5356 J. Agric. Food Chem., Vol. 58, No. 9, 2010
Ximenes et al.
to generate an alkyl radical (R•). In the presence of dissolved
molecular oxygen, this radical is converted to the corresponding
peroxyl radical (ROO•) (15). The mechanism of erythrocyte
hemolysis induced by thermolysis of AAPH is not completely
understood, but it has been correlated with lipid peroxidation and
oxidation of membrane proteins (16). The incubation of erythro-
cytes with AAPH also provokes the depletion of intracellular
GSH (17).
Because the above biological phenomenon is associated with
the disruption of the cell membrane and in view of increasing
evidence that the lipophilicity of gallic acid esters is decisive for
their beneficial effects, here, we aimed to synthesize selected
gallate esters and compare their efficacy as inhibitors of AAPH-
induced hemolysis and GSH depletion. The objective was to
retain the same oxidizable moiety while altering the lipophili-
city. Additionally, we studied and compared the antioxidant and
pro-oxidant effects of gallic acid and gallates in cell-free systems.
anhydrous MgSO
4
, and evaporated under reduced pressure. The crude
products were purified over a silica gel column eluted isocratically with
CHCl /MeOH (98:2). Structures of the semi-synthetic esters were estab-
3
1
13
lished by H and C NMR spectral analysis.
Erythrocyte Suspension. Human erythrocytes from healthy donors
were obtained from peripheral blood, centrifuged at 770g for 10 min, and
washed 3 times with phosphate-buffered saline (PBS) at pH 7.4. The
supernatant and buffy coat were removed by aspiration after each wash.
The cells were resuspended to 20% (v/v) in PBS. The blood samples were
taken from healthy volunteers. The study was approved by the faculty
research ethics committee.
Hemolysis Assays. The hemolysis studies were performed as pre-
viously described (19), with modifications. Equal amounts of erythrocytes
suspension and 100 mM AAPH in PBS were gently homogenized while
being incubated for 6 h at 37 °C (blood tube rotator). Aliquots (75 μL)
were removed at regular intervals, diluted 1:20 with PBS, and centrifuged
at 4000 rpm for 10 min. The degree of hemolysis was measured in the
supernatant by its absorbance at 540 nm. Reference values (100%
hemolysis) were determined with the same aliquot of erythrocytes but
diluted in 1500 μL of distilled water instead of PBS to provoke the total
lysis of the erythrocytes. By diluting the 100% hemolysis sample in PBS, a
calibration curve of percent hemolysis against absorbance was constructed
for conversion of the absorbance measurement to degrees of hemolysis.
The blank was PBS, and measurements were made in a UV-1240 spectro-
photometer (Shimadzu, Japan). When used, 10 μL aliquots of inhibitors in
ethanol were added at the beginning of the reaction. The same volume of
ethyl alcohol (10 μL) was added to the negative (without AAPH) and
positive (with AAPH) controls. In the studies where cumene hydroper-
oxide (Cu-OOH) was the hemolytic agent, this compound (500 μM) was
used instead of AAPH.
MATERIALS AND METHODS
Chemicals and Equipments. Gallic acid, vanillic acid, methyl vanillate,
(
()-6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid (trolox),
0
N-ethylmaleimide (NEM), (()-R-tocopherol (vitamin E), 2,2 -azobis(2-
amidinopropane) hydrochloride (AAPH), 5,5 -dithiobis(2-nitrobenzoic
0
acid) (DTNB), glutathione (GSH), 8-hydroxypyrene-1,3,6-trisulfonic acid
0
trisodium salt (pyranine), 2,2-diphenyl-1-picrylhydrazyl (DPPH), N,N -
dicyclohexylcarbodiimide, cumene hydroperoxide (Cu-OOH), horserad-
ish peroxidase (HRP) (EC 1.11.1.7), and o-phthalaldehyde (OPA) were
purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO). Hydrogen
Determination of Intracellular GSH. The intracellular concentra-
tion of GSH in erythrocytes was determined using the DTNB method (19),
with modifications. The 20% suspension of erythrocytes (100 μL) was
incubated with 100 μL of 50 mM AAPH at 37 °C for 2.0 h. After
incubation, the suspension was diluted to 1000 μL with PBS and
centrifuged at 6000 rpm for 10 min. The supernatant was removed, and
peroxide (H
2 2
O ) was prepared by diluting a 30% stock solution and calcul-
-1
ating its concentration from its absorption at 240 nm (ε240 =43.6 M
-1
cm ) (18). Column chromatography was carried out over 0.06-0.20 mm
silica gel (Acros Organics, Morris Plains, NJ). Gel permeation chromato-
graphy (GPC) was performed with Sephadex LH-20 (Pharmacia Biotech,
Uppsala, Sweden). Preparative high-performance liquid chromatography
2
the cell pellet was lysed by adding 700 μL of distilled H O. The protein in
(HPLC) was carried out in a Varian Prep-Star 400 system with a Pheno-
7
00 μL of lysate was precipitated by adding 500 μL of an aqueous solution
menex C-18 (250 ꢀ 21.2 mm) column. The monodimensional nuclear
of 10% trichloroacetic acid (TCA). After 5 min, the protein precipitate was
separated from the remaining solution by centrifugation at 14000 rpm for
magnetic resonance (NMR) spectra were recorded on a Varian Inova 500
1
13
spectrometer (11.7 T) at 500 MHz ( H) and 125 MHz ( C), using CDCl
and DMSO-d as solvents (Aldrich). All of the reagents used for buffers
and mobile phases were analytical-grade. Stock solutions of antioxidants
were prepared in ethyl alcohol. Ultrapure Milli-Q water from Millipore
was used for the preparation of buffers and solutions.
Gallic Acid Source. Gallic acid was extracted from Alchornea
glandulosa. The leaves were collected in the Biological Reserve and
Experimental Station at Mogi Gua c- u, S ~a o Paulo State, Brazil, in March
3
1
0 min. The supernatant (500 μL) was made alkaline by adding 187.5 μL
of Na HPO (300 mM) and 62.5 μL of NaOH (1 M). Next, 450 μL of
Na HPO (300 mM) and 100 μL of DTNB solution (0.2 mg/mL in 1.0%
6
2
4
2
4
sodium citrate) were added to 450 μL of the alkaline mixture. In the blank,
500 μL of supernatant was mixed with 250 μL of PBS instead of 187.5 μL
of Na HPO and 62.5 μL of NaOH, after which 450 μL of PBS and 100 μL
2 4
of DTNB were added to 450 μL of this mixture. Each sample test or
control had its blank. The absorbance was read at 412 nm. A standard
curve was constructed to measure the concentration of GSH. In this case,
700 μL of lysate was replaced by 700 μL of solutions of GSH at several
concentrations. The hemoglobin content (Hb) in an aliquot of the lysate
(prior precipitation with TCA) was determined by the cyanohemoglobin
method. The GSH values were expressed as μmol/g Hb (20).
Oxidation of GSH by AAPH. A solution of GSH (100 μM) was
incubated with 2.5 mM AAPH in PBS at 37 °C for 1.0 h (21). When
present, gallic acid and gallates (100 μM) were added before the addition of
AAPH. The remaining concentration of GSH was measured by the DTNB
method as follows: 0.45 mL of the reaction mixture was added to 0.45 mL
2005. A voucher specimen(SP319257) has beendeposited in the herbarium
of the Botanic Institute (S ~a o Paulo, SP, Brazil). The shade-dried plant
material (1.5 kg) was ground and defatted with n-hexane (3.5 L ꢀ 3, at
room temperature) and exhaustively extracted by maceration with MeOH
(
4.2 L ꢀ 3). The crude extract was concentrated under reduced pressure to
yield 3.8 g of a syrupy residue. The concentrate was then diluted with
MeOH/H O (4:1) and successively partitioned with EtOAc and n-BuOH.
After solvent removal in a rotary evaporator, the partition phases yielded
.5 and 0.8 g, respectively. The EtOAc residue (2.0 g) was chromato-
2
2
graphed by gel permeation over Sephadex LH-20, eluted with methanol, to
afford 11 fractions (A1-A11). Second-stage chromatographic purifica-
tion of fraction A2 (730 mg) by reverse-phase (RP)-HPLC (7.5:92:0.5
2 4
of 300 mM Na HPO and 0.1 mL of DTNB solution (0.2 mg/mL in 1.0%
MeOH/H
2
O/HOAc, UV detection at 265 nm, and flow rate at 15 mL/min)
sodium citrate). The absorbance at 412 nm was read against a blank
consisting of 0.45 mL of PBS, 0.45 mL of 300 mM Na HPO , and 0.1 mL
of DTNB. An analytical curve was produced using standard GSH
solutions, which were submitted to the DTNB methods.
1
yielded gallic acid (335 mg). The identification was based on analysis of H
2
4
13
and C NMR data, as well as by a comparison to authentic material
obtained from Sigma-Aldrich Chemical Co. (St. Louis, MO).
Synthesis of Esters (Methyl, Propyl, and Lauryl). A 3 mL solution
Oxidation of GSH by HRP/H O . A solution of GSH (100 μM)
2
2
0
of N,N -dicyclohexylcarbodiimide (DCC, 1.0 mmol) in p-dioxane was
was incubated with 100 μM H O and 0.1 μM HRP in PBS at 37 °C (21).
2
2
added to a cooled (5 °C) solution of 0.2 mmol of gallic acid and 20 mmol of
methyl, propyl, or lauryl alcohol in 6 mL of p-dioxane. After the solution
was stirred for 48 h, the solvent was removed under reduced pressure. The
residue was partitioned 3 times with EtOAc and filtered. The filtrate was
washed successively with saturated aqueous citric acid solution (3 times),
When present, gallic acid and gallates (100 μM) were added before the
addition of H . The reactions were stopped by adding 10 μg/mL
2 2
O
catalase. The remaining concentration of GSH was measured by the
DTNB method as above.
Measurement of GSH and GSSG by HPLC. A solution of GSH
saturated aqueous NaHCO
3
(3 times), and water (2 times), dried over
2 2
(100 μM) was incubated with 100 μM H O and 0.1 μM HRP in PBS at