Identification And Evaluation Of Bioactivity Of Small Molecule Functional Components From The Mushroom Pleurotus Nebrodensis

Pleurotus nebrodensis is one of medicinal fungus regarded as a valuable source of nutritional and bioactive components,but most researches on it are concentrated on its polysaccharide and optimization of culture conditions.Using the fruiting body of P.nebrodensis as material,this experiment explored the bioactivity and mechanism of functional molecules.The results provided theoretical basis for further development and utilization of P.nebrodensis.The main results were as follows:The methanol extract was successively partitioned into n-hexane,ethyl acetate and n-butanol fractions.The chemical simulation system showed that the ethyl acetate fraction had strong radical scavenging activity with a dose effect relationship on DPPH·,ABTS~+·,O~2 and OH than other parts.The components in the ethyl acetate fractions were obtained through a variety of separation and purification technologies and the color reaction.The monomers were reported,for the first time,as follows:ergosterol(1),uracil(2),ergosterol-3-O-?-D-glucopyranoside(3),cerevisterol(4),cerebroside B(5),5'-methylthioadenosine(6),adenosine(7),uridine(8)and hypoxanthine(9).The results of MTT assay showed that compound ergosterol,ergosterol-3-O-?-D-glucopyranoside,cerevisterol and cerebroside B exhibited moderate or strong tumor suppressive activities.Among them,ergosterol exhibited the most superior inhibitory activity against human breast cancer MCF-7 cells,and cell viability was as low as21.01%when the concentration was 250?M.Besides,the list followed by cerebroside B,ergosterol-3-O-?-D-glucopyranoside and cerevisterol.Compound 2 and 6-9 were shown to be inactive on cells.Hoechst 33342 staining method observed typical morphological alterations of cell apoptosis,including blue fluorescence enhancement and formation of apoptotic bodies.Agarose gel electrophoresis found DNA ladder belt appeared in cells treated with ergosterol,indicating DNA degradation occurred.Flow cytometry indicated that the apoptosis rate significantly increased from 0.02%in control group to 53.88%in ergosterol group.Cellular proportion reduced from 98.23%to 73.85%at G0/G1 phase but increased from 1.76%to 24.39%at S phase,indicating cell cycle was arrested in S phase.Real-time PCR showed that ergosterol up-regulated the expression of Bax and down-regulated Bcl-2 level,promoted the release of cyt C,activated caspase-9 and-3.In conclusion,these results demonstrated that ergosterol could inhibit the MCF-7 cell proliferation by blocking cell cycle and inducing mitochondria-mediated apoptosis pathway.