Article pubs.acs.org/jnp
Viniphenol A, a Complex Resveratrol Hexamer from Vitis vinifera Stalks: Structural Elucidation and Protective Effects against Amyloid-β-Induced Toxicity in PC12 Cells Yorgos Papastamoulis,†,‡ Tristan Richard,†,‡ Merian Nassra,† Alain Badoc,† Stéphanie Krisa,† Dominique Harakat,§ Jean-Pierre Monti,† Jean-Michel Mérillon,† and Pierre Waffo-Teguo*,† †
Université de Bordeaux, ISVV, GESVAB, EA 3675, F-33140 Villenave d’Ornon Cedex, France Institut de Chimie Moléculaire de Reims, Université de Reims Champagne-Ardenne, UMR 6229, CNRS, F-51687 Reims Cedex 2, France
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S Supporting Information *
ABSTRACT: Stilbenes have received much attention during the last two decades following the discovery of resveratrol in wine. Since then, there have been a growing number of papers reporting various biological activities of naturally occurring stilbenes. The aim of this study was to determine new minor stilbenes from Vitis vinifera stalks. Purification of these compounds was achieved by means of centrifugal partition chromatography, a versatile separation technique that does not require a solid stationary phase. Viniphenol A (1), a new resveratrol hexamer, was isolated along with five oligostilbenoids identified in V. vinifera for the first time, ampelopsin C, davidiol A, leachianol F, leachianol G, and E-maackin, a dimer with an unusual dioxane moiety, and 14 known hydroxystilbenes. The structure and stereochemistry of viniphenol A were determined on the basis of spectroscopic data analysis and molecular modeling under NMR constraints. Viniphenol A showed protective effects against amyloid-β-induced toxicity in PC12 cell cultures.
S
glucoside exhibiting potent activity.10,11 Thus, viniphenol A was evaluated for its neuroprotective activity against amyloid-β peptide (Aβ)-induced neurotoxicity in cultured PC12 cells using the MTT assay.
tilbenes occur naturally in several plant families such as the Cyperaceae, Dipterocarpaceae, Gnetaceae, and Vitaceae.1,2 Grapes (Vitaceae) and products manufactured from grapes are viewed as the most important dietary sources of these substances.3,4 Stilbenes have received considerable attention because of their potent health-promoting properties such as the prevention of cancers as well as cardiovascular and neurodegenerative diseases.5−7 The basic simple structure gives rise to a wide array of compounds, e.g., monomers varying in the number and position of their hydroxy groups, conjugation with sugar, methyl, methoxy, and other groups, complex oligomers resulting from the oxidative condensation of monomers, and the stereochemistry of the oligomers. More than a thousand stilbenes have been successfully identified in the plant kingdom.8,9 A limited number of high molecular weight resveratrol oligomers have been reported in the plant kingdom. These comprise two pentamers and a hexamer from the Vitis genus.8 In the present study, centrifugal partition chromatography (CPC) was used to purify stilbenoids from Vitis vinifera stalks. Viniphenol A (1), a new resveratrol hexamer, was isolated along with five oligostilbenoids identified in V. vinifera for the first time, i.e., ampelopsin C, davidiol A, leachianol G, leachianol F, and E-maackin, a dimer with an unusual dioxane moiety, and 14 known hydroxystilbenes. Most of the isolated compounds showed moderate antiamyloidogenic activities, the known scirpusin A and (+)-E-ε-viniferin © 2014 American Chemical Society and American Society of Pharmacognosy
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RESULTS AND DISCUSSION V. vinifera cv. Merlot vine stalks were collected from Domaine de Merlet in the Pessac-Leognan appellation. The compounds were purified by means of CPC, a versatile separation technique that does not require a solid stationary phase.12,13 The known compounds were identified as E-resveratrol, (+)-E-ε-viniferin, E-vitisin B, E-piceatannol, E-scirpusin A, E-miyabenol C, (+)-ampelopsin A, pallidol, ampelopsin H, (+)-E-ε-viniferin glucoside, (+)-E-piceid, quadrangularin A, hopeaphenol, and isohopeaphenol by comparison of their physicochemical data with reported values. E-Resveratrol, (+)-E-ε-viniferin, E-vitisin B,14 E-piceatannol,13 E-scirpusin A,15 E-miyabenol C,16 (+)-ampelopsin A,17 pallidol,12 (+)-E-piceid, hopeaphenol, and isohopeaphenol17 were previously reported in V. vinifera stalks. Ampelopsin H and quadrangularin A were previously isolated from V. vinifera leaves.18 (+)-E-ε-Viniferin glucoside was reported from V. vinifera cell suspension cultures.19 Five oligostilbenoids were identified in V. vinifera for the first time: Received: July 26, 2013 Published: February 12, 2014 213
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Table 1. 1H and 13C NMR Data of Viniphenol A (1) at 310 K
a
position
δC
1a 2a(6a) 3a(5a) 4a 7a 8a 9a 10a 11a 12a 13a 14a 1b 2b(6b) 3b(5b) 4b 7b 8b 9b 10b 11b 12b 13b 14b 1c 2c(6c) 3c(5c) 4c 7c 8c 9c 10c 11c 12c 13c 14c
133.4 129.1 115.5 157.3 92.8 52.6 140.0 124.1 156.1 101.4 155.6 105.8 133.8 130.5 113.1 154.0 41.6 48.9 141.3 117.6 158.6 93.7 157.6 104.7 135.3 128.8 113.3 153.6 32.5 49.9 141.5 117.0 158.7 94.4 156.6 110.0
δH (J in Hz) 7.31 d (8.6) 6.85 d (8.6) 5.62 d (8.3) 4.74 d (8.3)
6.22 d (1.8) 6.20 d (1.8) 6.60 d (8.6) 6.29 d (8.6) 4.61 s 4.29 d (12.0)
5.89 d (1.8) 4.91 d (1.8)
HMBC (C → H) 3a(5a), 7a, 8a 7a 2a(6a), 3a(5a) 2a(6a), 8a 7a, 14a 7a, 8a, 7b 8a, 11a, 12a, 14a, 7b, 8b 12a, 7b 14a 12a, 14a 8a, 12a 3b(5b), 7b, 8b 7b 2b(6b), 3b(5b) 2b(6b), 8b 7b, 14b, 7c, 8c 7b, 8b, 8a, 7c 8b, 12b, 14b, 8a 12b, 7a, 8a 14b 12b, 14b 8b, 12b 7c, 3c(5c)
5.97 brs 6.09 d (8.6) 5.03 dd (2.5, 12.0) 3.61 brs
5.73 d (1.7) 6.67 d (1.7)
3c(5c) 8c, 7b, 8b, 8e 7c, 14c, 8b, 7e, 8e 7c, 8c 8c, 12c, 14c, 8d 12c 14c 12c, 14c 8c, 12c
position
δC
1d 2d(6d) 3d(5d) 4d 7d 8d 9d 10d 11d 12d 13d 14d 1e 2e(6e) 3e(5e) 4e 7e 8e 9e 10e 11e 12e 13e 14e 1f 2f(6f) 3f(5f) 4f 7f 8f 9f 10f 11f 12f 13f 14f
129.7 129.9 115.2 157.2 88.7 47.9 143.5 119.9 157.2 101.7 156.3 107.6 138.5 129.1 114.4 153.9 49.3 53.0 144.1 119.9 161.0 94.0 157.5 112.7 133.2 128.2 115.3 157.2 94.7 56.0 147.4 n.o.a n.o. n.o. n.o. n.o.
δH (J in Hz) 6.88 d (8.6) 6.78 d (8.6) 5.11 d (12.0) 4.63 d (12.0)
6.89 d (1.9) 6.06 d (1.9) 6.43 d (8.6) 6.39 d (8.6) 4.68 d (12.0) 4.39 brd (12.0)
6.18 d (1.7) 7.64 d (1.7) 7.00 d (8.6) 6.68 d (8.6) 5.36 d (4.9) 4.65 d (4.9)
HMBC (C → H) 3d(5d), 7d, 8d 7d 2d(6d), 3d(5d) 2d(6d), 8d 7d, 14d 7d, 8d 8d, 12d, 14d, 7e 12d, 7e 14d 12d, 14d 12d 3e(5e), 7e 7e 2e(6e), 3e(5e) 2e(6e), 8e, 8c 7e, 14e, 8c 8e 8e, 12e, 14e, 7f, 8f 12e, 7f, 8f 14e 12e, 14e 8e, 12e 3f(5f), 7f, 8f 7f 2f(6f), 3f(5f) 2f(6f), 8f 7f 7f, 8f
n.o. n.o. n.o. n.o. n.o.
n.o., signal not observed.
The relative configuration of compound 1 was determined by ROESY experiments and by molecular modeling under NMR constraints. Cross-peaks between H-7a/H-14a, H-8a/H-2a(6a), H-7d/H-14d, H-8d/H-2d(6d), H-7f/H-14f, and H-8f/H-2f(6f) indicated the 2,3-trans configuration of the three dihydrobenzofuran rings. The relationship between the six aliphatic protons H-7b/H-8b/H-7c/H-8c/H-8e/H-7e was determined via NOE data. NOE correlations between H-8a/H-7b and H-8a/H-8b indicated that H-7b and H-8b on the dibenzocycloheptadiene ring (C-8a/C-9a/C-10a/C-7b/C-8b/C-9b/C-10b) were cofacial and β-oriented. The relationship between H-8b and H-7c is trans on the basis of the 12.0 Hz coupling constant.25 This relationship was confirmed by the NOE correlation between H-8b/H-2c(6c) and H-7c/H-14b. NOE correlations and J values of the dibenzocycloheptadiene protons were in agreement with those of pauciflorol C.25 The relationship between H-7e and H-8e is trans diaxial on the basis of the 12.0 Hz coupling constant.25 Such a relationship was confirmed by the NOE correlations between H-7e/H-14e and H-8e/H-2e(6e). NOE correlation between H-7e/H-8d indicated that these protons were cofacial and β-oriented. NOE correlations between H-7c/H-14e and H-8b/H-8d indicated
ampelopsin C, davidiol A, leachianol F, leachianol G, and maackin A. Ampelopsin C and davidiol A were identified in other Vitis species.20−22 Leachianol F and leachianol G were identified in Sophora leachiana roots.23 Maackin A was first discovered in Maackia amurensis wood.24 Compound 1 was obtained as a brown solid. Its molecular formula was determined to be C84H64O18 by HRESIMS in combination with 1H and 13C NMR data (Tables 1 and 2). The NMR data indicated that it is a hexamer consisting of resveratrol units (Figure 1). Following analysis of COSY and HSQC data at 310 K, the 1H and 13C NMR data showed the presence of six 4-hydroxyphenyl groups (rings A1 to F1), five 3,5-dioxygenated-1,2-disubstituted benzene rings (A2 to E2), three sets of dihydrobenzofuran protons (H-7a/H-8a, H-7d/H-8d, and H-7f/H-8f), and a set of aliphatic protons (H-7b/H-8b/ H-7c/H-8c/H-8e/H-7e). The connection of the partial structures was established by HMBC correlations (Table 1). The 1H NMR data of viniphenol A (1) recorded at 265 K showed resonances at δH 6.90, 6.33, and 6.01 for the protons of the F2 3,5-dihydroxyphenyl group (Table 2) indicative of the lack of free rotation about the C-1F2−C-8f bond. 214
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Table 2. 1H and 13C NMR Data of Viniphenol A at 265 K position
δC
1a 2a,6a 3a,5a 4a 7a 8a 9a 10a 11a 12a 13a 14a 1b 2b,6b 3b,5b 4b 7b 8b 9b 10b 11b 12b 13b 14b 1c 2c,6c 3c,5c 4c 7c 8c 9c 10c 11c 12c 13c 14c
133.0 129.3 115.6 157.5 92.9 52.6 139.8 124.0 156.2 101.0 155.3 105.4 133.7 n.o.a 113.0 153.9 41.3 48.9 141.4 117.2 158.2 93.8 157.8 104.8 n.o. n.o. 113.3 153.6 32.3 50 141.8 116.9 158.4 94.3 156.5 109.9
a
δH (J in Hz) 7.31 6.84 9.12 5.59 4.73
d d s d d
(8.6) (8.6)
8.05 6.21 8.55 6.17
s d (1.7) s d (1.7)
n.o. 6.28 8.12 4.61 4.25
d (8.6) s s d (12.0)
(8.6) (8.6)
5.90 d (1.8) 6.44 s 5.00 d (1.8) n.o. 6.09 7.96 5.03 3.61
brs s d (12.0) brs
5.73 d (1.7) 8.36 s 6.68 brs
position
δC
1d 2d,6d 3d,5d 4d 7d 8d 9d 10d 11d 12d 13d 14d 1e 2e,6e 3e,5e 4e 7e 8e 9e 10e 11e 12e 13e 14e 1f 2f,6f 3f,5f 4f 7f 8f 9f 10f 11f 12f 13f 14f
129.4 130.1 115.2 157.5 88.8 47.7 143.4 119.8 157.3 101.4 156.3 107.4 138.5 n.o. 114.4 153.9 49.2 53.0 143.8 120.0 160.7 94.1 157.6 112.1 133.6 128.2 115.4 157.4 94.5 55.6 148.0 107.8 158.4 101.0 159.5 106.1
small J7c,8c value and was confirmed by the cross-peaks between H-7c/H-8c and H-8c/H-8e. To elucidate the configuration of compound 1, molecular modeling under the above-mentioned NMR constraints was conducted to validate the 3D structure using the Accelrys software package.26 The relative configuration of viniphenol A (1) is in agreement with the 3D structure obtained by molecular modeling, taking into account all the NOE correlations observed in the ROESY spectra (Figure 2).
δH (J in Hz) 6.84 6.77 9.02 5.06 4.59
d d s d d
(8.6) (8.6)
8.60 6.90 8.60 5.99
s d (1.7) s d (1.7)
n.o. 6.31 8.04 4.67 4.43
d (8.6) s d (12.0) brd (12.0)
(12.0) (12.0)
6.14 d (1.7) 8.34 s 7.64 d (1.7) 6.98 6.65 8.89 5.35 4.66
d d s d d
6.90 8.93 6.33 8.61 6.01
brs s brs s brs
Figure 2. 3D model of viniphenol A (1) on the basis of NMR spectroscopic data and NMR restrained molecular modeling. Aromatic protons are not shown. The six contiguous aliphatic carbons (C-7b/C8b/C-7c/C-8c/C-8e/C-7e) are in magenta.
(8.6) (8.6)
To evaluate the antiaging effects of compound 1, its capacity to protect rat neural pheochromocytoma-derived PC12 cells from amyloid-β peptide induced toxicity was measured. Other resveratrol derivatives are known to exhibit protective effects against Aβ-induced toxicity in PC12 cells.27,28 An MTT assay was used to determine cell viability, and results were expressed as the percentage of control cells. Viniphenol A (1) significantly protected PC12 cells from the cytotoxic effect of Aβ25−35 (Figure 3). After exposure to Aβ25−35 alone, the viability of
(4.0) (4.0)
n.o., signal not observed.
Figure 3. Prevention of Aβ25−35-induced cell death by viniphenol A (13). Cell viability was determined by the MTT assay. The experiment was performed three times, and similar results were obtained. * differ significantly from each other.
PC12 cells was decreased to 44 ± 2%, and viniphenol A significantly increased this percentage in a dose-dependent manner. The results indicate that viniphenol A could be a candidate for novel neuroprotective strategies. Nevertheless, future studies evaluating the bioavailability and bioefficacy of this new phenolic compound need to be performed.
Figure 1. Structure of viniphenol A (1).
that H-8e and H-8c were also cofacial. This orientation was confirmed by the small J value between H-8e and H-8c. The relationship between H-8c and H-7c is cis on the basis of the 215
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(35−55 min), 38−50% B (56−60 min), 50−100% B (60−61 min), 100% B (61−70 min), 100−15% B (70−71 min), and 15% B (71−80). Fractions 3 and 4, containing mainly E-resveratrol and (+)-E-εviniferin, were submitted to a second CPC step using the M solvent system (n-heptane/EtOAc/MeOH/H2O, 5:6:5:6 v/v). E-Resveratrol could be separated in ascending mode, whereas (+)-E-ε-viniferin and E-vitisin B were separated in descending mode. Fraction 5 was fractionated with a second CPC step using the Arizona solvent system L (n-heptane/EtOAc/MeOH/H2O, 2:3:2:3 v/v). The major stilbenes E-piceatannol, E-maackin, and E-scirpusin A were purified in ascending mode, whereas E-miyabenol C (7) was separated in descending mode and purified by semipreparative HPLC. The elution program at 3 mL/min was 20% B (0−5 min), 20−26% B (5−10 min), 26% B (10−22 min), 26−50% B (22−52 min), 50% B (52−55 min), 50−100% B (55−56 min), 100% B (56−64 min), 100−20% B (64−65 min), and 20% B (65−72 min). Two dimers, (+)-ampelopsin A and pallidol, two trimers, ampelopsin C and davidiol A, and a tetramer, ampelopsin H, were purified from fraction 9 of the first CPC using the Arizona solvent system J (n-heptane/EtOAc/MeOH/H2O, 2:5:2:5 v/v) in ascending mode followed by HPLC as above. Fraction 13 of the descending mode fractions (1 g) of the first CPC was subjected to a second CPC purification using the F (n-heptane/ EtOAc/MeOH/H2O, 1:5:1:5 v/v) solvent system of the Arizona range and yielded 26 mg of viniphenol A (1). Viniphenol A was obtained with a high purity degree, completely avoiding the use of solid supports. (+)-E-ε-Viniferin glucoside and (+)-E-piceid were purified from the descending mode fraction 14 of the first CPC by semipreparative HPLC. The elution program at 3 mL/min was 15−20% B (0−5 min), 20−26% B (5−13 min), 26% B (26−35 min), 26−50% B (35−75 min), and 50% B (75−85 min). The descending mode fraction 17 obtained from the first CPC separation contained many phenolic compounds, especially stilbenes. The Arizona J system (n-heptane/EtOAc/MeOH/H2O, 2:5:2:5 v/v) proved to be the most adequate for purifying this fraction. A total of 131 fractions were collected, 117 in the ascending mode and 14 in the descending mode, and were grouped into 10 subfractions. Leachianols F and G, quadrangularin A, hopeaphenol, and isohopeaphenol were purified by semipreparative HPLC. The elution program at 3 mL/min was 5−10% B (0−5 min), 10% B (5−13 min), 10−20% B (13−26 min), 20% B (26−30 min), 20−26% B (30−37 min), 26% B (37−49 min), and 26−100% B (49−56 min). Viniphenol A (1): brown solid powder; [α]25D +67 (c 0.04, acetone); 1D and 2D NMR data, see Tables 1 and 2; HRESIMS m/z 1383.4005 [M + Na]+ (calcd for C84H64O18Na, 1383.3990). Molecular Modeling. Molecular modeling was conducted on a Silicon Graphics O2 workstation using the Molecular Simulation Incorporated software package (Accelrys, San Diego, CA, USA). Simulated annealing and energy minimization were done with Discover and NMR-Refine using the consistent-valence force field (cvff) model. Interproton distances were obtained from ROESY experiments. Quantitative determination of cross-peak intensities was calculated with Sparky. The NOE-distance constraints were estimated using a relation of the Braun model.30 The simulation annealing calculation protocol involved 14 steps as described in a previous work.26 The distance constraint was kept at 30 kcal/mol/Å2. Cell Culture and MTT Assay. Neural pheochromocytoma-derived PC12 were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). PC12 cells were maintained routinely in DMEM-Glutamax supplemented with 15% heat-inactivated horse serum, 2.5% fetal bovine serum, and 1% penicillin/streptomycin antibiotics at 37 °C in a humidified atmosphere of 10% CO2/90% air. All cells were cultured in poly-D-lysine-coated culture dishes. Cells were harvested from flasks and plated at a density of 10 000 cells per well in 96-well plates and incubated at 37 °C for 24 h. Aβ(25−35) preincubated with or without compound at 37 °C for 48 h was diluted with fresh DMEM-Glutamax and added to individual wells. The final concentration of Aβ was 10 μM. After 24 h of incubation, cell viability was determined by the conventional MTT reduction assay.
EXPERIMENTAL SECTION
General Experimental Procedures. The UV absorption was monitored with a Varian 345 dual-wavelength UV−vis detector simultaneously at 280 and 306 nm. Isolated compounds were analyzed by 1H NMR, 13C NMR, and 2D NMR spectroscopy (COSY, HSQC, HMBC, and ROESY) in acetone-d6 and/or methanol-d4. The spectra were recorded on an AC 300 MHz and Avance III 600 MHz Bruker spectrometers (Wissembourg, France). Mass spectra were acquired on a Thermo Finnigan LCQ Advantage ion-trap spectrometer equipped with an electrospray source (Thermo Electron, France). The following MS conditions were used for ionization, desolvation, focusing, and detection: spray voltage +4.5 kV, sheath gas flow rate 23, auxiliary gas flow rate 3 (ratio sheath gas/auxiliary gas), heated capillary temperature 220 °C, capillary voltage 26 V, tube lens offset 45 V, scan range m/z 150−2000, helium (He) used as the collision gas and nitrogen (N2) as the nebulizing gas. Fractionation was performed on an FCPC200 apparatus provided by Kromaton Technologies (Angers, France), fitted with a rotor made of 20 circular partition disks (1320 partition cells: 0.130 mL per cell; total column capacity of 204 mL; dead volume 32.3 mL). The content of the outgoing organic phase was monitored by an online UV absorbance measurement at 280 nm. Fractions were also analyzed by thin-layer chromatography on silica gel precoated 60 F254 plates (Merck). The plates were developed by spraying with an anisaldehyde sulfuric reagent containing 5 mL of p-anisaldehyde, 90 mL of ethanol, and 5 mL of sulfuric acid, and the compounds were detected at 254 and 366 nm. Compounds were purified from their mixtures obtained by CPC on a semipreparative Varian Prostar apparatus. A C18 reversed-phase column (Prontosil, 250 mm × 8.0 mm 5.0 μm, Bischoff) was used for purification. The analytical HPLC separations were conducted on a C18 column (Prontosil, 250 mm × 4.0 mm 5.0 μm, Bischoff) equipped with a guard column of the same nature. The UV−vis spectra of all compounds were recorded between 200 and 450 nm with a 2 nm step. The analyses were carried out on a Thermo Finnigan Surveyor chromatographic system (Thermo Electron, France). The HPLC effluent at 1 mL/min was introduced into the electrospray source in a postcolumn splitting ratio of 9:1 (100 μL/min). All the solvents were purchased from Scharlau (Sentmenat, Spain). Plant Material. Vitis vinifera cv. Merlot vine stalks were collected from Domaine de Merlet in the Pessac-Leognan appellation and identified by one of the authors (A.B.); a stem specimen was deposited in the herbarium of the Botanic Garden of Talence (France) under the voucher number TAL 20130710. Extraction and Isolation. Dried and finely ground stems of V. vinifera (3.15 kg) were extracted with acetone/water (6:4, v/v; 2 × 20 L, 12 h each) at room temperature under agitation. After filtration, the aqueous acetone solution was concentrated at 30−35 °C under reduced pressure. The residual aqueous phase was successively extracted with nheptane and MTBE. The MTBE extract was reduced under vacuum at 30−35 °C and lyophilized to yield 44 g of crude extract. After testing the “Arizona” Foucault et Chevolot solvent system, system K (n-heptane/EtOAc/MeOH/H2O, 1:2:1:2 v/v) was chosen for the first partition of the MTBE extract.29 The rotor was filled to capacity with the aqueous stationary phase in the ascending mode without rotating. For the first CPC step, MTBE extract was separated by four consecutive CPC runs. Therefore, an average of 1.8 g was injected in each run. For each run, the MTBE extract was dissolved in 8 mL of the organic/aqueous phase mixture (1:1). After the injection the organic (upper) mobile phase was pumped into the column in the ascending mode at a flow rate of 3 mL/min. Then, the rotation speed was increased from 0 to 1000 rpm. Fractions of 9 mL were collected (3 min/tube). After the CPC separation of MTBE extract (7.86 g), 16 fractions were obtained, eight in the ascending mode and eight in the descending mode. For HPLC experiments, ultrapure H2O was used, acidified with 0.025% TFA (solvent A) and MeCN (solvent B). The HPLC gradient at 1 mL/min used for optimal separation of the compounds contained in all MTBE extracts and further purified fractions was as follows: 15% B (0−5 min), 15−20% B (5−13 min), 20−26% B (13−26 min), 26% B (26−35 min), 26−38% B 216
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Journal of Natural Products
Article
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Cells were treated with MTT solution (final concentration, 0.5 mg/mL DMEM-Glutamax) for 3 h at 37 °C. The dark blue formazan crystals formed in intact cells were solubilized with DMSO for 0.5 h. The absorbance was measured at 595 nm with a microplate reader (Dynex, USA). Results were expressed as the percentage of MTT reduction in relation to the absorbance of control cells at 100%. All data represent the average of three tests. Data are shown as means ± SEM. For results, the analysis of variance ANOVA was used, and the degree of meaning of data was taken to probability p ≤ 0.05.
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ASSOCIATED CONTENT
S Supporting Information *
NMR spectra of viniphenol A (1) and NMR data of compounds maackin A, ampelopsin C, davidiol A, leachianol F, and leachianol G are available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*Tel: +33-55-757-5955. Fax: +33-55-757-5952. E-mail: pierre. waff
[email protected]. Author Contributions ‡
Y. Papastamoulis and T. Richard contributed equally.
Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS The authors thank G. Fondeville and E. Pedrot for technical assistance and R. Cooke for proofreading the manuscript. Research support was provided by the French Ministry of Research and the Aquitaine Regional Government. NMR and MS experiments were performed at the Plateforme Métabolome-Fluxome, Centre de Génomique Fonctionnelle de Bordeaux, Bordeaux, France.
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REFERENCES
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