Study On The Culture Conditions And Bioactivities Of Clavatol From Marine-Derived Fungi BYY-1

Marine Fungus has become an important part of bioactive natural products research with more and more attention been paid to the ocean discovery. Because of its unique living environment, marine fungus has produced special metabolites with unique chemical structures and vivid bioactivities. Thus, the metabolites from marine fungus may become the potential drugs in the clinic.Secondary metabolite Clavatol from one marine fungi strain BYY-1 was observed in this study. The entire region ITS1-5.8s-ITS2 of BYY-1 was amplified by PCR. Associated with morphological character, BYY-1 was identified as Aspergillμs clavatonanicμs. Production of Clavatol was optimized by orthogonal design. Submerged fermentation was tested. The result shows that the optimal condition for Clavatol production included glucose10g/L,peptone2.0g/L,sea water0～100mL/L,yeast0～1.0g/L ,(NH4)2SO43.0g/L. The production of Clavatol reached 44.6mg/L, and it equaled to 1.5 times of the production in customary half-seawater GPY medium. Meantime, the differences of Clavatol production and biomass were studied.Clavatol was studied for its bioactivities. The result showed that Clavatol has Inhibitory activity on hydrogen peroxide-induced chemiluminescence with IC₅₀ 6.25μg/mL and the anti-oxygen activity of IC₅₀ 7.15μg/mL with DPPH free radical-scavenging assay. Clavatol has the inhibition activity of trosinase and AchEI with the same IC₅₀ 6.94μmol/L .The inhibition kinetic and mechanism of Clavatol was studied. It showed that the tyrosinase inhibition by Clavatol was a reversible reaction. The inhibition mechanism of Clavatol on trosinase and AchEI belonged to competitive inhibitor and noncompetitive inhibitor, separately. The Michaelis constants(Km) was 0.166μmol/L and 0.037mmol/L. In this study, the fluorescence intensities of the emission peaks are inversely decreased with an increasing concentration of Clavatol. Although the decline in the fluorescence intensity is caused by quenching, there is no significantλcm shift with the accumulation Clavatol.Clavatol showed antitumor activity against Raji,Hela,MG-63,BGC-823,KB,HepG-2 cell lines with the IC₅₀ value were 24μg/mL,12μg/mL,32μg/mL, 28μg/mL,16μg/mL and 46μg/mL. Clavatol showed moderate antimicrobial activities against Bacillμs sμbtilis,Staphlococcμs aμreμs,Escherichia coli,Candida albicans and Aspergillμs niger with the MIC value were 156,160,85,26,38μg/mL respectively.Primary anti-tumor mechanism of Clavatol was also studied. The results of morphological observation,FCM,mitochondria membrane potential,,Annexin-V,ROS detection and Western blotting showed that the compound induces apoptosis of tumor cells. The exact mechanism of its anti-tumor activity needed to have further study.Strain BYY-1 was fermented by optimal medium in Submerged fermentation manner and Clavatol were gained. The in vivo test showed that the compound has the toxicity to mouse with LD50 1000-1250mg/kg, and the anti-tumor activity inhibited mice sarcoma s-180 cell line with ED50 111.87mg/kg. The therapy index was 10.61.Our study indicated that metabolite Clavatol from strain BYY-1 was worth to be researched in-depth. Compound Clavatol had the potentiality to be a new anti-tumor drug in clinic.