Plant material, extraction and phytochemical screening
The stem bark of E. abyssinica was collected in May 2007 in Menoua Division, West Cameroon. Botanical identification was done at the Cameroon National Herbarium in Yaounde by Mr Tadjouteu Fulbert, where a voucher specimen was kept under the reference number 44732/HNC. The stem bark was cut into pieces, air-dried under shade and ground into powder using an electric grinder. A mass of 375 g of powder was exhaustively extracted with 1 l of methanol. After filtration, the solvent was evaporated under reduced pressure in a rotary evaporator at 45°C to afford the methanol extract (47.50 g). An amount of 32.50 g of this extract was pre-dissolved in 100 ml of a mixture of methanol and water (1:9) and then 400 ml of n-hexane was added and shaken vigorously. After about 30 min, the n-hexane phase was collected and the process repeated thrice. Methanol was then evaporated from the polar phase and the aqueous residue treated sequentially with ethyl acetate and n-butanol. The n-hexane, ethyl acetate and n-butanol were evaporated under reduced pressure in rotary evaporator to afford 4.64, 15.78 and 2.63 g of fractions respectively. The aqueous residue (9.44 g) was obtained after drying the residual portion in the oven at 40°C for 48 h. The methanol extract and fractions were subjected to phytochemical screening using standard procedures .
Fractionation and isolation
A quantity of 10.5 g of the ethyl acetate fraction was subjected to silica gel 60 (0.20-0.500 mm) flash chromatography and eluted with mixtures of n-hexane (Hex) and ethyl acetate (EtOAc) of increasing polarity (0-100%) to yield a total of 9 fractions of 200 ml each. These fractions were combined on the basis of TLC profiles into four major fractions: F1 [4.77 g, Hex/EtOAc (100:0), (80:20), (70:30)], F2 [2.48 g, Hex/EtOAc (60:40), (50:50)], F3 [1.56 g, Hex/EtOAc (40:60)] and F4 [0.63 g, Hex/EtOAc (30:70), (20:80), (0:100)]. Fraction F1 was further dissolved in a mixture of Hex and EtOAc (60:40) and a whitish compound precipitated. It was filtered and rinsed with EtOAc to afford compound 1 (100 mg). The filtrate (4.5 g) was subjected to further silica gel column chromatography (0.063-0.200 mm) to afford compound 2 (113 mg). Fraction F3 afforded compound 3 (108 mg) as a yellowish powder from the mixture Hex/EtOAc (60:40). Fraction F4 was further subjected to column chromatography (0.063-0.200 mm) purification using Hex and EtOAc (40:60) to afford compound 4 (54 mg).
The n-hexane fraction was subjected to GC-MS using an Agilent 6890N Network GC system/5975 Inert × L Mass selective Detector at 70 eV and 20°C. The GC column was a CP-S-il 8 CB LB, fused silica capillary column (0.25 mm × 30 m, film thickness 0.25 μm). Helium was used as carrier gas at a flow rate of 1.2 ml/min. The injector port was maintained at 250°C; the oven temperature was programmed at 5°C/min from 70°C to 300°C. A solution of each fraction was prepared in chloroform at a concentration of 10% (wt/v). To 90 μl of this solution, 10 μl of trimethyl sulfonium hydroxide (TMSH) was added and 1 μl of the resulting mixture was injected into the GC -MS apparatus. The constituents were identified by comparing their mass spectra data with those stored in NIST05 and Wiley237 database libraries.
Aluminium sheet pre-coated with silica gel 60 GF254 (Merck) was used for thin layer chromatography (TLC). The spots were visualized under UV light (254 and 366 nm) with a UV lamp model 52-58 mineralight, and sprayed with 50% aqueous solution of H2SO4 followed by heating at 100°C.
IR spectra were measured with KBr disks using FT-IR-8400 S Shimadzu spectrophotometer. EI-MS were carried out on a GCT Premier CAB109 TOF mass spectrometer. 1H-, 13C-NMR and 2D-NMR (COSY 1H-1H, HMBC and HSQC) spectra were recorded in acetone-d
(500 MHz for 1H and 125 MHz for 13C) on a Brücker-Avance-500 MHz NMR spectrometer.
(5S,6R,8aR)-5-(carboxymethyl)-3,4,4a,5,6,7,8,8a-octahydro-5,6,8a-trimethylnaphthalenecarboxylic acid (1): White powder; 13C NMR (100 MHz, CDCl3 + CD3OD): δ 14.9 (5-Me), 17.8 (C-4), 20.7 (6-Me), 21.0 (8a-Me), 25.3 (C-7), 27.0 (C-3), 29.4 (C-8), 35.2 (C-6), 37.5 (C-8a), 38.5 (C-5), 43.6 (C-1'), 44.6 (C-4a), 136.9 (C-2), 142.6 (C-1), 169.3 (1-COOH), 175.4 (C-2'); FABMS: m/z 279 ([M - H]+, 98), 153 (100), 151 (44), 46 (13); HRFABMS: m/z 279.1592 (calcd. for C16H23O4: 279.1596).
Methyl 3,4,5-trihydroxybenzoate (2): Colorless needles; 13C NMR (100 MHz, CDCl3): δ 51.7 (OCH3), 109,8 (C-2/C-6), 125.2 (C-1), 148.4 (C-3/C-5), 138.9 (C-4), EIMS: m/z 184 ([M]+, 99), 153 (100), 125 (14), 107 (2), 79 (3); HREIMS: m/z 184.0370 (calcd. for C8H8O5: 184.0372).
Benzene-1,2,3-triol (3): White powder; 13C NMR (100 MHz, CDCl3): δ 110.2 (C-4/C-6), 124.6 (C-5), 138.5 (C-2), 148.8 (C-1/C-3); EIMS: m/z 126 ([M]+, 100), 108 (21), 80 (24), 52 (27); HREIMS: m/z 126.0312 (calcd. for C6H6O3: 126.0317).
2,3-dihydroxypropyltriacontanoate (4): Whitish gum; 13C NMR (125 MHz, CDCl3): δ 14.2 (CH3), 22.7-34.2 (28 CH2), 63.4 (C-3), 65.2 (C-1), 70.3 (C-2), 172.3 (COO); EIMS: m/z 43 (100), 57 (80), 71 (44), 83 (64), 85 (28), 97 (69), 99 (7), 111 (37), 125 (19), 127 (3), 139 (8), 141 (3), 153 (5).
Microorganisms and growth conditions
The microorganisms used in this study consisted of two Gram (+) bacteria (Enterococcus faecalis ATCC 10541 and Staphylococcus aureus ATCC 25922); six Gram (-) bacteria (Pseudomonas aeruginosa ATCC 27853, Escherichia coli ATCC 11775, Klebsiella pneumoniae ATCC13883, Salmonella typhi ATCC 6539, Proteus mirabilis and Shigella flexneri); and 10 yeasts (Candida albicans ATCC 9002, C. albicans ATCC 2091, C. albicans ATCC 24433, C. parapsilosis ATCC 22019, C. lusitaniae ATCC 200950, C. tropicalis ATCC 750, C. krusei ATCC 6258, C. guillermondi, C. glabbrata IP 35 and Cryptococcus neoformans IP 95026). The reference strains (ATCC) were obtained from American Type Culture Collection (Rockville, USA). The two clinical bacterial isolates were collected from "Centre Pasteur" (Yaoundé, Cameroon) and the two IP fungal strains were obtained from "Institute Pasteur" (Paris, France). The bacterial and fungal strains were grown at 35°C and maintained on nutrient agar (NA, Conda, Madrid, Spain) and Sabouraud Dextrose Agar (SDA, Conda) respectively.
The minimum inhibitory concentration (MIC) of the crude methanol extract, fractions and isolated compounds were determined through broth microdilution method in 96-well micro-titre plates as described by Zgoda and Porter . The 96-well plates were prepared by dispensing into each well 100 μl of Mueller Hinton broth for bacteria and Sabouraud Dextrose broth for yeasts. The test substances were initially prepared in 10% ethanol/tween 80 in broth medium at 3124.8 μg/ml (methanol extract and fractions), 1250 μg/ml (isolated compounds) and 50 μg/ml (reference antibiotics). A volume of 100 μl of each test sample was added into the first wells of the micro-titre plate. Serial two-fold dilutions of these test samples were made and 100 μl of inoculum standardized at 106 CFU/ml for bacteria or 2.5 × 105 CFU/ml for yeasts (at 600 nm, Jenway 6105 UV/Vis spectrophotometer- 50 Hz/60 Hz)  was then added into each well. The last wells (N°12) served as sterility controls (contained broth only) or negative control (broth plus inoculum). This gave final concentration ranges of 781.25-0.76 μg/ml, 312.50-0.30 μg/ml and 12.50-0.01 μg/ml for the methanol extract or fractions, isolated compounds and reference substances respectively. The plates were sealed with parafilm, then agitated with a plate shaker to mix their contents and incubated at 35°C for 24 h for bacteria and 48 h for yeast.
The MICs of each test sample was detected following addition of 50 μl (0.2 mg/ml) p-iodonitrotetrazolium chloride (INT, Sigma-Aldrich, South Africa) solution for bacteria. Viable bacteria reduced the yellow dye to a pink colour. For yeast, MICs were determined by visualising the turbidity of the wells. The MIC corresponded to the lowest well concentration where no colour or turbidity change was observed, indicating no growth of microorganism. The MBC or MFC was determined by adding 50 μl aliquots of the clear wells to 150 μl of freshly prepared broth medium and incubating at 35 °C for 48 h. The MBC or MFC was regarded as the lowest concentration of test sample which did not produce a colour or turbidity change as above. All tests were performed in triplicates.
DPPH radical scavenging activity
Radical scavenging activity of test samples (methanol extract, fractions and isolated compounds) was determined spectrophotometrically (Jenway, spectrophotometer model 1605) at 517 nm under UV/Visible light using DPPH radical . The methanol extract and fractions, isolated compounds, and L-ascorbic acid were prepared in methanol and tested at concentration ranges of 200 to 6.25 μg/ml, 64 to 0.25 μg/ml and 8 to 0.25 μg/ml respectively. A volume of 900 μl of DPPH· solution (20 mg/l) was mixed with 100 μl of test sample in a curve and the absorbance (Ab) was read immediately and after 30 min incubation at room temperature (As). The experiments were carried out in triplicate. The percentages of DPPH· scavenged (RSa %) by test samples were calculated as:
The radical scavenging activity fifty (RSa50) corresponding to the amount of sample necessary to decrease by 50% the amount of free radical DPPH was determined by plotting the scavenging activity against the logarithm of sample concentration .
The data on antioxidant activity were subjected to the one-way analysis of variance (ANOVA) and results were expressed (where appropriate) as mean ± standard deviation. Differences between means of samples were compared using Duncan's multiple range tests at P < 0.05.