A randomized controlled trial was conducted between October 2019 and March 2020 in Belgium. The study protocol was reviewed and cleared by the Ethics Committee of the Institutional Review Board (AZ Groeninge Kortrijk, Belgium). The study protocol and the purpose were explained orally to each participant. Oral and signed consent from each participant was obtained before the start of the study.
A total of 72 adults (38 women and 34 men), aged between 18 and 83 were chosen for the study from Euro-Dent clinic, Mortsel 2640, Belgium. For convenience, the study sample was chosen among the clinic patients who are eligible (criteria described below) and agreed to participate in the study with consent. Our study population represent all age group adults i.e., young (18–35; n = 33), middle aged (36–55; n = 28) and elderly (56–90; n = 11). The chosen 72 adults satisfied the following selection criteria for the study: (a) presence of a minimum number of teeth (seven), including one molar, (b) absence of dentures, (c) no recent history of antimicrobial therapy or other drug therapy, including immunosuppressive, and (d) no history of diabetes.
The periodontal score of the participants were scored on the basis of Dutch periodontal screening index (DPSI) as follows: 0, perfect gum and no bleeding; 1, inflammation and bleeding of gum (gingivitis); 2, conditions of category 1 and chalk hardened dental plaque; (− 3), conditions of category 2 with bone involvement (periodontitis); (+ 3) conditions of (− 3) with recessions of gum and root exposure; and 4, conditions of category (+ 3) with severe bone resorption and high tooth mobility. The number of individuals with DPSI score 0, 1 and 4 were extremely rare in Euro-Dent clinic during our study period, therefore we did not involve these three DPSI score categories in our study. Our study population involve 72 participants in total, which include DPSI score 2 (50 participants); DPSI score + 3 (10 participants) and DPSI score − 3 (12 participants). The chosen 72 participants were not taking any special treatments for dental plaque before or during the course of study.
Equipment and chemicals
The disposable sampling microbrush applicators were purchased from Microbrush International (Ireland). The plastic consumables like microtubes, petriplates, ready-to-use media of LB agar plates and DEV-Nutrient agar plates (90 mm) and paper discs were purchased from VWR International (Belgium). Product A, Product B and chlorhexidine 0.2% were purchased from local supermarket/medical store in Belgium. Pure isolates of CBD and CBG crystalline powders were purchased from PharmaHemp (Slovenia). The Synbiosis-aCOLyte 3 HD colony counter and Memmert incubator were purchased from Wilten Instrumenten (Netherlands). The sterile plate spreaders, 50 ml sterile conical tubes and Biosan orbital shaker-incubator were purchased from NOVOLAB (Belgium).
Dental plaque sampling
Prior to plaque sampling, saliva on the tooth surface was removed by water spray, and the sampling target area was dried with cotton. Plaque samples were collected from interdental spaces using disposable microbrush applicator and immediately dispensed into a 2 ml microtube containing 1 ml of phosphate buffer saline (PBS) and was processed for in vitro assay within 24 h from sampling.
Test material-mouthwash products
The test products i.e., CannIBite mouthwash products contain herbal ingredients including cannabinoids, wintergreen oil and stevia extract. Two test mouthwash (MW) products were used in this study i.e., CBD-MW containing (< 1% by weight) cannabidiol (CBD) and CBG-MW containing (< 1% by weight) cannabigerol (CBG). Most commonly available OTC mouthwashes, designated hereafter as Product A and Product B mouthwash were also used in this study to compare the efficacy of our test products. Product A represents alcohol-containing mouthwash with essential oils such as thymol, eucalyptol, and menthol as active ingredients. Product B represents alcohol-free mouthwash containing fluoride and potassium nitrate as active ingredients. In addition, the medical grade mouthwash containing 0.2% chlorhexidine (the so-called gold standard) was used as positive control. All the mouthwash products used in this study were directly used for in vitro assay without any dilutions.
In vitro assay
The dental plaque sample was mixed well using vortex and an aliquot of 100 μl of dental plaque sample was spread on the surface of LB agar plate or DEV-Nutrient agar plate using sterile spreader. For agar well diffusion method, agar well of 4 mm diameter were made (on plate pre-inoculated with dental plaque sample) using sterile agar well borer and 30 μl of undiluted MW was added to the well. In case of disc diffusion method, sterile paper disc of 5 mm diameter was placed on the surface (on plate pre-inoculated with dental plaque sample) and 15 μl of undiluted MW was gently added to the disc. To minimize the bias in data, we included three technical replicates for each sample. Each sample assay was performed in triplicates.
The petri dishes were sealed with thin layer parafilm (to reduce the evaporation of test products) and incubated at 37 °C for 36 h. After incubation, the diameter of zone of inhibition was measured manually using caliper. The pictures of plates were taken using automated colony counter. Zone of inhibition was not recorded using colony counter due to varying colony size, density, color and texture (within and between samples) because of the presence of multiple species of bacteria. Reducing the colony size threshold beyond a limit (< 0.2 mm) in colony counter leads to false reading. Moreover, the paper discs on the plate interfere with the contrast of colony vs. media background and affects the reading by colony counter. However the zone was clearly visible when the plate was observed in front of bright light source such as lamp and was easy to measure manually.
For minimum inhibitory concentration (MIC) assay, the test products were serially diluted with sterile LB broth media from 0 to 11 dilutions (0 represents undiluted test product) in 50 mL sterile conical tubes. After inoculating equal amount of dental plaque bacteria (fresh overnight culture pre-prepared a day before) in each tube containing 2 mL of respective serially diluted media, the samples were incubated in orbital shaker-incubator at 37 °C for 24 h. For comparison, we also tested the MIC assay for Product A, Product B and CHX 0.2%.
The liquid culture turbidity (for MIC) and plate readings (for zone of inhibition) were measured by the same person (unblind to test and control products used) who performed the assay, which was a major limitation in methodology as the samples were open-label.
The in vitro experiments were conducted in triplicates. The average values of zone of inhibition for each of the samples were calculated in Microsoft Excel (Table S1; and raw data provided as Table S2). The average values were used to represent in graphs and table. Student’s unpaired t-test was performed to compare the results between DPSI score groups for each test product (Table S3). Student’s unpaired t-test was also performed to compare the results between products irrespective of DPSI score grouping (Table S3).