Mechanism of antagonistic effects of Andrographis paniculata methanolic extract against Staphylococcus aureus.

Abstract

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 invitro. Antioxidative property of methanolic leaves extract of A.paniculata (0.06mg/mL). Minimum inhibitory concentration (MIC) was determined by its ability to reduce hydrogen peroxide (H2O2) toxicity against S.aureus ATCC 25923 [(3.8×108)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 O2- in S.aureus. Cells challenged with 7.5mmol/L H2O2 showed 0% survival in 30min whereas 25% survived after treatment with the extract and H2O2. 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 26.8U compared to untreated cells, 32.4U (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 1.828U compared to untreated cells which was 1.248U, (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 0.018U compared to untreated cells, 0.012U, (P<0.05). Extract-treated cells had significantly lower intra- and extracellular O2- levels with absorbance readings (A575nm) of 0.340 and 0.524 compared to untreated cells which were 0.516 and 0.928 (P<0.05), respectively. Taken together these results suggest that the low MIC of A.paniculata methanolic leaves extract (0.06mg/mL) reduce H2O2 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.