CISAK 2013 C4/P/42

Effect of Mangosteen (Garcinia mangostana L.)

Pericarp Extract On Biofilm Formation Of
Streptococcus Mutans On Orthodontic Wire
(In -Vitro)
Nurul A.R. Putranti
1
, Amalia R. Mufida
2
and Salma N.
3
Retno I.
4

1,2,3
Faculty of Dentistry, Airlangga University
Jalan Mayjen Prof.Dr.Moestopo No.47 Surabaya,Indonesia
4
Department of Oral Biology, Faculty of Dentistry, Airlangga University
Jalan Mayjen Prof.Dr.Moestopo No.47 Surabaya,Indonesia
1
rizkyputranti@yahoo.co.jp
2
amaliaramadhanimufida@yahoo.com.
3
salma.nurdamayanti@gmail.com
retno_in2007@yahoo.co.id
Abstract. Hard and soft surfaces in the oral cavity are coated with a dental biofilms. Orthodontic wire are considered to be a clinical risk
factor in terms enamel integrity because of biofilm accumulation on these surface. This study is to examine the effect of Mangosteen
(Garcinia Mangostana L.) pericarp extracts on the biofilm formation of Streptoccus mutans on orthodontic wire. First, the minimum
inhibitory concentration (MIC) was determined. The stainless steel orthodontic wire was incubated in BHI broth containing Streptococcus
mutans and 0,39% concentration of mangosteen (Garcinia mangostana L.) pericarp extract. The colony forming unit (CFU) of Streptococcus
mutans on orthodontic wire was counted after 48 hours incubation on TYC agar. The statistical analysis used paired t test with 0,05
significance degree level The result of minimum inhibitory concentration (MIC) of Mangosteen (Garcinia mangostana.L) pericarp extracts on
Streptococcus mutans growth was determined at 0.39%. There were significant differences (p=0.003 ; p < 0,05) on biofilm formation of
Streptococcus mutans between sample groups and control. These finding suggest that mangosteen (Garcinia mangostana L.) pericarp extracts
had antibacterial activity toward Streptococcus mutans and decreased biofilm formation of Streptococcus mutans on orthodontic wires.
Keywords: mangosteen (Garcinia mangostana L.)pericarp , orthodontic wire, streptococcus mutans adhesion
A. INTRODUCTION
Dental plaque is a biofilm of oral microorganisms on
the tooth surface that plays an important part in the development
of dental caries. Among bacteria in dental plaque, Streptococus
mutans is considered the most significant cariogenic bacteria.[1]
Bacterial adhesion to biomaterials and it ability to form biofilm
on bodies are well-known as steps in the pathogenesis of oral
infections [2].

Orthodontic appliances are considered to be a clinical
risk factor in terms of enamel integrity because of biofilm
accumulation on these surfaces [3]. Indeed, increased levels of
mutans streptococci and lactobacilli were detected in the oral
cavity folowing orthodontic treatments [4]. Orthodontic wire
which is used for long time during orthodontic treatment tends to
create new surfaces available for biofilm formation an therefore
to increase the level of microorganism in the oral cavity. It has
long been suggested that orthodontic wires lead to increased
plaque accumulation and elevated levels of streptococci and
lactobacilli. In addition, orthodontic patients with fixed
appliances frequently present and abundance of Streptococcus
mutans in plaque compared with untreated orthodontic patients.
Therefore, prevention of bacterial attachment to orthodontic
wires is a critical concern for orthodontists [5].

Garcinia mangostana L. commonly known as
mangosteen, is a tropical evergreen tree , presumed to have a
combination of appealing subjective characteristics, such as
taste, fragrance and visual qualities, nutrient richness,
antioxidant strength and potential impact for lowering risk of
human diseases [6]. Its pericarp contains a variety of xanthones,
such as -, -, -mangosteen which have remarkable biological
activities.Among these, -mangosteen has the most antibacterial
activity.
7
Extract from its pericarp has demonstrated antibacterial
activity againts a wide variety of microorganism including
Stphylococcus aereus (both normal and methicilin-resistant),
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Staphylococus epidermidis, Pseudomonas aeruginosa,
Salmonella typhimurium, Enterococcus species, Mycobacterium
tuberculosis and Propionibacterium acnes [8]-[11].

Some researchers carried out the effect mangosteen
pericarp extracts. for medicine. However, they mostly
concentrated on the effect of Mangosteen pericarp extract for
medicine. The effect a mangosteen pericarp extract as a
prevention on dental bioflm formation has not been well studied.
Since Streptococcus mutans exists almost exclusively in oral
bioffilms and is considered the primary etiologic agent of human
dental caries, we evaluated the effect of Mangosteen pericarp
extract on biofilm formation by Streptococcus mutans on
orthodontic wire in vitro.
B. MATERIAL AND METHODS
1. Preparation of Mangosteen pericarp extract
A fresh mangosteen pericarp was cleansed from dirt. Then
washed it into hot water, cut into pieces 0,5cm and the
mangosteen pericarp dried in the air flow for 2 hours of heat.
After the drying process, 300 gr of mangosteen pericarp was
inserted in to extractors machine, then 96% ethanol solvent was
added and shaken for 6 hours. The next process was filtered
using filter paper and put in a vacuum evaporator for 2-3 hours
until all the alcohol solvent separately, in order to obtain viscous
red-colored mangosteen pericarp extracts.
2. Antimicrobial activity test
Antimicrobial tests of Garcinia mangostana L. pericarp
extracts were carried out by disc diffusion. Streptococccus
mutans were evenly spread using sterilized cotton swab on TYC
plates, respectively.
Sterilized (autoclave at 121C for 15 minutes) Whatman AA
discs (6 mm in diameter) were placed on the plates and 20 L of
each extract at 2mL concentration was pipetted aseptically onto
the discs. The quantity of each extract was 2 mL/disc. Discs
prepared without extract were used as a negative control. The
discs were left to dry at room temperature before they were
placed on the surface of the TYC agar. The TYC plates were
then incubated for 48 hours. Each extract was tested against
each organism in triplicates. The cultures were examined for
areas of no growth around the disc (zone of inhibition). The
microorganisms that were susceptible to antimicrobial agents
were inhibited at a distance from the disc whereas the resistant
strains grew up to the edge of disc. Measurement of the
inhibition zones around the discs were done using rulers and
expressed in millimeter (mm) unit. Based on the antimicrobial
sensitivity test results, extracts that produced an inhibition zone
greater than 6 mm in the disc diffusion test were separated and
further examined for the minimum inhibitory concentration
(MIC) values.
3. Minimum inhibitory Concentration (MIC) Determination
MIC was determined by a broth dilution method. Thirty-six
well microtiter plates were used to identified minimum
inhibitory concentration (MIC), each mangosteen pericarp
extract concentration being tested in triplicate at serial dilutions
of mangosteen extract from 50%, 25%, 12.5%, 6.25%, 3.13% ,
1.56%, 0.78%, 0.39% , 0.19%. Colums 1 and 2 were used for
mangosteen pericarp extract as a negative control, and colomns
11 and 12 were used for positive controls. Each well was filled
with 5ml BHI broth containing 5ml Mangosteen extract and
Streptococcus mutans, incubated overnight at 37C.
Microbial suspension in sterile water containing 10
8
CFU/ml of bacteria was adjusted to McFarland no 0,5 standard
turbidity. After incubation 24 hours, a 20ml of bacterial
suspension was applied and spread on the TYC agar and
incubated for 24 hours.

4. Streptococcus mutans attachment on orthodontic wire
To evaluate the effect of mangosteen pericarp extract on
biofilm formation, we used sterile orthodontic wire (Ortho
Organizer Inc. Aston Avenue; stainless steel, round, 0.016) for
biofilm formation. The 2-cm orthodontic wires were coated with
freshly collected 1 ml of the unstimulate clarified whole saliva.
Following 1 hour of incubation at 37C, the saliva-coated
orthodontic wires were moved to Streptococcus mutans
containing sterille BHI broth. Samples were then incubated for
24 hours at anaerob condition. The orthodontic wire was
removed from BHI broth, coated with 0.39% of concentration of
mangosteen pericarp extract for 4 hours and washed with
phosphate buffered saline without calcium and magnesium
(PBS(-)). The orthodontic wire was vortex for 10 seconds. A
solution was plated on TYC agar by spreading technique. The
colony forming unit (CFU) of Streptococcus mutans on
orthodontic wire was counted on TYC agar after 48 hours
incubation at 37C
5. Statistical analysis
All statistical computations were performed by SPSS for
Windows (version 13.0;SPSS, Inc.,Chicago, IL,USA). Data from
bacterial attachment on orthodontic wire were analyzed by used
paired t test. Statistical significance was defined as P<0.05.
C. RESULT
The result shows, that extract of mangosteen pericarp showed
inhibitory activity againts Streptococcus mutans .The diameter
of maximum inhibition zone was determined 13.9 mm at
concentration 100% and the minimum zone of inhibition zone
was 9.4 mm at concentration 3.13%. Control show antibacterial
activity with zone of inhibition 6,13mm. Minimum inhibitory
concentration (MIC) of mangosteen pericarp extract againts
Streptococcus mutans was at concentration 0,39%. It shows that
mangosteen pericarp extract was active againts Streptococcus
mutans.

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Fig. 1. Test result for MIC after serial dilution of mangosteen extract from
100%, 50%, 25%, 12.5%, 6.25%, 3.13%, 1.56%, 0.78% (a) , until 0.39% , 0.19%
(b)









Fig. 3. Colony of Streptococcus mutans on TYC agar
In this study, the pericarp extract of Garcinia
mangostana L. shown inhibit Streptococcus mutans attachment
on the surface of orthodontic wire (p=0.003 ; p < 0,05) (Table 2).


Fig. 2. Colony of Streptococcus mutans on orthodontic wire with and without
mangosteen pericarp extract

Table 1. Statistical analysis of S. mutans attachment on orthodontic wire shown significant differences (p=0.003 ; p < 0,05)
D. DISCUSSION
The pericarp extract of garcinia mangostana linn has a
wide spectrum of antibacterial against several gram positive and
gram negative bacteria such as; Staphylococcus aureus,
Staphylococcus epidermidis, Bacillus subtilis,
Propionibacterium acnes, Pseudomonas aeruginosa, Salmonella
enteritidis, and Eschercia colli [8],[11]. Torrunruang K,et al
suggest that pericarp extract of Garcinia mangostana was
effective againts cariogenic Streptococcus mutans [12].

In the present study, we examined the effect of pericarp
extract of Garcinia mangostana L. on dental biofilm formation
using Streptococus mutans on the surface of orthodontic wire.
Biofilm formation begins with pellicle formation. The pellicle is
a thin coating of salivary proteins that attach to the tooth surface
or material surface within minutes after a professional cleaning.
The pellicle acts like double-sided adhesive tape, adhering to
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the tooth surface or material surface on one side and on the other
side, providing a sticky surface that facilitate bacterial
attachment to the tooth surface or on the surface of materials.
[13]-[15] The purpose using saliva in this study was to cover the
specimens with a pellicle.[2],[16]. The initial conditioning
salivary coat plays and important role in bacterial adsorption to
surfaces as the absorbed proteins can manipulate bacterial
adhesion to the conditioning film. Albumin , a protein found in
the saliva, is an inhibitor of hydrophobic interactions, and was
implicated in bacterial adhesion mediated by hydrophobic
interactions. Amylase, another salivary protein, has been shown
to promote bacterial adhesion by inducing specific interactions
with several types of Streptococci [17].

Firtsly, we identified the minimum bacteria concentration (MIC)
and founded that the MIC was at concentration 0,39%. Another
studies reports that the MIC of the pericarp extract for
Streptococcus mutans was 0.625 g/ml. [12]. We used DMSO
(Dimethyl Sulfoxide) for control to determined bacterial
inhibition activity. Another research say that DMSO has
antibacterial activity of fungal.
The biofilm formation on the surface of orthodontic
wire was examined by using viable counting method (CFU/ml)
and the result shown significance different between the samples
and control (p<0.003). The result suggested that pericarp extract
of Garcinia mangostana L. can inhibit biofilm formation on the
surface of orthodontic stainless steel wire.
Mangosteen pericarp extract containing several
xanthones such as , and mangosteen, gartinin and iso
mangosteen [7]. Chemical laboratory test result in this study was
shown the pericarp extract mangosteen containing xhanton
(10.70%) , saponin (3.82%) , tanin (5.92%) , mangostin
(2.82%), mangostin (7.88%), flavonoid (1.88%) and
mangostanin (11,88%). The chemical components of the extract
often vary depending upon the extraction protocol. When using
40% ethanol as solvent , the extract containing 10% mangostin
and 12% mangostin. Another study using ethyl acetate as
solvent reported that the extract was composed of 77,8% -
mangostin and 15,9% -mangostin. Among xanthone derivates
from mangosteen extract, -mangostin has been shown by
several study to exert the most potent antibacterial activity
[12].The possible explanation from this study is the mangostin
contained the pericarp extract of Garcinia mangostana L.
might be plays an important role to inhibit biofilm formation on
the surface of orthodontic wire. Further studies are still required
to clarify the mechanism inhibition of biofilm formation on the
surface of orthodontic wire by using pericarp extracts of
Garcinia mangostana.
E. CONCLUSION
In conclusion, this study showed that extract from
Garcinia Mangostana was effective againts antibacterial activity
of Streptococcus mutans and it also decreased biofilm formation
of Streptococcus mutans on orthodontic wires.
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