Studies On Secondary Metabolites And Bioactivities Of An Ocean-derived Fungi Alternaria Sp.114-1G

The ocean has a unique environment like dark,cold,low oxygen and high pressure,which makes marine microorganisms gradually evolve special physiological and metabolic systems.The ability of producing natural products with special structures makes marine microorganisms an important source of lead compounds for new drug development.Alternaria sp.,widely distributed in nature,is a producer for lots of active substances.Therefore,research on secondary metabolites of Alternaria sp.from marine sources has a high potential for discovery of compounds with high activities.In this study,we isolated an Alternaria sp.namely 114-1G from the lateral fins and body surface scrapings of Coryphaena hippurus as the research strain.First,the 114-1G strain was activated and fermented,and the obtained fermentation broth and the crude extract of mycelium were subjected to preliminary activity evaluation.Next,the fermentation conditions of the strain were optimized accordingly,and the crude extract of the fermentation product was separated and purified to obtain compounds,whose structures were then identified.Finally,the purified compounds were evaluated for anti-tumor,anti-oxidation and anti-inflammatory activities.The results are as follows:(1)The anti-proliferation activities of the fermentation extracts on three tumor cell lines were evaluated.The results showed that at 100 μg/m L concentration the extracts’ inhibition rates were 93.15% on human cervical cancer cell line Hela,79.74% on human ovarian cancer cell line Skov3,and 86.02% on human hepatocellular carcinoma cell line Huh7.(2)The optimized fermentation conditions were as follows: mannitol 25 g/L,maltose 15 g/L,glucose 10 g/L,monosodium glutamate 10 g/L,soybean peptone 5 g/L,yeast extract 3 g/L;60% sea water in whole medium,initial p H 7.5;and fermentation at 10 ℃ for 15 days under a 150 rpm shaking speed on a rotary shaker.(3)The crude extracts of this strains were separated and purified by multiple column chromatography methods.The gained compounds were analyzed by 1H-NMR,13C-NMR,HMBC,and 1H-1H COSY,14 compounds’ structures were finally determined,including 1 new compound(14).The structures include 5 cyclic dipeptides(compound 1,3,4,8,13),1 ester(compound 2),2 pyrimidines(compound 5,6),1 anthraquinone(compound 7),1 fatty acid(compound 9),2 bisphenol A and its esters(compound 10,11),1 pyridone(compound 12),and 1 hydroxyphenyl butanediol(compound 14).(4)The anti-oxidant,anti-inflammatory,and anti-tumor activities of the compounds obtained from 114-1G were evaluated.The results showed that at 100 μg/m L concentration,the inhibition rates of Compound 10 and 11 on Hela cell line reached 99.1% and 95%.In the DPPH free radical scavenging experiment,the free radical scavenging rates of compound 3,compound 4,compound 9,and compound 14 were 50.26%,49.85%,42.54% and 46.12% at a concentration of 0.5 mg/m L,respectively.The result showed that the compounds have certain antioxidant capacity.According to the anti-inflammatory activity experiments performed in BV-2 and Raw264.7 cell lines,compound 14 can reduce the concentration of NO in the cell culture system,indicating that compound 14 has capacity.The study optimized the fermentation conditions of 114-1G,obtained several compounds and explored their biological activities,providing a good foundation for the for the subsequent research on the application of active substances from marine microorganisms in medicine,food and other fields.