【作者】 吴晓萍；

【导师】 胡英杰；

【作者基本信息】 广州中医药大学 ， 中药学， 2007， 硕士

【摘要】 小叶榕是桑科榕属植物Ficus Microcarpa.L，主要分布在热带亚热带地区，在我国主要分布于广西、福建、广东、海南、台湾和浙江等地，为当地的主要植物品种之一。有研究表明，小叶榕叶中主要含三萜、脂肪族化合物和甾体化合物等，对治疗心血管疾病、抗炎和抑菌等方面都有显著的效果。民间习用于哮喘和慢性支气管炎等疾病。1985年以来，Higa M、郭悦雄等人分别对小叶榕气生根、树皮、心材和叶子进行了化学成分的研究，报道了七十多个化学成分，并且发现了在28位碳上含有羧基的三萜具有细胞毒等方面的生物活性（详见榕属化合物生物活性进展综述）。在国内，小叶榕入药主用其叶Folium Fici Microcarpae，但小叶榕叶的化学成分研究国内外的报道比较少。化学成分不明已成为制约小叶榕叶药材开发和含有小叶榕叶的中成药制剂研发的瓶颈问题。为了进一步寻找该植物的有效成分，本课题对该植物进行了化学成分研究。小叶榕叶乙醇提取物的乙酸乙酯萃取部分经过硅胶柱层析、反相硅胶柱层析、Sephadex LH-20凝胶柱层析、制备高效液相色谱仪分离，得到12个化合物。运用光谱分析(UV、¹HNMR、¹³CNMR、ESIMS、EI-MS、DEPT、HMQC、HMBC)鉴定了12个化合物的结构，这些化合物分别是：阿福豆素[（+）（2R，3S）-afzelechin，X-1]、表阿夫儿茶精[（-）（2R，3R）-epiafzelechin，X-2]、胡萝卜苷[daucosterol，X-3，]、儿茶素[（+）（2R，3S）-catechin，X-4]、β-谷甾醇[β-sitosterol，X-5]、phaseic acid（X-6）、苯甲酸[benzoic acid，X-7]、对羟基苯甲酸[4-hydroxy-benzoic acid，X-8]、[（-）（2R，3S）afzelechin-（4a-8）（2R，3S）afzelechin，X-9]、[（-）（2R，3S）afzelechin-（4a-8）（2R，3R）（-）epiafzelechin，X-10]、间苯三酚[phloroglucinol，X-11]、马斯里酸[maslinic acid，X-12]。除化合物X-4、5、12，其余化合物均是首次从该植物中分离得到的，其中化合物X-1、2、6、9、10为首次从该属植物中分离得到的。小叶榕叶及其制剂咳特灵在临床上主要用于治疗呼吸道疾病。呼吸道疾病是一种细菌或病毒引起的呼吸道感染。根据小叶榕叶的临床应用和民间使用的基础，我们初步考察了小叶榕叶的某些提取部位的抗病毒活性。结果表明，它们对抑制流感病毒，腺病毒，鼻病毒等病毒无效，但对疱疹病毒有效。鉴于此，我们在化学成分研究的基础上，对分得的单体化合物进行了细胞水平的活性筛选。抗病毒活性筛选采用细胞病变法观察某些单体化合物对HSV-1和HSV-2病毒的抑制作用。结果显示，以无环鸟苷（ACV）为阳性对照，phaseic acid对HSV-1和HSV-2病毒均有抑制作用；（+）（2R，3S）-afzelechin和（-）（2R，3R）-epiafzelechin对HSV-2也有一定的抑制作用，并呈现一定的剂量依赖关系。本研究填补了小叶榕叶抗病毒方面研究的空白。已有文献报道了小叶榕醇提物的抗菌效果，我们将会根据所分得的单体用于抗菌实验，这部分工作还在计划中。小叶榕叶药用价值较高，已被制成胶囊、片剂、颗粒剂等多种剂型供临床使用。但小叶榕药材及其制剂咳特灵在中成药标准上仍旧使用以芦丁为对照，硝酸铝显色的紫外分光光度测定总黄酮的方法作为质量控制标准。然而我们初步实验表明，用HPLC方法在小叶榕叶的甲醇提取液（浓度为0.8mg／mL）中检测不到芦丁。因此，以芦丁为对照的分析测定方法不科学。所以我们首次建立了小叶榕叶中黄烷类主要有效成分表阿夫儿茶精的定性定量分析方法：以硅胶G薄层作为固定相，优选出氯仿-甲醇（8：1）展开剂，可以清晰检出表阿夫儿茶精；HPLC法所用色谱柱为phenomenex C₁₈（250mm×4.6mm，5μm），以甲醇-水（22：78，v／v）为流动相，流速为1mL.min^-1，在208nm测定，表阿夫儿茶精在0.0176～0.3334μg呈良好的线性关系，相关系数0.9999。平均加样回收率100.6％，RSD2.9％。这可作为小叶榕叶药材的质量控制方法基础。更多还原

【Abstract】 Ficus microcarpa.L, is mainly distributed in South China. It is belonging to the Ficusfamily, and is widely distributed in the tropical and subtropical areas. In China it mainlygrows in Guangxi, Fujian, Guangdong, Hainan, Taiwan and Zhejiang provinces. Previousresearches showed that triterpenoids, acyclic compounds, steroid were found in the leaveswhich were of significant effect on treating cardiovascular disease, inflammation andbacteria. It is a folk medicine which is applied to treat asthma and chronic bronchitis. Since1985, Yogi M and Yueh-Hsiung Guo have studied the constituents from the aerial root,bark, heartwood and leaves of Ficus microcarpa. Nearly seventies compounds were isolatedincluding cytotoxic triterpenoids which had got a carboxyl at the C-28 respectively.The leaves of Ficus microcarpa is used as a drug in mainland. However, there are seldomreport about the constituents of it, which would restrict the development of it and itspreparation. In order to find out bioactive constituents, the ethyl acetate-soluble fraction ofethanol extract of Ficus microearpa was subjected to chromatographic columns filledrespectively with silica gel, reserved-phase （RP）-silica gel(C₁₈), Sephadex LH-20 gel,and/or separated by preparative HPLC. Twelve compounds were obtained. On the basis ofphysical and chemical properties as well as detail spectroscopic analysis (UV, ¹HNMR,¹³CNMR, EI-MS, ESI-MS, DEPT, HMQC, HMBC), these compounds have been identifiedas （+）（2R,3S）-afzelechin （X-1）, （2R,3R）（-）-epiafzelechin （X-2）, daucosterol （X-3）,（+）（2R,3S）-catechin（X-4）,β-sitosterol（X-5）, phaseic acid（X-6）, benzoic acid （X-7）,4-hydroxy-benzoic acid（X-8）, （-）（2R,3S） afzelechin-（4a-8）（2R,3S）afzelechin（X-9）, （-）（2R,3S） afzelechin-（4a-8）（2R,3R）epiafzelechin （X-10）, phloroglucinol （X-11）,maslinic acid（X-12）. Among these, compound X-1, 2, 3, 6, 7, 8, 9, 10, 11 were obtainedfrom this plant for the first time, while compound X-1, 2, 6, 9, 10 were obtained from ficusgenus for the this time.The leaves of Ficus microcarpa and its preparation are applied to treat chronic bronchitiswhich is infected by bacteria or virus. Because of its clinical application and civilian use,anti-virus activity was evaluated. Ethanol soluble part of its concentrated decoction showed obvious inhibitory effect to herpes simplex virus in vero cells, and no positive inhibition ofviral replication were observed to influenza virus, adenovirus, rhinoviruses in vitro.Therefore, the anti-herpes simplex virus activity was evaluated for the isolated compoundsby using assays of cytopathic effect inhibition. Our bioassay revealed for the first time thatphaseic acid （X-6） has effect on the both types of HSV, while （+）（2R,3S）-afzelechin（X-1）,（2R,3R）（-）-epiafzelechin（X-2） suppressed moderately the herpes simplex virus type 2（HSV-2） replication in vero cell culture in a dose-dependent fashion. It was also the firsttime to build up a relationship between isolated compounds of the leaves of Ficusmicrocarpa with antiviral activity for the Chinese materia medica. Pharmacologicalscreening of the other compounds for the anti-bacterial activity is in progress.Because of their higher medicinal value, the leaves of Ficus microcarpa.L are made intoseveral of dosage forms such as capsule, tablet, and granular formulation for clinical use.Current quality of Folium Fici Microcarpae and its preparation were still determined by aUV spectrophotometry and using rutin as a marker compound. But our experimentsdemonstrated that there was not any chromatographic peak of rutin in methanol extract ofvarious samples of the leaves of Ficus microcarpa.L when checked by HPLC. Therefore, inorder to analyze the constituents of Ficus microcarpa leaves, （-）-epiafzelechin, a principalconstituent of the heral drug, was assigned as a marker compound. It can be detected bysilica gel TLC with CHCl₃-MeOH （8:1） as developing solvent system and a 3％vanillin-6％HClO₄ solution as visualization reagent. The reversed phase HPLC, whichcarried out through a C-18 column Phenomenex BDS （250mm×4.6mm, 5μm）, with amobile phase methanol-water （22:78）, at a flow rate 1mL.min^-1 and detective wavelength208nm for assay of （-）-epiafzelechin, showed good linearity in the range of 0.0176～0.3334μg with a correlation coefficient 0.9999. The average recovery was 100.6％, RSD was 2.9％.Qualitative and quantitative analysis method for the bioactive constituent （-）-epiafzelechinin the leaves of Ficus microcarpa.L was built up for the first time, the results also provideda basis for the quality control of the Folium Fici microcarpae.更多还原