Two-stage Extraction Of Phenolics And Polysaccharides From Moringa Oleifera Leaf And Their In Vitro Hypoglycemic And Hypolipemic Activities

Hyperglycemia and hyperlipidemia are inducements and main clinical manifestations of chronic diseases such as type II diabetes and cardiovascular diseases,which can be effectively prevented or ameliorated by hypoglycemic and hypolipemic agents.Moringa oleifera leaf(MOL)is a kind of nutritious food.MOL phenolics and polysaccharides have been reported to possessing hypoglycemic and hypolipemic activities.However,the efficient extraction and directional preparation of MOL hypoglycemic and hypolipemic factors were lacked in existing studies,and the interaction and scientific compatibility of these factors were also ignored.In this study,MOL phenolics and polysaccharides with strong hypoglycemic and hypolipemic activities were prepared by two-stage extraction method,and their molecular composition and structural characteristics were analyzed.Comparing with MOL primary processed products,the effects and interactions of phenolics and polysaccharides on delaying the digestion and absorption of starch and lipids were studied based on the INFOGEST digestion method.This study aimed to obtain the scientific compatibility of MOL active factors and directionally develop MOL highly processed products.Theoretical and methodological supports will be provided for the development of plant-based functional food with hypoglycemic and hypolipemic activities.The main results were listed as follows:(1)The extraction rates ofα-glucosidase inhibitor(α-GI),total phenolics(TP)and total flavonoids(TF)were taken as orientations to optimize the preparation and purification process of phenolics from MOL powder.The results showed that the optimum extraction conditions were as follows:the solid-liquid ratio was 1:20,and 50%ethanol solution was added,refluxing at 50°C for 60 min,and the solid-liquid ratio of washing residue was 1:5.After optimization,the extraction rates ofα-GI,TP and TF were 722.84±18.02 mg AE/g MOL,19.01±0.28 mg GAE/g MOL and 7.57±0.03 mg QE/g MOL,increased by 27.81%,17.77%and 12.15%,respectively.XAD-16 macroporous resin was used to remove impurities.The phenolics with strongα-glucosidase inhibitory activity were enriched in the fractions of 50%ethanol solution,and the content of TP and TF were 272.49±2.77 mg GAE/g and 196.91±8.89 mg QE/g.Quercetin-3-O-glucoside and kaempferol-3-O-glucoside were important hypoglycemic factors in MOL,which content in the fraction was 6.86 g/100 g and 4.02 g/100 g,respectively.Theα-glucosidase inhibitory activity of the fraction was 12.49 times of acarbose.(2)The extraction rates of crude polysaccharide(CP)and bile acid(BA)sequestrant,and total sugar(TS)content were taken as orientations to optimize the preparation and purification process of polysaccharide from dephenolized MOL(DMOL)powder.The results showed that the optimum extraction conditions were as follows:the solid-liquid ratio was 1:10,and deionized water and 0.75%(w/w)pectinase were added,extracting at 55°C and p H 4.5 for 6 h followed by extracting at 105°C and p H 4.5 for 90 min.After optimization,the extraction rates of BA sequestrant and CP,and TS content were 83.64±3.27 mg CE/g DMOL,115.12±2.29mg DM/g DMOL and 672.99±11.67 mg GE/g CP,increased by 39.87%,22.48%and 37.16%,respectively.MOLP-CA was obtained by citric acid assisted extraction with protease hydrolysis,ultrafiltration,alcohol precipitation,dialysis and freeze-drying,which BA-binding capability was 70%of that of cholestyramine.Purified polysaccharides MOLP-CA2 and MOLP-CA3were isolated from MOLP-CA by ion exchange and gel column chromatography,and the BA-binding capabilities of them were 107%and 79%of that of MOLP-CA,and 80%and 58%of that of cholestyramine,respectively.MOLP-CA2 had molecular weight(MW)of 2.120×10~6Da,and its main glycan chain accounted for 71.7%,which was composed of→3)-β-D-Galp A-(1→,→3)-β-D-Galp-(1→,→3,6)-β-D-Galp-(1→,→3)-α-L-Rhap-(1→,→2)-α-L-Araf-(1→and→3)-α-L-Araf-(1→in the molar ratio of 1.01:5.04:1.04:2.69:1.59:1,and its branched chain was composed ofβ-D-Galp-(1→,→6)-β-D-Galp-(1 and→3)-α-L-Araf-(1→in the molar ratio of 1:1.67:2.56.MOLP-CA3 had MW of 3.086×10~4 Da,and its main glycan chain was composed of→3)-α-L-Rhap-(1→,→3,6)-β-D-Galp-(1→and→3)-β-D-Galp A-(1→in the molar ratio of 1.55:1:1,and its branched chain accounts for 82.52%,which was composed ofβ-D-Galp-(1→,α-D-Galp A-(1→,→6)-β-D-Galp-(1→,→3,6)-β-D-Galp-(1→,→3)-α-L-Rhap-(1→,→2)-α-L-Araf-(1→and→3)-α-L-Araf-(1→in the molar ratio of1:1.80:2.30:1.88:4.85:2.05:2.03.MOLP-CA2 was a kind of acidic polysaccharide with low branching degree,short branched chain and large MW.MOLP-CA3 was a kind of acidic polysaccharide with high branching degree,long branched chain and small MW.(3)Based on the INFOGEST digestion method,the capabilities of delaying starch hydrolysis and glucose diffusion and dialysis,binding BA,inhibiting cholesterol micelle solubility,and promoting the production of SCFAs were taken as orientations to select the best compatibility with strong hypoglycemic and hypolipemic activities.The results showed that the activities of highly processed products(phenolics,polysaccharide and their complexes)were significantly stronger than that of primary processed products(MOL and DMOL powder).For delaying starch hydrolysis,the best compatibility was phenolics and polysaccharide in the mass ratio of 25:75.For delaying glucose diffusion and dialysis,the best compatibility was phenolics and polysaccharide in the mass ratio of 75:25.For binding BA,the best compatibility was phenolics and polysaccharide in the mass ratio of 75:25 or 50:50.For inhibiting cholesterol micelle solubility,phenolics and polysaccharide showed antagonistic effect,therefore 100%polysaccharide was the best choice.For promoting the production of SCFAs,100%polysaccharide was the best choice.