Twenty-two volunteers of dental students were randomly divided into the intervention and control groups, each including 11 individuals of both genders.
The numbers one and two representing intervention and control groups respectively were written on twenty-two pieces of paper (11 each), and were put in a bowl. Students were asked to pull a piece of paper one by one, so that other students would not be aware of each other’s grouping. Their groups were assigned based on the random selection of the papers. Each student was given a two-digit code (01 to 22), and these 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 mouthwash 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 15 mL 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 mouth for almost 30 minutes . After these two weeks, they entered the 3-week washout phase (not using mouthwash) in order for their 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 again asked not to change their usual health care habits, but keep 15 mL 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 mountain 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 g of the powder was soaked in 2L of water for 48 hours and was filtered 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 °C using a rotary machine (Lab Tech EV 311-V, Rome, Italy). Eventually, concentrated extracts were dried by oven (Memmert UF-55, Frankfurt, Germany) at 42 °C for 3 days. Extraction yield was 10%. Dried extract was kept in a freezer at -22 °C for further experiment.
All components of the mouthwash except for T. polium extract were utilized for the preparation of the placebo. Distilled water (Zolal Teb Shimi, Tehran, Iran) was used instead of T. polium extracts to prepare the placebo.
Prepared mouthwashes were packed in matte plastic containers, and then were 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, 25% concentration of mouthwash was chosen for further experiment due to its adequate taste. The mouthwash ingredients were as below: 1L deionized water, 2 g T. polium extracts, 1 g artificial sweetener powder of 0.1% aspartame (Merck, Darmstadt, Germany), and 5 g powder of 0.5% coffee flavor (Nestle, Netherland, Holland).
To sampling the volunteers’ saliva, they were asked not take food or beverage for almost one hour prior to sampling. Sampling, however, was carried out every day at 10 a.m. in which 2 mL of their unprovoked saliva was collected in a sterile container .Unprovoked saliva sample of all individuals was taken at the beginning of the study, before using the mouthwash. The second test was arranged at last day of the first phase of experiment using a spitting method by which the volunteers were asked to take a minute's saliva in their mouth and then pour in a sterile falcon tub. Third and fourth tests were conducted before and after the second phase. During the experiment, the volunteers were asked not change their daily health care practices and brush their teeth using Crest toothpaste and Oral B toothbrush.
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 as mentioned in the standard protocol . Normal saline sterile (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 mL Agar TYCSB using a 0.01 ml standard loop (Lab Tron, Tehran, Iran) .The plates were placed in a CO2 incubator (Gallenkamp, Munich, Germany) at37 °Cfor 48 h.
Thereafter, 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 ° C. Based on previous recommendations [18, 19], colonies of positive mannitol, positive vogues-proskauer (VP), negative arginine, positive dextran, negative urease, and positive bile esculin were the target colonies. Eventually, Streptococcus mutans’ colonies were calculated and were multiplied by dilution ratio to determine the number of colonies per one mL of each participant’s saliva (CFU/mL).
Data was analyzed by SPSS software (Version 18.0). Quantitative data were reported as mean value and standard deviation. Qualitative data was reported as number and percentage. 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 number 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. Finally, 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, however, was considered to be 0.05 in this experiment.
Ethical issues approved by the Ethics Committee of the Rafsanjan University of Medical Sciences with the approval number of 937/9/31 and IRCT code No. IRCT2013121815842N1. An Informed consent was taken from participants and there was no compulsion to participation in the study. Attending or not attending had no effect on student education.
Consolidated standards of reporting trials (CONSORT) flow diagram
CONSORT flow diagram is seen in Fig. 2.