Preparation, Antitumor And Hepatoprotective Activities Of Potent Bioactive Polysaccharides Extracted From Talinum Triangulare

Talinum triangulare (Jacq.) Willd (Waterleaf) is a perennial herb from the family Portulacaceae. It is originally grown in rain forest regions of the tropical world, such as Afican, South America and India. T. triangulare, which is edible and has nutritional and medical values, has been used in the treatment and prevention of hepatic ailments and cancers in folk medicine. The extraction conditions of polysaccharides from T. triangulare were optimized and the antioxidant capabilities were evaluated in vitro. Hepatoprotective effect against CCl4-induced liver injury and anti-tumor activities against hepatoma H22-bearing mice in vivo were investigated by biochemical indexes analysis couple with histopathological examination. The mechanism of anti-tumor activities and immunoenhancing activities were further studied via flow cytometry and PCR technique. Finally, to elucidate structure-activity relationship, polysaccharide fractions were purified and characterized by GC, IR and NMR. The main results were as follows:(1) Optimal extraction of polysaccharides from T. triangulareThe extraction conditions of polysaccharides from T. triangulare were optimized by single factor test and orthogonal design with polysaccharide yield as index. The results showed that the optimal conditions of extraction were twenty-five volume of water was used at 100℃ and for 2.0 h. Account for the yield and subsequent concentration difficulty, the extraction process were repeated once and the TTP yield is 14.99%.(2) Assessment of antioxidant abilities of TTP in vitroThe antioxidant abilities of 40%,60%,80% ethanol precipitating TTP fractions and crude TTP were evaluated using three different models in vitro, including total reducing power, scavenging activities of hydroxyl radicals and superoxide anion radicals. The results showed different degrees of antioxidant activities in dose-dependent manners. The 40% TTP fraction possessed the strongest antioxidant activity, with the inhibition rate of hydroxyl radicals by 89.80% at 2.0 mg/mL and that of superoxide anion radicals by 63.23% at 5.0 mg/mL.(3) Effect of TTP on tumor growth and immune system of H22-bearing miceThe results indicated that TTP possessed the abilities of suppressing the growth of solid tumor of H22-bearing. H22 tumors were inhibited by 49.07% at the dose of 200 mg/kg·bw. Compared with the model group, the spleen index was significantly increased (P<0.001), and the levels of cytokines of TNF-a, IFN-y and IL-2 were also markedly ascended (P<0.01-0.001), which meant the immune system was ameliorated. Therefore, the anti-tumor effect of TTP is related to the immunomodulating activity of TTP.(4) Effect of TTP on murine macrophage RAW 264.7 cells and anti-proliferation capabilities against HepG2 cells of supernatant of RAW 264.7The results demonstrated that TTP could activate primary murine macrophage (PMM) cells. Compared with the control group, the PMM activation rate reached 135.39% at 200 μg/mL, and there was no significant difference between TTP and with the LPS (5 μg/mL) treated groups. After co-incubation with TTP for 48 h, the NO content in supernatant of RAW 264.7 was obviously elevated (P<0.001) and the cytotoxicity was also significantly enhanced, which was testified by MTT experiment and the flow cyometry results. The anti-proliferation rate of supernatant of RAW 264.7 treated with TTP was up to or even more than 60%. RT-PCR results showed that expression of iNOS gene was increased, which was in accordance with the trend of chemical index. Meanwhile, expression of TLR2/4 gene was also augmented, suggesting that TTP could activate macrophage via TLR2/4 receptors.(5) Hepatoprotective effect of TTP against CCl4-induced liver injuryThe results suggested that TTP had the abilities of restoring the activities of antioxidant substances SOD and GSH towards normalization and suppressing the production of lipid peroxidation product MDA. The hepatoprotective effect of TTP (200 mg/kg·bw) were close to that of silymarin (50 mg/kg·bw), which was revealed by biochemical indexes analysis and histopathological examination. It was inferred that TTP could protect liver from chemical damage by strengthening the activities of antioxidases, inhibiting the formation of free radical and the immunomodulating activity. (6) Purification and characterization of TTPTTP-1 and TTP-4 were acquired by successive column chromatography of DEAE-52 and Sephadex G-100. The molecular weight of TTP-4 was calculated as 87.4 kDa. Beta-digestion reaction proved that there was O-glycosidic bond in polysaccharides TTP-4. The monosaccharide composition analysis of TTP-4 revealed that it was a heteropolysaccharide consisted of rhamnose, arabinose, mannose and galactose, with a molar ratio of 1.22:1.00:1.05:1.51. A characteristic peak at 890 cm-1 in the infrared spectrum indicated the β-configuration of TTP-4, which was in accordance with the 1H-NMR spectrum. The result of 1H-NMR spectrum further confirmed that TTP-4 was composed by four monosaccharides.