Introduction

Dental plaque is a polymicrobial community that adheres to the tooth and other hard surfaces in the oral cavity as a sticky biofilm. These biofilms are a causative factor for diverse pathological conditions of the oral cavity such as caries, gingivitis, or periodontitis [1, 2]. Their volume increase in the gingival crevice due to poor oral hygiene practice, which may ultimately lead to chronic inflammation and the progressive destruction of the periodontal tissue supporting the teeth [3]. Hence, dental plaque control has been the most important measure to maintain good oral hygiene and keep periodontal diseases at bay. Both mechanical and chemical measures can help attain effective plaque removal. The mechanical plaque control involves using shear forces to remove the matrix-enclosed microbial biofilms from the tooth surface. This can be achieved through a deep cleaning procedure by a professional (e.g., scaling and root planing) or self‐performed such as twice-daily toothbrushing or using interdental cleaning products [4].

The chemical plaque control is usually used as an adjunct for mechanical cleaning to inhibit the growth and accumulation of microbiota. Two of the common vehicles to deliver various chemical (anti-plaque) agents include toothpaste and mouthwashes. A variety of organic and inorganic chemicals are available as anti-plaque agents including phenolic compounds, quaternary ammonium compounds, delmopinol, chlorhexidine gluconate (CHX), essential oils (e.g., methyl salicylate, menthol, and thymol), and herbal extracts [5]. Of these, CHX has been the most tested and potent anti-plaque agent. Its efficacy has been proven to be the gold standard against which other anti-plaque and anti-gingivitis agents are compared [6, 7]. CHX can exert both bacteriostatic and bactericidal properties depending on the applied concentration. However, a major side effect of CHX usage is the reduction in cell migration and survival [7, 8]. In addition, its long-term use is associated with a range of other side effects including teeth discoloration and staining, mucous membrane irritation, and taste disturbance. Hence, most dentists now recommend its use only under professional supervision [7-10].

Herbal mouthwashes have long been thought to be a suitable alternative to CHX for dental plaque and gingivitis reduction owing to their low side effect profile. Clinical trials conducted to determine the effectiveness of these mouthwashes have also demonstrated their efficacy as an adjunctive treatment [11]. In traditional medicine, myrrh (Commiphora myrrha), an oleo-gum-resin native to Middle Eastern and North African countries, was employed in the treatment of a wide range of inflammatory situations for hundreds of years [12]. Several studies have also found it promising in the management of various oral-related disorders, including inflamed gingiva, aphthous ulcers, and intramucosal wounds [13, 14]. However, of the various herbs, tested up to now as a mouthwash formulation, the potential for a myrrh-based mouthwash is comparatively less explored. In our previous pilot study, we compared the efficacy of myrrh-based mouthwash with CHX and found it to be slightly more effective than CHX in reducing plaque accumulation and gingival inflammation [15]. Similar findings were reported in earlier studies, where the efficacy of myrrh was found to be comparable to that of CHX [16, 17]. These studies, however, had several limitations in study design and sample selection.

Therefore, this study aims to establish the earlier findings on the efficacy of myrrh mouthwash in comparison to CHX using further laboratory tests, including the pro-inflammatory interleukin (IL)-1β biomarker, bleeding on probing (BOP), modified gingival index (MGI), and plaque index (PI). We hypothesize that there is no difference between 1% myrrh mouthwash and commercially available CHX 0.2% mouth rinse in relation to decreasing load of plaque, gingival inflammation control, and pro-inflammatory mediator (IL-1β) inhibition.

Materials and Methods

Study design

The study was a randomized controlled clinical trial done at the King Abdulaziz University, Faculty of Dentistry (KAUFD), Jeddah, Saudi Arabia. We adhered to the declaration of Helsinki for Bio-medical research which involves human subjects and the CONSORT 2010 Statement for reporting multi-arm trials. The ethical approval of the study was obtained from the Research Ethics Committee at KAUFD (protocol number: 058-15). The protocol for this trial is publicly available at ClinicalTrials.gov (NCT04723732). Each participant signed a written informed consent before inclusion. The study was conducted between August 2017 and April 2018.

Patient selection

Participants were selected from patients seeking treatment at the dental clinic of KAUFD. A poster was hung in the waiting room area inviting patients to take part in the study as volunteers. Interested patients were included in this research sticking to the inclusion/exclusion criteria. Inclusion criteria were: good periodontal health (i.e., absence of clinical attachment loss and less than ten percent BOP); the presence of more than 20 teeth with at least five teeth per quadrant; no history of systemic disease; absence of oral prophylaxis in the past six months. exclusion criteria were: more than 3mm pocket depth; severe malocclusion; the presence of braces or orthodontic wires; use of antibiotic and/or anti-inflammatory prescriptions in the last 6 months; tobacco consumption; lack of compliance with the study agenda; pregnant or women who breast-feed.

Due to the lack of previous studies, we calculated the sample size based on a pilot study conducted in our center on 12 participants [15]. The study showed a mean difference of 0.29 ± 0.17 between the test and the reference group in post-intervention values. We entered these pilot results in a statistical tool [18] named “Sample Size Calculator for Comparing Two Independent Means.” The tool estimated a sample size of 6 patients for each group based on 80% power and a 5% level of significance (P < 0.05, two-sided). We recruited 8 patients in each group considering possible loss to follow-up during the study. A total of 24 eligible patients, 12 male, and 12 female, with ages between eighteen and fifty-five years, were recruited.

Procedure

Patients were asked to complete a medical history questionnaire after the initial dental screening to confirm eligibility. The first visit included oral hygiene instructions and professional mouth cleaning processes such as oral prophylaxis or supra-gingival scaling-if needed. This was done 14 days before the study. At the second session, the examination of the periodontium was performed to confirm the good health of the gum and periodontium. Participants were then taught to refrain from cleaning teeth and any oral hygiene measures for two weeks to grow experimental gingivitis.

At the third session (experimental period, day 0), a periodontal examination was done to record the baseline values of gingival, plaque, bleeding, and inflammatory parameters. Participants were placed randomly into 1 of the 3 groups [1:1:1] through simple randomization technique (i.e., computer-generated random numbers): (a) normal saline, (b) 0.2% chlorhexidine gluconate mouthwash, and (c) 1% Commiphora myrrh mouthwash. Each group had eight participants. The myrrh mouthwash (1% g) was prepared following the same procedure described in the pilot study.15 Chlorhexidine gluconate 0.2% (Avalon Pharma, Riyadh) and normal saline 0.9% NaCl solution, 500 ml (Pharmaceutical solutions industry, Jeddah) were used as positive and negative controls, respectively.

Assignment of the mouthwashes to the groups was double-blind. The allocation of assigned interventions was concealed using anonymous, unlabeled opaque bottles. A general dentist (blinded at the baseline) performed the initial dental screening, oral hygiene procedures, and the distribution of bottles. Another dentist masked to the randomization list carried out the pre-and post-intervention periodontal examination. Patients were directed to persistently abstain from daily oral healthcare practices (e.g. tooth brushing or flossing) and use 15 ml of the given mouthwash two times every day for one minute. They were also instructed to use the given measuring cup, shake the bottle before using, refrain from the use other mouth rinses, and refrain from eating or drinking thirty mins following the use of the mouth rinse. We also asked them to report any complaints or side effects.

Patients’ compliance to the assigned mouthwashes was primarily evaluated using a follow-up sheet given to them. They were also repeatedly reminded by phone calls to use their mouthwashes correctly. Additionally, to double-check the appropriate use of mouthwashes, they were even asked to bring back the bottles to verify the amount of solution used. Afterward (day 14 later), the same examiner reevaluate the participants and recorded the final values of all clinical parameters. Professional scaling, oral prophylaxis, and fluoride application were done at the end of the study.

Outcome measures

Primary outcomes were assessed using Trombelli et al. [19] MGI, O'leary et al. [20] PI, Ainamo and Bay [21] BOP, and Human IL-1β ELISA kit (BioVendor R&D – Laboratorni medicine a.s., Karasek, Czech Republic). Throughout this study, all participants underwent a weekly check-up to monitor PI and MGI values. BOP and IL-1β were assessed at baseline and post-intervention (day 14) to determine the presence of gingival bleeding and active inflammation. A standardized periodontal probe with a 0.6 mm tip and 1 mm marking was used to assess BOP. A skilled examiner measured bleeding on probing by exerting probing forces going beyond 0.25 N (25 g).

Two teeth were selected for IL-1β sample collection: first premolar (#12) and third molar (#16). Supragingival plaques were removed from the teeth before gingival crevicular fluid (GCF) collection. Filter paper strips of 2×8mm were used to collect GCF. The strips were placed into the gingival crevice and left in that position for 30 seconds. They were then stored in Eppendorf tubes filled with 400μl phosphate-buffered saline (PBS). The tubes were subsequently placed on ice and moved to the laboratory where they were frozen at −20°c until assayed. Distilled water was used to elute GCF from the strips. The sample was removed following the manufacturer’s instructions. Human IL-1β ELISA kit by BioVendor was used to obtain the IL-1β level. Putting reagents in place, implementation of the test protocol, and calculation of results were done through abiding by the manufacturer’s Product Data Sheet enclosed with the kit. The absorbance of each strip was read on a spectrophotometer at 450 nm wavelength.

Intraexaminer reliability

We conducted intraexaminer reliability of sulcular depth in some patients. The evaluation was done on 2 visits with one week difference. Gingival index and plaque index assessment were performed by employing clinical scenarios and pictures on 2 different time points. The intraexaminer reliability using the intraclass coefficients were 0.88, 0.92, and 0.82 for gingival, plaque, and sulcular depth, respectively.

Data collection and analysis

Statistical analysis was done using dedicated software (SPSS 24, IBM Corp., Armonk, NY, USA). Both pre-and -post-intervention data were collected. Mixed ANOVA was utilized to perform the data analysis. One between-subject variable (i.e., the interventions) and one within-subject variable (i.e., the time, pre-mouthwash vs. post-mouthwash) were included in the analysis. The assumption of sphericity was assessed with Mauchly’s test. The violation of assumption was corrected with the Greenhouse-Geisser or Huynh-Feldt Corrections for Departure from Sphericity. Pairwise comparisons with Bonferroni adjustment were performed as a post-hoc follow-up test to determine the statistical difference between treatment groups at different time points. For all statistical analyses, P < .05 was considered statistically significant.

Results and Discussion

A sum of 24 individuals were initially involved in this study. 5 were later excluded for not showing up in the follow-up visits (Figure 1). The final study population included 19 subjects, 10 males and 9 females (myrrh group, n = 6; CHX group, n = 7; and saline group, n = 6). The mean age of the participants were 30 (±10.55) years. No major difference between the groups at baseline was seen. (Table 1) shows the mean values of MGI, BOP, PI, and IL-1β across groups at baseline and after the intervention.

(Table 2) presents mixed ANOVA tests carried out to evaluate the between and within-group differences. There was a statistically major difference in MGI and BOP scores (P = .014 and p <.001, respectively) when both intra- and inter-group variations were considered (time * treatment). No major changes in the clinical parameters of PI and IL-1β were found between treatment groups and time.

(Table 3) shows a pairwise comparison of outcome measures between all treatment groups at two-time points. No major difference in the mean MGI, BOP, PI, and average IL-1β between the treatment groups (P >.05 for all pairwise comparisons) at baseline (time point 1)was observed. After the intervention period (time point 2), the mean MGI and BOP scores were considerably lesser in the myrrh group in comparison to the control group (mean difference=1.121, P=.016 and mean difference =44.173, P <.001, respectively). The CHX group also had lower mean MGI and BOP scores compared to the control. However, the mean MGI difference between CHX and control was not statistically significant (P =.09). there was no major difference in the mean PI and average IL-1β scores between treatment groups at any of the two times (P >.05 for all comparisons).

Table 1. Descriptive statistics for various measurements

MGI = Modified gingival index, BOP = Bleeding on probing, PI = Plaque index, IL-1β = interleukin-1β,

SD = Standard deviation.

^a IL-1β averaged over teeth 12 and 16.

Table 2. Mixed ANOVA results for MGI, BOP, PI and IL-1β

a IL-1β averaged over teeth 12 and 16

* Statistically significant (p < .05)

Table 3. Pairwise Comparisons of MGI, BOP, PI and IL-1β Scores at Two Time Points

Based on estimated marginal means

*. The mean difference is significant at the .05 level.

b. Adjustment for multiple comparisons: Bonferroni.