HPLC法同时测定四物汤中8个成分及其在水提醇沉过程中的传递
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摘要
目的:建立HPLC波长切换法同时测定四物汤中5-羟甲基糠醛、绿原酸、咖啡酸、芍药苷、阿魏酸、毛蕊花糖苷、洋川芎内酯A、藁本内酯8个成分的含量,并分析其在水提醇沉过程中的传递情况。方法:按照四物合剂制法制备水煎液、醇沉上清液、醇沉沉淀液样品进行测定;采用Agilent HC-C_(18)色谱柱(250 mm×4.6 mm,5μm),流动相为乙腈(A)-0.5%磷酸水溶液(B),梯度洗脱,流速1.0 mL·min~(-1),柱温23℃;进样量10μL;检测波长为280 nm(0～10 min检测5-羟甲基糠醛,45～46 min,检测洋川芎内酯A)、327 nm(10～20 min检测绿原酸、咖啡酸)、230 nm(20～25 min检测芍药苷)、316 nm(25～29 min检测阿魏酸)、334 nm(29～45 min检测毛蕊花糖苷)、324 nm(46～50 min检测藁本内酯)。结果:5-羟甲基糠醛、绿原酸、咖啡酸、芍药苷、阿魏酸、毛蕊花糖苷、洋川芎内酯A、藁本内酯的质量浓度分别在0.61～19.50μg·mL~(-1)(r=1.000)、0.99～31.80μg·mL~(-1)(r=0.999 5)、0.18～5.88μg·mL~(-1)(r=0.999 7)、10.38～332.00μg·mL~(-1)(r=1.000)、0.91～29.10μg·mL~(-1)(r=0.999 7)、0.53～8.46μg·mL~(-1)(r=0.999 1)、 1.49～47.80μg·mL~(-1)(r=0.999 4)、0.71～22.60μg·mL~(-1)(r=0.999 1)范围内与峰面积呈良好线性关系;平均回收率分别为99.6%、100.1%、98.7%、98.1%、100.9%、99.2%、98.7%、97.0%,RSD分别为1.1%、1.1%、1.2%、0.90%、1.7%、1.9%、0.80%、1.5%。与水煎液相比,醇沉上清液样品中8个成分的损失比例为15%～20%,醇沉沉淀中残留了少量的有效成分。结论:所建含量测定方法合理可行,重复性好,可用于四物汤的质量控制;四物汤水提醇沉过程中伴随着有效成分"量"的变化,在工艺过程中应该注意对工艺参数的优选,以保留有效成分。
Objective:To establish an HPLC wavelength switching method for simultaneously determining the contents of 5-hydroxymethylfurfural,chlorogenic acid,caffeic acid,paeoniflorin,ferulic acid,acteoside,senkyunolide A and ligustilide in Siwu decoction. And to analyze transmitting of the components in water extraction and ethanol precipitation process. Methods:Siwu decoction sample,Siwu ethanol supernatant sample and Siwu ethanol sediment sample were prepared according to the preparation method of Siwu mixture,and the samples were determined by HPLC. Separation was performed on an Agilent HC-C_(18) column(250 mm× 4.6 mm,5 μm) by a gradient elution of acetonitrile(A)-0.5% phosphoric acid solution(B). The flow rate was 1.0 mL·min~(-1) and the column temperature was 23 ℃. The injection volume was 10 μL. The detection wavelengths were set at 280 nm(0-10 min for 5-hydroxymethylfurfural and,45-46 min for senkyunolide A),327 nm(10-20 min for chlorogenic acid and caffeic acid),230 nm(20-25 min for paeoniflorin),316 nm(25-29 min for ferulic acid),334 nm(29-45 min for acteoside),and 324 nm(46-50 min for ligustilide). Results:The 5-hydroxymethylfurfural,chlorogenic acid,caffeic acid,paeoniflorin,ferulic acid,acteoside,senkyunolide A and ligustilide showed good linearity in the ranges of 0.61~(-1)9.50 μg·mL~(-1)(r=1.000),0.99-31.80 μg·mL~(-1)(r=0.999 5),0.18-5.88μg·mL~(-1)(r=0.999 7),10.38-332.00 μg·mL~(-1)(r=1.000),0.91-29.10 μg·mL~(-1)(r=0.999 7),0.53-8.46μg·mL~(-1)(r=0.999 1),1.49-47.80 μg·mL~(-1)(r=0.999 4) and 0.71-22.60 μg·mL~(-1)(r=0.999 1),respectively.The average recoveries were 99.6%(RSD=1.1%),100.1%(RSD=1.1%),98.7%(RSD=1.2%),98.1%(RSD=0.90%),100.9%(RSD=1.7%),99.2%(RSD=1.9%),98.7%(RSD=0.80%) and 97.0%(RSD=1.5%),respectively. Compared with the Siwu decoction sample,the loss rate of eight active components in Siwu ethanol supernatant sample was 15%-20%. There were a few active components in the Siwu ethanol sediment sample.Conclusion:The HPLC wavelength switching method is reasonable,feasible and repeatable,which can be used for the quality control of Siwu decoction. In water extraction and ethanol precipitation process of Siwu decoction,the active components' content changes. So,we should pay attention to optimize the process parameters to retain the active components,in water extraction and ethanol precipitation process.
Objective:To establish an HPLC wavelength switching method for simultaneously determining the contents of 5-hydroxymethylfurfural,chlorogenic acid,caffeic acid,paeoniflorin,ferulic acid,acteoside,senkyunolide A and ligustilide in Siwu decoction. And to analyze transmitting of the components in water extraction and ethanol precipitation process. Methods:Siwu decoction sample,Siwu ethanol supernatant sample and Siwu ethanol sediment sample were prepared according to the preparation method of Siwu mixture,and the samples were determined by HPLC. Separation was performed on an Agilent HC-C_(18) column(250 mm× 4.6 mm,5 μm) by a gradient elution of acetonitrile(A)-0.5% phosphoric acid solution(B). The flow rate was 1.0 mL·min~(-1) and the column temperature was 23 ℃. The injection volume was 10 μL. The detection wavelengths were set at 280 nm(0-10 min for 5-hydroxymethylfurfural and,45-46 min for senkyunolide A),327 nm(10-20 min for chlorogenic acid and caffeic acid),230 nm(20-25 min for paeoniflorin),316 nm(25-29 min for ferulic acid),334 nm(29-45 min for acteoside),and 324 nm(46-50 min for ligustilide). Results:The 5-hydroxymethylfurfural,chlorogenic acid,caffeic acid,paeoniflorin,ferulic acid,acteoside,senkyunolide A and ligustilide showed good linearity in the ranges of 0.61~(-1)9.50 μg·mL~(-1)(r=1.000),0.99-31.80 μg·mL~(-1)(r=0.999 5),0.18-5.88μg·mL~(-1)(r=0.999 7),10.38-332.00 μg·mL~(-1)(r=1.000),0.91-29.10 μg·mL~(-1)(r=0.999 7),0.53-8.46μg·mL~(-1)(r=0.999 1),1.49-47.80 μg·mL~(-1)(r=0.999 4) and 0.71-22.60 μg·mL~(-1)(r=0.999 1),respectively.The average recoveries were 99.6%(RSD=1.1%),100.1%(RSD=1.1%),98.7%(RSD=1.2%),98.1%(RSD=0.90%),100.9%(RSD=1.7%),99.2%(RSD=1.9%),98.7%(RSD=0.80%) and 97.0%(RSD=1.5%),respectively. Compared with the Siwu decoction sample,the loss rate of eight active components in Siwu ethanol supernatant sample was 15%-20%. There were a few active components in the Siwu ethanol sediment sample.Conclusion:The HPLC wavelength switching method is reasonable,feasible and repeatable,which can be used for the quality control of Siwu decoction. In water extraction and ethanol precipitation process of Siwu decoction,the active components' content changes. So,we should pay attention to optimize the process parameters to retain the active components,in water extraction and ethanol precipitation process.
引文
[1]李泓涛,李胜涛.四物汤药理作用及其应用研究[J].光明中医,2016,31(6):897LI HT,LI ST.Study on the pharmacological effects and application of Siwu Tang[J].Guangming Tradit Chin Med,2016,31(6):897
[2]唐昆,吴希.四物汤的现代药理作用和临床应用[J].中国中医急症,2006,15(12):1397TANG K,WU X.Modern pharmacological effects and clinical application of Siwu Tang[J].J Emerg Tradit Chin Med,2006,15(12):1397
[3]胡琦,郭平.四物汤补血机制研究进展[J].山东中医杂志,2017,36(9):819HU Q,GUO P.Research progress on enriching blood mechanism of Siwu decoction[J].Shandong J Tradit Chin Med,2017,36(9):819
[4]中华人民共和国药典2015年版.一部[S].2015:786ChP 2015.VolⅠ[S].2015:786
[5]刘婷婷,何瑶,王丽娟,等.HPLC法同时测定四物汤中6种成分的含量[J].中药材,2016,39(7):1602LIU TT,HE Y,WANG LJ,et al.Simultaneous determination of six components in Siwu decoction by HPLC[J].J Chin Med Mater,2016,39(7):1602
[6]李思漫,林灿城,陈文青,等.高效液相色谱法测定四物汤中阿魏酸的含量[J].广东化工,2015,42(15):221LI SM,LIN CC,CHEN WQ,et al.Determination of ferulic acid in Siwu Tang by HPLC[J].Guangdong Chem Ind,2015,42(15):221
[7]黄月纯,刘翠玲,张琼丹,等.四物汤中四种成分的含量测定研究[J].现代中药研究与实践,2010,24(4):64HUANG YC,LIU CL,ZHANG QD,et al.Study on the content determination of four components in Siwu decoction[J].Res Pract Chin Med,2010,24(4):64
[8]郝乘仪,郭淑英,朱鹤云,等.HPLC法同时测定牛黄清感胶囊中6个成分的含量[J].药物分析杂志,2017,37(3):422HAO CY,GUO SY,ZHU HY,et al.Simultaneous determination of six compounds in Niuhuang Qinggan capsules by HPLC[J].Chin JPharm Anal,2017,37(3):422
[9]CUI HR,XU GH,WU MQ,et al.Simultaneous determination of eight active components in Liuwei Wuling tablet using HPLC[J].Chin Herb Med,2016,8(4):331
[10]袁强华,呼梅,宋英,等.HPLC-DAD波长切换法同时测定化扁汤中10种有效成分的含量[J].中国医院药学杂志,2017,37(1):30YUAN QH,HU M,SONG Y,et al.Simultaneous determination of ten active components in Huabian decoction by HPLC-DADwavelength switching method[J].Chin J Hosp Pharm,2017,37(1):30
[11]何瑶,王丽娟,刘婷婷,等.基于代谢组学技术分析四物汤治疗原发性痛经证的作用机制[J].中国实验方剂学杂志,2017,23(12):82HE Y,WANG LJ,LIU TT,et al.Analysis of therapeutic mechanism of Siwu Tang in primary dysmenorrhea syndrome based on metabolomics technique[J].China J Exp Tradit Med Form,2017,23(12):82
[12]曾柳庭,刘慧萍,杨凯麟,等.四物汤有效成分的关联性分析[J].中成药,2017,39(3):576ZENG LT,LIU HP,YANG KL,et al.Correlation analysis of active components in Siwu Tang[J].Chin Tradit Pat Med,2017,39(3):576
[13]ZHAO L,CHEN J,SU J,et al.In vitro antioxidant and antiproliferative activities of 5-hydroxymethylfurfural[J].J Agric Food Chem,2013,61(44):10604
[14]唐于平,黄美艳,张彦华,等.四物汤类方与组方药材及其所含主要芳香酸体外抗氧化活性比较与量效关系研究[J].中国中西医结合杂志,2012,32(1):64TANG YP,HUANG MY,ZHANG YH,et al.Comparison of in vitro anti-oxidative activities among Siwu decoction Serial Recipes,their composed crude herbs,and main aromatic acids,as well as their dose-effect correlation[J].Chin J Integr Tradit West Med,2012,32(1):64
[15]张海鸣,闫寒,彭娟,等.脂质体平衡透析-液质联用色谱法筛选四物汤药效物质的研究[J].药物分析杂志,2014,34(12):2107ZHANG HM,YAN H,PENG J,et al.Study on screening of effective substances of Siwu decoction by liposome equilibrium dialysis and LC-MS method[J].Chin J Pharm Anal,2014,34(12):2107
[16]高森,白雪,文柳静,等.HPLC-DVD变波长法同时测定跌打止痛散中6种指标成分[J].中草药,2016,47(16):2863GAO S,BAI X,WEN LJ,et al.Determination of six kinds of index components in Dieda Zhitong San by HPLC-DVD variable wavelength method[J].Chin Tradit Herb Drugs,2016,47(16):2863
[17]张梅,任爱农,杨玉兰,等.HPLC波长切换法同时测定清清颗粒中9个成分的含量[J].药物分析杂志,2015,35(1):29ZHANG M,REN AN,YANG YL,et al.Simultaneous determination of contents of nine components in Qingqing granules by HPLCwavelength switching method[J].Chin J Pharm Anal,2015,35(1):29
[2]唐昆,吴希.四物汤的现代药理作用和临床应用[J].中国中医急症,2006,15(12):1397TANG K,WU X.Modern pharmacological effects and clinical application of Siwu Tang[J].J Emerg Tradit Chin Med,2006,15(12):1397
[3]胡琦,郭平.四物汤补血机制研究进展[J].山东中医杂志,2017,36(9):819HU Q,GUO P.Research progress on enriching blood mechanism of Siwu decoction[J].Shandong J Tradit Chin Med,2017,36(9):819
[4]中华人民共和国药典2015年版.一部[S].2015:786ChP 2015.VolⅠ[S].2015:786
[5]刘婷婷,何瑶,王丽娟,等.HPLC法同时测定四物汤中6种成分的含量[J].中药材,2016,39(7):1602LIU TT,HE Y,WANG LJ,et al.Simultaneous determination of six components in Siwu decoction by HPLC[J].J Chin Med Mater,2016,39(7):1602
[6]李思漫,林灿城,陈文青,等.高效液相色谱法测定四物汤中阿魏酸的含量[J].广东化工,2015,42(15):221LI SM,LIN CC,CHEN WQ,et al.Determination of ferulic acid in Siwu Tang by HPLC[J].Guangdong Chem Ind,2015,42(15):221
[7]黄月纯,刘翠玲,张琼丹,等.四物汤中四种成分的含量测定研究[J].现代中药研究与实践,2010,24(4):64HUANG YC,LIU CL,ZHANG QD,et al.Study on the content determination of four components in Siwu decoction[J].Res Pract Chin Med,2010,24(4):64
[8]郝乘仪,郭淑英,朱鹤云,等.HPLC法同时测定牛黄清感胶囊中6个成分的含量[J].药物分析杂志,2017,37(3):422HAO CY,GUO SY,ZHU HY,et al.Simultaneous determination of six compounds in Niuhuang Qinggan capsules by HPLC[J].Chin JPharm Anal,2017,37(3):422
[9]CUI HR,XU GH,WU MQ,et al.Simultaneous determination of eight active components in Liuwei Wuling tablet using HPLC[J].Chin Herb Med,2016,8(4):331
[10]袁强华,呼梅,宋英,等.HPLC-DAD波长切换法同时测定化扁汤中10种有效成分的含量[J].中国医院药学杂志,2017,37(1):30YUAN QH,HU M,SONG Y,et al.Simultaneous determination of ten active components in Huabian decoction by HPLC-DADwavelength switching method[J].Chin J Hosp Pharm,2017,37(1):30
[11]何瑶,王丽娟,刘婷婷,等.基于代谢组学技术分析四物汤治疗原发性痛经证的作用机制[J].中国实验方剂学杂志,2017,23(12):82HE Y,WANG LJ,LIU TT,et al.Analysis of therapeutic mechanism of Siwu Tang in primary dysmenorrhea syndrome based on metabolomics technique[J].China J Exp Tradit Med Form,2017,23(12):82
[12]曾柳庭,刘慧萍,杨凯麟,等.四物汤有效成分的关联性分析[J].中成药,2017,39(3):576ZENG LT,LIU HP,YANG KL,et al.Correlation analysis of active components in Siwu Tang[J].Chin Tradit Pat Med,2017,39(3):576
[13]ZHAO L,CHEN J,SU J,et al.In vitro antioxidant and antiproliferative activities of 5-hydroxymethylfurfural[J].J Agric Food Chem,2013,61(44):10604
[14]唐于平,黄美艳,张彦华,等.四物汤类方与组方药材及其所含主要芳香酸体外抗氧化活性比较与量效关系研究[J].中国中西医结合杂志,2012,32(1):64TANG YP,HUANG MY,ZHANG YH,et al.Comparison of in vitro anti-oxidative activities among Siwu decoction Serial Recipes,their composed crude herbs,and main aromatic acids,as well as their dose-effect correlation[J].Chin J Integr Tradit West Med,2012,32(1):64
[15]张海鸣,闫寒,彭娟,等.脂质体平衡透析-液质联用色谱法筛选四物汤药效物质的研究[J].药物分析杂志,2014,34(12):2107ZHANG HM,YAN H,PENG J,et al.Study on screening of effective substances of Siwu decoction by liposome equilibrium dialysis and LC-MS method[J].Chin J Pharm Anal,2014,34(12):2107
[16]高森,白雪,文柳静,等.HPLC-DVD变波长法同时测定跌打止痛散中6种指标成分[J].中草药,2016,47(16):2863GAO S,BAI X,WEN LJ,et al.Determination of six kinds of index components in Dieda Zhitong San by HPLC-DVD variable wavelength method[J].Chin Tradit Herb Drugs,2016,47(16):2863
[17]张梅,任爱农,杨玉兰,等.HPLC波长切换法同时测定清清颗粒中9个成分的含量[J].药物分析杂志,2015,35(1):29ZHANG M,REN AN,YANG YL,et al.Simultaneous determination of contents of nine components in Qingqing granules by HPLCwavelength switching method[J].Chin J Pharm Anal,2015,35(1):29