Structural Analysis And Antioxidant Activities Of Neutral Polysaccharide Isolated From Epimedium Koreanum Nakai

Objective:The polysaccharide of isolated from Epimedium koreanum Nakai was extracted and purified according to the difference of electric charge,and a neutral polysaccharide was obtained.Its structural characteristics and antioxidant activity in vitro were analyzed,and then its structure-activity relationship was studied.Methods:1.EFPC was obtained by water extraction and alcohol precipitation,and the protein was removed by Sevage method and then purified by DEAE ion exchange column to obtain EFPN,EFPN was further purified by Sepharose CL-6B column to obtain EFPN-1;2.Physical and chemical properties analysis:the sugar content was determined bythe phenol-sulfuric acid method,the protein content was determined bythe Bradford’s method,and the monosaccharide composition was determined by HPLC of PMP;3.Structure analysis:FT-IR,NMR,GC-MS and HPLC was used to analyze the structure of EFPN-1;4.Antioxidation test in vitro:The antioxidation activities of EFPN and EFPN-1 in vitro were compared from three aspects:DPPH and hydroxyl radical scavenging,ferrous ion chelation and reducing antioxidant powder.Results:1.The total sugar content of EFPC、EFPN and EFPN-1 was 79.58%、87.14%and91.43%,respectively.The protein content of EFPC,EFPN and EFPN-1 was 0.05%、0.03%and 0.08%,respectively.The results of monosaccharide composition showed that EFPC was mainly composed of Man,Rha,GlaA,Glc,Gal,Ara,with a molar ratio of0.9:1.0:1.2:1.5:1.1:1.8.The EFPN was composed of Man,Rha,Glc,Gal,Ara with molarratio of 0.6:1.0:1.4:1.6:1.7;EFPN-1 is composed of Rha,Glc,Gal,Ara with moleratio of 1.0:6.8:1.8:0.7;2.By means of methylation analysis（GC-MS）,FT-IR analysis and NMR analysis,it is found that EFPN-1 indicating that they might form the backbone of EFPN-1.The side chains are mainly composed of（1→4,6）-linked-Glcp and（1→3,6）-linked-Galp,and the terminal residue is Ara,Gal,Rha;3.EFPN and EFPN-1 had a good scavenging effecton hydroxyl radical in the range of0-8 mg/mL,and the scavenging ability of EFPN-1 was better than that of EFPN,and the scavenging rates reached 90.05%;DPPH radical scavenging capacity of both EFPN and EFPN-1 was in a concentration dependent manner in the dosage ranging from 0.05 mg/mL to 0.4 mg/mL.and the scavenging rates of EFPN-1 reached 76.68%.The chelating ability of EFPN-1 in metalion chelating ability was better than that of EFPN,and the metal chelating ability of EFPN-1 reached 85.25%EFPN,EFPN-1 FRAP value in FRAP method was 4.40and 7.04 mmol FeSO₄/g,respectively.Conclusion:The structure and antioxidant activity of a neutral polysaccharide（named as EFPN-1）from Epimedium koreanum Nakai were studied.Based on the results from high performance gel permeation chromatography,methylation GC-MS and ¹³C nuclear magnetic resonance spectroscopy,EFPN-1 was a branched polysaccharide with a molecular weight of 21.9 kD and containing over 60%of glucose.Its backbone was composed of（1→4）-β-D-Glcp,branched partially at C-6 of（1→4）-linked Glc and C-6 of（1→3）-linked Gal,and terminated with almost equal Ara,Gal and Rha.Antioxidant tests showed that EFPN-1 exhibited a strong capacity on DPPH and hydroxyl radical scavenging,ferrous ion chelation and reducing antioxidant powder.EFPN-1 has potential to be developed as a natural antioxidant.