2.1. Plant extraction

The maceration extraction method was employed to obtain the EE extract from fresh rhizomes of E. pavieana (The Plant List Record 244738). The rhizomes were harvested from a local farm in Chantaburi province, Thailand, and authenticated by a plant taxonomist from the Department of Biology at Burapha University, Chonburi, Thailand. A voucher specimen (PI-SCBUU-001-2) was deposited at the Faculty of Science, Burapha University.

The rhizomes were cleaned, dried in a hot air oven, and ground into fine powder. The powdered rhizomes (300 g) were mixed with 3 L of food-grade ethanol (Union Chemical and Equipment Co. Ltd., Bangkok, Thailand) in a tightly sealed glass container and allowed to macerate for 5 days at room temperature, with intermittent shaking to ensure thorough extraction. Following maceration, the mixture was filtered using Whatman No. 1 filter paper (Millipore Sigma, MA, USA). The plant material underwent two additional extraction cycles to maximize yield. The combined filtrates were then concentrated using a rotary evaporator. The resulting viscous brown EE extract was freeze-dried, collected in a sterile tube, and stored at −20°C until further analysis.

The 4-methoxycinnamyl p-coumarate (MCC) was purified by the author following the protocol outlined in Srisook et al. (2017) and Iawsipo et al. (2018), while p-coumaric acid (PCA) was generously provided by Asst. Prof. Ekaruth Srisook from Burapha University (Srisook et al., 2017). The ¹H-NMR spectrum for the MCC compound is provided in the supplementary data.

2.2. Total phenolic content analysis

The richness of phenols in the EE extract was analyzed using the standard Folin–Ciocalteu method. Briefly, 25 µL of extracts at concentrations ranging from 0.5 to 4 mg/mL were mixed with 30 µL of Folin–Ciocalteu reagent (Merck, Darmstadt, Germany) and 100 µL of deionized water. The mixture was incubated in the dark for 6 min, followed by the addition of 250 µL of 7% sodium carbonate (Na₂CO₃) and 195 µL of deionized water. The resulting mixtures were allowed to stand for 30 min at room temperature. Absorbance was measured at 765 nm using a microplate reader. The concentrations of total soluble phenolic compounds (TPC) were determined using a standard curve of aqueous gallic acid solutions and were expressed as mg gallic acid equivalents per gram of extract (mg GAE/g).

2.3. Bacterial strains

Clinical isolates of C. acnes (DMST14916) and S. epidermidis (DMST4369) were obtained from the culture collection of the Department of Medical Sciences, Ministry of Public Health, Thailand. C. acnes was cultured in brain heart infusion (BHI) medium supplemented with 1% glucose under anaerobic conditions for all tests. S. epidermidis was maintained in tryptic soy broth (TSB) under normal atmospheric conditions, except when Mueller–Hinton (MH) medium was specified in the methods. All media were purchased from HiMedia Laboratories Pvt. Ltd., Maharashtra, India.

2.4. Agar well diffusion assay

The antimicrobial activity of the EE extract was assessed using the agar well diffusion method. BHI agar supplemented with 1% glucose was used for C. acnes, while MH agar plates were used for S. epidermidis. Each agar plate was inoculated by evenly swabbing the surface with a sterile cotton swab dipped in the bacterial culture, ensuring uniform distribution.

Wells with a diameter of 6 mm were created in the agar. To each well, 50 µL of EE extract or pure compounds (MCC or PCA) at various concentrations, dissolved in dimethyl sulfoxide (DMSO) (Sigma-Aldrich Corp., MO, USA), were added. Wells loaded with clindamycin or ampicillin served as positive controls, while those with DMSO served as negative controls. The plates were then incubated at 37°C for 72 h under anaerobic conditions for C. acnes and for 18–24 h under aerobic conditions for S. epidermidis. After incubation, the diameters of the inhibition zones surrounding the wells were measured in millimeters (mm).

2.5. Minimum inhibitory concentration and minimum bactericidal concentration determination

The minimum inhibitory concentration (MIC) of the EE extract against C. acnes and S. epidermidis was determined using the agar dilution method following the Clinical and Laboratory Standards Institute (CLSI) guidelines. Initially, 4 µL of bacterial suspensions, adjusted to appropriate turbidity levels, were inoculated onto MH agar plates. Serial two-fold dilutions of the extract were prepared in DMSO and applied in 5 µL aliquots atop the bacterial inoculum. Once the extract was dried, the plates were incubated at 37°C for 72 h under anaerobic conditions for C. acnes and 24 h for S. epidermidis. After incubation, the plates were inspected for visible bacterial growth inhibition, and the MIC was determined as the lowest concentration of the extract at which no bacterial growth was observed.

The minimum bactericidal concentration (MBC) of the EE extract was determined by subculturing aliquots from the MIC plates onto new agar plates. DMSO treatment was used as a negative control. Subsequently, the plates were incubated at 37°C under suitable conditions for each bacterium as described previously. The MBC was defined as the lowest concentration of the extract that resulted in no visible bacterial growth on the subculture plates.

2.6. Assessment of biofilm formation inhibition

The biofilm-forming potential of C. acnes under EE extract treatment was determined using a method slightly modified from Gannesen et al. (2019). Briefly, a starter inoculum (OD600 = 1.8) was diluted in 1:15 with BHI medium. Then, 100 µL of the diluted inoculum was mixed with 50 µL of EE extract at various concentrations, resulting in final concentrations ranging from 0.08 to 2.67 µg/µL, and the mixture was added to a 96-well plate. The plates were incubated at 37°C for 72 h under anaerobic conditions. After incubation, planktonic cultures were carefully removed by pipetting, and wells were washed with phosphate-buffered saline (PBS, pH 7.4). Plates were dried in a laminar flow cabinet. Biofilms were fixed with absolute ethanol for 15 min, after which the ethanol was removed. Then, plates were dried before staining with 0.1% crystal violet for 15 min. Excess crystal violet was washed off with PBS. Biofilm-bound crystal violet was extracted with absolute ethanol for 30 min at room temperature, and absorbance at 570 nm was measured using a microplate reader. Experiments were performed in triplicate and repeated twice. Additionally, post-treatment bacterial numbers were assessed by conducting parallel experiments with the same treatment conditions and measuring OD₆₀₀.

2.7. Evaluation of cell surface hydrophobicity

The effect of EE extract on bacterial cell surface adhesion to hydrocarbons was evaluated using the bacterial adhesion to hydrocarbon (BATH) assay, as modified from Lebeloane et al. (2024). Briefly, 1 mL of inoculum (OD₆₀₀ = 1) in BHI medium was treated with 200 µL of EE extract and incubated at 37°C for 48 h. Following incubation, cells were resuspended in PBS buffer (pH 7.4) to an OD₆₀₀ of 0.7–0.8 (A0) and mixed with 400 µL of toluene. The mixture was vortexed for 1 min and then incubated at 37°C for 15 min to allow phase separation. Absorbance of aqueous phase was subsequently measured at 600 nm (A1). The percentage of bacterial adherence to hydrocarbons (A) was calculated using Equation 1.

The mean results from duplicate experiments, repeated four times, were compared to the untreated control to assess the impact of EE extract on bacterial adhesion.

2.8. 2,2-Diphenyl-1-picrylhydrazyl scavenging assay

The antioxidant activity of the EE extract was assessed using the 2,2-Diphenyl-1-picrylhydrazyl (DPPH) scavenging assay according to Khatun et al. (2021), with some modifications. Various concentrations of the extract (0.25, 0.5, 1, 2, and 4 µg/µL) were mixed with DPPH solution (0.2 mM in methanol) (MedChemExpress, NJ, USA), with ascorbic acid (MilliporeSigma, MA, USA) used as a positive reference. After incubation in the dark at room temperature for 20 min, absorbance was measured at 517 nm using a microplate reader. The percentage of DPPH scavenged by the extract was calculated using the following formula:

Where A_DPPH is the absorbance of the control (DPPH solution without extract), and A_sample is the absorbance of the sample (DPPH solution with extract).

Additionally, the EC₅₀ value, which represents the concentration of the extract required to scavenge 50% of DPPH free radicals, was determined.

2.9. Bovine serum albumin denaturation assay

The anti-inflammatory activity of the EE extract was determined using the bovine serum albumin (BSA) denaturation assay adapted from Gunathilake et al. (2018). Various concentrations of the extract (0.03125–0.5 µg/µL) or ibuprofen (0.625–10 mM, as a positive control) were prepared in PBS. A mixture of 50 µL BSA solution (2% w/v in PBS) and 150 µL of the test solution was incubated at 37°C for 15 min and then heated at 70°C for 10 min. The reactions were performed in triplicate. After cooling, 150 µL of the mixture was transferred to a well of a 96-well plate, and absorbance was measured at 660 nm using a microplate reader. The percentage inhibition of BSA denaturation by the extract was calculated using the following formula:

Where A_control represents the absorbance of the control (BSA solution without extract), and A_sample represents the absorbance of the sample (BSA solution with extract).

2.10. Cytotoxicity assessment

The cytotoxicity of the EE extract was evaluated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay on primary human dermal fibroblasts (ATCC PCS-201-012^TM), as previously described by Silva et al. (2020), with slight modifications. Cells were seeded in 96-well plates at a density of 1 × 104 cells per well and allowed to adhere for 24 h in a CO₂ incubator under humidified atmosphere. Various concentrations of the EE extract in 0.3% DMSO (ranging from 50 to 400 µg/mL) were then added to the wells, and the cells were incubated for an additional 24 h. Following incubation, 10% MTT solution (AppliChem, Darmstadt, Germany) (5 mg/mL in PBS) was added to each well, and the plates were incubated for 4 h at 37°C in an incubator. The formazan crystals formed were dissolved in 100 µL of DMSO, and absorbance was measured at 570 nm using a microplate reader. Cell viability was expressed as a percentage relative to the untreated control, with 0.3% DMSO used as a negative control.

2.11. Quantitative HPLC analysis of phenolic compounds in the Etlingera pavieana extract

The quantitative analysis of phenolic compounds in the EE extract was performed using a validated HPLC method adapted from Srisook et al. (2021). Purified compounds previously identified in the plant served as reference standards (Srisook et al., 2017). An Agilent 1260 Infinity II HPLC system equipped with a UV-Vis diode array detector (DAD) was utilized for the analysis. Separation was achieved using a Phenomenex Luna C18 column (4.6 x 250 mm, 5 µm particle size). A mobile phase consisting of 100% water (solution A) and 100% methanol (solution B) was used with the following elution conditions: 0–15 min at 50% A and 50% B, 15–20 min at 20% A and 80% B, and 21–30 min at 50% A and 50% B, with a flow rate of 1.0 mL/min. Detection was performed at wavelengths of 254, 280, 310, and 320 nm, with the column temperature maintained at 35°C. 10 µL volume of each sample was injected, and chromatographic separation was conducted over a total run time of 30 min.

To quantify individual compounds in the EE extract, standard compounds were used to generate linear calibration curves by plotting peak areas against corresponding concentrations. Quantification was performed at the wavelengths that provided the strongest signal: MCA at 254 nm, PCA at 310 nm, and MCC and MCH at 320 nm. The chromatogram, however, was recorded at 280 nm to ensure all compound peaks were clearly visible. To confirm the accuracy of the quantification, a spiked sample containing 10 µg/mL of standards was also analyzed.

2.12. Statistical analysis

The data were presented as the mean ± standard deviation (SD) of at least six values. Graphical representations were constructed using Microsoft Excel 2021. To evaluate significant differences among groups, either a paired t-test or a one-way analysis of variance (ANOVA) was performed, followed by Tukey’s test using Minitab 18 software (Minitab Inc., PA, USA). Statistical significance was established at p < 0.05.

3.1. Etlingera pavieana extract and its total phenolic content

The ethanolic extract from E. pavieana (EE), obtained via maceration, displayed a viscous consistency, a brown coloration, and a distinctive anis-like aroma. The yield of the extract was 24.60 g, corresponding to an 8.2% yield. The richness of phenolics in the EE extract was confirmed using the Folin–Ciocalteu method, with gallic acid as the standard (y = 0.0016x + 0.0097; R² = 0.9981). The total phenolic content (TPC) was found to be 33.11 ± 0.01 mg GAE/g.

3.2. Antimicrobial activity of the Etlingera pavieana extract against Cutibacterium acnes and Staphylococcus epidermidis

The antimicrobial efficacy of the EE extract against C. acnes and S. epidermidis was clearly evident in the agar well diffusion assay. The extract displayed significant inhibitory effects on bacterial growth, as evidenced by the clear inhibition zones around the wells containing various extract concentrations (Figures 1A and 1B). The inhibition zones increased dose-dependently, reaching 24.00 ± 1.73 mm for C. acnes and 15.67 ± 0.58 mm for S. epidermidis at 256 µg/µL (Table 1). This dose-dependent response emphasizes the potency of the EE extract as a natural antimicrobial agent against common bacterial pathogens implicated in skin-related conditions. Notably, the extract demonstrated particularly pronounced activity against C. acnes, suggesting potential therapeutic benefits for managing acne vulgaris.

Figure 1

Antibacterial activity of EE extract and MIC/MBC determinations. Representative well diffusion assay plates showing the inhibitory effect of EE extract on C. acnes (A) and S. epidermidis (B) Representative plates for determining minimum inhibitory concentration (MIC) by agar dilution assay (C) and minimum bactericidal concentration (MBC) (D) for C. acnes.

The MIC values of the EE extract against C. acnes and S. epidermidis were determined using an agar dilution assay (Figures 1C and 1D). The MIC values were 4 µg/µL (0.02 mg/spot) for C. acnes and 64 µg/µL (0.32 mg/spot) for S. epidermidis (Table 2). The MBC of the EE extract was then determined by subculturing aliquots from the MIC plates onto fresh agar plates. The MBC values were 16 µg/µL (0.08 mg/spot) for C. acnes (Figure 1D) and 256 µg/µL (1.28 mg/spot) for S. epidermidis. The MBC/MIC ratio of 4 for both strains indicates a bactericidal effect (Patil & Patel, 2021). These findings further support the antimicrobial efficacy of the EE extract and its potential as a natural agent against bacterial infections.

3.3. Inhibition of biofilm formation and reduction in cell surface hydrophobicity by Etlingera pavieana extract

Given that C. acnes demonstrated a more pronounced response to EE extract compared to S. epidermidis in preliminary antimicrobial assessments, further experiments were focused exclusively on C. acnes. The objective was to elucidate the potential mechanisms underlying the antibacterial effects of EE extract, specifically targeting biofilm formation and cell surface hydrophobicity.

The effect of EE extract on biofilm formation by C. acnes was evaluated in conjunction with its impact on bacterial growth (OD600), using concentrations lower than the MIC (4 µg/µL). The results, illustrated in Figure 2A, indicate a significant reduction in biofilm formation starting at concentrations as low as 0.17 µg/µL, as evidenced by decreased absorbance values at A570 compared to the negative control. Additionally, approximately 50% biofilm formation was inhibited at a concentration of 0.67 µg/µL and higher. This reduction in biofilm formation was observed without a corresponding decrease in bacterial growth, highlighting the specific inhibitory effect of EE extract on biofilm production.

Figure 2

Inhibition of biofilm formation and reduction in cell surface hydrophobicity by EE extract in C. acnes. (A) Biofilm formation (A₅₇₀) was quantified using crystal violet staining, alongside monitoring bacterial growth (OD₆₀₀) after EE treatment for 72 h. Representative images of wells stained with crystal violet illustrate biofilm reduction at increasing EE concentrations. Images below depict bacterial aggregates in culture tubes, with visibly reduced aggregation upon EE exposure. (B) Cell surface hydrophobicity (%) of C. acnes was assessed using the BATH assay after exposure to various EE extract concentrations for 48 h. Representative images of solvent separation (toluene and water phases) show increased turbidity in the aqueous phase, indicating reduced hydrophobicity as EE concentration increases. Experiments were performed in replicates (A: n = 6, B: n =8), and the results are presented as Mean ± SD. Significant differences from the untreated control (Neg) are marked with an asterisk (*), at p < 0.05.

Furthermore, the effect of EE extract on cell surface hydrophobicity of C. acnes was assessed using the BATH assay. The results, presented in Figure 2B, showed a significant decrease in hydrophobicity at all tested concentrations of EE extract, starting from 0.25 µg/µL. The reduction in hydrophobicity likely precedes and contributes to the observed inhibition of biofilm formation, as decreased hydrophobicity impairs the initial bacterial adhesion necessary for biofilm development.

3.4. Antioxidant activity of the Etlingera pavieana extract determined by DPPH radical scavenging assay

The EE extract exhibited antioxidant activity in the DPPH radical scavenging assay (Figure 3A). At concentrations ranging from 0.25 to 4 µg/µL, the extract exhibited a dose-dependent increase in DPPH radical scavenging activity, reaching a maximum of 55.51%. Although its EC50 value (3.68 ± 0.19 µg/µL) was higher than that of ascorbic acid (14.84 ± 0.22 ng/µL), these findings highlight the potential of the EE extract as a natural antioxidant for combating oxidative stress-related conditions.

Figure 3

Antioxidant and anti-inflammatory activities of EE extract. (A) DPPH radical scavenging activity of EE extract was assessed at various concentrations (0.25, 0.5, 1, 2, and 4 µg/µL). Ascorbic acid was used as a positive reference. Error bars represent the standard deviation of triplicate measurements from three experiments. (B) Concentration-dependent anti-inflammatory activity of the EE extract assessed using the BSA denaturation assay. Dark gray bars represent the inhibition of BSA denaturation (%) by EE extract at concentrations ranging from 0.03125 to 0.5 µg/µL, while red bars represent the inhibition by the positive control Ibuprofen at concentrations ranging from 0.625 to 10 mM. Different letters (A-D) denote significant differences within the same treatment at p < 0.05.

3.5. Anti-inflammatory activity of the Etlingera pavieana extract assessed by the BSA denaturation assay

In the BSA denaturation assay, the EE extract demonstrated a concentration-dependent inhibitory effect on the denaturation of BSA (Figure 3B). At the lowest concentration tested (0.03125 µg/µL), the extract exhibited a modest inhibition of 20.72%, with inhibition increasing gradually with higher concentrations. Notably, at concentrations 0.25 µg/µL, the EE extract reached its maximum inhibitory effect, nearly completely inhibiting BSA denaturation and providing protection nearing 100%. Similarly, the positive control Ibuprofen exhibited a concentration-dependent inhibitory effect on BSA denaturation. At lower concentrations (<2.5 mM), Ibuprofen exhibited relatively low inhibition percentages, less than 10%. However, at concentrations of 2.5 mM and above, Ibuprofen demonstrated substantial inhibition, ranging from 32.71 to 94.01%. These results reveal the potential of the EE extract to prevent protein denaturation, highlighting its promising role as an anti-inflammatory agent.

3.6. Cytotoxicity of the Etlingera pavieana extract on human dermal fibroblasts

To ensure the safety of the EE extract, its cytotoxicity was evaluated on primary human dermal fibroblasts using the MTT assay. The results demonstrated that the EE extract did not adversely affect cell viability at concentrations ranging from 50 to 400 µg/mL (Figure 4). Cell viability remained above 90% across all tested concentrations, indicating that the EE extract is not detrimental to these dermal cells.

Figure 4

Cytotoxicity assessment of EE extract on primary human dermal fibroblasts using the MTT assay. Cells were treated with varying concentrations of the EE extract (50–400 µg/mL) for 24 h. (A) Cell viability was expressed as a percentage relative to the untreated control, with 0.3% DMSO serving as the solvent control. Error bars represent the standard deviation of triplicate measurements. NS denotes values not significantly different from non-treated cells (p <0.05). (B) The photographs were taken under inverted microscope at 20X objective lens magnification. The scale bar represents 100 µm. No significant morphological changes were observed in the treated cell groups compared to the control, supporting the conclusion that the extract is nontoxic to the tested cells.

3.7. Quantitative analysis of some phenolic compounds in the Etlingera pavieana extract using HPLC and their antibacterial activity

Four bioactive compounds, namely 4-methoxycinnamyl p-coumarate (MCC), trans-4-methoxycinnamaldehyde (MCA), 4-methoxycinnamyl alcohol (MCH), and p-coumaric acid (PCA), previously identified for their anti-inflammatory, anticancer, and antimicrobial properties in E. pavieana (Iawsipo et al., 2018; Poonasri et al., 2023; Srisook et al., 2017; Tachai & Nuntawong, 2016), were utilized as reference standards for the quantitative analysis of the EE extract. The HPLC chromatogram (Figure 5) displayed approximately 20 distinctive peaks, including four corresponding to the reference standards, two of which were predominant. MCC, the uncommon phenylpropanoids, exhibited the highest concentration at 3.67 ± 0.08 µg/mg, followed by PCA at 0.76 ± 0.03 µg/mg (Table 3).

Figure 5

HPLC chromatogram of EE extract and spiked EE extract detected at 280 nm. The chromatogram illustrates the separation of phenolic compounds in the EE extract (blue line) with four reference standards (dashed lines) in the background: 4-methoxycinnamyl p-coumarate (MCC), trans-4-methoxycinnamaldehyde (MCA), 4-methoxycinnamyl alcohol (MCH), and p-coumaric acid (PCA). The upper panel displays the chemical structures of these compounds. The middle panel shows the chromatogram of the EE extract alone, while the lower panel presents the chromatogram of the EE extract spiked with the four standards (10 µg/mL), highlighting the corresponding peaks for each compound.

The antibacterial activity of MCC and PCA against C. acnes and S. epidermidis was further assessed using the well diffusion method. MCC exhibited significant antibacterial activity against C. acnes, with inhibition zones of 18.67 ± 1.15 mm at 0.5 mg/mL and 14 ± 1.00 mm at 0.25 mg/mL (Figure 6). In comparison, PCA showed smaller inhibition zones of 12.5 ± 2.12 mm at 0.5 mg/mL and 7.25 ± 0.35 mm at 0.25 mg/mL. For S. epidermidis, MCC displayed antibacterial activity, while PCA showed no detectable inhibition zones at either concentration. These findings highlight the superior antibacterial efficacy of MCC relative to PCA and strengthen their significant contribution to the overall antibacterial activity observed in the EE extract.

Figure 6

Antibacterial activity of 4-methoxycinnamyl p-coumarate (MCC) and p-coumaric acid (PCA) against C. acnes and S. epidermidis, as determined by the well diffusion method. The bar graph presents the inhibition zone diameters (in mm) of MCC and PCA at concentrations of 0.5 µg/µL and 0.25 µg/µL. Insets show representative images of the agar diffusion assay MCC at 0.5 µg/µL (1) and PCA at 0.5 µg/µL (2). ND indicates no detectable inhibition.