General experimental procedures
Column chromatography were proceeded with Silica gel 60 F254 (70–230; Merck; Darmstadt, Germany). TLC developed on precoated silica gel Kieselgel 60 F254 plates (0.25 mm thick) and compounds were detected by spraying with 50% H2SO4 on it before being heated at 100 °C. Semi-preparative and preparative HPLC was performed using a Gilson FX-281322H2 High Performance Liquid Chromatography coupled to a DAD detector and an automatic fraction collector. ASunfire C18 column (10 μm, 10 × 250 mm) and (5 μm, 10 × 150 mm) were used in these separations. (+)-ESITOF-MS was performed as previously described [12]. We recorded NMR spectra on a Bruker Avance III spectrometer, equipped with a 1.7 mm TCI microcryoprobe, (500.0 and 125.0 MHz for 1H and 13C NMR, respectively). The chemical shifts are given in part per million (ppm) using the signal of the residual solvent as internal reference. The coupling constant (J) are in Hertz.
Plant material
The leaves of Albizia zygia (DC) J.F. Macbr were collected on the slopes of the cliff of Santchou, West Region of Cameroon in March 2013. It is a public and well known wild. Thus, access and collection of samples do not require any permission according to the legislation of Cameroon. These leaves were identified at the National Herbarium of Cameroun (NHC) by comparison to a voucher specimen under the number N° 43,969 HNC.
Extraction and isolation
Dried leaves of A. zygia were ground to a fine powder (0.77 Kg) and macerated with methanol (5 L) for 24 h (repeated 3 times) at room temperature. After filtration and removal of the solvent in vacuo, a crude extract of 42.0 g was obtained. The extract was subjected to silica gel column chromatography (CC) eluting with gradient of n-hexane-EtOAc and then EtOAc-MeOH to afford four major fractions (A-D). Fraction A was not further investigated, it contains mostly fatty material and fraction B (3.2 g) was separated by column chromatography over silica gel with a (5–30%) of n-hexane-EtOAc to give quercetin (6) (27.0 mg). Fraction C (12.6 g) was separated by column chromatography over silica gel using gradient (5–50%) of CH2Cl2-MeOH to give a mixture of compounds 2 and 3 (97.3 mg). Fraction D (20.8 g) was subjected to silica gel column chromatography eluted with gradient (5–40%) of EtOAc-MeOH to give phaseoloidin (1) (335.6 mg) and a mixture of 4 and 5 (9.8 mg). Further purification of the two above mentioned mixtures by semi-preparative HPLC eluted with a gradient of acetonitrile-water from 5 to 100% as mobile phase, afforded quercetin 3-O-α-L-rhamnopyranoside (2) (44.4 mg) and kampherol 3-O-α-L-rhamnopyranoside (3) (13.7 mg) from the first mixture, and quercetin 3,4′-di-O-α-L-rhamnopyranoside (4) (1.6 mg) and kaempferol 3,4′-di-O-α-L-rhamnopyranoside (5) (1.1 mg) from the second one.
Semi-synthetic compounds
Acetylation of 2-O-β-D-glucopyranosyl-4-hydroxyphenylacetic acid (1): 2-O-β-D-glucopyranosyl-4-hydroxyphenylacetic acid (10.0 mg, 3.03 10− 5 mol) was dissolved in 1 mL of pyridine, 0.25 mL of acetic anhydride (0.026 mol) were added, and the mixture was left to stand for 24 h. Extraction with CH2Cl2 and semi-preparative HPLC purification (ACN-H2O, 5–100) gave two new derivatives: compounds 7 (2.2 mg, yield:15%) and 8 (1.9 mg, yield: 11%).
2-O-β-D-glucopyranosyl-4-hydroxyphenylacetic acid (1): white powder;1H NMR (500 MHz, DMSO-d6): δH 6.60 (d, J = 2.6 Hz, H-3), 6.57 (dd, J = 2.6 and 8.7 Hz, H-5), 6.95 (d, J = 8.7 Hz, H-6), 3.58 (s, H-7), 4.53 (d, J = 6.7 Hz, H-1′), 3.51 (d, J = 16.5 Hz, H-2′), 3.67 (d, J = 11.9 Hz, H-3′), 3.61 (d, J = 15.9 Hz, H-4′), 3.13 (m, H-5′), 3.45 (m, H-6′); 13C NMR (125 MHz, DMSO-d6): δC 173.7 (C-8), 35.6 (C-7), 117.6 (C-4), 117.6 (C-5), 118.0 (C-3), 126.6 (C-1), 152.7 (C-2), 103.3 (C-1′), 73.9 (C-2′), 77.0 (C-3 ‘), 70.3 (C-4’), 77.5 (C-5 ‘), 61.5 (C-6’); (+)-HRESI-MS: m/z 348.1288 (calcd. For C14H22O9N, 348.1289).
Compound 7: colorless oil; 1H NMR (500 MHz, MeOD):δH 7.01(d, J = 2.6 Hz, H-3), 6.65 (dd, J = 8.6 and 2.6 Hz, H-5), 6.69 (d, J = 2.6 Hz, H-6), 3.62 (d, J = 16.4 Hz, H-7α), 3.46 (d, J = 16.4 Hz, H-7β), 5.35 (t, J = 7.4 Hz, H-1′), 4.33 (dd, J = 5.0 and 12.2 Hz, H-2′), 5.13 (m, H-3′), 4.18 (dd, J = 2.6 and 12.3 Hz, H-4′), 3.99 (m, H-5′), 5.17 (m, H-6’α), 5.11 (m, H-6’β), 2.10 (s, 3H), 2.08 (s, 3H), 2.05 (s, 3H), 2.01 (s, 3H); HRESI-MS (+): m/z 516.1708 (calcd for C22H30NO13, 516.1712).
Compound 8: colorless oil; 1H NMR (500 MHz, MeOD): δH 7.01 (d, J = 2.4 Hz, H-3), 6.99 (dd, J = 8.9 and 2.4 Hz, H-5), 7.17 (d, J = 8.9 Hz, H-6), 3.68 (d, J = 15.0 Hz, H-7), 3.48 (d, J = 15.9 Hz, H-7), 5.29 (d, J = 7.3 Hz, H-1′), 4.34 (dd, J = 5.5 and 12.3 Hz, H-2′), 5.21 (J = 2.1 and 7.5 Hz, H-3′), 4.17 (dd, J = 2.4 and 12.3 Hz, H-4′), 4.08 (m, H-5′), 5.16 (m, H-6’α), 5.12(m, H-6’β), 2.09 (s, 3H), 2.07 (s, 3H), 2.04 (s, 3H), 2.01(s, 3H), 2.26 (s, 3H); HRESI-MS (+): m/z 558.1814 (calcd for C24H32NO14, 558.1817).
Acetylation of quercetin 3-O-α-L-rhamnyranoside (2) Quercetin 3-O-α-L-rhamnyranoside (22.0 mg, 4.91 10− 5 mol) was dissolved in 2.5 mL of pyridine, and 0.75 mL of acetic anhydride (0.0079 mol) were added, the mixture was left to stand for 24 h. Extraction with CH2Cl2 and semi-preparative HPLC purification gave two new derivatives: compounds 9 (7.6 mg, yield 18%) and 10 (2.8 mg, yield 6%).
Quercetin 3-O-α-L-rhamnyranoside (2): yellow powder; 1H NMR (500 MHz, MeOD): δH 6.32 (s, H-6), 6.17 (s, H-8), 7.35 (s, H-2′), 7.29 (d, J = 7.9 Hz, H-6′), 6.92 (d, J = 7.9 Hz, H-5′), 5.36 (s, H-1″), 3.79 (d, J = 8.8 Hz, H-2″), 3.44 (m, H-3″), 3.37 (m, H-4″), 4.26 (m, H-5″), 0.91 (d, J = 6.1 Hz, H-6″); 13C NMR (125 MHz, MeOD): δC 134.8 (C-3), 178.1 (C-4), 156.9 (C-5), 93.5 (C-6), 164.7 (C-7), 98.6 (C-8), 157.9 (C-9), 104.3 (C-10), 121.6 (C-1′), 115.7 (C-2′), 144.9 (C-3′), 148.4 (C-4′), 115.1 (C-5′), 121.7 (C-6′), 102.2 (C-1″), 70.8 (C-2″), 70.6 (C-3″), 71.9 (C-4″), 70.5 (C-5″), 16.3 (C-6″); (+)-HRESI-MS: m/z 449.1076 (calcd. 449.1078 for C21H21O11).
Compound 9: yellow oil; 1H NMR (500 MHz, MeOD): δH 6.23 (d, J = 1.9 Hz, H-6), 6.41 (d, J = 1.9 Hz, H-8), 7.35 (d, J = 2.2 Hz, H-2′), 6.96 (d, J = 7.1 Hz, H-5′), 7.33 (dd, J = 2.2 and 7.1 Hz, H-6′), 5.60 (d, J = 1.6 Hz, H-1″), 5.63 (d, J = 3.3 Hz, H-2″), 5.28 (d, J = 3.3 Hz, H-3″), 4.88 (m, H-4″), 3.41 (m, H-5″), 0.87 (d, J = 6.3 Hz, H-6″), 2.13 (s, 11-Me), 2.02 (s, 13-Me), 1.99 (s, 15-Me); 13C NMR (125 MHz, MeOD): δC 133.1 (C-3), 161.9 (C-5), 93.3 (C-6), 164.1 (C-7), 98.6 (C-8), 157.2 (C-9), 104.5 (C-10), 120.9 (C-1′), 121.4 (C-2′), 145.4 (C-3′), 148.6 (C-4′), 114.9 (C-5′), 115.2 (C-6′), 97.8 (C-1″), 68.7 (C-2″), 69.2 (C-3″), 70.0 (C-4″), 68.1 (C-5″), 16.1 (C-6″), 170.0 (C-11), 18.9 (C-12), 170.6 (C-13), 19.2 (C-14), 170.3 (C-15), 19.0 (C-16); (+)-HRESI-MS: m/z 575.1388 (calcd. 575.1395 for C27H27O14).
Compound 10: yellow oil; 1H NMR (500 MHz, MeOD): δH 6.56 (d, J = 2.3 Hz, H-6), 6.82 (d, J = 2.5 Hz, H-8), 7.33 (d, J = 2.1 Hz, H-2′), 6.96 (d, J = 7.7 Hz, H-5′), 7.32 (dd, J = 2.0 and 7.1 Hz, H-6′), 5.46 (d, J = 1.3 Hz, H-1″), 5.29 (d, J = 3.6 Hz, H-2″), 5.27 (d, J = 3.6 Hz, H-3″), 4.77 (m, H-4″), 3.37 (m, H-5″), 0.87 (d, J = 6.1 Hz, H-6″), 2.13 (s, 11-Me), 2.02 (s, 13-Me), 1.98 (s, 15-Me), 2.37 (s, 17-Me); 13C NMR (125 MHz, MeOD): δC 133.1 (C-3), 161.9 (C-5), 108.7 (C-6), 163.8 (C-7), 100.3 (C-8), 157.2 (C-9), 104.5 (C-10), 120.9 (C-1′), 115.1 (C-2′), 145.4 (C-3′), 148.6 (C-4′), 114.9 (C-5′), 121.4 (C-6′), 97.9 (C-1″), 68.7 (C-2″), 69.2 (C-3″), 70.0 (C-4″), 68.1 (C-5″), 15.9 (C-6″), 170.0 (C-11), 19.1 (C-12), 170.4 (C-13), 19.0 (C-14), 170.3 (C-15), 19.0 (C-16), 169.9 (C-17), 19.5 (C-18); (+)-HRESI-MS: m/z 617.1497 (calcd for C29H29O15, 617.1501).
P. falciparum 3D7 lactate dehydrogenase assay: Parasites of the P. falciparum strain 3D7 were grown in fresh group 0 positive human erythrocytes, obtained from the Centro Regional de Transfusion Sanguınea-SAS (Granada, Spain). This assay was performed in duplicate for each compound using a sixteen (16) point dose response curve (½ serial dilutions) with concentrations starting from 50 μM until 1.5 nM to determine the IC50s of the compounds. Adding 25 μL of P. falciparum 3D7 parasite culture (per well) containing parasitized red blood cells at 0.25% parasitaemia and 2% haematocrit in RPMI-1640, 5% Albumax II, 2% D-sucrose 0.3% glutamine and 150 μM hypoxanthine and incubated at 37 °C for 72 h with 5% CO2, 5% O2 and 95% N2. For negative and positive growth controls, 10 μM chloroquine and complete parasite growth medium were respectively used. The final readouts of the assay was done by measuring the absorbance of the reactions at 650 nm in an Envision plate reader (Perkin Elmer, USA) and the results analysed by Genedata software (GenedataAG, Basel, Switzerland), parasite growth was measured by LDH assay as previously described [12, 13].
Anticancer assays: Five tumor cell lines (MiaPaca-2 (CRL-1420), a carcinoma pancreatic from 65 years adult; Hep G2 (HB-80665), a perpetual cell line which was derived from the liver tissue of a 15-year-old Caucasian American male with a well-differentiated hepatocellular carcinoma; A549 (CCL-185), a carcinoma lung from 58-year-old Caucasian made; A2058 (CRL-11147), Human skin melanoma from a 43 years Caucasian adult derived from lymph node and MCF-7 (HTB-22), a breast adenocarcinoma from 69 years woman) were obtained from ATCC. The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide) colorimetric assay, which measures mitochondrial metabolic activity, was employed to estimate the amount of living cells. According to the huge amount of celles to be plated, SelecT (TAP Biosystems, Royston, UK), a cell culture robotic system was used to process ten thousand cells per well (for 72 h assay). Cells were seeded at a concentration of 1× 104 cells/well in 200 μl culture medium and incubated at 37 °C in 5% CO2. After 24 h, the automated liquid-handling system Biomek FX (Beckman Coulter, Pasadena, CA, USA) was used to replace the medium with a final volume of 200 μL and 1 μL of compound (dilution 1/200) and to add controls to the plates and which were then be incubated for 72 h. The test compounds were examined in triplicate with serial two-fold dilutions. After incubation, MTT solution was prepared at 5 mg/mL in PBS 1X and then diluted at 0.5 mg/mL in MEM without phenol red. The sample solution in wells was removed and 100 μL of MTT dye was added to each well. The plates were gently shaken and incubated for 3 h at 37 °C in 5% CO2 incubator. The supernatant was removed and 100 μL of DMSO 100% was added. The plates were gently shaken to solubilize theoriginated formazan and absorbance at 570 nm was read in a Victor2 Wallac spectrofluorometer (PerkinElmer, Waltham, MA, USA). IC50 values were calculated as the concentration that decreases 50% of the cell viability using Genedata Screener software (Genedata AG, Basel, Switzerland). Curve fitting followed the Smart Fit strategy with Hill model selection.