1039 D3000 new imprint Word template Vol 13, No 1 (2025) ISSN 2167-8677 (online) DOI 10.5195/d3000.2025.1039 http://dentistry3000.pitt.edu ZnO Nanoparticles and Pepper Fruit Extract Effect on Streptococcus mutans and Staphylococcus aureus Sura A. Jaber College of Den*stry, University of Almaarif, Al Anbar, Iraq Abstract Objec.ves: To test the effect on Streptococcus mutans and Staphylococcus aureus isolates from dental caviHes of different amounts of zinc oxide (ZnO) nanoparHcles and Capsicum annuum L. extract. Methods: Zinc oxide nanoparHcles (ZnO, purity: 99.8%, D50: 10–30 nm) were synthesised from Skyspring Nanomaterials. Pepper fruits were procured from local mar- kets in Babylon. The fruits were cleansed using disHlled water to eliminate soil, air-dried, and subsequently pulverised using an electric grinder. The extract was produced with Soxhlet equipment. Results: The inhibiHon zones for S. mutans at doses of 0.2, 0.4, 0.6, and 0.8 mg/ml were 2.12, 2.27, 2.58, and 2.98 mm, respecHvely. The inhibitory zones for S. aureus measured 2.02, 2.2, 2.45, and 2.68 mm, respecHvely. Significant anHbacterial acHviHes of the pepper fruit extract were demonstrated against both S. mutans and S. aureus. The inhibiHon zones for S. mutans at 100, 200, 3000, and 350 mg/ml doses were 1.24, 1.46, 1.54, and 1.7 mm, respecHvely. The inhibitory zones for S. aureus measured 1.35, 1.56, 1.78, and 1.89 mm, re- specHvely. Conclusion: Both pepper fruit extract and ZnO nanoparHcles possess anHbacterial properHes. ZnO nanoparHcles at a dose of 1.0 mg/ml exhibited the most substanHal inhibiHon zone against S. mutans. In contrast, pep- per fruit extract at 300 mg/ml demon- strated the most significant inhibiHon zone against S. aureus. Open Access Cita%on: Jaber SA. (2025) ZnO Nanopar%cles and Pepper Fruit Extract Effect on Streptococcus mutans and Staph- ylococcus aureus. Den%stry 3000. 1:a001 doi:10.5195/d3000.2025.1039 Received: September 6, 2025 Accepted: September 24, 2025 Published: October 8, 2025 Copyright: ©2025 Jaber SA. This is an open access ar%cle licensed under a Crea%ve Commons AVribu%on Work 4.0 United States License. Email: Dr.sura.ameer@gmail.com Introduc)on For millennia, nature has served as a source of medicinal cures since the dawn of human- ity. Despite this, synthetic drugs often have adverse effects, and it has become more and more apparent that microbes are resistant to these substances. Consequently, numerous developing countries have begun to empha- sise herbal treatments and nanotechnology [1]. Zinc oxide nanoparticles (ZnO NPs) are important microelements that play im- portant roles in many bodily functions. Con- sumption of water and food leads to the ac- quisition of zinc, which the small intestine primarily absorbs and transports to the blood plasma. Many businesses and drug companies use zinc oxide nanoparticles be- cause they might be able to kill fungi, bacteria, diabetes, and inflammation, speed up wound healing, and protect cells from damage. Researchers have generated these nanoparticles using green synthesis tech- niques that use plants, fungi, bacteria, and al- gae [2,3]. The fruits of the pepper plant (Capsicum an- nuum L.) contain the alkaloid capsaicin, which imparts its spicy flavour. Capsaicin is present in elevated proportions in the fruits of spicy peppers. Alkaloids are essential mol- ecules in the process of making medicines and pharmaceuticals. They also have big ef- fects on the structure and function of cell membranes [4,5]. Paprika powder often comprises less than 0.5% essential oil. Pep- per fruits possess a volatile oil level that is comparatively modest, varying from 0.1% to 2.6%, contingent upon the cultivar and the maturity stage at harvest. The primary chemical classes of compounds present in capsicum oil encompass terpenes and their derivatives, alcohols, aldehydes, ketones, carboxylic acids, esters of carboxylic acids, benzene derivatives, naphthalene deriva- tives, hydrocarbons, sulphur and nitrogen- containing compounds, phenolic com- pounds, and carotenoid derivatives [6]. Dental caries (cavitation) is notably severe and persistent among numerous dental is- sues. It is an irreversible microbial illness that impacts the calcified tissues of the teeth, marked by the demineralisation of the inor- ganic component and the degradation of the organic matrix. Dental caries recognises Streptococcus mutans and other ZnO NanoparHcles and Pepper Fruit Extract Effect on Streptococcus mutans and Staphylococcus aureus Vol 13, No 1 (2025) DOI 10.5195/d3000.2025.1039 http://dentistry3000.pitt.edu 2 streptococci as the principal causative agents [7]. Dental plaque, which develops on the tooth surface, particularly in the cervical region, comprises bacteria encased within an organic matrix. This plaque is associated with gingivitis, periodontal disease, and tooth caries [8]. Previous studies have demonstrated the management of dental plaque through physical removal and antimi- crobial toothpaste and mouthwashes [9,10]. New types of bacteria, like Staphylococcus mutans and Staphylococcus aureus, are of- ten linked to cavities and other periodontal diseases in the mouth [11,12]. These bacte- ria are becoming resistant to antibiotics, which is a major global health problem. This is especially applicable in the context of oral infections. These bacteria cause tooth decay and are also linked to the development of systemic illnesses. This shows the im- portance of effective antimicrobial treat- ments in dental care. Even though conven- tional antibiotics have been utilised in the fight against these diseases, the growing re- sistance to these medications has made it necessary to investigate alternative treat- ments [13]. Nanomaterials, including zinc oxide (ZnO) nanoparticles, are among the most promis- ing options for antibacterial treatments. In oral health, zinc oxide (ZnO) has attracted significant attention due to its broad-spec- trum antibacterial properties. These include the ability to combat both gram-positive and gram-negative bacteria, making it a potential therapeutic agent [14]. Recent studies have demonstrated that ZnO nanoparticles can ef- fectively inhibit the growth of S. mutans and S. aureus. This could offer a new approach to treating oral microbial infections. However, the optimal concentration of ZnO nanoparti- cles needed for effective antibacterial activ- ity while minimizing harm to human cells re- mains under investigation [15]. Researchers have long recognised plant-de- rived chemicals for their antibacterial char- acteristics and nanomaterials. Numerous re- searchers are investigating the possibility of natural extracts serving as adjuncts or re- placements to traditional antibiotics [16,17]. Pepper fruit (Capsicum annuum), which is full of bioactive substances like capsaicin, flavonoids, and alkaloids, might be able to change the activity of microbes. This in- cludes antibacterial actions against a wide range of pathogens. Using ZnO nanoparticles in conjunction with plant extracts, such as pepper fruit extract, could increase antibac- terial action. This would reduce the require- ment for higher doses of either agent on its own, potentially lowering the likelihood of resistance development. This study aims to _ind out what happens to strains of Staphylococcus aureus and Staphylococcus mutans that have been taken from dental cavities when different amounts of ZnO nanoparticles and pepper fruit ex- tract are mixed together. The study aims to help the ongoing search for long-lasting al- ternatives to traditional antibiotics in dental care by looking at how well these agents kill bacteria on their own and when mixed to- gether. This research aims to provide in- sights into the potential of these agents as ef- fective treatments for oral infections. Materials and Methods We synthesised zinc oxide nanoparticles/na- nopowder (Speci_ications: ZnO, purity: 99.8%, D50: 10–30 nm, colour: white to light yellow) from Skyspring Nanomaterials (Fig. 1). The common contaminants in the nano- particles consist of Cu (25 ppm), Cd (25 ppm), Mn (25 ppm), Pb (20 ppm), and As (20 ppm). Figure 1. Zinc oxide nanoparticle. We obtained the pepper fruits from local markets in Babylon. We rinsed the samples with distilled water to remove dirt and then dried them in the shade. After drying, we pul- verised the samples using an electric mill [18]. The extraction was conducted via Soxhlet equipment. One hundred grams of the ground sample were placed in the thimble, and methanol at a 70% concentration was added to the round-bottom flask. The system was thereafter maintained at 60°C for 24 hours. Following the extraction process, the methanol was evaporated with a rotary evaporator to isolate the alcohol from the crude extract. The resultant extract was re- frigerated until utilised [19]. Analysis of the Impact of Varied Doses on the Proliferation of Specific Pathogenic Microorganisms We synthesised pepper fruit extract and ZnO nanoparticles using an MSA medium and in- cubated them anaerobically for 48 hours at 37°C before evaluating their antagonistic ac- tivity. Ten bacterial colonies were trans- ferred to create the bacterial inoculum. To get a 0.5 McFarland Standard turbidity, which is equal to 1.5 x 10⁸ cells/ml, the bac- terial suspension was put into a test tube with 5 ml of Brain Heart Infusion (BHI) broth in a clean environment. The infected plates with MSA media were prepared by dispers- ing 0.1 ml of the suspension onto the plates. The plates were maintained at room temper- ature for 15 minutes to facilitate inoculum absorption. A sterile cork borer with a diameter of 7 mm was employed to form wells in the inocu- lated agar plates. A micropipette was used to dispense 50 microliters of different concen- trations of the test chemicals into the wells (0.2, 0.6, 0.8, and 1.0 mg/mL for ZnO nano- particles; 100, 200, 300, and 350 mg/mL for the crude extract). Control plates were made by substituting the crude extract with 50 µl of sterilised distilled water in the wells. The plates were kept at 37°C for 48 hours, and the results were judged by measuring the size of the inhibition zone, which is the area around the well where bacteria can't grow [20]. Statistical Analysis The tests were executed as factorial experi- ments with three replications, employing a completely randomised design (CRD). The data were analysed with the least signi_icant difference (L.S.D.) at a 1% probability threshold (P ≤ 0.01) Results Table 1 demonstrates the strong impact of ZnO nanoparticles on Staphylococcus mutans and Staphylococcus aureus. At 0.3, 0.5, 0.7, and 0.9 mg/ml, the inhibition zones for S. mutans are 2.12, 2.27, 2.58, and 2.98 mm, and for S. aureus they are 2.02, 2.2, 2.45, and 2.68 mm. Table 2 indicates a substantial impact of pep- per fruit extract on S. mutans and S. aureus. At 100, 200, 300, and 350 mg/ml, the zones that stop S. mutans from growing are 1.24, 1.46, 1.54, and 1.7 mm, and the zones that stop S. aureus from growing are 1.35, 1.56, 1.78, and 1.89 mm. Discussion The study shows that ZnO nanoparticles can kill Staphylococcus mutans and Staphylococ- cus aureus bacteria, especially when 0.8 mg/ml of them are present. Nanoparticles may be responsible for this action. These na- noparticles may target subcellular compart- ments of the cell membrane, resulting in cel- lular damage and ultimately leading to cell death. Zinc oxide nanoparticles, also known as ZnO NPs, have demonstrated the ability to alter the construction of bacterial cell walls. ZnO NPs achieve this by influencing the link- age between N-acetylglucosamine and N- acetylmuramic acid in glycans. This, in turn, leads to the formation of pits and a weaken- ing of the cell wall. Along with this, ZnO ZnO NanoparHcles and Pepper Fruit Extract Effect on Streptococcus mutans and Staphylococcus aureus Vol 13, No 1 (2025) DOI 10.5195/d3000.2025.1039 http://dentistry3000.pitt.edu 3 nanoparticles can stick to the surface of pep- tides, which breaks down the glycan struc- ture of the bacterial cell wall [21,22]. The re- sults of this observation are similar to those of earlier research [23], which showed how important the concentration of ZnO nano- particles and the size of their particles are to antibacterial activity. Numerous studies show that the antibacte- rial activity of ZnO nanoparticles is concen- tration-dependent. These nanoparticles are better at killing microbes because they have a larger surface area and are more reactive. The increased surface area results in an in- crease in the number of reactive sites that are available for interactions with bacterial cells. Some people also think that ZnO nano- particles interact with the plasma membrane of bacteria, changing the surface chemistry and function of the membrane, lowering the amount of adenosine triphosphate (ATP), and messing up the cell's energy metabolism. In the end, these alterations have an effect on the stability of the bacterial cell membrane, which finally results in the membrane's dis- ruption [24]. Some types of bacteria, like Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa, can be killed by zinc oxide nano- particles (ZnO NPs). Researchers have found that 0.8%, 1%, and 1.5% of S. aureus bacteria survived at different concentrations of ZnO NPs; 1%, 1.5%, and 5% of P. aeruginosa bac- teria survived; and 2%, 3.7%, and 6% of E. coli bacteria survived [25]. Researchers found that the presence of ZnO NPs was associated with these percentages. Compared to Gram-positive bacteria, which have thicker and more resistant cell walls, Gram-negative bacteria, such as E. coli, have thinner cell walls, which makes them more sensitive to metal nanoparticles. This is be- cause Gram-negative bacteria are more sus- ceptible to metal nanoparticles. Because of this structural difference, nanoparticles can more easily penetrate Gram-negative bacte- ria cells [26]. Nanoparticles can target vari- ous bacterial structures in addition to their action on the cell membrane [27]. These structures include respiratory chain dehy- drogenases and bacterial chromosomes. It has also been shown that metallic nanoparti- cles like ZnO can make reactive oxygen spe- cies (ROS) that kill bacteria. These ROS are produced by creating oxidative stress and in- hibiting the oxidation of metal ions that have been freed [28]. This research also indicates that pepper fruit extract possesses powerful antibacterial characteristics. Since phenolic chemicals al- low the extract to penetrate bacteria cell walls, these properties may be linked to them. The findings of this study are con- sistent with those of other studies [29], which suggested that polyphenols are im- portant bioactive chemicals that are respon- sible for antibacterial activity. There are hy- droxyl groups or phenolic rings in these compounds that might interact with the pro- teins and membranes of bacteria. This could lead to the formation of complexes that stop the cell from working properly [30,31]. Researchers think that bioactive phytochem- icals like _lavonoids, alkaloids, and tannins, known to kill bacteria, make pepper extract bacteria-killing. These components are be- lieved to be responsible for what makes pep- per extract so effective. It has been observed in previous research [32, 33] that plant ex- tracts abundant in phenolic compounds are particularly effective against bacteria be- cause they disrupt their growth and metabo- lism. The active ingredients in pepper fruit extract, especially _lavonoids and alkaloids, help stop the growth of bacteria. This is more proof that pepper fruits can be used as a nat- ural antibacterial. Conclusion The conclusion is that ZnO nanoparticles and pepper fruit extract exhibit strong antibacte- rial effects. Microparticles of ZnO specifically target the cell walls and membranes of bac- teria. On the other hand, bioactive chemicals like phenols, flavonoids, and alkaloids in pepper fruit extract make it work. These re- sults show that ZnO nanoparticles and plant- based extracts may be useful in the fight against bacterial infections. They also offer a viable route for developing antimicrobial medicines based on natural materials and nanomaterials. References 1. Sakoulas, G., & Moellering, R. C. (2004). In- creasing antibiotic resistance among methicillin- resistant Staphylococcus aureus strains. Clinical Infectious Diseases, 46(Suppl), 360-367. 2. Skalniy, A. V., & Rudakov, I. A. 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Brantner, A., Males, Z., Papeljnjak, S., & An- tolic, A. (1996). Antimicrobial activity of Paliurus spina-christi Mill. Journal of Ethnopharmacology, 52, 119-122. Table 1. Effect of different concentrations of ZnO nanoparticles on S. mutans and S. aureus. Con- trol S. aureus S. mu- tans Concentrations mg/ml 0 2.02 2.12 0.2 0 2.2 2.27 0.4 0 2.45 2.58 0.6 0 2.68 2.98 0.8 1.25 1.50 LSD = Table 2. Effect of different concentrations of pepper fruit extract on S. mutans and S. aureus. Control S. auruas S. mutans Concentrations mg/ml 0 1.35 1.24 100 0 1.56 1.46 200 0 1.78 1.54 300 0 1.89 1.7 350 1.76 1.37 LSD =