指纹图谱联合多元统计分析的百合地黄汤物质基准质量评价及量值传递规律研究

Quality Evaluation and Quantity Transfer Law of Baihe Dihuang Decoction by Combining Fingerprint and Multivariate Statistical Analysis

目的:探索百合地黄汤(Baihe Dihuang decoction,BDD)物质基准制备工艺,建立BDD物质基准指纹图谱并进行质量评价,阐明BDD主要成分在药材-煎煮液-物质基准的量值传递规律。方法:优化百合物质基准制备工艺,从而确立BDD物质基准制备方法;采用高效液相色谱(high performance liquid chromatography,HPLC)法建立BDD物质基准指纹图谱;采用中药色谱指纹图谱相似度评价系统标定并指认共有峰和色谱峰;基于多元统计分析(聚类分析、偏最小二乘法判别分析)方法寻找引起不同批次BDD物质基准质量差异的主要化学成分,并进行含量测定;根据主要化学成分转移率研究BDD物质基准的量值传递规律。结果:(1)BDD物质基准制备方法为新鲜百合280 g浸泡10 h后去除浮沫,加水400 mL,全程不加盖文武火(武火煎煮至沸腾转文火保持微沸)煎至200 mL,后加200 mL鲜地黄汁煎至300 mL,过100目滤网得BDD煎煮液,浓缩(60℃水浴加热)后冷冻干燥。(2)建立了15批BDD物质基准HPLC指纹图谱(S1~S15),相似度均大于0.9,共标定21个共有峰,指认出4个色谱峰(8号峰梓醇、13号峰地黄苷D、17号峰王百合苷A、20号峰王百合苷B)。(3)多元统计分析结果显示,S1、S2、S3、S7、S8、S9、S10、S14和S15聚为一类,其他批次聚为一类,且8号峰梓醇、17号峰王百合苷A对聚类判别的影响较大。(4)15批BDD物质基准中,梓醇、地黄苷D、王百合苷A的含量范围分别为(21.77~46.41)mg·g^(-1)、(1.8~3.66)mg·g^(-1)、(0.34~1.09)mg·g^(-1)。(5)15批BDD物质基准中,梓醇、地黄苷D、王百合苷A的煎煮液-物质基准转移率范围分别为66.23%~97.64%、41.19%~89.06%、71.91%~94.01%。结论:本研究优化了BDD物质基准制备工艺,建立BDD物质基准HPLC指纹图谱,找到造成不同批次差异的主要化学成分,发现BDD物质基准的主要化学成分可在药材-煎煮液-物质基准中稳定传递,为研发BDD新型制剂奠定了坚实基础。

Objective:To explore the preparation process of Baihe Dihuang Decoction(BDD)substance reference,establish a BDD substance reference fingerprint,and conduct quality evaluation,elucidating the quantity transfer law of the main components of BDD in the drug material decoction substance reference.Method:Optimize the standard preparation process of lily substances to establish the standard preparation method for BDD substances;Establish a reference fingerprint of BDD substances using high-performance liquid chromatography(HPLC)method;Using a similarity evaluation system for traditional Chinese medicine chromatographic fingerprints to calibrate and identify common peaks and chromatographic peaks;Based on multivariate statistical analysis(cluster analysis,partial least squares discriminant analysis)methods,identify the main chemical components that cause differences in the baseline quality of BDD substances from different batches,and conduct content measurements;Study the quantity transfer law of BDD substance reference based on the transfer rate of main chemical components.Results:(1)The standard preparation method for BDD substances is to soak 280 g of fresh lilies for 10 hours,remove the floating foam,add 400 mL of water,and fry the whole process without covering the fire(boiling with fire until boiling,then turning to low heat and keeping slightly boiling)until 200 mL,then add 200 mL of fresh Rehmannia juice and fry to 300 mL,pass through a 100 mesh filter to obtain the BDD decoction,concentrate(heated in a 60℃water bath),and freeze dry.(2)Fifteen batches of BDD substance reference HPLC fingerprint spectra(S1-S15)were established,with similarity greater than 0.9.A total of 21 common peaks were calibrated,and four chromatographic peaks were identified(8 peaks Zichun,13 peaks Dihuang glycoside D,17 peaks Wangbaicalin A,and 20 peaks Wangbaicalin B).(3)The results of multivariate statistical analysis showed that S1,S2,S3,S7,S8,S9,S10,S14,and S15 clustered into one group,while other batches clustered into one group.Moreover,peak 8 Zichun and peak 17 Wangbaicalin A had a significant impact on clustering discrimination.(4)Among the 15 batches of BDD substance standards,the content ranges of Zichun,Dihuangoside D,and Wanglilianoside A are(21.77-46.41)mg·g^(-1),(1.8-3.66)mg·g^(-1),and(0.34-1.09)mg·g^(-1),respectively.(5)Among the 15 batches of BDD substance standards,the transfer rates of decoction substance standards for Zichun,Dihuang glycoside D,and Wanglilide A ranged from 66.23%to 97.64%,41.19%to 89.06%,and 71.91%to 94.01%,respectively.Conclusion:This study optimized the preparation process of BDD substance reference,established HPLC fingerprint of BDD substance reference,identified the main chemical components that caused differences in different batches,and found that the main chemical components of BDD substance reference can be stably transferred in the herbal decoction substance reference,laying a solid foundation for the development of new BDD formulations.