Research On Secondary Metabolites With Anti-Alzheimer's Disease And Other Activities From Marine Fungi And Seaweed Hizikia Fusifarme

The particularity of marine environment creates the biodiversity and chemodiversity of marine organisms,providing new resource for new drugs and functional foods.In this investigation,plasma mutagenesis and chemical induction were used to treat the marine fungi with remarkable anti-Alzheimer's disease?AD?activity,to activate the silent gene clusters and therefore obtain more new compounds.Bioautography was used as tracing approach to separate molecules with anti-AD related activity?anti-acetylcholinesterase and antioxidation?from marine fungi and seaweed.These molecules with special chemical structures provide templates for new drugs design and enrich the leads library for the development of marine drugs.The lead compound library provides a basis for the study of marine drugs and nutraceuticals in the prevention and treatment of AD.Compounds 1,3,4,7,9 and 10 were isolated from a marine fungus mutant strain Aspergillus unguis 6-20-6 by using vacuum liquid chromatography?VLC?,column chromatographies?CC?on silica gel,ODS,and Sephadex LH-20,preparative thin layer chromatography?pTLC?,preparative high performance liquid chromatography?pHPLC?and recrystallization.On the comprehensive elucidation of 1D NMR,2D NMR,MS and IR,compound 1 and compounds 3,4,7,9 were elucidated as a new depsidone derivative aspergillusidone G and four known depsidones,respectively,while compound 10 was identified to be a known cyclic peptide.Together with compounds 2,5,6 and 8,which were also previously isolated from a marine fungus Aspergillus unguis DLEP2008001?the origin strain of 6-20-6?under chemical induction,all compounds were tested for their anti-acetylcholinesterase?AChE?activity,scavenging DPPH free radicals activity,larvicidal activity and anti-microbials activity.Compound 9 inhibited AChE with IC₅₀0 of 56.75?M.Compounds 3–5 exhibited potent larvicidality against brine shrimp.Compounds 1,5,7,8,and 9 showed moderate to strong activity towards different microbes.In docking studies,higher negative CDOCKER interaction energy and richer strong interactions between AChE and 9 explained the greater activity of 9 compared to 1.Combined with the HPLC fingerprint and biosynthetic pathway of marine fungus Aspergillus unguis under different factors,the mechanism of its metabolite production was analyzed.Treatment with NaBr,procaine or plasma mutagenesis,can promote the biosynthetic steps 5,10,11 and other factors to produce compounds 1,4,and 7 more.And the production of compound 2 was enhanced by facilitating steps 8,9 and 14.Compound 5 was promoted by the addition of NaBr changing the bromo-chlorine competition in step 10.In addition,in the case when NaBr is used in combination with procaine,the formation of 1 can be promoted,so synergistic effect should exist between the two factors.A chemical induction experiment was performed using 5 concentrations?10 mM,1 mM,100?M,10?M,and 1?M?in 4 mediums?PSB,Malt,Soy and Rice?with 6inducers?SAHA,5-azaC,Pro,BuS,CuCl₂ and ZnCl₂?for strains Hypocrea lixii DLEN2008010,Trichoderma harzianum DLEN2008005 and Penicillium sp.C21-1.By using of TLC fingerprint,HPLC fingerprint and activity tracking,the combinational condition of DLEN2008010-Soy-Pro-10 mM was finally chosen for scale-up formation and further chemical investigation.A new isoflavone glycoside,compound 14,named as Lixiin A,was isolated together with 21 compounds,among which compounds 11–13 and 15–17 were identified as the known compounds.The structures of the new compound 14 was identified using 1D NMR,2D NMR and MS.Structural analysis of the remaining compounds and activity test of all compounds are ongoing.The marine fungus Trichoderma harzianum DLEN2008005 was fermented using PSB medium.Adenosine with 5 compounds and several near-pure alkaloids were isolated.Structural analysis of the remaining compounds and the activity test of all the compounds are ongoing.The drug likeness analysis of the compounds 1–9,11–18 showed that they are similar to the physicochemical properties of the predicted oral drugs.TLC and bioautography were used to compare the chemical diversity and active ingredients of Hizikia fusifarme from Dongtou Zhejiang province and Zhanjiang.The results showed that the ingredients of Hizikia fusifarme from Dongtou Zhejiang province is similar to that from Zhanjiang.A bioactive subfraction HFEO was obtained from Hizikia fusifarme from Dongtou Zhejiang province by using column chromatography.The main components of HFEO were revealed by GC-MS to be tetradecanoic acid,palmitic acid,?Z?-9-hexadecenoic acid,plant alcohol and arachidonic acid.The HFEO and the main component monomers were tested for their AChE inhibitory activity and DPPH free radicals scavenging activity.The results showed that the activity of HFEO mainly came from arachidonic acid.The cytotoxicity of HFEO to BV-2 was tested by MTT assay and almost no toxicity to BV-2 was observed within the dose of 20?g/mL.The NO test and ROS test showed that HFEO can inhibit NO production and reduce the level of ROS in BV-2 cells,suggesting that HFEO may have potential anti-inflammatory effects.Considering that this subfraction possesses AChE inhibitory activity,antioxidant activity and anti-neuroinflammatory activity,it may has research value as a functional food for enhancing memory function.The above studies indicate that marine fungi and seaweed Hizikia fusifarme can be used as sources of anti-AD and other active lead compounds,and this research is meaningfulfor developing anti-AD drugs or functional foods.