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IJMSInternational Journal of Molecular Sciences
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25 February 2026

24 Pages

Phytochemical Assessment, Evaluation of Antioxidant and Antibacterial Properties, and Molecular Docking to Elucidate the Regulation of Bacterial Biofilm Formation in an Herbal Formulation for the Treatment of Abscesses

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1
Faculty of Pharmacy, Srinakharinwirot University, Ongkharak, Nakhon Nayok 26120, Thailand
2
Faculty of Pharmacy, Chiang Mai University, Chiang Mai 50200, Thailand
3
Office of Research Administration, Chiang Mai University, Chiang Mai 50200, Thailand
4
Chula Pharmacokinetic Research Center, Faculty of Medicine, Chulalongkorn University, Bangkok 10330, Thailand
Int. J. Mol. Sci.2026, 27(5), 2145;https://doi.org/10.3390/ijms27052145
This article belongs to the Special Issue New Advances in Bioactive Compounds in Health and Disease
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Abstract

Abscess formation is commonly precipitated by bacterial infection. This study delineates the phytochemical composition and evaluates the antioxidant, antibacterial, and anti-biofilm activities of a Thai traditional anti-abscess herbal formulation comprising Curcuma zedoaria, Vitex trifolia, and Azadirachta indica. Validated high-performance liquid chromatography–photodiode array detection (HPLC–PDA) analysis of the ethanolic extract identified curcumin, demethoxycurcumin, bisdemethoxycurcumin, and vitexicarpin as principal constituents. Total phenolic and flavonoid contents were 32.08 ± 2.54 mg GAE/g and 17.52 ± 1.28 mg QE/g dry weight, respectively. Antioxidant assessment by 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay yielded an half maximal inhibitory concentration (IC50) of 53.46 ± 3.24 µg/mL, while reducing power corresponded to 383.97 ± 13.24 µg FeSO4/g dry weight. Molecular orbital analysis revealed a highest occupied molecular orbital and lowest unoccupied molecular orbital (HOMO–LUMO) gap for vitexicarpin (ΔE = 9.7710 eV), indicative of greater radical-scavenging potential relative to curcuminoids. Antibacterial assays demonstrated selective activity against Staphylococcus epidermidis (inhibition zone 1.48 ± 0.16 cm), with no observed inhibition of Staphylococcus aureus or Streptococcus pyogenes. Curcumin exhibited the highest activity against S. epidermidis (minimum inhibitory concentration (MIC) 62.5 µg/mL; minimal bactericidal concentration minimal bactericidal concentration (MBC) 125 µg/mL). Molecular docking showed curcumin binding to the teicoplanin-associated transcriptional regulator (TcaR) with a binding energy of −8.00 kcal/mol, comparable to methicillin (−8.16 kcal/mol), suggesting a potential mechanism for modulation of biofilm-associated regulatory pathways. Collectively, these findings indicate that the formulation has measurable antioxidant activity and targeted antibacterial efficacy against S. epidermidis, which may contribute to attenuation of abscess progression via interference with biofilm regulation.

1. Introduction

An abscess is a focal collection of purulent material within tissue that develops as a host defense to sequester infection. Its etiology is commonly polymicrobial, with anaerobic and facultative anaerobic organisms predominating, and in many instances dominated by Staphylococcus and Streptococcus species. Pathogenesis involves local immune response, neutrophil-rich necrotic tissue (pus), and often bacterial biofilm formation. It is the consequence of an acute inflammatory reaction to invading bacteria, parasites, or foreign bodies and is characterized by tissue necrosis, neutrophil infiltration, and eventual encapsulation by fibrous tissue [1]. Abscess formation occurs in discrete stages: (1) microbial entry into tissue provokes an acute inflammatory response; (2) the affected tissue becomes warm, erythematous, and edematous; (3) a marked neutrophilic influx occurs, with neutrophils recruited to the site to phagocytose and eliminate pathogens; (4) neutrophil-derived enzymes mediate collateral host tissue destruction, producing purulent exudate composed of dead cells, bacteria, and fluid; and (5) fibroblastic proliferation and collagen deposition encapsulate the purulent core, isolating the infection and limiting systemic dissemination [2].
Management of abscesses, characterized by a localized collection of pus due to infection, employs various approaches, including surgical intervention and medical therapy. Traditionally, the primary treatments involve incision and drainage (I&D) along with antibiotic administration [3]. In severe cases leading to complications like necrotizing mediastinitis, urgent surgical drainage coupled with intravenous antibiotics is crucial. Typically, empirical treatment begins with broad-spectrum antibiotics to counteract the polymicrobial nature of abscesses [4].
Beyond conventional therapies, there is growing interest in the role of herbal medicines in enhancing healing and reducing symptoms in abscess treatment. Phytotherapy, the use of medicinal plants, has historically been employed across cultures for the management of infections due to its anti-inflammatory and antibacterial properties [5]. Certain studies suggest that herbal products may possess inherent antibacterial properties that enhance their efficacy as adjunctive treatments in abscess management [6].
In Thai traditional medicine, an important herbal remedy for abscesses is a classic blend designed to address “Phi Hua Kwum” (a specific type of abscess), comprising equal parts of Curcuma zedoaria, Vitex trifolia, and Azadirachta indica. For oral administration, these three herbs are finely ground and mixed with 40-degree alcohol. The residue remaining after preparation is used as a poultice applied to the abscess three times daily [7]. Curcuma zedoaria, commonly referred to as white turmeric, is well-regarded for its antioxidant, anti-inflammatory, and antibacterial properties. The therapeutic effectiveness of C. zedoaria is well documented, particularly for skin infections, owing to its rhizomes, which are rich in curcuminoids that efficiently scavenge reactive oxygen species (ROS) and reduce oxidative stress [8,9]. These antioxidants play a vital role in mitigating oxidative damage and promoting skin health [10]. Furthermore, its anti-inflammatory properties, which reduce levels of pro-inflammatory cytokines such as tumor necrosis factor-alpha(TNF-α), are beneficial in managing inflammatory skin conditions such as dermatitis [11,12,13]. C. zedoaria also exhibits considerable antibacterial activity against prevalent pathogens, including Staphylococcus aureus and Streptococcus viridans, attributed to compounds that disrupt bacterial integrity [14,15]. The inclusion of C. zedoaria in topical products, such as soaps and creams, not only enhances their efficacy against skin ailments but also provides a safer alternative to conventional antimicrobial treatments [8,15].
Vitex trifolia contributes antioxidant benefits through its phenolic and flavonoid content, effectively neutralizing free radicals [16]. These antioxidants reduce oxidative stress, thereby preventing skin damage [17]. V. trifolia’s anti-inflammatory properties are linked to its modulation of cytokines and oxidative pathways, with compounds like vitexicarpin demonstrating protective effects against inflammation [18,19,20].
Azadirachta indica, or neem, is recognized for its multifaceted biological activities, including antioxidant, anti-inflammatory, and antibacterial properties [21]. These effects are attributed to its phytochemical diversity, including azadirachtin and various flavonoids [22]. Neem’s potent antibacterial properties are attributed to its active phytoconstituents, making it a valuable component of natural therapeutic formulations [23,24].
The bacterial teicoplanin-associated transcriptional regulator (TcaR) has emerged as a significant target for therapeutic inhibitors due to its role in regulating biofilm formation and antibiotic resistance in Staphylococcus species, particularly S. aureus and S. epidermidis. TcaR is classified within the multiple antibiotic resistance regulator (MarR) family of transcriptional regulators, which respond to various environmental stressors by modulating gene expressions. Given the growing concern about antibiotic resistance and biofilm-related infections, inhibiting TcaR offers an appealing strategy to enhance the efficacy of existing antimicrobial agents. Studies by Chang et al. have elucidated the structural characteristics of TcaR, confirming its interaction with various antibiotics and further detailing the conformational changes that occur upon ligand binding [25].
Frontier molecular orbitals (FMOs), notably the highest occupied molecular orbital (HOMO) and the lowest unoccupied molecular orbital (LUMO), are fundamental predictors of molecular reactivity and determinants of antioxidant activity. Key descriptors derived from FMOs include HOMO and LUMO energies and the HOMO–LUMO gap (ΔE). A smaller ΔE generally facilitates electron transfer and increases electron-donating capacity, thereby enhancing radical-scavenging efficacy. This mechanistic framework provides a basis for rationalizing observed antioxidant behavior and guiding structural modifications to improve activity [26].
This study builds on previous research highlighting the antioxidant, anti-inflammatory, and antimicrobial properties of the principal compounds found in individual herbs—C. zedoaria (bisdemethoxycurcumin, desmethoxycurcumin, and curcumin), V. trifolia (vitexicarpin and artemetin), and A. indica (rutin, isoquercitrin, nicotiflorin, astragalin, and quercitrin)—employed in the Thai Traditional Anti-Abscess Herbal Formulation, as illustrated in Table 1 and Figure 1. It is hypothesized that the synergistic effects of these three herbs will yield enhanced therapeutic outcomes. The research aims to perform a thorough analysis of total phenolic and flavonoid contents through validated colorimetric assays and to quantify prominent bioactive compounds, such as vitexicarpin and curcuminoids, using high-performance liquid chromatography (HPLC). Furthermore, the study will evaluate the antioxidant capacity utilizing DPPH and FRAP assays, and the antibacterial efficacy will be assessed against bacteria associated with skin infections, specifically Staphylococcus aureus, Staphylococcus epidermidis, and Streptococcus pyogenes. The frontier molecular orbitals (FMOs) were calculated to elucidate the antioxidant mechanisms of the major compounds. Additionally, molecular docking studies were performed to evaluate the binding affinity of these key compounds for the teicoplanin-associated transcriptional regulator (TcaR), providing evidence of their potential as natural antibacterial agents.
Table 1. Antioxidant, anti-inflammatory, and antibacterial activities of key compounds from C. zedoaria, V. trifolia, and A. indica (the symbol “✓” denotes a positive result for inhibition activities).
Figure 1. Chemical structure of key compounds in herbal formulations.

2. Results

2.1. Preparation of Herbal Extracts

The herbal extracts were obtained from the powdered forms of Curcuma zedoaria, Vitex trifolia, and Azadirachta indica, as well as from a mixed herbal formula, utilizing 95% ethanol as the solvent. The extraction yields for C. zedoaria, V. trifolia, A. indica, and the mixed herbal formula were 18.95 ± 2.16%, 11.40 ± 1.52%, 13.85 ± 1.75%, and 23.83 ± 3.64% dried weight, respectively.

2.2. Identification of Key Compounds in Mixed Herbal Formula Extracts

To accurately identify the key compounds in the mixed herbal formula extract samples, high-performance liquid chromatography with photodiode array detection (HPLC-PDA) was utilized. The results of the HPLC-PDA analysis, as illustrated in Figure 2 and Table 2, revealed the retention times of the key compound standards: (1) rutin, (2) isoquercitrin, (3) nicotiflorin, (4) astragalin, (5) quercitrin, (6) vitexicarpin, (7) bisdemethoxycurcumin, (8) demethoxycurcumin, (9) curcumin, and (10) artemetin with retention times of 8.01, 8.56, 8.87, 9.88, 10.19, 20.67, 22.67, 23.13, 23.56, and 24.14 min, respectively. The predominant compounds identified in the mixed herbal extract were vitexicarpin and three curcuminoids, which were subsequently validated for quantification.
Figure 2. Chromatograms of (a) the standard compounds (1) rutin, (2) isoquercitrin, (3) nicotiflorin, (4) astragalin, (5) quercitrin, (6) vitexicarpin, (7) bisdemethoxycurcumin, (8) demethoxycurcumin, (9) curcumin, and (10) artemetin), (b) the ethanol extracts of the mixed herbal sample and (c) blank sample (ethanol), detected at 354 nm using UV detection.
Table 2. Identification and quantification of key reference compounds in mixed herbal extracts.

2.3. Validation of HPLC Analytical Method for Determination of Vitexicarpin and Curcuminoids in Mixed Herbal Extract

The analytical methodology for assessing vitexicarpin and three curcuminoids (bisdemethoxycurcumin, demethoxycurcumin, and curcumin) in mixed herbal extracts has been developed, as outlined in Section 4.4. This method underwent rigorous system suitability testing and validation to ensure compliance with the International Council for Harmonisation (ICH) guidelines for the advancement of healthcare [47], in conjunction with the AOAC 2016 guideline [48].
The analyses were conducted on a 95% ethanol extract of the mixed herbal formulation, in six replicates, using High-Performance Liquid Chromatography (HPLC) under specific conditions with an injection volume of 10 µL. System suitability for the four analytes was confirmed to fall within acceptable parameters, as evidenced by relative standard deviations (% RSD) for retention time and peak area that remained below 2%. Each injection yielded theoretical plates exceeding 2500, with a tailing factor maintained within 1 ± 0.2. The number of theoretical plates serves as a direct indicator of the efficiency of the chromatographic column; a higher number correlates with superior separation performance, leading to sharper and more distinct peaks during the elution of analytes. Additionally, the resolution achieved was greater than 2.0. These results substantiate that the method meets the acceptance criteria established by the ICH guidelines; the comprehensive system suitability results are presented in Table 3.
Table 3. System suitability results of the analytical method.
Linearity was assessed using five-point calibration curves, with concentrations determined based on anticipated analyte quantities in mixed herbal formulation extracts, as inferred from preliminary studies. As illustrated in Table 4, the coefficient of determination (R2)—a measure of linearity—exhibited excellent linearity for all markers, ranging from 0.9997 to 0.9999 in the prepared calibration curves. The limits of detection (LODs) and limits of quantitation (LOQs) for the four marker components, calculated from three calibration curves, ranged from 0.0800 to 0.0993 µg/mL for LODs, and from 0.2494 to 0.3010 µg/mL for LOQs, respectively.
Table 4. The regression equation, linear range, coefficient of determination (R2), limit of detection (LOD), limit of quantification (LOQ), % recovery, and %RSD for the simultaneous analysis of four reference standards by HPLC (n = 3).
The mean percentage recoveries for all concentrations of reference standard solutions were 99.80–100.70% for vitexicarpin, 102.59–104.08% for bisdemethoxycurcumin, 98.51–101.23% for demethoxycurcumin, and 99.53–100.20% for curcumin. All recovery values were within the acceptable range of 95% to 105%, as established by the AOAC Guideline 2016. The percentage relative standard deviation (%RSD) for intra-day repeatability ranged from 1.14% to 2.73%, while the %RSD for inter-day intermediate precision ranged from 1.38% to 2.76%. Both %RSD values were below the acceptable threshold of 3.7%, as specified by the AOAC Guideline 2016. The method validation results demonstrated that the method met the acceptance criteria outlined in the AOAC Guideline 2016, as detailed in Table 4.

2.4. Analysis of Curcuminoids and Vitexicarpin in Mixed Herbal Remedy Ethanolic Extracts

Based on the chromatographic analysis depicted in Figure 2, the principal compounds quantified from the mixed herbal formulation extract include viexicarpin, curcumin, demethoxycurcumin, and bisdemethoxycurcumin. As shown in Table 2, the determined concentrations of these compounds were 0.52 ± 0.11 mg/g, 7.54 ± 0.13 mg/g, 3.76 ± 0.13 mg/g, and 4.45 ± 0.12 mg/g of dried weight, respectively.

2.5. Total Phenolics, Total Flavonoids, and Antioxidant Activities of Herbal Extracts

The data presented in Table 5 offer a comprehensive evaluation of the total phenolic content (TPC), quantified as milligrams of gallic acid equivalents per gram of dried weight (mg GAE/g), utilizing the calibration equation (Y = 0.1425 ln(X) + 0.1171) (R2 = 0.9987). In addition, the total flavonoid content (TFC) was assessed as milligrams of quercetin equivalents per gram of dried weight (mg QE/g), based on the calibration equation (Y = 0.041X + 0.0262) (R2 = 0.9986). The results indicate that the phenolic content in the ethanolic extracts of C. zedoaria, V. trifolia, A. indica, and the mixed herbal formulation was measured at 44.36 ± 2.98, 21.72 ± 2.47, 31.14 ± 2.04, and 32.08 ± 2.54 mg GAE/g, respectively. Similarly, the corresponding flavonoid contents were recorded as 18.97 ± 1.34, 14.19 ± 1.56, 18.99 ± 1.45, and 17.52 ± 1.28 mg QE/g, respectively.
Table 5. Total phenolic content (TPC), total flavonoid content (TFC), and antioxidant activities (measured by DPPH and FRAP assays) of C. zedoaria, V. trifolia, A. indica, and mixed herbal formulation extracts (n = 3).
The DPPH (2,2-diphenyl-1-picrylhydrazyl) assay is a commonly employed method for evaluating the free radical scavenging capacity of substances. In the presence of an antioxidant, electrons or hydrogen atoms are donated to DPPH radicals, thereby neutralizing them and resulting in a color change from purple to yellow. Antioxidant activity is quantified as the 50% inhibitory concentration (IC50), defined as the concentration of the sample required to inhibit 50% of DPPH radicals. The Ferric Reducing Antioxidant Power (FRAP) assay assesses the ability of a sample to reduce ferric ions (Fe3+) to ferrous ions (Fe2+). Results from this assay are expressed as the concentration of FeSO4 equivalent, with higher absorbance values indicating greater reducing (antioxidant) capacity. Spectrophotometric absorbance measurements reflect the sample’s electron-donating ability, which correlates with its overall antioxidant potential.
As shown in Table 5, the DPPH antioxidant activities of C. zedoaria, V. trifolia, A. indica, and the mixed herbal formulation, represented as IC50 values, were 67.64 ± 4.36, 61.45 ± 2.04, 25.38 ± 3.21, and 53.46 ± 3.24 µg/mL, respectively. In the FRAP assay, the ethanolic extracts of C. zedoaria, V. trifolia, A. indica, and the mixed herbal formulation exhibited reducing powers quantified as 248.64 ± 9.86, 360.52 ± 8.65, 572.24 ± 15.36, and 383.97 ± 13.24 µg/mL FeSO4/g dried weight, respectively.

2.6. Relationship Between Frontier Molecular Orbitals and Antioxidant Activities of Curcumin, Demethoxycurcumin, Bisdemethoxycurcumin, and Vitexicarpin

As shown in Table 6, vitexicarpin exhibited the lowest ΔE (9.7710 eV), consistent with its superior antioxidant performance (DPPH IC50 = 28.64 ± 4.32 µM; FeSO4/mM = 1399.88 ± 16.96 µg/mL). In contrast, bisdemethoxycurcumin presented the highest ΔE (10.2496 eV) and the weakest activity (DPPH IC50 = 66.04 ± 0.22 µM; FeSO4/mM = 603.32 ± 12.68 µg/mL). The observed inverse correlation between ΔE and antioxidant activity highlights the utility of frontier orbital energies in predicting the reactive potential of these compounds.
Table 6. Antioxidant activities and calculated HOMO and LUMO energies (in electron volts, eV) of curcumin, demethoxycurcumin, bisdemethoxycurcumin, and vitexicarpin.
Figure 3 shows that the highest occupied molecular orbital (HOMO) distributions for the three curcuminoids are localized on one half of each molecule, suggesting that their antioxidant behavior is largely dictated by partial conjugation of the molecular framework. Moreover, extensive delocalization of unpaired electrons across conjugated systems—as observed for vitexicarpin—stabilizes the radical species generated during antioxidant processes, thereby reducing their energy and improving radical-scavenging efficacy. Structural motifs such as extended conjugation and hydroxyl substituents substantially modulate electronic properties by narrowing the HOMO–LUMO gap and facilitating electron donation.
Figure 3. Diagrams depicting the frontier molecular orbitals of curcumin, dimethoxycurcumin, bisdemethoxycurcumin, and vitexicarpin. The highest occupied molecular orbital (HOMO) is represented in violet and blue shades, while the lowest unoccupied molecular orbital (LUMO) is displayed in light green and dark green shades.

2.7. Antibacterial Assessment of Herbal Extracts by Agar Plate Diffusion Assays

Disc diffusion assays (Table 7) were conducted to evaluate the antibacterial activity against S. aureus, S. epidermidis, and S. pyogenes. The results indicated that ethanolic extracts from individual plants, as well as mixed herbal extracts at a concentration of 5 mg/disc, did not exhibit inhibition zones on the inoculated plates for S. aureus and S. pyogenes. In contrast, the agar plates for S. epidermidis (Figure 4) demonstrated inhibition zones of 1.25 ± 0.03 cm, 1.39 ± 0.08 cm, 1.27 ± 0.02 cm, and 1.48 ± 0.16 cm for C. zedoaria, V. trifolia, A. indica, and the mixed herbal formulation extracts, respectively. These values were significantly lower than those of the positive control (amoxicillin, 500 µg/mL), which measured 2.25 ± 0.16 cm. Notably, the inhibition zones produced by the mixed herbal extract were larger than those from the individual plant extracts, suggesting a synergistic effect among the three plant constituents.
Table 7. Zone of inhibition (cm ± SD) of C. zedoaria, V. trifolia, A. indica, and mixed herbal formulation extracts, on plates inoculated with S. aureus, S. epidermidis and S. pyogenes.
Figure 4. Effect of plant extracts at a concentration of 5 mg/disc: (a) C. zedoaria, (b) V. trifolia, (c) A. indica, and (d) mixed herbal extract on plates inoculated with S. epidermidis.

2.8. Antibacterial Assessment of Key Compounds by Broth Dilution Assays

The antibacterial activity was evaluated using the agar disc diffusion method, revealing that all four extracts possess inhibitory effects against S. epidermidis. Consequently, the minimum inhibitory concentration (MIC) of key compounds from all plants was determined employing the broth microdilution method with S. epidermidis as the test bacterium (Figure 5a). Initial testing was conducted at final concentrations of 10, 5, 2.5, 1.25, and 0.625 mg/mL; however, no changes in solution clarity were observed. Therefore, 2,3,5-Triphenyltetrazolium chloride (TTC) dye was utilized to assess bacterial viability, wherein the presence of live bacteria resulted in a color change to pink (Figure 5b). Notably, upon staining with TTC, the extract solution of turmeric exhibited a significant decrease in pink color at a concentration of 10 mg/mL. Subsequently, the final concentrations were adjusted to 20, 10, 5, 2.5, and 1.25 mg/mL, but the extracts were found to be insoluble due to the excessive quantity of the dissolving agent. As a result, key compounds derived from C. zedoaria, V. trifolia, and A. indica, totaling eleven substances—such as curcumin, demethoxycurcumin, bisdemethoxycurcumin, vitexin, vitexicarpin, artemetin, rutin, isoquercitrin, nicotiflorin, astragalin, and quercitrin—were tested starting at final concentrations of 250, 125, 62.5, 32.25, and 16.125 µg/mL. As shown in Table 8, the MIC values for the key compounds were 62.5 µg/mL for curcumin and 125 µg/mL for demethoxycurcumin. In contrast, bisdemethoxycurcumin, vitexin, vitexicarpin, artemetin, rutin, isoquercitrin, nicotiflorin, astragalin, and quercitrin exhibited MIC values greater than 250 µg/mL. Following 24 h incubation, minimum bactericidal concentrations (MBCs) were 125 µg/mL for curcumin and 250 µg/mL for demethoxycurcumin. The elevated MBC values are attributable to persistent clear in the growth medium at all tested concentrations.
Figure 5. Illustration of a 96-well microplate used in the broth microdilution method for determining the minimum inhibitory concentrations (MICs) assay against S. epidermidis: (a) microplate without TTC dye, and (b) microplate stained with TTC dye.
Table 8. Minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) of key compounds against S. epidermidis.

2.9. Binding Interactions of Curcuminoids and Vitexicarpin with Bacterial Teicoplanin-Associated Transcriptional Regulator (TcaR)

An understanding of these interactions is critical for the design of effective TcaR inhibitors. As demonstrated in Table 8, the validated docking results, obtained by re-docking ligands into the PDB complex 3KP4, exhibited high accuracy, with a root-mean-square deviation (RMSD) of 1.726 Å.
Four principal compounds from a mixed herbal formulation—curcumin, demethoxycurcumin, bisdemethoxycurcumin, and vitexicarpin—were selected for docking simulations against the target protein (PDB ID: 3KP4) [25]. As shown in Table 9, the native ligand methicillin exhibited the lowest binding energy (−8.16 Kcal/mole), corresponding to an estimated inhibition constant (Ki) of 1.04 µM. Among the tested compounds, curcumin displayed the strongest affinity for TcaR, with a binding energy of −8.00 Kcal/mole, followed by demethoxycurcumin (−7.85 Kcal/mole), bisdemethoxycurcumin (−7.57 Kcal/mole) and vitexicarpin (−6.98 Kcal/mole). Three-dimensional interaction analysis (Figure 6) identified GLN31, SER41, HIS42, LYS60, and ARG110 as common contact residues for all compounds. Detailed analysis of the TcaR binding pocket (Figure 7) indicates that curcumin forms hydrogen bonds with GLU13, SER41, HIS42, LYS60, and ARG110; van der Waals contacts with LEU12, LYS14, ASN17, THR21, THR23, LEU27, GLN31, ALA38, GLU39, ASN45, MET46, and GLN61; pi–alkyl interactions with ILE16 and ALA24; an alkyl contact with HIS42; and a carbon–hydrogen interaction with ASN20. These docking results reveal that curcumin engages in many of the same interactions as methicillin, supporting its potential role in modulating S. epidermidis biofilm formation within the mixed herbal formulation.
Table 9. Results of Molecular Docking Studies for Methicillin, Curcumin, Demethoxycurcumin, Bisdemethoxycurcumin, and Vitexicarpin with Bacterial TcaR Protein.
Figure 6. Three-dimensional binding interactions of all docked compounds with TcaR protein.
Figure 7. Two-dimensional binding interactions of methicillin and curcumin with TcaR protein.

3. Discussion

The present study provides compelling evidence supporting the antioxidant and antimicrobial potential of the Thai Traditional Anti-Abscess Herbal Formulation composed of Curcuma zedoaria, Vitex trifolia, and Azadirachta indica. The identification and quantification of key phytochemicals, including curcumin, demethoxycurcumin, bisdemethoxycurcumin, and vitexicarpin, highlight the formulation’s chemical diversity and suggest that its biological activities arise from the combined, potentially synergistic actions of multiple plant-derived constituents rather than from a single active compound.
The substantial total phenolic and flavonoid contents observed in the formulation are consistent with its pronounced antioxidant capacity. This is supported by DPPH radical-scavenging activity (IC50 = 53.46 ± 3.24 µg/mL) and by strong reducing power measured by the FRAP assay. These antioxidant properties are biologically relevant, as oxidative stress plays a critical role in the pathogenesis and delayed healing of abscesses and infected tissues. By mitigating oxidative damage, the formulation may contribute to improved tissue recovery and enhanced host defense responses in inflammatory conditions. Frontier molecular orbital analysis suggests that the antioxidant properties of vitexicarpin are predominantly determined by its flavonoid core. The presence of conjugated π-systems and hydroxyl substituents promotes stabilization of radical species via extensive electron delocalization, concomitantly narrowing the HOMO–LUMO energy gap. Moreover, an elevated HOMO energy facilitates electron donation to reactive radicals, thereby enhancing radical-scavenging efficacy [49].
The mixed herbal formulation demonstrated a selective inhibitory effect against Staphylococcus epidermidis, indicating a targeted antimicrobial mechanism. This species-specific activity is notable because S. epidermidis is an opportunistic pathogen commonly implicated in skin and soft tissue infections and relies heavily on biofilm formation for persistence and antibiotic tolerance [50].
Among the phytochemicals assessed, curcumin demonstrated the greatest antibacterial potency, exhibiting a minimum inhibitory concentration (MIC) of 62.5 µg/mL and a minimum bactericidal concentration (MBC) of 125 µg/mL against S. epidermidis, in agreement with previous reports of curcumin’s antimicrobial and anti-biofilm activities [50,51,52]. Molecular docking simulations indicated that curcumin’s predicted binding affinity for TcaR was comparable to that of methicillin and exceeded those of demethoxycurcumin, bisdemethoxycurcumin, and vitexicarpin, consistent with the observed MIC values.
In S. epidermidis, polysaccharide intercellular adhesin (PIA) is a critical component of the biofilm extracellular matrix; PIA production is governed by the intercellular adhesion genes A, D, B, and C (icaADBC) operon, whose expression is regulated by the teicoplanin-associated transcriptional regulator TcaR [53,54,55]. TcaR is essential for biofilm formation, and disruption of the TcaR–DNA complex inhibits biofilm development, representing a potential therapeutic strategy. Molecular docking analysis indicated that curcumin binds TcaR with high affinity, exhibiting binding energy comparable to that of methicillin. Several antibiotics interact with TcaR at the winged helix–turn–helix DNA-binding motif, inducing conformational changes that diminish TcaR’s DNA affinity [25,56]. By an analogous mechanism, curcumin is proposed to induce conformational alterations in TcaR that prevent formation of the TcaR–DNA complex at the ica operon, thereby suppressing PIA synthesis and impairing biofilm formation (Figure 8). Collectively, these findings suggest that curcumin may modulate bacterial gene expression related to biofilm development and reduce bacterial persistence, rather than acting primarily through bactericidal activity [52].
Figure 8. Schematic representation of the proposed mechanism by which curcumin inhibits biofilm formation in Staphylococcus epidermidis.
The findings of this study provide molecular and biological evidence supporting the traditional use of this herbal formulation for treating abscesses, particularly those involving biofilm-forming bacteria. However, several limitations warrant acknowledgement. The antibacterial effects observed were moderate compared with conventional antibiotics, and the molecular docking results are predictive and require experimental validation. Accordingly, further investigations are needed to confirm the proposed mechanisms, including biofilm inhibition assays (e.g., crystal violet assay), gene expression profiling and qPCR of TcaR-regulated genes, in vitro TcaR inhibition assays, protein–ligand binding validation, and evaluation in relevant in vivo infection and inflammation models. Such studies are essential to clarify the formulation’s pharmacological potential, safety profile, and translational applicability. The synergetic effects of antibacterials should be confirmed by determining the Fractional Inhibitory Concentration Index (FICI), typically using a checkerboard assay. A synergistic effect is specifically defined when the FICI is less than 0.5. Other crucial methods include the time-kill curve assay, fractional biofilm inhibitory concentration (FBIC), and in vivo bacterial load reduction studies.

4. Materials and Methods

4.1. Materials

Plant materials were procured from a botanical shop in Bangkok, Thailand. Chromatography-grade acetonitrile and methanol were obtained from Merck (Darmstadt, Germany). Formic acid, DPPH, and FeSO4 were supplied by Sigma-Aldrich Co. (St. Louis, MO, USA).

4.2. Preparation of Crude Extract

The extraction process utilized a solid–liquid extraction method with 95% ethanol as the solvent. Ten grams of each sample were mixed with the solvent at a 1:10 (w/v) ratio and sonicated for 30 min. Following sonication, the mixture was filtered under vacuum to separate the solvent from the solid residue. The solid residue was re-extracted twice with fresh solvent. The resulting extracts were concentrated to dryness with a rotary evaporator. The dry weights of the extracts were documented, and the samples were stored at –20 °C for subsequent analyses.

4.3. HPLC Analysis of Vitexicarpin and Curcuminoids in Mixed Herbal Extracts

The HPLC system utilized in this research was an Agilent 1260 Infinity II series (Santa Clara, CA, USA). This system consisted of a quaternary pump, an autosampler, a multi-column thermostat, and a photodiode array detector. Chromatographic separation was achieved with an ACE 5 C18-AR column (4.6 × 250 mm, 5 µm) sourced from Aberdeen, Scotland, alongside a Phenomenex C18 guard column (4 mm × 3 mm × 5 µm) from Torrance, CA, USA. The mobile phase included 0.1% formic acid in ultra-purified water (referred to as phase A) and 0.1% formic acid in acetonitrile (referred to as phase B). The sample pretreatment was analyzed using a specific gradient protocol: starting with a gradient increase from 15% to 60% phase B over 30 min, followed by a rise to 90% phase B within 5 min, which was maintained for an additional 5 min, and concluding with a gradient return to 15% phase B over 3 min, held for 7 min. The analysis was performed at a flow rate of 1 mL/min, with detection wavelengths set at 354 and 420 nm, while the column temperature was kept at 25 °C and the injection volume was 10 µL. The analytical method developed underwent system suitability testing and method validation to ensure adherence to the standards set forth by the International Council for Harmonization (ICH) for improved healthcare, as well as the AOAC Guideline 2016.

4.4. Method Validation

The HPLC method was validated in accordance with AOAC guidelines, assessing linearity, limit of detection (LOD), limit of quantification (LOQ), accuracy, and precision.

4.4.1. Specificity

The specificity of the HPLC analysis was assessed by comparing the chromatograms of the standard solution with those of the sample solutions at a wavelength of 354 nm. The results showed that the standard peak was distinctly separated from other peaks in the sample chromatograms, as reflected by their corresponding retention times. Furthermore, the specificity was confirmed through three replicate analyses of both the standard and sample solutions.

4.4.2. Linearity

Six-point calibration curves were established using known concentrations of vitexicarpin ranging from 1.6 to 8 µg/mL, bisdemethoxycurcumin from 1.9 to 5.1 µg/mL, demethoxycurcumin from 1.9 to 5.1 µg/mL, and curcumin from 3.2 to 16 µg/mL, all demonstrating a correlation coefficient greater than 0.995, in accordance with AOAC guidelines. All measurements were performed in triplicate.

4.4.3. Limit of Detection (LOD) and Limit of Quantification (LOQ)

Using the data from the linear equations, the limit of detection (LOD) and limit of quantification (LOQ) were determined with the following formulas:
LOD = 3.3 × (Standard Deviation of Y-intercept)/(Mean of Slope)
LOQ = 10 × (Standard Deviation of Y-intercept)/(Mean of Slope)
The limit of detection (LOD) and limit of quantification (LOQ) values were determined to be 3.3 and 10 times the ratio of the standard deviation of the calibration curve to the average slope of the calibration curve, respectively.

4.4.4. Accuracy

The recovery performance of the developed HPLC method was assessed using spiked samples at three different concentration levels: 1.2, 1.6, and 2 µg/mL for vitexicarpin, bisdemethoxycurcumin, and demethoxycurcumin, and 2, 4, and 6 µg/mL for curcumin. Recovery percentages were determined by comparing the measured concentrations to the known actual concentrations of the spiked samples. The accuracy of the method was validated through triplicate analyses.

4.4.5. Precision

The precision of the method was evaluated by assessing repeatability across three consecutive injections performed on the same day at low, medium, and high concentrations. To determine intermediate precision, three separate injections were conducted on different days using the same concentration levels. The percentage relative standard deviation (%RSD) of the mean recovery percentage was calculated to quantify the method’s precision.

4.5. Determination of Total Phenolic Content

The total phenolic content (TPC) was measured using a modified Folin–Ciocalteu method suitable for the specific sample matrix. In this procedure, 1 mL of Folin–Ciocalteu reagent was combined with 300 µg/mL of the sample extract and allowed to react for 5 min. After this initial reaction, 1 mL of a 75 mg/mL sodium carbonate (Na2CO3) solution was added, and the mixture was brought to a final volume of 10 mL with distilled water. The reaction mixture was then incubated at room temperature for 90 min to allow for complete color development. Absorbance was subsequently measured at 765 nm against a blank. A standard curve for gallic acid was created using concentrations ranging from 10 to 40 µM, and TPC values were reported as milligrams of gallic acid equivalents (GAE) per gram of dried plant weight (mgGAE/g dried weight). Each sample was analyzed in triplicate to ensure statistical reliability.

4.6. Determination of Total Flavonoid Content

Total flavonoid content (TFC) was evaluated using a modified colorimetric method. In this assay, 1 mL of an 8 mg/mL aluminum chloride (AlCl3) solution was combined with 300 µg/mL of the sample extract, and the mixture was vortexed for uniformity. After an incubation period of 1 h, absorbance was measured at 415 nm against a blank to determine the flavonoid concentration. A calibration curve was created using quercetin standards at concentrations ranging from 15 to 80 µM. TFC values were expressed as milligrams of quercetin equivalents (QE) per gram of dried plant weight (mgQE/g dried weight). All analyses were performed in triplicate to ensure accuracy and consistency.

4.7. Evaluation of Antioxidant Activity

4.7.1. DPPH Radical Scavenging Activity

The antioxidant activity of the extracts was initially assessed using the DPPH (2,2-diphenyl-1-picrylhydrazyl) radical scavenging assay. In this procedure, 100 µL of the extract solution was mixed with 100 µL of a 0.6 mM ethanolic DPPH solution and stirred vigorously to ensure complete interaction. The mixture was then allowed to react for 30 min in a dark environment at room temperature. After the incubation period, the absorbance of the resulting solutions was measured at 520 nm using a spectrophotometer, with a control sample comprising the same DPPH concentration but without the extract.
Radical scavenging activity (RSA) was calculated as a percentage of DPPH discoloration using the following formula:
RSA (%) = [(Acontrol − Asample)/Acontrol] × 100%
where Acontrol represents the absorbance of the DPPH in methanol, and Asample refers to the absorbance of the DPPH solution mixed with the plant extract. The IC50 values were obtained using linear regression analysis to assess antioxidant capacity. All samples were analyzed in triplicate to ensure reliability and accuracy.

4.7.2. Ferric Reduction Ability-Antioxidant Power Test

For the Ferric Reducing Antioxidant Power (FRAP) test, the fresh working solution (FRAP reagent) was prepared by combining 300 mM acetate buffer (comprising 3.1 g of C2H3NaO2·3H2O and 16 mL of CH3COOH at pH 3.6), a 10 mM TPTZ (2,4,6-tripyridyl-s-triazine) solution in 40 mM HCl, and a 20 mM FeCl3·6H2O solution. FeSO4 served as the standard solution. In brief, 100 µL of the sample solution (or standards at 1 mg/mL) was mixed with 3 mL of the FRAP solution using a vortex mixer and allowed to react in the dark at room temperature for 5 min. The absorbance of the resulting-colored Fe-TPTZ complex was then measured at 593 nm. Absorbance values were substituted into the FeSO4 standard curve equation (μg/mL). The results were expressed as micrograms per milliliter equivalents of FeSO4 per gram of dry weight (µg/mL FeSO4/g DW).

4.8. Antibacterial Assay

4.8.1. Tested Microorganisms

A study was performed to explore the inhibitory effects of herbal extracts and key compounds on three bacterial strains associated with skin infections. The investigation focused on three bacterial strains from Department of Medical Sciences Thailand (DMST) Ministry of Public Health, Nonthaburi, Thailand, Year 2024: Staphylococcus aureus DMST 8013, Staphylococcus epidermidis DMST 15055, and Streptococcus pyogenes DMST 17020. The stock cultures were incubated on slant nutrient agar at 37 °C for 24 h and were subsequently stored in a refrigerator for future use.

4.8.2. Disc Diffusion Technique

A bacterial culture was introduced into a tube containing 4 to 5 mL of tryptic soy broth (TSB) obtained from the 24 h-incubated slant nutrient agar of each bacterial species. The broth culture was then incubated for 2 to 6 h at 35 °C until its turbidity level matched the standard 0.5 McFarland BaSO4. Turbidity was measured using a spectrophotometer set to 625 nm [57]. Nutrient agar (20 mL) was poured into Petri dishes, and bacterial cultures were collected from TSB using cotton swabs. The discs were carefully placed onto the seeded plates using sterile forceps. Dimethyl sulfoxide (DMSO) served as a negative control, and tetracycline at 500 µg/mL was used as a positive control. The inoculated plates were then incubated at 37 °C for 24 h. After incubation, the diameter of the clear zone around the discs was measured to determine the inhibition zones.

4.8.3. Determination of Minimum Inhibitory Concentrations (MICs) and Minimum Bactericidal Concentrations (MBCs)

The minimum inhibitory concentration (MIC) was determined using a tube dilution method [58]. The bacterial species under study were cultured for 24 h and then diluted in 10 mL of tryptic soy broth (TSB) to achieve an inoculum of 108 CFU mL−1, in accordance with the 0.5 McFarland standard. A series of culture tubes containing different concentrations of the sample was prepared in DMSO. A volume of 0.1 mL of the bacterial cell suspension was added to each tube, which were then incubated at 37 °C for 24 h. Bacterial growth in the broth was evaluated by measuring turbidity. The minimum inhibitory concentration (MIC) was defined as the lowest sample concentration that prevented visible growth of the test organism. Following 24 h incubation at 37 °C, the minimum bactericidal concentration (MBC) was determined as the lowest concentration yielding no detectable growth upon subculture (i.e., bactericidal effect). MBC determinations corresponded to wells that remained clear (no turbidity) at the tested concentrations.

4.9. Molecular Docking Analysis of Curcuminoids and Vitexicarpin with Bacterial TcaR

The X-ray crystallographic structures of bacterial TcaR (PDB: 3KP4) were retrieved from the RCSB Protein Data Bank [25]. Prior to saving in .pdb format, water molecules and ligands were removed from the structures. The chemical structures of curcuminoids and vitexicarpin were initially generated using ChemDraw Ultra version 8.0 and subsequently converted to three-dimensional (3D) structures in .pdb format using Chem3D Ultra version 8.0. To investigate receptor-ligand binding affinities, the AutoDock 4.2 program [59] was employed. Input files for AutoDock 4.2 were generated using AutoDockTools v1.5.6 (The Scripps Research Institute, La Jolla, CA, USA), which involved modifications such as changing atom types, removing water molecules, and adding polar hydrogen atoms and Gasteiger charges. The grid box dimensions were set with a default grid spacing of 40 × 40 × 40, with coordinates adjusted to −19.163, 74.452, and 33.837 for PDB 2AZ5; 39.787, 1.608, and 73.99 for PDB 5R8Q; −10.088, −16.571, and −51.639 for PDB 4JPF; and −26.255, −31.425, and 4.258 for PDB 3KP4, respectively. Structure files were saved in PDBQT format for molecular docking analysis. The docking analysis was conducted using AutoDock 4.2, and results with optimal binding conformations and energies were selected for further evaluation. Validation of docking results involved calculating the root-mean-square deviation (RMSD) by redocking the co-crystal ligand, with an acceptable RMSD threshold of ≤ 2.0 Å. Visualizations of the docked structures were generated using Discovery Studio (BIOVIA (Dassault Systèmes), San Diego, CA, USA).

4.10. Calculation of Frontier Molecular Orbitals and Visualization

Avogadro (v. 1.2.0) was employed to construct the initial molecular geometries [60]. The resulting optimized Cartesian (xyz) coordinates were exported and used to generate Orca input (.inp) files, with calculations specified at the restricted Hartree–Fock (RHF) level using the def2-SVP basis set. Orca [61] was then executed on these input files by opening an elevated command prompt, navigating to the folder containing the .inp files, and invoking the Orca executable (assuming Orca had been added to the system PATH) using the appropriate command.
Orca filename.inp > filename.out
From the same job folder, a Molden file was generated using the command:
Orca_2mkl filename –molden
Molden output files were visualized using IboView (v. 20211019) [62], which was utilized to perform intrinsic bond orbital (IBO) computations and to examine molecular geometries and molecular orbital (MO) visualizations.

4.11. Statistical Analysis

All data were expressed as mean ± standard deviation (SD) from at least three independent experiments. Statistical analysis was conducted using GraphPad Prism version 8.0 (GraphPad Software, San Diego, CA, USA). One-way ANOVA followed by Tukey’s post hoc test was used for multiple group comparisons, while unpaired t-tests were used for two-group comparisons. A p-value of < 0.05 was considered statistically significant.

5. Conclusions

The validated phytochemical and biological analyses demonstrate that the Thai Traditional Anti-Abscess Herbal Formulation—composed of Curcuma zedoaria, Vitex trifolia, and Azadirachta indica—contains significant levels of bioactive constituents (curcumin, demethoxycurcumin, bisdemethoxycurcumin, and vitexicarpin) and appreciable total phenolic (32.08 ± 2.54 mg GAE/g) and flavonoid (17.52 ± 1.28 mg QE/g) contents. The formulation exhibits measurable antioxidant activity (DPPH IC50 = 53.46 ± 3.24 µg/mL; FRAP equivalent 383.97 ± 13.24 µg FeSO4/g), with frontier molecular orbital analysis indicating vitexicarpin has a favorable electronic profile for radical scavenging. Antibacterial testing revealed selective activity against S. epidermidis (inhibition zone 1.48 ± 0.16 cm), with curcumin showing the strongest activity (MIC 62.5 µg/mL; MBC 125 µg/mL). Molecular docking suggests curcumin binds the teicoplanin-associated transcriptional regulator TcaR (binding energy −8.00 kcal/mol), comparable to methicillin, supporting a plausible mechanism whereby curcumin interferes with TcaR-mediated regulation of PIA production and biofilm formation. Together, these data support the formulation’s potential to attenuate abscess progression via combined antioxidant effects and targeted disruption of S. epidermidis biofilm regulatory pathways. Nevertheless, antibacterial potency was moderate versus conventional antibiotics and docking results remain predictive; confirmatory studies (biofilm inhibition assays, gene expression/qPCR of TcaR-regulated genes, biophysical protein–ligand validation, and in vivo efficacy and safety assessments) are warranted to substantiate mechanism and therapeutic applicability.

Author Contributions

Conceptualization, W.S. and S.T. (Sarin Tadtong); methodology, W.S., S.T. (Sarin Tadtong) and C.C.; software, W.S.; validation, W.S. and S.T. (Sarin Tadtong); formal analysis, W.S., S.T. (Sarin Tadtong), N.M., S.T. (Suttinee Techavijit) and C.C.; investigation, W.S., S.T. (Sarin Tadtong), N.M., S.T. (Suttinee Techavijit), S.S. and C.C.; resources, W.S., S.T. (Sarin Tadtong), and C.C.; data curation, W.S., S.T. (Sarin Tadtong), N.M., S.T. (Suttinee Techavijit), S.S. and C.C.; writing—original draft preparation, W.S., W.E.; writing—review and editing, W.S., S.T. (Sarin Tadtong), W.E. and C.C.; visualization, W.S.; supervision, W.S.; project administration, W.S.; funding acquisition, W.S. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by Srinakharinwirot University (grant number 457/2567).

Institutional Review Board Statement

Not applicable.

Data Availability Statement

The original contributions presented in this study are included in the article. Further inquiries can be directed to the corresponding author.

Acknowledgments

The authors would like to express their gratitude to the Faculty of Pharmacy at Chiang Mai University and the Faculty of Pharmacy at Srinakharinwirot University for providing the materials used in the experiments. Additionally, the authors extend their appreciation to Srinakharinwirot University for supporting the research grant (grant number 457/2567).

Conflicts of Interest

The authors declare no conflicts of interest.

Abbreviations

The following abbreviations are used in this manuscript:
AlCl3Aluminum chloride
AOACAssociation of Official Analytical Collaboration
BaSO4Barium sulfate
°CCelsius degree
cmCentimeter
2DTwo-dimension
3DThree-dimension
DMSODimethyl sulfoxide
DNADeoxyribonucleic acid
DPPH2,2–Diphenyl–1–picrylhydrazyl
DWDried weight
FBICFractional biofilm inhibitory concentration
FICIFractional inhibitory concentration index
FRAPFerric reducing antioxidant power
FeSO4Ferrous sulfate
gGram
GAEGallic acid equivalent
HClHydrochloric acid
HOMOHighest occupied molecular orbital
HPLCHigh Performance Liquid Chromatography
icaADBCIntercellular adhesion genes A, D, B, and C
IC50Half–maximal inhibitory concentration
ICHThe International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use
IL-1βInterleukin-1 beta
LLitter
LODLimit of Detection
LOQLimit of Quantitation
LPSLipopolysaccharide
LUMOLowest unoccupied molecular orbital
MarRMultiple antibiotic resistance regulator
mLMilliliter
MBCMinimum Bactericidal Concentration
MICMinimum Inhibitory Concentration
mMMillimolar
MolMole
MWMolecular weight
Na2CO3Sodium carbonate
QEQuercetin equivalent
PBDProtein data bank
PIAPolysaccharide intercellular adhesin
ROSReactive oxygen species
SDStandard deviation
TcaRTeicoplanin-associated transcriptional regulator
TFCTotal flavonoid content
TNF-αTumor necrosis factor-alpha
TPCTotal phenolic content
TPTZ2,4.6-tripyridyl-s-triazine
TSBTryptic soy broth
TTC2,3,5-Triphenyltetrazolium chloride
UVUltraviolet
µgMicro gram
µMMicro molar
%RSDPercentage relative standard deviation

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