本文選題：何首烏水提物 + 成分分析��； 參考：《北京中醫(yī)藥大學(xué)》2017年碩士論文

【摘要】：目的:對何首烏水提物正丁醇部位化學(xué)成分進(jìn)行分離鑒定;研究何首烏水提物及其萃取部位對肝細(xì)胞的毒性作用;研究大鼠肝微粒體代謝對何首烏水提物肝細(xì)胞毒性作用的影響;對何首烏水提物主要成分大黃素-8-O-β-D-葡萄糖苷(emodin-8-O-D-β-glucopyranoside,EG)進(jìn)行大鼠肝微粒體代謝初步研究,考察其在大鼠肝微粒體中代謝最佳孵育條件及其可能的代謝產(chǎn)物,探究其對大鼠肝微粒體五種細(xì)胞色素CYP450酶亞型的作用。意義:明確何首烏水提物中化學(xué)成分,為何首烏肝毒性研究奠定物質(zhì)基礎(chǔ);進(jìn)行何首烏水提物及其萃取部分成分分析與肝細(xì)胞毒性檢測,分析成分含量變化與其肝細(xì)胞毒性相關(guān)性;考察大鼠肝微粒體代謝對何首烏水提物肝細(xì)胞毒性的影響,以及成分與毒性之間的關(guān)系;大黃素-8-O-β-D-葡萄糖苷是何首烏水提物中主要的結(jié)合型蒽醌,關(guān)于其代謝研究仍比較缺乏,且相較體內(nèi)代謝研究而言,肝微粒體體外代謝簡單易操作,故對大黃素-8-O-β-D-葡萄糖苷進(jìn)行大鼠肝微粒體代謝研究,初步考察大黃素-8-O-β-D-葡萄糖苷在大鼠肝微粒體中的代謝行為,探究大黃素-8-O-β-D-葡萄糖苷對大鼠肝微粒體中多種CYP450酶活性的影響,旨在提示用藥過程中可能存在的藥物間相互作用,以防范藥物不良反應(yīng)。方法:采用傳統(tǒng)分離方法,對何首烏進(jìn)行提取,萃取,采用硅膠,聚酰胺,Sephadex LH-20等柱層析分離技術(shù)對何首烏正丁醇部位進(jìn)行系統(tǒng)分離,利用不同光譜分析方法鑒定化合物。建立HPLC分析方法,對何首烏水提物、乙酸乙酯萃取物、正丁醇萃取物的主要成分進(jìn)行含量測定,并采用MTT方法檢測各提取部位對肝源細(xì)胞(L02、HepG2)毒性,分析成分與肝細(xì)胞毒性的關(guān)聯(lián)性;利用大鼠肝微粒體對何首烏水提物進(jìn)行體外代謝;將何首烏水提物代謝各組溶液冷凍干燥,以不同濃度作用肝源L02及HepG2細(xì)胞,MTT法檢測其對兩種細(xì)胞的細(xì)胞活力的影響;HPLC測定12 mg生藥/ml溶液中三種主要成分(二苯乙烯苷、大黃素-8-O-β-D-葡萄糖苷、大黃素)的含量變化,成分與毒性之間的關(guān)系。利用HPLC檢測大黃素-8-O-β-D-葡萄糖苷經(jīng)大鼠肝微粒體的代謝消除率,確定其在大鼠肝微粒中最佳孵育條件;運(yùn)用cock-tail底物探針法檢測大鼠肝微粒體CYP450酶亞型特異性底物代謝消除率,探究大黃素-8-O-β-D-葡萄糖苷對大鼠肝微粒體CYP450酶亞型的影響。結(jié)果:從何首烏水提物正丁醇部位分離出10個化合物,鑒定出其中9個,分別為大黃素甲醚-8-O-β-D-葡萄糖苷、大黃素甲醚、大黃素、大黃素-8-O-β-D-葡萄糖苷、二苯乙烯苷、山奈酚、大黃素-8-甲醚、5-羧甲基-7-羥基-2-甲基色原酮、2,5-二甲基-7-羥基色原酮。MTT細(xì)胞實(shí)驗(yàn)檢測發(fā)現(xiàn),何首烏各提取物對兩種肝源細(xì)胞活力的抑制作用呈現(xiàn)一定劑量依賴性,毒性大小:水提物乙酸乙酯萃取物正丁醇萃取物。其中何首烏水提物對L02細(xì)胞IC50為4.187 mg/mL,對HepG2細(xì)胞IC50為3.786 mg/mL,何首烏水提物對HepG2細(xì)胞比L02細(xì)胞毒性更強(qiáng);濃度為12 mg生藥/mL的何首烏水提物未代謝組毒性大于何首烏水提物代謝組(P0.01);含量測定結(jié)果顯示,何首烏水提物代謝組與未代謝組相比,三種主要成分含量明顯降低。對大黃素-8-O-β-D-葡萄糖苷大鼠肝微粒體代謝進(jìn)行研究,確定大黃素-8-O-β-D-葡萄糖苷大鼠肝微粒體最佳孵育體系為:肝微粒體濃度1 mg/mL,大黃素-8-O-β-D-葡萄糖苷濃度0.05 mM,孵育時間60 min;發(fā)現(xiàn)一個大黃素-8-O-β-D-葡萄糖苷在大鼠肝微粒體中代謝相關(guān)峰;大黃素-8-O-β-D-葡萄糖苷對大鼠肝微粒體CYP1A2抑制作用IC50值為130μM,對大鼠肝微粒體CYP2C9抑制作用IC50值為0.887 μM。大黃素-8-O-β-D-葡萄糖苷對大鼠肝微粒體CYP2D1(相當(dāng)于人的CYP2D6)促進(jìn)作用EC50值為1.504 μM。大黃素-8-O-β-D-葡萄糖苷對大鼠肝微粒體CYP3A1(相當(dāng)于人的CYP3A4)、CYP2E1的作用未擬合出EC50或IC50值。結(jié)論:2,5-二甲基-7-羥基色原酮為首次從何首烏藥材中分離得到;何首烏水提物經(jīng)大鼠肝微粒體代謝后肝細(xì)胞毒性降低,三種主要成分含量均有所降低;大黃素-8-O-β-D-葡萄糖苷對大鼠肝微粒體CYP450多種亞型有抑制或促進(jìn)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.
【學(xué)位授予單位】：北京中醫(yī)藥大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R285.5

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