Antimicrobial activity and time-kill kinetics of Boesenbergia rotunda essential oil and geraniol alcohol against oral bacterial pathogens

Preparation of essential oil

Boesenbergia rotunda was sourced locally from the Muang district, Chiang Mai province. The authentication of plant rhizomes was conducted at the Faculty of Science, Maejo University, Chiang Mai, Thailand, with the assignment of a specimen voucher number MJU 2102-005. Cleaned rhizomes were chopped using a kitchen blender. The chopped rhizomes were then placed into a distillation flask along with distilled water and then heated. The steam with essential oil is cooled and condensed to produce a liquid mixture of water for 6 hours and essential oil was separated by using a Clevenger-type apparatus [16]. The obtained essential oils were dried over anhydrous sodium sulfate and subsequently stored at −20°C before analysis.

Bacterial strains and culture conditions

All tests were performed using S. pyogenes Department of Medical Science, Ministry of Public Health, Thailand (DMST) 30563, S. mutans DMST 18777, and S. sobrinus DMST 35719. The test strains were obtained from the DMST, cultured on brain heart infusion agar (BHA), and incubated at 37°C for 24 hours in an anaerobic jar, using a Gaspak system (Becton, Dickinson and Company) which limited oxygen to ≤1% and ensured a carbon dioxide level of ≥13%.

Disc diffusion assay

The disc diffusion method was performed [17] using Mueller–Hinton agar (MHA), and the bacteria were standardized to the 0.5 McFarland standard. The paper disks containing 10 μl BREO at a concentration of 0.863 g/ml were placed on the cultured plate. The diameter of the inhibition zone was determined following incubation at 37°C for a period of 24 hours. Commercially available antibiotic discs of tetracycline and erythromycin (30 μg/disc; Oxoid, UK) were designated as controls.

Broth microdilution method

The broth dilution method was performed as previously described [17]. Briefly, the minimum inhibitory concentration (MIC) of the BREO was examined using a 96-well microtiter plate assay. Broth microdilution was performed using Mueller–Hinton broth (MHB). BREO was prepared at concentrations ranging from 0.06 to 512 mg/ml in 10% dimethyl sulfoxide (DMSO). A solution containing 10% DMSO without BREO was used as the negative control. The last well of each row contained MHB without BREO as a positive control. The tested bacterial strains were adjusted to a 0.5 McFarland standard turbidity and diluted 1:100 in beta-hydroxybutyrate. A 50 μl bacterial suspension was added to each well of the microtiter plate. The plates were gently tapped at the corners to ensure thorough mixing of the solutions and then incubated at 37°C for 24 hours in an anaerobic jar. The lowest BREO concentration that showed no visible growth of the tested organisms was determined as the MIC. The MIC assay wells showing no observable proliferation were subjected to culturing on MHA, followed by a 24-hour incubation period at 37°C. The minimal bactericidal concentration (MBC) was identified as the lowest concentration of BREO that could inhibit microbial proliferation on MHA plates.

Thin layer chromatography-bioautography

The BREO active compounds were separated, and their antibacterial effect was evaluated using TLC plates. Previously described methods [18] were modified and performed in parallel on three TLC plates (I, II, and III) made of Silica Gel 60 F₂₅₄ (from MERCK, Germany). One microliter of BREO was added to each plate. All chemicals used for TLC were of analytical grade. BREO was separated using a mobile phase of toluene:ethyl acetate (95:5). To eliminate the solvent, the plates were left to dry in a fume hood for 15 minutes.

Plate I was visualized under ultraviolet (UV) light to identify the separated compounds. Plate II was sprayed with a 5% sulfuric acid spray, while Plate III was coated with bacteria-infused molten BHA for bioautography purposes. After a 24-hour humid incubation at 37°C, Plate III was treated with a 1% solution of thiazolyl blue tetrazolium bromide, followed by a 10-minute room temperature incubation. The presence of an antibacterial agent is indicated by clear spots on a purple background.

Plates I and II were compared to plate III to identify spots with the same retention factor (Rf) values as the antibacterial zone on plate III. These spots were scraped off and eluted from silica gel plates with dichloromethane. The eluted compounds were analyzed using gas chromatography–mass spectrometry (GC-MS).

Gas chromatography mass spectrometry

The BREO and active compounds extracted from the TLC plate were analyzed by GC-MS using an Agilent 6890 Plus instrument with an HP-5MSI column (0.25 mm × 30 m × 0.25 μm) and a Hewlett Packard 5973 detector. The procedures were modified from those previously described [19] with a slight alteration in the temperatures. The oven temperature was set to 60°C for the first 3 minutes, increased to 200°C at 3°C/minutes, and further increased to 280°C at 15°C/minutes. The temperature was maintained at 280°C for an additional 5 minutes. Helium was used as the carrier gas at a flow rate of 1 ml/minute, and 1 μl of BREO (diluted 1:100 with dichloromethane) was injected into the system. To extract the active compounds obtained from TLC, a 200 μl volume of dichloromethane was used for elution. The eluate was then filtered using a 0.2 μm membrane and subsequently injected into the GC-MS instrument. For the GC-MS analysis, the transfer line temperature was maintained at 150°C, while the ion source temperature was set to 230°C. To calculate the Kovats retention indices, a homologous series of C9–C20 n-alkanes were injected using identical conditions. Compound identification was verified by comparing the results with the Wiley 7 and Nist 05 libraries, as well as relevant literature data [20]. Geraniol (Sigma-Aldrich, St. Louis, MO) served as the standard compound.

Time-kill kinetics assay

In the time-kill assay, the antibacterial effects of BREO and geraniol were evaluated against S. pyogenes, S. mutans, and S. sobrinus. A suspension of bacteria was prepared in a normal saline solution to match the turbidity of a 0.5 McFarland standard. To the bacterial suspension, either BREO or geraniol was added, with final concentrations ranging from 0.5 to 4 times the MIC. Subsequently, the mixture was incubated at 37°C in a reciprocal shaker operating at 150 rpm. Viable counting was performed in triplicate at 0, 1, 5, 10, 30, and 60 minutes, and the cultures were additionally observed for 24 hours on BHA. The procedure was performed in triplicate, and the log₁₀ colony-forming units (CFU)/ml were plotted against time. The extract-free medium was used as a non-treated control [21]. The percentage kill was calculated as follows:

Table 1. Chemical constituents of the BREO by GC-MS analysis. [Click here to view]

Statistical analysis

The experiments were performed in triplicate, and the inhibition zones were analyzed using a one-way analysis of variance followed by Duncan’s multiple range test to identify significant differences. International Business Machines Statistical Package for the Social Sciences Statistics 2 software was used with a p-value < 0.05 regarded as significant.

Chemical composition analysis

The yield of BREO from fresh rhizomes was 4.5 ml per 1,000 g at a density of 0.863 g/ml. GC-MS revealed that BREO comprised 22 chemical components (Table 1). The major constituents were beta-ocimene (40.82%), geraniol (16.79%), and camphor (16.01%), which together accounted for more than 70% of the essential oil. These findings provide insights into the specific chemical compounds present in BREO and their potential role in its medicinal properties. A previous study [22] on the BREO also used GC-MS to analyze its chemical composition and identified nerol (39.6%) and L-camphor (36.0%) as the main constituents. While both essential oils contain high levels of camphor, there are notable differences in their chemical compositions, with BREO in this study having higher concentrations of beta-ocimene and geraniol.

Antibacterial activity

This study investigated the antibacterial activity of BREO against three oral pathogenic bacteria, S. pyogenes, S. sobrinus, and S. mutans. The agar disc diffusion assay revealed that the BREO had significant inhibitory effects on the growth of these bacteria (Table 2, Fig. 1). The MIC and MBC values for BREO (Table 3, Fig. 2) were 6.25–12.5 mg/ml, indicating the bactericidal effects of the extract on all tested bacteria. These findings are consistent with those of a previous study [23], demonstrating the antimicrobial properties of B. rotunda extracts against S. pyogenes and other oral pathogens with suggested mechanisms of action associated with the cell wall and cell membrane damage [24]. In addition to its antibacterial effects, B. rotunda extracts have inhibitory effects on biofilm formation by oral bacteria and anti-periodontitis activity. Furthermore, B. rotunda extracts have anti-inflammatory properties, inhibiting the synthesis of pro-inflammatory cytokines and the expression of certain enzymes involved in inflammation [25].

Figure 1. Inhibition zone of BREO against tested bacteria (1), tetracycline (2) and erythromycin (3) against S. pyogenes (A) S. mutans (B) and S. sobrinus (C) [Click here to view]

Table 2. Inhibition zone of the BREO against tested bacteria (statistical analysis shows differences in the activity for each species subjected to three different treatments). [Click here to view]

Figure 2. MIC microtiter plate of BREO against tested bacteria [S. pyogenes (a), S. mutans (b), S. sobrinus (c)] Row A-C (BREO) Row D-F (geraniol) Well 12A - 12F (positive control) Well 12G (negative control). [Click here to view]

TLC-bioautography and GC-MS

The TLC-bioautography analyses revealed that the crude extract of B. rotunda comprised a single antibacterial component with an Rf value of 0.28 against the tested bacteria (Fig. 4). This component was subsequently identified as geraniol through GC-MS analysis of BREO separation on a TLC plate (Fig. 3A–C). Although geraniol was not the most concentrated component in the BREO, the TLC-bioautography results demonstrated its antibacterial activity against all tested bacterial strains. A previous study [10] showed that B. rotunda possessed promising bioactive compounds exhibiting antioxidant and antibacterial properties. However, the specific active components responsible for these properties have not yet been identified. Our study suggests that geraniol is a key active antibacterial component of BREO against S. pyogenes, S. sobrinus, and S. mutans. These findings further support the promise of BREO as a source of antimicrobial substances and highlight the importance of investigating its potential medicinal uses.

Table 3. MICs and MBCs of the BREO against tested bacteria. [Click here to view]

Figure 3. GC-MS analysis of the BREO (A), components with antibacterial zone from TLC (B), and standard geraniol (C). [Click here to view]

Figure 4. TLC of BREO (A) and geraniol (B) on Silica Gel 60 F254 under UV 254. TLC of BREO (C) and geraniol (D) sprayed with 5% sulfuric acid. Bioautography of BREO (E) and geraniol (F) against S. pyogenes. Bioautography of BREO (G) and geraniol (H) against S. mutans. Bioautography of BREO (I) and geraniol (J) against S. sobrinus. [Click here to view]

Time-kill kinetics assay

A time-kill kinetics assay of BREO and geraniol against the tested bacteria was performed to characterize their bactericidal action (Fig. 5A–F). The cell survival patterns and reduction in cell numbers using varied concentrations of BREO, and geraniol were similar across the bacteria used in this study. BREO and geraniol displayed concentration-dependent kinetics. Higher concentrations resulted in rapid bacterial death, suggesting a dose-dependent action. The 1× MIC of BREO lowered the CFU count against all examined bacteria by ≥2 log₁₀ CFU/ml in 1 minute, whereas geraniol was not considered bactericidal at this time.

Figure 5. Time-kill kinetics curve of BREO against S. pyogenes (A), S. mutans (C), and S. sobrinus (E) and geraniol against S. pyogenes (B), S. mutans (D), and S. sobrinus (F) at different concentrations of BREO and geraniol. [Click here to view]

Table 4. Average percentage kill (mean ± SD) of tested bacteria in the presence of 4 × MIC of the BREO and geraniol. [Click here to view]

Table 4 shows the percentage kill of bacteria over 60 minutes using 4 × MIC of the tested bacteria. BREO reduced ≥99% of all tested bacteria in 1 minute. Geraniol reduced ≥99% of S. pyogenes and S. mutans in 10 minutes and ≥99% of S. sobrinus in 5 minutes. BREO exhibited bactericidal activity at 1 minute and demonstrated a higher percentage kill of all tested bacteria than geraniol at 5 and 10 minutes. In a previous study, Geraniol increased the rate of potassium leaked from intact cells and increased the membrane fluidity of C. albicans, as measured by fluorescence polarization [26]. The findings of our study demonstrate that BREO exhibits considerably greater efficacy in killing the tested bacteria than geraniol alone. The potential synergistic effects of other components in BREO must be further investigated as BREO contains several components that may contribute to its bactericidal activity.