柴胡饮片皂苷类成分变化及质量控制研究

目的：建立不同来源柴胡饮片及醋柴胡饮片的皂苷类成分高效液相色谱指纹图谱，为柴胡饮片及醋柴胡饮片质量标准的提高提供依据。方法：采用高效液相色谱串联电喷雾高分辨质谱仪（HPLC-ESI-MS/MS）对特征峰进行指认，测定柴胡饮片及醋柴胡饮片中柴胡皂苷c、a、b₂、d的含量，并对柴胡饮片及醋柴胡饮片的含5%氨水的80%甲醇提取液、5%氨水水煎液和纯水水煎液中的柴胡皂苷含量的变化进行了研究。选用Phenomenex Gemini 5 μ C₁₈（110A，250 mm×4.6 mm，5 μm）色谱柱，以乙腈（A）-水（B）为流动相，梯度洗脱（0~5 min，20%A；5~10 min，20%A→30%A；10~22 min，30%A→30%A；22~46 min，30%A→45%A；46~60 min，45%A→48%A），流速1 mL·min^-1，检测波长210 nm，柱温25 ℃。质谱采用高分辨离子阱负离子模式，鉴定皂苷类成分的结构。结果：建立了柴胡饮片及醋柴胡饮片指纹图谱检测方法和柴胡皂苷c、a、b₂、d 4个成分的含量测定法；对柴胡饮片及醋柴胡饮片含5%氨水的80%甲醇提取液、水煎液和碱水煎液中的柴胡皂苷含量的变化进行了测定研究。结论：所建立的方法可以用于柴胡饮片及醋柴胡饮片的质量控制与评价，对皂苷类成分变化规律的研究表明，柴胡饮片在炮制前后，以不同的溶剂提取，其皂苷成分的类型和含量差异较大，因此在柴胡的质量控制中，仅仅控制原生皂苷柴胡皂苷a和d尚不足，应考虑柴胡皂苷c和b₂的检测。

Objective: To establish the high-performance liquid chromatographic fingerprint of saikosaponins from the decoction pieces of Radix Bupleuri from different sources and vinegar-baked Radix Bupleuri in order to provide the foundation for the improvement of quality standard of the decoction pieces of Radix Bupleuri and vinegar-baked Radix Bupleuri. Methods: The HPLC-ESI-MS/MS was used to identify the chemical constituent of characteristic peaks in the fingerprint,determine the content of saikosaponins c,a,b₂,and d in the decoction pieces of Radix Bupleuri and vinegar-baked Radix Bupleuri and analyze the content variation of saikosaponons from the decoction pieces of Radix Bupleuri and vinegar-baked Radix Bupleuri in three different extraction conditions (sonication with 80% aqueous methanol solution containing 5% ammonia,decoction with 5% ammonia water solution and pure water). The analyses were performed on a Phenomenex Gemini 5 μ C₁₈ column(110A,250 mm×4.6 mm,5 μm);the mobile phase was acetonitrile(A)-water(B) with gradient elution mode(0-5 min,20%A;5-10 min,20%A→30%A;10-22 min,30%A→30%A;22-46 min,30%A→45%A;46-60 min,45%A→48%A) at the flow rate of 1 mL·min^-1;the detection was set at 210 nm;the column temperature was 25 ℃.The major constituents were separated within 60 min.The mass spectrometry detection was performed with electrospray ionization and negative ion mode.The high resolution ion trap detector was used for identification of the chemical constituent of characteristic peaks in the fingerprint. Results: The high-performance liquid chromatographic fingerprint of saikosaponins from the decoction pieces of Radix Bupleuri and vinegar-baked Radix Bupleuri was established.There were 6 characteristic peaks in the fingerprint.The chemical constituent of all characteristic peaks were identified by HPLC-ESI-MS/MS.The contents of saikosaponins c,a,b₂,and d was determined.The content variation of saikosaponons from the decoction pieces of Radix Bupleuri and vinegar-baked Radix Bupleuri in three different extraction conditions was analyzed. Conclusion: The method established in the present study could be used for the quality control of the decoction pieces of Radix Bupleuri and vinegar-baked Radix Bupleuri.The study on content variation of saikosaponons from the decoction pieces of Radix Bupleuri and vinegar-baked Radix Bupleuri in three different extraction conditions demonstrates that the types and content of saikosaponins have great differences before and after process when using different extract solvents.Therefore it is not enough just to control saikosaponins a and d in the quality standard of the decoction pieces of Radix Bupleuri.The detection of saikosaponins c and b₂ should also be considered.