玳玳果黄酮降脂提取物效应组分血浆蛋白结合特性研究
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摘要
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.
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.
引文
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[5]邱洪鑫,陈丹,刘永静,等.玳玳果黄酮滴丸对高脂血症大鼠的降血脂作用研究[J].中国现代应用药学,2011,28(7):597.
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[8]吴晓青,曾令军,陈丹.玳玳总黄酮提取物UPLC-MS体内外特征图谱[J].中国医院药学杂志,2014,34(20):1734.
[9]曾华平,陈红,陈丹,等.玳玳果黄酮降脂提取物效应组分大鼠肝肠微粒体代谢特性研究[J].中国中药杂志,2019,44(4):819.
[10]崔艳花,郭春梅,孙明忠,等.系列黄酮化合物与血清白蛋白结合及其结构相关性[J].中国生化药物杂志,2012,33(6):728.
[11]王萍,唐仕欢,苏瑾,等.基于整合药理学的中药现代研究进展[J].中国中药杂志,2018,43(7):1297.
[12]刘睿,谢跃生,潘桂湘,等.药物血浆蛋白结合率测定方法的研究进展[J].天津中医药,2007,24(6):526.
[13]黄勇,牟景丽,陈慧,等.平衡透析法、超滤法结合液质联用技术比较测定注射用复方荭草中黄酮类成分的血浆蛋白结合率[J].中国实验方剂学杂志,2012,18(20):91.
[14]李季泓.柚皮苷的大鼠血浆蛋白结合率研究[J].实用药物与临床,2015,18(5):578.
[15]方茹,冷小京,吴夏,等.三种蛋白和黄酮结合物中氢键与黄酮稳定性相关性分析研究[J].光谱学与光谱分析,2012,32(1):108.
[2]陈丹,刘永静.一种玳玳果总黄酮有效部位的制备方法:中国,CN201010556341. 9[P].2011-05-18.
[3]刘永静,陈丹,黄庆德,等.玳玳果中总黄酮提取工艺的研究[J].中国医院药学杂志,2009,29(21):1826.
[4]刘永静,陈丹,邱红鑫,等.玳玳黄酮有效部位提取物降血脂作用的研究[J].中国中医药科技,2013,20(6):622.
[5]邱洪鑫,陈丹,刘永静,等.玳玳果黄酮滴丸对高脂血症大鼠的降血脂作用研究[J].中国现代应用药学,2011,28(7):597.
[6]Zeng L J,Chen D,Huang Q D,et al. Isolation of a new flavanone from Daidai fruit and hypolipidemic activity of total flavonoids extracts[J]. Nat Prod Res,2015,29(16):1521.
[7]曾令军,陈丹,郑利,等.玳玳黄酮提取物特征活性成分不同生理状态的药动学特性比较[J].中国中药杂志,2014,39(2):309.
[8]吴晓青,曾令军,陈丹.玳玳总黄酮提取物UPLC-MS体内外特征图谱[J].中国医院药学杂志,2014,34(20):1734.
[9]曾华平,陈红,陈丹,等.玳玳果黄酮降脂提取物效应组分大鼠肝肠微粒体代谢特性研究[J].中国中药杂志,2019,44(4):819.
[10]崔艳花,郭春梅,孙明忠,等.系列黄酮化合物与血清白蛋白结合及其结构相关性[J].中国生化药物杂志,2012,33(6):728.
[11]王萍,唐仕欢,苏瑾,等.基于整合药理学的中药现代研究进展[J].中国中药杂志,2018,43(7):1297.
[12]刘睿,谢跃生,潘桂湘,等.药物血浆蛋白结合率测定方法的研究进展[J].天津中医药,2007,24(6):526.
[13]黄勇,牟景丽,陈慧,等.平衡透析法、超滤法结合液质联用技术比较测定注射用复方荭草中黄酮类成分的血浆蛋白结合率[J].中国实验方剂学杂志,2012,18(20):91.
[14]李季泓.柚皮苷的大鼠血浆蛋白结合率研究[J].实用药物与临床,2015,18(5):578.
[15]方茹,冷小京,吴夏,等.三种蛋白和黄酮结合物中氢键与黄酮稳定性相关性分析研究[J].光谱学与光谱分析,2012,32(1):108.
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