基于高效液相色谱指纹图谱的不同采收期虎杖叶的区分及多成分含量的测定

Differentiation of Polygonum Cuspidatum Leaves from Different Harvesting Periods and Determination of Multi-Components Based on Fingerprints of High Performance Liquid Chromatography

为研究不同采收期虎杖叶样品的品质差异,进行了题示研究。采收于3~10月的16批样品经烘干、粉碎后,分取0.1 g,加入80%(体积分数)甲醇溶液5 mL,超声45 min,离心10 min,取上清液,过0.22μm滤膜,滤液用高效液相色谱法(HPLC)测定。以不同体积比的乙腈和0.1%(体积分数)磷酸溶液的混合溶液为流动相,在Agilent HC-C_(18)(2)色谱柱上进行梯度洗脱分离。采集16批样品的色谱图,导入相似度评价系统进行相似度分析;以共有成分峰面积为变量,采用主成分分析(PCA)、系统聚类分析和正交偏最小二乘-判别分析(OPLS-DA)等化学模式识别法区分不同采收期的样品;以外标法对鉴定出的9种共有成分进行定量,并分析了这9种成分含量随季节变化的规律。结果显示:16批样品中含有17种共有成分,采收于3月的样品的相似度小于0.800;聚类分析将16批样品分为3类,和PCA分类结果一致;OPLS-DA筛选出5个潜在差异性质量标志物,可用于区分不同采收期样品的质量差异;不同采收期样品中芦丁、槲皮素、虎杖苷等成分随季节变化,二苯乙烯类、蒽醌类化合物在春季时含量最高,黄酮类化合物在秋季含量最高(槲皮素含量在7月达到峰值),酚酸类化合物含量变化较为平稳,可为虎杖叶的质量控制以及采收期的确定提供理论依据。

The study mentioned by the title was conducted to investigate the quality differences of samples of Polygonum cuspidatum leaves collected from different harvesting periods.After drying and crushing 16 batches of samples collected from March to October,an aliquot(0.1 g)was taken,and 5 mL of 80%(volume fraction)methanol solution was added.The mixture was ultrasonicated for 45 min,and centrifuged for 10 min.The supernatant was taken,and passed through a 0.22μm filter membrane.The filtrate was analyzed by high performance liquid chromatography(HPLC).Gradient elution separation was performed on Agilent HC-C_(18)(2)column with a mixture of acetonitrile and 0.1%(volume fraction)phosphoric acid solution at different volume ratios as the mobile phase.Chromatograms of 16 batches of samples were collected and imported into a similarity evaluation system for similarity analysis.Using common component peak areas as variables,chemical pattern recognition methods such as principal component analysis(PCA),systematic clustering analysis,and orthogonal partial least squares discriminant analysis(OPLS-DA)were used to distinguish samples from different harvesting periods.The 9 common components identified were quantified by the external standard method,and the seasonal variation patterns of the content of these 9 components were analyzed.It was shown that there were 17 common components in 16 batches of samples,and the similarity of the samples collected in March was less than 0.800,The 16 batches of samples were divided into 3 categories by systematic clustering analysis,which was consistent with the PCA classification result.The 5 potential quality difference biomarkers were screened by OPLS-DA,to distinguish the quality differences of samples from different harvesting periods.The content of components such as rutin,quercetin,and polydatin in samples from different harvesting periods varied with the seasons,with the content of styrene,and anthraquinone compounds highest in spring,flavonoids highest in autumn(quercetin content reached its peak in July),and phenolic compounds changing relatively steadily,which could provide theoretical basis for quality control of Polygonum cuspidatum leaves and determination of harvesting period.