Antibacterial Effects of ZnO Nanoparticles and Capsicum Annuum Extract on Staphylococcus Mutans and Staphylococcus Aureus Isolated from Dental Cavities

INTRODUCTION
The formation of biofilms and the growth of microorganisms such as Streptococcus mutans and Staphylococcus aureus are the main reasons for dental caries, making it one of the most widespread infectious diseases in the world. Dental caries is one of the most common oral contagious illnesses. It is caused by the demineralization of tooth enamel and dentin due to acids created by bacterial fermentation of dietary carbohydrates [1]. It is necessary to develop alternative antibacterial agents, particularly those derived from natural sources and nanotechnology, because of the increasing prevalence of antibiotic resistance [2]. Streptococcus mutans is believed to be the principal etiological agent among the important microbial agents involved. This assumption is based on its powerful acidogenic and aciduric capabilities and the capacity to manufacture extracellular polysaccharides that facilitate biofilm formation [3]. Meanwhile, Staphylococcus aureus, which has historically been associated with systemic infections, has been found in the oral cavity at high rate and is now recognized as a contributor for secondary caries and periodontal disease [4]. Zinc oxide (ZnO) nanoparticles have gained a lot of interest due to large surface area and strong antibacterial properties against many types of germs. Studies have indicated that ZnO nanoparticles are effective against both gram-positive and gram-negative bacteria, which makes them potential candidates for applications in the field of oral health [5]. Similarly, plant extracts, like Capsicum annuum, have been known for their healing and bacteria-fighting properties. The capacity of Capsicum annuum in degradation of bacterial membranes and interfere with quorum sensing has been demonstrated in a previous study [6]. Because of the biofilm-protected nature of dental plaque and the increasing prevalence of bacterial resistance towards traditional antibiotics, newly developed antimicrobial medicines must be produced [7]. Metal oxide nanoparticles, have emerged as potentially useful options due to high surface area-to-volume ratio, simple to functionalize, and distinctive modes of microbial suppression [8]. The safety profile, affordability, and extensive antibacterial efficacy against oral and systemic infections for ZnO nanoparticles have attracted the attention of scholars [9]. The rupture of bacterial cell membranes, the formation of reactive oxygen species (ROS), and interference with the enzymatic activities of microorganisms are all examples of the antibacterial mechanisms of ZnO nanoparticles [10]. Jones et al. reported that ZnO nanoparticles were highly efficient against gram-positive and gram-negative bacteria. The intensity of the nanoparticles’ activity increased in proportion to their size [5]. Botanical extracts are natural antibacterial agents. Pepper, known as Capsicum annuum, with medicinal and spice benefits has a high concentration of bioactive chemicals, including capsaicin, flavonoids, and phenolic acids, all contributing to its antibacterial capabilities [11]. It has been demonstrated that Capsicum annuum, the primary component responsible for the intense flavor of chili peppers, may disrupt the process of bacterial cell wall construction, the formation of biofilms, and the expression of virulence factors [12]. Previous research has proven that pepper extracts have modest antibacterial properties against oral and gastrointestinal infections. The effectiveness of pepper extracts is frequently impacted by the extract’s concentration and the solvent used during the extraction process [13]. Few studies have examined the antibacterial ability of ZnO nanoparticles and extracts of Capsicum annuum, especially against S. mutans and S. aureus produced from dental caries. This study evaluates and compares the antibacterial activity of ZnO nanoparticles and Capsicum annuum extract at various concentrations against S. mutans and S. aureus isolated from dental cavities.

MATERIALS AND METHODS
This investigation was conducted from January to April 2025 at the Department of Microbiology and Tissue Culture, College of Pharmacy, Mustansiriyah University, Baghdad, Iraq.

Collection and identification of bacterial isolates 
Dental plaque and carious specimens were active dental caries at local dental clinics in Baghdad. The specimens were obtained with sterile cotton swabs and covered in sterile tubes containing nutritional broth. Upon arriving at the laboratory, samples were cultivated on selected media: 

Mitis Salivarius Agar for Streptococcus mutans 
Mannitol Salt Agar for Staphylococcus aureus 
Plates were incubated at 37°C for 24 to 48 h under suitable conditions (CO2-enriched atmosphere for S. mutans). Colonies were characterized using morphological assessment, Gram staining, catalase and coagulase testing (for S. aureus), and biochemical profiling. Verification was further accomplished with commercial identification kits (API Strep and API Staph kits from bioMérieux, France).

Preparation of ZnO nanoparticle suspensions 
ZnO nanoparticles, which are 99.8% pure and have a size between 10 and 30 nm, were obtained from Sky Spring Nanomaterials (USA). The nanoparticles were measured using an analytical balance and dispersed in sterile distilled water via sonication to achieve homogenous suspensions at concentrations of 0.2, 0.6, 0.8, and 1.0 mg/mL. Before each experiment, we made all suspensions and stored them in amber bottles at 4°C to prevent deterioration.

Extraction of Capsicum annuum 
We obtained fresh pepper fruits (Capsicum annuum L.) from local marketplaces in Baghdad. The fruits were meticulously cleaned with distilled water to eliminate surface impurities, air-dried for five days at ambient temperature, and processed into a fine powder using an electric grinder. 

Soxhlet extraction procedure
100 g of fruit powders were placed in a thimble, and Soxhlet equipment was used to extract the ethanol for 6 h. We concentrated the extract at diminished pressure using a rotary evaporator at 45°C. The concentrated extract was diluted in ethanol to yield 100, 200, 250, and 300 mg/mL. Extracts were filtered using 0.22 µm syringe filters and preserved in sterile, amber glass vials at 4°C. 

Antibacterial assay 
The antibacterial efficacy was assessed utilizing the agar-well diffusion technique. Each isolate was produced in sterile saline using a standardized inoculum (0.5 McFarland standard, about 1.5 × 108 CFU/mL). The bacterial suspensions were inoculated onto Mueller-Hinton agar plates using a sterilized cotton swab. 
Wells (6 mm in diameter) were created in the agar with a sterile cork borer. 
100 µL of each ZnO suspension and Capsicum annuum extract was allocated into distinct wells. 
Positive control: Chlorhexidine (0.2%) 
Negative control: Sterile distilled water or ethanol, contingent upon the test group 
Plates were incubated at 37°C for 24 h. The inhibitory zones were quantified with a calibrated digital caliper.

Statistical analysis
All tests were duplicated, and the average inhibition zone and standard deviation were computed for each treatment. Statistical significance was assessed by one-way analysis of variance (ANOVA), followed by Tukey’s post hoc test for group mean comparisons. A p-value less than 0.05 was deemed statistically significant. Data were processed with IBM SPSS Statistics version 25.

RESULTS AND DISCUSSION
The antibacterial efficacy of ZnO nanoparticles and Capsicum annuum extract was assessed against S. mutans and S. aureus using the agar-well diffusion technique. The sizes of the inhibition zones were measured in millimeters, and the data are provided as the mean ± standard deviation from three repetitions.

Antibacterial activity of ZnO nanoparticles
ZnO nanoparticles exhibited a dose-dependent enhancement in antibacterial efficacy against both bacterial species. At the minimal dose (0.2 mg/mL), the inhibition zone for S. mutans measured 2.11 ± 0.10 mm, which incrementally reached 2.97 ± 0.16 mm at 1.0 mg/mL. The same pattern was seen against S. aureus, with inhibition zones ranging from 2.01 ± 0.11 mm to 2.66 ± 0.14 mm (Table 1, Fig. 1).
Antibacterial efficacy of Capsicum annuum extract 
The minimum dose of 100 mg/mL for S. mutans yielded a mean inhibition zone of 1.22 ± 0.08 mm, which reached 1.69 ± 0.11 mm at 300 mg/mL. S. aureus exhibited marginally greater sensitivity, with inhibitory zones measuring between 1.34 ± 0.09 mm and 1.87 ± 0.12 mm (Table 2, Fig. 2). 
The findings demonstrate that ZnO nanoparticles and Capsicum annuum extract display dose-dependent antibacterial properties against oral infections. ZnO nanoparticles demonstrated enhanced effectiveness, particularly at a concentration of 1.0 mg/mL, consistent with previous studies emphasizing the potent antibacterial properties of metal oxide nanoparticles attributed to oxidative stress and membrane disruption [8, 14]. Raghupathi et al. indicated that ZnO nanoparticles have greater efficacy against gram-positive bacteria owing to their robust peptidoglycan coating [15]. Sirelkhatim et al. also confirmed the significance of nanoparticle size in influencing antibacterial efficacy [16]. Furthermore, Rezaei et al. noted the antimicrobial properties of Capsicum extracts on oral flora [17]. Nevertheless, the extract exhibited worse efficacy than ZnO nanoparticles, perhaps owing to restricted diffusion or a decreased concentration of active compounds. The enhanced efficacy of ZnO nanoparticles at a concentration of 1.0 mg/mL can be ascribed to many established processes. ZnO nanoparticles engage with bacterial cell walls, rupturing the membrane and leaking intracellular contents [18]. They also produce ROS agents like hydrogen peroxide, which provoke oxidative stress in bacterial cells, resulting in cell death [19]. Raghupathi et al. [15] showed that ZnO nanoparticles have superior antibacterial efficacy due to high surface reactivity and penetrating capacity. Sirelkhatim et al. [16] observed that ZnO nanoparticles have greater effectiveness against gram-positive bacteria like S. aureus due to variations in cell wall composition. The antibacterial efficacy of Capsicum annuum extract was dose-dependent but inferior to that of ZnO nanoparticles. The observed inhibitory zones exhibited the most pronounced inhibition against S. aureus at 300 mg/mL. It assumed that Capsaicin, the primary active component, destabilize bacterial membranes and interfere with intracellular signaling pathways, including quorum sensing [11]. Although both treatments exhibited activity against the bacterial strains, ZnO has shown superior efficacy against S. mutans. Conversely, Capsicum annuum had marginally greater efficiency against S. aureus at its maximum dosage. S. mutans, with greater acid tolerance and a propensity for biofilm formation, may be more vulnerable to oxidative stress generated by ZnO, whereas S. aureus may demonstrate high sensitivity towards the lipid-soluble characteristics of capsaicin and its membrane-targeting effects [16]. This study is the lack of combination therapies, which might provide synergistic effects. Prior research indicated that the amalgamation of nanoparticles with plant extracts can enhance antibacterial efficacy through synergistic mechanisms [15]. Investigations should examine this synergy, assess cytotoxicity in oral cellular models, and evaluate these agents in vivo for prospective applications in mouthwashes, toothpastes, or coatings for dental materials. Several bioactivities of Capsicum annuum extract presents molecular docking studies to substantiate its potential medicinal uses [20]. The potential of ZnO nanoparticles can formulate novel therapies for antibiotic-resistant bacterial infections [21].

CONCLUSION
This research revealed that ZnO nanoparticles and Capsicum annuum fruit extract possess notable antibacterial properties against S. mutans and S. aureus derived from dental caries. The antibacterial effectiveness of both agents depended on their concentration. ZnO nanoparticles at a 1.0 mg/mL concentration had the most potent antibacterial activity, particularly against S. mutans. This corroborates the growing evidence that nanoscale ZnO compromises bacterial cell integrity and metabolic functions via oxidative stress and direct membrane contact. Capsicum annuum extract exhibited quantifiable and incremental suppression of bacterial proliferation with rising doses, with the most pronounced impact observed at 300 mg/mL. The antibacterial properties are due to active phytochemicals, including capsaicin and flavonoids, which may disrupt bacterial viability and biofilm formation. The findings endorse the prospective application of both ZnO nanoparticles and Capsicum annuum extract as alternative agents in oral healthcare, namely in preventing and managing tooth caries and related infections.

ACKNOWLEDGMENTS
The author gratefully acknowledges the support of Al-Maarif University and the College of Dentistry.

CONFLICT OF INTEREST
The authors declare that there is no conflict of interests regarding the publication of this manuscript.