General experimental procedures
A Schorpp Gerätetechnik (Germany) apparatus was used to take the melting points of different compounds.
The 1D (1H and 13C-NMR) and 2D (COSY, NOESY, HSQC and HMBC) spectra were performed in deuterated solvents (CD3OD) on Bruker Avance III 600 spectrometer at 600 MHz/150 MHz. All chemical shifts (δ) are given in ppm units with reference to tetramethylsilane (TMS) as internal standard and the coupling constants (J) are in Hz.
The mass spectra (HR-TOFESIMS) were carried out on Micromass Q-TOF micro instrument (Manchester, UK). Samples were introduced by direct infusion in a solution of MeOH at a rate of 5 μL/min.
Silica gel 60 Merck, 70–230 mesh and sephadex LH-20 were used to perform column chromatography while precoated silica gel 60 F254 (Merck) plates, were used to perform thin layer chromatography. 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 and heating at 100 °C for 5 min.
The aerial parts of G. grandulosum were harvested in a small village called Foto situated in the Menoua Division, Western region of Cameroon) in November 2015. The Plant was identified and authenticated by a Cameroonian Botanist (Mr. Fulbert Tadjouteu) at the National Herbarium where a voucher specimen was archived (No 65631/HNC).
Extraction and isolation
The extraction and isolation of compounds were done as previously described . Briefly, the aerial part of G. grandulosum was air-dried and powdered. The powder was macerated at room temperature with MeOH to afford the MeOH extract. Part of this extract (235 g) was suspended in water (300 mL) and successively partitioned with EtOAc and n-BuOH to yield 37 and 13 g of extracts, respectively. Column chromatography of the n-BuOH extract followed by purification of different fractions led to the isolation of five compounds.
Structural identification of the isolated compounds
The structures of isolated compounds were determined after interpretation of their physical, spectrometric and spectroscopic data summarized in this subsection.
Chrysoeriol-7-O-β-D-xyloside (1): yellow amorphous powder; molecular formula C21H20O10; 13C NMR (CD3OD, 150 MHz) δC: 165.3 (C-2), 104.3 (C-3), 184.1 (C-4), 161.7 (C-5), 99.6 (C-6), 163.1 (C-7), 95.8 (C-8), 158.5 (C-9), 107.0 (C-10), 123.1 (C-1′), 110.5 (C-2′), 148.2 (C-3′), 151.0 (C-4′), 116.7 (C-5′), 121.9 (C-6′), 55.2 (C-7′) for aglycone; 100.9 (C-1′′), 74.4 (C-2′′), 77.3 (C-3′′), 70.7 (C-4′′), 66.9 (C-5′′) for sugar moiety. 1H NMR data (CD3OD, 600 MHz) δH: 6.62 (1H, s, H-3), 6.38 (1H, d, J = 2.1 Hz, H-6), 6.71 (1H, d, J = 2.1 Hz, H-8), 7.44 (1H, d, J = 2.1 Hz, H-2′), 6.84 (1H, d, J = 8.4 Hz, H-5′), 7.47 (1H, dd, J = 8.4 and 2.1 Hz, H-6′), 3.87 (3H, s, H-7′) for aglycone; 4.95 (1H, d, J = 7.1 Hz, H-1′′), 3.37 (1H, m, H-2′′), 3.36 (1H, m, H-3′′), 3.49 (1H, m, H-4′′), 3.38 (1H, m, H-5′′a), 3.87 (1H, m, H-5′′b) for sugar moiety.
Luteolin-7-O-β-D-apiofuranosyl-(1 → 2)-β-D-xylopyranoside (2): yellow powder; molecular formula C25H26O14. m.p. = 203 °C. 13C NMR data (CD3OD, 150 MHz) δC: 165.3 (C-2), 103.5 (C-3), 182.5 (C-4), 162.9 (C-5), 99.7 (C-6), 163.1 (C-7), 94.9 (C-8), 157.4 (C-9), 105.5 (C-10), 121.7 (C-1′), 114.2 (C-2′), 146.4 (C-3′), 150.5 (C-4′), 116.6 (C-5′), 119.7 (C-6′) for aglycone; 99.0 (C-1′′), 76.0 (C-2′′), 77.0 (C-3′′), 70.0 (C-4′′), 66.1 (C-5′′), 109.3 (C-1′′′), 76.5 (C-2′′′), 79.7 (C-3′′′), 64.5 (C-4′′′), 74.4 (C-5′′′) for sugar moiety. 1H NMR data (CD3OD, 600 MHz) δH: 6.75 (1H, s, H-3), 6.40 (1H, d, J = 2.1 Hz, H-6), 6.75 (1H, d, J = 2.1 Hz, H-8), 7.44 (1H, d, J = 2.1 Hz, H-2′), 6.90 (1H, d, J = 8.4 Hz, H-5′), 7.47 (1H, dd, J = 8.4 and 2.1 Hz, H-6′) for aglycone; 5.18 (1H, d, J = 7.1 Hz, H-1′′), 3.52 (1H, dd, J = 9.0 and 7.1 Hz, H-2′′), 3.43 (1H, m, H-3′′), 3.41 (1H, m, H-4′′), 3.78 (1H, dd, J = 9.7 and 3.4 Hz, H-5′′a), 3.42 (1H, dd, J = 9.7 and 3.4 Hz, H-5′′b), 5.34 (1H, d, J = 1.3 Hz, H-1′′′), 3.75 (1H, m, H-2′′′), 3.30 (2H, d, J = 3.4 Hz, H-4′′′), 3.88 (1H, d, J = 9.3 Hz, H-5′′′a), 3.65 (1H, d, J = 9.3 Hz, H-5′′′b) for sugar moiety.
Chrysoeriol-7-O-β-D-apiofuranosyl-(1 → 2)-β-D-xylopyranoside (3): yellow powder; molecular formula C26H28O4; melting point = 181.8 °C. 13C NMR (CD3OD, 150 MHz) δC: 166.6 (C-2), 104.5 (C-3), 184.0 (C-4), 162.9 (C-5), 100.9 (C-6), 164.4 (C-7), 95.9 (C-8), 158.9 (C-9), 107.0 (C-10), 123.4 (C-1′), 110.4 (C-2′), 149.5 (C-3′), 152.3 (C-4′), 116.7 (C-5′), 121.9 (C-6′), 56.6 (C-7′) for aglycone; 100.6 (C-1′′), 78.6 (C-2′′), 77.9 (C-3′′), 70.9 (C-4′′), 66.9 (C-5′′), 110.0 (C-1′′′), 78.1 (C-2′′′), 80.7 (C-3′′′), 65.8 (C-4′′′), 75.4 (C-5′′′) for sugar moiety. 1H NMR data (CD3OD, 600 MHz) δH: 6.70 (1H, s, H-3), 6.45 (1H, d, J = 2.1 Hz, H-6), 6.77 (1H, d, J = 2.1 Hz, H-8), 7.52 (1H, d, J = 2.1 Hz, H-2′), 6.95 (1H, d, J = 8.4 Hz, H-5′), 7.56(1H, dd, J = 8.4 and 2.1 Hz, H-6′), 3.98 (3H, s, H-7′) for aglycone; 5.16 (1H, d, J = 7.1 Hz, H-1′′), 3.68 (1H, dd, J = 9.0 and 7.1, H-2′′), 3.63 (1H, m, H-3′′), 3.62 (1H, m, H-4′′), 3.98 (1H, m, H-5′′a), 3.48 (1H, t, J = 9.6, H-5′′b), 5.46 (1H, d, J = 1.7 Hz, H-1′′′), 3.98 (1H, m, H-2′′′), 3.56 (2H, brs, H-4′′′), 4.05 (1H, d, J = 9.4, H-5′′′a), 3.84 (1H, d, J = 9.4, H-5′′′b) for sugar moiety.
Chrysoeriol-7-O-α-L-rhamnopyranosyl-(1 → 6)-β-D-(4′′-hydrogenosulfate) glucopyranoside (4): yellow amorphous powder; molecular formula C28H31NaO18S. 13C NMR data (CD3OD, 150 MHz) δC: 166.8 (C-2), 104.5 (C-3), 184.1 (C-4), 163.1 (C-5), 101.1 (C-6), 164.0 (C-7), 96.2 (C-8), 159.0 (C-9), 107.2 (C-10), 123.6 (C-1′), 110.8 (C-2′), 149.6 (C-3′), 152.3 (C-4′), 116.9 (C-5′), 122.0 (C-6′), 56.7 (C-7′) for aglycone; 100.9 (C-1′′), 74.5 (C-2′′), 76.8 (C-3′′), 77.5 (C-4′′), 75.3 (C-5′′), 67.1 (C-6′′), 102.4 (C-1′′′), 71.9 (C-2′′′), 72.3 (C-3′′′), 74.2 (C-4′′′), 69.8 (C-5′′′), 17.9 (C-6′′′) for sugar moiety. 1H NMR data (CD3OD, 600 MHz) δH: 6.73 (1H, s, H-3), 6.56 (1H, d, J = 2.1 Hz, H-6), 6.84 (1H, d, J = 2.1 Hz, H-8), 7.55 (1H, d, J = 2.1 Hz, H-2′), 6.98 (1H, d, J = 8.4 Hz, H-5′), 7.59 (1H, dd, J = 8.4 and 2.1 Hz, H-6′), 3.99 (3H, s, H-7′) for aglycone; 5.15 (1H, d, J = 7.8 Hz, H-1′′), 3.61 (1H, dd, J = 9.1 and 7.8 Hz, H-2′′), 3.84 (1H, t, J = 9.1 Hz, H-3′′), 4.32 (1H, dd, J = 9.9 and 9.1 Hz, H-4′′), 3.89 (1H, m, H-5′′), 4.10 (1H, m, H-6′′a), 3.68 (1H, m, H-6′′b), 4.75 (1H, d, J = 1.3 Hz, H-1′′′), 3.95 (1H, dd, J = 3.4 and 1.3, H-2′′′), 3.72 (1H, dd, J = 9.5 and 3.4 Hz, H-3′′′), 3.32 (1H, t, J = 9.5 Hz, H-4′′′), 3.62 (1H, m, H-5′′′), 1.21 (3H, d, J = 6.2 Hz, H-6′′′) for sugar moiety.
Isorhamnetin-3-O-α-L-rhamnopyranosyl-(1 → 6)-β-D-glucopyranoside (5): yellow amorphous powder; molecular formula C28H32O15. 13C NMR data (CD3OD, 150 MHz) δC: 159.8 (C-2), 135.7 (C-3), 179.8 (C-4), 162.4 (C-5), 104.3 (C-6), 160.1 (C-7), 100.2 (C-8), 157.5 (C-9), 108.6 (C-10), 123.0 (C-1′), 114.5 (C-2′), 148.4 (C-3′), 151.1 (C-4′), 116.2 (C-5′), 124.4 (C-6′), 56.7 (C-7′) for aglycone; 104.0 (C-1′′), 75.9 (C-2′′), 78.1 (C-3′′), 71.8 (C-4′′), 77.4 (C-5′′), 68.7 (C-6′′), 102.6 (C-1′′′), 72.1 (C-2′′′), 72.3 (C-3′′′), 73.8 (C-4′′′), 69.8 (C-5′′′), 18.0 (C-6′′′) for sugar moiety. 1H NMR data (CD3OD, 600 MHz) δH: 6.71 (1H, d, J = 2.2 Hz, H-6), 7.09 (1H, d, J = 2.2 Hz, H-8), 7.91 (1H, d, J = 2.2 Hz, H-2′), 6.95 (1H, d, J = 8.5 Hz, H-5′), 7.72 (1H, dd, J = 8.5 and 2.2 Hz, H-6′), 3.98 (3H, s, H-7′) for aglycone; 5.30 (1H, d, J = 7.6 Hz, H-1′′), 3.49 (1H, dd, J = 9.1 and 7.6 Hz, H-2′′), 3.45 (1H, t, J = 9.1 Hz, H-3′′), 3.23 (1H, t, J = 9.1 Hz, H-4′′), 4.40 (1H, m, H-5′′), 3.84 (1H, dd, J = 12.1 and 1.9 Hz, H-6′′a), 3.42 (1H, m, H-6′′b), 4.53 (1H, d, J = 1.3 Hz, H-1′′′), 3.58 (1H, dd, J = 3.4 and 1.3, H-2′′′), 3.47 (1H, dd, J = 9.5 and 3.4 Hz, H-3′′′), 3.25 (1H, t, J = 9.5 Hz, H-4′′′), 3.41 (1H, m, H-5′′′), 1.10 (3H, d, J = 6.2 Hz, H-6′′′) for sugar moiety.
The microorganisms used in this study were consisted of five bacterial strains namely Staphylococcus aureus ATCC 25923, Vibrio cholerae NB2, PC2, SG24 (1) and CO6 . Also included were two fungi Candida albicans ATCC 9002 and Cryptococcus neoformans IP95026. These bacteria and yeasts were obtained from our local stocks.
Determination of minimum inhibitory concentration and minimum microbicidal concentration
The minimum inhibitory concentration (MIC) values were determined using the broth micro-dilution method as described earlier . The MIC values were defined as the lowest sample concentration that prevented the change in color indicating a complete inhibition of microbial growth. The lowest concentrations that yielded no growth after the subculturing were taken as the minimum microbicidal concentration (MMC) values . Ciprofloxacin (Sigma-Aldrich, Steinheim, Germany) and amphotericin B (Merck, Darmstadt, Germany) were used as positive controls for bacteria and yeast respectively.
Study on mode of action
MIC and MBC changes under osmotic stress condition
Osmotic stress was induced by adding 5% NaCl (w/v) to MHB. The MHB supplemented with 5% NaCl was then sterilized and used for the determination of a new MIC and MBC values of the samples as previously described . The incubation time was increased from 24 to 48 h at 37 °C.
Effect of isolated compounds on cell membrane
The alteration of cell membrane of V. cholerae NB2 was evaluated by measuring the optical densities at 260 nm of the bacterial suspensions in the presence and absence of compounds 1–5 using the method described by Carson et al. . For this purpose, the compounds were tested at their MIC using 1 mL of the bacterial suspension (approximately 108 CFU/mL). The mixture was then incubated at 37 °C at different time intervals (0: immediately after addition of the compound; 15; 30; 60 min), 50 μL of the mixture was taken and mixed with 1.95 mL of Phosphate Buffered Saline (PBS Buffer). The absorbance was measured on the spectrophotometer at 260 nm against the blank (PBS). For the negative control, 1 mL of bacterial suspension was incubated at 37 °C and 50 μL of the suspension was removed at the end of the various incubation times and mixed with 1.95 mL of Buffer. The optical densities were read in the same way.
The bacteriolytic activities of the isolated compounds were determined using the time-kill kinetic method as previously described  with slight modifications. Full growth of V. cholerae NB2 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 with shaking, then 100 μL was removed from each tube at 0, 15, 30, 60, and 120 min and the optical density measured at 600 nm. Vancomycin and tetracycline were used as positive controls and the tubes without isolated compounds served as negative controls.
Whole blood (10 mL) from albino rats was collected by cardiac puncture in EDTA tubes. 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 . The cytotoxicity was evaluated as previously described .
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.