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This is an open access journal, and articles are distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike 4.0 License, which allows others to remix, tweak, and build upon the work non-commercially, as long as appropriate credit is given and the new creations are licensed under the identical terms.
This study aimed to assess the effect of hydroalcoholic extract of the whole pomegranate fruit on Streptococcus mutans (S. mutans) and Lactobacillus acidophilus (L. acidophilus) and its clinical effect on dental plaque formation in 8–10-year-old children.
This study was conducted in two phases of in vitro and clinical trial. In the in vitro phase, the antibacterial effect of 50%–50% hydroalcoholic extract of whole pomegranate fruit on S. mutans and L. acidophilus was assessed by the disc diffusion test. In the double-blind cross-over clinical trial phase, 14 children between 8 and 10 years were randomly assigned to two groups of 38% pomegranate mouthwash and 0.12% chlorhexidine (CHX) after assessing their baseline dental plaque by oral hygiene index-simplified (OHI-S). The children were asked not to use any other plaque control measure during the study. Their OHI-S score was measured again after 5 and 14 days using disclosing tablets. Data were analyzed by t-test and paired t-test at 0.05 level of significance.
The hydroalcoholic extract of pomegranate showed a positive antibacterial effect on S. mutans and L. acidophilus. However, its inhibitory effect was significantly lower than that of 0.12% CHX (P < 0.05). None of the tested mouthwashes inhibited plaque formation, but pomegranate mouthwash and CHX decreased the OHI-S score by 34% and 36%, respectively (P < 0.05), with no significant difference between them (P > 0.05).
The whole pomegranate fruit hydroalcoholic extract showed significant inhibitory effects on S. mutans and L. acidophilus. Furthermore, 38% pomegranate mouthwash had a comparable efficacy to CHX in the reduction of dental plaque.
Elimination of dental plaque-containing microbial biofilm is a challenge in pediatric dentistry. Biofilm is composed of a complex accumulation of microorganisms entrapped in a hydrated polymer matrix.
Mouthwashes are among the most effective antimicrobial agents used for plaque control. Chlorhexidine (CHX) is the most commonly used antimicrobial mouthwash.
Herbal extracts have long been used for the treatment of infectious diseases.
Previous studies on the effects of pomegranate extract on plaque index evaluated only the effect of one component of P. granatum plant and not the whole fruit extract.
This study was conducted in two phases: an in vitro study and a clinical trial.
In vitro phase
Sample size
All tests for the assessment of the effect of hydroalcoholic extract of the pomegranate whole fruit were conducted in triplicate according to previous studies.
Extract preparation
To prepare the hydroalcoholic extract of the whole pomegranate fruit, 10 medium-size pomegranates were diced into small pieces and crushed in a mixer. Water/ethanol (50:50 ratio) was then added as solvent, and the mixture was filtered after 72 h and heated at 60°C to obtain a honey-like consistency. Discs containing 100% concentrated extract were then prepared.
Assessment of the antibacterial effect of the hydroalcoholic extract of the whole pomegranate fruit
Standard-strain S. mutans (ATCC 35668) and L. acidophilus (ATCC 4356) were obtained from the Iranian industrial bacteria and fungi culture collection. Viable bacteria were cultured in brain–heart infusion nutrient medium in a Gas-Pak jar under anaerobic conditions for 24 h at 37°C. They were then streak cultured on blood agar plate and subcultured three times a week. The disc diffusion test was used for the assessment of the antibacterial activity according to the guidelines of the Clinical and Laboratory Standards Institute.
Statistical analysis
Data were analyzed using the SPSS software version 15.5 (SPSS Inc., Chicago, IL, USA) using t-test and paired t-test at 0.05 level of significance.
Clinical trial phase
This study was conducted at the Pediatric Dentistry Department of School of Dentistry, Islamic Azad University of Medical Sciences in 2022. The study protocol was approved by the ethics committee of the university (IR.IAU.PS.REC.1401.424) and registered in the Iranian Registry of Clinical Trials (IRCT20230110057104N1).
Trial design
A double-blind crossover clinical trial was designed, in which one group used 0.12% CHX mouthwash (Nazhu Co, Iran) first and pomegranate mouthwash after a 7-day washout period, whereas this order was reverse for the second group. The results were reported in accordance with the Consolidated Standards of Reporting Trials.
Participants, eligibility criteria, and settings
The inclusion criteria were age between 8 and 10 years, systemic health, no antibiotic intake within the past 3 weeks before the study onset, absence of oral habits, no use of orthodontic or prosthetic appliances, no xerostomia, no periodontal disease, and absence of active caries in the buccal and lingual surfaces of maxillary right first molar, mandibular left first molar, maxillary left lateral incisor, mandibular right lateral incisor, maxillary right central incisor, and mandibular left central incisor teeth.
The sample consisted of 14 children between 8 and 10 years who were selected by targeted sampling.
Interventions
Preparation of mouthwash
The hydroalcoholic extract obtained in the in vitro phase of the study was used for the preparation of the mouthwash along with the mint extract in 7:2 ratio and edible dye. The mint extract and edible dye were also added to the CHX mouthwash for the purpose of standardization of the taste and color of both mouthwashes. Furthermore, both mouthwashes were delivered to patients in identical bottles.
Intervention
After obtaining written informed consent from the parents, all children underwent a clinical oral examination using a dental explorer and a dental mirror (Fatahteb, Iran) to ensure meeting the eligibility criteria. All participants underwent prophylaxis and scaling and polishing, if required. Interdental areas were cleaned by an unwaxed dental floss (Essential Floss, Oral B, Ireland) to ensure the absence of dental plaque, stains, and calculus. A disclosing tablet was used to ensure complete cleaning of the teeth. All sessions were scheduled for children between 9:30 and 10:30 am, and the next session was scheduled 2 days later. The children were requested not to use any oral hygiene measure (no toothbrushing, mouthwash, dental floss, or chewing gum) during this period. In the second treatment session, a disclosing tablet was used to reveal the baseline dental plaque of patients, which was quantified using the oral hygiene index-simplified (OHI-S) by a trained dental assistant. Accordingly, six teeth including four posterior teeth (#16, 26, 36, and 46) and two anterior teeth (#11 and 31) were evaluated. The lingual surfaces of the mandibular posterior teeth and the buccal surfaces of other teeth were examined. The scoring system was as follows:
Score 0: A surface free from dental plaque Score 1: Presence of plaque in less than one-third of the tooth surface Score 2: Presence of plaque in more than one-third and less than two-thirds of the tooth surface Score 3: Presence of plaque in more than two-thirds of the tooth surface.
The total OHI-S score was calculated by summing the scores of the examined buccal and lingual surfaces divided by the number of all tested surfaces.
All children underwent prophylaxis again, and the quality of cleaning was assessed using disclosing tablets (Eviplac, Biodinamica Co., Brazil) to ensure clean teeth free from dental plaque, stains, and calculus. Each child was allocated a code, and half of the children randomly received 40 mL labeled bottles containing CHX, whereas the other half received 40 mL nonlabeled bottles containing the extract mouthwash. The children and the examiner were not aware of the contents of the bottles. Each bottle was graded (4 grades) to indicate the volume to be used at each time of use. The participants were requested to rinse the mouthwash twice a day, once in the morning and once in the evening. They were asked to rinse 10 mL of the mouthwash for 30 s each time and then spit it out. Their next session was scheduled 2 days later, and they were asked not to use any other oral hygiene measures (toothbrush, dental floss, or chewing gum) during this 48 h period. In the third session, disclosing tablets were used to calculate the OHI-S score of the patients. Next, a 7-day washout period was considered,
Outcomes (primary and secondary)
The main objective of this study was to assess the effect of hydroalcoholic extract of the whole pomegranate fruit mouthwash on OHI-S score of children.
Sample size calculation
The minimum sample size was calculated to be 14 patients according to a previous study
Interim analyses and stopping guidelines
No interim analyses were performed, and no stopping guidelines were established.
Randomization
In this crossover study, children were randomly assigned to receive either 40 mL of pomegranate mouthwash (B) or CHX (A) in two different sequences (AB and BA) using a table of random numbers with the Rand option of Excel software.
Blinding
The mouthwashes were delivered to children in identical bottles, and the children and the examiner were not aware of the contents of the bottles.
Statistical analysis
Data were analyzed using SPSS version 15.5 (SPSS Inc., Chicago, IL, USA) by t-test and paired t-test at 0.05 level of significance.
Participant flow
The sample consisted of 8–10-year-old boys.
CONSORT flow diagram of patient selection and allocation to the study groups. OHI-S: Oral hygiene index-simplified, CHX: Chlorhexidine, MIH: Molar Incisor Hypomineralisation.
In vitro results
Clinical results
This study assessed the effect of hydroalcoholic extract of the whole pomegranate fruit on S. mutans and L. acidophilus in vitro and also the effect of this extract on dental plaque in children between 8 and 10 years. The first phase of the study which had an in vitro design showed that the hydroalcoholic extract of the pomegranate fruit had inhibitory effects on both S. mutans and L. acidophilus; however, its effects were significantly lower than those of CHX. Thus, the first null hypothesis of the study was rejected. Consistent with the present results, Naz et al.
The pomegranate fruit has been used for centuries with no toxic effects.
The second phase of the study assessed the effect of 38% hydroalcoholic extract of the pomegranate fruit on dental plaque in children between 8 and 10 years. The results showed that both mouthwashes significantly decreased the OHI-S score with no significant difference between them. Thus, the second null hypothesis of the study was accepted. Clinical studies on the effects of pomegranate mouthwash on plaque index are limited. Consistent with the present study, Bhadbhade et al.
In contrast to the present findings, Ahuja et al.
Conduction of the study in two phases of in vitro and in vivo was the main strength of this study. However, uncertainty about regular use of mouthwashes by children as instructed was a limitation of this study which might have affected the results. Furthermore, the constituents of the pomegranate fruit were not isolated to assess their individual effect in the present study. Future studies are recommended to assess the isolated fractions of the pomegranate fruit and evaluate their antibacterial effects on a wider range of oral pathogens, especially Gram-negative microorganisms. Moreover, the long-term cariostatic and anti-inflammatory effects of the pomegranate mouthwash in conjunction with mechanical plaque control measures should be investigated in both children and adults.
The whole pomegranate fruit hydroalcoholic extract showed significant inhibitory effects on S. mutans and L. acidophilus. Furthermore, 38% pomegranate mouthwash had a comparable efficacy to CHX in reduction of dental plaque.
Financial support and sponsorship
This study was derived from a research project approved and funded by Islamic Azad University of Medical Sciences, Tehran, Iran.
Conflicts of interest
The authors of this manuscript declare that they have no conflicts of interest, real or perceived, financial or nonfinancial in this article.