 
                 9.18 
                 6.13 
                 6.32 
                 9.23 
               
               
                   
                 obtained (g) 
               
               
                   
                 % yield 
                 1.26 
                 0.90 
                 1.05 
                 1.24 
                 0.86 
                 0.86 
                 1.24 
               
               
                   
                 % compound 
                 2.7 
                 2.0 
                 1.4 
                 1.0 
                 1.9 
                 1.4 
                 1.3 
               
               
                   
                 1 in LCO2 
               
               
                 SCO2 
                 Mass 
                 8.00 
                 7.60 
                 17.13 
                 14.20 
                 7.20 
                 10.70 
                 10.30 
               
               
                   
                 obtained (g) 
               
               
                   
                 % yield 
                 1.15 
                 1.43 
                 2.34 
                 1.91 
                 1.01 
                 1.46 
                 1.39 
               
               
                   
                 % compound 
                 5.4 
                 5.0 
                 5.2 
                 4.6 
                 4.5 
                 5.7 
                 5.6 
               
               
                   
                 1 in SCO2 
               
               
                   
               
             
          
           
               
                   
                 Experiment number 
               
             
          
           
               
                   
                   
                 47/100 
                 47/101 
                 47/102 
                 47/103 
                 47/104 
                 47/110 
               
               
                   
               
               
                 Plant 
                 Input weight 
                 736.3 
                 755.3 
                 733.1 
                 790.4 
                 785 
                 730 
               
               
                 material 
                 (g) 
               
               
                   
                 % compound 
                 0.16 
                 0.16 
                 0.20 
                 0.20 
                 0.06 
                 0.19 
               
               
                   
                 1 in input 
               
               
                 LCO2 
                 Mass 
                 7.98 
                 7.48 
                 8.2 
                 6.02 
                 5.61 
                 14.15 
               
               
                   
                 obtained (g) 
               
               
                   
                 % yield 
                 1.08 
                 0.99 
                 1.12 
                 0.76 
                 0.71 
                 1.94 
               
               
                   
                 % compound 
                 0.8 
                 1.4 
                 1.1 
                 1.7 
                 0.6 
                 0.7 
               
               
                   
                 1 in LCO2 
               
               
                 SCO2 
                 Mass 
                 11.00 
                 10.60 
                 14.14 
                 11.48 
                 5.27 
                 9.80 
               
               
                   
                 obtained (g) 
               
               
                   
                 % yield 
                 1.49 
                 1.40 
                 1.93 
                 1.45 
                 0.67 
                 1.34 
               
               
                   
                 % compound 
                 4.7 
                 4.6 
                 4.2 
                 5.2 
                 2.2 
                 3.2 
               
               
                   
                 1 in SCO2 
               
               
                   
               
             
          
         
       
     
       Example 4  
       [0035]    A series of extractions were performed using standardised conditions of extraction of milled  Hoodia gordonii  plant material with carbon dioxide at a pressure of 100 bar. Material was milled using a coffee grinder and sieved to between 106 and 600 μm. The ground material was dried at 70° C. for a minimum of 16 hours to remove any moisture. The series of experiments used plant material of 650-810 g. The extraction pressure was maintained at 100 bar with a CO 2  flow rate of 5 kg/h for an extraction period of 7 hours. The product was removed from the separator by washing with methanol. The methanol washings were evaporated to dryness to obtain an accurate value for the extraction mass yield. The extraction efficiency of compound 1 was obtained by HPLC analysis of the methanol washings and compared to the amount of compound 1 in the input material. Table 4 shows the quantity of input material per batch and the percentage of compound 1 in the extracts obtained for each of the extraction experiments where the extraction temperature was varied from 5 to 75° C. Compounds 3-13 were not detected in the product. 
         [0000]    
       
         
               
             
               
               
               
               
               
             
               
               
               
               
               
             
           
               
                 TABLE 4 
               
             
             
               
                   
               
               
                 The extraction efficiency for compound 1 obtained in the extraction of 
               
               
                 dried  Hoodia gordonii  plant material at a variety of temperatures with 
               
               
                 liquid or supercritical carbon dioxide at a fixed pressure of 100 bar 
               
             
          
           
               
                 Charge 
                   
                 Mass of 
                 Compound 
                 Extraction 
               
               
                 weight 
                 Temperature 
                 extract 
                 1 in 
                 efficiency 
               
               
                 (g) 
                 (° C.) 
                 (g) 
                 extract (%) 
                 for compound 1 (%) 
               
               
                   
               
             
          
           
               
                 807.6 
                 5 
                 9.69 
                 2.75 
                 18.33 
               
               
                 717.4 
                 15 
                 6.60 
                 2.71 
                 13.85 
               
               
                 707.4 
                 25 
                 8.77 
                 1.53 
                 10.54 
               
               
                 712.5 
                 35 
                 8.69 
                 0.46 
                 3.12 
               
               
                 725.7 
                 40 
                 11.97 
                 0.15 
                 1.65 
               
               
                 723.7 
                 45 
                 3.62 
                 0 
                 0.00 
               
               
                 657.6 
                 55 
                 0.59 
                 0.14 
                 0.07 
               
               
                   
               
             
          
         
       
     
       Example 5  
       [0036]    A series of extractions were performed on milled  Hoodia gordonii  plant material with carbon dioxide at a pressure of 300 bar. Material was milled using a hammermill passing through a 2 mm mesh sieve. The extraction pressure was maintained at 300 bar with a CO 2  feed ratio of 100 kg CO 2/ kg input for an extraction period of ca 7 hours. The extraction efficiency of compound 1 was obtained by HPLC analysis of the spent plant material and compared to the amount of compound 1 in the input material. Table 5 shows the quantity of input material per batch and the extraction efficiency of compound 1 for each of the extraction experiments where the extraction temperature was varied from 0 to 90° C. Compounds 3-13 were not detected in the product. 
         [0000]    
       
         
               
             
               
               
               
             
               
               
               
             
           
               
                 TABLE 5 
               
             
             
               
                   
               
               
                 The extraction efficiency for compound 1 obtained in the extraction of 
               
               
                 dried  Hoodia gordonii  plant material at a variety of temperatures with 
               
               
                 liquid or supercritical carbon dioxide at a fixed pressure of 300 bar 
               
             
          
           
               
                 Charge 
                 Temperature 
                 Extraction efficiency 
               
               
                 weight (g) 
                 (° C.) 
                 for compound 1 (%) 
               
               
                   
               
             
          
           
               
                 87.54 
                 0 
                 27 
               
               
                 88.55 
                 20 
                 31 
               
               
                 87.52 
                 40 
                 48 
               
               
                 85.92 
                 60 
                 53 
               
               
                 85.02 
                 80 
                 59 
               
               
                 84.75 
                 90 
                 60 
               
               
                   
               
             
          
         
       
     
       Example 6  
       [0037]    A series of extractions were performed on milled  Hoodia gordonii  plant material with supercritical carbon dioxide exploring the effects of high temperatures and pressures, the input particle size, and the presence of co-solvents. Material was milled using a hammermill passing through either a 0.7, 1 or 2 mm mesh sieve. The extraction pressure was maintained set at either 300 or 500 bar with a CO 2  feed ratio of 100 kg CO 2 /kg input for an extraction period of ca 7 hours. The extraction efficiency of compound 1 was obtained by HPLC analysis of the spent plant material and compared to the amount of compound 1 in the input material. Table 6 shows the quantity of input material per batch and the extraction efficiency of compound 1 for each of the extraction experiments. Compounds 3-13 were not detected in the product. 
         [0000]    
       
         
               
             
               
               
               
               
               
               
             
               
               
               
               
               
               
             
           
               
                 TABLE 6 
               
             
             
               
                   
               
               
                 The extraction efficiency for compound 1 obtained in the 
               
               
                 extraction of dried  Hoodia gordonii  plant material at a 
               
               
                 variety of temperatures, pressures, input particle size with 
               
               
                 supercritical carbon dioxide in the presence or absence 
               
               
                 of co-solvents 
               
             
          
           
               
                   
                   
                   
                   
                   
                 Extraction 
               
               
                   
                 Pre- 
                   
                   
                   
                 efficiency 
               
               
                   
                 treatment 
                   
                   
                 Presence 
                 for 
               
               
                 Charge 
                 sieve size 
                 Temperature 
                 Pressure 
                 of 
                 compound 1 
               
               
                 weight (g) 
                 (mm) 
                 (° C.) 
                 (bar) 
                 co-solvent 
                 (%) 
               
               
                   
               
             
          
           
               
                 85.02 
                 2 
                 80 
                 300 
                 None 
                 59 
               
               
                 84.92 
                 1 
                 80 
                 300 
                 None 
                 63 
               
               
                 84.41 
                 0.7 
                 80 
                 300 
                 None 
                 70 
               
               
                 42.08 
                 2 
                 80 
                 300 
                 None 
                 62 
               
               
                 85.76 
                 1 
                 80 
                 300 
                 5% EtOH 
                 80 
               
               
                 84.29 
                 1 
                 80 
                 500 
                 None 
                 81 
               
               
                 83.23 
                 0.7 
                 80 
                 500 
                 None 
                 83 
               
               
                 82.88 
                 0.7 
                 80 
                 500 
                 5% EtOH 
                 88 
               
               
                 83.86 
                 0.7 
                 80 
                 500 
                 None 
                 78 
               
               
                 84.08 
                 0.7 
                 80 
                 500 
                 5% EtOH 
                 84 
               
               
                 77.14 
                 0.7 
                 80 
                 500 
                 None 
                 84 
               
               
                 61.76 
                 0.7 
                 80 
                 500 
                 5% EtOH 
                 84 
               
               
                 59.34 
                 0.7 
                 100 
                 500 
                 None 
                 84 
               
               
                 78.33 
                 0.7 
                 80 
                 500 
                 5% EtOH 
                 86 
               
               
                 65.45 
                 0.7 
                 80 
                 500 
                 5% EtON 
                 80 
               
               
                   
               
             
          
         
       
     
         [0038]    The foregoing broadly describes the present invention, without limitation. Variations and modifications as will be readily apparent to those of ordinary skill in the art are intended to be included in the scope of this application and subsequent patent(s).