- Research article
- Open Access
Development of a novel, entirely herbal-based mouthwash effective against common oral bacteria and SARS-CoV-2
BMC Complementary Medicine and Therapies volume 23, Article number: 138 (2023)
Parallel to the growth of the oral healthcare market, there is a constantly increasing demand for natural products as well. Many customers prefer products that contain fewer toxic agents, therefore providing an environmentally friendly solution with the benefit of smaller risk to the user. Medieval and early modern medicinal knowledge might be useful when looking for natural, herbal-based components to develop modern products. Along with these considerations we created, tested, and compared an entirely natural mouthwash, named Herba Dei.
The manufacturing procedure was standardized, and the created tincture was evaluated by GC/MS analysis for active compounds, experimentally tested in cell-based cytotoxicity, salivary protein integrity, cell-free antioxidant activity, anti-bacterial and anti-viral assays, and compared with three market-leading mouthwashes.
Our tincture did not show significant damage in the cytotoxicity assays to keratinocyte and Vero E6 cells and did not disrupt the low molecular weight salivary proteins. Its radical scavenging capacity surpassed that of two tested, partly natural, and synthetic mouthwashes, while its antibacterial activity was comparable to the tested products, or higher in the bacterial aerobic respiratory assay. The active compounds responsible for the effects include naturally occurring phenylpropanoids, terpenes, and terpenoids. Our mouthwash proved to be effective in vitro in lowering the copy number of SARS-CoV-2 in circumstances mimicking the salivary environment.
The developed product might be a useful tool to impede the transmission and spread of SARS-CoV-2 in interpersonal contact and aerosol-generating conditions. Our mouthwash can help reduce the oral bacterial flora and has an antioxidant activity that facilitates wound healing and prevents adverse effects of smoke in the oral cavity.
Nowadays, besides the classical instruments of oral care, like toothbrushes and toothpaste, the use of supplementary tools and materials shows an increasing tendency. Dental floss, interdental toothbrush, and mouthwash can be used to improve oral hygiene .
The two most common dental diseases, caries, and periodontal diseases are bacteria-associated illnesses [2,3,4,5]. It is necessary to reduce the pathogenic oral flora, however, toothpaste might not contain sufficient antibacterial agents and in the oral cavity, the majority of bacteria can be found on the tongue and on the pharynx, which cannot be cleaned mechanically efficiently . Also, the matured biofilm on the teeth is a resistant plaque that can be effectively eliminated by rinsing after a mechanical cleaning [5, 7,8,9]. It is beneficial to use agents which decrease the bacterial populations. These agents can be added to mouthwashes thus, by rinsing, most of the surfaces in the oral cavity can be reached and cleaned. The optimal decrease in bacterial populations can be achieved by synthetic and natural active compounds [10,11,12]. The intended use of mouthwash solutions is to hinder bacterial growth, quorum sensing, and biofilm formation on the oral surfaces [13, 14]. The mechanisms of action include toxicity, membrane disruption, oxidative and acidic stress, interference with carbohydrate metabolism and protein translation [15,16,17]. Although, eradicating all the germs is impossible and would be also a blast because it could lead to opportunistic infections in the long term. Using a strong disinfection procedure before surgical or other invasive treatments are necessary but carrying it out frequently might be dangerous [18,19,20].
A mouthwash must be a versatile product; besides killing germs, it must provide fresh breath, anesthetize wounds, or help heal them with an astringent effect without discoloring the teeth or harming the salivary proteins [21,22,23,24]. Moreover, to decrease the toxic load of our body, it is beneficial to have a daily-used rinse without any coloring agents, preservatives, or additives that might be swallowed or taken up through the mucosa or wounds. An optimal mouthwash should help to prevent serious issues in the oral cavity and must be investigated precisely .
Accordingly, this study aimed at the production of a completely organic herbal-based mouthwash, considering its safety, anti-SARS-CoV-2, and both antibacterial and antioxidant activities.
Research of the current market and the traditional literature
To get a complete picture of the ingredients and active compounds of mouthwashes that are available over the counter, the Hungarian market was surveyed.
The recent statistics about personal care product usage in Hungary in 2021 was accessed at Statista, Ltd. . We also visited 22 pharmacies to trace down the best-selling products. All the selected mouthwashes which represent the market can be bought in pharmacies and drugstores in Hungary and Central Europe. The hazard of their additives was evaluated by the Environmental Working Group (EWG) hazard score system . EWG is a non-profit organization, their Skin Deep database about the cosmetics components combines product ingredient data with information from toxicity and regulatory databases . It uses a color-coded score system; 0–2 (green), 3–6 (yellow), and 7–10 (red) codes for low, moderate, and high hazard ingredients. An online survey was also performed to learn whether this is a real market gap in Hungary currently, or there is no need for a new natural alternative in oral care (Additional File 1). Since there were no entirely natural alternatives on the market, we aimed to process the monastic, folk, and traditional literature for possible ingredients. Four main works from the early modern Hungarian literature were studied to determine the possible composition of the prepared mouthwash. The processed works were the followings; Péter Melius Juhász: Herbárium , Ferenc Pápai Páriz: Pax Corporis , József Csapó: Új füves és virágos magyar kert , translated as “New grassy and flowered Hungarian garden”, a summary of medical botany, and Mihály Kováts military doctor’s book, Magyar Patika , translated as “Hungarian Pharmacy”.
In consideration of what requirements a mouthwash should fulfill, a list of plants was set up based on literature data, that could be suitable as ingredients. An optimal rinse is antibacterial, antifungal, has wound healing, breath freshening, antioxidant, and anti-inflammatory activity, contains only natural ingredients, has a nice taste and color, and the pH suites the oral environment without dissolving the enamel and harming the salivary proteins.
The mixtures were made using the following dried herbs that were purchased from a local producer, who has the appropriate permissions and/or licenses to produce and sell spice and herb products: 4 g Calendula officinalis (prod.nr.: H-110/19, Herbária Ltd., Budapest, Hungary), 1 g Hibiscus rosa-sinensis (prod.num.: H-056/20, Herbária Ltd., Budapest, Hungary), 1 g Hypericum perforatum (prod.num.: H-101/20, Herbária Ltd., Budapest, Hungary), 2 g Juglans regia (prod.num.: H-116/20, Herbária Ltd., Budapest, Hungary), 1.5 g Lavandula officinalis (prod.num.: H-136/20, Herbária Ltd., Budapest, Hungary), 2 g Mentha spicata (prod.num.: H-076/20, Herbária Ltd., Budapest, Hungary), 0.3 g Pimpinella anisum (prod.num.: H-161/19, Herbária Ltd., Budapest, Hungary), 0.2 g Quercus robur (prod.num.: H-145/03, Herbária Ltd., Budapest, Hungary), 2 g Sambucus nigra (prod.num.: H-139/13, Herbária Ltd., Budapest, Hungary). The plant material was reidentified by Bálint Bencze, dentist, oral surgeon, and phytotherapy specialist. No voucher specimen has been deposited in a publicly available herbarium. Plant materials used in this study can be purchased from the producer. Sample quality is ensured by a standardized management system operated by the producer which complies with the International Organization for Standardization (ISO) 9001 and with the International Featured Standards (IFS) requirements (Additional File 2). The characteristics and the applied parts of the selected plants were summarized in Table 1.
A total of 14 g of the prepared mixture was homogenized and deposited in dark glasses. We decided to use 50% ethanol as a solvent because it can extract most of the active ingredients but not the chlorophylls [56, 57]. 1000 mL of 50% ethanol was added to the mixture. The solution was shaken at room temperature for 4 × 30 min daily with ultrasound for 7 days . After centrifugation, the extract was filtered sequentially through an 8–20 μm (AF15), a 1.5–3 μm (AF71H), and a 0.2–0.4 μm (AFST140) pore size filters (Filtrox, St. Gallen, Switzerland). Finally, 250 mL distilled water was added to the solution that was shaken for 6 h . The ethanol content at the end was 40%. 1250 μL of Cinnamomum zeylanicum (prod.num.: P370, Aromax Ltd., Budapest, Hungary) and 1200 μL of Eugenia caryophyllata (prod.num.: P426, Aromax Ltd., Budapest, Hungary) essential oils (EOs) were added to 1 L of the previously prepared solution. Both essential oils were produced with the steam distillation method and have a quality license in point of active compound content in accordance with the Ph. Hg. VII (Additional File 3). The completed tincture was named Herba Dei (HD) mouthwash concentrate. The production of the mixture was standardized according to the above protocol. The concentrated tincture showed a pH of 5.5–6. The final developed product was registered at the European Commission cosmetic products notification portal (CPNP) under the reference number CPNP1760385. The tested samples were stored in the dark at 4 °C until the analyses.
The EWG scoring for the selected ingredients in our product is summarized in Table 2.
Gas chromatography/mass spectrometry (GC/MS)
The main oily compounds of the tincture were determined with GC/MS using an Agilent 6890 N analyzer with the 5973N mass-selective detector and an HP-5MS capillary column (30 m × 250 μm × 0.25 μm) (Agilent Technologies, Inc., Santa Clara, CA, USA). High-purity helium (6.0) served as carrier gas at a 1.0 mL/min constant flow rate. The GC oven temperature was programmed to increase from 60 °C (3 min isothermal) to 200 °C at 8ºC/min (2 min isothermal), from 200-230ºC at 10 ºC/min (5 min isothermal), and finally from 230–250 °C at 10 °C/min (1 min isothermal). Samples were introduced by direct injection (splitless); 1 μL (10 µL/mL sample in n-hexane) was injected at 0.7 mg/mL velocity with an Agilent 7683 autosampler. The injector temperature was 250 °C and the split ratio was 1:50 . Samples were compared to analytical standards (Sigma Aldrich, Munich, Germany) in three technical replicates for the following marker molecules: cinnamaldehyde, cinnamyl acetate, eugenol, linalool, beta-caryophyllene, 1,8-cineole, trans-anethole, alpha-pinene, beta-pinene, carvacrol, para-cymene, para-mentha-1,4-dien. References for the active oily compounds and their presence in the selected plants and related species are collected in Additional File 4.
Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) on salivary proteins
The effect of HD on salivary proteins was investigated by SDS-PAGE which was performed as described by Laemmli . The separated protein bands were visualized using silver staining . Unstimulated saliva was collected 8 h after eating and tooth brushing from healthy volunteers. The salivary protein investigation was the only experimental setup in this study that required ethical approval. The Regional and Local Research Ethical Committee approved this research under the registration number 5022—PTE 2015. This permission is valid until 31 January 2023. Immediately before collection, three times rinsing with sterile saline were performed. After centrifugation (5000 rpm, 5 min) the supernatant was homogenized and 400 µL of it was mixed with 400 µL of the mouthwash dilutions and other investigated substances for 60 s, as long as a normal rinsing maximally lasts. After another centrifugation step (13000 rpm, 5 min) 200 µL supernatant was separated for SDS-PAGE. 50 µL sample buffer (five times diluted) was added, then the mixture was boiled for 5 min at 100 °C. 10 µL processed sample was loaded to the gel. Gel concentration was set to 12.5%. As a marker, 10 µL bromophenol blue and low molecular weight marker set (Amersham Biosciences, Amersham, UK) was used. The running time was 45 min at constant 120 V, initial 20 mA, not exceeding 2W. The gel was fixed with glutaraldehyde and stained with colloidal silver. During the analysis, a calibration curve was created on the molecular weight markers using the relative migration distance (pixels) and log10 of the exact molecular masses (kDa).
Cytotoxicity test by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay
Cytotoxicity of the mouthwash was tested on human immortalized keratinocyte cell line, HaCaT (kindly provided by the laboratory of Prof. Tamás Bíró, Department of Immunology, Faculty of Medicine, University of Debrecen, Hungary). Cells were treated with the mouthwash in 96 well plates at 80–90% confluency with a dilution range of 1:4, 1:5, 1:6.67, 1:10 and 1:20 in complete Dulbecco's Modified Eagle Medium (DMEM, Merck, Burlington, MA, USA) for 60 s, along with untreated controls. All the cells were washed with Hanks’ buffer followed by the addition of 150 µL freshly prepared MTT stock in Hanks’ buffer, supplemented with glucose (Merck, Burlington, MA, USA) in each well. The treated plates were incubated for 4 h at 37 °C in 5% CO2. The MTT supernatant was removed and 100 µL dimethyl sulfoxide (DMSO, Merck, Burlington, MA, USA) was added to the cells followed by shaking on a plate shaker for 5 s. The absorbance of the dissolved formazan was measured at 570 nm on a Perkin Elmer EnSpire Multimode plate reader (Perkin Elmer, Waltham, MA, USA) in three technical replicates.
Antioxidant capacity measurement by 2,2-Diphenyl-1-Picrylhydrazyl (DPPH) radical scavenging assay
Three different, popular, commercially available mouthwashes were selected, which were Listerine Freshburst (Johnson & Johnson, New Brunswick, NJ, USA), Elmex Sensitive (GABA International Holding GmbH, Münchenstein, Switzerland), and Cserszömörcés (Herbária Zrt, Budapest, Hungary) to compare our developed product with. The appropriate concentration of HD was determined as 4-times dilution of the original tincture based on the previous cytotoxicity and SDS-PAGE experiments. To assess the antioxidant capacity, a radical scavenging assay was performed on the mouthwashes, which is based on the absorbance decrease of the stable organic radical DPPH [33, 63]. In brief, 50 µL acetate buffer (100 mM, pH 5.5) followed by 50 µL blank / standard / mouthwash sample dilutions (250 mg/mL – 0.1 mg/mL) and finally 100 µL of 200 µM DPPH (dissolved in 96% ethanol, Sigma-Aldrich, Munich, Germany) were pipetted in 96-well microplates. After 60 min incubation in the dark at room temperature, changes in the absorbance at 517 nm were measured on a Perkin Elmer EnSpire Multimode plate reader (Perkin Elmer, Waltham, MA, USA). The results were compared to serial dilutions of Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid, Sigma-Aldrich, Munich, Germany) water soluble vitamin E standard. All experiments were done in three technical replicates.
Antimicrobial activity tests
The tests were carried out with the most common oral pathogens, which have a proven role in caries, periodontal and inflammatory diseases, summarized in Table 3.
The tested mouthwashes were Listerine Freshburst, Elmex Sensitive, Cserszömörcés and HD. Main active ingredients of Listerine Freshburst are thymol, eucalyptol, menthol, and methyl salicylate. Elmex Sensitive contains sodium fluoride, amine fluoride, levulinic acid, sodium levulinate as main compounds. Active ingredients in the Cserszömörcés are tannin extracts with high tannic acid content and thyme. Each mouthwash was used in the recommended concentrations written in the product sheet. The appropriate concentration of HD was determined as 4-times dilution of the original tincture based on the previous cytotoxicity and SDS-PAGE experiments.
Disc diffusion assay
To determine the antimicrobial activity of the mouthwashes, first we performed a disc diffusion assay. Adequate agar medium, such as Mueller–Hinton (Sigma-Aldrich, Munich, Germany), blood agar (Sigma-Aldrich, Munich, Germany), Sabouraud agar (Sigma-Aldrich, Munich, Germany), Rogosa agar (Sigma-Aldrich, Munich, Germany) was used with 1.8% agar concentration.
Our controls were 40 mg/mL Gentamicin (Sandoz, Basel, Switzerland), 2% chlorhexidine-digluconate (CHX, Molar Chemicals, Halásztelek, Hungary), and 10% ethanol (Molar Chemicals, Halásztelek, Hungary). Experiments were performed in five technical replicates.
10 µL of the microbial suspensions with 0.4 optical density (measured at 590 nm) were dispensed respectively on the agar surface on a 92 × 16 mm dish. Following 30 min incubation, a single sterile paper disc was placed on the agars loaded with 10 µL of the test samples. After 72 h incubation at 37 °C, the plates were scanned and digitally evaluated with a densitometry software using a Kodak Image Station 2000R (Kodak, Rochester, NY, USA). The largest diameter of inhibition zone was determined .
To detect microbial aerobic capability, the fluorogenic agent resazurin was used. The irreversible reaction of resazurin to resorufin depends on the reduced form of nicotinamide adenine dinucleotide phosphate (NADPH) or nicotinamide adenine dinucleotide (NADH) which are the reductants in the presence of the adequate enzymes .
First, the optimal OD for the microbial suspensions and the resazurin concentration were determined on a Hitachi F-4500 Fluorescence Spectrophotometer (Hitachi High-Technologies, Tokyo, Japan) using 2.5 mL sterile cuvettes (BrandTech, Essex, CT, USA).
Based on the results of the optimization step, a fluorescence assay was performed at 540 nm excitation and 590 nm emission wavelengths using a Biotek Synergy HT plate reader (Santa Clara, CA, USA), on 96-well flat bottom plates. Into 200 µL broth 10 µL bacteria sample plus 5 µL resazurin (final concentration of 5 µM) were added. The mixture was supplemented up to 250 µL with sterile saline solution. The fluorescence of 200 µL samples was measured after 60 min. 10% ethanol was used as control. Absolute control contained inoculated broth without resazurin to exclude background fluorescence changes. Tests were carried out in five technical replicates.
Anti-Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) assay
Before the anti-viral tests, toxicity of the mouthwash on Vero E6 cells was inspected using CellTiter-Glo® adenosine triphosphate (ATP) quantification kit (Promega, Madison, WI, USA). Briefly, the cells were incubated with the mouthwash for 30 min, then it was replaced by DMEM (Lonza, Basel, Switzerland) with 2% heat-inactivated FBS (Gibco, Waltham, MA, USA), and 1% Pen-strep (Lonza, Basel, Switzerland). Following a two-day long incubation CellTiter-Glo reagent was added to the cells and the luminescence signal was measured on a Perkin Elmer EnSpire Multimode plate reader (Perkin Elmer, Waltham, MA, USA) .
To examine the anti-SARS-CoV-2 activity of the HD mouthwash, Vero E6 cells were infected with a Hungarian SARS-CoV-2 isolate (GISAID ID: EPI_ISL_483637). The mouthwash was diluted in a 1:3 ratio in sterile distilled water, as it is determined by the previous experiments. For each experiment the mouthwash was diluted freshly. The 4-times diluted mouthwash was mixed in a 1:1 ratio with 0.3% bovine serum albumin (BSA) solution as an interfering substance [75,76,77]. 25 µl aliquots were mixed with 25 µl SARS- CoV-2 virus isolate (MOI: 0.01). After 1 min of exposure time, activity was neutralized by tenfold dilution in DMEM (Lonza, Basel, Switzerland), containing 2% heat-inactivated fetal bovine serum (FBS) (Gibco, Waltham, MA, USA) and 1% Pen-strep (Lonza, Basel, Switzerland). The total volume of the viral-oral wash solution was then added to Vero E6 cells. 30 min later the viral-oral wash solution was replaced by DMEM (Lonza, Basel, Switzerland) supplemented with 2% heat- inactivated FBS (Gibco, Waltham, MA, USA) and 1% penicillin–streptomycin (Lonza, Basel, Switzerland) . Two days post infection, the cells were inspected under light microscope and ribonucleic acid (RNA) was extracted from the supernatant. Bio-Rad (Hercules, CA, USA) QX 200 Droplet- Digital PCR system was used to determine the viral copy number from the supernatant. The reaction was RNA-dependent RNA Polymerase (RdRp) gene specific, the primers and probes (Charité/Berlin assay) were ordered from Integrated DNA Technologies (Coralville, IA, USA). The RNA extracts were 100-times diluted prior to the droplet digital PCR. The experiments were done four times with 3 replicates.
Analysis and statistics
During the analysis of the SDS-PAGE experiments equation of the linear regression was used to define the molecular weight of the selected bands after measuring their relative migration distance on the gel digital photograph.
The IC50 value (the dilution of the mouthwash in percentage needed to scavenge 50% of the DPPH) in the antioxidant capacity measurements for the tested mouthwashes were calculated by linear regression using the scavenging activities vs. amount of the mouthwash in each sample. The lower the IC50 value of the sample, the higher its antioxidant activity. The radical scavenging capacity of the mouthwash in percentage of the blank was calculated by the following equation:
where A0 and A1 are absorbances of the blank and the samples, respectively .
100% dilution represented the recommended concentration of each mouthwash.
Pearson correlation and linear regression were used to assess the correlation between antioxidant capacity and antimicrobial activity.
One-way ANOVA and Dunnett’s post hoc test was used to compare whole lane densities in the SDS-PAGE experiments, to compare the results of the cytotoxicity assay, to analyze differences in the antioxidant capacity measurements, and to assess changes in viability of Vero E6 cells and copy numbers of SARS-CoV-2.
Two-way ANOVA with Dunnett’s post hoc test was used to compare band densities across the lanes in the SDS-PAGE experiments, to the statistical analysis in the disc diffusion assay, and to analyze the result of the resazurin assay.
Two-way ANOVA with Sidak's multiple comparisons test was used to compare the effectiveness of the mouthwashes against Gram positive and negative bacteria.
Level of significance was set at 0.05 for all the tests.
Integrated pixel density of the whole lanes and selected bands on the SDS-PAGE gel photograph were measured with Fiji (ImageJ2) software. Analysis and graphing were carried out in GraphPad Prism 8 (GraphPad Software, San Diego, CA, USA). Photoshop (Adobe Systems, San José, CA, USA) was used for creating figures and processing images.
According to the openly available results on Statista Global Consumer Survey on the personal care product usage in Hungary, 90% of the responders use oral and dental care products regularly . Our market survey was filled in by 381 volunteers (274 women, 108 man). More than half of the responders (60.70%) were between the age of 19–25. 54.86% of the total had university education or higher degree. 68.24% of the volunteers paid a visit not more than once a year at a dentist. Out of the total, 204 people were regular mouthwash users, who buy their favorite products mostly in hypermarkets and in cosmetic stores. The most popular product was Listerine: 44.12% of the regular users were applying it. 305 volunteers (80.05% of all responders) prefer the products from natural basis. All the regular users and 5 non-users stated that they would buy a mouthwash which has only natural ingredients and are even ready to visit herbal specialty stores to obtain it. The most important parameters based on which the responders choose the products are presented in Fig. 1.
Our online research on the ingredients included selected products from the Hungarian market. Researching the market supply, we found that all the rinses contain at least one synthetic compound, additive or preservative. The number of ingredients of the rinses’ ranges from 6 to 22 (Additional File 5).
Quantitative analysis of the active compounds of the ingredients using GC/MS
The quantitative assessment of the completely prepared mouthwash revealed that the main oily compounds responsible for biological effects are predominantly cinnamaldehyde and eugenol (Fig. 2).
SDS-PAGE on salivary proteins
Analyzing whole-lane densities (a-h on Fig. 3A), the SDS-PAGE experiments revealed that HD did not disturb the salivary protein integrity in either of the concentrations (Fig. 3C). Significant bands were also chosen to cover approximately all regions of the lanes in the low molecular weight range (1–7 in Fig. 3A). The investigated bands at the regions of 13–14 kDa (1), 18–19 kDa (2), 26–27 kDa (3), 39–40 kDa (4), 47–48 kDa (4), 54–55 kDa (5), 67–68 kDa (6), 78–79 kDa (7) did not show any statistical difference in their integrated pixel density compared to the native, untreated saliva (lane h) (Fig. 3A, D). Exact p values are listed in the Additional File 6. Original, uncropped gel photograph is presented in the Additional File 7.
HD mouthwash showed minimal cytotoxicity to keratinocyte cells
The viability of HaCaT cells did not show significant decrease following 60 s treatment with HD. All the concentrations were compared to the control, no statistical difference was found. At the 4-times dilution of the mouthwash, only a 5.32% decrease could be detected compared to the control samples (Fig. 4). P values are listed in the Additional File 6.
Herbal-based mouthwashes show high antioxidant capacity
Out of the four tested mouthwashes Cserszömörcés exerted the highest radical scavenging capacity with an IC50 value at the dilution of 0.795%. HD showed the second highest antioxidant activity with the IC50 value at the dilution of 5.005%. Listerine Freshburst was less effective in scavenging radicals and had an IC50 value at 62.087%. Elmex Sensitive did not show any antioxidant activity in our experimental setup; IC50 value could not be interpreted in this case (Fig. 5).
The antimicrobial activity of HD is comparable with that of the selected, commercially available mouthwashes in disc diffusion assay
HD in the recommended concentration showed lower antimicrobial activity against all the tested microbes compared to the positive controls, such as gentamicin (p < 0.0001), and CHX 2% (p < 0.0001) but had significantly higher effects than the 10% ethanol (p < 0.0001), which is the solvent of the mouthwash.
HD turned out to be more effective than Listerine Freshburst against all the tested bacteria (p < 0.0001), except for Lactobacillus plantarum, where Listerine Freshburst had the highest activity among all the tested mouthwashes.
HD showed similar effects without statistically significant difference as Elmex Sensitive when looking at Streptococcus mutans but had lower effect regarding Bacillus subtilis (p < 0.0001), Staphylococcus aureus (p < 0.0001), Streptococcus sanguinis (p < 0.0001). At the samples of Pseudomonas aeruginosa (p < 0.0001), Escherichia coli (p < 0.0001) and Lactobacillus plantarum (p < 0.05) HD was slightly more effective than Elmex Sensitive.
The activity of HD was significantly higher than that of the other herbal-based mouthwash, Cserszömörcés (p < 0.0001) at all the investigated bacteria (Fig. 6A). Mean diameter of inhibition zones for each bacterium and tested mouthwash are listed in Table 4. P values are listed in detail in the Additional File 6.
The aerobic respiration of bacteria shows significant decrease following HD treatment in resazurin assay
The bacterial aerobic respiration measured in suspension based on the irreversible reaction of resazurin to resorufin following 30 min treatment with HD was significantly lower compared to all the tested mouthwashes, to 10% ethanol and to the control sample. P value is less than 0.0001 in all the comparisons (Fig. 6B).
All the tested mouthwashes are effective against both Gram positive and negative bacteria
No significant difference could be detected in the activity of the tested mouthwashes against Gram positive or negative bacteria (Fig. 6C). Details of the statistical test and p values are listed in Additional File 6.
Antioxidant capacity of the mouthwashes did not correlate with their antibacterial activity
Results of the resazurin and the disc diffusion assay were correlated to the antioxidant capacity of the different mouthwashes measured in the DPPH assay. No correlation could be revealed between the antioxidant and antibacterial activity of the tested mouthwashes (Fig. 7).
HD mouthwash significantly lowered the SARS-CoV-2 copy number in vitro
A separate cytotoxicity test was performed on Vero E6 cells to assess the toxicity of the mouthwash exactly in the same conditions as the anti-SARS-CoV-2 experiments were carried out. HD in 4 × dilution itself and HD 4 × diluted + BSA did not lower the ATP content of the cells compared to the control.
Inhibition of SARS-CoV-2 replication is well-marked at the HD 4 × diluted + BSA samples compared to the control (p < 0.0001). Although the statistical probe did not show, the 4 × diluted HD itself (mean copy number: 481.5) also caused inhibition in the replication of the virus compared to the control (mean copy number: 705.5) and to the 10% ethanol (mean copy number: 712.2). This experiment also proved that it is not the 10% ethanol solvent of our mouthwash that might be responsible for the anti-viral effects, rather it is due to the herbal-based active compounds (Fig. 8).
In this work a novel, herbal-based mouthwash was created and evaluated using a combination of early pharmaceutical knowledge and modern chemical and biological testing methods. With this product, we aim to contribute to the oral healthcare market providing an entirely natural alternative to oral rinses and to contain the transmission of SARS-CoV-2. In the time of intensified science and vaccination denial , we hope to target those dental care patients with this mouthwash who oppose synthetic products, or even the SARS-CoV-2 vaccination.
The tight relationship between oral health, general well-being and life satisfaction is unquestionable. Oral health is not just an issue that can be approached from a subjective point of view but has exact economic and medical concerns in the western world, as well as in developing countries [79, 80]. Periodontal diseases and odontogenic infections can lead to secondary systemic consequences, such as cardiovascular problems, pneumonia, diabetes mellitus or chronic kidney disease, each of them exerting significant health-economic impact [81,82,83].
Herbal-based phytochemicals have gained an advantage in recent years in oral health care which is not only a result of the need for economic solutions, especially in developing countries, but also because they are an effective alternative to the problem of the multidrug-resistant pathogens and to the side effects of the synthetic products [25, 84]. Another important aspect is the environmental load of the cosmetics and healthcare solutions both at the level of manufacturing  and at the end-user . Natural, local, renewable, and herbal-based components offer an alternative in case the industry faces challenges because of the lack of raw materials . To support and establish sustainability and reduce water pollution it is wise to choose products based on their low carbon footprint and biodegradability.
In the recent decades, a concern has been raised about toxic chemicals in our body originating from cosmetics . Considering the tendency of health care product usage nowadays, we apply approximately up to 100 chemicals a day. Natural substances in this regard are less likely to have side effects . For example, essential oils are “Generally Recognized as Safe” (GRAS) by the United States Food and Drug Administration (FDA) to be used in food and cosmetics by experts without safety issues [33, 90].
Besides that HD is biodegradable, the toxicity and salivary protein integrity experiments showed that the toxic load exerted by the mouthwash in the oral cavity is very low. Moreover, considering the ethanol content of HD, which serves as a solvent for the non-polar compounds, a recent systemic review and meta-analysis revealed that there is no sufficient evidence to support the proposition that ethanol containing mouthwashes contribute to the development of oral cancer .
In case of oral care products, the importance of keeping the integrity of the oral protein environment must be emphasised, because the saliva contains proteins which are important parts of the local digestive and defence system. Selectively or entirely disrupting these factors every day can lead to just as serious consequences as the untreated periodontal diseases [92, 93]. Although, proteins were not defined by using antibodies, pixel density was measured in the SDS-PAGE experiments at regions which are reported to be around or near to the molecular weight of the major salivary proteins, such as lactoferrin and lactoperoxidases at 78–80 kDa [94, 95], amylase isoforms between 55–65 kDa [96, 97], defensins around 40–45 kDa , proline-rich protein isoforms in three or more major bands from 14 to 40 kDa , lysozyme, cathelicidin from 15 to 18 kDa [100, 101], salivary cystatins  and very low molecular weight proline-rich proteins  at 13–15 kDa. Our product performed very well at this key point, proving that efficient antibacterial activity can be reached using natural ingredients as well, without disrupting the oral environment the mouthwash must work in.
Oral care includes the treatment of any wounds that may occur in the oral cavity. A mouthwash besides the cleaning effect should facilitate the contraction and healing of wounds by mediating inflammation through antioxidant capacity . Moreover, antioxidants can also compensate for the local irritative agents like smoke, can help reduce halitosis , and are as effective in reducing bleeding as CHX solutions . Including herbal-based compounds with proven radical scavenging activity could be a possibility in this regard. Phenylpropanoids, terpenes, and terpenoids are present in significant quantities in plant extracts used to create our mouthwash and are already proven to have great potential in preventing oxidative damage [106, 107]. Bitter components, like tannins, and flavonoids in our product can reduce the amount of reactive oxygen species and induce angioneogenesis and epithelial cell proliferation, promoting wound healing . The anaesthetic effect must be mentioned as well, which is a primary requirement in the treatment of oral lesions, because of the rich innervation of the mouth.
The accumulation of microbial flora is responsible for plaque and biofilm formation on the oral surfaces [5, 6]. The capability of herbal-based extracts and their active components to inhibit the growth of these microorganism populations have been previously published [108, 109]. Our natural mouthwash formulation was found to be rich in terpenoids which include carvacrol, cineol, cymene, eugenol, linalool, and pinene. Previously reported data suggest that the bacterial cell wall and membrane disruption by these molecules along with ATP and ion leakage occur due to diversely positioned hydroxy groups around the phenolic rings of the terpenoids, which might be one of the major causes for the antimicrobial property of the herbal-based oily compounds [110, 111]. Cinnamaldehyde is proven to cause membrane disruption by forming lipid raft aggregates . Eugenol has dose-dependent inhibiting effect on bacterial growth in the log phase, and on ATP generation . β-caryophyllene is reported to inhibit bacterial efflux pumps , and to cause non-selective pore formation in bacterial membrane . Linalool interferes with the tricarboxylic acid cycle and glycolysis pathway . Trans-anethole was efficient in increasing susceptibility to other anti-microbial agents, and in hindering biofilm formation thus, proved to be able to protect against S. aureus recolonization . Carvacrol had effects on bacterial membrane ion gradients, even without causing significant ATP leakage . Alpha-pinene induced apoptosis by increasing the caspase-3 activity , but had significant synergistic interactions as well, which might be promising to various therapeutic fields .
The microbial susceptibility analysis done on the tested mouthwashes showed that our plant extract and essential oil-based natural mouthwash formulation successfully inhibited the growth of the usual Gram-positive and negative oral bacteria, which is consistent with previous studies made on herbal-based products [120,121,122]. Although the growth inhibition by HD was significant, it was also less robust than a treatment with antibiotics or CHX, which is not a drawback, because in everyday use it is not beneficial to completely eradicate the oral flora.
The significance of oral care regarding respiratory virus infections and transmission has been proven in the past few years during the COVID-19 pandemic more than ever . Oral health care products act at the front sites when it comes to upper respiratory pathogens thus, their importance in adding a level to our defence came to the focus recently [77, 124]. HD proved to be effective in lowering the viral copy number of SARS-CoV-2 under in vitro conditions, anticipating the possibility that it could reduce the viral load of saliva, contributing to infection control not only in the everyday interpersonal contact, but under aerosol-generating conditions, such as dental treatments, achieving all this in a natural way. Our mouthwash was performing best in the anti-viral assay at conditions which are mimicking the intraoral environment thus, are the closest to the intraoral usage. As for the treatment of COVID-19 related oral complications, the most important features of a mouthwash are the anti-viral, antibacterial, and anti-inflammatory activity. Since SARS-CoV-2 might bind to salivary mucins, and to the angiotensin-converting enzyme 2 (ACE2) receptor on the tongue, resulting in the distortion of gustatory sensation [123, 125], reducing the viral load of the saliva has a direct beneficial effect on the development of dysgeusia and oral lesions. In most cases a coinfection is present in the oral lesions and no bacterial sensitivity is provided to support treatment  thus, it might be beneficial to use a product which has an acceptable efficiency against a wide spectrum of Gram positive and negative bacteria but does not eradicate the oral flora entirely.
Our results support the idea of using herbal-based antioxidant, anti-bacterial and anti-viral components as the major elements of mouthwashes and oral cleansers. Moreover, these components based on their anti-halitotic and flavouring effects, have the potential to be applied as natural active ingredients in mouthwashes, making the use of additional flavouring and preservatives unnecessary . Following the licencing procedure and market appearance, our product gained a significant popularity among local oral health specialists and patients who were looking for natural alternatives which are experimentally evaluated. Local manufacturing ensures a cooperative control over production, as a beneficial consequence of which wasting could be reduced to zero.
Our product proved to be effective against the most common oral bacteria without disrupting the oral salivary environment, and in reducing SARS-CoV-2 copy number in vitro. Moreover, its antioxidant activity might help in healing wounds and to compensate for irritative agents.
Availability of data and materials
Data generated or analyzed during this study are included in this published article and its supplementary information files, except for the raw data of the market survey questionnaire, which is available upon reasonable request from the corresponding author.
Commission cosmetic products notification portal
Environmental Working Group
Gas chromatography/mass spectrometry
Sodium dodecyl sulfate–polyacrylamide gel electrophoresis
Human immortalized keratinocyte cell line
Dulbecco's Modified Eagle Medium
Reduced form of nicotinamide adenine dinucleotide phosphate
Reduced form of nicotinamide adenine dinucleotide
Severe Acute Respiratory Syndrome Coronavirus 2
Bovine serum albumin
Fetal bovine serum
United States Food and Drug Administration
Aspinall SR, Parker JK, Khutoryanskiy VV. Oral care product formulations, properties and challenges. Colloids Surf B Biointerfaces. 2021;200:111567.
Laudenbach JM, Kumar SS. Common dental and periodontal diseases. Dermatol Clin. 2020;38(4):413–20.
Zou J, Meng M, Law CS, Rao Y, Zhou X. Common dental diseases in children and malocclusion. Int J Oral Sci. 2018;10(1):7.
Laudenbach JM, Simon Z. Common dental and periodontal diseases: evaluation and management. Med Clin North Am. 2014;98(6):1239–60.
Curtis MA, Diaz PI, Van Dyke TE. The role of the microbiota in periodontal disease. Periodontol 2000. 2020;83(1):14–25.
Arweiler NB, Netuschil L. The oral microbiota. Adv Exp Med Biol. 2016;902:45–60.
Sterzenbach T, Helbig R, Hannig C, Hannig M. Bioadhesion in the oral cavity and approaches for biofilm management by surface modifications. Clin Oral Investig. 2020;24(12):4237–60.
Chałas R, Wójcik-Chęcińska I, Woźniak MJ, Grzonka J, Święszkowski W, Kurzydłowski KJ. Dental plaque as a biofilm - a risk in oral cavity and methods to prevent. Postepy Hig Med Dosw (Online). 2015;69:1140–8.
Veerachamy S, Yarlagadda T, Manivasagam G, Yarlagadda PK. Bacterial adherence and biofilm formation on medical implants: a review. Proc Inst Mech Eng H. 2014;228(10):1083–99.
Inácio Silveira DQ, Lia EN, Massignan C, Stefani CM. Natural products for the treatment of denture stomatitis: a systematic review. J Prosthet Dent. 2023;129(3):413–24. Epub 2021 Jul 8.
Kouidhi B, Al Qurashi YM, Chaieb K. Drug resistance of bacterial dental biofilm and the potential use of natural compounds as alternative for prevention and treatment. Microb Pathog. 2015;80:39–49.
Chinsembu KC. Plants and other natural products used in the management of oral infections and improvement of oral health. Acta Trop. 2016;154:6–18.
Husain FM, Ahmad I, Khan MS, Al-Shabib NA. Trigonella foenum-graceum (Seed) extract interferes with quorum sensing regulated traits and biofilm formation in the strains of pseudomonas aeruginosa and Aeromonas hydrophila. Evid Based Complement Alternat Med. 2015;2015:879540.
Takenaka S, Sotozono M, Ohkura N, Noiri Y. Evidence on the use of mouthwash for the control of supragingival biofilm and its potential adverse effects. Antibiotics (Basel). 2022;11(6):727.
Du GF, Yin XF, Yang DH, He QY, Sun X. Proteomic investigation of the antibacterial mechanism of. J Proteome Res. 2021;20(5):2319–28.
Walsh SE, Maillard JY, Russell AD, Catrenich CE, Charbonneau DL, Bartolo RG. Activity and mechanisms of action of selected biocidal agents on gram-positive and -negative bacteria. J Appl Microbiol. 2003;94(2):240–7.
Devi KP, Nisha SA, Sakthivel R, Pandian SK. Eugenol (an essential oil of clove) acts as an antibacterial agent against Salmonella typhi by disrupting the cellular membrane. J Ethnopharmacol. 2010;130(1):107–15.
Samaranayake L, Matsubara VH. Normal Oral Flora and the Oral Ecosystem. Dent Clin North Am. 2017;61(2):199–215.
Kosutic D, Uglesic V, Perkovic D, Persic Z, Solman L, Lupi-Ferandin S, Knezevic P, Sokler K, Knezevic G. Preoperative antiseptics in clean/contaminated maxillofacial and oral surgery: prospective randomized study. Int J Oral Maxillofac Surg. 2009;38(2):160–5.
Dammling C, Abramowicz S, Kinard B. Current concepts in prophylactic antibiotics in oral and maxillofacial surgery. Oral Maxillofac Surg Clin North Am. 2022;34(1):157–67.
Grohe B, Mittler S. Advanced non-fluoride approaches to dental enamel remineralization: the next level in enamel repair management. Biomater Biosyst. 2021;4:100029.
Sruthi S, Ashtami J, Mohanan PV. Biomedical application and hidden toxicity of Zinc oxide nanoparticles. Mater Today Chem. 2018;10:175–86.
Bilal M, Iqbal HMN. An insight into toxicity and human-health-related adverse consequences of cosmeceuticals - a review. Sci Total Environ. 2019;670:555–68.
Gasmi Benahmed A, Gasmi A, Menzel A, Hrynovets I, Chirumbolo S, Shanaida M, Lysiuk R, Shanaida Y, Dadar M, Bjørklund G. A review on natural teeth whitening. J Oral Biosci. 2022;64(1):49–58.
Moghadam ET, Yazdanian M, Tahmasebi E, Tebyanian H, Ranjbar R, Yazdanian A, Seifalian A, Tafazoli A. Current herbal medicine as an alternative treatment in dentistry: in vitro, in vivo and clinical studies. Eur J Pharmacol. 2020;889: 173665.
Personal care product usage by category [https://www.statista.com/global-consumer-survey/tool/10/gcs_hun_202100?index=0&absolute=0&missing=0&bars=1&heatmap=0&rows%5B0%5D=v0227_ecom_personalcare&tgeditor=0&pendo=0].
EWG Skin Deep Cosmetics Database Ratings [https://www.ewg.org/skindeep/understanding_skin_deep_ratings/].
EWG Skin Deep Cosmetics Database [https://www.ewg.org/skindeep].
Juhász PM. Herbarium: az faknac fuveknec nevekroel, természetekroel, és hasznairól / magyar nyelwre, és ez rendre hoszta az doctoroc koenyveiboel. 1578.
Páriz FP. Pax Corporis. 1747.
Csapó J. Új füves és virágos magyar kert. 1734.
Kováts M. Magyar Patika. Budapest: Kossuth Kiadó; 2005.
Das S, Gazdag Z, Szente L, Meggyes M, Horváth G, Lemli B, Kunsági-Máté S, Kuzma M, Kőszegi T. Antioxidant and antimicrobial properties of randomly methylated β cyclodextrin - captured essential oils. Food Chem. 2019;278:305–13.
Park SN, Lim YK, Freire MO, Cho E, Jin D, Kook JK. Antimicrobial effect of linalool and α-terpineol against periodontopathic and cariogenic bacteria. Anaerobe. 2012;18(3):369–72.
Prusinowska R, Śmigielski K. Composition, biological properties and therapeutic effects of lavender (Lavandula angustifolia L). A review. Herba Polonica. 2014;60:56.
Mahendran G, Verma SK, Rahman LU. The traditional uses, phytochemistry and pharmacology of spearmint (Mentha spicata L.): A review. J Ethnopharmacol. 2021;278:114266.
Snoussi M, Noumi E, Trabelsi N, Flamini G, Papetti A, De Feo V. Mentha spicata essential oil: chemical composition, antioxidant and antibacterial activities against planktonic and biofilm cultures of vibrio spp. Strains. Molecules. 2015;20(8):14402–24.
Liu Q, Meng X, Li Y, Zhao CN, Tang GY, Li HB. Antibacterial and antifungal activities of spices. Int J Mol Sci. 2017;18(6):1283.
da Costa JS, Barroso AS, Mourão RHV, da Silva JKR, Maia JGS, Figueiredo PLB. Seasonal and antioxidant evaluation of essential oil from. Biomolecules. 2020;10(2):328.
Saddiqe Z, Naeem I, Maimoona A. A review of the antibacterial activity of Hypericum perforatum L. J Ethnopharmacol. 2010;131(3):511–21.
de Araújo MRC, Maciel PP, Castellano LRC, Bonan PRF, Alves DDN, de Medeiros ACD, de Castro RD. Efficacy of essential oil of cinnamon for the treatment of oral candidiasis: a randomized trial. Spec Care Dentist. 2021;41(3):349–57.
Metwaly AM, Ghoneim MM, Eissa IH, Elsehemy IA, Mostafa AE, Hegazy MM, Afifi WM, Dou D. Traditional ancient Egyptian medicine: a review. Saudi J Biol Sci. 2021;28(10):5823–32.
Taheri A, Mirghazanfari SM, Dadpay M. Wound healing effects of Persian walnut. Eur J Transl Myol. 2020;30(1):8671.
Givol O, Kornhaber R, Visentin D, Cleary M, Haik J, Harats M. A systematic review of Calendula officinalis extract for wound healing. Wound Repair Regen. 2019;27(5):548–61.
Imran M, Khan AS, Khan MA, Saeed MU, Noor N, Warsi MH, Qadir A. Antimicrobial activity of different plants extracts against Staphylococcus aureus and Escherichia coli. Polim Med. 2021;51(2):69–75.
Shanbhag PP, Belatikar S, Sahane C. In vitro and in vivo evaluation of taste masked chlorhexidine-releasing oral films. J Drug Deliv. 2017;7:210–9.
Edwards SE, da Costa Rocha I, Williamson EM, Heinrich M. Phytopharmacy: an evidence-based guide to herbal medicinal products. 2015.
Da Porto C, Decorti D, Kikic I. Flavour compounds of Lavandula angustifolia L. to use in food manufacturing: comparison of three different extraction methods. Food Chem. 2009;112(4):1072–8.
Safiaghdam H, Oveissi V, Bahramsoltani R, Farzaei MH, Rahimi R. Medicinal plants for gingivitis: a review of clinical trials. Iran J Basic Med Sci. 2018;21(10):978–91.
Hayat L, Jacob DA. Dyeing wool and cotton fibres with acidic extract of Hibiscus rosa sinensis flower. Nat Prod Res. 2019;33(7):980–6.
Wölfle U, Seelinger G, Schempp CM. Topical application of St. John’s wort (Hypericum perforatum). Planta Med. 2014;80(2–3):109–20.
Dellafiora L, Galaverna G, Cruciani G, Dall’Asta C, Bruni R. On the mechanism of action of anti-inflammatory activity of hypericin: an in silico study pointing to the relevance of Janus Kinases inhibition. Molecules. 2018;23(12):3058.
Ho GT, Zou YF, Aslaksen TH, Wangensteen H, Barsett H. Structural characterization of bioactive pectic polysaccharides from elderflowers (Sambuci flos). Carbohydr Polym. 2016;135:128–37.
Shikov AN, Narkevich IA, Flisyuk EV, Luzhanin VG, Pozharitskaya ON. Medicinal plants from the 14. J Ethnopharmacol. 2021;268:113685.
Preethi KC, Kuttan G, Kuttan R. Anti-inflammatory activity of flower extract of Calendula officinalis Linn. and its possible mechanism of action. Indian J Exp Biol. 2009;47(2):113–20.
Ermi Hikmawanti NP, Fatmawati S, Asri AW. The effect of ethanol concentrations as the extraction solvent on antioxidant activity of Katuk (Sauropus androgynus (L.) Merr) Leaves Extracts. Publication: IOP Conference Series: Earth and Environmental Science. 2021;755(1):012060. Pub Date: April 2021. https://doi.org/10.1088/1755-1315/755/1/012060 Bibcode:
Mohamed EA, Lim CP, Ebrika OS, Asmawi MZ, Sadikun A, Yam MF. Toxicity evaluation of a standardised 50% ethanol extract of Orthosiphon stamineus. J Ethnopharmacol. 2011;133(2):358–63.
J Mason T, Chemat F, Vinatoru M. The extraction of natural products using ultrasound or microwaves. Curr Org Chem. 2011;15(2):237–47.
Abubakar AR, Haque M. Preparation of medicinal plants: basic extraction and fractionation procedures for experimental purposes. J Pharm Bioallied Sci. 2020;12(1):1–10.
Móricz Á, Horváth G, Böszörményi A, Ott PG. Detection and identification of antibacterial and antioxidant components of essential Oils by TLC-biodetection and GC-MS. Nat Prod Commun. 2016;11(11):1705–8.
Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970;227(5259):680–5.
Mortz E, Krogh TN, Vorum H, Görg A. Improved silver staining protocols for high sensitivity protein identification using matrix-assisted laser desorption/ionization-time of flight analysis. Proteomics. 2001;1(11):1359–63.
Csepregi R, Temesfői V, Das S, Alberti Á, Tóth CA, Herczeg R, Papp N, Kőszegi T. Cytotoxic, antimicrobial, antioxidant properties and effects on cell migration of phenolic compounds of selected transylvanian medicinal plants. Antioxidants (Basel). 2020;9(2):166.
Banu JN, Gayathri V. Preparation of antibacterial herbal mouthwash against oral pathogens. Int J Curr Microbiol App Sci 2016. 2016;5(11):205–21.
Cheung HY, Wong MM, Cheung SH, Liang LY, Lam YW, Chiu SK. Differential actions of chlorhexidine on the cell wall of Bacillus subtilis and Escherichia coli. PLoS ONE. 2012;7(5):e36659.
Zawadzki PJ, Perkowski K, Padzik M, Mierzwińska-Nastalska E, Szaflik JP, Conn DB, Chomicz L. Examination of oral microbiota diversity in adults and older adults as an approach to prevent spread of risk factors for human infections. Biomed Res Int. 2017;2017:8106491.
Souza LCD, Lopes FF, Bastos EG, Alves CMC. Oral infection by Pseudomonas aeruginosa in patient with chronic kidney disease - a case report. J Bras Nefrol. 2018;40(1):82–5.
Eduardo FP, Bezinelli LM, Gobbi MF, Santos VM, Maluf FC, Corrêa L. Severe oral infection caused by Pseudomonas aeruginosa effectively treated with methylene blue-mediated photodynamic inactivation. Photodiagnosis Photodyn Ther. 2019;26:284–6.
McCormack MG, Smith AJ, Akram AN, Jackson M, Robertson D, Edwards G. Staphylococcus aureus and the oral cavity: an overlooked source of carriage and infection? Am J Infect Control. 2015;43(1):35–7.
Abranches J, Zeng L, Kajfasz JK, Palmer SR, Chakraborty B, Wen ZT, Richards VP, Brady LJ, Lemos JA. Biology of oral Streptococci. Microbiol Spectr. 6(5):GPP3-0042-2018.
Rosan B, Lamont RJ. Dental plaque formation. Microbes Infect. 2000;2(13):1599–607.
Maidment C, Dyson A, Haysom I. A study into the antimicrobial effects of cloves () and cinnamon () using disc-diffusion assay. Nutr Food Sci. 2006;36(4):225–30.
Präbst K, Engelhardt H, Ringgeler S, Hübner H. Basic colorimetric proliferation assays: MTT, WST, and Resazurin. Methods Mol Biol. 2017;1601:1–17.
Bidra AS, Pelletier JS, Westover JB, Frank S, Brown SM, Tessema B. Comparison of in vitro inactivation of SARS CoV-2 with hydrogen peroxide and povidone-iodine oral antiseptic rinses. J Prosthodont. 2020;29(7):599–603.
Anderson DE, Sivalingam V, Kang AEZ, Ananthanarayanan A, Arumugam H, Jenkins TM, Hadjiat Y, Eggers M. Povidone-iodine demonstrates rapid in vitro Virucidal activity against SARS-CoV-2, the virus causing COVID-19 disease. Infect Dis Ther. 2020;9(3):669–75.
Eggers M, Koburger-Janssen T, Eickmann M, Zorn J. In vitro bactericidal and Virucidal efficacy of povidone-iodine gargle/mouthwash against respiratory and oral tract pathogens. Infect Dis Ther. 2018;7(2):249–59.
Meyers C, Robison R, Milici J, Alam S, Quillen D, Goldenberg D, Kass R. Lowering the transmission and spread of human coronavirus. J Med Virol. 2021;93(3):1605–12.
Lin YJ, Chou WJ, Chang YP, Yen CF. Denial of justification for vaccination: its multiple related variables and impacts on intention to get vaccinated against COVID-19. Vaccines (Basel). 2021;9(8):822.
Fiorillo L. Oral health: the first step to well-being. Medicina (Kaunas). 2019;55(10):676.
Gil-Montoya JA, de Mello AL, Barrios R, Gonzalez-Moles MA, Bravo M. Oral health in the elderly patient and its impact on general well-being: a nonsystematic review. Clin Interv Aging. 2015;10:461–7.
Tavares M, Lindefjeld Calabi KA, San Martin L. Systemic diseases and oral health. Dent Clin North Am. 2014;58(4):797–814.
Li X, Kolltveit KM, Tronstad L, Olsen I. Systemic diseases caused by oral infection. Clin Microbiol Rev. 2000;13(4):547–58.
Rautemaa R, Lauhio A, Cullinan MP, Seymour GJ. Oral infections and systemic disease–an emerging problem in medicine. Clin Microbiol Infect. 2007;13(11):1041–7.
Rajendiran M, Trivedi HM, Chen D, Gajendrareddy P, Chen L. Recent development of active ingredients in mouthwashes and toothpastes for periodontal diseases. Molecules. 2021;26(7):2001.
de Melo ED, Mounteer AH, Leão LH, Bahia RC, Campos IM. Toxicity identification evaluation of cosmetics industry wastewater. J Hazard Mater. 2013;244–245:329–34.
Juliano C, Magrini GA. Cosmetic ingredients as emerging pollutants of environmental and health concern a mini-review. Cosmetics. 2017;4(2):11.
Janakiram C, Venkitachalam R, Fontelo P, Iafolla TJ, Dye BA. Effectiveness of herbal oral care products in reducing dental plaque & gingivitis - a systematic review and meta-analysis. BMC Complement Med Ther. 2020;20(1):43.
Kim KB, Kwack SJ, Lee JY, Kacew S, Lee BM. Current opinion on risk assessment of cosmetics. J Toxicol Environ Health B Crit Rev. 2021;24(4):137–61.
Goyal N, Jerold F. Biocosmetics: technological advances and future outlook. Environ Sci Pollut Res Int. 2023;30(10):25148–69. Epub 2021 Nov 25.
Fung ES, Drechsel DA, Towle KM, Hoang MT, Novick RM, Poteete C, Paustenbach DJ, Monnot AD. Screening-level safety assessment of personal care product constituents using publicly available data. Cosmetics. 2018;5(2):38.
Aceves Argemí R, González Navarro B, Ochoa García-Seisdedos P, Estrugo Devesa A, López-López J. Mouthwash with alcohol and oral carcinogenesis: systematic review and meta-analysis. J Evid Based Dent Pract. 2020;20(2):101407.
Van Nieuw AA, Bolscher JG, Veerman EC. Salivary proteins: protective and diagnostic value in cariology? Caries Res. 2004;38(3):247–53.
Loo JA, Yan W, Ramachandran P, Wong DT. Comparative human salivary and plasma proteomes. J Dent Res. 2010;89(10):1016–23.
Zhang Y, Lu C, Zhang J. Lactoferrin and its detection methods: a review. Nutrients. 2021;13(8):2492.
Magacz M, Kędziora K, Sapa J, Krzyściak W. The significance of Lactoperoxidase system in oral health: application and efficacy in oral hygiene products. Int J Mol Sci. 2019;20(6):1443.
Peng Y, Chen X, Sato T, Rankin SA, Tsuji RF, Ge Y. Purification and high-resolution top-down mass spectrometric characterization of human salivary α-amylase. Anal Chem. 2012;84(7):3339–46.
Fisher SZ, Govindasamy L, Tu C, Agbandje-McKenna M, Silverman DN, Rajaniemi HJ, McKenna R. Structure of human salivary alpha-amylase crystallized in a C-centered monoclinic space group. Acta Crystallogr Sect F Struct Biol Cryst Commun. 2006;62(Pt 2):88–93.
Ghosh SK, Gerken TA, Schneider KM, Feng Z, McCormick TS, Weinberg A. Quantification of human beta-defensin-2 and -3 in body fluids: application for studies of innate immunity. Clin Chem. 2007;53(4):757–65.
Boze H, Marlin T, Durand D, Pérez J, Vernhet A, Canon F, Sarni-Manchado P, Cheynier V, Cabane B. Proline-rich salivary proteins have extended conformations. Biophys J. 2010;99(2):656–65.
Balekjian AY, Hoerman KC, Berzinskas VJ. Lysozyme of the human parotid gland secretion: Its purification and physicochemical properties. Biochem Biophys Res Commun. 1969;35(6):887–94.
Woo JS, Jeong JY, Hwang YJ, Chae SW, Hwang SJ, Lee HM. Expression of cathelicidin in human salivary glands. Arch Otolaryngol Head Neck Surg. 2003;129(2):211–4.
Dickinson DP. Salivary (SD-type) cystatins: over one billion years in the making–but to what purpose? Crit Rev Oral Biol Med. 2002;13(6):485–508.
Jasjit K, Mayur K, Soundarya S, Geetika A. Evaluation of healing and anti-plaque efficacy of antioxidant mouthrinse in patients with gingival inflamation – a single blind clinical study. International Journal of Oral Health Dentistry. 2021;7(1):14–9.
Alsaffar D, Alzoman H. Efficacy of antioxidant mouthwash in the reduction of halitosis: a randomized, double blind, controlled crossover clinical trial. J Dent Sci. 2021;16(2):621–7.
Abdulkareem AA, Al Marah ZA, Abdulbaqi HR, Alshaeli AJ, Milward MR. A randomized double-blind clinical trial to evaluate the efficacy of chlorhexidine, antioxidant, and hyaluronic acid mouthwashes in the management of biofilm-induced gingivitis. Int J Dent Hyg. 2020;18(3):268–77.
Korkina L, Kostyuk V, De Luca C, Pastore S. Plant phenylpropanoids as emerging anti-inflammatory agents. Mini Rev Med Chem. 2011;11(10):823–35.
Proshkina E, Plyusnin S, Babak T, Lashmanova E, Maganova F, Koval L, Platonova E, Shaposhnikov M, Moskalev A. Terpenoids as potential geroprotectors. Antioxidants (Basel). 2020;9(6):529.
Zomorodian K, Ghadiri P, Saharkhiz MJ, Moein MR, Mehriar P, Bahrani F, Golzar T, Pakshir K, Fani MM. Antimicrobial activity of seven essential oils from Iranian aromatic plants against common causes of oral infections. Jundishapur J Microbiol. 2015;8(2):e17766.
Fine DH, Furgang D, Barnett ML, Drew C, Steinberg L, Charles CH, Vincent JW. Effect of an essential oil-containing antiseptic mouthrinse on plaque and salivary Streptococcus mutans levels. J Clin Periodontol. 2000;27(3):157–61.
Ultee A, Bennik MH, Moezelaar R. The phenolic hydroxyl group of carvacrol is essential for action against the food-borne pathogen Bacillus cereus. Appl Environ Microbiol. 2002;68(4):1561–8.
Juven BJ, Kanner J, Schved F, Weisslowicz H. Factors that interact with the antibacterial action of thyme essential oil and its active constituents. J Appl Bacteriol. 1994;76(6):626–31.
Nowotarska SW, Nowotarski K, Grant IR, Elliott CT, Friedman M, Situ C. Mechanisms of Antimicrobial Action of Cinnamon and Oregano Oils, Cinnamaldehyde, Carvacrol, 2,5-Dihydroxybenzaldehyde, and 2-Hydroxy-5-Methoxybenzaldehyde against Mycobacterium avium subsp. paratuberculosis (Map). Foods. 2017;6(9):72.
Gill AO, Holley RA. Mechanisms of bactericidal action of cinnamaldehyde against Listeria monocytogenes and of eugenol against L. monocytogenes and Lactobacillus sakei. Appl Environ Microbiol. 2004;70(10):5750–5.
Santos EL, Freitas PR, Araújo ACJ, Almeida RS, Tintino SR, Paulo CLR, Ribeiro-Filho J, Silva ACA, Silva LE, Amaral WD, et al. Phytochemical characterization and antibiotic potentiating effects of the essential oil of Aloysia gratissima (Gillies & Hook.) and beta-caryophyllene. S Afr J Bot. 2021;143:1–6.
Moo CL, Yang SK, Osman MA, Yuswan MH, Loh JY, Lim WM, Lim SH, Lai KS. Antibacterial activity and mode of action of β-caryophyllene on. Pol J Microbiol. 2020;69(1):1–6.
Guo F, Chen Q, Liang Q, Zhang M, Chen W, Chen H, Yun Y, Zhong Q. Antimicrobial activity and proposed action mechanism of linalool against. Front Microbiol. 2021;12:562094.
Kwiatkowski P, Grygorcewicz B, Pruss A, Wojciuk B, Dołęgowska B, Giedrys-Kalemba S, Sienkiewicz M, Wojciechowska-Koszko I. The effect of Subinhibitory concentrations of. Microb Drug Resist. 2019;25(10):1424–9.
Kharazmkia A, Al-Abodi HR, Yadegari JG, Vahidi A, Mahmoudvand H. Potential effects of alpha-pinene, a monoterpene commonly found in essential oils against. J Parasit Dis. 2022;46(4):1055–61.
Allenspach M, Steuer C. α-Pinene: a never-ending story. Phytochemistry. 2021;190: 112857.
Charles CA, Lisante TA, Revankar R, Cortelli JR, Cortelli SC, Aquino D, Goyal CR, Amini P. Early benefits with daily rinsing on gingival health improvements with an essential oil mouthrinse–post-hoc analysis of 5 clinical trials. J Dent Hyg. 2014;88(Suppl 1):40–50.
Pilloni A, Pizzo G, Barlattani A, Di Lenarda R, Giannoni M, Guida L, Levrini L, Majorana A, Polimeni A. Perceived and measurable performance of daily brushing and rinsing with an essential oil mouthrinse. Ann Stomatol (Roma). 2010;1(3–4):29–32.
Jafri H, Ansari FA, Ahmad I, Ahmad Khan MS, Chattopadhyay D. Chapter 9 - prospects of essential oils in controlling pathogenic biofilm. In: New Look to Phytomedicine. edn.: Academic Press; 2019:203–236. Editor(s): Mohd Sajjad Ahmad Khan, Iqbal Ahmad, Debprasad Chattopadhyay. New Look to Phytomedicine. Academic Press. 2019;203-236. https://doi.org/10.1016/B978-0-12-814619-4.00009-4.
Iranmanesh B, Khalili M, Amiri R, Zartab H, Aflatoonian M. Oral manifestations of COVID-19 disease: a review article. Dermatol Ther. 2021;34(1): e14578.
Botros N, Iyer P, Ojcius DM. Is there an association between oral health and severity of COVID-19 complications? Biomed J. 2020;43(4):325–7.
Brandão TB, Gueiros LA, Melo TS, Prado-Ribeiro AC, Nesrallah ACFA, Prado GVB, Santos-Silva AR, Migliorati CA. Oral lesions in patients with SARS-CoV-2 infection: could the oral cavity be a target organ? Oral Surg Oral Med Oral Pathol Oral Radiol. 2021;131(2):e45–51.
Cox MJ, Loman N, Bogaert D, O’Grady J. Co-infections: potentially lethal and unexplored in COVID-19. Lancet Microbe. 2020;1(1):e11.
Sharmeen JB, Mahomoodally FM, Zengin G, Maggi F. Essential oils as natural sources of fragrance compounds for cosmetics and cosmeceuticals. Molecules. 2021;26(3):666.
Open access funding provided by University of Pécs. The research was financed by the Thematic Excellence Program 2021 Health and National Defense, National Security Sub-Programmes of the Ministry for Innovation and Technology in Hungary, within the framework of the EGA-10, EGA-13, NVA-07 projects of the University of Pécs, providing resources to the investigations; by the RRF-2.3.1–21-2022–00012 "National Laboratory on Human Reproduction", providing resources for laboratory consumables; by the RRF-2.3.1–21-2022–00010 "National Laboratory of Virology", making the virological studies possible by providing the opportunity to work in a BSL-4 laboratory; and by the ÚNKP-22-4-I new national excellence program of the Ministry for Culture and Innovation from the source of the National Research, Development and Innovation Fund.
Ethics approval and consent to participate
The study was approved by the Regional Committee for the Research Ethics at the University of Pécs (Ref No.: 5022—PTE 2015. This permission is valid until 31 January 2023).
Consent for publication
Healthy volunteers who donated saliva were fully informed and written consent was signed by each participant.
B.B. is the owner of Concordia Duo Ltd., the company which has the rights to produce and distribute the developed product. Although the evaluation of the mouthwash, the subject of this article, was carried out during the PhD studies of B.B. at the University of Pécs, Concordia Duo Ltd. has a signed agreement with the University of Pécs stating that the University waives all rights about the product. Other authors declare no conflict of interest.
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Additional file 1.
Questions of the market survey – translated. Translated questionnaire of our online market survey.
Additional file 2.
Herbal material quality assurance certificates. Description of data: International Organization for Standardization (ISO) 9001 and International Featured Standards (IFS) qualification of the herbal material provider.
Additional file 3.
Data sheet on quality assurance of the essential oils. Quality information and safety data sheet of the essential oils.
Additional file 4.
Summary table and references for the active oily compounds and their presence in the selectedplants and related species. A summary table, references and PubChem CID of the active oily compounds and their presence in the selected plants and related species.
Additional file 5.
Ingredients of different mouthwashes, categorized by the EWG numbers. Ingredients of different mouthwashes, categorized by the EWG numbers. *This ingredient’s score is higher if used in products intended for use around mouth and on lips due to increased risk of ingestion and absorption. †This ingredient’s score is higher if used in inhalable products (e.g., sprays, powders) because of respiratory concerns. ‡This ingredient’s score is higher if used in products intended for use around mouth, on lips, around the eyes and for damaged skin due to increased risk of ingestion and absorption .
Additional file 6.
Detailed statistical results and p values for the SDS-PAGE, cytotoxicity and disc diffusion. assays.
Additional file 7.
Original image of the silver stained SDS polyacrylamide gel for Fig. 2.
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Bencze, B., Temesfői, V., Das, S. et al. Development of a novel, entirely herbal-based mouthwash effective against common oral bacteria and SARS-CoV-2. BMC Complement Med Ther 23, 138 (2023). https://doi.org/10.1186/s12906-023-03956-3
- Herbal mouthwash
- Oral rinse
- Oral care
- Essential oil