【作者】 李丽；

【导师】 果德安； 毕开顺；

【作者基本信息】 沈阳药科大学 ， 药物分析学， 2005， 博士

【摘要】 吴茱萸(Evodia rutaecarpa Juss.Benth.)为著名中药复方吴茱萸汤中的君药，主要含生物碱类活性化合物，具有明显的温胃、止呕之功效。吴茱萸碱和吴茱萸次碱作为吴茱萸中最重要的两种生物碱，发现具有强心、抗炎、镇痛和抗肿瘤活性，因此引起了国内外的广泛关注。左旋四氢巴马亭为防己科植物华千金藤(Stephania ainica Diels)块根中提取出的一种生物碱，为延胡索(Corydalis yanhusuo W.T.Wang)中延胡索乙素的左旋体，中药制剂颅痛定中的主要成分，具有较强的镇痛作用。为了发现活性更强、副作用更小的活性化合物和鉴定其微量的体内代谢产物，人们一直在寻找有效的方法对这类化合物进行结构修饰和改造。对于药物的安全性和有效性评价中重要研究内容之一就是了解药物在体内的代谢过程。微生物转化方法已经成为天然产物结构修饰和体内代谢研究的重要手段之一，因其反应具有显著的位置选择性和立体选择性，可以催化生成自然界或化学界难以获得的，结构新颖的天然产物衍生物，同时也因其具有与人类类似的代谢酶等特点，被用来模拟药物在体内的代谢行为，并从中分离和鉴定人类或动物体内微量或痕量的代谢产物。为了探讨吴茱萸温胃、止呕作用的药效物质基础，并系统研究吴茱萸碱和吴茱萸次碱在微生物与大鼠体内的代谢，本论文对吴茱萸中不同提取部位进行了化学和药理学的初步研究，并采用LC／MS／MS与现代光谱技术对吴茱萸碱和吴茱萸次碱的微生物转化与体内代谢进行了深入系统的研究。同时，对左旋四氢巴马亭的体内外的代谢产物和代谢途径也进行了深入探讨。建立了HPLC方法测定吴茱萸中吴茱萸碱、吴茱萸次碱、吴茱萸苦素和柠檬苦素的含量。其中4个成分的平均含量分别为0.71％、0.72％、0.43％和0.57％。考察了吴茱萸中不同组分对大鼠离体胃底平滑肌条运动和对醋酸所致小鼠扭体的影响，吴茱萸的总提物(a)、吴茱萸中的总生物碱(d)和水溶性部分(e)对Ach刺激的大鼠胃条痉挛性收缩活动的影响与空白组比较具有非常显著性差异(P＜0.01)。除挥发油部分(b)外，其余提取部位对醋酸所致小鼠扭体的镇痛作用与空白组比较均具有非常显著性差异(P＜0.01)。系统研究了吴茱萸碱和吴茱萸次碱的微生物转化。通对20余株微生物进行转化筛选，结果表明Mucor，Cunninghamella和Penicillium等属的菌株对以上两种生物碱的转化作用较好。在筛选结果的基础上，选取微紫青霉Penicillium janthinellum AS 3.510和短刺小克银汉霉Cunninghamelal blakesleana AS 3.970分别对吴茱萸碱和吴茱萸次碱进行了放大生物转化试验。共分离得到8个产物，通过柱层析和半制备HPLC分离，其结构光谱鉴定为：10-羟基吴茱萸碱(J-3-1)、11-羟基吴茱萸碱(J-1)、3-羟基吴茱萸碱(J-3-2)、对羟基苯甲醇(J-2-1)、对羟基苯乙醇(J-2-2)、10-羟基吴茱次萸碱(C-1)、3-羟基吴茱萸次碱(C-2)和3，10-二羟基吴茱萸次碱(C-6)，其中除J-2-1和J-2-2外均为新化合物。转化反应的动态考察表明，Penicillium janthinellum AS 3.510和Cunninghamella blakesleana AS 3.970可以在7日内分别将底物转化成为羟基化代谢产物，但转化率比较低。采用MTT方法对吴茱萸碱和吴茱萸次碱的转化产物进行了体外细胞毒活性测试，结果表明J-1、J-3-1、J-3-2、C-2和C-6对BGC-823、Bel-7402、MCF-7和HL-60四个肿瘤细胞株均具有不同程度的细胞毒活性，其中J-1对各细胞株的活性均强于底物吴茱萸碱。考察了20余株微生物菌株对左旋四氢巴马亭的微生物转化，选用转化能力最强的微紫青霉Penicillium janthinellum AS 3.510对其进行放大转化研究。共分离得到3个转化产物，分别为左旋紫堇单酚碱(T-1，1-corydalmine)、3-去甲基左旋四氢巴马亭(T-2，1-corypalmine)和9-去甲基左旋四氢巴马亭(T-3)。转化反应的动态考察表明，Penicillium janthinellum AS 3.510能够缓慢催化O-去甲基化反应，7日后转化反应仍持续进行。采用LC／MS／MS方法，并结合微生物转化获得的单体对照品，系统研究了吴茱萸碱、吴茱萸次碱和左旋四氢巴马亭在大鼠体内的代谢产物和代谢途径。分别鉴定了2个吴茱萸碱在大鼠尿液和粪便中的代谢产物(J-3-1和J-3-2)和3个左旋四氢巴马亭在大鼠粪便中的代谢产物(T-1，T-1和Y-3)，并初步推测了3个左旋四氢巴马亭在大鼠粪便中的代谢产物的结构。并对此3个生物碱在微生物与及大鼠体内的代谢途径和代谢产物进行了对比。更多还原

【Abstract】 Evodia rutaecarpa Juss. Benth is the most important component of famous Wu-Zhu-Yu decoction of China. Previous studies showed that Evodia is effective in the treatment of stomach diseases. Evodiamine and rutaecarpine are the two major active indole alkaloids in the title plant. In recent years, they have been found to exhibit cardiotonic, anti-inflammation and antitumor activities and received more and more attention from scientists of various fields. 1-Tetrahydropalmatine （1-THP）, a naturally occurring neuroactive alkaloid from Stephania ainiaca Diels, has been widely used as analgetic medicine-Lu Tong Ding in China for many years. To find more effective compounds with low toxicity or to identify the in vivo metabolites, scientists have been searching for the approach to modify the structures by efficient methods.An important factor in the evaluation of safety and efficacy of any drug is the knowledge of how the drug is metabolized. Microbial metabolic studies have such advantages over chemical synthesis as high stereo- and regio-selectivity and are becoming a complementary tool in the study of drug metabolism in mammals for the reason that most families of human liver cytochrome P450 have been expressed in microorganisms as individual enzymes. For obtaining sizable amount of metabolites for pharmacological and toxicological studies, microbial metabolism is clearly useful.In efforts to study the chemical constituents responsible for antiemetic activity of Evodia and the metabolisms in microbial organism and rats of evodiamine and rutaecarpine, the chemical, pharmacological and metabolic studies were carried out. Metabolism of 1-THP in microbial organisms and rats were investigated at the same time.Two HPLC methods were set up to determinate the concentrations of evodiamine, rutaecarpine, rutaevine and evodin in Evodia, respectively, which get the results of 0.71%, 0.72%, 0.57% and 0.43%. Effects on stomach strips of different fractions of Evodia were evaluated. Fractions a, d and e showed obvious activity of antagoning contraction to stomach. Analgesic effect of different fractions of Evodia was also invesgated using acetic-acid induced twitching in mice and its analgesic potency was compared with ibuprofen. The fractions a, c, d and e all showed potential analgesic effect.Microbial transformations of evodiamine and rutaecarpine were carried out. Twenty strains of fungi were screened for their capabilities to transform the above two alkaloids. It was found that Mucor, Cunninghamella and Penicilliurn species could metabolize evodiamine and rutaecarpine to produce versatile products. Penicilliumjanthinellum AS 3.510 and Cunningharnella blakesleana AS 3.970 were found to be the most potent strain for evodiamine and rutaecarpine and were selected for the preparative-scale biotransformation. Eight products were isolated from the culture supernatant by silica gel column chromatography and preparative RP-HPLC. By means of extensive spectroscopic techniques, their structures were identified as 10-hydroxyevodiamine （J-3-1）, 11- hydroxyevodiamine （J-1）, 3-hydroxyevodiamine （J-3-2）, p-hydroxybenzyl alcohol （J-2-1）, p-hydroxyphenylethyl alcohol （J-2-2）, 10-hydroxyrutaecarpine （C-1）, 3-hydroxyrutaecarpine （C-2） and 3,10-dihydroxyrutaecarpine （C-6）, respectively, among which products J-3-1, J-3-2, J-1, C-1, C-2 and C-6 are new compounds. Two substrates were converted into hydroxyl metabolites in 7 days of incubation with low efficiency and the possible biotransforrnation pathways are proposed to account for the formation of the observed products. The in vitro cytotoxicities of the biotransformed products were determined by MTT method. J-1, J-3-1, J-3-2, C-2 and C-6 were found to exhibit potent inhibitory activities against human cancer cell lines （BGC-823, Bel-7402, MCF-7 and HL-60）, in which J-1 showed stronger inhibitory activities against four cell lines than evodiamine.A similar biotransformation process for 1-THP was carried out. Penicillium janthinellum AS 3.510 was selected as the strain for the preparative-scale biotransformation and 3 products were isolated. Their structures were identified as 1-corydalmine （T-1）, 1-corypalmine （T-2） and 9-demethyl-1-THP （T-3）, respectively. Time-course investigation revealed that Penicillium janthinellum AS 3.510 could slowly demethylate 1-THP at different -OCH₃ positions.LC/MS/MS analysis was used in the metabolism studies of evodiamine, rutaecarpine and 1-THP in rats. The structures of two metabolites （J-3-1 and J-3-2） of evodiamine and three metabolites of 1-THP （T-1, T-1 and T-3） in rat urine and feces were identified with the reference standards separated from biotransformation. Structures of three metabolites of 1-THP in rat feces were tentative deduced by MS and MS/MS analysis. The metabolisms of ebodiamine, rutaecarpine and 1-THP in microbial organisms and rats were compared.更多还原