Screening And Purifying Monoamine Oxidase Inhibiror From Metabolite Of Endophytic Fungi

Monoamine oxidase,a kind of flavin-containing and mitochondrial bound enzyme,which catalyzes the oxidative deamination of monoamine and keep the level of monoamine in our body.Monoamine is the most important and common kind of neurotransmitters,MAO abnormally rised will affect the level of monoamine in the body,they show the trend of negatively related.MAO include MAO-A and MAO-B,MAO abnormally rised can lead to Neuropsychiatric disease,such as depression,Parkinson's disease,alzheimer's disease,these desease bringing serious influence to the patients and their family.In recent years,the diseases caused by abnormal changes of monoamine oxidase is on the rise,and it is lack of ideal medicine for these diseases,monoamine oxidase inhibitors in the treatment play a groundbreaking role in the process of mental illness.The species of clinical monoamine oxidase inhibitors are little and the source of chemical synthesis,although treatment effect of the monoamine oxidase inhibitors is good,but its side effects in the treatment are liver toxicity,renal toxicity and so on.In order to develop novel and low side effects of natural sources of monoamine oxidase inhibitors,natural sources of monoamine oxidase inhibitors become a hot research topic in recent years.The source of most of them are medicinal plants,the research of it from endophytic fungi metabolites is rarely reported.This article screened natural compound from endophytic fungi's metabolites,which can inhibit MAO,in order to find a compound can inhibit MAO effectively as MAO inhibitirs or precursor of inhibitors.This study chose 23 strains fresh plant from changbai mountain,baicheng,liaoyuan jilin province and Ning Bo Zhejiang province,separating endophytic fungi 150 strains,through morphology and molecular identification,the endophytic fungi belonging to 14 genera.Choose aged males Wistar rat liver as the source of MAO,use differential velocity centrifugation method and density gradient centrifugation method get the raw MAO.determination protein content of the raw MAO is 0.85 mg/m L and specific activity of MAO is 23.18 U/mg.The enzyme extract of this experiment has accorded with reported literature,it could be used screening monoamine oxidase inhibitor.Regard these 150 kinds of metabolite as a research sample which extract with ethyl acetate,screening the monoamine oxidase inhibitior.When the concentration of fermentation product is 16mg/m L,the inhibitory rate of five strains endophytic fungi fermentation over 40%,there are two strains exceeded 75%,named CBS47-4 and NB62-2.According to the identification of morphology and molecular biology,CBS47-4 is Chaetomium aureum,its inhibitory is 77%,NB62-2 is Aspergillus terreus,its inhibitory is 78%.Optimizing the fermentation conditions of NB62-2,use the single factor test determine the carbon source and nitrogen source,use orthogonal design test determine the p H,temperature,fluid volume,inoculation,fermentation time.Regard the inhibitory rate and product dry weight as the main reference index.The optimized result is glucose provides carbon source,yeast extract provides nitrogen source,28?,p H 7,fluid volume is 40%,0.5% inoculated quantity,fermentation time is 7 days.Optimized the product of dry weight of 35.5 mg,the inhibition rate was 82.74%,compared with before optimization,the dry weight improved 18.87 mg,the inhibition rate increased 12.49%.Through large-scale fermentation to NB62-2,the raw product is 21.24 g,its inhibition rate is 82.35%,make use of silica gelcolumn chromatography,TLC,High Performance Liquid Chromatography and so on,we separated a single pure compound N7,the inhibition rate of N7 is 96.5%,its IC50 is 0.12mg/m L.Analysis N7 with LC-MS,molecular weight is 425.16,it may be a kind of Small Molecules.the inhibition to MAO-A is 67.25%,the inhibition to MAO-B is 89.53%.we can judge from the result that N7 inhibite both MAO-A and MAO-B,but its inhibitory for MAO-B more obvious.This research screening MAO inhibitor N7,it can restrain the monoamine oxidase effectively.MAO inhibitory activity of N7 better than that the same kind of fungus source of compounds which has been reported,added a potential new members to the source of fungus compound libraries of monoamine oxidase inhibitors.The active substances from nature brought the new skeleton to the chemical synthetic drugs,for the drug development laid an important foundation in the future.