Download antibacterial activity of bee propolis against clinical strains of

International Journal of Pharmaceutical Studies and Research
E-ISSN 2229-4619
Research Article
ANTIBACTERIAL ACTIVITY OF BEE PROPOLIS AGAINST
CLINICAL STRAINS OF STREPTOCOCCUS MUTANS AND
SYNERGISM WITH CHLORHEXIDINE
Arul Selvan K1, Rajendra Singh.C2 and Dr.Prabhu T3
Address for Correspondence
1
Asst. Professor of Microbiology, Krishnadevaraya College of Dental Sciences & Hospital Bangalore, Ph.D
Scholar, Dr. MGR University, Chennai.
2
Lecturer in Bio-Technology, Sir.M. Visvesvaraya Institute of Technology, Bangalore
3
Professor of Microbiology, formerly Principal, Bangalore Medical College & PhD Supervisor, Dr. MGR
University, Chennai
Email: [email protected]
ABSTRACT
Dental caries is a common oral infection prevalent in developing countries. Streptococcus mutans, a commensal bacteria of the
oral cavity, plays a major role in the etiology of caries. Dentists around the world use fluoride varnishes and pastes in the
prophylaxis and treatment of caries. Mouthrinses containing Chlorhexidine are also being used in the prevention of caries.
Fluoride is a highly toxic substance and has been observed to cause several side effects. Chlorhexidine has been recorded to be
less effective in controlling caries. Hence natural substances like Propolis, which possesses high anti-bacterial activity, have
attracted the attention of researchers in the prophylaxis of dental caries. The inhibitory effect of ethanolic extract of Propolis on
Streptococcus mutans has been well established in previous studies. In our study we prepared the ethanolic extract of Propolis
(EEP) in our laboratory and examined the inhibitory effect of EEP, Chlorhexidine and 1:1(v / v) combination of EEP &
Chlorhexidine against 50 strains of Streptococcus mutans isolated from patients with dental caries attending our dental hospital.
The disc diffusion method (Kirby-Bauer) was used to detect the inhibition of the growth of mutans streptococci. We have
observed that all the clinical strains were inhibited by EEP, the mean zone size of inhibition of growth being 26mm.
Chlorhexidine also inhibited all the strains, the mean zone size being 22.56mm. the combination of EEP & Chlorhexidine was
observed to be the most effective in the inhibition Strep. mutams, the mean zone size of inhibiton being 32.22mm. We are
reporting that Propolis exhibits synergistic activity with chlorhexidine in the inhibition of mutans streptococci.
KEY WORDS: Propolis, Chlorhexidine, Streptococcus mutans, Dental caries.
active agents to the teeth and gums. Many recent
INTRODUCTION
Streptococcus mutans is considered one of the most
studies have demonstrated antimicrobial activity
important cariogenic species of the human oral
against selected oral pathogens from natural sources.
microbial flora (8). There is ample evidence for the
Natural products have been used for folk medicine
association between S. mutants and dental caries
purposes throughout the world for thousands of
(15). The suppression of S. mutans by antimicrobial
years. Many of them have demonstrable
agents, especially locally administered chlorhexidine,
pharmacological properties, such as antimicrobial,
is consequently of clinical importance (13, 14).
anti-inflammatory and cytostatic, among others and
more recently propolis has been recognized as useful
Dental plaque, a film of microorganisms on the tooth
for human and veterinary medicine. Recently,
surface, plays an important part in the development
Propolis has been attracting the attention of
of caries and periodontal diseases (9). Some of the
researchers due to its various biological activities and
oral commensal bacteria such as the mutans
therapeutic properties. Propolis is a complex resinous
streptococci can colonize the tooth surface and
material collected by Apis mellifera honey bees from
initiate plaque formation by their ability to synthesize
various plant sources and mixed with secreted
extracellular polysaccharides from sucrose, mainly
beeswax. It is non-toxic and possesses multiple
water-insoluble glucan, using the enzyme
glucosyltransferase (3). Inhibiting the colonization of
pharmacological effects and a complex chemical
mutans Streptococci on the tooth surface is believed
composition (2). Propolis has been shown to reduce
to prevent the formation of dental plaque and
the incidence of dental caries in rats.(5). The
development of dental caries. The use of mouth
mechanism of antimicrobial activity of propolis is
rinses
containing
Chlorhexidine
has
been
complex and could be attributed to the synergistic
recommended by many dentists to prevent caries.
activity between phenolic and other compounds (7).
Chlorhexidine has been reported to reduce the
The combination of propolis with antibiotics can
accumulation of plaque. Mouthrinses are widely used
reduce drug dosages, minimize drug side effects, and
as adjuncts to oral hygiene and in the delivery of
decrease chances of drug resistance. The synergism
IJPSR/Vol. II/ Issue I/January- March, 2011/85-90
International Journal of Pharmaceutical Studies and Research
between EEP and antimicrobial drugs, especially
those agents that interfere on bacterial protein
synthesis such as choramphenicol, gentamicin ,
netilmicin , tetracycline ,and vancomycin has been
demonstrated. (4). In our study we have examined the
antimicrobial activity of ethanol extract of Propolis,
Chlorhexidine, a combination of Propolis and
Chlorhexidine
against
clinical
strains
of
Streptococcus mutans.
MATERIALS AND METHODS
PROPOLIS SAMPLES
Beehives were collected from different places in
Hunasamaranahalli village, Bangalore. The samples
were kept in a deep freezer at -200C for 3 days. The
hardened material was ground by a grinder and
ethanolic extraction was done as per the procedure
described by Bianchi, 1995(1). 30 grams of the
ground propolis was dissolved in 100 ml of 95%
Ethanol (A.R. grade, S.D. Fine Chemicals, Mumbai)
(Figure 1). This mixture was preserved for 2 days in a
bottle corked tightly and kept in an incubator at 300C
. After dissolving, it was filtered twice with Whatman
No.4 and No.1 filter papers. This solution was
evaporated by negative pressure at 50 0 C to get a
yellowish solid. This was called the ethanol extract of
propolis (EEP) and was preserved at +40C
Chlorhexidine diacetate was obtained from a
commercial source (Hi-Media, Mumbai).
Figure 1: Ethanol extraction of Propolis
Clinical isolates
Dimineralized portions of the decayed tooth were
obtained from 50 patients with caries attending the
out patient clinic of the Krishnadevaraya College of
Dental Sciences & Hospital, Bangalore. The patients
had dental caries at various stages. The specimens
were collected in 2 ml of sterile thioglycollate broth
in screw capped vials and incubated for 30 minutes at
370 C.
Primary isolation medium
Mitis – Salivarius Bacitracin agar (MSB agar) with
0.1 % Potassium tellurite was used for the primary
isolation of mutans streptococci from the clinical
IJPSR/Vol. II/ Issue I/January- March, 2011/85-90
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specimens. This medium contains the selective agents
- crystal violet, potassium tellurite and Bacitracin.
These agents inhibit most gram-negative bacilli and
most gram-positive bacteria except streptococci.
Sucrose is incorporated in a 5% concentration which
is utilized as an energy source by the streptococci.
The specimens were streaked onto a dry MSB agar
medium for isolation and incubated in a candle
extinction jar for 24 hours at 370C. Streptococcus
mutans forms raised, convex, undulate, opaque, pale
blue colonies, granular "frosted glass" appearance,
sometimes exhibiting a glistening bubble on surface
of colony due to excessive synthesis of glucan from
sucrose. (Figure 2).
Figure 2: Colonies of Streptococcus mutans on
Mitis-Salivarius-Bacitracin Agar
Identification of mutans streptococci
Strep. mutans was identified by Gram stain
morphology, culture morphology and biochemical
tests (mannitol / sorbitol fermentation positive,
esculin hydrolysis positive and arginine hydrolysis
negative)
Figure 3: Gram Stain appearance of S. mutans
Anti-microbial Susceptibility testing
Anti bacterial susceptibility testing for propolis was
done by the disk diffusion method as per the National
Council for Clinical Laboratory Standards guidelines
(NCCLS).
Disk diffusion test: (Kirby-Bauer method)
Reagents for the Disk Diffusion Test
1) Brain Heart Infusion agar (BHI agar)
BHI agar is a highly enriched medium for the growth of
fastidious bacteria, useful in the cultivation of
streptococci. The medium has been found effective in
the recovery of dental pathogens from clinical
International Journal of Pharmaceutical Studies and Research
specimens. The medium contains pancreatic digest of
casein,brain heart infusion, peptic digest of animal
tissue, sodium chloride,
disodium phosphate,
dextrose and agar. The brain heart infusion and
peptone provide the nutrients, organic nitrogen,
carbon, sulfur, vitamins and trace substances.
Dextrose provides the carbohydrate source for
fermentative microorganisms. Disodium phosphate is
added to the medium in order to maintain an optimal
pH. BHI agar was prepared in our laboratory from a
commercially available dehydrated base (Hi – Media,
Mumbai) according to the manufacturer's instructions.
Immediately after autoclaving, the medium was
allowed to cool in a 45 to 50°C water bath. The freshly
prepared and cooled medium was poured into glass
flat-bottomed petri dishes on a level, horizontal surface
to give a uniform depth of approximately 4 mm. This
corresponds to 25 to 30 ml for plates with a diameter of
100 mm. The agar medium was allowed to cool to
room temperature and unless the plates were used the
same day, they were stored in a refrigerator (2 to 8°C).
Plates were used within seven days after preparation. A
representative sample of each batch of plates was
examined for sterility by incubating at 30 to 35°C for
48 hours.
Preparation of anti-microbial agents
1) 0.2 % chlorhexidine digluconate solution was used
as the positive control (soln A)
2) 50 mg of the dried ethanolic extract of Propolis
was dissolved in 10 ml of sterile dimethyl
sulphoxide (DMSO) and was used as the test antimicrobial agent. (soln B)
3) A mixture of 1:1 (v / v) of solution A & B (soln C)
4) Sterile dimethyl sulphoxide was used as the
negative control (soln D)
Preparation of filter paper discs
Whatman filter paper No. 1 was used to prepare discs
approximately 6 mm in diameter, which are placed in a
petri dish and sterilized in a hot air oven at 1600 C for 2
hours.
Turbidity standard for inoculum preparation
To standardize the inoculum density for the
antimicrobial susceptibility test, a BaSO4 turbidity
standard, equivalent to a 0.5 McFarland standard was
used .
Procedure for Performing the Disc Diffusion Test
Inoculum Preparation
Growth Method
The growth method was performed as follows
1. At least three to five well-isolated colonies of
the same morphological type were selected from
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2.
3.
4.
5.
6.
7.
8.
9.
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an Blood agar plate culture. The top of each
colony was touched with a loop, and the growth
was transferred into a tube containing 5 ml of
BHI broth.
The broth culture was incubated at 370 C for 4
hours.
The turbidity of the actively growing broth
culture was adjusted with sterile saline to obtain
turbidity optically comparable to that of the 0.5
McFarland standards.
This results in a
suspension containing approximately 1.5 x 108
CFU/ml for strep mutans ATCC® 25175
Within 15 minutes of adjusting the turbidity of
the broth 50 µl of the broth was transferred to
the middle of a dry BHI agar and spread
uniformly over the entire agar surface using a
sterile L spreader.
4 sterile filter paper discs were placed at equal
distances on the inoculated medium using sterile
forceps.
The discs were impregnated with
1) 5µl of 0.2% chlorhexidine digluconate
solution (soln A) - Positive control
2) 5µl of ethanol extract of Propolis
dissolved in DMSO (soln B)
3) 5µl of combination of Propolis &
Chlorhexidine (soln C)
4) 5µl of sterile DMSOsolution (Soln D) –
Negative control
The plates were incubated in a candle
extinction jar for hours at 370C.
After 18 hours of incubation the plates were
observed for uniform lawn culture growth of
the bacteria and the zone of inhibition of
growth around the discs was measured.
The tests were performed in duplicate.
3
2
1
Figure 4: Zone of inhibition of growth of
Streptococcus mutans against
1) 0.2% Chlorhexidine diacetate (disc 1)
2) 0.5% Ethanol extract of Propolis EEP (disc 2)
3) Mixture of Chlorhexidine & EEP (disc 3)
International Journal of Pharmaceutical Studies and Research
RESULTS
All the strains of Streptococcus mutans isolated from
the patients (n = 50) were inhibited by the ethanolic
extract of Propolis, (Figure 1) the mean zone size of
inhibition of growth being 26.0 mm. (Graph 1) All
the strains were also inhibited by Chlorhexidine, the
mean zone size being 22.6 mm. The highest antimicrobial activity was observed in the combination of
Propolis & Chlorhexidine which inhibited the growth
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of Strep mutans, with the mean zone size of inhibiton
being 32.22 mm. DMSO did not inhibit the growth of
any strain of Streptococci. The actual zone sizes of
inhibition of growth Strep mutans against all tha
reagents used is presented in Table 1.
Chi square for trend was calculated to see the trend in
the anti-microbial activity of the different
antimicrobials used in the study and found significant
(P < 0.001).
Graph 1
Synergistic activity of Propolis &
Chlorhexidine
35
32.22
30
Zone size of
Inhibition (mm)
25
26
22.56
20
Mean zone size of
Inhibition
15
10
5
0
1
2
3
Anti-microbial agents
LEGEND:
1. Mean zone size of inhibition (mm) of growth for 0.2% chlorhexidine (Solution A)
2. Mean zone size of inhibition (mm) of growth for 0.5% Propolis (Solution A)
3. Mean zone size of inhibition (mm) of growth for 1:1 Propolis & Chlorhexidine
Table 1: Results of the antimicrobial susceptibility testing (Disc diffusion method) showing Size of the zone of
inhibition of growth
Clinical
Strain
1
2
3
4
5
6
7
8
9
10
0.2 %
Chlorhexidine
(Soln A)
26 mm
23 mm
22 mm
21 mm
26 mm
24 mm
22 mm
25 mm
21 mm
22 mm
0.5 % Propolis
(Soln B)
Combination
(Soln C)
DMSO
(Soln D)
28 mm
31mm
25 mm
25 mm
28 mm
27 mm
24 mm
28 mm
24 mm
25 mm
36 mm
41 mm
34 mm
32 mm
34mm
34 mm
28 mm
33 mm
31 mm
31 mm
0 mm
0 mm
0 mm
0 mm
0 mm
0 mm
0 mm
0 mm
0 mm
0 mm
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International Journal of Pharmaceutical Studies and Research
Clinical
Strain
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
0.2 %
Chlorhexidine
(Soln A)
24 mm
20 mm
23 mm
24 mm
24 mm
19 mm
23 mm
20 mm
22 mm
24 mm
21 mm
18 mm
19 mm
23 mm
25 mm
26 mm
21 mm
25 mm
17 mm
19 mm
23 mm
18 mm
22 mm
21 mm
24 mm
24 mm
21 mm
26 mm
25 mm
24 mm
22 mm
24 mm
20 mm
21 mm
23 mm
24 mm
26 mm
24 mm
23 mm
24 mm
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0.5 % Propolis
(Soln B)
Combination
(Soln C)
DMSO
(Soln D)
26 mm
22 mm
25 mm
26 mm
29 mm
22 mm
27 mm
23 mm
27 mm
26 mm
26 mm
23 mm
24 mm
24 mm
29 mm
28 mm
24 mm
28 mm
19 mm
23 mm
28 mm
21 mm
24 mm
22 mm
27 mm
28 mm
25 mm
28 mm
28 mm
27 mm
27 mm
28 mm
23 mm
24 mm
27 mm
31 mm
34 mm
29 mm
26 mm
27 mm
30 mm
29 mm
31 mm
31 mm
32 mm
28 mm
31 mm
29 mm
34 mm
30 mm
32 mm
26 mm
30 mm
29 mm
32 mm
33 mm
31 mm
34 mm
24 mm
27 mm
35 mm
39 mm
36 mm
28 mm
32 mm
36 mm
31 mm
33 mm
35 mm
31 mm
34 mm
34 mm
27 mm
26 mm
31 mm
40 mm
42 mm
38mm
34 mm
32 mm
0 mm
0 mm
0 mm
0 mm
0 mm
0 mm
0 mm
0 mm
0 mm
0 mm
0 mm
0 mm
0 mm
0 mm
0 mm
0 mm
0 mm
0 mm
0 mm
0 mm
0 mm
0 mm
0 mm
0 mm
0 mm
0 mm
0 mm
0 mm
0 mm
0 mm
0 mm
0 mm
0 mm
0 mm
0 mm
0 mm
0 mm
0 mm
0 mm
0 mm
DISCUSSION
Dental caries is one of the common oral infections
and inflicts a huge burden on the population and
economy of developing countries like India. Unlike
other infections, dental caries continues throughout
life, with new lesions in older people, an observation
consistent with the prevalence of caries in adults
where up to 95% have experienced one or more
carious lesions.(10). Currently, fluoride in various
preparations is the mainstay for caries prevention.
Fluoride exerts its major effects by reducing enameldentine
demineralization
and
enhancing
IJPSR/Vol. II/ Issue I/January- March, 2011/85-90
remineralization of early caries lesions. Rated by
degree of toxicity, fluorine would be placed
somewhere between lead and arsenic (6).
Research indicates that fluorides may cause allergies,
osteoporosis, and dental fluorosis, as well as
neurological damage (11). Nevertheless, as currently
used, fluoride offers incomplete protection against
dental caries. Previous studies have substantiated that
Propolis is highly active against pathogenic bacteria.
The minimal inhibitory concentrations (MICs) of
EEPs for C. jejuni isolates have been determined by
the agar dilution method with EEP showing MIC
International Journal of Pharmaceutical Studies and Research
values of 0.3125–0.156 mg/mL (12). Propolis being
non toxic has enormous medicinal scope. In our
study we have observed that bee propolis in
combination with chlorhexidine possess high antimicrobial activity against Streptococcus mutans.
Ethanol extract of Propolis has synergistic activity
with chlorhexidine in inhibiting the growth of Strep
mutans. Propolis in combination with chlorhexidine
can suppress the pathogenic potential of dental
plaque by inhibiting the adherence and accumulation
of cariogenic streptococci on the tooth surface. The
inhibition of growth of the clinical strains by trace
quantities of propolis in our study suggests that it can
be used in treatment of dental caries. Further, the
concentration of chlorhexidine in mouthwashes can
be reduced without lowering the anti-bacterial
potential.
ACKNOWLEDGEMENT
The authors are grateful to Sri Krishnadevaraya
Educational Trust for permitting to carry out this
study through departmental grants. We are also
thankful to Mr. Arun Kumar, Biostatiatician for
providing statistical help.
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10) National Institutes of Health. (2001). Diagnosis
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