何首乌水提物及EG大鼠肝微粒体代谢成分分析及肝细胞毒研究

发布时间：2018-05-29 03:52

本文选题：何首乌水提物 + 成分分析　；参考：《北京中医药大学》2017年硕士论文

【摘要】：目的:对何首乌水提物正丁醇部位化学成分进行分离鉴定;研究何首乌水提物及其萃取部位对肝细胞的毒性作用;研究大鼠肝微粒体代谢对何首乌水提物肝细胞毒性作用的影响;对何首乌水提物主要成分大黄素-8-O-β-D-葡萄糖苷(emodin-8-O-D-β-glucopyranoside,EG)进行大鼠肝微粒体代谢初步研究,考察其在大鼠肝微粒体中代谢最佳孵育条件及其可能的代谢产物,探究其对大鼠肝微粒体五种细胞色素CYP450酶亚型的作用。意义:明确何首乌水提物中化学成分,为何首乌肝毒性研究奠定物质基础;进行何首乌水提物及其萃取部分成分分析与肝细胞毒性检测,分析成分含量变化与其肝细胞毒性相关性;考察大鼠肝微粒体代谢对何首乌水提物肝细胞毒性的影响,以及成分与毒性之间的关系;大黄素-8-O-β-D-葡萄糖苷是何首乌水提物中主要的结合型蒽醌,关于其代谢研究仍比较缺乏,且相较体内代谢研究而言,肝微粒体体外代谢简单易操作,故对大黄素-8-O-β-D-葡萄糖苷进行大鼠肝微粒体代谢研究,初步考察大黄素-8-O-β-D-葡萄糖苷在大鼠肝微粒体中的代谢行为,探究大黄素-8-O-β-D-葡萄糖苷对大鼠肝微粒体中多种CYP450酶活性的影响,旨在提示用药过程中可能存在的药物间相互作用,以防范药物不良反应。方法:采用传统分离方法,对何首乌进行提取,萃取,采用硅胶,聚酰胺,Sephadex LH-20等柱层析分离技术对何首乌正丁醇部位进行系统分离,利用不同光谱分析方法鉴定化合物。建立HPLC分析方法,对何首乌水提物、乙酸乙酯萃取物、正丁醇萃取物的主要成分进行含量测定,并采用MTT方法检测各提取部位对肝源细胞(L02、HepG2)毒性,分析成分与肝细胞毒性的关联性;利用大鼠肝微粒体对何首乌水提物进行体外代谢;将何首乌水提物代谢各组溶液冷冻干燥,以不同浓度作用肝源L02及HepG2细胞,MTT法检测其对两种细胞的细胞活力的影响;HPLC测定12 mg生药/ml溶液中三种主要成分(二苯乙烯苷、大黄素-8-O-β-D-葡萄糖苷、大黄素)的含量变化,成分与毒性之间的关系。利用HPLC检测大黄素-8-O-β-D-葡萄糖苷经大鼠肝微粒体的代谢消除率,确定其在大鼠肝微粒中最佳孵育条件;运用cock-tail底物探针法检测大鼠肝微粒体CYP450酶亚型特异性底物代谢消除率,探究大黄素-8-O-β-D-葡萄糖苷对大鼠肝微粒体CYP450酶亚型的影响。结果:从何首乌水提物正丁醇部位分离出10个化合物,鉴定出其中9个,分别为大黄素甲醚-8-O-β-D-葡萄糖苷、大黄素甲醚、大黄素、大黄素-8-O-β-D-葡萄糖苷、二苯乙烯苷、山奈酚、大黄素-8-甲醚、5-羧甲基-7-羟基-2-甲基色原酮、2,5-二甲基-7-羟基色原酮。MTT细胞实验检测发现,何首乌各提取物对两种肝源细胞活力的抑制作用呈现一定剂量依赖性,毒性大小:水提物乙酸乙酯萃取物正丁醇萃取物。其中何首乌水提物对L02细胞IC50为4.187 mg/mL,对HepG2细胞IC50为3.786 mg/mL,何首乌水提物对HepG2细胞比L02细胞毒性更强;浓度为12 mg生药/mL的何首乌水提物未代谢组毒性大于何首乌水提物代谢组(P0.01);含量测定结果显示,何首乌水提物代谢组与未代谢组相比,三种主要成分含量明显降低。对大黄素-8-O-β-D-葡萄糖苷大鼠肝微粒体代谢进行研究,确定大黄素-8-O-β-D-葡萄糖苷大鼠肝微粒体最佳孵育体系为:肝微粒体浓度1 mg/mL,大黄素-8-O-β-D-葡萄糖苷浓度0.05 mM,孵育时间60 min;发现一个大黄素-8-O-β-D-葡萄糖苷在大鼠肝微粒体中代谢相关峰;大黄素-8-O-β-D-葡萄糖苷对大鼠肝微粒体CYP1A2抑制作用IC50值为130μM,对大鼠肝微粒体CYP2C9抑制作用IC50值为0.887 μM。大黄素-8-O-β-D-葡萄糖苷对大鼠肝微粒体CYP2D1(相当于人的CYP2D6)促进作用EC50值为1.504 μM。大黄素-8-O-β-D-葡萄糖苷对大鼠肝微粒体CYP3A1(相当于人的CYP3A4)、CYP2E1的作用未拟合出EC50或IC50值。结论:2,5-二甲基-7-羟基色原酮为首次从何首乌药材中分离得到;何首乌水提物经大鼠肝微粒体代谢后肝细胞毒性降低,三种主要成分含量均有所降低;大黄素-8-O-β-D-葡萄糖苷对大鼠肝微粒体CYP450多种亚型有抑制或促进作用。
[Abstract]:Objective: to isolate and identify the chemical components of n-butanol from the aqueous extract of Polygonum multiflorum; study the toxic effect of the extract and the extraction site of Polygonum multiflorum on liver cells; study the effect of liver microsomal metabolism on the toxicity of the liver cell of Polygonum multiflorum; the main component of the aqueous extract of Polygonum multiflorum -8-O- beta -D- glucoside (emodin-8 -O-D- beta -glucopyranoside, EG) in rat liver microsomal metabolism preliminary study, investigate its metabolic best incubating conditions and possible metabolites in rat liver microsomes, explore its effect on five cytochrome CYP450 subtypes of rat liver microsomes. Significance: the chemical constituents of the aqueous extract of Polygonum multiflorum, why the virulence of Polygonum multiflorum To establish the material basis, the analysis of the extracts of Polygonum multiflorum and its extraction components and the detection of hepatocyte toxicity, and the correlation between the changes of the content of the components and the toxicity of hepatocytes; the relationship between the hepatocyte toxicity of the liver microsomal metabolism of the rat liver and the relationship between the toxicity and the composition and toxicity; the -8-O- beta -D- glucose of emodin Glucoside is the main binding type anthraquinone in Polygonum multiflorum water extract, and its metabolism research is still relatively lack, and compared with metabolic research in vivo, liver microsomes are easy to operate in vitro metabolism. Therefore, the liver microsomal metabolism of emodin -8-O- beta -D- glucoside is studied in rat liver microsome, and a preliminary investigation of emodin -8-O- beta -D- glucoside in rat liver microparticles The effect of emodin -8-O- beta -D- glucoside on the activity of a variety of CYP450 enzymes in rat liver microsomes was investigated in order to indicate the possible interaction between drugs in the course of drug use in order to prevent adverse drug reactions. Methods: traditional separation method was used to extract and extract Polygonum multiflorum, silica gel, polyamide, Sephad Ex LH-20 column chromatography separation technique was used to systematically separate the n-butanol part of Polygonum multiflorum and identify the compounds by different spectral analysis methods. The HPLC analysis method was established to determine the content of the main components of Polygonum multiflorum water extract, ethyl acetate extract and n-butanol extract, and the MTT method was used to detect the liver source cells (L 02, HepG2) toxicity, analysis of the association between components and hepatotoxicity; using rat liver microsomes to metabolize the extract of Polygonum multiflorum in vitro; freeze drying of the aqueous extracts of Polygonum multiflorum water extract, the effect of different concentrations on the L02 and HepG2 cells of liver source, and the effect of MTT on the cell viability of two cells; HPLC determination of /ml in 12 mg raw drug /ml The changes in the content of three main components (two styrene glycosides, emodin -8-O- beta -D- glucoside, emodin) and the relationship between their components and toxicity. The optimal incubation conditions of -8-O- beta glucoside of emodin by rat liver microsomes were determined by HPLC, and the cock-tail substrate probe method was used to determine the optimal incubation conditions in rat liver microsome. The specific substrate metabolism elimination rate of rat liver microsomal CYP450 enzyme subtype was detected, and the effect of emodin -8-O- beta -D- glucoside on rat liver microsomal CYP450 subtype was investigated. Results: 10 compounds were isolated from the n-butanol part of Polygonum multiflorum water extract, and 9 were identified as emodin methyl ether -8-O- beta -D- glucoside and Rhein a Ethers, emodin, emodin -8-O- beta -D- glucoside, two styrene glucoside, anononol, emodin -8- methyl ether, 5- carboxymethyl -7- hydroxyl -2- methyl chromogenone, 2,5- two methyl -7- hydroxymethylacetone,.MTT cell test found that the extracts of Polygonum multiflorum showed a certain dose dependence on the activity of two kinds of hepatocyte, toxic size: water The extract of ethyl acetate extract was n-butanol extract. The IC50 of L02 cell was 4.187 mg/mL, IC50 was 3.786 mg/mL to HepG2 cells, and the water extract of Polygonum multiflorum was more toxic to HepG2 cells than L02 cells; the toxicity of unmetabolized Polygonum multiflorum water extract with 12 mg /mL was greater than that of Polygonum multiflorum (P0.01); Compared with the unmetabolized group, the content of the three main components of the water extract of Polygonum multiflorum was significantly lower than that of the unmetabolized group. The liver microsomal metabolism of the emodin -8-O- beta -D- glucoside rat was studied, and the best incubating body of the liver microsomes of the emodin -8-O- beta -D- glucoside was identified as the liver microsomal concentration of 1 mg/mL and the emodin -8-O- beta - D- glucoside concentration was 0.05 mM and incubation time was 60 min; the metabolic peak of a emodin -8-O- beta -D- glucoside in rat liver microsomes was found. The IC50 value of the -8-O- beta glucoside of emodin to rat liver microsomal CYP1A2 was 130 mu M. The IC50 value for CYP2C9 production of liver microsomes in rat liver microsomes was 0.887 Mu The effect of glucoside on rat liver microsomal CYP2D1 (equivalent to human CYP2D6) EC50 value is 1.504 mu M. emodin -8-O- beta -D- glucoside to rat liver microsomal CYP3A1 (equivalent to human CYP3A4), and CYP2E1's effect does not fit EC50 or IC50. Conclusion: 2,5- two methyl hydroxychromosterone is isolated from Radix Polygoni multiflorum for the first time. The hepatocyte toxicity of the aqueous extract of Radix Polygonum multiflorum was reduced after liver microsomal metabolism in rats, and the content of the three main components decreased. The emodin -8-O- beta -D- glucoside could inhibit or promote a variety of CYP450 subtypes of rat liver microsomes.
【学位授予单位】：北京中医药大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R285.5

【参考文献】