碱处理在微量黄芪药材质控指标成分含量测定中的应用研究

Application of alkali treatment in quantification of quality indexes in trace amount of Astragali Radix

目的:建立碱处理微量黄芪药材测定黄芪皂苷Ⅳ、毛蕊异黄酮葡萄糖苷和芒柄花苷含量的方法,表征碱处理提高上述指标成分收率的有关物质转化特征。方法:取传统采收期黄芪根0.15 g,按1∶30的比例加入70%乙醇(内含不同浓度的氨水),超声提取,进行碱处理体系的优化。利用UPLC-QTRAP-MS多反应离子监测模式,采用BEH C18(2.1mm×50 mm,1.7μm)色谱柱,以0.1%甲酸水(A)-乙腈(B)为流动相,梯度洗脱,流速:0.2 mL·min-1,柱温:30℃,测定黄芪皂苷IV含量;采用2015年版《中华人民共和国药典》黄芪含量测定项下毛蕊异黄酮葡萄糖苷色谱条件,HPLC法测定毛蕊异黄酮葡萄糖苷和芒柄花苷含量;UPLC-Q-TOF-MS法进行有关化合物的指认。结果:用含4%氨水的70%乙醇提取时,三质控指标成分收率最高,对应的检测线性范围分别为10.70~85.60μg·mL-1、1.039~72.73μg·mL-1和1.051~73.57μg·mL-1。该优化体系中,黄芪皂苷Ⅰ、酰基化毛蕊异黄酮葡萄糖苷和酰基化芒柄花苷转化较为完全,黄芪皂苷Ⅱ部分转化;此外,还存在一未知化合物向黄芪皂苷Ⅳ的明显转化。采用该优化体系分析发现,根施外源正己醛组黄芪药材中黄芪皂苷Ⅳ含量明显高于对照,但两异黄酮组分组间无显著差异。结论:建立了一种碱处理微量黄芪测定黄芪皂苷Ⅳ、毛蕊异黄酮葡萄糖苷和芒柄花苷含量的方法,该方法显著提高了黄芪药材中3个质控指标成分的收率,可应用于微量黄芪的质量评价与品质形成研究。

Objective：To establish a quantitative method of chemical indexes（astragaloside IV,calycosin-7-O-β-D-glucoside and ononin）for quality evaluation of trace amount of Astragali Radix by alkali treatment.To characterizethe conversion of the acylated metabolites to the three indexes caused by alkali treatment.Methods：The sample was prepared by ultrasonic extraction with 70% ethanol containing ammonium hydroxide with different concentration,to optimize the alkali level of the extracting solution.Astragaloside IV was quantified by UPLC-QTRAP-MS in multi-reaction monitoring mode,BEH C18 column（2.1 mm×50 mm,1.7 μm）was used and gradie-nt elution was carried out with 0.1% formic acid（A）-acetonitrile（B）as mobile phase at a flow of 0.2 m L·min1,the column temperature was maintained at 30 ℃;The quantification of calycosin-7-O-β-D-glucoside and ononin was performed by HPLC with the conditions for determination of the former in Astragali Radix described in ChP 2015.And the conversion was characterized by UPLC-Q-TOF-MS.Results：Th e extraction with 70% ethanol containing 4% ammonia resulted in the highest yields of the three indexes in trace amount of samples and the linearity ranges were 10.70-85.60 μg·m L-1,1.039-72.73 μg·m L-1 and 1.051-73.57 μg·m L-1,respectively.In the optimized conditions,the conversion of astragaloside I,acylated calycosin-7-O-β-D-glucoside and ononin was relatively complete,while astragaloside II was partially transformed.In addition,there was an obvious transformation from an unknown compound to astragaloside IV.Using the optimized method,the quantification of the trace amount of samples was performed and showed that the exposure to exogenous hexanal obviously increased astragaloside Ⅳ＇s content in the herb but had no obvious effect on the contents of calycosin-7-O-β-D-glucoside and ononin relative to the mock treatment.Conclusion：An optimized alkali treatment is established for quantitative microanalysis of the three chemical indexes in trace amounts of Astragali Radix,which simultaneously enhances their yields in the herb and can be used in the studies associated with the quality evaluation and quality formation of the herb.