Plant material
Acacia ataxacantha barks were obtained from Ouidah, department of atlantic, South Bénin. Specimens were authenticated by Dr. Yedomohan, Botanist from National Herbarium of University of Abomey-Calavi. Voucher specimen (AA 6509/ HNB) have been deposited at the same Herbarium. The collected material was dried for four weeks in laboratory (22 °C), grinded into fine powder, and subjected to extraction.
Extraction and isolation
Dry powdered bark of A. ataxacantha (250 g) was successively extracted three times (3 × 500 ml) for 72 h with hexane, dichloromethane, ethyl acetate and methanol by maceration at room temperature. The resulting extracts were filtered, concentrated under reduced pressure, and kept at 4 °C. The dichloromethane extract (2.5 g) was chromatographed by gradient elution on an open column (Silica gel Si 60, 0.063–0.200, mesh) using the mixture n-hexane/EtOAc and EtOAc-MeOH in increasing polarity to yield 36 fractions. These fractions were assembled into four fractions (A, B, C and D) according to the chromatographic profile obtained after thin layer chromatography (TLC) analysis. Fractions A (450 mg) and B (250 mg), soluble in dichloromethane, were recrystallized with methanol. The white precipitates obtained were purified by successive washing with methanol to obtain respectively compound 1 (25 mg) and 2 (32 mg). Purification of fraction C was done using preparative HPLC (Gilson VP 250/21, Nucleodur 100-5 C18ec, Macherey-Nagel, UV detection 220 and 254 nm) with a gradient elution 10:90 to 90:10 (solvent A: H2O + 0.1 trifluoroacetic acid (TFA), B: AcN + 0.1 TFA) to obtain 7 mg of compound 3.
Chemical elucidation of compounds
Structural determination of the isolated compounds was carried out by spectrophotometric methods (1D and 2D NMR, mass and UV spectrometry). 1D (1H, 13C) and 2D (COSY, NOESY, HSQC and HMBC) NMR spectrum were recorded at room temperature with a Bruker NMR spectrometer (400 MHz and 500 MHz), and mass spectra were recorded using LC-ESI-MS.
Microbial strains
Bacterial cultures used in this study included Staphylococcus aureus (ATCC 6538), Staphylococcus epidermidis (CIP 8039), Enterococcus faecalis (ATCC 29212), Methicillin-Resistant Staphylococcus aureus and Pseudomonas aeruginosa (CIP 82118), obtained from Laboratoire de Biophotonique et Pharmacologie, University of Strasbourg, France. Candida albicans (CIP 4872) culture used in the present study was obtained from national laboratory of drug control in Cotonou (Bénin). Bacterial were maintained on Mueller-Hinton agar (MHA) and yeast on Sabouraud Dextrose Agar (SDA) at 4 °C. Sub-culturing was done weekly. The cells were inoculated in MH broth for bacteria (37 °C, 18 h) or SD broth for yeast (30 °C, 48 h) prior to the test.
Bioautography and identification of antimicrobial compounds
This test was performed only on selected bacterial cultures which were remarkably inhibited by dichloromethane and ethyl acetate extracts, according to a modified version of the method of Srinivas et al., [11]. 10 μl of the extracts (20 mg/ml) were applied on a chromatographic plate (Pre-coated TLC-sheets ALUGRAM® silica gel 60 with fluorescent indicator UV254; layer thickness 0.20 mm for analytical TLC) followed by elution with a mixture of dichloromethane/methanol (98:2) and dried in air. The plates were run in duplicate. The first plate was used as the reference chromatogram. The spots in the chromatogram were visualized in UV chamber (wavelength 365 and 254 nm) and the plate was sprayed with sulfuric vanillin reagent. Other plates were used for the bioautography. The chromatograms were sprayed with bacterial culture (106 CFU/ml) of S. aureus, Methicillin-resistant S. Aureus, S. epidermidis, E. faecalis, P. aeruginosa and fungi culture (2 × 105 CFU/ml) of C. albicans. Each plate was incubated at (37 °C, 24 h) for bacteria and (30 °C, 48 h) for yeast. The inhibition zones were visualized by spraying the plates with p-iodonitrotetrazolium (INT, 2.0 mg/ml).
Disc diffusion assay
The experiment was performed according to the method described by Qaralleh et al [12] with some modifications. For the determination of antimicrobial activity, cultures were adjusted to 106 CFU/ml for bacteria and 2 × 105 CFU/ml for yeast using 0.5 McFarland standards. Subsequently, cultures were inoculated into MHA for bacteria or SDA for yeast by spreading. The stock solutions of tested compounds were prepared by solubilizing 1 mg of compound in 50 μl of dimethyl sulfoxide 2.5 % (DMSO 2.5 %). Then, these solutions were diluted in 950 μl of Mueller-Hinton broth for bacteria and Sabouraud broth for yeast strain to obtain 1 mg/ml. The sterile discs of 6 mm of diameter were impregnated with 100 μg (50 μl, 2 mg/ml) of each compound. Discs of gentamicin (30 μg) and fluconazole (25 μg) were used as standard antibacterial and antifungal controls respectively. The plates were incubated at 37 °C, 24 h for bacteria and 30 °C, 48 h for yeast. The diameters of inhibitory zones (including the diameter of the discs) were measured after the incubation period and values superior to 7 mm (D ˃7 mm) were considered as active against microorganisms. All experiments were performed in triplicate and the antimicrobial activity was expressed as the mean of inhibition zone diameters.
Minimum inhibitory concentration
The two-fold serial microdilution method was used to determine the minimum inhibitory concentration (MIC) values of isolated compounds against microorganisms [9]. 100 μl of isolated compound (100 μg/ml) and 50 μg/ml of antimicrobial standards (Gentamicin, Fluconazol) were serially diluted two-fold in triplicate with Mueller-Hinton broth for antibacterial test and Sabouraud broth for yeast test in 96-well microplates to make eight concentrations of isolated compound (0.78–100 μg/ml) and standards (0.39–50 μg/ml). 100 μl of fresh culture of bacteria (106 CFU/ml) and yeast (2 × 105 CFU/ml) were added to each well. DMSO (2.5 %) was used as negative control while gentamicin and fluconazole were used as positive controls.
Minimum bactericidal and fungicidal concentration
The minimum bactericidal (MBC) and minimum fungicidal concentration (MFC) of isolated compounds was determined according to the method of Escalona-Arranz et al [13]. 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 with yeast were incubated at 30 °C for 48 h. After incubation, the concentration at which there is no visible growth on the agar plate was recorded as the minimal bactericidal concentration (MBC) and minimal fungicidal concentration (MFC). The experiment was carried out in triplicate.
Determination of MIC index
The MIC index (MBC/MIC) was calculated for each isolated compound and positive control drug to determine whether a compound had bactericidal/fungicidal (MBC/MIC ≤4) or bacteriostatic/fungistatic (4 < MBC/MIC <32) effect [14].
In vitro antioxidant activity
The isolated compounds (2 mg) and quercetin (control) were dissolved in 1 ml of methanol HPLC-grade. Dilutions were performed to obtain a stock solution at 100 μg/ml. The antioxidant activity of isolated compounds on the stable radical 2,2-diphenyl-1-picrylhydrazyl (DPPH) was determined by the method developed by Danielle and Lall [15], with slight modifications. In this method 96-well plates were used. The stock solution (100 μl) of each isolated compound and quercetin was added separately to the wells in the top row. A two-fold serial dilutions was performed to obtain a concentration range from 1.56 to 100 μg/ml. Finally, 200 μl of methanolic solution of 2,2-diphenyl-1-picrylhydrazyl (2 %) were introduced in each well. The plates were allowed to develop in the dark for 30 min before the measurement of the absorbance at 517 nm using a Microplate Reader (Rayto-6500). The capability of each compound and the standard to scavenging the free radical was determined as inhibition percentage using the following formula:
$$ \mathrm{Inhibition}\ \mathrm{percentage}\ \left(\mathrm{I}\%\right) = \left[\left({\mathrm{A}}_{\mathrm{Blank}}\hbox{--}\ {\mathrm{A}}_{\mathrm{sample}}\right)/{\mathrm{A}}_{\mathrm{Blank}}\right]\kern0.5em \times \kern0.5em 100 $$
ABlank is the absorbance of the control reaction (containing all reagents except the test sample) Asample is the absorbance of sample/standard.
The concentration of compound reducing 50 % of free radical DPPH (IC50) was determined graphically. The assay was replicated three times and results are expressed as mean ± standard deviation.
Statiscal analysis
All experiments were conducted in triplicate and the results were expressed as means ± standard deviation. The graph was performed using the Graph Pad Prism 6.1 software (Microsoft, USA).