The Structural Characteristics、Selenium Nanoparticles Modification And Antitumor Activity Of Platycodon Grandiflorus Polysaccharides

Platycodon grandiflorus is an herbaceous perennial plant that belongs to the Campanulaceae family,which are related to many pharmacological properties,including anticancer,antiobesity,antiallergic,lipid-lowering,antigastric ulcer,and anti-oxidant properties.Platycodon grandiflorus contains many bioactive compounds,including polysaccharides.Polysaccharides obtained from various origins have a wide range of biological activities with lower toxicity,including antitumor activity and immune regulation.Meanwhile,a growing number of evidences indicated that polysaccharides are stabilizing agents which could modify the interface of nanoparticles in solution.At present,the extraction of Platycodon grandiflorus polysaccharide was relatively single,which caused the lower utilization of Platycodon grandiflorus raw materials.Therefore,in this study,the polysaccharides in the roots of Platycodon grandiflorus were extracted and purified by water extraction and then alkali extraction.Finally,two kinds of Platycodon grandiflorus polysaccharides were obtained and their structure and antitumor activity in vitro and in vivo were analyzed.Meanwhile,one of the polysaccharides was selected as the stabilizer to prepare selenium nanoparticles,and its antitumor activity was studied.Firstly,the structures of two Platycodon grandiflorus polysaccharides（PGP90 and PGP-AE）obtained by water extraction and alkali extraction were analyzed.The detection of chemical components suggested that the total carbohydrate of Platycodon grandiflorus polysaccharide PGP90 obtained by water extraction and PGP-AE obtained by alkali extraction were 93.36%and 92.78%respectively,with the absence of protein and uronic acid.HPGPC and monosaccharide composition analysis indicated that the molecular weight of PGP90 was about 3.1×10³Da and composed of fructose and glucose in a molar ratio of1:0.05;The molecular weight of PGP-AE was about 9.31×10⁵Da and mainly composed of arabinose,galactose and glucose in a molar ratio of about 1.06:1:0.14.Based on the analysis of methylation and NMR experiments,PGP90 is an inulin fructose,withβ-Fruf-（2→[→1）-β-Fruf-(2→]₁₆→1)-β-Fruf-（2→1）-α-Glcp structure;PGP-AE was branched and mainly composed of sugar residues→6)-β-Galp-（1→and→3,6）-β-Galp-（1→,and the branched chain is mainly composed of→3）-α-Araf-（1→,→5）-α-Araf-（1→and→3）-β-Galp-(1→.The significant structural differences between PGP90 and PGP-AE resulted in different antitumor activities in vitro.MTT assay suggested that PGP90 had no inhibitory effect on both MGC-803 and BGC-823 cells,while PGP-AE could notably inhibit the proliferation of gastric cancer cells.The IC50 value of PGP-AE on MGC-803 and BGC-823 cells were 432.12μg/m L and 475.54μg/m L,respectively.The observation of cell morphology and Hoechst 33258 staining showed that PGP-AE-treated cells displayed obvious apoptotic characteristics,including round shape and shrunken,incomplete membrane,unclear edge,lower adhesion ability,DNA condensation or fragmentation in the nucleus.Meanwhile,Annexin V-FITC/PI staining and cell cycle experiment confirmed that PGP-AE could change the membrane permeability and caused cell arrest at the G2 phase on both MGC-803 and BGC-823 cells,therefore inducing cells apoptosis.Furthermore,by detecting the levels of ROS,mitochondrial membrane potential and related apoptotic proteins,PGP-AE could cause excessive ROS,induce mitochondrial dysfunction,affect the expression of apoptosis related proteins and finally start apoptotic signals.PGP90 has low molecular weight and good water solubility.Compared with PGP-AE,the excellent dispersion performance of PGP90 makes the polysaccharide have better effect to modify selenium nanoparticles.PGP90 was selected to prepare selenium nanoparticles in the redox system composed of Na₂Se O₃and ascorbic acid solution.A Platycodon grandiflorus polysaccharide selenium nanoparticles complex（PGP90-Se NPs）was successfully prepared and its particle size,stability and morphology were characterized by corresponding detection methods.The results indicated that the average particle size of PGP90-Se NPs was 76.1 nm and its suspension could be stored stably at 4℃for at least 30days.Meanwhile,the antitumor activity of PGP90-Se NPs in vitro was also discussed.MTT results and morphological observation suggested that the IC50 value of PGP90-Se NPs on MGC-803 and BGC-823 cells were 213.45μg/m L and 238.18μg/m L,respectively and PGP90-Se NPs could inhibit the proliferation of cancer cells by inducing apoptosis.Further cell experiments indicated that PGP90-Se NPs could induce gastric cancer cells apoptosis through mitochondrial apoptosis pathway,which was mainly manifested in inducing cell DNA damage,changing mitochondrial membrane permeability,causing cell S phase arrest,promoting the production of excess ROS,inducing mitochondrial dysfunction and affecting the expression of corresponding apoptotic proteins such as Bax and Bcl-2 to promote apoptosis.Finally,the antitumor effects of PGP90,PGP-AE and PGP90-Se NPs in vivo were discussed by constructing S180 tumor bearing mice.The results suggested that PGP-AE had inapparent antitumor effect in vivo,while PGP90 and PGP90-Se NPs could inhibit the proliferation of tumor obviously with less side effects on mice.The tumor inhibition rates of those high-dose groups were 29.64%and 49.41%respectively.PGP90 and PGP90-Se NPs could protect the immune organs of tumor-bearing mice,improve the physical function,enhance the vitality of immune cells and increase the proportion of lymphocytes,thus improving the immune level of mice and changing the tumor microenvironment,so as to change the level of related apoptotic proteins of solid tumor cells and inhibit the growth of solid tumors.In this research,the structure,selenium nanoparticles modification and antitumor activity of two polysaccharides from Platycodon grandiflorus were systematically studied,which could provide a theoretical foundation for the utilization on natural active substance containing Platycodon grandiflorus polysaccharides.