Chemical Constituents And Biological Activities Of Secondary Metabolites From Stemona Sessilifolia And Dendrobium Officinale Endophytes

Although China is rich in medicinal plant resources,the slow growth cycle and excessive use has resulted in a shortage of medicinal plant.While the endophytic bacteria is rich in medicinal plants,and they can be fermentated in large scale to produce effective secondary metabolits after the optimization of fermentation conditions.Therefore,the study of plant endophytes has attracted more and more attentions.The endophytes have coexisted in their hosts for a long time.During their evolution,they can combined the genes with their hosts,which may result in the production of same or similar active components as the host did.Therefore,the discovery of active secondary metabolites from plant endophyte,especially from traditional Chinese medicine,has turned out to be an efficient manner to provide drug lead structure for diseases control and agricultural application,with important academic value and application prospect.In this thesis,we used the tissue-cutting method and the plate streaking culture method to isolate and purify the corresponding endophytes from Stemona sessilifolia and Dendrobium officinale.After taxonomical identification of strains by 16S rRNA technique,preliminary bioassay evaluation and chemical analysis by HPLC/LC-MS,two endophytic strains,Streptomyces sp.BS-1(from S.sessilifolia)and Streptomyces sp.SH-1.2-R-15(from D.officinale),were selected for further study.The crude extracts were prepared after scale-up fermentation and total 20 compounds were isoltate through silica gel column,Sephadex LH-20 chromatography,as well as high performance liquid chromatography(HPLC).Their structures were elucidated by MS and 1D/2D NMR(1H-1H COSY,HSQC,HMBC,ROESY)spectroscopic data,chemical communication and single crystal X-ray diffraction spectra.The bioactivities of isolated compounds were evaluated and some of them displayed potent insecticidal and cytotoxic activities.The research results are summarized as follows:.(1)25 strains were isolated from the fresh roots,stems and leaves of Stemona sessilifolia.The crude extracts of the strains were prepared by small amount of fermentation and extraction.One of the strain No.BS-1 was selected for further study based on bioassay evaluation(LC50:0.74 mg/L against Aphis gossypii)and LC-MS analysis.The strain BS-1 has been fermented and cultured in large scale,12 compounds were isolated by chromatographic separation and identified as followes:endostemonines A-J(1-10),3,4-dimethoxy cinnamic acid(11)and 3,4-dimethoxybenzoic acid(12).Compounds(1-10)were new pyrrole-2-carboxylic esters.Their absolute configurations was determined though chemical communication.Insecticidal activity results showed that six of tested new compounds displayed potent insecticidal activity against Aphis gossypii(LC50 range:3.55-32.00 mg/L)and moderate insecticidal activity against Tetranychus urticae(LC50 range:197.6-685.5 mg/L).(2)From the fresh roots,stems and leaves of Dendrobium candidum with different growth years,75 endophytic strains were cultured and purified.The crude extracts of the strains were prepared by small amount of fermentation and extraction.Strain No,SH-1.2-R-15 was selected also based on bioassay results(20.3%against Hep3B2.1-7 cells and 100%against S.aureus)and LC-MS analysis.The strain SH-1.2-R-15 has been fermented and cultured in large scale,8 compounds were isolated and identified in a same manner as followes:chartspiroton(15),peptidendrocins A(16)and B(17),chartreusin(18),(+)-streptazolin(19),strepchazolins A(20)and B(21),(R)-(E,E)-2-(1,3-pentadienyl)piperidine(22).Compound 15 was a novel tetracyclic spiro-naphthoquinone derivative and compounds 16-17 were new compounds.The structure and absolute configuration of 15 was confirmed by 13C NMR calculation method and single crystal X-ray diffraction.The main product 18 showed good cytotoxic activity(IC50=18.19 ?M against Hep3B2.1-7 cell,IC50=45.86 ?M against bladder cancer cell J82,and IC50=19.74 ?M against lung cancer cell H1299);as well as antibacterial activity against S.aureus(IC50=23.25 ?M).