Journal of Research in Medical and Dental Science 2018, Volume 6, Issue 5, Page No: 361-364 Copyright CC BY-NC 4.0 Available Online at: www.jrmds.in eISSN No. 2347-2367: pISSN No. 2347-2545

A Comparative Study on Antimicrobial Effect of Iranian Green and Tea on Growth of Oral Cariogenic Bacteria Streptococcus mutans PTCC 1683

AmirSina Golmohammadi*

Department of Restorative Dentistry, Dental Branch, Islamic Azad University of Medical Sciences, Tehran, Iran

ABSTRACT Tooth caries is a major health problem today. Tea with its polyphenolic compounds can show its anticariogenic effects by affecting the main Cariogenic bacteria in the oral cavity. The present study tended to examine the antimicrobial effect of two Iranian and Hibiscus Sabdariffa in comparison with chlorhexidine on Iranian type of Cariogenic Streptococcus mutans Bacteria. For this purpose, ethanolic extract of plants was first processed; then, the impregnated disk was placed on the bacterial culture medium and diameter of inhibition zones was calculated. After three iterations, the results showed the means 17, 14.16 and 16.83 for zone of inhibition diameter of green tee, hibiscus tea, and control chlorhexidine 0.2%, respectively. Moreover, ANOVA test showed a significant difference between zones of inhibition diameter of Hibiscus Sabdariffa and Camellia sinensis. Multiple comparison tests showed no significant difference between zones of inhibition diameter of these two teas and chlorohexidine control. Examining minimum bactericidal concentration (MBC) and minimum inhibitory concentration (MIC) of both hibiscus and green teas also showed that MBC was present in 12.5 mg/ml and 50 mg/ml hibiscus tea and MIC was present in 6.25 mg/ml green tea and 25 mg/ml hibiscus tea. According to findings, it is concluded that anti-microbial effects of Iranian green tea are higher than Iranian hibiscus tea. There is also no significant difference between antimicrobial effects of these three compounds. Accordingly, it is suggested to consider anti cariogenic effect of Iranian green tea and Iranian hibiscus tea and their daily intake.

Key words: Anticariogenic, Tooth decay, Antimicrobial effect, Tea

HOW TO CITE THIS ARTICLE: AmirSina Golmohammadi*, A comparative study on antimicrobial effect of iranian green tea and hibiscus tea on growth of oral cariogenic bacteria Streptococcus mutans PTCC 1683

, J Res Med Dent Sci, 2018, 6 (5):361-364 Corresponding author: AmirSina Golmohammadi from Hibiscus sabdariffa. Studies in Iran show high e-mail: [email protected] levels of DMFT index. DMFT index is referred to the Received: 06/10/2018 Accepted: 29/10/2018 total number of Decayed, Missed and Filled teeth that an individual can have. In 2006, a study was conducted INTRODUCTION on 8301 men and women in Iran; these people had an average of 2.7 ± 2.6 teeth with caries [4]. A study on Dental caries is one of the biggest and most important Oral health problems in children and adults today. province showed that these children had an average Among various factors of Caries, Cariogenic bacteria 3000 first-degree secondary students in Mazandaran ost important indicates the need to pay more attention to oral health bacteria affecting the Caries process is Streptococcus inof 2.93Iran. ±Using 4.98 decayed,healthy snacks,missed orsuch filled as teethtea, can[5]. helpThis mutansplay a . significantThis bacterium role. is One known of theas primary m bacterial reduce these indicators. A study conducted on children cause of dental Caries [1]. Mutans starts a tooth Caries has shown that children who took 1 to 3 glasses of tea daily had a lower Caries index than children who took forming a microbial plaque [2]. An attempt has been madeprocess to byidentify forming antibacterial a biofilm layer compounds on tooth which surface reduce and took gums containing polyphenol for 24 months had the amount of these bacteria in oral cavity. Meanwhile, lower1-2 glasses increased a week DMFT [6]. than In another the group study, that children did not whotake tea is known as an effective compound [3]. Tea with them [7]. Drinking tea in Iran is commonly associated its polyphenolic compounds has known antimicrobial with sweetening compounds such as sugar, chocolate, role. Most known teas, including green tea, originate from Camellia Sinensis. Hibiscus tea also originates More studies to evaluate the effect of concomitant tea which may reduce the anti-cariogenic effects of tea. Journal of Research in Medical and Dental Science | Vol. 6 | Issue 5 | October 2018 361 AmirSina Golmohammadi J Res Med Dent Sci, 2018, 6 (5):361-364 consumption with sweet compounds compared to tea RESULTS consumption without these compounds and its effects on dental caries can be remarkable. Descriptive results Camellia MATERIALS AND METHODS sinensis was recorded 18, 16 and 17 mm in culture According to Table 1, zone of inhibition for Herbal material Hibiscus sabdariffa The dried and fresh leaves of Hibiscus sabdariffa and inmedia culture No. 1,media, 2 and respectively.3, respectively. These Zone data of inhibition for control for Camellia sinensis were taken from a local market. These was recorded at 15, 12.5 and 15 mm leaves lacked any chemical processing and were made by chlorohexidineThe study of media were recordedcultured atafter 17, 17.5diluting and 16the mm. plant Iranian tea makers. extract and adding microbial compound showed that Microorganism First, Streptococcus mutans PTCC 1683 was taken from MBC was observed for hibiscus tea at a concentration of 1:2 (50 mg/ml) and for green tea at a concentration of 1:8 (12.5 mg/ml). MIC was also observed for hibiscus tea the Iranian Research Organization for Science and selective culture medium. at a concentration of 1:4 (25 mg/ml) and green tea at a Technology (IROST). The bacteria were cultured in a concentration of 1:16 (6.25 mg/ml) (Table 2). Herbal extract Table 1: Zone of inhibition diameter of plants by culture media Using the Maceration technique, 10 g powder of both Medium 1 Medium 2 Medium 3 Mean plants dissolved in 100 mg ethanolic solvent. The Studied plants (mm) (mm) (mm) (mm) Camellia sinensis 18 16 17 17 and 100 mg/ml solution prepared. Final powder Solved solution was filtered using Whatman No. 1 filter paper Hibiscus sabdariffa 14.16 Control 17 16 16.83 entire process of herbal extract is based on similar chlorohexidine 15 12.5 15 studieswith DMSO [8,9]. solvent and sterilized under UV rays. The w17.5 Disc diffusion test Table 2: MBC and MIC for the studied plants

Antimicrobial activity of ethanolic extract of both hibiscus Studied plants MBC MIC Camellia sinensis and Disk diffusion technique. Microbial suspension was Hibiscus sabdariffa and green tea was investigated using Kirby-Bauer test 1:8 (12.5 mg/ml) 1:16 (6.25 mg/ml) 1:2 (50 mg/ml) 1:4 (25 mg/ml) Agar Muller Hinton medium. The disks impregnated with Inferential results could be derived from Table 3 and Table extractprocessed of both using plants McFarland and the 0.5 control standard disks and impregnated cultured on with chlorohexidine 0.2% were transferred to culture media. The experiment was iterated three times; three 4. In Table 3, As P<0.05 in ANOVA, there is a significantCamellia disks were placed in each medium. sinensisdifference and in Hibiscus zone of sabdariffa inhibition diameter between two groups. By multiple comparison tests between All three culture media were placed in incubator at 37°C , a significant difference was found in zone of inhibition diameter between two measured in millimeters [8]. inhibition diameter between these two teas and control for 1 day. Finally, zone of inhibition of each disk was plants, while there was a significant difference in zone of MIC and MBC of green and hibiscus teas Chlorohexidine (Table 4). extracts were made Table 3: ANOVA of the difference in zone of inhibition diameter between two plants Using Muller Hinton Broth, herbal Sum of Mean of at concentrations 1:2 (50 mg/ml) and 1:4 (25 mg/ml) Df F-value P and 1:8 (12.5 mg/ml) and 1:16 (6.25 mg/ml) and 1:32 square square 2 (3.125 mg/ml). Then, 100 μl microbial suspensions inhibition all test tubes. A test tube containing control hibiscus 333.7 6 1.222 made by standard McFarland 0.5 were transferred to Zone of Between groups 167.15 7.583 tea, one containing control Camellia sinensis and one for Sum 8 6.205 0.035 control microbial suspension made. This test was also Within groups 5.22 performed for Hibiscus sabdariffa and the test tubes Table 4: Multiple comparison test between green tea and hibiscus were placed in an incubator at 37°C for 24 hours. test (Tukeys test)

After 1 day, contents of all 13 test tubes were separately Mean Studied sample Studied sample P Result difference media were incubated at 37°C for 1 day. The results Hibiscus sabdariffa 2.83333 0.046 cultured on 13 Muller Hinton Broth medium. The culture Camellia sinensis Control 0.16667 0.981 Significant bacterial colonies on culture media and Chlorohexidine were determinedevaluated in for the both form plants of growth [8]. or non-growth of None MBC and MIC Significant Journal of Research in Medical and Dental Science | Vol. 6 | Issue 5 | October 2018 362 AmirSina Golmohammadi J Res Med Dent Sci, 2018, 6 (5):361-364

Camellia sinensis 0.046 its antibacterial effect by binding to peptidoglycan of Hibiscus . Since the Streptococcus mutans cell sabdariffa Chlorohexidine -2.83333 Significant control wall is rich in peptidoglycan [16], the antimicrobial effect None bacterial wall [15] -2.66667 0.058 of green tea on this bacterium is explained in this way. On Camellia sinensis 0.981 Significant Chlorohexidine the other hand, studies have shown the effect of green None control -0.16667 Hibiscus sabdariffa 2.66667 Significant tea polyphenol on bacterial membrane [17]. It has been None observed that the polyphenol present in green tea has 0.058 DISCUSSION Significant been able to damage cell membrane of E. coli by altering the gene expression [18]. Antibacterial compounds of hibiscus tea also include polyphenolic compounds. many oral hygiene studies. Since tea is a major snack in These compounds interfere with functioning of cell Iran,Antimicrobial the importance effects of of microbiological tea have been studies confirmed of tea by membrane of the bacteria [19]. Antibacterial properties of this plant have been compared with Streptomycin; The present study tends to investigate the antimicrobial it has been effective against E. coli in contrast to propertiesshould not of be Iranian overlooked green in thetea fieldand ofhibiscus dental tea Caries. on streptomycin [20]. In addition to its anticariogenic role,

Streptococcus mutans, compared to chlorhexidine 0.2%. on Enterococcus faecalis, an important bacterium in the most important oral and dental Caries bacterium- theendodontic polyphenol infections content and in has tea shown has a significantits antimicrobial effect effect [21]. Properties of hibiscus tea are also not limited thanThe resultsthat of showed0.2% Chlorohexidine that the mean mouth zone ofwash inhibition (16.83 to antibacterial compounds. It has been shown in a study diameter of green growth (17 mm) was slightly higher that this herbal compound is also effective on Candida albicans, the most important oral and dental fungus [22]. mm). In their study, George et al., showed that the tea and 22 mm in chlorohexidine. In this study, unlike All of the above points to the fact that these two teas are mean length of inhibition zone was 14 mm in green not only effective in anticariogenic aspect, but also in difference between green tea and domestic mouthwash other oral hygiene areas. chlorohexidinethe present study, [10]. there In wasanother a statistically study in significantIran, the results were similar to the present study. Ranjbar et CONCLUSION

Considering the acceptable results of green tea al. claimed that there is no significant difference in samples compared to chlorhexidine control, as well as diameter of inhibition zone of green tea compared anticariogenic role of hibiscus tea against Streptococcus to chlorohexidine mouthwash (P=0.305) [11]. This mutans, it is suggested to use these herbal compounds polyphenoldifference in content diameter of ofthe inhibition teas used zone in these of green studies tea and or based on their anticariogenic properties. Green tea is the significant level can be attributedi in his to study the difference on Hibiscus in suggested in areas where this plant is common and sabdariffa process of samples. Al-Hashim easily found, including Iran. Compounds containing polyphenol, such as gums, toothpastes and mouthwashes in this study calculated [12]. Iranian the diameterindigenous of teas inhibition and Iranian zone can be used more extensively. Effective advertising can typeof alcoholic of Streptococcus extract (30 mutans mm). Thisused ratein this was study 14.16 as mm an be planned for this herbal compound through mass innovation can explain the difference in results of this study and other studies. The test results also showed be noted in textbooks. School advisers need to play an that green tea at low concentrations also has its own effectivemedia. Moreover, role in introducing the benefits these of two these teas two to students. teas can Since green tea is a product of northern Iran and bactericidal effect. MBC was observed in 1:8 (12.5 mg/ hibiscus tea is a product of the southern regions of Iran, ml) concentration of green tea, while it was observed the government can support the agricultural sector and direct the agricultural industry towards production of for hibiscus tea in a higher concentration (1:2 (50 mg/ these valuable products. ml)). The minimum concentrationHibiscus in which sabdariffa green at tea a could inhibit bacterial growth (MIC) was 1:16 (6.25 POTENTIAL PROBLEMS mg/ml),on compound which of was two observed green tea for and concentration of 1:4 (25 mg/ml). In a study in Portugal This study was done without any problems at the writing while MIC was slightly different [13].black Sulistyani tea, MBC et was al., and testing stages. reported at 12.5 mg/ml, similar to the present study, ACKNOWLEDGEMENT reported MBC for hibiscus tea at 57.6 mg/ml and MIC Differencesat 7.2 mg/ml, in polyphenol while above content study reportedof two plants MBC andcan beMIC attributed at 150 mg/ml to the anddifference 25 mg/ml, in their respectively antimicrobial [14]. The authors appreciate Dr. Zohreh Gholizadeh Siahmazgi Gallate are known as the most active type of polyphenols outand the knowledge-basedlaboratory and biochemical research center steps ofof Varna this study Paya properties. Epigallocatechin and Epigallocatechin-3- Project Maham (VPPM) for their assistance in carrying inJournal green of Research tea [14]. in Medical Epigallocatechin-3-Gallate and Dental Science | Vol. 6 | Issue exerts 5 | Octoberand 2018 Dr. Badri Abbasi, Assistant Professor of Higher363 Edu- AmirSina Golmohammadi J Res Med Dent Sci, 2018, 6 (5):361-364 cation Management at Free Islamic University, for tech isolated from dental plaque. J Mash Dent Sch nical and statistical editing of this study. - 12. REFERENCES activities2015; 39:335-42. of Hibiscus sabdariffa L. extracts. Afr J Al-Hashimi AG. Antioxidant and antibacterial 1. 13. InhibitoryFood Sci 2012; activity 6:506-11. of a green and blend JpnMatsumoto-Nakano Dent Sci Rev 2017. M. Role of Streptococcus onBarroso Streptococcus H, Ramalhete mutans. R, J Oral Domingues Microbial. A, 2018; et al. 2. mutans surface proteins for biofilmniak formation. D, et al. 10:1481322. The virulence of Streptococcus mutans and the 14. Sulistyani H, Fujita M, Miyakawa H, et al. Effect Krzyściak W, Jurczak A, Kościel of calyx extract on in vitro viability and

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