Endophytic Fungi From Annona Squamosa L.: Isolation, Identification, Bioactivity And Preliminary Study On The Secondary Metabolites

The nature products were the important sourse of the medicine.From the secondary metabolites of the endophytic fungi,a lot of compounds with new structures and great bioactive diversities could be found.So the study of endophytic fungi from some pharmaceutical plant was the effective method of the development of medicine finding.From the leaves,twigs,barks and roots of the medicinal plant Annona squamosa,131 endophytic fungi were isolated in different seasons from 2005 to 2006.Crude fermentation extraction of each strain was tested by six different bioactive screening programs including anti-microbial activity,anti-tumor activity,anti-oxidation activity,brine shrimp inhibitory activity,trypsin inhibitory activity and acetylcholinesterase inhibitory activity.The result of the bioactive tests showed that 71 strains of the endophytic fungi displayed one or more than one bioactivities.56 strains(42.8%) displayed anti-microbial activities on one or more than one of 5 indicator organisms.33 strains(25.2%) displayed anti-tumor activity on Raji,Hep G2 cells.The strains with anti-oxidation activity, brine shrimp inhibitory activity,trypsin inhibitory activity and acetylcholinesterase inhibitory activity were 20(15.3%),25(19.1%),7(5.3%) and 0(0.0%),respectively.The identification of 63 bioactive endophytic fungi by ITS rDNA assay indicated that the strains were distributed in at least 19 genera and a lot of the strains were Fusarium spp.(23.8%),Diaporthe spp.(15.9%),Phomopsis spp. (12.7%) and Aspergillus spp.(7.9%).Phylogenetic relationship of bioactive fungi isolated from Annona squamosa indicated that A10 and A26 were distributed in Basidiomycota and the other strains were distributed in 9 orders of Ascomycota,including Botryosphaeriales,Diaporthales,Hypocreales, Clavicipitales,Phyllachorales,Sordariales,Xylariales,Eurotiales and Pleosporales.The result of test showed great diversity of taxa.The diversity of polyketide synthase(PKS) gene was investigated using three pairs of primers,LC1-LC2c,LC3-LC5c,KS3-KS4c.The KS domain of 63 bioactive fungi was amplified.11 strains(17%) of them can be amplified and amplification products displayed 700bp DNA bands by 3 pairs of primers,13 strains(21%) by 2 pairs of primers,18 strains(29%) by only 1 pair of primers and 21 strains(33%) had none amplified product.The result showed the diversity of the PKS gene of these 63 endophytic fungi.Two bioactive endophytic fungi isolated from Annona squamosa,B27 (Phomopsis sp.) and B38(Hypoxylon sp.),were selected to study their secondary metabolites.5 compounds were isolated from B27,including one new compound(LX-1),which was ten-membered lactone,namely phomolide C and four known compounds(LX-2,LX-17,LX-6,LX-13),which were 1-methoxy-8-hydroxy-9,10-anthraquinone,1,8-Dihydroxy-9,10-anthraquinone, cytosporone C and Altiloxin A,respectively.Five known compounds(B38-3, B38-4,B38-1,B38-2,B38-5) were isolated from B38,too,which were Lachnellin C,Lachnellin B,Lachnellin D,Dienetriol 9 and 1,3-Benzodioxol-5-ylmethanol,respectively.In the later investigation of the antimicrobial and antitumor bioactivities,these ten compounds showed no antimicrobial bioactivity at 50μg/disk.Only compounds LX-1,B38-4 showed antitumor bioactivity on HeLa cell and their IC₅₀ were 42μg/mL and 49μg/mL, respectively.