Dental plaque is recognized as a primary etiological factor in the development of caries, gingivitis, and periodontal disease. Effective control of biofilm is essential to prevent these conditions. Various strategies are currently employed to combat biofilm formation by oral pathogens20. Among these, mouthwashes serve as adjuncts to mechanical plaque control methods, helping to reduce biofilm accumulation. Mouthwashes with antimicrobial properties exert their effects through three primary mechanisms: inducing apoptosis, inhibiting bacterial growth or cellular metabolism, and depending on their concentration, exhibiting either bactericidal or bacteriostatic properties1. Although CHX is widely used for plaque control due to its antimicrobial activity, its long-term use is limited by side effects such as taste alteration, supragingival calculus formation, oral mucosal desquamation, and contraindications in paediatric patients6,7.
Therefore, there is growing interest in safer, plant-based alternatives for effective plaque and biofilm control, such as essential oil-based nanoparticle formulations. Numerous studies have compared CHX with herbal mouthwashes. While herbal formulations may be less effective than chlorhexidine, they offer a safer alternative for oral prophylaxis without adverse effects. Our previous research demonstrated the efficacy of C. citratus EOs as natural anti-biofilm agents, particularly in preventing dental cavities17. The EOs showed significant antimicrobial activity against S. mutans biofilms, highlighting their potential as selective and non-invasive antimicrobial agents. Additionally, ZnONPs synthesized using lemongrass essential oil via green methods exhibited promising antimicrobial activity against oral pathogens. This study aims to formulate and evaluate an herbal mouthwash and compare its efficacy with a commercially available mouthwash. In this study, four mouthwash formulations were prepared, each containing varying concentrations of C. citratus ZnONPs: 5 µg/mL (F1), 10 µg/mL (F2), 50 µg/mL (F3), and 80 µg/mL (F4). The purpose of these variations was to determine the concentration with the most effective antibacterial activity and optimal consistency. The formulation process also involved the use of surfactants like Tween 80 and glycerol, which enhance solubility and reduce surface tension in oil-water solutions by stabilizing the interfacial layers, thus producing a clear and stable mouthwash.
The study evaluated the acidity (pH), stability, irritation potential, and antioxidant properties of the four mouthwash formulations, along with their antibacterial effects against three pathogens. The alcohol-free herbal formulations exhibited a transparent appearance, good water dispersion, and a pleasant aroma. The antioxidant activity, measured using the DPPH free radical scavenging assay, ranged from 34.56 to 55.78%, with the highest scavenging activity observed in the ZnONPs. However, this activity was lower than that reported in previous studies, such as Ulvan-silver nanoparticles at 50 µg/mL or 5% v/v, which exhibited significant antioxidative properties. It is worth considering that higher concentrations might yield enhanced activities21. Moreover, the herbal mouthwash showed greater antibacterial efficacy against S. mutans compared to S. aureus and E. coli, with the highest ZOI of 55 ± 2.5 mm observed against S. mutans across all formulations (F1, F2, F3, F4). This aligns with previous findings on the notable antibacterial activity of lemongrass ZnONPs against oral pathogens. The choice of lemongrass for nanoparticle synthesis reflects the increasing interest in utilizing natural plant properties for therapeutic purposes, as supported by prior studies22,23.
Biofilms serve as protective shelters for microorganisms, promoting pathogen survival. Antimicrobial agents are evaluated for their potency, bacteria-killing rate, and efficacy against biofilms, including their ability to kill bacteria or inhibit biofilm formation24. In this study, a biofilm assay was employed to assess the viability of cells within the biofilm. The F2 concentration of mouthwash effectively killed approximately 80% of biofilm biomass, with significant inactivation of S. mutans,S. aureus and E.coli cells within the biofilm. Furthermore, the highest concentration formulation, F4, nearly eradicated 96% of the biofilm produced by the highly cariogenic S. mutans, demonstrating its potent antibacterial activity. Several studies have reported the effects of different compounds on biofilms but not on planktonic cells, with sub-MIC levels of lemon essential oil and tea extract showing biofilm suppression with minimal impact on planktonic cell growth24,25.
Mouthwashes are a popular oral care product for routine use. This study also included pre-clinical test using zebrafish embryo to investigate potential toxicity. The results indicated that the ZnONPs-mediated mouthwash was less toxic than the commercially available chlorhexidine mouthwash, suggesting its suitability for regular use. The cytotoxic impact was evaluated using a brine shrimp mortality assay, where 80% of live embryo were observed at a concentration of 80 µg/mL, consistent with findings from other studies involving ZnONPs synthesized from coffee bean and xylitol26. In line with this study, research by Shanmugam et al. (2024) also found that ZnONPs synthesized using green methods from lemongrass and mint herbal formulations exhibited promising antimicrobial activity against oral pathogens22.
Bacterial adherence to tooth surfaces is primarily facilitated by sucrose-dependent adhesion, which significantly contributes to biofilm formation. This process is accelerated by the enzymatic conversion of sucrose into glucose, catalyzed by genes such as gtfB/C. The quorum sensing (QS) system further enhances biofilm maturation by enabling bacteria to optimize their physiological responses based on population density and environmental stimuli27. C. citratus EO combined with ZnONPs presents a potent antimicrobial herbal mouthwash, particularly effective against pathogens responsible for dental caries in the oral environment. The key bioactive compounds in CEO include carveol, pinene, and verbenyl acetate, each contributing distinctively to this antimicrobial effect17.
Although the molecular pathways were not directly validated in this study, the proposed mechanism illustrated in Fig. 4 is based on literature-supported roles of CEO-derived compounds and ZnONPs. Carveol disrupts bacterial cell membranes by integrating into the lipid bilayer, increasing membrane permeability, leading to the leakage of essential cellular contents, which ultimately results in cell death and inhibition of biofilm formation a critical factor in the development of dental caries28. Pinene enhances this antimicrobial action by further compromising membrane integrity and inhibiting bacterial metabolic processes. Additionally, pinene’s anti-inflammatory properties help mitigate the inflammation commonly associated with dental caries29. This compound specifically targets bacterial adhesion mechanisms and the production of glucosyltransferases (Gtfs), notably gtfB and gtfC. These enzymes synthesize glucans from sucrose, which are crucial components of the biofilm matrix. By inhibiting gtfB and gtfC, verbenyl acetate reduces glucan synthesis, weakening the biofilm structure and diminishing the virulence of S. mutans. Verbenyl acetate also inhibits bacterial enzyme activity and prevents pathogens from adhering to dental surfaces, thereby obstructing the initial stages of biofilm formation and subsequent caries development30.
Mechanistic insights into the prevention and inhibition of caries by the herbal mouthwash
This study concludes that the observed inhibition of bacterial growth by ZnONPs is attributable to their strong antibacterial efficacy. This remarkable antibacterial effect is largely due to the increased surface area of the smaller nanoparticles and the combined mechanisms described above31. The synergistic action of these compounds, together with ZnONPs, produces a comprehensive antimicrobial effect, making CEOZnONPs mouthwash a promising natural alternative for oral care. Further clinical trials will be required to confirm translational relevance.