Structure Analysis Of Ginseng Pectin RG-I-4 And Its Interaction With Galectin-3

Panax ginseng C.A.Meyer is a precious traditional Chinese medicineal plant and belongs to the Ginseng genus,which is famous for its bioactivities,such as relieve stress,anti-fatigue,antidepressant,hypoglycemic and immunomodulating.Ginseng polysaccharide is one of the effective active ingredients which are consisted of ginseng starch and ginseng pectin.Our previous researches focused the structure-activity relationship of ginseng pectin.In early stage,A fraction had been separated and purificated from a RG-I domain of pectin,named after RG-I-4.RG-I-4 was found to be an efficient inhibitor of Galectin-3,it showed great significance for anti-tumor development.The lack of detailed structural information on RG-I-4 was the impetus for the current study.The conclusions of this paper were as follows:Water-soluble Panax notoginseng polysaccharide(WGP)was obtained by hot water extraction and ethanol precipitation.WGP was separated and purificated by Diethylaminoethyl-cellulose and then ginseng pectin WGPA was obtained.WGPA was hydrolysed by Endo-PG,the production was purificated by ion exchange column that DEAE Sepharose Fast Flow and gel column that Sephacryl-100 to get RG-I-4 with yield of 2.0%,Its molecular weight was 80 KDa,It was composed of GalA:Gal:Rha:Ara:Glc:GlcA:Man= 28.7:24.9:20.4:20.5:1.4:1.3:0.7 and monosaccharide composition showed that Rha/GalA was 0.71.RG-I-4 had strong inhibitory activity on Galectin-3mediated hemagglutination,the minimum inhibition concentration was 0.25±0.05 ug/ml;RG-I-4 had strong affinity on bio-layer interferometry and the dissociation constant(KD)value for RG-I-4 was calculated to be 59.6 nM,were consistented with previous experimental results.The structural features of RG-I-4 was elucidated using periodate oxidation degradation and smith,enzymolysis,infrared spectroscopy and nuclear magnetic resonance.Analytical result showed that RG-I-4 has the following structure: RG-I-4 is composed by a backbone with repeating units of alternating Rha and GalA linked [?4)-?-D-GalA-(1?2)-?-L-Rha-(1?] and the degree of branching for Rha was 30%.The side chains,mainly linked to AG-I ? AG-II ? ?-1,4-Galactan and?-1,5-Arabinan.The activities of RG-I-4,RG-I-4-Re and enzymatic fragment was analysed by Galectin-3mediated hemagglutination and bio-layer interferometry.Analytical result shows that the RG-I-4after the GalA all reduction for Gal and the content of Gal in RG-I-4 did not affect its activity that binding to and inhibiting Galectin-3.The molecular weight was an important factor affecting the activity of RG-I-4,and the RG-I-4 fragment with molecular weight less than 6.9 KDa was weak.The length of the branched chain had a certain effect on the activity of RG-I-4,and the activity of RG-I-4was relatively high.