Plant material
The stem bark of A. adianthifolia was collected from Mbouda (West Region of Cameroon) in January 2010. The plant material was identified at the Cameroon National Herbarium in Yaoundé where a voucher specimen (N° 19778/SRFCam) was deposited.
Extraction, fractionation and isolation
The stem bark of A. adianthifolia was dried at room temperature (25 ± 2°C) for three weeks and crushed. Four kilograms of obtained powder was macerated into 15 l ethyl acetate (Merck) for two days and this process was repeated twice. After filtration, the filtrate was evaporated to dryness at 50°C under reduced pressure using a rotary evaporator. The dried crude extract (1.75% w/w) was stored at +4°C. A portion of 60 g of crude extract was then subjected to column chromatography (22 cm x 8 cm column) using 300 g of silica gel 40 (particle size 0.2-0.5 mm). The column was successively eluted with hexane (4200 ml), Hexane – EtOAc [19 : 1 (3900 ml), 4 : 1 (4200 ml), 7 : 3 (4800 ml), 3 : 2 (3300 ml), 1 : 1 (3300 ml) and 3 : 7 (6600 ml)] mixtures, ethyl acetate (4500 ml), ethyl acetate-methanol [19 : 1 (3000 ml), 17 : 3 (3000 ml) and 7 : 3 (1500 ml)] mixtures and methanol (3900 ml). One hundred and fifty four fractions of 300 ml each were collected and combined on the basis of their thin layer chromatography (TLC) profiles to afford nine main fractions. Fractions 1–17, 18–25, 26–40, 41–54, 55–78, 79–92, 93–124, 125–148 and 149–160 were referred to as F1, F2, F3, F4, F5, F6, F7, F8 and F9 respectively. These fractions were tested for their antimicrobial/antioxidant activities and the most active fractions were further subjected to purification in order to isolate the active principles. Fraction F2 (2.80 g) was loaded on a silica gel column (0.063-0.20 mm, 120 g) eluted with hexane-EtOAc gradients and 37 subfractions of 100 ml each were collected. Subfractions 1–7 obtained with hexane were purified on a sephadex LH-20 column eluted with CH2Cl2-MeOH (9:1) to afford lupeol (45 mg) as yellow crystal. Subfractions 8–20 obtained with hexane-EtOAc (9:1) were purified by CC on sephadex LH-20 gel eluted with hexane-EtOAc (8:2) to give the mixture of fatty acids B1 (33 mg): oleic acid and n-hexadecanoic acid as yellowish crystal. Subfractions 21–32 obtained with hexane-EtOAc (1:1) were purified on a sephadex LH-20 column eluted with hexane-EtOAc (7:3) to yield the mixture of fatty acids B2 (46 mg): n-hexadecanoic acid, octadecanoic acid and docosanoic acid as yellowish crystal. Aurantiamide acetate (30 mg) was obtained from fraction F4 (17.20 g, eluted with CH2Cl2-EtoAc 19:1) after purification by preparative TLC. Fraction F5 (10.30 g) yielded two individual minor compounds (detected only on TLC) and a complex mixture.
Identification of the isolated compounds
The structures of the isolated compounds were established using spectroscopic analysis, especially, NMR spectra in conjunction with 2D experiments and by direct comparison with published information [10, 11] and authentic specimens obtained in our laboratory for some cases. Melting points (uncorr.) were determined on a Kofler apparatus. 1 H, 2D 1 H-1 H COSY, 13 C, 2D HMQC and HMBC spectra were recorded with a Bruker Avance 500 MHz spectrometer. Optical spectra were recorded with a NICOLET 510 P FT-IR spectrometer, a UV-2101 PC spectrometer, and a Perkin- Elmer 241 polarimeter. Column chromatography was run on Merck silica gel 60 and gel permeation on sephadex LH-20, while TLC were carried out either on silica gel GF254 pre-coated plates (analytical TLC) or on silica gel 60 PF254 containing gypsum (preparative TLC), with detection accomplished by spraying with 50% H2SO4 followed by heating at 100°C, or by visualizing with an UV lamp at 254 and 366 nm.
The mixtures of fatty acids were identified by comparison of their mass spectra with those available from the equipment database (Wiley 7 Nist 05.L) and from the literature. Gas chromatography–mass spectrometry (GC-MS) data were obtained with an Argilent 6890 N Network GC system/5975 Inert x L Mass selective Detector at 70 eV and 20°C. The GC column was a CP- Sil 8 CB LB, fused silica capillary column (0.25 mm x 30 m, film thickness 0.25 μm). The initial temperature was 50°C for 1 min, and then heated at 10°C/min to 300°C. The carrier gas was helium at a flow rate of 1.20 ml/min. Mass spectral data were used to identify fatty acid fractions.
Determination of total phenol content
Total phenol content was determined spectrophotometrically in the extracts by using Folin–Ciocalteu method as previously described [12]. The Folin–Ciocalteu reagent was prepared by mixing 5 g sodium tungstate, 1.25 g sodium molybdate, 2.50 ml of 85% phosphoric acid, 10 ml 20% hydrochloric acid, 7.50 g lithium sulfate, two drops of bromine and deionized water to a final volume of 50 ml. Further, stock solutions of 20% sodium carbonate and 400 mg/l gallic acid were added. For each sample, 20, 10 and 1 μl of 10 mg/ml ethanolic extract or 20 μl of 1 mg/ml ethanolic isolated compounds were added to 640 μl distilled water and 200 μl freshly prepared Folin–Ciocalteu reagent, followed by incubation in the dark for 5 min.
Then, 150 μl of 20% sodium carbonate solution were added and samples were incubated in the dark for 30 min. The solution turned deep blue. The final concentration of the tested samples in the assayed solution was 100 μg/ml and 10 μg/ml for the extracts and isolated compounds respectively. At the same time, gallic acid standards of 6.25, 12.50, 25, 50 and 75 μg/ml final concentration solutions were reacted with the Folin–Ciocalteu reagent in the same way as the samples. The UV–vis spectra of all the samples were recorded against the reference solution (zero gallic acid) and the absorbance was monitored at 725 nm. The measurements were done in triplicate. For the gallic acid standards, a calibration curve (Pearson’s correlation coefficient: R
2 = 0.992) was constructed and the total level of phenolics for each sample was determined in terms of gallic acid equivalents.
Antimicrobial assay
Micro-organisms
The microorganisms used in this study consisted of six bacteria (Enterococcus faecalis ATCC10541, Staphylococcus aureus ATCC25923, Pseudomonas aeruginosa ATCC27853, Escherichia coli ATCC11775, Klebsiella pneumoniae ATCC13883, Salmonella typhi ATCC6539) and seven fungi (Candida albicans ATCC9002, ATCC2091 and 24433, Candida parapsilosis ATCC22019, C. lusitaniae ATCC200950, C. tropicalis ATCC750, C. krusei ATCC6258); all of which are reference strains obtained from American Type Culture Collection. Also, included were two clinical isolates of bacteria (Proteus mirabilis, Shigella flexneri) collected from Pasteur Centre (Yaoundé-Cameroon) and two fungal strains (C. glabbrata IP35, Cryptococcus neoformans IP95026) obtained from Pasteur Institute (IP, Paris-France). The bacterial and yeast strains were grown at 35°C and maintained on nutrient agar (NA, Conda, Madrid, Spain) and Sabouraud Dextrose Agar (SDA, Conda) slants respectively.
Determination of the minimum inhibitory concentration (MIC) and minimum microbicidal concentration (MMC)
MIC was determined by broth micro dilution method as previously reported [13]. The inocula of micro-organisms were prepared from 24 h old broth cultures. The absorbance was read at 600 nm and adjusted with sterile physiological solution to match that of a 0.5 McFarland standard solution. From the prepared microbial solutions, other dilutions with sterile physiological solution were prepared to give a final concentration of 106 colony-forming units (CFU) per millilitre for bacteria and 2x105 spores per millilitre for yeasts. Stock solutions of the extracts (crude extract and fractions) were prepared in 5% aqueous tween 20 (Fisher chemicals) at concentrations of 50 mg/ml (for crude extract and fractions) and 1.60 mg/ml (for pure compounds). The two-fold serial dilutions in concentrations of the extracts (25–0.048 mg/ml) and pure compounds (800–0.39 μg/ml) were prepared in Mueller Hinton Broth (MHB) (Conda, Madrid, Spain) for bacteria and Sabouraud Dextrose Broth (SDB) (Conda, Madrid, Spain) for yeasts. For every experiment, a sterility check (5% aqueous tween 20 and medium), negative control (5% aqueous tween 20, medium and inoculum) and positive control (5% aqueous tween 20, medium, inoculum and water-soluble antibiotics) were included. In general, the 24-macro well plates (Nunclon, Roskilde, Danmark) were prepared by dispensing into each well 880 μl of an appropriate medium, 100 μl of test substances and 20 μl of the inoculum (106 CFU/ml for bacteria and 5x105 spores/ml for yeasts). The content of each well was mixed thoroughly with a multi-channel pipette and the macro-well plates were covered with the sterile sealer and incubated at 35 °C for 24 h (for bacteria) and 48 h (for yeasts) under shaking by using a plate shaker (Flow Laboratory, Germany) at 300 rpm. Microbial growth in each well was determined by observing and comparing the test wells with the positive and negative controls. The absence of microbial growth was interpreted as the antibacterial or antifungal activities. The MIC was the lowest concentration of the test substances that prevented visible growth of micro-organisms. Minimum Bactericidal Concentrations (MBCs) or Minimum Fungicidal Concentrations (MFCs) were determined by plating 10 μl from each negative well and from the positive growth control on Mueller Hinton Agar (for bacteria) and Sabouraud Dextrose Agar (for yeasts). MBCs or MFCs were defined as the lowest concentration yielding negative subcultures or only one colony. All the experiments were performed in triplicate. Gentamicin and nystatin at the concentration ranging between 400 and 0.78 μg/ml served as positive controls for antibacterial and antifungal activities respectively.
Antioxidant assay
DPPH free radical scavenging assay
The free radical scavenging activity of the extracts as well as their isolated compounds was evaluated according to described methods [14]. Briefly, the test samples, prior dissolved in DMSO (SIGMA) beforehand, were mixed with a 20 mg/l 2,2-diphenyl-1-picryl-hydrazyl (DPPH) methanol solution, to give final concentrations of 10, 50, 100, 500 and 1000 μg/ml. After 30 min at room temperature, the absorbance values were measured at 517 nm and converted into percentage of antioxidant activity. L-ascorbic acid was used as a standard control. The percentage of decolouration of DPPH (%) was calculated as follows:
(1)
The percentage of decolouration of DPPH (%) was plotted against the test sample. Also, the percentage of decolouration of DPPH was converted in probits. The probit values were plotted against the logarithmic values of concentrations of the test samples and a linear regression curve was established in order to calculate the EC50 (μg/ml), which is the amount of sample necessary to decrease by 50% the absorbance of DPPH. All the analysis were carried out in triplicate.
Trolox equivalent antioxidant capacity (TEAC) assay
The TEAC test was done as previously described [15] with slight modifications. In a quartz cuvette, to 950 μl acetate buffer (pH =5.0, 100 mM), the following were added: 20 μl laccase (1 mM stock solution), 20 μl test sample, 10 μl ABTS (2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid)) (74 mM stock solution). The sample concentrations in the assay mixture were 200, 100, 10 μg/ml for the extracts and 20 μg/ml for the isolated compounds. The content of the generated ABTS●+ radical was measured at 420 nm after 230 s reaction time and was converted to gallic acid equivalents by the use of a calibration curve (Pearson’s correlation coefficient: R
2 = 0.997) constructed with 0, 4, 10, 14, 28, 56, 70 μM gallic acid standards rather than Trolox. Experiments were done in triplicate.
Statistical analysis
Statistical analysis was carried out using Statistical Package for Social Science (SPSS, version 12.0). The experimental results were expressed as the mean ± Standard Deviation (SD). Group comparisons were performed using One Way ANOVA followed by Waller-Duncan Post Hoc test. A p value of 0.05 was considered statistically significant.
