背景:黄芪桂枝五物汤,最初记录在金奎姚略的中国古典公式,用于治疗人类“血液障碍”(一种“Bi”综合征)。在临床实践中,HGWD已用于治疗类风湿性关节炎(RA)。
目的:同时反映药效和化学特性的化学标志物的表征对中药质量控制具有重要意义。以HGWD的抗RA作用为例,这项研究的目的是制定一个综合的策略,结合整体的化学概况和生物活性数据,以确定化学标记。
方法:首先,建立并验证了超高效液相色谱-二极管阵列检测器(UPLC-DAD)指纹图谱,以评估不同来源的HGWD的整体质量。通过UPLC-DAD指纹图谱和化学计量学方法相结合,筛选了与不同地理来源的HGWD相关的特征标记。第二,通过UPLC耦合到混合线性离子阱-Orbitrap质谱(UPLC-HRMS)对15批HGWD样品的化学特征进行表征。然后评价15个HGWD样品的体外抗RA活性。第三,进行了光谱-效应关系分析,以鉴定可能用作质量标志物的生物活性化合物.最后,优化并建立了UPLC-三重四极杆串联质谱方法,用于15批HGWD的特征和质量标志物的定量分析。
结果:总计,在UPLC-DAD指纹图谱中分配了30个常见峰。九个峰被识别并被认为是特征标记:原儿茶酸,香豆素,肉桂酸,氧合烟苷,芍药苷,calycosin,福蒙素,儿茶素,和albiflorin.此外,在UPLC-HRMS化学图谱中鉴定出95种常见化合物。药理学分析表明15个HGWD样品的抗RA活性有很大不同。谱效关系分析显示30种潜在的生物活性成分与抗RA活性正相关。其中,相对量>1%的五种化合物,芍药苷,黄芪甲苷,六氢姜黄素,formonetin和calycosin-7-葡萄糖苷,被选为质量标记,在LPS诱导的RAW264.7巨噬细胞中验证了它们的活性。最后,在15批HGWD样品中同时定量了上述12种代表性成分。
结论:将整体化学概况与代表性成分评估相结合,这种系统的策略可能是提高HGWD质量评价的可靠和有效的方法。
BACKGROUND: Huangqi Guizhi Wuwu Decoction (HGWD), a classical Chinese formula originally recorded in Jin Kui Yao Lue, was used for the treatment of human \"blood impediment\" (a type of \"Bi\" syndrome). In clinical practice, HGWD has been applied to treat rheumatoid arthritis (RA).
OBJECTIVE: The characterization of chemical markers reflecting both efficacy and chemical characteristics is of great significance for TCM quality control. With the anti-RA effects of HGWD as an example, the aim of this study was to develop a comprehensive strategy combining the overall chemical profile and biological activity data to identify chemical markers.
METHODS: First, an ultra-performance liquid chromatography-diode array detector (UPLC-DAD) fingerprint was established and validated to evaluate the holistic quality of HGWD of different origins. Characteristic markers associated with HGWD from different geographical origins were screened by a combination of UPLC-DAD fingerprint and chemometrics methods. Second, the chemical profiles of the 15 batches of HGWD samples were characterized by UPLC coupled tohybrid linear ion trap-Orbitrap mass spectrometry (UPLC-HRMS). The in vitro anti-RA activities of the 15 HGWD samples were then evaluated. Third, spectrum-effect relationship analysis was performed to identify bioactive compounds that could potentially be used as quality markers. Finally, a UPLC-triple quadrupole tandem mass spectrometry approach was optimized and established for quantitative analysis of the characteristic and quality markers in 15 batches of HGWD.
RESULTS: In total, 30 common peaks were assigned in the UPLC-DAD fingerprint. Nine peaks were recognized and considered characteristic markers: protocatechuic acid, coumarin, cinnamic acid, oxypaeoniflorin, paeoniflorin, calycosin, formononetin, catechin, and albiflorin. Furthermore, ninety-five common compounds were identified in the UPLC-HRMS chemical profile. The pharmacological analysis indicated that the anti-RA activities of the 15 HGWD samples were vastly different. The spectrum-effect relationship analysis revealed 30 potential bioactive constituents positively correlated with anti-RA activity. Among them, five compounds with relative amounts >1%, paeoniflorin, astragaloside IV, hexahydrocurcumin, formononetin and calycosin-7-glucoside, were selected as quality markers, and their activity was verified in LPS-induced RAW264.7 macrophages. Finally, the above 12 representative components were simultaneously quantified in the 15 batches of HGWD samples.
CONCLUSIONS: Combining a holistic chemical profile with representative component evaluation, this systematic strategy could be a reliable and effective method to improve quality evaluations of HGWD.