Study On Inhibition Of Acetylcholinesterase And Antioxidant Activity Of Metabolites From Plant Endophytic Fungi

Alzheimer’s disease（AD）is a chronic syndrome that causes progressive deterioration of the central nervous system,accounting for 60-70%of global dementia cases.The patients are mainly elderly,with memory impairment,executive dysfunction,personality and behavior changes,etc.,which seriously affect the life quality of patients and their families.The mechanism of AD is complicated which mainly includes cholinergic hypothesis,amyloid cascade hypothesis and so on.At present,acetylcholinesterase（ACh E）inhibitors are the most commonly used drugs for the treatment of AD.In addition,studies have shown that antioxidants can also improve the symptoms of AD to some extent.However,at present,these two types of drugs are very few,existing products are mainly chemical drugs with some disadvantages such as great side effects and short validity period.Therefore,the development of effective acetylcholinesterase inhibitors and antioxidants can have great significance for the treatment of AD.The active substances derived from natural products are of various types,novel structures,and rich in functions.They are one of the important sources of new drug research and development which can be used as new drugs or lead compounds.At present,certain components have been isolated from plants to improve AD symptoms including acetylcholinesterase inhibitors such as huperzine A and berberine hydrochloride,and antioxidants such as ursolic acid and ginkgolide.However,the growth cycle of plants is long and wild resources are relatively scarce.Plant endophytic fungi can produce metabolites with the same or similar structure as the host,as well as the biological activities.Compared with plants,Fungi have a shorter growth cycle,lower dependence on encironment,which make them more suitable for production.Therefore,Plant endophytic fungi could be an important resource bank for natural active substances.This study used Ellman’s method,DPPH removal experiment and cell experiment to screen endophytic fungal metabolites with activity of antioxidant and inhibiting acetylcholinesterase,aiming to discover new active ingredients as new drugs or lead compounds;Analysed the toxicity and reversibility of active metabolites;And used mass spectrometry to conducte a preliminary analysis of the structure of the active substance.The main research methods and results are as follows:1.In this study,193 plant endophytic fungi were isolated and collected purified from 50 plant specimens,belonging to 13 genera,including Aspergillus（20.7%）,Alternaria（16.1%）,Penicillium（14.0%）,Fusarium（12%）,Mucor（11.4%）,etc.2.The above-mentioned 193 fungi were fermented,then the fermentation products were extracted with obtain ethyl acetate and n-butanol.The extraction product were screened for ACh E inhibition and antioxidant activity using Ellman method and DPPH removal experiment.The results showed that the ethyl acetate extracts of strains Alt01,CL008,JTSQ-2 and HT-1 have ACh E inhibition and DPPH scavenging activities.Among them,the ACh E inhibition rate and DPPH clearance rate of the ethyl acetate extract of strain Alt01 were 82.13%and 79.39%,respectively,which were relatively high among the four candidates.Therefore,the strain Alt01 was selected for the further research.3.The strain Alt01 was identified as Alternaria alternata by morphology observation and molecular biology technology.4.It is preliminarily judged that the metabolite of strain Alt01 is a reversible acetylcholinesterase inhibitor by enzyme inhibition kinetic analysis,and it has a good activity under the condition of human p H（7.35-7.45）.The toxicity test results of strain Alt01 to Caenorhabditis elegans showed that its median lethal（LC50）concentration was 11.76 mg/m L,and it was non-toxic to nematodes at an IC50concentration for acetylcholinesterase inhibition,suggesting the prerequisites for becoming a clinical drug.5.In order to increase the output of active substances,the fermentation conditions were optimized.The results showed that the most suitable carbon source was potato extract,the most suitable nitrogen source was yeast extract,the most suitable inoculation spore volume was 8 m L×（1×10⁷）CFU/m L,the most suitable fermentation p H was 6.0,and the most suitable p H was 6.0.The optimum temperature is 28°C,and the optimum loading volume is 100 m L/250 m L conical flask.After optimization of the conditions,the dry weight of metabolites increased by 25%compared to the basal medium.6.In order to determine the effective components of Alt01 metabolites that inhibit ACh E,the obtained extraction products were separated by silica gel column chromatography,and a total of 19 components were obtained,of which components9 and 17 have high ACh E inhibitory activity.The inhibition rates were 91.08%and93.15%respectively.The molecular weights of components 9 and 17 were 569.1 and191.1 respectively,measured by LC-MS.GC-MS spectra shows that a main peak can be seen in the total ion chromatogram of component 9 with a retention time of 11.47min,which is inferred to be a single substance.According to the GC-MS library,it is preliminarily inferred that component 9 is a sterol alkaloid with 80%similarity.Component 17 is a multi-component mixture and needs to be further purified.7.In order to determine the active ingredients with antioxidant activity in the metabolites of Alt01,the DPPH inhibitory activity of the extracted products was tested by bioautography and TLC.There are four components with antioxidant activity,of which component g is the highest and may be the main active ingredient.The MTT method was used to detect the protective effect of component g on H₂O₂induced oxidative damage.The results show that the pretreatment of component g can effectively resist the oxidative damage of nerve cells induced by H₂O₂.The best concertration for protective effect is 100μg/m L.8.In order to further increase the expression level of the active product,the regulatory mechanism of its expression were discussed.The strains Alt01 and Alt02（Alternaria alternata with 79%ACh E inhibition rate obtained by the preliminary screening of this research group）and Alt03（Alternaria alternata without anti-ACh E activity）were selected for transcriptome sequencing analysis.The results showed that there were 1854 and 2473 differentially expressed genes in Alt01 and Alt02compared with the control strain Alt03,respectively,of which 789 were differentially expressed genes in common.KEGG pathway analysis showed that common differentially expressed genes are mainly involved in signaling pathways such as cysteine and methionine metabolism,glycolysis/gluconeogenesis,glyoxylic acid and dicarboxylic acid metabolism,and pyruvate metabolism.The pyruvate metabolism signal pathway is closely related to the synthesis of Steroidal alkaloids,and may be a key pathway that regulates the production of active substances from strain Alt01.In this study,a strain of Alternaria alternata was screened,and its metabolites have high ACh E inhibitory and antioxidant activities.The properties of its metabolites were analyzed,and the active components of its metabolites were separated.It was obtained that component 9 and component 17 had a higher inhibitory rate on ACh E,and component 9 was speculated to be a single substance,possibly a steroid.Somatic alkaloids,and explored the regulation mechanism of steroidal alkaloid synthesis may be related to the pyruvate metabolic pathway.The isolated antioxidant active component g has a good protective effect on H₂O₂induced oxidative damage.This study intends to screen new drugs and lead compounds that can be used as the treatment of AD to solve the current dilemma of disease treatment.