工业三七药渣中三七素的提取纯化及其止血药理活性研究
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摘要
为考察工业三七药渣中是否含有三七素,探究提取、分离纯化三七素的方法,并考察三七素的止血药理活性。实验以提取过三七总皂苷的工业三七药渣为原料,采用水提醇沉法,过滤沉淀得滤液,回收滤液中的乙醇得粗提液,粗提液经001×7阳离子树脂柱分离,薄层鉴别法检测洗脱液中是否含有三七素,收集与三七素对照品显相同斑点的洗脱液,经丙酮结晶,冷冻干燥得三七素供试品;高效液相色谱法测定三七素供试品中三七素的含量;玻片法测定三七素供试品的凝血时间。建立的高效液相色谱法测定三七素含量,该方法准确度、精密度、稳定性均良好,三七素供试品中三七素含量为59.03%;小鼠腹腔注射不同剂量的三七素供试品与阳性组比较,均能显著缩短凝血时间。建立的高效液相色谱法可用于三七素供试品含量的测定;三七素供试品能显著缩短凝血时间;三七素供试品中三七素含量反推到工业三七药渣中含量为0.72%;采用价格低廉的001×7阳离子交换树脂柱分离纯化三七素,此方法简单易行,成本低廉,适合大工业生产。综上,从工业三七药渣中提取纯化三七素具有良好的综合利用前景。
The purpose of this experiment was to investigate whether or not the industrial medicinal residue of Panax notoginseng contains dencichine,how to extract,isolate and purify the dencichine,and to investigate the hemostatic pharmacological activity of the dencichine.The industrial Panax notoginseng medicinal residue from which the total saponins of Panax notoginseng has been extracted is used as a raw material,using water extraction and alcohol precipitation method,filtering the precipitate to obtain a filtrate,the ethanol solution in the filtrate is recovered to obtain a crude extract,and the crude extract is separated by a 001 × 7 cationic resin column,the thin layer identification method was used to detect whether the eluate contained dencichine,and the eluate with the same spots as the reference substance of the dencichine was collected,crystallized by acetone,and lyophilized to obtain the sample of the dencichine.Determination of the content of dencichine in the samples of dencichine by high performance liquid chromatography and the clotting time of the samples of dencichine by slide method.The HPLC method was established for the determination of notoginseng content.The accuracy,precision and stability of the method were good.The content of dencichine in the samples of dencichine was 59.03%.The intraperitoneal injection of different doses of dencichine in the mice compared with the positive group can significantly shorten the clotting time.The established high performance liquid chromatography method can be used to determine the content of the test sample of dencichine;the test sample of dencichine can significantly shorten the clotting time.The content of dencichine in the sample of dencichine was reversed to 0.72% in the industrial Panax notoginseng medicinal residue.The low-cost 001 × 7 cation exchange resin column was used to separate and purify the notoginseng.This method is simple and easy,and the cost is low.Suitable for large industrial production.In summary,the extraction and purification of dencichine from industrial Panax notoginseng medical residue has a good comprehensive utilization prospect.
The purpose of this experiment was to investigate whether or not the industrial medicinal residue of Panax notoginseng contains dencichine,how to extract,isolate and purify the dencichine,and to investigate the hemostatic pharmacological activity of the dencichine.The industrial Panax notoginseng medicinal residue from which the total saponins of Panax notoginseng has been extracted is used as a raw material,using water extraction and alcohol precipitation method,filtering the precipitate to obtain a filtrate,the ethanol solution in the filtrate is recovered to obtain a crude extract,and the crude extract is separated by a 001 × 7 cationic resin column,the thin layer identification method was used to detect whether the eluate contained dencichine,and the eluate with the same spots as the reference substance of the dencichine was collected,crystallized by acetone,and lyophilized to obtain the sample of the dencichine.Determination of the content of dencichine in the samples of dencichine by high performance liquid chromatography and the clotting time of the samples of dencichine by slide method.The HPLC method was established for the determination of notoginseng content.The accuracy,precision and stability of the method were good.The content of dencichine in the samples of dencichine was 59.03%.The intraperitoneal injection of different doses of dencichine in the mice compared with the positive group can significantly shorten the clotting time.The established high performance liquid chromatography method can be used to determine the content of the test sample of dencichine;the test sample of dencichine can significantly shorten the clotting time.The content of dencichine in the sample of dencichine was reversed to 0.72% in the industrial Panax notoginseng medicinal residue.The low-cost 001 × 7 cation exchange resin column was used to separate and purify the notoginseng.This method is simple and easy,and the cost is low.Suitable for large industrial production.In summary,the extraction and purification of dencichine from industrial Panax notoginseng medical residue has a good comprehensive utilization prospect.
引文
1 Chinese Pharmacopoeia Commission.Pharmacopoeia of Peoples Republic of China:Vol Ⅰ(中华人民共和国:第一部)[M].Beijing:China Medical Science Press,2015.
2 Zhen GZ,Yang CR.Biology and application of Panax notoginseng(三七生物学及其应用)[M].Beijing:Science Press,1994:9-36.
3 Wang MX,Zhao JY,Sun DM,et al.Protective effects and underlying mechanisms of Panax notoginseng saponins against SH-SY5Y cell apoptosis induced by 6-hydroxydopamine[J].Acta Pharm Sin(药学学报),2016,51:898-906.
4 Liu XY,Ma J,Li CJ,et al.New triterpenoid saponins from the leaves of Panax notoginseng[J].Acta Pharm Sin(药学学报),2017,52:1432-1436.
5 Kosuge T,Yokota M,Ochiai A.Studies on antihemorrhagic priciples in the crude drugs for hemosatics.On antihemorrhagic principle in Sanchi ginseng Radix[J].Yakugku Zasshi,1981,101:629-632.
6 Lu Q,Li XG.Separation,identification and content determination of Panax notoginseng hemostatic components[J].Chin Tradit Patent Med(中成药),1988,9:34-35.
7 Xie GX,Qiu YP,Qiu MF.Analysis of dencichine in Panax notoginseng by gas chromatogrp-hy-mass spectrometry with ethyl chloroformate derivatization[J].J Pharm Biomed Anal,2007,43:920-925.
8 Jia W,Qiu MF,Xie GX,et al.Extraction of dencichine from Panax notoginseng(从三七中提取三七素的方法)CN1865232A[P].2006-11-22.
9 Tang ZY,Yuan HJ,You ZL,et al.Study on the separation of effective components in Panax notoginseng[J].Guid J Tradit Chin Med Pharm(中医药导报),2010,16(1):63-65.
10 Liu G,Liu YJ,Hu WY,et al.Determination of the content of dencichine in Panax notoginseng saponins extraction waste liquid by HPLC[J].Yunnan J Tradit Chin Med Mater Medica(云南中医中药杂志),2010,31(2):61-62.
11 Lan GH,Fang S,Chen P,et al.The synthetic method of high effective hemostatic drug dencichine(高效止血药三七素的合成制备方法)CN1292377A[P].2001-4-25.
12 Xie GX,Qiu MF,Zhao AH,et al.Separation puriffication and structutal analysis of dencichine from Panax notoginseng[J].Nat Prod Res Dev(天然产物研究与开发),2007,19:1059-1061.
13 Ju ZC,He CY,.Liu Q,et al.Determination of dencichine in Panax notoginseng tablet by HPLC[J].China J Chin Mater Med(中国中药杂志),2015,40:2594-2597.
14 Zhang YP,Yu Q,et al.Extraction abstraction and assaying of dencichine in Radix notoginseng[J].Shanxi J Tradit Chin Med(山西中医药杂志),2009,25(10):55-56.
15 Yang LJ,Gao EQ,Jiao CJ,et al.Determination of dencichine in Panax notoginseng by pre-column derivatization HPLC[J].Res Dev & Market(资源开发与市场),2015,31(1):1-3.
16 Li L,Wang CX,Qu Y,et al.Content determination of dencichine in Panax notoginseng by a reversed phase ion-pair chromatography[J].Yunnan J Tradit Chin Med Mater Medica(云南中医中药杂志),2015,40:4026-4030.
2 Zhen GZ,Yang CR.Biology and application of Panax notoginseng(三七生物学及其应用)[M].Beijing:Science Press,1994:9-36.
3 Wang MX,Zhao JY,Sun DM,et al.Protective effects and underlying mechanisms of Panax notoginseng saponins against SH-SY5Y cell apoptosis induced by 6-hydroxydopamine[J].Acta Pharm Sin(药学学报),2016,51:898-906.
4 Liu XY,Ma J,Li CJ,et al.New triterpenoid saponins from the leaves of Panax notoginseng[J].Acta Pharm Sin(药学学报),2017,52:1432-1436.
5 Kosuge T,Yokota M,Ochiai A.Studies on antihemorrhagic priciples in the crude drugs for hemosatics.On antihemorrhagic principle in Sanchi ginseng Radix[J].Yakugku Zasshi,1981,101:629-632.
6 Lu Q,Li XG.Separation,identification and content determination of Panax notoginseng hemostatic components[J].Chin Tradit Patent Med(中成药),1988,9:34-35.
7 Xie GX,Qiu YP,Qiu MF.Analysis of dencichine in Panax notoginseng by gas chromatogrp-hy-mass spectrometry with ethyl chloroformate derivatization[J].J Pharm Biomed Anal,2007,43:920-925.
8 Jia W,Qiu MF,Xie GX,et al.Extraction of dencichine from Panax notoginseng(从三七中提取三七素的方法)CN1865232A[P].2006-11-22.
9 Tang ZY,Yuan HJ,You ZL,et al.Study on the separation of effective components in Panax notoginseng[J].Guid J Tradit Chin Med Pharm(中医药导报),2010,16(1):63-65.
10 Liu G,Liu YJ,Hu WY,et al.Determination of the content of dencichine in Panax notoginseng saponins extraction waste liquid by HPLC[J].Yunnan J Tradit Chin Med Mater Medica(云南中医中药杂志),2010,31(2):61-62.
11 Lan GH,Fang S,Chen P,et al.The synthetic method of high effective hemostatic drug dencichine(高效止血药三七素的合成制备方法)CN1292377A[P].2001-4-25.
12 Xie GX,Qiu MF,Zhao AH,et al.Separation puriffication and structutal analysis of dencichine from Panax notoginseng[J].Nat Prod Res Dev(天然产物研究与开发),2007,19:1059-1061.
13 Ju ZC,He CY,.Liu Q,et al.Determination of dencichine in Panax notoginseng tablet by HPLC[J].China J Chin Mater Med(中国中药杂志),2015,40:2594-2597.
14 Zhang YP,Yu Q,et al.Extraction abstraction and assaying of dencichine in Radix notoginseng[J].Shanxi J Tradit Chin Med(山西中医药杂志),2009,25(10):55-56.
15 Yang LJ,Gao EQ,Jiao CJ,et al.Determination of dencichine in Panax notoginseng by pre-column derivatization HPLC[J].Res Dev & Market(资源开发与市场),2015,31(1):1-3.
16 Li L,Wang CX,Qu Y,et al.Content determination of dencichine in Panax notoginseng by a reversed phase ion-pair chromatography[J].Yunnan J Tradit Chin Med Mater Medica(云南中医中药杂志),2015,40:4026-4030.