Article
J. Agric. Food Chem., Vol. 58, No. 7, 2010 4127
systems. Chemical experimental systems are composed of redu-
cing peroxynitrite (ONOO-) and singlet oxygen (1O2), scaven-
ging 2,20-azinobis(3-ethylbenzothiazoline-6-sulfonate) cationic
radical (ABTS•þ), 2,20-diphenyl-1-picrylhydrazyl (DPPH) and
galvinoxyl radicals, bleaching β-carotene in linoleic acid
(LH)-Triton X-100 emulsion, and protecting methyl linoleate
against 2,20-azobis(2-amidinopropane hydrochloride) (AAPH,
R;NdN;R, R = -CMe2C(dNH)NH2)-induced oxidation.
Biological experimental systems include the protection of DNA
against the oxidation induced by AAPH, Cu2þ/glutathione
(GSH), and hydroxyl radical (•OH), respectively. Positive control
compounds such as Trolox or quercentin were not used because
the antioxidant effectiveness of Trolox or quercentin screened in
these experimental systems has been already reported elsewhere.
galvinoxyl solutions. The absorbance of the radical solutions was
recorded, and the decay rate for the radical was calculated on the basis
of the corresponding ε.
β-Carotene Bleaching Test and Protection of Methyl Linoleate
against AAPH-Induced Oxidation. An emulsion was prepared by
dissolving 5.0 mg of β-carotene, 40 mg of linoleic acid (LH), and 400 mg
of Triton X-100 in 5.0 mL of CHCl3. After CHCl3 was evaporated under
vacuum pressure, 100 mL of oxygen-saturated water was added, and the
mixture was shaken under ultrasonic vibration to form a homogeneous
β-carotene-LH emulsion (λmax = 460 nm) (18). The ethanol solutions of o-,
m-, and p-HBMC (0.1 mL) were mixed with 1.9 mL of β-carotene-LH
emulsion to make the final concentration of o-, m-, and p-HBMC at 400 μM.
The absorbance of the mixture was detected every 1 h and plotted versus time.
The protective effects of o-, m-, and p-HBMC on AAPH-induced
oxidation of methyl linoleate were investigated by detecting the decay of
the concentration of methyl linoleate (19). Methyl linoleate, methyl
palmitate (as the internal standard), AAPH, and o-, m-, or p-HBMC were
dissolved in tert-butanol/H2O (1:1, v/v) in a test tube with a final
concentration at 14.3 mM, 9.3 mM, 40 mM, or 500 μM, respectively.
The test tube was incubated at 37 °C to initiate the oxidation. Aliquots
were taken out every 100 min, and the concentration of methyl linoleate
was analyzed by GC (Hewlett-Packard 1890 equipped with an SE-54
30 m ꢀ 0.25 mm capillary column, 0.25 μm film thickness, N2). The
temperatures of the chromatograph chamber, injector, and hydrogen
flame ionization detector were 260, 280, and 300 °C, respectively (19).
Effects of o-, m-, and p-HBMC on the Oxidation of DNA
Mediated by Cu2þ/GSH. The oxidation of DNA mediated by Cu2þ
and GSH was carried out following the methodology described by Reed
et al. (20), with some modifications. Briefly, DNA, CuSO4, and GSH were
dissolved in phosphate-buffered solution (PBS1: 6.1 mM Na2HPO4,
3.9 mM NaH2PO4) with the final concentration at 2.0 mg/mL, 5.0 mM,
and 3.0 mM, respectively. Dimethyl sulfoxide (DMSO) solutions of o-, m-,
and p-HBMC were added with a final concentration at 0.6 mM. The
mixture was delivered into test tubes with each containing 2.0 mL. The test
tubes were incubated at 37 °C to initiate the oxidation of DNA. Three
tubes were taken out every 30 min and cooled immediately, to which
1.0 mL of PBS1 solution of EDTA (30.0 mM as the final concentration)
was added to chelate Cu2þ. The tubes were heated in a boiling water bath
for 30 min after 1.0 mL of TBA solution (1.00 g of TBA and 0.40 g of
NaOH dissolved in 100 mL of PBS1) and 1.0 mL of 3.0% trichloroacetic
acid aqueous solution were added. After the test tubes had cooled to
room temperature, 1.5 mL of n-butanol was added and shaken vigorously
to extract TBA reactive substance (TBARS). The absorbance of the
n-butanol layer was measured at 535 nm.
MATERIALS AND METHODS
Materials and Instrumentation. The diammonium salt of 2,20-
azinobis(3-ethylbenzothiazoline-6-sulfonate) (ABTS) and DPPH and gal-
vinoxyl radicals were purchased from Fluka Chemie GmbH, Buchs,
Switzerland. AAPH, the naked DNA sodium salt, methyl linoleate,
linoleic acid, and4-nitroso-N,N-dimethylaniline(NDMA) werepurchased
from Acros Organics, Geel, Belgium. Other agents were of analytical grade
and used directly. o-, m-, and p-HBMC were synthesized following the
methodology described by Siddaiah et al. (7) (see the Supporting Infor-
mation). The structures were identified by 1H and 13C NMR (Varian
Mercury 300 NMR spectrometer), and the purities of o-, m-, and p-HBMC
were identified by high-performance liquid chromatography and were
>98%.
Reducing Peroxynitrite Assay. ONOO- (ε302 = 1670 M-1 cm -1
)
was prepared following the methodology described by Uppu et al. (12),
with some modifications. Briefly, a solution of 2.2 mL of 30% H2O2 was
diluted to 50 mL with water and cooled in an ice/water mixture. Then,
4 mL of 5 M NaOH and 5 mL of 0.04 M diethylenetriaminepentaacetic
acid (DTPA, dissolved in 0.05 M NaOH) were added and diluted to
100 mL with water; then, 2.7 mL of isoamyl nitrite was added and
vigorously stirred for 5 h at room temperature. The aqueous phase was
washed with 6 ꢀ 200 mL of dichloromethane, and the surplus H2O2 was
decomposed by MnO2 to obtain ONOO- aqueous solution. To assess the
reduction capacity of the homoisoflavonoids, the o-, m-, and p-HBMC and
ONOO- were mixed in 0.1 M NaOH to 20.0 μM and 0.55 mM as the final
concentration, respectively. The absorbance of the mixture was scanned
from 250 to 550 nm every 15 min.
Quenching Singlet Oxygen. 1O2 was prepared following the methodo-
logy described by Moldonado et al. (13). Briefly, 10 mM histidine, 10 mM
sodium hypochlorite, 10 mM H2O2, and 50 μM 4-nitroso-N,N-dimethyl-
aniline (NDMA) were dissolved in 45 mM sodium phosphate buffer
(pH 7.1) to generate 1O2. The scavenging abilities of o-, m-, and p-HBMC
were tested by adding various concentrations of the homoisoflavonoids to
the aforementioned mixture to a final volume of 2.0 mL. The mixture was
incubated at 30 °C for 40 min, and then the absorbance was measured at
440 nm. The percentage of 1O2 quenched by o-, m-, and p-HBMC was
calculated as (Adetect - Aref)/(A0 - Aref) ꢀ 100, where A0 and Aref were the
absorbance before and after the incubation in the control experiment,
respectively, whereas Adetect was the absorbance after the incubation in the
presence of o-, m-, and p-HBMC.
Effects of o-, m-, and p-HBMC on OH-Induced Oxidation of
•
DNA. •OH was generated from the reaction between tetrachlorohydro-
quinone (TCHQ) and H2O2 (21). DNA and H2O2 were dissolved in
phosphate-buffered solution (PBS2: 8.1 mM Na2HPO4, 1.9 mM NaH2-
PO4, 10.0 μM EDTA) to a final concentration of 2.0 mg/mL and 2.0 mM,
respectively, to which TCHQ and o-, m-, and p-HBMC (dissolved in
DMSO as the stock solutions) were added with a final concentration at
4.0 mM and 0.6 mM, respectively. Then, the above mixture was delivered
into test tubes with each containing 2.0 mL. The test tubes were incubated
at 37 °C for 30 min and cooled immediately. The following operation was
the same as for Cu2þ/GSH-mediated oxidation of DNA. The absorbances
of TBARS in the control experiment and in the presence of HBMC were
assigned as A0 and Adetect. The protectiveeffects ofo-, m-, and p-HBMC on
•OH-induced oxidation of DNA were expressed by Adetect/A0 ꢀ 100.
Effects of o-, m-, and p-HBMC on AAPH-Induced Oxidation of
DNA. AAPH-induced oxidation of DNA was carried out following our
previous method (22). Briefly, DNA and AAPH were dissolved in PBS2
with a final concentration at 2.0 mg/mL and 40 mM, respectively. Various
concentrations of o-, m-, and p-HBMC (dissolved in DMSO as the stock
Scavenging ABTS•þ, DPPH, and Galvinoxyl Radicals. The
ABTS•þ radical was derived from the oxidation of ABTS salt. Two
milliliters of 4.0 mM ABTS aqueous solution was oxidized by 1.41 mM
K2S2O8 for 16 h, and then 100 mL of ethanol was added to make the
absorbance of ABTS•þ around 0.70 at 734 nm [εABTS = 1.6 ꢀ 104
•þ
M-1 cm-1 (14)]. DPPH and galvinoxyl were dissolved in ethanol to make
the absorbance around 1.00 at 517 nm [εDPPH = 4.09 ꢀ 103 M-1
cm-1 (15)] and at 428 nm [εgalvinoxyl = 1.4 ꢀ 105 M-1 cm-1 (16)],
respectively. o-, m-, and p-HBMC were mixed with ABTS•þ, DPPH,
and galvinoxyl radical solutions, respectively, to test the abilities of
homoisoflavonoids to scavenge radicals (17). The ethanol solution of o-,
m-, or p-HBMC was added to the aforementioned radical solutions at
room temperature. The final concentrations of o-, m-, or p-HBMC were
1.0 mM to trap ABTS•þ solution and 2.0 mM to trap DPPH and
solution) were added. The following operation was the same as for Cu2þ
/
GSH-mediated oxidation of DNA except that the heating period was
15 min after TBA and trichloroacetic acid were added.
Statistical Analysis. All of the data were the average value from at
least three independent measurements with the experimental error within
10%. The equations and the data in figures were analyzed statistically via
one-way ANOVA-Dunnett by using Origin 6.0 professional software,
and p < 0.001 indicated a significant difference.