Study On The Secondary Metabolites Produced By Two Endophytes

Compared with synthetic drugs,natural medicines have unexpected advantages in not only structural novelty but also biological activity.Co-evolved with host plants for a long time,endophytes had developed special secondary metabolic pathways and could produce compounds with novel structures and remarkable activities.Studies have showed that endophytes were able to produce active chemical constituents same as or similar to the host plant,and also produce new compounds different completely from the host plant.Therefore,searching for active pharmaceutical constituents from endophytes has become an important direction in the field of natural medicines,which also could solve the problems of medicinal sources such as slow growth and lackless of medicinal plants to some extent.Streptomyces rochei SR-1102 and Myrothecium roridum IFB-E091 were endophytes isolated from the rhizosphere of cucumber and the root of Artemisia annua,respectively.Researches on separation and purification and structure identification of their secondary metabolites were carried out in this dissertation.S.rochei SR-1102 was cultured in liquid medium with fermenter,and the fermentation broth was extracted with ethyl acetate three times and provided 33 g of crude extract after removing solvent in vaccum.M.roridum IFB-E091 was cultured in solid medium and the solid fermentation was extracted with mixed solvent(chloroform-methanol,1:1)three times and gave 38 g of crude extract after removing solvent in vaccum.Above-mentioned crude extracts of two endophytes were fractionated by silica gel column chromatography and provided several different polarity fractions,respectively.Guided by 'H-NMR spectrum and TCL,further separation and purification were performed through silica gel column chromatography,gel filtration on Sephadex LH-20,reversed phase chromatography,and HPLC etc.Seven compounds(1-7)were isolated from S.rochei SR-1102 and one compound(8)was obtained from M.roridum IFB-E091.Structures of these compounds were determined using modern spectral techniques such as high-resolution mass spectrometry(HR-MS),1D and 2D nuclear magnetic resonance spectroscopy(NMR),and maldi-tof mass spectrometry,and by comparing with literatures.They were identified as benzoic acid(1),daidzein(2),dihydrodaidzein(3),genistein(4),dakkatin(5),N,N'-diferuloylputrescine(6),N-p-doumaroyl-N'-feruloylputrescine(7),and halovir F(8),respectively.Among them,compound 8 was a linear peptide compound similar to halovirs A-E structurally,and it was a new compound that has not been reported yet.Compounds 2-5 were isoflavone compounds,and compounds 6 and 7 were dimers of hydroxycinnamic acid amide.Compound 3 was isolated from Streptomyces sp.for the first time,and compound 7 was obtained from microorganisms for the first time.S.rochei SR1102 showed significant inhibitory effect to a variety of plant pathogens such as Fusarium oxysporum f.Sp.Cucumebrium.Benzoic acid,isoflavones and hydroxycinnamic acid amide dimers obtained from the fermentation broth of SR-1102 in this study all showed different degrees of antibacterial activity,indicating that they might be the main basic chemicals for the antibacterial activity of the strain.This result provided an experimental basis for the further development and utilization of the strain.Streptomyces sp.is a high potential antibiotic-producing strain,however,no novel antibacterial compounds had been found in the present study probably because of the gene silencing phenomenon.Therefore,further relevant experiments will be carried out in our group to explore the potential of SR-1102 to produce new antibacterial compouns and provide new lead compounds for developing antibacterial drugs.M.roridum IFB-E091 could produce various types of secondary metabolites.A new characteristic secondary metabolite,namely trichothecene macrolide roritoxin E,and a new indole alkaloid had been isolated from its solid culture.A new linear peptide halovir F(8)was obtained again in the present study.Peptide compounds have rich biological activities.The result could enrich the peptide library,even provide a potential lead compound for drug development.The bioactivities screening for halovir F(8)would be conducted in our group for finding out its potential bioactivity,which will lay the foundation for subsequent research.