Purification And Structural Analysis Of Pectin From Eucommia Ulmoides Oliver

Eucommia ulmoides Oliver is a traditional Chinese medicinal plant,which has the functions of strengthening muscles and bones,tonifying liver and kidney and lowering blood pressure.Polysaccharide is one of the main active components of Eucommia ulmoides Oliver,which has many functions such as improving immunity,anti-oxidation,lowering blood sugar and so on.At present,studies on Eucommia ulmoides Oliver polysaccharides mainly focus on the activity.There are few studies on the composition and structure of Eucommia ulmoides Oliver polysaccharides,and the structural characteristics are still unclear.In order to further understand the structural characteristics of Eucommia ulmoides Oliver polysaccharides,we designed the research content of this thesis.The specific research results are as follows.Eucommia ulmoides Oliver polysaccharide(WEUP)were extracted from Eucommia ulmoides Oliver through boiling water extraction and ethanol precipitation,with the yield of3.7%.WEUP contains 53.6% total carbohydrates,32.7% uronic acid and 6.2% protein.The analysis of monosaccharide composition showed that WEUP was mainly composed of galacturonic acid(Gal A),glucose(Glc),galactose(Gal),arabinose(Ara)and rhamnose(Rha).WEUP was purified by DEAE-cellulose column,and the yield of d H2 O eluted neutral sugar grade WEUP-N and 0.5 M Na Cl solution eluted acid sugar grade WEUP-A were 41.1%and 21.2%,respectively.The neutral sugar grade was mainly composed of starch,arabinosan and galactan,This thesis did not study it further.WEUP-A was further purified by DEAE-cellulose and Sepharose CL-6B gel chromatography to obtain relatively uniform charge and molecular weight WEUP-A3 a and WEUP-A3 b.WEUP-A3 b is the main fraction,its yield was 74.1%,the molecular was 44 k Da,and the proportion of main monosaccharides was Gal A: Rha: Gal: Ara = 43.4: 21.0: 11.5: 19.2.The results of methylation analysis and NMR spectra showed that WEUP-A3 b contained many pectin domains,such as RG-I,HG and RG-II.The HG structure in WEUP-A3 b was decomposed by endo-polygalacturonase,and the products were separated by gel chromatography to obtain WEUP-A3b-E1(45.5%),WEUP-A3b-E2(24.7%)and WEUP-A3b-E3(17.1%).Methylation,NMR spectroscopy and TBA assay showed that WEUP-A3b-E1 had RG-I structure and contained arabinosan,galactan or AG-type side chains.WEUP-A3b-E2 was RG-II type pectin.WEUP-A3b-E3 was the oligo-galacturonic acid after enzymatic hydrolysis of HG pectin.The ratio of the three pectin domains of RG-I,HG and RG-II in Eucommia ulmoides Oliver pectin WEUP-A3 b was about 55:17:14.The structure of Eucommia ulmoides Oliver RG-I pectin(WEUP-A3b-E1)was analyzed by methylation,nuclear magnetic resonance spectroscopy(NMR)and enzymatic hydrolysis.The results showed that the main chain of Eucommia ulmoides Oliver RG-I pectin was composed of α-1,4-Gal A and α-1,2-Rha,and the branching degree was 22.3%.The side chain of RGI contains AG-II,AG-I,arabinosan,galactosan.AG-II has a more complex structure and higher molecular weight.AG-I has high molecular weight and is closely substituted in the main chain with other kinds of side chains.The side chains of arabinan and galactoglycan contain both high degree of polymerization and low degree of polymerization.According to the results of mass spectrometry,the short side chains of galactoglycan generally have 1-6 degrees of polymerization,while the degree of polymerization of the short side chains of arabinan is 1-7,among which the shorter side chains may be dispersed in the main chain.The side chain B and side chain A of RG-II structure were obtained by acid hydrolysis of WEUP-A3B-E2.ESI-MS analysis showed that the side chain B is a nonamer with 0～2 acetyl groups,and its structure was Api-Rha-Ace A-Gal(Ara-(Rha)(Rha-Ara))-2Me Fuc,and side chain A was an 8-carbohydrate structure with 0～4 methyl ester groups,the structure was Api-Rha(α-Gal A)(β-Gal A)-Fuc(2Me Xyl)-Glc A-Gal.In this thesis,polysaccharides from Eucommia ulmoides Oliver pectin were extracted and purified by physical,chemical and enzyme methods.The structure of pectin was analyzed,which enriched the structural information of pectin and laid a foundation for further research on structure-activity relationship and drug development of pectin.