HPLC法同时测定积雪草中2个黄酮类成分的含量.pdf

HPLC法同时测定积雪草中2个黄酮类成分的含量87HPLC法同时测定积雪草中2个黄酮类成分的含量伍蕴璐1，吴宇婷1，苏晨薇1，李君羽1，柳贤福1，李数霞2*（1.广西中医药大学赛恩斯新医药学院南宁 530222；2.广西中医药大学研究生院 南宁 530222）项目基金：广西大学生创新创业训练计划项目（编号：S202113643057）、广西壮瑶药重点实验室（桂科基字201432号）、广西中医药大学大学生科研训练项目（编号：2021DXS25）、壮瑶药协同创新中心（桂教科研201320号）、广西壮族自治区民族药资源与应用工程研究中心（桂发改高技函20202605号）第一作者：伍蕴璐，药学专业，广西中医药大学赛恩斯新医药学院在校生*通讯作者：李数霞，硕士，讲师，从事中药、民族药质量分析研究【摘 要】目的：建立HPLC法同时测定积雪草中2个黄酮类成分含量的方法。方法：采用HPLC法，色谱柱为AgilentC18柱（250mm4.6mm，5 m）；流动相为甲醇：0.1%磷酸水（6040）；检测波长为360nm；流速1.0mLmin-1；柱温30；进样量10 L。结果：得到图谱与标准物质对照，确定其2个成分分别为槲皮素，山柰酚（以下简称2个成分）；2个成分在测定范围内线性关系良好，平均加样回收率为99.54%和99.25%，RSD均小于3.0%，4个不同批次样品中2个成分含量分别为0.029%0.038%、0.072%0.091%。结论：该方法简便准确、重复性好，可用于积雪草2个黄酮类成分的含量测定。【关键词】积雪草；HPLC；含量测定；槲皮素；山柰酚Simultaneous determination of two components in Centella asiatica L.Urb.by HPLCWu Yunlu1，Wu Yuting1，Su Chenwei1，Li Junyu1，Liu Xianfu1，Li Shuxia2*（Guangxi University of Chinese Medicine，1.Science Medical College，2.Graduate School Nanning，530222）【Abstract】Objective：To establish an HPLC method for simultaneous determination of two components for Centella asiaticaL.Urb.Methods：The HPLC assay was performed on a Agilent C18（250 mm4.6 mm，5 m）；The mobile phase was methanol：0.1%Phosphoric acid（60：40），The flow rate was 1 mLmin-1，the detection wavelength was 360 nm and the column temperature was 30 and the injection volume was 10 L.Results：Two constituents of quercetin and kaempferol were determined by comparing the spectra with standard substances.The linear relationship of the two components was good within the range of determination.The average recovery of sample addition is 99.54%and 99.25%（RSD3.0%）.The contents of two components in four batches of samples were 0.029%0.038%and 0.072%0.091%.Conclusions：The method is simple，accurate and reproducible.It can be used for the determination of two components in Centella asiaticaL.Urb.【Keywords】Centella asiatica L.Urb.；HPLC；Content determination；Quercetin；Kaempferol积雪草为伞形科植物积雪草（Centella asiatica L.Urb.）的干燥全草，具有清热利湿、解毒消肿的功效，本文采用HPLC法对积雪草中槲皮素、山柰酚的含量进行同时测定，为积雪草质量评价提供参考依据。1 材料1.1 仪器Agilent 1200高效液相色谱仪（美国Agilent公司），DAD检测器；KQ-100DE型数控超声波清洗仪（昆山市超声波仪器有限公司）；BP211D万分之一电子分析天平（德国赛多利斯）；Millipore Simplicity-185超纯水仪（美国密里博公司）等。质量标准Identification&SourceGendingSwhyenz 881.2 试药对照品槲皮素（批号：100081-201610，纯度HPLC99.9%）购于中国食品药品检定研究院；山柰酚（批号：3 532，纯度HPLC99.8%）购于上海诗丹德标准技术服务有限公司；积雪草药材经广西中医药大学赛恩斯新医药学院药用植物学教研室鉴定为伞形科植物积雪草（Centella asiaticaL.Urb.）的干燥全草，采集信息见表1；甲醇（色谱纯，美国Fisher科学世界公司）；其余试剂均为分析纯；水为超纯水。表1 积雪草中药饮片信息样品采集地采集时间积雪草广西贵港2021.09广西钦州2021.10广西南宁2021.10广西柳州2021.102.5 线性关系考察精密吸取“2.2”项下对照品混合溶液2 L、4 L、6 L、10 L、16 L、20 L注入液相色谱仪，按“2.1”项下色谱条件进行分析，以进样量为横坐标（X），峰面积为纵坐标（Y）绘制标准工作曲线，线性关系考察结果见表2。2 方法与结果2.1 色谱条件色谱柱为Agilent C18柱（250 mm4.6 mm，5 m）；流动相为甲醇-0.1%磷酸水溶液（6040）；检测波长为360 nm；流速为1.0 mLmin-1；柱温35；进样量为10 L。2.2 对照品溶液的制备精密称取槲皮素、山柰酚对照品适量，加甲醇制成每1 mL分别含槲皮素0.032 mg、山柰酚0.054 mg的混合对照品溶液。2.3 供试品溶液的制备取积雪草药材粉末约1 g，精密称定，精密加入100%甲醇溶液20 mL，称重，加热回流提取1 h，放冷，用100%甲醇溶液补足减失的重量，摇匀，过0.45 m微孔滤膜，精密吸取10 L注入高效液相色谱仪测定1，2。2.4 专属性试验分别精密吸取“2.2”项下对照品混合溶液和积雪草供试品溶液各10 L进样分析，结果见图1。图1 混合对照品（A）和积雪草供试品（B）色谱图表2 线性关系考察结果成分回归方程r线性范围/g槲皮素y=3 256.1x-5.276 90.999 80.0640.64山柰酚y=3 026.3x-10.4850.999 90.1081.082.6 精密度试验精密吸取“2.2”项下对照品混合溶液10 L，连续进样6次，计算2个成分峰面积的RSD分别为0.22%和0.23%。表明仪器精密度良好，结果见表3。A 檞皮素1250403020100mAU2612410814t/minB 山柰酚12250200150100500mAU2612410814t/minHPLC法同时测定积雪草中2个黄酮类成分的含量89表3 精密度考察结果成分123456RSD/%槲皮素峰面积1 020.11 0221 022.91 024.71 026.11 025.10.22山柰素峰面积1 616.91 621.11 622.21 624.91 627.61 624.70.232.7 重复性试验取同一批样品（广西贵港），按“2.3”项下方法分别制备6份供试品溶液，按“2.1”项下色谱条件进行测定，记录峰面积，按回归方程分别计算2个成分含量的RSD分别为1.94%和1.63%。表明方法重复性良好，结果见表4。表4 重复性考察结果成分123456平均含量RSD/%槲皮素（mg/g）0.389 80.386 90.371 70.373 90.3 7850.375 40.379 41.94山柰（mg/g）0.885 10.910 70.922 20.912 10.9 1570.890 60.906 11.632.8 稳定性试验取同一份供试品（广西贵港），按“2.1”项下色谱条件分别在0 h、2 h、6 h、10 h、16 h、24 h进样10 L，计算2个成分峰面积的RSD分别为0.45%和0.22%。表明供试品溶液在24 h内稳定，结果见表5。表5 稳定性考察结果成分026101624RSD/%槲皮素峰面积548.2544.6546.4550551.3546.80.45山柰素峰面积1 312.61 310.91 316.41 313.31 317.21 309.90.222.9 加样回收率试验取同批号（广西贵港）已知含量的样品9份，每份约0.5 g，精密称定，分别精密加入一定量的2个成分对照品，按“2.3”项下方法制备供试品溶液，按“2.1”项下色谱条件进样10 L，计算2个成分的平均回收率，结果见表6。表6 加样回收率的实验结果（n=9）化合物样品重量/g已知量/mg加入量/mg测得量/mg加样回收率/%平均加样回收率/%RSD/%槲皮素0.501 2 0.190 2 0.15 0.345 1 101.94 99.54 1.26 0.503 2 0.190 9 0.15 0.340 8 98.61 0.501 5 0.190 3 0.15 0.342 2 99.95 0.500 6 0.189 9 0.19 0.380 9 100.51 0.501 1 0.190 1 0.19 0.378 9 99.36 0.501 8 0.190 4 0.19 0.376 6 98.01 0.500 9 0.190 0 0.22 0.410 7100.30 0.507 3 0.192 5 0.22 0.408 898.33 0.505 5 0.191 8 0.22 0.409 398.87 质量标准Identification&SourceGendingSwhyenz 90化合物样品重量/g已知量/mg加入量/mg测得量/mg加样回收率/%平均加样回收率/%RSD/%山奈素0.501 2 0.454 1 0.36 0.810 2 98.91 99.25 1.15 0.503 2 0.455 9 0.36 0.810 3 98.43 0.501 5 0.454 4 0.36 0.812 8 99.55 0.500 6 0.453 6 0.45 0.902 2 99.69 0.501 1 0.454 0 0.45 0.909 6 101.23 0.501 8 0.454 7 0.45 0.905 8 100.25 0.500 9 0.453 9 0.54 0.981 797.75 0.507 3 0.459 7 0.54 0.987 8 97.80 0.505 5 0.458 0 0.54 0.995 9 99.60 续表2.10 样品的测定取4个不同批次的积雪草样品，按“2.3”项下方法制备样品溶液，测定5个成分的含量，结果见表7。表7 含量测定结果（%，n=3）产地槲皮素山柰酚广西贵港0.0380.091广西钦州0.0290.072广西南宁0.0320.081广西柳州0.0340.0803 讨论3.1 供试品溶液制备方法考察本文考察了超声提取和加热回流提取两种提取方法，结果显示加热回流提取法对待测成分的提取率更高，加热回流提取2 h和1 h无明显差异，结合文献资 料4，5，故选择回流提取1 h。考察甲醇、乙醇两种提取溶剂，结果显示甲醇提取率更高且色谱基线平稳，峰型对称，继续考察60%、70%、80%、90%和100%甲醇提取，经HPLC法测定，比较其色谱图，结果表明以100%甲醇作为提取溶剂提取率较高，且干扰少。故最终确定以100%甲醇为提取溶剂，回流提取1 h。3.2 色谱条件考察通过全波长扫描得到混合对照品的3D图谱，结果显示在波长为360 nm处2个待测成分均有较大吸收且灵敏度较好。考察了不同比例的乙腈-0.1%磷酸水、甲醇-0.1%磷酸水、乙腈-水、甲醇-水等流动相系统，考察25 和30 柱温以及0.5 mLmin-1和1 mLmin-1的流速，结果显示以甲醇-0.1%磷酸水溶液（6040）为流动相，柱温为30，流速为1 mL/min时分离度较好，保留时间适中，峰形较对称。3.3 小结含量测定结果显示，4个不同产地的积雪草不同成分含量测定结果说明不同产地的积雪草样品中的槲皮素和山柰素含量存在一定的差异。本文所建立的同时测定积雪草中2个成分的方法简便准确、精密度高、稳定性和重复性好，加样回收率符合相关规定，可用于积雪草的质量评价。参考文献1柳贤福，丁聪，黄春燕，等.HPLC法同时测定刘寄奴中5个成分的含量J.药物分析杂志，2019，39（11）：1 998-2 002.2柳贤福，黄红泓，覃日宏，等.白苞蒿提取工艺优化及槲皮素含量测定J.湖南师范大学自然科学学报，2018，41（5）：43-47.309soujdon cujyau hawj de gemjnoix gijdoeg dem ak yaugoj.Bien faenzcieng neix yaek doenggvaq diemjra bwnjcauj vwnzyen、cazyawj cingjleix yienhdaih ywyauq doegleix daengj swhliu damqra gij fazcanj baenaj yenzgiu gofuswj，aeu neix daeuj vih gya gvangq wngqyungh gofuswj daezhawj hagsib caeuq canhgauj.【Aenswz Gvanhgen】Gofuswj；vayoz cwngzfwn；ywleix cozyung；doxboiq；gemjnoix gijdoeg dem ak yaugojGij Cingzgvang Seizneix Damqgouz Ywcuengh Ndoksienggoh（骨伤科壮药的研究现状）Liengz Sez，Cangh Cauhyi，Huz Gyauhgyauh，Denz Vei*（Gvangjsih Cunghyihyoz Dayoz，Gvangjsih Nanzningz 530200）【Cwzyau】Cangyih ndoksieng yihyoz dwg youq gij lijlun gihcuj cangyih cijdauj laj，youz cangyih gunghcozcej ginggvaq ciengzgeiz saedhengz caeuq cungjgez ndaej cugbouh cauxbaenz，gij danyw gapbaenz caeuq gij fuengfap daj baihrog yw de haemq lai，cujyau doiq yw laemxsieng、ndokraek、gvanhcezyenz daengj bingh ndoksienggoh ndaej yw ndei caeuq miz gaijliengz cozyungh haemq ndei，doenggvaq vwnzyen yenzgiu，gvinab cungjgez ok cangyih yw bingh ndoksienggoh gij daegsaek caeuq youhsi de，caiqlij gyoebgangj doiq danyw ciengzyungh guh vayoz cwngzfwn、ywleix cozyung caeuq linzcangz wngqyungh sam fuengmienh gij yenzgiu de，vih linzcangz yw bingh ndoksienggoh caeuq fuengzyw daezhawj lohnaemj caeuq roenloh.【Aenswz Gvanhgen】Ndoksieng cangyih；ywcuengh；ywdoj fuzfangh；gij yenzgiu canggvang seizneixGij Fazcanj Baenaj Gyawj Sam Bi Daeuj Damqgouz Gogihguzcauj Cang-Yauzyoz（壮瑶药鸡骨草近三年研究进展）Cinz Giz1，Cinh Danhdanh2*（1.Gvangjsih Hozbuj Yen Yinzminz Yihyen，Gvangjsih Hozbuj 536100；2.Gvangjsih Cunghyihyoz Dayoz，Gvangjsih Nanzningz 530001）【Cwzyau】Bien faenzcieng neix doiq gij fazcanj baenaj gyawj sam bi daeuj damqgouz gogihguzcauj guh cunghhozsing yenzgiu.Gogihguzcauj youq caetliengh gaemhanh fuengmienh、youq lwleix fuengmienh，doiq saedniemhsingq aendaep sienghaih gij yingjyangj de、ganhyi gipsingq ciujcinghsing aendaep sienghaih、yw yizhingz ganhyenz gij cozyung gihci、gang yangjva hozsing、daise cwngzfwn、doiq gueng gaeqokgyaeq gij hezcih cijbyauh dih yingjyangj；linzcangz wngqyungh fuengmienh，yw binghdaep、yw baenz binghveiaiz gaxgonq gij binghbienq de、yw bingh sinhgonhfeiyenz cungj miz baudauj.Bien faenzcieng neix vih gogihguzcauj youq caetliengh gaemhanh fuengmienh、ywleix fuengmienh caeuq linzcangz wngqyungh fuengmienh caenh itbouh damqgouz daezhawj canhgauj.【Aenswz Gvanhgen】Gogihguzcauj；caetliengh；linzcangz wngqyunghDamqgouz Cingqgyau Sawqniemh Youhva Dungfunghlizanh Gojliz Gij Daezaeu Gunghyi De（正交试验优化痛风立安颗粒的提取工艺研究）Liz Li，Suh Genlungz，Lij Fungcinh，Cinz Sungh，Lij Yinyungz*（Gvangjsih Gozci Cangyih Yihyen，Gvangjsih Nanzningz 530201）【Cwzyau】Muzdiz：Senjndei ywcuengh dungfungh-lizanh gojliz gij daezaeu gunghyi de.Fuengfap：Yungh L9（34）cingqgyau sawqniemh sezgi，aeu ywyicinzbizding ndaw cungcezfungh、hujcangganh ndaw gohujcang、majcenzganh ndaw gaeuyinjdunghdwngz gij conjyizliz caeuq ganhgauhliz guh gaujcaz cijbyauh，aeu gyaraemxliengh、cienqyw baez soq、cienqyw seizgan guh gaujcaz yinhsu，senjndei dungfunghlizanh gojliz gij daezaeu gunghyi de caiqlij guh gunghyi niemhcingq.Gezgoj：Gij daezaeu gunghyi senjndei dwg danywliengh gya 12 boixliengh raemx cienq sam dangq，moiq dangq 40 min，niemhcingq sawqniemh gezgoj biujmingz，yicinzbizding、hujcangganh、majcenzganh bingzyaenx conjyizliz faenbied dwg 55.89、111.99、47.69（RSD faenbied dwg 3.68、2.47、0.74，n=3），bingzyaenx ganhgauhliz dwg 17.59（RSD=1.68，n=3）.Gietlwnh：Gij daezaeu gunghyi senjndei dungfunghlizanh gojliz onjdingh ndaejguh、cungzfuksingq ndei，ndaej vih ngoenzlaeng damqgouz swnghcanj gunghyi daezhawj baengzgawq.【Aenswz Gvanhgen】Dungfunghlizanh gojliz；cingqgyau sawqniemh；daezaeu gunghyiHPLC Fap Doengzseiz Raudingh Ndaw Gocizsezcauj 2 Aen Vuengzdoengzlei Cwngzfwn Gij Hamzliengh De（HPLC法同时测定积雪草中2个黄酮类成分的含量）Vuj Yinlu1，Vuz Yijdingz1，Suh Cinzveih1，Lij Ginhyij1，Liuj Yenzfuz1，Lij Suyaz2*（Gvangjsih Cunghyihyoz Dayoz，1.Saiwnhswh Sinhyihyoz Yozyen，2.Yenzgiuswnghyen，Gvangjsih Nanzningz 530222）壮文摘要Identification&SourceGendingSwhyenz 310【Cwzyau】Muzdiz：Laebguh aen fuengfap HPLC faz doengzseiz raudingh ndaw gocizsezcauj 2 aen vuengzdoengzlei cwngzfwn gij hamzliengh de.Fuengfap：Yungh HPLC faz，swzbujci dwg Agilent C18 ci（250 mm4.6 mm，5 m）；riuzdoengsieng dwg gyazcunz：0.1 raemxlinzsonh（60：40）；niemhdingh bohcangz dwg 360 nm；riuzdoengh suzdu 1.0 mLmin-1；civwnh 30；haeujyienghliengh 10 L.Gezgoj：Ndaejdaengz duzbuj caeuq biucinj vuzciz doiqciuq，doekdingh 2 aen cwngzfwn de，faenbied dwg huzbizsu，sanhnaifwnh（lajneix genjdanh heuhguh 2 aen cwngzfwn）；2 aen cwngzfwn youq ndaw gvaengxlaengx raudingh sensing gvanhaeh ndei，bingzyaenx gyayiengh soudauqlwd dwg 99.54 caeuq 99.25，RSD cungj noix gvaq 3.0，gij yangbinj 4 buek mbouj doengz ndaw de 2 aen cwngzfwn hamzliengh faenbied dwg 0.0290.038、0.0720.091.Gietlwnh：Cungj fuengfap neix gienjbienh cinjdeng、cungzfuksing ndei，ndaej yungh youq raudingh gocizsezcauj 2 aen vuengzdoengzlei cwngzfwn gij hamzliengh de.【Aenswz Gvanhgen】Gocizsezcauj；HPLC；raudingh hamzliengh；huzbizsu；sanhnaifwnhDamqgouz Yungh Swjvai-Gojgen gvanghbujfaz Doiq Rag Gomingzyizyez Guh Genbez（采用紫外-可见光谱法对明日叶根鉴别的研究）Cinh Danhdanh，Veiz Ginhlingz，Liuz Li，Loz Siengzyez，Guj Baujgin，Cangh Myauj，Giuh Ginz*（Gvangjsih Cunghyihyoz Dayoz，Gvangjsih Nanzningz 530001）【Cwzyau】Muzdiz：Vih dingh ok rag gomingzyizyez gij gvanghbuj genbez fuengfap de daezhawj canhgauj.Fuengfap：Yungh gij fuengfap swjvai-gojgen gvanghbuj daeuj genbez.Gezgoj：Rag gomingzyizyez mbouj doengz yungzci、mbouj doengz daezaeu fuengfap ndaejdaengz gij dizcijyiz，gij dwzcwngh gizsouhfungh de gak miz daegdiemj.Gietlwnh：Baez saedniemh neix vih doiq rag gomingzyizyez guh gvanghbuj genbez gaemhanh fuengfap daezhawj le gij baengzgawq canhgauj.【Aenswz Gvanhgen】Gomingzyizyez；rag；swjvai-gojgen gvanghbujYawhsawqniemh Gij Vayoz Cwngzfwn Godwngzvangzdanz Ywcuengh（壮药藤黄檀的化学成分预试验）Cinh Danhdanh1，Lij Vwnzsiu*1，2，Giuh Ginz1，Veiz Ginhlingz1，Liuz Yenzyenz1，Cinz Cunhbingz1，Cinh Hanhsinh*（1.Gvangjsih Cunghyihyoz Dayoz，Gvangjsih Nanzningz 530001；2.Hwzlungzgyangh Cunghyihyoz Dayoz Gyahmuzswh Yozyen，Hwzlungzgyangh Gyahmuzswh，154007）【Cwzyau】Muzdiz：Vih caenh itbouh damqgouz godwngzvangzdanz ywcuengh gij caetliengh gaemhanh fuengfap de daezhawj canhgauj.Fuengfap：Yungh ywdoj vayoz hidungj yawhsawqniemh fuengfap.Gezgoj：Doenggvaq doiq faenjco gij mbaw godwngzvangzdanz guh vayoz yawhsawqniemh，cobouh nyinhnaeuz ndaw gij mbaw godwngzvangzdanz aiq hamzmiz youzcizlei、youjgihsonhlei、vuengzdoengzlei、fwnhlei、ganhlei daengj vayoz cwngzfwn.Gietlwnh：Baez saedniemh neix vih caenh itbouh damqgouz godwngzvangzdanz guh sawqniemh gihcuj caeuq baengzgawq.【Aenswz Gvanhgen】Godwngzvangzdanz；yawhsawqniemh；vayoz cwngzfwnYungh HS-GC-MS Faensik Gocinzyangh Cwx Caeuq Vunzgoeng Ndaem Gij Veihfazsing Cwngzfwn De（野生和人工种植沉香挥发性成分的HS-GC-MS分析）Hanz Sah1，Vangz Swngcauh2，Cinz Veiminj2，Lij Hungzlieng2，Lij Sanghsangh2，Lij Ban2，Gungh Ci Gyangz1，2*（1.Gvangjsih Cunghyihyoz Dayoz Yauzyihyoz Yozyen，Gvangjsih Nanzningz 530002；2.Gvangjsih Cunghyihyoz Dayoz Saiwnhswh Sinhyihyoz Yozyen，Gvangjsih Nanzningz 530222）【Cwzyau】Yungh gij gisuz dingjgunggisieng swzbuj cizbuj liemzyungh（HS-GC-MS）doiq gij yangbinj gocinzyangh cwx caeuq vunzgoeng ndaem Haijnanz ok gij veihfazsing cwngzfwn de guh faensik，gezgoj byaujmingz，gocinzyangh cwx caeuq vunzgoeng ndaem gij veihfazsing cwngzfwn de cungj dwg vahozvuz fanghyanghcuzlei caeuq beibandezlei guhcawj，veihfazsing cujfaen cungjloih caeuq siengdoiq hamzliengh cungj miz itdingh cengca，ndaw de gocinzyangh cwx faenbied bingzdingh ok 21 aen cujfaen，ciemq cungj veihfazsing cwngzfwn 59.38，ndaw de dwg cinzyangh fuhnanz（9.15）、benjgihbingjdungz（7.63）、duigyazyangjgih benjgihbingjdungz（6.42）、bwzmuzcunz（5.74）、bwzmuzyanghcenz（4.56）daengj guh cujyau cwngzfwn.Gocinzyangh vunzgoeng ndaem faenbied bingzdingh le 17 aen cujfaen，ciemq cungj veihfazsing cwngzfwn 44.76，dwg cinzyangh fuhnanz（11.35）、benjgihbingjdungz（9.21）、bwzmuzyanghcenz（7.62）、-muzyanghcunz（4.87）guh cujyau veihfazsing cwngzfwn，ndaw 2 aen yangbinj gungh miz 8 cungj cwngzfwn，faenbied dwg benjgihbingjdungz、cinzyangh fuhnanz、-muzyanghcunz、bwzmuzyanghcenz、-faznizhih、