基于一测多评与化学计量分析的滇重楼及其近缘种质量评价方法

Quality Evaluation Method for Dianchonglou(Paris polyphylla var.yunnanensis)and Its Related Species Based on One Measurement Multiple Evaluation and Chemometrics Analysis

目的 建立一测多评法(quantitative analysis of multi-components by a single marker, QAMS)与HPLC指纹图谱及化学计量分析相结合的方法对滇重楼及其近缘种的质量进行评价。方法 建立滇重楼及其近缘种的指纹图谱并进行相似度分析、聚类分析(cluster analysis, CA)以及主成分分析(principal component analysis, PCA)。以重楼皂苷VII为内标,建立重楼皂苷II、重楼皂苷III、重楼皂苷VI、重楼皂苷I、重楼皂苷H、重楼皂苷D、伪原薯蓣皂苷的相对校正因子,并考察QAMS的可行性。色谱条件:采用Agilent ZORBAX SB C18色谱柱(4.6 mm×250.0 mm, 5μm);乙腈(A)和水(B)为流动相进行梯度洗脱;流速0.8 mL/min;柱温30℃;检测波长203 nm;进样量5μL。结果 建立了不同种重楼样品的指纹图谱,相似度分析显示不同种重楼样品的相似度介于0.646~0.913,表明不同种重楼间的化学成分及含量差异性较大;以重楼皂苷VII为内标,所建立的滇重楼QAMS含量测定方法,与外标法(external standard method, ESM)得到的结果进行比较无显著差异,确定了QAMS在含量测定中的可行性,将QAMS应用于滇重楼近缘种的含量测定,只有长柱重楼的全部样品(CZCL01~04)和TCCL03符合《中华人民共和国药典》的质控标准;进一步以重楼样品中的8个皂苷类成分的峰面积作为变量进行聚类分析和主成分分析,聚类分析结果显示滇重楼及其近缘种样品可以聚为4类,长柱重楼、大理重楼和腾冲重楼均可自成一类,其余种重楼无法进行区分。主成分分析中46批重楼样品大体可以分为5类,大理重楼、长柱重楼、腾冲重楼各为一类,大部分滇重楼为一类,毛重楼、矮重楼、独龙重楼样品在主成分分析中无分类趋势。结论 QAMS与指纹图谱及化学计量分析相结合的方法可以完成对滇重楼及其近缘种中8种皂苷类成分的同时快速定量。并对其质量进行评价。实验建立的分析方法准确可行,可以在保护中药资源、节省时间的前提下对滇重楼及其近缘种样品的质量进行综合评价。

Objective To establish a method combining quantitative analysis of multi-components by a single marker(QAMS)with HPLC fingerprint and stoichiometry to evaluate the quality of Dianchonglou(Paris polyphylla var.yunnanensis)and its relatives.Methods The fingerprint maps of Dianchonglou(Paris polyphylla var.yunnanensis)and its relatives were established,and similarity analysis,cluster analysis(CA)and principal component analysis(PCA)were carried out.Taking polyphyllin VII as the internal standard,the relative correction factors of polyphyllin II,polyphyllin III,polyphyllin VI,polyphyllin I,polyphyllin H,polyphyllin D and pseudoprotodioscin were established,and the feasibility of QAMS was investigated.Agilent ZORBAX SB C18 column(4.6 mm×250.0 mm,5μm)was used.Acetonitrile(A)and water(B)were gradient eluted for the mobile phase.The flow rate was 0.8 mL/min.The column temperature was 30℃.The detection wavelength was 203 nm.The injection volume was 5μL.Results Fingerprint maps of different heavy building samples were established,and similarity analysis showed that the simi-larity of different Chonglou(Paris polyphylla)samples was between 0.646 and 0.913,indicating that the chemical composition and content of different Chonglou(Paris polyphylla)were quite different.Taking Polyphyllin VII as the internal standard,the QAMS content determination method of Dianchonglou(Paris polyphylla var.yunnanensis)established was not significantly different from the results obtained by the external standard method(ESM),and the feasibility of QAMS in content determination was determined,and QAMS was applied to the content determination of relatives of Dianchonglou(Paris polyphylla var.yunnanensis),and only all samples of Changzhu Chonglou(Paris forrestii)(CZCL01~04)and TCCL03 met the quality control standards of the Chinese Pharmacopoeia.Furthermore,the peak area of the 8 saponin components in the Chonglou(Paris polyphylla)samples was used as variables for cluster analysis and principal component analysis,and the cluster analysis results showed that the samples of Dianchonglou(Paris polyphylla var.yunnanensis)and its relatives could be clustered into 4 categories,and the Changzhu Chonglou(Paris forrestii),Dali Chonglou(Paris daliensis)and Tengchong Chonglou(Paris tengchongensis)all could form their own categories,and the other heavy buildings could not be distinguished.In the principal component analysis,the 46 batches of Chonglou(Paris polyphylla)samples can be roughly divided into 5 categories,each of Dali Chonglou(Paris daliensis),Changzhu Chonglou(Paris forrestii)and Tengchong Chonglou(Paris tengchongensis)belongs to one category,most of Dianchonglou(Paris polyphylla var.yunnanensis)is one category,and the samples of Maochonglou(Paris mairei),Aichonglong(Paris polyphylla var.nana)and Dulong Chonglong(Paris dulongensis)had no classification trend in principal component analysis.Conclusion The combination of QAMS and fingerprinting and stoichiometric analysis can complete the simultaneous and rapid quantification of 8 saponin components in Dianchonglou(Paris polyphylla var.yunnanensis)and its relatives and can evaluate its quality.The analytical method established in this experiment is accurate and feasible,which can comprehensively evaluate the quality of samples from Dianchonglou(Paris polyphylla var.yunnanensis)and its relatives under the premise of protecting Chinese medicine resources and saving time.