ABSTRACT
OBJECTIVE@#To investigate the effects of Andrographis paniculata (Burm.f.) Wall. Ex Nees (A. paniculata) on expressions and activities of catalase, superoxide dismutase and alkylhydroperoxide reductase C in Staphylococcus aureus (S. aureus) with respect to its survival in vitro.@*METHODS@#Antioxidative property of methanolic leaves extract of A. paniculata (0.06 mg/mL). Minimum inhibitory concentration (MIC) was determined by its ability to reduce hydrogen peroxide (HO) toxicity against S. aureus ATCC 25923 [(3.8 × 10) cfu/mL]. Effects of the extract on expressions of katA (encoding catalase), sodA and sodM [encoding superoxide dismutases (SODs)], and ahpC [encoding alkylhydroperoxide reductase C (AhpC)] in S. aureus were determined by RT-qPCR and corresponding enzyme activity assays were performed. Nitroblue tetrazolium reduction (NBT) assay was performed to determine effects of the extract on intracellular and extracellular levels of O in S. aureus.@*RESULTS@#Cells challenged with 7.5 mmol/L HO showed 0% survival in 30 min whereas 25% survived after treatment with the extract and HO. Cells that were treated with the extract alone had 43% survival in the same exposure period. Expressions of sodA and sodM genes in extract-treated cells were lowered 0.8-fold and 0.7-fold, respectively with decrease in total SOD activity of 26.8 U compared to untreated cells, 32.4 U (P < 0.05). In contrast, extract-treated S. aureus cells showed 3.3-fold increase in katA expression with corresponding increase in catalase activity of 1.828 U compared to untreated cells which was 1.248 U, (P < 0.05). More profoundly, ahpC expression was increased 61-fold in extract-treated cells, (P < 0.05) with corresponding increase in AhpC activity of 0.018 U compared to untreated cells, 0.012 U, (P < 0.05). Extract-treated cells had significantly lower intra- and extracellular O levels with absorbance readings (A) of 0.340 and 0.524 compared to untreated cells which were 0.516 and 0.928 (P < 0.05), respectively.@*CONCLUSIONS@#Taken together these results suggest that the low MIC of A. paniculata methanolic leaves extract (0.06 mg/mL) reduce HO toxicity and more importantly, was in itself effectively inhibitory against S. aureus. Further, our observations suggest that a probable mode of its inhibitory mechanism against S. aureus is by reducing total SOD activity through downregulation of sodA and sodM expressions.
ABSTRACT
Objective To investigate the effects of Andrographis paniculata (Burm.f.) Wall. Ex Nees (A. paniculata) on expressions and activities of catalase, superoxide dismutase and alkylhydroperoxide reductase C in Staphylococcus aureus (S. aureus) with respect to its survival in vitro. Methods Antioxidative property of methanolic leaves extract of A. paniculata (0.06 mg/mL). Minimum inhibitory concentration (MIC) was determined by its ability to reduce hydrogen peroxide (H
ABSTRACT
<p><b>OBJECTIVE</b>This study investigated the effects of allylpyrocatechol (APC), the major component in ethanolic extract of Piper betle, on key oxidative stress resistance enzymes important for the survival of Staphylococcus aureus, a major pathogen in the human host.</p><p><b>METHODS</b>Effects of APC on expressions of genes encoding catalase (katA), superoxide dismutases (SODs), including sodA and sodM, and alkyl hydroperoxide reductase (ahpC) in Saureus were quantitated by RT-qPCR in reference to gyrA and 16S rRNA. Corresponding activities of the enzymes were also investigated. The Livak analysis was performed for verification of gene-fold expression data. Effects of APC on intracellular and extracellular reactive oxygen species (ROS) levels were determined using the nitroblue tetrazolium (NBT) reduction assay.</p><p><b>RESULTS</b>APC-treated Saureus cells had higher sodA and sodM transcripts at 1.5-fold and 0.7-fold expressions respectively with corresponding increase in total SOD activity of 12.24 U/mL compared to untreated cells, 10.85 U/mL (P<0.05). Expression of ahpC was highest in APC-treated cells with 5.5-fold increased expression compared to untreated cells (P<0.05). Correspondingly, ahpC activity was higher in APC-treated cells at 0.672 (A) compared to untreated cells which was 0.394 (A). In contrast, katA expression was 1.48-fold and 0.33-fold lower respectively relative to gyrA and 16S rRNA. Further, APC-treated cells showed decreased catalase activity of 1.8 ×10(U/L or μmol/(min·L)) compared to untreated cells, which was 4.8 ×10U/L (P<0.05). Absorbance readings (A) for the NBT reduction assay were 0.709 and 0.695 respectively for untreated and treated cells, which indicated the presence of ROS. APC-treated Saureus cells had lower ROS levels both extracellularly and intracellularly, but larger amounts remained intracellularly compared to extracellular levels with absorbances of 0.457 and 0.137 respectively (P<0.05).</p><p><b>CONCLUSION</b>APC induced expressions of both sodA and sodM, resulting in increased total SOD activity in Saureus. Higher sodA expression indicated stress induced intracellularly involving O, presumably leading to higher intracellular pools of HO. A concommittant decrease in katA expression and catalase activity possibly induced ahpC expression, which was increased the highest in APC-treated cells. Our findings suggest that in the absence of catalase, cells are propelled to seek an alternate pathway involving ahpC to reduce stress invoked by Oand HO. Although APC reduced levels of ROS, significant amounts eluded its antioxidative action and remained intracellularly, which adds to oxidative stress in treated cells.</p>