Abstract Examining the impact of AKBA-loaded silver nanoparticles (AKBA-AgNPs) on enamel surface morphology, demineralization and remineralization processes, and enamel microhardness. There were five groups of human teeth that had been extracted to be tested in an in vitro study an “in vitro experimental laboratory study” was conducted using 40 sample size enamel specimens randomly allocated into five groups (n = 8 per group) thus 80 samples for MH and EDX experiment. Group 1 was a negative control, group 2 was a positive control treated with NaF, group 3 was AKBA + AgNPs, group 4 was AKBA + NaF, and group 5 was AKBA + NaF + AgNPs. Prior to artificial demineralization and therapy, baseline measurements  were conducted. A Vickers microhardness tester was utilized to measure the surface microhardness, and energy-dispersive X-ray spectroscopy (EDX) was applied to examine the calcium and phosphorus content. A significance level of P < 0.05 was used for statistical analysis, which comprised ANOVA and post hoc testing. No significant differences were found at baseline or after demineralization (P > 0.05). After treatment, all experimental groups showed significant improvement in microhardness and phosphorous levels compared to the negative control (P < 0.001), which demonstrated no remineralization. AKBA, especially when combined with NaF and AgNPs, effectively enhanced enamel remineralization and surface microhardness. This combination may be a promising bioactive line for the management of early enamel caries.

Keywords: Boswellic acid, Enamel microhardness, Initial caries lesions, Remineralization, Silver nanoparticles

Citation:  Abdulameer, R.J. and Radhi, N.J.M.H. 2026. Effect of boswellic acid–silver nanoparticles on microhardness of initial caries lesions. Natural and Life Sciences Communications. 25(4): e2026095.

Graphical Abstract:

INTRODUCTION

Enamel hardness and density vary across the tooth crown, decreasing from the surface toward the dentinoenamel junction (DEJ). Although enamel has relatively low tensile strength, its high modulus of elasticity reduces the risk of fracture (Abdulkareem et al., 2026). Enamel thickness varies across different regions of the tooth, with greater thickness at the incisal edge and a gradual decrease toward the cervical area near the cementoenamel junction (CEJ), reflecting functional and structural adaptation (Jánosi et al., 2024). Its unique mechanical properties are attributed to the high content of hydroxyapatite crystallites organized into enamel prisms, forming a three- dimensional structure that enhances strength and hardness (Hornby et al., 2014).

Several species of streptococci, including Streptococcus mutans, Actinomyces, and others, are essential etiological factors in the development of caries. Atopobium, low-pH non-S. mutans streptococci, Veillonella, Lactobacillus, Bifidobacterium, Propionibacterium, and Actinomyces are among the other species that are important in caries formation (Aas et al., 2008; Talib and Mohammed, 2026).

Healthy adult enamel contains about 95 wt% calcium phosphates, formed through mineralization of a protein matrix regulated by enamel-specific proteins and proteases (Gil-Bona and Bidlack, 2020).

Conservative and preventative methods of treating dental caries are utilized to assess the risk of the disease in each individual patient, detect it early on, and try to stop or reverse dental caries in its path so that the tooth structure is preserved (Fontana and González-Cabezas, 2016, 2019; Fontana et al., 2018).

One of the most popular remineralizing agents for early detection and prevention of dental caries is fluoride (Selwitz et al., 2007). Hydroxyapatite and fluoride can react to produce fluorapatite or fluoridated hydroxyapatite as a byproduct (Selwitz et al., 2007; Ozsvath, 2009).  The remineralizing agents are available in semisolid or liquid formulations, making them easy to give and ensuring good approval among patients. Suboptimal therapeutic concentration and outcome are the results of poor retention in the oral environment, which is the principal disadvantage and obstacle (Mizrahi and Domb, 2008). Toothpaste, gels, pills, and mouth rinses are the most typical forms of administration for medical products that prevent dental caries (Walsh et al., 2019).

According to Kesharwani et al. (2018), structures, devices, and systems can be controlled in terms of size and shape at the nanoscale, enabling for their design, characterization, and application in the field of nanotechnology. It is commonly described as “technology on the nanoscale”; however, this definition has evolved with the growing emphasis on precisely defining the nanoscale range (1–100 nm). Accordingly, nanotechnology has also been described as an “atomically precise technology” or “engineering with atomic precision” (Nasrollahzadeh et al., 2019).

Recent progress in nanotechnology has greatly contributed to the introduction of innovative and cost-effective dental materials and treatment methods. In addition, nanotechnology has improved our understanding of the structural, physical, biomechanical, biological, and chemical properties of these advanced dental materials (Foong et al., 2020; Mohammed and Talib, 2026).

By regulating plaque and assisting in the remineralization of initial caries, nanotechnology can be beneficial to the management and prevention of dental caries (Hannig and Hannig, 2010; Cheng et al., 2015).

Silver nanoparticles (Ag NPs) have been used for caries prevention in several studies (Ahmadian et al., 2018; Ahmed et al., 2019). These studies utilized silver nanoparticles in the form of silver nanocomposites, dentifrices, coated orthodontic brackets, nanosilver fluoride solutions, sealants, and glass ionomer cement with Ag NPs. In vitro research using Ag NPs and silver nanocomposites was done to treat and prevent secondary caries (Zhang et al., 2013; Wu et al., 2018). Ag NPs were also incorporated into the resin of orthodontic materials (adhesives, elastomeric ligatures, and removable retainers) for caries prevention (Hernández-Gómora et al., 2017; Metin-Gürsoy et al., 2017). Nanosilver fluoride solution was effective in remineralizing early enamel caries and arresting dentinal caries (Teixeira et al., 2018; Tirupathi et al., 2019).

Dental sealants containing Ag NPs may be more effective than conventional sealants in preventing enamel caries in first permanent molars (Salas-López et al., 2017). and clinical trials have demonstrated that orthodontic brackets incorporating Ag NPs can prevent enamel caries (Metin-Gürsoy et al., 2017).

Silver nanoparticles possess strong antimicrobial properties against bacteria, fungi, and certain viruses due to their high surface area and boosted surface reactivity at the nanoscale. Owing to their strong antibacterial properties, silver nanoparticles have been widely investigated in dentistry as promising agents for controlling cariogenic microorganisms and arresting dental caries (Kadhem and Al Haidar, 2023).

Silver nanoparticle-based formulations have demonstrated promising remineralization potential, supporting their use in the management of early carious lesions (Kadhem and Al Haidar, 2023). In addition to nanotechnology-based materials, natural bioactive compounds have attracted increasing attention for their potential role in enamel remineralization. Acetyl-11-keto-β-boswellic acid (AKBA), the most active constituent of Boswellia serrata, has demonstrated anti-inflammatory and regenerative properties. Therefore, combining AKBA with silver nanoparticles may enhance bioactivity and improve enamel remineralization through synergistic effects (Efferth and Oesch, 2022).

Therefore, the aim of the present in vitro study was to evaluate the effect of AKBA-loaded silver nanoparticles, alone and in combination with sodium fluoride, on the remineralization of initial enamel caries lesions using microhardness testing, FESEM, and EDX analysis.

MATERIALS AND METHODS

Materials

As authorized via the health research committee (Ref No. 970, 18/11/2024), the roots of the collected, permanently, and caries-free human premolars (mainly stored in deionized water in a cold cabinet (+4°C) and were used within a month from the extraction time (Yaman et al., 2014), prior to orthodontic treatment were sectioned (Isomet 1000, Buehler, Lake Bluff, IL, USA) at the Cemento-Enamel Junction (CEJ) consuming a water-cooled diamond blade (330-CA/RS-70300, Struers, Detroit Rd. Westlake, LLC, Cleveland, OH, USA). Every crown was split into two halves, and only one of the halves, the buccal half, was retrieved. Molds made of epoxy resin were filled with three slabs cut from each surface, with dimensions of 4.0 mm × 4.0 mm × 2.0 mm. After being polished under water-cooled silicon carbide by Lariee Technology CO.LTD of China for 10 seconds at P1200, 10 seconds at P2500, and 4 minutes at P4000, the specimens were ultrasonically cleaned for 4 minutes to eliminate surface debris (Al-Shareefi et al., 2022; Shubbar et al., 2023). Afterwards, these slabs were let to undergo pH-cycling for a duration of 10 days, ranging from 4.5 to 7.0 (ten Cate and Duijsters, 1982).

Preparation of the AKBA solutions

Boswellic acid was selected due to its potential remineralizing and anti-inflammatory properties. Acetyl-11-keto-β-boswellic acid (AKBA) powder (Cat. No. HY-N0892R, CAS No. 67416-61-9, MW 512.72 g/mol) was purchased from MedChemExpress (MCE, USA) and stored at 4°C in a sealed, light- and moisture-protected container. A 2 mM stock solution was prepared by accurately weighing the required amount of AKBA using an analytical balance (KERN ALS 220-4N, KERN & Sohn GmbH, Balingen, Germany) and dissolving it in 5 mL of deionized water with continuous stirring until a clear solution was gained. Working solutions of 1 µM, 0.1 µM, and 0.01 µM were prepared from the stock via serial dilution using deionized water and a micropipette. The concentration of 0.1 µM was selected for the subsequent experiments based on the results of our preliminary study, in which it demonstrated the highest enamel microhardness and the most favorable surface morphology compared with the other tested concentrations. All solutions were freshly prepared, properly labeled, stored in sealed containers, and handled under aseptic conditions at room temperature (Aldandan et al., 2024).

Boswellic acid was selected for its potential remineralizing and anti-inflammatory properties. To ensure precise solution preparation, the material was centrifuged after being received in powder form to create uniform particle distribution, settling small particles and producing a homogeneous powder.

Preparation of silver nanoparticles/loading AKBA-AgNPs

Silver nanoparticles (AgNPs) were purchased in powder form (10 g; purity 99.9%; average particle size (APS) 80 nm; Gray-black powder) from Hongwu International Group Ltd. The required amount of AgNP powder was accurately weighed using an analytical balance (KERN ALS 220-4N, KERN & Sohn GmbH, Balingen, Germany) and dispersed in deionized water to prepare an AgNP suspension with a final concentration of 0.02 mg/mL, following previously reported protocols (Kongyodsueb et al., 2024). For AKBA loading, the prepared AKBA working solution (0.1 µM) was combined with the AgNP suspension at a 1:1 ratio, as adopted in previously reported drug-loading approaches (Todorova et al., 2023). The mixture was gently stirred under aseptic conditions at room temperature to obtain AKBA-loaded silver nanoparticles (AKBA-AgNPs). The suspension was homogenized using an ultrasonic cleaner to ensure uniform dispersion, as ultrasonication is known to reduce particle agglomeration and improve suspension homogeneity (Zhang et al., 2024).

The final formulation was freshly prepared, properly labeled, stored in sealed containers, and used according to the experimental timeline.

Preparation of stock solutions

A stock solution of acetyl-11-keto-β-boswellic acid (AKBA) (0.395 mM) was prepared by accurately weighing the required amount of AKBA powder and dissolving it in deionized water under continuous stirring until a clear solution was obtained. The stock solution was stored in a sealed, light-protected container at 4°C and used for the preparation of working dilutions (Aldandan et al., 2024).

A stock suspension of silver nanoparticles (AgNPs, 0.1 mg/mL) was prepared by dispersing the required amount of AgNP powder in deionized water. The suspension was ultrasonicated to enhance dispersion and ensure uniformity prior to dilution.

Preparation of working solutions

From the AKBA stock, a working solution of 0.1 µM was prepared using deionized water. A sodium fluoride solution (NaF, 0.05%) was prepared by dissolving the required amount of NaF powder in deionized water, following a previously reported protocol (Niazy et al., 2024).

The AgNPs working suspension was prepared by diluting the stock suspension to achieve a final concentration of 0.02 mg/Ml (Kongyodsueb et al., 2024).

Preparation of treatment formulations

The treatment formulations were freshly prepared under aseptic conditions at room temperature. AKBA-loaded silver nanoparticles (AKBA–AgNPs) were obtained by mixing AKBA working solution (0.1 µM) with the AgNPs working suspension (0.02 mg/mL) until a homogeneous formulation was achieved.

For the combined formulation (AKBA–AgNPs–NaF), NaF solution (0.05%) was added to the AKBA–AgNP mixture and gently homogenized to ensure uniformity. In addition, an AKBA–NaF formulation was prepared by combining AKBA (0.1 µM) with NaF (0.05%) under gentle mixing.

Experimental groups and treatment formulations

In accordance to the applied treatment protocol, the prepared enamel specimens were manually  randomly allocated into five experimental groups (n = 8 each group), Group 1 (negative control) received no material application; Group 2 (positive control) was treated with sodium fluoride (NaF, 0.05%); Group 3 was treated with AKBA (0.1 µM) combined with silver nanoparticles (AgNPs, 0.02 mg/mL); Group 4 was treated with AKBA (0.1 µM) combined with NaF (0.05%); and Group 5 was treated with a combined formulation of AKBA (0.1 µM), AgNPs (0.02 mg/mL), and NaF (0.05%). Per prior reports, all treatments were applied to the enamel surfaces for a standardized contact period of 3 minutes at room temperature in an aseptic environment (Zhao et al., 2017; Sayed et al., 2020).

A sodium fluoride solution (NaF, 0.05%) was prepared by dissolving the required amount of sodium fluoride powder in deionized water, as previously described (Niazy et al., 2024).

Statistical analysis

Statistical analysis was performed using SPSS version 26. Data were expressed as mean ± standard deviation (SD). Normality of data distribution was assessed prior to analysis. One-way analysis of variance (ANOVA) was used to compare differences among the experimental groups at different stages (baseline, demineralization, and post-treatment). Repeated measures ANOVA was applied to evaluate intra-group differences across the three phases. A P-value of less than 0.05 was considered statistically significant.

RESULTS

Enamel microhardness

The results of the ANOVA test exhibited that surface microhardness values were not significantly different across all groups during the demineralization and baseline phases (P > 0.05), while there were significant statistical differences among groups in the remineralization phase, as revealed in Table 1.

The descriptive and statistical analysis of surface microhardness values across the experimental groups and phases is presented in Table 1. At baseline (MHB), no statistically significant difference was noted among the groups (P = 0.062), indicating comparable initial enamel microhardness values. Similarly, after demineralization (MHD), no significant differences were detected between the groups (P = 0.422), confirming uniform demineralization of the enamel specimens, Table 1.

Following treatment (MHT), a highly statistically significant difference was detected among the groups (P < 0.001). All treated groups showed higher microhardness values than the negative control group. Group 3 demonstrated the highest post-treatment microhardness, whereas Group 1 showed the lowest values (Table 1).

Table 1. Descriptive and statistical test repeated measure one-way ANOVA of surface microhardness in (kg/mm²) at the three time periods for every remineralizing agent.

Note: ±SD: Standard Deviation, Min.: Minimum, Max.: Maximum, F-statistic (Analysis of Variance, ANOVA).

Microscopic feature of the enamel surface by using filed emission scanning electron microscopy (FESEM)

The baseline specimens showed no signs of mineral deposition and had a surface morphology that was both uneven and rough, according to scanning electron microscopy (SEM) (Figure 1 (a)). Specimens revealed significant surface irregularities, porosity, and structural collapse after demineralization, as illustrated in Figure 1 (b), demonstrating effective mineral loss compared to the baseline group. As shown in Figure 1 (c), the absence of observable mineral deposition and persistent surface imperfections in the negative control group indicate that spontaneous remineralization did not occur. Surface remineralization was suggested by the presence of crystalline deposits and a more homogenous morphology in the NaF-treated group compared to the demineralized group (Figure 1 (d)). Figure 1 (e) shows that the group treated with AKBA and silver nanoparticles had a crystalline structure resembling a dense needle with spherical nano-sized particles embedded in the matrix. Figure 1 (f) reveals that as compared to the NaF alone group, the AKBA + NaF group exhibited improved remineralization due to the formation of thick, clustered crystalline deposits that formed a nearly continuous surface layer with significantly reduced porosity. A synergistic remineralization effect was suggested by high-magnification imaging, which showed densely packed needle-like crystals interspersed with nano-sized particles, and by the fact that the triple-combination group (AKBA + NaF + AgNPs) exhibited full surface coverage with dense crystalline deposits and nearly complete occlusion of surface porosities (Figure 1 (g).

Figure 1. Scanning electron micrographs (SEM) of enamel surfaces; Sound enamel (baseline, with de-ionized water only) specimen with a complicated surface shape (a), A demineralized enamel after pH cycling specimen with a rougher and more porous surface (b), A negative control (no treatment, with artificial saliva only) group that had surface flaws that didn't go away and no mineral deposition (c), the group that was treated with (0.05% NaF) had crystalline layers and fewer pores (d), the (0.1 µM AKBA+ 0.02 mg/mL  AgNPs group) showed needle-like crystal structures with nanoparticles inserted in them (e), the ( 0.1 µM AKBA + 0.05%  NaF) group has dense crystallized layers(f), the (0.1 µM AKBA + 0.05% NaF +0.02 mg/mL  AgNPs) group has a thick crystalline structure that covers a large area of the surface (g).

Energy-dispersive X-ray spectrometry (EDX)

The data obtained by EDX analysis for every group regarding the weight percentage of calcium (Ca) phosphate (PO₄). A lower mean weight percentage for Ca and PO₄ was observed during demineralization. Both elements' weight percentages increased following remineralization in the treatment group. Descriptive and statistical tests of Ca and PO₄ in the AKBA group.

The EDX analysis illustrated no statistically significant differences in phosphorous content among the experimental groups at baseline (PB) (P = 0.704), indicating comparable initial mineral composition. Similarly, following demineralization (PD), phosphorous levels did not vary significantly among groups (P = 0.310), confirming that the demineralization process affected all specimens uniformly (Table 2).

Table 2. Descriptive and statistical test of phosphorous among groups and phases.

Note: ±SD: Standard Deviation, Min.: Minimum, Max.: Maximum, F-statistic (Analysis of Variance, ANOVA).

After treatment (PT), a highly statistically significant difference in phosphorus content was detected among the groups (P < 0.001). The negative control group exhibited the lowest phosphorus levels after treatment, while all treated groups demonstrated a marked increase in phosphorus content. The highest post-treatment phosphorus values were recorded in Groups 4 and 5, followed closely by Groups 2 and 3.

One-way ANOVA demonstrated no significant differences among groups at baseline (P = 0.159) or after demineralization (P = 0.569), indicating comparable initial conditions. However, a highly significant difference was observed after treatment (P < 0.001), confirming variation in remineralization potential among the experimental groups. Within-group analysis also revealed significant differences among the three phases in all groups (P < 0.001) (Table 3).

The significant increase in calcium content after treatment, particularly in the AKBA + AgNPs group, suggests that the incorporation of bioactive boswellic acid with silver nanoparticles enhances mineral deposition and surface recovery. A possible explanation for this is the synergistic effect of nanoparticle-mediated mineral nucleation and the bioactive properties of AKBA, resulting in improved calcium uptake compared with fluoride alone (Table 3).

Table 3. Descriptive and statistical test of calcium among groups and phases.

Note: ±SD: Standard Deviation, Min.: Minimum, Max.: Maximum, F-statistic (Analysis of Variance, ANOVA).

DISCUSSION

The current study estimated the effect of different treatment formulations on enamel surface microhardness following demineralization and treatment. It is confirmed that all specimens had similar beginning conditions and experienced uniform mineral loss because there were no significant variations among groups at baseline and following demineralization. All experimental groups displayed significant enhancement in surface microhardness after treatment compared to the control group, indicating that the treatment was successful in remineralizing the enamel. There was a clear difference in the groups treated with fluoride and those that were not, lending credence to the idea that fluoride helps strengthen enamel's defenses against acid attacks. There was an increase in post-treatment microhardness values in the combination treatment group compared to the untreated or single-agent groups. Fluoride, silver nanoparticles, and AKBA may work together synergistically to improve mineral recovery by combining their antibacterial and bioactive characteristics.

All treated groups showed substantial recovery after treatment and a notable decrease in microhardness following demineralization; however, the negative control group showed no improvement. According to these results, the reported improvements were caused by the treatment itself. The in vitro design does not completely mimic oral circumstances, which limits the outcomes; they are promising, nonetheless. The clinical and long-term efficacy of these formulations needs additional research. The current research showed that after applying AKBA, enamel microhardness increased significantly. This confirms the results of the study by Khan et al., who found that SEM analysis verified their claim that loading AKBA onto silver nanoparticles increased their bioactivity and surface interaction. It is possible that the enhanced surface properties observed in both studies are due to bioactive materials' capacities to encourage mineral deposition and stabilize surfaces (Khan et al., 2019).

There is evidence that demineralization, in the form of different treatment methods, significantly increases enamel microhardness, which may contribute to improved enamel resilience (Rheima et al., 2024).

EDX analysis confirmed that all groups had similar beginning circumstances and mineral loss, showing that their calcium and phosphorus levels were equivalent at baseline and after demineralization. After the treatment, the calcium and phosphorus content of the experimental groups were significantly higher than the negative control, suggesting that the enamel had been effectively remineralized. Improved mineral recovery was seen in fluoride-containing formulations, and a synergistic impact involving AKBA, silver nanoparticles, and fluoride was suggested by the combination treatments' higher performance. The remineralization capability of the applied formulations was supported by these findings, which were compatible with the surface microhardness data.

Treatment with AKBA + AgNPs resulted in a much higher calcium content than treatment with either boswellic acid alone or silver nanoparticles alone, suggesting that the combination improves mineral deposition and surface healing. This could be because, when combined with the bioactive characteristics of AKBA, nanoparticle-mediated mineral nucleation improves calcium uptake compared to fluoride alone.

Although the exact mechanism of AKBA in enamel remineralization has not yet been fully elucidated, its bioactive properties may contribute to creating a favorable environment for mineral deposition. AKBA has been reported to exhibit anti-inflammatory, antioxidant, and regenerative activities, which may enhance crystal formation and stabilization on the demineralized enamel surface. These properties may facilitate the deposition of calcium and phosphate ions, resulting in improved enamel microhardness and remineralization. (Khan et al., 2019; Aldandan et al., 2024).

Sodium fluoride and silver nanoparticles did not outperform the other treatment modalities in terms of remineralizing effect, according to the study's results. In a study done by Zhao et al. (2020), it was shown that a mixture of 2.5% sodium fluoride and PEG-coated silver nanoparticles had a remineralizing effect in simulated dentine caries that was similar to that of 12% silver diamine fluoride (SDF) (Zhao et al., 2020). This finding is in partial accord with their findings. Possible explanations for the observed results discrepancies include variations in dental substrate, fluoride content, and nanoparticle composition (Zhao et al., 2020).

Furthermore, the current findings are in line with those of Kongyodsueb et al. (2024), who discovered that silver nanoparticles failed to inhibit the advancement of caries when sodium fluoride varnish was applied afterwards (Kongyodsueb et al., 2024). These results point to the possibility that the combined antimicrobial and remineralizing effects of SDF account for its greater efficacy, whereas the application timing, concentration, or physicochemical interactions between silver nanoparticles and fluoride ions may explain why AgNPs and fluoride do not have a synergistic effect (Kongyodsueb et al., 2024).

The superior microhardness observed in Group 3 may be attributed to the synergistic interaction between AKBA and silver nanoparticles. Silver nanoparticles provide a high surface area that promotes mineral nucleation and facilitates the formation of a more compact mineral layer, while AKBA may enhance crystal growth and stabilize the newly formed mineral deposits through its bioactive properties. This synergistic effect may explain the higher enamel surface microhardness observed in this group.

Different experimental groups showed distinct morphological differences when analyzed using scanning electron microscopy. Mineral deposition was absent from the baseline specimens, which had a surface morphology that was very uneven (Figure 1 (a)). The process of demineralization is displayed in Figure 1 (b), marked surface roughness and pronounced porosity were evident, confirming effective mineral loss and structural breakdown.

The lack of surface imperfections and observable mineral deposition in the negative control group (Figure 1 (c)) suggests that spontaneous remineralization did not occur. Surface crystalline precipitates with partial reduction in porosity, showing fluoride-induced remineralization, were observed in the NaF-treated group (Figure 1 (d)). Nevertheless, the deposits appeared to be moderately distributed.

The combination of AKBA and NaF resulted in a more impressive remineralizing impact, as seen in Figure 1 (f), where clustered crystalline aggregates covered a large portion of the surface with less porosity than when NaF was used alone.

At increased magnification, the AKBA + AgNPs group exhibited a more ordered and denser needle-like crystalline morphology (Figure 1 (e)). The particles contained within the mineral matrix were uniformly distributed and nano-sized. The crystals seemed densely packed and well-formed, suggesting that the crystal development and mineral deposition were both aided by nanotechnology. This group's crystal dispersion was more uniform, and its nano-structural organization was more noticeable than that of the other treatment groups.

Crystal morphology of the AKBA + AgNPs group seemed more structurally defined and evenly ordered at the nano-scale, despite the fact that the triple-combination group (AKBA + NaF + AgNPs) (Figure 1 (g)) showed wide surface coverage with dense crystalline deposits.

The potential of this formulation in promoting effective surface remineralization was highlighted by SEM findings that indicated strong nano-structured mineral deposition when silver nanoparticles were combined with AKBA.

LIMITATIONS

The study is conducted in an in vitro experimental laboratory study using artificial saliva and controlled conditions, which may not fully replicate the complexities of the oral environment. Factors such as salivary composition, pH fluctuations, and the presence of bacteria, which influence enamel remineralization in vivo, are not considered.

CONCLUSION

Within the limitations of this in vitro experimental laboratory study, AKBA combined with silver nanoparticles and/or sodium fluoride demonstrated promising potential for enhancing enamel remineralization and improving surface microhardness. These findings suggest that this bioactive formulation may represent a promising adjunctive approach for the management of early enamel caries. Further in vivo and clinical studies are required to confirm its clinical effectiveness.

ACKNOWLEDGEMENTS

The authors gratefully acknowledge the Faculty of Agro-Industry, Chiang Mai University, for providing instrumental support. Special thanks are also extended to the Botany Departments of Alagappa University and Periyar University, as well as the Department of Life Science at Gachon University, for their valuable facilities and assistance in conducting the biological activity studies.

AUTHOR CONTRIBUTIONS

Roa'a Jamal Abdulameer: Conceptualization (Lead), Data Curation (Lead), Software (Lead), Validation (Lead), Writing – Original Draft (Lead); Nada Jafer Mohammed Hassan Radhi: Formal Analysis (Equal), Funding Acquisition (Lead), Investigation (Lead), Supervision (Lead), Validation (Equal), Visualization (Equal), Writing – Review & Editing (Equal).

CONFLICT OF INTEREST

The authors declare that they have no conflicts of interest.

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