Study On The Scondary Metabolites Of Endophytic Fungi Of Paeonia Ostii From Genuine Medicinal Meterials In Anhui And Evaluated Its Biological Activity

Objective: The dominant strains in root bark of Paeonia ostii were identified by studying the diversity of endophytic fungi,and then the antifungal activity of these strains were evaluated.Response surface analysis was used to explore how to make the process in fermentation of endophytic fungi easier and time-saving,and further research to these dominant strains with chemical diversity to pursue novel,biologically active natural products from endophytic fungi.The correlation between heavy metal elements and endophytic fungi provides important information for revealing the genuineness of P.ostii.Methods: The endophytic fungus isolated from the fresh root bark of P.ostii were identified by molecular biological technique,and the diversity of these strains in P.ostii were analyzed by statistical methods;The response surface methodology was used to optimize the fermentation process of a dominant endophytic fungus from P.ostii;Various chromatography and spectrum were used to study the chemical diversity of three strains from P.ostii and then the P.ostii activity of new compounds were evaluated through SRB method;The growth of endophytic fungi in the medium containing heavy metal elements was observed,and the endophytic fungi in the root bark of P.ostii were analyzed with resistance to different species and different levels of heavy metal ions.Results: 1.A total of 157 strains were isolated from the root bark of P.ostii in 5 different growing areas,belonging to 16 genera and 32 species.The Sorenson similarity index and cluster analysis showed there is a significant difference of the endophytic fungi in the five different growing areas.And the endophytic fungus Fusarium oxysporum,Alternaria alternate,Fusarium acutatum,Climacocystis montana,Aspergillus sp.MBL1612,Penicillium corylophilum and Penicillium guanacastense were dominant species in the root bark of P.ostii.2.The optimum fermentation conditions for the endophytic fungus F.oxysporum were obtained by response surface methodology: sucrose content was 20 g/L,yeast extract content was 5 g/L,KH2PO3 content was 1.00 g/L,and K2HPO3 content was 1.00 g/L.Under these conditions,the predicted biomass of mycelium was 1.661 g,and the reagent verification value was 1.647 g.3.The activity of seven dominant strains against one pathogenic fungus to P.ostii and six other phytopathogenic fungus showed the inhibition rate to these seven pathogenic fungus of the endophytic fungus A.sp.MBL1612 beyond 80%,especially the inhibition rate to R.solani is as high as 100%.4.Eighty compounds isolated from three dominant strains which from P.ostii.Nineteen compounds were isolated from A.sp.MBL1612(have high inhibition activity to pathogenic fungus)contained: aspergilolide(1),aspergiline B(2),aspergilol A(3),aspergilol B(4),aspergilol C(5),aspergilol D(6),aspergilol E(7),2S,4S-spinosate(8),2,4-dihydroxy-3-methylacetophenone(9),verruculogen(10),tryprostatin B(11),helvolic acid(12),13-oxofumitremorgin B(13),fumitremorgin C(14),demethoxy fumitremorgin C(15),terezine D(16),aszonalenin(17),12,13-dihydroxy-fumitremorgin C(18),and aspterric acid(19).Twenty-seven compounds isolated form C.montana(one of the most frequent isolation endophytic fungus from Paeonia ostii)included: climacomontaninate A(20),climacomontaninate B(21),climacomontanetate(22),-[Me-Phe-D-Hiv-Me-Phe-D-Hiv]-(23),methylester of9,10-dehydrofusaric acid(24),fusaric acid(25),9,10-dehydrofusaric acid(26),cordysinin B(27),methyl 2-(1H-indol-3-yl)acetate(28),N-acetyltryptamine(29),3-decanoxyl(30),indole-3-carboxylic acid(31),N-(4-hydroxyphenethyl)acetamide(32),N-benzene ethylacetamide(33),2-{4-[(3-hydroxybutan-2-yl)oxy]phenyl}acetic acid(34),cyclo(S-pro-S-leu)(35),cyclo-(D-pro-L-leu)(36),cyclo(ala-ile)(37),cyclo(ala-leu)(38),cyclo-(trans-4-hydroxy-L-prolyl-L-phenylalanine)(39),cyclo(R-pro-S-phe)(40),cyclo-(trans-4-hydroxy-L-prolyl-L-leucine)(41),cyclo(L-pro-L-phe)(42),1H-indole-3-acetic acid(43),cyclo(L-phe-D-tyr)(44),cyclo(2-hydroxy-pro-R-leu)(45).Thirty-four compounds isolated from F.oxysporum(one of the most frequent isolation endophytic fungus from P.ostii)contained:1-[4’-(1"-hydroxyethyl)phenyl]ethanol(46),methoxy benzene(47),p-toluic acid(48),4-(3-méthyl-2-butenyl)oxy 1-phényl acétique(49),3,4-dihydroxybenzoic acid(50),p-aminobenzoic acid(51),2,3,4-trihydroxybenzenepropanoic acid(52),1-phenylpropan-2-one(53),2-piperidone(54),2-pyrrolidinone(55),(2S)-proline(56),cyclo-[gly-ala](57),uracil(58),parabanic acid(59),thymidine(60),(22E,24R)-ergosta-7,22-diene-3β,5α,6β-triol(61),(5S)-5-[(4-hydroxyphenyl)methyl]-2,4-imidazolidinedione(62),(3S,6S)-3-(butan-2-yl)-6-(1-hydroxyethyl)piperazine-2,5-dione(63),cyclo(S-pro-S-leu(64),cyclo(L-ala-L-pro)(65),cyclo-(val-pro)(66),cyclo(ala-leu)(67),cyclo(L-pro-L-phe)(68),cyclo(R-pro-S-phe)(69),cyclo(tyr-pro)(70),cyclo(L-phe-L-tyr)(71),cyclo(D-cis-hyp-L-phe)(72),cyclo-(trans-4-hydroxy-L-prolyl-L-phenylalanine(73),cyclo(gla-tyr)(74),cyclo-(trans-4-hydroxy-L-prolyl-L-leucine)(75),L,L-cyclo(tryptophanylprolyl)(76),cyclo-[L-leu-gly](77),N-(3-(1H-indol-3-yl)propyl)acetamide(78),1H-indole-3-acetic acid(79).5.The strains FD4 and FD11 were obtained under culture conditions with Cu and were not obtained under the other two elements culture conditions.At the same time,the strain FD13 obtained under the condition of Ti and the strains FD6 and FD7 obtained under the condition of Mn were not obtained from the other two elements.Besides,the diversity of endophytic fungi in medium that contain metal elements is higher than the medium without metal elements.Conclusion: The dominant population of endophytic fungi in five areas were significantly different and showed regional specialization;The biomass of endophytic fungi in the fermentation process can be effectively increased by optimizing the fermentation conditions;The dominant strain A.sp.MBL1612 from P.ostii has broad spectrum effect to against pathogenic fungus;Seventy-nine compounds isolated from the fermentation products of three strains from P.ostii and which belonged to sixty-nine different compounds,the 1-7 and 20-22 were new compounds;There is difference in endophytic fungi under the culture conditions with different elements and concentration.