Extraction And Grading Of Polysaccharides From Mulberry(Morus Alba L.)Leaves And Their Effects Against Radiation-induced Oxidative Stress

Mulberry leaf polysaccharides?MLPs?,the main active components of mulberry leaves,have multiple biological activities such as antioxidant,anti-diabetic,anti-tumor,anti-inflammatory and immune-stimulatory.In this study,mulberry leaves were used to optimize the extraction process,isolate polysaccharide fractions,study physicochemical properties and investigate the anti-radiation effects of polysaccharide?MLP80%?.SEM analysis of mulberry leaf powder residues after ultrasonic assisted extraction and hot water extraction showed that ultrasonic assisted extraction has positive effects on increasing the rate of mass transfer for bioactive compounds.Therefore,the ultrasonic assisted extraction of mulberry leaf polysaccharides was optimized by single factor experiments and response surface methodology?RSM?.The optimal extraction conditions with an extraction yield of 6.86±0.15%for crude MLP were determined as follows:extraction temperature 60?,ultrasonic power 60 W,extraction time 30 min and ratio?v/w,mL/g?of extraction solvent?water?to raw material was 30:1.The extracted mulberry leaf polysaccharide was isolated into 4 mulberry leaf polysaccharide fractions?MLP20%,MLP40%,MLP 60%and MLP 80%?using graded ethanol precipitation.In vitro antioxidant activity screening showed that MLP80%has better antioxidant activity.Analytical and physical methods were used to determine the physicochemical properties of MLP80%and crude MLP.The polysaccharide content of MLP80%was significantly higher than that of crude MLP?P<0.05?,while the content of protein,uronic acid,and total polyphenols in crude MLP was significantly higher than that of MLP80%?P<0.05?.The difference in the content of sulfate radical in MLP80%and crude MLP was not significant?P>0.05?.The molecular weights of crude MLP and MLP80%were determine by HPLC-GPC method as 322.78 kDa and 137.87 kDa respectively,and the MLP 80%molecular weight distribution was relatively uniform.The structural properties of the polysaccharides were analysed by infrared spectroscopy,ultraviolet spectroscopy and scanning electron microscopy.The UV spectra of crude MLP and MLP80%showed maximum absorption peaks at 200 nm,indicating that they have characteristic absorption peaks of polysaccharides.Fourier transform-infrared spectroscopy indicated that crude MLP and MLP80%were polysaccharides with O-H,C-H,C=O,C-O,S=O,C-O-S functional groups.The anti-radiation effect of MLP80%was studied using an in ovo model.MLP80%was injected into the air sac of chicken ova for three consecutive days,after which the ova were given whole body ⁶⁰Co-?radiation?5.0 Gy,1.0 Gy/min?on day 20 of incubation.Chickens were sacrificed on the day of hatch and bodyweight,organ indexes,activity of superoxide dismutase?SOD?,glutathione peroxidase?GSH-Px?,catalase?CAT?,and the content of malondialdehyde?MDA?in serum were determined.When compared with the normal group,the organ indexes were significantly reduced?P<0.01?,the activity of antioxidant enzymes?CAT,GSH-Px and SOD?were significantly decreased?P<0.01?and the MDA content was increased significantly?P<0.01?in the model group,indicating that organ atrophy,redox imbalance and lipid peroxidation occurred due to exposure to whole body ⁶⁰Co-?radiation.However,in ovo administration of MLP80%significantly increased the organ indexes?P<0.05?,significantly increased?P<0.05?the activity of antioxidant enzymes?CAT,GSH-Px and SOD?and significantly decreased?P<0.05?the MDA content in the model+MLP80%groups,suggesting that MLP80%could restore the redox balance and relieve radiation induced oxidative stress.The results also showed that MLP80%could enhance the immune function by significantly?P<0.01?increasing cytokines such as IL-2 and IL-12.The results in the present study suggested that MLP80%has promising radiation protective and immune stimulatory properties and could be explored as a natural immune stimulant.