Study On Marine Fungal Diversity And Activity Of Two Fungi In The Coastal Waters Of Haikou

In recent years,marine fungus has become an important source of active marine natural products.Because the marine fungi live in the oligotrophic,alkalescent seawater environment,it may form a unique metabolic mechanism to produce secondary metabolites which are different from the inland fungi.The research of the marine fungi has become a hot area gradually.162 strains of marine fungi are isolated and purified from various marine biological samples collected in coastal regions in Haikou(the west coast of Haikou niche,along the Nandu River into Haikou niche and YanFeng mangrove niche).After liquid fermentation,inhibition effects of four pathogenic bacteria as indicators(Staphylococcus aureus,Klebsiella pneumoniae,Pseudomonas aeruginosa,Escherichia coli)are measured using Kirby-Bauer disc diffusion method(short for K-B method),and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylterazolium bromide colorimetry(short for MTT colorimetry)method were taked to test ferments liquid to inhibit the proliferation of B-16 melanoma cells and measuring their anti-tumor activity.Antibacterial experiment results showed that only 28 strains of marine fungi had no inhibitory effect on the four indicator bacteria,accounting for 17.28%of the total isolates strains.42 strains of marine fungi had certain inhibitory effect on the four indicator bacteria,which made up 25.92%of the total amount.4 strains of marine fungi had strong inhibit effect against the four pathogenic bacteria,7 strains of marine fungi had strong inhibit effect on at least two indicator bacteria.Research showed that isolation from the niche of mangrove had stronger antibacterial activity than separation from west the niche of coast in Haikou,isolations from the estuarial niche of Nandu River had less antibacterial activity compared with the two above.MTT experimental results showed that 123 strains of metabolites isolated from the marine fungi all showed some inhibitory effect on the proliferation of B-16 cells,taking 75.92%in the total isolation.The 50%inhibiting concentration(IC50)of melanoma cells to 76 strains of metabolic extract was between 500 ?g/mL and 100?g/mL.The IC50 metabolic extract of 25 strains which inhibited B-16 cell proliferation was less than 50 ?g/mL,that accounted 23.58%for the total of separated stains.Research results indicated that the strains separated from habitat of the Haikou west coast had more antitumor activity than fungi separated from the entrance of Nandu River habitat,isolation from the mangrove forest habitat had the least antitumor activity.According to observed their colony structure,conidium birth location,spore morphology characteristics of active strain M-22 and M-40,and through analysis of their DNA sequences of the internal transcribed spacer(ITS1-5.8S-ITS4)regions of thier ribosomal RNA gene.Used their ITS sequence to matched with other fungi genes from GenBank.They two marine fungi were then identified as Penicillium sclerotiorum,and Cladosporium cladosporioides.The strains of M-22 and M-40 which have anti-tumor activity are plentiful fermented,and then isolated by modern separation technology(column chromatography,preparative TLC,preparative HPLC,etc.),use spectrum to learn knowledge,finally from the strains of M-22 were isolated from compounds 1(18.9 mg)identified as penicilazaphilone C,its production was 1.575 mg/mL;from M-40 were isolated Compounds 2(52.3 mg)identified as Ergosterol,its output was 2.906 mg/mL.The penicilazaphilone C is a new compound,and ergosterol also is first separated from the Cladosporium genus.Antitumor activity and antibacterial test of the penicilazaphilone C and compound ergosterol found that penicilazaphilone C have stronger inhibit effect against four indicator bacteria(S.aureus,K.pneumoniae,P.aeruginosa,E.coli)and their minimum inhibitory concentration(MIC)were 31.25,62.5,15.63,15.63 ?g/mL,respectively.But the compound ergosterol showed weaker inhibit effect against four indicator bacteria,its MIC just only were 250 ?g/mL and 125 ?g/mL to this bacteria.The two compounds all exhibited good inhibitory effect on the proliferation of melanoma cells B-16 and human gastric carcinoma cell SGC-7901,and the IC50 of B-16 cell and SGC-7901 cells to penicilazaphilone C were 27.11 ?g/mL and 303.70?g/mL,respectively.The IC50 of two cancer cells to compound ergosterol were 80.48?g/mL and 149.05 ?g/mL,respectively.But the two compounds are not showed obvious inhibitory effect for normal breast epithelium cells M-10.