Antibacterial Compounds from Propolis of Tetragonula laeviceps and Tetrigona melanoleuca (Hymenoptera: Apidae) from Thailand

This study investigated the chemical composition and antimicrobial activity of propolis collected from two stingless bee species Tetragonula laeviceps and Tetrigona melanoleuca (Hymenoptera: Apidae). Six xanthones, one triterpene and one lignane were isolated from Tetragonula laeviceps propolis. Triterpenes were the main constituents in T. melanoleuca propolis. The ethanol extract and isolated compounds from T. laeviceps propolis showed a higher antibacterial activity than those of T. melanoleuca propolis as the constituent α-mangostin exhibited the strongest activity. Xanthones were found in propolis for the first time; Garcinia mangostana (Mangosteen) was the most probable plant source. In addition, this is the first report on the chemical composition and bioactivity of propolis from T. melanoleuca.


Introduction
Propolis is a resinous material collected by bees from various plant exudates. Bees use propolis to narrow the nest entrances, seal cracks and embalm dead organisms inside the hive. The antibiotic properties of propolis provide a healthy hive environment for the honeybee colony. Propolis is an apicultural product that has been used for its biological properties, as an alternative medicine and for disease prevention, in different parts of the world. The chemical composition of propolis depends on the collection site, available plant sources and bee species [1], [2]. Several species of bees produce propolis, including Apis mellifera and stingless bees (Meliponini) [3], [4].
Stingless bees are widespread over tropical and some subtropical regions of the world [5], [6]. They are the major visitors of many flowering plants in the tropics. Propolis from stingless bees is well known for its therapeutic properties, including antimicrobial, antitumor and antioxidant activities [7], [8]. In Thailand, Tetragonula laeviceps is widely distributed and important because it is kept by local population and produces a large amount of propolis [9].
Research on the composition and biological activities of native Thai stingless bee propolis is scarce, although information on its chemical composition and bioactive compounds would be highly beneficial. This study investigated the chemical composition and antimicrobial activity of propolis of two native Thai stingless bee species, Tetragonula laeviceps and Tetrigona melanoleuca. Here we report, for the first time, information about T. melanoleuca propolis.

Ethics Statement
No specific permits were required for the described field studies. All field work was conducted on private land and with owner permission. The field studies did not involve endangered or protected species.

Propolis samples
Three Tetragonula laeviceps propolis samples were collected from Trat Province in eastern Thailand (12°21 0 N, 102°25 0 E) in December 2009. The Tetrigona melanoleuca propolis sample was collected from Chiang Mai Province in northern Thailand (18°48 0 N, 98°57 0 E) in February 2012. The propolis samples were collected from honeypots and scraping from the nests.

GC/MS analysis
Propolis samples (three of Tetragonula laeviceps and one of Tetrigona melanoleuca) were extracted with 70% ethanol (1:10, w/v) at room temperature for 24 h (3 times). (see supplement S1 Fig). The propolis extracts were evaporated to dryness and silylated using N,O-Bis(trimethylsilyl)trifluoroacetamide (BSTFA). Five milligrams of dry ethanol extract were mixed with 50 μl of dry pyridine and 75 μl of BSTFA, heated at 80°C for 20 min and analyzed by GC/ MS. The GC/MS analysis was performed with a Hewlett Packard Gas Chromatograph 5890 Series II Plus linked to a Hewlett Packard 5972 mass spectrometer system equipped with a 23 m long, 0.25 mm id and 0.5 μm film thickness HP5-MS capillary column. The temperature was programmed from 100 to 310°C at a rate of 5°C/min. Helium was used as the carrier gas with a flow rate 0.7 ml/min, split ratio of 1:80, injector temperature of 280°C and ionization voltage of 70 eV.

Extraction and isolation
NMR spectra: 1 H NMR (600 MHz) and 13 C NMR (150 MHz), Bruker AV 600. The NMR solvents are indicated in the Supplementary files together with the corresponding MNR spectra.
All structures were elucidated using NMR (1D and 2D) spectral data (S2-S14 Figs) and compared with the literature.

Antibacterial assay
The antibacterial activity of propolis ethanolic extracts and isolated compounds were investigated. The antibacterial assay was determined by dilution method, measuring the minimal inhibitory concentration (MIC) value in a 96-well microtiter plate [24]. Eleven test microorganisms; Listeria monocytogenes DMST 17303, Micrococcus luteus DMST 15503, Pseudomonas aeruginosa ATCC 9027, Staphylococcus epidermidis DMST 15505, Streptococcus pyogenes DMST 17020, methicillin-resistant Staphylococcus aureus (MRSA) DMST 20625, Serratia marcescens DMST 21632, Salmonella typhimurium DMST 562, Bacillus cereus TISTR 687, Escherichia coli ATCC 25922 and S. aureus TISTR 517 were used to test antimicrobial activity. All isolated compounds were dissolved by Dimethyl Sulfoxide (DMSO) for the antimicrobial test. Tested bacteria were cultured in Mueller Hinton broth (MHB) and incubated at 37°C for 24 hours. Bacteria were suspended in MHB by adjusting to 0.5 McFarland, yielding a final density of 10 8 cfu/ml. The ethanol extracts of propolis were prepared in concentrations ranging from 0.25 mg/ml to 128 mg/ml. In addition, pure compounds were prepared in concentrations ranging from 0.39 μg/ml to 25 μg/ml for this assay. The two fold serial dilutions of propolis extract or isolated compounds (180 μl) and test strain solution (20 μl) were added into each well of the microtiter plate (Cell Culture Plates, metric volume 0.36 ml). Positive (broth and inoculum) and negative (sterile broth) growth controls were used to compare. The MICs were determined as the lowest concentrations of compounds preventing visible bacteria growth. The minimum bactericidal concentrations were determined by subculturing 10 μl of inoculum from the MIC wells onto Mueller Hinton agar plates. The MBCs were determined as the lowest concentration that prevented visible growth of bacteria subcultures on the agar plate. Each sample was tested in triplicate. Gentamicin was used as positive control. The MICs and MBCs of gentamicin ranged from 0.02-0.78 mg/ml and 0.02-1.56 mg/ml, respectively.

Statistical analysis
Statistical significance was evaluated using one way analysis of variance (ANOVA) by SPSS version 16 (SPSS Inc.).

Chemical composition
The chemical profiles of propolis ethanol extracts were studied by GC-MS (after silylation). All three samples of T. laeviceps propolis displayed identical profiles, while T. melanoleuca propolis was different from them (Total Ion Chromatograms: S15 Fig). Moreover, the GC-MS profiles for propolis of both species did not coincide with any known propolis type and demonstrated the lack of plant secondary metabolites previously found in propolis. For this reason, it was necessary to isolate and identify individual compounds in order to reveal the specific chemistry and, if possible, the plant origin of the studied stingless bee propolis.
The petrol ether fraction of the ethanol extract of T. laeviceps propolis was subjected to repeated chromatographic separation and six individual compounds were isolated and characterized (Fig 1), among which the prenylated xanthones: α-mangostin 1, mangostanin 2, 8-deoxygartanin 3, gartanin 4, γ-mangostin 6 and the dammarane triterpene dipterocarpol 5. From the ethyl acetate fraction of the ethanol extract, a further xanthone garcinone B 7 and the furofurane lignane methylpinoresinol 8 were also isolated and identified. It is important to note that the xanthones are new propolis constituents and the first xanthones to be isolated from the propolis. Prenylated xanthones have been recognized as major secondary metabolites of Garcinia mangostana (Mangosteen), and all the xanthones (1-4, 6, 7) have been previously isolated from the pericarp and young fruit of mangosteen [10], [25], [26]. As it is well known that bees collect resinous material from the surfaces of young leaves, fruits and buds, G. mangostana is the most probable plant source of T. laeviceps propolis. The mangosteen trees are widespread across India, Myanmar, Malaysia, the Philippines, Sri Lanka and Thailand. The pericarp has been used in Thai indigenous medicine for the treatment of trauma, diarrhea and skin infections for a long time [27], [28]. Previous studies have demonstrated antibacterial activity of xanthones and extracts obtained from Mangosteen [29].
From petrol ether fraction of the ethanol extract of T. melanoleuca propolis, the triterpenes 3-O-acetyl ursolic acid 11, dipterocarpol 5, ocotillone I 12, ocotillone II 13, and two mixtures: of ursolic and oleanolic aldehydes 9-10, and of cabralealactones 14-15, were isolated after repeated chromatographic procedures. Their structures were confirmed by comparison of their NMR spectra with literature data. (S10-S14 Figs) All of these triterpenes are new propolis constituents (Fig 2). Their presence in this propolis provides valuable chemotaxonomic information about the plants from which the stingless bees T. melanoleuca collected resin. The simultaneous occurrence of dammarane (5, 12-15), ursane and oleanane derivatives (9, 10) has been described as an indicator of the presence of dammar in the mixture [30]. Dammar is a triterpenic resin produced by trees belonging to the family Dipterocarpaceae. Dammar was reported to possess antiviral activities and to be protective against in vitro low density lipoprotein (LDL) oxidation [31].
A further confirmation of origin of T. melanoleuca propolis from dammar resin was the identification in its GC-MS profile of other known dammar components: 2,3-dihydroxyolean-12-en-28-oic (maslinic) acid and 2,3-dihydroxyurs-12-en-28-oic (corosolic) acid were identified by comparison of the spectra of their silylated derivatives (S16 Fig) with literature data [32]. Two other acids were tentatively identified as 2,3-dihydroxyoleanadien-28-oic acid and 2,3-dihydroxyursadien-28-oic acid, based on comparison of the mass spectra of their TMS derivatives (S17 Fig) with the mass spectra of underivatized 2,3-dihydroxyoleanadien-28-oic acid and 2,3-dihydroxyursadien-28-oic acid [30], mass spectra of 2,3-diacetyloxyoleanadien-28-oic acid and 2,3-diacetyloxyursadien-28-oic acid [33] and mass spectra of silylated maslinic and corosolic acids [30]. The major peak in the TIC chromatogram (23% of TIC) belonged to 2,3-dihydroxyursadien-28-oic acid and this is characteristic for the specific chemical profile of Dipterocarpaceae resins, which has been previously demonstrated by Burger et al. [30]. Actually, different stingless bee species are known to collect resin from dipterocarp trees [34]; stingless bees are even called "dammar bees" in some parts of India [35]. Nonetheless, the reported triterpenes (5,(9)(10)(11)(12)(13)(14)(15) have not previously been found in stingless bee propolis.  [36]. The ethanol extract of T. melanoleuca propolis suppressed the development of S. aureus, methicillin-resistant Staphylococcus aureus and E. coli. The MICs and MBCs ranged from 2-16 mg/ml and 16-128 mg/ml, respectively. In general, the MIC of the total extracts were close to or above the value of 1 mg/ml, accepted as the highest relevant value in studies of the antibacterial activity of natural product extracts [37] The results demonstrated that, of all tested organisms, S. epidermidis was the most sensitive and S. marcescens the least sensitive (MIC = 16 mg/ml; MBC = 128 mg/ml). As can be seen, propolis displayed both bacteriostatic and bactericidal actions depending on the concentration, type of propolis, type of bacteria tested and methodologies to determine antimicrobial activity [38]. The ethanol extract of propolis from T. melanoleuca showed less activity against tested microorganism compared with T. laeviceps.
Furthermore, in search of the active principles, isolated pure compounds from both propolis types were tested for their antibacterial activity against several bacteria. The constituents of T. laeviceps propolis showed good activity (Table 1), especially against S. pyogenes (MIC = 0.78-25 μg/ml; MBC = 1.30-25 μg/ml), followed by L. monocytogenes (MIC = 0.78-25 μg/ml; MBC = >25 μg/ml). Concerning statistical analysis results, α-mangostin 1 was the most important antibacterial compound among the eight active compounds identified in the T. laeviceps propolis samples (p < 0.05). It is well known that the mangostins 1 and 6 are the major bioactive compounds in the mangosteen [39]. The antibacterial activities of T. laeviceps propolis extract could be attributed to the xanthones, especially 1 and 6.
The triterpenes isolated from T. melanoleuca propolis exhibited MIC 25 μg/mL against both Gram-positive and Gramm-negative bacteria. The only exception was the mixture of oleanolic and ursolic aldehides (9 and 10) with MIC 6.35 μg/mL against S. aureus. Previous studies have reported the antibacterial activity of these two compounds [40], [41]. The MBC were over 25 μg/mL in all cases, only 25 μg/mL for ocotillone I, ocotillone II and the mixture of cabralealactones against S. aureus.

Conclusions
The results of our study have revealed new data about the chemical composition and plant origin of stingless bee propolis from Thailand. They indicate for the first time the plant source, based on taxonomic markers, of the T. laeviceps propolis in Trat Province: the mangosteen Garcinia mangostana. They also indicated for the first time, based on our chemical study of T. melanoleuca propolis, that these stingless bees collect resin from dipterocarp trees. The antibacterial tests demonstrated some potential of the propolis extract from T. laeviceps against S. epidermidis, confirming its use in traditional medicine. The antibacterial activity of individual constituents of the studied propolis has also been proved.
Supporting Information