The second type of extracellular enzyme responsible for metabolizing sugars is FTF, encoded by the ftf gene, which is responsible for fructan synthesis [ 12 , 13 ].

A biofilm is a community of microbial cells, which has been attached to a surface, enwrapped in a matrix of polysaccharide material [ 14 ].

The oral biofilms consist of various non-cariogenic and cariogenic bacteria embedded in an extracellular matrix composed of bacterial enzymes e. Currently, common preventive means to inhibit oral diseases include aggressive chemical agents, such as chlorhexidine and antibiotics, which have various undesired side effects, including tooth staining, mucosal erosion, taste disturbance and bacterial resistance [ 16 , 17 , 18 ].

Therefore, naturally occurring compounds, such as green tea polyphenols, have attracted much attention. Due to divers health benefits, there has been a significant increase in consumption of green tea among various cultures, making it one of the most popular beverage in the world [ 19 ].

Green tea, produced from Camellia sinensis C. sinensis leaves, has a high concentration of polyphenols, in particular catechins, which possess anti-oxidant properties.

The leaves of C. sinensis undergo minimal oxidation during processing and thus preserve their anti-oxidant and anti-bactericidal properties [ 17 ]. The major tea catechins include epigallocatechin 3-gallate EGCG , epigallocatechin EGC , epicatechin EC , epicatechin 3-gallate ECG , and catechin C [ 20 , 21 ].

These polyphenols were found to have anti-microbial traits and can inhibit a wide range of gram-positive and gram-negative bacteria in vitro [ 22 , 23 ]. EGCG has been shown to disrupt EPS and biofilm formation of S.

mutans , by suppressing gtfB , gtfC and gtfD genes [ 24 , 25 ]. Green tea polyphenols, especially EGCG, have the ability to interfere with quorum sensing QS , which is essential for biofilm formation by different bacteria [ 26 , 27 ]. Quorum sensing is considered a potential target of anti-microbial compounds.

One of the mechanisms of tea catechins to damage bacteria is binding to the bacterial cell membrane, which prevents the ability of the bacteria to bind to each other and to form biofilm [ 28 ]. In vivo studies have shown that green tea mouthwash has the ability to inhibit S.

mutans biofilm formation on tooth surface when given to dental population [ 29 , 30 , 31 ]. These studies raised the importance of green tea polyphenols as natural anti-microbial compounds, which can be safe for use and prevent dental diseases.

Since EGCG is a major polyphenol of tea extracts and EGCG tablets are provided as a natural supplement, we wanted to study the effect of this EGCG source on S.

mutans viability, EPS production and biofilm formation in vitro. It is also worthwhile to assay the membrane potential since it regulates metabolism, bacterial cell division, pH homeostasis, and membrane transport [ 32 ]. The aim of the present study was to examine the action mechanisms of EGCG on S.

mutans with specific emphasize on planktonic growth and biofilm formation. Here we show that EGCG has both growth inhibitory and anti-biofilm activities. The minimum biofilm inhibitory concentration MBIC was lower than the minimum growth inhibitory concentration MIC , suggesting a direct anti-biofilm effect.

Some mechanistic insights are presented. One EGCG tablet Source Naturals, Scotts Valley, CA, USA containing mg EGCG, was dissolved in 10 ml of DDW by a 1 h shaking at 4 °C. Then serial dilution was done in BHI to achieve final concentrations of 0.

Since the effective concentrations were in the range of 0. The working solutions were used fresh. Control bacteria received the same incubation conditions without EGCG see below. mutans UA from the stock of the Biofilm Research Laboratory, was grown as monospecies culture. Before each experiment, a frozen stock of S.

For planktonic growth, the overnight S. mutans cultures were diluted in BHI in the absence or presence of various concentrations of EGCG 0. The percentage of bacteria in planktonic phase was calculated by dividing the OD of treated samples by OD of control samples, multiplied by , after subtracting the background OD of an EGCG solution in BHI in the absence of bacteria.

For biofilm formation, the overnight S. In parallel, BHI without bacteria in the absence or presence of EGCG was used to measure any background signals caused by EGCG in the assays used see below. The setup for biofilm formation was as follows: µl of BHIS containing different concentrations of EGCG and 20 µl of S.

mutans were added to each well of a 96 flat-bottomed well tissue culture plate Corning, NY, USA. Each EGCG concentration was tested in triplicates.

The biofilm biomasses were quantified using the crystal violet assay as described [ 36 ], with slight modifications. The biofilms formed in the well tissue culture plates after treatment with EGCG were carefully washed twice with PBS to remove unbound bacteria and to obtain a clean biofilm.

After 20 min incubation at room temperature RT , the stained biofilms were washed twice with DDW and left to dry overnight at RT. After dissolving the stain, µl were transferred to a new well tissue culture plate and quantified spectrophotometrically by measuring the absorbance at nm using the M plate reader.

The percentage biofilm formation was calculated by dividing the OD of treated samples by OD of control samples, multiplied by To label the EPS in the biofilms, 1 µl of a 1 mM Alexa Fluor labeled Concanavalin A ConA conjugate solution Molecular Probes, Life Technologies, Carlsbad, California, USA was added to the samples during the incubation period with or without EGCG.

Live bacteria showed green fluorescence, while dead bacteria emitted red fluorescence. The stained biofilms were inspected under a Nikon Spinning Disk microscope Nikon Corporation, Tokyo, Japan connected to Yokogawa W1 Spinning Disk Yokogawa Electric Corporation, Tokyo, Japan [ 37 ].

Optical sections were acquired at spacing steps of 5 μm intervals from the surface through the depth of the biofilm. A three-dimensional image of the microbes and EPS distribution within the biofilms was constructed using the Nikon Imaging Software NIS- Elements.

The NIS elements software was used to quantify the fluorescence intensity in each biofilm layer. The assay was performed similarly as described [ 38 , 39 ]. Biofilms were allowed to form in 6-well tissue culture plates Corning.

Each sample consisted of µl of an overnight culture of S. After a 24 h incubation, 1 ml of Tri-Reagent Sigma-Aldrich, St. Louis, MO, USA was added to the washed biofilms to extract the total RNA from the biofilms. The biofilms were scraped into the Tri-Reagent solution with the help of a sterile cell scraper, and the fluid was transferred into 2 ml sterile screw tubes containing µl acid-washed glass beads followed by cell disruption in a Fast Prep Cell Disrupter Bio , Savant Instruments, Inc.

After centrifugation to remove the glass beads, the supernatant was transferred to a new Eppendorf tube and µl of chloroform Bio-Lab, Jerusalem, Israel were added to each sample, followed by vigorous vortex for 15 s.

After 15 min at RT, the samples were centrifuged at 13, rpm for 15 min at 4 °C, and the upper phase µl was transferred to a new tube. The samples were allowed to stand at RT for 30 min before centrifugation at 13, rpm for 30 min at 4 °C.

The dried RNA was resuspended in ultrapure water UPW. The purity and concentration of the RNA were determined using a Nanodrop ND Instrument Wilmington, DE, USA. PCR conditions included an initial heating at 50 °C for 2 min, an activation step at 95 °C for 10 min, followed by 40 cycles of amplification 95 °C for 15 s, 60 °C for 1 min.

Gene expression was expressed in relative values, setting the expression level of the control samples to one. The membrane potential of untreated and EGCG-treated planktonic S.

DiOC 2 3 exhibits green fluorescence in all bacterial cells, but the fluorescence shifts toward red emission at higher membrane potential values. Briefly, an overnight culture of S.

mutans was resuspended in PBS to an OD of 0. The bacterial suspension was divided into 1 ml aliquots with different concentrations of EGCG and stained with 10 µl of 3 mM DiOC 2 3 for 30 min in the dark. In addition, we measured the forward scatter FSC and the side scatter SSC of the untreated and EGCG-treated bacteria.

FSC detects light scatter along the path of the laser and its intensity is proportional to the diameter of the cell.

SSC measures light scatter at a ninety-degree angle relative to the laser and provides information about the granularity of the cell. Untreated and EGCG-treated planktonic S.

Thereafter, the samples were coated with iridium and visualized using a Magellan L High Resolution Scanning Electron Microscope HR-SEM at 10,×—50,× magnifications [ 33 ]. We first studied the effect of EGCG on the planktonic growth of S. mutans and observed that EGCG reduced the planktonic growth of S.

mutans in a dose-dependent manner, with a significant growth inhibition at an EGCG concentration of 1. Therefore, the MIC 50 and MIC 80 of EGCG was 1.

The effect of epigallocatechin gallate EGCG on planktonic growth of S. Planktonic growth of S. mutans treated with different concentrations of EGCG for 24 h as measured by optical density OD at nm.

We next studied the effect of EGCG on biofilm formation. To this end, S. mutans was incubated for 24 h with increasing concentrations of EGCG, and the biofilm biomass was quantified by CV staining. It is evident from Fig.

mutans in a dose-dependent manner, starting from 1. Therefore, the MBIC 50 and MBIC 95 of EGCG was 1. The effect of epigallocatechin gallate EGCG on biofilm biomass of S. Biofilm biomass of S.

mutans after a 24 h incubation with different concentrations of EGCG, as determined by crystal violet CV staining measured at an optical density OD of nm. The reconstructed CSLM 3D images show that EGCG reduced the number of live bacteria and the amount of EPS in a dose-dependent manner Fig.

An increase in dead bacteria was observed in the samples treated with 1. Epigallocatechin gallate EGCG reduced the number of live bacteria and the amount of exopolysaccharides EPS in a dose-dependent manner.

Computerized 3D reconstruction of the biofilm layers after treating S. mutans with different concentrations of EGCG for 24 h, as recorded by CLSM and generated by the Nikon Imaging Software NIS-elements.

a Control. A representative sample of each treatment is shown. Green color represents live cells, red color represents dead cells and blue color represents the EPS.

The three fluorescence intensities in the different layers of the images in Fig. According to Fig. Only remnants of live bacteria were seen in the samples treated with 2. We observed a higher PI staining of dead bacteria in the 1. There was no significant EPS signal in the 2.

Quantification of the fluorescence intensity of each biofilm layer after treating S. mutans with increasing concentrations of EGCG for 24 h.

A Live bacteria stained by SYTO 9. B Dead bacteria stained by PI. C Exopolysaccharides EPS production stained by Alexa Fluor - conjugated ConA. The anti-biofilm effect of EGCG was further demonstrated by determining the amount of DNA in the resulting biofilms.

The DNA content of S. mutans biofilm formed after treatment with EGCG was found to be significantly decreased at concentrations of 2. The effect of epigallocatechin gallate EGCG on the DNA content of S. mutans biofilm. Quantitative polymerase chain reaction qPCR analysis of the DNA content in S.

mutans biofilms after treatment with different concentrations of EGCG for 24 h. Since we observed that EGCG reduced the biofilm formation of S. mutans , we questioned whether this compound affects gene expression of biofilm-related genes.

Biofilms of S. mutans were treated with a sub-inhibitory concentration of EGCG 0. Our data Fig. On the other hand, there was a significant upregulation of the virulence gene spaP and of the stress response heat-shock protein genes groEL and dnaK 1.

The effect of epigallocatechin gallate EGCG on gene expression in S. Real-time PCR analysis of various genes involved in biofilm formation and oxidative stress after a 24 h treatment of S. mutans with EGCG 0.

The relative expression levels of the genes analysed by real-time PCR was normalized against 16S rRNA and 23S rRNA that served as internal standards. Since the membrane potential affects many of the bacterial functions [ 32 ], it was prompting to analyze the effect of EGCG on this parameter.

The membrane potential of planktonic S. mutans was measured 30 min and 2 h after exposure to EGCG using the DiOC 2 3 reagent on flow cytometry. The green fluorescence is an indication for the amount of dye taken up by the bacteria, while the red fluorescence is increased upon higher membrane potential.

The latter observation suggests that EGCG induces hyperpolarization of the membrane potential. There was no significant difference between the two different incubation times, suggesting that the immediate hyperpolarization of the membrane by EGCG was maintained over time.

Notably, EGCG caused a significant shift in the side scatter SSC on flow cytometry Fig. The increase in SSC suggests that EGCG leads to an irregular structure of the bacteria.

The effect of epigallocatechin gallate EGCG on membrane potential of planktonic S. DiOC 2 3 staining of S. mutans treated with 1.

Red fluorescence 30 min. B Red fluorescence 2 h. C Green fluorescence 30 min. D Green fluorescence 2 h. E Forward scatter area FSC-A 30 min. F FSC-A 2 h. G Side scatter area SSC-A 30 min. H SSC-A 2 h. Similar results were obtained with 2.

We, therefore, performed HR-SEM imaging on planktonic S. mutans incubated in the absence control or presence of EGCG for 2 h. The EGCG-treated bacteria appeared with nano-scale dotted structures on their surfaces, in contrast to the smooth surface of control bacteria Fig. It could be that these are EGCG-induced protein precipitated aggregates.

High resolution scanning electron microscope HR-SEM images of epigallocatechin gallate EGCG -treated planktonic S. Planktonic S. mutans was exposed to different concentrations of EGCG for 2 h, fixed and processed for HR-SEM imaging.

A , D Control. A — C x10, magnification. D—F x50, magnification. Oral biofilm is associated with a variety of oral diseases, inflicting the dental population by inducing caries, gingivitis, and periodontal diseases. Conventional treatment and prevention of oral diseases include chemical agents such as chlorhexidine and antibiotics, which have various undesired side effects such as teeth discoloration and bacterial resistance [ 16 , 17 , 18 ].

Recently, natural compounds have been proposed as novel treatment options for oral diseases, in an effort to avoid side effects derived from common drug delivery means [ 29 , 41 ]. Green tea, which is one of the most popular beverages in the world, contains high concentrations of anti-oxidants, one of them is EGCG.

Taylor et al. However, no significant difference in clinical parameters was found between the treatments of SRP plus EGCG and SRP alone at 3-month follow-up. During the next 3 months, the PD and CAL continued to be improved in the SRP plus EGCG group, while the scenario was different in sites treated SRP alone that these two clinical parameters tended to rebound, thus presenting a significant difference of PD reduction as 0.

Meanwhile, the clinical significance of two treatment strategies was tested with the 2 mm threshold, which was regarded as a clinical level to monitor the disease progression and evaluate the treatment success, and also avoid examination bias of the periodontal probe with 1 mm increment [ 9 ].

Within sites treated with SRP alone in this study, the longitude tendency of PD and CAL was to rebound, which was also observed in previous studies related to mechanical treatment on chronic periodontitis [ 30 ], while the efficacy was maintained with an additional EGCG medication.

Among previous studies, only Rattanasuwan et al. Although both sustained-release medication and the new-type scaler tip could deliver drugs to the bottom of deep pockets, even the longer acting duration within the former, the combined effect of EGCG and ultrasonic cavitation might be one of the reasons on clinical difference between the study above and ours.

However, the analysis of the red complex and P. forsythia showed a significant difference favored the combined EGCG medication. Hattarki et al. forsythia at 5-week and P.

gingivalis at 1-week. The purified EGCG exhibited a potent anti-microbial effect on P. gingivalis biofilms in vitro, while no additional significant difference was found in our in vivo study, which might be ascribed to the relatively shorter exposure time of EGCG solution in the subgingival environment [ 13 ].

However, through repeated medication at 3-month follow-up, an additional microbial effect on T. forsythia was found at month-6, which was also observed in the previous study [ 17 ].

The antimicrobial effect of purified EGCG in vivo needs more clinicomicrobiological studies to verify in the future. In the present study, the additional PD reduction mainly ascribed to the gain of CAL, which might indicate that the anti-inflammatory effects of EGCG on periodontal tissues and the new-type scaler ejecting the drug towards the bottom of deep pockets could contribute the formation of epithelial attachment [ 14 , 15 ].

This study focuses on the long-term efficacy of purified EGCG delivered by the new-type scaler tip, while its effect within 3 months also deserves to be investigated. Furthermore, as another liquid drug, chlorhexidine could exert antibacterial effect and has been applied in periodontal treatments for decades, which also has the potential to be delivered by the new-type scaler tip.

Based on our primary results and previous studies, multicenter trial with large sample size should be performed to verify the efficacy of purified EGCG or green tea catechins as periodontal medication and compare with other drugs such as chlorhexidine in the future.

For the rational comparison within similar subjects, this study recruited patients with generalized chronic periodontitis which was consistent with previous studies [ 16 , 17 , 18 ]. As the new international classification of periodontal diseases has been proposed in [ 31 ], 18 patients into the final analysis could be diagnosed as generalized periodontitis stage II or III, grade B based on their baseline examination, which could provide a reference for future studies using this new classification system.

The purified EGCG showed the potential to improve outcome of non-surgical periodontal treatment and the new-type scaler tip provided an alternative vehicle for subgingival medication.

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J Periodontol. Lindhe J, Nyman S. Long-term maintenance of patients treated for advanced periodontal disease. Sherman PR, Hutchens LH, Jewson LG, Moriarty JM, Greco GW, Mcfall WT. The effectiveness of subgingival scaling and root planning.

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Local anti-infective therapy: pharmacological agents: a systematic review. Ann Periodontol. Killoy WJ. The clinical significance of local chemotherapies. PubMed Google Scholar. Bonito AJ, Lux L, Lohr KN. Impact of local adjuncts to scaling and root planing in periodontal disease therapy: a systematic review.

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. 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.

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Thermo-reversible green tea catechin gel for local application in chronic periodontitis: a 4-week clinical trial. Hattarki SA, Pushpa SP, Bhat K. Evaluation of the efficacy of green tea catechins as an adjunct to scaling and root planing in the management of chronic periodontitis using PCR analysis: a clinical and microbiological study.

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Reynolds MA, Lavigne CK, Minah GE, Suzuki JB. Clinical effects of simultaneous ultrasonic scaling and subgingival irrigation with chlorhexidine.

Mediating influence of periodontal probing depth. Taggart JA, Palmer RM, Wilson RF. A clinical and microbiological comparison of the effects of water and 0. Guarnelli ME, Franceschetti G, Manfrini R, Trombelli L.

Adjunctive effect of chlorhexidine in ultrasonic instrumentation of aggressive periodontitis patients: a pilot study. Armitage GC. Development of a classification system for periodontal diseases and conditions. Liu G, Luan Q, Chen F, Chen Z, Zhang Q, Yu X. Shift in the subgingival microbiome following scaling and root planing in generalized aggressive periodontitis.

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Papapanou PN, Sanz M, Buduneli N, Dietrich T, Feres M, Fine DH, Flemmig TF, Garcia R, Giannobile WV, Graziani F, et al. The most important compounds of green tea are the polyphenols. Polyphenols exist in many plants such as fruits, vegetables, teas and cocoa.

Flavonoids are a major group of polyphenols. The main flavonoids in green tea are catechins flavoaols , such as epicatechin, epigallocatechin EGC , epicatechin gallate ECG , epigallocatechin gallate EGCG , gallocatechin GC , gallocatechin gallate GCG , catechin, and catechin gallate CG.

Besides catechins, apigenin, apigenin- 7-O-glucoside Api-G , myricetin, kaempferol, and vitechin are reported as green tea flavonoids [4]. This catechin has a strong potential against carcinogenesis, angiogenesis, and tumor metastasis [3]. However, Scholl et al. reported that green tea polyphenols can have various beneficial or adverse health effects depending on the plasma levels of catechin [5].

The keywords used for this review were as follows: Green Tea Extract, Polyphenol, Oral Health, Preventive Dentistry. These keywords were searched in PubMed and among the papers found, 39 English papers from to which were most related to the subject were selected and reviewed.

Green tea catechins have a bitter taste. They are water soluble and their biological activities affect cell membrane functions such as signaling, cell cycle, and mitochondrial activity.

Catechins have inhibitory effect against S. mutans and S. Antibacterial effects of green tea against mutans streptococcus is reported in previous studies. Rasheed et al. indicated the bactericidal effect of catechins against Escherichia coli, Streptococcus salivarius and Streptococcus mutans [6].

It is suggested that EGCG damages the cytoplasmic membrane of the bacteriae by generation of hydrogen peroxide [7]. The antibacterial property of Camellia Sinensis extract against Streptococcus mutans and Lactobacillus acidophilus is also reported by Anita et al.

Tannin and catechins of green tea are able to inhibit enzymatic activity of amylase which is responsible for caries incidence by hydrolysis of starch in foods to lower molecular weight carbohydrates [10].

Tea catechins also prevent the attachment of oral streptococci to tooth surfaces and inhibit streptococcal glucosyl transferase. EGCG in specific concentration and application interval, can prevent acid production by cariogenic bacteria via inhibition of lactate dehydrogenase LDH , and increases the minimum pH of the oral cavity from 4.

LDH converts pyruvic acid to lactic acid. Although fluoride existing in green tea is a useful component for tooth caries resistance, it is suggested that the main component responsible for anti-caries properties of green tea are polyphenols and tannins [12].

Daneshyar et al. suggested green tea varnish to prevent root surface caries [13]. In a recent human study, the antimicrobial effects of green tea against Streptococcus mutans, Lactobacilli spp. and Candida albicans was compared with the gold standard antibacterial material, chlorhexidine CHX.

It was concluded that green tea was more effective than CHX for inhibition of Streptococcus mutans and less effective about Lactobacilli spp. Neither CHX nor green tea were sufficiently effective against Candida albicans. The authors suggested green tea as a costeffective material for caries prevention [14].

Green tea catechins have also been studied for their effects on periodontal status. Due to the wide range of antibacterial effects of green tea against gram positive and gram-negative microorganisms, it is suggested as a useful antiplaque agent. Catechins keep the salivary and plaque pH at about neutral, so they prevent the colony growth and activity of streptococcus mutans.

EGCG may inhibit the activity of matrix metalloproteinase-9 MMP-9 which helps the formation of osteoclasts in periodontal disease, and therefore prevents alveolar bone resorption [15]. Kaur et al. compared the antiplaque effect of green tea catechin mouthwash on patients and concluded that 7 day application of this mouthwash had comparable anti-plaque efficacy with chlorhexidine, and moreover, it did not have the bitter taste and side effects of CHX, including tooth discoloration and supra-gingival calculus formation related its long-term use [16].

Lagha et al. reported the efficacy of green tea catechins to protect the gingival epithelium against invasion by Porphyromonas gingivalis, so they have a promising effect on prevention from periodontal disease [17].

MMPs in dentin and saliva are responsible for degradation of the organic matrix of dentin. They activate when the oral cavity pH drops by the acids produced during the cariogenic challenge.

MMPs help the progression of dentin caries. MMPs responsible for the organic matrix degradation of dentin are MMPs 2, 8 and 9 [18,19]. Using materials that inhibit MMPs, such as CHX, can be helpful for caries prevention.

The proposed mechanism of action for MMP inhibitors is maintenance of the demineralized organic matrix on dentin surface [20]. EGCG extract in green tea is reported as an MMP inhibitor [21,22]. Kato et al. studied the effect of green tea on dentin erosion and abrasion.

They observed the protective effect of green tea. They also reported, in contrary to previous studies, that a delay of 30 minutes for tooth brushing after an erosive challenge did not reduce the amount of tooth wear, and it was the same as brushing immediately after erosion [23].

Barbosa et al. reported the effectiveness of supplementation of soft drinks with green tea extract on their reduced erosive potential. They suggested green tea as a natural supplement that does not any side effects or negative effects on taste of the drink [20]. Green tea polyphenols are antioxidant agents and free radical scavengers.

One of the major side effects of bleaching is impairment of the immediate bond strength of composite resin to the bleached tooth, due to the oxygen molecules remained in tooth structure [24].

Postponing the adhesive restorative treatment for at least one week is the most acceptable method for restoring the bond strength [25]. Flavonols of green tea leaves, especially EGCG, have antioxidant property [28,29].

Polyphenols prevent formation of free radicals, and neutralize the existing free radicals by exchanging electrons, via their trihydroxy and dihydroxy groups of B ring [30]. Khamverdi et al. suggested the application of EGCG as an antioxidant agent for reversal of the decreased bond strength to bleached enamel.

They tested different concentrations and application times of EGCG and concluded that green tea catechins can be used for removal of free radicals from tooth structure, instead of two weeks delay between bleaching and adhesive restoration [30]. Berger et al. also confirmed green tea as an alternative antioxidant for adhesive restorations after bleaching [31].

However, Sharafeddin et al. did not report any improvement in bond strength of bleached teeth by application of green tea and some other natural materials [26]. evaluated the effect of EGCG on bond strength and bond durability of self-etch adhesives.