新疆孕马尿提取物中伴随物质的研究
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摘要
目的:利用精细分离填料MCI GEL CHP20P ,Sephadex LH-20对孕马尿中除结合雌激素外的其他成分进行分离及结构鉴定,对所分离的化合物进行含量测定,并对孕马尿提取物生产工艺过程中的各中间体以及经MCI GEL CHP20P柱分离纯化后的孕马尿提取物进行成分监测。
方法:1)以MCI GEL CHP20P为分离材料,以甲醇-水为洗脱溶剂对孕马尿提取物进行梯度洗脱,得到各不同馏分。2)以Sephadex LH-20为分离材料,以甲醇-水和甲醇-氯仿为洗脱溶剂对所得的各馏分进行再分离。3)对所分离得到的化合物以核磁共振的氢谱、碳谱及二维谱为依据,进行结构解析。4)对分离得到的两个化合物进行含量测定。5)对孕马尿提取物生产工艺过程中的各中间体以及经MCI GEL CHP20P柱分离纯化后的孕马尿提取物成分进行检测。
结果:1)以MCI GEL CHP20P为分离材料,以甲醇-水为洗脱溶剂,得到各不同馏分,并在80%部分得到芒柄花素。2)以Sephadex LH-20为分离材料,以甲醇-水为洗脱溶剂,在20%部分分离得到苯甲酸,在100%部分分离得到三个化合物,即:鸡豆黄素A、大豆苷元、染料木素。3)芒柄花素的线性范围为0.08～0.56μg/ml,鸡豆黄素A的线性范围为0.09～0.62μg/ml,平均回收率分别为99.5%,99.6%,RSD分别为0.56%,0.78%。4)孕马尿提取物生产工艺过程中的各中间体,除B样品外,其他样品均含有大豆苷元和染料木素,C样品与G样品含有芒柄花素,只有G样品含有鸡豆黄素A;经MCI GEL CHP20P柱分离纯化后的孕马尿提取物基本除去了我们所分离鉴定的这四种成分。
结论:1)MCI GEL CHP20P ,Sephadex LH-20作为一种精细分离材料,对孕马尿中除结合雌激素外的其他成分进行分离,通过对不同洗脱溶剂的筛选,确定甲醇-水和甲醇-氯仿为最佳洗脱溶剂;采用梯度洗脱的方法,从中分离出苯甲酸、芒柄花素、鸡豆黄素A、大豆苷元、染料木素。结果表明,Sephadex LH-20对孕马尿中的其他成分有很好的分离作用,达到了较好的分离效果。2)我们对芒柄花素和鸡豆黄素A进行了含量测定,其重现性好、稳定性、精密度高,为今后检测孕马尿提取物中的伴随物提供依据。3)我们对孕马尿提取物生产工艺过程中的各中间体以及经MCI GEL CHP20P柱分离纯化后的孕马尿提取物进行了检测,得到了较为满意的结果,证实使用MCI GEL CHP20P柱分离孕马尿提取物的分离方法切实可行,为今后孕马尿的分离纯化工艺提供依据。
Objective: Use fine separation fill MCI GEL CHP20P and Sephadex LH-20 to separate other compositions from pregnant mare urine, except the conjugated estrogens. Identify the structure of those compositions and determine the content of the isolated compounds that was the extract from pregnant mare urine which was through MCI GEL CHP20P.
Methods: 1)Use MCI GEL CHP20P as separation materials and methanol - water as the solvent elution with pregnant mare urine and got the different fractions. 2)Use Sephadex LH-20 as the separation materials and methanol - water as well as methanol - the chloroform as solvent elution fractions to make a re-separation of each fraction of distillate. 3)The isolated compounds in the NMR spectra of hydrogen, carbon and two-dimensional spectroscopy spectra were analysised on the structural aspect. 4)The two compounds isolated from the pregnant mare urine were assayed. 5) Monitored extract from mare urine of the progesterone production process of the various components of intermediate.
Results: 1) Use MCI GEL CHP20P as separation materials and methanol - water as eluting solvent, formononetin was separated in part of 80% . 2)By Sephadex LH-20 for the separation materials and methanol - water as eluting solvent, in 20% part of was isolated benzoic acid, in 100% part was isolated three compounds, namely: biochanin A, daidzein, genistein. 3)The linear range of formononetin was 0.08 to 0.56μg/ml , linear range of biochanin A was 0.09 to 0.62μg/ml, the average recoveries were 99.5% and 99.6%, respectively RSD is 0.56% and 0.78%. 4)The various intermediates in the production process of the extract from pregnant mare urine, exception of B samples, other samples contained daidzein and genistein, C and G samples contained samples of formononetin, only G samples containing biochanin A;these four components was removed through the MCI GEL CHP20P column.
Conclusion: 1)MCI GEL CHP20P, Sephadex LH-20 as a fineness separation materials, in other components except pregnant mare urine combination of conjugated estrogens were separated by the different solvent elution screening to determine methanol -A water and Alcohol - chloroform is the best eluting solvent gradient elution, isolated the benzoic acid, formononetin,biochanin A, daidzein, genistein of the extract from pregnant mare urine . The results showed that Sephadex LH-20 on the other components of pregnant mare urine have a good separation, which can achieve a better effect. 2)We have assayed formononetin and biochanin A, reproducible, high stability, high precision, provide the basis for concomitant components of the extract from pregnant mare urine. 3)Refined the various intermediates of different stages of pregnant mare urine extract in the production process as well as detected the MCI GEL CHP20P column extract of the pregnant mare urine, have been more satisfied with the results confirmed and practical separation method for the industrial production in the future of the intermediates and products lay the foundation for the ingredients.
方法:1)以MCI GEL CHP20P为分离材料,以甲醇-水为洗脱溶剂对孕马尿提取物进行梯度洗脱,得到各不同馏分。2)以Sephadex LH-20为分离材料,以甲醇-水和甲醇-氯仿为洗脱溶剂对所得的各馏分进行再分离。3)对所分离得到的化合物以核磁共振的氢谱、碳谱及二维谱为依据,进行结构解析。4)对分离得到的两个化合物进行含量测定。5)对孕马尿提取物生产工艺过程中的各中间体以及经MCI GEL CHP20P柱分离纯化后的孕马尿提取物成分进行检测。
结果:1)以MCI GEL CHP20P为分离材料,以甲醇-水为洗脱溶剂,得到各不同馏分,并在80%部分得到芒柄花素。2)以Sephadex LH-20为分离材料,以甲醇-水为洗脱溶剂,在20%部分分离得到苯甲酸,在100%部分分离得到三个化合物,即:鸡豆黄素A、大豆苷元、染料木素。3)芒柄花素的线性范围为0.08～0.56μg/ml,鸡豆黄素A的线性范围为0.09～0.62μg/ml,平均回收率分别为99.5%,99.6%,RSD分别为0.56%,0.78%。4)孕马尿提取物生产工艺过程中的各中间体,除B样品外,其他样品均含有大豆苷元和染料木素,C样品与G样品含有芒柄花素,只有G样品含有鸡豆黄素A;经MCI GEL CHP20P柱分离纯化后的孕马尿提取物基本除去了我们所分离鉴定的这四种成分。
结论:1)MCI GEL CHP20P ,Sephadex LH-20作为一种精细分离材料,对孕马尿中除结合雌激素外的其他成分进行分离,通过对不同洗脱溶剂的筛选,确定甲醇-水和甲醇-氯仿为最佳洗脱溶剂;采用梯度洗脱的方法,从中分离出苯甲酸、芒柄花素、鸡豆黄素A、大豆苷元、染料木素。结果表明,Sephadex LH-20对孕马尿中的其他成分有很好的分离作用,达到了较好的分离效果。2)我们对芒柄花素和鸡豆黄素A进行了含量测定,其重现性好、稳定性、精密度高,为今后检测孕马尿提取物中的伴随物提供依据。3)我们对孕马尿提取物生产工艺过程中的各中间体以及经MCI GEL CHP20P柱分离纯化后的孕马尿提取物进行了检测,得到了较为满意的结果,证实使用MCI GEL CHP20P柱分离孕马尿提取物的分离方法切实可行,为今后孕马尿的分离纯化工艺提供依据。
Objective: Use fine separation fill MCI GEL CHP20P and Sephadex LH-20 to separate other compositions from pregnant mare urine, except the conjugated estrogens. Identify the structure of those compositions and determine the content of the isolated compounds that was the extract from pregnant mare urine which was through MCI GEL CHP20P.
Methods: 1)Use MCI GEL CHP20P as separation materials and methanol - water as the solvent elution with pregnant mare urine and got the different fractions. 2)Use Sephadex LH-20 as the separation materials and methanol - water as well as methanol - the chloroform as solvent elution fractions to make a re-separation of each fraction of distillate. 3)The isolated compounds in the NMR spectra of hydrogen, carbon and two-dimensional spectroscopy spectra were analysised on the structural aspect. 4)The two compounds isolated from the pregnant mare urine were assayed. 5) Monitored extract from mare urine of the progesterone production process of the various components of intermediate.
Results: 1) Use MCI GEL CHP20P as separation materials and methanol - water as eluting solvent, formononetin was separated in part of 80% . 2)By Sephadex LH-20 for the separation materials and methanol - water as eluting solvent, in 20% part of was isolated benzoic acid, in 100% part was isolated three compounds, namely: biochanin A, daidzein, genistein. 3)The linear range of formononetin was 0.08 to 0.56μg/ml , linear range of biochanin A was 0.09 to 0.62μg/ml, the average recoveries were 99.5% and 99.6%, respectively RSD is 0.56% and 0.78%. 4)The various intermediates in the production process of the extract from pregnant mare urine, exception of B samples, other samples contained daidzein and genistein, C and G samples contained samples of formononetin, only G samples containing biochanin A;these four components was removed through the MCI GEL CHP20P column.
Conclusion: 1)MCI GEL CHP20P, Sephadex LH-20 as a fineness separation materials, in other components except pregnant mare urine combination of conjugated estrogens were separated by the different solvent elution screening to determine methanol -A water and Alcohol - chloroform is the best eluting solvent gradient elution, isolated the benzoic acid, formononetin,biochanin A, daidzein, genistein of the extract from pregnant mare urine . The results showed that Sephadex LH-20 on the other components of pregnant mare urine have a good separation, which can achieve a better effect. 2)We have assayed formononetin and biochanin A, reproducible, high stability, high precision, provide the basis for concomitant components of the extract from pregnant mare urine. 3)Refined the various intermediates of different stages of pregnant mare urine extract in the production process as well as detected the MCI GEL CHP20P column extract of the pregnant mare urine, have been more satisfied with the results confirmed and practical separation method for the industrial production in the future of the intermediates and products lay the foundation for the ingredients.
引文
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[3]张兰兰,赵文军,吴雪萍.孕马尿中甾体成分的研究[J].中草药, 2003, 34(增刊):92-93.
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[1] Benjamin S, Marrian GF. The isolation of estrone sulfate from the urine of pregnant mare[J]. BiolChem, 1938, 126 :663-666.
[2] Rusell E, Elmer J, Eugern L, et al. Pregnanediols in pregnancy urine of mares[J]. A m Chem Soc, 1937, 59 : 2297-2298.
[3] Klyne W, Schachter B, Marrian GF. Extraction of steroid sulfates and isolationof 16-allopreganen-3(β)-ol-20-one sulfate [J]. Biochem, 1948, 43 :231-234.
[4] Paterson J YF, Klyne W. Isolation of 16-allopregan-3 (β)- ol-20-one sulfate[J]. Biochem, 1948, 43 :614-616.
[5] Klyne W ( Edinburgh univ scot). Isolation of uranediol sulfate [J]. Biochem, 1948, 43 :611-614.
[6] Brooks RV, Klyne W, Eiller E, et al. ( Postgraduate Med school, London). Fractionation of the neutral steroids. Examination of some nonketonicfractions [J]. Biochem, 1952, 51 :694-707.
[7] Heard RDH. New ketone from the urine of pregnant mares [J]. A m Chem Soc, 1948, 60 :493-494.
[8] Oppenauer Z. Isolation of dehydroandrosterone and of other hydroxyketones fromPMU [J]. Physiol Chem, 1941, 270 :97-105.
[9] Ludomir I, Jozef A, Rudolf S (Agr coll Kosice Czech). Pa2 per electrophoresisgonadoctropic substancesin pregnant mares urine [J]. Endokrynol Polska, 1965, 16 :167-176.
[10]Olivio O. Gonadoctropic substances in PMU and donkeys [J]. BOLL Soc Sci Univ camerino, 1953, 46 :83-95.
[11]Prado JL, Eline S. Some properties of highly purified horse urinary kallikrein[J]. A nn N Y Acad Sci, 1963, 104 :186-189.
[12]Frederick O, Gemmill Jr, Chester E, et al. Treating cardiac disorders with△9(11)-dehydro-8-isoestrone [P]. WO:1995, 3(7):224. 849.
[13]Poole Salwa K, Poole Colin F. Separation of pharmaceutically important estrogens by micellar electrokinetic chromatography [J]. J Chromatogr, 1996, A749:247-255.
[14]Novakovic J, Agbaba D, Vladimirov S, et al. Densitometric determination of conjugated estrogens in the raw material and in pharmaceutical preparations [J]. J Pharm Biomed Anal, 1990, 8(3):253-257.
[15]Gatti R, Gioia MG, Cavrini V. HPLC analysis of pharmaceutical estrogens inraw materials and dosage form [J]. J Pharm Biomed Anal, 1998, 17 :337-347.
[16]Hill, Edward N, Thomas W. Pharmaceutical compositions of conjugated estrogens and methods of analyzing mixtures containing estrogenic compounds [P]. WO:05/6, 855, 703, 2005-02-15.
[17]赵文军,吴雪萍.孕马结合态混合雌激素的提取方法[P]. (CN:ZL 01104852. 2, 2001-08-15. ).中国, 01104752. 2[P]. 2001-08-15.
[18]张兰兰,赵文军,吴雪萍.孕马尿中甾体成分及其活性的研究现状[J].天然产物研究与开发, 2003, 15 (4):354-358.
[19]张兰兰,赵文军,吴雪萍.孕马尿中甾体成分的研究[J].中草药, 2003, 34(增刊):92-93.
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[1] Benjamin S, Marrian GF. The isolation of estrone sulfate from the urine of pregnant mare[J]. BiolChem, 1938, 126 :663-666.
[2] Rusell E, Elmer J, Eugern L, et al. Pregnanediols in pregnancy urine of mares[J]. A m Chem Soc, 1937, 59 : 2297-2298.
[3] Klyne W, Schachter B, Marrian GF. Extraction of steroid sulfates and isolationof 16-allopreganen-3(β)-ol-20-one sulfate [J]. Biochem, 1948, 43 :231-234.
[4] Paterson J YF, Klyne W. Isolation of 16-allopregan-3 (β)- ol-20-one sulfate[J]. Biochem, 1948, 43 :614-616.
[5] Klyne W ( Edinburgh univ scot). Isolation of uranediol sulfate [J]. Biochem, 1948, 43 :611-614.
[6] Brooks RV, Klyne W, Eiller E, et al. ( Postgraduate Med school, London). Fractionation of the neutral steroids. Examination of some nonketonicfractions [J]. Biochem, 1952, 51 :694-707.
[7] Heard RDH. New ketone from the urine of pregnant mares [J]. A m Chem Soc, 1948, 60 :493-494.
[8] Oppenauer Z. Isolation of dehydroandrosterone and of other hydroxyketones fromPMU [J]. Physiol Chem, 1941, 270 :97-105.
[9] Ludomir I, Jozef A, Rudolf S (Agr coll Kosice Czech). Pa2 per electrophoresisgonadoctropic substancesin pregnant mares urine [J]. Endokrynol Polska, 1965, 16 :167-176.
[10]Olivio O. Gonadoctropic substances in PMU and donkeys [J]. BOLL Soc Sci Univ camerino, 1953, 46 :83-95.
[11]Prado JL, Eline S. Some properties of highly purified horse urinary kallikrein[J]. A nn N Y Acad Sci, 1963, 104 :186-189.
[12]Frederick O, Gemmill Jr, Chester E, et al. Treating cardiac disorders with△9(11)-dehydro-8-isoestrone [P]. WO:1995, 3(7):224. 849.
[13]Poole Salwa K, Poole Colin F. Separation of pharmaceutically important estrogens by micellar electrokinetic chromatography [J]. J Chromatogr, 1996, A749:247-255.
[14]Novakovic J, Agbaba D, Vladimirov S, et al. Densitometric determination of conjugated estrogens in the raw material and in pharmaceutical preparations [J]. J Pharm Biomed Anal, 1990, 8(3):253-257.
[15]Gatti R, Gioia MG, Cavrini V. HPLC analysis of pharmaceutical estrogens inraw materials and dosage form [J]. J Pharm Biomed Anal, 1998, 17 :337-347.
[16]Hill, Edward N, Thomas W. Pharmaceutical compositions of conjugated estrogens and methods of analyzing mixtures containing estrogenic compounds [P]. WO:05/6, 855, 703, 2005-02-15.
[17]赵文军,吴雪萍.孕马结合态混合雌激素的提取方法[P]. (CN:ZL 01104852. 2, 2001-08-15. ).中国, 01104752. 2[P]. 2001-08-15.
[18]张兰兰,赵文军,吴雪萍.孕马尿中甾体成分及其活性的研究现状[J].天然产物研究与开发, 2003, 15 (4):354-358.
[19]张兰兰,赵文军,吴雪萍.孕马尿中甾体成分的研究[J].中草药, 2003, 34(增刊):92-93.
[20]巴恩,格尔林,米勒.结合型亲脂化合物被贫化的天然牝马结合型雌激素混合物的获取方法[P]. WO:035. 067, 2004-04-29.