黄芩素在溃疡性结肠炎模型大鼠肝、肠微粒体中酶促反应动力学研究

Enzyme kinetics of biacalein in the liver and intestinal microsomes of ulcerative colitis rats

目的考察黄芩素在正常和溃疡性结肠炎大鼠肝、肠微粒体中的酶促反应动力学差异。方法采用3%葡聚糖硫酸钠连续自由饮用10 d,诱导溃疡性结肠炎大鼠模型,分别制备正常和溃疡性结肠炎大鼠肝、肠微粒体,通过建立大鼠肝、肠微粒体体外孵育体系对黄芩素代谢进行研究,采用HPLCDAD检测方法对代谢产物黄芩苷进行定量测定,应用Graph Pad Prism 5.0软件分析数据,对比黄芩素在正常和溃疡性结肠炎模型中的酶促反应动力学常数最大反应速率(Vmax)和米氏常数(Km)。结果黄芩素(5~150μmol·L^(-1))在正常和溃疡性结肠炎大鼠肝微粒体中的代谢属于经典的米氏方程,Km分别为(59.13±7.756)、(57.45±6.699)μmol·L^(-1),Vmax分别为(6.086±0.3234)、(7.447±0.347)μmol/(min·mg),而在肠微粒体中符合底物抑制动力学特征,抑制速率(Ki)分别为(166.4±58.31)、(141.7±41.17)μmol·L^(-1),Vmax分别为(4.130±0.6035)、(5.797±0.7903)μmol/(min·mg)。不管是正常组还是模型组,黄芩素在肝脏中的代谢速率要显著高于肠微粒体;与正常组相比,溃疡性结肠炎大鼠肝、肠微粒体中黄芩素葡萄糖醛酸化反应的速率显著升高,同时发现了黄芩素的一个新代谢产物,可能为黄芩苷的同分异构体。结论溃疡性结肠炎疾病状态能够显著升高葡萄糖醛酸化转移酶的活性,可能是导致黄芩苷在疾病状态下体内暴露强度增加的因素之一。

Objective To determine the difference in metabolism and enzymatic kinetics of baicalein in the liver and intestinal microsomes of normal and ulcerative colitis（UC） rats, respectively. Methods The UC rat model were established by using 3% dextran sulfate sodium（DSS） for 10 days. The normal rats were given water randomly, and then the liver and intestinal microsomes from the normal and the UC rats were prepared for incubation of baicalein in vitro. HPLC-DAD method was used to determine the glucuronides of baicalein. The enzyme kinetics parameters Vmax and Km of the normal and the UC group were calculated and compared by Graph Pad Prism 5.0 software. Results The metabolic behaviors of baicalein metabolized in the liver microsomes followed the typical Michaelis-Menten kinetics, either in the normal group [Vmax and Km were（6.086±0.3234） μmol/（min·mg） and（59.13±7.756） μmol·L^-1] or in the UC group [Vmax and Km were（7.447±0.347） μmol/（min·mg）and（57.45±6.699） μmol·L- 1]. The metabolic behaviors of baicalein metabolized in the intestinal microsomes followed the characteristics of substrate inhibition, either the normal group [Vmax and Ki were（4.130±0.6035） μmol/（min·mg） and（166.4±58.31） μmol·L- 1] or in the UC group [Vmax and Ki were（5.797±0.7903） μmol/（min·mg） and（141.7±41.17） μ mol·L- 1]. The metabolism of baicalein in the liver microsomes was stronger than that in the intestinal microsomes, regardless of the normal or UC rats. Compared with the normal rat, the rate of glucuronides of baicalein was significantly enhanced, both in the liver or the intestinal microsomes. Conclusion UC can improve glucuronosyl transferase（UGT） activity in rat, and it may be one reason why exposure intensity of baicalin was markedly increased in UC.