In Vitro Assessment of Antibacterial Activity of Pomegranate Vinegar and Rose Water Compared with Persica Mouthwash Against Oral Bacteria

Original Article

In Vitro Assessment of Antibacterial Activity of Pomegranate Vinegar and Rose Water Compared with Persica Mouthwash against Oral Bacteria

F. Ramezanalizadeh 1, M. Rabbani 2
, M. Khoroushi 3, A. Aliasghari 4

1 Graduate Student, Department of Microbiology, School of Biology, Isfahan University of Medical Sciences, Isfahan, Iran 2 Associate Professor, Department of Microbiology, School of Biology, Isfahan University of Medical Sciences, Isfahan, Iran 3 Professor, Department of Operative Dentistry, School of Dentistry, Isfahan University of Medical Sciences, Isfahan, Iran 4 Postgraduate Student, Department of Microbiology, School of Biology, Isfahan University of Medical Sciences, Isfahan, Iran

Abstract Background and Aim: Mutans streptococci are the main cause of tooth decay. Application of natural materials as mouthwash has been effective in reducing the bacterial count. This study aimed to assess the antimicrobial effects of rose water and pomegranate vinegar in comparison with Persica mouthwash on two oral bacteria responsible for tooth decay. Materials and Methods: Strongly adherent strains of Streptococcus mutans and Streptococcus sobrinus were selected for this in vitro study. Antimicrobial effects of pomegranate vinegar and rose water on microbial count in the biofilm and adhesion potential of bacteria were evaluated by microtiter plate method. Also, the well-plate technique was used to assess the effect of rose water and pomegranate vinegar in comparison with Persica mouthwash on bacterial growth and proliferation. The obtained resulted were analyzed by one-way ANOVA and Tukey’s post hoc test using Graph Pad Prism version 5 software. Level of significance was set at P=0.05 with 95% confidence interval. Results: Pomegranate vinegar, rose water and Persica decreased plaque formation by S. mutans by 93%, 80% and 68%, respectively. These values for the S. sobrinus were 92%, 57% and 48%, respectively (all Ps<0.001). Pomegranate vinegar was more effective than the other two materials (P<0.001). However, none of these materials eliminated the Downloaded from jidai.ir at 7:31 +0330 on Tuesday September 28th 2021 biofilm. Pomegranate vinegar and Persica mouthwash inhibited the growth of the afore-mentioned bacteria. Conclusion: Within the limitations of this study, it seems that pomegranate vinegar and
Corresponding author: rose water have the potential to prevent or control the proliferation of S. mutans and S. M. Rabbani, Associate s o b r i n u s . Professor, Department of Key Words: Dental Caries, Mouthwashes, Streptococcus mutans, Streptococcus Microbiology, School of sobrinus Biology, Isfahan University of Medical Sciences, Isfahan, Iran

[email protected]

Received: 19 March 2015 Accepted: 15 May 2015 Journal of Islamic Dental Association of IRAN (JIDAI) Summer 2015 ;27, (3)

Introduction 500,000 plant species worldwide; out of which, Increased public demand for natural substances only 1% have been photo-chemically evaluated for treatment of diseases has resulted in a search and there is a great potential for finding bioactive for herbal medications. There are approximately materials [1]. Several articles are available on the

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antimicrobial efficacy of plant extracts; however, production of moisturizing lotions or cleansers the effects of medicinal plants on oral pathogenic [8]. If its antimicrobial efficacy is confirmed, it microorganisms have been less commonly can be effectively used in mouthwashes for caries evaluated. prevention. Thus, this in vitro study aimed to Tooth decay is a common infectious disease with assess the effect of rose water on the most higher prevalence among low socioeconomic common cariogenic bacteria. groups [2]. Treatment of caries is time Vinegar has also been used in the Iranian consuming and costly. Considering the high traditional medicine. Its production dates back to prevalence of caries, increased bacterial 4000 B.C. Its production process is sort of an resistance to antibiotics, adverse effects of incomplete oxidation reaction. In the process of chemical and synthetic drugs as well as their high vinegar production, yeasts first convert sugar to cost, there is a clear need for natural, cost alcohol and then acetobacters convert alcohol to effective alternatives for prevention of caries. citric acid [9]. Vinegar increases the The currently available chemical medications accumulation of glycogen, prevents hypertension, change the oral microbiota and have stimulates the uptake of calcium and decreases complications such as diarrhea, vomiting and total serum cholesterol and triacylglycerol as tooth staining [1,3]. Streptococcus mutans and S. indicated in animal studies [10]. Use of vinegar sobrinus are the main causes of caries due to their for infection control and treatment of acute high potential for biofilm formation. These conditions dates back to the time of Hippocrates, bacteria adhere to tooth enamel and produce who recommended vinegar for wound cleansing glucans following the consumption of sucrose in and faster healing [9]. Different types of foods and result in accumulation of glucans in the vinegars are available. This study aimed to assess biofilm and eventually cause enamel destruction the antimicrobial effect of pomegranate vinegar viaacid production [4]. Since bacterial adhesion on oral bacteria. The effects of pomegranate is the first step for bacterial prolifertaion and extract on oral bacteria have been previously tooth decay, adhesion is the new target for the evaluated but no study has evaluated the effect of modalities aiming to inhibit bacterial colonization pomegranate vinegar on oral bacteria. [3]. In another study, Brazilian researchers evaluated In this regard, medicinal plants have also been the effect of pomegranate extract on adhesion of evaluated. Studies in this regard are divided into oral bacteria (which is the first step in develop- two groups. Group one studies assess the activity ment of caries) [11]. of natural compounds, plant extracts and pure Persica mouthwash contains Salvadora, Persica, Downloaded from jidai.ir at 7:31 +0330 on Tuesday September 28th 2021 photochemical agents against oral pathogenic mint and yarrow extracts. Several studies have microorganisms. Group two studies assess the reported antimicrobial, antiplaque and efficacy of these products for inhibition of oral anti-cariogenic effects of Salvadora (Miswak) biofilm formation via decreasing microbial [12]. Since Persica mouthwash contains attachment to tooth surfaces [5]. Materials that natural substances and is commercially available have a potential to inhibit bacterial adhesion and in the market, it was used for the purpose of growth would be ideal for prevention of tooth comparison in the current study. caries. Rose water and vinegar have been recommended Rose water and vinegar have been used in the for caries prevention in the Iranian traditional Iranian traditional medicine to prevent tooth medicine. Since the antimicrobial effects of these decay. Rose water is derived from Rosa two substances on oral streptococci have not been damascena Mill. From the Rosacea family [6]. evaluated before, this study aimed to assess the Rose water is produced in Iran in large amounts antimicrobial effects of these substances on and is exported to different countries worldwide. cariogenic oral microorganisms in comparison It has several applications and is used for with Persica (a commonly used botanical different conditions such as skin lesions [7]. It is mouthwash). also used in cosmetic industries and for the

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Materials and Methods wavelength. Bacterial strains were then classified In this in vitro study, five strains of S. mutans based on their OD as follows [15]: were isolated from the dental caries and dental OD: The mean light absorbance of bacteria plaque of candidates using a sterile curette. ODc: The mean light absorbance of control wells Standard strains of S. mutans (ATCC35668) and OD≤ODc: No adhesion S. sobrinus (ATCC27607) were also obtained ODc

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Effect of rose water, pomegranate vinegar and plates were incubated for 16-20 hours and the Persica mouthwash on attachment of bacteria diameter of the growth inhibition zone was then with strong adhesion: measured. Overnight culture of strongly adherent The data were analyzed using SPSS version 16. streptococci in TSB medium supplemented with One-way ANOVA and Tukey’s post hoc test 1% sucrose was done. To assess the effect of were used for analysis of the data related to S. these materials on bacterial adhesion, two mutans, and the Kruskal Wallis test and Mann methods were used: Whitney U test were used to analyze the data In the first method, 200µL of a mixture with regarding S. sobrinus. Level of significance was similar portions of antimicrobial agent and set at P=0.05. bacteria was transferred to the wells while the control wells only contained microbial Results suspension. In the second method, 100µL of rose Assessment of the quantity of biofilm formation water, pomegranate vinegar and Persica by streptococci: mouthwash were poured into wells and To assess the effect of rose water, pomegranate streptococcal suspension was added 30 minutes vinegar and Persica on bacterial adhesion, first later. In the control wells, first 100µL of the quantity of formed biofilm was assessed in phosphate buffered saline (PBS) and after 30 order to select strains with greater adhesion to minutes, 100µL of streptococcal suspension were assess the effect of these antimicrobial agents on added. After 24 hours of incubation, the solutions adhesion of bacteria with strong adhesion. and nutrients were removed from the wells and Diagram 1 shows the mean of three after three times of rinsing with PBS, staining measurements of OD of bacteria. Standard S. was performed using 2% crystal violet for five mutans and S. sobrinus strains (bacteria #1 and 2) minutes. After addition of 33% acetic acid, OD were the strongly adherent strains. Bacteria #4-7 of the solvent was read using an ELISA reader. isolated from the mouth of candidates had poor Assessment of the effect of rose water, adhesion and bacterium #3 was non-adherent. pomegranate vinegar and Persica on bacterial Effect of understudy materials on S. mutans and growth and proliferation: S. sobrinus biofilm: The well-plate technique was used to assess the The results showed that rose water, pomegranate effect of antibacterial agents in this respect [7]. vinegar and Persica were able to prevent biofilm After 18-20 hour culture of S. mutans and S. formation by these bacteria. The percentage of sobrinus in brain heart infusion broth, a microbial reduction in biofilm formation in presence of Downloaded from jidai.ir at 7:31 +0330 on Tuesday September 28th 2021 suspension with 0.5 McFarland standard pomegranate vinegar, rose water and Persica was concentration was prepared of the bacteria and 0.089%, 0.38% and 0.089%, respectively for S. cultured on Mueller Hinton agar supplemented mutans and 0.061%, 0.37% and 0.062%, with 5% defibrinated sheep blood using a sterile respectively for S. sobrinus; no statistically swab in three directions with a 60° angle. significant difference was noted in this respect Inoculated plates were placed on a smooth (P=0.514). surface for three to five minutes and then, Effect of understudy materials on strongly equal wells measuring 6mm in diameter were adherent bacteria: created in the medium using the tip of a sterile The effects of rose water, pomegranate vinegar Pasteur pipette. One drop of melted Mueller and Persica on adhesion of S. mutans and S. Hinton agar was poured into each well in order to seal the bottom of the wells. Next, 100 µL of the sobrinus are presented in Table 1. All three antimicrobial agents were added to each well. materials significantly decreased the adhesion of Distilled water was used as the negative control. S. mutans and S. sobrinus compared to the The plates were refrigerated for one hour in order control group (P<0.001). As seen in Table 1, for the antimicrobial agents to be able to diffuse rosewater resulted in 80 and 56% reduction in in the medium before bacterial proliferation. The adhesion of S. mutans (in the first and second

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methods, respectively) and 57% and 60%, decreased the adhesion of both bacteria to the respectively for the adhesion of S. sobrinus, same extent (P=0.105). which indicate no significant difference in the Effect of medicinal plants on growth of two methods for the effect of rose water on S. streptococci: sobrinus (P=0.451). Also, the effect of rosewater Table 2 shows the mean diameter of the growth on adhesion of S. mutans in the second method inhibition zones of S. mutans and S. sobrinus. As had no significant difference with that of Persica seen in Table 2, rosewater could not inhibit the (P=0.319). However, the second method was growth of S. mutans and S. sobrinus and caused effective for S. sobrinus (P=0.003). no growth inhibition zone. Pomegranate vinegar Pomegranate vinegar inhibited the adhesion of created considerable growth inhibition zones with both bacteria and had the greatest effect on 16 and 18mm diameters around S. mutans and S. bacterial adhesion compared to other materials in sobrinus, respectively. The growth inhibition both methods (P<0.001); no significant zone of S. mutans was 11mm in presence of difference was observed between the two Persica, which was significantly smaller than that methods in this respect (P=0.514) caused by pomegranate vinegar (P<0.001); this Persica significantly decreased bacterial adhesion value was 14mm for S. sobrinus, which was also compared to the control group (P<0.001). No smaller than that caused by pomegranate vinegar significant difference was found between the two (P=0.003). methods for S. sobrinus, and both methods

2/0 Streptococcus Motanes 1/5 Streptococcus Motanes 2 2 9 9 4 1/5 4 D D 1/0 O O 1/0 0/5 0/5

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Diagram 1. The trend of reduction in biofilm formation in presence of rose water, pomegranate vinegar and Persica mouthwash

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Table 1. The percentage of reduction in adhesion of S. mutans and S. sobrinus in presence of rosewater, pomegranate vinegar and Persica

Percentage of Percentage of Standard Standard Material Bacteria reduction in reduction in deviation deviation adhesion (method 1)* adhesion (method 2)** S.mutans 80 0.06 56 0.08 Rosewater S.sobrinus 57 0.10 60 0.16 Pomegranate S.mutans 93 0.01 95 0.02 vinegar S.sobrinus 92 0.03 93 0.03 S.mutans 68 0.11 52 0.06 Persica S.sobrinus 48 0.10 32 0.12

*Antimicrobial agents and bacteria were transferred to wells simultaneously **Antimicrobial agents were transferred to wells 30 minutes earlier than the bacteria

Table 2. The mean diameter of growth inhibition zones for S. mutans and S. sobrinus in presence of pomegranate vinegar, rose water and Persica

Material Mean diameter of growth inhibition Bacteria Standard deviation zone in well-plate method (mm) S.mutans 16 0.89 Pomegranate vinegar S.sobrinus 18 0.95 S.mutans 0 0 Rose water S.sobrinus 0 0 S.mutans 11 0.79 Persica S.sobrinus 14 1

Discussion 2010 evaluated the antimicrobial and disinfecting Several studies have assessed the antimicrobial effects of 0.12% chlorhexidine, 0.50% white efficacy of Persica herbal mouthwash for vinegar and two other materials on S. mutans, S.

Downloaded from jidai.ir at 7:31 +0330 on Tuesday September 28th 2021 reduction of dental plaque. This material has pyogenes, Staphylococcus aureus and Candida antimicrobial effects on oral pathogenic bacteria albicans on toothbrush and showed that vinegar [12]. The current study showed the efficacy of decreased the count of S. mutans, S. pyogenes Persica in decreasing the adhesion of oral and S. aureus [18]. streptococci; this result is in agreement with that A few studies are available on the antimicrobial of other studies on the effect of this mouthwash effects of compounds derived from pomegranate on oral bacteria. On the other hand, inability of on oral pathogenic microorganisms. Persica to eliminate biofilm or inhibit the growth Loo et al, in 2010 reported the effect of lactic and proliferation of oral bacteria was noted as acid on oral bacteria [19]. Pomegranate extract well. contains about 40% lactic acid. Also, The antimicrobial effects of vinegar have been Vasconcelos et al, in 2006 showed the previously confirmed. Ismael evaluated the effect antimicrobial effects of pomegranate extract on of several types of vinegar on the biofilm of S. mutans [11]. Streptococcus pyogenes isolated from patients Subramaniam et al. compared the antimicrobial and showed that vinegar eliminated a minimum effects of pomegranate extract with those of aloe of 90% of the biofilm [17]. Komiyama et al, in Vera and sorbitol and showed that pomegranate

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extract had greater antimicrobial efficacy effects of vinegar and rose water in effective compared to the other two against S. mutans [20]. formulations on dental caries in clinical trials. The current results showed that pomegranate vinegar significantly inhibited the adhesion and Conclusion proliferation of bacteria; however, after biofilm Considering the optimal antimicrobial effects of formation, it could not eliminate it. Vinegar may pomegranate vinegar on proliferation of be suitable for elimination of oral bacteria since it cariogenic streptococci and the optimal efficacy can inhibit adhesion and proliferation of bacteria of pomegranate vinegar and rose water in at the same time. An important issue with regard decreasing the adhesion of these bacteria, future to vinegar is its acidic pH. Although mutans studies are recommended to assess the streptococci are among the acid-producing application of these materials in different bacteria (acid production is a mechanism for pharmaceutical formulations for caries them to compete with other pathogenic prevention. microorganisms in the oral cavity), they cannot survive vinegar exposure. Iranian traditional Acknowledgement medicine has also pointed to the anticariogenic The authors would like to thank the Biology effects of vinegar; however, this property of Group of Isfahan University of Science for their vinegar has been neglected due to the existing scientific cooperation and the Research Deputy concerns regarding the adverse effects of acids on of Isfahan University of Medical Sciences for tooth enamel. But, further studies are required to financially supporting this project. focus on the constituents of vinegar and purify the extract effective on cariogenic bacteria. References Search of the literature yielded no study on the 1. Palombo, E.A.Traditional medicinal plant effect of rose water on plaque producing oral extracts and natural products with activity against bacteria. Thus, the current study was the first to oral bacteria: potential application in the assess the effect of rose water on oral bacteria. prevention and treatment of oral diseases. Evid The results showed that rose water had a Based Compliment Alternat Med, 2011; 2011: significant effect on decreasing the adhesion of 680354:15 pages. bacteria but had no effect on their growth and 2. Curzon ME, Preston AJ. Risk groups: nursing could not eliminate biofilm after its formation. bottle caries/caries in the elderly. Caries Res. This finding shows that the mechanism of rose 2004 Jan;38(1):24-33. water in decreasing biofilm formation is different 3. Lamont RJ, and HF. Jenkinson. Oral Downloaded from jidai.ir at 7:31 +0330 on Tuesday September 28th 2021 from its bactericidal and growth inhibition microbiology at a glance. Wiley Black well mechanisms; however, a definite conclusion in publication,England. 2010,Chapter 13. this regard requires further studies. 4. Taubman MA, Nash DA. The scientific and The results of this study showed that public-health imperative for a vaccine against pomegranate vinegar, Persica and rosewater were dental caries. Nat Rev Immuno. 2006 July; 6(7): all effective against adhesion of oral bacteria; 555-563. however, they were not effective for elimination 5. Steinberg D, Feldman M, Ofek I, and Weiss, of biofilm. This finding may be due to strong EI. Effect of a high-molecular weight component adhesion of dental biofilm due to the activity of of cranberry on constituents of dental biofilm. J mutans streptococci. They first attach to plates by Antimicrob Chemother. 2004 Jul; 54(1):86-89. sucrose-independent adsorption and then 6. Mirz M, Najafpour Navae M. The effect of sucrose-dependent adherence occurs following distillation method on extracted compounds from the synthesis of insoluble glucans [21]. rose water. IJMAPR. 2007 fall;23(3):375-381. Therefore, these colonization mechanisms may 7. Tofighi Z, Molazem M, Doostdar B, Taban P, be sointense that they prevent the effect of Shahverdi AR, Samadi N, et al. Antimicrobial antimicrobial agents on elimination of biofilm. activities of three medicinal plants and Future studies are required to further assess the investigation of flavonoids of Tripleurospermum

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