Safety Evaluation And Anti-tumor Mechanism Of Solid Fermentation Products Of Fomitopsis Pinicola

Fomitopsis pinicola?Sw.?P.Karst is a kind of macrofungi distributed worldwide,and also one of the most common wood-rooting fungi in the northern hemisphere.The chemical composition of F.pinicola are mainly polysaccharides,steroids,terpenoids and alkaloids,etc;With anti-tumor,anti-bacterial,anti-obesity,anti-fungal,anti-inflammatory analgesic,immune regulation,liver protection,anti-oxidation,treatment of diabetes and other effects.F.pinicola were taken to treat various diseases in In Chinese folk,but not yet developed and utilized.In view of the needs of industrial production,the solid fermentation process has been initially established by our group and the fermentation product has been obtained.In this paper,the fermentation process of F.pinicola was optimized,and the safety evaluation was carried out,in order to lay the foundation for the development of new resource foods.The extraction process of FPOA,which is the active component of solid fermentation products,was further studied and compared with the extraction process of F.pinicola.Combined with the previous research results,the anti-tumor mechanism of FPOA was further studied in order to provide a scientific basis for the development of innovative drugs.Based on the previous studies,primary medium is designed as follows:yeast extract powder6 g/L,glucose 20 g/L,peptone 5 g/L,MgSO4 0.5 g/L,KH2PO4 1 g/L and vitamin B1 0.1 g/L.Corn is used as medium for solid fermentation,then the culture medium was placed in a 27?culture biochemical incubator and cultured continuously for 25 days,the mycelium grows most intensively at that time.Acute toxicity test in rats was carried out to evaluate the safety of solid fermentation products of F.pinicola.This experiment was carried out every morning and 7.19g/kg was taken as the maximum dose for oral administration.During the observation of the whole test,there was no death or other abnormal symptoms in the administration group of the male and female animals after the drug was given.The results of body weight showed a normal growth trend at different times,and the related toxic symptoms were not observed with the solid fermented product of the red margin.The experimental results show that the LD50 of solid fermentation products of F.pinicola is greater than 7.19g/kg.On the basis of acute toxicity test,the toxicity test of SD rats was carried out for 1 months.The experiment was carried out every morning and the dose of oral administration was 1.275g/kg,2.550g/kg,5.100g/kg.At the end of the dosing period,the remaining animals were observed for4 weeks of recovery,the reversibility,persistent,and delayed toxicity of the toxic response were observed.The safety was evaluated by clinical observation,ophthalmologic examination,weight change,hematological examination,blood biochemical examination,coagulation index,urine routine examination,gross anatomy and histopathological examination.During the whole test,there was no death or abnormality in the subjects;The results of ophthalmologic examination showed that all the animals were normal in the eyes,cornea,iris,optic disc and fundus;The average body weight of the control group and the administration group was on the rise;The average feeding of the male and female group did not detect the difference of drug related compared with the control group.At the end of the administration period and the recovery period,the animals were tested for hematology,blood biochemistry,hemagglutination,and routine urine tests.Compared with the same sex control group at the same time,the total number of WBC in the high dose male group was higher than that of the control group?p<0.05?,the content of AST in the middle dose female group was higher than that of the control group?p<0.05?,the content of T-BIL in the low dose female group was higher than that of the control group?p<0.05?.However,fluctuations in the normal reference range of animals are not toxicologically significant.There was no statistical difference between the blood coagulation index and the routine urine test.The results of organ weight showed that although there was a significant difference between the individual index and the control group,but because of lack of dose response relationship or lack of consistency of the three,the overall consideration was not related to the drug factors.At the end of the administration and recovery period,anatomical and histopathological examinations of animals in the control and high-dose groups showed liver vacuolar degeneration,focal necrosis,hepatic hemorrhage,renal tubular epithelial cell regeneration,uterine dilatation etc.However,there was no statistically significant difference between the two groups,and the histological findings were also common lesions in SD rats,with no statistical difference.Based on the above results,under the experimental conditions,the solid fermentation products of F.pinicola.was given to SD rats for 5 weeks by gavage,and the recovery period was 4 weeks,the index of body weight,food intake,hematology,blood biochemistry,hemagglutination,urine and ophthalmology were not significantly affected.The level of no visible harmful effect?NOAEL?was 5.1 g/kg·bw in SD rats,it is preliminarily identified that the solid fermentation products of F.pinicola.is safe.The extraction process of FPOA,which is the active component of solid fermentation products,was further studied and compared with the extraction process of F.pinicola.The solid fermentation products were extracted with petroleum ether and ethyl acetate,although the yield is lower than that of F.pinicola,however,solid fermentation products can be rapidly obtained in large quantities to ensure the sustainability of raw materials,and reagents can be reused many times to avoid wastage and reduce costs.The stability of the solid fermentation products is further studied,the experimental results showed that there was no significant change in the appearance,color and content of FPOA after 6 months.Combined with the previous research results,the anti-tumor mechanism of FPOA was investigated using human cervical cancer Hela cells.The purpose of our experiment was to elucidate the mechanism of action of FPOA in Hela cervical cancer cells.Cell viability was examined using an MTT assay,and the morphological detection of apoptosis was conducted using DAPI staining.The rate of apoptosis was examined via Annexin V-FITC/PI double staining,and the expression of apoptosis-associated proteins was determined by western blot analysis.FPOA was observed to inhibit HeLa cell proliferation,with IC₅₀ values of 25.28,15.30 and 11.79?g/mL at 24,48 and 72 h,respectively.Nuclear chromatin condensation,nuclear condensation and blue fluorescence enhancement were observed in the HeLa cells following treatment with FPOA,as revealed by DAPI staining.The percentage of apoptotic cells was 3.00,3.12,6.18 and 32.28%following treatment with FPOA at concentrations of 0,7.5,15 and 30?g/mL,respectively.Western blot analysis showed that caspase-3 and-9 were cleaved more frequently and PARP was cleaved after treatment with FPOA.Furthermore,the expression of Bax was increased,while that of Bcl-2 was decreased,after treatment with FPOA.The results show an increase in ROS with increasing doses of FPOA.These results suggest that FPOA can induce HeLa cell apoptosis through mitochondrial pathway.