基于Box-Behnken设计和主成分分析法研究当归-苦参药对的提取工艺及不同比例配伍对急性心肌缺血大鼠的影响

Based on Box-Behnken design and principal component analysis method to explore extraction process of Angelicae Sinensis Radix-Sophorae Flavescentis Radix herb pair and effects of different compatibilities on rats with acute myocardial ischemia

目的 优化当归-苦参提取工艺并测定其不同配比的化学成分含量,研究不同配比药对对急性心肌缺血大鼠的影响。方法 利用Box-Behnken设计对当归苦参药对的提取工艺进行考察,确定最佳提取工艺;通过HPLC法测定当归-苦参药对不同配比(1∶0、1∶1、1∶2、1∶3、2∶1、3∶1、0∶1)下的绿原酸、阿魏酸、洋川芎内酯I、藁本内酯、欧当归内酯A、槐果碱、苦参碱、氧化槐果碱、槐定碱、氧化苦参碱、苦参酮11种成分的含量;利用酶标仪进行血清生化指标的检测,再将药效学指标与化学成分含量用主成分分析(principal component analysis,PCA)法整合,通过化学和药效2个层面综合评价药对的最佳配比。结果 最佳提取工艺为30倍量70%乙醇,回流提取3次,每次1 h。与当归-苦参0∶1比较,当归-苦参不同配比(1∶1、2∶1、3∶1)样品中随着当归比例的升高,苦参中6种成分提取量呈“U”型分布;与当归-苦参1∶0比较,当归-苦参不同配比(1∶1、1∶2、1∶3)样品中随着苦参比例的升高,当归中5种成分提取量亦呈“U”型分布。心肌缺血大鼠各治疗组中当归-苦参1∶2组疗效最明显,单味药组改善程度最不明显。结论 研究方法简单可行,通过化学层面和药效学角度分析两者不同配比的成分变化及对急性心肌缺血大鼠的影响,可为当归-苦参药对开发为治疗心血管疾病方面的新复方提供理论依据。

Objective To optimize the extraction process of the Danggui(Angelicae Sinensis Radix, ASR)-Kushen(Sophorae Flavescentis Radix, SFR) herb pair and determine the content of chemical components in different compatibility ratios, and study the effects of different ratios on rats with acute myocardial ischemia in rats. Methods The Box-Behnken design was used to investigate the extraction process of ASR-SFR herb pair to determine the best extraction process. The chemical contents of the 11 components in different ratios(1:0, 1:1, 1:2, 1:3, 2:1, 3:1, 0:1) of ASR-SFR, including chlorogenic acid, flerulic acid, senkyunolide I, ligustilide,levistilide A, sophocarpine, matrine, oxysophocarpine, sophoridine, oxymatrine and kurarinone, were determined by high performance liquid chromatography. The serum biochemical indexes were measured with a microplate reader, and then the pharmacodynamic indicators and the content of chemical components were integrated with principal component analysis, and the optimal ratio of herb pairs were comprehensively evaluated through two aspects of chemistry and pharmacodynamics. Results The best extraction process was as follow: 30 times amount of 70% ethanol, reflux extraction for three times, 1 h each time. Compared with ASR-SFR with ratio of 0:1, the extracted amounts of the six components of Kushen showed a “U” shaped distribution with increasing the proportion of Danggui in different ratios(1:1, 2:1, 3:1) of ASR-SFR herb pair. And compared with ASR-SFR with ratio of 1:0, the extracted amounts of the five components of Danggui showed a “U” shaped distribution with increasing the proportion of Kushen in different ratios(1:1,1:2, 1:3) of ASR-SFR herb pair. Among the treatment groups of myocardial ischemia rats, the ASR-SFR herb pair(1:2) group had the most obvious effect, and the improvement degree of the single-drug group was the least obvious. Conclusion The method of this study is simple and feasible. Analyzing the changes in the composition of different ratios of ASR-SFR herb pair from the perspective of chemistry and pharmacodynamics can provide a theoretical basis for the development of a new compound for the treatment of cardiovascular diseases.