Coptis chinensis Extract-Loaded Mouthwash: Antimicrobial Efficacy, Biocompatibility, and Clinical Benefits for Periodontal Health

Abstract

This study investigated the antimicrobial potential of Coptis chinensis rhizome extract against key oral pathogens and evaluated the safety and clinical efficacy of a CCE-loaded mouthwash. CCE exhibited broad-spectrum bactericidal activity, with low minimum inhibitory concentrations (0.002–0.008%) and minimum bactericidal concentrations (0.004–0.016%) against Streptococcus mutans, Aggregatibacter actinomycetemcomitans, and Porphyromonas gingivalis. Time-kill kinetics revealed that CCE promptly eradicated Porphyromonas gingivalis. To balance antimicrobial potency and sensory acceptability, specifically the extract’s bitterness, we established the CCE concentrations in the mouthwash at 0.01% and 0.02% (w/v). Preclinical safety evaluations in animal models, including oral mucosal irritation and skin sensitization tests, confirmed the biocompatibility of 0.02% CCE, yielding “None” and “Non-sensitizer” ratings, respectively. Furthermore, a four-week, randomized, double-blind clinical trial (n = 73) revealed that 0.02% CCE mouthwash substantially reduced halitosis-inducing volatile sulfur compounds (hydrogen sulfide by 59.5% and methyl mercaptan by 50.0%). Significant improvements were also observed in the Plaque Index (55.2% reduction), Gingival Index (52.0% reduction), and Bleeding on Probing (77.3% reduction), with no adverse effects. These findings provide preliminary evidence that CCE mouthwash improves halitosis-related parameters and gingival indices in adults with self-reported halitosis, though further research is required to evaluate its long-term impact on broader periodontal disease states.

1. Introduction

Maintaining oral hygiene is essential for preventing dental caries, periodontitis, and halitosis—conditions largely driven by the proliferation of oral pathogens [1]. Synthetic antimicrobial agents such as chlorhexidine (CHX) have long been incorporated into mouthwash formulations because of their high efficacy [2]. However, their long-term clinical utility is severely restricted. Prolonged use of CHX is associated with significant side effects, most notably extrinsic tooth staining and a persistent bitter aftertaste (dysgeusia), which can reduce patient compliance. Moreover, CHX may cause desquamative lesions of the oral mucosa and exhibits cytotoxicity toward gingival fibroblasts and osteoblasts, raising concerns about its impact on long-term periodontal tissue regeneration [3,4]. These drawbacks have stimulated growing interest in safer, naturally derived alternatives. A wide range of botanical extracts and bioactive compounds—such as essential oils (e.g., Boesenbergia rotunda, Melaleuca alternifolia), green tea polyphenols, propolis, and antimicrobial peptides such as poly-L-lysine—have been investigated for oral care applications [5,6,7]. Despite promising laboratory findings, few natural-product-based oral rinses have undergone clinical validation for efficacy and safety.

Among the botanical candidates, Coptis chinensis (Goldthread) has emerged as particularly promising because of its rich alkaloid profile, which includes berberine, coptisine, and palmatine. Coptis chinensis rhizome extract (CCE) is well-documented for diverse biological activities, including antioxidant, antidiabetic, and neuroprotective effects, making it a versatile therapeutic agent in traditional medicine [8,9]. The alkaloids within CCE are also effective as broad-spectrum antimicrobials, disrupting bacterial cell membranes and inhibiting DNA synthesis [10,11]. In addition, CCE demonstrates substantial anti-inflammatory effects, suppressing proinflammatory cytokines such as TNF-α and IL-6 and modulating the NF-κB signaling pathway [12]. Based on their potent antimicrobial and anti-inflammatory properties, formulations such as berberine gelatin and CCE syrup have been clinically investigated for the management of recurrent aphthous stomatitis and acute bronchitis, respectively, with their therapeutic efficacy and safety being validated in clinical trials [13,14]. Given its dual antimicrobial and anti-inflammatory properties, we expected that CCE represents a potential candidate for next-generation oral hygiene products.

The development of a successful oral care formulation requires a multi-tiered validation process that bridges the gap between laboratory efficacy and clinical applicability. Antibacterial potency must first be quantified through minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), and time-kill assays against key oral pathogens [15]. Because oral care products contact sensitive tissues, safety assessments, including oral mucosal irritation and skin sensitization tests, are essential to establish biocompatibility. Finally, clinical trials are necessary to confirm therapeutic utility, with endpoints such as reductions in volatile sulfur compounds (VSCs) and improvements in the Plaque Index (PI), Gingival Index (GI), and Bleeding on Probing (BOP) serving as critical indicators of efficacy [16].

In this study, we aimed to evaluate the antimicrobial activity of CCE against major oral pathogens and subsequently assess the safety and efficacy of a CCE-loaded mouthwash through both animal models and clinical trials. The choice of a mouthwash delivery system of CCE over other oral care formats, such as toothpaste or gels, is strategically based on its superior pharmacokinetic accessibility to various ecological niches in the oral cavity. A liquid rinse format ensures a more comprehensive distribution of active ingredients across the entire oral mucosa, including the posterior tongue dorsum and interproximal areas, which are major reservoirs for VSC-producing anaerobic pathogens [17]. Furthermore, the low viscosity of a mouthwash allows for better penetration into the gingival crevices and periodontal pockets, ensuring that the antimicrobial CCE reaches the deep-seated pathogens like Porphyromonas gingivalis (P. gingivalis) and Aggregatibacter actinomycetemcomitans (A. actinomycetemcomitans) more effectively than highly viscous formulations. To quantify antimicrobial potency, we conducted MIC, MBC, and time-kill assays against key oral pathogens. The concentrations of CCE for clinical evaluation were selected to balance potent antimicrobial efficacy with sensory palatability, specifically considering the extract’s characteristic bitterness. We established the in vivo safety profile using oral mucosa irritation and skin sensitization tests. Finally, we performed a four-week, double-blind, randomized, placebo-controlled clinical trial to evaluate the efficacy of mouthwash in improving halitosis, gingivitis, periodontitis, and gingival bleeding, alongside a comprehensive safety assessment. By integrating these multifaceted findings, this research provides a robust scientific basis for applying CCE as a safe and effective ingredient in next-generation oral hygiene products.

2. Materials and Methods

2.1. Materials

The CCE utilized in this study was supplied by Borak Co., Ltd. (Hwaseong, Republic of Korea). Briefly, 30 kg of dried rhizomes of Coptis chinensis were pulverized or sliced into appropriate sizes and placed in an extraction vessel. The material was extracted with five volumes of distilled water at 100 °C for 2 h under sealed conditions. After filtration, four volumes of methanol were added, and the mixture was cooled to 4 °C to induce precipitation and crystallization. Following the removal of the supernatant, the residue was further concentrated under reduced pressure and dried to yield 1.01 kg of the final dry extract. The obtained CCE was characterized as containing 80% berberine chloride, 17.6% worenine, and less than 2% each of coptisine and palmatine. Quality control assessments were performed to monitor batch-to-batch consistency, confirming that the variability in the berberine content across all batches used in the experiments was within 5%. Streptococcus mutans (S. mutans) (ATCC 25175), P. gingivalis (ATCC 33277), and A. actinomycetemcomitans (ATCC 33384) were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). Brain Heart Infusion (BHI) broth, Tryptic Soy Broth (TSB), BHI agar, and Tryptic Soy Agar (TSA) were purchased from BD Biosciences (Franklin Lakes, NJ, USA). Hemin and vitamin K1 were obtained from Sigma-Aldrich (St. Louis, MO, USA). Eugonic LT 100 broth was purchased from Remel (Lenexa, KS, USA). D-sorbitol solution was purchased from PKC (Seoul, Republic of Korea). Polyoxyl 40 hydrogenated castor oil was obtained from BASF (Ludwigshafen, Germany). Citric acid and sodium benzoate were purchased from Shinwon Industrial (Eumseong, Republic of Korea) and Daebong LS (Incheon, Republic of Korea), respectively. Phosphate-buffered saline (PBS) was purchased from Bandio Co., Ltd. (Pocheon, Republic of Korea).

2.2. Antimicrobial Efficacy of Natural Botanical Extracts Against Streptococcus mutans

S. mutans was cultured in Brain BHI broth at 37 °C under anaerobic conditions to simulate the oral microenvironment [18]. Natural extracts were prepared as 1.0% (w/v) stock solutions in BHI broth supplemented with 3.0% (w/v) polyoxyl 40 hydrogenated castor oil to ensure solubilization of hydrophobic phytochemicals MICs were determined using a standardized broth microdilution assay in 96 well microtiter plates. Following incubation, the bacterial cultures were centrifuged to obtain a cell pellet. The pellet was then resuspended and diluted in sterile physiological saline to standardize the bacterial density to approximately 1–9 × 10⁸ CFU/mL, serving as the working bacterial suspension. The antibacterial efficacy of the test samples was evaluated using the broth microdilution method in 96-well plates. Extracts were subjected to twofold serial dilutions across a range of 1.000% to 0.001% (w/v) BHI broth, and 200 μL of each dilution was dispensed into the wells. Each well was then inoculated with the prepared bacterial suspension to achieve a final concentration of 1 × 10⁶ CFU/mL. After 48 h of anaerobic incubation, bacterial growth was quantified by optical density at 600 nm (OD₆₀₀) [19]. A vehicle control containing only polyoxyl 40 hydrogenated castor oil exhibited no intrinsic antimicrobial activity. All assays were performed in triplicate to ensure reproducibility.

2.3. Antimicrobial and Bactericidal Efficacy of CCE Against Oral Pathogens

Three oral pathogens were utilized in this study: S. mutans, A. actinomycetemcomitans, and P. gingivalis. S. mutans and A. actinomycetemcomitans were cultured in BHI broth at 37 °C for 24 h under anaerobic and microaerophilic conditions, respectively. P. gingivalis was cultured in Supplemented TSB (ATCC Medium 2722) at 37 °C for 48–72 h under anaerobic conditions. To prepare the working inocula, the bacterial cultures were centrifuged, and the resulting pellets were resuspended in sterile physiological saline (S. mutans and P. gingivalis) or sterile phosphate-buffered saline (A. actinomycetemcomitans). The bacterial densities were standardized to 1–9 × 10⁸ CFU/mL for S. mutans, A. actinomycetemcomitans, and P. gingivalis.

The MIC of CCE against S. mutans, A. actinomycetemcomitans, and P. gingivalis was determined using the broth microdilution method. Test samples were serially diluted in appropriate culture medium and dispensed into 96-well plates (200 μL per well). The inoculum densities were standardized according to the Clinical and Laboratory Standards Institute (CLSI) guidelines, with modifications optimized for the growth characteristics of fastidious oral pathogens. Each well was inoculated with the standardized bacterial suspension to achieve a final concentration of 1 × 10⁶ CFU/mL; for P. gingivalis, the inoculum volume did not exceed 1% of the total assay volume. The plates were incubated at 37 °C under the respective atmospheric conditions. Bacterial growth was monitored by measuring the OD₆₀₀ at 0, 24, and 48 h post-inoculation for S. mutans and A. actinomycetemcomitans, and at 0 and 48 h for P. gingivalis. To determine the MBC, aliquots from wells showing no visible growth at 48 h were subcultured onto BHI agar plates for S. mutans and A. actinomycetemcomitans or supplemented TSA plates for P. gingivalis. These plates were incubated at 37 °C for 24–48 h (S. mutans), 24–72 h (A. actinomycetemcomitans), and 48–96 h (P. gingivalis) to evaluate the degree of bacterial killing.

2.4. Preparation of Mouthwash Formulations and Sensory Evaluation

A sensory evaluation was conducted to assess palatability, with a specific focus on bitterness, which represents the primary organoleptic drawback of alkaloid-rich extracts. The study protocol was reviewed and approved by the Institutional Review Board (IRB No. IRB-240925T002; approved on 14 October 2024) and was conducted in accordance with the ethical principles of the Declaration of Helsinki and Korean Good Clinical Practice (KGCP) guidelines.

Five experimental mouthwash formulations were prepared containing 0.005%, 0.01%, 0.02%, 0.04%, and 0.06% (w/v) of CCE. At first, CCE were dissolved in hot purified water at 60 °C for 30 min. D-sorbitol (4 g/100 mL) was then incorporated as a sweetening agent to mitigate potential bitterness, followed by the addition of sodium benzoate (60 mg/100 mL) as a preservative. To prevent the precipitation of CCE, polyoxyl 40 hydrogenated castor oil (0.6 g/100 mL) was employed as a non-ionic surfactant. The pH of the solution was subsequently adjusted to a physiologically compatible range using citric acid (500 mg/100 mL). Finally, the mixture was brought to the target volume with purified water and stirred for 30 min, for complete dissolution.

The Sensory evaluation study panel consisted of 23 healthy adult volunteers. To ensure the reliability and standardization of the assessment, all panelists participated in a mandatory orientation and calibration session prior to the evaluation. During this session, reference solutions were used to align the participants’ understanding of the 5-point Likert scale, specifically defining the intensity levels for bitterness. In a controlled setting, participants were instructed to rinse their mouths with 15 mL of a randomly assigned formulation for 30 s and then expectorate. To prevent taste fatigue and carry-over effects, adequate intervals were maintained between samples, during which participants rinsed their mouths thoroughly with distilled water. Bitterness was rated on a 5-point Likert scale (1 = Very weak, 5 = Very strong). A score of 3.0 (“Moderate”) was defined as the upper limit of consumer acceptability [20].

2.5. Time-Kill Kinetic Assay

Bactericidal kinetics of 0.01% and 0.02% CCE were assessed against S. mutans and A. actinomycetemcomitans. Stock solutions (0.01% and 0.02%) were prepared in purified water, with purified water used as the control. S. mutans and A. actinomycetemcomitans were grown in BHI broth at 37 °C for 24 h under anaerobic and microaerophilic conditions, respectively [21]. Cell pellets were harvested, resuspended, and adjusted to the working inoculum densities described in Section 2.3. Test suspensions were inoculated to achieve 1 × 10⁶ CFU/mL Aliquots were withdrawn at 0, 0.5 min, 1 min, 5 min, 10 min, 30 min, 1 h, 2 h, 3 h, and 4 h. Reactions were immediately neutralized using Eugonic broth containing lecithin, polysorbate 80, and Triton X-100 [22]. Surviving bacteria were quantified by plating serial dilutions on BHI agar and incubating at 37 °C for 48 h under anaerobic and microaerophilic conditions for S. mutans and A. actinomycetemcomitans, respectively.

The antimicrobial efficacy against P. gingivalis was evaluated using a suspension contact time assay [23,24]. The standardized inoculum was added to 15 mL of control and test samples to achieve a final bacterial concentration of 1 × 10⁷ CFU/mL. The mixtures were maintained at room temperature for predetermined contact times: 0, 0.5 min, 1 min, 5 min, 10 min, 30 min, 1 h, 2 h, 3 h, and 4 h. Following each contact period, the samples were neutralized using Eugonic broth supplemented with lecithin, polysorbate 80, and Triton X-100. The neutralized suspensions were serially diluted in phosphate-buffered saline and 100 μL of each dilution was spread onto Supplemented TSA in duplicate. The plates were incubated invertedly at 37 °C for 120 h under anaerobic conditions.

2.6. Oral Mucosa Irritation Test

To evaluate the localized biocompatibility of the mouthwash, an oral mucosa irritation test was performed following ISO 10993-23 guidelines [25]. The study protocol was approved by the Institutional Animal Care and Use Committee (IACUC) of Biotoxtech Co., Ltd., Cheongju-si, Republic of Korea (Approval No. 240900000037). All experimental procedures were conducted in accordance with the Animal Protection Act of the Republic of Korea (Act No. 4379, enacted on 31 May 1991; as amended by Act No. 19486, 20 June 2023). Six male Golden hamsters (8–10 weeks old) were acclimatized for one week and randomly assigned to a test group (G1, n = 3; 0.02% CCE) and a control group (G2, n = 3; purified water). The 0.02% CCE concentration, representing the maximum intended clinical dose, was selected for pre-clinical safety evaluations based on a ‘worst-case scenario’ strategy. The substances were administered via the cheek pouch, a highly sensitive model for human oral mucosa. A cotton wool pellet (diameter of 5 mm) saturated with the formulation was inserted into the left cheek pouch for 5 min, 5 times daily at 1 h intervals for 7 consecutive days (35 total applications), simulating frequent clinical use. On day 8, the animals were euthanized via carbon dioxide inhalation. The cheek pouch tissues were harvested for macroscopic and microscopic evaluation. The Irritation Index was calculated based on four histopathological parameters: (1) Epithelium (integrity, degeneration, or erosion), (2) Leukocyte infiltration (presence and density of inflammatory cells), (3) Vascular congestion (vessel dilation or disruption), and (4) Edema (fluid accumulation). Each parameter was scored from 0 (Absent/Normal) to 4 (Severe/Marked). The final Irritation Index was derived by averaging the cumulative scores and categorized as None (0), Minimal (1–4), Mild (5–8), Moderate (9–11), or Severe (12–16). Histopathological scoring and qualitative evaluations were performed by an independent, board-certified pathologist who was blinded to the treatment groups. To mitigate observer bias, all tissue sections were anonymized and identified via random numerical codes during the examination.

2.7. Skin Sensitization Test in Guinea Pig

To evaluate the potential of 0.02% CCE to induce delayed-type hypersensitivity (type IV), a Guinea Pig Maximization Test (GPMT) was performed in accordance with ISO 10993-10 guidelines [26,27]. Fifteen healthy male Hartley guinea pigs were acclimatized for 7 days and randomly assigned to three groups (n = 5 per group): a test group (G1, CCE 0.02%), a negative control group (G2, purified water), and a positive control group (G3, CDNB). The experimental procedure consisted of the following three phases:

Intradermal Induction (Day 0): The intrascapular region of each animal was clipped. Three pairs of 0.1 mL intradermal injections were administered:

• Site A: A 1:1 (v/v) mixture of Freund’s Complete Adjuvant (FCA) and physiological saline.
• Site B: The test substance (CCE 0.02% for G1), negative control (purified water for G2), or positive control (0.1% CDNB for G3).
• Site C: A 1:1 (v/v) emulsion of the substance used in Site B and FCA.

Topical Induction (Day 7): To enhance skin permeability, the injection sites were pre-treated with 10% sodium dodecyl sulfate (SDS) on Day 6, as the test item was expected to be non-irritating. On Day 7, a filter paper patch (2 × 4 cm) saturated with 0.5 mL of the respective induction material (0.02% CCE for G1, purified water for G2, or 1% CDNB for G3) was applied to the site and secured with an occlusive dressing for 48 h.

Challenge Phase (Day 21) and Evaluation: Following a 2-week rest period, the flanks of the animals were clipped. Occlusive patches containing the challenge materials were applied for 24 h. G1 and G2 were challenged with both 0.02% CCE and purified water on separate sites, while G3 was challenged with 0.1% CDNB or olive oil. Skin reactions, including erythema and edema, were macroscopically graded at 24 and 48 h after patch removal according to the Magnusson and Kligman scale (0: No reaction to 3: Intense erythema and swelling). The sensitization rate was calculated as the percentage of animals exhibiting a score of ≥1.

2.8. Clinical Evaluations of CCE-Loaded Mouthwash Formulation

2.8.1. Participant Selection

A four-week, double-blind, randomized, placebo-controlled clinical trial was conducted to evaluate the efficacy and safety of the CCE mouthwashes (Vehicle, F0.01%, and F0.02%,

Table 1. Detailed composition of the mouthwashes with different CCE concentrations (0%, 0.01%, or 0.02% (w/v)) employed in clinical evaluations.

Functions	Ingredient (mg)	Vehicle	F0.01%	F0.02%
Active ingredient	CCE	-	10	20
Sweetening agent	D-sorbitol solution	4000	4000	4000
Solubilizer	Polyoxyl 40 hydrogenated castor oil	600	600	600
Buffering agent	Citric acid	500	500	500
Preservative	Sodium benzoate	60	60	60
Solvent	Purified water	q.s. (a)	q.s.	q.s.
Total volume (mL)		100	100	100

^(a) q.s indicates the quantity sufficient of purified water required to reach the final volume of 100 mL.

). Although the trial was not prospectively registered in a public database, the study protocol was approved by the Institutional Review Board of Ellead (IRB No. IRB-240925T002; approved on 14 October 2024) and conducted in accordance with the Declaration of Helsinki and Korean Good Clinical Practice (KGCP) guidelines [28,29]. Written informed consent was obtained from all participants prior to the commencement of the study. CONSORT flow diagram of the clinical trial was depicted in Figure 1.

Healthy adults aged 20–60 years who complained of self-reported halitosis were screened. Inclusion required baseline VSCs levels to exceed the following thresholds via gas chromatography (OralChroma™, CHM-2, FIS Inc., Osaka, Japan): hydrogen sulfide (H₂S) ≥ 112 ppb and methyl mercaptan (CH₃SH) ≥ 26 ppb [30]. Furthermore, all subjects were required to provide written informed consent, demonstrate an understanding of the study protocol, and commit to the scheduled follow-up visits. Exclusion criteria included systemic diseases (e.g., uncontrolled diabetes, hepatic/renal disorders, gastrointestinal diseases, diabetes, or Sjögren’s syndrome), severe periodontitis, orthodontic treatment, pregnancy, lactation, or known hypersensitivity to oral care products. Individuals with dental restorations or implants that could interfere with clinical assessment were also ineligible. Medication-related exclusions included the use of antibiotics within two weeks prior to screening or the concurrent use of antidepressants or antihistamines.

2.8.2. Randomization and Treatment Protocol

Participants were randomly assigned via a blocked sequence into three groups: Group A (vehicle, n = 25), Group B (F0.01%, n = 25), and Group C (F0.02%, n = 25). To maintain double-blind conditions, all mouthwashes were identical in appearance and flavor. To ensure allocation concealment, an independent researcher who was not involved in recruitment or clinical assessment prepared opaque, sequentially numbered, sealed envelopes containing the group assignments. After the baseline examination, a separate study coordinator enrolled the participants and assigned them to their respective interventions. Both the participants and the examining dentist remained blinded to the treatment assignments; the mouthwashes were provided in identical, coded containers to preserve the mask throughout the four-week trial. Participants rinsed with 15 mL of the assigned solution for 0.5 min four times daily (post-meal and pre-bedtime) for 4 weeks. To ensure the accuracy of clinical measurements, participants were required to adhere to a standardized protocol. They were instructed to perform their final oral hygiene 12 h prior to the clinic visit and to refrain from brushing on the morning of the assessment. Consumption of aromatic foods, alcohol, and tobacco was prohibited for 24 h preceding each VSC measurement. Throughout the study period, subjects were restricted from using any supplementary halitosis-suppressing products or medications known to induce xerostomia. Furthermore, participants were advised to avoid excessive smoking, alcohol consumption, and the intake of VSC-producing or halitosis-inducing foods—such as dairy, garlic, onions, caffeine, and meat—while maintaining their baseline dietary and oral hygiene habits. To ensure treatment adherence, participants were required to maintain a daily compliance log, recording the frequency and timing of each mouthwash use. During the study period, one participant in the vehicle group discontinued after initiating dental treatment, and one participant in the F0.02% group withdrew consent due to difficulty attending follow-up visits. Ultimately, 73 participants (mean age 48.8 ± 8.5 years; 65 females, 8 males) completed the trial.

2.8.3. Clinical Evaluation and Safety Monitoring

Clinical assessments were conducted at baseline, immediately post-treatment, and at weeks 2 and 4. To evaluate the efficacy against halitosis, the absolute concentrations (ppb) of H₂S and CH₃SH were quantified from oral gas samples using the OralChroma™ system.

Concurrently, periodontal clinical indices were assessed at baseline, week 2, and week 4 by a single, experienced dentist throughout the study. To ensure diagnostic consistency, the examiner completed an intensive pre-study orientation to standardize scoring across all clinical parameters. These evaluations included the PI to measure biofilm accumulation, the GI to assess mucosal erythema and edema, and BOP to detect active subgingival inflammation.

PI: Biofilm accumulation was quantified according to the criteria of Loe and Silness [31]. PI was evaluated on six index teeth (16, 21, 24, 36, 41, and 44). Four surfaces per tooth (mid-buccal margin, distobuccal, mesiobuccal, and lingual) were examined using a periodontal probe, and each surface was scored from 0 to 3 (0: No plaque; 1: A film of plaque adhering to the free gingival margin, detectable only by probing; 2: Moderate accumulation of soft deposits visible to the naked eye; 3: Abundance of soft matter within the gingival pocket and/or on the tooth margin). The PI was calculated as the mean score of all examined surfaces.

GI: The severity of gingival inflammation was assessed based on the Loe and Silness criteria [32]. GI was evaluated on the same six index teeth and four surfaces per tooth. Each of the four gingival areas per tooth was scored on a 0–3 scale based on visual signs of erythema and edema (0: Normal gingiva; 1: Mild inflammation, slight change in color and edema; 2: Moderate inflammation, redness, and edema; 3: Severe inflammation, marked redness, edema, and ulceration). The mean GI was derived by averaging the scores of all sites.

BOP: Active subgingival inflammation was detected by recording the Presence (1) or Absence (0) of bleeding within 10 s after gentle periodontal probing [33]. The BOP value was calculated as the mean score across all examined sites.

Clinical safety was monitored throughout the study period. At every visit, the investigating dentist performed visual intraoral inspections encompassing the mucosa, gingiva, and tongue to identify any objective signs of localized irritation, tooth staining, or allergic reactions. This professional examination was complemented by the continuous recording of any spontaneous adverse events, such as dysgeusia or general discomfort, reported by the participants to ensure a comprehensive safety profile.

2.9. Statistical Analysis

Statistical analyses were performed using IBM SPSS Statistics version 26.0 (SPSS Inc., Chicago, IL, USA), and the significance level (p) was set at 0.05. The null hypothesis states that there is no significant difference in the mean change of clinical indicators between the test group and the control group. Analyses were conducted on the per-protocol set. The Shapiro–Wilk test was employed to verify the normality assumption of the data. Parametric statistical methods were used when the normality assumption was satisfied (p ≥ 0.05), while non-parametric methods were utilized otherwise (p < 0.05). Baseline homogeneity between groups was assessed; groups were considered homogeneous if p ≥ 0.05. Specifically, the GI and BOP (Group A vs. Group C) were analyzed using the independent t-test. The level of VSCs and clinical indices PI, BOP (Group A vs. Group B, Group B vs. Group C)] were analyzed using the Mann–Whitney U test. Within-group comparisons (longitudinal changes) were performed as follows: For level of VSCs and clinical indices [PI, GI (Group A), BOP], the Friedman test was utilized. Significant differences were further analyzed using the Wilcoxon signed-rank test to identify differences between time points, with p-values adjusted using the Holm–Bonferroni method. For GI (Group B and Group C), Repeated Measures ANOVA was performed. As the sphericity assumption was satisfied via Mauchly’s test, tests of within-subjects effects were conducted to identify longitudinal changes. Significant differences were further evaluated using a contrast test to determine differences between specific time points. Between-group comparisons across time points were analyzed using generalized estimating equations (GEE).

3. Results and Discussions

3.1. Antimicrobial Activity of Botanical Extracts Against S. mutans

We evaluated the antimicrobial efficacy of a diverse library of botanical extracts against S. mutans, the primary etiologic agent of dental caries, to identify potent bioactive candidates for oral therapeutic applications [34,35,36]. Among the screened extracts, CCE exhibited the strongest inhibitory activity, with an MIC of 0.008% (w/v; 0.08 mg/mL). This potency was substantially higher than that of other well-known natural antimicrobials; for instance, CCE was twofold more effective than Salvia rosmarinus (rosemary; 0.16 mg/mL) and Phyllanthus emblica (Indian gooseberry; 0.16 mg/mL) extracts, and over 60-fold more potent than Curcuma longa (turmeric; 5.00 mg/mL) extract (Figure 2). In contrast, green tea and ginkgo extracts exhibited no inhibitory effects within the tested range, highlighting the unique antimicrobial profile of CCE. The superior antimicrobial activity of CCE is primarily attributed to its high content of protoberberine alkaloids, which include berberine, coptisine, and palmatine. These alkaloids act through a multitargeted mechanism that goes beyond simple bacteriostasis [37,38,39]. Specifically, berberine intercalates into bacterial DNA and RNA, inhibiting the synthesis of proteins and nucleic acids [38]. Furthermore, it disrupts bacterial cell division by inhibiting the polymerization of FtsZ, a crucial protein for Z-ring formation during cytokinesis [39]. The pronounced susceptibility of S. mutans to CCE can be further explained by its specific cell wall architecture. As a Gram-positive bacterium, S. mutans lacks the outer lipopolysaccharide membrane found in Gram-negative bacteria, which often acts as a barrier to hydrophobic or bulky alkaloid molecules [40]. The absence of an outer membrane allows protoberberines to efficiently accumulate intracellularly, enabling them to reach their cytoplasmic targets (such as FtsZ and DNA) more readily.

The MIC of CCE observed in this study (0.08 mg/mL) is substantially lower than values reported in the literature, indicating higher antimicrobial potency [41]. Earlier studies generally documented MIC values for CCE or purified berberine against S. mutans in the range of 0.125–0.5 mg/mL [42]. This enhanced efficacy is suggestive of potential synergistic interactions among the protoberberine alkaloids concentrated via our distinct extraction protocols. While berberine is often considered the primary active constituent, the concurrent presence of coptisine and palmatine in CCE might exert a multitargeted synergistic effect, potentially by inhibiting bacterial efflux pumps or increasing membrane permeability [43]. However, future research should fully elucidate the precise molecular interactions and the optimal synergistic ratios among these protoberberine alkaloids.

3.2. Minimum Inhibitory and Bactericidal Concentrations of CCE Against Oral Pathogens

Following the initial screening, we performed a quantitative evaluation to determine the inhibitory and bactericidal thresholds of CCE against major oral pathogens. In antimicrobial testing, the MIC denotes the lowest concentration that prevents visible bacterial growth, whereas the MBC represents the lowest concentration that achieves a ≥99.9% reduction in viable cells [44,45]. Evaluating both parameters is crucial in dental pharmacology, as bactericidal action is preferred for effectively eradicating pathogenic biofilms rather than merely suppressing their growth [45]. The quantitative assay revealed that CCE exhibits potent broad-spectrum bactericidal activity (Figure 3). Against S. mutans, the primary etiologic agent of dental caries [46], the extract demonstrated an MIC of 0.08 mg/mL (0.008% (w/v)) and an MBC of 0.16 mg/mL (0.016% (w/v)). The extract exhibited even greater potency against Gram-negative periodontopathogens. For A. actinomycetemcomitans, a key pathogen strongly associated with severe periodontal inflammation [47], the extract yielded identical MIC and MBC values of 0.04 mg/mL (0.004% (w/v)). Furthermore, P. gingivalis—which is intrinsically linked to periodontitis and halitosis [48]—showed significant susceptibility to the extract, with an MIC of 0.02 mg/mL (0.002% (w/v)) and an MBC of 0.04 mg/mL (0.004% (w/v)). The heightened potency of CCE against Gram-negative anaerobes is primarily attributed to the multifaceted antibacterial mechanisms of berberine, its major quaternary ammonium alkaloid. Berberine enhances outer membrane permeability and simultaneously inhibits multidrug efflux pumps, thereby increasing its intracellular accumulation [49,50]. Once internalized, berberine disrupts bacterial proliferation by interfering with FtsZ-mediated cell division [51]. In addition, the bactericidal efficacy of CCE against P. gingivalis is further amplified through the induction of oxidative stress. Berberine has been shown to stimulate the intracellular accumulation of reactive oxygen species (ROS), including superoxide radicals and hydrogen peroxide [52]. Because P. gingivalis possesses a deficient antioxidant defense system, berberine-induced ROS production results in a catastrophic redox imbalance, leading to irreversible oxidative damage to essential cellular components [53,54]. From a clinical perspective, these variations in MBC provide a robust microbiological basis for the observed outcomes. The pronounced bactericidal effect against P. gingivalis—a primary producer of VSCs—correlates directly with the significant reduction in H₂S and CH₃HS levels observed in the clinical trial. Moreover, the antibacterial activity against S. mutans and A. actinomycetemcomitans suggests potential preventive and therapeutic benefits in reducing dental caries, periodontal inflammation, and the generation of VSCs.

The clinical relevance of these findings is underscored by the narrow margin between the MIC and MBC values across all tested strains. Because the MBCs are identical to or merely twofold higher than their corresponding MICs, we confirm that CCE exerts a direct, lethal bactericidal action rather than a temporary bacteriostatic one. This lethality is primarily attributed to the rich protoberberine alkaloid content of the extract—particularly berberine—which fatally disrupts bacterial cell membrane integrity and metabolic pathways [55]. From these findings, we hypothesize that a CCE-loaded mouthwash formulation exceeding 0.005% (w/v) would exert substantial bactericidal effects against critical inflammation- and halitosis-inducing periodontopathogens.

3.3. Sensory Evaluation and Determination of Concentration of CCE in Mouthwash

Despite the potent bactericidal efficacy of CCE, its high alkaloid content—specifically berberine—imparts an inherently intense bitterness. This sensory attribute poses a substantial challenge to patient compliance [56,57]. To address this, we conducted a preliminary sensory evaluation with 23 participants to assess the organoleptic properties of different CCE concentrations and determine a suitable threshold for clinical application. Figure 4 summarizes the results of the sensory evaluation for the five formulations. As anticipated, we observed a dose-dependent increase in bitterness intensity, showing a strong positive correlation with CCE concentration. The coefficient of variation (CV) for bitterness scores was relatively high (25–35%) at low concentrations (0.005–0.02%), but decreased significantly to below 15% at higher CCE concentrations (0.04–0.06%). This improved consistency at higher doses indicates an acceptable level of inter-rater agreement among the 23 panelists.

Formulations containing 0.005% and 0.01% CCE exhibited favorable palatability, with mean bitterness scores of 1.7 and 1.9, respectively. These values suggest a reasonable level of initial acceptability for oral care use. These values indicate a negligible to mild aftertaste that is unlikely to compromise user adherence. The 0.02% formulation yielded a score of 2.7, which is within the predetermined upper acceptance threshold of 3.0. In contrast, concentrations of 0.04% and 0.06% exceeded the sensory limit with scores of 3.1 and 4.0, respectively, suggesting they might be less tolerated by users. Consequently, within the limited scope of this pilot assessment, 0.02% CCE was determined as the maximum concentration feasible from a sensory perspective.

Determining the final CCE concentration required a strategic equilibrium between antimicrobial potency and sensory acceptability. Although preliminary in vitro assays indicated that a minimum concentration of 0.005% is required to inhibit key periodontal pathogens, such as P. gingivalis and A. actinomycetemcomitans, relying solely on the MIC is insufficient for clinical applications. To ensure robust therapeutic efficacy in the dynamic oral environment—where active ingredients are subject to constant salivary dilution and limited contact time—a safety margin above the MIC is essential. Based on these integrated criteria, we established the CCE concentrations at 0.01% and 0.02%, providing sufficient antimicrobial activity against oral pathogens while maintaining a sensory profile that ensures high patient compliance.

3.4. Time-Kill Kinetics Against Oral Pathogens

We conducted in vitro time-kill kinetic assays to evaluate the bactericidal efficacy of 0.01% and 0.02% CCE against three major oral pathogens. The formulations provided distinct and potent antimicrobial profiles depending on the target bacterial species, exhibiting rapid and complete bactericidal activity against P. gingivalis (Figure 5a), a keystone pathogen implicated in chronic periodontitis [58]. In the control group, P. gingivalis maintained a high viable count of approximately 7.1 log₁₀ CFU/mL during the initial phase. In contrast, both 0.01% and 0.02% CCE treatments exhibited significant bactericidal activity within 0.5 min under the tested in vitro conditions. Because typical mouthwash usage lasts approximately 30–60 s, the ability of these formulations to eliminate P. gingivalis almost instantaneously underscores their strong therapeutic potential in preventing and managing periodontal disease.

Beyond their rapid action against P. gingivalis, the CCE treatments also exhibited effective antimicrobial activity against S. mutans (associated with dental caries) and A. actinomycetemcomitans (associated with aggressive periodontitis) (Figure 5b,c). The reduction in viable counts for these strains followed a time-dependent kinetic profile. Despite the inherent resilience of these pathogens, the formulations successfully reduced bacterial viability, leading to a progressive decline in viable counts over the incubation period. Specifically, 0.02% CCE induced a pronounced reduction in S. mutans and A. actinomycetemcomitans, lowering the bacterial load from baseline levels of 6.0–6.8 log₁₀ CFU/mL to approximately 2.9–4.5 log₁₀ CFU/mL within the first hour of contact. Collectively, although the sample size was limited to n = 2, the high level of reproducibility and the dose-dependent bactericidal trend suggest that these data provide reliable supporting evidence for the formulation’s antimicrobial activity. The observed inhibitory trends against P. gingivalis, S. mutans, and A. actinomycetemcomitans in vitro are expected to contribute to the improvements in halitosis-related parameters and gingival inflammatory indices observed during the 4-week clinical trial.

3.5. Oral Mucosal Irritation Test

The clinical applicability of oral care products is contingent upon their localized safety profiles. Conventional mouthwashes often rely on high concentrations of alcohol or surfactants such as sodium lauryl sulfate, which are frequently associated with mucosal sloughing, burning sensations, and localized irritation [59,60]. To address these concerns, we assessed the safety of 0.02% CCE using a seven-day repeated-exposure hamster cheek pouch model, conducted in accordance with ISO 10993-23 standards [25]. We monitored the cumulative dose, recording a mean absorption of 0.156 g per pellet.

Throughout the seven-day administration period, macroscopic examination revealed no signs of erythema, swelling, or eschar formation in any of the subjects (Figure 6a). As summarized in Figure 6b, both the test group (0.02% CCE) and the control group (purified water) recorded an Irritation Index of 0, classified as “None” under ISO 10993-23 criteria. These findings indicate that the CCE formulation did not induce visible irritation or tissue damage under repeated exposure conditions. Histopathological evaluation further substantiated these observations. The squamous epithelium of the CCE-treated group remained intact and morphologically indistinguishable from that of the control, with no evidence of focal erosion, cellular degeneration, or inflammatory infiltration. The absence of leukocyte accumulation and vascular congestion indicates that the alkaloid constituents of CCE—particularly berberine—do not elicit a proinflammatory response in the submucosa at the therapeutic concentration tested [61].

3.6. Skin Sensitization Test

To evaluate the potential of 0.02% CCE to induce delayed-type (Type IV) hypersensitivity, we employed the GPMT [26]. This cell-mediated immune response occurs when chemical constituents act as haptens, triggering allergic contact dermatitis upon repeated exposure [62]. To validate the sensitivity and reliability of the assay system, we included a positive control group treated with 1-chloro-2,4-dinitrobenzene (CDNB) formulated in olive oil. We utilized olive oil as the vehicle for the positive control because its lipophilic nature substantially enhances the epidermal penetration and systemic absorption of the dissolved compound [63]. CDNB is a well-established, potent contact allergen. Mechanistically, CDNB is a highly reactive electrophile that readily undergoes nucleophilic aromatic substitution, covalently binding to nucleophilic groups (such as sulfhydryl and amino groups) on endogenous skin proteins. This newly formed hapten-carrier protein complex is phagocytosed by epidermal Langerhans cells, processed, and presented to T-lymphocytes in the draining lymph nodes, thereby eliciting a robust Type IV hypersensitivity reaction [64,65].

As expected, the challenge phase in the positive control group resulted in severe dermal reactions (Figure 7). At both 24 and 48 h post-challenge, all animals in the CDNB group exhibited marked erythema and edema (Magnusson and Kligman scores of 2–3), yielding a 100% sensitization rate. This robust response confirmed the sensitivity and optimal function of the experimental model (

Table 2. Results of skin sensitization test in guinea pigs.

Test Group	Item for Sensitization		Item for Challenge	Score of Skin Reaction (a)	No. of Animals		No. of Animals with Positive Reactions	Sensitization Rate (%)	Sensitization Grade
	1st (Conc.)	2nd (Conc.)			24 h (b)	48 h (b)
G1 Test	CCE 0.02%	CCE 0.02%	CCE 0.02%	0	5	5	0/5	0	I (Negative)
				1	0	0
				2	0	0
				3	0	0
				Mean score	0	0
			Purified water	0	5	5	0/5	0	I (Negative)
				1	0	0
				2	0	0
				3	0	0
				Mean score	0	0
G2 Negative control	Purified water	Purified water	CCE 0.02%	0	5	5	0/5	0	I (Negative)
				1	0	0
				2	0	0
				3	0	0
				Mean score	0	0
			Purified water	0	5	5	0/5	0	I (Negative)
				1	0	0
				2	0	0
				3	0	0
				Mean score	0	0
G3 Positive control	CDNB 0.1%	CDNB 1%	CDNB 0.1%	0	0	0	5/5	100	V (Extreme)
				1	0	0
				2	5	5
				3	0	0
				Mean score	2	2
			Olive oil	0	5	5	0/5	0	I (Negative)
				1	0	0
				2	0	0
				3	0	0
				Mean score	0	0

^(a) Magnusson and Kligman grading scale; ^(b) Observation after patch removal for challenge. Conc.: Concentration; CDNB: 1-chloro-2,4-dinitrobenzene (used as a potent allergen for positive control). Note: Skin reactions were graded at 24 h and 48 h post-challenge according to the Magnusson and Kligman scale: 0 (No visible change), 1 (Discrete or patchy erythema), 2 (Moderate and confluent erythema), and 3 (Intense erythema and swelling).

). In contrast to the positive control, both the 0.02% CCE group and the negative control group (purified water) recorded a sensitization rate of 0% and were assigned a grade of 0 (Figure 7, Table 2). The test group treated with 0.02% CCE exhibited complete immunological inertness. During the induction phase, we administered FCA to deliberately potentiate the immune response by maximizing the recruitment and activation of antigen-presenting cells [66]. Even under these stringent, maximized sensitization conditions designed to mimic a “worst-case scenario,” none of the 10 guinea pigs in the test group displayed any visible signs of erythema or edema at any observation time point. Consequently, the test formulation was classified as a “Non-sensitizer” according to ISO 10993-10 criteria [26]. Although certain botanical alkaloids can occasionally serve as sensitizers, the optimized 0.02% concentration of CCE remains well below the threshold for immunological activation.

3.7. Clinical Evaluations of CCE Mouthwashes

Clinical evaluation of 0.01% and 0.02% CCE mouthwashes (F0.01%, and F0.02%) demonstrated substantial therapeutic efficacy across all measured periodontal parameters, confirming the translation of in vitro antibacterial activity into clinical benefits. The sample size was determined based on the primary efficacy variable, the change in H₂S levels. To estimate the effect size, we referenced a previous clinical study [67] that evaluated the inhibitory effects of a 0.1% chlorine dioxide mouthwash on oral malodor over a two-week period. Based on the mean differences and pooled standard deviations of H₂S concentrations from the reference data, the Cohen’s d effect size was calculated to be 0.4017. For the power analysis, we utilized G*Power software (version 3.1.9.4). For the Repeated Measures ANOVA (the primary statistical model for comparing three groups across multiple time points), the effect size f was set at 0.20085 (d/2). With a significance level (α) of 0.05, a statistical power (1 − β) of 0.85, and a total of three groups, the initial power analysis indicated that a minimum of 60 participants (20 per group) was required to detect a significant difference. To ensure statistical robustness despite potential attrition, we applied a 20% predicted dropout rate, and the final target sample size was adjusted to 75 participants (25 per group). Both F0.01% and F0.02% formulations remained sensory adaptable and well-tolerated, with high participant compliance and no reports of taste fatigue.

Halitosis is primarily caused by VSCs, such as H₂S and CH₃SH, which result from the anaerobic degradation of sulfur-containing amino acids by periodontal pathogens [68,69,70]. Whereas H₂S is often linked to tongue biofilm, CH₃SH is a more cytotoxic byproduct and a critical indicator of active periodontal disease [69]. In this study, both test formulations (F0.01% and F0.02%) achieved an immediate and marked reduction in VSCs after the first use (Figure 8a,b). Specifically, the F0.02% reduced H₂S and CH₃SH levels by 85.3% and 87.8%, respectively, whereas the F0.01% yielded similar reductions of 84.2% and 91.0% (p < 0.001 for both). This rapid onset of action aligns with the potent bactericidal effect of CCE against P. gingivalis observed in time-kill assays. Furthermore, this suppression was sustained throughout the 4-week period. At week 4, the F0.02% maintained reduction rates of 59.5% for H₂S and 50.0% for CH₃SH, whereas the F0.01% yielded 65.7% and 58.8% reductions (p < 0.001). VSC reduction did not differ significantly between the F0.01% and F0.02% in VSC reduction (p > 0.05), indicating that a concentration as low as 0.01% is sufficient to achieve optimal malodor control.

This antimicrobial pressure was further evidenced by the substantial decrease in the PI (Figure 9a). By the end of the four-week trial, patients treated with F0.02% exhibited a 55.2% reduction in plaque accumulation, compared with an 18.2% reduction in the vehicle-treated group. Dental plaque is a complex biofilm that initiates the inflammatory cascade in gingival tissues [71]. The ability of CCE—specifically its primary alkaloid, berberine—to interfere with the adhesion of cariogenic and periodontopathic bacteria to the enamel surface likely accounts for this clinical anti-biofilm effect [72]. By preventing plaque maturation, the CCE formulation effectively removes the primary etiological factor for gingival inflammation.

Gingivitis is characterized by gingival tissue inflammation, manifesting as redness and swelling [73]. The clinical improvement in the GI provides direct evidence of the therapeutic role of CCE. In the F0.02%-treated group, the mean GI score decreased from 1.25 at baseline to 0.60 at the final 4-week follow-up (Figure 9b). This 52% improvement indicates a substantial resolution of mucosal inflammation. The marked drop in absolute GI values reflects a transition from moderate gingival inflammation to a state of clinical health, facilitated by the dual action of CCE in reducing bacterial triggers and attenuating the host tissue response. Statistical analysis revealed no significant difference between the 0.01% and 0.02% CCE concentrations in GI improvement (p = 0.396).

Moreover, BOP—the most objective indicator of active subgingival inflammation [74]—showed a marked decrease in both test groups (Figure 9c). In F0.02% group, the BOP rate decreased by 77.3%, whereas the F0.01% group exhibited a reduction of 69.7% over the 4-week period (p < 0.001). In contrast, the vehicle-treated group showed a minimal improvement of 19.0%. The sharp decline in BOP indicates that CCE not only reduces the bacterial load of key pathogens such as P. gingivalis but also potentially mitigates the host inflammatory response by inhibiting proinflammatory cytokines within the gingival sulcus. BOP score reduction did not differ significantly between F0.01% and F0.02% groups (p > 0.05).

Notably, the clinical efficacy of 0.01% and 0.02% CCE was comparable across most clinical endpoints, including VSCs, GI, and BOP. This suggests a ceiling effect, indicating that 0.01% CCE provides sufficient pharmacological activity to reach a therapeutic plateau. These findings have significant implications for formulation optimization and cost-effectiveness; adopting 0.01% as the target concentration ensures maximum clinical efficacy while simultaneously reducing raw material costs and mitigating potential bitterness or sensory fatigue associated with long-term use. On the other hand, the vehicle control group exhibited modest improvements in clinical parameters such as PI (18.2%) and BOP (19.0%). These changes are likely attributable to the Hawthorne effect, where participants improve their oral hygiene behaviors due to trial monitoring, as well as the mechanical rinsing effect provided by the vehicle [75]. Specifically, the surfactant in the vehicle (Polyoxyl 40 hydrogenated castor oil) assists in the physical detachment of dental biofilm, while D-sorbitol and citric acid may enhance natural oral clearance by stimulating salivary flow [76,77].

Clinical safety records indicated that no adverse events, whether localized to the oral cavity or systemic, were reported by any of the 73 participants who completed the trial. Expert visual inspections of the oral mucosa, including the gingiva, tongue, and buccal membranes, revealed no signs of erythema, ulceration, or desquamation in either the 0.01% or 0.02% CCE-treated groups. Furthermore, the CCE-loaded mouthwash demonstrated a favorable safety profile with no reported incidents of staining or sensory impairment. Collectively, these clinical results validate the CCE-loaded mouthwash as a safe and effective alternative for comprehensively managing halitosis and periodontal health.

The clinical relevance of our findings is further contextualized by comparing them with existing literature on CHX, the gold standard in oral antisepsis. CHX is widely recognized for its high substantivity and its capacity to reduce VSCs by 80–90% in short-term applications. Previous clinical meta-analyses have revealed that CHX treatment markedly decreases PI scores—for instance, from a baseline of 2.45 to 1.02 (a 58.4% reduction)—and GI scores from 1.88 to 1.15 (a 38.8% reduction), effectively mitigating gingival inflammation and BOP [3,78]. Despite its efficacy, CHX is frequently associated with localized adverse effects; extrinsic tooth staining is reported in approximately 35% of participants, while 22% of subjects experience transient dysgeusia (taste alteration), typically manifesting as a persistent bitter aftertaste [79]. In contrast, the CCE-based formulations showed significant therapeutic benefits and an acceptable safety profile, although these results should be interpreted within the context of the study’s four-week duration and limited sample size. Following four weeks of intervention of F0.02%, H₂S and CH₃SH levels were reduced by 59.5% and 50.0%, respectively. Furthermore, PI and GI scores exhibited significant declines of approximately 55% and 52%, respectively. Notably, throughout the 4-week trial period, there were no observed signs of erythema, ulceration, mucosal desquamation, tooth staining, or sensory impairment. The antimicrobial and VSC-neutralizing properties of CCE, combined with its safety profile, suggest its significant utility as an adjunctive therapy in the clinical management of gingivitis, periodontitis, and chronic halitosis (Table S1). By effectively suppressing key periodontopathogens at low concentrations, CCE-loaded mouthwash can be integrated into routine periodontal maintenance to reduce the inflammatory load and prevent the progression of tissue destruction. Furthermore, its ability to sustainably lower oral VSC levels provides a promising long-term solution for patients with refractory malodor.

Despite the encouraging outcomes, this study has several limitations. First, as some of the authors are affiliated with the manufacturer of the tested formulation, a potential for interpretation bias could be perceived. To mitigate this risk, the trial was designed and executed by an independent third-party clinical research organization, and a blinded data analysis was performed. Regarding the safety evaluation of the CCE-loaded mouthwash, it is important to note that the current findings are derived from preliminary animal models and a 4-week clinical observation. Although no signs of mucosal irritation, staining, or adverse sensory effects were observed during the trial, further long-term clinical studies with extended follow-up periods are required to fully establish the biocompatibility and safety of CCE-loaded mouthwash in diverse populations. A notable limitation of the present study is the marked imbalance in the gender distribution of the participants, with a significant predominance of females (n = 65, 89.0%) over males (n = 8, 11.0%). This demographic skew was an unintentional result of the voluntary recruitment process and may reflect higher levels of proactive oral health-seeking behavior or a greater interest in clinical research within the female population during the study period. From a physiological perspective, this gender imbalance warrants careful interpretation of the clinical outcomes. Periodontal tissues are highly responsive to fluctuations in female sex steroid hormones, such as estrogen and progesterone, which are known to modulate the inflammatory response and alter the vascularity of the gingiva [80,81]. Consequently, the observed improvements in the GI and BOP within our predominantly female cohort may be influenced by these specific biological factors, potentially limiting the direct extrapolation of the findings to the general male population. Nonetheless, the primary antimicrobial mechanism of the CCE—specifically its ability to target key oral pathogens such as P. gingivalis—is considered biologically consistent across sexes. Furthermore, as this study was conducted over a four-week follow-up period and focused on adults with self-reported halitosis (excluding severe periodontitis), the results may not be generalized to the long-term management of chronic periodontal diseases.

To build upon these preliminary findings, future research should focus on longitudinal studies (e.g., 6–12 months) involving a larger cohort with a more balanced and diverse demographic profile. In addition, more comprehensive sensory acceptability evaluations will be conducted to further refine the formulation, thereby effectively overcoming the inherent sensory limitations of CCE and ensuring high patient adherence. Furthermore, future investigations into combined therapies—such as CCE paired with other natural compounds like glycyrrhizin or xylitol—or its incorporation into diverse oral hygiene delivery systems, may offer synergistic benefits in terms of enhanced efficacy and bitterness masking, necessitating further validation through detailed mechanistic biomarker studies. Finally, metagenomic analysis of the oral microbiome following CCE use would provide a more comprehensive understanding of its impact on the microbial ecosystem beyond the targeted pathogens.

4. Conclusions

The present findings provide short-term clinical evidence for the potential of CCE as a natural antimicrobial agent to improve halitosis-related parameters and gingival inflammatory indices in adults with self-reported halitosis. In vitro antimicrobial assays revealed that CCE exhibits potent inhibitory activity against key oral pathogens. Furthermore, preclinical safety evaluations—including the oral mucosal irritation and skin sensitization tests—confirmed the biocompatibility of the CCE-loaded mouthwash, yielding an Irritation Index of 0 and categorizing it as “Non-sensitizer” (Grade I). The four-week clinical evaluation revealed that 0.01% and 0.02% CCE mouthwashes achieved a marked reduction in VSCs (H₂S and CH₃SH), effectively addressing the causes of halitosis. Furthermore, the treatments significantly improved clinical indices, including the PI, GI, and BOP. Our findings suggest that CCE-loaded mouthwash holds significant clinical promise as a safe and effective adjunctive therapy for the management of periodontal disease and chronic halitosis. Building upon these short-term improvements in halitosis-related parameters and gingival inflammatory indices, future research should prioritize longitudinal clinical trials to evaluate the long-term clinical efficacy and safety of CCE across diverse stages of periodontal destruction, thereby extending the scope beyond the preliminary evidence provided by this four-week trial.