Antibacterial and cytotoxicity testsMain reagents

Heme chloride and vitamin K1 were purchased from Qingdao Hi-tech Industrial Park Hope Bio-technology Co., Ltd, sterile defidrinated sheep blood was purchased from Guangzhou Hongquan Biotechnology Co., Ltd. The stock solution of heme chloride was prepared according to the ratio of heme chloride: distilled water: sodium hydroxide = 1:5:40, filtered and sterilized, and stored at 4 ℃ in the dark.

Brain Heart Infusion (BHI) medium, purchased from Becton, Dickinson and Company. According to the instructions, the solid medium was added with 1.5–2% agar, autoclaved. When the solution was cooled to about 50 ℃, 5% sterile defiber sheep blood, 0.05% vitamin K1, and 0.1% hemin chloride stock solution were added, mixed, and divided into plates, sealed after cooling into solid, and stored in the dark at 4 ℃ for later use.

The combination of three antibacterial ingredients: ε-PL (150 µg/mL), FP (0.25 µg/mL) and domiphen (300 µg/mL).

Test strains: F. nucleatum ATCC 25,586, P. gingivalis ATCC 33,277, S. mutans NBRC 13,955, and A. actinomycetemcomitans HK 1651. All four strains were cultured using BHI medium, F. nucleatum and P. gingivalis were cultured under anaerobic conditions, and S. mutans and A. actinomycetemcomitans were cultured under aerobic conditions.

Determination of minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC)

The two-fold broth micro dilution method was used to detect the MIC of the mixed antibacterial solution of the three ingredients against the four pathogens. At first, sterile 96-well culture plates were prepared, and the antibacterial solution was added in a final concentration gradient from 25% (v/v) to 0.10% (v/v) in 1st to 9th wells, and the chlorhexidine (0.5%) was added in 10th wells as a positive control, no drug in 11th wells as a negative control, and only medium in 12th wells as a blank control. Then, the bacterial broth in the logarithmic growth phase was diluted with BHI liquid medium and added to 96-well plates to achieve a final concentration of 1 × 108 CFU/mL. Three parallel groups were prepared for each strain. Subsequently, the 96-well plates containing bacterial and antimicrobial solution were placed in a constant temperature incubator at 37 °C for 20 to 24 h. The turbidity of the medium in each well was visually assessed at the end of the incubation period, and the lowest drug concentration at which the medium remained clear was designated as the MIC for the antimicrobial solution.

The bacterial solution from the well exhibiting the MIC and its four adjacent above wells were individually transferred and inoculated on culture agar plate. Following 24 h of incubation in a 37 °C incubator, the MBC was the concentration of the tested drug that completely eliminated the tested bacteria by showing no colony appearance.

Cell viability detection using a cell counting kit 8 assay

The effect of the mouthwash on the viability of human gingival fibroblasts (HGFs) was evaluated by a CCK-8 cell counting kit (CCK-8; Biosharp, China). HGFs were seeded into 96-well plates with high-glucose DMEM medium (BasalMedia Technologies Co., Shanghai, China), and transferred to a CO2 incubator for 24 h at 37 °C. Cells were then stimulated with the different concentrations of test mouthwash for 30 s, and a mouthwash containing 0.12% chlorhexidine and 0.02% metronidazoleand as the positive control, only medium as the negative control. Afterwards, the cells were incubated in a medium containing 10 µL test reagents for 1 h at 37 ℃. The optimal absorbance at 450 nm was determined using a microplate reader.

Clinical trials on halitosis and supragingival plaque reductionGeneral information

This study was approved by the Ethics committee of School of Stomatology Shandong University (Stomatological Hospital of Shandong University) (No.20,230,601). A randomized, double-blind and parallel controlled trial was registered on Chinese Clinical Trial Registry (No. ChiCTR2300073816, Date: 21/07/2023) and carried out. A total of 80 subjects who met the inclusion criteria without any exclusion criteria were recruited. They were randomly divided into the test and the control group, 40 subjects in each group. The specific operation process was shown in Fig. 1.

Fig. 1

Flow chart of clinical trial

Inclusion criteria: (1) ages from 18 to 65 years old with good general health without serious systemic diseases; (2) having more than 20 detectable teeth with an appropriate degree of plaque or gingivitis; (3) agreeing not to use any non-test mouthwash during the trial period; (4) avoiding to consume any food producing oral odor such as garlic, leeks, stinky tofu; (5) keeping a good oral hygiene habits by brushing twice a day; (6) having VSCs levels ≥ 125 examined by the Halimeter; (7) signing an informed consent form.

Exclusion criteria: (1) concurrently joining other clinical studies; (2) using other oral health care products containing phenols or fragrances during the days of testing; (3) having the history of allergy to the tested product and its ingredients; (4) having these conditions including AIDS, insulin-dependent diabetes mellitus, anticancer chemotherapy within 6 months, immunodeficiency, autoimmune disease, or other serious medical conditions; (5) having severe gingivitis, periodontitis or oral ulcers, wearing partial or full dentures; (6) smoking or using tobacco products; (7) using antihistamines in the last week or immunosuppressants or antibiotics in the last month; (8) females with pregnancy, during breastfeeding or menstruation; (9) the presence of any disease or condition that may interfere with the examination procedure and the smooth completion of the test.

The usage of mouthwash

The researchers primarily explained the purpose and process of the study in detail to the recruited subjects before their informed consents were given. The researchers collected the subjects’ personal information including the medical history and dietary habits.

εAll recruited subjects underwent an oral examination followed by training on standardized brushing methods and mouthwash use. After brushing their teeth twice a day, the individuals were told to rinse their mouths with either a 20 mL BOP® mouthwash (test mouthwash) added with ε-PL, FP, and domiphen, or a non-supplemented one (control mouthwash) for 30 s, which was designated as the test group and the control group.

Baseline examination

Assessment of halitosis: The degree of halitosis was assessed by measuring the levels of VSCs using Halimeter [22]. Before measurement, the subject closed the mouth for 3 min. Then, the subject breathed through the nose with the mouth opening slightly, and the collector is placed 0.5 cm above the middle and posterior third of the dorsum of tongue to read the peak value on the display screen. The subjects with a mean VSCs level above 125 across the three tests were eligible for inclusion and were randomly assigned to the test and control groups.

Assessment of supragingival plaque: To determine supragingival plaque formation, six marked teeth (upper right 6, upper left 1 and 4, lower left 6 and lower right 1 and 4) were stained with the dye using cotton swab. After 1 min, each tooth was examined on four tooth surfaces, namely mesial buccal, mid-buccal, distal-buccal and lingual. Based on the PLI scoring standards [23] (Table 1), each tooth was scored as the sum of the four tooth surface scores divided by four, and the individual score was counted as the sum of the fractions of each tooth divided by the number of teeth examined.

Table 1 PLI scoring standards The examination of trial outcome

The clinical trial lasted for 7 days. The VSCs levels were measured at the baseline, 0, 10, 24 h, and 7 days following the first mouthwash application, and the amount of plaque was only tested at baseline and day 7. Both the detection items and methods were identical to the baseline. In addition, the oral soft and hard tissue examination and compliance statistics were performed at 0, 10, 24 h and 7 days after the first use of mouthwash to evaluate the potential side effects of the new formula.

Statistical analysis

Quantitative data were described as mean, standard deviation, minimum, median and maximum. Qualitative indicators were described by frequency table, percentage or constituent ratio. Quantitative data were visualized using QQ plots, and a normal distribution compliance was assessed by the Shapiro-Wilk normality test. Homogeneity of variance was tested by Levene’s test. Quantitative data, data in line with normal distribution were compared between the two groups by t test, the multiple groups with equal variance were compared by ANOVA analysis, and the multiple groups with unequal variance were compared by Welch’s ANOVA test. The Wilcoxon rank-sum test was used to confirm the skewed distribution. Qualitative data were analyzed by chi-square test, Fisher exact test or Knuskal-wallis test. In addition, to evaluate the effect of the mouthwash on the breath index, a stratified analysis was performed, and severe breath was defined as those with a breath score greater than the median.

All data were analyzed using the R 15.6.0 platform. All tests were two-sided, and the confidence level was 0.05.