乳香主要活性成分AKBA和KBA的体外代谢稳定性研究

发布时间：2018-06-14 12:12

本文选题：AKBA + 抗炎　；参考：《大连医科大学》2017年硕士论文

【摘要】：目的:本论文选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)进行体外代谢稳定性研究,主要目的是研究其代谢特点,鉴定代谢产物的化学结构,明确其主要代谢途径,揭示参与代谢的单酶亚型,阐明动力学特点和相关参数(Vm,Km,CLint),并进一步考察主要代谢通路的种属间差异,并评价AKBA和KBA主要代谢产物的生物活性并明确其作用机制,揭示主要代谢通路对其生物活性的影响。方法:(1)利用生物合成法制备代谢产物,并结合天然药物化学的分离、纯化方法和波谱学手段(LC-MS、NMR)鉴定代谢产物的化学结构,确定其主要代谢位点;(2)运用体外代谢显型技术,使用人原代肝细胞、人肝微粒体、人肠微粒体、各类型酯酶、重组单酶及常用实验动物肝微粒体对AKBA和KBA的代谢通路和种属间差异进行系统筛选、对代谢酶归属、动力学模型、表观动力学参数和单酶贡献率进行确定;(3)选用LPS刺激RAW264.7细胞建立炎症模型,利用Griess法,MTT法,western blotting以及激光共聚焦免疫法来考察AKBA和KBA及其主要代谢产物的抗炎活性和细胞毒性并研究其抗炎机制。结果:本课题通过系统地研究AKBA和KBA的代谢稳定性,确定了AKBA的主要代谢通路为酯酶水解为KBA,KBA的主要代谢通路为I相CYP代谢;首先生物合成了KBA的四个主要代谢产物,并通过NMR鉴定了其结构,确定四种代谢产物分别为KBA的21位,16位,30位,20位单羟基化产物,其中有三个化合物为首次报道的新化合物;其次由动力学实验得到不同生物样本中AKBA和KBA代谢的动力学参数(Km,Vmax、CLint、Ki);接着AKBA在人原代肝细胞的代谢实验中发现有大量KBA(46.7%)和KBA单羟基化产物(50.8%)生成,而仅残余极少量AKBA(2.5%),这与其代谢稳定性和动力学实验中AKBA的快速水解和KBA单羟基化反应结果一致,最后确定了羧酸酯酶介导的AKBA水解反应和CYP450介导的KBA单羟基化反应是AKBA和KBA主要的代谢通路;通过单酶归属、化学抑制实验、单酶贡献率研究结果可知,羧酸酯酶2(CE2)选择性催化AKBA脱乙酰化生成KBA;单酶CYP3A4、CYP3A5及CYP3A7均能催化KBA氧化反应,其中CYP3A4起主要的催化作用。值得注意的是,AKBA的脱乙酰反应和KBA的羟基化反应均表现出很大的种属差异性,AKBA的水解反应只在人肝微粒体和人原代肝细胞中发生,在多种种属肝微粒体中未检测到水解产物;而KBA的21位和20位单羟基化产物主要在人肝微粒体、猴肝微粒体中检测到,16位和30位单羟基化产物主要在其他种属中检测到。另外,四个KBA的单羟基化产物均有一定的抗炎活性,且与原型药物相比细胞毒性有所降低,通过深入研究抗炎机制,发现KBA在人中的主要代谢产物21位和20位单羟基化产物是通过下调LPS刺激的RAW264.7细胞中i NOS以及phospho-Ik Bα和phospho-p65的蛋白水平,并显著抑制NF-κB p65由细胞质向细胞核的转移,从而发挥其抗炎作用的。结论:本论文首次对AKBA和KBA在不同生物样本中的代谢稳定性进行了系统地研究,阐明了其主要代谢通路、相关的动力学模型和表观动力学参数等,为中药乳香及其主要活性成分AKBA和KBA的临床用药提供参考;首次发现经CYP450代谢后的KBA主要代谢产物具有相对母体化合物较低的细胞毒性和较好的抗炎活性,这为乳香中主要活性成分AKBA和KBA开发低毒的小分子非甾体抗炎药,提供一定的参考;此外,AKBA和KBA的代谢存在较大的种属差异性,脱乙酰化反应是AKBA在人体中的特殊反应,并未在其他种属中观察到此现象;并且发现人和其他种属间存在KBA的代谢位点的差异,这些研究结果为AKBA和KBA的临床前研究中,模型动物的选择提供重要的指导。
[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.
【学位授予单位】：大连医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R285.5

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