Comparative Evaluation of Antimicrobial Properties of Pomegranate Peel Extract Against Streptococcus Mutans And Lactobacillus - An In Vitro Study

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International Dental & Medical Journal of Advanced Research (2018), 4, 1 6 ORIGINAL ARTICLE Comparative Evaluation of Antimicrobial Properties of Pomegranate Peel Extract Against Mutans And

University Dental College and Hospital, Pune, Maharashtra, India Keywords: Chlorhexidine, Hiora, Lactobacillus acidophilus, pomegranate peel extract,, zone of inhibition Correspondence: Nikita Kadam,

com Received 31 March 2018; Accepted 06 May 2018 doi: 10.15713/ins.idmjar.88 Abstract Background: Dental caries is an infectious disease influenced by cariogenic microorganisms.

Chlorhexidine is used as a gold standard mouthwash but with some side effects such as staining on long-term use. Nature itself is the best physician; it helps in treating oral health.

One of them is pomegranate. It is the oldest edible fruit and considered as a pharmacy unto itself. Pomegranate has phenolic acids, flavonoids, and tannins in its different parts.

1 International Dental & Medical Journal of Advanced Research (2018), 4, 1 6 ORIGINAL ARTICLE Comparative Evaluation of Antimicrobial Properties of Pomegranate Peel Extract Against Mutans And Lactobacillus - An In Vitro Study Sanket Kunte, Nikita Kadam, Alok Patel, Preetam Shah, Rahul Lodaya, Laxmi Lakde Department of Pediatric and Preventive Dentistry, Bharati Vidyapeeth Deemed University Dental College and Hospital, Pune, Maharashtra, India Keywords: Chlorhexidine, Hiora, Lactobacillus acidophilus, pomegranate peel extract,, zone of inhibition Correspondence: Nikita Kadam, Department of Pediatric and Preventive Dentistry, Bharati Vidyapeeth Deemed University Dental College and Hospital, Pune, Maharashtra, India. Phone: nikita.kadam90@gmail.com Received 31 March 2018; Accepted 06 May 2018 doi: /ins.idmjar.88 Abstract Background: Dental caries is an infectious disease influenced by cariogenic microorganisms. is associated with the initiation of caries, while lactobacilli with disease progression. Chlorhexidine is used as a gold standard mouthwash but with some side effects such as staining on long-term use. Nature itself is the best physician; it helps in treating oral health. Medicinal plants such as neem, tulsi, clove oil, triphala, and many more have been used in dentistry since ages to treat oral health problems such as bleeding gums, halitosis, and caries. One of them is pomegranate. It is the oldest edible fruit and considered as a pharmacy unto itself. Pomegranate has phenolic acids, flavonoids, and tannins in its different parts. Hydrolysable tannins called punicalagins which have free scavenging properties are the most abundant polyphenols found in pomegranate containing mouthwash. It has anticariogenic, anti-inflammatory, and antimicrobial properties. Aim: The aim of this study is to evaluate the antimicrobial efficacy of pomegranate peel extract, Hiora, and chlorhexidine. Materials and Methods: In this study, three different mouthwashes were used and divided into four groups as follows: Group A as chlorhexidine mouthwash (Hexidine), Group B as herbal mouthwash (Hiora), Group C as pomegranate peel extract 100% concentrated, and Group D as pomegranate peel extract 75% concentrated. Reference strains of S. and Lactobacillus acidophilus were selected as it plays an important role in caries initiation and progression. S. and Lactobacillus were grown in blood agar plates. For agar well diffusion method, wells of approximately 6-mm diameter and 4 mm in depth were made using a cork. In those wells, mouthwashes were added. Plates were incubated for 48 h at 37 C. The zone of inhibition was measured with the help of Vernier caliper. Results: The zone of inhibition of the solutions against S. Mutans and Lactobacillus shows a statistically significant P < 0.001** with f value of and , respectively. Conclusion: The pomegranate peel extract and Hiora show antimicrobial activities, and hence, they can be replaced for the long-term use to avoid the side effects of chlorhexidine mouthwash. Clinical significance: The pomegranate peel extract is a natural product with the antibacterial property; hence, it can be easily incorporated into oral hygiene measures such as mouthwashes or toothpastes. Introduction The tooth surface is unique in that it is the only body part that is not subjected to metabolic turnover. It is, however, subjected to various infections due to factors that favour microbial growth. [1] This microbial growth leads to one of the most widespread diseases of the tooth, i.e., dental caries. Dental caries is an infectious disease influenced by factors such as the relationship between diet and cariogenic microorganisms in the oral cavity and the characteristics of the host. [2] Caries begins in childhood and eventually affects 90% of adults. Even so, dental decay s effect on low-income individuals is disproportionate, leading to earlier onset, more affected teeth, complications, and ultimately teeth lost during adulthood due to caries. [3] International Dental & Medical Journal of Advanced Research Vol

2 Antimicrobial properties of pomegranate peel extract Kunte, et al. bacteria are mainly responsible for the initial phase of the caries lesion, especially in the enamel (initiation), whereas Lactobacillus is more involved with the progression of caries. Targeting forms the most important measure for the prevention of dental caries. [4] Current methods of caries management are limited to traditional preventive approaches in combination with restorative treatments. These methods have proved inadequate to control the disease. [2] Commercial chemical mouthwashes are available in larger number. One of them is chlorhexidine, which is considered as a gold standard. [5] It is the most effective anti-plaque agent, [6] but it is not a magic bullet. [5] However, chlorhexidine has some side effects such as tooth staining on long-term use, taste disturbance, enhanced supragingival calculus formation, and desquamation of the oral mucosa. [7] Hence, considering all the side effects, it suggests that there is a need for further research and introduction of new antibacterial agents that are strongly against S. with minimal side effects on the oral tissues, especially in children. Herbal medicines are an important source of nutrients that promote health. Various herbal products such as Propolis and Azadirachta indica have shown significant advantages in reducing signs of gingival and periodontal inflammation. The use of plants and their derivatives which possess preventive and therapeutic effects could contribute to the oral health. [8] The pomegranate is also called as Punica granatum L. It is an age-old, distinguishing, and highly unique fruit. Pomegranate is considered as a member of two different species consisting of the Punicaceae decent. [9] The pomegranate is widely found in Himalayas from India to Iran, but it is cultivated since primordial times in Mediterranean region. [9] In Ayurvedic medicine, the pomegranate is considered a pharmacy unto itself. [9-11] Numerous studies have been conducted on the antimicrobial properties of pomegranate peel and its curative effects. Among the different parts of the fruit, 50% of weight corresponds to the peel. The phenolic group found in the peel is the main contributors of the functional properties of pomegranate fruit. [12] Pomegranate has some compound such as phenolic punicalagins, gallic acid, and fatty acids; catechin, epigallocatechin 3-gallate, quercetin, rutin; and other flavonols, flavones, flavanones, and anthocyanidins. [9] Tannins from pomegranate fruit have been found to inhibit human salivary alpha-amylase, which catalyzes the hydrolysis of starch to oligosaccharides and binds to viridians streptococci and enamel, therefore, providing a substrate for cariogenic microbes. [12] Thus, the aim of this study is to determine and compare the efficacy of pomegranate peel extract as a mouthwash against S. and Lactobacillus. Materials and Methods The study protocol was approved by the Research Committee of Bharati Vidyapeeth Deemed University dental college and Hospital, Pune, Maharashtra, India. The study was carried out on two different microorganisms and three different mouthwashes. Pomegranate mouthwash was used in two different concentrations. The three mouthwashes were divided into four groups (n = 40) as follows: Group A (n = 10) as 0.12% chlorhexidine mouthwash (Hexidine by ICPA) Group B (n = 10) as an herbal mouthwash (Hiora mouthwash, The Himalaya Drug Company, Bengaluru, India) Group C (n = 10) as pomegranate peel extracts (100% concentration) [Figure 1] Group D (n = 10) as pomegranate peel extracts (75% concentration) [Figure 1]. 0.12% chlorhexidine was considered as control and other three as test groups. Pomegranate peel extract mouthwash was used at a concentration of 100 ml and 75 ml/100 ml. Preparation of pomegranate peel extract Pomegranate peels were dried in sunlight and overnight in a hot air oven at 60 C for 7 days. The dried peels were powdered. The obtained powder was used to prepare an aqueous extract in Soxhlet extractor. After the use of Soxhlet, the obtained extract was used that is of 100% concentration and to obtain 75% of concentration, from 100% of pomegranate peel extract, 75-ml extract is taken, and in it, 25-ml distilled water was added. Herbal mouthwash (Hiora) The key ingredients of herbal mouthwash (Hiora) are Belleric Myrobalan (Bibhitaki) is an antimicrobial and antifungal agent that keeps infections away. Another ingredients include Betel (Nagavalli) leaf effectively stops halitosis, and it has mild stimulating properties which are beneficial for toothaches. Meswak (Salvadora persica) is widely accepted teeth cleaning agent. Pilu is anticariogenic and eliminates toothache and halitosis. Two different strains S. (MTCC No. 497) [Figure 2] and Lactobacillus acidophilus (MTCC No ) [Figure 2] were grown in blood agar plates. For agar well diffusion method, four wells of approximately 6-mm diameter and 4-mm depth were made in the medium using a cork. Then, mouthwashes were added in those plates. Plates were incubated at 37 C for 48 h. The zone of inhibition was measured with the help of Vernier caliper in mm [Figure 3]. The experiment was performed and repeated under strict aseptic conditions to ensure the consistency of all findings. Results All the data were tabulated and subjected to the statistical analysis using ANOVA and two-sample t-tests. When treated with chlorhexidine, herbal mouthwash, pomegranate peel extract 100%, and pomegranate peel extract 75%; the mean zone of inhibition against S. is mm, mm, mm, and mm, respectively, as shown in Table 1 and Graph 1; and against lactobacillus, the mean zone of inhibition of these mouthwashes was mm, mm, 2 International Dental & Medical Journal of Advanced Research Vol

microorganisms Table 1: Comparison of the zone of inhibition regarding the mean (SD) against when treated with different solutions using ANOVA test Solutions n Mean±SD F value P value Chlorhexidine 0.

: Figure 2: and Lactobacillus strains 19.92 mm, and 17.31 mm, respectively, as shown in Table 2 and Graph 2. Herbal mouthwash had demonstrated a significant reduction in S. and lactobacillus count.

3 Kunte, et al. Antimicrobial properties of pomegranate peel extract Figure 1: Pomegranate peel extracts of 75% and 100% of concentration Figure 3: The zone of Inhibition of different mouthwashes against two microorganisms Table 1: Comparison of the zone of inhibition regarding the mean (SD) against when treated with different solutions using ANOVA test Solutions n Mean±SD F value P value Chlorhexidine 0.12% ± <0.001** Hiora ±0.476 Pomegranate extract 100% ±0.551 Pomegranate extract 75% ±0.698 SD: Standard deviation, S. : Figure 2: and Lactobacillus strains mm, and mm, respectively, as shown in Table 2 and Graph 2. Herbal mouthwash had demonstrated a significant reduction in S. and lactobacillus count. Herbal mouthwash had shown a significant reduction in S. and lactobacillus count when compared to all other mouthwashes used for this study. Pomegranate peel extract 100%, followed by chlorhexidine and pomegranate peel extract 75% had shown a significant reduction in colony count of S.. Chlorhexidine, followed by pomegranate peel extract 100% and pomegranate peel extract 75% had shown a significant reduction in lactobacillus count. When both microorganisms were treated with different solutions, the comparison of the zone of inhibition regarding the mean was performed using two-sample t-tests as presented in Table 3 and Graph 3. Discussion The increase of antibiotic resistance as well as undesirable side effects of synthetic drugs such as tooth staining on long-term use, unpleasant taste, and also they are higher in cost. Hence, there is a need for new antimicrobial agents of plant origin which is safe, preventive, and economical as well. In the present study, pomegranate peel extract has been tested against S. and L. acidophilus. S. bacteria are involved in the initial phase of caries and lactobacillus leads the progression of caries. Chlorhexidine is considered as a control group in this study Table 2: Comparison of the zone of inhibition regarding the mean (SD) against Lactobacillus when treated with different solutions using ANOVA test Solutions N Mean±SD F value P value Chlorhexidine 0.12% ± <0.001** Hiora ±0.639 Pomegranate extract 100% ±1.124 Pomegranate extract 75% ±0.570 SD: Standard deviation as it is the gold standard. When treated against S. and lactobacillus, it shows the effective zone of inhibition against lactobacillus. Herbal mouthwash (Hiora) is an herbal preparation which includes natural herbs with their useful properties such as anticariogenic, anti-plaque and anti-inflammatory, immunity booster effects, and natural flavouring agents. It has extracts of Belleric Myrobalan (Bibhitaki) which is an antimicrobial and antifungal agent that keeps infections at bay. Betel (Nagavalli) leaf effectively tackles halitosis, and its mild stimulating properties are beneficial for toothaches. A meswak (S. persica) tree twig, known as meswak, is popular teeth-cleaning agents. Pilu prevents tooth decay and eliminates toothache and bad breath. [13] It has powders of peppermint satva acts as a natural mouth freshener (Mentha piperita) and Yavani satva (Trachyspermum ammi), and oils of gandhapura tailum (Wintergreen oil) and Ela which are aromatic essential oil. Kaim et al. suggested that there are certain ingredients in herbal oral rinses that exhibit the evidence of antiinflammatory and antifungal therapeutic effects. [14] International Dental & Medical Journal of Advanced Research Vol

Antimicrobial properties of pomegranate peel extract Kunte, et al.

with different solutions (n=40) using ANOVA test Graph 2: Comparison of the zone of inhibition regarding

test Graph 3: Comparison of the zone of inhibition regarding (mean [standard deviation]) against both

4 Antimicrobial properties of pomegranate peel extract Kunte, et al. Graph 1: Comparison of the zone of inhibition regarding (mean standard deviation) against when treated with different solutions (n=40) using ANOVA test Graph 2: Comparison of the zone of inhibition regarding mean (standard deviation) against lactobacillus when treated with different solutions (n=40) using ANOVA test Graph 3: Comparison of the zone of inhibition regarding (mean [standard deviation]) against both organisms when treated with different solutions (n=40) using two-sample t-tests 4 International Dental & Medical Journal of Advanced Research Vol

5 Kunte, et al. Antimicrobial properties of pomegranate peel extract Table 3: Comparison of the zone of inhibition regarding the mean (SD) against both organisms when treated with different solutions using two sample t tests Solutions Group N Mean±SD t value P value Chlorhexidine 0.12% Hiora Pomegranate extract 100% Pomegranate extract 75% ± <0.001** Lactobacillus ± ± <0.001** Lactobacillus ± ± <0.001** Lactobacillus ±1.124 S. : ± <0.001** Lactobacillus ±0.570 About 50% of the total pomegranate fruit weight corresponds to the peel, which is an important origin of bioactive compounds such as phenolics, flavonoids, ellagitannins, and proanthocyanidin compounds; minerals mainly potassium, nitrogen, calcium, phosphorus, magnesium, sodium, and complex polysaccharides; [15] and polyphenolic flavonoids are antimicrobial and antifungal. Tannins from pomegranate fruit have been found to inhibit human salivary alpha-amylase, which catalyzes the hydrolysis of starch to oligosaccharides and binds to viridans streptococci and enamel, therefore, providing substrate for cariogenic microbes. [12] According to Lee et al., (2005) pomegranate extracts inhibits sucrose digesting enzyme and the organisms are responsible for plaque formation by competitive and noncompetitive inhibition. Polyphenolic flavonoids present in pomegranate are effective in maintaining good oral health, thereby eliminating the development of gingivitis. [16] In 2016, Hajifattahi et al., assessed the effect of hydroalcoholic extract of P. granatum Linn. petal on sanguinis, S., salivarius, sobrinus, and Enterococcus faecalis. They investigated that hydroalcoholic extract of P. granatum had a significant antibacterial effect on oral bacteria. The maximum effect on S. was seen. [17] Asimilar study done by, Ferrazzano et al. studied the antimicrobial activity of hydroalcoholic extracts of pomegranate peel extract and juice against S. and Rothia dentocariosa Rd1. They concluded that pomegranate peel extract and juice are able to differentiate the main cariogenic bacteria. [18] Vasconcelos et al., in 2006, investigated the antimicrobial effect of pomegranate phytotherapeutic gel and miconazole (Daktarin oral gel) against S., S. sanguis, and mitis strains. They have shown the greater efficiency of pomegranate phytotherapeutic gel in inhibiting microbial adherence than miconazole. [19] According to Bhate et al., there was a statistically significant reduction in the proportion of gingival index scores and plaque index scores in the CHX group. [14] In another study by, Mahajan et al., studied the efficacy and antimicrobial properties of five herbal mouth rinses with chlorhexidine gluconate mouthrinse. They collected 20 plaque samples from periodontitis patients and healthy patients. Then, they were streaked on blood agar plate. Well diffusion method was used to compare 0.2% chlorhexidine gluconate and different herbal mouthrinses such as Hiora, pomegranate, neem, cloves, tulsi, and distilled water. The plate was incubated at 37 C for 24 h and examined for the zones of inhibition. After the examination, they concluded that Hiora, pomegranate, and chlorhexidine mouthrinse were equally effective, and hence, they can be easily used to inhibit the growth of oral microorganisms. [20] In this study, herbal mouthwash had shown a significant reduction in S. and lactobacillus count. Pomegranate peel extract 100% followed by chlorhexidine and pomegranate peel extract 75% had shown a significant reduction in colony count of S.. Chlorhexidine followed by pomegranate peel extract 100% and pomegranate peel extract 75% had shown a significant reduction in lactobacillus count. Hence, herbal mouthwash and 100% pomegranate peel extract can be used in case of initial carious lesions. Conclusion The mouthwash used in this study revealed the antibacterial activity against oral microorganisms. Pomegranate peel extract or herbal mouthwash can be used in place of commercially available mouthwash such as chlorhexidine to avoid its side effects. However, further clinical research will be required. Limitations Results of this study cannot be match up with clinical situation. In vitro study does provide uniform and reliable means for evaluating and comparing the antimicrobial activity of different mouthrinses. References 1. Ophori EA, Eriagbonye BN, Ugbodaga P. Antimicrobial activity of propolis against. Afr J Biotechnol 2010;9: Leite SJ, Oliveira FM, Almeida LM, Gusmão MA, Chedier LM, Vasconcelos EG, et al. In vitro assessment of the antimicrobial effects of pomegranate (Punica granatum L.) peel decoction on saliva samples. Rev Ciênc Farm Básica Appl 2014;35: Lewis CW. Fluoride and dental caries prevention in children. Pediatr Rev 2014;35: Venkataraghavan K, Shah S, Choudhary P, Shah S, Trivedi K. Effectiveness of a new generation anti cavity mouthwash on and Lactobacillus acidophilus Count: A Microbiological Study. World J Dent 2014;5: Dabholkar CS, Shah M, Kathariya R, Bajaj M, Doshi Y. Antimicrobial activity of pomegranate mouthwash. J Clin Diagn Res 2016;10:ZC Mathur S, Mathur T, Srivastava R, Khatri R. Chlorhexidine: The gold standard in chemical plaque control. Natl J Physiol Pharm International Dental & Medical Journal of Advanced Research Vol

6 Antimicrobial properties of pomegranate peel extract Kunte, et al. Pharmacol 2011;1: Addy M, Moran J. Chemical supragingival plaque control. In: Lindhe J, Karring T, Lang NP, editors. Clinical Periodontology and Implant Dentistry, 5 th ed. Copenhagen: Blackwell Mumksgaard; p Subramaniam P, Dwivedi S, Uma E, GirishBabu KL. Effect of pomegranate and Aloe vera extract on : An in vitro study. Dent Hypotheses 2012;3: Jurenka JS. Therapeutic applications of pomegranate (Punica granatum L.): A review. Altern Med Rev 2008;13: Deswal H, Singh Y, Grover HS, Bhardwaj A, Verma S. Pomegranate as an curative therapy in medical and dental sciences: A review. Innov J Med Sci 2016;4: Bhandari PR. Pomegranate (Punica granatum L). Ancient seeds for modern cure? Review of potential therapeutic applications. Int J Nutr Pharmacol Neurol Dis 2012;2: Ramesh KS, Shamin SF. Role of pomegranate in preventive dentistry. Int J Res Ayurveda Pharm 2012;3: Kaim JM, Gultz J, Do L, Scherer W. An in vitro investigation of the antimicrobial activity of an herbal mouthrinse. J Clin Dent 1998;9: Bhate D, Jain S, Kale R, Muglikar S. The comparative effects of 0.12% chlorhexidine and herbal oral rinse on dental plaqueinduced gingivitis: A randomized clinical trial. J Indian Soc Periodontol 2015;19: Sreekumar S, Sithul H, Muraleedharan P, Azeez JM, Sreeharshan S. Pomegranate fruit as a rich source of biologically active compounds. Biomed Res Int 2014;2014: Hajifattahi F, Moravej-Salehi E, Taheri M, Mahboubi A, Kamalinejad M. Antibacterial effect of hydroalcoholic extract of Punica granatum Linn. Petal on common oral microorganisms. Int J Biomater 2016;2016: Ferrazzano GF, Scioscia E, Sateriale D, Pastore G, Colicchio R, Pagliuca C, et al. In vitro antibacterial activity of pomegranate juice and peel extracts on cariogenic bacteria. Biomed Res Int 2017;2017: Vasconcelos LC, Sampaio FC, Sampaio MC, Pereira MD, Higino JS, Peixoto MH. Minimum inhibitory concentration of adherence of Punica granatum Linn (pomegranate) gel against S., S. mitis and C. albicans. Braz Dent J 2006;17: Bhate D, Jain S, Kale R, Muglikar S. The comparative effects of 0.12% chlorhexidine and herbal oral rinse on dental plaqueinduced gingivitis: A randomized clinical trial.j Indian SocPeriodontol2015; 19: Mahajan R, Khinda PK, Gill AS, Kaur J, Saravanan SP, Shewale A, et al. Comparison of efficacy of 0.2% chlorhexidine gluconate and herbal mouthrinses on dental plaque: An in vitro comparative study. Eur J Med Plants 2016;13:1-11. How to cite this article: Kunte S, Kadam N, Patel A, Shah P, Lodaya R, Lakde L. Comparative evaluation of antimicrobial properties of pomegranate peel extract against and Lactobacillus - An in vitro study. Int Dent Med J Adv Res 2018;4:1-6 This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit Kunte S, Kadam N, Patel A, Shah P, Lodaya R, Lakde L International Dental & Medical Journal of Advanced Research Vol

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