多糖类组分对大鼠肝微粒体酶CYP1A2的体外活性抑制作用

Inhibitory Effects of Polysaccharides on the Activity of CYP1A2 Enzyme in Rat Liver Microsomes in Vitro

目的建立液相色谱-串联质谱法(LC-MS/MS)测定大鼠肝微粒体中对乙酰氨基酚含量,研究人参多糖、贻贝多糖、黄原胶对大鼠肝微粒体细胞色素P450(cytochrome P450,CYP)1A2的体外抑制作用。方法以格列齐特为内标,建立LC-MS/MS测定大鼠肝微粒体中对乙酰氨基酚含量方法。以α-萘黄酮为阳性对照药,分别将系列多糖溶液、CYP1A2酶的特异性探针底物非那西丁及大鼠肝微粒体进行孵育,LC-MS/MS测定代谢产物对乙酰氨基酚的含量,计算半数抑制浓度(IC_(50)),评价人参多糖、贻贝多糖、黄原胶对大鼠肝微粒体CYP1A2酶的抑制活性。结果对乙酰氨基酚在10~1000 ng·mL^(-1)内线性良好,精密度试验RSD均<8.24%,提取回收率为92.53%~103.23%,稳定性试验RSD均<10.77%。该试验条件下,在大鼠肝微粒体温孵体系中,人参多糖、贻贝多糖、黄原胶对CYP1A2的IC_(50)值均>100μmol·L^(-1)。结论在正常剂量下,人参多糖、贻贝多糖、黄原胶对CYP1A2酶亚型均无抑制作用,可以与其底物联合用药。

OBJECTIVE To establish an LC-MS/MS method for the content determination of acetaminophen in rat liver microsomes and evaluate the inhibitory effects of several polysaccharides includes ginseng polysaccharide,mussel polysaccharide and xanthan gum on the activity of cytochrome P450(CYP)1A2 of rat liver microsomes in vitro.METHODS LC-MS/MS analytical method was eatablished to determine the concentration of acetaminophen in rat liver microsomes while taking gliclazide as the internal standard.α-Naphthalene flavone was used as the positive control drug,a series of polysaccharide solutions,phenacetin(as the specific probe substrate of CYP1A2 enzyme)and rat liver microsomes were incubated in vitro respectively.The content of metabolite acetaminophen was measured by LC-MS/MS and the half inhibitory concentration(IC_(50))was calculated,so as to evaluate the inhibitory activity of ginseng polysaccharide,mussel polysaccharide and xanthan gum on CYP1A2 enzyme in rat liver microsomes.RESULTS The linear range of acetaminophen was 10−1000 ng·mL^(-1),RSD of precision test was<8.24%,extraction recoveries were 92.53%−103.23%,and RSD of stability test was<10.77%.In mixture metabolic system of rat liver microsome enzymes,the IC_(50) of ginseng polysaccharide,mussel polysaccharide and xanthan gum were all>100μmol·L^(-1) under this condition.CONCLUSION There is no inhibitory effect of ginseng polysaccharide,mussel polysaccharide and xanthan gum on CYP1A2 enzyme subtypes at normal dose,and they can be used in combination with its substrate.