Selenized Modification, Characterization And Evalution Of Antitumor Activities Of Three Polysaccharides In Vitro

Se（Selenium）,an essential microelement in life,plays an irreplaceable role in delaying senility,improving immunity,preventing cardiovascular disease and treating Keshan disease.There are inorganic and organic selenium in nature.The inorganic selenium have cumulative and mutagenic effect.Usually,the organic selenium with low toxicity is selected to achieve the goal of selenium supplementation in humans.The selenized polysaccharides played a very important role in selenium-supplement reagent.However,content（0.5-20μg/g）of selenium is pretty low and limited by geographical restrictions.Reagents in selenized moddification were involved in HNO₃-Na₂SeO₃,CH₃COOH-Na₂SeO₃ and other at present in order to gain biological activity.In many aprotic solvents,content（500-1.2×10⁴μg/g）of selenium of synthetic selenized polysaccharides is low due to polysaccharides poor solubility.It is crucial to solve the solubility of polysaccharides and improve the esterification ability of selenite therefore.In recent years,ionic liquids（IL）instead of conventional solvents,have been a major breakthrough in the solvent and catalyst for polysaccharides.Ionic liquids provided a new thought in the development and preparation in new functional material of polysaccharides.In this paper,acidic ionic liquids were syntheized as solvent（or cosolvent）and catalyst catalyst to prepare selenized polysaccharides with higher selenium.The effect of ionic liquids on the dissolution of polysaccharides was investigated.The selenized derivatives were structurally characterized by FT-IR,XPS,¹³C NMR and SEC methods.Selenized polysaccharides with different selenium content and molecular weight were employed to preliminary study of antitumor activity in vitro.The main contents of this paper are as follows:1.Preparation and characterization of varieties of acidic ionic liquidsVarious acidic ionic liquids were synthesized with imidazole（Im）,methyl imidazole（MIm）,pyridine（Py）and nitrogen methyl pyrrolidone（NMP）as cation,sulfuric acid（H₂SO₄）,nitric acid（HNO₃）,hydrochloric acid（HCl）,phosphoric acid（H₃PO₄）,and methyl sulfonic acid（CH₃HSO₃）as anion and characterized by FT-IR and ¹H NMR.2.Effects of anion and cation on synthesis of selenized artemisia sphaerocephala polysaccharide in acidic ionic liquidsBased on the two kinds of reaction systems of ionic liquid-formamide and Cl^-acid ionic liquids,selenized artemisia sphaerocephala polysaccharide（SeASP）with different selenium content were synthesized.The effect of anion and cation change on synthesis of SeASP was studied.For four kinds of cations,the catalytic capacity followed by[HNMP]H₂PO₄（selenium content 26769μg/g）,[HPy]Cl（selenium content 25898μg/g）,[HIm]NO₃（selenium content 20438μg/g）and[HMIm]CH₃SO₃（selenium content 18267μg/g）.The acidity of anions is directly relate to content of selenium,and the acidity was high or low both to be unbeneficial to the synthesis reaction.Acidic ionic liquids containing Cl^-have ability to dissolve ASP.FT-IR showed thatυ_Se=O andδ_C-O-Se appeard new peak at 1100 cm^-1 and 870 cm^-1.XPS analysis showed that Se existed as Se⁴⁺in SeASP.The ¹³C NMR result confirmed that the weak C-6 substitution was observed in SeASP.The results of molecular weight determination showed addition of acidic ionic liquids resulted in degradation of polysaccharides chain,but the degradation was much lower than reported in the literature.3.Selenium modification of three polysaccharides under formamide-[HNMP]HSO₄ systemSynthesis of selenized Fenugreek gum polysaccharide（SeFG）,selenized Locust bean gum polysaccharide（SeLBG）and SeASP catalyzed at room temperature ionic liquids（[HNMP]HSO₄）.[HNMP]HSO₄ can effectively improve the activity of selenite,esterifying capacity of polysaccharide and the selenium content of SeASP.The selenium content in SeFG,SeLBG and SeASP increased from 4060μg/g,4428μg/g and 5612μg/g to 31317μg/g,28821μg/g and 33933μg/g.FT-IR,XPS shows that Se existed as Se⁴⁺in selenized polysaccharide.The reaction between selenized derivatives and MB can confirmed that the introduction of selenite groups can change the conformation of polysaccharide chains.4.Preliminary study on antitumor activity of SeASP in vitroIn vitro cell models of human lung cancer cells（H1299）,human lung bronchogenic carcinoma cells（H1650）and human hepatoma cells（HepG-2）were established.The inhibitory effects of SeASP with different selenium content and molecular weight ofon tumor cells in vitro were studied.SeASP₃₈（selenium content was 27607μg/g,the molecular weight was 5.884×10⁴Da）acting on the H1650,H1299 and HepG2 of tumor cells IC50 values were 71.818,579.063 and 302.87μg/mL.Low selenium content（27607μg/g）and moderate molecular weight(5.884×10⁴ Da)samples showed stronger inhibitory activity on tumor cells in vitro.SeASP₃₈ has the ability of inhibiting cell cycle and apoptosis,and can arrest H1299cell cycle in G1 and G2 phases.The early apoptosis rate and late apoptosis rate of 24h and 48 h were 2.81%,5.29%and 4.54%,3.92%,respectively.In summary,acidic ionic liquids consisting of various of anions and cations,as solvents and catalysts,can effectively increase the dissolution and capacity of selenium and improve the selenium content of polysaccharides.Cell experiments in vitro confirmed that selenized derivatives can inhibit the growth of tumor cells and achieve anti-tumor effect.In this paper,the sample of tumor cell activity is relatively less,so more samples are necessary to screen for further research.The research can provide reference for the synthesis and activity study of selenized polysaccharide.