WO2001027134A1 - Process for extracting estrogens from pregnant mare urine (pmu) - Google Patents
Process for extracting estrogens from pregnant mare urine (pmu) Download PDFInfo
- Publication number
- WO2001027134A1 WO2001027134A1 PCT/US1999/023730 US9923730W WO0127134A1 WO 2001027134 A1 WO2001027134 A1 WO 2001027134A1 US 9923730 W US9923730 W US 9923730W WO 0127134 A1 WO0127134 A1 WO 0127134A1
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- Prior art keywords
- pregnant mare
- mare urine
- process according
- salt
- urine
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07J—STEROIDS
- C07J9/00—Normal steroids containing carbon, hydrogen, halogen or oxygen substituted in position 17 beta by a chain of more than two carbon atoms, e.g. cholane, cholestane, coprostane
Definitions
- the present invention relates to a process for extracting estrogens from pregnant mare urine (PMU).
- Conjugated estrogens are effective drugs for treating the symptoms of menopause.
- the natural source of conjugated estrogens may come from the extraction of pregnant mare urine (PMU).
- PMU pregnant mare urine
- the method primarily utilizes a solid-liquid extraction to separate the desired conjugated estrogens from pregnant mare urine.
- the disadvantages of this method lie in the use of a column separation.
- the Amberlite resin packed in the column has limited life and high cost. Also, the process is time- consuming. In addition, the amount of pregnant mare urine which can be treated is quite limited.
- the present invention provides a process for extracting estrogens from pregnant mare urine (PMU).
- PMU pregnant mare urine
- the operation of the present invention is simple and efficient. Moreover, the extraction efficiency is high, which enables convenient transportation of pregnant mare urine, and is economical. Finally, the amount of conjugated estrogens recovered from the extract of pregnant mare urine is high.
- the present invention provides a process for extracting estrogens from pregnant mare urine (PMU). It involves the addition of a salt and a liquid-liquid extraction using an organic solvent which is immiscible with pregnant mare urine.
- the present invention provides a process for extracting estrogens from pregnant mare urine (PMU) comprising the following steps:
- step (b) conducting a liquid-liquid extraction of the pregnant mare urine of step (a) by using an organic solvent which is immiscible with the pregnant mare urine, and
- the main principles of the present invention essentially lie in the addition of a salt to lower the solubility of conjugated estrogens in an aqueous phase, and the liquid-liquid extraction to partition the majority of conjugated estrogens into the organic phase. Therefore, the conjugated estrogens are separated with proteins and phenolic materials abundant in the aqueous phase and a primary purification is accomplished. The ionic strength of the aqueous phase is changed due to the presence of salt. This- leads to the formation of chemical bonds between conjugated estrogens and cations.
- the pregnant mare urine may be pre-treated in a known manner to remove solid impurities, such as by a filtration or a centrifugation.
- the salt suitable for use in step (a) can be any organic or inorganic salt which is known to persons skilled in the art.
- the salt when the salt is an organic salt, it can be selected from the group consisting of terra ammonium salts and mixtures thereof; when the salt is an inorganic salt, it can be, for example, one expressed in the formula M x A y , wherein M is selected from the group consisting of alkali metals and earth alkali metals; A is selected from the group consisting of halogen, hydroxyl, a carbonate ion, a sulfate ion, a sulfite ion, a phosphate ion and a nitrate ion; each of x and y represents a positive integer of 1, 2 or 3.
- the salt is selected from the group consisting of sodium chloride, potassium chloride, magnesium chloride, sodium hydroxide, potassium hydroxide, magnesium hydroxide, sodium sulfate, potassium sulfate, magnesium sulfate, tetramethyl ammonium chloride, tetraethyl ammonium chloride and mixtures thereof.
- Step (b) is a liquid-liquid extraction step carried out by using an organic solvent which is immiscible with the pregnant mare urine.
- the organic solvent suitable for use in step (b) can be any one known to person skilled in the art, such as that selected from the group consisting of C 4 -C 6 alkanes, C ⁇ -C 6 haloalkanes, C -C 8 ethers, C 3 -C 8 ketones, C 2 -C 6 esters, C 4 - C 8 alcohols, cyclic C 4 -C 8 alcohols, cyclic C 3 -C 8 ketones, C C 8 amides, C 6 - C 12 aryl hydrocarbons, furans, pyridines, nitriles and mixtures thereof.
- the organic solvent is selected from the group consisting of ethyl acetate, acetone, tetrahydrofuran (THF), acetonitrile, cyclohexanol and mixtures thereof.
- the amount of the salt in step (a) can be that which allows its concentration in pregnant mare urine to reach the range of 0.5 M to a saturated concentration, preferably 1 M to a saturated concentration.
- Step (a) can be conducted at a temperature range of 5 ° C to the temperature at which the solubility of the salt is optimized.
- step (a) is carried out at a temperature of 5°C to 55 ° C .
- Step (b) can be carried out under neutral to basic conditions.
- the pH value of the solution is adjusted to a range between 7 and 9.
- the ratio of the organic solvent and the pregnant mare urine of step (b) can be within the range of 1 : 1 to 8: 1, preferably 1 : 1 to 6: 1.
- a surfactant conventionally known to persons skilled in the art can be incorporated into step (b) to eliminate the foaming condition and the emulsion layer which occurs during the liquid-liquid extraction.
- Surfactants suitable for use in the process of the present invention can be cationic, anionic or amophoteric surfactants.
- Preferred surfactants are quaternary ammonium salts or sulfonic salts.
- the extract of pregnant mare urine obtained from the process of the present invention can be subjected to a series of purification and separations by conventional methods to separate the conjugated estrogens contained therein.
- the recovery rate of the conjugated estrogens from the concentrate of pregnant mare urine in accordance with the process of the present invention can be from 30% to 90%.
- An advantage of the present invention is that its operation is easy and efficient.
- the extraction efficacy is good, which enables convenient transportation of pregnant mare urine and is economical.
- the amount of conjugated estrogens recovered from the extract of pregnant mare urine is high.
- 2 M sodium chloride solution is prepared by adding 116 g of sodium chloride into 1 liter of pregnant mare urine at 25°C . The solution is well stirred. When the precipitation is balanced, 2 liter of ethyl acetate is added into the solution. The ratio of organic phase to aqueous phase is 2: 1. A liquid-liquid extraction is conducted. The organic phase is separated and the organic solvent is removed. Then, the residue is dried. The dried material is dissolved in a mixed solution of acetonitrile and water. HPLC analysis reveals that the dried material contains 73% of conjugated estrogens originally contained in the pregnant mare urine.
- Example 1 The procedure of Example is repeated under the conditions shown in Table 1 recited below.
- the recovery rate is also given in Table 1.
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- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)
- Steroid Compounds (AREA)
Abstract
The present invention relates to a process for extracting estrogens from pregnant mare urine (PMU) comprising the following steps: (a) adding a salt to the pregnant mare urine, (b) conducting a liquid-liquid extraction of the pregnant mare urine of step (a) by using an organic solvent which is immiscible with the pregnant mare urine, and (c) separating the organic extract and drying it to form an extract. The operation of the present invention is easy and efficient. The concentrating efficiency is high, which enables convenient transportation of pregnant mare urine and is economical. In addition, the amount of conjugated estrogens recovered from the extract of pregnant mare urine is high.
Description
PROCESS FOR EXTRACTING ESTROGENS FROM PREGNANT
MARE URINE fPMin
FIELD OF THE INVENTION
The present invention relates to a process for extracting estrogens from pregnant mare urine (PMU).
BACKGROUND OF THE INVENTION
Conjugated estrogens are effective drugs for treating the symptoms of menopause. The natural source of conjugated estrogens may come from the extraction of pregnant mare urine (PMU). U.S. Patent No. 2,834,712, issued to American Home Products
Company in 1958, discloses a process for preparing substantially pure estrogenic mixtures with low toxicity. Said process primarily utilizes the principle that substances will exhibit different solubilities in different solutions under different pH values to carry out the liquid-liquid extraction. The purification of pregnant mare urine thereby separates estrogenic mixtures from pregnant mare urine. The procedure disclosed in this patent is quite complicated and the solvents added in the process (such as benzene and hexane) are not suitable for pharmaceutical formulations. In addition, the amount of estrogenic mixtures recovered from pregnant mare urine is limited.
U.S. Patent Nos. 5,723,454 and 5,814,624, issued to Solvay Deutschland GmbH in 1998, discloses a method for obtaining a natural mixture of conjugated estrogens from pregnant mare urine. The method primarily utilizes a solid-liquid extraction to separate the desired conjugated estrogens from pregnant mare urine. The disadvantages of this method lie in the use of a column separation. The Amberlite resin packed in the column has limited life and high cost. Also, the process is time-
consuming. In addition, the amount of pregnant mare urine which can be treated is quite limited.
To overcome the problems encountered in the art as delineated in the above, the present invention provides a process for extracting estrogens from pregnant mare urine (PMU). The operation of the present invention is simple and efficient. Moreover, the extraction efficiency is high, which enables convenient transportation of pregnant mare urine, and is economical. Finally, the amount of conjugated estrogens recovered from the extract of pregnant mare urine is high.
SUMMARY OF THE INVENTION
The present invention provides a process for extracting estrogens from pregnant mare urine (PMU). It involves the addition of a salt and a liquid-liquid extraction using an organic solvent which is immiscible with pregnant mare urine.
DETAILED DESCRIPTION OF THE INVENTION
The present invention provides a process for extracting estrogens from pregnant mare urine (PMU) comprising the following steps:
(a) adding a salt to the pregnant mare urine,
(b) conducting a liquid-liquid extraction of the pregnant mare urine of step (a) by using an organic solvent which is immiscible with the pregnant mare urine, and
(c) separating the organic extract and drying it to form an extract. The main principles of the present invention essentially lie in the addition of a salt to lower the solubility of conjugated estrogens in an aqueous phase, and the liquid-liquid extraction to partition the majority of conjugated estrogens into the organic phase. Therefore, the conjugated estrogens are separated with proteins and phenolic materials abundant in
the aqueous phase and a primary purification is accomplished. The ionic strength of the aqueous phase is changed due to the presence of salt. This- leads to the formation of chemical bonds between conjugated estrogens and cations. A small amount of covalent materials precipitate in the aqueous solution, while the majority of conjugated estrogens selectively enter the organic phase which is immiscible with pregnant mare urine and is separated from proteins and phenolic compounds. After the organic phase is separated from the aqueous phase, the organic solvent is evaporated. An extract of pregnant mare urine with an extremely high concentration of conjugated estrogens is thus obtained.
In the process of the present invention, the pregnant mare urine may be pre-treated in a known manner to remove solid impurities, such as by a filtration or a centrifugation.
The salt suitable for use in step (a) can be any organic or inorganic salt which is known to persons skilled in the art. When the salt is an organic salt, it can be selected from the group consisting of terra ammonium salts and mixtures thereof; when the salt is an inorganic salt, it can be, for example, one expressed in the formula MxAy, wherein M is selected from the group consisting of alkali metals and earth alkali metals; A is selected from the group consisting of halogen, hydroxyl, a carbonate ion, a sulfate ion, a sulfite ion, a phosphate ion and a nitrate ion; each of x and y represents a positive integer of 1, 2 or 3. Preferably, the salt is selected from the group consisting of sodium chloride, potassium chloride, magnesium chloride, sodium hydroxide, potassium hydroxide, magnesium hydroxide, sodium sulfate, potassium sulfate, magnesium sulfate, tetramethyl ammonium chloride, tetraethyl ammonium chloride and mixtures thereof.
Step (b) is a liquid-liquid extraction step carried out by using an organic solvent which is immiscible with the pregnant mare urine. The
organic solvent suitable for use in step (b) can be any one known to person skilled in the art, such as that selected from the group consisting of C4-C6 alkanes, Cι-C6 haloalkanes, C -C8 ethers, C3-C8 ketones, C2-C6 esters, C4- C8 alcohols, cyclic C4-C8 alcohols, cyclic C3-C8 ketones, C C8 amides, C6- C12 aryl hydrocarbons, furans, pyridines, nitriles and mixtures thereof. Preferably, the organic solvent is selected from the group consisting of ethyl acetate, acetone, tetrahydrofuran (THF), acetonitrile, cyclohexanol and mixtures thereof.
The operating conditions for carrying out the process of the present invention can be determined and adjusted by persons skilled in the art based on their own knowledge in this field under the guidelines of published patents and the disclosure of the present invention. For example, the amount of the salt in step (a) can be that which allows its concentration in pregnant mare urine to reach the range of 0.5 M to a saturated concentration, preferably 1 M to a saturated concentration. Step (a) can be conducted at a temperature range of 5°C to the temperature at which the solubility of the salt is optimized. Preferably, step (a) is carried out at a temperature of 5°C to 55 °C .
Step (b) can be carried out under neutral to basic conditions. Preferably, the pH value of the solution is adjusted to a range between 7 and 9. Moreover, the ratio of the organic solvent and the pregnant mare urine of step (b) can be within the range of 1 : 1 to 8: 1, preferably 1 : 1 to 6: 1. Optionally, a surfactant conventionally known to persons skilled in the art can be incorporated into step (b) to eliminate the foaming condition and the emulsion layer which occurs during the liquid-liquid extraction. Surfactants suitable for use in the process of the present invention can be cationic, anionic or amophoteric surfactants. Preferred surfactants are quaternary ammonium salts or sulfonic salts.
The extract of pregnant mare urine obtained from the process of the
present invention can be subjected to a series of purification and separations by conventional methods to separate the conjugated estrogens contained therein. Surprisingly, the inventor found that the recovery rate of the conjugated estrogens from the concentrate of pregnant mare urine in accordance with the process of the present invention can be from 30% to 90%.
An advantage of the present invention is that its operation is easy and efficient. The extraction efficacy is good, which enables convenient transportation of pregnant mare urine and is economical. In addition, the amount of conjugated estrogens recovered from the extract of pregnant mare urine is high.
The following embodiments are provided to demonstrate the feasibility of the present invention, to substantiate the technical contents of the invention, and to allow persons skilled in the art to understand the contents of the invention and to practice it. They are not intended to limit the reasonable scope of protection. Various improvements and obvious modifications that can be accomplished easily by persons skilled in the art based on the teachings of conventional techniques are all within the scope of the present invention.
EXAMPLE
Example 1
2 M sodium chloride solution is prepared by adding 116 g of sodium chloride into 1 liter of pregnant mare urine at 25°C . The solution is well stirred. When the precipitation is balanced, 2 liter of ethyl acetate is added into the solution. The ratio of organic phase to aqueous phase is 2: 1. A liquid-liquid extraction is conducted. The organic phase is separated and the organic solvent is removed. Then, the residue is dried. The dried
material is dissolved in a mixed solution of acetonitrile and water. HPLC analysis reveals that the dried material contains 73% of conjugated estrogens originally contained in the pregnant mare urine.
Examples 2 to 19
The procedure of Example is repeated under the conditions shown in Table 1 recited below. The recovery rate is also given in Table 1.
Table 1
Claims
1. A process for extracting estrogens from pregnant mare urine (PMU) comprising the following steps:
(a) adding a salt to the pregnant mare urine, (b) conducting a liquid-liquid extraction of the pregnant mare urine of step (a) by an organic solvent which is immiscible with the pregnant mare urine, and
(c) separating the organic extract and drying it to form an extract.
2. A process according to claim 1, wherein the salt of step (a) is an inorganic salt of the formula MxAy, in which M is selected from the group consisting of alkali metals and earth alkali metals, A is selected from the group consisting of halogen, hydroxyl, a carbonate ion, a sulfate ion, a sulfite ion, a phosphate ion and a nitrate ion, and x and y each represents a positive integer which is 1, 2 or 3.
3. A process according to claim 1, wherein the salt of step (a) is an organic salt, it can be selected from the group consisting of tetra ammonium salts and mixtures thereof.
4. A process according to claim 2 or 3, wherein the salt of step (a) is selected from the group consisting of sodium chloride, potassium chloride, magnesium chloride, sodium hydroxide, potassium hydroxide, magnesium hydroxide, sodium sulfate, potassium sulfate, magnesium sulfate, tetramethyl ammonium chloride, tetraethyl ammonium chloride and mixtures thereof.
5. A process according to claim 1, wherein the amount of the salt in step (a) is that which allows its concentration in pregnant mare urine to reach the range of 0.5 M to a saturated concentration.
6. A process according to claim 4, wherein the amount of the salt in step (a) is that which allows its concentration in pregnant mare urine to- reach the range of 1 M to a saturated concentration.
7. A process according to claim 1, wherein the organic solvent which is immiscible with the pregnant mare urine of step (b) is selected from the group consisting of C -C6 alkanes, C C6 haloalkanes, C2-C8 ethers, C3-C8 ketones, C2-C6 esters, C -C8 alcohols, cyclic C4-C8 alcohols, cyclic C3-C8 ketones, C C8 amides, C6-C12 aryl hydrocarbons, furans, pyridines, nitriles and mixtures thereof.
8. A process according to claim 7, wherein the organic solvent which is immiscible with the pregnant mare urine of step (b) is selected from the group consisting of ethyl acetate, acetone, tetrahydrofuran (THF), acetonitrile, cyclohexanol and mixtures thereof.
9. A process according to claim 1, wherein step (b) is carried out under a neutral to basic condition.
10. A process according to claim 1, wherein the ratio of the organic solvent and the pregnant mare urine of step (b) is in the range of 1 : 1 to 8: 1.
11. A process according to claim 1, which further comprises an additional step, so as to further purify conjugated estrogens from the extract.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| AU64269/99A AU6426999A (en) | 1999-10-13 | 1999-10-13 | Process for extracting estrogens from pregnant mare urine (pmu) |
| PCT/US1999/023730 WO2001027134A1 (en) | 1999-10-13 | 1999-10-13 | Process for extracting estrogens from pregnant mare urine (pmu) |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| PCT/US1999/023730 WO2001027134A1 (en) | 1999-10-13 | 1999-10-13 | Process for extracting estrogens from pregnant mare urine (pmu) |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| WO2001027134A1 true WO2001027134A1 (en) | 2001-04-19 |
Family
ID=22273791
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| PCT/US1999/023730 WO2001027134A1 (en) | 1999-10-13 | 1999-10-13 | Process for extracting estrogens from pregnant mare urine (pmu) |
Country Status (2)
| Country | Link |
|---|---|
| AU (1) | AU6426999A (en) |
| WO (1) | WO2001027134A1 (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2009513561A (en) * | 2003-07-17 | 2009-04-02 | ゾルファイ ファーマスーティカルズ ゲゼルシャフト ミット ベシュレンクテル ハフツング | Process for obtaining equine-bound estrogen natural mixture |
| JP4778233B2 (en) * | 2002-10-11 | 2011-09-21 | ゾルファイ ファーマスーティカルズ ゲゼルシャフト ミット ベシュレンクテル ハフツング | Method for obtaining a natural mixture of conjugated equine estrogens with few unbound lipophilic compounds |
| US8349819B2 (en) | 2002-10-09 | 2013-01-08 | Dr. Reddy's Laboratories New York, Inc. | Steroid extraction process from urine sources |
| US8815299B2 (en) | 2002-10-11 | 2014-08-26 | Abbott Products Gmbh | Method for obtaining a natural mixture of conjugated equine estrogens depleted in non-conjugated lipophilic compounds |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US2711988A (en) * | 1950-06-02 | 1955-06-28 | American Home Prod | Estrogenic sulphates extraction process |
-
1999
- 1999-10-13 WO PCT/US1999/023730 patent/WO2001027134A1/en active Application Filing
- 1999-10-13 AU AU64269/99A patent/AU6426999A/en not_active Abandoned
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US2711988A (en) * | 1950-06-02 | 1955-06-28 | American Home Prod | Estrogenic sulphates extraction process |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US8349819B2 (en) | 2002-10-09 | 2013-01-08 | Dr. Reddy's Laboratories New York, Inc. | Steroid extraction process from urine sources |
| JP4778233B2 (en) * | 2002-10-11 | 2011-09-21 | ゾルファイ ファーマスーティカルズ ゲゼルシャフト ミット ベシュレンクテル ハフツング | Method for obtaining a natural mixture of conjugated equine estrogens with few unbound lipophilic compounds |
| US8815299B2 (en) | 2002-10-11 | 2014-08-26 | Abbott Products Gmbh | Method for obtaining a natural mixture of conjugated equine estrogens depleted in non-conjugated lipophilic compounds |
| JP2009513561A (en) * | 2003-07-17 | 2009-04-02 | ゾルファイ ファーマスーティカルズ ゲゼルシャフト ミット ベシュレンクテル ハフツング | Process for obtaining equine-bound estrogen natural mixture |
| JP4691028B2 (en) * | 2003-07-17 | 2011-06-01 | ゾルファイ ファーマスーティカルズ ゲゼルシャフト ミット ベシュレンクテル ハフツング | Process for obtaining equine-bound estrogen natural mixture |
| US7964586B2 (en) | 2003-07-17 | 2011-06-21 | Abbott Products Gmbh | Method for obtaining a natural mixture of conjugated equine estrogens |
Also Published As
| Publication number | Publication date |
|---|---|
| AU6426999A (en) | 2001-04-23 |
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