Plant materials, β-lactam antibiotics, bacterial strains, and cell line
The dried fruit hulls of mangosteen were purchased locally in Nakhon-Ratchasima, Thailand. The samples were identified by Dr. Paul J. Grote, Suraneree University of Technology. The voucher specimens (SGM0804U) were deposited in the School of Pharmacology, Institute of Science, Suranaree University of Technology, Nakhon-Ratchasima, Thailand. The mature fruit was cleaned. The fruit rinds were cut into small pieces, dried in a hot oven at 50 °C for 72 h and ground into powder, passed through a sieve (20 mesh). The powdered sample was kept in an airtight container protected from light until used.
All clinical isolates of S. saprophyticus were obtained from the Department of Medical Science, National Institute of Health, Ministry of Public Health, Bangkok, Thailand. The susceptible strain S. aureus ATCC 29213, a reference strain, was obtained from the American Type Culture Collection (ATCC). Oxacillin, Nisin, o-nitrophenol-β-D-galactoside (ONPG), α-mangostin standard, and β-lactamase type IV isolated from E. cloacae were obtained from Sigma-Aldrich, UK. Meuller-Hinton broth (MHB) and Mueller-Hinton agar (MHA) were purchased from Oxoid (Basingstoke, UK).
The 3T3-L1 mouse embryonic fibroblasts and bovine calf serum (CBS) were purchased from the American Type Culture Collection (ATCC, USA). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), Dulbecco’s Modified Eagle’s medium (DMEM), Fetal bovine serum (FBS), Penicillin-Streptomycin, N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid (HEPES) were obtained from Gibco Invitrogen (Grand Island, NY, USA).
Isolation and purification of α-mangostin
α-mangostin from the pericarp of GML was isolated and purified according to previous methods with some modifications [12, 14]. Briefly, 1 kg of dried pericarp powder of GML was extracted successively with n-hexane, dichloromethane (CH2Cl2), ethanol, and acetone using a Soxhlet extractor. The filtered extracts were then concentrated using a rotatory evaporator to provide a yellowish power for n-hexane (84 g) and dichloromethane (106 g), brown sticky semi-solid for ethanol (262 g) and acetone (130 g) respectively.
The CH2Cl2 crude extract was further separated by silica gel column chromatography to yield 11 fractions. These fractions were subjected to HPLC (C18 column, a mobile phase of methanol-water (85:15) and a flow rate of 1.0 ml/min, a photodiode array detector) and purified using preparative thin layer chromatography to obtain isolated α-mangostin. The α-mangostin was analyzed by HPLC and its chemical structure was elucidated by 1H NMR and 13C NMR. The spectrum structure data of this compound was compared with those previously reported .
Standardised bacterial suspensions
To select bacterial suspensions with a known viable count, the method of Liu et al.  was followed with little modifications. MHA and Cation-adjusted Mueller-Hinton broth (CAMHB) were used as a medium.
Minimum inhibitory concentration (MIC) and checkerboard determinations
The antibacterial activity and drug interaction of isolated α-mangostin from the pericarp of GML with oxacillin were performed by MIC and checkerboard assays, respectively using broth macrodilution procedure. These assays were conducted following the methods of Clinical and Laboratory Standard Institute guidelines [16, 17]. In summary, 0.25 ml of 5 × 106 cfu/ml bacterial suspensions was added to a series of 2.25 ml CAMHB plus 1 in 10 serial dilutions of the α-mangostin plus oxacillin combinations to give 5x105 cfu/ml. Tubes of the broth without antimicrobialsl were used as the control. The cultures were incubated for 24 h at 37 °C. The tests were carried out in triplicate. MICs were determined for each antibacterial combination and the isobolograms were plotted. The interaction between the two agents was calculated by the fractional inhibitory concentration (FIC) index of the combination. The FIC of each agent was calculated by the complete growth inhibition of microorganism in the combination tube. The following formula was used for FIC index calculation: FIC of α-mangostin = MIC α-mangostin in the combination/MIC of α-mangostin alone; FIC of oxacillin = MIC of oxacillin in the combination/MIC of oxacillin alone; therefore, FIC index = FIC of α-mangostin + FIC of oxacillin. When the FIC index of the combination is equal to or less than 0.5, the combination is defined as synergistic; when the FIC index falls between 0.5 and 4.0, it indicates ‘no interaction’ between the agents and a value above four is considered to show antagonism between the two compounds . S. aureus ATCC 29213 was used as positive control. The MICs and FIC index is presented as the median values obtained in duplicates from three independent experiments.
Kill curve determinations (Viable counts)
The experiment was carried out to confirm antibacterial and synergistic activities of isolated α-mangostin from the pericarp of GML when used singly and in combination with oxacillin as previously described by Mun et al. and Richards et al. [19, 20]. Compounds were used at the half minimal inhibitory concentration (1/2-MICs) when each compound was assessed alone. However, to study the effect of the compounds in combination, each compound was used at the MIC that yielded synergism.
The ability of isolated α-mangostin from the pericarp of GML to inhibit the activity of β-lactamase type IV isolated from E. cloacae was determined in accordance with the methods of Eumkeb et al. and Richards et al. [21, 22]. Concisely, benzylpenicillin, a substrate for β-lactamase type IV, was adjusted to concentrations sufficient to hydrolyze 50-60 % substrate within 5 min, β-lactamase at 100 μg/ml was used. The α-mangostin at 1, 2, 4 and 8 μg/ml were preincubated with the enzyme in 50 mM sodium phosphate buffer (pH 7.0) at 37 °C for 5 min before adding a substrate. A time - course assay was performed at 0, 5, 10, 15 and 20 min using methanol/acetic acid (100:1) as a stopping agent. Aliquots (10 μl) of each sample were injected onto a reverse-phase HPLC (Ascentis C18 column) to analyse the remaining benzylpenicillin. The mobile phase consisted of 10 mM ammonium acetate (pH 4.5 acetic acid): acetonitrile (75:25) with a flow rate of 1 ml/min, UV detection of peaks was at 200 nm,, and the column maintained at 35 °C. The quantity of remaining benzylpenicillin was calculated by comparing the area under the chromatographic curve.
Transmission electron microscopy (TEM)
To determine the ultrastructure morphology of bacteria after treatment with isolated α-mangostin from the pericarp of GML either alone or in combination with oxacillin, the method of Richards et al.  was followed. To investigate the mechanism of action of these agents, the half-MICs of both compounds used alone and Sub-FICs of the combination, were chosen for examination. To confirm the effects of these agents either used singly and in combination on cell size, the cell area from micrographs were analyzed by measuring cell width multiplied by cell length (nm2). The experiment was performed in triplicate, and the cell areas are displayed as mean ± SEM .
Immunofluorescence staining and confocal microscopy
The disruption of peptidoglycan after exposure to α-mangostin either used singly or in conjunction with oxacillin was performed using immunofluorescence and visualized under a confocal laser scanning microscope following the method of Teethaisong et al. . Shortly, after the FIC index was obtained from checkerboard, the half-MICs value of isolated α-mangostin or oxacillin alone and the 3/4 FIC of this combination that showed synergistic FIC index was chosen for examination. The cells grown without any antibacterial agent were employed as control .
Cytoplasmic membrane (CM) permeabilization assays
Two methods were used to assess CM permeabilization. Firstly; the CM permeabilization experiment was performed, with some modifications, to confirm results as previously described by Shen et al. and Zhou et al. [26, 27]. Shortly after the FIC index was determined by the checkerboard assay, the half-MIC values for isolated α-mangostin or oxacillin alone, and the 3/4 MIC values for this combination that indicated synergistic FIC index were selected against ORSS to measure CM permeability. This method was performed by measuring the release of UV-absorbing material (Varian’s Cary 100 UV-Vis spectrophotometer, Varian, Inc., California, USA) .
Secondly, the α-mangostin-induced permeabilization of the CM of ORSS was determined essentially as recently described . In brief, to assay CM permeabilization, the wells contained 50 μl ONPG plus either half-MIC values for isolated α-mangostin or oxacillin alone and the 3/4 MIC values for this combination that indicated synergistic FIC index were prepared shortly before the experiment. Finally, 50 μl of cell suspension (OD 0.3) was added to the wells to give a final concentration of 100 μg/ml ONPG. After warming to 37 °C the plates were positioned in the plate reader at 37 °C. ONPG uptake and cleavage by β-galactosidase within the cytoplasm was characterized by monitoring absorption over a period of 120 min at 420 nm. Complete permeabilization was induced in the presence of 0.5 μg/ml Nisin as a positive control and wells lacking drugs or isolated α-mangostin test served as a negative control [29, 30].
In vitro cytotoxicity test (MTT assays)
The 3T3-L1 preadipocytes were cultured in Dulbecco’s modified Eagle’s medium (DMEM) with high glucose, supplemented with 10 % CBS, 1.5 mg/ml sodium bicarbonate, 100 U/ml penicillin and 100 μg/ml streptomycin until confluent. The cells were maintained at 37 °C in 5 % CO2 and 95 % humidity. The cytotoxic effect of α-mangostin, oxacillin, either alone or in combination on cell proliferation was determined using a tetrazolium dye (MTT) in a colorimetric assay . Briefly, the cells were seeded in a 96-well plate at a density of 5 × 103 cells/well. The cells were allowed to adhere for 48 h and then were treated with various concentrations of three compounds for 24 h. After incubation, the cultured medium was removed, and 0.5 mg/ml of MTT was added. Then, cells were further incubated for 4 h at 37 °C. Formazan crystals formed by viable cells were dissolved in DMSO and absorbance was measured at 540 nm with a microplate spectrophotometer (Benchmark Plus, Bio-Rad, Japan).
The experiments were carried out in triplicate; data were expressed as mean ± standard error of the mean (SEM). Significant differences in the enzyme assay among each of treated groups at the same time, the cell area of each treated group, CM permeabilization, and MTT assays were analysed by one-way ANOVA followed by Scheffe’s posthoc test. The p < 0.01 was considered as the statistically significant difference.