Studies On The Optimization Of Submerged Fermentation Process,and Hypoglycemic And Anti-tumor Effects Of Grifola Frondosa

Grifola frondosa?also called Chestnut Mushroom or Polyporus frondosus in China?is a kind of rare edible and medical fungi,which belongs to Polyporales bacteria.A large number of studies found that G.frondosa has various pharmacological bioactivities including anti-tumor,enhance immunity,hypoglycemic,anti-virus,anti-radiation and scavenging free radicals etc.In recent years,many scholars at home and abroad have researched deeply and widely from its nutritional value,food value,medicinal value,cultivation techniques and new product processing or other aspects.At present,the chemical composition and pharmacological activity of G.frondosa were mainly studied in fruiting bodies and polysaccharides.However,the research on the biological function of the deep fermentation process of the mycelium of G.frondosa and the biological function of hypoglycemia and anti-tumor are relatively few,the depth and width need to be further improved.Although the yield of G.frondosa have been increasing year by year,but due to used to export mainly,resulting in a serious shortage of domestic market demand.Therefore,it is a great significance to improve the yield of G.frondosa,establish industrial production process and continue to carry out G.frondosa mycelium nutritional and pharmacological active substances study for the rational application of G.frondosa fruiting body and mycelia.In this study,chemometrics methods were employed in the optimization of the fermentation medium and condition.Plackett-Burman experiment and Box-Behnken design?BBD?experiment were used to obtained the best fermentation medium and conditions based on the single-factor optimization strategy.The optimum components of nutrient medium comprised?g/L?:glucose 19.44,yeast extract 10,peptone 10,KH₂PO₄ 1.70,MgSO₄ 1.53,VB₁ 0.10,under the condition of fermentation culture for5 days,initial pH 6,temperature 26?,rotating speed 125 r/min.The content of total sugar in G.frondosa mycelia was 9.23%,the reducing sugar was 3.23%,the triterpenoid compound was 2.49%,the flavonoid compound was 0.19%,adenosine was 0.1%,mannitol was 3.62%,crude fat was 28.16%and total protein was 31.55%.Hypoglycemic and antitumor activities of Grifola frondosa mycelia were studied under the optimum conditions of liquid submerged fermentation,which provided the theoretical basis for the better development and utilization of this fungus resource.Type II diabetic rats that established by eight-week high-fat diet administration followed by one-week tail intravenous injection of 25 mg/kg streptozotocin?STZ?were used to evaluate the hypoglycemic effects of the G.frondosa mycelium water extract?GFE?.After four-week GFE and metformin?Met?orally treatment,results showed that GFE increased the body weight,glucose tolerance?OGTT?and decreased the fasting blood glucose level in diabetic rats compared with the model group,which indicated that GFE have a better hypoglycemic activity.Creatinine?Scr?,urea nitrogen?BUN?,Albuminuria?ALB?and?-N-acetylglucosaminidase?NAG?are the markers of diabetic nephropathy,we found that diabetic rats have a higher level of Scr,BUN,ALB and NAG.After 4-week of GFE treatment,blood biochemical indicates showed that GFE significantly decreased the levels of Scr,BUN,NAG in serum and ALB compared with the model rats,which further demonstrated that GFE have a certain degree of protection and mitigation for kidney.The levels of Interleukin-2?IL-2?,interleukin-2R?IL-2R?,Interleukin-6?IL-6?,tumor necrosis factor-??TNF-??and interferon-??IFN-??in the serum were notablely reduced than those of rats from model group after 4-week of GFE and Met treatment,however increased the content of matrix metalloproteinase-9?MMP-9?.At the same time,GFE significantly increased the levels of Superoxide dismutase?SOD?,Glutathione peroxidase?GSH-Px?and Catalase?CAT?in serum,decreased Methane dicarboxylic aldehyde?MDA?and Reactive Oxygen Species?ROS?levels to normal compared with the model rats indicating that effective antioxidant activity.Interesting,GFE can effectively inhibit the level of nuclear factor-kappa B?NF-?B?in rat serum and kidney tissue compared with the model group.Therefore,we hypothesized that GFE possesses antidiabetic and antinephritic effects which may be mediated by NF-?B-related signalingThe an-titumor activity of GFE was investigated by in vitro cell experiment and in vivo experiment of nude mice.The effects of GFE on breast cancer and liver cancer were evaluated by using MCF-7 and MDA-MB-231,PLC/PRF/5 and HepG2 together with MCF-7 and PLC/PRF/5 induced tumor ectopic model respectively.The results showed that GFE significantly inhibited the viability of tumor cells,promoted the release of lactate dehydrogenase?LDH?,increased the content of ROS in tumor cells,decreased the mitochondrial membrane potential,and activated the mitochondrial apoptosis pathway,inducing tumor cells apoptosis.In vivo experiments,GFE effectively inhibited the growth of tumor tissue.In addition,GFE increased the levels of Bax,cleaved caspase-3 and cleaved caspase-8,and reduced the expression of Bcl-2and Bcl-x L protein in tumor tissue.All above datas further confirmed that AKT/GSK-3?and ERK-dependent mitochondrial apoptotic pathways are involved in GFE-mediated anti-tumor activity.