Method for obtaining a natural mixture of conjugated equine estrogens depleted in non-conjugated lipophilic compounds

Abstract

A method for obtaining an extract containing the natural mixture of conjugated equine estrogens by liquid-liquid extraction of the mixture of conjugated equine estrogens, wherein the mixture obtained is depleted in non-conjugated lipophilic compounds selected from the group consisting of non-conjugated flavonoids, non-conjugated isoflavonoids, non-conjugated norisoprenoids, non-conjugated steroids, in particular androstane and pregnane steroids, and comparable non-conjugated compounds.

What is claimed is:  
     
         1 . A method for obtaining a natural mixture of conjugated equine estrogens depleted in non-conjugated lipophilic compounds, said method comprising: 
 a) subjecting an aqueous initial phase, selected from the group consisting of 
 (i) an aqueous solution of a natural mixture of conjugated estrogens from pregnant mares' urine already depleted in phenolic urine contents;  
 (ii) an aqueous concentrate of a natural mixture of conjugated estrogens from pregnant mares' urine already depleted in phenolic urine contents;  
 (iii) a concentrate of a urine liquid, and  
 (iv) a urine concentrate, optionally pre-purified by filtration, to a liquid-liquid extraction with a sufficient quantity of an organic solvent extracting agent which is suitable for extracting non-conjugated lipophilic compounds and is at most only slightly miscible with water, and subsequently separating a resulting aqueous phase;  
   b) optionally repeating method step (a) with the resulting aqueous phase, and    c) recovering an aqueous phase containing the natural mixture of conjugated estrogens.    
     
     
         2 . A method according to  claim 1 , further comprising concentrating the recovered aqueous phase containing the natural mixture of conjugated estrogens.  
     
     
         3 . A method according to  claim 1 , wherein the non-conjugated lipophilic compounds are selected from the group consisting of non-conjugated flavonoids, non-conjugated isoflavonoids, non-conjugated norisoprenoids, non-conjugated steroids, and comparable non-conjugated compounds.  
     
     
         4 . A method according to  claim 1 , wherein the non-conjugated lipophilic compounds comprise steroids selected from the group consisting of androstane and pregnane steroids.  
     
     
         5 . A method according to  claim 1 , wherein said extracting agent is immiscible with water.  
     
     
         6 . A method according to  claim 1 , wherein the aqueous initial phase in method step a) comprises (i) an aqueous solution of a natural mixture conjugated estrogens from pregnant mares' urine already depleted in phenolic urine contents, or (ii) an aqueous concentrate of a natural mixture of conjugated estrogens from pregnant mares' urine already depleted in phenolic urine contents.  
     
     
         7 . A method according  claim 1 , wherein the extracting agent is selected from the group consisting of straight-chain, branched or cyclic C 4 -C 10  alcohols, C 2 -C 10  esterified acids, C 3 -C 10  aldehydes, C 4 -C 10  ketones, C 2 -C 10  ethers, C 3 -C 6  nitrites, C 1 -C 3  haloalkanes, and mixtures two or more of the aforementioned solvents.  
     
     
         8 . A method according to  claim 7 , wherein the extracting agent is selected from the group consisting of C 1 -C 4 -alkyl acetates, hexanol, diethyl ether, methylene chloride, methyl tert.-butyl ether and mixtures of two or more of the aforementioned solvents.  
     
     
         9 . A method according to  claim 8 , wherein the extracting agent comprises a C 1 -C 4 -alkyl acetate.  
     
     
         10 . A method according to  claim 9 , wherein the extracting agent is ethyl acetate.  
     
     
         11 . A method according to  claim 1 , wherein in method step (a) the aqueous initial phase is adjusted to a pH value in the range from 4 to 12.  
     
     
         12 . A method according to  claim 11 , wherein the pH value in step (a) is in the range from 4.0 to 7.0.  
     
     
         13 . A method according to  claim 12 , wherein the pH value in step (a) is in the range from 4.0 to 6.0.  
     
     
         14 . A method according to  claim 13 , wherein the pH value in step (a) is in the range from 4.7 to 5.3.  
     
     
         15 . A method according to  claim 1 , wherein in method step (a) the volume ratio of the aqueous initial phase to the extracting agent lies in the range from 5:1 to 1:3.  
     
     
         16 . A method according to  claim 15 , wherein the volume ratio of the aqueous initial phase to the extracting agent lies in the range from 2:1 to 1:2.  
     
     
         17 . A method according to  claim 1 , wherein in method step (b) the volume ratio of the aqueous initial phase obtained from step (a) to the extracting agent lies in the range from 20:1 to 1:1.  
     
     
         18 . A method according to  claim 17 , wherein in step (b) the volume ratio of the aqueous initial phase obtained from step (a) to the extracting agent lies in the range from 10:1 to 2:1.