Method for obtaining a natural mixture of conjugated equine estrogens
A method for obtaining an extract containing the natural mixture of conjugated equine estrogens in which a mixture of conjugated estrogens obtained by solid-phase extraction from pregnant mares' urine is depleted in phenolic urine contents and 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.
1. A method for obtaining a natural mixture of conjugated estrogens from pregnant mares' urine, comprising:
contacting a urine with an amount of a polymeric adsorption resin sufficient to adsorb the mixture of conjugated estrogens contained in the urine, and separating a resulting polymeric adsorption resin loaded with the mixture of conjugated estrogens from the rest of the urine;
washing the polymeric adsorption resin loaded with the mixture of conjugated estrogens with a washing water which has been adjusted to a pH value of at least 12.0;
contacting the washed adsorption resin with a sufficient amount of an elution liquid to desorb the mixture of conjugated estrogens loaded thereon,
wherein the elution liquid is a one-phase or two phase mixture containing:
(i) water,
(ii) at least one water-immiscible or only slightly water-miscible organic solvent suitable for the elution of the non-conjugated lipophilic compounds selected from the group consisting of non-conjugated flavonoids, non-conjugated isoflavonoids, non-conjugated norisoprenoids, and non-conjugated steroids, and which organic solvent is selected from the group consisting of straight-chain, branched or cyclic C 4 -C 10 alcohols, C 1 -C 10 esterified acids, C 3 -C 10 aldehydes, C 4 -C 10 ketones, C 3 -C 10 ethers, C 3 -C 6 nitriles, C 1 -C 3 haloalkanes, and mixtures of two or more of the foregoing solvents; and
(iii) at least one water-miscible organic solvent selected from the group consisting of water-miscible ethers, lower alkanols, lower aliphatic ketones, and mixtures of two or more of the foregoing solvents;
separating an eluate containing the natural mixture of conjugated estrogens from the adsorption resin;
wherein the natural mixture of conjugated equine estrogens obtained is depleted in phenolic urine contents and in non-conjugated lipophilic compounds selected from the group consisting of non-conjugated flavonoids, non-conjugated isoflavonoids, non-conjugated norisoprenoids, and non-conjugated steroids;
and if the separated eluate is a two-phase mixture, further comprising separating the phases and recovering an aqueous phase containing the natural mixture of conjugated estrogens.
2. A method according to claim 1 , wherein the starting urine is a urine freed of mucilaginous substances and solids, a reduced concentrate of such a urine, or a reduced urine retentate obtained by membrane filtration of such a urine.
3. A method according to claim 1 , wherein the polymeric adsorption resin loaded with the mixture of conjugated estrogens subjected to an intermediate washing with water between the washing with water which has been adjusted to a pH of at least 12.0 and the contacting with the elution liquid.
4. A method according to claim 1 , wherein the water in the elution liquid is adjusted to a pH value in the alkaline range.
5. A method according to claim 1 , further comprising reducing the volume of the separated eluate.
6. A method according to claim 1 , wherein the organic solvent in (ii) is selected from the group consisting of C 1 -C 4 -alkyl acetates, butanol, cyclohexanol, hexanol, diethyl ether, methylene chloride, methyl tert, butyl ether, and mixtures of two or more of the forgoing.
7. A method according to claim 6 , wherein the organic solvent in (ii) is selected from the group consisting of C 1 -C 4 -alkyl acetates and mixtures thereof.
8. A method according to claim 7 , wherein the organic solvent in (ii) is ethyl acetate, isopropyl acetate, or a mixture thereof.
9. A method according to claim 1 , wherein the water-miscible organic solvent is selected from the group consisting of acetone, ethanol, tetrahydrofuran, and mixtures of two or more of the foregoing.
10. A method according to claim 9 , wherein the water-miscible organic solvent is acetone, ethanol, or a mixture thereof.
11. A method according to claim 10 , wherein the water-miscible organic solvent is acetone.
12. A method according to claim 1 , wherein the organic solvent in (ii) is selected from the group consisting of C 1 -C 4 -alkyl acetates and mixtures thereof, and wherein the water-miscible organic solvent in (iii) is acetone, ethanol or a mixture thereof.
13. A method according to claim 12 , wherein the organic solvent in (ii) is ethyl acetate, isopropyl acetate, or a mixture thereof.
14. A method according to claim 12 , wherein the organic solvent in (ii) is ethyl acetate, and the water-miscible organic solvent in (iii) is acetone.
15. A method according to claim 1 , wherein the elution liquid comprises water and water-immiscible or only slightly water-miscible organic solvent in a volume ratio of water to organic solvent of from 5:1 to 1:5.
16. A method according to claim 15 , wherein the volume ratio of water to water-immiscible or only slightly water-miscible organic solvent is from 2:1 to 1:2.
17. A method according to claim 1 , wherein the elution liquid comprises water and water-miscible organic solvent in a water to water-miscible organic solvent volume ratio of from 4:1 to 1:5.
18. A method according to claim 17 , wherein the water to water-miscible organic solvent ratio is from 2:1 to 1:3.
19. A method according to claim 1 , wherein the water, water-immiscible or slightly water-miscible organic solvent, and water-miscible organic solvent in the elution liquid are present in amounts such that the ratio of the sum of the volumes of the water and water-immiscible or slightly water-miscible organic solvent to the volume of water-miscible organic solvent is from 5:1 to 1:5.
20. A method according to claim 19 , wherein the ratio of the sum of the volumes of the water and the water-immiscible or slightly water-miscible organic solvent to the volume of the water-miscible organic solvent is from 2:1 to 1:2.
21. A method according to claim 12 , wherein the elution liquid comprises water; at least one C 1 -C 4 -alkyl acetate; and acetone, ethanol or a mixture thereof in a volume ratio of from 1:1:1 to 1:1:2.
22. A method according to claim 12 , wherein the volume ratio of water; at least one C 1 -C 4 -alkyl acetate; and acetone, ethanol or a mixture thereof is about 1:1:1.4.
23. A method according to claim 1 , wherein the polymeric adsorption resin is a semipolar adsorption resin.
24. A method according to claim 23 , wherein the polymeric adsorption resin is a non-ionic, semipolar adsorption resin.
25. A method according to claim 1 , wherein the elution of the mixture of conjugated estrogens from the washed adsorption resin is carried out at temperatures in the range from 20° C. to 80° C.
26. A method according to claim 25 , wherein the elution is carried out at temperatures in the range from 30° C. to 50° C.