Study On The Preparation,Structural Characterization And Antitumor Mechanism Of Dandelion Polysacchatides

Dandelion is a widely distributed medicinal and edible plant with excellent medicinal value.At present,a variety of bioactive components have been isolated and identified from dandelion.As one of the bioactive substances of dandelion,polysaccharides have attracted more and more attention.Dandelion polysaccharides have significant anti-inflammatory,hypoglycemic and immunomodulatory effects.The structure and activity of polysaccharides are influenced by many factors,especially the variation of extraction temperature.Temperature not only affects the yield of polysaccharides but also is closely related to the structure of polysaccharides,which in turn determines their complex biological activities.The extraction temperature of dandelion polysaccharides is almost below 100℃,and the research on the structure and biological activity of polysaccharides above 100℃is still limited.In addition,in recent years,polysaccharides extracted from cold water have also received attention because of their good biological activity.Therefore,this study focused on three temperatures of 4℃,80℃and 120℃to extract dandelion polysaccharide and to investigate its relationship between structural characteristics and anti-tumor activity in vitro and in vivo.Firstly,the extraction conditions of three groups of crude polysaccharides were optimized by single factor and response surface experiments to obtain higher yield.The results showed that the optimal extraction conditions of c DLP4 were the ratio of water to material of 20:1,extraction time of 16 hours,extraction times of 3 times,and the yield of polysaccharide was 2.89%±0.13%.The optimal extraction conditions of c DLP80 were as follows:the ratio of water to material 20:1,extraction time of 3 hours,extraction times of 2times,and the yield of polysaccharide was 4.55%±0.17%.The optimal extraction conditions of c DLP120 were the ratio of water to material 20:1,the extraction time of 2 hours,and the extraction times of 2 times.The yield of c DLP120 was 8.49±0.23%.Then,the three groups of polysaccharides were purified by deproteinization,dialysis and column chromatography to obtain DLP4,DLP80 and DLP120.Next,the structure of the three groups of polysaccharides was characterized.The results of HPLC showed that the molecular weight of DLP4,DLP80 and DLP120 decreased with increasing temperature,which were 2.19×10~6Da,2.08×10~6 Da and 1.64×10~6 Da,respectively.Ion chromatography results showed that DLP4 and DLP80 were mainly galactose,arabinose,glucose and mannose.DLP120 was dominated by galactose,arabinose,rhamnose and galacturonic acid.The methylation results showed that the sugar residues of DLP4 were mainly T-Araf,T-Glcp,6-Galp,3,6-Galp.The sugar residues of DLP80 were mainly T-Araf,5-Araf,T-Glcp,6-Galp,3,6-Galp.The sugar residues of DLP120 were mainly 4-Galp,4-Galp A,T-Galp,5-Araf,3,5-Araf and T-Araf.Combined with IC,FT-IR,methylation and NMR data,DLP120 is a pectic polysaccharide composed of rhamnogalacturonansⅠ(RG-Ⅰ)and arabinogalactanⅡ(AG-Ⅱ)。MTT and cell morphology assays revealed that DLP120 have better antitumor activity compared with DLP4 and DLP80。Next,the mechanism of DLP120 inhibiting HepG2 cell proliferation in vitro was investigated.It was observed by cell morphology that HepG2 cells treated with DLP120 showed a typical apoptotic morphology with cell crumpling and formation of apoptotic vesicles.Apoptosis was also observed by Hoechest33258staining.DLP120 caused cell cycle arrest in S phase and early and late apoptosis in HepG2cells by cell cycle and Annexin V-FITC/PI double staining assays.In addition,DLP120treatment also contributed to a decrease in mitochondrial membrane potential and a significant increase in intracellular reactive oxygen species(ROS)levels in HepG2 cells.The excessive accumulation of ROS induced oxidative stress and activated the p53 signaling pathway.Furthermore,the expression of mitochondrial and oxidative stress-related proteins was examined by western blot(WB).The results showed that the level of Bax increased,while the expression of Bcl-2 decreased dramatically.Moreover,the expressions of cytochrome C,caspase-3,caspase-9,p53,Fas and Fas L were also increased.WB confirmed that the excessive accumulation of ROS activated the p53 signaling pathway.In conclusion,DLP120 induces apoptosis of HepG2 cells through mitochondrial pathway and p53 signaling pathway.Finally,H22 tumor-bearing mice model was established to research the antitumor effect and mechanism of DLP120 in vivo.The results showed that DLP120 could not only significantly inhibit tumor growth,but also effectively protect the immune organ damage caused by tumor transplantation.DLP120 can alleviated liver and spleen enlargement and attenuated thymus atrophy.In terms of immune cells of H22 tumor-bearing mice,DLP120could not only significantly improve the killing ability of NK cells and phagocytosis ability of macrophages,but also stimulate lymphocyte proliferation and increase the proportion of lymphocyte subsets CD3+T,CD4+T,CD8+T and CD19+B.Thus,DLP120 can effectively protect the immune system dysfunction caused by tumors,thereby inhibiting the growth of tumors in the body.To further explore the mechanism of DLP120 inhibiting tumor growth,HE staining was used to directly observe the morphology of tumor tissues and cells.The morphological characteristics of apoptosis of tumor cells were observed.Especially,ROS levels of tumor cells in DLP120 group were significantly increased in a dose-dependent manner.The excessive accumulation of ROS would destroy the redox balance of the microenvironment and cause cell damage.P53 signaling pathway is one of the important pathways that regulate many cellular functions such as DNA repair,apoptosis,cell senescence and cell cycle.WB results showed that the protein expressions of Bax,cytochrome c,caspase-3,caspase-9,p53,Fas and Fas L were increased,while the expression of Bcl-2 was down-regulated,indicating that DLP120 could simultaneously trigger mitochondrial and p53 signaling pathways to induce tumor cell apoptosis and inhibit tumor proliferation.