Experimental
Melting points were recorded with a Reichert microscope and are uncorrected. 1H NMR (500 MHz) and 13C NMR (125 MHz) were recorded at room temperature in CD3OD or (CD3)2SO, on a Bruker Avance DRX-500 spectrometer. Chemical shifts (δ) are reported in parts per million (ppm) with the solvent signals as reference relative to TMS (δ = 0) as internal standard, while the coupling constants (J values) are given in Hertz (Hz). COSY, ROESY, TOCSY, HSQC and HMBC experiments were recorded with gradient enhancements using sine shape gradient pulses. The IR spectra were recorded with a Shimadzu FT-IR-8400S spectrophotometer. ESI-MS experiments were performed using a Micromass Q-TOF micro instrument (Manchester, UK) with an electrospray source. Column chromatography was run on Merck silica gel 60 (70–230 mesh) and gel permeation on Sephadex LH-20 while TLC was carried out on silica gel GF254 pre-coated plates with detection accomplished by spraying with 50% H2SO4 followed by heating at 100°C, or by visualizing with an UV lamp at 254 and 365 nm.
Plant material
The leaves of O. spinosa Forssk. were collected at Dschang, West Region, Cameroon, in May 2007. Authentication was done at the Cameroon National Herbarium, Yaoundé, where the voucher specimen (No. 21975 HNC) is deposited.
Extraction and isolation
The air-dried and powdered leaves of O. spinosa (2 kg) were extracted by percolation in methanol for 3 days at room temperature. Evaporation of solvent under reduced pressure yielded 40 g of extract. Part of the MeOH extract (35 g) was subjected to column chromatography (silica gel 60, 70–230 mesh) and eluted with hexane followed by hexane-EtOAc gradient. Sixty fractions of 200 ml each were collected and combined on the basis of TLC analysis to afford four major fractions: A (9 g; hexane-EtOAc 100:0 and 9:1), B (8.5 g, hexane-EtOAc 4:1 and 7:3), C (11 g, hexane-EtOAc 1:1 and 0:100) and D (6.7 g, EtOAc-MeOH 19:1 and 9:1). Fraction A, mainly oil, was not further investigated in this work. Fraction B was further purified on silica gel column chromatography eluted with hexane-EtOAc 17:3, 4:1 and 3:1, respectively, to afford 30 sub-fractions (B1 and B2). Sub-fraction B2 (17–30) was purified through Sephadex LH-20 column chromatography eluted with CH2Cl2-MeOH 1:1 to yield kaempferol (1) (2.5 mg), quercetin (2) (5 mg) and apigenin-7-O-β-D-glucuronopyranoside (3) (11 mg). Silica gel column chromatography of fraction C, eluted with EtOAc-MeOH-H2O 9:0.5:0.5 yielded apigenin-7-O-β-D-glucuronopyranoside (3) (25 mg) and quercetin 3-O-β-D-galactopyranoside (4) (33 mg). Fraction D was purified through Sephadex LH-20 gel permeation eluted with MeOH to give 25 sub-fractions (10 ml each). Re-crystallization of these sub-fractions yielded quercetin 3-O-β-D-galactopyranoside (4) (6 mg), quercetin 3-O-α-L-rhamnopyranosyl (1 → 6) β-D-glucopyranoside (5) (24.5 mg) and complex mixtures.
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Kaempferol (1): yellow crystals from hexane-EtOAc; m.p. 275–277°C; C15H10O6.
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Quercetin (2): yellow needles from hexane-EtOAc; m.p. > 300°C; C15H10O7.
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Apigenin-7-O-β-D-glucuronopyranoside (3): yellow powder from EtOAc; m.p. > 300°C; C21H18O11.
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Quercetin 3-O-β-D-galactopyranoside (4): yellow needles from EtOAc-MeOH; m.p. 230–232°C; C21H20O12.
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Quercetin 3-O-α-L-rhamnopyranosyl (1 → 6) β-D-glucopyranoside (5): yellow powder from EtOAc-MeOH, m.p. 213–216°C; C27H30O16.
Antimicrobial assay
Bacterial and fungal strains
The studied microorganisms were both reference (from the American Type Culture Collection) and clinical (from Pasteur Institute Paris, France) strains of Enterobacter aerogenes, Escherichia coli, Klebsiella pneumoniae, Candida albicans, and Cryptococcus neoformans. Also, included were two clinical isolates of Candida parapsilosis and Staphylococcus aureus collected from Pasteur Centre (Yaoundé-Cameroon). The bacterial and fungal species were grown at 37°C and maintained on nutrient agar (NA, Conda, Madrid, Spain) and Sabouraud Dextrose Agar (SDA, Conda) slants respectively.
Preparation of microbial inoculum
The inocula of yeasts and bacteria were prepared from overnight cultures by picking numerous colonies and suspending them in sterile saline (NaCl) solution (0.90%). Absorbance was read at 530 nm for yeasts or at 600 nm for bacteria and adjusted with the saline solution to match that of a 0.50 McFarland standard solution. From the prepared microbial solutions, other dilutions with saline solution were prepared to give a final concentration of 106 yeast cells/ml and 106 CFU/ml for bacteria [14,20].
Antimicrobial assay
The antimicrobial activity was investigated by determining the minimum inhibitory concentrations (MICs), minimum bactericidal concentrations (MBCs) and minimum fungicidal concentrations (MFCs).
MICs were determined by broth micro dilution [12,21]. Stock solutions of the tested samples were prepared in 10% v/v aqueous dimethylsulfoxide (DMSO) solution (Fisher chemicals, Strasbourg, France) at concentration of 4096 μg/ml. This was two-fold serially diluted in Mueller-Hinton Broth (MHB) for bacteria and Sabouraud Dextrose Broth (SDB) for fungi to obtain a concentration range of 2048 to 0.25 μg/ml. For every experiment, a sterility check (10% aqueous DMSO and medium), negative control (10% aqueous DMSO, medium and inoculum) and positive control (10% aqueous DMSO, medium, inoculum and water-soluble antibiotics) were included. One hundred microliters of each concentration was introduced into a well (96-wells microplate) containing 90 μl of SDB or MHB and 10 μl of inoculum was added to obtain a final concentration range of 4096 to 0.125 μg/ml. The plates were covered with a sterile lid, and incubated on the shaker at 37°C for 24 h (bacteria) and 48 h (yeasts). MICs were assessed visually after the corresponding incubation period and were taken as the lowest sample concentration at which there was no growth or virtually no growth. The assay was repeated thrice.
For the minimum microbicidal concentration (MMC) determination, 10 μl aliquots from each well that showed no growth of microorganism were plated on Mueller-Hinton Agar or Sabouraud Dextrose Agar and incubated at 37°C for 24 h (bacteria) and 48 h (yeasts). The lowest concentration that yielded no growth after the sub-culturing was taken as the MBCs or MFCs. Chloramphenicol (Sigma-Aldrich, Steinheim, Germany) for bacteria and nystatin (Sigma-Aldrich, Steinheim, Germany) for yeasts were used as positive controls.
Antioxidant assay
DPPH free radical scavenging assay
The free radical scavenging activity of the MeOH extract as well as some of its isolated compounds was evaluated according to described methods [22,23] with slight modifications. 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:
$$ \%\ \mathrm{decolouration}\ \mathrm{of}\ \mathrm{DPPH}=\frac{\left(\mathrm{Absorbance}\ \mathrm{of}\ \mathrm{control}\ \hbox{-}\ \mathrm{Absorbance}\ \mathrm{of}\ \mathrm{test}\ \mathrm{sample}\Big) \times 100\right)}{\mathrm{Absorbance}\ \mathrm{of}\ \mathrm{control}} $$
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 [11]. All the analyses were carried out in triplicate.
Trolox equivalent antioxidant capacity (TEAC) assay.
The TEAC test was done as previously described [24] 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 laccase were purified from Sclerotinia sclerotiorum according to the protocol described [25]. The sample concentrations in the assay mixture were 400, 200, 100, 10 μg/ml for the MeOH extract 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 = 0.998) constructed with 0, 4, 10, 14, 28, 56, 84 μM gallic acid standards rather than Trolox [22,23]. Experiments were done in triplicate.
Hemolytic assay
Whole blood (10 ml) from a healthy man was collected into a conical tube containing heparin as an anticoagulant (blood group O was used). Authorization for the collection of blood was obtained from the Medical and Ethical Committee (in Yaoundé-Cameroon). The written informed consent for participation in the study was obtained from a parent of 39 years old. Erythrocytes were harvested by centrifugation for 10 min at 1,000 × g and room temperature and washed three times in PBS solution. The top layer (plasma) and the next, milky layer (buffy coat with a layer of platelets on top of it) were then carefully aspirated and discarded. The cell pellet was resuspended in 10 ml of PBS solution and mixed by gentle aspiration with a Pasteur pipette. This cell suspension was used immediately.
For the normal human red blood cells, which are in suspension, the cytotoxicity was evaluated as previously described [26]. MeOH extract (at concentrations ranging from 64 to 2048 μg/ml) and compounds 2–5 (32 to 512 μg/ml), were incubated with an equal volume of 1% human red blood cells in phosphate buffered saline (10 mM PBS, pH 7.4) at 37°C for 1 h. Ampicillin and chloramphenicol were tested simultaneously. Non-hemolytic and 100% hemolytic controls were the buffer alone and the buffer containing 1% Triton X-100, respectively. Cell lysis was monitored by measuring the release of hemoglobin at 595 nm with a spectrophotometer (Thermo Scientific, USA). Percent hemolysis was calculated as follows:
$$ \frac{\left[\left(\mathrm{A}595\mathrm{of}\ \mathrm{sample}\ \mathrm{treated}\ \mathrm{with}\ \mathrm{compound}\hbox{--} \mathrm{A}595\ \mathrm{of}\ \mathrm{sample}\ \mathrm{treated}\ \mathrm{with}\ \mathrm{buffer}\right)\right]}{\left[\left(\mathrm{A}595\ \mathrm{of}\ \mathrm{sample}\ \mathrm{treated}\ \mathrm{with}\ \mathrm{Triton} \times \hbox{--} 100\ \hbox{--} \mathrm{A}595\mathrm{of}\ \mathrm{sample}\ \mathrm{treated}\ \mathrm{with}\ \mathrm{buffer}\right)\right]}\times 100 $$
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.