An AB/BA randomized, crossover, double-blind clinical trial study was designed (Fig 1).The Ethics Committee of the Rafsanjan University of Medical Sciences approved the ethical issues. The approval number is 937/9/31 and with the reference code of IRCT2013121815842N1 from the Iranian Registry of Clinical Trials (IRCT).
Inclusion included four criteria: Not using antibiotics in the last month, Not having active caries, Plaque index equal to or less than 20% (demographics aging from eighteen to twenty-five years old) and signing a consent form before participating in this study.
Samples were selected from dental students of Rafsanjan School of Dentistry. Sample size was calculated based on a previous study . The following formula was used to estimate the sample size:
See formula 1 in the supplementary files.
Twenty-two dental students, who volunteered to participate, were randomly chosen and were put into two categories: Intervention group and control group. Each group included 11 individuals of both genders.
Number 1 (representing the intervention group) and number 2 (representing the control group) were written on twenty-two pieces of paper (11 each), and were placed in a bowl. We asked the participants to each take a number so they can be assigned to the group the number represented. Their groups were assigned based on the random selection of the papers. Each student was given a two-digit code (01 to 22). The codes were used during the trial, instead of their names.
The study had two phases. In each phase, one group was chosen as intervention, while the other group was the control group. Intervention and control groups were given the mouthwashes with and without T. polium, respectively. The codes of the students were written on mouthwash bottles and were given to the students, so that neither the students nor the laboratory personnel were aware of the type of mouthwash (double-blinded).
The volunteers were asked not to change their usual health care practices, but keep 15mL of the mouthwash in their mouth for 30 seconds twice a day for two weeks. They, however, were requested to neither have food or beverages nor wash their mouthsfor almost 30 minutes . After two weeks, they entered a 3-week washout phase (not using mouthwash). This exercise wasfor saliva’s S. mutans value to return to initial levels . In this phase, opposite to the first phase, placebo and T. polium mouthwashes were given to the first and second groups. Then, they were asked not to change their usual health care habits again, but keep 15mL of the mouthwash in their mouth for 30 seconds twice a day for two weeks.
The extract of T. polium was prepared at the Faculty of Pharmacy, Kerman University of Medical Sciences.T. polium was collected from the mountainous area around Kerman, the capital city of Kerman province in the South East of Iran, in June. Plant flowers were cleaned, then washed with cold water and deionized water (Zolal Teb Shimi, Tehran, Iran), and were dried in a place away from direct sunlight. The dried samples were grounded with an electric mill (Moulinex 1043, Paris, France), and passed through a sieve with a mesh size of 32. Next, 250 grams of the powder was soaked in 2 liters of water for 48 hours and wasfiltered with a vacuum pump (Eyela A-35, Tokyo, Japan), a Buchner funnel (Isolab, Hannover, Germany), and a filter paper (Munktell, Bavaria, Germany). Obtained extracts were concentrated by vacuum distillation method at 52° Celsius, using a rotary machine (Lab Tech EV 311-V, Rome, Italy). Eventually, concentrated extracts were dried in an oven (Memmert UF-55, Frankfurt, Germany) at 42°Celsiusfor 3 days. Extraction yield was 10%. Dried extract was kept in a freezer at -22° Celsius for further experimentation.
All components in the mouthwash, except for T. polium extract, were utilized to prepare the placebo. Distilled water (Zolal Teb Shimi, Tehran, Iran) was used instead of T. polium extracts to make the placebo.
Prepared mouthwashes were packed in matte plastic containers, and thenwere tagged with specific codes that were only known to the researchers, and were given to the volunteers. Each compound was prepared at the earliest time to its consumption to ensure the highest drug stability and the highest amount of active ingredients. After extract preparation, 0.2% concentration of mouthwash was chosen for further experiment due to its adequate taste. The mouthwash ingredients were as below: 1 Liter deionized water, 2 grams of T. polium extracts, 1gram of artificial sweetener powder of 0.1% aspartame (Merck, Darmstadt, Germany), and 5grams powder of 0.5% coffee flavor (Nestle, Netherland, Holland).
To sample the volunteers’ saliva, they were asked not take food or beverage for almost one hour prior to sampling. Sampling was done every day at 10 AM while 2 milliliter of their unprovoked saliva was collected in a sterile container (spitting method) . Unprovoked saliva sample of all individuals was taken at the beginning of the study, before using the mouthwash. The second test was conducted at the last day of the first phase of experiment using a spitting method. Participants were asked to take a minute's saliva in their mouth, and then pour in a sterile falcon tub.The third and fourth tests were done before and after the second phase. During the experiment, the volunteers were asked not to change their daily health care practices and brush their teeth using Crest toothpaste and Oral B toothbrushes.
To determine the number of Streptococcus mutans colonies, samples were sent to the Laboratory of Microbiology, University of Rafsanjan, Iran. One day before saliva sampling, the culture medium was prepared. The TYCSB (Tryptone-Yeast-Cysteine-Sucrose-Bacitracin) medium of S. mutans was prepared in accordance with the standard protocol . Standard 0.9% NaCl (Zolal Teb Shimi, Tehran, Iran) was used to make a 1:1000 dilution of the samples, and then they were transferred to the plates containing 20 milliliter agar TYCSB using a 0.01 milliliter standard loop (Lab Tron, Tehran, Iran) . The plates were placed in a CO2 incubator (Gallenkamp, Munich, Germany) at 37° Celsius for 48 hours.
Accordingly, Gram's method was used to distinguish between gram negative and positive bacteria. Gram-positive colonies (cocci) were selected to perform the catalase test. Next, catalase-negative cocci were incubated under biochemical tests for 48 hours at 37°Celsius. Based on previous recommendations [20, 21], colonies of positive mannitol, positive vogues-proskauer (VP), negative arginine, positive dextran, negative urease, and positive bileesculin were the target colonies. Eventually, Streptococcus mutans’ colonies were calculated and were multiplied by dilution ratio to determine the number of colonies per one milliliter of each participant’s saliva (CFU/mL).
Data was analyzed by SPSS software (Version 18.0). Quantitative data was reported via the Mean value and Standard Deviation (numbers and percentages). Paired t-test was used to determine the effect of each mouthwash on the Streptococcus mutans’s colonies. Moreover, an independent two-sample t-test was used to compare the Mean of Streptococcus mutans’s colonies after mouthwash consumption in each phase of the experiment. Besides, crossover analysis was used to compare the mean of Streptococcus mutans’s colonies after using T. polium and placebo mouthwashes during the study. As a final point, a standard AB/BA crossover model was used to find the results of treatment and the carryover effect on the residual biological effects after using the mouthwash until the final phase . The significant level was 0.05 in this experiment.
Ethics Committee of the Rafsanjan University of Medical Sciences approved the research’s ethics. Approval number is 937/9/31 with IRCT code number of IRCT2013121815842N1. All the participants signed an informed consent form and we observed zero resistance in participation from the volunteers. Attendance in this study was not compulsory and the participants did not receive any course credits for attending.
Consolidated standards of reporting trials (CONSORT) flow diagram
CONSORT flow diagram is seen in fig 2.