清热明目茶HPLC特征指纹图谱研究及多成分含量测定

Study on the characteristic fingerprint of Qingre Mingmu tea by HPLC and determination of multi-component

目的:建立清热明目茶HPLC指纹图谱并测定清热明目茶中绿原酸、橙黄决明素、大黄素、大黄酚、大黄素甲醚、大黄酸、芦荟大黄素的含量,为清热明目茶的质量控制提供参考。方法:采用Hypersil Gold C18(250mm×4.6 mm,5μm)色谱柱;以乙腈(A)-0.5%磷酸水溶液(B)为流动相,梯度洗脱(0~12 min,15%A;12~20min,15%A→28%A;20~36 min,28%A→40%A;36~50 min,40%A→50%A;50~60 min,50%A→55%A;60~68 min,55%A→62%A;68~74 min,62%A→85%A;74~90 min,85%A→15%A),流速:1.0 mL·min-1;检测波长为327 nm(0~20 min,检测绿原酸)和287 nm(20~80 min,检测橙黄决明素、大黄素、大黄酚、大黄素甲醚、大黄酸、芦荟大黄素);柱温28℃,进样量10μL。通过相似度评价结合聚类分析对12批次清热明目茶指纹图谱进行质量评价,并对指认的7个指标成分进行定量测定研究。结果:建立了清热明目茶的指纹图谱,标定了21个共有峰,12批清热明目茶与对照图谱的相似度均≥0.94,聚类分析将其分为3类。绿原酸、橙黄决明素、大黄素、大黄酚、大黄素甲醚、大黄酸、芦荟大黄素的线性范围分别为0.041 6~0.416 4μg、0.386 2~3.862 2μg、0.180 6~1.805 9μg、0.209 8~2.097 7μg、0.040 1~0.400 8μg、0.100 1~1.001 3μg、0.108 1~1.080 5μg,线性关系良好(r≥0.999 4);重复性试验RSD<1.5%,平均加样回收率在98.9%~104.2%。12批样品中上述7个成分的含量范围分别为0.410 3~0.451 3、2.314 1~2.365 6、1.186 3~1.216 9、1.890 1~1.959 1、0.410 8~0.423 4、0.631 8~0.653 7和0.588 9~0.616 8 mg·g-1。结论:所建立的清热明目茶HPLC指纹图谱检测和定量分析方法准确可靠,专属性强,灵敏度高,能全面反映其内在质量,可用于清热明目茶的质量控制。

Objective:To establish the HPLC fingerprint and determine the content of chlorogenic acid,aurantio-obtusin,emodin,chrysophanol,physcion,rhein and aloe-emodin in Qingre Mingmu tea simultaneously,which provides the reference for quality control of Qingre Mingmu tea.Methods:HPLC analysis was performed on Hypersil Gold C18(250 mm×4.6 mm,5 μm).The mobile phase consisted of acetonitrile(A)-0.5% acetic acid solution(B)with gradient elution(0-12 min,15%A;12-20 min,15%A → 28%A;20-36 min,28%A → 40%A;36-50 min,40%A → 50%A;50-60 min,50%A → 55%A;60-68 min,55%A → 62%A;68-74 min,62%A → 85%A;74-90 min,85%A → 15%A).The detection wavelengths were set at 327 nm for chlorogenic acid and 287 nm for aurantio-obtusin,emodin,chrysophanol,physcion,rhein and aloe-emodin and the flow rate was 1.0 m L·min-1.The column temperature was 28 ℃ and the injection volume was 10 μL.Similarity evaluation combined with cluster analysis(CA)was used to evaluate the fingerprint of 12 batches of Qingre Mingmu tea and seven markers were quantified.Results:The HPLC fingerprint of Qingre Mingmu tea was constructed with 21 common chromatographic peaks.The similarities of 12 batches of samples were all above 0.94 comparing with the reference fingerprint.All samples were classified into 3 categories by clustering analysis.The linear relationship for chlorogenic acid,aurantio-obtusin,emodin,chrysophanol,physcion,rhein and aloe-emodin were observed in the ranges of 0.041 6-0.416 4 μg,0.386 2-3.862 2 μg,0.180 6-1.805 9 μg,0.209 8-2.097 7 μg,0.040 1-0.400 8 μg,0.100 1-1.001 3 μg and 0.108 1-1.080 5 μg,respectively with r ≥0.999 4.The RSDs of repeatability tests were less than 1.5% and the average recoveries varied from 98.9% to 104.2%.The contents of hlorogenic acid,aurantio-obtusin,emodin,chrysophanol,physcion,rhein and aloe-emodin in 12 batches of Qingre Mingmu tea were 0.410 3-0.451 3 mg·g-1,2.314 1-2.365 6 mg·g-1,1.186 3-1.216 9 mg·g-1,1.890 1-1.959 1 mg·g-1,0.410 8-0.423 4 mg·g-1,0.631 8-0.653 7 mg·g-1 and 0.588 9-0.616 8 mg·g-1,respectively.Conclusion:The established method of fingerprint and quantitative determination of Qingre Mingmu tea by HPLC is accurate,reliable,specific and sensitive.It can fully reflect the internal quality and can be used for quality control of Qingre Mingmu tea.