Study On The Preparation,structure Characterization And In Vitro Bioactivities Of Sulfated Schizophyllan

Schizophyllan,a neutral?-glucan polysaccharide,exhibits various biological activities,including anti-tumor,immunomodulatory,anti-inflammatory and moisturizing activities.However,the large-scale application of schizophyllan in industry is limited due to its large molecular weight,high viscosity and poor resolubility.To provide theoretical basis for the application of sulfated schizophyllan(S-SPG)in medicine,cosmetics and other industries,this study aimed to investigate the sulfated modification of schizophyllan and its structure characterization via infrared,ultraviolet and nuclear magnetic resonance(NMR)methods,and further evaluate its?-glucosidase inhibitory activity,anticoagulant activity and moisturizing activity.Based on the single factor method,response surface method was utilized to optimize the process of surfated modification,where the sulfur trioxide-pyridine method was used to prepare sulfated schizophyllan using N,N-Dimethylformamide(DMF)as reaction solvent.The obtained optimal reaction condition was as followed:the mass ratio of sulfur trioxide-pyridine complex to schizophyllan was 4:1,the reaction temperature was 70^°C,the reaction time was 3h,and the maximum degree of substitution was 1.85.The result of solubility experiment showed that the sulfated schizophyllan possessed stronger water solubility compared to schizophyllan.Afterwards,four sulfated schizophyllan with different substitution degrees were obtained by changing diverse preparation conditions:S-SPG1(DS=0.70),S-SPG2(DS=1.00),S-SPG3(DS=1.25)and S-SPG4(DS=1.85).The structure of the prepared sulfated schizophyllan was characterized.The molecular weight of schizophyllan,S-SPG1,S-SPG2,S-SPG3 and S-SPG4 were 3.79×10⁷ Da,1.67×10⁷Da,9.32×10⁶ Da,3.04×10⁶ Da,and 8.04×10⁵ Da,respectively,indicating the molecular weight of schizophyllan reduced remarkably during the modification.The UV and FT-IR spectras proved that the sulfated group was successfully inserted into schizophyllin.The ¹³C NMR spectroscopy data showed that during the sulfation of schizophyllan,the C-6 might be the first position to be substituted by sulfate,followed by C-2 and C-4.In addition,only C-6 substitution was occurred in S-SPG1 and S-SPG2 withnot fully sulfation,C-6 substitution was the dominant substitution in S-SPG3 with slight C-2 substitution,C-6 substitution was the predominant substitution in S-SPG4 with different degrees of substitutions of C-2 and C-4.The SEM results suggested that the modified schizophyllan had different morphological surface than before.The congo red experiment indicated that sulfated modification destoried the triple helix structure of schizophyllan,and modified schizophyllan showed irregular linear structure in aqueous solution.The inhibitory activity of sulfated schizophyllan on?-glucosidase was studied,and the results showed the IC₅₀ of S-SPG1,S-SPG2,S-SPG3,and S-SPG4 were>2 mg/m L,1.74mg/m L,0.85 mg/m L and 0.80 mg/m L,respectively;at the concentration of 2 mg/m L,their inhibition rates were 38.68%,56.48%,78.81%and 84.66%,respectively,and the inhibitory activity of S-SPG3 and S-SPG4 on?-glucosidase exceeded that of acarbose.The kinetic experiment confirmed sulfated schizophyllan was a reversible mixed type I inhibitor on?-glucosidase.The anticoagulant activity of sulfated schizophyllan was evaluated.Results showed that S-SPG1,S-SPG2,S-SPG3 and S-SPG4 exhibited potential anticoagulant activity via the endogenous coagulation pathway including prolonging APTT and TT compared with schizophyllan.The values of APTT and TT were positively correlated with the concentration and degree of substitution of sulfated schizophyllan.S-SPG4 with the highest degree of substitution showed the strongest anticoagulant activity.At the concentration of 0.2 mg/m L,the APTT values of S-SPG1,S-SPG2,S-SPG3,and S-SPG4 were 76.40±1.70 s,86.40±1.30 s,98.70±2.40 s,184.35±1.55 s,respectively;while that of TT were 43.85±3.95 s,70.75±2.75s,77.10±0.10 s,>90 s,respectively,however,there was no prolongation effect on PT.The moisturizing activity of sulfated schizophyllan in vitro was investigated.When the relative humidity of environment was 43%and 81%,the moisture absorption of sulfated schizophyllan was 1.3-2 times than that of schizophyllan;concretely,the moisture absorption of S-SPG4 at 72 h was 77.93%(RH=43%)and 52.54%(RH=81%),respectively,which just inferior to glycerin.In the dry environment,the moisture retention of sulfated schizophyllan was slightly weaker than that of sodium hyaluronate.When the relative humidity was 43%,the moisture retention of S-SPG4 was only inferior to glycerin,but slightly stronger than that of schizophyllan.Therefore,sulfated schizophyllan can be utilized as a potential and promising moisture retention reagent.