358
E. Santa-Helena et al. / Biomedicine & Pharmacotherapy 92 (2017) 356–364
sulfamic acid (30 mol%, as a catalyst) were added to a round-
bottom flask equipped with a magnetic stir bar and reflux
condenser. The mixture was subjected to reflux for 24 h. The
crude product was then cooled to ambient temperature, concen-
trated in vacuum and purified through column chromatography
using hexane/ethyl acetate (80:20 ratio) as an eluent, resulting in a
pure yellow paste compound.
PBS once and resuspended. For each sample, aliquots of 160 mL
were placed in triplicate in a 96-well plate, and the fluorescence
intensity was determined for 90 min at 37 ꢁC in a Victor 2
fluorometer (Perkin-Elmer), with excitation wavelengths of
485 nm and emission at 520 nm. The ROS levels were expressed
in fluorescence units.
Dihexadecyl 2,6-dimethyl-4-(2-nitrophenyl)-1,4-dihydropyridine-
3,5-dicarboxylate (DPN):
2.1.6. In vitro analyses
MW 767 g molꢀ1; Yellow paste; Yield: 75%. NMR 1H (300 MHz,
CDCl3) d 0.88 (t, 6H, J = 6.0 Hz), 1.26 (m, 52H), 1.54 (m, 4H), 2.31 (s,
6H), 3.94 (m, 2H), 4.06 (m, 2H), 5.82 (s, 1H), 5.97 (bs, 1H), 7.23 (m,
1H), 7.45 (t, 1H, J = 7.5 Hz), 7.53 (d, 1H, J = 9.0 Hz), 7.72 (d, 1H,
J = 9.0 Hz); 13C (75 Mz, CDCl3) d 14.5 (2C), 19.9 (2C), 23.0 (2C), 26.2
(3C), 28.9 (4C), 29.6 (3C), 29.7 (3C), 29.9 (4C), 30.0 (6C), 32.2 (3C),
35.0, 64.6 (2C), 104.1 (2C), 124.3, 127.2, 131.6, 133.0, 142.9, 144.9
(2C), 148.2, 163.3 (2C); IR(film, nmaxcmꢀ1): 758, 1215, 1306, 1354,
1489, 1531, 1689, 2854, 2926, 3018, 3346, 3437.
2.1.6.1. DPPH photometric assay. The effect of DPN and NIF on
DPPH (2,2-diphenyl-1-picrylhydrazyl) radicals was measured
using the modified method of Sharma and Bhat [19]. The
compounds were diluted to final concentrations of 1, 10, 100
and 200
incubated for 30 min at 30 ꢁC in the dark, and the absorbance was
measured at 517 nm against a blank. The IC50 value ( M) is the
mM. The reaction mixture was shaken thoroughly and
m
effective concentration at which DPPH radicals were scavenged by
50%. The radical scavenging activity was calculated using the
following equation:
2.1.2. Cell cultures
Wistar rat H9c2 cardiomyoblast embryo cells were obtained
from the Rio de Janeiro Cell Bank (Rio de Janeiro, Brazil) and
maintained in culture bottles at 37 ꢁC in Dulbecco’s Modified
Eagles Medium (DMEM). This was supplemented with sodium
bicarbonate (1.5 g/L), L-glutamine (4 mM), Hepes (25 mM), glucose
(4.5 g/L), 1% antibiotic (penicillin 100 U/ml and streptomycin
100 mg/ml) and an antimycotic agent (amphotericin 0.25 mg/ml),
and 10% fetal bovine serum was added. Cells were maintained in a
manner that did not exceed 80% confluence.
Scavenging effect (%) = (Acontrol ꢀ Asample/ Acontrol) ꢂ 100
Where Acontrol is the absorbance of the control (without
compound).
2.1.6.2. ABTS radical scavenging. The determination of the radical
scavenging effect of DPN and NIF on ABTS (2,20-azino-bis-(3-
ethylbenzthiazoline-6-sulfonic acid)
–
ABTSꢃ+) radicals was
performed according to the method of [20], with some
modifications. Briefly, ABTS radical was added to a medium
containing DPN and NIF (1, 10, 100 e 200 mM). The media were
2.1.3. Experimental design
incubated for 30 min at 25 ꢁC. The decrease in absorbance was
measured at 734 nm, depicting the scavenging activity of
compounds against the ABTS radical. Results are expressed as
percentage of the blank (without compound). The radical
scavenging activity was calculated following the same equation
used in the DPPH assay (see Section 2.1.6.1).
The H9c2 cells (1 ꢂ10 cells/mL) were placed in 24- or 96-well
plates (according to the test performed), with a DMEM culture
medium at 37 ꢁC for 24 h for adhesion. At the end of this period, the
cells were distributed in the following groups: control (in DMEM
culture medium), I/R (in culture medium DMEM + induction of
hypoxia and reoxygenation), starvation (in PBS), starvation + I/R (in
PBS + induction hypoxia and reoxygenation), starvation + vehicle
(in PBS + 1% dimethyl sulfoxide, DMSO), anandamide (AEA + star-
vation), Anandamide + I/R (AEA + starvation + vehicle + induction of
hypoxia and reoxygenation), nifedipine (NIF + starvation), nifedi-
pine + I/R (NIF + starvation + induction of hypoxia and reoxygena-
tion), 3,5-dipalmitoyl-nifedipine (DPN + starvation) and 3,5-
dipalmitoyl-nifedipine + I/R (DPN + starvation + induction of hyp-
oxia and reoxygenation). All groups were tested with the induction
of hypoxia and reoxygenation (groups I/R) or in the absence of this
condition. The molecules were administered at concentrations of
2.1.6.3. Ferric ion reducing antioxidant power (FRAP) assay. The
FRAP assay was carried out as described by Benzie et al. [21], with
slight modifications. The FRAP reagent was prepared by mixing
38 mM sodium acetate (anhydrous) in milli Q water pH 3.6, 20 mM
FeCl3 6H2O in milli Q water and 10 mM 2,4,6-tri(2-pyridyl)-s-
ꢄ
triazine (TPTZ) in 40 mM HCl in proportions of 10:1:1. This reagent
was freshly prepared before each experiment. Different
concentrations of DPN and NIF and FRAP reagent were added to
each sample, and the mixture was incubated at 37 ꢁC for 40 min in
the dark. The absorbance of the resulting solution was measured at
593 nm by a spectrophotometer. FRAP values were expressed as
absorbance.
10 and 100 mM, always 24 h after assembling the experimental
plates and immediately before the I/R induction.
2.1.4. Induction of ischemia and reoxygenation (I/R)
The ischemia conditions were obtained by replacing the culture
medium with the experimental medium, which consisted of a PBS
buffer (pH 6.8 at 37 ꢁC), and the plates were incubated in a hypoxic
chamber with nitrogen passage coupled to a vacuum pump to
decrease the oxygen pressure. After 30 min of hypoxia, PBS was
replaced with DMEM, and normoxic conditions were restored by
subjecting the plates to the reoxygenation period for another
30 min.
2.1.7. Cell viability
Cell viability was evaluated using the MTT method (3-[4,5-
dimethylthiazol-2-yl]-2,5-difeniltetrazolium). Briefly, cells were
rinsed with PBS and added to a DMEM, with 10% MTT, and then
incubated for 3 h at 37 ꢁC. The DMEM was then removed, and
formazan crystals were dissolved in 200
its absorbance values were immediately read at 490 nm in a
spectrophotometer with plate reader (800 ELX Universal
mL of DMSO (Sigma), and
a
Microplate Reader, Bio-TEK). The values were expressed as
arbitrary values.
2.1.5. Generation of reactive oxygen species (ROS)
After reoxygenation, the cardiomyoblast and controls were
suspended using trypsin and then placed in eppendorf tubes and
incubated at 37 ꢁC for 30 min in PBS with diacetate 20,70-
2.1.8. Analysis of apoptosis and necrosis
Apoptosis and necrosis were evaluated according to Ribble [22]
(modified). To analyze cell apoptosis and necrosis, after reoxygen-
diclorofluorescein (H2DCF-DA) in a final concentration of 40
mM.
After being incubated with H2DCF-DA, the cells were washed with
ation, 2 mL of a working solution composed of PBS, 100 mg/ml