Isolation,Purification,and Structural Characterization Of Pectic Polysaccharides From Six Medicinal Plants

Pectin is an important active ingredient in medicinal plants.It is a kind of acidic polysaccharide rich in galacturonic acid(Gal A).It has many functions,such as immunoregulation,anti-tumor,antioxidant,hypoglycemic,anti-inflammatory and so on.The diverse functions of pectin are closely related to its complex structure.According to the structural differences,pectin is mainly divided into four types including homogalacturonan(HG),type I rhamnogalacturonan(RG-I)and type II rhamnogalacturonan(RG-Ⅱ)domains.The structures of pectin from different medicinal plants are different,which makes these pectins may have different functions.In order to deeply study the structure-activity relationship of pectin from medicinal plants,it is necessary to prepare pectin from different medicinal plants and analyze their structures.In this thesis,pectins were extracted and purified from six medicinal plants.The structural features of these pectins were compared and analyzed at the overall molecular level.Then,different types of domains were prepared by degrading these pectin molecules with enzymes,and the fine structures of different types of pectin domains were further compared and analyzed.The results are as follows:Water-soluble polysaccharides were extracted from six medicinal plants by hot water extraction and alcohol precipitation.The yields were 5.8%,9.2%,3.5%,5.3%,13.0%and 14.7%respectively.These water-soluble polysaccharides were separated by anion-exchange chromatography to remove neutral polysaccharides and prepare acidic polysaccharides.Monosaccharide composition analysis showed that Gal A was the main component in the acidic polysaccharides of Lycopus lucidus,Stachys japonica,Caulis Polygoni Multiflori,Polygonum orientale and Radix isatidis,while the acidic polysaccharides of Folium isatidis mainly contained Gal A,Rha,Gal and Ara.These acidic polysaccharides were further separated and purified by gel filtration chromatography and six major pectin fractions were obtained.The molecular weights of six pectin fractions were determined by high performance gel permeation chromatography to be 26.4 k Da-81.3 k Da.Monosaccharide composition analysis showed that Gal A,Rha,Gal and Ara were major monosaccharides in pectin from Folium isatidis,while Gal A was a major monosaccharide in other homogeneous pectin fractions,with the content of Gal A higher than 60%.The degree of methyl-esterification(DM)of pectin was in the range of 9.3%to 34.3%determined by infrared spectroscopy(FT-IR).Qualitative analysis by thiobarbituric acid(TBA)assay showed that these pectins contained a certain proportion of RG-Ⅱdomain.NMR analysis showed that these pectin molecules containedα-1,4-Gal A,α-1,2/1,2,4-Rha,α-1,5/1,3,5-Ara,β-1,3/1,6/1,3,6-Gal characteristic signals,suggesting these pectins also contained HG and RG-I domains.In order to further analyze the structural characteristics of different domains in six pectin fractions,endo-polygalacturonase(Endo-PG)was used to degrade these pectins,and the enzymolysis products were purified by size exclusion chromatography.RG-I domain,RG-Ⅱdomain and HG domain were prepared from six pectin molecules,with different yields.It was found that the proportion of RG-I,RG-Ⅱand HG domains in the six pectin molecules was different.Among them,the proportion of RG-I domain in Folium Isatidis pectin was the highest,the proportion of RG-Ⅱdomain in Caulis Polygoni Multiflori pectin was the highest,and the proportion of HG domain in Radix isatidis was the highest.As the structure of RG-Ⅱdomain is relatively conserved and its fine structure has been clearly reported,the structure of it will not be further analyzed.The oligosaccharide fragments of HG domains were analyzed by UPLC-HILIC-MS~n.It was found that these oligosaccharides contained 1-9 Gal A and could be substituted by 0-5 methyl groups.Among the 16 oligosaccharide fractions detected,9 oligosaccharides were common to six HG domains.The distribution of methyl ester groups on oligogalacturonic acids was further analyzed by tandem secondary mass spectrometry.It was found that the number of adjacent methyl ester groups in oligosaccharides was up to 4.There were both continuous and relatively dispersed methyl ester groups in the HG domains.NMR and methylation analysis showed that six RG-I domains were composed ofα-1,4-Gal A andα-1,2/1,2,4-Rha in the main chain,and the degree of branching was 34%-64%.The side chains were composed of arabinan,galactan,type I and type II arabinogalactan(AG)in different proportions.The rhamnogalacturonan lyase(Bh Rgl)was used to degrade these RG-I domains.The hydrolysates were separated by size exclusion chromatography to obtain three types of structural fragments with different molecular weights.These structural fragments were RG-I structural units with long side chains,medium length side chains and short side chains,respectively.These RG-I domains mainly contained short side chain structural units,except that RG-I from Radix isatidis contained a high proportion of long side chain structural units.In conclusion,pectin molecules were isolated and purified from six medicinal plants in this thesis.The structures of these pectins were systematically analyzed and compared by using a variety of structural analysis methods.The results enrich the structural information of these medicinal plant pectins and provide a structural basis for the study of the structure-activity relationship of medicinal plant pectins.This study also provides the theoretical foundation for the development and utilization of these pectins.