Antimicrobial And Antitumor Activities And Secondary Metabolites Of Endophytic Fungi From Cephalotaxus Hainanensis Li

The endophytic fungi from plants have been the important resource for screening for biologically active substances, because endophytic fungi can produce the same or similar secondary metabolites as their hosts. As medicinal plant, Cephalotaxus hainanensis Li. has been paid much attention as it can produce homoharringtonine and harringtonine compounds with antitumor activity. This present study, first of all, investigated the antimicrobial activity of 179 endophytic fungal strains from C. hainanensis Li sampled from three areas in Hainan Province, then classified highly antimicrobial active endophytic fungal strains, and investigated the antitumor activity of highly antimicrobial active endophytic fungal strains. In order to determine the target strain, the study also analyzed the chemical composition of the secondary metabolites of these strains. After that, purified secondary metabolites of the target strain, and identified the structure of monomeric compounds. In the end, this study investigated the antitumor activity of these monomeric compounds. The main contents are as follows:The antimicrobial activity of 179 endophytic fungal strains from C. hainanensis Li against seven pathogens including Escherichia coli ATCC25922?Staphylococcus aureus ATCC25923?Bacillus subtilis ATCC6633?Streptococcus agalactiae HYXY08?Rhizoctonia solani ZLL101? Sclerotinia sclerotiorum XJS01 and Fusarium oxysporum RKY102 was investigated by fungal plugs method.15 endophytic fungal strains showed highly antimicrobial activity.The highly antimicrobial active endophytic fungal strains were classified by morphological and molecular biological (18SrDNA-ITS) methods. These 15 strains were classified as Trametes polyzona A8?Diaporthe kyushuensis A24?Diaporthe kyushuensis A25?Penicillium copticola A29?Albonectria rigidiuscula B18? Corynespora cassiicola B29?Nemania diffusa B33?Colletotrichum tropicicola D4? Colletotrichum thailandicum E4?Phomopsis asparagi F26?Periconia sp. F31? Colletotrichum magnisporum F32?Diaporthe actinidiae F45?Phomopsis fukushii F75 and Phomopsis fukushii F91. Among them, it is the first report of isolation of endophytic fungi such as A. rigidiuscula B18 and C. magnisporum F32 were isolated from Cephalotaxus plants.The antitumor activity of 15 highly antimicrobial activity endophytic fungal strains against five cancer cell lines including the human K562 chronic myeloid leukemia?the NB4 acute promyelocytic leukemia and the human HL-60 acute myeloid leukemia cell lines?Hep-G-2 hepatic, and colon LoVo cancer cell lines was investigated by MTT method.15 strains all had cytotoxic activity against at least one tumor cell line. It is worth emphasizing that strain Penicillium copticola A29 demonstrated the highest activities against K562, NB4, and HL-60. Then the study analyzed the chemical compositions of the secondary metabolites of these strains. The results showed that the secondary metabolites of Penicillium copticola A29 were rather diverse. So the strain Penicillium copticola A29 was determined target strain.After large-scale fermentation of Penicillium copticola A29, the secondary metabolites of Penicillium copticola A29 were purified by silica gel column?HPLC and PLC methods. Ten monomeric compounds were isolated. Then the ten compounds were identified by spectral analysis (ESI-MS?1H-NMR ?13C-NMR) as 3-Chloro-4-methoxyphenylacetic acid (1)?Vanillic Acid (2)?dihydrosporogen-AO 1 (3)?3-chloro-4-hydroxyphenylacetic acid (4)?6-dehydropetasol (5)?phomenone (6)? 3:4di-hydroxy-5-methoxyphenylacetic (7)?sporogen-AO 1 (8)?7-hydroxypetasol (9)? Dibutyl phthalate (10).The antitumor activity of ten monomeric compounds was investigated by MTT method. Results showed that compound 9 exhibited moderate cytotoxicity against HL-60, compound 5 exhibited certain cytotoxicity against K562 and HL-60. compound 3 exhibited certain cytotoxicity against K562?NB4 and HL-60 with IC50 values of 19.61,59.51 and 1.93 ?g/mL, respectively. compound 6 exhibited considerable cytotoxicity against K562?NB4 and HL-60 with IC50 values of 2.87, 0.12 and 2.33 ?g/mL, respectively. And compound 8 exhibited considerable cytotoxicity against K562?NB4 and HL-60 with IC50 values of 1.76,0.21 and 1.91 ?g/mL, respectively. In addition, compound 6 and 8 exhibited certain cytotoxicity against Hep-G-2.Summarily, the strain Penicillium copticola A29 with highly antimicrobial and antitumor activities was determined by fungal plugs and MTT methods. And ten monomeric compounds were identified from the secondary metabolites of Penicillium copticola A29. Among them, dihydrosporogen-AO 1?phomenone and sporogen-AO 1 exhibited considerable cytotoxicity against K562?NB4 and HL-60. The study provided reliable experimental basis for exploring natural highly antimicrobial and antitumor active compositions from endophytic fungi of Cephalotaxus hainanensis Li.