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 Table of Contents  
ORIGINAL ARTICLE
Year : 2017  |  Volume : 4  |  Issue : 1  |  Page : 18-21

Effect of concentration and brewing time on the antimicrobial activity of Camellia sinensis (green tea) against oral bacteria


1 Department of Periodontics, Faculty of Dental Sciences, SGT University, Gurgaon, Delhi-NCR, Haryana, India
2 Department of Microbiology, Faculty of Medicine and Health Sciences, SGT University, Gurgaon, Delhi-NCR, Haryana, India

Date of Web Publication14-Feb-2017

Correspondence Address:
Veenu Madaan Hans
Department of Periodontics, Faculty of Dental Sciences, SGT University, Gurgaon, Delhi-NCR, Haryana
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/1658-6816.200138

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  Abstract 

Background and Aim: Green tea, a rich source of flavonoids, has tremendous oral health potential. This study evaluates the effect of brewing green tea at varying concentrations and duration on its antimicrobial activity against oral bacteria.
Materials and Methods: Green tea was brewed in 15 concentration and time combinations. Paper disc diffusion test was carried out with these infusions on Tryptic Soy Agar plates inoculated with oral bacteria. Zone of inhibition after 72 h of incubation at 37°C was measured in millimeters.
Results: Higher zone of inhibition was observed in concentration brewed for 15 min than 30 and 60 min. Zone of inhibition was directly proportional to the concentration of green tea brewed. Most effective concentration was 80 mg/ml with a mean of 2.83 mm which was significantly higher than mean zone of inhibition for 60 mg/ml (P < 0.05).
Conclusion: Increasing the concentration and decreasing the brewing time of green tea results in a higher inhibitory effect on growth of oral bacteria.

Keywords: Antibacterial, Camellia sinensis, green tea, oral health


How to cite this article:
Grover HS, Deswal H, Hans VM, Devi L S. Effect of concentration and brewing time on the antimicrobial activity of Camellia sinensis (green tea) against oral bacteria. Saudi J Oral Sci 2017;4:18-21

How to cite this URL:
Grover HS, Deswal H, Hans VM, Devi L S. Effect of concentration and brewing time on the antimicrobial activity of Camellia sinensis (green tea) against oral bacteria. Saudi J Oral Sci [serial online] 2017 [cited 2017 Jun 25];4:18-21. Available from: http://www.saudijos.org/text.asp?2017/4/1/18/200138


  Introduction Top


Tea, world's second most consumed beverage, is a product of leaf and bud of a shrub Camellia sinensis.[1] Since its origin in China dating back to 2700 BC, tea has come a long way from being an everyday drink to becoming a therapeutic aid in many illnesses. Tea has been classified into three main types: nonfermented green tea, semi-fermented oolong tea, and fermented black tea, depending on their manufacturing process. Green tea is produced by drying and steaming the fresh leaves of the shrub, so as to stop the oxidation process by deactivating polyphenol oxidase.[2] Although black tea is the most consumed among all types of tea, green tea's popularity has increased around the world because of its therapeutic benefits. The bulk of green tea is composed of proteins, fibers, and phenolic compounds making up to 65%–75% of dry weight together.[3] Amino acids such as theanine (5-N-ethylglutamine), tryptophan, and glutamic acid along with phenolic acids such as chlorogenic acid and caffeic acid form a part of green tea. It is a rich source of minerals and trace elements such as calcium, magnesium, chromium, manganese, zinc, selenium, sodium, and potassium.[3] Green tea is also high in flavonoids which are phenol derivatives. The main flavonoids present in green tea are catechins (flavan-3-ols) which include epigallocatechin-3-gallate (EGCG), epigallocatechin, epicatechin-3-gallate, and epicatechin. Along with flavonoids, green tea also contains gallic acid.[1] Studies have shown that there may be 90 mg of EGCG in a cup of green tea brewed with approximately 2.5 g of green tea leaves in 200 ml of water.[4] The health benefits of green tea not only depend on the amount consumed but also there is variability in the bioavailability of the consumed catechins. In humans, EGCG has been found to have less bioavailability than other catechins.[5]

Since ancient times, green tea has been used as a medicinal agent in traditional Chinese medicine for treatment of headaches, as a detoxification agent and an energizer. Several studies have demonstrated its antibacterial and anti-inflammatory properties.[6] It has been confirmed that EGCG is an effective antibacterial polyphenol and is believed to act through cell membrane disruption and preventing DNA supercoiling leading to bacterial cell death.[6] The beneficial effects of green tea in prevention of dental caries and periodontal disease have also been investigated.[7],[8] Green tea is known to prevent the adhesion of Streptococcus mutans, Streptococcus sobrinus, and Porphyromonas gingivalis as well as inhibiting their growth.[4],[9] Since the antibacterial effect of green tea is already known, this article aims to investigate the effect of brewing green tea at various concentration and duration combinations on its antimicrobial activity against oral bacteria.


  Materials and Methods Top


Preparation of oral culture

For preparation of oral culture, Tryptic Soy Agar plates with 5% sheep's blood were prepared in a sterile environment. To obtain the samples, oral cavity of two human patients was swabbed around the gingival margin. The human patients selected for sample collection had moderate to severe gingivitis (80% of sites had positive bleeding on probing [BOP]) with a substantial plaque accumulation at gingival margin (Silness and Loe plaque score of >2). The obtained specimens were inoculated immediately onto prepared agar plates. The swab was rolled over a small area on the edge of the agar plate and then streaked in a zigzag fashion to cover the entire agar surface through a sterile loop. The culture plates were then incubated at 37°C for 72 h. Several colonies of pure culture were obtained from the mixed flora swab. These cultures were then used for inoculating agar plates prepared for paper disc diffusion test.

Preparation of green tea concentration and brewing time combination

To obtain green tea concentration with various brewing time, commercially available green tea leaves (Tetley®, India) were crushed and brewed at 90°C for varying times. Green tea leaves were measured with an electronic measure and concentration of 0, 20, 40, 60, and 80 mg/ml were achieved upon addition to distilled water. These concentrations were then brewed for 15, 30, and 60 min to obtain fifteen concentration time combinations. The concentration of 0 mg/ml acted as a control, wherein only distilled water was boiled.

Paper disc diffusion test

To carry out paper disc diffusion test, Tryptic Soy Agar plates were poured under strict sterile conditions. These plates were then inoculated by spreading a lawn of oral culture on each plate using a sterile loop. Paper discs were infused with green tea by placing the paper disc in the brewed concentration for approximately five minutes. Ten discs of each concentration duration combination were used. These discs were then placed on the inoculated agar plates which were thereafter incubated for 72 h at 37°C. Afterward, the zone of inhibition around the discs was measured with a standardized metallic ruler. The measurements were rounded off to 0.5 mm.


  Results Top


Zone of inhibition around all concentration duration combinations was calculated by taking the mean of zone of inhibition around all ten discs used for each combination. The zone of inhibition around the disc was proportional to the concentration of green tea infused [Figure 1]. The control discs with 0 mg/ml concentration exhibited no zone of inhibition and an increased growth of bacteria. Similar results were obtained for a concentration of 20 mg/ml brewed for any time duration. Compared to 15 min of brewing time, the zone of inhibition decreased when the tea was brewed for 30 min. It further went down, when the duration was increased to 60 min [Table 1]. Most effective concentration against bacteria was 80 mg/ml with an average zone of inhibition of 2.83 mm [Table 2]. Comparing the mean zone of inhibition around the concentrations of 60 mg/ml and 80 mg/ml with a two-sample t-test, P = 0.024 was obtained, which was statistically significant (P < 0.05).
Figure 1: Variation in zone of inhibition with varying green tea concentration and brewing time

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Table 1: Zones of inhibition for varying green tea concentrations and brewing time

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Table 2: Mean zone of inhibition for various green tea concentrations

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  Discussion Top


Green tea is known worldwide for conferring health benefits on consumption because of its antioxidant and anti-inflammatory properties. Green tea has also been reported to have antibacterial and antiviral activity.[7],[8],[10] Oral diseases such as caries and periodontitis have been established to be of infectious origin with host and environment playing an important role. Several studies have been conducted on the oral health benefits of green tea. Experiments in both animals and humans have concluded that green tea consumption can significantly reduce caries formation even when there are sugars present in the diet.[7] The catechins present in green tea have also been investigated for their effect on P. gingivalis. It was observed that these catechins inhibited the cysteine proteinases (gingipains) produced by P. gingivalis. The inhibitory effect was more against R-gingipain compared to K-gingipain.[8] Apart from this, green tea is a natural source of fluoride which has a significant role in maintaining the health of the oral cavity. In an experimental study, it was deduced that approximately 34% of the fluoride was retained in the oral cavity when rinsed with tea.[11] Green tea polyphenols are believed to inhibit the growth and metabolism of dental plaque bacteria. The purpose of our study was to confirm the effect of concentration and brewing time on antimicrobial properties of green tea against oral bacteria.

This investigation was carried out on a nonspecific mix of oral bacteria obtained on a swab from oral cavity. Since it was not determined in the mix whether the organism was Gram-negative or Gram-positive, it cannot be said conclusively whether green tea was more effective against Gram-positive or negative bacteria. Although the results suggest that green tea is effective in reducing the bacterial load in the oral cavity. Several studies have been carried out that confirm the inhibitory effect of green tea against oral bacteria. The bactericidal activity of green tea catechins at a concentration of 1 mg/ml has been demonstrated against Prevotella and P. gingivalis.[12]

The results of the present study revealed that the zone of inhibition steadily increased with increase in concentration. The concentration of 20 mg/ml produced no zone of inhibition, probably because of insufficient polyphenol content. These results suggest that a higher concentration of green tea is necessary for exhibiting its antibacterial property. In another study, EGCG at a concentration of 250–500 µg/ml completely inhibited the growth of three strains of P. gingivalis, whereas for Prevotella Melaninogenica, the minimum inhibitory concentration (MIC) was 200 µg/ml.[9] It was also observed that higher the concentration of EGCG more was the inhibitory effect, conforming to the results of our study.

The required amount of green tea to be consumed to attain its beneficial effects is debatable. There are experiments that depict that a cup of tea containing an average of 50–100 mg of polyphenols may be effective in preventing bacterial adherence and glucan synthesis.[13] This also exceeds the MIC of 50 µg/ml for S. mutans and 250–500 µg/ml for P. gingivalis.[9],[14] Green tea is known to inhibit the growth and adherence of known periopathogens, P. gingivalis, and Prevotella.[9] Daily intake of green tea was found to be associated with periodontal health status assessed by BOP, probing depth (PD), and clinical attachment loss (CAL). More the number of cups, lesser were the mean BOP, PD, and CAL.[15] Therefore, a single cup of tea may still reduce the plaque formation in oral cavity over a period of time. Further, the results of this study infer that the tea brewed for 15 min was most effective in inhibiting the bacteria. The possible explanation of lesser activity at 30 and 60 min could be oxidation and inactivation of polyphenols on continued heating.

The results of our study demonstrate that daily intake of green tea brewed for 15 min at 90 degree centigrade could help in plaque control by its inhibiting effects on bacteria. Green tea has known health benefits for which it is consumed in various parts of the world. Encouraging its consumption will also help in oral hygiene and health maintenance. Its regular consumption may replace the need of a chemical mouthwash which has several side effects. Thus, green tea provides a natural remedy for maintenance of oral health.

There are certain drawbacks of the study which could be a potential source of error. First, only one brand of tea was tested. Since the catechin content of the tea is dependent on the geographical location and growing conditions of tea and the how the leaves are processed; that is the fermentation and heating before drying, different brands may produce different results. Second, the measurement was taken in millimeters with rounding off to 0.5 mm. Better measurement techniques could have provided more accurate results. Finally, the investigation was carried out against a mix of bacteria. Many of the oral bacteria could not have grown on the culture medium used. Furthermore, the effect of green tea on other organisms such as fungus and viruses which play a role in many oral diseases could not be deduced. Although there remains no doubt about the antibacterial activity of the green tea against the oral bacteria, long-term clinical studies are required to establish the beneficial effect of green tea in oral cavity.


  Conclusion Top


The results of this study conclude that higher the concentration of green tea, greater was the inhibitory effect against the growth of oral bacteria. Furthermore, prolonging the brewing time may hamper the antibacterial activity of green tea.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

 
  References Top

1.
Rietveld A, Wiseman S. Antioxidant effects of tea: Evidence from human clinical trials. J Nutr 2003;133:3285S-92S.  Back to cited text no. 1
    
2.
Zuo Y, Chen H, Deng Y. Simultaneous determination of catechins, caffeine and gallic acids in green, Oolong, black and pu-erh teas using HPLC with a photodiode array detector. Talanta 2002;57:307-16.  Back to cited text no. 2
    
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Venkateswara B, Sirisha K, Chava VK. Green tea extract for periodontal health. J Indian Soc Periodontol 2011;15:18-22.  Back to cited text no. 3
[PUBMED]  Medknow Journal  
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Wu CD, Wei GX. Tea as a functional food for oral health. Nutrition 2002;18:443-4.  Back to cited text no. 4
    
5.
Manach C, Scalbert A, Morand C, Remesy C, Jimenez L. Polyphenols: Food sources and bioavailability. Am J Clin Nutr 2004;79:727-47.  Back to cited text no. 5
    
6.
Toda M, Okubo S, Ohnishi R, Shimamura T. Antibacterial and bactericidal activities of Japanese green tea. Nihon Saikingaku Zasshi 1989;44:669-72.  Back to cited text no. 6
    
7.
Elvin-Lewis M, Vitale MK, Opjas T. Anticariogenic potential of commercial teas. J Prosther Dent 1980;6:273-6.  Back to cited text no. 7
    
8.
Okamoto M, Sugimoto A, Leung KP, Nakayama K, Kamaguchi A, Maeda N. Inhibitory effect of green tea catechins on cysteine proteinases in Porphyromonas gingivalis. Oral Microbiol Immunol 2004;19:118-20.  Back to cited text no. 8
    
9.
Sakanaka S, Aizawa M, Kim M, Yamamoto T. Inhibitory effects of green tea polyphenols on growth and cellular adherence of an oral bacterium, Porphyromonas gingivalis. Biosci Biotechnol Biochem 1996;60:745-9.  Back to cited text no. 9
    
10.
Weber JM, Ruzindana-Umunyana A, Sicar S, Cowan J. Adenovirus infection is inhibited in vitro by green tea catechins. J Clin Virol 2003;28:S91.  Back to cited text no. 10
    
11.
Simpson A, Shaw L, Smith AJ. The bio-availability of fluoride from black tea. J Dent 2001;29:15-21.  Back to cited text no. 11
    
12.
Hirasawa M, Takada K, Makimura M, Otake S. Improvement of periodontal status by green tea catechin using a local delivery system: A clinical pilot study. J Periodontal Res 2002;37:433-8.  Back to cited text no. 12
    
13.
Sakanaka S, Sate T, Kim M, Yamamoto T. Inhibitory effects of green tea polyphenols on glucan synthesis and cellular adherence of cariogenic streptococci. Agric Biol Chem 1990;54:2925-9.  Back to cited text no. 13
    
14.
Sakanaka S, Kim M, Taniguchi M, Yamamoto T. Antibacterial substances in Japanese green tea extract against Streptococcus mutans, a cariogenic bacterium. Agric Biol Chem 1989;53:2307-11.  Back to cited text no. 14
    
15.
Kushiyama M, Shimazaki Y, Murakami M, Yamashita Y. Relationship between intake of green tea and periodontal disease. J Periodontol 2009;80:372-7.  Back to cited text no. 15
    


    Figures

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    Tables

  [Table 1], [Table 2]



 

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