Development and Microstructural Analysis of Rapid-Release Acanthus montanus Effervescent Mouthwash Tablets for Oral Care

Abstract

The translation of underutilized botanical resources into advanced therapeutic dosage forms represents a critical frontier in pharmaceutical sciences. This study systematically characterizes the phytochemical and biological profiles of three Acanthus species (Acanthus ebracteatus, Acanthus ilicifolius, and Acanthus montanus) to develop a functional, rapid-release effervescent mouthwash tablet for oral mucosal repair. Phytochemical screening demonstrated that ethanolic extraction selectively concentrated bioactive phenolics and saponins compared to conventional aqueous methods. The ethanolic A. montanus extract emerged as the premier candidate, exhibiting the highest total phenolic content, excellent biocompatibility with human gingival fibroblasts, and significant promotion of in vitro cellular migration. Furthermore, it demonstrated robust, broad-spectrum antimicrobial efficacy against key cariogenic (Streptococcus mutans) and opportunistic (Candida albicans) oral pathogens. To transition this botanical discovery into a viable delivery system, the optimized extract was integrated into an effervescent base via wet granulation utilizing absolute ethanol, followed by blending with extragranular excipients and single-stroke compression. The resulting optimized matrix, incorporating 10% sodium starch glycolate, achieved an ideal mechanical-kinetic balance, yielding exceptional structural toughness (0.23% friability) and rapid disintegration (2.02 min). Advanced 3D synchrotron X-ray tomographic microscopy revealed that this specific super-disintegrant ratio generates a highly interconnected, porous internal architecture driving accelerated capillary-mediated breakdown. Ultimately, this study successfully translates a botanical extract into a mechanistically optimized, functionally validated dosage form for targeted oral therapies.

1. Introduction

Oral mucosal lesions, ranging from aphthous ulcers to denture-induced stomatitis, represent prevalent clinical challenges that significantly impair patient quality of life [1]. Standard management of these conditions necessitates a multifaceted approach, such as reducing localized inflammation, eradicating opportunistic microbial infections, and accelerating soft tissue regeneration [2]. As the demand for sustainable and naturally derived therapeutics grows, botanical resources rich in bioactive secondary metabolites have garnered significant attention as compelling alternatives to synthetic drugs [3,4,5]. Among these, plant-derived phenolic compounds are extensively documented for their robust antioxidant, antimicrobial, and tissue-repairing properties [6,7]. When operating synergistically with other metabolites like saponins, these phytochemicals offer a highly effective platform for oral microbial regulation and mucosal healing [8].

The genus Acanthus (Family Acanthaceae) represents a highly promising, yet underexploited, reservoir of these bioactive phytochemicals [9]. Distributed across tropical and subtropical zones, these spiny shrubs have a strong ethnomedical history in both Asia and Africa for managing inflammatory and infectious diseases [10,11]. In Southeast Asia, two amphibious mangrove species, Acanthus ilicifolius L. and Acanthus ebracteatus Vahl, are officially recognized in Thai Herbal Pharmacopoeia as effective topical anti-inflammatory agents [12,13,14].

However, Acanthus montanus (Nees) T. Anderson presents a compelling terrestrial alternative that remains vastly underutilized in advanced bioproduct development [15,16]. Distinct from its amphibious Thai counterparts, A. montanus is a drought-tolerant shrub native to Africa, characterized by a potent phytochemical profile rich in alkaloids, flavonoids, tannins, and saponins [17,18,19]. While preliminary in vitro screening has highlighted its antioxidant capacity and general cytocompatibility, translating these basic chemical assays into targeted, physiologically relevant therapies remains a challenge [20,21,22]. Specifically, a direct functional comparison evaluating the oral mucosal healing and antimicrobial potential of this terrestrial species against the established mangrove Acanthus species is notably absent from the current literature.

Furthermore, translating these botanical extracts into viable clinical therapies requires an optimized, patient-centric delivery system. Traditional aqueous mouthwashes frequently suffer from inherent formulation drawbacks, including physicochemical instability, hydrolytic degradation of labile phytochemicals, and dosage variability [23]. Engineering the enriched ethanolic extract into a solid effervescent mouthwash tablet effectively overcomes these limitations [23,24]. By maintaining the active constituents in a dry, solid state until the point of care, this delivery format ensures enhanced shelf-life, precise dose uniformity, and superior portability [25,26].

Functionally, these specialized dosage forms are designed to be dropped into a small volume of water within an external vessel prior to administration. Upon hydration, the acid-base effervescent reaction rapidly liberates carbon dioxide, facilitating the immediate and uniform macroscopic dispersion of the botanical matrix. The freshly prepared solution is then immediately utilized as an oral rinse, enabling the targeted, localized delivery of the therapeutic compounds across the compromised oral mucosa. In recent years, this robust bioprocessing platform has been successfully adapted for various botanical therapeutics, with notable examples including effervescent formulations of Echinacea purpurea for immune support, as well as green tea catechins and Mentha species specifically engineered for targeted oral hygiene [27,28].

The preparation of botanical effervescent dosage forms typically relies on non-aqueous wet granulation. By utilizing absolute ethanol as the granulating fluid, the integrity of moisture-sensitive and thermolabile bioactive secondary metabolites, particularly phenolics and saponins, is preserved during the agglomeration of the effervescent base (citric acid, tartaric acid, and sodium bicarbonate) [29]. This methodology effectively entraps the concentrated bioactive phytochemicals within an anhydrous microenvironment, significantly enhancing their long-term physicochemical stability [23,30]. To ensure clinical viability, these formulations undergo rigorous characterization of their mechanical friability, internal matrix porosity, and dissolution kinetics [30,31]. To further optimize these functional parameters, modern effervescent systems frequently incorporate super-disintegrants, such as sodium starch glycolate. These specialized excipients function via extensive capillary wicking and massive volumetric swelling. By drawing water rapidly into the tablet core, they physically disrupt the matrix and expose a maximized surface area, thereby accelerating both the chemical effervescent reaction and the complete structural fragmentation of the dosage form [32,33].

While the ethnomedical use of mangrove Acanthus species is well-documented, translating these botanical resources into optimized solid dosage forms remains a significant gap. Furthermore, the therapeutic potential of the terrestrial A. montanus for oral mucosal repair has yet to be systematically evaluated. Bridging pharmacognosy with pharmaceutical engineering, this study aims to comparatively analyze A. ebracteatus, A. ilicifolius, and A. montanus to identify the optimal bioactive candidate via phytochemical quantification (phenolics and saponins), oral pathogen antimicrobial screening, and human gingival fibroblast (HGF) migration assays. Moving beyond conventional in vitro characterization, the premier extract is subsequently engineered into a stable effervescent mouthwash tablet. Finally, 3D Synchrotron Radiation X-ray Tomographic Microscopy (SR-XTM) is utilized to mechanistically elucidate how super-disintegrant excipients dictate the internal capillary architecture, disintegration kinetics, and biophysical performance of this novel therapeutic delivery system.

2. Materials and Methods

2.1. Materials and Botanical Authentication

The raw botanical materials evaluated in this study comprised the leaves of Acanthus ebracteatus Vahl (Ae), Acanthus ilicifolius L. (Ai), and Acanthus montanus (Nees) T. Anderson (Am). To ensure strict reproducibility and traceability in accordance with standard pharmacognostic guidelines [34,35], the plant materials were acquired from a certified botanical supplier. The supplier harvested the respective plant species from distinct growing regions across Thailand—specifically, Nakhon Pathom, Ranong, and Bueng Kan provinces—between March and May 2023. To eliminate phytochemical variability associated with developmental stages or seasonal shifts, the supplier verified that the collected materials were harvested at a mature vegetative stage. Furthermore, a single, standardized batch (lot) of each species was procured and utilized across all extraction and formulation experiments to ensure absolute chemical consistency.

Upon receipt at the laboratory, a rigorous secondary authentication was conducted to definitively confirm species identity. This taxonomic authentication was performed by plant taxonomist Piyanut Thongphasuk, utilizing standard macroscopic and morphological identification keys in alignment with the Forest Herbarium (BKF) of Thailand. Comprehensive provenance details were documented, and corresponding reference voucher specimens were deposited at the Department of Pharmacognosy Herbarium, College of Pharmacy, Rangsit University, Thailand (Voucher No. RxRSU_AE040221, RxRSU_AI060421, and RxRSU_AM020422, respectively).

All chemicals, reagents, and reference standards utilized in this study were of analytical or pharmaceutical grade. The effervescent base and tablet excipients—including citric acid, tartaric acid, sodium lauryl sulfate (SLS), talcum, xylitol, polyvinylpyrrolidone (PVP K50), and volatile flavoring oils (lemon and peppermint)—were purchased from Aketong Chemipun (Bangkok, Thailand). Sodium starch glycolate (Explotab^®) was obtained from JRS Pharma, Rosenberg, Germany). Sodium bicarbonate and ascorbic acid were supplied by Ajax Finechem (Sydney, Australia). The pharmaceutical vehicles utilized for pre-formulation solubility screening—comprising propylene glycol (PG), polyethylene glycol 400 (PEG 400), glycerin, sodium carboxymethyl cellulose (SCMC), Polysorbate 80 (Tween^® 80), Poloxamer 407, and dimethyl sulfoxide (DMSO)—were similarly procured from Aketong Chemipun (Bangkok, Thailand).

For the phytochemical and antioxidant evaluations, 2,2-Diphenyl-1-picrylhydrazyl (DPPH) and standard gallic acid were obtained from Sigma-Aldrich (Darmstadt, Germany), while Folin–Ciocalteu reagent was supplied by Merck (Darmstadt, Germany). For the antimicrobial evaluations, culture media (agar) and the positive reference standards (chlorhexidine and clotrimazole) were procured from standard commercial sources. All extraction and bioprocessing solvents, including 95% (v/v) ethanol and absolute ethanol, were of analytical grade.

2.2. Extract Preparation

Following taxonomic authentication, the selected Acanthus leaves were thoroughly washed to remove surface impurities and dried in a forced-air convection oven. The continuous mechanical air circulation facilitated efficient moisture removal, while the temperature was strictly maintained at a mild 50 °C to prevent the thermal degradation of thermolabile secondary metabolites. Drying was sustained under these parameters until a constant weight was achieved. The desiccated leaves were subsequently pulverized and passed through a No. 40 mesh sieve (425 μm). While a formal particle size distribution analysis was not performed, this sieving step was utilized to establish a strict maximum particle size threshold. This ensured the elimination of oversized botanical fragments and maintained a uniform, high surface-area-to-volume ratio across all batches, a critical prerequisite for optimal and reproducible solvent penetration during extraction. The resulting fine powders were sealed in polyethylene bags and stored in a desiccator at 25 ± 0.5 °C, strictly protected from light and moisture, until required for extraction.

Extraction was performed utilizing either distilled water or 95% (v/v) ethanol to evaluate the recovery of varying phytochemical classes. These benign, environmentally sustainable solvents were selected to align with green bioprocessing principles, ensuring the resulting botanical extracts remained non-toxic and suitable for downstream formulation into oral delivery systems [36]. Distilled water was utilized to target highly polar constituents, whereas 95% (v/v) ethanol was selected to isolate moderately polar bioactive secondary metabolites, specifically phenolics and saponins [20,37].

The extraction followed a standardized thermal decoction protocol. Briefly, a 10 g sample of the pulverized plant material was accurately weighed and transferred into an Erlenmeyer flask containing 100 mL of the selected solvent (either purified water or 95% (v/v) ethanol), establishing a 1:10 (w/v) solid-to-solvent ratio. This specific ratio was empirically selected to ensure complete hydration and submersion of the bulky, highly porous botanical matrix. Maintaining this fluid volume prevents the formation of a viscous slurry, thereby facilitating uniform heat distribution and preserving the thermodynamic concentration gradient required for efficient mass transfer during thermal processing. (While this 1:10 ratio provides a robust, high-yield baseline for bench-scale prototype development, it is acknowledged that future industrial scale-up will likely necessitate bioprocessing optimization, such as counter-current extraction, to minimize total solvent consumption and improve downstream evaporation economics).

The mixture was heated in a water bath until the solvent reached its boiling point, after which the temperature was maintained for precisely 15 min. To ensure the complete removal of both macroscopic cellular debris and fine suspended particulate matter, the resulting extract was subjected to a two-stage sequential filtration process. The liquid was first passed through a tightly packed cotton wool plug, followed immediately by filtration through high-retention, fine-pore pleated filter paper. This sequential clarification effectively eliminated fine particulates without the necessity of supplementary centrifugation. To ensure exhaustive phytochemical recovery, this entire extraction cycle (boiling and filtration) was repeated three times using fresh solvent. The clarified filtrates from all three cycles were pooled, and the solvent was subsequently eliminated via evaporation over a water bath. The concentrated crude extract was cooled in a desiccator and weighed in a pre-tared evaporating dish. The final extraction yield was calculated and expressed as a percentage on a dry weight basis (% w/w).

2.3. Extract Characteristics and Properties

2.3.1. Yield and Physical Appearance

The extraction yield was calculated as a percentage of the recovered crude extract relative to the initial dry weight of the pulverized plant material (% w/w). Organoleptic characteristics, encompassing physical state, color, odor, and texture, were visually and texturally documented to establish a macroscopic baseline profile of each extract.

2.3.2. Solubility Determination

To identify optimal vehicles for downstream dosage form development, the equilibrium solubility of the extracts was evaluated across a targeted spectrum of pharmaceutical solvents and surfactant systems. The screening panel was specifically selected to represent diverse solubilization mechanisms commonly utilized in oral mucosal formulations (such as dielectric cosolvents, micellar surfactants, and polymeric suspending agents). The vehicles and their specific concentrations were formulated to reflect physiologically acceptable thresholds for oral preparations: propylene glycol (PG), polyethylene glycol 400 (PEG 400) as primary neat cosolvents; glycerin, 1% (w/v) sodium carboxymethyl cellulose (SCMC) as a standard cellulosic suspending agent; 5% (w/v) Polysorbate 80 (Tween^® 80) to evaluate non-ionic micellar solubilization; 2% (w/v) Poloxamer 407 to assess amphiphilic block-copolymer compatibility below its critical gelation point; and 10% (v/v) dimethyl sulfoxide (DMSO) as an aggressive organic cosolvent limit.

Briefly, a targeted mass of exactly 5.0 mg of each crude extract was accurately weighed utilizing a calibrated analytical balance (precision ± 0.1 mg) and dispersed in 1.0 mL of the respective solvent vehicle within a sealed glass vial. This precisely quantified 5 mg/mL threshold was established to evaluate formulation feasibility for the targeted final dosage form. To ensure complete thermodynamic interaction, the mixtures were continuously agitated in an isothermal shaking water bath strictly maintained at 25 ± 0.5 °C for 24 h.

To prevent the potential induction of kinetic supersaturation and subsequent overestimation of solubility, the continuous agitation was halted, and the vials were allowed to equilibrate undisturbed in the isothermal environment (25 ± 0.5 °C) for an additional 2 h. This critical settling phase allowed the matrices to reach true thermodynamic equilibrium. The suspensions were subsequently centrifuged at 500 rpm for 5 min to fully sediment any precipitated or undissolved particulate matter.

Because crude botanical extracts constitute complex multi-component matrices with overlapping chromophores that render UV-Vis spectrophotometric saturation analysis highly inaccurate, solubilization capacity was evaluated via rigorous macroscopic inspection. Visual assessment of the supernatant clarity and the presence or absence of a sediment pellet was conducted under controlled laboratory lighting against both matte black and stark white contrasting backgrounds to effectively detect trace opalescence or fine suspended particulates.

Because this assay was designed as a targeted pre-formulation feasibility screen rather than an absolute thermodynamic saturation profiling study, calculating exact numerical solubility limits was deemed unnecessary. The primary objective was to confirm whether the selected vehicles could completely solubilize the botanical matrix at the specific, therapeutically relevant concentration required for the final dosage form (5 mg/mL). Therefore, based on this precise optical thresholding, vehicle compatibility at this targeted limit was categorized using standardized United States Pharmacopeia (USP) descriptive terms (freely soluble, sparingly soluble, or insoluble). This categorical approach provided a robust, functionally sufficient ‘go/no-go’ metric for selecting the optimal liquid vehicle for downstream formulation.

2.3.3. Total Phenolic Content (TPC) Quantification

TPC was quantified utilizing the established Folin–Ciocalteu colorimetric assay. Extract solution (100 mg/mL) and a serial dilution of gallic acid standards (0.001–0.06 mg/mL) were prepared for analysis. In a 96-well microplate, aliquots of the samples and standards were reacted with Folin–Ciocalteu reagent and neutralized with 7.5% (w/v) sodium bicarbonate. Following a 2-h incubation period in the dark at ambient temperature, the absorbance of the resulting complex was measured at 760 nm using a microplate reader. Measurements were performed in triplicate. The TPC was extrapolated from the gallic acid calibration curve and expressed as milligrams of gallic acid equivalents per gram of crude extract (mg GAE/g extract).

2.3.4. Determination of Total Saponin Content

Total saponin content was determined via the vanillin-sulfuric acid colorimetric assay [38]. A 10-point standard calibration curve was generated using serially diluted aescin (starting from a 100 mg/mL stock in DMSO). For the assay, 50 µL of the sample extract (10 mg/mL) or standard was mixed with 250 µL of 8% (w/v) vanillin solution, 250 µL of distilled water, and 2.5 mL of 72% (v/v) sulfuric acid. The mixtures were vigorously vortexed to ensure homogeneity and incubated in a 60 °C water bath for 15 min to drive the acid-catalyzed colorimetric reaction, marked by a transition from yellow to purple.

After cooling, 200 µL aliquots of the reacted solutions were transferred into a 96-well microplate, and absorbance was recorded at 560 nm. The aescin calibration curve was constructed from quintuplicate measurements (n = 5), ensuring a coefficient of determination (R² ≥ 0.999). Sample extracts were analyzed in triplicate (n = 3). Total saponin content was quantified using the linear regression equation derived from the standard curve and expressed as both milligrams of aescin equivalents per gram of crude extract (mg AE/g extract) and per gram of dry plant material (mg AE/g dry weight).

2.4. HPLC Quantification of Verbascoside

2.4.1. Instrumentation and Chromatographic Conditions

Quantitative analysis of verbascoside was performed using an Agilent 1260 Infinity HPLC system, controlled via Agilent OpenLab CDS software (EZChrom Edition, v. A.04.04). Chromatographic separation was achieved on a Luna C18(2) 100 Å analytical column (250 × 4.6 mm, 5 µm particle size) maintained at 30 °C [39,40]. The mobile phase consisted of a binary gradient of acetonitrile (Solvent A) and 1% (v/v) aqueous acetic acid (Solvent B) delivered at a flow rate of 1.0 mL/min. The gradient elution program was executed as follows: 0–2 min, 0% A; 2–35 min, linear increase from 0% to 50% A; 35–36 min, linear return to 0% A; and 36–40 min, isocratic 0% A for column re-equilibration. The injection volume was set to 10 µL, and the eluent was monitored at a detection wavelength of 330 nm.

2.4.2. Preparation of Standard and Sample Solutions

A primary stock solution of the verbascoside reference standard (1000 µg/mL) was prepared in HPLC-grade methanol. This stock was diluted to a working concentration of 100 µg/mL, which was subsequently serially diluted (two-fold) to generate an eight-point calibration curve ranging from 0.78 to 100 µg/mL (specifically: 0.78, 1.56, 3.13, 6.25, 12.5, 25, 50, and 100 µg/mL). To prepare the test samples, the aqueous and 95% ethanolic botanical extracts were fully dissolved in distilled water and methanol, respectively, achieving a final concentration of 2 mg/mL. All standards and samples were prepared in triplicate and filtered through a 0.45 µm nylon syringe filter prior to HPLC injection.

2.4.3. Method Validation

The analytical method was validated in accordance with the International Council for Harmonization (ICH) guidelines [41], including limit of detection (LOD), limit of quantification (LOQ), precision, and accuracy.

Specificity: Confirmed by comparing the UV-Vis spectral profiles and retention times of the target peaks in the crude extracts against the verbascoside reference standard, ensuring peak purity and accurate identification.

LOD and LOQ: Determined utilizing the signal-to-noise (S/N) ratio approach. The LOD and LOQ thresholds were established at S/N ratios of 3:1 and 10:1, respectively.

Precision: Evaluated by calculating the relative standard deviation (%RSD) of the standard solutions at three concentration levels (3.125, 25, and 50 µg/mL). Intraday precision was assessed through triplicate analyzes within a single day, while interday precision was evaluated over three consecutive days.

Accuracy: Determined via a spike-recovery assay. Briefly, a 0.5 mL aliquot of the 95% ethanolic extract (2 mg/mL) was spiked with the verbascoside standard at three distinct concentration levels (3.125, 25, and 50 µg/mL) within a 5 mL volumetric flask, and the volume was adjusted accordingly. Percentage recovery was calculated from triplicate analyses (n = 3).

2.5. Antimicrobial Activity

2.5.1. Microbial Strains and Culture Media

The antimicrobial efficacy of the aqueous and ethanolic extracts was evaluated against a targeted panel of reference microorganisms. This panel included the primary cariogenic pathogen Streptococcus mutans, the opportunistic oral yeast Candida albicans, and two commensal Gram-positive bacteria, Staphylococcus aureus and Bacillus subtilis.

To ensure optimal viability, the microbial strains were maintained on agar suitable for their specific metabolic requirements. C. albicans was cultured on Sabouraud Dextrose Agar (SDA), while S. aureus and B. subtilis were maintained on Tryptic Soy Agar (TSA), and S. mutans was maintained on Brain Heart Infusion (BHI) agar. Prior to the diffusion assay, all strains were subcultured and incubated overnight at 37 °C. Direct colony suspensions were prepared by transferring morphologically identical colonies into sterile saline solution (0.85% w/v NaCl). The optical density of each microbial suspension was strictly adjusted to match a 0.5 McFarland turbidity standard (representing approximately 1.5 × 10⁸ CFU/mL) to ensure a standardized inoculum size.

2.5.2. Agar Well Diffusion Assay

Antimicrobial screening was performed utilizing a standardized agar well diffusion protocol. A sterile cotton swab was used to uniformly streak the adjusted microbial suspensions across the surface of the respective agar plates in three directions, establishing a confluent lawn of growth. Once the inoculated agar surfaces were visibly dried, uniform wells (8 mm in diameter) were aseptically excised into the agar matrix. Subsequently, 50 µL aliquots of the test extracts, prepared at a specified concentration of 1.0 mg/mL, were carefully dispensed into each well (delivering a specific total dose of 50 µg of extract per well).

To validate the sensitivity of the assay, chlorhexidine (a broad-spectrum antibacterial) and clotrimazole (a targeted antifungal) were utilized as positive controls. The respective vehicles used to dissolve the extracts (10% DMSO, sterile water, and 95% ethanol) were included as negative controls to confirm that the solvents imparted no baseline inhibitory effects.

Plates inoculated with S. aureus, B. subtilis, and C. albicans were incubated aerobically at 37 °C for 24 h. To accommodate its specific microaerophilic growth requirements, S. mutans was incubated at 37 °C for 24 h in a humidified, anaerobic atmosphere. Following the incubation period, antimicrobial efficacy was quantified by measuring the diameter of the clear zones of inhibition surrounding each well utilizing a precision digital caliper. All assays were executed in independent triplicate (n = 3), with the final results expressed as the mean inhibition zone diameter ± standard deviation (SD) in millimeters.

2.6. Cell Culture and Biological Assays

2.6.1. Maintenance of Human Gingival Fibroblasts (HGF)

Human gingival fibroblasts (HGF ATCC no. 5001118, Manassas, VA, USA) were maintained in high glucose Dulbecco’s Modified Eagle’s medium (DMEM, Corning^® Life Sciences, Union City, CA, USA) supplemented with 10% (v/v) fetal bovine serum (FBS; Gibco, CA, USA), 2 mM L-glutamine, 100 U/mL penicillin, 0.1 mg/mL streptomycin, and 0.025 mg/mL amphotericin B (Invitrogen, Union City, CA, USA). The cell cultures were incubated at 37 °C in a humidified, 5% CO₂ atmosphere (Shel Lab, Cornelius, OR, USA). To ensure optimal metabolic activity and assay reliability, all biological evaluations were conducted utilizing cells strictly within the logarithmic growth phase [42,43].

2.6.2. Cytocompatibility Assessment (MTT Assay)

The general cytocompatibility of the botanical extracts was evaluated utilizing a standardized MTT colorimetric assay. HGFs were seeded into 96-well microplates at a density of 1 × 10⁴ cells/well and incubated overnight to facilitate monolayer attachment. The culture medium was subsequently aspirated and replaced with fresh medium containing the test extracts at concentrations ranging from 0.001 to 1.00 mg/mL. Untreated cells cultured in complete medium served as the negative (100% viability) control, whereas cells treated with 0.1 mg/mL hydrogen peroxide (H₂O₂) served as the positive (cytotoxic) control [43].

After 24-h incubation period, the treatment medium was discarded, and the cells were incubated with MTT solution (400 µg/mL) for 4 h in the dark to allow for the mitochondrial reduction of the tetrazolium dye. The supernatant was carefully aspirated, and the resulting intracellular formazan crystals were solubilized by adding 100 µL of dimethyl sulfoxide (DMSO) per well. Absorbance was quantified at 570 nm utilizing a microplate reader (arioscan, Thermo Fisher, Waltham, MA, USA). Cell viability was calculated using the following equation:

$$\mathrm{Cell}\mathrm{viability}(\%) = \frac{{\mathrm{A}}_{\mathrm{sample}}}{{\mathrm{A}}_{\mathrm{control}}} \times 100$$

where A_extract and A_control represent the absorbance values of the extract-treated cells and the untreated negative control, respectively. All assays were performed in independent triplicates (n = 3).

2.6.3. In Vitro Scratch Wound Assay (Cellular Migration)

The tissue-regenerative potential of the extracts was evaluated via an in vitro scratch wound assay, optimized to monitor HGF motility [4,44]. Cells were seeded into 24-well plates at a density of 4 × 10⁴ cells/well and cultured overnight to yield a fully confluent monolayer. A standard, linear mechanical scratch (wound gap) was introduced across the center of the cellular monolayer utilizing a sterile 200 µL pipette tip. Detached cellular debris was meticulously removed via gentle washing with sterile phosphate-buffered saline (PBS).

The denuded monolayers were subsequently incubated in fresh medium supplemented with the targeted extracts at non-cytotoxic concentration (as predetermined by the MTT assay) or an untreated control medium [4]. The progression of cellular migration into the wound gap was continuously monitored and photographically documented at 0, 16, 20, and 24 h post-scratching using an inverted phase-contrast microscope at 10× magnification. The wound gap area was quantitatively analyzed using ImageJ software (version 1.54g), and the percentage of wound closure was calculated to determine migratory efficacy relative to the initial gap area at hour zero.

2.7. Effervescent Mouthwash Tablet Design and Preparation

2.7.1. Tablet Formulation and Bioprocessing

The optimized ethanolic A. montanus (Am) extract was formulated into three distinct effervescent mouthwash tablet batches (F1, F2, F3). Each batch was scaled to produce 100 tablets, with a target weight of 500 mg per tablet containing exactly 50 mg of the bioactive botanical extract. This specific 50 mg dose was calculated so that upon dissolution in a standard 10 mL volume of water prior to rinsing, the final mouthwash solution achieves a targeted therapeutic concentration of 5.0 mg/mL, a parameter strictly aligning with our validated solubility and in vitro biological efficacy thresholds.

The bioprocessing followed established non-aqueous wet granulation methodologies suitable for moisture-sensitive effervescent systems [27,28]. Briefly, the crude botanical active pharmaceutical ingredient (API) and the effervescent base (citric acid, tartaric acid, sodium bicarbonate) were homogenized. To initiate granulation without triggering premature effervescence, absolute ethanol was added incrementally quantum satis (q.s.), utilizing just a sufficient quantity to bind the dry powders into a cohesive, damp mass.

Following the incorporation of volatile flavoring oils, this wet mass was dried in a hot air oven at 40 °C for 1 h to fully volatilize the residual ethanol. The dried granules were subsequently passed through a No. 40 mesh sieve (425 µm) to achieve a uniform particle size distribution. These granules were geometrically blended with the remaining extragranular excipients (xylitol, polyvinylpyrrolidone (PVP), Explotab^®, talcum, and sodium lauryl sulfate (SLS)) utilizing a tumbling technique. Finally, the uniform blends were compressed utilizing a single-stroke tablet press. The specific compositional variations among the three formulations are detailed in

Table 1. Key Variable Excipients in Formulations F1-F3 (per 100-tablet batch).

Formulation	API (g)	PVP (Binder)	Explotab® (Disintegrant)
F1	5.0	2% (0.975 g)	0% (0 g)
F2	5.0	3% (1.0 g)	6% (2.5 g)
F3	5.0	2% (1.0 g)	10% (5.0 g)

.

2.7.2. Tablet Quality Control Evaluation

The compressed effervescent mouthwash tablets from each batch underwent comprehensive physicochemical characterization in accordance with standard pharmacopeial guidelines. The evaluated parameters included general appearance, thickness, hardness, weight variation, friability, disintegration time, and dissolution rate. Tablet thickness and hardness (n = 6 per batch) were quantified utilizing a Vernier caliper and a Monsanto hardness tester, respectively. Assessments for weight variation and friability were conducted using a precision analytical balance and a Roche Friabilator (type KF, K.S.L Engineering Co., Ltd., Bangkok, Thailand), adhering strictly to the USP 34/NF 29 protocols.

Furthermore, the in vitro disintegration time was evaluated in accordance with standard pharmacopeial methodologies for effervescent tablets. Briefly, individual tablets (n = 6) were dropped into beakers containing 200 mL of purified water maintained at ambient room temperature (25 ± 1 °C), accurately simulating the real-world preparation conditions of the mouthwash solution. The disintegration process was visually monitored, and the time required for complete cessation of effervescence and total macroscopic dispersion of the matrix was recorded against a predefined acceptance threshold of ≤5 min.

2.7.3. Microstructural Analysis by Synchrotron Radiation X-Ray Tomographic Microscopy (SR-XTM)

To visualize and quantify the 3D internal architecture of the dosage forms, SR-XTM was conducted at Beamline 1.2 W, operating at 1.2 GeV and 150 mA. To prevent motion artifacts during acquisition, the tablet samples were firmly immobilized on the goniometer stage utilizing a cotton pad and Kapton tape [45]. The X-ray beam, featuring a mean energy of 11.5 keV, was attenuated through a 350 µm aluminum foil filter to minimize ring artifacts. Radiographic projections were captured across a 180° continuous rotation at 0.1° increments. The optical detection system comprised a 200 µm YAG:Ce scintillator (Crytur Ltd., Turnov, Czech Republic) coupled to an optical X-ray microscope (Optique Peter, Lentilly, France) and a 16-bit pco.edge 5.5 sCMOS camera (Excelitas Technologies Corp., Pittsburgh, PA, USA).

Raw radiographic data pre-processing, background normalization, and tomographic reconstruction were executed utilizing Octopus Reconstruction software v.8.9.1 (TESCAN, Gent, Belgium) [46]. The resulting spatial data was used to extract quantitative internal porosity metrics, and the tomograms were subsequently rendered into high-resolution 3D microstructural models utilizing Drishti software v.2.6.4 [47].

2.8. Statistical Analysis

All quantitative experiments were conducted in triplicate (unless otherwise specified), and the resulting data are presented as the mean ± standard deviation (SD). To determine statistical significance among multiple formulation groups, a one-way analysis of variance (ANOVA) was performed, followed by Fisher’s Least Significant Difference (LSD) post hoc test for pairwise comparisons. Prior to correlation analyzes, the assumption of data normality was verified utilizing the Shapiro–Wilk test. The linear relationship between internal tablet porosity (quantified via Octopus Analysis; TESCAN, Gent, Belgium) and physical disintegration time was evaluated using the Pearson correlation coefficient. All statistical computations were executed utilizing SPSS version 13.0 for Windows (SPSS Inc., Chicago, IL, USA). A two-tailed p-value of <0.05 was considered statistically significant.

3. Results and Discussion

3.1. Extract Yields and Physical Properties

The bioprocessing of the three Acanthus species yielded dark brown, odorless crude extracts. As detailed in

Table 2. The extracted weights and percentage yields (% w/w) of Acanthus ebracteatus (Ae), Acanthus ilicifolius (Ai), and Acanthus montanus (Am) using ethanolic (EtOH) and aqueous (Water) solvents. Data are expressed as mean ± SD (n = 3).

Plant Extracts	Solvent	Weight (g)	Yield (%)
Acanthus ebracteatus (Ae)	EtOH	0.61 ± 0.09	6.08 ± 0.10
	Water	1.57 ± 0.07	15.80 ± 0.05
Acanthus ilicifolius (Ai)	EtOH	0.81 ± 0.08	8.08 ± 0.06
	Water	1.83 ± 0.03	18.37 ± 0.04
Acanthus montanus (Am)	EtOH	0.77 ± 0.05	7.75 ± 0.04
	Water	1.61 ± 0.03	16.09 ± 0.05

, the overall mass recovery was profoundly solvent-dependent, with aqueous extraction consistently recovering a higher gross crude mass than 95% ethanol (EtOH). The maximum extraction yield was achieved with the aqueous extract of A. ilicifolius (18.37 ± 0.04% w/w), followed by A. montanus (16.09 ± 0.05% w/w) and A. ebracteatus (15.80 ± 0.05% w/w). Conversely, ethanolic extraction resulted in significantly lower gross yields across all species, reaching a minimum in A. ebracteatus (6.08 ± 0.10% w/w).

This pronounced variation highlights the polarity-driven solubility of the phytoconstituents within these specific plant matrices. The markedly higher aqueous yields strongly suggest that the predominant extractable mass in these Acanthus leaves is composed of highly polar, water-soluble components. These likely include primary metabolites, ubiquitous structural carbohydrates, and specific polar glycosides [13,48]. While broader phytochemical literature frequently reports that ethanolic solvents can yield both higher mass efficiencies and richer bioactive profiles in various other plant families [49], our data reveals a distinct bioprocessing dynamic for the Acanthus genus. In this context, 95% ethanol, acting as a moderately polar solvent, appears to selectively partition a targeted subset of secondary metabolites without co-extracting the massive abundance of polar bulk materials, thereby resulting in a lower gross mass recovery.

However, it is critical to contextualize these yield parameters. While gross mass recovery provides a macroscopic overview of extraction efficiency, it is an imperfect proxy for true therapeutic potential. High aqueous yields frequently encompass substantial quantities of inactive, non-target bulk materials [20]. Therefore, evaluating bioprocessing efficiency solely on mass recovery can be misleading.

These initial extraction profiles establish a foundational baseline for our subsequent formulation strategy. Although aqueous extraction provides superior quantitative mass yield, identifying the optimal bioactive agent for downstream product development requires balancing this gross recovery against the actual concentration of functional metabolites [50]. Consequently, our extract selection process was not dictated by mass yield, but rather by a systematic evaluation of targeted bioactive constituents (phenolic and saponin contents) and their corresponding functional biological activity. This multiparametric approach, correlating chemical profiles with targeted antimicrobial efficacy and HGFs biocompatibility, ensures that the final engineered delivery system is optimized for true therapeutic potency rather than mere raw extraction mass.

3.2. Equilibrium Solubility and Pre-Formulation Profiling

To identify the optimal vehicle for the final dosage form, the solubilization capacity of the dried aqueous and ethanolic extracts, which function collectively as the crude botanical active pharmaceutical ingredients (APIs), was systematically screened. To reflect realistic formulation strategies, the screening panel compared two distinct physicochemical approaches: the use of neat, pure cosolvents (PEG 400, PG, and glycerin) intended to alter bulk polarity, and the use of structured aqueous systems (1% SCMC, 5% Tween 80, 2% Poloxamer 407, and 10% DMSO) intended to provide localized micellar or polymeric stabilization. While direct quantitative measurement of the vehicles’ dielectric constants was outside the scope of this pre-formulation screen, the divergence in solubility behavior can be qualitatively interpreted through the well-established polar and structural properties of these respective microenvironments.

As detailed in

Table 3. Solubility testing of Acanthus ebracteatus Vahl (Ae), Acanthus ilicifolius (Ai), and Acanthus montanus (Am). The concentration of the extract sample is 5 mg/mL.

Solvent/ Sample	PG	PEG400	Glycerin	1% SCMC	5% Tween80	2% Poloxamer	10% DMSO
Water extract
Ai	x	+++	xx	x	x	xx	xx
Ae	+++	+++	xx	x	xx	xx	xx
Am	+++	+++	xx	++	++	++	xx
Ethanol extract
Ai	+++	+++	xx	++	x	xx	x
Ae	+++	+++	xx	++	x	x	x
Am	+++	+++	xx	+++	++	+++	x

Notice: Very soluble: +++, Soluble: ++, Precipitating: x, Insoluble: xx.

, the solubility behavior revealed a distinct dependence on the fundamental thermodynamic mechanism of the vehicle. Among the pure solvents, PEG 400 emerged as the most universally effective cosolvent, facilitating the complete macroscopic dissolution of all six extract variants at the targeted 5 mg/mL threshold. PG similarly demonstrated robust solubilizing capacity, with the singular exception of the aqueous A. ilicifolius (Ai) extract. The near-universal success of these neat cosolvents is mechanistically attributed to their ability to significantly lower the bulk dielectric constant of the medium. By providing an intermediate polarity, PEG 400 and PG effectively solvate the highly complex, multi-component mixture of polar glycosides, moderately polar phenolics, and hydrophobic aglycones inherent to crude botanical APIs. Conversely, the pronounced insolubility observed in glycerin and 10% DMSO suggests highly unfavorable solvent-solute interactions, likely driven by competitive hydrogen-bonding networks that thermodynamically hinder the dissolution of these specific plant matrices.

In contrast, solubility was severely restricted when screened against the structured aqueous vehicles. Because these systems are dominated by bulk water, which possesses a very high dielectric constant, the thermodynamic penalty for dissolving non-polar phytochemicals is immense. In these environments, successful solubilization relies entirely on the hydrophobic botanical constituents partitioning into the limited lipophilic cores of surfactant micelles (Tween 80, Poloxamer 407) or being sterically stabilized by a cellulosic network (SCMC). The mangrove species extracts (Ai and Ae) predominantly precipitated or remained entirely insoluble in these complex structured environments. This suggests that their phytochemical profiles are dominated by rigidly hydrophobic constituents or bulky resins that exceed micellar encapsulation capacity, with the singular notable exception being the ethanolic Ae extract, which successfully maintained macroscopic solubility in 1% SCMC.

Notably, the A. montanus (Am) extracts exhibited exceptional, broad-spectrum compatibility across both the pure cosolvents and the complex aqueous systems. Both the aqueous and ethanolic Am extracts achieved complete dissolution within the hydrophilic polymeric network of 1% SCMC and demonstrated moderate to high solubility within the non-ionic micellar environments of 5% Tween 80 and the thermoreversible block-copolymer 2% Poloxamer 407. This unique formulation versatility is fundamentally linked to the distinct phytochemical architecture of Am. As demonstrated in the subsequent phytochemical profiling, A. montanus is uniquely enriched in amphiphilic secondary metabolites, specifically saponins. These native amphiphiles likely function as co-surfactants, interacting synergistically with the aqueous vehicles to form highly stable dispersions without relying exclusively on pure organic cosolvents. Crucially, this robust phase behavior provides strong empirical evidence that A. montanus is uniquely suited among the tested species for incorporation into advanced delivery systems, such as oral mucosal liquids, bioadhesive hydrogels, or in situ forming matrices, as it demonstrates a significantly reduced thermodynamic risk of active ingredient precipitation during extended storage.

3.3. Total Phenolic Content and Formulation Synergy

The total phenolic content (TPC) of the Acanthus extracts was quantified utilizing a standardized Folin–Ciocalteu colorimetric assay, establishing a critical baseline for the botanical raw materials. The analysis revealed a significant, species-dependent variation in phenolic accumulation. The ethanolic Am extract exhibited the highest phenolic density at 1.73 ± 0.09 mg GAE/g extract, outperforming both Ai (1.14 ± 0.06 mg GAE/g) and Ae (1.04 ± 0.02 mg GAE/g). While we acknowledge that colorimetric assays provide a macroscopic estimation of total phenolics rather than the single-molecule resolution achievable via chromatographic techniques such as HPLC or LC-MS [51], this targeted screening successfully validated A. montanus as the most efficient phenolic accumulator among the evaluated terrestrial and mangrove species.

Transitioning these phytochemical findings to the engineered bioproduct, a single 500 mg effervescent mouthwash tablet formulated with 50 mg of the Am crude extract is calculated to deliver an estimated 0.0865 mg of GAE per unit dose. When benchmarked against systemic botanical supplements derived from high-phenolic matrices, such as Echinacea or Mentha species, this absolute phenolic mass is quantitatively modest [52]. However, the therapeutic rationale of the effervescent delivery system relies on localized, rather than systemic, bioavailability. Upon immediate disintegration, the effervescent matrix releases these phenolic compounds directly into the oral mucosal microenvironment. Here, they function as immediate hydrogen donors, neutralizing the localized reactive oxygen species (ROS) that are frequently implicated in gingival inflammation and mucosal tissue degradation.

Crucially, the relatively modest absolute mass of phenolics delivered per tablet suggests that the formulation’s functional efficacy, particularly its documented antimicrobial and wound-healing capacity, does not rely solely on this specific fraction. In complex plant matrices, therapeutic bioactivity is rarely driven by a single isolated compound. Rather, we hypothesize that the efficacy of the Am bioproduct stems from synergistic interactions between the phenolics and the extract’s other predominant secondary metabolites, most notably saponins. The amphiphilic, membrane-disrupting properties of saponins are well-documented to increase microbial cell wall permeability. This natural surfactant-like action likely facilitates the intracellular influx of phenolic compounds, thereby potentiating their targeted antimicrobial and tissue-repairing effects [8]. Therefore, the functional success of the effervescent mouthwash tablet is driven not by an overwhelming phenolic mass, but by leveraging the synergistic, multi-component phytochemical architecture unique to the A. montanus extract.

3.4. Total Saponin Content

The total saponin content of the aqueous and ethanolic extracts was quantified via the vanillin-sulfuric acid colorimetric assay and expressed as milligrams of aescin equivalents per gram of dry weight (mg AE/g dry weight). As depicted in Figure 1, the recovery of saponins was profoundly dictated by the choice of extraction solvent. Across all three species, extraction with 95% ethanol consistently and substantially outperformed the aqueous extraction. The ethanolic extract of Ai exhibited the highest total saponin concentration (70.70 ± 4.23 mg AE/g dry weight), followed by the ethanolic extracts of Am (50.14 ± 3.01 mg AE/g) and Ae (38.70 ± 2.92 mg AE/g). Conversely, aqueous extraction resulted in remarkably poor saponin recovery. The aqueous Am extract yielded the highest amount within the water group at only 15.79 ± 2.39 mg AE/g, while the aqueous Ae extract contained negligible saponin levels (0.92 ± 0.43 mg AE/g).

The pronounced disparity in saponin recovery, most notably the 4.5-fold higher concentration observed in the Ai ethanolic extract compared to the most efficient aqueous extract, highlights the critical role of solvent polarity in targeted bioseparation. Saponins are complex, amphiphilic secondary metabolites comprising a non-polar triterpenoid or steroid aglycone (sapogenin) conjugated to highly polar sugar moieties. While their glycosidic chains confer a degree of water solubility, the bulky hydrophobic aglycone core dictates that intermediate-polarity solvents, such as 95% ethanol, are significantly more effective at penetrating the plant matrix and solubilizing the intact molecules [38]. While this standard ethanolic extraction successfully recovered high bioactive yields, future bioprocessing optimizations utilizing techniques such as ultrasound-assisted extraction (UAE) or deep eutectic solvents (DES) could be explored to further accelerate extraction kinetics [53,54].

Crucially, these quantitative findings validate the extraction dynamics hypothesized in Section 3.1. While aqueous extraction provided a substantially higher gross mass yield (16.09% w/w for Am), the resulting extracts were fundamentally dilute, yielding only 15.79 mg/g of saponins. In contrast, 95% ethanol functioned as a highly selective solvent. Despite yielding a lower overall crude mass (7.75% w/w for Am), the ethanolic extraction selectively enriched the targeted matrix, concentrating the saponin content up to 50.14 mg AE/g. This stark quantitative contrast confirms that the bulky aqueous yields are predominantly composed of non-target, highly polar bulk constituents, whereas ethanol effectively isolates the functional secondary metabolites.

For the downstream formulation of advanced bioproducts, such as the target effervescent delivery system, this enrichment factor is paramount. While the ethanolic Ai extract exhibited the absolute peak saponin concentration, the ethanolic Am extract demonstrated a highly robust saponin recovery (50.14 mg AE/g) that perfectly complements its peak phenolic content established previously. Therefore, the ethanolic A. montanus extract represents the crude botanical active pharmaceutical ingredient (API), providing a concentrated, multi-component phytochemical matrix rich in natural surfactants and antioxidants to maximize the therapeutic potential of the final dosage form.

3.5. HPLC Validation and Targeted Quantification of Verbascoside

To chemically standardize the extracts and correlate their biological efficacy to a specific bioactive marker, the concentration of verbascoside, a potent, tissue-repairing phenylethanoid glycoside, was quantified via HPLC-DAD.

The specificity of the analytical method was verified through diode-array detection. The UV absorption spectra of the target peaks in the crude plant extracts perfectly matched the verbascoside reference standard, confirming peak purity and the absence of co-eluting interference from the complex botanical matrix. The optimized binary gradient successfully resolved the verbascoside peak at a retention time of 21.13 min [39,40]. The method exhibited exceptional sensitivity, establishing an LOD and LOQ at 9 ng/mL and 27 ng/mL, respectively. Method reliability proved highly robust; intraday precision (%RSD of 0.10–1.39%) and interday precision (%RSD of 1.38–4.31%) remained well below the stringent 5% acceptable thresholds for complex biological matrices [40]. Furthermore, spike-recovery assays yielded an accuracy range of 95–105%, confirming the method’s precision and suitability for targeted phytomatrix quantification.

As detailed in

Table 4. Quantification of Verbascoside in Acanthus Extracts (n = 3).

Sample	Extraction Solvent	Verbascoside Content (%)
Acanthus ilicifolius (Ai)	Water	ND
	95% Ethanol	0.14 ± 0.02
Acanthus ebracteatus (Ae)	Water	ND
	95% Ethanol	0.46 ± 0.02
Acanthus montanus (Am)	Water	ND
	95% Ethanol	6.38 ± 0.18

ND = not detected.

, the quantitative analysis revealed a stark dichotomy driven by the extraction solvent. Verbascoside was completely undetectable (ND) in the aqueous extracts across all three Acanthus species. While existing literature notes that verbascoside is intrinsically water-soluble [55], its molecular structure is notoriously susceptible to hydrolytic cleavage and thermal degradation. The extended high-temperature decoction utilized during the aqueous bioprocessing likely catalyzed its degradation to levels below the analytical detection limit.

In striking contrast, extraction with 95% ethanol successfully preserved the active metabolite. Ethanol rapidly denatures endogenous degradative plant enzymes and provides a thermodynamically stabilizing environment for labile glycosides during thermal processing [56]. Within this ethanolic cohort, A. montanus (Am) emerged as an exceptionally rich source, yielding a verbascoside content of 6.38 ± 0.18% (w/w). This was overwhelmingly superior to the ethanolic extracts of Ae (0.46 ± 0.02%) and Ai (0.14 ± 0.02%).

This targeted quantification provides a crucial molecular rationale for the biological observations detailed earlier. Verbascoside is well-documented for its antioxidant, antimicrobial, and wound-healing bioactivities [57,58]. The profound enrichment of this specific bioactive marker in the ethanolic A. montanus extract directly correlates with its superior performance in the human gingival fibroblast migration and oral pathogen inhibition assays. Ultimately, this chemical validation firmly establishes the ethanolic Am extract as the premier candidate for downstream translation into the effervescent delivery system.

3.6. Biological Efficacy: Cytocompatibility and Mucosal Repair

3.6.1. In Vitro Cytocompatibility Profile

Establishing the in vitro safety profile of botanical bioproducts is a fundamental prerequisite for their translation into clinical delivery systems. The cytocompatibility of the Acanthus extracts was evaluated on HGFs using the MTT colorimetric assay across a concentration gradient (0.001 to 1.0 mg/mL), as shown in Figure 2. The assay parameters were validated internally; the untreated negative control maintained 100% baseline viability, whereas the positive cytotoxic control (H₂O₂) effectively reduced viability to approximately 56%.

Across all three evaluated species, the extracts exhibited a mild, dose-dependent influence on HGF viability. At lower concentrations (0.001 and 0.01 mg/mL), the extracts demonstrated exceptional biocompatibility, consistently maintaining cell viability above 90%. Notably, treatment with the Am extract at 0.001 mg/mL yielded a calculated viability exceeding 100%. While this may suggest a minor proliferative stimulus, plant extracts rich in redox-active secondary metabolites (such as phenolics) can occasionally interact with the tetrazolium reduction chemistry of the MTT assay, potentially yielding slightly inflated absorbance values [59]. As the concentration escalated to the maximum tested dose (1.0 mg/mL), a moderate reduction in cell viability was recorded. Nonetheless, the Ae and Ai extracts maintained viabilities above 70%, while the Am extract demonstrated superior cellular tolerance, sustaining viability at approximately 85%.

According to the ISO 10993-5 guidelines for the biological evaluation of medical and pharmaceutical formulations, a reduction in cell viability exceeding 30% (viability < 70%) is generally classified as cytotoxic [60]. Based on this standard threshold, all three Acanthus extracts are classified as non-cytotoxic to HGFs across the entire tested concentration range [61,62]. We hypothesize that the mild viability decline observed at the 1.0 mg/mL apex does not indicate acute toxicity, but rather reflects localized cellular stress induced by the high osmotic load and dense accumulation of complex secondary metabolites inherent to crude botanical matrices in a static in vitro environment. Ultimately, the superior biocompatibility of the Am extract correlates directly with our previous quantification of its peak phenolic content, compounds highly documented for maintaining intercellular homeostasis and mitigating oxidative stress. These findings provide a robust safety justification for utilizing the Am extract as the primary crude botanical active pharmaceutical ingredient (API) in the downstream formulation.

3.6.2. In Vitro Mucosal Repair and Cellular Migration

The biotherapeutic capacity of the Acanthus extracts to actively promote mucosal repair was evaluated utilizing an in vitro scratch wound assay. To ensure precise comparability and eliminate toxicity-induced variables, all extracts were administered at a standardized, non-cytotoxic concentration of 0.01 mg/mL (Figure 3), benchmarked against a serum-free medium negative control [63].

Quantitative analysis of the wound closure (Figure 3) revealed a highly treatment-specific and time-dependent migratory response. Over the 24-h observation period, the untreated control group exhibited only a basal migration rate, achieving 53% wound gap closure. In stark contrast, all three Acanthus treatments significantly accelerated HGF motility. Cells treated with the ethanolic Am extract demonstrated the most pronounced acceleration in tissue repair, achieving 62.07 ± 3.46%, 75.35 ± 3.08%, and 79.41 ± 4.81% wound closure at 16, 20, and 24 h, respectively. The Ae and Ai extracts also robustly promoted closure, reaching 75.25 ± 3.87% and 68.47 ± 3.11% at 24 h, easily outperforming the basal control.

Fibroblast migration is a fundamental physiological prerequisite for the proliferative phase of gingival wound healing and oral mucosal repair [44,64]. Because the standard two-dimensional scratch assay cannot definitively isolate cell motility from localized cell division, the accelerated gap reduction observed here is likely attributable to a combined effect of enhanced actin-driven cellular motility and localized proliferation [65]. This aligns perfectly with the established MTT data; because the extracts are non-toxic at 0.01 mg/mL, the fibroblasts remain fully viable and capable of executing the complex, high-energy cellular mechanics required to seal the denuded area.

The pronounced wound closure observed in the Am-treated group provides biological validation for the phytochemical synergy hypothesized in earlier sections. The concurrent presence of highly concentrated phenolics and saponins in the ethanolic A. montanus extract likely neutralized oxidative stress, providing a favorable physiological microenvironment that simulates the intracellular signaling cascades governing fibroblast motility. While future transcriptomic profiling will be required to map these specific molecular pathways, these current biological data definitively establish the exceptional potential of the A. montanus extract as a functional, tissue-repair active ingredient for targeted oral delivery systems.

3.7. Targeted Antimicrobial Activity and Pathogen Inhibition

The antimicrobial efficacy of the Acanthus extracts was evaluated against a targeted panel of clinical microorganisms, specifically, Staphylococcus aureus, Bacillus subtilis, Streptococcus mutans, and Candida albicans, utilizing a standardized agar well diffusion assay. The negative solvent control (DMSO) yielded no zones of inhibition (ND), confirming that the observed antimicrobial activity was driven exclusively by the botanical matrices.

As detailed in

Table 5. Antimicrobial activity of the aqueous and ethanolic extracts of Acanthus species, expressed as the diameter of the inhibition zone (mm). Data are presented as mean ± standard deviation (n = 3). ND indicates not detected (no zone of inhibition).

Extract	Inhibition Zone ± S.D. (mm)
	S. aureus ATCC 6538	B. subtilis ATCC	S. mutans ATCC	C. albicans ATCC 10231
Aqueous
A. ebracteatus	10.00 ± 1.41	ND	ND	ND
A. ilicifolius	ND	ND	ND	ND
A. montanus	10.51 ± 0.71	ND	ND	ND
Ethanolic
A. ebracteatus	12.67 ± 0.21	8.75 ± 0.77	ND	ND
A. ilicifolius	13.50 ± 0.12	8.50 ± 0.70	ND	ND
A. montanus	13.67 ± 0.31	9.50 ± 0.72	9.92 ± 0.12	10.10 ± 0.25
DMSO	ND	ND	ND	ND

, the choice of extraction solvent profoundly dictated the antimicrobial profile. Corroborating the phytochemical data, the ethanolic extracts demonstrated substantially broader and stronger inhibitory effects than their aqueous counterparts. S. aureus proved to be the most universally susceptible microorganism, inhibited by all ethanolic extracts (zones ranging from 12.67 ± 0.21 to 13.67 ± 0.31 mm) and weakly inhibited by the aqueous extracts of Ae and Am. However, the aqueous extracts entirely failed to inhibit any of the other tested strains, rendering them largely ineffective for complex oral applications.

Notably, the ethanolic extract of Am emerged as the most potent biotherapeutic candidate. It was the only formulation to exhibit robust, broad-spectrum activity against all four pathogens. Beyond generating the largest inhibition zones against the common commensal bacteria S. aureus (13.67 ± 0.31 mm) and B. subtilis (9.50 ± 0.72 mm), the Am extract uniquely demonstrated significant targeted activity against the primary cariogenic bacterium S. mutans (9.92 ± 0.12 mm) and the opportunistic oral yeast C. albicans (10.10 ± 0.25 mm). While crude botanical matrices inherently produce smaller inhibition zones than purified commercial antibiotics [66], the capacity of the Am extract to simultaneously suppress both bacterial and fungal etiological agents positions it as a highly valuable, multi-target active ingredient for oral health.

This pronounced biological disparity between the aqueous and ethanolic extracts perfectly validates the bioprocessing dynamics established earlier in this study. The superior pathogen inhibition of the ethanolic extracts correlates directly with their selective enrichment of active secondary metabolites [67]. Conversely, the aqueous extraction, despite yielding a higher gross mass, primarily recovered inactive bulk primary metabolites (such as structural carbohydrates), resulting in a functionally dilute and largely ineffective antimicrobial fraction [68].

Mechanistically, the robust antimicrobial response of the ethanolic Am extract is likely driven by profound phytochemical synergy. The highly concentrated saponins function as natural surfactants, permeabilizing the complex microbial cell membranes and fungal walls. This structural disruption facilitates the intracellular influx of the enriched phenolic compounds (including the validated verbascoside marker), which subsequently precipitate vital microbial proteins and inhibit crucial enzymatic pathways [67].

By effectively managing these key drivers of the oral microbiome, while concurrently maintaining excellent cytocompatibility with gingival fibroblasts [16], the ethanolic A. montanus extract overcomes the traditional limitations of crude extracts. Ultimately, this multiparameter biological success definitively establishes the Am extract as the optimal active pharmaceutical ingredient for downstream engineering into the final effervescent delivery system.

3.8. Bioproduct Formulation: Microstructural and Kinetic Evaluation

The optimized ethanolic A. montanus extract was successfully engineered into three effervescent mouthwash tablet formulations (F1–F3). To systematically evaluate the impact of super-disintegrant concentration on kinetic performance, these batches were formulated with 0%, 6%, and 10% Explotab^®, respectively. The post-compression physicochemical properties, encompassing weight variation, hardness, friability, and disintegration time [30], are summarized in

Table 6. Physical properties and quality control evaluation of the formulated effervescent mouthwash tablets (F1–F3) containing the ethanolic A. montanus extract. Data are expressed as mean ± standard deviation.

Parameter	F1 (0% Explotab)	F2 (6% Explotab)	F3 (10% Explotab)	Acceptance Criteria
Weight Variation (g) (n = 15)	0.48 ± 0.01	0.52 ± 0.02	0.50 ± 0.01	±10% of 500 mg
Thickness (mm) (n = 15)	2.63 ± 0.10	2.86 ± 0.34	3.16 ± 0.27	N/A
Hardness (kg) (n = 15)	1.22 ± 0.09	1.10 ± 0.54	1.36 ± 0.13	1.0–2.0 kg (In-house)
Porosity (%) (n = 3)	2.79 ± 0.18	3.38 ± 0.37	4.62 ± 0.39	N/A
Friability (%) (n = 10)	7.59% (Fail)	0.42% (Pass)	0.23% (Pass)	<1.0%
Disintegration (min) (n = 6)	4.27 ± 0.08	3.06 ± 0.26	2.02 ± 0.23	<5.0 min

.

All three formulations successfully met the established pharmacopeial acceptance criteria for weight uniformity (target 500 mg) and achieved the targeted mechanical hardness specification (1.0–2.0 kg). However, a critical divergence was observed in the mechanical integrity of the compressed matrices. Formulation F1 (0% super-disintegrant) exhibited severe failure during testing, sustaining an unacceptable mass loss of 7.59%. Conversely, the incorporation of Explotab^® profoundly enhanced inter-particulate cohesion. Formulations F2 and F3 yielded exceptional friability values of 0.42% and 0.23%, respectively, comfortably surpassing the stringent < 1.0% pharmacopeia threshold. While sodium starch glycolate (Explotab^®) is primarily recognized for its extensive volumetric swelling rather than plastic deformation [69], its incorporation into the dry powder blend evidently facilitated improved particle rearrangement and bonding during direct compression, thereby conferring the robust structural integrity observed in F2 and F3.

The physical evaluation of the effervescent mouthwash tablets underscores the functional impact of the formulation matrix on the kinetic performance of the dosage form. It must be noted that formulation F2 contained a slightly higher concentration of polyvinylpyrrolidone (3% PVP) to optimize intermediate powder compressibility, meaning its excellent friability and altered disintegration kinetics are likely driven by a synergistic combination of the polymeric binder and the super-disintegrant.

However, by establishing a direct, unconfounded comparison between the control formulation F1 (0% Explotab) and the optimized formulation F3 (10% Explotab), both of which rigorously maintained a constant 2% PVP binder concentration, the distinct mechanistic role of the super-disintegrant becomes explicitly clear. In this controlled comparative pair, the severe friability failure of F1 indicates that the baseline effervescent powder blend lacked sufficient inter-particulate bonding. The addition of 10% Explotab^® in F3 not only rescued the mechanical structural integrity of the matrix (achieving 0.23% friability) but simultaneously accelerated the disintegration kinetics by more than two-fold (2.02 min). Because the binder concentration remained static between these two extremes, the profound microstructural expansion and subsequent kinetic acceleration observed in F3 can be definitively attributed to the massive volumetric swelling and capillary wicking action of the sodium starch glycolate.

Beyond mechanical strength, the critical performance metric for an effervescent delivery system is rapid and complete aqueous dissolution. The concentration of the super-disintegrant directly governed both the internal microstructural architecture and the resulting disintegration kinetics. Quantitative 3D SR-XTM analysis revealed a dose-dependent increase in internal tablet porosity, escalating from 2.79% in F1 to 4.62% in F3. These values align with established pharmaceutical literature, where directly compressed tablet porosity spans a broad spectrum depending on compression force and excipient geometry [70]. Crucially, this heightened porosity demonstrated a strong inverse correlation with the disintegration time. Formulation F3 achieved complete disintegration in just 2.02 ± 0.23 min, representing a more than two-fold kinetic acceleration compared to the control F1 (4.27 ± 0.08 min).

Mechanistically, this accelerated breakdown is driven by a synergistic, dual-action disintegration model. First, the enhanced porosity generated by the higher Explotab^® concentration (10% in F3) acts as a highly interconnected internal capillary network. This porous architecture facilitates immediate, aggressive water wicking into the tablet core. Upon hydration, the sodium starch glycolate undergoes massive volumetric swelling. Simultaneously, the penetrating water triggers the classic acid-based effervescent reaction (between citric acid, tartaric acid, and sodium bicarbonate), generating a rapid, localized release of carbon dioxide gas. The combined internal pressure from both the physical polymer swelling and the chemical gas generation aggressively ruptures the structural matrix.

Ultimately, formulation F3 represents the functionally optimized bioproduct. By meticulously tailoring the microstructural architecture, F3 successfully pairs the requisite mechanical toughness required for large-scale bioprocessing, packaging, and transport (0.23% friability) with the highly accelerated, capillary-driven disintegration kinetics (2.02 min) necessary for a highly efficacious, user-friendly oral mucosal delivery system.

3.9. Effervescent Mouthwash Tablet Morphology and Porosity

To visualize the internal architecture and quantify the spatial porosity of the engineered bioproducts, three-dimensional Synchrotron Radiation X-ray Tomographic Microscopy (3D SR-XTM) was employed [71]. Figure 4 presents the reconstructed 3D tomograms of formulations F1, F2, and F3, which have been digitally segmented to distinguish the solid constituent phases from the internal void spaces (rendered in purple).

The tomographic imaging revealed striking microstructural variations driven entirely by the super-disintegrant concentration. Formulation F1 (0% Explotab^®) exhibited a highly dense internal architecture characterized by minimal, isolated air pockets. Conversely, the incorporation of 6% Explotab^® in F2 physically disrupted this dense packing, introducing larger void spaces. Formulation F3, containing the highest concentration of super-disintegrant (10%), displayed a highly porous, profoundly heterogeneous microstructure. Quantitative tomographic analysis validated these visual observations, revealing a total porosity increase from 2.79% in F1 to 4.62% in F3, characterized by an expansive, continuous network of voids permeating the entire tablet core [72].

These microtomographic findings provide a definitive, quantifiable structural basis for the physical disintegration kinetics observed in Section 3.8. The 3D SR-XTM reconstructions explicitly elucidate the biophysical mechanism underlying the accelerated tablet breakdown [45]. By expanding the internal architecture, the super-disintegrant establishes a highly interconnected capillary network. This specific structural porosity is critical; it facilitates rapid and pervasive water wicking deep into the tablet core. Consequently, the massive internal surface area exposed to the infiltrating solvent immediately triggers the acid-base effervescent reaction uniformly throughout the matrix, rather than confining it to the dissolving outer surface.

Furthermore, upon hydration, the dispersed Explotab^® particles undergo massive volumetric expansion, exerting severe multidirectional internal stress against the surrounding matrix [73,74]. In formulation F3, the synergistic convergence of maximal capillary wicking, aggressive polymer swelling, and widespread internal gas generation drives the immediate, multi-point fragmentation of the tablet, yielding the fastest recorded disintegration time.

The ability to visualize and quantify this internal architecture represents a significant advancement over traditional, purely empirical formulation methods. By utilizing SR-XTM, this study provides definitive mechanistic evidence that the rapid disintegration of botanical effervescent systems is not driven solely by the chemical effervescent reaction but is fundamentally governed by the engineered physical porosity of the solid matrix [45,75,76]. These microstructural metrics conclusively validate F3 as the optimal bioproduct. The 10% super-disintegrant concentration achieves a perfect bioprocessing balance. It maintains the necessary mechanical integrity for manufacturing and transport (confirmed by the <1.0% friability score) while establishing the extensive internal capillary networks required for immediate breakdown. Ultimately, mastering this physical mechanism ensures the rapid, predictable release of the highly active A. montanus extracts, maximizing its therapeutic bioavailability in the oral mucosal environment.

While this study successfully establishes the microstructural and in vitro biological framework for the A. montanus effervescent delivery system, the transition from a laboratory prototype to a clinical dosage form necessitates further rigorous safety validation. Future pre-clinical developmental phases will mandate comprehensive pharmacopeial testing, specifically evaluating heavy metal limits, pesticide residues, and microbiological purity, to ensure strict regulatory compliance and safety prior to human in vivo application.

4. Conclusions

This study successfully demonstrates the valorization of an underutilized botanical resource, Acanthus montanus, into a highly functional, engineered bioproduct for oral mucosal care. By systematically evaluating extraction dynamics, we established that while aqueous bioprocessing yields a higher gross crude mass, thermal ethanolic extraction is strictly required to selectively enrich the targeted bioactive secondary metabolites, specifically phenolics, saponins, and the validated marker verbascoside. This selective phytochemical enrichment proved fundamental to the extract’s biological efficacy. In direct contrast to the inactive aqueous yields, the ethanolic A. montanus extract delivered robust, broad-spectrum antimicrobial inhibition against critical cariogenic and opportunistic oral pathogens (Streptococcus mutans and Candida albicans). Concurrently, the extract maintained excellent cytocompatibility with human gingival fibroblasts and significantly accelerated cellular motility, achieving robust in vitro wound closure indicative of profound mucosal repair potential. Bridging these biological validations with advanced formulation science, the optimized botanical extract was successfully engineered into a solid effervescent delivery system. By utilizing 3D Synchrotron Radiation X-ray Tomographic Microscopy (SR-XTM), we provided definitive mechanistic evidence linking formulation chemistry to physical kinetic performance. The precise incorporation of a 10% sodium starch glycolate engineered a highly interconnected internal capillary network. This microstructural optimization successfully resolved mechanical friability limitations while facilitating an aggressive, capillary-driven disintegration mechanism that releases the bioactives in under three minutes. Ultimately, this multidisciplinary approach, integrating targeted phytochemical extraction, cellular validation, and advanced microstructural engineering, provides a rigorous biotechnological foundation for utilizing A. montanus in targeted oral therapies, strongly justifying its progression into future in vivo and clinical investigations.