The anti-staphylococcal activity (planktonic and biofilm) of Cnestis ferruginea is due to benzoquinone, the oxidation product of hydroquinone

Abstract

Introduction: Cnestis ferruginea is used frequently in African traditional medicine for treating infectious diseases. Previous bioassay-guided purification has identified hydroquinone as the major bio-active compound in the aforementioned plant, responsible for its antibacterial activity against Staphylococcus aureus. While the phenol hydroquinone can be directly extracted from the plant, it may undergo (reversible) oxidation under mild conditions to yield benzoquinone, a compound with known antimicrobial activity against i.a. S. aureus.

Methods: We, examined whether hydroquinone or its oxidation product, benzoquinone, is the active compound against bacteria such as S. aureus. To achieve this we performed broth microdilution (planktonic) and biofilm activity tests against two different strains of S. aureus. The inhibitory concentrations (IC₅₀) of benzoquinone and hydroquinone under various circumstances were compared, assessing their stability, and examining their effectiveness against two strains of S. aureus (Rosenbach and USA 300) in both planktonic and biofilm environments.

Results: Benzoquinone demonstrated antibacterial activity against S. aureus Rosenbach and USA 300 with IC₅₀ of 6.90 ± 2.30 mM and 7.72 ± 2.73 mM, respectively, while the corresponding values for hydroquinone were 15.63 ± 2.62 mM and 19.21 ± 4.84 mM, respectively. However, when oxidation was prevented by the addition of antioxidants such as ascorbic acid or glutathione, hydroquinone lost its antibacterial property, while benzoquinone retained activity. Comparing conditions in which hydroquinone could convert into benzoquinone against conditions in which this conversion was inhibited, showed that hydroquinone alone did not inhibit bacterial growth of S. aureus, while benzoquinone alone did.

Discussion: These results prove that the oxidation product benzoquinone is responsible for the antimicrobial activity previously ascribed to hydroquinone.

Keywords: Cnestis ferruginea; S. aureus; benzoquinone; biofilm; hydroquinone; oxidation.

Figure 1

Bacterial growth in the presence of two-fold dilution series of the antioxidants ascorbic acid (top) and glutathione (bottom) in TSB medium pH 7.0. The X-axis shows the concentration of the added compound, the Y-axis shows the % bacterial inhibition compared to the solvent control. The error bars show the range of values in all data sets used; the top cap is the maximum value, and the bottom cap is the minimum value. The graphs on the left show inhibition percentage of the compound against S. aureus USA 300 (planktonic) and the ones on the right against S. aureus Rosenbach (planktonic).

Figure 2

Bacterial growth in the presence of two-fold dilution series of hydroquinone (top) and benzoquinone (bottom) in TSB medium pH 7.0. X-axis shows the concentration of the added compound and Y-axis shows the % growth inhibition compared to solvent control. The error bars show the range of values in all data sets used; the top cap is the maximum value, and the bottom cap is the minimum value. The left panels show the results for S. aureus USA 300 (planktonic), the right ones for S. aureus Rosenbach (planktonic).

Figure 3

Bacterial growth in the presence of two-fold dilution series of hydroquinone with ascorbic acid (top) and hydroquinone with glutathione (bottom) in TSB medium pH 7.0. X-axis shows the concentration of the added compound, and Y-axis the % growth inhibition compared to solvent. The error bars show the range of values in all data sets used; the top cap is the maximum value, and the bottom cap is the minimum value. The left panels show results for S. aureus USA 300 (planktonic), the right ones for S. aureus Rosenbach (planktonic).

Figure 4

Absorbance (Y-axis) at wavelengths from 230 to 620 nm (X-axis). The color code at the bottom right in each panel indicates the concentration of either hydroquinone or benzoquinone, depending on the graph. S. aureus strain USA300 planktonic cells in TSB medium pH 7.0 were used for all experiments. Panels on the left show measurements at time point 0 hour, those on the right after an incubation period of 24 hours at 37°C. Compounds tested were hydroquinone (A, A’), with benzoquinone (B, B’), hydroquinone plus ascorbic acid (C, C’), and hydroquinone plus glutathione (D, D’).