Dental caries is one of the most major oral health problems in the world [1]. The people are susceptible to dental caries throughout their lifetime and approximately 36% of world population are experiencing tooth decay in their permanent teeth [2]. Microorganisms especially mutans streptococci have a major role in the initiation of dental caries. In early stage of dental plaque formation, Streptococcus sanguis is colonized on the tooth surface [3].

Due to increased resistance to antibiotics and chemotherapeutics, it is needed to safe and effective products for prevention and management dental caries [4]. Natural products with herbal, animal or microbial origin have good potential in this area. Healing herbs has long history for use in gum and tooth problems [5].

Vinegar is a sour liquid [6] and includs acetic acid, vitamins, mineral salts, amino acids, polyphenolic compounds, nonvolatile organic acids. Vinegar with anti-infective properties, antitumor activity, antiglycemic effect [7] has various medicinal uses such as prevention of hypertension, reduction serum total cholesterol and triacylglycerol [8]. Vinegar in the Iranian traditional medicine used in prevention of dental caries [9].

Rose water is major product of Rosa damascena in Iran. It contains 10-50% rose oil [10]. In folk medicine of Iran rose water has been used for various skin problems [11] and as antispasmodic for abdominal pain [12] and is important ingredient in cosmetic industries [11]. Rose water have traditionally been used in to prevent tooth decay [9]. It is reported that rose water has antimicrobial effects and can inhibit Candida albicans mycelial growth and has bactericidal activity against methicillin-resistant Staphylococcus aureus (MRSA) [13].

Green tea (Camellia sinensis ) contains proteins, amino acids, carbohydrates, minerals, trace elements, lipids, vitamins B, C and E, caffeine, theophylline, polyphenolic compounds. It has various effects such as antiviral, antibacterial and antidiabetic effects, and may be used for skin treatment, also recommended for prevention of dental caries [14].

The aim of this study was to determine antibacterial effects of vinegar, rose water and green tea against oral streptococci including Streptococcus mutans , Streptococcus salivarius , Streptococcus sanguis and Streptococcus salivarius .

Test organisms

The antibacterial activity of the vinegar, rose water and green tea was evaluated against four strains of bacteria which was purchased from Iranian Research Organization for Science and Technology (IROST) including: Streptococcus mutans (ATCC 35668), Streptococcus salivarius (ATCC 27607), Streptococcus sanguis (PTCC1449) and Streptococcus salivarius (PTCC 1448).

All The bacterial strains were cultured in Tryptic Soy Broth (TSB), Blood Agar and Mitis Salivarius Agar (MSA) at 37°C in a atmosphere containing 5% CO₂. Then, these bacteria were confirmed by Gram staining, catalase test and bacitracin tests. The bacteria kept in freezer at -80°C until used in the study.

at -80°C until used in the study.

Material preparation

For this study, homemade grape vinegar was prepared from Isfahan, Rose water purchased from Kashan and Green tea was purchased from Medicinal Plants Market of Isfahan, Iran.

The dried powder of green tea was mixed with 50% ethanol solution. After 48 hours it was filtered through Whatman (No.1) filter paper. Then extract was placed in the rotary evaporator (Heidolph Company, Germany) and finaly put in the oven at 40°C to dry completely.

Determination of antibacterial activity

Bacterial suspensions with 1 × 10⁸ bacteria, were prepared from tryptic soy agar; (TSA; Quelab, Montreal, Canada). They were cultured on the Muller-Hinton agar medium with 5% defibrinated sheep blood using sterile swabs. Then, 50 μL of the vinegar, rose water and green tea solutions were poured into the wells (6 mm in diameter). These plates were kept at 4°C until the materials in the wells were completely diffused into the agar, and the plates were incubated anaerobically at 37°C for 24 h. The diameter of the inhibition zone was measured [15].

Determination of MIC and MBC

The MIC (minimum inhibitory concentration) of vinegar, rose water and green tea solutions determined by micro-dilution broth method. Overnight culture of bacteria in tryptic soy broth (TSB; Quelab, Montreal, Canada) were adjusted to 10⁶ colony-forming units (CFU/mL). Two-fold serial dilutions of test compounds were prepared in broth. In 96-well plates, 100 μL of each dilution of test solutions were placed into the well containing 100 μL of bacterial suspension.

Triplicate samples were performed for each test concentration [3]. Negative control wells were containing culture medium and bacteria. After 24 h anaerobically incubation at 37°C the turbidity was measured by micro-plate reader (AWARENESS, Technology INC, Stat fax 2100).

MBC (minimum bactericidal concentration) was considered as the lowest concentration of test solutions that did not seen any visible bacterial growth on the tryptic soy agar (TSA) plates after 24 h incubation under anaerobic condition at 37°C [16].

Determination of anti-adhesion effect

Anti-adhesion effect of test solutions measured by the micro-titer plate method as described previously by Di et al. with some modifications [17]. Briefly, the overnight grown streptococci in TSB with 1% sucrose, were adjusted to reach a concentration of 10⁶ CFU/ mL. Then 10 μL serial dilutions of test solutions were added to 1 mL of each bacterial suspension. Then 200 μL of them were transferred into each well. Blank wells only contained buffer and control wells contained bacteria without treatment. After 24 h of incubation at 37°C at 5% CO₂, wells were washed 3 times with 200 μL of sterile phosphate-buffered saline (PBS), to remove unattached cells. Then the adherent bacteria were stained with 200 μL of 2% crystal violet for 5 min, then the excess stain was rinsed off using placing the plates under running tap water.

Then 200 μl of 33% (v/v) glacial acetic acid was added and the level of biofilm formation was evaluated via measuring the absorbance of the solution at 492 nm by an ELISA reader of the adherence reduction was calculated using equation:

B=absorbance of blank, C=absorbance of control and T=absorbance of test [18].

Statistical analysis

The data were statistically analyzed using Graf Pad Prism5 software (GraphPad Software Inc., CA, USA) by one way ANOVA and Tukey’s post hoc tests.

Measurement of inhibition zone diameter

The means of three separate measurements of inhibition zone diameter of vinegar, rose water and green tea against Streptococcus mutans , S. salivarius , S. sobrinus and S. sanguis are summarized in Table 1.

Table 1: The mean of diameters of inhibition zones of vinegar, rose water and green tea against four oral streptococci *Green tea 500-62.5 mg/ml.

Vinegar compare to rose water and green tea showed the highest antibacterial activity against all test bacteria (P<0.05). Rose water did not showed zone of inhibition against mentioned bacteria.

The largest zones of inhibition were measured as 24.2 and 22 mm as diameters of zones of inhibition of vinegar against Streptococcus salivarius and Streptococcus salivarius respectively.

The largest zones of inhibition by green tea were found at the first dose (500 mg/ml) and a decrease in concentration resulted in a decrease in the zone of growth inhibition. Green tea in concentration of 62.5 mg/ml did not express zone of inhibition.

Rose water did not show antibacterial activity against S. mutans , S. sobrinus , S. sanguis and S. salivarius .

Determination of MIC and MBC

The MIC and MBC of vinegar, rose water and green tea against Streptococcus mutans , Streptococcus salivarius, Streptococcus sanguis, Streptococcus salivarius, are presented in Table 2. The MIC and MBC of vinegar against S. salivarius was 0.0312 and 0.0625 respectively which was significantly less than that for the other three bacteria (P<0.05), indicating higher antibacterial activity of vinegar against S. salivarius.

Table 2: Values of MIC and MBC for the four groups of oral streptocci bacteria. *Green tea: mg/ml.

The MIC and MBC of green tea against Streptococcus mutans and Streptococcus salivarius was 7.81 and 31.25 mg/ml respectively which was significantly less than that for Streptococcus sanguis and Streptococcus salivarius: 15.625 and 62.5 mg/ml, respectively (P<0.05).

The MIC of rose water against Streptococcus salivarius, Streptococcus sanguis, Streptococcus salivarius was 1 and for Streptococcus mutans was not seen any inhibition zone, Also, the rose water did not express MBC against all test bacteria (Figure 1).

Figure 1: The effect of various concentrations of vinegar, rose water and green tea on the growth of four oral streptococci.

Determination of anti-adhesion effect

The anti-adhesion effect of vinegar, rose water and green tea was evaluated for each streptococci species and the results are presented in Table 3 and Figure 2.

Figure 2: The effect of various concentrations of vinegar, rose water and green tea on adherence of four studied oral streptococci.

Table 3: Percentage of adherence reduction of various concentrations of vinegar, rose water and green tea on oral streptococci *Green tea 500-125 mg/ml.

All three materials significantly decreased the adhesion of test streptococci compared to the control group (P<0.05).

Vinegar and green tea showed highest antibacterial activity against test streptococci. Rose water showed lowest adherence reduction (53.27%, 60.75% and 65.90%) for Streptococcus mutans , Streptococcus salivarius and Streptococcus sanguis respectively, compare with green tea and vinegar.

In this study, antimicrobial activity of vinegar, rose water and green tea was evaluated on four oral streptococci, including S. mutans , S. sobrinus , S. sanguis and S. salivarius . Various concentrations of vinegar have expressed maximum inhibition zone against streptococci compare to rose water and green tea.

Maruyama et al. evaluated the effects of rose water against Candida albicans and methicillin resistant Staphylococcus aureus (MRSA) and showed rose water decreased MRSA 99.99% within 1 h, and inhibited Candida albicans mycelial growth 50% [13].

Ismael showed that types of Date, Apple, and Grape vinegars eradicated biofilm of Streptococcus pyogenes (100%), (95.5%), and (90.9%), respectively [6].

Komiyama et al. evaluated the antimicrobial and disinfecting effects of 0.12% chlorhexidine, 0.50% white vinegar and two other materials on S. mutans , S. pyogenes , Staphylococcus aureus and Candida albicans on toothbrush and showed that vinegar decreased the count of S. mutans , S. pyogenes and S. aureus [19].

Ramezanalizadeh et al. studied effect of pomegranate vinegar and rose water in comparison with Persica mouthwash on Streptococcus mutans and Streptococcus salivarius and showed reduced plaque formation by S. mutans 93%, 80% and 68%, and for S. sobrinus were 92%, 57% and 48% respectively, and reported pomegranate vinegar was more effective than the other two materials.

And showed inhibition zone for S. mutans and S. sobrinus was respectively and dont seen inhibition zone for rose water [9].

The result of this study shows that vinegar and green tea decreased >70% that adhesion of S. mutans , S. sobrinus , S. sanguis and S. salivarius . And rose water decreased >70% that adhesion of S. salivarius , >60% of S. sobrinus , S. sanguis and >50% S. mutans .

El-Shamy et al. was evaluate the antimicrobial effectiveness of vinegar against Streptococcus mutans, Lactobacillus salivarius , Staphylococcus aureus, Enterococcus faecalis, and Candida albicans and showed vinegar was effective against S. mutans , S. aureus , E. faecalis , and C. albicans as compared to oradex mouthrinse. The mean inhibition zones of vinegar for Streptococcus mutans was 31.60 ± 0.5 [20]. In our study inhibition zone diameter vinegar for Streptococcus mutans was 18 mm.

Our results revealed that rose water had a effect on decreasing the adhesion of bacteria but had no effect on their growth and had not bactericidal effect, that these findings are consistent with the results of previous study.

Further studies are required to determine bioavailability active these compounds and understand of their mechanism.

Grover et al. studied effect of antimicrobial activity green tea at varying concentrations against oral bacteria and showed with increased the concentration inhibitory effect increased and highest mean zone of inhibition was 2.83 mm at 80 mg/ml [21].

The results of the present study showed that zone of inhibition green tea decreased with decrease in concentration and the highest zone of inhibition green tea was for S. mutans at 500 mg/ml that these findings are in agreement with the results of previous study.

Naderi et al. evaluated antibacterial effect Iranian green and black tea on Streptococcus mutans and showed they have an antibacterial effect on 100 to 400 mg/ml. And MIC green tea was 150 mg/ml, mean diameter of inhibition zone was 9.5 mm [22].

In this study MIC for green tea for Streptococcus mutans was 7.81 mg/ml and zone of inhibition was 13.67 mm at 500 mg/ml. And for S. sobrinus , S. sanguis and S. salivarius were 11.5, 11.5, 12.5 mm.

Comparison of vinegar, green tea and rose water showed that effect of vinegar on growth bacteria was significantly higher than that of green tea and rose water (P<0.05). In summary, the present study shows that these substances have anti-adhesion effects in vitro .

The current study showed that vinegar and green tea expressed inhibitory effect against oral streptococci and vinegar, rose water and green tea decreased adhesion of oral streptococci .

Although this materials are regarded as safe and affordable, but additional qualitative and quantitative efforts are needed to get the most effective and safe protocol for using of them for prevention of dental caries.