Isolation And Antioxidant Activity Of Inhibitory Activity Of Subtances On Enzymes Producing By Inonotus Obliquus Under Submerged Fermentation

Inonotus obliquus is a rare large basidiomycete fungus of the family phellinus,which mainly lives in eastern Russia,northeast China,Europe and North America,etc(45°N-50°N altitude).It has been used in the field of medical and health care because of its widely biological activity and no reported side effects.Bioactive ingredients of I.obliquus are abundant,including polysaccharides,polyphenols,triterpenoids and etc.However,the rare wild resources of I.obliquus and its slow growth under natural conditions have greatly limited its research and development and utilization.The submerged fermentation technology is an alternative.I.obliquus could produce more metabolites during the fermentation with the supplementation of lignocellulose.Polysaccharides and polyphenols are the main substances in the liquid broth and have drawn wide attention due to hypoglycemic,antioxidant,anti-tumor,anti-aging activities and etc.In this paper,the dynamic effects of the addition of wheat straw and sugarcane bagasse on the production of exo-polysaccharides and exo-polyphenols were studied under submerged fermentation.Substances with dual activity of inhibiting a-glucosidase and antioxidant were screened out,and the compositions of the substances were analyzed using UPLC-MS.The results were as follows:(1)Dynamic fermentation:The dynamic effects of wheat straw and bagasse on the production of polysaccharides and polyphenols by Inonotus obliquus were studied by liquid fermentation.The yield of the two strains increased rapidly at first,entered a slow growth period,reached a maximum and entered a decay period,and the maximum time of the polysaccharides(1.35±0.39 g/L)and polyphenols(102.61±3.69 mgGAE/L)was the same.Therefore,in this study,the optimal yield of polysaccharides and polyphenols in submerged fermentation of Inonotus obliquus was determined to be on the 9th day.(2)Screening of substances with enzyme inhibition activity:Extracts from polysaccharides and different solvent fractions of polyphenols eluted from D101 resin chromatography were screened with a-amylase and a-glucosidase inhibition activity.Among them,water-soluble polyphenols effectively inhibited the a-amylase activity(IC50?40.31±1.43 mg/mL)and n-butanol-soluble polyphenols effectively inhibited the a-glucosidase activity(IC50=12.77 ± 0.47 mg/mL).The IC50 values of acarbose as the positive control against the two enzymes were 6.82±0.03 mg/mL and 2.13 ±0.02 mg/mL,respectively.Then,the water extract and n-butanol extract were further separated by D101 macroporous resin column to give five subfractions.The inhibitory activity of a-amylase was not significantly increased in the water extract after separation.The inhibitory activity of ?-glucosidase was remarkable with the 4th subfraction(IC5O=5.39±0.10 mg/mL)from n-butanol extract.(3)Evaluation of antioxidant activity:The antioxidant activity of polysaccharides and the 4th subfraction of n-butanol extract was further evaluated with the IC50 of DPPH,ABTS and hydroxyl radical scavenging activity.The results showed that the IC50 values of polysaccharides were 2.82±0.01 mg/mL,6.40 ±0.50 mg/mL and 0.82±0.04 mg/mL,respectively.The values of the 4th subfraction were 0.29±0.001 mg/mL,0.35±0.003 mg/mL and 0.32 ±0.001 mg/mL,respectively,indicating that it had strong antioxidant activity.(4)Component analysis:The conpounds of the 4th subfractionwere analyzed by TLC,HPLC and UPLC-MS.The results showed that the subfraction was composed of two lignans(C23H30O3 and C23H32O2).In conclusion,the submerged fermentation of L obliquus effectively improved its growth rate and made the extraction process easier.The 4th subfractionof n-butanol extract produced by I.obliquus under submerged fermentation with sugarcane bagasse had dual activities of ?-glucosidase inhibition and antioxidant.