INTRODUCTION
The two most common types of dental diseases, dental caries and
periodontal disease, are plaque-related infections. Dental caries involves
demineralization, cavitation and breakdown of calcified dental tissue
and is caused by microorganisms that ferment dietary carbohydrates, notably
sucrose, to produce acids; these acids initiate dissolution of the tooth
enamel. Strains of Streptococcus mutans, Streptococcus mitis
and Streptococcus sanguis have long been implicated in the formation
of dental plaque and cariogenicity and are often responsible to bacteremias
following dental manipulations (Tom á s et al., 2005). Natural
products are now preferred by a large proportion of the population and
have been reported to possess antimicrobial activity (Hammer et al.,
1999). Plant extracts have recently been shown to be a good alternative
to synthetic chemical substances for caries prevention (Hamilton-Miller,
1995). Extracts from Celastrus scandens, Chamaebatia foliolosa,
Digitaria sanguinalis, Ginkgo biloba, Juniperous virginiana
(Heisey and Gorham, 1992), Anacardium occidentale L. (Muroi and
Kubo, 1993) and Ilex paraguayensis (Kubo et al., 1993) have
proved to be effective against Streptococcus mutans.
Tea is prepared from the young shoots of tea plant Camellia sinensis.
Five thousand years ago Chinese identified this plant and used it for
patient treatment and staining textile (Koo and Cho, 2004). Extracts of
leaves from the tea plant Camellia sinensis contain polyphenolic
components with activity against a wide spectrum of microbes. Studies
conducted over the last 20 years have shown that the green tea polyphenolic
catechins can inhibit the growth of a wide range of Gram-positive and
Gram-negative bacterial species with moderate potency. Toda et al.
(1989) found that extract of tea inhibited and killed Staphylococcus
aureus, Staphylococcus epidermidis, Salmonella typhi,
S. typhimurium, S. enteritidis, Shigella flexneri,
Shigella dysenteriae and Vibrio species including Vibrio cholerae.
Tea extracts have been found to be active against Clostridium spp.
and phytopathogens such as Erwinia spp. and Pseudomonas spp.
(Ahn et al., 1991).
The aim of this study was to evaluate the antimicrobial activity and
bactericidal concentrations of non fermented and semi fermented Camellia
sinensis extracts (green and black tea) on oral Streptococci and compare
their effects on biofilm formation and adherence to surfaces using a variety
of in vitro test systems.
MATERIALS AND METHODS
Extraction of samples: Fresh grade 1 black (semi fermented) and green (non fermented) tea
that were essence free, quickly after production purchased from traditional
producer company in Lahijan city in the north of Iran. Tea samples were
stored in plastic bags at 4 ° C and transported to the microbiology
laboratory of Science and Research campus of IAU in Tehran. Samples powdered
in a blender and then extracted with methanol: water mixture (62.5:37.5
v/v) in the Clevenger extractor as follows: powdered dry material (500
mg) was weighed into a test tube. A total of 10 mL of 62.5% aqueous methanol
was added and the suspension was stirred slightly. Tubes were sonicated
5 min and centrifugated for 10 min (1500 g) and supernatants were collected.
The materials were re-extracted twice. The extracts were concentrated
by further evaporation to one-fifth volume, filter sterilized and stored
at 4 ° C.
Gas chromatography: GC analyses were performed with a Shimadzu 17A gas chromatograph
(Shimadzu, Japan) equipped with a flame ionization detector and a 60 m
x 0.25 mm (I.D.) DB-WAX (J and W Scientific, Folsom, CA) fused-silica
capillary column. The operating conditions were as follows: oven temperature,
40, 3 ° C min-1 rise to 220 ° C; carrier gas, helium and
flow rate, 1.0 mL min-1. The retention times and peak areas
of the eluted volatile components were integrated with a Chromatopack
integrator.
GC-MS analysis: GC-MS analysis was carried out on a Hitachi M-808 double-focusing
instrument equipped with a Hewlett-Packard 5890 gas chromatograph. The
GC conditions were identical to those of the above analytical GC runs.
Mass spectral data were acquired and processed by a built-in computer
system (M-0101) developed by Takasago International. The components of
the distillate were identified by comparing their GC retention times and
MS fragmentation with those of the authentic samples.
Bacterial strains and cultures: Streptococcus mutans ATCC 25175, Streptococcus mitis
ATCC 9811 and Streptococcus sanguis ATCC 10556 that purchased from
Microbiological Reference Centre of Iran, were grown on brain heart infusion
broth, (BHI Difco, Detroit, USA) at 37 ° C with 5% defibrinated sheep
blood in an atmosphere contain 5% CO2 and for antimicrobial
susceptibility test, Mueller Hinton Agar (MHA, Difco, France) was used.
Antimicrobial activity tests: For this examination Well diffusion and broth dilution methods were
used separately. Standardized bacterial suspensions containing 106
cells mL-1 were obtained by spectrophotometry (Shimadzo UV
120-01), then 100 μ L of this suspension cultured onto Mueller Hinton
Agar with cotton Swab, then 30 μ g of black and green tea extracts
(100 mg mL-1) were added separately to 5 mm wells in a Mueller
Hinton Agar supplemented with 5% defibrinated sheep blood and maintained
48 h in 37 ° C incubator with 5% CO2 atmosphere and then
their MIC were assessed. Ten microgram discs of Penicillin used for positive
control and distilled water used for negative control.
In broth dilution method 50 μ g of black and green tea extracts added
to 10 mL BHIB containing 5% defibrinated sheep blood that inoculated with
106 cells mL-1 and incubated in 5% CO2.
After 5 min of contact at room temperature, surviving Colony Forming Units
(CFU) were counted by inoculating of 0.5 mL treated sample cultures on
BHI agar at 37 ° C with 5% defibrinated sheep blood in an atmosphere
contain 5% CO2 after 48 h. Each experiment was carried out
twice, duplicates and controls with no black and green tea extracts were
always included.
Bacterial adherence to glass beads: Nine milliliters mixtures of each tea extract (Semi fermented and
non fermented Persian Camellia sinensis at concentration of 1,
2 and 3 mg mL-1) with dehydrated culture medium (BHI broth)
were distributed into 15 tubes. Then, standardized glass slides (diameter
= 2 mm, length = 5 cm) and glass beads (Spherical beads with 5 mm diameter)
were added to the separate tubes and submitted to autoclave sterilization.
For evaluation of bacterial adherence, one milliliter of standardized
bacterial suspension (106 cfu mL-1) obtained by
spectrophotometry was inoculated into each tube (final volume 10 mL) and
then incubated for 90 min at 37 ° C in atmosphere of 5% CO2.
After the period of incubation, the glass slides and glass beads were
transferred into tubes containing buffered phosphate saline pH 7.2 (PBS,
Sigma, USA). Tubes containing glass beads were submitted to agitation
(Phoenix AP56, Brazil), from this initial suspension; dilutions of 10-1
and 10-2 were obtained in sterilized NaCl 0.85% saline solution.
Then, aliquots of 0.1 mL of each dilution were plated in duplicate on
BHI agar and incubated for 48 h at 37 ° C in atmosphere of 5% CO2.
After this period, the number of colonies was counted and the value of
logarithm of colony forming units per milliliter was calculated (log cfu
mL-1). The results obtained were analyzed statistically by
ANOVA test (p < 0.01).
On the other hand, the glass slides that obtained from control tube and
treated with different concentration of tea extracts were directly examined
under phase contrast microscopy and the adherence pattern of the treated
and non treated bacteria was observed and compared with each other.
RESULTS
GC mass spectroscopy results: Extracts of non-fermented (green) and semi-fermented (Black) teas
prepared from Camellia sinensis were compared by means of GC (Table
1). The amounts of almost all volatile components, including alcoholic
aroma constituents hydrolyzed by enzyme, increased as a result
of semi-fermentation.
Antimicrobial activity: In comparison between semi and non fermented tea shoots, growth
inhibitory concentration was lower for semi fermented Camellia sinensis
extract and its antimicrobial activity was better as compared to green
tea. The diameter of inhibitory zone in concentration of 3 mg mL-1
of black tea extract was 25.5 mm for Streptococcus mutans, 28 mm
for Streptococcus mitis and 29.5 mm for Streptococcus sanguis
and for the same concentration of green tea extract the MICs were 24,
26.5 and 27.5 mm orderly. This is a noticeable result if compared with
positive control (Penicillin) that has 33, 34 and 33 mm inhibitory zone
in order for these bacteria. The results are shown in Table
2.
Bactericidal effect of both extracts was clear and significant different
exist between extracts effects. In comparison between non fermented and
semi fermented Camellia sinensis extracts, the later has faster
effect on inhibition growth of oral streptococci. In case of 3 mg mL-1
of black tea extract after 30 min the count of viable cells was below
1 log of cfu mL-1 and this amount achieved by same amount of
green tea extract in 40 min (Fig. 1-3).
Oral Streptococci`s adherence to glass: ANOVA test results showed significantly higher counts for control
group (p < 0.01) and the lowest counts were obtained for semi fermented
Camellia sinensis extract (p < 0.01) (Fig. 4).
| Table 1: |
The content of volatile components in Persian (Lahijan)
Black and Green teas (Camellia sinensis) |
 |
| The content of each component expressed by the ratio
of each peak area of the internal standard of the gas chromatogram
|
| Table 2: |
In vitro susceptibility of S. mutans ATCC
25175, S. mitis ATCC 9811 and S. sanguis ATCC 10556
to semi fermented and non fermented Camellia sinensis extracts |
 |
 |
| Fig. 1: |
Growth inhibition of Streptococcus mutans ATCC
25175 to semi fermented Camellia sinensis |
Phase contrast microscopy: Microscopic examinations showed that untreated oral streptococci
were able to attach each other and produce a clump on the slide surface
(Fig. 5) that is a basic and initial step for biofilm
formation but 1 mg mL-1 of semi fermented and 1.5 mg mL-1
of non fermented Camellia sinensis extracts inhibited the attachment
of these bacteria and bacterial
 |
| Fig. 2: |
Growth inhibition of Streptococcus sanguis
ATCC 10556 to semi fermented Camellia sinensis |
 |
| Fig. 3: |
Growth inhibition of Streptococcus mitis
ATCC 9811 to semi fermented Camellia sinensis |
 |
| Fig. 4: |
The numbers of oral streptococci treated with 2 mg mL-1
of non fermented and semi fermented Camellia sinensis on glass
beads. The bacterial strains shown with (1) S. mutans, (2)
S. mitis and (3) S. sanguis |
cell were washed easily and crowded on some areas. This result confirms
biofilm inhibitory effects of these extracts especially semi fermented extract.
 |
| Fig. 5: |
Phase contrast microscopy images of Streptococcus
mutans ATCC 25175 before treatment (Left image) and after treatment
with 1 mg mL-1 of semi fermented Camellia sinensis
extract (Right image) taken from glass slide surfaces |
DISCUSSION
Recent investigations are focusing on the nutritional effects of
tea on the human body and health. In this study among the two types of
tea extracts that tested on oral streptococci, semi fermented Camellia
sinensis (black tea) seems to be the more effective extract against
streptococcal growth and biofilm formation (Table 2).
Epidemiological studies revealed a reduction in caries formation in tea
drinking populations and school kids from tea planted areas in Japan (Onisi,
1993).
Black tea has many more components than green tea, to some extent because
of the oxidation processes that occur during fermentation. Further reactions
take place when the dried finished tea leaves are extracted into water,
increasing the complexity of the chemical mix in a cup of tea. In addition,
further chemical changes occur when a cup of tea is left to stand. Results
of GC-MS shows much higher amounts of volatile component in Camellia
sinensis after exposure to fermentation (Table 1).
Volatile flavor components make up a very small fraction of flush and
tea leaf (10 to 20 ppm) but play an important part in providing taste
and antibacterial activity in our study. More than 300 such components
have been reported in black tea leaf (Gutman and Ryu, 1996) and more than
100 such components have been reported in green tea. Kubo et al.
(1993) found some of these volatile components to be microbiologically
active, but not at cup-of-tea concentrations (Ahn et al., 1990).
Minimum inhibitory concentrations and Biofilm inhibitory concentrations
of semi fermented Camellia sinensis were lower for tested oral
streptococci in comparison to non fermented Camellia sinensis (Table
2) that shows a direct relationship with amount of volatile components
of extracts (Table 1). Green tea consumption has been
reported to increase the acid resistance of teeth to damage by cariogenic
bacteria (Gutman and Ryu, 1996; Hamilton-Miller, 2001). It has been demonstrated
that tea can inactivate glucosyltransferase and dextran sucrase, thus
it can inhibit the formation of water insoluble glucan and lactic acid,
respectively (Otake et al., 1991).
Among the studied oral bacteria, S. mutans ATCC 25175 was more
resistant (Fig. 1) and S. sanguis ATCC 10556 was
more sensitive to the both tea extracts (Fig. 2).
Combinations of the flavor compounds, especially indole with some of
the sesquiterpenes, displayed marked bactericidal synergy (Hamilton-Miller,
1995). Bacterial adherence to glass surface is the model system chosen
because the adherence is mediated by glucan as well as the in vivo
situation and the glass adherence assay is still used in some recent studies
(Koo et al., 2000; Mattos-Graner et al., 2000; Carter et
al., 2001; Tao and Tanzer, 2002).
The numbers of oral streptococci cells that treated with 2 mg mL-1
of non fermented and semi fermented Camellia sinensis after 30
min on glass beads revealed that semi fermented Camellia sinensis
has more biofilm inhibitory effect (Fig. 4) that confirms
the results of MICs by Well diffusion plates. Complementary test on the
slide surface observed under phase contrast microscope showed significant
reduction in attachment of these bacteria together (Fig.
5) while 30 min after treatment of bacteria with 1 mg mL-1
of semi fermented and 1.5 mg mL-1 of non fermented Camellia
sinensis extracts the bacteria were not able to attach each other
and have poor colonization for biofilm formation. These bacteria didn`t
have successful colonization and were more vulnerable to antibacterial
agents.
The effectiveness of antimicrobial agents decreases with increasing age
of the biofilm. Intact biofilms are also more resistant than disrupted
communities (Wilson, 1996). The reasons for such differences are related
to the community`s properties and the bacterial cell changes that they
induce.
Significant reduction in adherence of S. mutans, S. mitis
and S. sanguis to glass surface, represent decrease in the
cariogenic activity caused by adhesion of these bacteria specially
S. mutans because this ability is considered as an essential step
in the initiation and development of dental caries. Elvin-Lewis and Steelman
(1968) claimed to have noted statistically improved dental health in children
who drank at least one cup of tea daily compared to the dental health
of those whose intake was less than 3 cups per week (Kubo et al.,
1993).
Although antimicrobial activity was expected based on the presence of
some common components with tea like volatile components that exhibit
this property and possible anti-cariogenic activity related to the reduction
of oral streptococci`s adherence. These data are very promising considering
that adherence is one of the main virulence factor of this species. Other
studies including anti-adhesive activity on dental enamel and dentine
and also in vivo studies are essentially necessary to highlight
the clinical applications of these findings.
In conclusion, semi fermented Camellia sinensis inhibits the adherence
of Streptococcus mutans, Streptococcus mitis and Streptococcus
sanguis in vitro much better than non fermented Camellia
sinensis extract at the same concentrations employed in this study
due to rich content of volatile components exist in semi fermented extracts
that can be very useful for controlling dental caries.
ACKNOWLEDGMENTS
We thank Miss Behin Omidi the chief of Microbiology Laboratory in
Science and Research branch of IAU for her helpful comments and providing
the materials.
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