中藥提取物羧酸酯酶抑制劑篩選及對緩解小鼠伊立替康遲發(fā)型腹瀉作用的研究
發(fā)布時間：2018-10-22 07:43
【摘要】：1.羧酸酯酶(CEs)是α/β-水解酶超家族的一員,負(fù)責(zé)催化水解各種內(nèi)源性和外源性物質(zhì)包括脂肪酸、環(huán)境毒素和藥物。通過抑制腸道腫瘤細(xì)胞中高含量的羧酸酯酶(hCE2)可以緩解伊立替康導(dǎo)致的嚴(yán)重腹瀉癥狀。本文采用羧酸酯酶2特異性熒光探針-熒光素乙二酸酯(FD)作為底物構(gòu)建同源模型,在體外肝微粒體水平下對hCE2具有抑制活性的中藥提取物進行了高通量的篩選試驗。結(jié)果顯示,補骨脂乙醇提取物對hCE2表現(xiàn)很強的抑制作用。通過構(gòu)建超高速液相色譜(UFLC-UV)指紋圖譜將補骨脂提取物成分分離,每隔30秒對LC洗脫液片段進行收集,并對每一個分離的LC片段進行抑制活性檢測。試驗證明有5個主要成分表現(xiàn)出強抑制活性,借助液相色譜-二極管陣列檢測器-電噴霧離子化串聯(lián)質(zhì)譜法(LC-DAD-ESI-MS/MS)通過LC保留時間、紫外光譜和高分辨質(zhì)譜與標(biāo)準(zhǔn)品對照確定強效抑制活性化合物包括新補骨脂異黃酮、補骨脂乙素、補骨脂二氫黃酮甲醚、補骨脂甲素A、補骨脂酚�；旌先烁挝⒘ｓw(HLM)與底物FD在37。C條件下孵育一定時間后,用酶標(biāo)儀檢測探針底物的代謝產(chǎn)物的生成率,計算IC50值。進一步,用Lineweaver-Burk作圖和Dixon作圖來判斷可逆抑制類型,并計算抑制動力學(xué)參數(shù)Ki。結(jié)果顯示,5個化合物對hCE2呈現(xiàn)濃度依賴性抑制； 5個化合物的兩種作圖形式交點都交于第二象限橫坐標(biāo),表明補骨脂5種主要成分對hCE2的抑制類型為非競爭性抑制;Ki值范圍0.62μM-3.89μm,證實補骨脂提取物中5個主要成分對hCE2具有較強抑制活性。5個活性成分總量占補骨脂提取總量的50%,并在小鼠遲發(fā)型腹瀉模型上進行了藥效學(xué)評價。本研究在國內(nèi)外首次證實了補骨脂及其化學(xué)單體成分作為天然產(chǎn)物具有hCE2強效抑制活性,揭示補骨脂及其單體成分可作為hCE2強效抑制劑和其在減輕CPT-11誘發(fā)嚴(yán)重延遲性腹瀉的療效有良好的應(yīng)用前景。2.在已證實黃芩素可作為β-D-葡萄糖醛酸苷酶抑制劑的基礎(chǔ)上,本研究通過試驗證明其亦是人羧酸酯酶2特異性抑制劑,說明黃芩素具有特異性抑制羧酸酯酶2和p-D-葡萄糖醛酸苷酶雙靶點作用機制。黃芩素對hCE2的抑制呈現(xiàn)濃度依賴性抑制,Ki值為6.2μM：用Lineweaver-Burk作圖和Dixon作圖來判斷其抑制類型為非競爭性抑制。動物試驗、腸道組織切片、HE染色、AB-PAS染色和免疫組化試驗表明：黃芩素組腹瀉程度顯著下降(P0.05),并能夠降低CPT-11誘發(fā)的鼠遲發(fā)性腹瀉及腸粘膜損傷的程度,明顯減少小鼠腸道組織含有的3種促炎性因子TNF-α,IL-6和IL-1β的生成。
[Abstract]:1. Carboxylesterase (CEs) is a member of the 偽 / 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- 偽, IL-6 and IL-1 尾 in the intestinal tissues of mice was significantly reduced.
【學(xué)位授予單位】：山西農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：S853.7
[Abstract]:1. Carboxylesterase (CEs) is a member of the 偽 / 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- 偽, IL-6 and IL-1 尾 in the intestinal tissues of mice was significantly reduced.
【學(xué)位授予單位】：山西農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：S853.7
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