玳玳果黄酮降脂提取物效应组分血浆蛋白结合特性研究

Binding characteristics of plasma protein in active parts of Daidai lipid-lowering flavonoid extract

该文研究玳玳果黄酮降脂提取物效应组分与血浆蛋白的结合能力,并探讨通过改进传统超滤法计算血浆蛋白结合率的公式,提高试验结果的稳定性及可靠性。研究采用UPLC-MS/MS建立同时测定超滤液中提取物效应组分新橙皮苷和柚皮苷含量的定量分析方法;在优化的离心条件及孵育条件下,以超滤法传统公式计算血浆蛋白结合率,另引入校正因子(F)计算,比较经校正因子计算所得的血浆蛋白结合率结果的异同,评价提取物与大鼠血浆蛋白的结合能力。所建立的UPLC-MS/MS定量分析方法具备良好的专属性,标准曲线与线性范围、方法准确度与精密度、定量下限均符合有关规定,该方法能够满足大鼠血浆在经超滤管滤过后超滤液中的效应组分的定量检测需要。实验发现,效应组分新橙皮苷及柚皮苷与大鼠血浆蛋白结合率均在90%以上,且二者血浆蛋白结合表征出浓度依赖性,同时二者与大鼠血浆蛋白的结合能力无显著性差异。此外,在传统超滤法计算蛋白结合率时引入校正因子,可以较为有效地反映中药提取物多组分群影响误差,这一校正方式可为超滤法测定中药血浆蛋白结合能力方法学改进提供一个可参考的思路与实践方法。

To study the binding capacity of active ingredients of Daidai lipid-lowering flavonoid extract and plasma protein,investigate the ways to improve the traditional formula for calculating protein binding rates based on ultrafiltration,and increase the stability and reliability of the experimental results. UPLC-MS/MS was used to establish a quantitative analysis method for simultaneous determination of active ingredients( neohesperidin and narngin) in ultrafiltrate. The protein binding rates were calculated by the traditional ultrafiltration formula. The correction factors( F) were introduced later,and the binding rates calculated with the correction factors were compared with those without the correction factors. The binding capacity of the extract and plasma protein was evaluated. The quantitative analysis method established by UPLC-MS/MS had a good specificity. The standard curve and linear range,method accuracy,precision and lower limit of quantitation all met the requirements. The method met the requirement for quantitative detection of the active ingredients in ultrafiltrate after the rat plasma was filtrated in the ultrafiltration tube. Under the experimental conditions,the binding rates of both active ingredients( neohesperidin and narngin) were higher than 90%. The active ingredients and rat plasma protein were bound in a concentration-dependent manner,with statistically significant differences( P<0. 01). There was no statistically significant difference between the protein binding abilities of the two active ingredients with rat plasma protein. Therefore,the active ingredients of Daidai lipid-lowering flavonoid extract had a relatively strong binding strength with rat plasma protein,and they were bound in a concentration-dependent manner. Additionally,when calculating protein binding rates by the traditional ultrafiltration formula,the correction factors could be introduced to effectively reflect the errors of multiple ingredient groups in traditional Chinese medicine extracts.This correction method could provide a reference thinking and practical reference for the improvement of the determination method of the traditional Chinese medicine plasma protein binding ability based on ultrafiltration.