华蟾酥毒基和酯蟾毒配基与不同血浆蛋白结合率的测定

Determination of protein binding rates of cinobufagin and resibufogenin with different plasmas by HPLC

建立测定华蟾酥毒基和酯蟾毒配基与人、比格犬和大鼠血浆蛋白结合率的方法;采用平衡透析法,并以高效液相色谱法进行测定,计算华蟾酥毒基和酯蟾毒配基与不同血浆的蛋白结合率。华蟾酥毒基和酯蟾毒配基在3种不同透析内液中的线性范围均为0.50～40.00μg.mL 1,透析外液均为0.25～4.00μg.mL 1。在3种不同透析内液和透析外液中,华蟾酥毒基提取回收率为(68.73±2.16)%～(79.27±1.62)%,酯蟾毒配基为(71.59±4.31)%～(83.47±2.63)%。华蟾酥毒基在人、大鼠和比格犬血浆中的平均血浆蛋白结合率分别为85.63%、80.21%和70.10%,酯蟾毒配基分别为84.51%、75.11%和70.60%。结果表明,华蟾酥毒基和酯蟾毒配基与3种血浆蛋白结合率较高,且结合率人>大鼠>比格犬。

The aim of this paper is to report the development of a method for the determination of the binding rates of cinobufagin （CBG） and resibufogenin （RBG） with different plasma proteins. Equilibrium dialysis method was used to imitate the binding process between cinobufagin, resibufogenin and plasma proteins. HPLC was employed to determine the concentration of cinobufagin and resibufogenin inside and outside of the dialysis membrane, and then on the basis of which protein binding rates were calculated. The calibration curves of both cinobufagin and resibufogenin with human, rats＇ and Beagle dogs＇ plasma were linear in the range of 0.50 - 40.00 μg mL-1, while the buffer solution was 0.25 - 4.00 μg.mL-1. The extract recovery of cinobufagin was in the range of （68.73±2.16）% - （79.27± 1.62）%, while that of resibufogenin was （71.59±4.31）% - （83.47± 2.63） %. The average plasma protein binding rates with cinobufagin were 85.63%, 80.21% and 70.10% in human, rats＇ and Beagle dogs＇ plasma, whereas those binding rates with resibufogenin were 84.51%, 75.11% and 70.60%, respectively. The results suggested that the protein binding rates of cinobufagin and resibufogenin were of middle strength, which in rats＇, Beagle dogs＇ and human plasma were decreased in the following order： human plasma 〉 rats＇ plasma 〉 Beagle＇ dogs plasma.