Research And Antitumor Activity Of Secondary Metabolites Of Pholiota Adiposa

In Asian dietary habits and traditional Chinese medicine,medicinal fungi(mushrooms)have been used as foods,health products,and even medicines for preventing and treating disease,as food supplements and treatments for diseases about hypertension,diabetes,inflammation,and cancer.Pholiota adiposa are medicinal fungi.With further research,it has been found that Pholiota adiposa secondary metabolites can be used to improve the immune system function of mice and may be used for the treatment of tumors.In this paper,the chemical constituents and antitumor research progresses of Pholiota adiposamedicinal fungi were studied.The effects of the new anti-tumor active substances of Pholiota adiposaon tumor cell proliferation inhibition,apoptosis induction and signal pathway were analyzed in depth,Provides the basis for deep research.This article aims to study the crude extractseffect of the Pholiota adiposa on non-small cell lung cancer(A549),human cervical cancer cells(Hela),mouse macrophage(RAW264.7)and Wistar rat cardiac fibroblasts(CF),the cytotoxicity and apoptosis of these cell strains were measured,and the components affecting the metabolites of the Pholiota adiposa of the above cells were further separated and purified by high performance liquid chromatography(HPLC)to decide a single component.By means of the double extraction method,crude extracts and spin-steaming of fresh Pholiota adiposa fruit bodies,re-extraction of methanol and spin-steaming methods are firstly performed using ethyl acetate to obtain Pholiota adiposasecondary metabolite extracts,and in Hela,CF,A549 and RAW264.7 were incubated with different concentrations of the secondary metabolites of Pholiota adiposaand incubated for 12 h,24 h,and 48 h,respectively.MTT assay was used to determine the cytotoxicity of different cells,and the mitochondrial membrane potential(JC-1)and Hoechst33258 staining was used to detect the apoptosis of different cells.The proportion of mobile phase methanol and water was adjusted separately in the optimized HPLC separation process,and the effluent was collected every 2 min.The isolates were obtained by Rotary evaporation and weighing,and 11 different components were detected by MTT assay oneffect of proliferation of A549 cells,respectively.We observed the effects of treatment with different concentrations of secondary metabolites of Pholiota adiposa on the morphology of cell strains.The results showed that the cell volume and nucleus were significantly reduced and gradually rounded,and the activity and adhesion of cells decreased significantly.Immediately afterwards,we examined the effects of different concentrations of secondary metabolites of Pholiota adiposa on the apoptosis of cell strains.Hoechst33258 staining showed that the normal cell nucleus was blue.In the other cell strains,the control group showed blue nucleus.In the apoptosis cell strains,there were obvious dense staining of the nucleus.The detection results of mitochondrial membrane potential showed that in Wistar rat cardiac fibroblasts(CF),the red and green fluorescence were distributed uniformly in the control and experimental groups,while in the other three cells,the cells were treated by the Pholiota adipose after the incubation of the material,the red fluorescence in the cells gradually decreased,and the green fluorescence gradually increased,thereby judging that the mitochondrial membrane potential gradually decreased in the cells.Since is a kind of semi-adherent cells,after treatment with secondary metabolites of Pholiota adiposa,it is easy to cause cell sheddingthis cell strain are not tested.In summary,the secondary metabolites of the Pholiota adiposa have the effect of inducing apoptosis.The results showed that the secondary metabolites of Pholiota adiposa all inhibited A549 and Hela cells,showing time and dose dependence,and had less inhibitory effect on RAW264.7 and CF cells,and had a promoting effect in short time of treatment.The detection of mitochondrial membrane potential(JC-1)and Hoechst 33258 staining indicated that secondary metabolites of Pholiota adiposa could induce apoptosis of A549 and Hela cells to varying degrees,whereas CF cells in normal Wistar rats had no such effect in a certain concentration and time range.In HPLC separation and purification,the concentration of the mobile phase methanol was set in the range of 20%-40%,40% methanol was used as the mobile phase,and the components were collected according to the separation time period;while 20% methanol was used as the mobile phase,the components were collectedaccording to peak.The results showed that the extract of Pholiota adiposa had a promoting apoptotic effect on A549 at 2-4min and 4-6min;the experimental results with 20% methanol as a mobile phase were at 5-6.5min and 6.5-10 min.The Pholiota adiposa extract has a promoting apoptotic effect on A549.According to the experimental data,secondary metabolites of the Pholiota adiposa play an important role in inhibiting proliferation and inducing apoptosis in cells.The main content of this paper is divided into six chapters: Chapter one is the introduction,mainly reviewing the progress of research on the metabolites of the Pholiota adiposa,and the effects of different components on the proliferation and apoptosis of tumor cells.Chapter II,CF isolation and purification and cryopreservation,resuscitation,and culture of cancer cells.Chapter 3,crude separation of metabolites from Pholiota adiposa.The fourth chapter,the study of the secondary metabolites of the Pholiota adiposa on cell proliferation and inhibition,mainly introduces the detection of MTT-cell activity and semi-inhibitory concentration.The fifth chapter,the primary study of apoptosis and apoptotic pathways of secondary metabolites of the Pholiota adiposa,mainly for RAW264.7,A549,Hela and CF five kinds of cell strains,using mitochondrial membrane potential and Hoechst33258 staining method to detection apoptosis of cell.In chapter 6,the secondary metabolites of the Pholiota adiposa were separated and purified by HPLC.Different sections were collected and the effects on the proliferation and apoptosis of A549 cells were detected.The individual components were separated as far as possible.Chapter VII summarizes and forecasts,summarizes the main conclusions of the experiment,and looks forward to the follow-up work.