Purification and characterization of pectin methylesterase isoenzymes from orange peel

Pectin methylesterase [PME] isoenzymes from Citrus sinensis [Balady orange] peel have been purified and characterized. The purification procedure included CM-cellulose, DEAE-cellulose and Sephacryl S-200 columns. PMEl, PME2 and PME4 were purified to homogeneity. The molecular weights of PMEl, PME2 and PME4 were determined using Sephacryl S-200 column and estimated to be 23,000,·47,400 and 79,500 Da, respectively. PME1, PME2 and PME4 had isoelectric points of 5.4, 5.9 and 5.6, and pH optima of 6.0, 6.0 and 5.5, respectively. Maximum PME activity was reached at 50 mM NaCl for PMEl and PME4, and 25 mM NaCl for PME2. Km values of PMEl, PME2 and PME4 using pectin with degree of esterification [DE] of 8% were 0.55, 1.0 and 3.33 mg pectin/ml, respectively. The affinity of PMEs was increased with the increasing of the DE of pectin. The time-dependent heat-inactivation curves for the three isoforms exhibited exponential behaviour and showed that PMEl was more heat resistant than PME2 and PME4. Effect of pH on enzyme stability, kinetic properties [Vmax and Keat] of isoenzymes, and effect of different metal cations on enzyme activity were investigated. A study of the inhibitory effect of ferrous sulfate [FeSO[4]] on the PME activity in freshly prepared Balady orange juice revealed that 1 mM FeSO[4] was the best concentration that maintained the homogeneity and stability of the juice

Bee honey and Nigella sativa inhibit nitric oxide mediated cytochrome C release and down regulation of connexin 43 induced by methylnitrosourea in hepatic tissues of Sprague Dawley rats

We investigated the protective effect of bee honey and Nigella sativa against methylnitrosourea [MNU] induced oxidative stress, apoptosis and down-regulation of the gap junction protein connexin 43 [Cx43] in hepatic tissues. Our results showed that MNU induced hepatic dysplasia and oxidative stress through decreasing reduced glutathione [GSH] level, decreasing the activities of glutathione reductase [GRase] and superoxide dismutase [SOD], enhancing glutathione peroxidase [GPx] activity and elevating nitric oxide [NO] level in liver tissues as compared to control. Apoptosis, as indicated by significant release of cytochrome c [cyto c] from mitochondria into the cytosol, was observed in MNU injected rats and was positively correlated with the elevation of N0 level. Bee honey and Nigella protected against the previous deleterious changes by significant elevation of GSH level, increasing the activities of GRase and SOD, decreasing the activity of GPx, decreasing NO level and stopping release of cyto c from mitochondria as compared to MNU injected rats. Moreover, down-regulation of Cx43 by MNU was prevented in 50% and 75% of animals that received Nigella alone or both Nigella and bee honey, respectively. These data illustrated the consequences of MNU induced hepatocellular dysplastic changes and oxidative stress and shed the light about the possible sites targeted by bee honey and Nigella in their protective effect against the earlier stages of liver carcinogenesis