Pharmaceutical Biology

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In vivo antibacterial activity of Garcinia mangostana

pericarp extract against methicillin-resistant
Staphylococcus aureus in a mouse superficial skin
infection model

Nitima Tatiya-aphiradee, Waranya Chatuphonprasert & Kanokwan

Jarukamjorn

To cite this article: Nitima Tatiya-aphiradee, Waranya Chatuphonprasert & Kanokwan

Jarukamjorn (2016) In�vivo antibacterial activity of Garcinia�mangostana pericarp extract
against methicillin-resistant Staphylococcus�aureus in a mouse superficial skin infection model,
Pharmaceutical Biology, 54:11, 2606-2615, DOI: 10.3109/13880209.2016.1172321

To link to this article: https://doi.org/10.3109/13880209.2016.1172321

Published online: 14 May 2016.

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PHARMACEUTICAL BIOLOGY, 2016
VOL. 54, NO. 11, 2606–2615
http://dx.doi.org/10.3109/13880209.2016.1172321

RESEARCH ARTICLE

In vivo antibacterial activity of Garcinia mangostana pericarp extract against

methicillin-resistant Staphylococcus aureus in a mouse superficial skin infection
model
Nitima Tatiya-aphiradeea,b, Waranya Chatuphonprasertb,c and Kanokwan Jarukamjorna,b
a
Faculty of Pharmaceutical Sciences, Khon Kaen University, Khon Kaen, Thailand; bResearch Group for Pharmaceutical Activities of Natural
Products using Pharmaceutical Biotechnology (PANPB), Khon Kaen University, Khon Kaen, Thailand; cFaculty of Medicine, Mahasarakham
University, Mahasarakham, Thailand

ABSTRACT ARTICLE HISTORY

Context: Garcinia mangostana Linn. (Guttiferae) (GM) pericarp has been shown to exhibit good in vitro Received 11 September 2015
antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA); however, there is currently Accepted 27 March 2016
no available information regarding its in vivo antibacterial activity. Published online 13 May 2016
Objective: To examine in vivo antibacterial activity of G. mangostana extract against MRSA.
KEYWORDS
Materials and methods: GM pericarp was extracted by ethanol (GM-EtOH) and methanol (GM-MeOH). The Mangosteen; a-mangostin;
crude extracts were examined for in vitro antibacterial activity against MRSA using broth microdilution tape stripping model
assay. The in vivo antibacterial activity of 10% GM-EtOH against MRSA was determined in a tape stripping
mouse model of superficial skin infection for 9 days by evaluating transepidermal water loss (TEWL) and
performing colony counts from cultured swabs.
Results: GM-EtOH showed greater in vitro activity against MRSA than GM-MeOH in broth microdilution
assay with minimum inhibitory concentration 17 versus 20 lg/mL and minimum bactericidal concentration
30 versus 35 lg/mL, respectively. The GM-EtOH (13.20 ± 0.49%) contained a-mangostin more than the GM-
MeOH (9.83 ± 0.30%). In the tape stripping mouse model, 10% GM-EtOH reduced the number of MRSA col-
onies (0–1) recovered from infected wounds (>100 colonies) on the first day of treatment, restored TEWL
to normal levels on the fourth day, and had completely healed the wounds by day 9.
Conclusion: GM-EtOH showed promising in vivo antibacterial activity against MRSA in a superficial skin
infection model in mice. It is of interest to develop a topical formulation of GM-EtOH to further study its
potential as a novel antibacterial agent.

Introduction 1996; Suksamrarn et al. 2003; Chomnawang et al. 2009;

The fruit of Garcinia mangostana Linn. (Guttiferae) (GM), com-
monly called mangosteen, is a tropical plant known as the
‘‘queen of fruit’’ due to its pleasant taste and health benefits.
The pericarp of mangosteen has been used for a long time in
Ayurvedic medicine for the treatment of diarrhea, dysentery and
abdominal pain (Balasubramanian & Rajagopalan 1988) and it
has been reported to possess anti-inflammatory (Gopalakrishnan
et al. 1980; Nakatani et al. 2002; Chen et al, 2008), antioxidant
(Yoshikawa et al. 1994; Moongkarndi et al. 2004; Jung et al.
2006), anticancer (Ho et al. 2002; Matsumoto et al. 2003), anti-
malarial (Riscoe et al. 2005; Mahabusarakam et al. 2006), anti-
parasite (Keiser et al. 2012), antifungal (Sundaram et al. 1983;
Gopalakrishnan et al. 1997) and antibacterial activities (Iinuma
et al. 1996; Suksamrarn et al. 2003; Chomnawang et al. 2005;
Rassameemasmaung et al. 2007). The pericarp of G. mangostana
is a rich source of bioactive xanthones, namely a-, c- and
b-mangostin, garcinone E and gartanin (Gutierrez-Orozco &
Failla 2013). Both a- and c-mangostin have been shown to
exhibit antibacterial activity against a range of pathogens includ-
ing Propionibacterium acnes, Staphylococcus aureus,
Staphylococcus epidermidis, Mycobacterium tuberculosis and
Streptococcus mutans (Mahabusarakam et al. 1986; Iinuma et al.
Koh et al. 2013; Torrungruang et al. 2013).
S. aureus is a Gram positive coccoid bacteria that is a major
cause of infections of the integument, soft tissue, bone and cardio-
vascular system (Lowy 1998). Superficial skin infections caused by
S. aureus result in abscess wounds and a purulent exudate (Daum
2007). The drugs of choice for the treatment of S. aureus infection
are b-lactam antibiotics, but there is an increasing incidence of S.
aureus strains resistant to b-lactam compounds, so-called methi-
cillin-resistant S. aureus (MRSA) (Haddadin et al. 2002). Hence,
development of a new effective antibacterial compound against
MRSA is required (Iinuma et al. 1996; Haddadin et al. 2002;
Chomnawang et al. 2009).
Several in vitro studies have shown activity of G. mangostana
pericarp extract against MRSA. Minimum inhibitory concentra-
tions (MICs) have been reported in the range of 0.78–1.25 mg/mL
and minimum bactericidal concentrations (MBCs) are 0.78–5 mg/
mL (Sutabhaha et al. 1997; Voravuthikunchai & Kitpipit 2005;
Chomnawang et al. 2009). To our knowledge, there is currently
no available information on the activity of G. mangostana extract
against MRSA in vivo. Therefore, the aim of this study was to
examine the antibacterial effect of G. mangostana pericarp crude
extract against MRSA in a superficial skin infection using the tape
stripping mouse model.

CONTACT Assoc. Prof. Dr. Kanokwan Jarukamjorn, Ph.D. kanok_ja@kku.ac.th Faculty of Pharmaceutical Sciences, Khon Kaen University, Khon Kaen 40002,
Thailand
ß 2016 Informa UK Limited, trading as Taylor & Francis Group

Materials and methods
Chemicals and reagents
Folin-Ciocalteu reagent, quercetin, dimethylsulphoxide (DMSO),
gallic acid, gentamicin, erythromycin and oxacillin were the prod-
ucts of Sigma-Aldrich Chemical (St. Louis, MO). Aluminium
chloride and sodium acetate were supplied by Ajax Finechem Pty
Ltd. (Auckland, New Zealand). Mueller Hinton agar (MHA),
Mueller Hinton broth (MHB) and mannitol salt agar (MSA) were
from Himedia (Mumbai, India). a- and c-Mangostin were
obtained from Chengdu Biopurify Phytochemicals Ltd. (Sichuan,
China). Propylene glycol was a product of Srichand United
Dispensary Co., Ltd. (Bangkok, Thailand). Other chemicals were
obtained from commercial suppliers with high purity and quality.
Preparation of G. mangostana pericarp crude extract
Mangosteens were purchased from a fruit market in Khon Kaen
province, Thailand in April 2014. Reference specimen (PANPB-
GM 2014-001) was deposited at the Herbarium of the Faculty of
Pharmaceutical Sciences, Khon Kaen University, Thailand. The
fruits were washed and the pericarps were collected and dried in
an oven at 50–60  C prior to grinding. The powdered G. mangos-
tana pericarp was subjected to Soxhlet extraction at 80–90  C for
3 h using ethanol (GM-EtOH) and methanol (GM-MeOH) as sol-
vents. The extracts were then filtered before rotary evaporation
and freezing at 20  C overnight. The frozen extracts were
lyophilized using a freeze-dryer and kept at 20  C for further
study.
Determination of chemical markers in the G. mangostana
crude extract a- and c-mangostin contents in the G.
mangostana crude extract
The a- and c-mangostin contents was determined using high-per-
formance liquid chromatography (HPLC). The G. mangostana
extracts were diluted in ethanol and filtered through a 0.22-lm
membrane before being subjected to HPLC using a C18 column
(Hypersil ODS, 5 lm, 4.0 mm 250 mm, Agilent Technologies,
Santa Clara, CA). The isocratic mobile phase consisted of 70%
acetonitrile in distilled water at a flow rate of 1 mL/min. The
detection wavelength was set at UV 244 nm. The a- and c-man-
gostin contents of the G. mangostana crude extracts were calcu-
lated from standard curves of either a- or c-mangostin standards
(Figure 1).
Total phenolic content in the G. mangostana crude extract
The total phenolic content was determined using the Folin-
Ciocalteu method (Chatuphonprasert & Jarukamjorn 2012).
Briefly, a mixture of the G. mangostana extract with Folin-
Ciocalteu reagent was spectrophotometrically measured at a wave-
length of 700 nm. Total phenolic content was calculated as the
weight unit equivalent to the dry weight of the gallic acid standard.
Total flavonoid content in the G. mangostana crude extract
Determination of the total flavonoid content was performed using
the aluminium chloride colorimetric method (Chatuphonprasert
& Jarukamjorn 2012) with some modifications. The G. mangos-
tana extract was mixed with a reaction mixture of aluminium
chloride–sodium acetate–distilled water (10:10:160) and incubated
PHARMACEUTICAL BIOLOGY 2607
at room temperature for 30 min before spectrophotometry at a
wavelength of 405 nm. Total flavonoid content was determined as
the weight unit equivalent to the dry weight of the quercetin
standard.
Anthocyanin pigment content in the G. mangostana crude
extract
The anthocyanin pigment content was determined using the pH
differential method of Wrolstad (1993). The G. mangostana
extract was adjusted to pH 1.0 and another to pH 4.5. The pro-
portion of the G. mangostana extract absorbance at different
wavelengths (500–540 nm) was calculated to the anthocyanin con-
tent using cyanidin-3-glucoside as the standard.
Percent contribution of tannin in the G. mangostana crude
extract
The principle of polymeric tannin pigment resistance to bisulphite
bleaching (Wrolstad 1993) was employed to analyse the tannin
content in the G. mangostana extract. First, the maximal absorb-
ance of G. mangostana extract at 500–540 nm was used to calcu-
late total colour density. Bisulphate solution was then added to
the sample and the maximal absorbance at 500–540 nm was used
to calculate the polymeric colour fraction.
polymeric colour fraction
% contribution of tannin ¼  100:
total colour density
In vitro antibacterial activity of the ethanolic G. mangostana
extract and a-mangostin
Preparation of bacterial suspension
The microorganisms employed in this study were methicillin-sen-
sitive S. aureus (MSSA) ATCC 23235 and MRSA DMST 20651
(kind gift from Dr. Suttiwan Thammawat, Mahasarakham
University, Thailand). To prepare inocula, bacteria were incubated
on MHA plates at 37  C for 12–18 h before colonies were selected
and resuspended in 0.9% normal saline solution (NSS) to a con-
centration of 1  106 CFU/mL.
Agar well diffusion assay
Four hundred microlitres of a 1  106 CFU/mL bacterial inoculum
(MSSA or MRSA) in NSS was evenly spread on a MHA plate
before 6 mm wells were made in the agar using a Kochborer drill
(No. 3). Fifty microlitre aliquots of the ethanolic G. mangostana
extract (GM-EtOH, 5 mg/well), a-mangostin (0.66 mg/well,
equivalent to a-mangostin content in the GM-EtOH), erythro-
mycin (10 lg/well), gentamicin (15 lg/well) and control (DMSO)
were loaded into the wells and the plates were incubated at 37  C
for 24 h. The diameters of the inhibition zones (mm) were meas-
ured and are presented as the average from four replicates.
Determination of MIC and MBC
MIC was determined by the broth microdilution method.
Bacterial inoculum (2 mL) (1  106 CFU/mL) was mixed with
8 mL of MHB and 100-lL aliquots were pipetted into 96-well
microplates. Then, 100-lL aliquots of the G. mangostana extracts,
2608 N. TATIYA-APHIRADEE ET AL.

Figure 1. HPLC chromatogram. (A) Ethanol solvent (blank), (B) a-mangostin standard solution (10 lg/mL) with retention time (tR) 6.016 min, (C) c-mangostin standard
solution (10 lg/mL) with retention time (tR) 3.207 min, (D) GM-EtOH, the ethanolic G. mangostana extract with the peak of a- and c-mangostin and (E) GM-MeOH, the
methanolic G. mangostana extract with the peak of a- and c-mangostin.

a-mangostin, erythromycin or gentamicin in MHB were added
and serially diluted two-fold across the microplate. The micro-
plates were incubated at 37  C for 24 h and bacterial growth was
measured as absorbance at 625 nm in a plate reader. The lowest
concentration of the G. mangostana extract to inhibit the growth
of the target organism was the MIC value. To examine MBC, all
wells from the MIC value to higher concentrations were sampled
onto MHA plates using a simple streak technique, and incubated
at 37  C for 24 h. The lowest concentration of the extract with no
colonies of the target organism was the MBC value.

In vivo antibacterial effect of the ethanolic G. mangostana
extract and a-mangostin
Preparation of bacterial suspension
MRSA DMST 20651 was incubated on MHA plates at 37  C for
12–18 h. Colonies were directly suspended in 0.9% NSS to a con-
centration of 1  108 CFU/mL.
Preparation of antibacterial agents
The commercial topical antibiotics were purchased from a local
R
drugstore namely 0.1% gentamicin cream (SkinfectV, Bangkok
Lab and Cosmetic, Bangkok, Thailand) and 4% erythromycin gel
(ErazitTM, Zyg Pharma Pvt., Pithampur, India). Ten percent GM-
EtOH, 1.32% a-mangostin (equivalent to a-mangostin in the 10%
GM-EtOH) and 1.32% erythromycin were prepared in a 10%
ethanol in propylene glycol.
The tape stripping mouse model
Adult male ICR mice (7 weeks old) were obtained from the
National Laboratory Animal Center, Mahidol University, Nakhon
Pathom, Thailand. All animals were housed in polysulphone cages
with wood chip bedding in the Northeast Laboratory Animal
Center, Khon Kaen University, Khon Kaen, Thailand, under the
supervision of a certified laboratory veterinarian. Commercial
regular diet and water were supplied ad libitum. The research
protocol was approved by the Animal Ethics Committee of the
Northeast Laboratory Animal Center (AEKKU-NELAC001/2558).
The mice were anaesthetized by intraperitoneal injection of
R
pentobarbital sodium (NembutalV, Libourne, France) at a dose of
approximately 50–80 lg/kg and the back was shaved with a sterile
blade. Small pieces of elastic bandage tape were applied to and
stripped from a 2  2 cm2 dorsal area 10–20 times, until the skin
glistened and reddened with no bleeding. The degree of skin dam-
age was standardized to a transepidermal water loss (TEWL,
DermaLabVCombo, Hadsund, Denmark) of 70–75 g/m2 h before
R
10 lL MRSA (1  108 CFU/mL) was applied. A one-hundred
microlitre aliquot of each topical agent, 10% GM-EtOH in a 10%
ethanol in propylene glycol, 10% ethanol in propylene glycol
(Base), 4% commercial erythromycin gel and 0.1% commercial
gentamicin cream, was applied to the wound surface (n ¼ 10) at
24 h intervals. The control (no MRSA) and the no treatment con-
trol (MRSA infected) groups were simply left untreated. The
TEWL was measured every other day for 9 days and the wounds
were swabbed daily for culture on MSA plates and determination
of the number of S. aureus colonies.
Moreover, in vivo antibacterial activity of a-mangostin was fur-
ther examined. The tape stripping model was performed as previ-
ous examination. A one hundred microlitre aliquot of each topical
preparation, 10% GM-EtOH, 1.32% a-mangostin (Alpha-MGS) or
1.32% erythromycin in 10% ethanol in propylene glycol, was
applied to the wound surface everyday (n ¼ 10) at 24 h intervals.
The TEWL was measured every other day for 9 days (three repli-
cates) and the wounds were swabbed daily for culture on oxacil-
lin-contained mannitol salt agar plates (Oxa-MSA, oxacillin 6 lg/
mL MSA) (Isenberg 1998) and determination of the number of
MRSA colonies.
Statistics
The results were analysed by one-way analysis of variance
(ANOVA) followed by LSD post hoc test (SPSS ver. 17.0, Chicago,
IL). p  0.05 was considered statistically significant.
PHARMACEUTICAL BIOLOGY 2609
Table 1. Contents of chemical markers in the G. mangostana crude extract.
Chemical markers GM-EtOH GM-MeOH
Phenolic contents (mg/g) equivalent to gallic acid 62.88 ± 3.68 69.42 ± 5.10
Flavonoid contents (mg/g) equivalent to quercetin 36.95 ± 0.03 33.18 ± 3.83
Anthocyanin (mg/g) equivalent to 98.20 ± 22.04 11.70 ± 0.69
cyanidin-3-glucoside
Percentage of tannin contribution (%) 64.56 ± 1.08 71.49 ± 1.57
a-Mangostin (% of dry weight) 13.20 ± 0.49 9.83 ± 0.30
c-Mangostin (% of dry weight) 2.34 ± 1.01 2.96 ± 0.28
GM-EtOH: the ethanolic G. mangotana crude extract; GM-MeOH: the methanolic
G. mangostana crude extract.
Results
Contents of chemical markers in the G. mangostana crude
extract
There were only small differences in the phenolic and flavonoid
content of the extracts (Table 1). Total phenolic content (as gallic
acid equivalents) was 62.88 ± 3.68 mg/g for GM-EtOH and
69.42 ± 5.10 mg/g for GM-MeOH and total flavonoid content (as
quercetin equivalents) was 36.95 ± 0.03 mg/g for GM-EtOH and
33.18 ± 3.83 mg/g for GM-MeOH. The GM-EtOH extract had a
higher anthocyanin content (as cyaniding-3-glucoside equivalents)
than GM-MeOH (98.20 ± 22.04 versus 11.70 ± 0.69 mg/g) but the
percentages of tannin contribution were similar (64.56 ± 1.08 ver-
sus 71.49 ± 1.57%, respectively).
The G. mangostana extracts were examined for a- and c-
mangostin contents using HPLC (Figure 1). The quantitative
analysis was validated with a linearity range of 1–100 lg/mL for
a-mangostin (R2 ¼ 0.9999) and 10–100 lg/mL for c-mangostin
(R2 ¼ 0.9997). The coefficients of variation within the linearity
range of a-mangostin were 1.07 ± 0.38% (within-day) and
1.51 ± 0.50% (between-day), and c-mangostin were 1.20 ± 0.48%
(within-day) and 0.75 ± 0.13% (between-day). The accuracy of a-
and c-mangostin was 100.42 ± 0.23% and 97.74 ± 0.63%, respect-
ively. The retention time of a-mangostin was 6.016 min (Figure
1B) and c-mangostin was 3.207 min (Figure 1C). The GM-EtOH
extract had a higher a-mangostin content than the GM-MeOH
extract (13.20 ± 0.49 versus 9.83 ± 0.30% dry weight, respectively),
whereas the c-mangostin content was similar (2.34 ± 1.01 and
2.96 ± 0.28% dry weight, respectively; Table 1). The 10% GM-
EtOH in a 10% ethanol in propylene glycol preparation that was
used for the in vivo experiments was also analysed for its a- and
c-mangostin contents and the % recoveries were 92.17 ± 6.30 and
92.39 ± 8.04%, respectively.
In vitro antibacterial activity of G. mangostana crude extract
and a-mangostin against MSSA and MRSA
In the agar diffusion assay, the in vitro antibacterial activity of the
GM-EtOH and GM-MeOH extracts was examined in comparison
with two standard antibiotics, gentamicin and erythromycin, at
the standard doses for antibiotic susceptibility testing, and its
active constituent, a-mangostin. The GM-EtOH (5 mg), GM-
MeOH (5 mg) and a-mangostin (0.66 mg, the equivalent content
to the GM-EtOH) produced inhibition zones against both MSSA
(12.75 ± 1.04, 13.25 ± 0.65 and 11.17 ± 0.29 mm, respectively) and
MRSA (13.17 ± 1.15, 12.50 ± 0.00 and 10.00 ± 0.00 mm, respect-
ively; Table 2), while gentamicin (10 lg) and erythromycin (15 lg)
did inhibition zones against MSSA only (16.50 ± 2.29 and
22.83 ± 0.29, respectively; Table 2). Neither gentamicin nor
erythromycin produced inhibition zone against MRSA. For the
broth microdilution assay, the GM-EtOH produced lower MIC
2610 N. TATIYA-APHIRADEE ET AL.

values compared to GM-MeOH against both MSSA (14 and groups had significantly higher TEWL values on day 4 compared
17 lg/mL, respectively) and MRSA (17 and 20 lg/mL, respect- to the no MRSA control. On day 6, the no MRSA control, no
ively). MBC values were 25 lg/mL for both extracts against MSSA treatment, base and GM-EtOH treatment groups all showed a
but the GM-EtOH extract produced a lower MBC against MRSA reduction in TEWL from day 4, but the GM-EtOH group was the
compared to GM-MeOH (30 versus 35 lg/mL; Table 2). MIC val- only treatment to not have a significantly higher TEWL than the
ues of a-mangostin were 6.25 lg/mL against both MSSA and no MRSA control. On day 9, only the gentamicin group showed
MRSA and better than either GM-EtOH or GM-MeOH extracts, significantly higher TEWL compared to the no MRSA control.
while those of MBC against MSSA and MRSA (100 lg/mL) were Despite TEWL levels returning to baseline on day 9 for most
weaker than both extracts. These findings suggested to select the treatment groups, the MRSA-infected mice with high TEWL levels
GM-EtOH for the in vivo wound healing study and its antibacter- for 6 days exhibited a worsening of the wound-appearance with
ial activity based on its higher a-mangostin content (Table 1) and purulent lesions noted on the last day of observation (day 9). The
superior in vitro antibacterial activity against MRSA (Table 2). gentamicin-treated MRSA-infected mice showed the highest
TEWL values (63–117 g/m2 h) for the duration of the experiment
and these were associated with incurable purulent wounds
In vivo wound healing and antibacterial activity of the throughout the period of observation.
ethanolic G. mangostana extract and a-mangostin in the To examine the wound healing property of a-mangostin, the
tape stripping mouse model tape stripping mouse model of superficial skin infection was car-
ried out. TEWL was measured to indicate skin barrier function of
Wound healing the wounds before initiation of the wound (day 0), after initiation
We used TEWL as an indicator of skin barrier function in the of the wound (day 0.5), and on days 2, 4, 6 and 9 of treatment
tape stripping mouse model of superficial skin infection. The (Figure 3). After shaving, TEWL was in a range of 15–20 g/m2 h
TEWL values were determined before initiation of the wound for all groups, and it was increased to 75–80 g/m2 h after tape
(day 0), after initiation of the wound (day 0.5), and on days 2, 4, stripping. On day 4, TEWL values of the no treatment (NT) and
6 and 9 of treatment (Figure 2). Baseline TEWL was approxi- the a-mangostin topical treated MRSA-infected groups were sig-
mately 10–20 g/m2 h after shaving for all groups. After tape strip- nificantly higher than the no MRSA control, and the TEWL levels
ping for 10–20 times (day 0.5), skin damage was observed and of these two groups were persistently high till day 9. Consistent to
TEWL ranged from 70 to 90 g/m2 h. There was no difference in the previous independent experiment, TEWL values of the GM-
TEWL values 2 days after treatment for any groups. On day 4, EtOH and the erythromycin topical treated MRSA-infected
the control (no MRSA) group showed a marked reduction in groups were returned to the levels similar to that of the control
(no MRSA) group on day 4.
TEWL, indicating wound healing as did the GM-EtOH extract
and erythromycin topical treated MRSA-infected groups. In con-
trast, the no treatment control, base and gentamicin treatment Antibacterial activity
Table 2. Inhibition zone, MIC and MBC values of the G. mangostana crude To determine the antibacterial effect of the treatments, wounds
extract against MSSA and MRSA.
were swabbed daily and colony counts were performed on the
Inhibition zone (mm) MIC (lg/mL) MBC (lg/mL) MSA plates. Only yellow pigmented colonies representing S. aur-
MSSA MRSA MSSA MRSA MSSA MRSA eus were counted. The no treatment (NT) group had high number
GM-EtOH 12.75 ± 1.04 13.17 ± 1.15 14 17 25 30 of S. aureus colonies from the wounds on the starting day (day 1)
GM-MeOH 13.25 ± 0.65 12.50 ± 0.00 17 20 25 35 and persistence along the period of study (9 days, Figure 4). The
a-Mangostin 11.17 ± 0.29 10.00 ± 0.00 6.25 6.25 100 100 treatment of GM-EtOH ultimately reduced the number of S. aur-
Gentamicin 16.50 ± 2.29 0.00 ± 0.00 1.56 >10,000 25 >10,000
Erythromycin 22.83 ± 0.29 0.00 ± 0.00 0.78 >10,000 >25 >10:000 eus colonies to the level comparable to those of the control (with-
out MRSA infection) group since the first to the last day of
MIC: minimum inhibitory concentration; MBC: minimum bactericidal concentra-
tion; MSSA: methicillin-sensitive S. aureus ATCC 23235; MRSA: methicillin-resistant application. The erythromycin treated MRSA-infected group
S. aureus DMST 20651; GM-EtOH: the ethanolic G. mangotana crude extract; GM- showed the constant reduction in the number of S. aureus colo-
MeOH: the methanolic G. mangostana crude extract. nies at the fifth day. The base (10% ethanol in propylene glycol)

Figure 2. Effects of GM-EtOH on TEWL in the tape stripping model in mice. TEWL was measured on the back of the mice as described in the method (n ¼ 9–10).
Control, non-infected mice with the tape stripping induced wound; NT, MRSA-infected wound in mice with no treatment; Base, MRSA-infected wound in mice treated
with 100 lL of a 10% ethanol in propylene glycol solution; GM-EtOH, MRSA-infected wound in mice treated with 100 lL of a 10% GM-EtOH in a 10% ethanol in propyl-
ene glycol solution; Erythromycin, MRSA-infected wound in mice treated with 100 lL of a 4% commercial erythromycin gel; Gentamicin, MRSA-infected wound in mice
treated with 100 lL of a 0.1% commercial gentamicin cream. *p < 0.001 versus control on the same day using one-way ANOVA followed by LSD post hoc test.
 PHARMACEUTICAL BIOLOGY 2611

Figure 3. Effects of GM-EtOH and a-mangostin on TEWL in the tape stripping model in mice. TEWL was measured on the back of the mice as described in the method
(n ¼ 7–10). Control, non-infected mice with the tape stripping induced wound; NT, MRSA-infected wound in mice with no treatment; GM-EtOH, MRSA-infected wound in
mice treated with 100 lL of a 10% GM-EtOH in a 10% ethanol in propylene glycol solution; Alpha-MGS, MRSA-infected wound in mice treated with 100 lL of a 1.32% a-
mangostin in a 10% ethanol in propylene glycol solution; Erythromycin, MRSA-infected wound in mice treated with 100 lL of a 1.32% erythromycin in a 10% ethanol in
propylene glycol solution. *p < 0.001 versus control on the same day using one-way ANOVA followed by LSD post hoc test.

Figure 4. The effect of GM-EtOH on** the number of S. aureus colonies. MRSA was swabbed from the wound of the mouse back to culture on the MSA plate and num-
ber of colonies were counted at 24 h after the incubation (n ¼ 9-10). Control, non-infected mice with the tape stripping induced wound; NT, MRSA-infected wound in
mice with no treatment; Base, MRSA-infected wound in mice treated with 100 lL of a 10% ethanol in propylene glycol solution; GM-EtOH, MRSA-infected wound in mice
treated with 100 lL of a 10% GM-EtOH in a 10% ethanol in propylene glycol solution; Erythromycin, MRSA-infected wound in mice treated with 100 lL of a 4% commer-
cial erythromycin gel; Gentamicin, MRSA-infected wound in mice treated with 100 lL of a 0.1% commercial gentamicin cream. *p < 0.001 versus control and #p < 0.001
versus GM-EtOH on the same day using one-way ANOVA followed by LSD post hoc test.

and the gentamicin treated MRSA-infected group did not exhibit
any efficient reduction in number of S. aureus colonies through-
out the course of the study compared with the NT and control
groups, respectively.
To quantify the exact number of MRSA colonies that were
present on the wound after the treatments, a more specific select-
ive media, oxacillin-contained mannitol salt agar (Oxa-MSA),
which might restrict the growth of MSSA, was further considered
to examine antibacterial activity of GM-EtOH and a-mangostin.
The wounds were swabbed daily and cultured on Oxa-MSA plates
where only MRSA were grown, and the number of MRSA colo-
nies was counted. The treatment of GM-EtOH consistently
reduced the number of MRSA colonies to the level comparable to
those of the control (without MRSA infection) group since the
first to the last day of application (Figure 5). The no treatment
(NT) group showed high number of MRSA colonies from the
wounds on the starting of the application and significantly dif-
fered from the control (without MRSA infection) group for 5
days, while the a-mangostin and the erythromycin treated MRSA-
infected group showed the constant reduction in the number of
MRSA colonies to the comparable level as the control (without
MRSA infection) group at the third day.
Photographs of the mouse skin wounds on the last day (day 9)
of observation are shown in Figures 6 and 7. The non-infected
wound (control) was completely healed within the period of
observation (Figure 6A). The MRSA-infected skin without treat-
ment (NT) or treated with the base only resulted in purulent
wounds on day 9 (Figure 6B and C), which was consistent with
the delayed return of TEWL values to baseline (Figure 2) and the
high number of colonies from their swabs (Figure 4).
The treatments of MRSA-infected mice with GM-EtOH
(Figure 6D) or erythromycin (Figure 6E) resulted in completely
healed wounds on day 9. Treatment with gentamicin did not
appear to improve the condition of the wound on MRSA-infected
mice (Figure 6F) with the wounds remaining purulent with corre-
sponding high TEWL values (75.67 ± 13.14 g/m2 h in day 9,
Figure 2) and large number of bacterial colonies from the swab
(Figure 4).
The mouse skin wounds on the last day (day 9) of observation
after the treatments of GM-EtOH and a-mangostin were further
compared (Figure 7). The non-infected wound (control) was com-
pletely healed within the period of observation (Figure 7A) as the
previous independent examinations. Likewise, the treatments of
MRSA-infected mice with GM-EtOH (Figure 7C) or erythromycin
2612 N. TATIYA-APHIRADEE ET AL.

Figure 5. The effect of GM-EtOH and a-mangostin in the number of MRSA colonies. MRSA was swabbed from the wound of the mouse back and diluted to 2 mL of
NSS before culturing on the MSA containing oxacillin (6 lg/mL) plate. The number of colonies was counted at 24 h after the incubation (n ¼ 7–10). Control, non-infected
mice with the tape stripping induced wound; NT, MRSA-infected wound in mice with no treatment; GM-EtOH, MRSA-infected wound in mice treated with 100 lL of a
10% GM-EtOH in a 10% ethanol in propylene glycol solution; Alpha-MGS, MRSA-infected wound in mice treated with 100 lL of a 1.32% a-mangostin in a 10% ethanol
in propylene glycol solution; Erythromycin, MRSA-infected wound in mice treated with 100 lL of a 1.32% erythromycin in a 10% ethanol in propylene glycol solution.
*p < 0.05 versus control and #p < 0.05 versus GM-EtOH on the same day using one-way ANOVA followed by LSD post hoc test.

Figure 6. The effect of GM-EtOH on the wound-appearance. The photograph of the wound was recorded daily (n ¼ 9–10) after the treatments. The figure showed the
wound-appearance on the last day of observation (the ninth day). Control, non-infected mice with the tape stripping induced wound; NT, MRSA-infected wound in mice
with no treatment; Base, MRSA-infected wound in mice treated with 100 lL of a 10% ethanol in propylene glycol solution; GM-EtOH, MRSA-infected wound in mice
treated with 100 lL of a 10% GM-EtOH in a 10% ethanol in propylene glycol solution; Erythromycin, MRSA-infected wound in mice treated with 100 lL of a 4% commer-
cial erythromycin gel; Gentamicin, MRSA-infected wound in mice treated with 100 lL of a 0.1% commercial gentamicin cream.

(Figure 7E) resulted in completely healed wounds on day 9. The
MRSA-infected skin without treatment (NT) resulted in purulent
wounds on day 9 (Figure 7B), which was consistent with the
delayed return of TEWL values to baseline (Figure 3) and
the high number of MRSA colonies from their swabs (Figure 5).
The treatment with a-mangostin improved condition of the
wounds on MRSA-infected mice (Figure 7D), however, the
wounds were not completely healed with remaining mild purulent
on the last day of observation (day 9) and corresponding high
TEWL values (66.67 ± 21.08 g/m2 h in day 9, Figure 3).
 PHARMACEUTICAL BIOLOGY 2613

Figure 7. The effect of GM-EtOH and a-mangostin on the wound-appearance. The photograph of the wound was recorded daily (n ¼ 7–10) after the treatments. The fig-
ure showed the wound-appearance on the last day of observation (the ninth day). Control, non-infected mice with the tape stripping induced wound; NT, MRSA-infected
wound in mice with no treatment; GM-EtOH, MRSA-infected wound in mice treated with 100 lL of a 10% GM-EtOH in a 10% ethanol in propylene glycol solution;
Alpha-MGS, MRSA-infected wound in mice treated with 100 lL of a 1.32% a-mangostin in a 10% ethanol in propylene glycol solution; Erythromycin, MRSA-infected
wound in mice treated with 100 lL of a 1.32% erythromycin in a 10% ethanol in propylene glycol solution.

Discussion
Despite a major cause of nosocomial infection, endocarditis,
osteomyelitis, pyoarthrosis, Staphylococcal scalded skin syndrome
(Brewer et al. 2008) and toxic shock syndrome (Iwatsuki et al.
2006), S. aureus is a common cause of superficial skin infections
(Chiller et al. 2001). The increasing incidence of multidrug-resist-
ant S. aureus strains (Lowy 1998) is resulting in the treatment of
S. aureus infection becoming more difficult and complicated
(Price et al. 1998; Cohen & Kurzrock 2004; Iyer & Jones 2004).
The resistance to methicillin was related to mecA gene encoded
penicillin-binding protein 2a (PBP2a), which had low binding
affinity for b-lactams antibiotics (Henze & Berger-B€achi 1995;
Chambers 1997). Thus, there is a great need for the development
of new compounds that are active against antibiotic-resistant
strains, in particular, MRSA. Previous reports have indicated that
a compound isolated from mangosteen pericarb, a-mangostin, has
potent antibacterial activity against MRSA, however, to our know-
ledge there has been no in vivo study of its effectiveness.
In the present study, a-mangostin in the GM-EtOH was
13.20 ± 0.49% dry weight. Corresponding with the study of
Pothitirat, Chomnawang, Supabphol, et al. (2009) that reported
the content of a-mangostin at 13.63 ± 0.06% dry weight in the
ethanolic extract of mature fruit, whereas that contained in the
young fruit extract was 8.07 ± 0.11% dry weight. The content of
a-mangostin in the G. mangostana extract varied by several fac-
tors, such as stage of maturity, cultivated area, harvesting period
and extraction method. G. mangostana from South Thailand had
little higher a-mangostin than those of the Eastern (17.64 ± 3.89
versus 16.43 ± 3.69% w/w in the extract and 4.85 ± 0.83 versus
4.35 ± 0.60% w/w in the dried powder, respectively) (Pothitirat,
Chomnawang and Gritsanapan 2009).
Voravuthikunchai and Kitpipit (2005) reported that the MIC of
ethanolic G. mangostana extract against MRSA (PSU 0201-PSU
0235) was 0.05–0.4 mg/mL with the MBC of 0.1–0.4 mg/mL. We
presently found the lower MIC and MBC values of the GM-EtOH
against MRSA (DMST 20651) of 17 and 30 lg/mL, respectively. In
addition, MIC and MBC values against several strains of MRSA
were reported. Sutabhaha et al. (1997) demonstrated MIC and
MBC of petroleum ether G. mangostana extract against S. aureus
ATCC 25923 of 3.12 and 6.25 lg/mL, respectively, while both val-
ues against MRSA (a local strain of Thailand) were of 0.78 lg/mL.
Chomnawang et al. (2009) reported antibacterial screening of etha-
nolic G. mangostana extract against 16 clinical MRSA strains but
only the susceptibility values against MRSA T1301 were considered
effective with MIC of 0.039 mg/mL and MBC of 0.312 mg/mL. The
antibacterial effect of G. mangostana varied upon several factors,
i.e., bacterial strain, stage of maturity, cultivated area, and harvest-
ing period including solvent, and method of extraction.
Previous studies have reported that a-mangostin possessed
antibacterial activity (Mahabusarakam et al. 1986; Suksamrarn
et al. 2003). In our hands, the GM-EtOH and GM-MeOH extracts
showed higher MIC values against MSSA and MRSA compared to
the equivalent dose of pure a-mangostin (Table 2); however,
MBC values against MSSA and MRSA of either GM-EtOH or
2614 N. TATIYA-APHIRADEE ET AL.
GM-MeOH were lower than those of a-mangostin.
Furthermore, the ranges of MIC and MBC of a-mangostin
against either MSSA or MRSA were markedly different than
those values of the extracts. These observations possibly referred
to bacteriostatic property of a-mangostin. Hence, to get to the
bactericidal level, a-mangostin should be applied to the wound
more than once a day. In addition, the in vitro and the in
vivo findings on antibacterial activity were inconsistence. The
GM-EtOH effectively reduced the number of MRSA colonies to
the level comparable to those of the control (without MRSA
infection) group since the first day of application (Figure 5)
and resulted in completely healed wounds on the last day of
observation (day 9), while the a-mangostin showed the constant
reduction in the number of MRSA colonies to the comparable
level as the control group at the third day and the wounds
with remaining mild purulent on the last day of observation
(Figure 7). These observations might be explained by the exist-
ence of other constituents in the GM-EtOH extract that
additionally possess antibacterial properties, such as c- and
b-mangostin (Suksamrarn et al. 2003; Koh et al. 2013). These
might additionally underline that only in vitro examination is
insufficient to assure in vivo antibacterial activity.
Gentamicin and erythromycin, two antibacterial agents that act
by inhibiting protein synthesis in the bacteria, were selected as the
comparison drugs for this study as they are commonly found in
topical antibacterial preparations and can be purchased easily at a
relatively low price from drugstores. Although some strains of
MRSA have been reported to be susceptible to gentamicin (Riley
et al. 1995; Turnidge & Bell 1999; Nimmo et al. 2000), in micro-
dilution method, as expected, gentamicin and erythromycin were
less susceptible (Table 2)
The tape stripping mouse model represents an abrasion-wound
similar to that possibly occurring in daily life (Dai et al. 2011).
This model is performed easily with an uncomplicated procedure
and is suitable for the superficial S. aureus skin infection
(Kugelberg et al. 2005). The infection is established by using the
tape stripping technique to disrupt the epidermal layer until a
TEWL of 70–75 g/m2 h is achieved and inoculating with bacteria
at 1  108 CFU/mL (Kugelberg et al. 2005). The wounds and the
number of recovered MRSA from MRSA infected-mice with no
treatment (NT) were persistent until the last day of the study
(Figure 4). The base treatment slightly decreased the number of
colonies, but the wound was not better improved (Figure 4 & 6).
These observations supported that the base did not exert antibac-
terial effect. The wound of gentamicin-treated group appeared
worsely with deficient healing (Figure 6F). These observations
partly resulted from inappropriate formulation type of the genta-
micin cream for the treatment of this superficial MRSA infection.
For culturing S. aureus from the wound, MSA was employed
due to its selective characteristic media to S. aureus (Flournoy et al.
1990; Smyth & Kahlmeter 2005). Only S. aureus grows on the MSA
with mannitol fermentation and produces yellow zone surrounding
the colony to facilitate for S. aureus colony-counting (Kateete et al.
2010). Furthermore, oxacillin (6 lg/mL) was added into MSA
(Oxa-MSA) to restrict the growth of MSSA to quantify the exact
number of MRSA colonies on the wound (Isenberg 1998).
The 10% ethanol solution was additionally mixed with GM-
EtOH to increase solubility of the extract before further mixing
with propylene glycol. Propylene glycol was applied as a co-solvent
for GM-EtOH because it was expected to be a componential agent
in a topical formulation of GM-EtOH for further development.
Erythromycin is commonly ineffective against MRSA, but it
might be active in some strains of MRSA (Riley et al. 1995).
Although erythromycin shows less inhibitory property against
MRSA in the in vitro study of the present study (Table 2), cor-
responding to the previous study (Rayner & Munckhof 2005),
manifestation of the wound after the treatment of erythromycin
was almost completely healed (Figures 6E and 7E), comparable
to the control (Figures 6A and 7A). Erythromycin decreased the
TEWL values as GM-EtOH did (Figures 2 and 3), and showed
the satisfied wound-healing effect for the MRSA superficial
infection by the tape stripping in mice, albeit its effect in the
number of MRSA colonies was weaker than GM-EtOH (Figures
4 and 5). These controversial outcomes of erythromycin between
in vitro and in vivo might result from its low water solubility
property to compatible with the water phase of MHA to show
the antibacterial efficiency. On the other hand, the commercial
erythromycin product tested in the present study was present as
a gel which is suitable for the treatment of an abrasion wound.
Moreover, erythromycin might diffuse through the bacterial
membrane due to the co-solvent effect in this commercial
preparation.
The GM-EtOH exhibited the strongest antibacterial activity for
the superficial MRSA infection in mice. The appearance of the
wound was completely healed (Figures 6D and 7C), comparable
to the control (Figures 6A and 7A). Moreover, the GM-EtOH
properly decreased the number of colonies in the wound
(Figures 4 and 5).
This is the first time to simultaneously report the efficiency of
GM-EtOH against MRSA in the in vitro agar well diffusion fol-
lowed by broth microdilution assay and revealed the antibacterial
activity of G. mangostana against the superficial MRSA infection
in the tape stripping mouse model representing the abrasion skin
infection. Therefore, it is worthwhile for further study on efficacy
of G. mangostana in other skin infection models and its molecular
mechanism against MRSA including the antibacterial activity
against other bacterial strains. These findings suggested G. man-
gostana as a promising antibacterial candidate for the superficial
MRSA infection.
Conclusions
The present study ultimately assured superior antibacterial activity
of GM-EtOH against MRSA in the in vitro agar well diffusion
and microdilution examinations, and in the superficial MRSA
infection in mice than a commercial antibiotic erythromycin.
Therefore, GM-EtOH is a promising antibacterial candidate to be
of interest to further develop as an alternative topical formulation
against MRSA.
Acknowledgments
Dr. Suttiwan Thammawat, Mahasarakham University, Thailand, for
kindly provided MSSA ATCC 23235 and MRSA DMST 20651 cul-
tures. Dr. Glenn Borlace is kindly acknowledged for English editing
and critical evaluation of the manuscript preparation.
Disclosure statement
The authors have no conflict of interest.
Funding information
Nitima Tatiya-aphiradee sincerely thanks the Graduate School of
Khon Kaen University, Thailand, for a scholarship (571H117)

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