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Secondary Metabolites From Four Plant Endophytic Fungi Along With Their Biological Activities

Posted on:2018-06-10Degree:DoctorType:Dissertation
Country:ChinaCandidate:Z L MaoFull Text:PDF
GTID:1480305147484954Subject:Plant pathology
Abstract/Summary:PDF Full Text Request
The plant endophytic fungi can defined as the fungi which spend the whole or part of their lifecycle colonizing inter-or intr-cellualrly inside the healthy tisues of the host plants,typically causing no apparent symptoms of disease.This study is about the isolation and identification of the secondary metabolites produced by two endophytic fungi Ponipodef12(Hyalodendriella sp.)and Ponipodef09(Botryosphaeria dothidea)from Populus deltoides × P.nigra and two endophytic fungi Sigrf05(Pleosporales sp.)and Sigrf10(Lophiostoma sp.)from Siraitia grosvenorii along with their biological activities.Some structure-activity relations were discussed.The separation and preparation for the two main secondary metabolites(botrallin and TMC-264)with bioactivities were also investigated.The main results are as follows.(1)Twenty-one secondary metabolties were isolated and purified from the ethyl acetate crude extract from the culture of the fungus Ponipodef12 by VLC,MPLC,Sephadex LH-20 chromatography,semi-preparative HPLC and recrystallization methods.The structures of these compounds were deduced by analyses of the NMR spectroscopic and mass spectrometry data,in combination with chemical conversion.Eleven compounds belonged to dibenzo-?-pyrones among which P12-14,P12-20 and P12-23 were new compounds,the known compound P12-10 was reported as the natural product for the first time.Eight compounds(P12-17,P12-18,P12-21,P12-24,P12-25,P12-26,P12-28 and P12-29)belonged to 14-membered resorcylic acid lactones which were all new compounds.In addition,two amidic alkaloids(P12-11 and P12-12)were identified,and P12-11 was a new one.Among the secondary metabolties isolated from the fungus Ponipodef12,compounds P12-9,P12-14,P12-15 and P12-16 had antibacterial activity against four plant pathogenic bacteria.Compounds P12-9,P12-14 and P12-16 had inhibitory activity on spore germination of Magnaporthe oryzae,and P12-16 particularly displayed the strongest activity the same as the positive control(carbendazim).Compounds P12-1,P12-9 and P12-23 had inhibitory activity on acetylcholinesterase.Only P12-25 showed inhibitory activity on two nematodes Caenorhabditis elegans and Meloidogyne incongnita.Only P12-9 displayed strong antitumor activity against five human tumor cell lines,meanwhile,this compound showed strong antiviral activity against two herpes simplex virus the same as the positive control(acyclovir).Nine compounds were screened to have larvicidal activity on fourth-instar larvae of Aedes aegypti,P12-11 was the best with LC50 and LC90 values as 5.93 and 20.99 ?g/mL,respectively.(2)Ten secondary metabolties were isolated and purified from the ethyl acetate crude extract of Ponipodef09.Compounds P09-1,P09-2,P09-3 and P09-6 belong to benzo-?-pyrones.Compounds P09-4 and P09-5 were benzofuranones,and P09-4 was a new compound,and P09-5 was reported as the natural product for the first time.In addition,P09-7 was glyoxylic acid,and P09-8,P09-9 and P09-10 were adenosines.Among the secondary metabolties isolated from Ponipodef09,compounds P09-4 and P09-5 had moderate antibacterial activity against four plant pathogenic bacteria.Compounds P09-2,P09-3 and P09-4 showed inhibitory activity on spore germination of M.oryzae.Compound P09-4 exhibited antitumor activity against the human colon cancer cells HCT-116 with IC50 value as 1.80 ?M,it displayed the strongest larvicidal activity against fourth-instar larvae of A.aegypti with LC50 and LC90 values as 3.56 and 10.69 ?g/mL,respectively,the same as the positive control(rotenone).(3)Seven secondary metabolties were isolated and purified from the ethyl acetate crude extract of Sigrf05 and they all belonged to ?-pyrones.Compounds S05-3,S05-4,S05-5a,S05-5b,S05-6 and S05-8 were new compounds.S05-7 was a known compound clearanol A.Among the secondary metabolties isolated from the fungus Sigrf05,compound S05-8 showed moderate antibacterial activity against four plant pathogenic bacteria with IC50 values ranging from 44.29 to 63.06 ?M.On the other hand,compounds S05-3,S05-4,S05-5a and S05-5b had inhibitory activity on spore germination of M.oryzae,in which S05-5a showed the best IC50 as 47.77 ?g/mL,and the IC50 values of the others were ranging from 51.08 to 111.50 ?g/mL.Compounds S05-4 and S05-8 exhibited strong antitumor activity against the five human cancer cells,and the IC50 of S05-4 against gastric carcinoma cells BGC-823 was 1.26 ?M then the IC50 of S05-8 against the colon cancer cells HCT-116 was 1.17 ?M.(4)Six secondary metabolties were isolated and purified from the ethyl acetate crude extract of Sigrf10.Compounds S10-3 and S10-4 were ?-pyridones and S10-5,S10-6,S10-12 and S10-14 were new ?-pyrones.Among the secondary metabolties isolated from the fungus Sigrf10,compound S10-3 and S10-4 showed antibacterial activity against four plant pathogenic bacteria with IC50 values ranging from 35.68 to 44.85 and 35.66 to 40.44 ?M,respectively.On the other hand,compounds S10-5 and S10-6 had moderate inhibitory activity on spore germination of M.oryzae with IC50 values as 86.61 and 81.67?g/mL,respectively.(5)Two dibenzo-a-pyrones,botrallin and TMC-264 were obtained from extracts of the endophytic fungus Ponipodef12,using a two-step method that high-speed counter-current chromatography(HSCCC)and semi-preparative HPLC.The two-phase solvent system of n-hexane-ethyl acetate-methanol-water at the volume ratio of 1.2:1.0:0.9:1.0(v/v)was selected as the best one for further isolate and purify the two target compounds.Botrallin(17.0 mg)and TMC-264(14.8 mg)were successfully obtained from the ethyl acetate extract(300 mg)of endophytic fungus Ponipodef12.(6)A rapid and cost-effective method of macroporous resin SP825L for the enrichment of botrallin and TMC-264 as major bioactive dibenzo-?-pyrones in the endophytic fungus Ponipodef12 was established.The optimal conditions for resin SP825L adsorption of botrallin and TMC-264 were processing pH value as 5.0,bed volumes(BV)as 28 BV,flow rate as 3 BV/h,and temperature as 25?.Those for desorption were ethanol-water elution system at the ratio of 50:50(v/v),flow rate as 4 BV/h and eluent volume as 7 BV.After one round treatment with resin SP825L,the contents of botrallin and TMC-264 were increased 9.48 and 25.51 times,respectively.The recoveries for botrallin and TMC-264 were 88.85%and 94.90?,respectively.
Keywords/Search Tags:endophytic fungi, fungus Ponipodef12, fungus Ponipode09, fungus Sigrf05, fungus Sigrf10, secondary metabolites, dibenzo-?-pyrones, 14-membered resorcylic acid lactones, high-speed counter-current chromatography, macroporous resin, biological activity
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