黄连解毒汤中9种主要成分在大鼠脑组织中分布特征研究

目的建立灵敏、准确、可靠的小檗碱、小檗红碱、药根碱、巴马汀、黄芩苷、汉黄芩素、千层纸素A、京尼平苷和栀子黄素B在大鼠海马、皮层、纹状体和脑脊液组织中高效液相色谱-串联质谱联用(HPLC-MS-MS)分析方法,用于黄连解毒汤中9种主要成分在大鼠脑组织中分布研究。方法优化黄连解毒汤中9种主要成分及内标克拉霉素和氯霉素的质谱检测条件,确定分析条件并对方法学进行考察。正常大鼠ig给予黄连解毒汤,末次给药1 h后采集脑脊液、海马、皮层和纹状体样品,经处理后将该方法用于9种成分的测定。结果小檗碱、药根碱、巴马汀、黄芩苷、汉黄芩素、千层纸素A、栀子黄素B在1～250 ng/mL呈现良好的线性关系(r~20.99),小檗红碱在1～500 ng/mL具有良好的线性关系(r~2=0.991 2),京尼平苷在5～500 ng/mL具有良好的线性关系(r~2=0.999 5);各成分的日内、日间精密度RSD均小于12.94%,准确度均在-12.71%～6.91%;提取回收率均在88.02%～106.70%,基质效应均在88.92%～105.10%;短期、冻融循环和长期稳定性RSD均小于12.51%。各成分在海马、皮层和纹状体中的含量为小檗碱黄芩苷京尼平苷小檗红碱巴马汀汉黄芩素药根碱千层纸素A栀子黄素B;各成分在脑脊液中的含量为京尼平苷小檗碱小檗红碱巴马汀黄芩苷栀子黄素B药根碱汉黄芩素千层纸素A。结论该方法可用于大鼠脑组织中小檗碱、药根碱等9种成分含量的同时检测,并可用于这9种成分在大鼠脑组织分布研究,为探究黄连解毒汤治疗脑部疾病的物质基础提供依据。

Distribution characteristics of nine main components of Huanglian Jiedu Decoction in rat brain

Objective To establish a sensitive, accurate and reliable LC-MS-MS analysis method for the nine major components (berberine, berberrubine, jatrorrhizine, palmatine, baicalin, wogonin, oroxylin A, geniposide and gardenin B) in Huanglian Jiedu Decoction (HJD) in rat hippocampus, cortex, striatum, and cerebrospinal fluid, and to study the distribution characteristics of the components in brain tissues of rats. Methods The mass spectrometry detection conditions and chromatographic analysis conditions of nine main components and internal standards (clarithromycin and chloramphenicol) was optimized, and then the methodological investigations were performed. After HJD was administrated for 1 h, the samples of cerebrospinal fluid, hippocampus, cortex and striatum tissues were collected, and the contents of nine components in these samples were measured. Results Berberine, jatrorrhizine, palmatine, baicalin, wogonin, oroxylin A and gardenin B showed a good linear relationship in the range of 1-250 ng/mL (r² > 0.99). Berberrubine had a good linear relationship in the range of 1-500 ng/mL (r²=0.991 2), and geniposide had a good linear relationship in the range of 5-500 ng/mL, respectively (r²=0.999 5). The intra-day and inter-day precision of each component was less than 12.94%, the accuracy was from −12.71% to 6.91%; The extraction recovery rate was from 88.02% to 106.7%; The matrix effect was from 88.92% to 105.10%; And the short-term, freeze-thaw cycle and long-term stability were less than 12.51%. The content of each component in the hippocampus, cortex and striatum was berberine > baicalin > geniposide > berberrubine > palmatine > wogonin > jatrorrhizine > oroxylin A > gardenin B; And the content of each component in cerebrospinal fluid was geniposide > berberine > berberrubine > palmatine > baicalin> gardenin B > jatrorrhizine > wogonin > oroxylin A. Conclusion The method can be used for the simultaneous detection of the concentration of nine active components in brain tissues of rats, and successfully applied to the study of brain tissue distribution, which provides a reference for HJD to treat brain diseases.