| Niu Dali is also called Millettia Speciosa Champ(M.speciosa),which is a Chinese traditional medicinal and food homologous herb.Due to the rich nutrient contents and muliti-bioactivities(e.g.anti-inflammatory and anti-fatigue),it has extremely high nutritional and health care value.Polysaccharide(MSCP)is one of the most important functional factors in M.speciosa,but the precise structure,immune regulation mechanism and protective effect on Parkinson’s disease(PD)model of MSCP are still unclear.In addition,there is little information about other physicochemical and functional properties of MSCP,such as interfacial activity and emulsifying properties,and related research is still in the blank.In view of the above situation,this dissertation firstly prepared the crude polysaccharide by water extraction-alcohol precipitation,and carried out preliminary characterization and identification of its chemical composition and structure.Two high-purity M.speciosa polysaccharide components were prepared by separation and purification technologies.Secondly,their molecular structure was analyzed based on analytical methods such as nuclear magnetic resonance;the RAW264.7 cell model was used to explore their immunomodulatory activity and the potential mechanisms,and the protective effect on PC12 cells injured by 6-hydroxydopamine(6-OHDA)was studied.Thirdly,in order to explore the potential as an emulsifier,the adsorption characteristics of MSCP at the oil-water interface were investigated,and its emulsifying performance and underlying mechanisms were analyzed.Finally,the strategy of covalently grafting acetyl groups was adopted to solve the problem of poor emulsifying ability of MSCP,and the emulsifying stabilization mechanisms of acetylated MSCP with different scale structure were explored.The main findings of this paper are listed as follows.(1)After water extraction-alcohol precipitation,decolorization,deproteinization and other steps,the light yellow powdery crude polysaccharide MSCP was prepared;FTIR showed that MSCP had the characteristic functional groups of general polysaccharides;the total sugar,protein,uronic acid and water content in MSCP were 80.25%,9.89%,0.07%and 5.33%,respectively;the average molecular weight of MSCP was 1.56×10~4 Da;XRD results showed that MSCP mainly exists in an amorphous structure;SEM showed that the microscopic morphology of MSCP was mainly flake-like aggregates,and the surface was smooth but contained some small voids.AFM observation revealed that the MSCP was a cylindrical-like structure with a diameter of about 50～90 nm and a height of about 4～6 nm.MSCP was purified by DEAE-52 and Sephadex G-100 column chromatography to obtain two components,MSCP1and MSCP2.(2)The average molecular weight of MSCP1(M_w=0.94×10~4 Da,PDI=1.47)was significantly smaller than that of MSCP2(M_w=2.85×10~4 Da,PDI=1.82),but its molecular weight distribution is more concentrated.The monosaccharide compositions and glycosidic bond types of MSCP2 were more abundant than those of MSCP1.MSCP2 consisted of five monosaccharides,such as fucose,arabinose,galactose,glucose and xylose(molar ratio 0.03:0.04:0.05:1.16:0.06),and was made up of seven kinds of glycosidic bonds,including T-Glcp,1,4-Glcp,1,4-Xylp,1,6-Glap,T-Araf,1,3,4-Fucp and 1,4-Glcp A;while MSCP1 was only composed of glucose and xylose(molar ratio 1.31:0.01),which contained 1,4-Glcp,T-Glcp,1,4,6-Glcp and 1,4-Xylp four glycosidic bonds;MSCP1 and MSCP2 are also quite different in microscopic morphology.MSCP1 presents some fragmented and cylindrical-like structures,while MSCP2 mainly presents a rod-like shape with different lengths;in addition,neither MSCP1 nor MSCP2 has a triple helix conformation.(3)In the RAW264.7 cell model,both MSCP1 and MSCP2 could enhance the pinocytosis ability of macrophages,stimulate the cells to secrete ROS and increase the secretion levels of NO,TNF-αand IL-6 by upregulating the expression of corresponding mRNA.These results indicates that both MSCP1 and MSCP2 have immunomodulatory activities,but the activity of MSCP2 is significantly higher than that of MSCP1.The results of antibody blocking experiments showed that MSCP1 could induce cell activation through the recognition of TLR4and GR receptors on the surface of macrophage membranes,while MSCP2 could activate immune responses through the recognition of TLR4,SR and GR receptors.In addition,MSCP1and MSCP2 have a significant protective effect on 6-OHDA-induced PC12 cell injury,and the mechanism of action is related to improving the survival rate of damaged PC12 cells,reducing ROS secretion and the proportion of early apoptosis,inhibiting the decrease of mitochondrial membrane potential(MMP),and enhancing the activity of intracellular antioxidant enzymes.(4)The interfacial tension test showed that MSCP can significantly reduce the interfacial tension between oil and water.The test results of interface layer thickness indicated that MSCP formed multilayer adsorption at oil-water interface and constructed a thick interface layer.The effective control of emulsion droplet size,microstructure,rheological properties,and stability could be achieved by changing the MSCP concentration(c)or the oil-phase ratio(Φ).Over a wide range of ionic strength(0～500 m M)and heat treatment(50～90°C),the emulsions prepared by MSCP had better stability than those prepared by polysaccharides such as gum arabic(GA)or octenyl succinic anhydride(OSA)modified starch.In addition,MSCP could significantly reduce the production of hydroperoxides(LH)and malondialdehyde(MDA),suggesting that MSCP could inhibit lipid oxidation in emulsions.(5)Acetylated polysaccharide AC-MSCP samples with different scale structure were successfully prepared by covalent modification of MSCP with acetyl groups.Although the thermal stability and free radical scavenging ability of AC-MSCP decreased,the ability to reduce interfacial tension was significantly improved,and the emulsifying ability and stability of AC-MSCP were stronger than those of MSCP;among them,the emulsifying ability of AC-MSCP2 and AC-MSCP3 were better than commercial polysaccharide emulsifier gum arabic(GA).Compared with other emulsions prepared by AC-MSCP,the emulsion prepared by AC-MSCP3 had the smallest changes in particle size after 30 days of storage,which showed better stability.AC-MSCP1～2 showed similar LH and MDA inhibitory ability to MSCP,but AC-MSCP3 showed slightly weaker MDA inhibitory ability than MSCP.To sum up,MSCP is not only a new type of immune-enhancing adjuvant and neuroprotectant,but also is a natural emulsifier,which has great application potential in functional foods,medicines or cosmetics.In addition,covalent modification of acetyl groups was proved to be an effective strategy to improve the emulsification performance of hydrophilic polysaccharides,and AC-MSCP with different scale structures can be applied to emulsion systems with different requirements.This study has very important academic value and practical significance for filling the blank of relevant research on M.speciosa,broadening its application scope and promoting the development of M.speciosa industry. |
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