双峰驼CYP1A酶体外活性及其对探针药物代谢的影响
发布时间：2018-08-05 15:30
【摘要】：双峰驼具有耐饥渴、耐酷暑严寒、耐粗饲料等生物学特点,而且还喜欢采食戈壁、荒漠草原上的盐碱含量较高的植物和某些有毒植物,且不出现任何不良反应。然而,有关双峰驼能够在极端恶劣的环境下生存和对外源性化学物质的生物转化能力方面的研究却处于空白状态。CYP 1A酶作为参与机体内源性和外源性物质代谢的关键酶之一,在维持机体内环境的相对平衡以及与外环境之间的相互作用上均起着至关重要的作用。为探明双峰驼CYPlA酶的体外活性及其对外源性药物代谢的影响,开展了本项研究。首先,采用差速离心法制备双峰驼肝微粒体,并运用BCA法测定其蛋白浓度；CO还原差示光谱法分别测定CYP总酶含量和细胞色素b5含量；并通过CYP1A酶对7-乙氧基香豆素的脱烃基作用初步评价其体外活性。结果表明,所制备的双峰驼肝微粒体悬液蛋白浓度为1.652±0.341mg/g、CYP总酶含量为0.08±0.014nmol/mg、细胞色素b5(Cybts)含量为0.113±0.036nmol/mg,且其中的CYP1A酶具有降解其特异性底物-7-乙氧基香豆素的活性。在此基础上,为研究CYP1A2酶的体外活性,首先建立了适宜的CYP1A2酶体外孵育体系,并以该酶的特异性底物非那西丁与肝微粒体反应体系共同孵育一定时间,而后用混有内标物的冰甲醇终止反应,使用高效液相色谱紫外检测法(HPLC-UV)测定反应产物-对乙酰氨基酚的含量。流动相为甲醇：水=25：75,流速为0.5mL/min,检测波长为235nm,柱温为25℃,进样量为20μL,等浓度洗脱30min。产物对乙酰氨基酚、内标物间乙酰氨基酚、底物非那西丁的保留时间分别为5.046、6.304、23.452min,各峰分离良好。对肝微粒体孵育体系进行优化后,得到最适肝微粒体浓度为4.95mg/mL,最适孵育时间为30min,最适底物浓度为200μg/mL。通过Lineweaver-Burk双倒数作图法,计算出最大反应速度Vmax为0.2268±0.0418nmol/min/mg,米氏常数Km为2.8205 ±0.5198μmol/mL,内在代谢清除率CLint为0.0804 ± 0.0231μL·min/mg。最后进行了α-萘黄酮对CYP1A2酶活性的影响实验。结果显示,它对双峰驼CYP1A2具有较强的、不可逆的竞争性抑制作用,其半抑制浓度IC50为0.5411±0.0405μmol/mL。因此,通过本实验成功制备出能够满足双峰驼CYP1A酶体外活性研究的肝微粒体悬浮液,建立了测定CYP1A2酶特异性探针药非那西丁及其代谢产物的HPLC-UV检测方法,初步阐明了CYP1A酶体外活性及其特异性抑制剂的影响。研究结果可为全面揭示双峰驼CYP酶系的体内活性奠定基础,提供科学依据。
[Abstract]:Bactrian camel has the biological characteristics of hunger and thirst tolerance to heat and cold and forage and so on. It also likes to feed on Gobi desert steppe plants and some poisonous plants without any adverse reactions. However, studies on the ability of Bactrian camels to survive in extremely harsh environments and biotransformation of exogenous chemicals remain blank. CYP1A is one of the key enzymes involved in the metabolism of endogenous and exogenous substances in the body. It plays an important role in maintaining the relative balance of the internal environment and the interaction with the external environment. In order to investigate the in vitro activity of CYPlA enzyme and its effect on exogenous drug metabolism in Bactrian Camel, this study was carried out. Firstly, the liver microsomes of bactrian camels were prepared by differential centrifugation, and the total enzyme content of CYP and the content of cytochrome b _ 5 were measured by BCA method. The dealkylation of 7-ethoxy coumarin by CYP1A enzyme was evaluated in vitro. The results showed that the concentration of hepatic microsomal protein was 1.652 卤0.341mg / g, the total enzyme content was 0.08 卤0.014nmol / mg, the content of cytochrome B5 (Cybts) was 0.113 卤0.036nmol / mg, and the CYP1A enzyme had the activity of degrading its specific substrate -7-ethoxycoumarin. On this basis, in order to study the activity of CYP1A2 enzyme in vitro, a suitable incubation system of CYP1A2 enzyme in vitro was established, and the specific substrate of the enzyme, phenacetin, was incubated with liver microsomal reaction system for a certain time. Then the ice methanol with internal standard was used to terminate the reaction and the content of paracetamol was determined by high performance liquid chromatography ultraviolet detection (HPLC-UV). The mobile phase consisted of methanol: water 25: 75, the flow rate was 0.5 mL / min, the detection wavelength was 235 nm, the column temperature was 25 鈩，
本文编号：2166218
[Abstract]:Bactrian camel has the biological characteristics of hunger and thirst tolerance to heat and cold and forage and so on. It also likes to feed on Gobi desert steppe plants and some poisonous plants without any adverse reactions. However, studies on the ability of Bactrian camels to survive in extremely harsh environments and biotransformation of exogenous chemicals remain blank. CYP1A is one of the key enzymes involved in the metabolism of endogenous and exogenous substances in the body. It plays an important role in maintaining the relative balance of the internal environment and the interaction with the external environment. In order to investigate the in vitro activity of CYPlA enzyme and its effect on exogenous drug metabolism in Bactrian Camel, this study was carried out. Firstly, the liver microsomes of bactrian camels were prepared by differential centrifugation, and the total enzyme content of CYP and the content of cytochrome b _ 5 were measured by BCA method. The dealkylation of 7-ethoxy coumarin by CYP1A enzyme was evaluated in vitro. The results showed that the concentration of hepatic microsomal protein was 1.652 卤0.341mg / g, the total enzyme content was 0.08 卤0.014nmol / mg, the content of cytochrome B5 (Cybts) was 0.113 卤0.036nmol / mg, and the CYP1A enzyme had the activity of degrading its specific substrate -7-ethoxycoumarin. On this basis, in order to study the activity of CYP1A2 enzyme in vitro, a suitable incubation system of CYP1A2 enzyme in vitro was established, and the specific substrate of the enzyme, phenacetin, was incubated with liver microsomal reaction system for a certain time. Then the ice methanol with internal standard was used to terminate the reaction and the content of paracetamol was determined by high performance liquid chromatography ultraviolet detection (HPLC-UV). The mobile phase consisted of methanol: water 25: 75, the flow rate was 0.5 mL / min, the detection wavelength was 235 nm, the column temperature was 25 鈩，
本文编号：2166218
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