Isolation And Identification Of Anti-tumor Active Substances In Secondary Metabolites Of Pholiota Adiposa

As an important part of natural medicine resources,edible and medicinal fungiplay an important role.Nowadays,a variety of physiological active substances,such as polysaccharides,peptides,proteins,terpenoids,steroids,lectins,etc.,have been isolated from the edible and medicinal fungal fruit bodies,and the biological functions such as antioxidant,anti-inflammatory,anti-tumor,hypoglycemic,blood pressure lowering and immunomodulatory have been verified by experiments.As a kind of medicinal fungus,Pholiota adiposa is the most potential food medicinal fungus at present,and it has abundant edible value and medicinal value.With the further research,some studies have found that the secondary metabolites of P.adiposa can improve the body immune system and inhibit certain tumor cells,which provides a favorable evidence for the further study of P.adiposa.The purpose of this paper is to extract the anti-tumor active substances in the secondary metabolites of P.adiposa,and to isolate and identify them,which provides a new idea for the research and development of natural metabolites.The secondary metabolite in the liquid nitrogen-milled fresh P.adiposa fruit body powder is extracted by ethyl acetate extraction method,and after repeated filtration,rotary steaming,methanol dissolution,drying and so on,the secondary metabolite of P.adiposa were finally obtained.The anti-tumor activity was determined by MTT colorimetric assay,and the secondary metabolite of P.adiposa were isolated by the combination of in vitro anti-tumor activity tracking and chemical separation.High Performance Liquid Chromatography(HPLC)was used to separate antitumor active components from secondary metabolites,and chemical structures were identified based on physicochemical properties and spectral data,mainly by Gas Chromatography-Mass Spectrometry(GC-MS),Liquid Chromatography-Mass Spectrometry(LC-MS)and Nuclear Magnetic Resonance(NMR).The results of the specific study are as follows:1.Extracted from ethyl acetate shaker to finally obtain secondary metabolite extract of P.adiposa.The liquid nitrogen-milled fresh P.adiposa fruit body powder was added with1.5 to 2 times of ethyl acetate solution 72 h,finally the weight of secondary metabolite extract of P.adiposa was 7.59 g through steaming by rotary evaporator.2.The secondary metabolite of P.adiposa was separated by C18 semi-preparative column and C18 analytical column,which obtains 5 peak spectra and 3 active peak spectra,respectively.The secondary metabolite extract of P.adiposa was fully dissolved by methanol and then separated by HPLC,firstly,5 peak spectra was obtained by C18semi-preparative column according to the ratio of methanol:ultrapure water=20%:80%,the time period is 2~4 min,4~4.5 min,4.5~7 min,7~10 min and10~14 min;Secondly,two highly active peak spectra in the semi-preparative column according to the results of the MTT method was separated by the C18 analytical column,three active peak spectra were obtained,the first peak spectrum was in the range of 6.8 to 9.8 min,the second peak was 4.2 to 8 min,and the third peak was 18 to 20.5 min.3.Anti-tumor activity of secondary metabolites of P.adiposa was measured by MTT in vitro,it was finally determined that a total of 5 of all the peak spectra obtained by HPLC separation had higher antitumor activity.MTT can be reduced to a crystalline dark purple product formazan by some dehydrogenases in the mitochondrion,which it can be completely dissolved in a specific solvent,and then the Optical Density is measured near the wavelength of 570 nm using an enzyme-labeled instrument.The peaks obtained by two HPLC separations were subjected to MTT assay,and five of them were found to have high antitumor activity.4.The pure product obtained by HPLC was identified,and the possible relative molecular mass and structural formula of the active material in the peak No.3 were obtained.The pure cotton with antitumor activity isolated by HPLC was identified by Gas Chromatography-Mass Spectrometry(GC-MS),Liquid Chromatography-Mass Spectrometry(LC-MS)and Nuclear Magnetic Resonance(NMR),the characteristics of its molecular formula and chemical structure were obtained,the relative molecular mass and structural formula of the active material in the peak No.3 were initially determined by the above method.