Antistaphylococcal and biofilm inhibitory activities of acetyl-11-keto-β-boswellic acid from Boswellia serrata

Abstract

Background: Boswellic acids are pentacyclic triterpenes, which are produced in plants belonging to the genus Boswellia. Boswellic acids appear in the resin exudates of the plant and it makes up 25-35% of the resin. β-boswellic acid, 11-keto-β-boswellic acid and acetyl-11-keto-β-boswellic acid have been implicated in apoptosis of cancer cells, particularly that of brain tumors and cells affected by leukemia or colon cancer. These molecules are also associated with potent antimicrobial activities. The present study describes the antimicrobial activities of boswellic acid molecules against 112 pathogenic bacterial isolates including ATCC strains. Acetyl-11-keto-β-boswellic acid (AKBA), which exhibited the most potent antibacterial activity, was further evaluated in time kill studies, postantibiotic effect (PAE) and biofilm susceptibility assay. The mechanism of action of AKBA was investigated by propidium iodide uptake, leakage of 260 and 280 nm absorbing material assays.

Results: AKBA was found to be the most active compound showing an MIC range of 2-8 μg/ml against the entire gram positive bacterial pathogens tested. It exhibited concentration dependent killing of Staphylococcus aureus ATCC 29213 up to 8 × MIC and also demonstrated postantibiotic effect (PAE) of 4.8 h at 2 × MIC. Furthermore, AKBA inhibited the formation of biofilms generated by S. aureus and Staphylococcus epidermidis and also reduced the preformed biofilms by these bacteria. Increased uptake of propidium iodide and leakage of 260 and 280 nm absorbing material by AKBA treated cells of S aureus indicating that the antibacterial mode of action of AKBA probably occurred via disruption of microbial membrane structure.

Conclusions: This study supported the potential use of AKBA in treating S. aureus infections. AKBA can be further exploited to evolve potential lead compounds in the discovery of new anti-Gram-positive and anti-biofilm agents.

Figure 1

Effect of AKBA at different concentrations (8, 16 and 32 μg/ml) on the cell viabilty of S. aureus ATCC 29213. S. aureus cells without AKBA served as control. The effect of AKBA was observed bacteriostatic at all tested concentrations when compared with non treated control (P < 0.05) over a period of 24 h study. Each time point represents the mean log₁₀ standard deviations (±SD) of three different experiments performed in duplicate. *, P < 0.05; (Student's t test).

Figure 2

Effect of AKBA on the biofilm formation (A) and preformed biofilm (B) by S. aureus ATCC 29213 and S. epidermidis ATCC 12228. After incubation, the biofilms were stained with crystal violet and the optical density of stained adherent bacteria was determined with a multidetection microplate reader at a wavelength of 595 nm (OD₅₉₅). The results are expressed as average optical density readings for crystal violet assays compared to growth control. The biofilm of S. aureus and S. epidermidis were significantly inhibited (A) and reduced (B) compared with those of bacteria without AKBA (P < 0.01). Values are mean (±SD) from four independent determinations. *, P < 0.01 (Student's t test).

Figure 3

Uptake of propidium iodide in cell of S. aureus ATCC 29213. Cells of S. aureus were treated with AKBA at 64 μg/ml for 60 and 120 min. Control group included cells untreated with AKBA. AKBA treated cells significantly increases the fluorescence compared with untreated control (P < 0.05). Data represent the mean and standard deviations (±SD) of two different experiments performed in triplicate. *, P < 0.05 (Student's t test).

Figure 4

Effect of AKBA on the leakage of 260 and 280 nm absorbing materials in S. aureus ATCC cells. Control group (treated with lytic enzymes and considered as 100% leakage) and treated with AKBA at 64 μg/ml for 90 and 120 min. No compound added served as untreated control. Values are means (±SD) from three independent determinations. *, P < 0.05 (Student's t test), AKBA treated group compared to untreated control group.