芩连润肺汤的HPLC指纹图谱建立、含量测定及多元统计分析
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篇名: 芩连润肺汤的HPLC指纹图谱建立、含量测定及多元统计分析
TITLE: HPLC Fingerprint Establishment ,Content Determination and Multivariate Statistical Analysis of Qinlian Runfei Decoction
摘要: 目的:建立芩连润肺汤的指纹图谱,测定其中11种成分的含量,并进行聚类分析、正交偏最小二乘判别法(OPLS-DA)分析。方法:采用高效液相色谱法(HPLC)。色谱柱为ZORBAXEclipsePlusC18,流动相为乙腈-0.05%磷酸溶液(梯度洗脱),检测波长为260nm,柱温为30℃,流速为1.0mL/min,进样量为10μL。以汉黄芩苷为参照,采用《中药色谱指纹图谱相似度评价系统(2012版)》绘制10批芩连润肺汤的HPLC指纹图谱并进行相似度评价,确定共有峰;同法测定芩连润肺汤中11种成分的含量;采用SPSS21.0软件进行聚类分析,采用SIMCA14.0软件进行有监督模式的OPLS-DA分析,并筛选影响其质量的标志物。结果:10批芩连润肺汤共有21个共有峰,与对照指纹图谱的相似度均大于0.98;共指认了11个共有峰,分别为芦丁、连翘酯苷A、连翘酯苷B、鸢尾苷、野鸢尾苷、黄芩苷、连翘苷、汉黄芩苷、黄芩素、次野鸢尾黄素、汉黄芩素。上述11种成分检测进样量的线性范围分别为9.9600~49.8000、1.9740~9.8700、0.6720~3.3600、0.9600~4.8000、0.5490~2.7450、5.0400~25.2000、1.3740~6.8700、0.6150~3.0750、0.7599~3.7950、0.1620~0.8100、0.0420~0.2100μg(r均大于0.999);精密度、稳定性(48h)、重复性试验的RSD均小于2%;平均加样回收率为95.81%~100.29%(RSD为0.43%~1.73%,n=6);含量分别为8.9244~12.8208、0.3522~0.8687、0.4356~0.7112、0.3898~1.3090、0.3358~0.5301、1.6805~4.5423、0.7018~1.5842、2.2402~5.4425、2.3510~5.5589、0.1060~0.1822、0.0768~0.1289mg/g。聚类分析结果显示,当类间距为10时,10批样品可聚为两类,S1~S3聚为一类、S4~S10聚为一类;当类间距为5时,第2类又可聚为两类,S6、S7、S9聚为一类,S4、S5、S8、S10聚为一类。OPLS-DA分析结果显示,S6、S7、S9位于得分图的上方,S4、S5、S8、S10位于得分图的左下侧,S1~S3位于得分图的右下侧,与聚类分析结果一致;黄芩苷、鸢尾苷、连翘酯苷A、黄芩素、汉黄芩苷的变量重要性投影值均大于1。结论:所建指纹图谱、含量测定方法的精密度较高、稳定性较好,结合多元统计分析可用于芩连润肺汤的质量控制。黄芩苷等5种成分为影响芩连润肺汤的质量标志物。
ABSTRACT: OBJECTIVE:To establish the f ingerprint of Qinlian runfei decoction,determine the contents of 11 components, and conduct cluster analysis and orthogonal partial least squares discriminant analysis (OPLS-DA). METHODS :HPLC method was used. The determination was performed on ZORBAX Eclipse Plus C 18 column with mobile phase consisted of acetonitrile- 0.05% phosphoric acid solution (gradient elution )at the flow rate of 1.0 mL/min. The detection wavelength was set at 260 nm, and column temperature was 30 ℃. The sample size was 10 μL. Using wogonoside as reference,HPLC fingerprints of 10 batches of Qinlian runfei decoction were drawn and the similarity evaluation was conducted with Similarity Evaluation System of TCM Chromatographic Fingerprint (2012 edition),the common peaks were also confirmed ;the contents of 11 components in Qinlian runfei decoction were determined by the same method. SPSS 21.0 software was used for clus ter analysis ,and SIMCA 14.0 software was used for OPLS-DA to screen marker components affecting quality. RESULTS :There were 21 common peaks in 10 batches of Qinlian runfei decoction ,and the similarity with control fingerprint was greater than 0.98. A total of 11 common peaks were identified , which were rutin , forsythiaside A , forsythiaside B , iris, irigenin, baicalin, forsythiaside, wogonoside, baicalein, irisflorentin and wogonin. The line ar ranges of 11 components were 9.960 0-49.800 0,1.974 0-9.870 0,0.672 0-3.360 0,0.960 0-4.800 0,0.549 0- 2.745 0,5.040 0-25.200 0,1.374 0-6.870 0,0.615 0-3.075 0,0.759 9-3.795 0,0.162 0-0.810 0,0.042 0-0.210 0 μg(all r> 0.999); RSDs of precision , stability (48 h) and repeatability tests were less than 2% ; the average recoveries were 95.81%-100.29% with RSDs of 0.43%-1.73%(n=6);the contents were 8.924 4-12.820 8,0.352 2-0.868 7,0.435 6-0.711 2, 0.389 8-1.309 0,0.335 8-0.530 1,1.680 5-4.542 3,0.701 8-1.584 2,2.240 2-5.442 5,2.351 0-5.558 9,0.106 0-0.182 2,0.076 8- 0.128 9 mg/g,respectively. The results of cluster analysis showed that when class spacing was 10,it could be divided into two groups,S1-S3 and S 4-S10;when the class spacing was 5,the second class could be divided into two categories ,S6,S7,S9 were clustered into one category ,and S 4,S5,S8,S10 were clustered into one category. The results of OPLS-DA analysis showed that S6,S7 and S 9 were at the top of the figure ,S4,S5,S8 and S 10 were at the lower left side of the figure ,and S 1-S3 were at the lower right side of the figure ,which was consistent with the cluster analysis results ;VIP values of baicalin ,iris,forsythiaside A , baicalein and wogonoside were all greater than 1. CONCLUSIONS :Established fingerprint and content determination methods have high precision and good stability. Combined with multivariate statistical analysis ,it can be used for the quality control of Qinlian runfei decoction. Five components as baicalin are the marker components affecting the quality of Qinlian runfei decoction.
期刊: 2021年第32卷第16期
作者: 闫凯莉,尹程程,刘梦瑶,崔长升,汪莹,孙晓舟,孙丽平,齐滨,刘莉
AUTHORS: YAN Kaili, YIN Chengcheng,LIU Mengyao,CUI Changsheng,WANG Ying,SUN Xiaozhou,SUN Liping,QI Bin,LIU Li
关键字: 芩连润肺汤;高效液相色谱法;指纹图谱;含量测定;聚类分析;正交偏最小二乘判别法
KEYWORDS: Qinlian runfei decoction ;HPLC;Fingerprint;Content determination ;Cluster analysis ;Orthogonal partial least
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