Study On Anti-tumor Effect,protection And Treatment Of Deafness Of Active Components From Two Mangrove Endophytic Fungi

Malignant tumors are major chronic diseases that endanger human health.They have become a leading cause for morbidity and mortality worldwide.Most of platinum drugs used to treat malignant tumors have ototoxicity,so it is absolutely essential to develop new drugs to prevent and treat ototoxicity while looking for anti-tumor drugs.Mangrove endophytic fungi,occurring at the tropical and subtropical zones,are widely recognized as one of the important sources of new biologically active compounds with novel structures and unique skeletons.In this study,the secondary metabolites of two endophytic fungi isolated from the leaves of mangrove plant Sonneratia caseolaris in Nansha Mangrove Nature Reserve of Guangzhou,Guangdong Province,were studied in order to obtain potential anti-tumor compounds with prevention and treatment of drug-induced deafness.The research contents are mainly as follows:In first part,strain Pseudopithomyces sp.1512101 was fermented in PYG liquid medium.The culture broth was extracted with ethyl acetate and concentrated.After naturally dried,the mycelia were extracted with methanol and concentrated under reduced pressure to afford an aqueous solution,which was then extracted with ethyl acetate.All the ethyl acetate fraction was concentrated under reduced pressure.Under the guidance of PLA₂ modified Fe₃O₄@PLGA-PEG magnetic nanoparticles?PLA₂-MNPs?combined with LC-MS established by our research group,the ligand compounds in strain Pseudopithomyces sp.1512101 were isolated and purified.Thirteen compounds were separated by silica gel column chromatography and Sephadex LH-20 column chromatography.Their structures were identified by NMR,MS and physicochemical properties,including 6 cyclic peptides,3sterols,2 alkaloids,1 lactone and 1 alkane:ergosterol?WGM1-1?,ergosterol peroxide?WGM1-2?,multiplolide A?WGM1-3?,cerevisterol?WGM1-4?,cyclo-?4-hydroxyl-Pro-Leu??WGM1-5?,cyclo-?Pro-Val??WGM1-6?,cyclo-?Pro-Leu??WGM1-7?,cyclo-?Pro-Gly??WGM1-8?,cyclo-?Gly-Ala??WGM1-9?,fusaristatin C?WGM1-10?,n-tetracosane?WGM1-11?,thymine?WGM1-12?,uracil?WGM1-13?.By comparing LC-MS information,WGM1-1,WGM1-2,WGM1-4,WGM1-5,WGM1-6 and WGM1-10 were identified as potential ligand compounds.Compounds WGM1-12 and WGM1-13 were primary metabolites obtained from endophytic fungus Pseudopithomyces sp.1512101.Many cyclic dipeptides and sterols were isolated from the secondary metabolites.Among them,cyclic dipeptides had a wide range of biological activities and were of great significance in finding drug targets.And sterols played an important role in the physiological functions of fungi.For example,WGM1-1 was an important component of fungal cell membrane,which played an irreplaceable role in maintaining the structure and function of cell membrane.It can be seen that studying the biological activity of the secondary metabolites of Pseudopithomyces sp.1512101 is of great significance for the discovery of active lead compounds.In the second part,endophytic fungus 1512111 was cultivated.The fermentation product was immersed with methanol,and concentrated under reduced pressure,which was then extracted with ethyl acetate.Six compounds were isolated and purified from the strain and their structures were identified by NMR and MS,including 2 diterpenes,1 dihydroisocoumarins,1 cyclic peptide,1 alkaloid,and 1 carboxylic acid:5-hydroxymellein?WGM2-1?,clavatustide C?WGM2-2?,dilactone 3?WGM2-3?,botryosphaerin A?WGM2-4?,succinic acid?WGM2-5?,uracil?WGM2-6?.Through the study of the chemical composition of the solid fermentation product of fungus 1512111,it was found that the chemical structures of the metabolites of this strain were rich.Combined with the relevant literature,WGM2-1 had a good antifungal activity.When the concentration of WGM2-1 was 1 mg/mL,the radius of zone of inhibition against Microbotryum violaceum was 5 mm.And WGM2-3had good anti-tumor activity(DU-145,GI₅₀=0.34?g/mL)and antibacterial activity?C.neoformans,MICs=16?g/mL?.In the third part,the s PLA₂ inhibitory activity screening kit and MTT method were used to investigate the anti-tumor activities of isolated ligand compounds.The experimental results revealed that WGM1-2,WGM1-5,WGM1-6,and WGM1-10 showed certain inhibition activities to sPLA₂ at 100?M.The inhibition rates were 21.5%,18.5%,21.2%,and 19.6%respectively.In addition,all the ligands had different anti-tumor activities through literature search.In this study,WGM1-10 was found to have better inhibitory activity on human lung cancer cell A549?10?M,73.3%?by MTT method for the first time.The results further illustrated that PLA₂-MNPs combined with LC-MS can be used to screen anti-tumor drugs from the secondary metabolites of endophytic fungi.In the fourth part,gentamicin and cisplatin mediated HEI-OC1 cochlear hair cell injury model and zebrafish hair cell injury model were used to investigate the effect of some compounds on the prevention and treatment of drug-induced deafness.The results showed that WGM1-1,WGM1-2,WGM1-3,WGM1-4,WGM1-8 and WGM2-5 had potential prevention and treatment effects on drug-induced deafness.And the literature found that WGM1-1,WGM1-2,WGM1-4 and WGM1-8 also had different degrees of anti-tumor activities.To sum up the results,it can be seen that sterols not only have good anti-tumor activity,but also have good prevention and treatment effect on drug-induced deafness.It is speculated that the hydroxyl active site at the C-3 position of sterol compounds can be structurally modified to form a series of related active derivatives.At the same time,the active site for preventing and treating deafness can be further investigated.In summary,the secondary metabolites of two mangrove endophytic fungi Pseudopithomyces sp.1512101 and 1512111 were studied and 19 compounds were isolated and identified.WGM1-10 was found to have better inhibitory activity on tumor cell A549 for the first time.At the same time,it was found that WGM1-1,WGM1-2,WGM1-4 and WGM1-8 not only had anti-tumor activities,but also had potential prevention and treatment effects on drug-induced deafness.The results of this study not only enriched the research content of marine natural products,provided a certain theoretical basis and scientific basis for the development and utilization of mangrove endophytic fungal resources,but also provided a certain research for finding anti-tumor compounds with prevention and treatment of drug-induced deafness.