The essential oil from the roots of Bupleurum longiradiatum, obtained by hydro-distillation was analyzed by gas chromatography/mass spectrometry (GC/MS) and evaluated for antimicrobial activity and antioxidant activity. Fifty-one compounds were identified, representing 99.3% of the total oil. The major constituents were thymol (7.0%), butylidene phthalide (6.8%), 5-indolol (5.6%), heptanal (5.3%), 4-hydroxy-2-methylacetophenone (5.3%), 4,5-diethyl-octane (5.3%), bormeol (5.1%) and hexanoic acid (5.1%) .The oil was tested against 4 bacteria at different concentrations using disc diffusion and 96-well dilution methods. The inhibition zones and minimum inhibitory concentration values for bacterial strains were in the range of 7.0-18.0 mm and 250-500 μg/mL, respectively. The in vitro antioxidant activity was assessed by DPPH radical scavenging and inhibition of lipid peroxidation methods. The oil showed a potent free radical scavenging activity, as evidenced by the low IC 50 value for DPPH radical (566.2μg/mL) and inhibition of lipid peroxidation (induced by FeSO 4 , H 2 O 2 and CCl 4) with IC 50 values of 275.2 μg/mL, 296.9 μg/mL and 118.7 μg/mL, respectively.
In previous studies, tobacco protoplasts were transformed with the bacterial gene encoding neomycin phosphotransferase II (NPT II). Transformed calluses lost neomycin phosphotransferase II activity after several subcultures. Treatment of calluses with 5-azacytidine, a demethylating agent, restored enzyme activity, suggesting that methylation of npt H sequences might be responsible for loss of NPT II activity. Studies presented here were designed to test that hypothesis. Results indicated that the effect of 5-azacytidine could not be blocked by the DNA replication inhibitor, hydroxyurea, nor by the 5-azacytidine analogue, cytidine as would be expected with a DNA demethylation mechanism. The level of NPT II mRNA was not increased by 5-azacytidine. Treatment with cycloheximide, a protein synthesis inhibitor, had no effect on 5-azacytidine-increased NPT II activity. There was no increase of NPT II protein caused by 5-azacytidine, whereas 5-azacytidine increased activity of NPT II. In contrast, the auxin 2,4-D increased both the NPT II protein and activity. Assays for malate dehydrogenase demonstrated that the effect of 5-azacytidine and hydroxyurea on NPT II was not due to an overall effect on callus metabolism. In vitro studies involving standard bacterial NPT II enzyme and crude extracts from untreated and 5-azacytidine-or hydroxyurea-treated calluses showed that the activity of NPT II added to the untreated extracts was lower than the activity of NPT II added to the extracts from calluses treated with 5-azacytidine or hydroxyurea, indicating that there was an unknown factor (or factors) in callus extracts which affected the activity of NPT II and itself was affected by 5-azacytidine and hydroxyurea treatment. These results suggested that one effect of 5-azacytidine in increasing NPT II activity was posttranslational.
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