酒炙车前子防治奶牛胎衣不下中有效物质分离体系的确定
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摘要
酒炙车前子防治奶牛胎衣不下是验方,本实验室验证酒炙车前子在防治奶牛胎衣不下方面有着显著的疗效,并确定了有效部位。但其防治奶牛胎衣不下的有效物质基础尚不明确。因此本试验目的是确定一个能够将其有效部位中所包括的化合物按照极性由低到高分离成三部分的体系。通过预实验确定了分离二氯甲烷部位和乙酸乙酯部位的洗脱剂和展开剂体系。使用相同内径的层析柱,其中硅胶的高度设置成3个梯度:15,25,35cm,使用洗脱剂进行梯度洗脱后,利用薄层色谱法进行检测,由色谱图观察化合物是否能成功分离成三部分。二氯甲烷部位和乙酸乙酯部位的结果显示,柱高15cm条件下无法有效地分离化合物,柱高25cm条件下化合物回收比例要高于柱高35cm条件下的化合物回收比例。因此确定了二氯甲烷部位分离化合物的洗脱剂体系:石油醚∶乙酸乙酯为1∶2;石油醚∶乙酸乙酯为1∶30;二氯甲烷∶甲醇为30∶1;展开剂体系:石油醚∶乙酸乙酯为4∶1;柱体高度为25cm。乙酸乙酯部位分离化合物的洗脱剂体系:石油醚∶乙酸乙酯为100∶1;石油醚∶乙酸乙酯30∶1;二氯甲烷∶甲醇为30∶1;展开剂体系:石油醚∶乙酸乙酯为10∶1;柱体高度为25cm。采用干法上样,上样量应适当增加。该分离体系的确定对研究酒炙车前子中防治奶牛胎衣不下的有效物质基础提供了技术支撑,也为研制高效、安全、廉价的防治奶牛胎衣不下的中药制剂奠定了基础。
Semen plantaginis treated by stir-frying with wine has been proved to be remarkable effect in the prevention and treatment of cow retained fetal membranes,and the effective parts has been maken by our laboratory,but the active ingredients in the effective parts have not been reported.Therefore,the purpose of this experiment is to determine a system that can separate the active ingredients in the effective parts into three parts by polarity from low to high.The eluant and developing systems for the separation of dichloromethane parts and ethyl acetate parts were determined by pre-experiment.The same diameter chromatographic column was used,the height of silica gel in the column was set as three gradients:15,25 and 35cm.After gradient elution,thin layer chromatography was performed to observe whether the compound can be successfully separated into three parts.The results of dichloromethane parts and ethyl acetate parts showed that the active ingredients could not be effectively separated at 15 cm column height and the recovery rate at25 cm column height was higher than that at 35 cm.The eluent and developing system of the active ingredients separated at the dichloromethane parts were determined that:PET∶EAC,1∶2;PET∶EAC,1∶30;DCM∶MT,30∶1.The column height was 25 cm.The eluent and developing system of the active ingredients separated at the ethyl acetate parts:PET∶EAC,100∶1;PET∶EAC,30∶1;DCM∶MT,30∶1.Dry loading method was used,and the weight of sample should be increased.The determination of the separation system provided technical support for the study on the effective substances of preventing and controlling of the retaining fetal membranes in Semen plantaginis by stir-frying wine,and also laid a practical foundation for the development of high efficiency,safe and cheap Chinese medicine preparation for preventing and controlling the retained fetal membranes in the cows.
Semen plantaginis treated by stir-frying with wine has been proved to be remarkable effect in the prevention and treatment of cow retained fetal membranes,and the effective parts has been maken by our laboratory,but the active ingredients in the effective parts have not been reported.Therefore,the purpose of this experiment is to determine a system that can separate the active ingredients in the effective parts into three parts by polarity from low to high.The eluant and developing systems for the separation of dichloromethane parts and ethyl acetate parts were determined by pre-experiment.The same diameter chromatographic column was used,the height of silica gel in the column was set as three gradients:15,25 and 35cm.After gradient elution,thin layer chromatography was performed to observe whether the compound can be successfully separated into three parts.The results of dichloromethane parts and ethyl acetate parts showed that the active ingredients could not be effectively separated at 15 cm column height and the recovery rate at25 cm column height was higher than that at 35 cm.The eluent and developing system of the active ingredients separated at the dichloromethane parts were determined that:PET∶EAC,1∶2;PET∶EAC,1∶30;DCM∶MT,30∶1.The column height was 25 cm.The eluent and developing system of the active ingredients separated at the ethyl acetate parts:PET∶EAC,100∶1;PET∶EAC,30∶1;DCM∶MT,30∶1.Dry loading method was used,and the weight of sample should be increased.The determination of the separation system provided technical support for the study on the effective substances of preventing and controlling of the retaining fetal membranes in Semen plantaginis by stir-frying wine,and also laid a practical foundation for the development of high efficiency,safe and cheap Chinese medicine preparation for preventing and controlling the retained fetal membranes in the cows.
引文
[1]BEAGLEY J C,WHITMAN K J,BAPTISTE K E,et al.Physiology and treatment of retained fetal membranes incattle[J].J Vet Intern Med,2010,24(2):261-268.
[2]LEBLANC S J.Postpartum uterine disease and dairy herd reproductive performance:a review[J].Vet J,2008,176(1):102-114.
[3]黄美凤,罗朝夫,黄国胜.母牛胎衣不下的防治措施[J].兽医导刊,2017(4):133.
[4]LIANG D,ARNOLD L M,STOWE C J,et al.Estimating US dairy clinical disease costs with a stochastic simulation model[J].J Dairy Sci,2017,100(2):1472-1486.
[5]王宝宝.奶牛胎衣不下的手术剥离及药物疗法[J].中兽医学杂志,2017(3):62.
[6]王存礼,王俊.治疗牛胎衣不下的一种方剂[J].中国兽医杂志,1982(3):30.
[7]刘乐和,魏忠伟.车前子治疗牛胎衣不下[J].黑龙江畜牧兽医,1993(1):44.
[8]鲁文赓,王瑶,孙宏亮,等.酒炙车前子治疗奶牛胎衣不下有效部位的筛选[J].黑龙江畜牧兽医,2018(7):176-180.
[9]杜会茹,黄文杰,蒋翠岚,等.厚朴酚中压硅胶柱层析纯化工艺的优化[J].中成药,2017,39(4):835-837.
[10] PAGKALIWANGAN M,HUMMEL J,GJOKA X,et al.Optimized continuous multicolumn chromatography enables increased productivities and cost savings by employing more columns[J].Biotechnol J,2018,:doi:10.1002/biot.201800179.
[11]俞作仁,周印锁.车前子及其混伪品的鉴别研究[J].中医药学报,2000(5):36-37.
[12]国家药典委员会.中华人民共和国药典3部[M].北京:中国医药科技出版社,2010.
[13]谢明,杨爽爽,王亮亮,等.中药车前子的研究进展[J].黑龙江医药,2015(3):474-476.
[14] CHOI S,JUNG S,LEE H,et al.Glycation inhibitory activity and the identification of an active compound in Plantago asiatica extract[J].Phytotherapy Res,2010,22(3):323-329.
[15]谢小梅,付志红,吴娟,等.车前多糖对小鼠免疫功能的影响[J].免疫学杂志,2009(2):232-233.
[16] CHIANG L C,CHIANG W,CHANG M Y,et al.Antiviral activity of Plantago major extracts and related compounds in vitro[J].Antiviral Res,2002,55(1):53-62.
[2]LEBLANC S J.Postpartum uterine disease and dairy herd reproductive performance:a review[J].Vet J,2008,176(1):102-114.
[3]黄美凤,罗朝夫,黄国胜.母牛胎衣不下的防治措施[J].兽医导刊,2017(4):133.
[4]LIANG D,ARNOLD L M,STOWE C J,et al.Estimating US dairy clinical disease costs with a stochastic simulation model[J].J Dairy Sci,2017,100(2):1472-1486.
[5]王宝宝.奶牛胎衣不下的手术剥离及药物疗法[J].中兽医学杂志,2017(3):62.
[6]王存礼,王俊.治疗牛胎衣不下的一种方剂[J].中国兽医杂志,1982(3):30.
[7]刘乐和,魏忠伟.车前子治疗牛胎衣不下[J].黑龙江畜牧兽医,1993(1):44.
[8]鲁文赓,王瑶,孙宏亮,等.酒炙车前子治疗奶牛胎衣不下有效部位的筛选[J].黑龙江畜牧兽医,2018(7):176-180.
[9]杜会茹,黄文杰,蒋翠岚,等.厚朴酚中压硅胶柱层析纯化工艺的优化[J].中成药,2017,39(4):835-837.
[10] PAGKALIWANGAN M,HUMMEL J,GJOKA X,et al.Optimized continuous multicolumn chromatography enables increased productivities and cost savings by employing more columns[J].Biotechnol J,2018,:doi:10.1002/biot.201800179.
[11]俞作仁,周印锁.车前子及其混伪品的鉴别研究[J].中医药学报,2000(5):36-37.
[12]国家药典委员会.中华人民共和国药典3部[M].北京:中国医药科技出版社,2010.
[13]谢明,杨爽爽,王亮亮,等.中药车前子的研究进展[J].黑龙江医药,2015(3):474-476.
[14] CHOI S,JUNG S,LEE H,et al.Glycation inhibitory activity and the identification of an active compound in Plantago asiatica extract[J].Phytotherapy Res,2010,22(3):323-329.
[15]谢小梅,付志红,吴娟,等.车前多糖对小鼠免疫功能的影响[J].免疫学杂志,2009(2):232-233.
[16] CHIANG L C,CHIANG W,CHANG M Y,et al.Antiviral activity of Plantago major extracts and related compounds in vitro[J].Antiviral Res,2002,55(1):53-62.