Study On The Secondary Metabolites And Their Functions Of Endophytes In Three Medicinal Plants

There are various medicinal plant resources in China,and bout over one thousand kinds of medicinal plants are commonly used.However,the long growth cycle and the severe felling cause the shortage of medicinal plant resources.The low extraction rate of medicinal components and the low availability of extracted residues,which greatly restricts the development of clinical drugs.The endophytic bacteria of medicinal plants has attracted more and more attention because of its advantages of abundant resources,diverse species,short growth cycle,easy metabolism control,easy breeding of strains and industrial production through large-scale fermentation.In the process of long-term coexistence with the host,its gene recombines with the host,which can produce the same or similar medicinal active ingredient as the host plant.Therefore,it has important ecological and economical significance to select active substances and novel compounds from their secondary metabolites.In this thesis,20 endophytic bacteria were isolated from the leaves of the medicinal plants of Dioscorea opposita and Chinese yam.After comparing their secondary metabolites,three strains(SY6,SL3,and SL9)were screened out as our research objects.The three strains of endogenous bacteria were respectively subjected to liquid and solid fermentation,then extracted to obtain crude extracts.The crude extracts were separated and purified by using silica gel and Sephadexephadex LH-20 dextran gel to obtain monomer compounds.Various spectrums were utilized to identify the planar and steric structures of the compounds.The optimization of the production process and the physiological activity of the secondary metabolite produced by SY6,penicillic acid,were investigated.The main research contents are as follows:1.Comprehensive application of the modern technology,the three strains for biological species were identified.SY6 is an endophytic fungus.The primers were designed by using the conserved sequence of rDNA.The ITS region was amplified by PCR and sequenced.The Blast alignment was performed by using the sequence in Genbank.Sequence similarity analysis was performed on the sequence data of the gene.SY6 was identified as Aspergillus ostianus.SL3 and SL9 are endophytic bacterias.The primers were designed by using the conserved sequence of 16S rRNA.The 16S rDNA were amplified by PCR and sequenced.The Blast alignment was performed by using known sequences from Genbank.Sequence similarity analysis was performed on the gene sequence data.It was determined that SL3 belongs to Bacillus licheniformis and SL9 belongs to Bacillus velezensis.2.The fermentation broth of SY6,SL3 and SL9 strains was treated to obtain a crude fermentation product.The crude fermentation product was isolated and purified by means of silica gel,Sephadex LH-20sephadex,and semi-preparative HPLC.A total of 9 monomeric compounds were obtained.The structures of the five compounds identified were penicillic acid,ergosterol,dihydropenicillic acid,thymine,and indole-3-carboxylic acid.3.In this dissertation,the herbicidal activities of penicillic acid and SY6 were investigated.Eleven field weeds were selected.The results showed that the penicillic acid and SY6 SY6 extract solution have a phytotoxic effect on 6 species of weeds,Amaranthus retroflexus L.,Echinochloa crusgalli(L.)Beauv.,Convolvulus arvensis L.,Setaria viridis(L.)Beauv.,Eleusine indica(L.)Gaertn.,and Solanum nigrum L.The concentration of penicillic acid was negatively correlated with the seed germination rate.When the concentration of penicillic acid reached 1.25 mg/mL,the budding rate of the six seeds was 0.The concentration of SY6 extract solution also showed a negative correlation with the germination rate of weed seeds.When the concentration of SY6 extract solution reached 1.00 mg/mL,the budding rate of alfalfa grass,field bindweed and solanine seeds was 0.When the concentration of SY6 extract solution was 1.25 mg/mL,the budding rate of the seeds of Amaranthus retroflexus,Setaria viridis,and Eleusine barbara was 0.By comparing the herbicidal effects of penicillic acid,SY6 extract and the reference acetochlor,it was evident that the herbicidal effect of SY6 extract was the same as the reference acetochlor.And penicillic acid and the reference acetochlor were superior to penicillic acid.At the same time,the antibacterial properties of penicillic acid were preliminarily examined by the method of punching.The representative strains of Staphylococcus aureus and Escherichia coli were selected.The results showed that penicillic acid had a certain inhibitory effect on gram-positive and negative bacteria.As the concentration of penicillic acid decreases,the inhibitory concentration decreases.When the concentration penicillic acid of 2.50 mg/mL is low sensitive.When the concentrations of penicillic acid of 5.00 mg/mL,7.50 mg/mL,10.00 mg/mL are moderately sensitive.The gram-negative bacteria E.coli and the gram-positive bacteria SA have the same antibacterial activities,and the gram-negative bacteria is slightly better than the gram-negative bacteria.4.The secondary metabolites of SY6 strain contain a large amount of penicillic acid,which was increased by optimizing the fermentation conditions during the experiment.The optimized fermentation conditions were that seed liquid was loading 61.35 mL,pH 5.24,30.19?,and rotating speed was 180 rpm for 24 h.The fermentation medium was that seeded with 10%inoculum with the same volume as the seed medium,pH 5.24,30.19?,and rotating speed is 180 rpm for 120 h.Penicillium acid has maximum yield.The yield increased from 65.1179 mg/L to 183.6325 mg/L and the yield increased by 182%.