Study On Structural Feature, Antitumor Activity Of Rice Bran Polysaccharide And Their Sulfates

Rice bran is the important by-product of the rice milling industry, it was considered as the rich and reproducible resource. Although the yield of rice bran in our country was primacy in the world, the low-level of study on rice bran has limit the intensively utilization potential value of rice bran. In this paper, we studied the isolation, purification and structural feature of RBPS2a, the sulfated rice bran polysaccharide SRBPS2a showed the potent antitumor activities, therefore, the mechanism of antitumor activity on SRBPS2a both in vitro and in vivo and the the molecular mechanism on apoptosis of tumor cells were also investigated. There is limited reported on mechanism of antitumor activity and apoptosis of tumor cells of sulfated rice bran polysaccharide. The present study could improve the utilize value and enrich the basic theory of rice bran polysaccharide.In order to maximize the yield of rice bran polysaccharide, response surface methodology (RSM) and RBF neural network were employed to optimize the ultrasound-assisted extraction condition. The RSM model showed that the yield of rice bran polysaccharide was related to temperature(X1), ultrasonic power(X2), interactions of temperature(X1×X1) and quadratic of temperature and ultrasonic power(X1×X2).According to the RBF- QPSO, the optimum extraction conditions were temperature of 82.5℃, ultrasonic power of 335 W, extraction time of 37.5 min and Water/solid ratio of 18:1. RBF-QPSO was better than RSM in ultrasonic-assisted extraction of rice bran polysaccharide.Based on the bioactivity-directed test, a novel heteropolysaccharide RBPS2a with anti-tumor activity was obtained from rice bran by hot water extraction, ethanol precipitation, and purified by Sepharose CL-6B gel chromatography after Q-Sepharose big beads chromatography. RBPS2a was eluted as a single symmetrical narrow peak on high-performance gel-permeation chromatography (HPGPC) and the average molecular weight was 9×105 Da. Gas chromatography of absolute acid hydrolysate of RBPS2a suggested that it was composed of arabinose, xylose, glucose and galactose with a molar ratio of 4:2:1:4. The Fourier-transform infrared spectra (FT-IR) and 1H, 13C NMR spectroscopy analysis revealed that RBPS2a had a backbone consisting ofβ- (1→3)- linked D- galacopyranosyl residues substitured at O-2 with glycosyl residues composed ofα- D- xylose–(1→4)-α- D- arabinose (1→andα- D- glucose–(1→4)-α- D-arabinose (1→linked residues.Sulfated rice bran polysaccharides (SRBPS), were prepared by chlorosulfonic acid-pyridine (CSA-Pyr) method, according to the antitumor activities test. The optimum modification conditions were reaction temperature of 70℃, the ratio of chlorosulfonic acid to pyridine of 1:4 and the reaction time of 2h. Under this condition, sulfated derivatives SRBPS2a exhibit relatively strong antitumor activity in vitro. The average molecular weight of SRBPS2a was 3.5×105 Da and the degree of sulfation (DS) was 1.29. The Fourier-transform infrared spectra (FT-IR) and 13C NMR spectroscopy analysis revealed that SRBPS2a was mainly consist ofβ- (1→3)- D- galacopyranosyl residues and the sulfate substitution site is on C-2 and C-4, the side chains were cut off during the sulfated reaction. Furthermore, SRBPS2a exhibited evident growth inhibition on Mouse mammary tumor EMT-6 cells both in vitro and in vivo.The antitumor activity and its mechanism of SRBPS2a were examined and evaluated with the mice transplanted EMT-6 tumor in vivo. At the dosage of 75 mg/kg.d and 50 mg/kg.d, SRBPS2a had strong antitumor activity with 55.95% and 44.05%. Compared with the 5-FU group, SRBPS2a could increase both the thymus index and the spleen index. The histopathology of tumors from the various groups indicated that the tumor cells of untreated mice grew vigorously, however the tumor cells from the different SRBPS2a treated groups had clear nucleus pycnosis and necrosis areas in different degree. The expression of tumor gene Bax and Bcl-2 in tumor tissues were also detected with immuno-histochemistry. After the expression between the experimental and control groups, there was obvious difference in the quantities of Bax and Bcl-2 genes expression between the experimental and control group, which indicated that SRBPS2a could control growth of tumor and accelerate the tumor cell apoptosis by influencing the expression related tumor genes.HepG-2 cell line was used to investigate the mechanism of SRBPS2a in vitro. Results showed that the tumor cells treated with SRBPS2a for 48 h showed significant morphological changes, such as cell shrinkage, membrane blebbing and volume reduction. Scanning electron microscope (SEM) photos showed that the villus of the HepG-2 cell was disappeared at higher concentration and the surface of membrane was smooth and shrinkage to form apoptotic bodies. Moreover, typical apoptotic morphological features (volume reduction, chromatin condensation, nuclear fragmentation) were also observed by fluorescent microscopy (Hoechst 33342). Flow cytometric analysis showed that the HepG-2 cell cycle was arrested in the G2/M phase, and the subdiploid peak of DNA characteristic of apoptotic was observed, and the apoptosis rate was about 23.4%. Further investigation results showed that the apoptotic machinery of HepG-2 induced by SRBPS2a was associated with a decrease in Bcl-2/Bax ratio, drop in mitochondrial trans-membrane potential, and upgrade the protein expression of caspase-3 by immunocytochemical staining.