Process Optimization,characterization And Bioactivities Of Hedysarum Polysaccharides Extracted By Complex Enzyme Combined With Ultrasonication

Hedysarum polysaccharides(HPS)constitute the principal active fraction of Radix Hedysari.The obtaining HPS through traditional water extraction has its disadvantages,including a low extraction rate,poor solubility,difficult purification,and the high molecular weight of natural structure polysaccharides,and greatly limits their bioavailability.Therefore,there is an urgent need to develop a simple,effective and environmentally friendly new extraction method.This molecular modification technique involving enzymatic treatment and ultrasonic degradation can change the spatial structure,reduce the molecular weight,and improve the solubility of polysaccharides,which affects their biological activities.To date,there has not yet been reported on the optimization process,physicochemical properties and biological activity of HPS extracted by a method involving extraction combined with modification.In this study,complex enzyme were combined with ultrasonication extraction method(MC),the yields and contents of HPS as a comprehensive index,to select optimal extraction combinations by single factor and orthogonal test.The physicochemical properties and bioactivities of polysaccharides extracted by complex enzyme combined with ultrasonication extraction(HPS-MCs: HPS-MC?HPS-MC(50%)and HPS-MC(80%))and hot water extraction(HPS-R)were compared.In order to investigate the multiple relationships between the structural characteristics and immunomodulatory activity by a more comprehensive way,HPS-MC(80%)was fractionated into three major homogeneous polysaccharide fractions(HPS-MC(80%)s: HPS-MC(80%)-1,HPS-MC(80%)-2,and HPS-MC(80%)-3)by DEAE-52 column chromatography.HPS-MC(80%)s were characterized by chemical analysis,gas chromatography(GC),high-performance gel permeation chromatography coupled with multi-angle laser light scattering(HPGPC-MALLS),Fourier transform infrared spectroscopy(FT-IR),and scanning electron microscopy(SEM).The effects of HPS-MC(80%)s on the proliferation of macrophages and splenocytes were investigated either by themselves or in combination with lipopolysaccharide(LPS).The results show that: The optimum technological parameters were compound enzyme ratio 1:1,ultrasonic power 105 W,ultrasonic time 60 min and enzymatic hydrolysis of pH=5,the yields and contents of polysaccharides were respectively 14.01±0.64% and 92.45±1.47%,the yields and total carbohydrate contents of HPS-MC were significantly higher,and the molecular weights,absolute viscosity and protein contents of HPS-MC decreased.In vitro antioxidant system,the concentration of polysaccharide is within the range of 1-7 g/L,the antioxidant activity of HPS-MC was higher than that of HPS-R,and the lowest molecular weight of HPS-MC(80%)showed a significant dose effect relationship with the increase of the experimental concentration.Under the immunosuppressive condition induced by cyclophosphamide(CP)in mice,compared with HPS-R,the dosage of polysaccharide was 100~400 mg/kg·d HPS-MCs could improve the immune organ index,the content of Interleukin 4(IL-4),Tumor necrosis factor ?(TNF-?)and Interferon ?(IFN-?)in serum,decreased the content of malondialdehyde(MDA)in liver homogenate,and increased the activities of total superoxide dismutase(T-SOD)and total antioxidative capacity(T-AOC)in liver homogenate.In particular,HPS-MC(80%)had the strongest ability to reverse immunosuppressive injury induced by CP in a dose-dependent manner in mice.Consequently,three homogeneous polysaccharide fractions,HPS-MC(80%)-1,HPS-MC(80%)-2,and HPS-MC(80%)-3,were further isolated from HPS-MC(80%)by DEAE-52 column chromatography.These three polysaccharide fractions had different structural characteristics,including monosaccharide compositions,molecular weights,glycosidic linkage configurations,and chain conformations in saline solution.Furthermore,the three homogeneous polysaccharide fractions,especially HPS-MC(80%)-2 and HPS-MC(80%)-3,significantly stimulated splenocyte and macrophage proliferation,which contributed to their monosaccharide composition and molecular weight,etc.To sum up,compared with conventional hot water extraction,MC is an economical,environmentally friendly and efficient method.The HPS extracted by this method has higher yields,purity,antioxidant activity and immunomodulatory activity,especially HPS-MC(80%).