Screening And Secondary Metabolite Research Of Seaweeds And Marine Fungi With Anti-Alzheimer’s Disease Related Activity

Alzheimer’s disease(AD)is the main type of senile dementia.AD is found to possess complex and diversepathogenesis mechanisms,among them,the oxidative stress hypothesis and cholineric hypothesis are closely related to the occurrence and symptoms of AD.Based on these two hypothesis,current natural anti-AD drugs mainly come from plants and terrestrial microbes.However,studies on seaweed-derived acetylcholinesterase inhibitors and marine fungal anti-AD reagents are relatively rare or lagged.As important parts of marine biological groups,seaweeds and fungi provide novel resources for anti-AD leading compounds and are urgently needed to be exploited.This study was based on the above two hypotheses.A combinational acetylcholinesterase(ACh E)inhibitory-2,2-diphenyl-1-picrylhydrazyl(DPPH)scanvenging activity screening model was applied.TLC screening method,96-well plate screening method and Artemia lethality method were used to screen and evaluate 10 species of seaweeds and 35 marine fungal strains from waters including Zhanjiang’s.The results show that 5 marine fungal strains and 2 seaweeds’ extracts exhibited ACh E inhibition ratios greater than the positive control tacrine,for which the ACh E inhibition ratio at 200 μg/m L was 89.8 %;6 marine fungal strains and 4 seaweeds displayed both ACh E inhibition(inhibition ratios at 200 μg/m L were greater than 20 %)and DPPH scanvenging activity(DPPH scanvenging ratios at 200 μg/m L were greater than 30 %);the Artemia mortality ratios arelower than 12 % for marine fungal extracts and lower than 50 % for seaweed extracts,respectively.It is the first report that Padina sp.showed anti-AD related activity.The 4 marine fungal strains(C21-1,Penicillium sp.;C23-3,Aspergillus sp.;DLEN2008005,Trichoderma harzianum;TBG1-17,Penicillium corylophilum)and 2 seaweeds(Hizikia fusifarme and Padina sp.)with high diversity or difference and low toxicity were analyzed by bioactivity-coupled HPLC,which providedguidance for the subsequent separation and purification.The screening results showed that strain C23-3 has the strongest activity on ACh E inhibition and DPPH scanvengingand high diversity of active TLC spots.The strain C21-1also has strong ACh E inhibitory spots distinctly different from other strains.Their activity was significantly higher than the seaweeds,so the strains C23-3 and C21-1 were selected for fermentation.Three pure compounds were obtained from the fermented crude extracts of C23-3 by means of Sephadex LH-20 column,ODS column and preparative HPLC,including an ACh E inhibitor territrem B,an antioxidant butyrolactone I and an anticancer agent terrein.Two pure compounds were obtained from the fermented crude extracts of C21-1,including an ACh E inhibitor homodimeric WIN 64821 and a broad-spectrum antimicrobial agent(R)-(-)-hydroxysydonic acid.At the same time,three components with strong ACh E inhibitory activity,F-5,F-7 and F-8 were isolated from C23-3 fermentations with IC50 of 0.5,13.1 and 0.63 μg/m L,respectively.Three DPPH scanvenging active fractions F-6,F-8 and F-9,with EC50 of less than 100 μg/m L,were also obtained.Though strain C23-3 owned highly diverse active spots have high diversity,their composition was complex and the yields were low,which limited the obtainment of enough amount of active compounds.Considering that some small molecule chemoinducers can increase the yield and diversity of metabolites by activating certain silent genes,in this study,histone deacetylase inhibitors(sodium butyrate and SAHA),DNA methylation inhibitors(5-azacytidine and procaine),and inorganic salts(Zn Cl2,Cu Cl2)were taken as chemical inducers to further enhance the yield and diversity of valuable metabolites.The morphological changes of C23-3 before and after induction were analyzed,and the metabolites were analyzed by TLC and HPLC.The results showed that the yield of possible active components were increased when induced by Zolinza(SAHA,100 μM),Zn Cl2(100 μM),sodium butyrate(100 μM),and SAHA and Zn Cl2-induced groups produced new substances with anti-AD-related activity.In this paper,we obtained 6 marine fungal strains and 4 seaweeds displayed both ACh E inhibition and DPPH scanvenging activity and low toxicity of Artemia.There are three monomeric compounds with anti-AD-related activity were isolated and purified fromthe fermentation products of strains C21-1 and C23-3,including two ACh E inhibitors(sebctoid B and homodimeric WIN 64821)and one antioxidant(butyrolactone I).At the same,the method of epigenetic modification induced by small molecule chemistry significantly increased the yield of butyrolactone Ⅰ in secondary metabolites of strain C23-3.