Research On Anti-Alzheimer’s Disease Related Bioactive Constituents From Rhodiola Rosea L.,Polygala Tenuifolia Willd.and Marine Fungus C23-3

Alzheimer’s disease（AD）is a serious threat to the health of the elder peoples;oxidative stress,low acetylcholine levels and inflammation are closely related to AD.Terrestrial and marine pharmaceautial organisms are important resources for drugs and functional foods to cope with sorts of healthy challenges.Traditional Chinese medicine has always been an important resource for the treatment of Alzheimer’s disease.Rhodiola rosea L.and Polygala tenuifolia Willd.are edible traditional Chinese medicinal herbs and they contain abundant anti-Alzheimer’s active ingredients with antioxidant activity,activity of improving the function of cholinergic system and neural protective activity.In recent years,a large number of secondary metabolites from marine fungi have been reported with antioxidant activity and acetylcholinesterase inhibitory activities.These secondary metabolites provide templates and leads library for the development of anti-AD drugs.This project focused on anti-Alzheimer’s active ingredients including applicable research using edible traditional Chinese medicinal herbs as materials and basic research using marine fungus as material.The main contents of application research on edible traditional Chinese medicinal herbs is to determine the optimal ultrasonic extraction conditions of anti-Alzheimer’s active ingredients with low or medium polarity for Rhodiola rosea L.and Polygala tenuifolia Willd.Through orthogonal experiment using ethyl acetate,petroleum ether and mixed solvent of ethyl acetate with petroleum ether as extracting solvents.The bioautography was used to analyze the diversity of bioactive substances.The antioxidant activity and acetylcholinesterase inhibitory activities of the extracts from Rhodiola rosea L.and Polygala tenuifolia Willd.of each factor and level were evaluated using DPPH method and Ellman method,respectively.The optimum extracting conditions are obtained by the single factor experiment combined with orthogonality experiment.It was found that the optimizing process conditions of taking antioxidant active substances of Rhodiola rosea L.as an indicator are:ultrasonic power 300W,solid-liquid ratio 1:20（g/mL）,ethyl acetate-petroleum ether（9:1）,extraction times of 3 times.The optimizing process conditions of taking acetylcholinesterase inhibitory active substances of Rhodiola rosea L.as an indicator are:ultrasonic power 300W,solid-liquid ratio 1:40,ethyl acetate-petroleum ether（9:1）,extraction times of 4 times.The optimizing process conditions of taking antioxidant active substances of Polygala tenuifolia Willd.as an indicator are:ultrasonic power 300W,solid-liquid ratio1:30（g/mL）,ethyl acetate-petroleum ether（9:1）,extraction times of 4 times.The optimizing process conditions of taking acetylcholinesterase inhibitory active substances of Polygala tenuifolia Willd.as an indicator are:ultrasonic power 300W,solid-liquid ratio 1:30（g/mL）,ethyl acetate-petroleum ether（9:1）,extraction times of 3 times.The verification experiments prove that these optimizing process conditions are stable and can attain optimization purposes.The main contents of basic research of taking marine fungi as materials are cultivation of marine fungus Aspergillus terreus C23-3 derived from corals of the South China Sea by Zn Cl₂revulsant（terminal concentration:10m M）and extraction separation of its secondary metabolites.Ten compounds LJY-1～LJY-13 were isolated from marine fungus Aspergillus terreus C23-3 by using TLC,vacuum liquid chromatography,column chromatographies on silica,ODS column,preparative thin layer chromatography,gel chromatography,preparative liquid chromatography and recrystallization.On the means of 1D NMR technique,2D NMR technique,ultraviolet spectrum,TLC and literature report,compound LJY-1 and compounds LJY-3,LJY-4,LJY-6,LJY-8,LJY-9,LJY-10,LJY-11,LJY-13 were elucidated as lactones and butyrolactone I;territrem B;epi-aszonalenin A;aszonalenin;benzaldehyde,4-hydroxy-3-（3-methyl-2-buten-1-yl）-;mannitol;territrem C;benzoic acid,3,4,5-trimethoxy-2-[[2-[(2-[（3-pyridinylcarbonyl）amino]benzoyl]amino]-,methyl ester respectively;LJY-7 is a new compound.Structural elucidation of the remaining 4 compounds and biological tests are on going.The marine fungus Aspergillus terreus C23-3 were co-cultured with Bacillus subtilis,Rhodotorula mucilaginosa and Vibrio parahaemolyticus,respectively.It was found that the autoclaved thallus co-cultured with strain C23-3 groups could stimulate the marine fungus C23-3 to produce more butyrolactone I when cultured for 12 days.The co-culture method can stimulate strain C23-3 to produce some originally silenced active secondary metabolites by TLC,biological autoradiography and HPLC.The above studies indicate that the optimizing process conditions of anti-Alzheimer’s active ingredients of Rhodiola rosea L.and Polygala tenuifolia Willd.can be determined by comprehensive application of low or medium polarity,ultrasonic extraction,single factor experiment and orthogonal experiment.The marine fungus C23-3 contained abundant anti-Alzheimer’s disease related active compounds;Rhodiola rosea L.,Polygala tenuifolia Willd.and marine fungi C23-3 are important resources of seeking and developing anti-AD health products,functional foods or lead compound.