|Year : 2013 | Volume
| Issue : 3 | Page : 120-124
An in vitro comparative antibacterial study of different concentrations of green tea extracts and 2% chlorhexidine on Enterococcus faecalis
Leena P Martina, Ambrose Vedamanickam Rajesh Ebenezar, Mohamed Fayas Ghani, Ashwin Narayanan, Meenakshi Sundaram, Ajit George Mohan
Department of Conservative Dentistry and Endodontics, Sri Ramaswamy Memorial Dental College, SRM University, Chennai, Tamil Nadu, India
|Date of Web Publication||20-Nov-2013|
Leena P Martina
B 13, 5th floor, Brown Stone Apts, Mahalingapuram, Chennai 600 034, Tamil Nadu
Source of Support: None, Conflict of Interest: None
Aim : The purpose of this study was to comparatively evaluate the antibacterial property of various concentrations of green tea extracts and 2% chlorhexidine (CHX) against Enterococcus faecalis 0(E. faecalis) in vitro. Materials and Methods: The activity of green tea extract and CHX on E. faecalis was measured on agar plates using the agar diffusion method. In addition, the tube dilution method was used to determine the minimum inhibitory concentration (MIC) and minimum bactericidal concentration of green tea extract against E. faecalis. Results: The MIC of green tea extract was found to be 3.5%. The zone of inhibition was found to be concentration dependent. The 2.5% concentration of green tea extract showed 20 mm zone of inhibition, while the 3% concentration demonstrated a 30 mm zone of inhibition. Two percent CHX showed 30 mm zone of inhibition. Conclusion: Three percent concentration of green tea extract showed an antibacterial activity equivalent to 2% CHX against E. faecalis.
Keywords: Catechins, chlorhexidine, E. faecalis, green tea extract
|How to cite this article:|
Martina LP, Ebenezar AV, Ghani MF, Narayanan A, Sundaram M, Mohan AG. An in vitro comparative antibacterial study of different concentrations of green tea extracts and 2% chlorhexidine on Enterococcus faecalis. Saudi Endod J 2013;3:120-4
|How to cite this URL:|
Martina LP, Ebenezar AV, Ghani MF, Narayanan A, Sundaram M, Mohan AG. An in vitro comparative antibacterial study of different concentrations of green tea extracts and 2% chlorhexidine on Enterococcus faecalis. Saudi Endod J [serial online] 2013 [cited 2019 Sep 15];3:120-4. Available from: http://www.saudiendodj.com/text.asp?2013/3/3/120/121504
| Introduction|| |
Microorganisms are the major causative factors associated with endodontic treatment failures and hence it is important to find methods and materials to predictably eradicate the root canal infection. Adequate cleaning and shaping in conjunction with effective irrigation protocol is an absolute necessity in eliminating microorganisms from the complex root canal system.  Various irrigating solutions have been used during root canal preparation to remove debris and necrotic pulp tissue and to eliminate microorganisms that cannot be reached by mechanical instrumentation.  Clinical studies have demonstrated that chemomechanical preparations and use of antimicrobial medicaments are effective in reducing the bacterial load in root canal systems. 
Chlorhexidine (CHX) is one of the most widely used root canal irrigant and is a bactericidal agent. Its interaction and uptake by bacteria were studied initially by Hugo and Longworth, who found that uptake of CHX by Escherichia More Details coli and Staphylococcus aureus was very rapid and dependent on CHX concentration and pH.  Its property of substantivity enables it to prevent bacterial colonization within the root canal system and hence is used as an intracanal irrigant.  Oncag et al., evaluated the antibacterial properties of 5.25% sodium hypochlorite, 2% CHX, and 0.2% CHX plus 0.2% cetrimide (cetrixidin) after 5 min and 48 h on extracted tooth infected by E. faecalis and found that 2% CHX and cetrixidin were significantly more effective against E. faecalis. 
E. faecalis is found to be the most prevalent microorganism in root canals with therapy-resistant lesions.  This microorganism can even survive in an environment with scant available nutrients and in which commensality with other bacteria is minimal. Its mode of growth is through the formation of a biofilm, an adaptive process that enables the microorganism to endure in severely harsh conditions like obturated root canals.  It can proficiently invade dentinal tubules and is therefore probable that cells within dentinal tubules surviving chemomechanical instrumentation and intracanal medication could colonize the tubules and reinfect the obturated root canal. 
Many natural foods present antibiotic functions that can reduce or limit the growth of bacteria in the human body. Among those natural foods, green tea has been valued for its antimicrobial property for thousands of years in Asian cultures.  Green tea extract is obtained from the leaves of the plant Camellia sinensis which are rich in flavonoids in the form of catechins. Catechins are reported to exert antibacterial activity against many pathogens like E. coli, Streptococcus mutans, Shigella dysenteriae, Vibrio cholera, etc. 
Green tea extracts have shown to completely eradicate E. faecalis in 6 min. Catechins present in green tea extracts have excellent antioxidant, anti-inflammatory, and radical scavenging ability. These properties along with ease of availability, economical, longer shelf-life, little or minimal toxicity, and lack of microbial resistance makes it an excellent alternative to the currently available endodontic irrigants. 
Hence this study was undertaken to evaluate the antibacterial property of various concentrations of green tea extract and 2% CHX on E. faecalis.
| Materials and Methods|| |
Preparation of the green tea extract dilutions
Green tea extract was supplied by Tetrahedron Beverages Pvt. Ltd., Kakkalur, Tamil Nadu, India. It was supplied in the form of powder which is readily soluble in water. Green tea extract dilutions were prepared in the concentrations ranging from 0.5 to 6% with difference of 0.5 serial dilutions by weighing the required amount of powder necessary over an electronic weighing balance and mixing it with 100 ml of sterile boiling distilled water for 5 min and filter sterilized. The dilutions were kept for sterility check overnight at 37°C. For the agar diffusion method 1-3% concentrations were used.
Agar diffusion test
Pure strain of E. faecalis from American Type Culture Collection (ATCC #24212, Institute of Basic medical sciences, Chennai, India) subcultured in Brain Heart Infusion (BHI) culture media (Institute of Basic medical sciences, Chennai, India) and under gaseous conditions to confirm their purity. Meuller-Hinton agar was prepared on sterile Petri dish More Detailses and kept for sterility check at 37°C for 24 h. After sterility check, the inoculae of ATCC 24212 strains were used to make lawn culture on Meuller-Hinton agar plates (Institute of Basic Medical Sciences, Chennai, India). A total of 6 wells were punched in the plate using a sterile punch and the test materials (1-3%) of green tea extract solutions and 2% CHX solution (Asep-RC, Steadman Pharmaceuticals Pvt. Ltd., Thiruporur, Tamil Nadu) were placed in the well using a sterile micropipette, and the plates were incubated at 37°C for 24 h. The diameter of the zone of inhibition was recorded in millimeters, using an inhibition zone measuring scale. The plates were duplicated in all the experiments. The results were statistically analyzed using one-way analysis of variance (ANOVA) and Student's t-test.
Minimum inhibitory concentration
E. faecalis bacterial strain was grown in BHI broth (Institute of Basic Medical Sciences, Chennai, India) for 24 h at 37°C. Serial dilution of the green tea extract was performed as follows: 100 μl of each dilution was added to 100 μl of Mueller-Hinton broth. To this broth, 5 μl of bacterial suspension which was adjusted to 0.5 McFarland was added and this mixture was incubated at 37°C for 24 h. After 24 h the tubes were visually checked for turbidity (bacterial growth). The lowest dilution inhibiting the growth was taken as MIC. A loopful of the broth dilutions were taken and streaked on Meuller-Hinton agar plates. The growth of bacteria was checked after incubation for 24 h at 37°C. The lowest dilution which showed no growth of bacteria was taken as minimum bactericidal concentration (MBC).
Bacterial suspension added to Meuller-Hinton broth without the addition of green tea extract served as positive control and distilled water was taken as the negative control.
| Results|| |
Agar diffusion method
The mean values of the zone of inhibition for green tea extract and CHX. The zone of inhibition for 1 and 1.5% green tea extract was 9 mm, 2.5% was 12 mm, 2.5% was 20 mm, and that for 3% was 30 mm. The zone of inhibition for 2% CHX was 30 mm. From the results it is clear that 3% green tea extract has got the same zone of inhibition as that of 2% CHX.
Minimum inhibitory concentration
Green tea extract serial dilutions of 0.5-6% were tested. It was found that from 3.5 to 6% there was no growth of the organism and 0.5-3% showed growth of organisms on agar plates. Positive control showed growth of the organism and there was no growth seen in negative control.
| Discussion|| |
Enterococcus faecalis (E. faecalis), facultative anaerobic gram positive cocci highly prevalent in cases with post treatment disease associated with virulence factors (aggregation substance, enterococcal surface proteins, gelatinase, cytolysin toxin, extracellular superoxide production, capsular polysaccharides, and antibiotic resistance determinant) can facilitate the adherence of host cells and extracellular matrix, tissue invasions, immunomodulation effect, and cause toxin mediated damage.  Several studies have demonstrated its resistance to calcium hydroxide, the most common intracanal medicament used in endodontic treatment.  This has drawn attention to the use of other medicaments like CHX.
Catechins present in green tea possess inhibitory action for S. mutans and S. sobrinus, with MICs ranging between 50 and 1,000 μg/ml. It prevents the attachment of oral streptococcal pathogens to surfaces. The catechins also inhibit streptococcal glucosyl transferase, and is an effective inhibitor of the enzyme thereby inhibiting the carious process. Hence, green tea was selected for this study. 
The agar diffusion method was used in this study as a preliminary assessment as it is one of the most often used methods for antimicrobial activity assessment. This method is accepted as an adequate way of comparing the antibacterial effect of different dental materials, medicaments, and root canal irrigants. The diffusion capacity of materials in agar are dependent on several factors like contact between material and agar, molecular weight, concentration of test material, and agar gel viscosity.  Furthermore, control and standardization of inoculation density, evaluation of results, selection of agar medium, incubation temperature of plates, and reading point of inhibition haloes are also restricting factors affecting the dynamics and variability of diffusion tests in an agar medium.  In this study using agar diffusion method, a zone of inhibition of 30 mm was seen for 3% green tea extract dilution, 20 mm for 2.5% green tea extract dilution, and 30 mm for 2% CHX.
CHX is a synthetic cationic bisguanide that consists of two symmetric 4-chlorophenyl rings and two bisguanide groups, connected by a central hexamethylene chain.  According to the results of this study, the antibacterial property of 2% CHX could be because, CHX a positively charged hydrophobic and lipophilic molecule interacts with the negatively charged phospholipids and lipopolysaccharides on the cell membrane of bacteria, thereby altering the cells osmotic equilibrium. This increases the permeability of the cell wall, which allows the CHX molecule to penetrate into the bacteria. The results of this study are in accordance with the previous study done by Basson and Tait  who compared the ex vivo effectiveness of calcium hydroxide, iodine potassium iodide (IKI), and a CHX solution in disinfecting root canal systems that were infected with A. israelli and found that CHX was the only disinfectant that was able to eliminate A. israelli.  CHX at low concentrations (0.2%) causes the leakage of potassium and phosphorous out of the cell and act as a bacteristatic agent but at higher concentration (2%) CHX is bactericidal as precipitation of the cytoplasmic contents occurs, which results in cell death  and confirms with the results of this study.
Though CHX is being considered as one of the ideal intracanal irrigants available today, it has its own drawbacks like tissue irritation and the reactive oxygen species (ROS) formation.  In order to overcome these drawbacks and to obtain an irrigant with antibacterial property equivalent to CHX, green tea extract was chosen for this study to evaluate its efficacy against E. faecalis as green tea is a common beverage being consumed and can be safely tried as a root canal irrigant.
The main varieties of tea are green, black, Oolong, and Puerh tea; the difference being in their method of processing.  Green tea is made from unfermented leaves and contains the highest concentration of polyphenols. Polyphenols are present in tea in the form of catechins. The antibacterial property of green tea in this present study was obtained at the concentration of 2.5 and 3% which was comparable to that of CHX. This antibacterial property may be attributed to the catechins present in green tea. 
Green tea contains catechin compounds like: Catechin, epicatechin, epigallocatechin, epicatechin gallate, and epigallocatechin gallate (also known as EGCG). EGCG is found to be the most active component in green tea.  These catechins are water soluble and have a bitter taste. Catechins have wide biological activities which affect membrane dependent cellular processes such as signaling, cell cycle, arachidonic acid metabolism, and mitochondrial functionality. Bacterial strains are susceptible to tea extracts due to differences in cell wall components. Bacterial cell membrane has a lipid bilayer structure. EGCG binds to the lipid bilayer and causes aggregation of lipid vesicles and leaks the contents from a suspension of vesicles.  It causes membrane expansion leading to membrane thinning, loss of cell structure, and finally death.  It also interferes with DNA replication process by inhibiting bacterial DNA gyrase enzyme. Molecular docking calculations suggest that the benzopyran ring of EGCG penetrates deep into the active site and the galloyl moiety anchors it to the cleft through interactions with its hydroxyl groups. This explains the higher activity of catechins present in green tea extracts. 
MIC is the lowest concentration of an antimicrobial that will inhibit the visible growth of a microorganism after overnight incubation and are important in diagnostic laboratories to confirm resistance of microorganisms to an antimicrobial agent and also to monitor the activity of a new antimicrobial agent.  MIC was determined for green tea extract alone as the bactericidal concentration for CHX is already known. According to our results, 3.5% of green tea extract was the minimum concentration that inhibited the growth of E. faecalis. According to the results of this study, 3.5% green tea extract has shown to have antibacterial action.
Though the mechanism of action of catechins is still obscure, structure-activity relationship studies indicated that their antibacterial activities were predominantly related to gallic acid moiety and the number of hydroxyl groups.  Hence, the results obtained in this study regarding the antibacterial action of green tea extract seem to be very promising and it can be considered to be used as an alternative to other root canal irrigants.
| Conclusion|| |
Within the limitations of this study, it can be concluded that green tea extract did show antibacterial activity against E. faecalis which was similar to that of the 2% CHX. Further research needs to be done on its contact time, surface tension, and pH to use it as a root canal irrigant.
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