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RESEARCH ARTICLE
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Antimicrobial activities of stingless bee propolis from mangosteen
orchard
Boonyadist Vongsak1*, Kulwara Poolpol2, Sumet Kongkiatpaiboon3,
Sasipawan Machana1
1
Faculty of Pharmaceutical Sciences, Burapha University Chonburi, 20131,
Thailand
2
Faculty of Allied Health Sciences Burapha University Chonburi, 20131, Thailand
3
Drug Discovery and Development Center, Thammasat University, Pathum Thani,
12120, Thailand
Abstract
Propolis has been relied on numerous therapeutic purposes such as treatment
of cold, ulcer and inflammation. The biological activities and chemical constituents
of each type of propolis mainly depend on geographical regions and on the bee
species. In the study, the propolis from three stingless bee species, Tetragonula
pagdeni Schwarz, Lepidotrigona ventralis Smith, and Lepidotrigona terminata
Smith, which are commercially cultivated in artificial hives in fruit gardens was
collected in the same orchard and compared antimicrobial activity. The active
compound from the species that demonstrated the strongest activity were identified.
Gram-positive bacteria: Staphylococcus aureus, gram-negative bacteria: Escherichia
coli, Enterobacter sp., Proteus mirabilis, anaerobic bacteria: Propionibacterium
acnes and pathogenic yeast: Candida albicans were tested. All extracts revealed
antimicrobial activity of S. aureus and P. acnes. The extract of T. pagdeni propolis
exhibited the highest antimicrobial activity with 8 mm and 9 mm of inhibition zone
of S. aureus and P. acnes, respectively at concentration 100 µg/disc. While, the
propolis extracts of L. ventralis and L. terminata at concentration 10 mg/disc
demonstrated comparable size of inhibition zone of S. aureus and P. acnes. The
minimal inhibition concentrations of T. pagdeni extract were 31.25 µg/ml against S.
aureus and 15.625 µg/ml against P. acnes. Thus, the extract of T. pagdeni propolis
was selected to separate and identify the active compound. By chromatographic
method, alpha-mangostin was elucidated and evaluated as the active compound. In
conclusion, T. pagdeni propolis extract could be suggested as good source for
utilizing as antimicrobial agent and for further development as pharmaceutical
products.
Keywords: Antimicrobial, stingless bees, Propolis, mangosteen
Address correspondence and reprint request to: Boonyadist Vongsak, Faculty of Pharmaceutical
Sciences, Burapha University Chonburi 20131, Thailand. E-mail address: boonyadist@buu.ac.th.
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Introduction
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In Thailand and India, stingless bee propolis is widely applied for the treatment of
maladies such as acne, toothache and inflammation.7,8 Antimicrobial,
antiproliferative and antioxidant activities of some species of stingless bee propolis
were investigated.9 However, the study of propolis from Thai stingless bees,
(Tetragonula pagdeni Schwarz, Lepidotrigona ventralis Smith, and Lepidotrigona
terminata Smith), which are commercially cultivated in artificial hives in fruit
gardens and used in several preparations in Thailand, is limited. Thus, the objective
of the present work was to compare antimicrobial effect of propolis from three
stingless bee species in the same area of Thai mangosteen orchard. The active
compound from the species that established the strongest activity were identified.
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Propolis of Tetragonula pagdeni Schwarz, Lepidotrigona ventralis Smith, and
Lepidotrigona terminata Smith were collected from the mangosteen garden in
December from Makham district, Chanthaburi province, Thailand in 2014, and kept
in the dark at 0 °C until use. The stingless bees were identified by Dr.Chama Inson,
Department of Entomology, Faculty of Agriculture, Kasetsart University. The
voucher specimens (Tetragonula pagdeni No.1214003, Lepidotrigona ventralis
No.1214001 and Lepidotrigona terminata No.1214002) were deposited at Faculty of
Pharmaceutical Sciences, Burapha University, Thailand.
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Propolis from different bee species (20 g) was separately cleaned and cut into small
pieces and was then sonicated with 80% ethyl alcohol (400 ml) at 40 °C for 30 min.
The suspension was centrifuged at 3,000 g for 5 min at 20 °C. The supernatant was
kept while the pellet was re-extracted using the same procedure. The supernatants
were pooled together and evaporated in a rotary evaporator. Each extract was
dewaxed by sonication with 100 ml of hexane at 40°C for 20 min and centrifuged at
2,000 g for 5 min at 20°C. The supernatant was discarded while the crude residue
was kept and stored in the dark at 0 °C.
Propolis is one of natural products that exhibits extensive pharmacological activity
such as antiviral, antibacterial, antifungal, antioxidant, anti-inflammatory, antitumor
activities and is also listed in the Chinese Pharmacopoeias and London
Pharmacopoeias.1,2 Nevertheless, the biological activities and chemical of propolis
diverge depending on bee species and the flora at site of bee collection. For example,
propolis from North America and Europe regions’ main compounds contain mostly
cinnamic acids and their esters, flavones, flavanones, while that from Brazil mainly
consist of diterpenic acids, prenylated p-coumaric acids.3,4 In addition, different
races of honeybees collected at the similar area established varying potency.
Stingless bees: Trigona incisa, Timia apicalis, Trigona fusco-balteata and Trigona
fuscibasis, from Indonesia revealed different degree of cytotoxicity. Apis mellifera
caucasica showed a greater antibacterial activity to Apis mellifera carnica and Apis
mellifera anatolica.5,6
Materials and Methods
Propolis sample and preparation
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Microorganisms
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The pathogenic microorganisms used in this study were: gram-positive bacteria
including Staphylococcus aureus, Propionibacterium acnes and gram-negative
bacteria including Escherichia coli, Enterobacter sp., and Proteus mirabilis.
Important pathogenic yeast, Candida albicans was also used in this study. The
bacteria and yeast were obtained from Faculty of Allied Health Sciences, Burapha
University, Thailand. All strains were confirmed by cultural and biochemical
characteristics and stored at 4oC for further use. Anaerobic jar was used for the
growth of P. acnes in all experiment of the study.
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Antimicrobial activities test
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Antimicrobial activity of each extract was determined using a modified Kirby-Bauer
disc diffusion method. Briefly, the microorganisms were cultured in nutrient broth
(Criterion, Hardy Diagnostics) at 35-37 oC for 18-24 h, and then collect the cells by
centrifugation and wash with PBS. The cell suspensions were adjusted with
McFarland standard no. 0.5 density to obtain a final concentration of approximately
108 CFU/ml by using densitometer (DEN-1 McFarland Densitometer). After that
they were spread onto Muller Hinton Agar (Criterion, Hardy Diagnostics). The
extracts were tested using 6 mm sterilized filter paper discs (GE Healthcare Life
Sciences). Discs were impregnated with 10 μl of the extract in different
concentrations. Incubation was done at 35 oC for 18 – 24 h. Inhibition zone of each
extract was observed and indicated its antimicrobial activity of the extracts. The
diluent of the extracts (1% DMSO) was used as a negative control. Antimicrobial
testing with standard antibiotics discs, ampicillin (10 µg/disc), gentamycin (10
µg/disc), penicillin G (10 Units/disc), chloramphenicol (30 µg/disc), kanamycin (30
µg/disc), and tetracycline (30 µg/disc) (Oxoid) were served as positive controls for
antimicrobial activity.
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Broth microdilution test
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The minimum inhibitory concentration (MIC) of the extracts was determined using
conventional broth microdilution method according to the CLSI guideline. The
extracts were prepared in Mueller-Hinton broth in different concentrations, and then
added into each well of sterile 96-well microtiter plate (50 μl/well). The adjusted
bacterial suspension in Mueller-Hinton broth was added to each well (50 μl/well).
Finally, final concentrations of the extract of 1 mg/ml to 0.98 µg/ml and final
inoculum concentration of 1 × 105 CFU/ml was obtained. The plate was incubated
for 18-24 hours at 35-37°C. A control well containing the growth medium, the
bacteria, and the extract was also set-up. The minimum inhibitory concentration
(MIC) of the extracts was regarded as the lowest concentration of the extract that
inhibits the visible bacterial growth.
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Separation of active compounds
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The propolis extract from Tetragonula pagdeni that exhibited the strongest activity
was selected to separate the bioactive compounds. The crude residue was subjected
to column chromatography (3 × 20 cm, Silica gel (0.063 – 0.200 mm) with 20% ethyl
acetate in hexane as a mobile phase monitored by thin-layer chromatography. The
fraction was applied to pTLC with dichloromethane (triple run) as a mobile phase.
The pure compound was dissolved in 99.98% CDCl3 (ca. 5 mg in 0.7 ml) and
transferred into 5 mm NMR sample tube (Promochem, Wesel, Germany). Spectra
were recorded by the Bruker Topspin software on a Bruker AVANCE 400
spectrometer (Bruker, Rheinstetten, Germany). The NMR data was compared with
the previous study and reported as alpha-mangostin.
Table 1. Antimicrobial activity of T. pagdeni propolis extract against microorganisms
Concentration of the T. pagdeni propolis extract (per disc)
Microorganisms
Size of inhibition zone (mm)
S. aureus
Propionibacterium acnes
E. coli
Enterobacter sp.
Proteus mirabilis
Candida albicans
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100 µg
8 mm
9 mm
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50 µg
7 mm
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10 µg
-
1% DMSO
-
Table 2. Antimicrobial activity of L. ventralis propolis extract against microorganisms
Concentration of the L. ventralis propolis extract (per disc)
Microorganisms
S. aureus
Propionibacterium acnes
E. coli
Enterobacter sp.
Proteus mirabilis
Candida albicans
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20 mg
Size of inhibition zone (mm)
10 mg
1 mg
100 µg
8 mm
11 mm
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7 mm
9 mm
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-
-
1%
DMSO
-
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Table 3. Antimicrobial activity of L. terminata propolis extract against microorganisms
Concentration of the L. terminata propolis extract (per disc)
Microorganisms
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20 mg
Size of inhibition zone (mm)
10 mg
1 mg
100 µg
S. aureus
11 mm
8 mm
-
-
1%
DMSO
-
Propionibacterium acnes
12 mm
8 mm
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-
-
E. coli
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-
-
-
-
Enterobacter sp.
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-
-
-
-
Proteus mirabilis
-
-
-
-
-
Candida albicans
-
-
-
-
-
Results
The antimicrobial effect of the extracts
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Determination of antimicrobial activity of each extract against S. aureus, P. acnes,
E. coli, Enterobacter sp., P. mirabilis, and C. albicans by disc agar diffusion method
was measured by measuring the zone of inhibition. One hundred microgram, 50 and
10 µg per disc of T. pagdeni propolis extract were tested with those microorganisms.
At the concentration of 100 µg of T. pagdeni propolis showed inhibition effect
against S. aureus and P. acnes, indicated by the inhibition zone of 8 and 9 mm,
respectively. Moreover, at the concentration of 50 µg of the extract can inhibit S.
aureus (Table 1).
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L. ventralis propolis extract at the concentrations of 20 mg, 10 mg, 1 mg and 100 µg
were tested. The extract showed antibacterial activity against S. aureus at the
concentration of 20 and 10 mg and against P. acnes at the same concentration (Table
2). Antibacterial activity of L. terminata propolis was presented against S. aureus.
Inhibition zone of 11 and 8 mm were showed at the concentration of 20 and 10 mg,
respectively. P. acnes was also inhibited by L. terminata propolis at the same
concentrations and showed inhibition zone of 12 and 8 mm, respectively (Table 3).
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Only T. pagdeni propolis was investigated for minimal inhibition concentration
(MIC) because the species exhibited strongest activity and another extracts could not
dissolve in the culture media. MIC value for the T. pagdeni propolis extract was
tested against the S. aureus and P. acnes by 2-fold serial dilution method ranging
from 1 mg/ml to 0.98 µg/ml. The extract showed MIC of 31.25 µg/ml against S.
aureus whereas it has greater activity against P. acnes by showing MIC of 15.625
µg/ml. Alpha-mangostin, an active compound that separated from T. pagdeni
propolis extract, has antibacterial effect against S. aureus (inhibition zone of 10 mm)
and P. acnes (inhibition zone of 9 mm). The microbial activities of the extracts were
dependent on the dose of the test material. As the concentration increased the
inhibition zone was also increased.
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Discussion
Propolis is utilized for medicinal and nutraceutical purposes. Most of studies
describe the antimicrobial activity of propolis extract collected by European honey
bee, Apis mellifera. Stingless bee propolis is a kind of propolis collected by stingless
bee species, a large group of eusocial insect that play a part in plant pollination in
tropical regions and the lack of studies on its propolis pharmacological activity.8,9
The antibacterial activity of propolis of some stingless bee species has been revealed
against Gram-positive bacteria, particularly with Staphylococcus strains, whereas
weaker activity against Gram-negative bacteria.7 Our study indicated that Thai
stingless bee propolis from mangosteen orchard has antibacterial activity for grampositive bacteria, S. aureus and P. acnes, but could not against some gram-negative
bacteria and fungi at concentration 20 mg/disc. T. pagdeni propolis extract
demonstrated the strongest activity. Alpha-mangostin was elucidated as active
compound.
Conclusion
The study provides data to support the usage of different stingless bee propolis
cultivated in mangosteen orchard as raw material for antimicrobial agents. Based on
this study, the propolis extract of T. pagdeni should be utilized as a better source of
raw material than others. The alpha-mangostin could possibly be applied as markers
for standardization.
Acknowledgements
This work was supported by the Research Grant of Burapha University through
National Research Council of Thailand (Grant no. 07/2559 and 08/2559).The authors
also would like to thank Faculty of Pharmaceutical Sciences, Burapha University for
facility support and Mr. Wisit Tanooard as well as Thai local bee keeper of Kon
Chan Channarong (stingless bee) in Chantaburi province, Thailand for propolis
collection.
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