Abstract The present study describes an eco-friendly way to produce nanochitosan–zinc oxide nanocomposite. It uses byproducts from Sepia prashadi as a renewable biopolymer source. This biogenic method turns marine waste into useful materials. It also helps create multifunctional nanomaterials in an environmentally responsible way. Fourier Transform Infrared Spectroscopy (FTIR) confirmed key functional groups. This shows the composite formed through hydrogen bonding and electrostatic interactions. Scanning Electron Microscopy (SEM) revealed the nanocomposites had various shapes, ranging from spherical to irregular. Their sizes ranged from nanometres to micrometers. X-ray Diffraction (XRD) analysis indicated ZnO was embedded in the chitosan matrix, making it appear absent. The nanocomposites showed strong antimicrobial activity against oral pathogens, including Streptococcus mutans and Candida albicans. Their inhibition was better than their individual components. These results highlight the promise of S. prashadi-based nanochitosan–zinc oxide nanocomposites as biocompatible antimicrobial agents for oral health. They offer a sustainable alternative to traditional synthetic materials.

Keywords: Nanochitosan- Zinc oxide, Sepia prashadi, Oral pathogen, Dental health, nanochitosan, Antimicrobial activity

Citation:  Thirumal, K., Pitchai, A., Ganapathy, D., and Ramasamy, P. 2026. Green-synthesis of nanochitosan- zinc oxide nanocomposite from Sepia prashadi as a promising antimicrobial agent for oral healthcare. Natural and Life Sciences Communications. 25(4): e2026086.

Graphical Abstract:

INTRODUCTION

Antimicrobial resistance (AMR) is a leading global health threat, as recognized by the World Health Organization. Oral pathogens are increasingly tolerant to conventional agents like chlorhexidine and triclosan. While widely used and effective, these antimicrobials present issues such as cytotoxicity, environmental persistence, and the risk of resistance. These concerns highlight the urgent need for safer, biocompatible alternatives in oral healthcare. Nanomaterials from natural biopolymers have emerged as promising options. They offer both therapeutic benefits and environmental sustainability. Chitosan is a deacetylated derivative of chitin, obtained from marine organisms. It is extensively studied in biomedical research. Its biocompatibility, biodegradability, non-toxicity, low immunogenicity, and mucoadhesivity make it versatile for pharmaceutical and dental uses (Vlaia et al., 2021; Yu et al., 2021). Chitosan serves as both a coating and a drug carrier (de Lapena et al., 2022). It also has broad-spectrum biological activities such as antimicrobial, antiviral, antitumor, lipid-lowering, and immunomodulatory effects (Vaidya et al., 2024; Kanchanomai et al., 2025; Venkatachalam et al., 2025). Its positive charge enables electrostatic interactions with negatively charged microbial and tumor cell membranes. This results in membrane disruption, apoptosis, and inhibition of angiogenesis. At the nanoscale, these actions are amplified. Nanochitosan shows greater bioactivity, drug-loading efficiency, and targeted delivery, making it especially valuable for oral and biomedical use.

To harness these amplified properties, nanochitosan is commonly synthesized via ionic gelation. This is a process where chitosan interacts with polyanions (negatively charged molecules) such as tripolyphosphate (TPP), forming nanoparticles. This method is simple, rapid, and adaptable. It produces nanoparticles with tunable size and morphology (controllable shape and structure) depending on TPP concentration, agitation speed (rate of stirring), and ultrasonic treatment (use of sound waves to mix) (Ghobadi-Oghaz et al., 2022). The resulting nanoparticles are smooth, spherical, and capable of encapsulating diverse therapeutic agents (drugs), expanding their utility in drug delivery and antimicrobial applications (Sivabalan et al., 2025). Characterization of nanochitosan—including measurements of dimensions, surface charge, morphology, and drug-loading capacity (amount of drug a nanoparticle can carry)—is essential to optimize formulations and ensure reproducibility for biomedical use (Sultan et al., 2022).

Nanotechnology-based interventions using zinc oxide (ZnO) nanoparticles are promising due to strong antimicrobial activity and the ability to generate reactive oxygen species (ROS). These disrupt microbial membranes and cells (Premkumar et al., 2025; Vigneshwaran et al., 2025). ZnO is also effective at remineralizing dental tissues. It shows strong antibacterial action, disrupts biofilm formation, and promotes enamel repair. However, concerns remain about its cytotoxicity and tendency to aggregate. Combining ZnO with chitosan addresses these issues. The biopolymer stabilizes ZnO nanoparticles, improves their dispersion, and adds to their antimicrobial effect. Chitosan–ZnO nanocomposites thus show improved antibacterial performance, maintaining biocompatibility and biodegradability (Rana et al., 2021; Rashki et al., 2021; Desai et al., 2026).

Further integrating sustainability into these advances, green synthesis strategies have elevated the relevance of these nanocomposites. Green synthesis uses biological resources such as marine-derived chitosan as both reducing agents (helping form nanoparticles by donating electrons) and stabilizing agents (preventing nanoparticles from clumping together), eliminating the need for toxic chemicals. This aligns with sustainability goals and reduces environmental impact (Kasi et al., 2025). Recent studies confirm that green-synthesized chitosan–ZnO nanocomposites show enhanced antimicrobial activity against oral pathogens and favorable biocompatibility, making them next-generation solutions for oral healthcare (Aouadi et al., 2024; Kachare et al., 2024; Pitaksuteepong et al., 2024).

While previous studies have reported chitosan–ZnO nanocomposites, these have mainly focused on chemically synthesized materials and general antimicrobial applications. In direct response to this shortcoming, the current study integrates Sepia prashadi-derived chitosan into a green synthesized ZnO nanocomposite system specifically tailored for oral healthcare. By combining sustainable biopolymer sourcing with eco friendly synthesis and demonstrating enhanced antimicrobial activity against oral pathogens, this work directly addresses the existing gap and advances knowledge in oral nanotherapeutics. This study aims to establish the potential of a sustainable nanochitosan–zinc oxide (CS–ZnO) nanocomposite from S. prashadi by-products, synthesized through an eco-friendly method, as a biocompatible oral nanotherapeutic. The extracted nanochitosan–zinc oxide (CS–ZnO) nanocomposite was characterized to determine its physicochemical properties, and the synthesis conditions were optimized to produce stable nanocomposites. The resulting materials were subsequently evaluated for antimicrobial activity against oral pathogens using the well diffusion method.

MATERIALS AND METHODS

Extraction of chitosan

Chitosan has been extracted from the internal cuttlebone of S. prashadi through a series of steps that included demineralisation, deproteinization, and deacetylation of chitin. To make nanochitosan using the ionic gelation method, chitosan was first dissolved in acetic acid and then crosslinked with sodium tripolyphosphate while being stirred constantly. To confirm effective nanoscale modification and structural integrity, the synthesised nanochitosan underwent comprehensive assessment through XRD for crystalline structure analysis, SEM for surface morphology evaluation, and FTIR for functional group identification (Palaniappan et al., 2025).

Synthesis of nanochitosan- ZnO nanocomposite

To prepare the nanochitosan–zinc oxide (ZnO) nanocomposite, 0.5 g of nanochitosan was dispersed in 100 mL of distilled water. An equal amount (0.5 g) of commercially obtained ZnO (analytical grade) was added, and the mixture was continuously stirred to achieve uniform dispersion, and a 5% (v/v) acetic acid solution was added dropwise to initiate the reaction, which was maintained for 2–4 h. Sodium tripolyphosphate was then introduced to facilitate crosslinking, and the suspension was stirred for an additional 12 h to promote composite formation. Following this, the mixture was centrifuged for 30 min to separate the solid fraction, which was subsequently dried in a hot air oven at 100°C. The dried material was finely ground using a mortar and pestle and subjected to calcination at 800°C, resulting in the nanochitosan–ZnO nanocomposite with enhanced structural integrity and functional properties. (Eswaran et al., 2023).

Fourier transform infrared spectroscopy

Perkins-Elmer spectrometer (FITR - Perkin Elmer Spectrum Two, UK) was used to record the Fourier Transform Infrared (FT-IR) spectra of solid chitosan and nanochitosan–zinc oxide nanocomposites that were taken from the cuttlebone of S. prashadi.

X ray diffraction

An XRD - Bruker D8 Advance, Germany was used to look at the structural features of nanochitosan–zinc oxide nanocomposites. The intensity of the diffracted X rays was determined based on the orientation of the specimen and the diffraction angle (2θ). Using this diffraction pattern, you can find out the material's structural properties, as well as the size and direction of the crystallites (tiny parts of the crystalline structure).

Scanning electron microscopy

Scanning electron microscopy (SEM, JEOL model 6390) was employed to examine the morphological characteristics of the nanochitosan–zinc oxide nanocomposites. In SEM, a focused electron beam scans a defined area of the specimen in a raster pattern. The beam interacts with the sample, creating signals that can be detected, such as secondary electrons, internal currents, and photon emissions. These signals are used to make detailed images of the surface morphology.

Antimicrobial activity by zone of inhibition

The antimicrobial activity of the synthesized nanochitosan–zinc oxide nanocomposites was evaluated using the agar well diffusion method (Ali et al., 2025). A stock solution of 10 mg/mL was prepared, from which test concentrations of 25%, 50%, and 100% (v/v) were obtained. Overnight cultures of Streptococcus mutans, Staphylococcus aureus, and Pseudomonas aeruginosa were adjusted to the 0.5 McFarland standard, corresponding to approximately 1.5 × 10⁸ CFU/mL, to ensure standardized inoculum density. The standardized bacterial suspensions (100 μ) were evenly spread on Mueller–Hinton (MH) agar plates, which had been sterilized by autoclaving at 12°C for 15 min under 15 lb pressure. For antifungal assays, Candida albicans was cultured in Czapek Dox broth (Hi Media) sterilized under identical autoclaving conditions, incubated at 37°C for 72 h, and subsequently swabbed onto sterile Petri dishes to prepare uniform inoculation.

Agar wells of 6 mm diameter were aseptically bored into the inoculated plates, and 50 μL of each test concentration of the nanocomposite was dispensed into the wells in triplicate. Plates inoculated with bacterial strains were incubated at 37°C for 24 h, while those inoculated with fungal strains were incubated at 37°C for 72 h. Zones of inhibition were measured in millimeters to quantify antimicrobial efficacy. Amikacin, Levofloxacin, and Ciprofloxacin (1 mg/mL) served as positive controls for bacterial strains, Fluconazole (1 mg/mL) was used as the positive control for
C. albicans, and distilled water was included as a negative control to confirm specificity of the antimicrobial response.

Minimum inhibitory concentration (MIC)

The minimum inhibitory concentration (MIC) of nanochitosan–zinc oxide nanocomposite was determined following a standard protocol (Ganesan et al., 2025). A 0.5 McFarland inoculum, equivalent to approximately 1.5 × 10⁸ CFU/mL, was prepared for each bacterial strains (S. mutans, C. albicans) by diluting 0.5 mL of overnight cultures in sterile medium. Nanochitosan–zinc oxide nanocomposite was obtained from a 10 mg/mL stock solution and tested at concentrations ranging from 2.0 to 10 mg/mL. Two control conditions were included: one containing 10 μL of nanochitosan–zinc oxide nanocomposite without inoculum, and another in which nanochitosan–zinc oxide nanocomposite were replaced with sterile water while maintaining the inoculum. All test and control tubes were incubated at 37°C for 18 h. Following incubation, bacterial growth was assessed visually by examining turbidity. The MIC was defined as the lowest concentration of nanochitosan–zinc oxide nanocomposite that inhibited visible bacterial growth. Each assay was performed in triplicate to ensure reproducibility.

Statistical analysis

The inhibitory effect of the nanochitosan–zinc oxide nanocomposite was analysed using one-way ANOVA in SPSS-23. Subsequently, we utilized Duncan’s multiple range test (DMRT). P-values of 0.05 or lower were deemed significant. All experiments were conducted in duplicate. Results are expressed as mean ± standard deviation (SD).

RESULTS

FTIR spectroscopy

FTIR analysis was conducted to identify functional groups and confirm the formation of the composite. Spectra were recorded in the 4,000–400 cm⁻¹ range using a Perkin-Elmer spectrometer. As shown in Figure 1a, pure ZnO powder displayed a strong absorption band at approximately 490 cm⁻¹, indicating Zn–O stretching vibrations in the ZnO lattice and confirming the presence of oxide groups. A broad band at around 3,379 cm⁻¹ was attributed to O–H stretching vibrations of adsorbed water molecules. For pure chitosan, characteristic absorption bands were observed at ~3,408.53 cm⁻¹ (O–H and N–H stretching), and ~1,544.40 cm⁻¹ (amide II, N–H bending) (Figure S1). In contrast, the nanochitosan–ZnO nanocomposite exhibited distinctive peaks at 3,225 cm⁻¹ (N–H and O–H stretching) (Figure 1b), 2,888 cm⁻¹ (asymmetric CH₃/CH₂ stretching), and 1,378 cm⁻¹ (O–H bending). Importantly, a strong absorption band at ~454 cm⁻¹ corresponded to Zn–O stretching vibrations, confirming the incorporation of ZnO nanoparticles. The observed spectral shifts and additional bands collectively validate robust coordination between Zn²⁺ ions and chitosan functional groups, thereby confirming successful composite formation and structural integrity. These spectral characteristics collectively indicate robust coordination between Zn²⁺ ions and the functional groups of nanochitosan, thereby validating successful composite formation. These spectral characteristics, corroborated by supplementary analyses, validate the successful synthesis and structural integrity of nanochitosan–zinc oxide nanocomposite.

Figure 1 a. FT-IR spectral analysis of pure zinc oxide (ZnO).

Figure 1 b. FT-IR spectral analysis of nanochitosan–zinc oxide nanocomposites from Sepia prashadi.

X-ray diffraction

The crystalline structure of the synthesized nanochitosan–zinc oxide nanocomposites was analyzed using X ray diffraction (XRD). Diffraction patterns were recorded within the 2θ range of 10°–40° (Figure 2). A prominent peak was observed at 26.64°, which differed from the diffraction profile of pure chitosan, while additional peaks appeared with lower intensity. Distinct peaks with Full Width at Half Maximum (FWHM) values were observed at 9.07°, 26.64°, and 44.53°, as well as at 0.547°, 0.221°, and 0.369°, respectively (Table 1S) confirmed the formation of the nanochitosan–zinc oxide nanocomposite. The reduced intensity of certain peaks indicated lower crystallinity compared to native chitosan, which is consistent with the amorphous nature of chitosan and the presence of CaCO₃, a natural constituent of cuttlebone. The combined diffraction spectrum exhibited characteristic features of ZnO incorporation, thereby confirming successful integration of zinc oxide into the nanochitosan matrix. All diffraction peaks are indexed in accordance with the hexagonal phase of the ZnO wurtzite crystal structure, featuring principal planes (100), (002), (101), (102), (110), (103), (200), and minor planes (201) and (112). No supplementary peaks indicative of impurity phases was detected in any of the analysed samples.

Figure 2. XRD image of nanochitosan–zinc oxide nanocomposites from Sepia prashadi.

SEM and EDX

Scanning electron microscopy (SEM) and energy-dispersive X-ray spectroscopy (EDX) were employed to examine the surface structure, particle size, and structural properties of the synthesized nanochitosan–zinc oxide nanocomposite. SEM images (Figure 3A) revealed that the nanochitosan–zinc oxide nanocomposite was distributed across the surface, forming a textured landscape conducive to ZnO binding. The nanocomposite formed clusters of predominantly spherical particles that aggregated irregularly. EDX elemental mapping (Figure 3B) confirmed the composition, which consisted primarily of carbon (C), nitrogen (N), oxygen (O), and phosphorus (P).

Figure 3. SEM (A) & EDX (B) image of nanochitosan–zinc oxide nanocomposite from Sepia prashadi.

Antimicrobial activity

Figure 4 and Table 1 show the efficacy of nanochitosan–zinc oxide nanocomposite against selected bacterial and yeast strains. At 50% and 100% (v/v), the nanocomposite produced inhibition zones of approximately 13 ± 1.75 mm against S. mutans and C. albicans. At 50% (v/v), there was no inhibitory effect on S. aureus or P. aeruginosa. At 25% (v/v), no antimicrobial activity was observed against any tested pathogens. Antifungal activity was present at both 50% and 100% (v/v). Statistically significant differences (P < 0.05) in inhibition levels were found between the two concentrations for all microbial species.

Figure 4. Antimicrobial activity of nanochitosan–zinc oxide nanocomposites from Sepia prashadi.

Table 1. Antimicrobial activity of nanochitosan–zinc oxide nanocomposites from Sepia prashadi.

Minimum inhibitory concentration (MIC)

The MIC values of nanochitosan–Zinc oxide nanocomposites against microbial strains, including Streptococcus mutans and Candida albicans, were recorded as 2 mg/ml, 4 mg/ml, 6 mg/ml, 8 mg/ml and 10 mg/ml (w/v) respectively (Table 2). S. mutans exhibited bacterial growth inhibition at a concentration of 6 mg/ml, while C. albicans inhibited bacterial growth at a concentration of 8 mg/ml.

Table 2. MIC of nanochitosan–zinc oxide nanocomposites from cuttlebone of Sepia prashadi.

Note: *MIC concentration, - No growth, + Cloudy solution (slight growth), ++ Turbid solution (strong growth), +++ Highly turbid solution (dense growth).

DISCUSSION

Nanomaterials and nanoparticles are very useful in material science and biomedicine, especially because their tiny size allows them to get inside cells and organelles for targeted drug delivery (Singh and Lillard, 2009). There are many ways to make chitosan micro- and nanoparticles. Drug molecules are usually held together by hydrogen bonding, electrostatic interactions, or hydrophobic linkages. There are many ways that drugs can be released from chitosan nanoparticles, such as polymer swelling (Liu et al., 2018), drug diffusion, polymer erosion or degradation, or a combination of erosion and degradation (Al-Naamani et al., 2016). Polymer swelling happens when water is absorbed until it dissolves. The solubility of chitosan in water or biological media has a big effect on this process (Fonseca-Santos and Chorilli, 2017).

The broad absorption band at 3,462 cm⁻¹ was due to –OH stretching vibrations, and the peak at 1,639 cm⁻¹ was due to C–O stretching of primary amides, which is in line with what we found. (Gamboa-Solana et al., 2021) also found similar results. The FTIR spectrum of chitosan nanoparticles also shows several unique peaks that show off their structural features. (Xu and Du, 2003) identified the absorption band at 1,636 cm⁻¹ as indicative of C=O stretching vibrations, and the N–H bending vibration was observed at 1,544 cm⁻¹. Their finding that at the band 1,553 cm⁻¹ was due to N–H bending of secondary amide groups and that the signal at 1,412 cm⁻¹ was due to C–N stretching of amine groups. A significant peak at 1,017 cm⁻¹ was linked to the symmetric and asymmetric stretching of the PO₃ group, while the presence of PO₄²⁻ was further supported by previous research conducted by (Dounighi et al., 2012). This band was thought to be due to the C=O stretching of amino groups.  Also, the peak at 889 cm⁻¹, which is linked to PO₄²⁻, set the spectral profile of nanochitosan apart from that of native chitosan. Also, the different vibration range between 400 and 500 cm⁻¹ confirmed the Zn–O bond, which proved that zinc oxide was part of the composite structure (Re et al., 1999).

The peaks detected in ZnO nanoparticles and CS-ZnO composite corresponded well with the hexagonal ZnO particle database (JCPDS No. 36-1451) (Prokhorov et al., 2020). The standard XRD pattern of chitosan (#) (Figure 2) frequently exhibits diffraction peaks that correspond to its crystalline structure and associated regions (JCPDS card No. 00–039–1894) (Kumar et al., 2026). Comparable cubic structures were identified in chitosan-based nanomaterials, validating the structural integrity and hybrid characteristics of the synthesized composite. The XRD patterns of chitosan nanoparticles showed structural features that weren't readily apparent, with a strong peak at 22.75° (Palaniappan et al., 2025). This observation suggests that the nanoparticles exhibit an amorphous structure, a trait frequently regarded to be beneficial for biomedical applications. In line with previous research, diffraction peaks at 2θ = 17° and 24° further validated the non-crystalline structure of chitosan (Rostami et al., 2022), aligning with our results. These findings collectively corroborate prior evidence indicating that chitosan nanoparticles produced through ionic gelation maintain an amorphous structure (Palaniappan et al., 2025). The XRD profile of the nanochitosan–zinc oxide nanocomposite exhibited distinct chitosan peaks at 17.8° and 22.6° (Al-Rajhi et al. 2024), alongside characteristic ZnO diffraction peaks at 31.9° and 47.26°. The presence of these distinct peaks verifies the effective incorporation of ZnO into the chitosan matrix and underscores the elevated phase purity of the nanocomposite (Serouti et al., 2024).

The SEM pictures showed that the surface was rough and uneven, which showed that the composite had a heterogeneous structure. To improve the texture of the surface and make sure that the active ingredients were spread out evenly, chitosan was added (Thanh et al., 2020; Zhang et al., 2022). EDX supports the material's stability in terms of its composition. The chitosan matrix, which keeps the nanoparticles stable, was the main source of the carbon signal. We made detailed elemental mapping images (Al-Rajhi et al., 2024) to make sure our results were as accurate as possible. Overall, these results show that nanochitosan synthesis works and that it has a lot of potential in medicine, biotechnology, and materials science (Palaniappan et al., 2025).

Regarding antibacterial activity, the statistical analysis indicated that the antibiotic controls exhibited significantly greater inhibitory effects compared to the CSNP–ZnO nanocomposite treatments. For C. albicans, although inhibition zones were observed (13 mm), the statistical differences again favoured the antibiotic control, and no significant difference was detected between the nanocomposite treatment and the control groups. These findings suggest that while the CSNP–ZnO nanocomposite demonstrated measurable antimicrobial activity, its efficacy was comparatively lower than that of the standard antibiotics tested. The interpretation of results must therefore be carefully revised to reflect that the superiority lies with the antibiotic controls rather than the nanocomposite treatment. There were notable variations in inhibitory levels (P < 0.05) among all microbial species evaluated at three different doses with a nanochitosan-zinc oxide nanocomposite.

The primary goal of nanoparticle based antimicrobial strategies is to achieve selective disruption of pathogenic organisms without adversely affecting normal host cells (Toropova et al., 2017). The observed inhibition of C. albicans demonstrates the potential of ZnO based nanocomposites; however, the statistical outcomes necessitate cautious interpretation. Previous studies have shown that the antimicrobial efficacy of ZnO nanocomposites is highly dependent on the Zn content ratio (Gordon et al., 2011), which may account for variability in performance across different microbial strains. In the present study, the antibacterial activity results indicate that statistically significant differences favor the antibiotic control rather than the nanocomposite treatment. A similar pattern is evident for C. albicans, where the statistical significance again highlights the superiority of the antibiotic.

To effectively employ zinc oxide (ZnO) nanoparticles in applications such as food preservation and the development of non toxic antimicrobial derivatives, it is essential to understand their underlying mechanisms of action. At the nanoscale, ZnO particles exhibit pronounced antimicrobial activity through interactions with bacterial cell surfaces and intracellular components, thereby activating specific bactericidal pathways. Proposed mechanisms include the generation of reactive oxygen species (ROS), which induce membrane lipid peroxidation, leakage of reducing sugars, DNA and proteins, and a consequent decline in cell viability (Giovino et al., 2013; Cerchiara et al., 2015). Antifungal mechanisms have also been documented, encompassing structural alterations, suppression of protein synthesis, DNA damage, disruption of the cell wall, mitochondrial dysfunction, and destabilization of antioxidant defenses via ROS and Zn²⁺ mediated pathways (Mills et al., 2003; Quiñones et al., 2018). Furthermore, ZnO nanoparticles can reduce glutathione (GSH) levels by inhibiting enzymes responsible for GSH biosynthesis, thereby lowering the antioxidant capacity of fungal cells (Loh et al., 2010).

In this study, the nanoscale dimensions of the synthesised nanochitosan–zinc oxide (ZnO) nanocomposite enabled infiltration into bacterial cells, resulting in cellular damage, respiratory disruption, and ultimately cell death. Higher levels of ZnO nanoparticles were linked to stronger antimicrobial activity, primarily due to the release of Zn²⁺ ions, which interact with the negatively charged bacterial cell wall and cause its breakdown. Additionally, ZnO nanoparticles generate reactive oxygen species (ROS), leading to oxidative stress and interactions with proteins, DNA, enzymes, and lipids, thereby inhibiting growth or inducing cell death. The differences in how Gram positive and Gram negative bacteria respond to antimicrobials can be explained by variations in cell wall structure (Kumar et al., 2020, Selvanathan et al., 2022; Chan et al., 2024) (Figure 5).

Figure 5. Antimicrobial mechanism of nanochitosan–zinc oxide nanocomposite.

In the present study, the absence of activity against S. aureus and P. aeruginosa at lower concentrations may be attributed to differences in cell wall architecture and resistance mechanisms. Gram positive S. aureus possesses a thick peptidoglycan layer, while P. aeruginosa exhibits robust efflux systems and outer membrane barriers, both of which likely require higher ROS flux or Zn²⁺ ion release to achieve inhibition. In contrast, the observed antifungal activity against C. albicans aligns with prior reports indicating that ZnO nanostructures disrupt fungal cell membranes through oxidative stress and ion mediated enzyme inactivation, processes enhanced by reduced crystallite size and lattice distortion. Table 3, summarizes the antibacterial activities that have been reported for green-synthesized ZnO NPs.

Table 3. Summarizes the antimicrobial activities that have been reported for ZnO NPs.

CONCLUSION

The main objective of this study was to synthesize and characterize nanochitosan–zinc oxide (ZnO) nanocomposites derived from S. prashadi, and to evaluate their antimicrobial efficacy against oral pathogens. Successful synthesis was confirmed by FTIR, SEM, and XRD, indicating a uniform fine structure with well‑integrated ZnO particles in the chitosan matrix. The composite exhibited significantly enhanced antimicrobial activity compared to chitosan or ZnO alone, underscoring the synergistic effect of the biopolymer–metal oxide combination. By valorizing marine resources, this approach presents an eco‑friendly alternative to synthetic agents, with considerable potential for incorporation into oral healthcare formulations such as toothpaste and mouthwash. Future work should address cytotoxicity evaluation, quantitative in vivo validation, formulation stability, and regulatory pathways to advance these findings into sustainable dental health products.

LIMITATIONS OF THE STUDY

This study shows that S. prashadi-derived nanochitosan–zinc oxide nanocomposites can be made using green methods. They have antimicrobial potential. However, there are limitations. The antimicrobial tests included only four oral pathogens and were done in vitro. No in vivo validation was performed. A basic biocompatibility test was done. More detailed studies on cytotoxicity and long-term safety in oral tissues are needed. The physicochemical analysis was not comprehensive. Particle size distribution, crystallite size, lattice strain, and detailed morphology were not fully examined. More research is also needed to understand the nanocomposite's mechanism against microbes and its drug release pattern. The study did not cover large-scale synthesis, batch consistency, or process optimization. Because of these gaps, future work should include broader antimicrobial testing, advanced material analysis, thorough cytotoxicity and in vivo studies, and strategies for scalable production to support clinical use.

ACKNOWLEDGEMENTS

The authors are thankful to the Department of Prosthodontics & Implantology, Saveetha Dental College and Hospitals, Saveetha Institute of Medical and Technical Sciences (SIMATS), Saveetha University Chennai, Tamil Nadu, for providing the necessary facilities and support.

AUTHOR CONTRIBUTIONS

Kiruthigha Thirumal and Annathai Pitchai: Conceptualization (Supporting), Methodology (Equal), Software (Lead), Validation (Equal), Formal Analysis (Equal), Investigation (Equal), Resources (Lead), Data Curation (Equal), Writing – Original Draft (Lead), Visualization (Lead); Dhanraj Ganapathy: Conceptualization (Supporting), Methodology (Supporting), Writing – Review & Editing (Supporting); Pasiyappazham Ramasamy: Conceptualization (Lead), Methodology (Equal), Software (Supporting), Validation (Supporting), Formal Analysis (Equal), Investigation (Lead), Writing – Review & Editing (Lead), Supervision (Lead), Project Administration (Lead), Funding Acquisition (Lead).

CONFLICT OF INTEREST

The authors declare no competing financial interests or personal relationships that could influence the work reported in this study.

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Supplementary

Figure S1. FT-IR spectral analysis of chitosan from Sepia prashadi (Allapitchai et al., 2024).

Table S1. XRD pattern of nanochitosan–zinc oxide nanocomposites from Sepia prashadi with Full width at half maximum (FWHM).