Study On Acetylcholinesterase And Monoamine Oxidase Inhibitory Activity By Metabolites Of Endophytic Fungi

Endophytic fungi are fungi lived in the whole or part growth cycle of healthy plants with harmony and resulted no obvious disease of the plant.It can produce lots kinds of metabolite,and may produce substance with the same or similar activity of the host plant.So it can replace the plant to produce natural active products or be a sustainable source of them.Metabolites of endophytic fungi have wide bioactivities,which can be research as lead compounds of antibiotic,antiviral,anticarcinogen,antioxidant,hypolycemic agent,thrombolytic,pesticide,immunosuppressor and drugs for cardiovascular and cerebrovascular diseases.Alzheimer disease(AD)is a neurodegenerative disease of the central nervous system.The number of patients surge increase sharply with aging population.However,the category of drugs is less with poor effect.Acetylcholinesterase(ACHE)is the key enzyme of central and peripheral cholinergic nerve transmission.The loss of cholinergic neurons and synapses and impaired cholinergic nerve transmission in cerebral cortex and certain subcortical areas usually lead to the symptoms as cognitive deficiencies and memory loss of AD patients.Acetylcholine(Ach)can be increased and the cholinergic transmissioncan be improved by AChE inhibitors which can inhibit cholinesterase,and then the symptoms of cholinergic deficiency can be relieved.Inhibit the cholinesterase(mainly the AChE)is main strategy of curing severe ACh loss in central nervous system.Monoamine Oxidase(MAO)is a mitochondria enzyme containing flavin adenine dinucleotide(FAD)with a catalytic ability to deamination of neurotransmitters and biogenic amines.MAO includes MAO-A and MAO-B.Clinically MAO-A inhibitors used to treat depression,while MAO-B inhibitors mainly used in the treatment of Parkinson's disease(PD).Because of the important role in the nervous system,MAO is always the research focus of new drug developers and scholars at home and abroad.Nevertheless there is still short of inhibitors with high efficiency and selectivity.It is desiderated to discover a new MAO inhibitor with high selectivity and fewer side effects.In this research,we collected 50 plant specimens and isolated 216 endophytic fungi which belong to 13 genera.We extracted the metabolites with ethyl acetate and butyl alcohol respectively,and then tested AChE inhibitory activity.From them,we screened out two fungi which can produce metabolite with high AChE inhibitory activity.The Inhibition rate is 79.2% and 70.0%.After morphological and molecular identification,we thought the two fungi are Alternaria alternate and Aspergillus terreus.We fermented them on a large scale with solid fermentation,and then used ethyl acetate to extract the metabolite.Firstly,we study the elution polarity range of active component with 200 mL silica gel column,and then eluted the polarity range emphatically with 3400 mL silica gel column.After purification by recrystallization,component C-B-17 was obtained.Through UV-Vis,HPLC and LC-MS analysis,we know it's maximum absorption wavelength was 320 nm,the content was 89.2%,and the molecular weight was 464.2.Then we verified the activity with A? model of AD,CL4176 strain of Caenorhabditis elegans,the model organism.The result is the sample C-B-17-1 can prolong paralysis time of the C.elegans,which proved the sample C-B-17-1 has the potential to be a new drug of AD with further study.In addition,we separated and purified two metabolites stored in my laboratory.And then we tested MAO inhibitory activity of components by enzyme label method,picked out 20 components with MAO inhibition in total.The study was aim to screen fungi with high AChE inhibition and MAO inhibition in metabolites,which can be the lead compounds for new drugs treated diseases of the nervous system(such as AD,Parkinson's Disease,depression and so on),and end the dilemma of cure the diseases.