Nebal F.doc J Bagh College Dentistry Vol. 25(3), September 2013 Antibacterial effect Pedodontics, Orthodontics and Preventive Dentistry158 Antibacterial effect of cardamom and black tea aqueous extract on mutans streptococci in comparison to chlorhexidine (in vitro study) Nebal Th. Shaker, B.D.S., M.Sc. (1) ABSTRACT Background: Antimicrobial agents have been considered as having potential for the prevention of dental caries. This study aimed to test the effect of different concentrations of cardamom and black tea extracts on the sensitivity and growth of salivary mutans streptococci in comparison to chlorhexidine gluconate (0.2%) in vitro. Materials and methods: In this study. Mutans streptococci were isolated from saliva of 34 healthy people (aged between 22-40yrs). The bacteria was isolated, purifiedand diagnosed according to morphologicalcharacteristic and biochemical tests. Aqueous extracts of cardamom and black tea were prepared. Different concentrations of extracts were prepared and estimated in gm/ 100ml deionized water. The agar diffusion technique was used to determine the antibacterial activity of cardamom and tea extracts in which the inhibition of bacteria growth by different concentrations of extracts was measured by diameter of inhibition zone in millimeter. The viable count was measured in different concentrations for both types of extracts on comparison to chlorhexidine 0.2%. Results: The result showed that the mutans streptococci is more sensitive to tea extract than cardamom one, where the mean value of diameter of inhibition zone was higher by tea extract than cardamom type in all concentrations and chlorohexidine0.2% is more effective than both extracts. For viable count no statistical significant difference between two extract types at concentration of 40% but there are a high statistical significant difference for other concentrations, where the chlorhexidine is moreeffective than tea type and the last one is more effective than cardamom type with p value 0.05 . Conclusions: Cardamom and black tea have antibacterial effect against Mutans streptococci; the accused factor of dental caries. Key words: Cardamom, black tea, Mutans streptococci, antibacterial. (J Bagh Coll Dentistry 2013; 25(3):158-164). INTRODUCTION Finding healing powers in plants is an ancient idea. People on all continents have long applied poultices infusions of hundreds, if not thousands of indigenous plants, dating back to prehistory (1). Cardamom is the third most expensive spice in the world (after saffron and vanilla), and the high price reflects the high reputation of this most pleasantly scented spice (2). It refers to several plants of the genera Elettaria and Amomum in the ginger family Zingibreaceae. Both genera are native to India; they are recognized by their small seed pod, triangular in cross-section and spindle shaped, with a thin papery outer shell and small black seeds. Elettaria pods are light green while Amomum pods are larger and dark brown commonly known as black cardamom (3, 4). The content essential oil in the seeds is strongly on storage conditions, but may be as high as 8%. In oil were found α-terpineol 45%, myrcene 27%, limonene 8%, menthone 6%, β-phenllandrene 3%, 1,8-cineol 2% and heptane 2% (5).Cardamom has a strong, unique taste, with an intensely aromatic, resinous fragrance. Both forms of cardamom are used as flavorings in food and drink, as cooking spices and as a medicine. It is a common ingredient in Indian cooking, and is often used in backing in Nordic countries such as in the Finnish (1)Assistant lecturer, Department of Preventive Dentistry. College of Dentistry. Al- Mustansyria University sweet bread pulla or in Scandinavian bread Julekake. 60% of the world production is exported to Arab countries, where the large part used to prepare coffee and tea. Individual seeds are sometimes chewed, in much the same way as chewing gum to neutralize the toughest breath odors (6). In traditional medicine it is used to treat infections in teeth and gums, to prevent and treat throat troubles, congestion of the lungs and pulmonary tuberculosis, inflammation of eyelids and totreat digestive disorders such as stomach- aches, constipation, and dysentery. It also used to break up kidney stones and gall stones, and was reportedly used as an antidote for both snake and scorpion venom (7). Cardamom is best stored in pod form because once the seeds are exposed or ground they quickly lose their flavor. The seeds show a loss of about 40% of the essential oil per year (8). One of the most popular, widely used plants all over the world is the tea plant (Camellia sinesis; Fam; Teacea) (9). The tea plant is a shrub or semi tree belonging to the transtroemia with alternative evergreen leaves. The fresh leaves pickets from the tea plant are processed into varicose kind of tea according to the means of manufacturing methods but the three main types of tea can be classified as black, green and oolong tea.There are more than 500 various chemical elements contained in tea. Black tea has many J Bagh College Dentistry Vol. 25(3), September 2013 Antibacterial effect Pedodontics, Orthodontics and Preventive Dentistry159 more components than green tea partly because of the oxidation process that occur during fermentation (10). Tea consists of caffeine 1%_4%, tannin 10_24%; fluoride, cadimium, cobalt, vitamins and volatile oil have been identified among the elements in tea (11). Tea plant is considered to be one of the most important medicinal plants due to its various beneficial effects. Tea has antioxidant effect, antimutagenic activity; antimicrobial effects, anticarcinogenic property, reduce blood pressure and the possibility of coronary heart disease. Tea improves the cognitive and psychomotor performance (12). The antibacterial activity (especially anticariogenic activity of cardamomand tea) is going to take place in this study aiming tobenefit from tea and cardamom extract to control and prevent dental caries in the future. Dental caries is a microbial disease affecting mineralized tissues of teeth (13). Mutans streptococcus (MS) is considered to be the most accused factors to the initiation and progression of dental caries (14). MATERIALS AND METHODS Preparation of aqueous extracts Cardamom was purchased from the market. Ground into fine powder in an electrical mixer. 100g of the finely powdered cardamom mixed with one liter of de- ionized water and kept in a water bath at 60 C for five hours, then filtered through filter paper. The extract was then left to dry at 40º C in hot air oven for evaporation of water. The extract was preserved in a refrigerator until use (15). The commercial type of tea was used in this project supplied from local supermarket. The aqueous extract was prepared by 100g of dried leaves of black tea were infused in 500ml. of boiling distilled water, and left to cool at room temperature. Agitation of the infusion with magnetic stirrer had been done alternatively. Then the infusion was filtered by filter paper (wattman No. 1) and the residue discarded. The extract left to dry in a petridish at room temperature, the resulted powder kept in tightly closed glass container in refrigerator until used for different concentrations(1). Isolation of mutans streptococcus Stimulated saliva was collected from 34 healthy individual (aged 22-40yrs). Saliva was homogenized by vortex mixer for 2 minutes and serial dilutions were prepared by using normal saline then the appropriate dilutions from saliva for each microbial type was inoculated on Mitis- Salivaris Bacitracin Agar (MSB agar), selective media for MS; by taking 100ml from dilution (10- 2,10-4) and spread on plates of MSB agar by sterile microbiological spreader after thatincubated anaerobically by using gas pack supplied in anaerobic jar for 48 hours at 37 C followed by aerobic incubation 24 hours at 37 C(16). The colonies on MSB agar were studied under microscope and gram stain was used followed by biochemical test to identify the isolates by catalase test and carbohydrate fermentation test(17).Different concentrations of cardamom and black tea extracts were prepared, 40%, 80%, 120% and 160%. The agar diffusion technique was applied to study the antimicrobial effect of these concentrations on thegrowth of MS and colony counts. Statistical analyses Data were analyzed using SPSS program version 20.Data were present in simple measures of mean and standard deviation, the significance of difference between studied variables was tested using student t- test for comparing between two means of independent groups. ANOVA used to test the difference among groups and Dunitti test used to compare the results of two groups with control one. P value less than0.05 was used as the level of significance. RESULTS The result revealed a high statistical significant difference by independent t- test between the two types of extracts. (p value ≤ 0.05). The result showed that the micro-organism is more sensitive to tea extract than cardamom one, where the mean value of diameter of inhibition zone was higher by tea extract than cardamom type in all concentrations as seen in Table 1and figure 1. The result of ANOVA test illustrates a high statistical significant differences (p value 0.05) regarding the mean diameter of inhibition induced by three types of extracts at all concentrations ,where the result showed that the chlorohexidine (CHX) is more effective than tea extract and the latter one is more effective than cardamom one, as seen in Table 2. When a comparison of inhibition zone induced by two types of extracts (tea and cardamom) and control group (CHX) by Dunetti test revealed high statistical significant differences in all tested concentrations (p value 0.05) where the chlorhexidine is more effective than tea and cardamom extracts in all concentrations as seen in Table 3. The figures 2 and 3 showed a positive linear correlation between the diameter of inhibition zone and different concentration of each extract, where the mean of diameter of inhibition zone increased by increasing the concentration. J Bagh College Dentistry Vol. 25(3), September 2013 Antibacterial effect Pedodontics, Orthodontics and Preventive Dentistry160 Counts of M.S were tested in the presence of different concentrations of cardamom and tea aqueous extract. The result revealed no statistical significant difference between two extract types at concentration of 40% but there are a high statistical significant difference for other concentrations with p value less than 0.05 as seen in Table 4. The result demonstrated a high statistical significant difference among three groups at different concentrations regarding the mean viable count of MS, where the result revealed that the chlorhexidine is more effective than other groups with p value 0.05 .When the Dunneti test to compare the result of viable count by two extract types with the control group (chlorhexidine),the result revealed a high statistical difference between each type of extract and control group in different concentrations with p value 0.05.Table 5 and 6. DISCUSSION Scientific analysis of plant components follows a logical pathway. Initial screening of plants for possible antimicrobial activities typically being by using crude aqueous or alcoholic extraction and sometimes can be followed by various organic extraction methods. The aqueous extract of plants is considered to be the best way for the extract activity, in addition , the amateur herbalist will be able to prepare this extract easily(1). The sensitivity of mutans streptococci to different concentrations of cardamom and black tea aqueous extract in comparison to 0.2% chlorhexidine gluconate antimicrobial agent was tested. The zone of inhibition were found to increase when the concentration of cardamom and tea extract increased; but for tea extract zone of inhibition were higher than that seen for cardamom one. This may indicate that the sensitivity of Mutans streptococci to higher concentrations of aqueous extract of tea was more than that of cardamom extract. It could due to that the antimicrobial constituents of tea was in much more amount than that obtained from cardamom extract. In the present study effects of aqueous extract of cardamom and tea on the viable count of mutans streptococci were tested. Both types had antibacterial activity against mutans streptococci because they were able to reduce the viable cell count profoundly. This is a desirable property for anticariogenic agent. The results showed that no statistical significant difference between two extract types at concentration of 40% but there are a high statistical significant difference for other concentrations. CHX had the greatest effect in comparison to the both. Mouth washes are used to clean and deodorize the oral cavity. Generally, they contain antibacterial agents and are most often used for their deodorizing, refreshing and antiseptic effects (9). The results of this study indicate that cardamom and tea had highly significant antimicrobial activity. Such antimicrobial property is a desirable property for mouth washes. These findings were in coincidence with other studies (9,18-21). REFERENCES 1. Cowan M. Plant products as antimicrobial agents. Clinical Microbiology Reviews 1999; 12: 564-82. 2. Buckingham JS, Petheram RJ. Cardamom cultivation and forest biodiversity. Agricultural Research, London, 2004. 3. Norajit K, Lua N, Orapin K. Antibacterial effect of five Zingiberaceae essential oils. Molecules 2007; 12(8): 2047-60. 4. Aubertine C. Cardamom in Lao PDR: the hazardous future of an agroforest system product. International Forest Research. Jakarta, Indonesia, 2004. 5. Kaskoos KN, Mir SR. Essential oil composition of fruits of AmomumsudulatumRoxb. J Essential Oil Bearing Plants 2008; 11: 184-7. 6. Sabulal B, Dan M, Pradeep NS. Composition and antimicrobial activity of essential oil from cardamom. Acta Pharm 2006; 56:473-80. 7. Kumar GP, Chaturvedi A. Antimicrobial activity of some medicinal plants of Euphorbiaceae. India Drugs 2006; 43:156-9. 8. Gurudutt KN, Naik JP, Srinivas P. Volatile constituent of large cardamom (AmomumsubulatumRoxb). FlavFragr J 1996; 11:7-9. 9. Esimone C, Adikwu M, Nwafor S, Okoklo C. Potential Use of tea extract as a complementary mouthwash: comparative evaluation of two commercial samples. J alternative and complementary medicine 2001; 7(5): 523-7. 10. Tingli L. Mechanism and clinical study on the anti- caries effect of green tea polyphenols. Information of Chinese Traditional Medical Science. 2002; 6: 22-3. 11. McKay D, Blumberg J. the role of tea in human health. Journal of the American College of Nutrition 2002; 21(1):1 -13. 12. Turkemen N. Effect of extraction conditions on measured total polyphenol contents and antioxidant and antibacterial activities of black tea. Molecules. 2007; 12(3): 484-96. 13. Kidd E, Jouyston-Bechal S. Essentials of dental caries. The disease and its management. Oxford. Hong Kong 2002. p.1-20. 14. Touys L, Amsel R. anticariogenic effects of black tea (camellia sinensis) in caries-prone rate. Quintessence- Int 2001; 32(8):647-50. 15. Rout PK, Sahoo D, Jena KS, Rao YR. Analysis of the oil of large cardamom. J Essent Oil Res 2003; 15: 265- 6. 16. Koneman EW, Schreckenberge PC, Allens SD, Jr.WCW, Janada WM. Diagnostic Microbiology. 4th ed. J.B. Lippincott Co., USA. 1992. J Bagh College Dentistry Vol. 25(3), September 2013 Antibacterial effect Pedodontics, Orthodontics and Preventive Dentistry161 17. Fingold C, BaronE. Methods for identification of etiologic agents of infectious disease. In: Bialey and Scotts diagnostic microbiology 7thedt. CV Mosby Co. St. Louis 1986, 382-422. 18. Supriya A, Wakode S. Antimicrobial activity of essential oil and various extracts of fruits of greater Cardamom. Indian J Pharmaceutical Sciences 2010; 72(5): 657-659. 19. Gilani SR, Shahid I, Javed M, Mehmud S, Ahmed R. Antimicrobial activities and physio-chemical properties of the essential oil from Amomumsubulatum. Int J Appl Chem 2006; 2:81-6. 20. Al-Izzy M. Antibacterial effects of black and green tea extract on mutans streptococci and lactobacilli(in vitro and vivo study).M.Sc. thesis. College of Dentistry, Baghdad University, 2005. 21. Sasaki H, Masumoto M, TanakaT, Maeda M. Antibacterial activity of polyphenol components in oolong tea extract against streptococcus mutans. Caries Res 2004; 38: 2-8. Table1. Mean diameter of inhibition zone/mm by two types of extracts. Conc. Type of extract No. Mean diameter of inhibition zone/mm ±S.D. t- test p-value 40% Tea 34 6.6 .7851 7.186 HS Card 34 5.3 .6974 80% Tea 34 7.1 .7634 8.022 HS Card 34 5.7 .6599 120% Tea 34 7.6 .7855 9.041 HS Card 34 6.1 .6213 160% Tea 34 8.1 .6937 11.157 HS Card 34 6.4 .5841 HS=highly significant Figure 1: 1Mean diameter of inhibition zone of MS/mm by two types of extracts in different concentrations. Table 2. Mean diameter of inhibition zone by different types of extracts at different concentrations Concentration /extract type No. Mean Of inhibition zone/mm ±S.D. 95% Confidence Interval for Mean F test p-value Lower Bound Upper Bound 40% Tea 34 6.6 .7851 6.403 6.950 318.061 HS Card 34 5.3 .6974 5.139 5.626 CHX 34 10.6 1.1500 10.258 11.060 80% Tea 34 7.1 .7634 6.904 7.437 275.080 HS Card 34 5.7 .6599 5.552 6.013 CHX 34 10.6 1.1500 10.258 11.060 120% Tea 34 7.6 .7855 7.385 7.933 234.952 HS Card 34 6.1 .6213 5.889 6.323 CHX 34 10.6 1.1500 10.258 11.060 160% Tea 34 8.1 .6937 7.899 8.383 217.456 HS Card 34 6.4 .5841 6.202 6.610 CHX 34 10.6 1.1500 10.258 11.060 HS=highly significant 0 2 4 6 8 10 conc.40% conc.80% conc.120% conc.160% Chart Title Tea ex Card ex. J Bagh College Dentistry Vol. 25(3), September 2013 Antibacterial effect Pedodontics, Orthodontics and Preventive Dentistry162 Table 3. Compares of mean of inhibition zone between two types of extracts and control group (CHX) Dependent Variable (I) Type of extract (J) Type of extract (Control group) Mean Difference (I-J) Sig. 95% Confidence Interval Lower Bound Upper Bound 40% Tea CHX -3.9824* .001 -4.472 -3.493 Card CHX -5.2765* .001 -5.766 -4.787 80% Tea CHX -3.4882* .001 -3.969 -3.007 Card CHX -4.8765* .001 -5.357 -4.396 120% Tea CHX -3.0000* .001 -3.479 -2.521 Card CHX -4.5529* .001 -5.032 -4.074 160% Tea CHX -2.5176* .001 -2.978 -2.057 Card CHX -4.2529* .001 -4.713 -3.793 CHX=chlorhexidine Figure 2. Correlation between different concentrations of cardamom extract and mean diameter of inhibition zone Figure 3. Correlation between different concentrations of tea extract and mean diameter of inhibition zone 4 5 6 7 8 0% 50% 100% 150% 200% Inhibition zone/mm by card ex. for different conc. Linear (Inhibition zone/mm by card ex. for different conc ! 5 5% 6 5% 7 5% 8 5% 0% 50% 100% 150% 200% Mean ofInhibition zone in mm by Tea ext.at diff.conc. Mean ofInhibition zone in mm by Tea ext.at diff.conc. Linear ( Mean ofInhibition zone in mm by Tea ext.at diff.conc ! J Bagh College Dentistry Vol. 25(3), September 2013 Antibacterial effect Pedodontics, Orthodontics and Preventive Dentistry163 Figure 4: Mean diameter of inhibition zone by different concentrations of two types of extracts and control group. Table 4: Mean count of MS by different concentrations for two types of extracts ConcentrationsType of extract N0. Mean count of MS ±S.D. t- test P value zero time Tea 34 76.59 11.004 0.358 0.721 Card 34 75.65 10.657 40% Tea 34 67.00 9.851 -1.684 0.09(NS) Card 34 70.50 9.740 60% Tea 34 61.53 8.999 -2.587 0.012 Card 34 67.29 9.373 120% Tea 34 58.00 9.036 -2.587 0.002 Card 34 64.74 9.571 160% Tea 34 52.68 9.550 -3.365 0.001 Card 34 60.32 9.187 Table 5: Mean count of MS in different concentration for three types of extracts Concentrations/extracts NO. Mean of viable count of MS ±S.D. 95% Confidence Interval for Mean F test P value Lower Bound Upper Bound zero time Tea 34 76.59 11.004 72.75 80.43 128.465 HS Card 34 75.65 10.657 71.93 79.37 CHX 34 44.59 5.349 42.72 46.45 40% Tea 34 66.50 9.851 63.06 69.94 90.006 HS Card 34 70.50 9.740 67.10 73.90 CHX 34 44.59 5.349 42.72 46.45 60% Tea 34 61.53 8.999 58.39 64.67 71.961 HS Card 34 67.29 9.373 64.02 70.56 CHX 34 44.59 5.349 42.72 46.45 120% Tea 34 57.56 9.036 54.41 60.71 52.691 HS Card 34 64.74 9.571 61.40 68.07 CHX 34 44.59 5.349 42.72 46.45 160% Tea 34 52.68 9.550 49.34 56.01 30.925 HS Card 34 60.32 9.187 57.12 63.53 CHX 34 44.59 5.349 42.72 46.45 J Bagh College Dentistry Vol. 25(3), September 2013 Antibacterial effect Pedodontics, Orthodontics and Preventive Dentistry164 Table 6. Comparison of mean viable count between two extract types and control group Concentrations (I) Type of extract (J)Type of extract (CONTROL) Mean Difference in mean of viable count of MS (I-J) P value. 95% Confidence Interval Lower Bound Upper Bound zero time Tea CHX 32.000* .001 26.90 37.10 Card CHX 31.059* .001 25.96 36.16 40% Tea CHX 21.912* .001 17.25 26.58 Card CHX 25.912* .001 21.25 30.58 60% Tea CHX 16.941* .001 12.53 21.36 Card CHX 22.706* .001 18.29 27.12 120% Tea CHX 12.971* .001 8.51 17.44 Card CHX 20.147* .001 15.68 24.61 160% Tea CHX 8.088* .001 3.60 12.58 Card CHX 15.735* .001 11.24 20.23 Figure 5. Mean of viable count of MS by different concentrations of two types of extracts and control group