夏枯草HPLC-ECD指纹图谱建立及抗氧化谱效关系研究

Establishment of HPLC-ECD Fingerprinting of Prunella Vulgaris L. and Study on Its Antioxidant Spectrum-Effect Relationship

关键词:  夏枯草  电化学检测法  抗氧化  指纹图谱  谱效关系

Key words:  Prunella vulgaris L.  electrochemical detection  antioxidation  fingerprint  spectrum-effect relationship

摘要:

目的 建立高效液相色谱-电化学检测法(HPLC-ECD)夏枯草指纹图谱，测定其体外抗氧化活性并进行谱效关系的研究。方法 样品经80%甲醇提取，采用XBridge BEH shield RP₁₈色谱柱(3.0 mm×150 mm，2.5 μm)为固定相，乙腈-柠檬酸缓冲溶液(50 mmol·L^–1，pH 2.8)为流动相，梯度洗脱，体积流量为0.6 mL·min^–1；ECD检测电压为600 mV，柱温为40 ℃；通过DPPH自由基清除能力、ABTS自由基清除能力和铁离子还原能力评价其抗氧化活性，Folin-Ciocalteau法测定总酚含量，结合相似度评价对21批夏枯草进行质量分析，进一步采用灰色关联分析和偏最小二乘法回归分析对抗氧化谱效关系进行研究。结果 HPLC-ECD指纹图谱共标定19个共有峰，指认了其中的14个成分，21批样品相似度均>0.9；夏枯草具有较强的ABTS、DPPH自由基清除能力和铁离子还原能力。HPLC-ECD指纹图谱可以特异性体现样品中抗氧化成分的信息，且色谱图总峰面积同总酚含量、抗氧化活性之间呈显著线性相关。灰色关联分析结果表明19个共有峰与抗氧化活性的关联度>0.6，偏最小二乘法回归分析的结果表明迷迭香酸、咖啡酸、芦丁等化合物对抗氧化活性的贡献较大。结论 HPLC-ECD的指纹图谱结合体外抗氧化法可用于夏枯草的质量评价和抗氧化物质基础研究。

Abstract:

OBJECTIVE To establish the fingerprint of Prunella vulgaris L. by high performance liquid chromatography with electrochemical detection(HPLC-ECD), and to evaluate the antioxidant activity in vitro and study the spectrum-effect relationship. METHODS The samples were extracted with 80% methanol. The XBridge BEH shield RP₁₈ column(3.0 mm× 150 mm, 2.5 μm) was selected as the stationary phase, acetonitrile-citrate solution(50 mmol·L^-1, pH 2.8) was selected as the mobile phase with gradient elution at the flow rate of 0.6 mL·min^-1. The detection potential of ECD was 600 mV, the column temperature was 40℃. The antioxidant activity of Prunella vulgaris L. was evaluated by DPPH radical scavenging activity, ABTS radical scavenging activity and ferric reducing antioxidant power. The content of total phenolics was measured by the Folin-Ciocalteau method. The quality of 21 batches of Prunella vulgaris L. were analyzed by similarity evaluation. Furthermore, the spectrum-effect relationship of antioxidation was studied by gray relational analysis and partial least square regression analysis. RESULTS A total of 19 common peaks were obtained by HPLC-ECD fingerprinting, among which 14 components were identified. The similarity of the 21 batches of Prunella vulgaris L. were >0.9. At the same time, Prunella vulgaris L. had superior ability of ABTS, DPPH radical scavenging activity and ferric reducing antioxidant power values. HPLC-ECD fingerprint could specifically reflect the information of antioxidant components in the sample, and there was a significant linear correlation among the total peak area of chromatogram, total phenolics content and antioxidant activity. The results of gray relational analysis showed that there was a high correlation between 19 common peaks and antioxidant activity with the relational degree >0.6. The results of partial least square regression analysis showed that the compounds have the greatest contribution to antioxidative activity were rosmarinic acid, caffeic acid and rutin, etc. CONCLUSION The HPLC-ECD fingerprint combined with antioxidant method can be used to evaluate the quality of Prunella vulgaris L. and investigate the material base of antioxidant activity.