| Alfalfa(Medicago sativa L.)is a leguminous plant that is often referred to as the "king of forages" due to its high nutritional value.Alfalfa polysaccharide is a polysaccharide extracted from alfalfa plant,which is a macromolecule with certain biological activity.A variety of human diseases are closely related to oxidative stress and immunity,and polysaccharides have antioxidant,anti-aging,hypolipidemic,immune regulation and other activities.Therefore,the development and utilization of natural,effective and safe antioxidants such as plant polysaccharides have become the focus of current research.In this thesis,alfalfa root polysaccharide was extracted,isolated and purified,and its structure was characterized,and then its in vitro activity was studied.The main contents are as follows:1.In this thesis,the crude polysaccharide was obtained by extracting the roots of alfalfa by hot water reflux method;it was deproteinized by the combination of enzymatic hydrolysis method and Sevage method.The crude polysaccharides after deproteinization of alfalfa were further separated and purified by macroporous resin column chromatography,DEAE cellulose column chromatography and Sephadex G-200 column chromatography to obtain three polysaccharides,named RAPS-1,RAPS-2 and RAPS-3.The purities of the three polysaccharides were 91.80 %,87.05 %,and 87.91 %,respectively.2.In this thesis,the molecular weights of RAPS-1,RAPS-2 and RAPS-3 were determined by high performance gel permeation chromatography and were 22.60 k Da,26.95 k Da and 38.25 k Da,respectively.Determination of RAPS-1 by gas chromatography-mass spectrometry(GC-MS)from rhamnose(Rha),arabinose(Ara),xylose(Xyl),glucuronic acid(Glc A),glucose(Glc),galactose(Gala),its mole percentages were: 10.25 %,12.53 %,6.58 %,4.56 %,15.74 %,36.30 %;RAPS-2 was composed of rhamnose(Rha),arabinose(Ara),xylose(Xyl),glucose(Glc),galactose(Gala),the mole percentages were: 24.07 %,14.48 %,1.24 %,15.05 %,39.37 %;RAPS-3was composed of rhamnose(Rha),arabinose(Ara),xylose(Xyl),glucuronic acid(Glc A),mannose(Man),glucose(Glc),galactose(Gala),the mole percentages were: 31.61 %,11.78 %,1.93 %,5.49 %,3.18 %,14.56 %,24.63 %.According to Fourier transform infrared spectroscopy and 1D(2D)spectra nuclear magnetic resonance spectroscopy data,RAPS-1 and RAPS-2 both had α-pyranose-type structures,while RAPS-3 hadα-furanose-type structures.In addition,its surface morphology and thermal stability were investigated by scanning electron microscopy(SEM)and thermogravimetric analysis(TG).The results showed that with the increase of the molecular weight of the polysaccharide,the surface structure of the polysaccharide became denser and the thermal stability was also enhanced.3.In this thesis,the in vitro antioxidant and lipid-lowering activities of the alfalfa polysaccharides were studied.1)From the results of the free radical scavenging experiments in vitro,the three polysaccharides all had potential antioxidant activities;the maximum free radical scavenging efficiency of RAPS-1 can reach 87.0 %.2)Through the in vitro model of non-alcoholic fatty liver in HepG2 cells,it can be seen from the experimental results of the above-mentioned polysaccharide-lowering activity:(1)The experimental results showed that the three polysaccharides could reduce the lipid accumulation in HepG2 cells induced by oleic acid to varying degrees;(2)Taking RAPS-1as an example,the lipid-lowering activity of alfalfa polysaccharide was further discussed.The results showed that at a concentration of 1000 μg/m L,RAPS-1 could significantly enhance the activity of intracellular SOD antioxidant enzymes,reduce the content of MDA,and improve the total antioxidant capacity(T-AOC)of cells,thereby reducing oleic acid-induced lipid accumulation in HepG2 cells.In conclusion,RAPS-1 had inhibitory effect on oleic acid-induced intracellular lipid accumulation in HepG2 cells,revealing that alfalfa polysaccharide may play its role mainly by restoring its antioxidant system. |
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