Study On Active Components Of Two Mangrove Symbiotic Actinomycetes For Preventing Deafness

Deafness is the most common sensory disability in the world,which refers to hearing loss or even disappearance in varying degrees.Drug-induced deafness is one of the main causes of deafness.With the extensive use of chemotherapeutic drugs and antibiotics in recent years,nearly 100 kinds of drugs can cause drug-induced deafness in clinic.Therefore,it is necessary to develop new drugs to prevent and treat ototoxicity.Scientists believe that drug-induced deafness is caused by excessive reactive oxygen species in vivo,which can cause ear inflammation and damage cochlear hair cells,leading to deafness.At present,models for investigating drug ototoxicity have been reported,which provide a new platform for the discovery of active ingredients for the prevention and treatment of drug-induced deafness.Marine natural products with novel structure and diverse biological activities are important resources for drug research and development.Among them,mangrove ecosystem has abundant microbial resources,which are important sources of marine natural products.A large number of actinomycetes with COX-2inhibitory activity have been screened out from animal samples in this ecosystem.Therefore,the discovery of leading compounds with the activity of preventing and curing deafness from microbial metabolites in mangrove ecosystem provides a new idea for the study of active drugs for preventing and curing deafness.In this study,the symbiotic actinomycetes isolated from the epidermis of Phascolosoma esculenta in the mangrove ecosystem of Zhanjiang,Guangdong Province,were studied.Strains 1624105 and1624107 were selected for further study by combining COX-2 inhibitory activity with chemical composition analysis by HPLC.Two different screening techniques were used to guide the isolation and purification of secondary metabolites.Twenty-seven compounds were obtained and identified.The COX-2 inhibitory and prevention of ototoxicity of some compounds were evaluated.The results were as follows:In the first part,liquid culture of strain 1624107 was carried out,and the fermentation product was obtained by ethyl acetate extraction.Under the guidance of the fixed-point fingerprint profiles predicted by OPLS-DA model established by COX-2 inhibition activity and UPLC-MS fragmentation information,nineteen compounds were obtained by silica gel column chromatography,Sephadex LH-20 column chromatography,HPLC,ODS and recrystallization.Their chemical structures were identified by NMR,MS and physicochemical properties.And the absolute configuration was determined by Marfey's analysis.They were identified as phenylacetic acid?YZC1-1?,pentadecanoic acid?YZC1-2?,hexadecanoic acid?YZC1-3?,4-hydroxy-3-methoxybenzoic acid?YZC1-4?,cyclo-?L-Pro-L-Thr??YZC1-5?,cyclo-?Pro-Ile??YZC1-6?,nicotinic acid?YZC1-7?,ethyl 3,4-dihydroxybenzoate?YZC1-8?,ethyl gallate?YZC1-9?,3-phenyllaclaclactic acid?YZC1-10?,p-hydroxbenzoic acid?YZC1-11?,succinic acid?YZC1-12?,thymine?YZC1-13?,cyclo-?Pro-Val??YZC1-14?,uracil?YZC1-15?,dihydrouracil?YZC1-16?,2-furoic acid?YZC1-17?,daidzein?YZC1-18?,genistein?YZC1-19?.Among them,the MS information of compounds YZC1-3,YZC1-6,YZC1-10 and YZC1-19 were corresponded to the MS fragments of fixed-point fingerprint profiles.In the second part,the liquid fermentation of strain 1624105 was carried out.The three-phase extracts were extracted by petroleum ether,ethyl acetate and n-butanol from the fermentation broth and mycelium respectively.The activity of the extract was investigated by using the HEI-OC1 cell damage model induced by gentamicin and cisplatin.The results showed that ethyl acetate extract had protective effect in cisplatin model group.Under the guidance of bioassay-guided fractionation,eight compounds from ethyl acetate extract were obtained,and their chemical structures were identified by NMR,MS and physicochemical properties as follows:11-methyl-?^1,5-diene-3,7,10-tricarbonyl-2,4,8,9-tetranitrogen-cycloundecandien?YZC2-1?,4-hydroxybenzylcyanide?YZC2-2?,2,3-dihydroxybenzoic acid?YZC2-3?,4-hydroxyphenylacetic acid?YZC2-4?,cyclo-?Pro-Ile??YZC2-5?,phenylacetic acid?YZC2-6?,stigmasterol?YZC2-7?,cyclo-?Phe-Ala??YZC2-8?.Compound YZC2-1was isolated from secondary metabolites of actinomycetes for the first time.In the third part,COX-2 inhibitory activity of the target compounds were investigated by using COX inhibitor screening kit to validate the accuracy of OPLS-DA analysis model established.The blank control group,the positive drug group and the sample group were set up respectively.Celecoxib was used as the positive control group.The results showed that compounds XY-1,YZC1-6,and YZC1-10 showed certain inhibition activity at 50?M.The inhibition rates were 31.7%,41.5%and 42.1%respectively.In the fourth part,the effects of some compounds on the prevention and treatment of deafness were investigated by using HEI-OC1 cells damage model and zebrafish hair cells damage model induced by gentamicin and cisplatin.Blank control group,model group,positive drug group and sample group were set up respectively.Edaravone and N-acetylcysteine were used as positive control group.The results showed that compounds YZC2-4 and YZC1-6 had significant protective effects on HEI-OC1 cells in the cisplatin model group,while compounds YZC1-5,YZC2-1,YZC2-2,YZC2-3 and YZC2-6 had no significant activity in the two model groups.Two Compounds were further evaluated by zebrafish model.The results showed that compound YZC1-6 has significant protective effects on zebrafish hair cells in the cisplatin model group.In summary,the secondary metabolites of two marine symbiotic actinomycetes 1624105 and 1624107 were studied by two different natural product screening techniques,and twenty-seven compounds were obtained and identified.COX-2 inhibitory activity and ototoxicity of some compounds were investigated by COX inhibitory activity screening kit,HEI-OC1 cells damage model induced by gentamicin and cisplatin and zebrafish hair cells damage model.Different from the traditional screening methods,the fixed-point fingerprint profiles based on OPLS-DA analysis model and bioassay-guided fractionation can quickly obtain the active components from natural extracts.These findings show a certain application value for the research of the active components which were used to prevent and treat the deafness.