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Table of Contents   
ORIGINAL RESEARCH  
Year : 2021  |  Volume : 32  |  Issue : 2  |  Page : 226-229
Green tea catechins showed antibacterial activity on streptococcus mutans –An in vitro study


1 Department of Periodontology, Maratha Mandal's NGH Institute of Dental Sciences and Research Centre, Belagavi, Karnataka, India
2 Department of Central Research Laboratory, Maratha Mandal's NGH Institute of Dental Sciences and Research Centre, Belagavi, Karnataka, India

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Date of Submission26-May-2021
Date of Decision27-Jul-2021
Date of Acceptance20-Aug-2021
Date of Web Publication22-Nov-2021
 

   Abstract 


Background: The main bacterial aetiological agents in caries formation are the α-haemolytic Streptococcal species Streptococcus mutans, which has been found to be the initiator of most dental caries. The leaves of Camellia sinensis known as green tea, has properties, such as antibacterial and anti-cariogenic. Epigallocatechin-3-gallate (EGCG) one of the most abundant catechins found in green tea is known to contribute to these effects. Aim: To evaluate the antibacterial effect of green tea catechins namely EGCG on S. mutans with two different methods at different concentrations. Objectives: 1) To assess the antimicrobial efficacy of EGCG by disc diffusion test at concentrations of 100, 75, and 50 μg/mL. 2) To assess the antimicrobial efficacy of EGCG by Minimum inhibitory concentration (MIC) test at concentrations ranging from 0.2 to 100 μg/mL. Methodology: Commercially available purest form of green tea polyphenol EGCG was used in the study. Disc diffusion test on agar medium and MIC test was used to determine the susceptibility of the S. mutans to green tea catechins EGCG. Results: The results of the agar well diffusion method showed that the EGCG extract has shown zones of inhibition against S. mutans at concentrations of 100 μg/mL (28.67 mm), 75 μg/mL (15.33 mm), 50 μg/mL (10.33 mm) while that of MIC test of EGCG extract of concentrations ranging from 0.2 to 100 μg/mL against S. mutans shows that the mean MIC value was 1.07. Conclusion: Catechins in the tea are potentially anti-cariogenic agents which can reduce bacterial presence in the oral cavity and have the potential to be further used for the preparation of dentifrice and mouthwash.

Keywords: Camellia sinensis, caries, dental decay, epigallocatechin-3-gallate, Streptococcus mutans

How to cite this article:
Hattarki SA, Bogar C, Bhat KG. Green tea catechins showed antibacterial activity on streptococcus mutans –An in vitro study. Indian J Dent Res 2021;32:226-9

How to cite this URL:
Hattarki SA, Bogar C, Bhat KG. Green tea catechins showed antibacterial activity on streptococcus mutans –An in vitro study. Indian J Dent Res [serial online] 2021 [cited 2021 Dec 8];32:226-9. Available from: https://www.ijdr.in/text.asp?2021/32/2/226/330856



   Introduction Top


Dental decay and periodontitis are perhaps the most common bacterial infections in dentistry. The data indicate that among the 200–300 species present in human dental plaque only a few may be considered as pathogens in caries and periodontitis, as there has been evidence implicating Streptococci mutans being responsible for a majority of human dental decay and periodontitis.[1] Among the organisms present in the dental plaque, S. mutans often gets the most attention in dental-related studies because it has been previously shown to favour attachment to tooth enamel[2],[3] as plaque formation occurs through various mechanisms on the tooth surface and the morphology of the tooth surface dictates the composition of the plaque ecosystem. Among the tooth colonisers, the primary etiologic agent for dental caries is the  Streptococcus mutans Scientific Name Search ns). Several studies have shown that high levels of S. mutans are at high risk for tooth decay and exacerbation of the periodontal disease.[1],[4]

Green tea is a popular drink around the world made from the infusion of dried Camellia sinensis leaves. Studies have shown that Camellia sinensis has numerous medicinal advantages, such as anti-bacterial, anti-spore, anti-cariogenic, and anti-viral properties (Nakayama et al., 2015; Steinmann et al., 2013). Green tea contains polyphenols which are composed of catechins. Epigallocatechin-3-gallate (EGCG) is the most abundant catechin and a major component that contributes to these effects. EGCG has a greater effect on gram-positive bacteria than gram-negative bacteria. The incorporation of EGCG at increasing concentrations has been shown to decrease bacterial numbers (Sakanaka et al., 2000). Hence, polyphenols have the ability to interfere with biofilm formation. The previous literature reported that green tea extracts have short-term anti-plaque antibacterial capabilities.[5] Therefore, in order to determine the EGCG's effect on S. mutans, Minimum inhibitory concentration (MIC) and disc diffusion tests were performed to observe the catechin effect on the growth the inhibition of S. mutans.


   Materials and Methods Top


Culturing and maintenance of bacterial cultures

A standard strain of S. mutans (ATCC no. 25175) was procured from CSIR-IMTech Chandigarh, India. The strain was then inoculated in the brain heart infusion (BHI) broth (HIMEDIA ©) and incubated at 37°C for 24–48 h and examined for growth. A 100 uL of BHI broth containing colonies was inoculated on BHI agar and incubated for 24–48 h. After 48 h, the BHI agar plate was examined for growth and pure colonies of S. mutans were picked up and subjected for further studies. Approval from the ethics committee is obtained taken in June 16, 2020.

Preparation of EGCG

The purest form of green tea polyphenol EGCG was purchased from Ambe NS Agro products private limited, Vasundhara, Ghaziabad, Utter Pradesh. The EGCG stocks were prepared by dissolving in dimethyl sulfoxide (DMSO) to stock concentrations of 100 mM. EGCG was then further diluted in phosphate buffered saline (PBS) to varying concentrations of 100, 75, 50, 25, and 10 μg/mL before using it on the S. mutans.[6]

Disc diffusion test

The disc diffusion test on the agar medium was used to determine the susceptibility of the S. mutans. In this procedure, the media used was the BHI agar. Pure colonies of S. mutans were transferred to the BHI broth to adjust the turbidity to equal that of 0.5 McFarland turbidity standard.[7] Within 15 min of adjusting the inoculum to a McFarland 0.5 turbidity standard, a dip of a sterile cotton swab into the inoculum was done and rotated against the wall of the tube above the liquid to remove excess inoculum. The entire surface of the agar plate was swabbed three times, rotating the plates approximately 60° between streaking to ensure even distribution. Hitting the sides of the Petri plate and creating aerosols was avoided. The inoculated plate was allowed to stand for at least 3 min but no longer than 15 min before making wells. A hollow tube of 5 mm diameter was heated and pressed above the inoculated agar plate and removed immediately by making a well into the plate. Dilutions of 100, 75, 50, 25 and 10 μg/mL in PBS were made from the EGCG/ciprofloxacin powder. Ciprofloxacin which is effective against gram-positive bacteria was used as a positive control in the experiment.

About 2.5 μL of these dilutions were added in each of these wells. Five concentrations of EGCG were used and compared with the positive control ciprofloxacin. These plates were then incubated within 15 min, at a temperature of 37°C for 18–24 h. The plates were read-only if the lawn of growth was confluent or near confluent. The diameter of the inhibition zone was measured to the nearest whole millimetre by holding the measuring device.

Minimum inhibitory concentration test

Nine dilutions of EGCG were done with the BHI broth in 10 microcentrifuge tubes. In the initial tube, 20 mL of EGCG was added into the 380 mL of BHI broth. For further dilutions, 200 mL of BHI broth was added into the next nine tubes separately. Then from the initial tube, 200 mL was transferred to the first tube containing 200 mL of BHI broth. This was considered as 10-1 dilution. From 10-1 diluted tube, 200 mL was transferred to the second tube to make 10-2 dilution. The serial dilution was repeated up to 10-9 dilution of EGCG. From the maintained stock cultures of S. mutans, 5 μL was taken and added into 2 mL of the BHI broth. In each serially diluted tube, 200 mL of the above culture suspension was added. The tubes were incubated for 24 h and observed for turbidity. The effect of the different concentrations of EGCG on the growth of S. mutans was examined and noted [Figure 1].
Figure 1: Shows epigallocatechin-3-gallate (EGCG) powder used on S. mutans at different concentrations on agar diffusion plates

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   Results Top


The antimicrobial activity of EGCG extract against S. mutans was studied by MIC and agar well diffusion methods.

Minimum inhibitory concentration

The results of the MIC test of EGCG extract of concentrations ranging from 0.2 to 100 μg/mL against S. mutans shows that the mean MIC value was 1.07 with a standard deviation of 0.46 while standard ciprofloxacin was 1.67 (mean MIC) and 0.58 (standard deviation) thereby indicating a close effect of EGCG on S. mutans when compared with the ciprofloxacin values as shown in [Table 1].
Table 1: Mean zones of inhibition and standard deviation obtained by agar well diffusion method on S.mutans when treated with EGCG and ciprofloxacin

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Agar well diffusion

The agar well diffusion method or well plate method was used in this study as it is the most commonly used method of antimicrobial activity for determination especially of newer substances like plant extracts and new drug formulations. This technique is a well-accepted way of comparing the antibacterial effect of different dental materials, medicaments, etc.[8] The results of agar well diffusion indicated as the zone of inhibition around the well containing the EGCG extract/ciprofloxacin was considered if the inhibition zone was greater than 6 mm (disc diameter). The experiments were performed in triplicates and the mean values of the zones of inhibition with ± standard deviations were calculated. For comparison between the zone of inhibition between the test material (EGCG) and standard (ciprofloxacin), the Mann–Whitney U test was applied. GraphPad prism 5.01 version was used for the analysis. The P value < 0.05 was taken as statistically significant.

[Table 2] shows the results of the agar well diffusion method. The EGCG extract has shown zones of inhibition against S. mutans at concentrations of 100 μg/mL (28.67 mm), 75 μg/mL (15.33 mm), 50 μg/mL (10.33 mm), whereas at concentrations of 25 and 10 μg/ml, the extract has not shown any activity against S. mutans. The comparison between the extract and ciprofloxacin has shown a significant difference with the P value of 0.0119.
Table 2: Mean MIC value (μg/mL) of EGCG against S. Mutans

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   Discussion Top


The cariogenic microorganisms such as Streptococcus mutans, Streptococcus sanguis, Streptococcus salivarius, Streptococcus mitis,  Streptococcus oralis Scientific Name Search Lactobacillus acidophilus present in the biofilm play a vital role in the aetiology of dental caries.[9] Streptococcus mutans being one of the major causative agents plays an important role in cariogenic biofilm formation.[10],[11] Medicinal plants have been used for therapeutic purposes for thousands of years and recent clinical studies have revealed physiological responses to tea extracts.

In our study, green tea polyphenol EGCG was used to determine its effectiveness as an antimicrobial agent against bacterial growth. Different concentrations of EGCG were used to study the effect on S. mutans using the MIC and disc diffusion methods. EGCG was able to inhibit the growth at a concentration of 50–100 μg/mL and was able to kill S. mutans at a concentration of 100 μg/mL.

Chlorhexidine (CHX) is considered the gold standard antimicrobial in dentistry[12] yet there have been few studies which have compared the antimicrobial effect of EGCG and CHX.[13],[14],[15],[16],[17] The result showed that both CHX and EGCG were effective in inhibiting microbial growth. In our study, the effect of EGCG showed inhibition against Streptococcus mutans. One more study compared the antimicrobial activity of EGCG with black, oolong and Pu-erh tea against S. mutans[18] which showed antimicrobial activity against Streptococcus mutans.

Different methods have been adopted and used to evaluate the effect of EGCG in different forms against the microorganisms and methods like counting colony-forming units, counting of microorganisms, and biofilm formation were used. In this study, we have used the disc diffusion method and MIC method to check the efficacy of EGCG.

There is one study investigating EGCG as a potential anti-cariogenic agent which showed that EGCG can inhibit the growth of S. mutans.[19] EGCG has been shown to interfere with the bacterial adhesion on the enamel thereby interfering and preventing the proliferation of S. mutans by suppressing the glucosyltransferase and amylase activity, thus, reducing the acid production in the dental plaque.[19],[20],[21] Dental caries can be prevented by proper tooth brushing, flossing and using different mouth rinses; though using a mouthwash plays a pivotal role in the prevention of dental caries because of its antibacterial properties. Mouthwashes such as chlorhexidine, sodium hypochlorite, cetylpyridinium chloride and amine fluoride are commonly used in practice though their potential side effects and disadvantages need to be considered. There is always a need for less toxic and irrigating agents to replace the chemical-containing substances. Many investigators have reported that catechins are inhibitory for S. mutans with the MIC ranging from 50 to 1,000 μg/mL.[20],[22] Xu et al.[23] reported that EGCG inhibited the growth of S. mutans at an MIC of 31.25 μg/mL and a minimum bactericidal concentration of 62.5 μg/mL. Our study showed MIC inhibition of S. mutans by EGCG extract of concentration ranging from 0.2 to 100 μg/mL with the mean MIC value of 1.07 μg/mL.


   Conclusion Top


Though there have been different methods used to evaluate the efficacy of EGCG against microorganisms, the results suggested that catechins is a stable substance and with antimicrobial properties capable to inhibit the S. mutans. This property can, therefore, be made use of in the preparation of mouthwashes or dentifrices for the prevention of dental caries and periodontal diseases.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
   References Top

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Correspondence Address:
Dr. Sanjeevini A Hattarki
Department of Periodontology, Maratha Mandal's NGH Institute of Dental Sciences and Research Centre Belagavi, Karnataka - 590 019
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ijdr.ijdr_512_21

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