| Emblic is a medicinal and edible tropical fruit,and emblic polysaccharide is one of effective component in it.It has activity of antioxidant,anti-tumor and immunity-enhancement.The principal work of this paper consists of three aspects:the extraction process optimization of emblic polysaccharide(EPS),the influence of EPS on the proliferation of HepG-2 cells and carboxymethyl modification of EPS.1.Response Surface Optimization of Microwave-assisted Extracting(MAE)EPSResponse surface methods and Box-Benhnken central composite design were employed to optimize EPS extraction process.The optimal conditions of microwave-assisted extracting of EPS were follows:the microwave time 70.06 s,the microwave power 489.48 W,leaching time 3.86 h,and leaching temperature 100℃.The corresponding condition were modulate into 70 s,480 W,3.85 h,and 100℃ respectively,considering practical conditions.As a result,the experimental extraction rate was 8.11%.In addition,the correlation coefficient of the model R2=0.9683,Adj R2=0.9366,Pred R2=0.8387,indicating that the optimization model was established successfully.2.The Influence of EPS on the Proliferation of HepG-2 CellsMTT proliferation assay indicated that the proliferation of HepG-2 cells was suppressed under the treatment of 1000μg·mL-1 EPS for 48 h,the inhibition rate could be 29.68 ± 3.26%;HepG-2 cells number decreased and the cells appearance turned shrunken,rounded off,showing apoptosis phenomenon with the treatment of 1000 μg · mL-1 EPS for 48 h;The detection of Annexin V-PE showed that the EPS induced the apoptosis of HepG-2 cells,and the apoptosis rate was highest(13.73%±1.25)under the treatment of 1000 μg·mL-1 EPS for 72 h,showing a tendency of both time-and dose-dependent.After the dyed of CDCFH-DA(6-carboxy-2,7-dichlorodihydrofluorescein diacetate),it was observed that that the content of reactive oxygen species(ROS)in cells increased sharply and the cells were injured after 48 h of the EPS treatment;After the treatment of EPS,the content of nitric oxide(NO),malonaldehyde(MDA),lactic dehydrogenase(LDH),glutathione peroxidase(GSH-PX)increased and the superoxide dismutase(SOD)content decreased,suggesting that EPS impaired HepG-2 cells by increasing intracellular ROS content.The results of quantitative RT-PCR(qRT-PCR)showed that oxidative stress relative genes such as Nrf2(nuclear related factor),NQO-1(quinone oxidoreductase 1)and HO-1(heme oxygenase-1)increased with the prolonging of EPS processing,suggesting an injury mechanism on HepG-2.The apoptosis related genes such like p21(cell cycle related protein)and Caspase-3(cysteine protease gene family)were also increased with the prolonging of EPS processing;The expression of p53(tumor suppressor gene encoding 53kDa protein),Bak(B cell lymphoma/leukemia-2 gene family gene)were markedly higher than that of the control;The ratio of Bcl-2/Bax decreased with the prolonging of time.The results of qRT-PCR indicated that EPS has suppressing effect on HepG-2 cells.3.Antioxidant and Antineoplastic Activities of Carboxymethyled Polysaccharide from Fruits of Phyllanthus emblic L.EPS was carboxymethylated in the system of sodium hydroxide-isopropanol sodium monochloroacetate and the activity of both of the polysaccharide about scavenging free radical,NO2-and inhibiting proliferation effect of HepG-2 as well as cell apoptosis were Comparative investigated for the sake of evaluating CM-EPS activity.Characteristics of the spectrum(FT-IR)showed the modification was successfully,Carboxymethylation of the polysacchride from emblica(CM-EPS)was prepared.CM-EPS exhibited an outstanding ability on scavenging NO2-and enhanced inhibitory effect against the proliferation of HepG-2 cells remarkable.In addition,it also revealed a declined scavenging effect on ABTS radical but exhibited a markedly enhanced scavenging effect on DPPH in high concentrations compared with the native polysaccharide. |
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