Studies On Secondary Metabolites Of Bacterial Blight PXO99~A

The genus of Xanthomonas is ubiquitous in nature and it has been reported to do a great harm to both plants and animals.The validity periods of medicines that aim to prevent and cure diseases through killing pathogens are becoming shorter and shorter.The emergence of this issue has become a huge threat to the life and health of all human beings.Numerous studies have demonstrated that quorum sensing(QS)exist in both intra-specific and inter-specific microorganisms,and this sensing capability helps to perceive concentration variations of specific signaling molecules released by these microorganisms,through which they could regulate themselves to follow the group behaviors,QS plays a role in regulation of a variety of biological functions in human beings,animals and plants,in particular with the expression of various pathogenic factors from pathogenic bacteria.Therefore,QS system interference has becoming an attractive method in controlling infectious diseases.In recent years,a series of biologically active substances have been found in microorganisms,and these promising substances can be used as the leading compounds for drug design.At present,the research on QS of Xanthomonas are mainly focused on genetic information,while fewer study are conducted about the secondary metabolites of Xanthomonas,and for this reason,we selected model strain PXO99A as our research object and expanded the following research:Part I,Literature reviewIn order to collect information about microbial secondary metabolites,QS,anti-tumor effects and anti-infection effects of microorganisms,76 pulished literatures were studied.The information was summarized which could serve as a reference guide for our further research.Part II,Study on the secondary metabolites of Xanthomonas oryzae pv.Oryzae PXO99AFirstly,bacterial fermentation broth was extracted by Pet,CHCl3,EtOAc,and n-BuOH,concentrated under normal atmospheric pressure.Four types of extracts that harbored different chemical polarity were collected.Secondly,GC-MS was used to analyze the chemical compositions in these four layers,and then by utilizing recrystallization and column chromatography(normal phase silica gel column chromatography,gel filtration chromatography,macroporous resin column chromatography,and MCI column chromatography,etc.)these compositions were separated and purified.Thirdly,we used the spectroscopic techniques(ESI-MS,1H-NMR,13C-NMR,TOCSY,HMBC,and HMQC,etc.),physical and chemical methods to verify their structures.More than 20 compounds were isolated from the supernatant of PXO99A fermentation broth,among which,the structures of 17 compounds have been confirmed by NMR data analysis and other substance analysis tools,besides,one mixed crystal(compound X)also has its candidate structure.There are 3 cyclic dipeptides:cyclo-(L-pro-L-tyr)(1),cyclo-(L-pro-L-Leu)(2),cyclo-(L-trp-L-tyr)(3);4 esters:Dimethyl phthalate(4),Methyl-3-indolylacetata(5),1,3-Dioctadecanoyl-racglycerol(6),Triethyl citrates(7);2 Alcohols:2,3-butanediol(8),Phenethyl alcohol(9);2 amides:Erucamide(10),Oleic acid amide(11);2 Monosaccharides:?-D-fructopyranose(12),?-glucose(13);1 fatty acid:palmiticacid(14);1 Ketones:Diacetone alcohol(15);2 Phenols:P-hydroxyphenyl ethanol(16),phenol(17).The compound X might be composed of cyclo-(asn-ileasp)(1'),cyclo-(asn-val)(2'),cyclo-(asn-leu)(3'),Uracil(4').However,we can't separate this mixed compounds because it was only soluble in DMSO,and it has no response when treated with cromogenic agent.Among them,the compounds 1-11,14-17 were first discovered in PXO99A.The main compounds isolated from the PXO99A are cyclic dipeptide(1,2,3),esters(4,5,6,7)and alcohols(8,9).Wherein cyclo-(L-pro-L-tyr)(1),cyclo-(L-pro-L-leu)(2),cyclo-(L-trp-L-tyr)(3)have been reported to have anti-tumor and anti-infective activities.P-hydroxyphenyl ethanol(8)showed a weak cytotoxic effect and QS activity.Phenyl ethanol(9)is a QS signal molecules with specificity and Palmitic acid(14)exhibited anti-tumor activity and apoptosis-inducing effects in endothelial cells and pancreatic ? cells.Innovation points:The material basis of the secondary metabolites of PXO99A was explored for the first time in this project.Four different polar solvent extractions and separations of the PXO99A fermentation broth were analyzed in order to lay the foundation for discovery of new compounds which are also biologically active substances,also our work give some implications for study of relationship between Xanthomonas metabolites and their pathogenesis.