黄芪异黄酮对照提取物的制备及其在黄芪药材含量测定中的应用

目的：制备黄芪异黄酮对照提取物，探讨选择对照提取物替代单体成分对照品测定黄芪药材中异黄酮类成分的可行性。方法：提取、制备黄芪异黄酮对照提取物，并进行毛蕊异黄酮葡萄糖苷、芒柄花素葡萄糖苷、毛蕊异黄酮、芒柄花素4个异黄酮成分的定值；采用HPLC法测定黄芪药材中4个异黄酮成分的含量，使用色谱柱为Phenomenex C₁₈（250 mm×4.6 mm，5 μm），以乙腈-0.3%甲酸水溶液为流动相，梯度洗脱（0~20 min，10%A→30%A；20~25 min，30%A；25~35 min，30%A→60%A；35~45 min，60%A→10%A），流速1.0 mL·min^-1，检测波长260 nm，柱温25 ℃。结果：黄芪异黄酮提取物中4个主成分毛蕊异黄酮葡萄糖苷、芒柄花素葡萄糖苷、毛蕊异黄酮、芒柄花素浓度分别在4.93~394.4 μg·mL^-1（r=0.9997）、2.68~214.4 μg·mL^-1（r=0.9992）、3.09~247.2 μg·mL^-1（r=0.9999）、1.53~122.4 μg·mL^-1（r=0.9999）范围内与色谱峰面积值呈良好的线性关系；平均回收率（n=6）分别为100.0%、99.8%、99.6%、100.1%，RSD均小于2.0%。结论：黄芪异黄酮对照提取物可替代相应的对照品，用以同时测定黄芪中毛蕊异黄酮葡萄糖苷、芒柄花素葡萄糖苷、毛蕊异黄酮、芒柄花素4个黄芪异黄酮成分的含量。

Objective: To prepare isoflavone reference extract of Astragali Radix,and explore the feasibility in the place of single component reference in assay. Methods: Extract and prepare isoflavone reference extract of Astragali Radix was extracted and prepared,and the contents of calycosin glycoside and other 3 components in the extract.The HPLC analysis was performed,and a Phenomenex C₁₈ column(250 mm×4.6 mm,5 μm)with gradient elution used acetonitrile and 0.3%formic acid solution as the mobile phase(0-20 min,10%→30%A;20-25 min,30%A;25-35 min,30%→60%A;35-45 min,60%→10%A).The flow rate was 1.0 mL·min^-1,the column temperature was 25 ℃,and the detection wavelength was 260 nm. Results: The method had a good linearity in the ranges of 4.93-394.4 μg·mL^-1(r=0.9997)for calycosin-7-O-β-D-glycoside,2.68-214.4 μg·mL^-1(r=0.9992)for formononetin-7-O-β-D-glycoside,3.09-247.2 μg·mL^-1(r=0.9999)for calycosin,and 1.53-122.4 μg·mL^-1(r=0.9999)for formononetin.The average recoveries of four components were 100.0%,99.8%,99.6%,100.1%with RSDs of less than 2.0%. Conclusion: The isoflavone reference extract of Astragali Radix can be used as the substitute to determine the four isoflavones in Astragali Radix instead of corresponding reference substances.