Abstract Dental caries remains a major public health concern in Indonesia, with a reported prevalence of 82.8%. One promising preventive strategy is the application of bioactive therapeutic materials, including nano-hydroxyapatite (n-HAp) reinforced with natural biopolymers. In this study, an n-HAp gel was formulated using tiger snail shells as a calcium carbonate (CaCO₃) precursor and enriched with Ajwa dates/miswakpropolis biocomposite to enhance remineralization and antibacterial performance. The tiger-snail shells, containing approximately 97% CaCO₃, were converted into n-HAp via the sol–gel method. The Fourier Transform Infrared Spectroscopy (FTIR) spectra confirmed the presence of PO₄³⁻ and CO₃²⁻ groups, indicating successful organic–inorganic hybridization, while X-Ray Diffractometer (XRD) analysis verified the formation of a pure hydroxyapatite phase with crystallite sizes of 13–15 nm. Although the n-HAp structure remained stable, crystallite size reduction was observed at 30 wt% due to the inhibitory effects of organic constituents. Microstrain remained constant from 0–20 wt% and increased at 30 wt%. The Scanning Electron Microscopy (SEM) imaging revealed agglomerated gel particles with uniform granules averaging 0.252 ± 0.003 µm. Antibacterial assays against Streptococcus mutans, Lactobacillus acidophilus, and Streptococcus sanguinis demonstrated enhanced inhibition zones with increasing n-HAp content, with the highest activity recorded at 30 wt% against S. mutans (8.1 mm). Overall, the n-HAp gel derived from tiger-snail shells and natural bioactive additives shows strong potential as an eco-friendly biocomposite for dental enamel remineralization and antibacterial protection.

Keywords: Dental caries, Nano-Hydroxyapatite, Tiger-snail shells, Ajwa dates/ Miswak/Propolis, Gel, Enamel remineralization

Funding: We would like to express our sincere gratitude to the Directorate General of Higher Education, Research, and Technology through the Student Creativity Program (PKM), Directorate of Vocational Education, Directorate of Higher Education and Ministry of Higher Education, Science and Technology, Republic of Indonesia through Regular Fundamental Research 2025 Grant for financially supporting this research.

Citation:  Arum, E.D.P., Meliasari, L.P., Setiati, R.Y., Dayani, M.S.P., Saputro, R.A., and Sari, M. 2026. Nano-hydroxyapatite gel derived from tiger-snail shells (Babylonia spirata, L): A biocomposite incorporating Ajwa date/miswak /propolis for enamel remineralization. Natural and Life Sciences Communications. 25(3): e2026059.

Graphical Abstract:

INTRODUCTION

Dental and oral health play a very important role in the health of the body. Speech function, chewing, and facial structure are greatly influenced by dental and oral health. In Indonesia, dental health still requires serious attention due to the high prevalence of periodontal disease and the possibility of cavities forming when teeth are damaged (caries) (Sari et al., 2025a). Dental health in Indonesia still requires serious attention due to the high prevalence of periodontal disease and dental caries, which can form cavities after the tooth becomes decayed (Sari et al., 2025a). Data from the 2023 Indonesian Health Survey (SKI) indicate that the prevalence of dental caries among 12-year-old children in Indonesia is approximately 82.8% (Hasan et al., 2024). Dental caries can be caused by food, drink, and bacterial infections. Bacteria that cause dental caries include Streptococcus mutans, Streptococcus sanguinis, and Lactobacillus acidophilus (Mazurel et al., 2025; Sari et al., 2025a). Dental caries is primarily associated with the consumption of fermentable carbohydrates, particularly sugars present in foods and beverages such as candies, chocolates, sugar-sweetened soft drinks, and fruit juices. These sugars can be metabolized by cariogenic bacteria, including Streptococcus mutans, Streptococcus sanguinis, and Lactobacillus acidophilus, leading to acid production and subsequent enamel demineralization. (Mazurel et al., 2025; Sari et al., 2025a). Severe and untreated dental caries can lead to complications such as gingivitis, dental nerve infections, abscesses in the gums, and can lead to death (Neal and Schlieve, 2022). Severe and untreated dental caries can cause complications and worsen periodontal conditions such as gingiva, dental nerve infections, gum abscesses, and lead to death (Neal and Schlieve, 2022). Moreover, untreated dental caries may progress to pulp necrosis and periapical abscess formation, which in some cases can spread to surrounding periodontal tissues or drain through the gingiva (Mady et al., 2022).

One promising approach for managing dental caries is the application of therapeutic biomaterials such as nano-hydroxyapatite (n-HAp) incorporated with natural biopolymers, including Ajwa dates and miswak. The n-HAp, a calcium phosphate–based compound, is recognized for its osteoconductivity, biocompatibility, and close structural and chemical resemblance to the inorganic components of human teeth (Koonrungsesomboon et al., 2022; Kurzyk et al., 2023; Sari et al., 2025b). This material can be synthesized using both chemical precursors and natural calcium carbonate (CaCO₃) sources. A wide range of biogenic materials—such as snail shells, eggshells, and animal bones—contain high levels of CaCO₃ and have therefore been widely explored as sustainable raw materials for n-HAp production (Sekar and Lee, 2022). Various methods have been developed to synthesize n-HAp, including precipitation (Sari et al., 2023), nanoemulsion (Ezekiel et al., 2018), sol-gel (Sari et al., 2025b, and hydrothermal (Jeong et al., 2024). In this study, the n-HAp synthesis process was carried out using the sol-gel method, a modification from previous research (Sari et al., 2025b).

The sol–gel technique is a wet-chemical synthesis approach in which fine particles are dispersed within a liquid medium. Compared with other fabrication methods, it offers several advantages, including the formation of highly homogeneous precursor solutions, operation at relatively low processing temperatures, and the ability to yield products with high purity and crystallinity. Additionally, the sol–gel process minimizes unwanted reactions with residual compounds, making it a reliable route for producing controlled ceramic and biomaterial structures. Additionally, the sol–gel process minimizes unwanted reactions with residual compounds, making it a reliable approach for producing homogeneous and nanostructured ceramic matrices, such as calcium phosphate or n-HAp networks, commonly used in biomaterial applications. This approach also minimizes material loss during processing and can contribute to lower emission levels, supporting more environmentally responsible manufacturing practices (Bokov et al., 2021). The sol-gel process mechanism begins with the formation of the sol phase, which then undergoes a transformation into the gel phase. The resulting gel then undergoes a phase change into a solid and, after experiencing a heating process, forms a bioceramic with good mechanical properties and bioactivity (Owens et al., 2016). The sol-gel process mechanism begins with the formation of the sol phase, which then undergoes transformation into the gel phase. The resulting gel subsequently undergoes a phase change into a solid, through solvent evaporation and gelation via a controlled heating process at high temperatures, transforming the gel network into a bioceramic with good mechanical properties and bioactivity. Therefore, the gel network undergoes a sol–gel transition to form a solid matrix, which can subsequently be converted into bioceramic materials through crystallization or thermal treatment (Owens et al., 2016). N-HAp produced through the sol-gel method generally has a fine particle size and a large surface area, making it highly potential for application in the biomedical field, particularly as an enamel remineralization agent (Fiume et al., 2021). However, pure n-HAp still has limitations in its biological properties, especially in terms of antibacterial activity, which plays an essential role in preventing the growth of bacteria that cause dental caries (Anil et al., 2022). To improve its biological and antimicrobial performance, it is important to note that pure n-HAp exhibits limited intrinsic antibacterial activity. This is primarily due to its near-neutral pH (7.2–7.6), which unlike highly alkaline materials does not create an environment hostile enough to significantly inhibit cariogenic bacterial growth. Therefore, the incorporation of bioactive components with known antimicrobial properties, such as those found in natural biopolymers, is necessary to enhance its efficacy against pathogens like Streptococcus mutans (Ghosh et al., 2022; Abbasi et al., 2025).

Previous work by Ramadhanti et al. (2025) reported the synthesis of nano-hydroxyapatite (n-HAp) from Asian moon shells for dental remineralization. However, that study was limited to physicochemical characterization and did not incorporate additional bioactive components or evaluate antibacterial performance. Previous work by Ramadhanti et al. (2025) reported the synthesis of nano-hydroxyapatite (n-HAp) from Asian moon shells (Amauropsis islandica) for dental remineralization. However, unlike Asian moon shells used in that study, the current research employs tiger snail (Tigersa sp.) shells used in the current study represent a distinct biogenic source with different habitat origins and structural characteristics. That previous study was limited to physicochemical characterization and did not incorporate additional bioactive components or evaluate antibacterial performance. Although the synthesized n-HAp showed promising remineralization potential, its efficacy against cariogenic bacteria remained unexplored. In the present study, n-HAp derived from tiger-snail shells (Babylonia spirata, L.) was combined with natural biopolymers—Ajwa dates, miswak, and propolis—to enhance antibacterial activity while maintaining remineralization capability. Tiger-snail shells were selected due to their abundance in eastern Indonesia, where population densities range from 206,700 to 640,000 individuals per km², and because their shells contain approximately 97% CaCO₃. This high calcium content makes tiger-snail shells a valuable biogenic source for n-HAp synthesis, while calcium (Ca) itself is an essential mineral for maintaining dental health (Sari et al., 2025b).

The combination of n-HAp and biopolymers such as ajwa dates, miswak, and propolis can increase antibacterial activity in dental caries. Ajwa dates (Phoenix dactylifera) are rich in phytochemicals such as tannins and alkaloids that work as antibacterials (Alsuhaymi et al., 2023; Aryati et al., 2023). Miswak (Salvadora persica) has been shown to reduce cariogenic bacteria such as Streptococcus mutans and decrease plaque accumulation, supporting its traditional use for tooth cleaning and oral hygiene (Nordin et al., 2020; Sabbagh et al., 2020). Propolis (Apis mellifera) is a natural antimicrobial agent that can be incorporated into toothpaste to help prevent plaque formation (Song and Ge, 2019). Dental caries arises from bacterial infection and acidic oral conditions that gradually demineralize tooth structures. The n-HAp, a biomaterial chemically similar to the inorganic matrix of teeth, has strong potential for dental remineralization. Environmentally friendly n-HAp can be synthesized from biogenic waste such as snail shells rich in calcium carbonate. Using a sol-gel method, high-purity n-HAp can be produced and subsequently combined with natural biopolymers such as Ajwa date extract, miswak, and propolis to develop an antibacterial biocomposite for oral health applications (Ahmed et al., 2022).

Gel-based formulations are considered advantageous over conventional toothpaste or mouthwash due to their superior spread ability, ease of application, and prolonged retention on the tooth surface, which enhances the interaction between active ingredients and enamel (Sari et al., 2025a). This prolonged contact time supports more effective remineralization, particularly in areas undergoing demineralization (Sari et al., 2022). Furthermore, gel preparations offer better user comfort and enable optimal delivery of bioactive agents such as n-HAp and natural antibacterial biocomposites. In this study, the n-HAp gel was prepared using a homogenization approach to achieve a uniform dispersion of particles within the gel matrix (Juntavee et al., 2021). The homogenization process is a critical determinant of the gel’s stability, viscosity, and final microstructure. Effective homogenization promotes uniform particle incorporation, smooth gel consistency, and favorable rheological characteristics suitable for biomedical applications. Additionally, this method facilitates enhanced interaction between n-HAp and the incorporated natural biopolymers, thereby improving both remineralization performance and antibacterial efficacy (Asadi et al., 2024).

This study aims to develop an n-HAp gel biocomposite derived from tiger-snail shells and incorporating Ajwa date, miswak, and propolis extracts as natural antibacterial agents for tooth enamel applications. The influence of n-HAp concentration (0, 10, 20, and 30 wt%) on gel characteristics was evaluated through physicochemical analyses to determine the optimal formulation. In addition, the antibacterial performance of the developed gels was assessed by measuring the inhibition zones to investigate the contribution of natural biopolymers to enhancing antimicrobial activity against cariogenic bacteria. In this study, n-HAp concentrations of 0, 10, 20, and 30 wt% were selected to evaluate the effect of different n-HAp concentrations on the physicochemical and antibacterial properties of the resulting biocomposites. These concentration variations are expected to provide an overview of the optimum composition between n-HAp and natural biocomposite materials in producing gels with maximum morphology, crystallographic properties, chemical functional groups, and antibacterial properties of dental material candidate for enamel remineralization applications (Sari et al., 2025).

MATERIALS AND METHODS

The fabrication method of the n-HAp gel based on Ajwa dates/miswak/propolis biocomposite was divided into five main stages: (1) preparation of the CaO precursor from tiger-snail shells, (2) synthesis of n-HAp, (3) preparation of Ajwa date/miswak/ propolis biocomposite, (4) fabrication of n-HAp gel, and (5) sample characterization using X-ray Diffractometer(XRD) (Rigaku Miniplex 600, Tokyo, Japan), Fourier Transform Infrared Spectroscopy (FTIR) (Shimadzu QATR-S, Kyoto, Japan), Scanning Electron Microscope (SEM) (JEOL Ltd. IT-210, Japan) and antibacterial activity testing using the Kirby–Bauer disk diffusion method.

Materials

The n-HAp derived from tiger-snail shells was adapted from previous study (Sari et al., 2025b). Food-grade antibacterial agents were sourced from verified local suppliers in Indonesia: date extracts from the Town Farm Official Store (Depok), miswak from the Official Herbal Wholesale Center (East Jakarta), and propolis from the Melia Propolis brand (Malang, East Java). The precursors of diammonium hydrogen phosphate ([NH₄]₂HPO₄) and ammonium hydroxide (NH₄OH) 25% solution were purchased from Merck (Kenilworth, NJ, USA) and used without further purification (Sari et al., 2022; Sari et al., 2025b). Food-grade basis gel was sourced from verified local suppliers in Indonesia: Guar gam from Mitra Jaya Chemical (Bekasi) and Na CMC from Raja Kimia (West Jakarta). Glycerin (A-1043) analysis grade was obtained from Smartlab Brand, Indonesia.

Preparation of Ajwa date/miswak/propolis biocomposites

Ajwa dates and miswak were thoroughly washed and oven-dried for 48 h at 60°C, then ground into fine powder. Ajwa date powder and miswak powder with a concentration of 10 wt% were prepared by dissolving 10 g of each powder in 90 mL of distilled water using a magnetic stirrer at 450 rpm for 4 h until homogeneous. The date and miswak solutions were then mixed at a 1:1 volume ratio at room temperature and stirred at 450 rpm until fully homogenized. Propolis with a concentration of 10 wt% was prepared by dissolving 11.1 mL of propolis in 88.9 mL of distilled water. The propolis solution was stirred using a magnetic stirrer (Thermo Fisher Scientific, Waltham, MA, USA) for 4 h at 60°C and 450 rpm, then mixed with the date/miswak biocomposite solution at a 1:1 ratio under the same conditions. The preparation of Ajwa date/miswak/propolis biocomposite can be seen in Figure 1. The concentration of Ajwa date/miswak/propolis extract at 10 wt% was chosen based on studies showing that herb extracts added at this concentration level provide optimal antibacterial performance while maintaining the stability and homogeneity of the gel. The study (Ahmadi et al., 2022) reported that extract concentrations in the range 5-15 wt% can enhance antibacterial activity without compromising the integrity of the gel network. In contrast, higher concentrations may negatively impact viscosity and phase stability. The Ajwa date, miswak, and propolis components were incorporated as bioactive extracts dispersed within the gel matrix; therefore, their particle size was not specifically determined. In this formulation, n-HAp serves as the primary remineralizing agent due to its nano-scale particle size, which facilitates mineral ion deposition and apatite crystal formation on demineralized enamel. The herbal extracts mainly contribute to antibacterial activity, indirectly supporting the remineralization process. N-HAp particles with sizes below 100 nm are reported to effectively penetrate enamel defects and promote remineralization due to their similarity to the size of natural enamel crystallites (Hemalatha et al., 2020).

Figure 1. Fabrication process of the Ajwa date/miswak/propolis biocomposite: Extraction of each Ajwa date, miswak, propolis (A), and homogenization process of ajwa date/miswak/propolis biocomposite (B).

Figure 2. Bio-assisted synthesis of n-HAp/Ajwa date/miswak/propolis biocomposite gel (A), and the mixing process between n-HAp solution and date/miswak/propolis biocomposite gel (B).

The base gel was prepared using a mixture of Na-CMC, guar gum, and glycerine. Na-CMC and guar gum functioned as thickening agents. At the same time, glycerine acted as a binder and humectant within the gel formulation. Na-CMC and guar gum were diluted by dissolving each material at 2 wt% in distilled waters. The solutions were stirred for 1 h until homogeneous, wt% indicated by a uniform viscosity without any clumps. The homogenized Na-CMC solution was then slowly poured into the guar gum solution while stirring, and the homogenization continued for 1 h. The Na-CMC and guar gum mixture was subsequently combined with diluted glycerine, and all three components were stirred again using a magnetic stirrer for 1 h until an entirely homogeneous base gel was obtained.

Bio-assisted synthesis of n-HAp/Ajwa date/miswak/propolis biocomposite gel

The n-HAp was synthesized using a bio-assisted synthesis approach, incorporating natural extracts and aqueous processing conditions to reduce the use of hazardous chemicals. Additionally, the synthesis was carried out under relatively mild processing conditions, including oven drying at 100°C, which is considered a moderate energy requirement. The n-HAp/Ajwa date/miswak/propolis nano-biocomposite gel was prepared by mixing the base gel with the Ajwa date/miswak/propolis biocomposite solution at a 1:1 ratio, followed by homogenization using a stirrer for 1 h until a uniform mixture was obtained. Subsequently, the n-HAp derived from tiger snail shells was incorporated into the base gel-biocomposite mixture at varying concentrations of 0, 10, 20, and 30 wt%. The wt% values refer to the mass percentage of dry n-HAp relative to the total dry solid content of the final gel formulation. Therefore, the n-HAp content varied between 0, 10, 20, and 30 wt% relative to the total mass of the composite formulation. The corresponding masses of dry n-HAp powder used in each formulation were 0 g, 0.3221 g, 0.6442 g, and 0.9663 g, respectively. The mixture was then homogenized for 1 hour to form a uniform nano-biocomposite gel. Each homogeneous n-HAp/biocomposite gel variation was then dried in an oven at 100°C for 45 min. The gel was dried in an oven at 100°C for 45 min to remove residual moisture and stabilize the nano-biocomposite structure. This temperature was chosen to guarantee effective drying while avoiding thermal deterioration of the beneficial ingredients in ajwa date/miswak/propolis. The temperature of 100°C was applied to promote efficient extraction of bioactive compounds and solvent evaporation, thereby improving the concentration of antibacterial constituents in the extract. The bio-assisted synthesis process of n-HAp/Ajwa date/miswak/propolis biocomposite gel can be seen in Figure 2.

Physicochemical characterization of n-HAp/Ajwa date/miswak/propolis biocomposite gels

Crystallographic properties

Crystallographic properties, such as crystallite size and microstrain of the n-HAp/Ajwa date/miswak/propolis nano-biocomposite gel with concentration variation of 0, 10, 20, and 30 wt% were determined by XRD ((Rigaku Miniplex 600, Tokyo, Japan). The XRD data were recorded in the range 2θ:10-80° using Cu-Kα radiation at λ=0.154 nm, step size (0.02°), scanning speed (2°/min), voltage (40 kV), and current (25 mA). The obtained diffraction profiles were then compared with reference standards from the Joint Committee on Powder Diffraction Standards (JCPDS) to confirm phase composition and structural attributes (Sari et al., 2025b).

Chemical composition

FTIR (Shimadzu QATR-S, Kyoto, Japan) was employed to identify the chemical structure of the nano-biocomposite gel samples. The characteristic absorption bands obtained from the spectra were used to confirm the functional groups associated with each component within the formulation. Prior to analysis, the powdered samples were finely ground, homogenized with potassium bromide (KBr) at a ratio of 1:100 (w/w), and subsequently compressed into pellets (Sari et al., 2025b). FTIR measurements were recorded across the wavenumber range of 400–4,000 cm^-1.

Morphology, particle size distribution, and percentage of minerals element of samples

The morphology of the best samples was examined using SEM (JEOL Ltd. IT-210, Japan). The particle grain size distribution of the samples was calculated according to the measurements of 100 randomly selected particles using ImageJ software version 2006 (National Institutes of Health (NIH), Bethesda, MD, USA) (Sari et al., 2022). EDS, integrated with the FE-SEM system, was used to determine the elemental composition of the samples. This analysis was performed to quantify the mineral content and calculate the Ca/P molar ratio in the best samples The Ca/P ratio was calculated according to the following equation (Sari et al., 2025):

Antibacterial analysis

Antibacterial assessment was performed to determine the capability of the developed materials to suppress bacterial growth under conditions relevant to oral biological environments (Sari et al., 2025a). The test was carried out against selected Gram-positive bacteria, namely Streptococcus mutans, Lactobacillus acidophilus and Streptococcus sanguinis. The evaluation followed the Kirby–Bauer disk diffusion assay to measure the zones of inhibition produced by each sample (Khan et al., 2019). 

The bacterial strains Streptococcus mutans and Streptococcus sanguinis were cultured in Mueller–Hinton Agar (MHA) agar plates and incubated at 37°C for 24 h under aerobic conditions. The bacterial suspension was adjusted to approximately 1×10⁸ CFU/mL (0.5 McFarland standard) before inoculation. Sterile Mueller–Hinton Agar (MHA) agar plates were prepared, and 100 µL of the bacterial suspension was evenly spread on the agar surface using a sterile spreader to obtain a uniform bacterial lawn. The disks were carefully placed on the surface of the inoculated agar plates. Sterile paper disks (6 mm in diameter) were used as carriers in the agar diffusion assay. Each disk was impregnated with 20 µL of the diluted gel sample (10 wt%) to reduce viscosity and facilitate diffusion of antimicrobial components. The impregnated disks were then placed on agar plates inoculated with the test microorganisms to evaluate antimicrobial activity by measuring the inhibition zone. A positive control disk containing Clindamycin, with an inhibition zone of 13 mm, was used as a reference. In contrast, a blank disk containing sterile distilled water (aquadest) was used as the negative control, showing no inhibition (0 mm) for comparison. The antibacterial test was performed in a single experimental run, and the inhibition zone diameters were measured and reported as the average value of the replicates. The inhibition zone was measured exclusively as the width of the clear halo beyond the edge of the disk, excluding the disk diameter itself.  The bacterial cultures were grown on Mueller-Hinton Agar (MHA). All plates were incubated at 37°C for 24 h under microaerophilic conditions. After incubation, the inhibition zones surrounding the disks were visually examined and measured using a digital caliper. Measurements were performed using a digital caliper with a precision ±0.01 mm to ensure accuracy and consistency in the recorded values (Balouiri et al., 2016; CLSI, 2021). 

RESULTS

Chemical composition of n-HAp/Ajwa date/miswak/propolis nano-biocomposite gels

The functional groups were analyzed based on the FTIR results. The results of FTIR characterization of the n-HAp/Ajwa date/miswak/propolis nano-biocomposite gel with concentrations of 0, 10, 20, and 30 wt% showed a typical spectrum for each of its constituent components (Figure 3 and Table 1). The absorption band at 2,943–2,947 cm⁻¹ indicates the presence of a C–H group in the Ajwa date content, according to Abdillah et al. (2017), who attribute it to flavonoids, thereby supporting the results of this research. In the sample, an absorption band at 1,450-1,100 cm⁻¹ indicated aromatic C–C and secondary alcohol (C–O) groups, corresponding to the flavonoid content in propolis. The presence of propolis in the gel provides an antibacterial effect through flavonoid compounds. It strengthens organic–inorganic interactions with the n-HAp matrix.

Although positive and negative controls were not included in the FTIR and XRD analyses, the structural deviation of the fabricated nano-biocomposite gels can still be evaluated by examining the preservation of the characteristic features of n-HAp. The FTIR spectra showed the typical phosphate (PO₄³⁻) and carbonate (CO₃²⁻) bands of n-HAp, indicating that the main chemical structure of n-HAp remained preserved after incorporation into gel matrix. Only slight shifts in peak position and intensity were observed, which are likely due to intermolecular interactions between n-HAp and the organic components in the gel. Consistently, the XRD results showed that the crystallite size remained within the nanoscale range and no additional crystalline phases were detected, suggesting that the incorporation of the gel matrix did not significantly alter the crystal structure of n-HAp. The FTIR spectra and XRD diffraction patterns of the fabricated composites were consistent with the characteristic features of n-HAp reported in previous studies. The presence of Ajwa date, miswak, and propolis extracts did not significantly modify the crystalline structure of n-HAp, although slight variations in peak intensity were observed due to the incorporation of organic components. Any minor variations observed in peak intensity are likely due to the presence of organic components from the natural extracts rather than structural changes in the n-HAp phase.

Meanwhile, in miswak, a typical absorption band of C–H stretching was observed around at 1,041-1,043 cm⁻¹, related to the presence of phytochemical components such as alkaloids, and organic sulfur. Miswak extract contains flavonoids and alkaloids, as evidenced by the absorption of aromatic and hydroxyl groups in the FTIR spectrum; thus, this result is consistent with the phytochemical profile of miswak as a natural antibacterial agent (Akbar et al., 2022). In addition, the typical inorganic peaks of n-HAp were identified as phosphate groups (PO₄³⁻) at 991-993 cm⁻¹ and carbonates (CO₃²⁻) at 1,413-1,419 cm⁻¹.

Figure 3. FTIR spectra of n-HAp/Ajwa date/miswak/propolis nano-biocomposite gel with concentration: 0 wt% (A), 10 wt% (B), 20 wt% (C), and 30 wt% (D).

Table 1. Chemical composition and wavenumbers (cm^-1).

In this study, the analysis focused on the characteristics of the final gel biocomposite as a potential agent for enamel remineralization. Although single controls for each raw material (such as Ajwa dates, miswak, n-HAp, propolis, and CMC) are not shown separately in Figure 3, comparisons to standard data or characteristics of these materials in the literature have been used as baselines. This was done to confirm that the incorporation of active ingredients into the gel matrix was successful, where the characteristic peaks of each raw material remained detectable in the final product without any significant chemical phase degradation. The FTIR spectrum of the composite gel was interpreted by comparing the observed peaks with reference spectra reported in previous studies for n-HAp and natural organic extracts, since the objective of this analysis was to confirm the functional groups present in the final composite formulation.

The XRD and FTIR analyses focused on the fabricated biocomposite gels to assess their chemical composition and crystallographic properties following the incorporation of Ajwa dates, miswak, and propolis extracts. Although individual raw material controls were not independently plotted, the n-HAp was synthesized from baseline. The results indicated no formation of extraneous crystalline phases or significant shifts in characteristic peaks, confirming that the structural integrity of the n-HAp lattice remained preserved within the composite. Minor variations in peak intensity and broadening were observed, attributed to dilution effects from the amorphous CMC matrix and organic extracts rather than to a chemical transformation of the n-HAp phase. The characterization results of the composite gel were interpreted by comparing the observed spectral features with previously reported data for n-HAp and natural organic extracts. The presence of characteristic peaks confirms the successful incorporation of the components within the composite matrix.

Crystallographic properties of n-HAp/Ajwa date/miswak/propolis nano-biocomposite gels

XRD analysis confirmed that all nano-biocomposite formulations, regardless of Ajwa, miswak, or propolis additive concentration, predominantly exhibited the characteristic diffraction peaks of n-HAp, with no indication of additional crystalline phases (Figure 4). The XRD profiles of the formulated gels indicated that the incorporation of polymeric components such as Na-CMC, glycerin, and guar gum altered the crystalline arrangement of n-HAp (Nisa et al., 2022). As a result, distinct n-HAp diffraction peaks were not clearly observed in the n-HAp/Ajwa date/miswak/propolis nano-biocomposite gel. This suggests that the interaction between n-HAp and the organic constituents of the gel matrix may disrupt or reduce its crystalline structure, as n-HAp is prone to structural modification when integrated with biopolymers such as propolis and miswak, as well as other ingredients in the gel formulation. The characterization results of the composite gel were interpreted by comparing the observed features with reference data reported in the literature for n-HA and organic extracts, as the primary objective of this analysis was to confirm the structural characteristics of the final composite formulation.

Table 2. Crystallographic properties of samples.

As shown in Table 2, the calculated crystallite sizes ranged from 13 to 15 nm and remained relatively consistent at concentrations up to 20 wt%. However, at 30 wt%, a reduction in crystallite size to approximately 13.1 nm was observed, suggesting that higher levels of organic components may restrict crystal growth. The observed particle morphology is mainly attributed to the n-HAp particles present in the composite. The Ajwa date, miswak, and propolis components were incorporated as extracts within the gel matrix; therefore, their individual particle sizes are not distinctly observable in the composite structure. Additionally, microstrain values remained relatively stable within the 0–20 wt% range, suggesting that the biopolymer content at these levels does not significantly affect the composite's crystal lattice. However, at 30 wt%, an increase in microstrain was observed, indicating greater lattice distortion, likely due to stronger interactions between the higher biopolymer content and the n-HAp structure.

Figure 4. XRD pattern of n-HAp/Ajwa date/miswak/propolis nano-biocomposite gel with concentration: 0 wt% (A), 10 wt% (B), 20 wt% (C), and 30 wt% (D).

As shown in Table 2, the calculated crystallite sizes ranged from 13 to 15 nm and remained relatively consistent at concentrations up to 20 wt%. However, at 30 wt%, a reduction in crystallite size to approximately 13.1 nm was observed, suggesting that higher levels of organic components may restrict crystal growth. The crystallite size of n-HAp estimated from XRD analysis (13–15 nm) corresponds to the size of individual crystalline domains. Additionally, microstrain values remained stable across the 0–20 wt% range. Still, they demonstrated an increase at 30 wt%, indicating a greater degree of lattice distortion in samples with higher biopolymer content. The biopolymer components were maintained at similar weight percentages in all formulations to ensure consistent gel-forming properties and to minimize variations in the matrix structure. By keeping the biopolymer composition constant, any observed differences in the physicochemical characteristics or performance of the samples can be primarily attributed to the variation in n-HAp content rather than changes in the polymer matrix. This approach allows a more reliable evaluation of the influence of n-HAp concentration on the structural and functional properties of the nano-biocomposite gel.

Morphology, particle size distribution, and percentage of minerals element of n-HAp/Ajwa date/miswak/propolis nano-biocomposite gels

The 30 wt% formulation was selected for SEM/EDS analysis as a representative sample with the highest n-HAp content, allowing clearer observation of the composite morphology and elemental distribution. To determine morphology, particle size, and mineral elements, SEM-EDS was used for further characterization. In the observation results at a magnification of 1 µm, Figure 5(A) shows the surface morphology of the particles, which tend to form clumps with uniform grain sizes. In contrast, Figure 5(B) shows a histogram of the particle size distribution, with an average of 0.252 ± 0.003 µm indicates that the SEM measurement represents agglomerated particles or granules formed by the aggregation of multiple n-HAp crystallites within the gel matrix. The spherical particle appearance within the gel matrix confirms the presence of n-HAp, although the quantity is expected to be limited due to the strong interaction with the biopolymer components. As shown in Figure 5 (C), the mass percentages of calcium (Ca) and phosphate (P) are 24.26 ± 0.38 and 11.99 ± 0.24, respectively. Therefore, by using Eq (1), n-HAp/Ajwa date/miswak/propolis nano-biocomposite gels 30 wt% exhibited a Ca/P molar ratio of 1.55–1.60, which approaches the stoichiometric ratio of natural n-HAp. The Ca and P mass percentages yield a Ca/P ratio of approximately 1.57 ± 0.03, calculated from measurements collected at multiple regions of the sample surface. This value is close to the stoichiometric Ca/P ratio of n-HAp.

Figure 5. SEM-EDS results of n-HAp/Ajwa date/miswak/propolis nano-biocomposite gel 30 wt%: morphology (A), particle grain size distribution (B), and EDS spectrum (C).

The EDS-derived Ca and P mass percentages correspond to a Ca/P ratio of 1.57. The observed range originates from replicate measurements across different EDS spectra. In n-HAp-polymer systems, increasing the content of organic components may result in peak broadening and decreased crystallinity. Conversely, certain compositions may exhibit relatively sharper peaks, reflecting a more stable crystalline domain, which is often used to select representative samples for further morphological characterization (El-Rafei et al., 2025). To examine surface morphology and elemental composition, SEM/EDS was used to verify the formation of n-HAp and assess the Ca/P ratio within the optimized matrix; the 30 wt% sample was selected as a representative formulation.

Antibacterial analysis of n-HAp/Ajwa date/miswak/propolis nano-biocomposite gels

Antibacterial tests were conducted on three gram-positive bacteria that cause dental caries: Streptococcus mutans, Lactobacillus acidophilus, and Streptococcus sanguinis (Hu et al., 2021). The inhibition zone width was measured from the edge of the disk to the outer boundary of the clear zone, rather than the total diameter including the disk. Therefore, smaller values (e.g., 2.5 mm) represent the radial inhibition distance beyond the disk margin, not the full inhibition diameter. The increase in the inhibition zone diameter indicates that the addition of n-HAp enhances the antibacterial activity of the biocomposite gel. The antibacterial evaluation demonstrated that increasing the n-HAp concentration in the biocomposite gel resulted in a progressive improvement in antibacterial performance (Dewi et al., 2025). For Streptococcus mutans (Figure 6(A)), the inhibition zones increased from 3.57 mm at lower concentrations to 6.25 mm and then to 8.10 mm at 30 wt%. A similar trend was observed for Lactobacillus acidophilus (Figure 6(B)), with inhibition values of 2.50 mm, 6.72 mm, and 4.66 mm, respectively. Streptococcus sanguinis (Figure 6(C)) exhibited no inhibitory response at the lowest concentration; however, a clear inhibition zone appeared at moderate concentration (6.09 mm) and increased further to 6.88 mm at the highest n-HAp loading. Overall, the antibacterial performance of the formulated biocomposite gels falls within the moderate category (5–10 mm) (Table 3), with the most effective activity observed at 30 wt%, particularly against Streptococcus mutans and Streptococcus sanguinis.

The inhibition zones produced by several experimental groups were relatively small compared with the positive control; therefore, they are less clearly visible in the photographic images presented in Figure 6. The inhibition width was measured radially from the disk margin rather than as the total diameter, resulting in numerically smaller values. However, the inhibition zones were identifiable during the experiment and measured directly using a digital caliper; the corresponding values are presented in Table 3.

Figure 6. The test results of antibacterial activity of n-HAp/Ajwa date/ miswak/propolis nano-biocomposite gels with concentration variation of 0,10,20, and 30 wt% against Streptococcus mutans (A), Lactobacillus acidophilus (B), and Streptococcus sanguinis (C).

Table 3. Diameter inhibition rate of n-HAp/Ajwa date/miswak/propolis nano-biocomposite gels.

An increase in n-HAp concentration provides a larger active surface area for interaction with bacterial cells. At higher loading levels (30 wt%), the n-HAp/Ajwa date/miswak/propolis nano-biocomposite gel contains a greater density of nanoparticles, which can facilitate enhanced release of calcium and phosphate ions into the surrounding medium. The availability of calcium and phosphate ions is widely reported to support enamel remineralization and may also contribute to antibacterial effects, such as disruption of bacterial cell membranes, interference with metabolic processes, and reduced bacterial adhesion and biofilm formation, as described in previous studies (Sari et al., 2025a).

DISCUSSION

FTIR spectroscopy (Figure 3) was also used to observe the chemical changes during the fabrication process of n-HAp/Ajwa date/miswak/propolis nano-biocomposite gels. The increased absorption intensity observed at 30 wt% may indicate stronger interactions between the n-HAp inorganic phase and the surrounding biopolymer matrix. However, since no comparison with raw materials or control samples was performed in this study, the specific contribution of each component to the antibacterial and remineralization permormance cannot be conclusively determined. Additionally, the FTIR spectra displayed characteristic bands corresponding to PO₄^3-, CO₃^2-, and OH^- functional groups associated with the n-HAp phase, indicating the presence of n-HAp within the nano-biocomposite gel matrix (Menon et al., 2025).  The absence of harmful chemical signatures and the predominantly low-crystallinity nature of the material further indicate that the synthesized nano-biocomposite gels as a remineralization agent are suitable for use in dental applications (Juntavee et al., 2021). Further studies using advanced analytical techniques, such as ICP-OES, ICP-MS, or GC-MS/HPLC, may provide additional insight into the elementalcom position and bioactive compounds in the composite system.

As presented in Figure 4, the XRD patterns indicate that the incorporation of Na-CMC, glycerin, and guar gum alters the crystalline structure of n-HAp within the gel matrix (Nisa et al., 2022; Alkaron et al., 2024). Consequently, the XRD patterns did not exhibit distinct crystalline reflections of n-HAp in the gels containing Ajwa date, miswak, and propolis. The reduced intensity of diffraction peaks may be associated with the presence of organic components within the gel matrix. However, due to the absence of comparison with control materials such as pristine n-HAp or base gel formulations, the specific effect of each component on the crystalline structure cannot be conclusively determined. The incorporation of Na-CMC, glycerin, and guar gum into the n-HAp gel matrix led to broadening and reduced intensity of diffraction peaks, making the characteristic crystalline reflections of n-HAp more difficult to distinguish. Comparable behavior has been observed in other n-HAp–polymer composite systems, where a higher proportion of organic components can inhibit crystal growth and diminish detectable crystallinity (Alkaron et al., 2024).

The calculated crystallite size and microstrain values are presented in Table 2. The crystallite size of n-HAp was estimated using the Scherrer equation based on the full width at half maximum (FWHM) of the (211) diffraction peak. Microstrain values were also calculated from the peak broadening to evaluate lattice distortion in the composite samples. The characteristic diffraction peaks of n-HAp are still detectable in the composite samples. The results show that the crystallite size of n-HAp in the composite gels remains within the nanoscale range 13–15 nm, while the microstrain slightly increases with increasing biocomposite concentration. The increase in microstrain indicates lattice distortion resulting from the interaction between n-HAp nanoparticles and organic molecules in the gel matrix. These interactions may modify the crystal structure and generate internal strain within the lattice. Such structural modification can enhance the surface reactivity of n-HAp, which may contribute to its potential application in tooth enamel remineralization (Anil et al., 2022; Bristy et al., 2025).

The presence of spherical particles indicates that the n-HAp and Ajwa date/miswak/propolis biopolymers have been well homogenized in the gel extract, which can be seen in SEM analysis, as shown in Figure 5. The SEM images indicate that the n-HAp particles tend to form agglomerates rather than remain dispersed as individual particles. This phenomenon is commonly reported for n-HAp due to its high surface energy, which promotes particle-particle interactions and aggregation, particularly at high temperatures (Kusnieruk et al., 2016; Méndez-Lozano et al., 2022; Tang et al., 2024).

The antibacterial activity observed in this study was evaluated using the agar diffusion method, which provides a comparative indication of bacterial growth inhibition. As shown in the antibacterial analysis (Figure 6 and Table 3), different bacteria exhibited different inhibition responses to the tested biocomposite gel. Streptococcus mutans and Lactobacillus acidophillus showed relatively smaller inhibition zones compared to other tested bacteria. This observation may be related to their known ability to form biofilms, which has been reported in previous studies (Neilands and Svensäter, 2007). In contrast, Streptococcus sanguinis showed inhibition only at hinger concentrations of the biocomposite gel, indicating a lower sensitivity compared to other tested bacteria. This observation is consistent with previous studies reporting that Streptococcus sanguinis can exhibit greater tolerance to environmental change (Puccio et al., 2021; Usuda et al., 2023). 

Despite the promising findings of this study, several limitations should be acknowledged. First, the antibacterial evaluation was performed using the agar diffusion method, which provides a qualitative assessment of bacterial growth inhibition. Quantitative analyses, such as minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC), were not conducted in this study. They may be considered in future investigations to more precisely determine the antibacterial potency of the developed composite. Second, pH analysis and ion release measurements were not conducted; therefore, the potential contributions of pH variation and calcium or phosphate ion release to the antibacterial and remineralization mechanisms were inferred from previously reported studies rather than directly measured. Advanced analytical techniques, such as ICP-OES or ICP-MS, could provide further insight into the material's ion release behavior. In addition, in vitro bioactivity tests in simulated body fluid (SBF) were not performed in the present work, which could further confirm the apatite-forming ability and remineralization potential of the composite system. Moreover, the developed composite may have potential for dental applications, although further biocompatibility and bioactivity studies are necessary to confirm its suitability. Therefore, future studies should therefore include these analyses, along with more detailed characterization of individual raw materials and quantitative antibacterial assays, to provide a more comprehensive understanding of the material's physicochemical and biological performance. Moreover, the developed composite gel demonstrates promising structural and antibacterial properties and may serve as a candidate for enamel remineralization applications. However, further studies involving direct enamel remineralization analysis are required to confirm its clinical effectiveness.

CONCLUSION

This study successfully developed an n-HAp-based biocomposite gel using calcium precursors obtained from tiger-snail shells combined with natural antibacterial biopolymers—Ajwa dates, miswak, and propolis—to enhance tooth enamel remineralization potential. FTIR analysis verified the coexistence of characteristic n-HAp bands with organic functional groups, demonstrating effective integration of inorganic and biopolymer components within the gel structure. Adjusting the n-HAp content influenced the physicochemical characteristics of the material, where the 30 wt% formulation exhibited improved crystalline features and structural uniformity as evidenced by XRD results. The crystallite size ranged between 13 and 15 nm. At the same time, the increased microstrain at higher loading levels suggested enhanced interaction between the mineral phase and the organic matrix. SEM imaging further confirmed nanoscale morphology with particle agglomeration commonly observed due to the high surface energy of n-HAp. Moreover, antibacterial assays revealed that higher n-HAp concentrations correlated with enhanced antibacterial performance, particularly against Streptococcus mutans and Streptococcus sanguinis, with the most potent inhibition at 30 wt%. Overall, the integration of n-HAp derived from tiger-snail shells with bioactive natural extracts produced a biocomposite gel exhibiting favorable structural and antibacterial characteristics. However, the rheological properties of the gel system were not evaluated in the present study. Therefore, further investigation, including rheological analysis and additional physicochemical assessments, is required to determine its suitability for dental applications better.

ACKNOWLEDGEMENTS

We would like to express our sincere gratitude to the Directorate General of Higher Education, Research, and Technology through the Student Creativity Program (PKM), Directorate of Vocational Education, Directorate of Higher Education, and Ministry of Higher Education, Science and Technology, Republic of Indonesia through Regular Fundamental Research 2025 Grant for financially supporting this research. In addition, the authors would like to thank the Biomaterial Physics Laboratory at the Faculty of Mathematics and Natural Sciences, Universitas Negeri Yogyakarta, Indonesia, Integrated Laboratory for Research and Testing, Universitas Gadjah Mada, Yogyakarta, Indonesia, and Testing Laboratory, Universitas Muhammadiyah Yogyakarta, Indonesia for the facilities, technical assistance, and antibacterial analysis of samples.

AUTHOR CONTRIBUTIONS

Eliana Diah Puspita Arum: Conceptualization (Lead), Data Curation (Lead), Software (Lead), Formal Analysis (Equal), Validation (Lead), Writing – Original Draft (Lead); Lutfi Puspita Meliasari: Software (Equal), Data Curation (Equal), Formal Analysis (Equal), Validation (Equal), Visualization (Equal), Writing – Original Draft (Equal); Ratri Yulina Setiati: Software (Equal), Data Curation (Equal), Formal Analysis (Equal), Software (Equal), Validation (Equal), Visualization (Equal), Writing – Review & Editing (Equal); Melisa Sekarlina Putri Dayani: Software (Equal), Data Curation (Equal), Formal Analysis (Equal), Validation (Equal), Writing – Review & Editing (Supporting); Rizky Amin Saputro: Data Curation (Equal), Formal Analysis (Equal), Validation (Equal), Writing – Review & Editing (Supporting); Mona Sari: Conceptualization (Lead), Data Curation (Lead), Formal Analysis (Equal), Funding Acquisition (Lead), Investigation (Lead), Supervision (Lead), Validation (Equal), Visualization (Equal), Writing – Review & Editing (Lead).

CONFLICT OF INTEREST

The authors declare that they have no conflicts of interest.

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