UPLC同时测定4种酶定向炮制黄芪中的6种黄酮类成分含量

Simultaneous Determination of Six Flavonoids in Astragali Radix Directional Processed with Four Enzymes by UPLC

目的:建立黄芪中6种黄酮类成分的含量测定方法,研究4种酶(复合酶、植物纤维素酶、蜗牛酶及β-葡萄糖苷酶)定向炮制对黄芪中黄酮苷类成分及其苷元含量的影响。方法:采用ACQUITY UPLC HSS T3色谱柱(2.1 mm×100 mm,1.8μm),流动相0.1%甲酸水溶液-0.1%甲酸乙腈溶液梯度洗脱,检测波长260 nm,流速0.3 mL·min^-1,柱温30℃,进样量2μL。结果:6种成分毛蕊异黄酮苷、毛蕊异黄酮、芒柄花苷、芒柄花素、黄芪紫檀烷苷、美迪紫檀素的分离度良好,在各自线性范围内线性关系良好(R2≥0.9985),精密度、稳定性、重复性试验的RSD均<5.0%;平均加样回收率97.62%~101.13%,RSD 1.4%~2.7%。在0.5 g·L^-1酶溶液水平,毛蕊异黄酮苷、毛蕊异黄酮、芒柄花苷、芒柄花素、黄芪紫檀烷苷、美迪紫檀素在复合酶制黄芪中的质量分数分别为0.0820,0.3359,0.0559,0.1049,0.0150,0.0097 mg·g^-1,在纤维素酶制黄芪中的质量分数分别为0.1057,0.3642,0.0702,0.1174,0.0208,0.0125 mg·g^-1,在蜗牛酶制黄芪中的质量分数分别为0.0314,0.5100,0.0435,0.2109,0.0130,0.0130 mg·g^-1,在β-葡萄糖苷酶制黄芪中的质量分数分别为0.0853,0.3124,0.0615,0.1108,0.0058,0.0096 mg·g^-1。结论:黄芪经4种酶炮制后,除黄芪紫檀烷苷外,毛蕊异黄酮苷、芒柄花苷的含量均降低,但这3种黄酮苷类成分所对应苷元的含量均显著增加。该方法操作简便、准确、重复性好,适用于黄芪中6种黄酮类成分的含量测定,可为黄芪的酶定向炮制研究提供参考。

Objective:To establish an UPLC method for the simultaneous determination of 6 flavonoids,and to research for the effect of Astragali Radix directional processed with four enzymes(complex enzyme,plant cellulase,snail enzyme,andβ-glucosidase)on the contents of flavonoid glycosides and their aglycones in this herb.Method:Chromatographic separation was carried out on ACQUITY UPLC HSS T3 column(2.1 mm×100 mm,1.8μm)with the mobile phase of 0.1 mol·L^-1 formic acid solution-0.1 mol·L^-1 formic acid acetonitrile solution for gradient elution.The detection wavelength was set at 260 nm,the flow rate was 0.3 mL·min^-1,the column temperature was 30℃,and the injection volume was 2μL.Result:Calycosin-glucoside,calycosin,ononin,formononetin,9,10-dimethoxy-pterocarpan-3-O-β-D-glucoside and 3-hydroxy-9,10-dimethoxypterocarpan showed good linear relationships within their own ranges(R2≥0.9985),the relative standard deviations(RSDs)of precision,stability and repeatability were all<5.0%,and the average recovery was97.62%-101.13%with RSDs of 1.4%-2.7%.In 0.5 g·L^-1 level of enzyme solution,the contents of calycosinglucoside,calycosin,ononin,formononetin,9,10-dimethoxy-pterocarpan-3-O-β-D-glucoside and 3-hydroxy-9,10-dimethoxy-pterocarpan in Astragali Radix processed with complex enzyme were 0.0820,0.3359,0.0559,0.1049,0.0150,0.0097 mg·g^-1,the contents of them in Astragali Radix processed with plant cellulase were0.1057,0.3642,0.0702,0.1174,0.0208,0.0125 mg·g^-1,their contents in Astragali Radix processed with snail enzyme were 0.0314,0.5100,0.0435,0.2109,0.0130,0.0130 mg·g^-1,and their contents in Astragali Radix processed withβ-glucosidase were 0.0853,0.3124,0.0615,0.1108,0.0058,0.0096 mg·g^-1,respectively.Conclusion:After the processing of Astragali Radix by four enzymes,in addition to 9,10-dimethoxy-pterocarpan-3-O-β-D-glucoside,the contents of calycosin-glucoside and ononin are reduced,but the contents of their three corresponding aglycones are significantly increased.The established method is simple,accurate and reproducible,and is suitable for the simultaneous determination of 6 flavonoids in Astragali Radix,which can provide a reference for this herb directional processed with enzymes.