Purification,Modification,Structural Characterization And Antioxidant Activities Of Polysaccharides From Rhododendron Dauricum

Rhododendron dauricum L.belongs to the Rhododendron genus and family Ericaceae with cough-expectorant properties,which is widely distributed in Northeast China,Japan,North Korea and Russia.At present,studies on the chemical components of R.dauricum is mainly focused on small molecule compounds,such as flavonoids,terpenoids,coumarins and phenolic acids,while polysaccharides,as one of macromolecules,have rarely been reported.Therefore,in this thesis,dried leaves of R.dauricum were used as raw materials,and refined R.dauricum polysaccharide fractions were obtained by extraction,separation and purification,and their acetylated structural modification,characterization and antioxidant activity were investigated.·The crude polysaccharides from the leaves of R.dauricum（RDP）were prepared by water extraction and alcohol precipitation.D101 resin was screened as the most suitable resin for RDP by static adsorption method.Then,the optimum decolorization conditions of polysaccharides from RDP were obtained by analytical hierarchy process combined with response surface methodology.The optimum decolorization conditions were as follows:polysaccharides concentration 7.88 mg/m L,decolorization time 70.12 min and liquid-solid ratio 6.06 m L/g and decolorization temperature 52.72℃,and the maximum predicted comprehensive score was 3.74.Under these conditions,the comprehensive score of actual decolorization effect was3.55±0.15（n=3）.Furthermore,two R.dauricum polysaccharide fractions（RDP-1and RDP-2）were separated using DEAE-52 cellulose anion exchange chromatography.The polysaccharide contents of two fractions（RDP-1 and RDP-2）were determined by phenol-sulfuric acid method,and the results showed that the polysaccharide contents of RDP-1 and RDP-2 were 93.12±3.34%and 90.67±3.84%,respectively.The structural characterization of RDP-1 and RDP-2 was investigated by a series of modern analytical instruments,including UV-Vis spectrophotometry,fourier infrared spectroscopy（FTIR）,high performance liquid chromatography（HPLC）,high performance gel permeation chromatography（HPGPC）,gas phase mass spectrometry（GC-MS）coupling and nuclear magnetic resonance spectroscopy（NMR）.The analysis results demonstrated that both fractions had the characteristic groups of polysaccharides,and were heteropolysaccharides composed of glucose,mannose,galactose and arabinose with different monosaccharide molar ratios;The molecular weights of RDP-1 and RDP-2 were 1.0948×10⁴k Da and 1.1899×10⁵k Da,respectively,with triple helix conformation.The results of methylation analysis and NMR spectroscopy indicated that both fractions were composed of→5)-Araf-（1→,→6）-Glcp-（1→,→2,4）-Manp-(1→,Glcp-（1→and→6）-Galp-(1→glycosidic bonds in different linkage sequences.Based on the single-factor experiments,the acetylation modification process of RDP was optimized by response surface methodology.The optimum conditions for the acetylation modification were as follows:reaction time 3.15 h,reaction temperature 49.98℃,liquid-to-solid ratio 15.96 m L/g,and the maximum predicted degree of substitution（DS）was 0.472.Under these conditions,the acetyl degree of substitution（DS）of the acetylated modified crude polysaccharide（Ac RDP）and R.dauricum polysaccharides（Ac RDP-1,Ac RDP-2）were 0.455,0.419 and 0.445,respectively.The results of FTIR spectroscopy revealed that the characteristic peaks of Ac RDP-1 and Ac RDP-2 were significantly enhanced at 1251.89 cm^-1,indicating the successful completion of the acetylation modification.The monosaccharide content of Ac RDP-1 and Ac RDP-2 was varied,and the molecular weight was reduced to 9.3525×10³k Da and 4.7016×10³k Da,and the three-stranded helix structure was disrupted.The evaluation of antioxidant activity suggested that RDP and Ac RDP had different scavenging effects on the DPPH,OH and ABTS free radicals.The scavenging ability of Ac RDP,Ac RDP-1 and Ac RDP-2 on ABTS and OH radicals was significantly higher than that of RDP,RDP-1 and RDP-2 by One-Way ANOVA comparison,indicating that the acetylated structural modification could promote the antioxidant effect of RDP.In this thesis,the decolorization process,structural characterization,acetylated structure modification and antioxidant activities of R.dauricum polysaccharides were systematically investigated,which laid the theoretical foundation for the development and utilization of R.dauricum and its industrial production.