Antimicrobial effect of green tea extract on cariogenic microorganisms isolated from high caries risk patients: A clinical study

Mohamad G Kucharia , Mostafa A Hassanb and Motaz A Ghulmanc Abstract: Nowadays, there is a great concern in alternative and complementary medicine; especially in antimicrobial agents extracted from natural plant sources. This clinical study was conducted to evaluate the anticariogenic potentials (if any) of green tea extract mouthwash on salivary cariogenic bacteria isolated from high caries risk patients. Twenty high caries risk patients (10 males and 10 females) were selected for this study. Stimulated saliva was collected before and after mouthrinsing with green tea extract, then the levels of salivary mutans streptococci and lactobacilli were measured using a commercial chair-side caries risk test kit (CRT bacteria, Ivoclar Vivadent). The density of the mutans streptococci and lactobacilli colonies were compared with the corresponding model chart provided by manufacture. The colonies density was then expressed in colony forming units (cfu) guided by the manufacturer chart. The results of the present investigation showed a marked reduction of both mutans streptococci and lactobacilli after mouthrinsing with green tea extract. Student t-test showed a significant difference (P<0.05) between the recorded mean scores for both bacterial species before and after green tea extract mouthrinsing. This study provides an additional rationale for the inclusion of green tea extract mouthrinse as an adjunct to daily oral hygiene procedures especially for high caries risk patients. Introduction: Dental caries is a multifactorial infectious disease caused by cariogenic (acidogenic and aciduric) bacteria colonizing in the oral cavity (1, 2). Different bacteria were isolated from saliva and teeth of cariogenic patients and identified by a variety of morphological and biochemical tests (3). Yet; streptococcus mutans and lactobacillus acidophilus are generally regarded as the most cariogenic bacteria (4-6). Evidence-based studies reported that mutans streptococci microorganisms are strongly associated with initiation of dental caries, while lactobacilli are mainly associated with further development of carious lesion (7, 8). In addition to cariogenic microorganisms in the dental plaque; the susceptible tooth surfaces and availability of fermentable carbohydrates are essential interacting factors for development and progression of carious process (9,10). Several studies have reported a direct correlation between presence of cariogenic microorganisms in plaque and that in saliva (i.e. whenever the saliva for a given patient demonstrated high bacterial counts of these microorganisms, the dental plaque did too) 11,12. The increased numbers of mutans streptococci and lactobacilli microorganisms in saliva were used to identify -----------------------------------------------------------------------------------------------------------a: Associate Professor of Microbiology, Faculty of Science, King Abdul-Aziz University, Jeddah, Saudi Arabia b: Associate Professor of Operative Dentistry, Faculty of Dentistry, King Abdul-Aziz University, Jeddah, Saudi Arabia c: Assistant Professor of Operative Dentistry, Faculty of Dentistry, King Abdul-Aziz University, Jeddah, Saudi Arabia

subjects at increased risk for dental caries (13-16). Levels of these cariogenic bacteria in the mouth can be assessed by selective media culturing either in a microbiological laboratory or in the dental office through commercially available chair-side caries risk assessment kits. These kits permit the semi-quantitative determination of mutans streptococci and lactobacilli microorganisms in saliva (17-19). The CRT bacteria from Vivadent is one of these chair-side available kits; thoroughly evaluated and showed reliable results when compared with laboratory methods (17,20). Recently, there is a shift in the paradigm of treatment of dental caries from surgical to medical approach to cope with the infectious nature of the disease (21). Antimicrobial mouthwashes are considered essential measures used to control cariogenic microorganisms in both plaque and saliva. Nowadays, there is a great concern in alternative medicine; especially antimicrobial agents extracted from natural plant sources (22-24). Tea is an aqueous infusion of dried leaves of the plant Camellia Sinensis (family Theaceae) and is the most popular beverage consumed by human society worldwide, second only to water (25). Depending on the manufacturing process, teas are classified into three major types: the non-fermented green tea, the semi-fermented Oolong tea, and the fermented black tea. Green and Oolong teas are commonly consumed in China, Japan and Eastern Asia, while black tea is mainly brewed in European countries, Middle East and India (26, 27). It was reported that, Green tea exerted the strongest antimicrobial activity followed by Oolong tea and black tea respectively (27). Previous experimental studies on rats reported that tea extract drinks could reduce the incidence of dental caries in their teeth even if they fed on high cariogenic diets (28-30). Similarly, several in vivo and vitro studies on humans have documented the antibacterial effects of green tea extract on cariogenic bacteria and its inhibitory action on dental plaque formation (31-37). There are limited studies on green tea extract when used as mouthwash for the prevention of dental caries (25, 26, 38). Therefore, this study was proposed to evaluate the anticariogenic potentials (if any) of green tea extract mouth wash on salivary cariogenic bacteria isolated from high caries risk patients. Materials and Methods: Subject selection The subjects of this study were 20 patients (10 males and 10 females) between the age of 18 and 23 years selected from screening clinics at Faculty of Dentistry, King Abdul-Aziz University, Jeddah, Saudi Arabia. The selected patients were medically healthy with no systemic diseases and had not used any antibiotics or antiseptic mouthwashs during the last two weeks. Smokers were not included in the study. One examiner carried out clinical examinations using a mouth mirror and a probe according to the criteria of the World Health Organization (39). All subjects selected were assessed as high caries risk patients on the basis of high recorded DMFT (decayed, missed and filled teeth) scores and past caries experience as a more reliable caries predictor (40, 41). Preparations of green tea extract mouthrinse The green tea extract was prepared following the simple technique described by Das et al (42) by adding 2.5 g of green tea leaves to 100 ml boiling water and then steeped for 15 min. The infusion was cooled to room temperature and then filtered to obtain a 2.5% aqueous tea extract. This extract was kept in refrigerator at 4C to be used within two

days at the subsequent procedure. Each patient was asked to rinse with 20 ml from prepared green tea extract solution of for 5 minutes Collection of Saliva Before collecting saliva for bacteriologic testing, the patients were asked not to eat or drink for at least one hour. Stimulated saliva was collected at baseline before and after mouthrinsing with green tea extract. Each patient was given one of the paraffin wax pellet (supplied with the kit) and asked to chew it for a total period of six minutes and saliva was collected in a sterile plastic container at two minute intervals. Afterward, each patient was asked to rinse with 20 ml from the prepared green tea extract solution for 5 minutes then spit all saliva. Similarly, saliva was collected again as mentioned before to be used in the subsequent bacteriologic testing. Determination of salivary levels of Mutans Streptococci and Lactobacilli: The levels of mutans streptococci and lactobacilli were measured using a commercial chair-side caries risk test kit (CRT bacteria, Ivoclar Vivadent AG, FL-9494 Schaan/Liechtenstein). The kit is comprised of a slide attached to the cover of the vial. The commercial product had one side of the slide coated with a solid selective culture medium (mitis salivarius agar enriched with sucrose) for the cultivation of Mutans streptococci, while the medium on the other side of the slide (Rogosa agar) was for the cultivation of lactobaccili. The agar carrier was removed from the test vial, and the sodium bicarbonate tablet (included with the kit) was placed at the bottom of the vial; as it releases CO2 when it comes into contact with moisture. This creates favorable conditions for bacterial growth. The protective foils were removed carefully from the two agar surfaces without touching the agar. Holding the agar carrier slightly oblique, both agar surfaces were thoroughly wetted with saliva using a pipette allowing excess saliva to drip off. The agar carrier was slide back into the vial, which was closed tightly. Using a waterproof pen, the name of the patient and date were written on the vial at this step. The test vial was then placed in the upright position in the Cultura incubator (Ivoclar Vivadent) at 37C for 48 hours to allow the bacterial colonies to grow. After removal of the vial from the incubator, the density of the mutans streptococci and lactobacilli colonies were compared with the corresponding evaluation picture in the enclosed model chart provided with the kit by the manufacture. The agar carrier was held slightly oblique under a light source to facilitate the evaluation. The colonies density was then expressed in colony forming units (cfu) guided by the manufacturer chart as follows (43): Mutans Streptococci and Lactobacilli Scoring (Fig. 1): 0 = Very low colonies are detected 1 = Low, colonies growth were ranging from102 to 103 cfu 2 = Medium, colonies growth were ranging from103 to 105 cfu 3 = High, colonies growth were > 105 cfu The data were collected and statistically analysed using statistical SPSS computer program (version 10, SPSS Inc., Chicago, IL, USA). Student t-test was used for comparison between recorded mean scores of each bacterial specie (P<0.05) before and after mouthrinsing with green tea extract. Results: The results of the present investigation showed a marked reduction of both streptococci and lactobacilli after mouthrinsing with green tea extract ranging from score 3 to zero

(Tables I and II). The reduction in cfu scores was more with mutans streptococci than lactobacilli group. Using student t-test for comparison between the recorded mean scores of cfu of mutans streptococci and lactobacilli microorganisms before and after mouthrinsing with green tea extract, there was significant difference between the recorded mean scores for each given bacterial specie (P<0.05), tables III and IV. Tables I: Frequency distributions of mean scores of cfu of Streptococcus Mutans before and after green tea extract mouthrinsing Streptococcus Mutans group score total 0 1 2 3 Before (control) 0 2 3 15 20 After 1 12 5 2 20

Tables II: Frequency distributions of mean scores of Lactobacilli before and after green tea extract mouthrinsing Lactobacilli group score total 0 1 2 3 Before (control) 1 3 6 10 20 After 2 8 4 6 20

Tables III: Student t-test for Streptococcus Mutans mean scores before and after green tea extract mouthrinsing Paired Samples Statistics Pair 1 BEFORE AFTER Paired Samples Correlations Mean 5 5 N 4 N 4 4 Std. Deviation 6.782329983 4.966554809 Std. Error Mean 3.391164992 2.483277404

Pair 1 BEFORE & AFTER * Significant at P<0.05

Correlation Sig. -0.306765341 0.693234659 *

Tables IV: Student t-test for Lactobacilli mean scores before and after green tea extract mouthrinsing Paired Samples Statistics Mean N Std. Deviation Std. Error Mean Pair 1 BEFORE 5 4 3.915780041 1.957890021 AFTER 5 4 2.581988897 1.290994449 Paired Samples Correlations N Correlation Sig. Pair 1 BEFORE & AFTER 4 0.329690237 0.670309763 * * Significant at P<0.05

Fig. (1): Representative samples of colonies growth of mutans streptococci and lactobacilli on CRT bacteria culture media, before and after green tea extract mouthrinsing Discussion There is increasing interest in plant extracts as potential therapeutic agents for treatment of certain oral diseases and to control dental caries. This era of alternative and complementary medicine has a number of interesting outcomes (44, 45). Green tea extract is known to pack a strong antioxidant cocktail made up of polyphenolic compounds called catechins. Two in particular, epigallocatechin gallate (EGCG) and epicatechin gallate (ECG), have been shown to combat oral plaque and bacteria (25, 36). Green tea extract mouthrinsing evaluated in this study resulted in immediate antimicrobial effect against both mutans streptococci and lactobacilli microorganisms. This finding was in agreement with Esimone et al how attributed their finding to direct bactericidal effect of tea polyphenols. Meanwhile, the immediate reduction of mutans streptococci cfu scores after green tea extract mouthrinsing reported in this study was in agreement with Rasheed & Haider (25), Sakanaka et al (46) and Kawamura & Takeo (47). Previous studies have reported that, anticariogenic activity of green tea extract was due to tea catachines and not fluorides and include the following: a direct bactericidal effect against mutans streptococci (25, 46, 47), prevention of bacterial adherence to teeth (34, 36); inhibition of glucosyl transferase enzyme, thus limiting the biosynthesis of sticky glucan (48, 49) and inhibition of salivary and bacterial amylases (50, 51). Contrarily, the immediate reduction of lactobacilli cfu scores after green tea extract mouthrinsing reported in this study might also be explained by the direct bactericidal effect of green tea polyphenols on lactobacillus microorganisms. However, this finding was not consistent with findings reported by Koo & Cho (52) and Jin et al (53) who reported immediate increase and no change of lactobacillus counts from control, respectively. The results inconsistency might be due to the different methodology applied concerning green tea extract concentration, technique of sample collection and bacteriologic culturing kits. The previous reports described above suggest that tea drinking or the use of specific extract of tea may prevent or slow down the progression of caries. However, without clinical evidence, the information remains of academic interest only. As antimicrobial mouthrinses are most frequently recommended to patients whose mechanical oral hygiene procedures are not adequate for the control of supragingival plaque and gingivitis,
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consequently; this study provides an additional rationale for the inclusion of aqueous extract of green tea mouthrinse as an adjunct to daily oral hygiene procedures. Conclusion: Within the conditions of this study it could be concluded that: 1- Aqueous extract of green tea possessed antimicrobial effect against mutans streptococci and lactobacilli cariogenic microorganisms 2- There is a rationale for inclusion of green tea extract mouthrinse as an adjunct to daily oral hygiene procedures especially for high caries risk patients. 3- Further clinical investigations are required to evaluate the effects of green tea extract on oral and dental tissues when used as a routine mouthwash on daily basis. Acknowledgement: The authors thank Dr. Nada Bin-Madi, Faculty Dentistry, King Abdul-Aziz University for her assistance at the clinical part of this assessment study. References: 1. Loesche W.: Role of streptococcus mutans in human dental decay. Microbiol Rev 1986: 50: 353380 2. Houte J. Role of microorganisms in caries etiology. J Dent Res 1994: 73: 672 681. 3. Sugars and dental caries, Riva Touger-Decker R and Loveren C.: Am. J. Clin. Nutr. 2003; 78 (suppl):881S892S 4. Berkowitz R, Acquisition and transmission of mutans streptococci. J. Cal. Dent. Assoc. 2003; 31:135-138 5. Featherstone J, The caries balance: Contributing factors and early detection. J. Cal. Dent. Assoc. 2003; 31:129-133 6. Epstein-JB; McBride-BC; Stevenson-Moore-P; Merilees-H; Spinelli. The efficacy of chlorhexidine gel in reduction of streptococcus mutans and lactobacillus species in patients treated with radiation therapy. J Oral-Surg-Oral-Med-Oral-Pathol. 1991; 71: 172-178 7. Zickert I, Emilson C, Krasse B.: Streptococcus mutans, lactobacill and dental health in 13-14 year old Swedish children. Community Dent. Oral Epidemiol. 1982; 10: 7781 8. Kingman A, Little W, Gomez I, Heizetz SB, Driscoll WS, Sheats R, et al. Salivary levels of streptococcus mutans and lactobacilli and dental caries experiences in a U.S. adolescents population. Community Dent. Oral Epidemiol. 1988; 16: 98-103 9. Hardie J.: Oral microbiology: current concepts in the microbiology of dental caries and periodontal disease. Br Dent J. 1992; 172: 271-278 10. Marsh P.: Microbial ecology of dental plaque and its significance in health and disease. Advances in Dental Research 1994; 8, 263-271 11. Mundorff S, Eisenberg A, Leverett D, Espeland M, Proskin H: Correlation between numbers of microflora in plaque and saliva; Caries Res. 1990; 24: 312-317 12. Sullivan A, Borgstrm M, Granath L, Nilsson G: Number of mutans streptococci or lactobacilli in a total dental plaque sample does not explain the variation in caries better than the numbers in stimulated saliva; Community Dent. Oral Epidemiol. 1996; 24: 159-163 13. Bjarnason S, Kohler B.: Caries risk assessment in adolescents. Swed. Dent. J 1997, 21: 4148.

14. Rethman J.: Trends in preventive care: Caries risk assessment and indications for sealants. J. Am. Dent. Assoc., 2000; 131: 8S-12S 15. Ozawa Y, Chiba J, Sakamoto S.: HLA class II alleles and salivary numbers of mutans streptococci and lactobacilli among young adults in Japan. Oral Microbiol. Immunol. 2001, 16: 353357 16. Vehkalahti M, Nikula-Sarakorpi E, Paunio I.: Evaluation of salivary tests and dental status in the prediction of caries increment in caries-susceptible teenagers. Caries Res. 1996, 30: 2228. 17. Jordan H, Laraway R, Snirch R and Marmel M.: A simplified diagnostic system for cultural detection and enumeration of streptococcus mutans. J Dent. Res. 1987, 66(1):57-61. 18. Jensen B and D. Bratthall D.: A new method for estimation of mutans streptococci in human saliva; J. Dent. Res. 1989, 68: 468-472 19. Featherstone J, Adair S, Anderson M, Berkowitz R, Bird W, Crall J, Den Besten P, Donly K, Glassman P, Milgrom P, Roth J, Snow R, Stewart R.: Caries management by risk assessment: consensus statement. J Calif. Dent. Assoc. 2003; 31:257-269 20. Kneist S, Laurisch L, Heinrich-Weltzien R, StsserL.: A modified mitis salivarius medium for a caries diagnostic test; J. Dent. Res. 1998, 77: 970 (Abstr. 2712) 21. Steinberg S.: Paradigm shift in the treatment of caries. Gen Dent. 2002; 50: 333-338 22. Tichy J, Novak J. Extraction, assay, and analysis of antimicrobials from plants with activity against dental pathogens (Streptococcus sp.) J Altern. Complement. Med. 1998, 4:39-45 23. Cowan M.: Plant Products as Antimicrobial Agents. Clinical Microbiology Reviews, 1999; 12: 564582 24. Morgan T, Beezer A, Mitchell J and Bunch A.: A microcalorimetric comparison of the anti-streptococcus mutans efficacy of plant extracts and antimicrobial agents in oral hygiene formulations, Journal of Applied Microbiology 2001; 90: 53-58 25. Rasheed A; Haider M. Antibacterial activity of Camellia sinensis extracts against dental caries. Arch. Pharm. Res. 1998; 21: 348-352 26. Wu C. and Wei G.: Tea as a Functional Food for Oral Health Nutrition 2002; 18:443 444 . 27. Hamilton-Miller J.: Antimicrobial Properties of Tea (Camellia sinensis L.) Antimicrobial Agents and Chemotherapy. 1995; 39: 23752377 28. Touyz LZ, Amsel R.: Anticariogenic effects of black tea (Camellia sinensis) in caries prone-rats. Quintessence Int. 2001; 32: 647-650 29. Linke H, LeGeros R.: Black tea extract and dental caries formation in hamsters. Int J Food Sci. Nutr. 2003; 54: 89-95 30. Ooshima T, Minami T, Aono W, Izumitani A, Sobue S, Fujiwara T, Kawabata S, Hamada S.: Oolong tea polyphenols inhibit experimental dental caries in SPF rats infected with mutans streptococci. Caries Res. 1993; 27: 124-129 31. Saito N.: Anti-caries effects of polyphenol compound from Camellia sinensis Nichidai Koko Kagaku. 1990; 16: 154-163 32. Hamilton-Miller J.:.Anti-cariogenic properties of tea (Camellia sinensis). J. Med. Microbiol. 2001; 50:299-302

33. Sasaki H, Matsumoto M, Tanaka T, Maeda M, Nakai M, Hamada S, Ooshima T. Antibacterial activity of polyphenol components in oolong tea extract against streptococcus mutans. Caries Res. 2004; 38: 2-8 34. Matsumoto M, Minami T, Sasaki H, Sobue S, Hamada S, Ooshima T.: Inhibitory effects of oolong tea extract on caries-inducing properties of mutans streptococci. Caries Res; 1999; 33: 441-445 35. Matsumoto M, Hamada S, Ooshima T.: Molecular analysis of the inhibitory effects of oolong tea polyphenols on glucan-binding domain of recombinant glucosyltransferases from streptococcus mutans MT8148. FEMS Microbiol. Lett. 2003; 7: 73-80 36. Otake S, Makimura M, Kuroki T. Anticaries effects of polyphenolic compounds from Japanese green tea. Caries Res. 1991; 25: 438 443 37. Ooshima T, Minami T, Aono W, Tamura Y, Hamada S.: Reduction of dental plaque deposition in humans by oolong tea extract. Caries Res. 1994; 28: 146-149 38. Esimone C., Adikwu M, Nwafor S, and Okolo C., Potential Use of Tea Extract as a Complementary Mouthwash: Comparative Evaluation of Two Commercial Samples The Journal of Alternative and Complementary Medicine 2001; 7: 523527 39. Ismail A.: Visual and Visuo-tactile Detection of Dental Caries J. Dent. Res. 2004; 83(Spec Iss C): C56-C66 40. Powell L. Caries risk assessment: relevance to the practitioner. J Am. Dent. Assoc. 1998, 129: 349353. 41. Stookey G and Gonzlez-Cabezas C, Emerging Methods of Caries Diagnosis Journal of Dental Education 2001; 65: 1001-1006 42. Das D, Mukherjee S, Mukherjee M, Das A and Mitra C.: Aqueous extract of black tea (Camellia sinensis) prevents chronic ethanol toxicity. Current Science. 2005; 88: 952-961 43. Almas K, Al-Zeid Z. The Immediate Antimicrobial effect of a toothbrush and Miswak on cariogenic bacteria: A Clinical Study J. Contemp. Dent. Pract. 2004; 5:105-114. 44. Hammer K, Carson C and Riley T, Antimicrobial activity of essential oils and other plant extracts. Journal of Applied Microbiology 1999; 86: 985990 45. Gibbons S.: An overview of plant extracts as potential therapeutics. Expert Opinion on Therapeutic Patents 2003; 13: 489-497 46. Sakanaka S, Kim M, Yamamoto T.: Antibacterial substance in Japanese green extract against streptococcus mutans, a cariogenic bacterium. Agr. Biol. Chem. 1989; 53: 2307-2311 47. Kawamura J, Takeo T.: Antibacterial activity of tea catechin to streptococcus mutans. J. Jap. Soc. Food Sci. Technol. 1989; 36: 463-467 48. Hattori M; Kusumoto IT; Namba T; Ishigami T; Hara Y.: Effect of tea polyphenols on glucan synthesis by glucosyltransferase from streptococcus mutans. Chem. Pharm. Bull. 1990; 38: 717-720 49. Nakahara K, Kawabata S, Ono H, Ogura K, Tanaka T, Ooshima T, Hamada S. Inhibitory effect of oolong tea polyphenols on glycosyltransferases of mutans streptococci. Appl Environ Microbiol. 1993; 59: 968-973 50. Kashket S, Paolini VJ.: Inhibition of Salivary Amylase by water-soluble extract of tea. Arch. Oral Biol. 1988; 3: 845-846

51. Zhang J., S. Kashket.: Inhibition of Salivary Amylase by Black and Green Teas and Their Effects on the Intraoral Hydrolysis of Starch, Caries Research 1998; 32: 233238 52. Koo M and Cho C.: Pharmacological effects of green tea on the gastrointestinal system, A review. European Journal of Pharmacology 2004; 500: 177 185 53. Jin B, Lee J, Kim H, and Paik D.: The Effect of Green Tea drinking on Dental Caries Prevention., June 2006 IADR Brisbane General Session & Exhibition