RP-HPLC法同时测定茵陈蒿汤中14个成分

目的：建立反相高效液相色谱法同时测定茵陈蒿汤（茵陈、栀子、大黄）中滨蒿内酯、儿茶素、绿原酸、东莨菪内酯、金丝桃苷、秦皮乙素、京尼平苷、槲皮素、京尼平、大黄酸、芦荟大黄素、大黄酚、大黄素、大黄素甲醚的含量。方法：采用Eclipse Plus C₁₈（250 mm×4.6 mm，5 μm）色谱柱，以乙腈（A）-0.1% 磷酸水溶液（B）为流动相，梯度洗脱，流速1.0 mL·min^-1，柱温30 ℃，检测波长210 nm（儿茶素、绿原酸、秦皮乙素、京尼平、东莨菪内酯、滨蒿内酯、槲皮素）、238 nm（京尼平苷、金丝桃苷）、254 nm（芦荟大黄素、大黄酸、大黄素、大黄酚、大黄素甲醚），进样量25 μL。结果：14 个成分可实现完全分离，在一定的线性范围内呈良好的线性关系（r>0.999 8）、精密度（RSD ≤ 2.0%）、稳定性（RSD ≤ 1.9%）和重复性（RSD ≤ 1.8%）良好，平均回收率（n=6）在94.4%~98.0% 范围内，RSD 在0.86%~2.8% 范围内。3 批茵陈蒿汤中滨蒿内酯、儿茶素、绿原酸、东莨菪内酯、金丝桃苷、秦皮乙素、京尼平苷、槲皮素、京尼平、大黄酸、芦荟大黄素、大黄酚、大黄素、大黄素甲醚14 个成分的含量测定结果分别为0.136~0.168、0.080~0.104、0.403~0.470、0.008~0.009、0.061~0.068、0.020~0.027、0.269~0.306、0.036~0.048、0.005~0.006、0.122~0.164、0.037~0.044、0.030~0.037、0.011~0.014、0.007~0.010 mg·g^-1。结论：该方法经方法学验证，可用于茵陈蒿汤中14 个化学成分的含量测定。

Objective: To establish a RP-HPLC method for simultaneous determination of scoparone,catechin, chlorogenic acid,scopoletin,hyperoside,esculetin,geniposide,quercetin,genipin,rhein,aloe-emodin, chrysophanol,emodin and physcion in Yin-Chen-Hao-Tang(Artemisia capillaris Thunb.,Gardenia jasminoides Ellis and Rheum Palmatum L.). Methods: The separation was carried out on an Eclipse Plus C₁₈(250 mm×4.6 mm,5 μm)column eluted with the mobile phase of acetonitrile(A)-0.1% phosphoric acid aqueous solution (B)in a gradient elution manner at a flow rate of 1.0 mL·min^-1. The detection wavelength was set at 210 nm for scoparone,catechin,chlorogenic acid,scopoletin,esculetin,quercetin and genipin,at 238 nm for geniposide and hyperoside,and at 254 nm for rhein,aloe-emodin,chrysophanol,emodin and physcion. The column temperature was set at 30℃ and the injection volume was 25 μL. Results: Satisfactory separation was obtained for the 14 compounds. In the given concentration range,all the compounds showed good linear correlation(r>0.999 8). The RSD values of precision,stability and repeatability were lower than 2.0%,1.9% and 1.8%,respectively. The average recoveries of the 14 compounds were 94.4%-98.0% with the RSD values of 0.86%-2.8%(n=6). The contents in 3 samples were in the ranges of 0.136-0.168 mg·g^-1 for scoparone,0.080-0.104 mg·g^-1 for catechin,0.403-0.470 mg·g^-1 for chlorogenic acid,0.008-0.009 mg·g^-1 for scopoletin,0.061-0.068 mg·g^-1 for hyperoside,0.020-0.027 mg·g^-1 for esculetin,0.269-0.306 mg·g^-1 for geniposide,0.036-0.048 mg·g^-1 for quercetin,0.005-0.006 mg·g^-1 for genipin,0.122-0.164 mg·g^-1 for rhein,0.037-0.044 mg·g^-1 for aloe-emodin, 0.030-0.037 mg·g^-1 for chrysophanol,0.011-0.014 mg·g^-1 for emodin and 0.007-0.010 mg·g^-1 for physcion. Conclusion: This method was proved by methodology validation that it is suitable for the determination of those 14 constituents in Yin-Chen-Hao-Tang.

[1] 邓中甲. 方剂学[M]. 北京:中国中医药出版社,1998:293 DENG ZJ. Prescriptions[M]. Beijing:China Press of Traditional Chinese Medicine,1998:293
[2] WANG S,LI X,NIU Y,et al. Identification and screening of chemical constituents with hepatoprotective effects from three traditional Chinese medicines for treating jaundice[J]. J Sep Sci, 2016,39(19):3690
[3] 王永宏,赵晨曦,陈本美,等. 茵陈蒿汤对二甲基亚硝胺诱导大鼠 肝纤维化的逆转作用[J]. 中国中药杂志,2014,39(8):1473 WANG YH,ZHAO CX,CHEN BM,et al. Reverse effect of Yinchenhao decoction in dimethyl nitrosamine-induced hepatic fibrosis in rats[J]. China J Chin Mater Med,2014,39(8):1473
[4] 李娟. 茵陈蒿汤治疗急性黄疸型肝炎的观察[J]. 中外医学研究, 2014,12(7):37 LI J. The observation of the acute jaundice hepatitis after the treatment by Yinchenhao Tang[J]. Chin Foreign Med Res,2014, 12(7):37
[5] LEE HI,LEE MK. Coordinated regulation of scopoletin at adipose tissue-liver axis improved alcohol-induced lipid dysmetabolism and inflammation in rats[J]. Toxicol Lett,2015,237(3):210
[6] SUBRAMANIAM SR,ELLIS EM. Umbelliferone and esculetin protect against N-nitrosodiethylamine-induced hepatotoxicity in rats[J]. Cell Biol Int,2016,40(7):761
[7] 谭桢,刘爱明,罗敏,等. 茵栀黄注射液抗胆汁淤积药效成分的筛 选及其作用机制研究[J]. 中国中药杂志,2016,41(6):1113 TAN Z,LIU AM,LUO M,et al. Screening of effective ingredients of Yinzhihuang injection against cholestasis and their mechanism[J]. China J Chin Mater Med,2016,41(6):1113
[8] 史秀玲,高银辉. 绿原酸对小鼠急性肝损伤的保护作用[J]. 中国 实验方剂学杂志,2011,17(19):199 SHI XL,GAO YH. Protective effects of chlorogenic acid on injury in mice[J]. Chin J Exp Tradit Med Form,2011,17(19):199
[9] ZHANG JQ,SHI L,XU XN,et al. Therapeutic detoxification of quercetin against carbon tetrachloride-induced acute liver injury in mice and its mechanism[J]. J Zhejiang Univ Med Sci,2014,15 (12):1039
[10] CHOI JH,KIM D W,YUN N,et al. Protective effects of hyperoside against carbon tetrachloride-induced liver damage in mice[J]. J Nat Prod,2011,74(5):1055
[11] CHEN P,CHEN Y,WANG Y,et al. Comparative evaluation of hepatoprotective activities of geniposide,crocins and crocetin by CCl4-induced liver injury in mice[J]. Biomol Ther,2016,24(2):156
[12] NAKATAKE R,TSUDA T,MATSUURA T,et al. Genipin inhibits the induction of inducible nitric oxide synthase through the inhibition of NF-kappaB activation in rat hepatocytes[J]. Drug Metab Lett, 2016,10(4):
[13] KIM J,KIM HY,LEE SM. Protective effects of geniposide and genipin against hepatic ischemia/reperfusion injury in mice[J]. Biomol Ther,2013,21(2):132
[14] 覃鲁珊,赵海平,赵艳玲,等. 大黄蒽醌与鞣质对大鼠肝脏的保护 和损伤双向作用[J]. 中国中西医结合杂志,2014,34(6):698 QIN LS,ZHAO HP,ZHAO YL,et al. Protection and bidirectional effect of rhubarb anthraquinone and tannins for rat's liver[J]. Chin J Integr Tradit West Med,2014,34(6):698
[15] 葛建华,窦志华,罗琳,等. HPLC 法测定茵陈蒿汤中绿原酸和栀 子苷的含量[J]. 江苏中医药,2009,41(1):56 GE JH,DOU ZH,LUO L,et al. Simultaneous determination of chlorogenic acid and geniposide in Yin-Chen-Hao-Tang by HPLC[J]. Jiangsu Tradit Chin Med,2009,41(1):56
[16] 田书霞,阎姝,王竹云,等. 高效液相法同时测定茵陈蒿汤中7 种 成分[J]. 时珍国医国药,2013,24(2):399 TIAN SX,YAN S,WANG ZY,et al. Simultaneous determination of seven constituents in Yin-Chen-Hao-Tang by HPLC[J]. Lishizhen Med Mater Med Res,2013,24(2):399
[17] 窦志华,孟萍,姜晓燕,等. 10 家医院饮片组方的茵陈蒿汤中11 种成分的含量测定[J]. 中国实验方剂学杂志,2015,21(23):44 DOU ZH,MENG P,JIANG XY,et al. Determination of 11 constituents in Yinchenhao Tang composed by decoction pieces from hospitals of traditional Chinese medicine[J]. Chin J Exp Tradit Med Form,2015,21(23):44
[18] ZHANG H,WANG X,HU P,et al. Serum metabolomic characterization of liver fibrosis in rats and anti-fibrotic effects of Yin-Chen-Hao-Tang[J]. Molecules,2016,21(1):E126
[19] 中国药典2015 年版. 一部[S]. 2015:23,239,248 ChP 2015.Vol Ⅰ[S]. 2015:23,239,248