Artificial drugs have unpleasant side effects, on the other hand, the number
of drug resistant microorganisms is increasing. So researchers are trying to
pay more attention to herbal drugs. Research centers and world health organization
prepare lots of programs to make use of plants extracts.
Mouthwashes for example, chlorhexidine, have several adverse effects despite
good plaque control and an antimicrobial effect (Gurgan
et al., 2006; Jenabian et al., 2008).
Finding plants that have antimicrobial effects and using them as mouthwashes
have advantages, such as, a decrease of side effects and also they are more
Most studies have been done on skin, respiratory and urinary system pathogens
and there has been little research about oral pathogens (Ahmad
and Beg, 2001).
Researchers have evaluated antimicrobial effects of some plants with different
methods. Among them, one study showed that persica mouthwash cannot alter oral
microbiota especially Streptococcus mutans, so it cannot prevent dental
caries (Jajarm et al., 2009).
Bozin et al. (2007) detected the antibacterial
activity of rosemary and sage on Escherichia coli, Salmonella typhi,
S. enteritidis and Shigella sonei.
De et al. (1999) evaluted antimicrobial activities
of 35 different Indian spices traditionally used. This study indicated that
clove, cinnamon, bishops weed, chilli, horseraddish, cumin, tararind,
black cumin, pomegranate seeds, nutmeg, garlic, onion, tejpat, celery, cambodge,
have potent antimicrobial activities against the test organisms such as Bacillus
subtilis, Escherichia coli and Saccharomyces cerevisiae.
Few research studies are available on antibacterial effects of traditional
plants on Streptococcus mutans. One research revealed the crude extract
endophytic Streptomyces sp. ST8 decrease bacterial adherence of Streptococcus
mutans (Taechowisan et al., 2008).
In Xavier and Vijayalakshmi (2007) study, ethanol extracts
of Allium sativum bulb and Azadirachta indica leaf extracts exhibited high degree
of inhibition effect on Streptococcus mutans (Xavier
and Vijayalakshmi, 2007) Crude aqueous extract of Piper betle L.
exhibited reduced effect towards the growth, adhering ability, glucosyltransferase
activity against Streptococcus mutans (Nalina and
Rahim, 2007). Because this microorganism has been implicated as one of the
oral bacteria that cause dental caries, this study was initiated to complete
previous studies on the antimicrobial effects of some plants that are traditionally
used as medicine against Streptococcus mutans. To the best of our knowledge,
antimicrobial activity of these selected plants has not evaluated on Streptococcus
mutans. In this study, the antimicrobial effects of ten herbal extracts
on Streptococcus mutans were compared with chlorhexidine whose antimicrobial
activity has been demonstrated in some previous studies (Gurgan
et al., 2006; Van der Vyver et al., 2009).
These studies could prove useful for providing herbal mouthwash with antibacterial
activity against Streptococcus mutans.
MATERIALS AND METHODS
The test organism; Streptococcus mutans was a standard strain ATCC-1683 that
was prepared from the Scientific and Industrial Research Center in Tehran, Iran
in April 2008. For this test, 0.5 Mc Farland standard microbial suspension was
used which contains 108 bacteria mL-1 nutrient broths.
Streptococcus mutans was cultured on blood agar which was used as a
culture media. The medicinal plants traditionally used in medicines for oral
and gingival disease were selected. These 10 plants were: Matricaria chamomillathyme
(chamomile), Mentha arvensis (mint), Allium vineale (garlic),
Cinnamomum zeylanicum (cinnamon), Melaleuca alternifolia (tea
tree), Eugenia aromatica (clove), Mentha spicata (spearmint),
Salvia officinalis (sage) Rosemaryinus officinalis (rosemary)
and Thymus serpyllum (thyme).
For Preparation of plant extracts, the method of Alade and
Irobi (1993) was adapted with little modifications. Thirty grams of each
powdered plant material were dissolved in 100 mm of pure methanol and placed
on a shaker for 48 h (Alade and Irobi, 1993). Each mixture
was stirred every 24 h using a sterile glass rod and then was passed through
a Whatman filter paper No. 1 (Whatman, UK). They were kept in an incubator at
37°C for 48 h to produce 0.5 to 2.5 mg mL-1 concentrates.
The antimicrobial sensitivity of test extracts was determined by the standard
Disc diffusion method.
One hundred needle tips containing 10 disks were placed on a medical vial and
were sterilized with autoclave. In sterile conditions, every needle consisting
of 10 disks was placed into a glass containing one of the plant methanol extracts
After an hour the discs were removed and put in a 40°C incubator for 20
min to be dried.
On each plate, one plant extract disc, one 0.2% chlorhexidine disc which was
used as a positive control and one methanol and one blank disc which were used
as negative controls were placed. These disks were placed at a distance of 15
mm from the edge of the plate and 24 mm from the center of the next disk. The
plates were incubated at 37°C for 24 h.
After 24 h, the antimicrobial activity was evaluated by measuring the inhibitory
zone diameter observed with a pair of calipers. This test was repeated 10 times
for each plant extract.
The zone of inhibition of each test plant against Streptococcus mutans
was compared with chlorhexidine using T test analysis.
After 24 h, in all plates, no zone indicative of the lack of growth around
the methanol and the empty discs which were used as negative controls was observed.
In all plates, the inhibition zone around the chlorhexidine discs which were
used as positive control was observed. The mean of inhibition zone was 14.06
mm; S.D. = 1.70 mm.
Among test plants, there was a halo indicative of the lack of growth around
rosemary and clove discs. The mean of the inhibitory zone related to rosemary
and clove were 11.52 mm; SD = 0.96 mm and 15.26 mm; SD = 1.55 mm, respectively
(Fig. 1, 2 and Table 1).
A sample from the zone of inhibition around each plant was taken. In a gram
staining sample taken from the zone of inhibitory around the clove disks, Streptococcus
mutans was observed by microscope.
||Comparison of the mean the inhibitory zone of the plant extracts
discs with chlorhexidine
|*The hemolysis zone around the clove disc
|| The inhibitory zone around the rosemary disc
|| The hemolysis zone around the clove disc
Most probably, the acidic pH of clove led to lyses of blood agar culture media.
Clove pH which was determined with chromatograph paper was found to be between
2.0 to 2.5 which demonstrate that the clove didnt have antimicrobial activity.
These halos are related to the ability of clove for hemolysis. Then, this test
was repeated for clove extract in empty blood agar plates in which the hemolysis
zone, was also observed.
The mean of the inhibitory zone related to rosemary was 11.52±0.96 and
it was significantly less than chlorhexidine (p = 0.001) (Table
The result of the screening of antibacterial activity of 10 plant extracts
against Streptococcus mutans is shown in Table 1. As
the diameter of every disc was 0.6 mm, according to this table, except rosemary
and clove discs, halo indicative of the lack of growth wasnt seen around
other plant discs.
In this study, the antimicrobial activity of 10 traditional plant extract against
Streptococcus mutans was evaluated by the standard Disc Diffusion method.
The results of this study showed that only rosemary had antimicrobial activity
against Streptococcus mutans.
Researchers have studied the antimicrobial effects of some plant extracts but
little research has been done on Streptococcus mutans.
These studies showed antimicrobial activity of other plants species on Streptococcus
mutans, but other kinds of herbal extracts such as ethanol or aqueous extracts
were evaluated (Nalina and Rahim, 2007).
To the best of our knowledge, antimicrobial activity of herbal extracts which
are tested in present study have not evaluated against Streptococcus mutans.
Some test plants had antimicrobial effects in previous research but in the present
study, antimicrobial activity was not seen.
For example, Groppo et al. (2002) studied antimicrobial
activity of garlic, and tea tree oil. In this study, dishes containing blood
agar and Mitis Salivavius Bacitaci agar were incubated with subjects' saliva
total microorganisms. The tea tree oil group showed antimicrobial activity against
Streptococcus mutans and other oral microorganisms but the garlic group
showed antimicrobial activity, just against Streptococcus mutans (Groppo
et al., 2002).
In other research, the inhibitory effect of garlic was determined (Fani
et al., 2007; Groppo et al., 2007).
Fani et al. (2007) studied inhibitory activity
of the aqueous extract of garlic on multidrug-resistant strains of Streptococcus
mutans isolated from human carious teeth. This study showed inhibitory activity
of garlic Streptococcus mutans comparable with chlorhexidine by disc
sensivity test and broth dilution methods.
Despite the antibacterial effects of garlic extract, side effects such as unpleasant
taste, halitosis and nausea were reported (Groppo et al.,
In Bozin et al. (2007) study the essential oils
of rosemary and sage was analyzed by means of gas chromatography-mass spectrometry
for their antimicrobial activity against 13 bacterial strains. Similar to present
study the essential oils of rosemary and sage showed antimicrobial activity
against some bacteria.
Also, Cervenka et al. (2006) studied the antimicrobial
activity of seventeen spices and medicinal plant extracts against Arcobacter
butzleri by disc diffusion methods. They determined cinnamon, barberry,
chamomile, sage and rosemary extracts had strong antimicrobial activity toward
the arcobacter strains tested. The methanol extracts of these plants, except
cinnamon and rosemary, showed higher antimicrobial activity than the chloroform
In contrast to the present study, other studies showed the antimicrobial effect
of some plants. The study of the effect of aromatic oils against Pseudomonas
aeruginosa by the cup-palate methods, showed that cinnamon water possesses
profound activity against Pseudomonas aeruginosa. But, clove, dill and
peppermint waters exhibited no significant preservative actions (Ibrahim
and Ogunmodede, 1991).
In another study, mixed extracts were prepared from Chinese chive, cinnamon,
and corni fructus, which exhibited antimicrobial activity against Escherichia
coli. These extracts had excellent stability to heat, pH, and storage (Mau
et al., 2001).
Ankri and Mirelman (1999) showed that garlic allicin
exhibits antibacterial activity against a wide range of Gram-negative and Gram-positive
bacteria including multidrug-resistant enterotoxic genetic strains of Escherichia
coli. Also, garlic allicin have antifungal activity, antiparasitic activity
and antiviral activity. The main antimicrobial effect of allicin is due to its
chemical reaction with thiol groups of various enzymes, e.g. alcohol ehydrogenase,
thioredoxin reductase, and RNA polymerase. It can affect metabolism of proteinsase
involved in the virolence of E. coli.
Sasaki et al. (1999) detected the antibacterial
activity of garlic and showed that the usage of fresh garlic powder was more
effective than old garlic powder.
Weseler et al. (2005) studied the antibacterial
activity of chamomile against Helicobacter pylori; chamomile inhibited
bacterial growth. Another study demonstrated that chamomile has moderate antimicrobial
activities (McKay and Blumberg, 2006). These results
are different from present research in which the antibacterial activity of chamomile
Feres et al. (2005) showed that the antimicrobial
effects of clove and sage were less marked in comparison to chlorhexidine. This
result was similar to the present results in which neither plants exhibited
In contrast, other studies proved the antimicrobial effects of these plants
(Bozin et al., 2007; De et
al., 1999). These different results can be due to variant bacterial
strains. Also, some plants such as, thyme and sage have different strains in
different countries. On the other hand, differences in extract type and concentration
could lead to differences in results.
For example, Mahasneh and El-Oqlah (1999) studied the
antimicrobial activity of petroleum ether, ethanol, butanol, and aqueous crude
extracts of nine plants against four bacterial and three fungal species. Methanol
and hexane extracts did not show any activity. But, the butanol extracts Ononis
Spinosa (OS), Bryonia Syriaca (BS) had high moderate antifungal activity.
Ahmad et al. (1998) studied 82 Indian medicinal
plants traditionally used in medicines, by using well dioffusion method and
determined that alcoholic extracts showed greater activity than their corresponding
aqueous and hexane extracts (Van der Vyver et al.,
To the best of our knowledge, as few studies have been done on antimicrobial
effects of medical plants against oral pathogens, it is better that the effect
of herbal extracts on other oral bacteria that have cariogenic activity be studied.
Because of the antimicrobial effects of some medical plants, which have minimal
side effects in comparison with chemical drugs, more in vivo and in
vitro investigations about oral cavity flora should be recommended. It is
suggested that more research should be carried out to find plants with antimicrobial
activity for producing herbal mouthwashes.
This study demonstrated that rosemary has antibacterial activity against Streptococcus
mutans. If similar results are confirmed in clinical trials, this plant
extracts can be used to produce new, useful and economic antimicrobial mouthwashes.