生物测定法用于中药质量评价的探索研究——以夏枯草抗氧化活性与总酚酸含量相关性的研究为例

Exploration on feasibility of introducing bioassay method into quality evaluation of Chinese herbal medicines by studying on the correlation between antioxidant activity of Prunella vulgaris and its total phenolic acids content for example

研究不同来源及不同部位夏枯草的抗氧化活性,并对其所含原儿茶酸、原儿茶醛、咖啡酸、异迷迭香酸苷和迷迭香酸5种酚酸类成分含量进行测定,探讨夏枯草抗氧化活性与总酚酸含量间的相关性,为制定科学合理的质量标准服务。以夏枯草50%甲醇提取液为研究对象,分别采用DPPH和HPLC测定夏枯草的抗氧化活性及原儿茶酸、原儿茶醛、咖啡酸、异迷迭香酸苷和迷迭香酸5种酚酸类成分的含量。DPPH采用夏枯草50%甲醇提取液0.5 m L与0.1 mmol·L^(-1)DPPH·乙醇溶液反应60 min,于517 nm波长处测定吸光度,用清除率及IC50进行抗氧化活性评价。HPLC采用Epic C18色谱柱,乙腈-0.1%甲酸水溶液为流动相,梯度洗脱,280 nm进行检测。采用偏最小二乘法对多批不同产地和不同部位夏枯草的抗氧化能力与其中5种酚酸类成分的总含量间的相关性进行分析。夏枯草50%甲醇提取液与0.1 mmol·L^(-1)DPPH·乙醇溶液反应的量效范围为0.300~1.65 g·L^(-1)(药材),原儿茶酸、原儿茶醛、咖啡酸、异迷迭香酸苷和迷迭香酸的进样量分别在0.007 84~0.980,0.011 5~1.44,0.008 64~1.08,0.080 0~1.00,0.079 8~0.998μg与各自峰面积积分值成良好的线性关系。5种成分的平均加样回收率分别为97.76%,96.88%,100.3%,102.1%,104.5%,RSD分别为1.8%,1.6%,1.7%,0.62%,0.75%。夏枯草各部位的抗氧化能力与总酚酸含量和在一定的药材质量范围内具有良好的线性相关性。因此,在确定的药材质量范围内,采用DPPH生物测定法结合HPLC含量测定法共同用于夏枯草药材的质量控制,可考虑作为一种新的尝试用于夏枯草质量标准的修订。

This paper aims to investigate the correlation between the antioxidant activity of Prunella vulgaris and its total phenolic acids content by measuring the antioxidant activity of different sources and different organs of P. vulgaris and the total contents of proto- catechuic acid, protocatechuic aldehyde, caffeic acid, salviaflaside and rosmarinic acid in these samples. Using the 50% methanol ex- tract of P. vulgaris samples as the research object, DPPH method and HPLC method were used respectively to determine the antioxi- dant activities and the total contents of the above-mentioned five analytes in P. vulgaris samples. 0. 5 mL of 50% methanol extract of P. vulgaris reacts with 0. 1 mmol L -1 DPPH ethanol solution for 60 min, then the absorbance of the reaction solution was measured at 517 nm, scavenging rate and ICs0 values were calculated by the absorbance and the sample concentration for evaluating the antioxidant activity. HPLC analysis was made on a Cls Epic column, with acetonitrile-0. 1% formic acid aqueous solution as mobile phase （gradi- ent elution）, and the detection wavelength was set at 280 nm. The correlation between the antioxidant capacity of different habitats and different organs of P. vulgaris and the total contents of five kinds of phenolic acids was analyzed by partial least squares method. The reaction dose-response range of 50% methanol extract of P. vulgaris with 0. 1 mmol L- 1 DPPH ethanol solution was 0. 300-1.65 g L- 1 When the quantities of potocatechuic acid, protocatechuic aldehyde, caffcic acid, salviaflaside and rosmarinic acid were respec- tively in 0. 007 84-0. 980, 0. 011 5-1.44, 0. 008 64-1.08, 0. 080 0-1.00 and 0. 079 8-0. 998 txg range, their quantities were in good linear relationship with the corresponding peak areas. The average recovery of 5 components were 97.76% , 96. 88% , 100. 3% , 102. 1%, 104. 5%, with RSD of 1.8%, 1.6%, 1. 7%, 1.6% and 1.7%, respectively. In a certain range of crude drug quantity, the antioxidant activity of each organ of P. vulgaris and total phenolic acids content inside has a good linear correlation. Therefore, in certain quality range of crude drug, DPPH bioassay combined with HPLC content determination can be used for the quality control of P. vulgaris, as is a new method for the quality control of P. vulgaris.