Isolation,Purification,Structure Characterization,Immunological Activity And Its Functionalization Of Larch Arabinogalactan

Arabinogalactan(AG)is a water-soluble polysaccharide that is widely found in plant cell walls.Larch(Larix spp.)is a deciduous tree of the genus Larix of the Pinaceae family.It is the main forest component tree species of the alpine coniferous forests in northeast,Inner Mongolia,north china and douthwest of china,and rich in arabinogalactan.In order to fully develop and utilize the resources of Larch polysaccharides,systematic studies on the separation and purification,structure analysis and activity evaluation of larch arabinogalactan were carried out in this paper.The influence of polysaccharide branches on the biological activity of arabinogalactan was also investigated.On this basis,the biological function of larch arabinogalactan was expanded,and its anticancer activity was improved by functionalization and modification of polysaccharides.The results can provide a theoretical basis for the development,functional modification and activity evaluation of plant polysaccharides.The main results are summarized as follows:The water-soluble arabinogalactans were extracted from Larix principis-rupprechtii wood powder by hot water extraction method,and the crude polysaccharides were separated and purified by polyamide decolorization,DEAE-Sepharose Fast Flow anion exchange resin and Superdex 75 gel chromatography to obtain two polysaccharide components(AGW and AGS),the yields of AGW and AGS were 3.0% and 6.6%(w/w),respectively.The structure characterization results of polysaccharides showed that the average molecular weights of AGW and AGS were 15.3 k Da and 18.4 k Da,respectively.AGW and AGS were pure polysaccharide components,no protein or amino acid components in the molecule structure.They were composed of rhamnose,arabinose,galactose and glucuronic acid,but the molar ratio of sugar groups is slightly different.The primary structure of the two polysaccharides is similar,and both are the main chain formed by galactose through ?-1,3-glycosidic bonds,and side chains is connected to the O-6 position of the galactosyl group in the main chain by ?-1,6-linkage.?-L-Araf residue is connected to the O-6 position of ?-D-Galp side chain,T-Araf residue is connected to the O-3 position of ?-L-Araf residue through ?-1,3-linkage.The T-Arap residue is directly connected to the O-6 position of the ?-D-Galp side chain.AGS has a polyelectrolyte effect and exhibits different solution behaviors in salt ion solutions.Mouse macrophage RAW 264.7 as an in vitro model was used to evaluate the immune activity of arabinogalactan.The results showed that AGW and AGS were biosafe and non-toxic to cells,and could induce the differentiation of RAW 264.7 cells with significant immunomodulatory activity.They could specifically activate potential signal transduction pathways to activate immune response and promote the secretion of immune factors NO,TNF-?,IL-1? and IL-6 and the expression of related mRNA of immune factors.The AGW side chains was partially removed by Smith degradation method to investigate the role of the side chains on the activity,and the partially side chains degraded polysaccharides DAGW1 and DAGW2 were obtained.The results of polysaccharide structure analysis showed that the arabinose and galactose of DAGW1 and DAGW2 were not compeletly degraded,however the contents of arabinose and galactose in DAGW1 and DAGW2 were 5.05% and94.95%,2.16% and 97.84%,respectively.The conformation of DAGW1 and DAGW2 changed from disorder to a random coil state.DAGW1 and DAGW2 do not produce toxic side effects and still have immunostimulatory activity.However,DAGW1 has a stronger ability to promote cytokine secretion in RAW 264.7 cells than that of DAGW2,indicating that the polysaccharide side chain group has a huge impact on its biological activity.The chlorosulfonic acid-pyridine method was used to sulfate AGS to obtain four sulfated polysaccharides with degree of substitution of 0.80,0.72,0.67 and 0.61,and molecular weights of 22.03,20.89,19.96,19.24 k Da,respectively.Using Human lung cancer cell A549,human hepatoma cell HepG-2 and human breast cancer cell MCF-7 as in vitro models,it was found that S-AGSs had a significant inhibitory effect on the growth of three cancer cells in a dose-dependent manner.Flow cytometry results showed that S-AGSs promoted cell apoptosis to inhibit cell growth,eventually.The results showed that the sulfation modification could endow arabinogalactan with anticancer activity,and the anticancer effect with DS of 0.72 was the best.Using AGS as a stabilizer,AGS-SeNPs were prepared by the redox reaction of sodium selenite and ascorbic acid.Four selenium nanocomposites were obtained by controlling the ratio of sodium selenite and AGS,with sizes of 94.24,151.90 and 173.2 nm,respectively.SEM and TEM showed that the particles of AGS-SeNPs all showed similar uniform spherical shape and were uniformly dispersed.AGS-SeNPs had obvious anti-tumor activity,inhibiting the growth of tumor cells A549,HepG-2 and MCF-7 in a dose-dependent manner.A large number of apoptotic bodies appeared under the intervention of AGS-SeNPs.The flow cytometry results showed that AGS-SeNPs inhibit tumor cell proliferation by mediating cell apoptosis.The results showed that arabinogalactan could stabilize the elemental selenium to form nanoparticles,and endow the arabinogalactan selenium nanoparticles with anti-tumor activity.In summary,arabinogalactan is a good water-soluble immune enhancer,which can be used as a health food or an auxiliary treatment of diseases.At the same time,the structure modification of arabinogalactan and the preparation of polysaccharide nanoparticles can further enrich the functions of polysaccharides,provide new ideas for the design of new anti-tumor drugs,and provide a theoretical basis for the high-value utilization of forestry source polysaccharides.