CN1133639C - Extraction method of combined mixed estrin of pregnant mare - Google Patents
Extraction method of combined mixed estrin of pregnant mare Download PDFInfo
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- CN1133639C CN1133639C CNB011048522A CN01104852A CN1133639C CN 1133639 C CN1133639 C CN 1133639C CN B011048522 A CNB011048522 A CN B011048522A CN 01104852 A CN01104852 A CN 01104852A CN 1133639 C CN1133639 C CN 1133639C
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Abstract
The present invention relates to a method for extracting combined mixed pregnant mare estrogen. Combined mixed pregnant mare estrogen is directly extracted from the urine of a pregnant mare or enriched liquid containing pregnant mare estrogen by utilizing polar macroreticular adsorption resin containing cyano. The macroreticular absorption resin is polar absorption resin prepared by polymerizing divinylbenzene, vinyl cyanide, etc., and has the characteristics of preferable acid and alkali stability, difficult conglomeration, large adsorption quantity of combined pregnant mare estrogen, good acid and alkali tolerance, easy regeneration, etc. The method has the advantages of simple technology, low cost, high yield coefficient and easy industrialized production.
Description
The present invention relates to a kind of from the urine of pregnant mare (abbreviation pregnant horse urine) extract a kind of method of natural combined mixed estrin.
Oestrogenic hormon medically is being applied to Hormone Replacement Therapy, and osteoporosis and coronary heart disease can be prevented and treat to the climacteric syndrome that the combined mixed estrin is used for the treatment of and prevents female pathology or artificial menopause after date to occur.Natural combined mixed estrin has obtained permanent application with its good curative effect and tolerance.
Contain natural combined estrogen mixture in the Gestation period mare urine, be that form with multiple estrogenic sulfuric acid monoester salt is dissolved in the pregnant horse urine, usually contain dry-matter 40-90 gram in every liter of the pregnant horse urine, remove urea, urobenzoic acid and other common inorganic salt, outside organism and the viscous material, the content of general combined mixed estrin is counted the 80-300 mg/litre with sodium salt.
The patent of the combined oestrogenic hormon extracting method of early stage report has United States Patent (USP): US2429398, US 2519516, US 2696265, US 2711988, US 2834712, its extracting method is earlier with low-carbon alcohol, ester, as n-hexyl alcohol, hexalin, propyl carbinol, ethyl acetate, butylacetate etc., or the low-carbon (LC) hydrochloric ether obtains the pregnant Sterones of combined with the purifying removal of impurities more than five to ten steps again as organic solvent extractions such as chloroform, methylene dichloride, ethylene dichloride.Because the problems that occur in liquid-liquid extraction process as phenomenons such as emulsion layer formation, precipitations, cause extracting operational difficulties, the difficulty that particularly is separated causes yield to reduce, and the rapid abstraction purification removal of impurities of multistep also causes the oestrogenic hormon loss, makes total recovery very low.The dissolved extraction agent easily causes environmental pollution in the waste in addition.
Begin from U.S. Pat 2551205 report active carbon absorption technologies, successively there are US3769401, US 5723454, US 5814624 to report that ion exchange resin, polyacrylic ester polymeric adsorbent, reverse phase silica gel are applied to the estrogenic extraction of combined, wherein therefore ion exchange resin used very difficult because active principle is difficult to wash-out and the very difficult regeneration of resin from resin.Reverse phase silica gel causes industrial cost's height because of having regeneration difficulty and material expensive.Though Semi-polarity polyacrylate(s) polymeric adsorbent can be avoided the problems referred to above, but exist chemical stability not high, wash-out, regenerated acid, base strength there is requirement, if the improper hydrolysis of ester group that causes of condition, this resin is easy to conglomeration and becomes piece in application in addition, thereby in application, have the resin absorption degradation, cause yield to reduce.The present invention is directed to present existing problem, and, develop polar macroporous netted polymeric adsorbent that a kind of utilization contains cyano group and be used for directly or from the pregnant solution that contains pregnant Sterones, extract the method for the pregnant Sterones of combined from pregnant horse urine through a large amount of experimental study.
The object of the invention is, the extraction method of combined mixed estrin of pregnant mare of developing is to utilize the polar macroporous netted polymeric adsorbent contain cyano group to be used for from pregnant horse urine directly or extract the pregnant Sterones of combined from the pregnant solution that contains pregnant Sterones.This macro-reticular adsorbing resin is the Polar Adsorbent Resin of being come by polymerization such as divinylbenzene, propylene cyanogen, have ph stability preferably, be difficult for conglomeration and become piece, to the pregnant horse combined of effective constituent oestrogenic hormon adsorptive capacity height, resin is easy to characteristics such as regeneration to the tolerance degree height of soda acid.This method technology is simple, with low cost, yield is high, be easy to suitability for industrialized production.
Extraction method of combined mixed estrin of pregnant mare of the present invention follows these steps to carry out:
A, pregnant horse urine liquid is contacted with the Polar Adsorbent Resin that contains cyano group, dynamically or Static Adsorption all can, the resin isolation of combined mixed estrin is arranged with load then; Pregnant horse urine liquid is not for containing or contain the clarification urine or half suspension liquid of mucosubstance and solid matter;
B, use through to overregulate PH be the water of 12.0-14.0 or contain the water-soluble low-carbon alcohol of weight percent 0-15% or ketone aqueous organopolysiloxane wash load resin;
C, washed Polar Adsorbent Resin is contacted with the elutriant that can resolve the combined mixed estrin that is adsorbed on the resin, dynamically or static the parsing; This elutriant is a kind of lower alcohol, ketone polar organic solvent aqueous solution composition that can be miscible with water, and its aqueous solution is the alkaline solvent of PH 9-13, and desorption temperature is below 60 ℃;
D, elutriant concentrate, are drying to obtain the pregnant Sterones mixture of combined after PH to 6-8.5 is regulated in acid.
Among the step a used polarity polymerization polymeric adsorbent be contain cyano group with divinylbenzene, the polymerization of propylene cyanogen and the Polar Adsorbent Resin of coming; Polymeric adsorbent is the macroporous netlike structure; The volume multiple of the resin absorption pregnant horse urine of a volume is 20-100.
Eluent is that pH value is the water of 12.0-14.0 or contains Diluted Alcohol or acetone soln below 15% among the step b.
Elutriant among the step c be a kind of can with the miscible lower alcohol of water, the solution of ketone polar organic solvent, this solution PH is 9-13, the volume ratio of its organic solvent and water is 50: 50 to 20: 80.
Extraction method of combined mixed estrin of pregnant mare of the present invention, pregnant horse urine can be with filtering or settled method is removed the crude urine of suspension viscous material and solids wholly or in part or through spissated pregnant horse urine concentrate in this method.
In method steps a, use and contain organic non-ionic polymers that the cyano group Polar Adsorbent Resin is a macroporous netlike, possess hydrophilic property structure, its surface-area 300-900m
2/ g, mean pore size is between the 40-100 dust.This resin detects proof through thin-layer chromatography when at room temperature on average female mother's pregnant horse urine carries out static post absorption about 8 months with pregnancy period of 100 times of column volumes, does not obviously detect combined oestrogenic hormon and leaks.
Aldehydes matter washings consumption in method steps b in the wash load resin is 2-7 a times of resin volume.
Elutriant is a kind of can the composition with the aqueous solution of the miscible lower alcohol of water, ketone polar organic solvent in method steps c, and this aqueous solution is inorganic alkali solution, and as sodium hydroxide or potassium hydroxide, its pH value is between the 9-13; The elutriant consumption is 3-8 a times of resin volume.
With diluted acid elutriant is neutralized between the 6-8.5 before concentrating in method steps d, acid can be selected hydrochloric acid, sulfuric acid, phosphoric acid or acetic acid; Evaporation is no more than 60 ℃ with drying temperature.
Embodiment 1
At first that 50 milliliters of polar macroporous adsorption resins that contain cyano group are (commercially available, the HPD-500 resin of producing in chemical plant, Cangzhou, Hebei) being contained in post footpath * post high is in the post of 2.0cm * 25cm, with 2 liters of pregnant horse urines (not containing or contain the clarification urine or half suspension liquid of mucosubstance and solid matter) at room temperature with 300 milliliters/hour flow velocity upper prop, see through liquid after testing, its method is: the effluent liquid of 2 liters of pregnant horse urines is detected through thin-layer chromatography (TLC) with the concentrated sample behind the ethyl acetate extraction for last 50 milliliters, do not see that combined oestrogenic hormon leaks;
Then the load post is used through to overregulate PH be the water of 12.0-14.0 or contain the water-soluble low-carbon alcohol of 0-15% or 250 milliliters of wash load resins of ketone sig water, after testing, its method is: in the washings and back is detected through thin-layer chromatography (TLC) with the concentrated sample behind the ethyl acetate extraction of equal volume, do not seen obviously that combined oestrogenic hormon washes out;
Again washed Polar Adsorbent Resin is contacted static the parsing with the elutriant that can resolve the combined mixed estrin that is adsorbed on the resin; This elutriant be a kind of can with 250 milliliters of the miscible lower alcohol of water or ketone isopolarity aqueous solutions of organic solvent, PH 9-13, desorption temperature is below 60 ℃, wash-out under the column temperature, earlier collecting 25 milliliters (attached column volumes) is first section of elutriant, and then to collect every part of volume respectively be 50 milliliters second section, the 3rd section, the 4th section, the 5th section; The 1-3 experiment that the concentration that 1-5 section combined oestrone detects through high performance liquid chromatography (HPLC) sees Table:
| The 5th section concentration | ?0.102mg/ml | 0.019mg/ml | 0.010mg/ml | 0.035mg/ml |
Elutriant with the 2-5 section merges at last, with diluted acid elutriant is neutralized to (acid can be selected hydrochloric acid, sulfuric acid, phosphoric acid or acetic acid) between the 6-8.5, through concentrating, be drying to obtain the pregnant Sterones mixture of combined.This mixture can directly or be used for medicine after being further purified composite.
Embodiment 2
At first that 50 milliliters of polar macroporous adsorption resins that contain cyano group are (commercially available, the HPD-500 resin of producing in chemical plant, Cangzhou, Hebei) being contained in post footpath * post high is in the post of 2.0cm * 25cm, contain the pregnant horse urine crude extract aqueous solution that combined oestrone is 95.2mg/L with 2 liters, at room temperature with the flow velocity upper prop of 300ml/ hour (5 times column volume/hour), adopt thin-layer chromatography (TLC) to detect, do not see that combined oestrogenic hormon leaks;
Then the load post being used through overregulating PH is 250 milliliters of wash load resins of diluted alkaline water of 14.0, adopts thin-layer chromatography to detect, and does not see that combined oestrogenic hormon obviously washes out;
Again washed Polar Adsorbent Resin is contacted static the parsing with the elutriant that can resolve the combined mixed estrin that is adsorbed on the resin; This elutriant is an ethanol/water, its volume ratio is 35: 65, PH is 250 milliliters of 12.5 the aqueous solution, 60 ℃ of desorption temperatures, wash-out under the column temperature, earlier collecting 25 milliliters (attached column volumes) be first section of elutriant, and then to collect every part of volume respectively be 50 milliliters second section, the 3rd section, the 4th section, the 5th section, and every section concentration sees Table tests 4;
Elutriant with the 2-5 section merges at last, with diluted acid elutriant is neutralized to 8.0 (acid can be selected hydrochloric acid or sulfuric acid or phosphoric acid or acetic acid), through concentrated, the dry pregnant Sterones mixture of combined that gets.This mixture can directly or be used for medicine after being further purified composite.
Embodiment 3
At first that 50 milliliters of polar macroporous adsorption resins that contain cyano group are (commercially available, the HPD-500 resin of producing in chemical plant, Cangzhou, Hebei) puts into the pregnant horse urine dynamic adsorption that 2 liters of combined oestrone are housed is 37.5mg/L, stir and leach loaded resin after 30 minutes, this resin washs with the sig water gradation of 3 * 60ml PH 13.0, the final load resin is used 60 milliliters of PH12.5 ethanol respectively: the volume ratio of water is that 35: 65 Diluted Alcohol is resolved respectively under 50 ℃ three times, the each stirring 20 minutes, leach elutriant, three elutriants are merged, behind 7-8, concentrate the combined mixed estrin extract that draws 1.043 grams through regulating pH value, through high performance liquid chromatography (HPLC) test combined oestrone content is 6.1%, and this mixture can directly or be used for medicine after being further purified composite.
Resin regeneration: the 50 milliliters of polymeric adsorbents of will waiting to regenerate wash with water to neutrality more earlier with 70% ethanol elution of 250 milliliters of PH 13.0, and this resin can be repeatedly used.
Claims (6)
1, a kind of extraction method of combined mixed estrin of pregnant mare is characterized in that following these steps to carrying out:
A, pregnant horse urine liquid is contacted with the Polar Adsorbent Resin that contains cyano group, dynamically or Static Adsorption all can, the resin isolation of combined mixed estrin is arranged with load then; Pregnant horse urine liquid is not for containing or contain the clarification urine or half suspension liquid of mucosubstance and solid matter;
B, use through to overregulate PH be the water of 12.0-14.0 or contain the water-soluble low-carbon alcohol of weight percent 0-15% or ketone aqueous organopolysiloxane wash load resin;
C, washed Polar Adsorbent Resin is contacted with the elutriant that can resolve the combined mixed estrin that is adsorbed on the resin, dynamically or static the parsing; This elutriant is a kind of lower alcohol, ketone polar organic solvent aqueous solution composition that can be miscible with water, and its aqueous solution is the alkaline solvent of PH 9-13, and desorption temperature is below 60 ℃;
D, elutriant concentrate, are drying to obtain the pregnant Sterones mixture of combined after PH to 6-8.5 is regulated in acid.
2, extraction method of combined mixed estrin of pregnant mare according to claim 1, it is characterized in that polarity polymerization polymeric adsorbent used among the step a be contain cyano group with divinylbenzene, the polymerization of propylene cyanogen and the Polar Adsorbent Resin of coming.
3, extraction method of combined mixed estrin of pregnant mare according to claim 2 is characterized in that polymeric adsorbent is the macroporous netlike structure.
4, extraction method of combined mixed estrin of pregnant mare according to claim 1, the multiple that it is characterized in that the resin absorption pregnant horse urine of a volume among the step a is 20-100.
5, extraction method of combined mixed estrin of pregnant mare according to claim 1 is characterized in that eluent is that pH value is water or 15% following Diluted Alcohol or the acetone soln of 12.0-14.0 among the step b.
6, extraction method of combined mixed estrin of pregnant mare according to claim 1, it is characterized in that elutriant among the step c be a kind of can with the miscible lower alcohol of water, the solution of ketone polar organic solvent, this solution PH is 9-13, and the volume ratio of its organic solvent and water is 50: 50 to 20: 80.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB011048522A CN1133639C (en) | 2001-02-19 | 2001-02-19 | Extraction method of combined mixed estrin of pregnant mare |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB011048522A CN1133639C (en) | 2001-02-19 | 2001-02-19 | Extraction method of combined mixed estrin of pregnant mare |
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| Publication Number | Publication Date |
|---|---|
| CN1308083A CN1308083A (en) | 2001-08-15 |
| CN1133639C true CN1133639C (en) | 2004-01-07 |
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| Application Number | Title | Priority Date | Filing Date |
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| CNB011048522A Expired - Fee Related CN1133639C (en) | 2001-02-19 | 2001-02-19 | Extraction method of combined mixed estrin of pregnant mare |
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Families Citing this family (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20040072814A1 (en) | 2002-10-11 | 2004-04-15 | Solvay Pharmaceuticals Gmbh | Method for obtaining a natural mixture of conjugated equine estrogens depleted in non-conjugated lipophilic compounds |
| CN1332973C (en) * | 2003-03-06 | 2007-08-22 | 深圳市海达克实业有限公司 | Extraction process of conjugated female hormone |
| MXPA06000645A (en) | 2003-07-17 | 2006-03-30 | Solvay Pharm Gmbh | Method for obtaining a natural mixture of conjugated equine estrogens. |
| CN1325062C (en) * | 2005-12-06 | 2007-07-11 | 新疆特丰药业股份有限公司 | Method for obtaining binded estrogen mixture from pregnant horse urine, and its uses of big pore adsorption resin in the method |
| CN114588179A (en) * | 2020-12-03 | 2022-06-07 | 中国科学院大连化学物理研究所 | Method for separating and purifying conjugated estrogens from pregnant mare urine by using preparative chromatography |
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2001
- 2001-02-19 CN CNB011048522A patent/CN1133639C/en not_active Expired - Fee Related
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