目的:基于指纹图谱和网络药理学方法,对曼地亚红豆杉质量标志物(Q-Marker)进行预测,建立基于Q-Marker的曼地亚红豆杉药材评价方法.方法:采用Waters SymmmetryShieldTM RP 18(250 mm ×4.6 mm,5 µm)色谱柱,以乙腈-0.1％三氟乙酸水为流动相,梯度洗脱,流速1 mL·min-1,柱温30 ℃,检测波长254 nm,进样体积10 μL,建立8个年限24批曼地亚红豆杉药材指纹图谱并标定共有峰,采用层次聚类分析(hierarchical clustering analysis,HCA)进行分类评价,借助正交偏最小二乘法-判别分析(orthogonal partial least square-discriminant analysis,OPLS-DA)筛选不同年限曼地亚红豆杉的主要差异性标志物;结合网络药理学,通过相应数据库筛选差异性标志物的核心靶点和关键通路,构建"成分-靶点-通路"网络图,以此预测曼地亚红豆杉的Q-Marker;采用HCA及主成分分析(principal component analysis,PCA)进一步验证曼地亚红豆杉的Q-Marker,综合评价其质量.结果:建立了 24批曼地亚红豆杉药材指纹图谱,标定了 25个共有峰,指认出18个化合物,包括紫杉烷类、黄酮类、生物碱类、甾体类、酚类,相似度均＞0.900;HCA的结果显示不同年限曼地亚红豆杉存在一定的差异性;经OPLS-DA筛选出11个差异性标志物,分别为紫杉醇、10-脱乙酰基巴卡亭Ⅲ(10-DAB)、巴卡亭Ⅲ、三尖杉宁碱、10-去乙酰紫杉醇(10-DAT)、阿魏酸、山柰素、芦丁、金松双黄酮、穗花杉双黄酮、对羟基苯甲醛;基于差异性标志物,运用网络药理学从有效性角度对其进行分析,初步预测紫杉醇、10-DAB、巴卡亭Ⅲ、三尖杉宁碱、10-DAT为曼地亚红豆杉的Q-Marker,主要通过作用于PIK3R1、AKT1、EGFR、HRAS、MAPK1等14个核心靶点,调控癌症通路、AGE-RAGE信号在糖尿病并发症中的作用等信号通路,发挥消肿散结、通经利尿的功效;HCA结果验证了预测的Q-Marker的合理性,PCA综合评价结果表明十年生春季采收的曼地亚红豆杉质量较佳.结论:基于指纹图谱和网络药理学方法筛选出紫杉醇、10-DAB、巴卡亭Ⅲ、三尖杉宁碱、10-DAT为曼地亚红豆杉的Q-Marker,结合化学识别模式方法对不同年限曼地亚红豆杉的质量进行综合排序,其中十年生春季采收的得分最高,质量较好,为曼地亚红豆杉的质量标准的研究及资源评价提供了新思路.
Analysis of quality markers of Taxus media based on fingerprint and network pharmacology
Objective:To predict potential quality markers(Q-Marker)of Taxus media Rehd.based on finger-print and network pharmacology methods,and establish an evaluation method of Taxus media Rehd.based on Q-Marker,as so to provide basis for the establishment of quality standard and quality evaluation system of Taxus media Rehd..Methods:Waters SymmmetryShieldTMRP18(250 mm ×4.6 mm,5 μm)chromatographic column was used for separation,and the wavelength was 254 nm.The mobile phase was acetonitrile-0.01％trifluoroacetic acid aqueous solution for gradient elution at a flow rate of 1.0 mL·min-1,and the column temperature was 30 ℃.The injection volume was 10 μL The fingerprints of 24 batches of Taxus media Rehd.with 8 kinds of cultivation years were established and the common peaks were identified.Hierarchical clustering analysis(HCA)was used for classification,orthogonal partial least square-discriminant analysis(OPLS-DA)was used to screen out the main marker compo-nents that cause differences between different years.Combined with network pharmacology,the core targets and key pathways were constructed a"component-target-pathway"network map through corresponding databases.Q-Mark-ers were further verified and quality of Taxus media Rehd.comprehensively evaluated by HCA and PCA.Results:A total of 25 common peaks were obtained in the HPLC fingerprint of Taxus media Rehd.,18 compounds were identified,and their similarities were all above 0.900,including taxanes,flavonoids,alkaloids,steroids and phenols.The results of HCA showed that there were some differences among samples with different cultivation years.Eleven different mark-ers were screened by OPLS-DA,and they were paclitaxel,10-deacetylbaccatin Ⅲ(10-DAB),baccatin Ⅲ,cepha-lomannine,deacetyltaxol(10-DAT),ferulic acid,kaempferide,rutin,amentoflavone-4',4",7-trimethyl ether,skyrin,4-hydroxybenzaldehyde.Based on the difference markers,network pharmacology was applied to analyze them from the perspective of effectiveness.Paclitaxel,10-DAB,bakatine Ⅲ,cephalomannine and 10-DAT were prelimin-stly predicted as Q-Markers of Taxus media Rehd..They could regulate cancer pathway,AGE-RAGE signaling pathway in diabetic complications and other signaling pathways by PIK3R1,AKT1,EGFR,HRAS and MAPK1 targets,and play the role of reducing swelling and menstruation diuresis clearing.The results of HCA verified the rationality of Q-Marker,and the comprehensive evaluation results of PCA showed that sample which was cultivated for ten years and harvested in spring bore the best quality.Conclusion:Paclitaxel,10-DAB,bakatine Ⅲ,cephalomannine and 10-DAT are selected as Q-Markers based on fingerprint and network pharmacological methods.The quality of Taxus media Rehd.in different cultivation years is sorted comprehensively by combining with chemical recognition pattern method.The 10-year-old sample harvested in spring gets the highest score and the quality is good.The study provides new method for establishing of quality standard and the quality evaluation of Taxus media Rehd..
Taxus media Rehd.fingerprintnetwork pharmacologychemical recognition patternpaclitaxel10-DABbakatine Ⅲcephalomannine10-DATquality markerquality evaluation
万方数据
维普
