Studies On The Bioactive Constituents And Quality Control Of Folium Fici Microcarpae

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 3ï¼…vanillin-6ï¼…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 100.6ï¼…, RSD was 2.9ï¼….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.