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Table of Contents   
ORIGINAL RESEARCH  
Year : 2012  |  Volume : 23  |  Issue : 1  |  Page : 43-48
Effect of different types of tea on Streptococcus mutans: An in vitro study


Department of Pedodontics and Preventive Dentistry, The Oxford Dental College, Hospital and Research Center, Bangalore, India

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Date of Submission13-Dec-2010
Date of Decision17-Jan-2011
Date of Acceptance08-Nov-2011
Date of Web Publication26-Jul-2012
 

   Abstract 

Context: If tea can be shown to have an inhibitory effect on the growth of Streptococcus mutans there can be a basis for using it as an agent for reducing caries.
Aims: The aim of the study was to determine the effect of aqueous and organic extracts of three types of tea (green, oolong, and black tea) on the growth of S. mutans.
Settings and Design: In vitro study.
Material and Methods: Qualitative and quantitative phytochemical analysis of the three types of tea was done. Organic extracts of methanol and ethanol and aqueous extracts (50% and 100%) of tea were prepared. Fifty microliters of these extracts were inoculated into wells prepared on Mueller-Hinton agar plates that had been previously smeared with S. mutans. The agar plates were incubated at 37΀C for 24 hours. A similar procedure was followed using 0.2% chlorhexidine, which served as the positive control.
Statistical Analysis Used: Analysis of variance (ANOVA), post hoc Tukey test, Student's 't ' test (two-tailed, dependent), and Student's 't' test (two-tailed, independent) were used for analysis of the data.
Results: All the phytochemicals were found to be higher in oolong tea. Both aqueous and organic extracts of oolong tea showed greatest zones of inhibition, followed by green tea and black tea. Aqueous extracts of oolong and green tea showed greater zone of inhibition than chlorhexidine. All the three types of tea inhibited growth of S. mutans. The greatest inhibition was observed with aqueous extract of oolong tea.
Conclusions: Oolong tea extracts (aqueous and organic) showed a greater inhibitory effect on the growth of S. mutans than the other tea extracts .

Keywords: Black tea, flavonoids, green tea, oolong tea, phytochemicals, Streptococcus mutans, tannins, tea

How to cite this article:
Subramaniam P, Eswara U, Maheshwar Reddy K R. Effect of different types of tea on Streptococcus mutans: An in vitro study. Indian J Dent Res 2012;23:43-8

How to cite this URL:
Subramaniam P, Eswara U, Maheshwar Reddy K R. Effect of different types of tea on Streptococcus mutans: An in vitro study. Indian J Dent Res [serial online] 2012 [cited 2014 Jul 23];23:43-8. Available from: http://www.ijdr.in/text.asp?2012/23/1/43/99037
Tea, which originated in China, has conquered the world's taste over the last 2000 years. It is obtained from the leaf and bud of the plant Camellia sinensis. [1],[2] Depending on the manufacturing process, tea can be 'non-fermented' green tea, 'semifermented' oolong tea, or 'fermented' black tea. [3],[4] Consumption of tea is a daily routine in many parts of the world. It is the second most commonly consumed beverage in the world after water. The per capita mean consumption of tea in the world has been reported to be 120 ml/day. [4] In India, the per capita consumption of tea annually is 706 g. [5]

Green tea polyphenols have demonstrated significant antioxidant, anticarcinogenic, anti-inflammatory, thermogenic, probiotic, and antimicrobial properties in numerous human, animal, and in vitro studies. [6],[7] While there is an extensive literature suggesting medical health benefits associated with drinking tea (i.e., C sinensis), evidence-based information regarding the effects of tea on dental health is quite limited. Japanese folklore has it that drinking green tea 'makes the mouth clean.' [8]

One of the most prevalent and expensive bacterial infections in the world is dental caries, which is primarily caused by Streptococcus mutans. [9] The etiology of dental caries and variations in its prevalence are ascribed to differences in dietary habits (especially the consumption of sugar), variations in the patterns of oral hygiene, changes in the virulence of oral and dental plaque microflora, and alterations in the oral protective mechanisms (including the immune status). Glucans is an extracellular slime layer produced by many oral streptococci in the presence of sucrose that promotes adhesion and the formation of a dental plaque biofilm. The formation of dental plaque leads to localized demineralization due to the accumulation of acids. Theoretically, the inhibition of each step in the process of caries formation contributes to the prevention of dental caries. [10] Besides cleaning of teeth, the use of an antimicrobial agent to limit growth of cariogenic microorganisms in the oral cavity can also contribute to prevention of caries. In the last few years, there has been an increased interest in the properties of some plant-derived stimulant beverages, particularly chocolate, coffee, and tea, which have demonstrated anticariogenic activity both in vitro and in vivo.

Three commonly used and available tea varieties were selected for study in the present research. Black tea is consumed the world over and, in India, is favored the most. Green tea has been well researched. Though the beneficial effects of oolong tea have been reported, relatively little research has been done on it as it consumed only in a few parts of Asia, especially in eastern China and Taiwan. This study was therefore undertaken to assess whether crude extracts (organic and aqueous) of three types of tea (green, oolong, and black tea) have any effect on the growth of S. mutans and to compare the effect of these extracts with that of 0.2% chlorhexidine mouthwash.


   Materials and Methods Top


Phytochemical analysis

Both qualitative and quantitative phytochemical analysis was done for all the types of tea, using the method followed by Edeoga et al.[11]

Prior to conducting the antibacterial tests to assess the effect of tea on the growth of S. mutans, the freeze-dried bacteria were cultured and crude organic and aqueous extracts of the tea were prepared.

Culturing of freeze-dried S. mutans

The freeze-dried S. mutans (MTCC 497) obtained from IMTECH (Institute of Microbial Technology, Chandigarh) was first transferred to brain heart infusion (BHI) broth and incubated for 24 hours to make the S. mutans viable. Then, this suspension was smeared on Mueller-Hinton Agar (MHA) plate. After 24 hours of incubation at 37°C, five colonies were transferred into 10 ml of BHI broth for use the next day to test the effect of tea on the growth of S. mutans. [12]

Preparation of tea extracts

Three different types of tea leaves of the same brand (Infinitea) were used for the study; they included black tea (Assam Orthodox variety), green tea (Elixir variety), and oolong tea (Enigma variety). Organic and aqueous extracts of all the three types of tea were freshly prepared on the day of the antibacterial test.

Organic extracts of tea

For each variety of tea, ethanol (100% w/v) and methanol (100% w/v) were used to prepare crude organic extracts. Fifty milliliters of each solvent was added to 5 g of the dry powdered tea sample. A mortar and pestle was used to grind the tea leaves with the solvent, and this mixture was filtered using Whatman filter paper to obtain an extract. The extract obtained was reduced to approximately 5 ml over a water bath to obtain a 100% (w/v) concentrated organic extract of each tea.

Aqueous tea extracts

Distilled water was used to prepare aqueous extracts with concentrations of 50% and 100%. Ten grams of each variety of tea was added to 100 ml of boiling distilled water and further boiled for 30 minutes. The extract obtained was reduced to 10 ml to obtain 100% (w/v) concentration. This was diluted with distilled water at a ratio of 1:1 to obtain aqueous extract with 50% (w/v) concentration.

The extracts were cooled to room temperature prior to antibacterial testing.

Antibacterial test

The bacterial sensitivity test was carried out in laminar air flow equipment using the diffusion technique. A suspension of BHI broth containing cultivated S. mutans was placed on the center of the MHA plate and evenly spread on the plate using a sterile cotton swab. [12] Five wells were then punched on the MHA using the tip of a 1-ml micropipette. An additional well was prepared at the center in the plates used for organic extracts only; this well served as the control, where 50 μl of the respective solvent was used. Since an earlier study showed that distilled water produces no inhibition of S. mutans growth,no control well was made for aqueous tea extracts. [12]

The antibacterial effect of aqueous and organic tea extracts was compared to that of 0.2% chlorhexidine. Fifty microlitres of each organic tea extract and 0.2% chlorhexidine was inoculated into the wells using a micropipette. Using this method, the experiment was done in triplicate for each type of tea extract and a total of 18 plates were prepared for all the three types of tea.

The aqueous extracts were tested in the same manner, with a total of 18 plates for the three types of tea.

After 24 hours of incubation, the  Petri dish More Detailses were observed for zones of inhibition, i.e., areas without growth of S. mutans. The zone of inhibition was measured as the maximum width from the edge of the well to the periphery of the inhibition zone with the help of Vernier calipers.

Statistical procedures

Data obtained were tabulated and subjected to descriptive statistical analysis. Analysis of variance (ANOVA) was used to find the significance of study parameters between three or more groups of samples and post hoc Tukey test was used to find the pair-wise significance. Student's 't ' test (two-tailed, dependent) was used to find the significance of study parameters on continuous scale within each group. Student's 't ' test (two-tailed, independent) was used to find the significance of study parameters on continuous scale between two groups. The statistical software namely SPSS® 15.0, Stata® 8.0, MedCalc® 9.0.1 and Systat® 11.0 were used for the analysis of the data and Microsoft® Word and Excel were used to generate graphs, tables, etc.


   Results Top


The qualitative analysis showed the presence of alkaloids, tannins, saponins, steroids, flavonoids, and cardiac glycosides in all the three types of tea. Terpenoids were present only in green and black tea and phlobatannins were absent in all the three types of tea [Table 1]. Amongst the three types of tea, oolong tea showed the highest amount of phytochemicals quantitatively [Table 2].
Table 1: Qualitative analysis of the three types of tea

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Table 2: Quantitative analysis showing constituents per gram of tea

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After an incubation period of 24 hours, all the extracts of the three types of tea showed inhibitory effect on the growth of S. mutans [Table 3]. The zones of inhibition with the various extracts of three types of tea were compared in the following order: (a) methanol extracts, (b) ethanol extracts, (c) aqueous extracts, (d) aqueous extracts vs organic extracts and (e) chlorhexidine vs organic and aqueous extracts.
Table 3: Overall comparison of organic and aqueous extracts of the three types of tea on S. mutans growth

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Amongst the methanol extracts both oolong tea and green tea showed significantly greater inhibition zones than black tea extract. Ethanol extract of oolong tea showed greater inhibition than extract of green and black tea; the difference was moderately significant. Aqueous extracts (i.e., 50% and 100%) of both oolong and green tea showed significantly greater zones of inhibition when compared to that of black tea [Table 3].

In comparison to methanol extracts, both aqueous extracts of green tea showed significant inhibition of S. mutans growth. Aqueous extracts (50% and 100%) of oolong tea and green tea showed significantly greater zones of inhibition than their respective ethanol extracts. There was no significant difference between the organic and aqueous extracts of black tea [Table 4] and [Table 5].
Table 4: Comparison of methanol extract with aqueous extracts of tea on S. mutans growth

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Table 5: Comparison of ethanol extract with aqueous extracts of tea on S. mutans growth

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The methanol extract and aqueous extract (100%) of oolong tea showed significantly greater inhibition of S. mutans than chlorhexidine (0.2%) [Table 6] and [Table 7].
Table 6: Comparison between organic extracts of tea and chlorhexidine on S. mutans growth

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Table 7: Comparison between aqueous extracts of tea and chlorhexidine on S. mutans growth

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   Discussion Top


The chemical composition of tea is complex; tea contains polyphenols, alkaloids (caffeine, theophylline, and theobromine), amino acids, carbohydrates, proteins, chlorophyll, volatile compounds, minerals, trace elements, and other unidentified compounds. [13] Among these, polyphenols constitute the most interesting group and are the main bioactive molecules in tea [14] and, in consequence, tea can be considered an important dietary source of polyphenols, particularly flavonoids. Flavonoids are phenol derivatives synthesized in substantial amounts (0.5%-1.5%) and variety (more than 4000 identified) and are widely distributed among plants. [15] The main flavonoids present in tea are the polyphenolic catechins (flavon-3-ols), namely epigallocatechin-3-gallate (EGCG), epigallocatechin (EGC), epicatechin-3-gallate (ECG), and epicatechin (EC). The tea catechins, in particular, are major constituents of fresh tea leaves. [16] These constituents are oxidized during fermentation to yield a complex mixture of secondary polyphenols, including theaflavins, theasinensins, and oolongtheanins (derived from epigallocatechins and/or epigallocatechin gallate via theasinensins by enzymatic oxidation). [17] However, most of the secondary polyphenols in black tea have not yet been chemically characterized because of their complexity and the difficulties associated with their separation and purification. Oolong tea also contains considerable amounts of catechins and oligomerized catechins. [18]

To the best of our knowledge, most of the previous studies have been done with purified organic extracts of green tea, oolong tea, and black tea. In our study only crude extracts of the different types of tea, which can be obtained with simple laboratory procedures, were prepared. Since tea is consumed regularly in the aqueous form (tea infusion) this study was carried out to determine the effect of aqueous extracts of tea on S. mutans growth and to compare the effect with that of organic extracts of tea. A high concentration (50% and 100%) of crude aqueous extracts of all the three types of tea were prepared, as a pilot study conducted earlier showed no effect on S. mutans growth with 1% and 2% aqueous extracts of all three types of tea. We used 0.2% chlorhexidine as a positive control in this study due to its known antibacterial action. [19]

It has been earlier suggested that fluoride is an important element in tea contributing to caries inhibition. But the low levels of fluoride in tea (0.38-0.70 ppm) suggest that tea may also contain other substances that inhibit caries. [20] Qualitative analysis of all the three types of tea revealed the presence of various phytochemicals. Quantitatively, alkaloids, tannins, saponins, and flavonoids were highest in oolong tea, which is consistent with the spectrophotometry results reported by van Het Hof et al.[21] These phytochemicals have various actions. The alkaloids are said to interfere with microbial cell division, [22] whereas flavonoids possess anti-glucosyltransferase activity and inhibit bacterial adherence. [23] Tannins, on the other hand, inhibit bacterial growth with their strong iron-binding capacity and also inhibit glucosyltransferase activity and bacterial adhesion. [24] It has been reported that tea polyphenols have no effect on de/remineralization of enamel blocks, suggesting that tea polyphenols exert an anti-caries effect via an antimicrobial mode of action. [25]

In the present study the crude extracts (organic and aqueous) of all three types of tea showed an inhibitory effect on the growth of S. mutans. However, oolong tea extracts (organic and aqueous) showed greater inhibitory effect on S. mutans growth than green tea and black tea. This may be due to the presence of greater amounts of phytochemicals in oolong tea as compared to green and black tea. This is in accordance with the findings of Sasaki et al.[26] who reported that the bactericidal activity of oolong tea extract is due to the synergistic effect of monomeric polyphenols. Bratcher has also shown that oolong tea extracts cause a significant reduction in S. mutans levels. [27] Oolong tea extract when given at a concentration of

500 μg/ml in experimental animals showed significant reduction of dental caries, and the cariostatic activity was considered to be mainly caused by anti-glucosyltransferase activity. [28] Black tea and green tea contain similar amounts of flavonoids; however, green tea contains more catechins (simple flavonoids), while black tea contains mostly polymerized catechins (theaflavins and thearubigens) due to the fermentation process. [29],[30],[31] This may be the reason why black tea exhibits less inhibitory action on S. mutans than oolong and green tea.

In this study, boiling the tea leaves and further concentrating the extract seems to have succeeded in obtaining significant amount of phytochemicals of all the three types of tea. In the case of oolong tea, the amount of phytochemicals obtained with methanol and distilled water was probably the same and therefore the effects of the methanol and aqueous extracts were similar. In the case of green tea, the amount of phytochemicals obtained after boiling with distilled water might have been more than that obtained by using methanol and ethanol as solvent. This implies that concentrated aqueous extracts of tea have a potential role in dentistry.

At low concentrations, chlorhexidine tends to alter bacterial cell membrane integrity by binding to the inner bacterial cell membrane, leading to leakage of low-molecular-weight components, especially potassium. This bacteriostatic mechanism of chlorhexidine is reversible in nature. [32] However, it is interesting to observe that oolong tea and green tea aqueous extracts showed greater inhibitory effect than chlorhexidine. This may be due to their irreversible effect on the bacterial membrane. An alcohol-free mouthwash can probably be prepared from aqueous extracts of green and oolong tea.

Although the varieties of tea used in this study were manufactured from the leaves of the same plant species, C sinensis, the glucosyltransferase-inhibitory polyphenols are produced only in oolong tea or black tea. Unlike green tea, oolong tea and black tea are semifermented and fermented, respectively, during the manufacturing process. The fermentation and heating of tea leaves result in polymerization of monomeric polyphenolic compounds like catechins. Our findings clearly indicate that the conformational changes due to polymerization of catechins can critically affect the inhibitory action of the glucosyltransferases on mutans streptococci. [33] It is not known how tea extracts exert their antibacterial and other biological effects, though EGCG and EC have been reported to disrupt reconstituted bacterial membranes. [34] Tea polyphenols exert different actions by acting as a slow-release source of catechins and theaflavins, which inhibit Streptococcus growth and also inhibit the adherence of S. mutans to the tooth surface. [35]

The results of the present study strongly suggest that certain components of tea exert a significant anticariogenic effect by virtue of their inhibitory activity against S. mutans. Green tea has been recommended for its various health benefits, including its anticariogenic effects. The present study suggests that drinking oolong tea is more effective than green and black tea in the prevention of dental caries. Studies on quantitative estimation of the phytochemicals in tea extracts (aqueous and organic) need to be done. Further evaluation of individual ingredients of tea extracts, would probably corroborate these findings.

Though tea is not consumed at a concentration of 50% and 100% (the concentrations used in this study), drinking tea at lower concentrations over prolonged periods might also be effective in bringing about a reduction in dental caries. Also, tea extracts can possibly be incorporated into chewing gums, toothpastes, mouthwashes, and dental floss for its preventive actions.


   Acknowledgments Top


This research was done at Wingene Biotech Research Labs, Bangalore, India. We are grateful to Mr. Srigiri Srinivas (Director) and Ms. Shaista Anjum (Head of Academics) of Wingene Biotech Research Labs for their assistance during this experiment. We also thank Mr. KP Suresh (Statistician) for his assistance in statistical analysis.

 
   References Top

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Correspondence Address:
Priya Subramaniam
Department of Pedodontics and Preventive Dentistry, The Oxford Dental College, Hospital and Research Center, Bangalore
India
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DOI: 10.4103/0970-9290.99037

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    Tables

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6], [Table 7]

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