【作者】 梁高林；

【导师】 胡昌奇；

【作者基本信息】 复旦大学 ， 药物化学， 2005， 博士

【摘要】 锦鸡儿（Caragana sinica）系豆科锦鸡儿属植物,在我国大多数省区均有分布。其根或根皮（中药名:金雀根）民间主要用于治疗虚损劳热、咳嗽、高血压、关节痛风、跌打损伤等。我们研究发现金雀根乙醇浸膏的乙酸乙酯部位主要化学成分是二苯乙烯类化合物,具有多样的生物活性。其中,kobophenol A（1）是主要组分之一,约占乙酸乙酯部位的20%。1是二苯乙烯类四聚体。应用改良的E-screen实验筛选发现1是一个具有很强雌激素活性的植物雌激素。MTT法表明1体外刺激成骨细胞增殖率高达25.6%,可望研发成治疗骨质疏松症的新药。基于此,我们对1进行了动物体内外代谢产物及其机理研究,以便搞清其在动物的体内行为。为新药临床前研究提供扎实的理论基础。 从口服1的大鼠粪便中分离纯化出四个1的主要代谢产物:koboquinone A-C（2,3,4）和carasinol B（5）。其中2,3,4是新化合物,是1的Ⅰ相氧化代谢物;5是1的Ⅰ相异构化代谢物。在分离代谢产物的过程中,我们从大鼠的尿中分离得到2个大鼠内源性Ⅱ相代谢物indoxyl sulfate（IS,6）和indole-3-acetylglycine（IAA-Gly,7）。其中,化合物6是尿毒素;7是生长素的甘氨酸结合物,首次被我们报道从大鼠尿中分离、纯化得到。此外,我们还从大鼠的粪便中分离得到大豆素（daidzin）的最终代谢产物（-）-（S）-equol（8）并首次报道了该化合物的单晶X-衍射。 为研究1在动物的体内行为并验证所得代谢产物的合理性,我们进行了1在动物体内的药代动力学等研究。考察的甲醇+乙酸乙酯法提取的不同组织样品中的1在1、5、25 μg/ml三种浓度下绝对加样回收率在78.7-98.6%之间,可满足血浆药物浓度-时间曲线实验和组织分布实验;1在5、10、25μg/ml三种浓度下的血浆蛋白结合率分别为66.0、75.4和85.9%;大鼠分别以4、10、20 mg/kg静脉给药后,药物浓度-时间曲线用3P87统计软件处理,符合二室模型（P<0.05）。AUC_0-∞呈现良好线性关系（R²=1）;表明药物在大鼠体内的动力学不依赖于剂量;t_1/2分别为0.64、0.58、0.83 h;表明药物进入大鼠体内能很快被吸收和分布。大鼠以250 mg/kg灌胃给药后,符合二室模型。C_max为1.84 mg/L,T_max为0.57 h,AUC_0-∞为22.41 h·mg·L^-1;计算的绝对生物利用度为2.0%;t_1/2为5.78 h,药物浓度-时间曲线出现双峰,提示可能形成肝肠循环。静脉和灌胃给药后原形药物大部分经粪排泄。组织分布实验表明,药物主要分布在肝脏。以上药代动力学数据表明:以口服形式进入大鼠体内的化合物1应该主要被消化道菌丛和肝脏所代谢,并且以前者为主。 由于肝脏是药物的主要代谢器官,为验证代谢路径,我们首先进行了化合物更多还原

【Abstract】 Caragana sinica (Buc’hoz) Rehd. (Fabaceae) is widely distributed in China. Its dried roots (Chinese name: Jinquegen) have been used in China as a folk medicine for the treatment of asthenia syndrome, vascular hypertension, leukorrhagia, bruises and contused wounds. In our previous study, we found that the EtOAc extract of the roots contained many oligostilbenes which had multi-faceted bioactivities. Kobophenol A (1) is a stilbene tetramer, accounts for nearly 20% of the EtOAc extract. Using improved E-screen assay, we found that 1 is a phytoestrogen and has strong estrogenic activity. MTT assay showed that 1 is a phytoestrogen and can strongly stimulate the proliferation of osteoblasts cultured in vitro up to 25.6%. It can be likely developed as a new drug to cure and prevent osteoporosis. Based on this, we conducted studies on its metabolites in vivo and thereafter their mechanism so that uncovered its behavior in vivo. These studies might supply the preclinical research of 1 with sound theoretic foundation.Four main metabolites were isolated and purified from rats’ feces orally administered with 1, koboquinone A-C (2, 3, 4) and carasinol B (5). 2. 3 and 4 are new compounds, phase I oxidative metabolites of 1. 5 is the isomeric metabolite of 1. During the isolation of metabolites, we also purified two phase II endogenous metabolites, indoxyl sulfate (IS, 6) and indole-3-acetylglycine (IAA-Gly, 7). from rats’ urine. Compound 6 is a harmful uremic toxin while 7 is the phase II metabolite of indole-3-acetic acid (IAA. Auxin). Compound 7 has not been heard to be isolated from rats’ urine, to our knowledge. Additionally, we isolated and purified the terminal metabolite of phytoestrogen daidzin, (-)-(S)-equol (8), from rats’ feces and reported its X-ray single crystal analysis for the first time.To investigate the behavior of 1 and validate the rationality of the metabolites of 1 in animals, we conducted pharmacokinetic experiments of 1 in animals. The absolute rate of adding sample recovery of three concentrations (1, 5. 25 μg/ml) of 1 were between 78.7-98.6% which can meet needs of the pharmacokinetic and tissue distribution experiments; The serum protein binding rates of 1 at the concentrations of 5. 10, 25μg/ml were 66.0. 75.4 and 85.9%, respectively; After iv administration of 1 at the dose 4, 10, 20 mg/kg, the Drug Concentration-Time Curves were assayed by 3P87 software and best fit to a two-compartmental model (P<0.05). AUC and Dose relationship shows an excellent linearity (R~2 = 1); The pharmacokinetics are notdose-dependent, ti/2 values were 0.64^ 0.58 n 0.83 h, respectively; This shows quick absorption and distribution for 1 in rats. Kinetic process of 1 in rats after ig administration at dose of 250 mg/kg also was fitted to a two-compartmental model. Parameters values for 1 were Cmax 1.84 mg/L, Tmax 0.57 h, AUCo-c 22.41 h?mg?L’\ t1/2 5.78 h, respectivly; The absolute oral bioavailability of 1 was calculated to be 2.0%; There is a slightly second absorption peak on the Drug Concentration-Time Curve. This suggests a formation of a hepatoenteral circulation. ’Primary parent drug was eliminated with feces after iv and ig administration. Mice tissue distribution experiments show that 1 was prominently concentrated in livers. These pharmacokinetic parameters indicated that 1 be metabolized by gastrointestinal tract microflora and liver microsomes when orally administered. The former is primary.Liver is an important organ in which a majority of drug metabolism takes place. In order to validate the pathway of the metabolites of 1 in vivo, we incubated 1 with liver microsomes in vitro. Within the range 5-200 (xg/ml of 1 we tested, we found that 1 was conjugated with coenzyme I (J3-NADPH) and thereafter no metabolite was detected. To exclude the possibility of a high concentration of coenzyme I . we incubated 1 with hepatocyte and still we did not get the metabolite.So we began the study on the metabolism of 1 by intestinal bacteria. After 1 being incubated with human intestinal bacterium Escherichia coli, there were very slight differences between the HPLC chromatograms of incubation solution and the negative contrast. Fortunately, after 1 being incubated with human intestinal bacterium klebsiella pneumoniae. the HPLC peak of the primary metabolite 2 in the chromatogram was so obvious that we identified it by LC-ESI-MS/MS analysis. Results in this study indicated that oxidative metabolites 3 and 4 might also be the products of 1 by the action of intestinal bacteria.Since compound 5 is one of the isomers of 1, there is possibility that synthetic strategy can be used to find the relationship between compound 1 and its isomeric metabolite 5. 1 has two benzofuran moirties and one tetrahydrofuran. Provided the carbon atoms that connect to the oxygen atom were substituted by electron withdrawing group, they are ready to take the isomerization reaction when catalyzed by acid. To avoid protonation effect, lewis acid boron trifluoride etherate was used and the isomeric metabolite 5 was successfully synthesized from 1 in an anhydrous condition. Therefore, ingested 1 in rats might be transformed to 5 by the acidic gastric juice.更多还原