Antitumor And Immune Activities Of Rice Bran Polysacchairde And Sulfated Polysacchairde Based On Ionic Liquid

As a by-product of rice processing, rice bran was considered as a substance withdifferent biological functions, and contained polysaccharides, protein, phytic acid, oryzanol.Lastest studies manifested that bioactive polysaccharides could inhibit tumor cellsproliferation, improve the immune activity, lower blood pressure. This provides a newdirection for intensively processing of rice bran. In this study, we studied the sulfatedmodification of rice bran polysaccharides (RBP) based on green ionic liquids(IL), andcompared inhibitory activity and immune activity of polysaccharides and sulfatedpolysaccharides. In addition, the mechanism of antitumor activity against tumor cells, wasalso investigated.Firstly, the antitumor and immune effects of RBP and fractions by Sepharose Big Beads andSepharose CL-6B gel column were evaluated. RBP failed to inhibit B16and HepG-2cellsgrowth in vitro. However, Raw264.7macrophages stimulated with RBP exerted cytotoxiceffects on B16cells in vitro. This cytotoxicity was associated with the stimulation of nitricoxide (NO) production and tumor necrosis factor-α (TNF-α) secretion by macrophages in adose-dependent manner. RBP2a, a fraction of RBP, notably enhanced the inhibition of B16andHepG-2cells and boosted the immunepotentiation effect compared with RBP.Secondly, based on inhibition rate against B16and HepG-2cells proliferation, a suitableIL was screened and sulfated modification conditions were determined. The results show that[BMIM]BF4was more suitable for sulfated modification than other IL. And the optimummodification conditions were reaction temperature of80°C,and the reaction time of1h in the[BMIM]BF4and DMF reaction system (the ratio of20:10). Under this condition, IC50of sulfatedderivatives against B16and HepG-2cells was124.2,741.17μg/mLThirdly, RBP2a was sulfated modification on the above optimal reaction conditions, andSRBP2a-B was acquired. SRBP2a-B could suppress B16and HepG-2cells growth inconcentration-dependent, and IC50inhibition rates were97.31,482.41μg/mL. In addition,SRBP2a-B could stimulate NO and TNF-α production, The NO and TNF-α productioninduced by250μg/ml of SRBP2a-B was nearly3.67times and321.0times higher thannormal. However, SRBP2displayed a cytotoxic effect on Raw264.7at higher concentration,and survival ratio was only79.78%at500μg/mL.Finaly, the antitumor mechanism of SRBP2a-B against B16cells was preliminarilystudied. The morphological changes of B16cells were assessed by inverted microscope,confocal microscopy and scanning electron microscopy. B16cells showed a typical apoptoticshape: cells gradually shrinkaged, the nuclei gradually concentrated, coiled, cohesion, andsurface villi cells gradually disappeared, apoptotic bodies even gradually increased with theSRBP2a-B concentration increasing. In addition, the cell cycle distribution showed that lowconcentration SRBP2a-B could induce B16cells arrested in S phase, while the highconcentrations SRBP2a-B induce B16cells arrested in G1phase. The apoptosis rates weredetermined by Annexin V-Fluos/PI, and the proportion of viable apoptotic cells were4.16%,11.61%,18.98%at50,250,500μg/mL. What’s more, orange and green fluorescence ratio showed a trend of decline with the increase of SRBP2a-B concentrations. The results showedthat the mitochondrial membrane potential was decreased, and the B16cells apoptosis inducedby SRBP2a-B is mediated by the mitochondrial pathway.