ORIGINAL Ardakani ARTICLE et al
Antibacterial Effect of Iranian Green-Tea-containing Mouthrinse vs Chlorhexidine 0.2%: An In Vitro Study Mohammad Reza Talebi Ardakania/Shima Golmohammadib/Sara Ayremlouc/ Soudabeh Taherid/Sedighe Daneshvare/Mansour Meimandif Purpose: Considering the antioxidant, anti-inflammatory and antimicrobial properties of green tea, this study aimed to evaluate the antibacterial effect of mouthrinses containing green tea extract vs 0.2% chlorhexidine on selected microorganisms in vitro. Materials and Methods: The antibacterial activity of both mouthrinses and the pure green tea extract was assessed by using disk diffusion and the minimal inhibitory concentration (MIC) methods against five microorganisms: Streptococcus mutans, Streptococcus sanguis, Enterococcus faecalis, Pseudomonas aerogenosa and Escherichia coli. Growth inhibition zones were measured in mm after 24 h of incubation at 37°C. The two mouthrinses were assessed at concentrations of 1, 2, 4, 8, 16, 32, 64, 128, 256 and 512 mg/ml to determine the MIC, which was interpreted as the lowest concentration of the agent that completely inhibited the growth of the test species. Results: 0.2% chlorhexidine produced a larger zone of growth inhibition than did the mouthrinse made of green tea extract (P < 0.01). Paradoxically, the growth inhibition zones of the tested bacteria were significantly larger in pure extract of green tea than in 0.2% chlorhexidine (P < 0.01). The chlorhexidine mouthrinse inhibited the growth of all tested species and exhibited significantly lower MICs than did the green tea mouthrinse (P < 0.01). Conclusions: Even though the mouthrinse made with green tea extract presented an in vitro antimicrobial activity inferior to 0.2% chlorhexidine, the pure extract had considerable bactericidal effect. Key words: antibacterial effect, 0.2% chlorhexidine, green tea, mouthrinse Oral Health Prev Dent 2014;2:157-162
Submitted for publication: 03.05.12; accepted for publication: 22.02.13
doi: 10.3290/j.ohpd.a31663
O
ral bacterial plaque is the most common cause of the caries and periodontal disease, which can lead to the destruction of tooth structure and early loss of periodontal supporting tissues (Bahn, a
Associate Professor, Iranian Center for Dental Research (ICDR), Dental School, Shahid Beheshti University of Medical Sciences, Tehran, Iran.
b
Assistant Professor, Department of Periodontics, Dental School, Lorestan University of Medical Sciences, Khoramabad, Iran.
c
Graduate Student, Department of Prosthodontics, Dental School, Azad University of Medical Sciences, Tehran, Iran.
d
Research Fellow, Department of Microbiology, Medical School, Shahid Beheshti University of Medical Sciences, Tehran, Iran.
e
Graduate Student, Department of Periodontics, Dental School, Dalhousie University of Medical Sciences, Halifax, Canada.
f
Assistant Professor, Department of Periodontics, Dental School, Shahid Beheshti University of Medical Sciences, Tehran, Iran.
Correspondence: Dr. S. Golmohammadi, Department of Periodontics, Dental School, Lorestan University of Medical Sciences, Khoramabad, Iran. Tel: +98-661-321-1037, Fax: +98-661-324-3753. Email:
[email protected]
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1970; Mislowsky and Mazzella, 1974). The production of various destructive metabolites by Grampositive and Gram-negative bacteria residing in oral bacterial plaque induces gingival inflammation, which might play a key role in the progression from gingivitis to periodontal disease (Madianos et al, 2005). Studies have clearly shown that preventing the onset or progression of periodontal diseases is facilitated by regular plaque-control practices. However, a lack of manual dexterity may decrease the efficacy of daily mechanical plaque removal in the majority of the population (Löe, 2000). Moreover, chemical antiseptics and antibiotics are also adjunctive therapeutic regimes effective in the control of dental-plaque related disease, but side effects such as staining, bitter taste, burning sensation, desquamative mucosal reactions, ulceration, subjective dryness of the oral cavity, nonselective bacterial sensitisation, allergic reactions, resistance mechanisms, toxicity and difficulties in
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controlling the standard concentration of these agents in the oral cavity are also problems that may occur (Hepsø et al, 1988). Similarly, alcohol used in oral care products can be irritating to the cheeks, teeth and gums. Excessive use of products that contain alcohol may also weaken the immune system’s natural ability to fight bacteria and illness (Romeo et al, 2007). Therefore, consumer awareness and concerns over the potential risks that synthetic materials pose to human health have renewed the interest in using natural alternatives. Tea as a beverage made from the fresh leaves of Camellia sinensis has been safely consumed worldwide for centuries. Green tea and its extract in particular have shown many health benefits including antioxidant, anticarcinogenic and antimicrobial activities (Hamilton-Miller 1995; Adhami et al, 2003; Cooper et al, 2005). Antibacterial activities of tea polyphenols and tea extract have been reported against human and animal disease-related (Ryu, 1980) phytopathogenic (Fukai et al, 1991), cariogenic (Tahir and Moeen, 2010), food-borne (Kim et al, 2004) and also thermophilic spore-forming bacteria (Sakanaka et al, 2000). There are some inconsistent results regarding which specific bacterial species are significantly reduced by tea (Hara et al, 1989; Toda et al, 1991). Presumably, these differences are attributable to strain variations, different varieties of tea and different processing and extraction procedures. Besides, the mechanism of anti-microbial or immunostimulating induced cell death by active ingredients of green tea is not clearly understood. Thus, the purpose of this in vitro study was to evaluate the antimicrobial efficacy of a mouthrinse containing green-tea pure extract against five different bacteria in comparison with 0.2% chlorhexidine, which is considered the gold standard for its clinical efficacy in chemical plaque control. The other objectives of this investigation were to determine the phagocytic activity of the neutrophils by nitroblue tetrazolium (NBT) in both green tea extract and 0.2% chlorhexidine.
MATERIALS AND METHODS Bacterial culture and control tests The standard strains of S. mutans, S. sanguis, E. coli, E. fecalis, and P. aeruginosa were purchased from the Iranian Organisation of Science and Technology (IROST) in Tehran, Iran (Table 1).
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The bacteria were subcultured on agar media specific for each bacterium and then incubated at 37°C in the presence of 5% CO2 for 24 h. Overnight cultures were prepared in Trypticase Soy Broth (Merck; Darmstadt, Germany) and Brain Heart Infusion (Merck) by transferring a few colonies grown on agar media. The bacterial suspensions were then diluted in broth to an optical density of McFarland No. 0.5 (approximate numbers 1.5 x 108 bacteria ml -1). Finally, 50 μl of this suspension was added to each sample tube and then incubated at 37°C in the presence of 5% CO2 for 24 h. In order to ensure the accuracy, reliability and reproducibility of the microorganism quality, control procedures were performed. A standard titanium loop was inserted into the sample tubes to obtain a portion of the specimen for subculturing on agar media before each test modality. After 24-h incubation, the plates were examined to evaluate the size and the shape of the colonies grown. The catalase test was performed to confirm species identification. Finally, direct light-microscopic examination of Gram-stained smears showed the presence of the organism.
Preparation of green tea mouthrinse The air-dried leaves of green tea (Camellia sinensis) were collected from Lahijan, Iran. The leaves were cut into pieces and pulverised using a sterile electric grinder. The soluble ingredients in the ground plant part were then extracted by using 95% ethyl alcohol (Merck) and water as different solvents. The resulting extract was centrifuged and the ethanol was evaporated. After taking the extract, green tea mouthrinse was made by mixing 10 mg of pure extract with 0.4 ml of ethyl alcohol and 199 ml of distilled water.
Disk diffusion method A 24-h broth culture (McFarland No. 0.5) of the bacteria was aseptically subcultured and evenly spread using sterile swabs on the surface of MuellerHinton agar plates. The culture was allowed to adhere to the plate for 3–5 min. Blank disks were impregnated in 0.5 ml of either green tea mouthrinse or 0.2% chlorhexidine mouthrinse (Behsa Pharmaceutical; Tehran, Iran) for 10 s. All mouthrinse impregnated disks were placed on the agar plates. Antibiotic-impregnated (positive con-
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Table 1
Bacterial species used
Species
ATCC No
Growth condition
Culture medium
Gram-staining
S. mutans
ATCC 35668, PTCC 1683
Aerobic
Mitis salivarius
positive
S. sanguis
ATCC 10556, PTCC 1449
Aerobic
Blood agar
positive
E. coli
ATCC1395
Aerobic
Mac Cankey agar
negative
E. faecalis
ATCC 29199
Aerobic
Bile eschulin
positive
P. aeruginosa
ATCC 1310
Aerobic
Nutrient agar
negative
trol) and saline-impregnated (negative control) disks were placed on either side of the bacterial plates using sterile forceps. All plates were then incubated at 37°C for 48 h. All experiments were done in quadruplicate. The zone of growth inhibition (the distance between the edge of the disk and the edge of the bacterial colony) was measured in mm.
of 0.1% NBT (Sigma Chemical; St Louis, MO, USA). After adding 0.1 ml of green tea extract with different concentrations (0.5, 1, 1.5, 2, 2.5, 3, 3.5 and 4 mg/ml), the solution was incubated at 37°C for 10 min. Afterwards, 50 μl of mixture was transferred onto a glass slide and stained with Wright Stain. Then, the neutrophils that contained dark deposits of formazan (reduced NBT) were observed under light microscope and compared between different concentrations.
Minimum inhibitory concentration According to the agar diffusion method as described by Verpoorte et al (1982), 10 test tubes at concentration of 1, 2, 4, 8, 16, 32, 64, 128, 256 and 512 mg/ml for each mouthrinse and for pure green tea extract were inoculated with 48-h bacterial cultures. For each bacterial species, a positive control tube containing the growth medium and the bacteria in suspension (McFarland turbidity standard No: 0.5) was used. Then, the mixtures were incubated at 37°C for 24 h. The minimum inhibitory concentration (MIC) was interpreted as the lowest concentration of the agents that completely inhibited the growth of the test species. In order to determine the purity of the bacterial content and rule out the possibility of contamination in the MIC test tubes, an inoculum of the first tube that showed bacterial growth was cultured in specific media as shown in Table 1.
Nitroblue tetrazolium (NBT) test The nitroblue tetrazolium (NBT) in vitro test was used to demonstrate the ability of green tea extract to stimulate the phagocytic activity of neutrophils. 0.2 ml of heparinised venous blood (10 U of sodium heparin/ml; Upjohn; Kalamazoo, MI, USA) from a healthy adult was reacted with an equal volume
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Statistical analysis In order to assess the differences between groups, analysis of variance (ANOVA) was performed at the significance level of P < 0.05 using SPSS 17.00 statistical software (SPSS; Chicago, IL, USA).
RESULTS The results of the disk diffusion method are presented in Table 2. The inhibition zones of each bacterium were measured on four plates, demonstrating that the degree of agreement between the four parallel series of plates was acceptable (the differences were not statistically significant). The green tea mouthrinse produced a significantly larger inhibition zone than the negative control for all tested microorganisms (P < 0.01). However, this diameter was significantly smaller than that of the chlorhexidine mouthrinse or pure green tea extract (P < 0.01). This shows a weak antibacterial effect of the green tea mouthrinse compared to chlorhexidine or pure extract of the green tea. Table 3 gives the MIC results by mg of tested antibacterial agent per ml. According to the results of the inhibition zone tests, the Gram-positive bacteria were significantly
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Table 2 Mean diameter of inhibition zones induced by two mouthrinses and pure extract on five tested microorganisms (shown in mm) S. mutans
S. sanguis
E. faecalis
P. aeruginosa
32.1
33.5
31.8
30.7
33.2
Negative control (sterile normal saline solution)
0.0
0.0
0.0
0.0
0.0
0.2% chlorhexidine mouthrinse
24.6
24.8
20.3
22.9
19.7
Green tea mouthrinse
11.5
11.6
7.5
10.3
7.6
Pure extract of green tea
30.5
30.9
25.2
29.8
24.5
Positive control (antibiotics)
E. coli
Table 3 Minimum inhibitory concentrations for the 0.2% chlorhexidine and green tea mouthrinses and green tea extract (shown in mg/ml) 0.2% chlorhexidine
Green tea extract
S. mutans
8
128
8
S. sanguis
4
64
8
E. coli
8
256
16
E. faecalis
8
128
8
P. aeruginosa
16
256
16
more susceptible to both mouthrinses and green tea extract than were the Gram-negative bacteria (P < 0.05). However, the intragroup difference was not significant, neither in the Gram-positive nor the Gram-negative group. The results of the nitroblue tetrazolium (NBT) test showed that the 1.5 mg/ml-concentration of green tea extract provoked phagocytic activity of neutrophils wheh added to the blood-cell-containing suspension. In extract concentrations 3 mg/ml did not cause activation of neutrophils.
DISCUSSION The direct antibacterial effect of green tea was examined in this study in order to develop an effective mouth rinse containing green tea that does not exhibit the side effects of chlorhexidine. Not surprisingly, chlorhexidine 0.2% had the strongest an-
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tibacterial effect in comparison with green tea mouthrinse. However, the pure green-tea extract compared favourably with CHX 0.2%. Polyphenols, catechins, gallic acid and aflavins are proposed as the antibacterial agents in tea (Battino et al, 1999) which may act by directly damaging the bacterial plasma membrane (Ikigai et al, 1993) and yield an anticariogenic potential. The latter characteristic has been attributed to the fact that green tea is a natural source of fluoride and an effective vehicle for fluoride delivery to the oral cavity (Fung et al, 1999; Yang and Landau, 2000; Simpson et al, 2001). However, it has also been demonstrated that green tea polyphenols (GTP) through growth inhibition of oral bacteria, such as E. coli, S. salivarius, and S. mutans, rather than fluoride account for this property (Otake et al, 1991; Wu and Wei 2002). On the other hand, green tea constituents have the potential to reduce periodontal breakdown by inhibiting the activity of proteinase of Porphyromonas gingivalis and collagenase of the host tissues (Makimura et al, 1993).
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In a previous study by Jalayer Naderi et al (2011), it was shown that Iranian green tea has anti-S. mutans activity with a minimum inhibitory concentration of 150 mg/ml. The results of our study illustrated the antibacterial effect of pure green tea extract and mouthrinse against both Gram-negative and Gram-positive bacteria, with the antibacterial effect being more pronounced in Gram-positive than Gram-negative bacteria. The superiority of Gram-negative bacteria in terms of resistance to some antibacterial agents might be by virtue of their outer membrane that acts as a barrier preventing access of the enzyme (Masschalck and Michiels, 2003). We chose S. mutans, S. sanguis and E. faecalis as test microorganisms for our study because they have been implicated in oral diseases. P. aerogenosa and E. coli were tested as two Gram-negative bacteria for observing the efficacy of green tea against the lipopolysaccharide layer or endotoxin that might induce an immune response. According to the results of this study, the extract of Iranian green tea is effective against the tested microorganisms. This antibacterial activity is similar to that of Chinese and Japanese tea, as reported in several studies and patent literature (Otake et al, 1991; Toda et al, 1991; Tsunoda et al, 1991; You, 1993). In this study, pure green tea leaves without any aromatic or additive materials were used. Similar to studies by Hamdi et al (2008) and Jalayer Naderi et al (2011), the tea leaves were selected from Lahijan province; due to the superiority of ethanol extract of Camellia sinensis in comparison with water extract found by those authors, we chose to apply ethanol extract to prepare the green tea mouthrinse. Here, it is important to bear in mind that through the process of preparing the ethanol extract of green tea, which is based on the volatility of different ingredients, at the final stage, ,the ethanol was evaporated completely before the final extract was condensed (Tahir and Moeen, 2010). Therefore, no antibacterial effect of the final product can be attributed to alcohol. A point to be emphasised is that the lower antibacterial potency of green tea mouthrinse in comparison with its pure extract indicates a need for further modification of the proposed formulation or even adding some other herbal ingredients with synergistic effects (Lauten et al, 2005). With regard to the limitations of our study, it should be pointed out that bacterial pathogenicity is a multifactorial process, involving microbial virulence and host response, along with genetic and
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environmental factors such as saliva buffering and diet (Bagg et al, 1999). The results of this in vitro test are not directly transferable to the oral cavity and do not translate into clinical effectiveness. In addition, bacteria organised in a biofilm have a decreased sensitivity to antibacterial agents and topical antimicrobial agents, such as mouthrinses. The mouthrinse must be able to penetrate the biofilm matrix and deliver the active agents quickly (Lee et al, 2004). In addition, results of the disk diffusion test should be interpreted with caution; the mean inhibition zone of one mouthrinse may not be directly comparable with that of another mouthrinse because they may diffuse at different rates. Nevertheless, the in vitro method is a common and useful technique used for screening the antimicrobial efficacy of mouthrinses before their in vivo testing. The nitroblue tetrazolium (NBT) reduction test was first introduced by Park et al (1968) with the aim of detecting bacterial infections in patients with fever of unknown origin. The presence of extrinsic elements as a contributory factor for reducing NBT might enhance or inhibit the function of the immune system through increasing phagocytosis (Durak et al, 1993). In this study, there were no signs of neutrophils containing dark deposits of formazan (reduced NBT) at concentrations of less than 1.5 ml/mg or more than 3 mg/ml. Based on this result, an optimal concentration of green tea extract that enhances the phagocytic activity of neutrophils might be 1.5 to 3 mg/ml.
CONCLUSION The formulation of green tea mouthrinse used in this study is less effective than chlorhexidine 0.2% against the tested Gram-negative and Gram-positive bacteria. However, pure extract of Iranian green tea is an effective antibacterial agent comparable to chlorhexidine 0.2%. Both the green tea extract and chlorhexidine 0.2% were more efficacious against Gram-positive than Gram-negative bacterial species.
ACKNOWLEDGEMENTS This study was supported in part by The Dental Research Centre, Dental School of Shahid Beheshti University of Medical Sciences. The authors wish to thank the members of microbiology department, Medical School of Shahid Beheshti University of Medical Sciences.
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