中药提取物羧酸酯酶抑制剂筛选及对缓解小鼠伊立替康迟发型腹泻作用的研究
发布时间：2018-10-22 07:43
【摘要】：1.羧酸酯酶(CEs)是α/β-水解酶超家族的一员,负责催化水解各种内源性和外源性物质包括脂肪酸、环境毒素和药物。通过抑制肠道肿瘤细胞中高含量的羧酸酯酶(hCE2)可以缓解伊立替康导致的严重腹泻症状。本文采用羧酸酯酶2特异性荧光探针-荧光素乙二酸酯(FD)作为底物构建同源模型,在体外肝微粒体水平下对hCE2具有抑制活性的中药提取物进行了高通量的筛选试验。结果显示,补骨脂乙醇提取物对hCE2表现很强的抑制作用。通过构建超高速液相色谱(UFLC-UV)指纹图谱将补骨脂提取物成分分离,每隔30秒对LC洗脱液片段进行收集,并对每一个分离的LC片段进行抑制活性检测。试验证明有5个主要成分表现出强抑制活性,借助液相色谱-二极管阵列检测器-电喷雾离子化串联质谱法(LC-DAD-ESI-MS/MS)通过LC保留时间、紫外光谱和高分辨质谱与标准品对照确定强效抑制活性化合物包括新补骨脂异黄酮、补骨脂乙素、补骨脂二氢黄酮甲醚、补骨脂甲素A、补骨脂酚。混合人肝微粒体(HLM)与底物FD在37。C条件下孵育一定时间后,用酶标仪检测探针底物的代谢产物的生成率,计算IC50值。进一步,用Lineweaver-Burk作图和Dixon作图来判断可逆抑制类型,并计算抑制动力学参数Ki。结果显示,5个化合物对hCE2呈现浓度依赖性抑制； 5个化合物的两种作图形式交点都交于第二象限横坐标,表明补骨脂5种主要成分对hCE2的抑制类型为非竞争性抑制;Ki值范围0.62μM-3.89μm,证实补骨脂提取物中5个主要成分对hCE2具有较强抑制活性。5个活性成分总量占补骨脂提取总量的50%,并在小鼠迟发型腹泻模型上进行了药效学评价。本研究在国内外首次证实了补骨脂及其化学单体成分作为天然产物具有hCE2强效抑制活性,揭示补骨脂及其单体成分可作为hCE2强效抑制剂和其在减轻CPT-11诱发严重延迟性腹泻的疗效有良好的应用前景。2.在已证实黄芩素可作为β-D-葡萄糖醛酸苷酶抑制剂的基础上,本研究通过试验证明其亦是人羧酸酯酶2特异性抑制剂,说明黄芩素具有特异性抑制羧酸酯酶2和p-D-葡萄糖醛酸苷酶双靶点作用机制。黄芩素对hCE2的抑制呈现浓度依赖性抑制,Ki值为6.2μM：用Lineweaver-Burk作图和Dixon作图来判断其抑制类型为非竞争性抑制。动物试验、肠道组织切片、HE染色、AB-PAS染色和免疫组化试验表明：黄芩素组腹泻程度显著下降(P0.05),并能够降低CPT-11诱发的鼠迟发性腹泻及肠粘膜损伤的程度,明显减少小鼠肠道组织含有的3种促炎性因子TNF-α,IL-6和IL-1β的生成。
[Abstract]:1. Carboxylesterase (CEs) is a member of the 伪 / 尾 -hydrolase superfamily, which is responsible for the catalytic hydrolysis of various endogenous and exogenous substances, including fatty acids, environmental toxins and drugs. Inhibition of high levels of carboxylesterase (hCE2) in intestinal cancer cells can relieve the severe diarrhea caused by Iritecan. In this paper, the homologous model of carboxylesterase 2 specific fluorescence probe, fluorescein adipic acid ester (FD), was used as the substrate to construct the homologous model. High throughput screening experiments were carried out on the Chinese traditional medicine extracts with inhibitory activity of hCE2 at the level of liver microsomes in vitro. The results showed that the ethanol extract of psoralen showed a strong inhibitory effect on hCE2. The components of psoralen extract were separated by constructing a UFLC-UV fingerprint, and the fragments of LC elution were collected every 30 seconds, and the inhibitory activity of each LC fragment was detected. Five main components showed strong inhibitory activity. The retention time of LC was obtained by means of liquid chromatography-diode array detector (LC-DAD-ESI-MS/MS) and electrospray ionization tandem mass spectrometry (LC-DAD-ESI-MS/MS). UV spectrum and high resolution mass spectrometry (HRMS) were used to determine the strong inhibitory activity of psoralen isoflavone, psoralen B, psoralen dihydroflavone methyl ether, psoralen A and psoralen phenol. After mixed human liver microsomal (HLM) and substrate FD were incubated at 37.C for a certain time, the production rate of metabolites of the probe substrate was detected by enzyme labeling instrument, and the IC50 value was calculated. Furthermore, the reversible inhibition type was determined by Lineweaver-Burk and Dixon diagrams, and the inhibition kinetic parameter Ki. was calculated. The results show that the five compounds exhibit concentration-dependent inhibition on hCE2, and the intersection points of the two mapping forms of the five compounds are intersected in the second quadrant. The results showed that the inhibition type of psoralen on hCE2 was non-competitive, and the Ki value was 0.62 渭 M-3.89 渭 m, which confirmed that the five main components of psoralen had strong inhibitory activity on hCE2, and the total amount of five active components accounted for the total amount of psoralen extract. The pharmacodynamics was evaluated on mice model of delayed diarrhea. In this study, it was first confirmed that psoralen and its chemical monomers had strong hCE2 inhibitory activity as natural products. The results showed that psoralen and its monomers could be used as potent inhibitors of hCE2 and had a good application prospect in alleviating severe delayed diarrhea induced by CPT-11. 2. Baicalin has been proved to be a 尾 -D-glucuronidase inhibitor, and has been proved to be a specific inhibitor of human carboxylesterase 2. The results suggest that baicalin has the mechanism of specific inhibition of carboxylesterase 2 and p-D-glucuronidase. Baicalin inhibited hCE2 in a concentration-dependent manner, with a Ki value of 6.2 渭 m. Lineweaver-Burk and Dixon were used to determine the type of inhibition as non-competitive inhibition. Animal experiments, intestinal tissue sections, HE staining, AB-PAS staining and immunohistochemical tests showed that the diarrhea degree of baicalin group was significantly decreased (P0.05), and the degree of delayed diarrhea and intestinal mucosal injury induced by CPT-11 was decreased in rats. The production of TNF- 伪, IL-6 and IL-1 尾 in the intestinal tissues of mice was significantly reduced.
【学位授予单位】：山西农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S853.7
[Abstract]:1. Carboxylesterase (CEs) is a member of the 伪 / 尾 -hydrolase superfamily, which is responsible for the catalytic hydrolysis of various endogenous and exogenous substances, including fatty acids, environmental toxins and drugs. Inhibition of high levels of carboxylesterase (hCE2) in intestinal cancer cells can relieve the severe diarrhea caused by Iritecan. In this paper, the homologous model of carboxylesterase 2 specific fluorescence probe, fluorescein adipic acid ester (FD), was used as the substrate to construct the homologous model. High throughput screening experiments were carried out on the Chinese traditional medicine extracts with inhibitory activity of hCE2 at the level of liver microsomes in vitro. The results showed that the ethanol extract of psoralen showed a strong inhibitory effect on hCE2. The components of psoralen extract were separated by constructing a UFLC-UV fingerprint, and the fragments of LC elution were collected every 30 seconds, and the inhibitory activity of each LC fragment was detected. Five main components showed strong inhibitory activity. The retention time of LC was obtained by means of liquid chromatography-diode array detector (LC-DAD-ESI-MS/MS) and electrospray ionization tandem mass spectrometry (LC-DAD-ESI-MS/MS). UV spectrum and high resolution mass spectrometry (HRMS) were used to determine the strong inhibitory activity of psoralen isoflavone, psoralen B, psoralen dihydroflavone methyl ether, psoralen A and psoralen phenol. After mixed human liver microsomal (HLM) and substrate FD were incubated at 37.C for a certain time, the production rate of metabolites of the probe substrate was detected by enzyme labeling instrument, and the IC50 value was calculated. Furthermore, the reversible inhibition type was determined by Lineweaver-Burk and Dixon diagrams, and the inhibition kinetic parameter Ki. was calculated. The results show that the five compounds exhibit concentration-dependent inhibition on hCE2, and the intersection points of the two mapping forms of the five compounds are intersected in the second quadrant. The results showed that the inhibition type of psoralen on hCE2 was non-competitive, and the Ki value was 0.62 渭 M-3.89 渭 m, which confirmed that the five main components of psoralen had strong inhibitory activity on hCE2, and the total amount of five active components accounted for the total amount of psoralen extract. The pharmacodynamics was evaluated on mice model of delayed diarrhea. In this study, it was first confirmed that psoralen and its chemical monomers had strong hCE2 inhibitory activity as natural products. The results showed that psoralen and its monomers could be used as potent inhibitors of hCE2 and had a good application prospect in alleviating severe delayed diarrhea induced by CPT-11. 2. Baicalin has been proved to be a 尾 -D-glucuronidase inhibitor, and has been proved to be a specific inhibitor of human carboxylesterase 2. The results suggest that baicalin has the mechanism of specific inhibition of carboxylesterase 2 and p-D-glucuronidase. Baicalin inhibited hCE2 in a concentration-dependent manner, with a Ki value of 6.2 渭 m. Lineweaver-Burk and Dixon were used to determine the type of inhibition as non-competitive inhibition. Animal experiments, intestinal tissue sections, HE staining, AB-PAS staining and immunohistochemical tests showed that the diarrhea degree of baicalin group was significantly decreased (P0.05), and the degree of delayed diarrhea and intestinal mucosal injury induced by CPT-11 was decreased in rats. The production of TNF- 伪, IL-6 and IL-1 尾 in the intestinal tissues of mice was significantly reduced.
【学位授予单位】：山西农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S853.7
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