本文選題：AKBA + 抗炎��； 參考：《大連醫(yī)科大學(xué)》2017年碩士論文

【摘要】：目的:本論文選用乳香中的主要活性成分乙酰-11-羰基-β-乳香酸(AKBA)和11-羰基-β-乳香酸(KBA)進(jìn)行體外代謝穩(wěn)定性研究,主要目的是研究其代謝特點(diǎn),鑒定代謝產(chǎn)物的化學(xué)結(jié)構(gòu),明確其主要代謝途徑,揭示參與代謝的單酶亞型,闡明動(dòng)力學(xué)特點(diǎn)和相關(guān)參數(shù)(Vm,Km,CLint),并進(jìn)一步考察主要代謝通路的種屬間差異,并評(píng)價(jià)AKBA和KBA主要代謝產(chǎn)物的生物活性并明確其作用機(jī)制,揭示主要代謝通路對(duì)其生物活性的影響。方法:(1)利用生物合成法制備代謝產(chǎn)物,并結(jié)合天然藥物化學(xué)的分離、純化方法和波譜學(xué)手段(LC-MS、NMR)鑒定代謝產(chǎn)物的化學(xué)結(jié)構(gòu),確定其主要代謝位點(diǎn);(2)運(yùn)用體外代謝顯型技術(shù),使用人原代肝細(xì)胞、人肝微粒體、人腸微粒體、各類(lèi)型酯酶、重組單酶及常用實(shí)驗(yàn)動(dòng)物肝微粒體對(duì)AKBA和KBA的代謝通路和種屬間差異進(jìn)行系統(tǒng)篩選、對(duì)代謝酶歸屬、動(dòng)力學(xué)模型、表觀動(dòng)力學(xué)參數(shù)和單酶貢獻(xiàn)率進(jìn)行確定;(3)選用LPS刺激RAW264.7細(xì)胞建立炎癥模型,利用Griess法,MTT法,western blotting以及激光共聚焦免疫法來(lái)考察AKBA和KBA及其主要代謝產(chǎn)物的抗炎活性和細(xì)胞毒性并研究其抗炎機(jī)制。結(jié)果:本課題通過(guò)系統(tǒng)地研究AKBA和KBA的代謝穩(wěn)定性,確定了AKBA的主要代謝通路為酯酶水解為KBA,KBA的主要代謝通路為I相CYP代謝;首先生物合成了KBA的四個(gè)主要代謝產(chǎn)物,并通過(guò)NMR鑒定了其結(jié)構(gòu),確定四種代謝產(chǎn)物分別為KBA的21位,16位,30位,20位單羥基化產(chǎn)物,其中有三個(gè)化合物為首次報(bào)道的新化合物;其次由動(dòng)力學(xué)實(shí)驗(yàn)得到不同生物樣本中AKBA和KBA代謝的動(dòng)力學(xué)參數(shù)(Km,Vmax、CLint、Ki);接著AKBA在人原代肝細(xì)胞的代謝實(shí)驗(yàn)中發(fā)現(xiàn)有大量KBA(46.7%)和KBA單羥基化產(chǎn)物(50.8%)生成,而僅殘余極少量AKBA(2.5%),這與其代謝穩(wěn)定性和動(dòng)力學(xué)實(shí)驗(yàn)中AKBA的快速水解和KBA單羥基化反應(yīng)結(jié)果一致,最后確定了羧酸酯酶介導(dǎo)的AKBA水解反應(yīng)和CYP450介導(dǎo)的KBA單羥基化反應(yīng)是AKBA和KBA主要的代謝通路;通過(guò)單酶歸屬、化學(xué)抑制實(shí)驗(yàn)、單酶貢獻(xiàn)率研究結(jié)果可知,羧酸酯酶2(CE2)選擇性催化AKBA脫乙酰化生成KBA;單酶CYP3A4、CYP3A5及CYP3A7均能催化KBA氧化反應(yīng),其中CYP3A4起主要的催化作用。值得注意的是,AKBA的脫乙酰反應(yīng)和KBA的羥基化反應(yīng)均表現(xiàn)出很大的種屬差異性,AKBA的水解反應(yīng)只在人肝微粒體和人原代肝細(xì)胞中發(fā)生,在多種種屬肝微粒體中未檢測(cè)到水解產(chǎn)物;而KBA的21位和20位單羥基化產(chǎn)物主要在人肝微粒體、猴肝微粒體中檢測(cè)到,16位和30位單羥基化產(chǎn)物主要在其他種屬中檢測(cè)到。另外,四個(gè)KBA的單羥基化產(chǎn)物均有一定的抗炎活性,且與原型藥物相比細(xì)胞毒性有所降低,通過(guò)深入研究抗炎機(jī)制,發(fā)現(xiàn)KBA在人中的主要代謝產(chǎn)物21位和20位單羥基化產(chǎn)物是通過(guò)下調(diào)LPS刺激的RAW264.7細(xì)胞中i NOS以及phospho-Ik Bα和phospho-p65的蛋白水平,并顯著抑制NF-κB p65由細(xì)胞質(zhì)向細(xì)胞核的轉(zhuǎn)移,從而發(fā)揮其抗炎作用的。結(jié)論:本論文首次對(duì)AKBA和KBA在不同生物樣本中的代謝穩(wěn)定性進(jìn)行了系統(tǒng)地研究,闡明了其主要代謝通路、相關(guān)的動(dòng)力學(xué)模型和表觀動(dòng)力學(xué)參數(shù)等,為中藥乳香及其主要活性成分AKBA和KBA的臨床用藥提供參考;首次發(fā)現(xiàn)經(jīng)CYP450代謝后的KBA主要代謝產(chǎn)物具有相對(duì)母體化合物較低的細(xì)胞毒性和較好的抗炎活性,這為乳香中主要活性成分AKBA和KBA開(kāi)發(fā)低毒的小分子非甾體抗炎藥,提供一定的參考;此外,AKBA和KBA的代謝存在較大的種屬差異性,脫乙�；磻�(yīng)是AKBA在人體中的特殊反應(yīng),并未在其他種屬中觀察到此現(xiàn)象;并且發(fā)現(xiàn)人和其他種屬間存在KBA的代謝位點(diǎn)的差異,這些研究結(jié)果為AKBA和KBA的臨床前研究中,模型動(dòng)物的選擇提供重要的指導(dǎo)。
[Abstract]:Objective: the main purpose of this thesis is to study the metabolic stability of acetyl -11- - beta - frankincense acid (AKBA) and 11- carbonyl - beta - frankincacic acid (KBA), the main active ingredient in the frankincense. The main purpose is to study its metabolic characteristics, identify the chemical structure of the metabolites, clarify its main metabolic pathways, reveal the single enzyme subtypes involved in metabolism and clarify the kinetics. Characteristics and related parameters (Vm, Km, CLint), and further examine the differences between species and genera of major metabolic pathways, and evaluate the biological activities of the main metabolites of AKBA and KBA, and clarify their mechanisms of action, and reveal the effects of the main metabolic pathways on their biological activities. Methods: (1) use biosynthesis method to prepare metabolites and combine natural drug chemistry. The chemical structure of metabolites was identified by separation, purification and spectroscopy (LC-MS, NMR), and the main metabolic sites were identified. (2) the metabolic pathways and species of AKBA and KBA were used in vitro metabolic developing techniques, using human primary hepatocytes, human liver microsomes, human intestinal microsomes, various types of esterases, recombinant single enzymes and common experimental animal liver microsomes. The difference between the metabolic enzymes belonging, the kinetic model, the apparent kinetic parameters and the single enzyme contribution rate were determined. (3) LPS was used to stimulate RAW264.7 cells to establish an inflammatory model, and the anti-inflammatory activity and fine of AKBA and KBA and its main metabolites were examined by Griess, MTT, Western blotting and laser confocal immunoassay. Cytotoxicity and its anti-inflammatory mechanism. Results: by systematically studying the metabolic stability of AKBA and KBA, the main metabolic pathway of AKBA and KBA was determined by esterase hydrolysis to KBA, and the main metabolic pathway of KBA was I phase CYP metabolism. First, four major metabolites of KBA were biosynthetic, and its structure was identified by NMR, and four kinds of metabolism were identified. The products were 21, 16, 30, and 20 mono hydroxylated products of KBA, of which three compounds were new compounds reported for the first time. Secondly, kinetic parameters (Km, Vmax, CLint, Ki) were obtained by kinetic experiments (Km, Vmax, CLint, Ki) in different biological samples. Then AKBA found a large number of KBA (46.7%) in the metabolic experiments of human primary hepatocytes. The mono hydroxylation product (50.8%) of KBA was generated, but only a small amount of AKBA (2.5%) remained, which was consistent with the rapid hydrolysis of AKBA and the result of KBA mono hydroxylation in the metabolic stability and kinetics experiments. Finally, the main metabolic pathways of AKBA and KBA mediated by the AKBA hydrolysis mediated by carboxylesterase and the CYP450 mediated KBA mono hydroxylation reaction were the main pathway of the metabolic pathway of AKBA and KBA. The results of single enzyme belonging, chemical inhibition experiment, single enzyme contribution rate studies show that carboxyleesterase 2 (CE2) selectively catalyzes the deacetylation of AKBA to produce KBA; single enzyme CYP3A4, CYP3A5 and CYP3A7 can catalyze the reaction of KBA oxidation, and CYP3A4 plays a major catalytic role. It is worth noting that both AKBA deacetylation and KBA hydroxylation are all shown. Many species are different. The hydrolysis of AKBA occurs only in human liver microsomes and human primary hepatocytes. The hydrolysates are not detected in the liver microsomes of various species, while the 21 and 20 mono hydroxylated products of KBA are detected mainly in human liver microsomes and in monkey liver microsomes. The 16 and 30 mono hydroxylated products are mainly in other species. In addition, the four KBA mono hydroxylated products had some anti-inflammatory activity, and compared with the prototype drug, the cytotoxicity decreased. Through the in-depth study of the anti-inflammatory mechanism, it was found that the 21 and 20 mono hydroxylated products of the main metabolites of KBA in human were I NOS, phospho-Ik B A and pH through the down regulation of LPS prickled RAW264.7 cells. Ospho-p65 protein level, and significantly inhibit the transfer of NF- kappa B p65 from the cytoplasm to the nucleus, thus exerts its anti-inflammatory effect. Conclusion: the metabolic stability of AKBA and KBA in different biological samples was systematically studied in this paper for the first time, and the main metabolic pathways, related kinetic models and apparent kinetic parameters were elucidated. It provides a reference for the clinical use of Chinese herbal medicine and its main active components AKBA and KBA; it is discovered for the first time that the main metabolites of KBA after CYP450 metabolism have lower cytotoxicity and better anti-inflammatory activity relative to the parent compound, which is a small molecular nonsteroidal anti-inflammatory drug, which is the main active ingredient of the frankincense, AKBA and KBA, to develop low toxic molecules. A certain reference; in addition, there is a large difference in the metabolism of AKBA and KBA. Deacetylation is a special reaction in the human body and is not observed in other genera; and the differences in the metabolic sites of KBA are found between human and other genera. These results are the models of the preclinical study of AKBA and KBA. The choice of things provides important guidance.
【學(xué)位授予單位】：大連醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：R285.5

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1 崔永蕾;乳香主要活性成分AKBA和KBA的體外代謝穩(wěn)定性研究[D];大連醫(yī)科大學(xué);2017年

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本文編號(hào)：2017336