女贞子中齐墩果酸的定性分析及其与人血清白蛋白相互作用的电喷雾质谱研究

Qualitative Analysis of Oleanolic Acid from Ligustri Lucidi Fructus and Study on the Interaction of Oleanolic Acid with Human Serum Albumin by Electrospray Ionization Mass Spectrometry

目的：对女贞子中主要药用成分进行定性分析,用电喷雾质谱法探究人血清白蛋白与齐墩果酸相互作用。方法：定性分析采用高效液相色谱电喷雾高分辨质谱（HPLC-ESI-MS）法,色谱柱使用Agilent ZORBAX Eclipse XDB-C18柱（2.1×150 mm,5-Micron）,流动相为甲醇（A）-0.1%乙酸水溶液（B）,梯度洗脱：0-30 min 10%-90%A,30-40 min 90%A,40-60 min 90%-10%A,柱温：30℃,进样量：1μL,流速：0.2 m L·min^-1,女贞子乙醇提取物采用HPLC-ESI-MS负离子检测模式。结果：采用HPLC-ESI-MS定性地检测了女贞子乙醇超声提取液,鉴定出9种有效成分（齐墩果酸、红景天苷、特女贞苷、橄榄苦苷、芹菜素、委陵菜酸、2α-羟基熊果酸、2α-羟基齐墩果酸和19α-羟基-3-乙酰乌索酸）。电喷雾质谱观测到齐墩果酸能与人血清白蛋白形成较为稳定的1：1蛋白质复合物,其结合常数为1.63×10^4L·mol^-1。结论：该方法定性分析了女贞子乙醇提取物中主要药用成分,首次用电喷雾质谱法研究了人血清白蛋白与齐墩果酸的相互作用,从分子水平上揭示了齐墩果酸与人血清白蛋白相互作用的质谱信息。

Objective： To qualitatively analyze the alcohol extract of Ligustri Lucidi Fructus, and to analyze the interaction between oleanolic acid （OA） and human serum albumin （HSA）. Methods： The LC column used for separation was Agilent ZORBAX Eclipse XDB-C18 （2.1×150 mm,5-Micron）, and column temperature was 30℃. Mobile phases were methanol （A） and 0.1% acetic acid aqueous solution （B）, and the gradient elution was programmed as 10%-90% A in 0～30min, 90% A in 30～40min, and 90%～10%A in 40-60 min. Injection vol- ume was 1 μL, and the flow rate was 0.2 mL·min^-1. Alcohol extract of Ligustri Lucidi Fructus was analyzed by HPLC-ESI-MS in the negative ion mode. Results： Nine effective components （oleanolic acid, salidroside, specnuezhenide, oleuropein, apigenin, tormentic acid, 2α-hydroxy-ursolic acid, 2α-hydroxy-oleanolic acid and 19α- hydroxy-3-acetyl-ursolicacid） were identified by HPLC-ESI-MS. ESI-MS observed that the oleanolic acid formed a stable 1：1 HSA-OA complex, and the binding constant was measured to be 1.63×10^4L·mol^-1. Conclusion： High performance liquid chromatography electrospray ionization high resolution mass spectrometry （HPLC-ESI-MS） method was established to qualitatively analyze the alcohol extract of Ligustri Lucidi Fruetus. The interaction between oleanolic acid （OA） and human serum albumin （HSA） was first analyzed by electrospray ionization mass spectrometry （ESI-MS）, demonstration the MS information of their binding at the molecular level.