白扁豆总皂苷的含量测定与成分分析

Content Determination and Component Analysis of Total Saponins in Lablab Semen Albums

[目的]对白扁豆进行提取分离纯化以及含量测定与成分分析。[方法]用石油醚对粉碎的白扁豆脱脂,80%乙醇进行回流提取,正丁醇萃取,浓缩正丁醇得白扁豆总皂苷。白扁豆总皂苷粗提物通过硅胶柱、中压C_(18)色谱柱进行进一步纯化,用薄层监测收集富集皂苷类成分的洗脱液,得到需要成分。采用紫外可见分光光度计(UV)测定,以0.5%香草酸-冰醋酸溶液和高氯酸为显色剂,测定波长为540 nm,并采用四极杆-静电场轨道肼高分辨质谱仪(UHPLC-Q-Exactive Orbitrap MS)在负离子模式下对总皂苷进行成分分析。[结果]白扁豆总皂苷通过硅胶柱、中压C_(18)色谱柱纯化得到较纯的总皂苷。总皂苷UV测定的线性范围为0.0096~0.0192 mg/mL(R^(2)=0.9981);平均回收率为98.15%。白扁豆总皂苷共鉴定出18个化合物。[结论]采用硅胶柱干法上样及中压C_(18)色谱柱分离纯化,该方法纯化度高。UHPLC-Q-Exactive Orbitrap MS技术与UV法可用于白扁豆总皂苷的定性与定量分析,为后续白扁豆总皂苷生物活性研究提供数据支撑。

[Objective]To extract,separate and purify the lablab semen albums,and conduct the content determination and component analysis.[Method]The pulverized lablab semen albums were degreased using petroleum ether,followed by reflux extraction with 80%ethanol and extraction with n-butyl alcohol,and then the n-butyl alcohol was concentrated to obtain total saponins in the lablab semen albums.The total saponins were separated and purified via silica gel column and medium-pressure C_(18) chromatographic column,the eluent of thin-layer red spots was collected through thin-layer monitoring,and the components needed were obtained.The content determination was realized by the colorimetric method.To be more specific,0.5%vanillic acid-glacial acetic acid solution and perchloric acid were used as the color developing agents,the determination wavelength was 540 nm,and the component analysis of total saponins was implemented using the quadrupole rod-electrostatic field orbitrap high-resolution mass spectrometer(UHPLC-Q-Exactive Orbitrap MS)under the negative ion mode.[Result]The total saponins were extracted from the lablab semen albums and purified through the silica gel column and medium-pressure C_(18) chromatographic column.The linearity range in the colorimetric determination of total saponins was 0.0096-0.0192 mg/mL(R^(2)=0.9981);the average recovery rate was 98.15%.A total of 18 chemical compounds were identified from the total saponins in the lablab semen albums.[Conclusion]The separation and purification were carried out through the silica gel column dry-type sample loading and medium-pressure C_(18) chromatographic column,with high degree of purification.The UHPLC-Q-Exactive Orbitrap MS technology and colorimetric method are applicable to the qualitative and quantitative analysis of total saponins in the lablab semen albums,and will provide a data support for the subsequent research on their bioactivity.