Protective Effects Of Polysaccharides Obtained From Paecilomyces Hepiali Fermentation Mycelium In Experimental Diabetic Animal Model

Paecilomyces Hepiali is an entophyte fungi separated from natural Cordyceps sinensis with significant pharmacological activities.According to the announcement of the Ministry of Health,Paecilomyces Hepiali has been approved as a fungal species that can be used for health food products.A great deal of researches have shown that the compositions and activities of Paecilomyces Hepiali mycelia are similar to that of Cordyceps sinensis.Jinshuibao capsule whose main active component is Paecilomyces Hepiali mycelia is widely used for benefit the lung and kindey.However,there are seldom researches about the antidiabetic and antinephritic activities of polysaccharides separated from Paecilomyces Hepiali mycelium.The study aims to evaluate the antidiabetic and antinephritic effects of polysaccharide separated from mutant strain of Paecilomyces Hepiali.This study will provide a theoretical support for the development of Paecilomyces Hepiali as a new antidiabetic and antinephritic drugs.Mutation breeding and optimization of fermentation technology are two efficient methods used in microbiology to obtain new high yield.In the present study,mutant Paecilomyces Hepiali PH40 which possesses higher mycelia was obtained via chemical mutagenesis combined with a series of screening process.The Paecilomyces Hepiali PH40 were continuously cultured for 10 generations,and data revealed that PH40 displayed a good genetic stability and the best fermentation period was 96 h.Furthermore,various chemometrics methods were applied to optimize the fermentation medium of Paecilomyces Hepiali PH40 progressively.Three parameters(glucose,yeast extract powder and zinc chloride)selected via Plackett-Burman designwere further optimized by response surface methodology.Optimum fermentation medium consisted of(g/L): glucose 21.9,yeast extract powder 15.74,peptone 10,monobasic potassium phosphate 0.5,magnesium sulphate 0.5,sodium chloride 0.5,zinc chloride 0.011 and vitamin B1 0.1.The mycelium yield of PH40 was 25.24 g/L,1.4-fold as high as that obtained from the original medium.Type ? 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 antidiabetic and antinephritic effects of the Paecilomyces hepiali mycelium water extract(PHC).After four-week metformin and PHC treatment,an increment of body weight,a decrement of plasma glucose,low levels of total cholesterol,and low density lipoprotein cholesterol in diabetic rats were observed.PHC promoted glucose metabolism by enhancing insulin,pyruvate kinase activity,and increasing the synthesis of glycogen.PHC normalized the disturbed levels of SOD,MDA,and GSH-Px in kidney.The inhibitory effects on the levels of interleukin(IL)-2,IL-6,IL-10,and tumor necrosis factor-? in serum and kidney revealed the protection of PHC against diabetic nephropathy.Compared with nontreated diabetic rats,four-week PHC treatment resulted in a decrement on nuclear factor kappa B(NF-?B)expression in kidney.These results show that Paecilomyces hepiali possesses antidiabetic and antinephritic effects which are related to the modulation of NF-?B activity.Polysaccharide is an important component of fungal mycelia with significant pharmacological activities.In this study,chemometrics methods were used to optimize the aqueous extract of polysaccharide from Paecilomyces hepiali mycelium.Three parmeters(temperature,time,solvent ratio)were optimized by response surface methodology and the optimum extration process were described as: Paecilomyces hepiali mycelium was extrated with 46.56 volumes of double distilled water at79.18? for 3.94 h.The average rates of polysaccharide is 13.08%.The candidate component PHEA was purified from Paecilomyces hepiali mycelium water extract by Sevag reagent,ethanol precipitation,DEAE-52 cellulose anion exchange column,and superdex G200 gel permeation chromatography system.According to the HPLC,PHEA had a molecular weight of 3011.47 k Da,and contained D-arabinose,D-mannitol,and D-glucose in a molar ratio of 2.65:1:1.04.No absorbance peak was caught at wavelength of 260 nm and 280 nm in UV spectra,suggesting the absence of nucleic acid and proteins.According to the TIR spectra,structure of PHEA was characterized by a strong –OH absorption peak,and C-H,C=O characteristic absorption peak.There are also C-O-C,C-O-H,pyran ring skeleton structure existing in PHEA.Oxidation-Smith degradation suggested that PHEA comprises(1?2),(1?4),(1?6)linkages.The antidiabetic and antinephritic effects of PHEA were further detected in db/db mice.After 8 weeks administration of 120 mg/kg Met and PHEA at doses of 10,20,40 mg/kg,a decrement of plasma glucose,glycated hemoglobin,body weight,food and water take were observed.The hypolipidemic activities of PHEA were observed by the normalization of T-CHO,TG,and HDL-C.PHEA promotes glucose metabolism by enhancing insulin,pyruvate kinase levels,and suppressing glycogen synthetase kinase activity.PHEA treatment significantly prevented blood glucose levels from shooting up after glucose feeding and the glycogen levels have a significant increment in the PHEA treatment mice.Compared with the nontreated db/db mice,PHEA normalized the disturbed serum and kidney levels of SOD,CAT,GSH-Px,MDA.The suppression of ICAM-1,frizzled-6,MCP-5,IL-1?,G-CSF,phosphor-AKT,phosphor-GSK-3?,phosphor-NF-?B,phosphor-STAT3 levels and the enhancement of CAT,HO-1,Nrf2 activities in kidney showed the protection of PHEA against diabetic nephropathy.Histologic and morphology examinations showed that PHEA repaired the damage to pancreas,liver,and kidney tissues in diabetic mice.These results show that PHEA possesses antidiabetic and renal protective effects and thus further support the use of Paecilomyces hepiali in patients with diabetes.