Pilot Study On The Antioxidation Activity In Vitro And Mechanism Of Feruloylated Oligosaccharides Extracted From Maize

ObjectiveThis study to observe the antioxidant activity in vitro of Feruloyl oligosaccharides (FOs) extracted from Maize on injury Pheochromocytoma cells (PC 12) induced by hydrogen peroxide (H2O2) and the change of related indicators in this process. Moreover, the possible mechanisms of the antioxidant activity of FOs were discussed.Methods1. Establishment of oxidatie damage model with PC12 cells in vitroPC 12 cells were cultured in vitro. They were divided into normal control group and seven oxidative groups which were injured by H2O2 with different concentration (0.25,0.4,0.5,0.8,1.0, 1.6,2.0 mmol/L, respectively). After 4 hours, the absorbance (A) changes in MTT in each group was measured. Moreover, the apoptosis rate, the contents of MDA and the activity of LDH were detected in normal control group and H2O2-L, M, H groups (0.4,0.8,1.6 mmol/L, respectively) and the morphological changes were observed by the light microscope, respectively.2. Pilot Study on the antioxidation activity in vitro and mechanism of FOsPC 12 cells were divided into 9 groups. They were normal control group, H2O2 injury group (0.8 mmol/L), FOs groups with different concentration (25,50,100,200,400,800,1600μmol/L). Cells were injured by H2O2 for 4 hours after they were incubated with different concentration FOs for 0,6,12,24 hours. Then the absorbance (A) changes in MTT in each group was measured. And the apoptosis rate, the contents of MDA and the activity of LDH and SOD were detected in normal control group, H2O2 injury group and FOs-L, M, H groups (50,200, 800μmol/L) and the morphological changes were observed, respectively.Result 1. Establishment of oxidatie damage model with PC12 cells in vitro1) The absorbance (A) changes in MTT were significantly lower than those in the normal control group (P<0.05), which implies the effect is in dose dependent manner. And the 50% inhibitory concentration is 0.8 mmol/L.2) The morphological changes of PC12 cells of the 0.4 mmol/L group were similar to that of the normal control group, while changes of the 0.8 and 1.6 mmol/L groups were significantly different. The cells of 0.8 mmol/L group became distorted, at the same time, the synapse became shorter while the cell border was still clear. The cells of 1.6 mmol/L group became distorted apparently and the border was not clear. Beside those, the cells fall off from the bottom of wells.3) The rate of the apoptosis of H2O2-L, M and H groups were higher than that of the normal control group (P<0.01), and the effect is in dose dependent manner.4) The contents of MDA and the activity of LDH were significantly higher than that of the normal control group (P<0.01).2. Pilot Study on the antioxidation activity in vitro and mechanism of FOs 1) The absorbance (A) changes in MTT were lower than those in the H2O2 injury group (P< 0.05), which implies the effect is dependent on concentration. And the cell viability comes to the highest point in group with FOs 800μmol/L.2) The antioxidant activity in vitro of FOs is not only in dose but also in time dependent manner. The cell viability of PC12 incubated with FOs at least 50μmol/L for 6,12,24 hours, is higher than the 0 hour group (P<0.05). When the FOs concentrations were 200, 400 or 800μmol/L, the cell viability of PC12 incubated for 12 or 24 hours, is higher than that of the 6 hours group (P<0.05). But the difference of the cell viability between 12 and 24 hours groups is not statistically significant (P>0.05).3) The cells shape of FOs-L, M and H groups returned to normal condition. With the increasing of FOs concentration, the amount of cells adhered on the bottom of wells increased, and the synapse elongated also.4) The rate of the apoptosis of FOs-L, M and H groups were lower than that of the H2O2 injury group (P<0.01), and the effect is in dose dependent manner. 5) The MDA's contents and the LDH's activity of FOs-L, M and H groups were significantly lower than that of the H2O2 injury group (P<0.01). But the activity of SOD was obviously increased (P<0.01).ConclusionIn this experiment, PC 12 cells can be injured by H2O2 and show antiproliferation effect and apoptosis in varying degrees.0.8 mmol/L and 4 hours is the optimum condition for this research. The FOs has best protective effect on PC 12 cells injured by H2O2 in condition of 800μmol/L and 12 hours. The mechanisms of the antioxidant activity of FOs include holding the free radicals in check and terminating its chain reaction; improving the activity of antioxidase such as SOD and enhancing the anti-oxidation capacity of PC 12 cells, etc.