General experimental procedures
MS data were measured on JEOL MS Station JMS-700 spectrometer or JEOL 600 MS Route spectrometer. 1H NMR (500 and 400 MHz) and 13C NMR (125 and 100 MHz) were recorded using JEOL spectrometers or Bruker Avance AV-400 spectrometer. The chemical shifts were reported in parts per million (ppm) with TMS as internal standard. Deuterated solvents, methanol (CD3OD), dimethyl sulfoxide (DMSO-d
6
), pyridine (C5D5N) and chloroform (CDCl3) were used as solvents for the NMR experiments. CC was performed on silica gel 60 F254 (70–230 mesh; Merck) and gel permeation on Sephadex LH-20. TLC was carried out on precoated silica gel Kieselgel 60 F254 plates (0.25 mm thick), and spots weredetected with UV lights (254 and 365 nm) and further sprayedwith 20% H2SO4 reagent followed by heating to 100 °C.
Sample collections
Plant materials were collected in two locations of the Western Region of Cameroon: the whole plant of Dissotis senegambiensis (Guill. & Perr.) Triana in Bansoa (January 2013) and the roots of Amphiblemma monticola Jacq.-Fél. in Bamena (May 2016). Their identification was done by Mr. Fulbert Tadjouteu, a botanist of the Cameroon National Herbarium in Yaoundé, where voucher specimens, No 24736/SRF/Cam (D. senegambiensis) and No 45094/HNC (A. monticola), were deposited.
Extraction
The powdered material of D. senegambiensis (1.8 kg) was extracted three times (72 h for each time) by maceration with ethanol (8 L) at room temperature. Evaporation of solvent under vacuum afforded 78 g of crude extract. A portion of this extract (76 g) was successively triturated with n-hexane, EtOAc and n-butanol. TLC analysis showed that the n-hexane and EtOAc extracts (19.5 and 20.5 g, respectively) were qualitatively the same. They were thus combined to afford 40 g of extract called “EtOAc extract”.
Dried and pulverized roots (1.5 kg) and aerial part (0.08 kg) of A. monticola were respectively macerated with ethanol (5 L with roots and 1 L with aerial part) for 24 h (3 times) at room temperature. Evaporation of solvent under reduced pressure afforded 49 g and 4.28 g of crude extracts, respectively.
Phytochemical analysis
The extracts were screened for secondary metabolites using standard procedures as previously described [19,20,21,22]. The plant extracts were screened for the presence of different classes of compounds including triterpenoids, steroids, flavonoids, phenols, glycosides, tannins and alkaloids.
Isolation of constituents
A portion (38 g) of “EtOAc extract” of D. senegambiensis was subjected to silica gel (70 to 230 mesh) column chromatography (CC) eluted with gradient of n-hexane-EtOAc (100:0, 9:1, 4:1, 7:3, 3:2, 1:1 and 0:100) followed by gradient of EtOAc-MeOH (19:1, 9:1, 4:1, 7:3, 1:1 and 0:100). Fifty-five fractions of 300 mL each were collected and combined into six major fractions on the basis of their TLC profiles: A (1–6; 4.0 g), B (7–12; 4.5 g), C (13–17; 3.6 g), D (18–26; 4.7 g), E (27–36; 5.5 g), and F (37–55; 9.1 g). Fraction A crystallized to afford a mixture of two compounds. This mixture was subjected to silica gel CC and eluted with n-hexane- EtOAc (49:1) to yield β-amyrin palmitate (4.2 mg; 1) and α-amyrin acetate (3.5 mg; 2). Fraction C crystallized to afford ursolic acid (15.0 mg; 3). Fraction E was subjected to silica gel CC and eluted with CH2Cl2 –MeOH mixture of increasing polarity to yield sitosterol-3-O-β-D-glucopyranoside (35.1 mg; 4) and vitexin (28.5 mg; 5). Similarly as with fraction E, fraction F afforded trans-tiliroside (25.0 mg; 6). A portion (18 g) of the n-BuOH extract was also subjected to silica gel CC eluted with gradient of CH2Cl2-MeOH (100:0, 19:1, 9:1, 4:1and 0:100). Twenty-two fractions of 300 mL each were collected and combined into four major fractions on the basis of their TLC profiles: G (1–7; 2.7 g), H (8–12; 3.5 g), I (13–18; 3.6 g) and J (19–22; 3.7 g). Fraction G was subjected to silica gel CC and eluted with CH2Cl2 –MeOH mixture of increasing polarity to yield vitexin (15.1 mg; 5) and trans-tiliroside (13.1 mg; 6). An attempt to purify fractions B, D, H, I and J failed.
A portion (47 g) of EtOH extract of the roots of A. monticola was fractionated on silica gel CC eluted with CH2Cl2-MeOH of increasing polarity to give 25 fractions of 300 mL each. After comparative TLC, they were combined into 4 major fractions: A (1–8; 7.6 g), B (9–16; 11 g), C (17–21; 5.1 g) and D (22–25; 5.8 g). Fraction A was chromatographed on a silica gel column eluted with a continuous gradient of n-hexane-EtOAc to afford lupeol (9, 120.8 mg) and a mixture of sterols. Similarly, fractions B and C were eluted with CH2Cl2-MeOH of increasing polarity yielding four (B1-B4) and three (C1-C3) sub-fractions, respectively. B2 (1.9 g), B3 (2.3 g), C2 (1.9 g) and C3 (1.2 g) were passed separately on LH-20 Sephadex CC eluted with CH2Cl2-MeOH (1:1) to give 3,4′-di-O-methylellagic acid (20.0 mg; 7) from B2, dimethyl 4,4′,5,5′,6,6′-hexahydroxybiphenyl-2,2′-dicarboxylate (15.0 mg; 8) from B3, ellagic acid (23.0 mg; 10), 3-hydroxy-4,5-dimethoxybenzoic acid (4.0 mg; 11) and a mixture of 10 and 12 (7.0 mg) from C2, and 3-O-methylellagic acid 4′-O-β-D-xylopyranoside (2.3 mg; 12) from C3. Re-crystallization of B4 (0.7 g) in EtOAc afforded a mixture (31.9 mg) of ursolic acid (3) and oleanolic acid (13). Fraction D was subjected to silica gel CC eluted with a gradient mixture of CH2Cl2-MeOH to afford four sub-fractions (D1-D4). Repeated silica gel CC of D2 (0.8 g), eluted with CH2Cl2-MeOH (from 49:1 to 9:1) gave sitosterol-3-O-β-D-glucopyranoside (45.0 mg; 4) and amphiblemmone A (9.7 mg; 14).
Due to the small quantity of plant material, the aerial part of A. monticola (4.28 g of crude EtOH extract), compared to the roots (same collection in the field), was not further studied in this work.
Antimicrobial activity of extracts and compounds
Tested microorganisms
The microorganisms used in this study include four bacterial (Staphylococcus aureus ATCC25923, methicillin sensitive S. aureus MSSA1, methicillin resistant S. aureus MRSA3 and methicillin resistant S. aureus MRSA4) and three yeast strains (Candida albicans ATCC10231, Candida tropicalis PK233 and Cryptococcus neoformans H99). These microorganisms were taken from our laboratory collection. The fungal and bacterial strains were grown at 37 °C and maintained on Sabouraud Dextrose Agar (SDA, Conda, Madrid, Spain) and nutrient agar (NA, Conda) slants respectively.
Inocula preparation
The inocula of bacteria and yeasts were prepared from overnight cultures as previously described [23]. Absorbance was read spectrophotometrically at 530 nm and 600 nm for yeasts and bacteria respectively. The final concentrations of microbial suspensions were 2.5 × 105 cells/mL for yeasts and 106 CFU/mL for bacteria.
Antimicrobial assay
The antimicrobial activity was evaluated by determining the minimum inhibitory concentrations (MICs). MICs of extracts and compounds were determined by broth micro dilution [24]. Each test sample was dissolved in 10% v/v aqueous dimethylsulfoxide (DMSO) to give a stock solution. This was serially diluted two-fold in Mueller-Hinton Broth (MHB) for bacteria and Sabouraud Dextrose Broth (SDB) for fungi to obtain a concentration range of 4096 to 0.25 μg/mL. Then, 100 μL of each sample concentration was added to respective wells (96-well micro plate) containing 90 μL of SDB/ MHB and 10 μL of inoculum to give final concentration ranges of 2048 to 4 μg/mL (for extracts) and 256 to 0.125 μg/mL (for compounds). Dilutions of nystatin (Sigma-Aldrich, Steinheim, Germany) and ciprofloxacin (Sigma-Aldrich, Steinheim, Germany) were used as positive controls for yeasts and bacteria respectively. Broth with 10 μL of DMSO was used as negative control. The cultured micro plates were covered; then, the contents of each well were mixed thoroughly using a plate shaker (Flow Laboratory, Germany) and incubated at 37 °C for 24 h (bacteria) and 48 h (yeasts) under shaking. After the incubation period, MICs were assessed visually and were taken as the lowest sample concentration at which there was no growth or virtually no growth. The lowest concentration that yielded no growth after the sub-culturing was considered as the minimum microbicidal concentrations (MMCs). All the tests were performed in triplicate.
Antioxidant assay
Ferric reducing antioxidant power (FRAP) assay
The FRAP was determined by the Fe3+-Fe2+ transformation in the presence of extracts and compounds as previously described [25]. The Fe2+ was monitored by measuring the formation of Perl’s Prussian blue at 700 nm. Butylated hydroxytoluene (BHT) was used as a positive control. All the tests were performed in triplicate.
Diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging assay
The free radical scavenging activity of extracts and compounds was evaluated according to described methods [26]. The EC50 (μg/ml), which is the amount of sample necessary to inhibit by 50% the absorbance of free radical DPPH was calculated [26]. Vitamin C was used as a standard control. All the analyses were carried out in triplicate.
Hemolytic assay
Whole blood (10 mL) from albino rats was collected by cardiac puncture into a conical tube containing EDTA as an anticoagulant. The study was conducted according to the ethical guidelines of the Committee for Control and Supervision of Experiments on Animals (Registration no. 173/CPCSEA, dated 28 January, 2000), Government of India, on the use of animals for scientific research. Erythrocytes were harvested by centrifugation at room temperature for 10 min at 1000 x g and were washed three times in PBS buffer [27]. The cytotoxicity was evaluated as previously described [27].
Statistical analysis
Data were analyzed by one-way analysis of variance followed by Waller-Duncan Post Hoc test. The experimental results were expressed as the mean ± Standard Deviation (SD). Differences between groups were considered significant when p < 0.05. All analyses were performed using the Statistical Package for Social Sciences (SPSS, version 12.0) software.