HPLC-DAD-ESI-MS/MS 法对黄连解毒汤四个极性部位化学成分的定性定量研究及其相关的神经保护作用

doi:10.5246/jcps.2013.04.048

HPLC-DAD-ESI-MS/MS 法对黄连解毒汤四个极性部位化学成分的定性定量研究及其相关的神经保护作用

杨阳, 赵海誉, 王宏洁, 宋建芳, 杨健, 司南, 边宝林^*

中国中医科学院中药研究所, 北京 100700

收稿日期:2013-01-06 修回日期:2013-02-22 出版日期:2013-07-10 发布日期:2013-07-10
通讯作者: 边宝林*

Qualitative and quantitative analysis of four different polarity fractions
from Huang-Lian-Jie-Du-Decoction by HPLC-DAD-ESI-MS/MS and their related neuroprotective effects

Yang Yang, Haiyu Zhao, Hongjie Wang, Jianfang Song, Jian Yang, Nan Si, Baolin Bian^*

Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing, 100700, China

Received:2013-01-06 Revised:2013-02-22 Online:2013-07-10 Published:2013-07-10
Contact: Baolin Bian*

摘要/Abstract

摘要：

为了阐明黄连解毒汤神经保护作用与化学成分的相关性, 本实验将黄连解毒汤水提取物分为四个不同极性段, 运用HPLC-DAD-ESI-MS/MS 鉴定了四个极性段的17个主要化学成分, 并运用HPLC-DAD对其中11个化合物进行了定量研究。方法学考察显示线性良好(r², 0.9994-0.9999), 重复性和稳定性的RSD值分别为1.66%-3.67%, 1.26%-4.77%, 回收率(95.24%-105.41%, RSD, 0.29%-2.69%)。在化学研究的基础上, 运用PC12细胞核元代神经元细胞对黄连解毒汤及四个不同极性段神经保护作用进行了考察。结果表明水提取物和正丁醇部分比其他极性段的神经保护作用更强。根据化学成分的定性和定量分析, 可以推断在水提物和正丁醇部位含量明显较高的环烯醚萜和生物碱与神经保护作用相关。

关键词: 黄连解毒汤, 定性定量分析, 神经保护

Abstract: Huang-Lian-Jie-Du-Decoction (HLJDD) has been widely used for the treatment of Alzheimer’s disease (AD) in clinic. However, the relationship between its chemical profile and neuroprotective bioactivity was not clearly clarified yet. In present study, the water extract of HLJDD and subsequent three polarity fractions divided by different reagents were investigated. A total of 17 chromatographic peaks were confirmed by comparison with standards and their UV, MS spectra. Among them, 11 major compounds were determined by HPLC-DAD method with good linear regression relationship (r², 0.9994-0.9999), precisions (inter-day precision RSD, 0.79%-1.07%; intra-day precision RSD, 1.59%-2.10%), repeatability (RSD, 1.66%-3.67%), stability (RSD, 1.26%-4.77%) and recovery (95.24%-105.41%, RSD, 0.29%-2.69%). Furthermore, PC12 cells and primary neurons cells were used for the neuroprotective effective assessment of aforementioned four samples from HLJDD. The total aqueous extract and n-butanol extract of HLJDD presented more significant effects than the other two parts. According to their quality and quantity determination results, iridoids and alkaloids have a positive correlation with the neuroprotective effectiveness of HLJDD.

Key words: Huang-Lian-Jie-Du-Decoction, Qualitative and quantitative analysis, Neuroprotective effects

中图分类号:

R284.1

Supporting:

Foundation items: Key Projects in the National Science & Technology Pillar Program during the 11^th Five-Year Plan Period (Grant No. 2008BAI51B02), National Natural Science Fund Project of China (Grant No. 81202904).
^#These authors contributed equally to this work.*Corresponding author. Tel.: 86-10-84041249

杨阳, 赵海誉, 王宏洁, 宋建芳, 杨健, 司南, 边宝林^*. HPLC-DAD-ESI-MS/MS 法对黄连解毒汤四个极性部位化学成分的定性定量研究及其相关的神经保护作用[J]. , DOI: 10.5246/jcps.2013.04.048.

Yang Yang, Haiyu Zhao, Hongjie Wang, Jianfang Song, Jian Yang, Nan Si, Baolin Bian^*. Qualitative and quantitative analysis of four different polarity fractions
from Huang-Lian-Jie-Du-Decoction by HPLC-DAD-ESI-MS/MS and their related neuroprotective effects[J]. , DOI: 10.5246/jcps.2013.04.048.

参考文献

[1] Wenk, G.L. J. Clin. Psychiatry. 2003, 64, 7-10.
[2] Bai, H. Hebei Tradit. Chin. Med. Pharm. 2008, 30, 658.
[3] Xiao, S. Kampo Med. 2003, 27, 15-18.
[4] Watanabe, K.; Matsuura, K.; Gao, P.F.; Hottenbacher, L.; Tokunaga, H.K.N. Evid. Based Complement. Alternat. Med. 2011, 2011, 1-19.
[5] Fang, Q.; Zhan, X.P.; Mo, J.L. Chin. J. Chin. Mater. Med. 2004, 29, 575-578.
[6] Dou, S.; Liu, L.; Jiang, P.; Zhang, W.; Liu, R. Chromatographia. 2009, 69, 659-664.
[7] Sun, J.; Ma, J.; Jin, J.; Wang, H.; Wen, Q.; Zhang, H. Acta Pharm. Sin. 2006, 41, 380-384.
[8] Wang, A.; Ge, G.; Qi, X.; Hu, Y.; Liang, S.; Yang, L. Fitoterapia. 2010, 81, 662-667.
[9] Zhang, Y.; Xu, H.; Chen, X.; Chen, C.; Wang, H.; Meng, F. J. Pharm. Biomed. Anal. 2011, 56, 497-504.
[10] Ma, Z.; Yang, X. Chin. J. Chin. Mater. Med. 2008, 33, 2080-2085.
[11] Xu, D.; Chen, P.; Zhang, L.; Cao, Y.; Ding, A. Chin. J. Exp. Tradit. Med. Form. 2011, 17, 78-80.
[12] Wang, M.; Ji, T.; Yang, J.; Su, Y. J. Chin. Mater. Med. 2009, 32, 208-210.
[13] Yu, Y.; Gao, H.; Dai, Y.; Yao, X. Chin. Tradit. Herb. Drugs. 2010, 41, 148-153.
[14] Wu, Y.; Sun, J.; Shi, R. J. Chin. Pharm. Univ. 2005, 36, 59-62.
[15] Zhang, Q.; Ye, Y.; Yan, Y.; Zhang, W.; Chu, L.; Wei, E. J. Zhejiang Univ. 2009, 38, 75-80.
[16] Zheng, Y.; Feng, D.; Cheng, X.; Zhou, W.; Zhang, Y. Pharm. Res. 2008, 17, 6-9.
[17] Xu, J.; Yu, Q.; Cai, S.; Wang, S. J. Shenyang Pharm. Univ. 2003, 20, 132-134.
[18] Yuan, C.; Zhu, L.; Pang, H.; Shan, Z.; Wang, S.; Gao, Y. Chin. J. Chin. Mater. Med. 2007, 32, 249-252.
[19] Wu, Y.; Sun, J.; Yu, S.; Shi, R. J. Beijing Univ. TCM. 2004, 27, 50-52.
[20] Yu, C.J.; Zheng, M.F.; Kuang, C.X.; Huang, W.D.; Yang, Q. J. Alzheimers Dis. 2010, 22, 257-266.
[21] Durairajan, S.S.K.; Liu, L.F.; Lu, J.H.; Chen, L.L.; Yuan, Q.; Chung, S.K.; Huang, L.; Li, X.S.; Huang, J.D.; Li, M. Neurobiol. Aging. 2012, 33, 2903-2919.
[22] Zhu, F.; Qian, C. BMC Neurosci. 2006, 7, 78.
[23] Yin, F.; Liu, J.; Ji, X.; Wang, Y.; Zidichouski, J.; Zhang, J. Neurosci. Lett. 2011, 492, 76-79.
[24] Li, N.; Liu, B.; Dluzen, D.E.; Jin, Y. J. Ethnopharm. 2007, 2007, 458-463.
[25] Sheng, G.; Zhang, J.; Pu, X.; Ma, J.; Li, C. Eur. J. Pharmacol. 2002, 2002, 119-124.
[26] Wang, J.Y.; Shum, A.Y.C. Neurosci. Lett. 2002, 322, 187-191.
[27] Wang, J.Y.; Shum, A.Y.C.; Ho, Y.J. J. Neurosci. Res. 2003, 72, 508-519.
[28] Tirosh, O.; Sen, C.; Roy, S.; Packer, L. Neuroscience. 2000, 97, 531-541.
[29] Justesen, U. J. Mass Spectrom. 2001, 36, 169-178.
[30] Lehner, A.F.; Craig, M.; Fannin, N.; Bush, L.; Tobin, T. J. Mass Spectrom. 2004, 39, 1275-1286.
[31] Essafi, N.E.; Kerhoas, L.; Ducrot, P.H. Rapid Commun. Mass Spectrom. 2007, 21, 1165-1175.