General experimental procedures
1D and 2D NMR spectra were recorded using Bruker 400 MHz spectrometer. The chemical shifts (δ) are reported in parts per million (ppm) relative to tetramethylsilane (TMS δ = 0). The coupling constants (J) are given in Hz. Deuterated methanol (CD3OD) was used as solvent in the NMR experiments. Mass spectral data ESI-MS, were recorded on Agilent technology instrument Accurate Mass Q-ToF spectrometer. The material used for chromatographic separation was Sephadex LH-20. Pre-coated silica gel 60 F254 TLC plates (Merck, Germany) were used for monitoring fractions and spots were detected with UV light (254 and 365 nm). Mueller Hinton (Agar and Broth) and Sabouraud Dextrose (Agar and Broth) were used for preparation of culture media for the antibacterial and antifungal activities respectively. Gentamicin, 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical, and quercetin were purchased from Sigma-Aldrich Co., USA. All solvents used were of analytical grade.
Collection and preparation of plant material
Fresh barks of Acacia ataxacantha were collected in September 2012 from Ouidah, Department of Atlantic, South of République du Bénin. Botanical identification was performed by a botanist from the Herbier National du Bénin at University of Abomey-Calavi and a specimen with voucher number AA 6509/HNB was deposited at the same Herbarium. The collected plant material was chopped into small pieces and air-dried in the laboratory (22 °C), under shade for two weeks. The dry plant material obtained was ground to a fine powder using an electric grinder (Excella mixer grinder).
Preparation of plant extract
Dry powdered barks of A. ataxacantha (250 g) were successively extracted with hexanes (3 × 500 ml), dichloromethane (3 × 500 ml), ethyl acetate (3 × 500 ml) and methanol (3 × 500 ml) by maceration with continuous shaking (at 24 °C for 72 h) using an orbital shaker (Ika Ks 260 basic). The extracted matter from each solvent was filtered first using a cotton plug followed by Whatman No 1 filter paper. The resulting extracts were concentrated using rotary evaporator (BUCHI Rotavapor RII, Switzerland) under reduced pressure and extracts were stored in refrigerator at 4 °C.
Isolation of bioactive compound
The best solvent system for column chromatography was selected for elution after carrying out the TLC of the ethyl acetate extract by the combinations of different solvents such as dichloromethane, ethyl acetate and methanol alternatively. Among all combination of solvents, dichloromethane: methanol combination showed the best resolution of the components of the extract on TLC plate.
The ethyl acetate extract (1 g) was dissolved in 1 ml of a mixture of CH2Cl2/CH3OH (50:50, v/v) and fractionated using Sephadex LH-20 column (diameter: 4 cm; Sephadex height: 25 cm). The elution solvent was the mixture CH2Cl2/CH3OH (50:50 and 20:80 v/v), to afford a total number of 31 fractions. A monitoring with TLC of these fractions yielded four main fractions (I-IV). Fraction III that consisted of major compounds in the extract was subjected to further purification. Purification of this fraction was done using preparative liquid chromatography (Gilson VP 250/21, Nucleodur 100-5 C18ec, Macherey-Nagel, UV detection 220 and 254 nm) by gradient elution (flow rate 20 ml/min) using water/acetonitrile (85:15 to 00:100 v/v) with 0.1% trifluoroacetic acid as mobile phase, to obtain compound 1 (9 mg).
Structural elucidation of isolated compound
Structural determination of the isolated compound was carried out by spectrophotometric methods (1D and 2D NMR, mass and UV spectrometry). 1D and 2D NMR spectrum were recorded at room temperature with a Bruker NMR spectrometer 400 and 500 MHz in CD3OD. The 2D experiments (COSY, NOESY, HSQC, HMBC) were performed using standard Bruker programs.
Acid hydrolysis of isolated compound
The isolated compound (1 mg) was dissolved in 6% HCl (1 ml) and heat at 80 °C for 2 h. The mixture was then extracted with CHCl3 (3 × 5 ml). The H2O phase was evaporated and dried to obtain the monosaccharide residue. This residue was identified as galactose by comparison with standards (galactose and glucose) on TLC in CHCl3/CH3OH/H20 (8:5:1) [23].
Microbial strains
The microorganisms used in this study included Gram-positives bacteria such as Staphylococcus aureus (ATCC 6538), Staphylococcus epidermidis (CIP8039), Enterococcus faecalis (ATCC 29212), Staphylococcus aureus Methicillin Resistant (SAMR) and Gram-negative Pseudomonas aeruginosa (CIP 82118). The microorganisms were obtained from Laboratoire de Biophotonique et Pharmacologie, University of Strasbourg, France. Candida albicans (CIP 4872) strain was obtained from national laboratory of drug control in Cotonou (Bénin). Bacterial cultures were maintained on Mueller-Hinton agar (MHA) and yeast cultures were maintained in Sabouraud Dextrose Agar (SDA) at 4 °C. Sub-culturing was done weekly. The bacteria were inoculated in Mueller-Hinton broth (MHB) for 18 h at 37 °C and yeast in Sabouraud Dextrose broth (SDB) for 48 h at 30 °C, prior to the test.
Minimum inhibitory concentration
The two-fold serial microdilution method was used to determine the minimum inhibitory concentration (MIC) values of isolated compound against microorganisms [21]. The stock solution (1 mg/ml) was prepared by solubilizing 1 mg of isolated compound in 50 μl of dimethyl sulfoxide (DMSO 2.5%) followed by 950 μl of MHB. Briefly, 100 μl of isolated compound (100 μg/ml), gentamicin and fluconazole (50 μg/ml) were two-fold serially diluted with Mueller-Hinton broth for antibacterial assay and Sabouraud broth for yeast assay in 96-well microplates to make eight concentrations of isolated compound (100-0.78 μg/ml) and control (50-0.39 μg/ml). 100 μl of freshly culture of bacteria (106 CFU/ml) and yeast (2 × 105 CFU/ml) was added to each well. DMSO (2.5%) was used as negative control while gentamicin and fluconazole were used as positive controls. The microplates were covered and incubated at 37 °C. After 18 h of incubation, 40 μl of 0.2 mg/ml solution of p-iodonitrotetrazolium (INT) which is an indicator solution for determination of bacterial growth were added to each well and microplates were further incubated at 37 °C. The minimal inhibitory concentration was determined 30 min after addition of INT.
Minimum bactericidal and fungicidal concentration
The minimum bactericidal concentration (MBC) and minimum fungicidal concentration (MFC) of isolated compound was determined according to the method of Escalona-Arranz et al. [24]. To determine the MBC and MFC, aliquots of 20 μl from all dilutions not showing any growth of bacteria and yeast were inoculated on sterile MHA plates (for bacteria) and SDA (for yeast) by spreading using swab sticks. Inoculated plates were incubated at 37 °C for 24 h for all bacteria while those inoculated with fungi were incubated at 30 °C for 48 h. After incubation, the concentration at which there is no visible growth of the organisms on the agar plates was recorded as the minimal bactericidal concentration (MBC) for bacteria and minimal fungicidal concentration (MFC) for yeast. The experiment was carried out in triplicate.
Determination of MIC index
The MIC index (MBC/MIC) was calculated for isolated compounds and positive controls to determine whether a compound had bactericidal/fungicidal (MBC/MIC ≤4) or bacteriostatic/fungistatic (4 < MBC/MIC <32) effect on the growth of bacteria or fungi [25].
Time-kill kinetic index
The time-kill kinetic index of isolated compound was determined as described by Miyasaki et al. [26] with slight modifications. The objective of this test is to know the duration of bactericidal or fungicidal effect of isolated molecule. Briefly, bacterial and yeast overnight cultures were diluted to the 106 CFU/mL with MH and SD broth respectively. Equal volume of each diluted inoculum and tested compound were mixed at their respective predetermined MBC and MFC values and incubated with shaking at respective temperature of 37 °C for bacteria and 30 °C for yeast. At different time intervals viz. 0, 1, 4, 8, 12, … 36 h, 0.1 mL of the mixed suspension was spread on suitable agar petri dishes in triplicate and incubated for 36 h at suitable temperature. After 18 h incubation, viable colonies were enumerated. The results were recorded in terms of log10 CFU and plotted vs. time for each microbial tested.
In vitro antioxidant activity
The antioxidant activity of isolated compound on the stable radical 2,2-diphenyl-1-picrylhydrazyl (DPPH) was performed using the method developed by Danielle and Lall, [27] with slight modifications. In this assay, 96 wells plates were used. For this assay, the stock solution was prepared by disolving 2 mg of isolated compound in 1 ml of methanol HPLC-grade to obtained 100 μg/ml. Quercetin was also prepared at the same concentration as the standard reference. Briefly, 200 μl of stock solution and quercetin was added separately to the wells in the top row. This was followed by the addition of 100 μl of methanol to the remaining wells. From the wells of the first row of the plate, the two-fold serially dilutions were performed to obtain a concentration ranges from 1.56 to 100 μg/ml. Finaly, 200 μl of methanolic solution of 2,2-diphenyl-1-picrylhydrazyl (2%) were introduced in each well of a microplate. The plates were allowed to develop in the dark room for 30 min before the measurement of the absorbance at 517 nm using a Microplate Reader (Rayto-6500). The capability of the compound and the reference standard to scavenging the free radical was determined following formula bellow:
$$ \mathrm{Inhibition}\ \mathrm{percentage}\ \left(\mathrm{I}\%\right) = \left[\left({\mathrm{A}}_{\mathrm{Blank}}\hbox{--}\ {\mathrm{A}}_{\mathrm{sample}}\right)\ /\ {\mathrm{A}}_{\mathrm{Blank}}\right]\ \mathrm{x}\ 100, $$
Where, ABlank is the absorbance of the control reaction (containing all reagents except the test sample) and Asample is the absorbance of sample (isolated compound or quercetin).
The concentration of samples reducing 50% of free radical DPPH (IC50) was determined using the regression line representing the inhibition percentage of DPPH versus the sample concentration. The assay was replicated three times and results are expressed as mean ± standard deviation.
Statistical analysis
The results were expressed as means of triplicate determination ± standard deviation (SD). The graphical was performed using the Graph Pad Prism 6.1 software (Microsoft, USA).