General experimental procedures
NMR analysis
The 1H and 13C-NMR spectra were recorded on a Bruker Avance III 600 spectrometer equipped with a cryo-platform (1H at 600 MHz and 13C at 150 MHz). 2D NMR experiments were performed using standard Bruker microprograms (Xwin-NMR version 2.1 software). All chemical shifts (δ) are reported in parts per million (ppm) with the solvent signal as reference relative to TMS (δ = 0) as internal standard, while the coupling constants (J) are given in Hertz (Hz). Deuterated solvents, methanol (CD3OD), dimethyl sulfoxide (DMSO-d6), and chloroform (CDCl3) were used as solvents for the NMR experiments.
Chromatographic methods
Column chromatography was run on Merck silica gel (VWR, France) 60 (70–230 mesh) and gel permeation on Sephadex LH-20 (VWR, France), while TLC was carried out on silica gel GF254 pre-coated plates and the spots were visualized by an UV lamp multiband UV-254/365 nm (ModelUVGL-58 Upland CA 91786, U.S.A) followed by spraying with 50% H2SO4 followed by heating at 100 °C.
Sample collection
The whole plant Trifolium baccarinii Chiov was collected on January 2016 in Dschang, western region of Cameroon. The botanical identification was carried out at the National Herbarium of Cameroon after comparison with the samples deposited at the reference number 2976/HNC.
Extraction and Fractionation
The plant material was air-dried at room temperature and ground into fine powder. The dried powder of T. baccarinii (4.5 kg) was extracted at room temperature with methanol (3 × 20L, 72h) to yield 281 g of crude methanol extract after evaporation of solvent under reduced pressure. A part of crude extract (271 g) undergoes a differential solubilization with H2O/EtOAc (300 mL / 500 mL) followed by H2O/n-BuOH (300 mL / 500 mL). After evaporation of each solvent under reduced pressure, we obtained 70 g of EtOAc and 54 g of n-BuOH extracts.
Isolation of Compounds
A part of EtOAc extract (65 g) of T. baccarinii was subjected to silica gel column chromatography using n-hexane-EtOAc (100:0 → 0:100) followed by EtOAc-MeOH (90:10 → 80:20) gradient graduated elution. Sixty-six fractions of 400 mL were collected and combined on the basis of their TLC profiles to give fourteen major fractions: A (1), B (2-6), C (7-10), D (11-14), E (15-20), F (21-25), G (26-31), H (32-34), I (35-40), J (41-46), K (47-52), L (53-58), M (59-60) and N (61-66). Purification of fraction G (8.0 g) on silica gel column chromatography with n-hexane-EtOAc (90:10) as eluent yielded compound 1 (27 mg) and 2 (35 mg). Silica gel column chromatography of fraction H (10.0 g) eluted with n-hexane-EtOAc (80:20) gave compound 3 (10 mg). Fraction K (5 g) was subjected to silica gel column chromatography eluted with EtOAc to afford seven sub-fractions (K1-K7). Compound 4 (10 mg) was obtained from sub-fraction K3 (250 mg) after Sephadex LH-20 column chromatography using MeOH as eluent. Fraction D (4.2 kg) was subjected to silica gel column chromatography eluted with n-hexane-EtOAc (93:7) to yield four sub-fractions (D1-D4). Further purification of sub-fraction D2 on silica gel column chromatography eluted with n-hexane-EtOAc (95:5) yield compound 5 (30 mg). The purification of fraction J (3.5 g) on Sephadex LH-20 column chromatography affords two sub-fractions J1 and J2. Fraction J2 (600 mg) was further purified on silica gel column chromatography with n-hexane-EtOAc (20:80) to give compound 6 (20 mg).
Like the EtOAc extract, a part of n-BuOH extract (50 g) of T. baccarinii was subjected to a silica gel column chromatography using the mixture EtOAc-MeOH (100:0 → 40:60) gradient graduated elution. Seventy-six fractions of 400 mL were collected and combined on the basis of their TLC profiles in four major fractions: A (1-7), B (8-23), C (24-41) and D (42-68). Purification of fraction B (5 g) on silica gel column chromatography with EtOAc as eluent mainly leads to compounds 6 (580 mg), 7 (20 mg) and 8 (16 mg). Fraction C (4 g) was purified over a silica gel column chromatography eluted with EtOAc-MeOH-H2O (90:10:5) to give compound 9 (20 mg). Fraction D (3 g) was subjected to multiple chromatography separation over silica gel using EtOAc-MeOH-H2O (90:10:5) and (80:20:10) as eluents to give two sub-fractions D1 and D2. The latter (D2,1 g) was further purified on silica gel column chromatography using EtOAc-MeOH-H2O (80:20:10) as system and also gave other two sub-fractions D2-1 and D2-2. Purification of sub-fraction D2-2 (200 mg) on Sephadex LH-20 column chromatography using MeOH as eluent yielded compound 10 (20 mg).
Antimicrobial assay
Microorganisms
The antimicrobial activity was performed against five bacterial and two fungal species. The selected microorganisms were the Gram-positive (Staphylococcus aureus ATCC25923, methicillin resistant S. aureus MRSA03 and methicillin resistant S. aureus MRSA04) and Gram-negative (Pseudomonas aeruginosa ATCC27853 and Escherichia coli S2(1)) bacteria and yeast strains of Candida albicans ATCC10231 and Cryptococcus neoformans H99. These microorganisms were taken from our laboratory collection. The fungal and bacterial strains were maintained on Sabouraud Dextrose Agar (SDA, Conda, Madrid, Spain) and nutrient agar (NA, Conda) slants respectively.
Determination of minimum inhibitory concentration (MIC) and minimum Microbicidal concentration (MMC)
The antimicrobial activity was investigated by determining the minimum inhibitory concentrations (MICs), minimum bactericidal concentrations (MBC) and minimum fungicidal concentrations (MFCs). MICs of extracts/compounds were monitored using the broth microdilution method16. Test sample was dissolved in dimethylsulfoxide (DMSO, Fisher chemicals) to give a stock solution that was twofold serially diluted 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. 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 inoculums (at 1 x 106 CFU/mL for bacteria and 1 x 105 spores/ mL for yeasts) were added to obtain a final concentration range of 2048 to 0.125 µg/mL. Plates were covered and incubated on the shaker at 37 °C, 30 °C and 30 °C during 24 h, 48 h and 72 h for bacteria, Candida albicans and Cryptococcus neoformans, respectively. 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.
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 as described above. The lowest concentration that yielded no growth after the sub-culturing was taken as the MBCs or MFCs. Oxacillin (Sigma-Aldrich, Steinheim, Germany) for bacteria and nystatin (Sigma-Aldrich, Steinheim, Germany) for yeasts were used as positive controls; while broth with 20 µL of DMSO was used as negative control. The assay was carried out in triplicate and repeated three times with similar results.
Antibacterial Mechanism Studies
The mechanisms of antibacterial action were determined by lysis, salt tolerance assays and antioxidant enzyme activities.
Bacteriolytic assay
The bacteriolytic activity of methanol extract and compound 5, which exhibited the highest antimicrobial activities, was performed against P. aeruginosa and S. aureus using the time-kill kinetic method as previously described17 with some modifications. Full growth of bacterium in MHB was diluted 100 times and incubated at 37 °C to produce an OD600 of 0.8 as starting inoculum. Sample solutions were added to the starting bacterial suspension to give a final concentration of 2 × MIC and incubated at 37 °C under agitation at 150 rpm. After the incubation period corresponding to 0, 15, 30, 60, 120 and 240 min, 100 µL was removed from each tube and the optical density was recorded at 600 nm using BIOBASE UV-VIS spectrophotometer. Corresponding dilutions of test samples were used as blanks. Oxacillin was used as positive controls and the tubes without extract/compound served as negative controls. All the measurements were done in triplicate and repeated three times with similar results.
Loss of Salt Tolerance in Staphylococcus aureus.
The ability of S. aureus ATCC25923 and methicillin resistant S. aureus MRSA03 cells treated with methanol extract and compound 5 to grow on Mueller Hinton agar (MHA) supplemented with NaCl was investigated. In preliminary experiments, untreated suspensions of S. aureus were plated on MHA supplemented with NaCl from 40 to 100 g/L. Plates were incubated and upon incubation, the resulting colonies were counted. Concentrations of NaCl, 50, 60 and 70 g/L, which modestly compromised the colony-forming abilities of untreated microorganisms were selected. For further experimentation steps, suspensions of bacteria were prepared as previously described and treated with MeOH extract or compound 5 at 1/2x MIC, MIC, and 2x MIC. After 1 h incubation, samples were removed, serially diluted, and inoculated onto MHA and MHA-NaCl (50, 60 and 70 g/L). Bacterial culture without sample was used as control for each MHA-NaCl plate. Upon incubation, the numbers of CFU per milliliter on each MHA-NaCl plate were compared to those on the MHA plate, and the result was expressed as a percentage18.
Antioxidant enzyme activities
For evaluation of catalase and superoxide dismutase (SOD) activities, S. aureus ATCC25923 and methicillin resistant S. aureus MRSA03 (1.5 × 108 CFU/ml, 500 μl) cultures from the late exponential growth phase were treated with MIC and 1/2xMIC of methanol extract (500 μl), compound 5 (500 μl) and oxacillin (500 μl) solutions and incubated at 37 °C for 24 h. The suspension was centrifuged at 3000 rpm for 5 min to separate the supernatant. Pellet was washed twice with PBS and re-suspended in 500 μl of cell lysate buffer (1 mM EDTA, 10 Mm Tris-HCl, 0.1% Triton-X-100 and 150 mM NaCl)19 and kept for incubation at 37 °C for 1 h. Contents were then centrifuged at 3000 rpm for 5 min and the supernatant was collected for enzyme activity assays.
Catalase activity was assessed by using kit (Sigma, catalogue no. CAT100) in the cell lysate Briefly, 750 μl of assay buffer (50 mM) was mixed with 25 μl of 50 mM H2O2 and 10 μl of cell lysate. The mixture was incubated for 5 min. After that, reaction terminated by the addition of 900 μl of stop solution (15 mM sodium azide) and content was thoroughly mixed. Then, 10 μl of reaction mixture was taken into separate tube and mixed with 1 ml of colour reagent (2 mM 3,5-dichloro-2-hydroxybenzenesulfonic acid and 0.25 mM 4-aminoantipyrine) and incubated for 15 min. The absorbance was monitored at 520 nm and the catalase activity was calculated based on the following equation: [Δμ moles (H2O2) × d × 100) / V × t], where Δμ moles (H2O2) = difference in amount of H2O2 added to the reaction mixture between blank and given sample, d = dilution of original sample for catalase reaction, V = sample volume in catalase reaction and t = reaction duration (min).
Superoxide dismutase (SOD) activity was determined using kit (Sigma, Catalogue No. 19160) in the cell lysate. Cell lysate (20 μl) was mixed with working solution of water soluble tetrazolium salt (WST, 200 μl) and enzyme solution (20 μl). The reaction mixture was incubated in dark at 37 °C for 20 min and the absorbance was read at 450 nm on a BioTek Synergy 2 multiplate reader. The SOD activity was calculated based on the following formula: [(ABlank1–ABlank3) - (ASample–ABlank2) / (ABlank1–ABlank3) × 100], where Blank 1 contains ultrapure water, WST solution and enzyme solution; Blank 2 contains sample solution, WST solution and dilution buffer whereas Blank 3 contains ultrapure water, WST solution and dilution buffer
Antioxidant assay
Gallic acid equivalent antioxidant capacity (GEAC) assay.
The GEAC test was done as previously described20 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'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (74 mM stock solution). The laccase were purified from Sclerotinia sclerotiorum according to the protocol described21. The sample concentrations in the assay mixture were 800, 400, 200, 100, 10 µg/mL for the extracts and 200, 100, 50, 25, 12.5 µg/mL for the isolated compounds. The content of the generated ABTS●+ radical was measured at 420 nm after 240 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. Experiments were done in triplicate and repeated three times with similar results.
Diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging assay
The free radical scavenging activity of extracts and compounds was evaluated according to described method22. The EC50 (µg/ml), which is the amount of sample necessary to inhibit by 50% the absorbance of free radical DPPH was calculated22. Vitamin C was used as a standard control. All the analyses were carried out in triplicate and repeated three times with similar results.
Cytotoxicity assay
Wistar rats (Rattus novergicus) aged 10–12 weeks and weighing 220 to 250 g were randomly selected from our colony. All the procedures and protocols involving animals and their care were conducted in conformity with the institutional guidelines and approved by the Cameroon National Ethical Committee (Reg. No. FWA-IRB00001954) and in compliance with the ARRIVE guidelines. Efforts were also made to minimize animal suffering and to reduce the number of animal used in the experiment. All the rats were sacrificed by intraperitoneal injection of the mixture of ketamine (50 mg/ kg) and xylazine (10 mg /kg) for anaesthesia. Whole blood (10 mL) from albino rats was collected by cardiac puncture into a conical tube containing Ethylene Diamine Tetra Acetic Acid (EDTA) as an anticoagulant. Erythrocytes were harvested by centrifugation at room temperature for 10 min at 1,000 x g and were washed three times in PBS buffer23. The cytotoxicity was evaluated as previously described23.
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.