Evaluation of antioxidant activity of vanillin by using multiple antioxidant assays

Article abstract of DOI:10.1016/j.bbagen.2010.11.004

Background: Vanillin, a compound widely used in foods, beverages, cosmetics and drugs, has been reported to exhibit multifunctional effects such as antimutagenic, antiangiogenetic, anti-colitis, anti-sickling, and antianalgesic effects. However, results of studies on the antioxidant activity of vanillin are not consistent. Methods: We systematically evaluated the antioxidant activity of vanillin using multiple assay systems. DPPH radical-, galvinoxyl radical-, and ABTS⁺-scavenging assays, ORAC assay and an oxidative hemolysis inhibition assay (OxHLIA) were used for determining the antioxidant activity. Results and conclusion: Vanillin showed stronger activity than did ascorbic acid and Trolox in the ABTS⁺-scavenging assay but showed no activity in the DPPH radical- and galvinoxyl radical-scavenging assays. Vanillin showed much stronger antioxidant activity than did ascorbic acid and Trolox in the ORAC assay and OxHLIA. In the ABTS⁺-scavenging assay, ORAC assay and OxHLIA, vanillin reacted with radicals via a self-dimerization mechanism. The dimerization contributed to the high reaction stoichiometry against ABTS ⁺ and AAPH-derived radicals to result in the strong effect of vanillin. Oral administration of vanillin to mice increased the vanillin concentration and the antioxidant activity in plasma. These data suggested that antioxidant activity of vanillin might be more beneficial than has been thought for daily health care. General significance: Based on the results of the present study, we propose the addition of antioxidant capacity to the multifunctionality of vanillin.

Full text of DOI:10.1016/j.bbagen.2010.11.004

Biochimica et Biophysica Acta 1810 (2011) 170–177
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Biochimica et Biophysica Acta
journal homepage: www.elsevier.com/locate/bbagen
Evaluation of antioxidant activity of vanillin by using multiple antioxidant assays
a,
a
a
b
Akihiro Tai ⁎, Takeshi Sawano , Futoshi Yazama , Hideyuki Ito
a
Faculty of Life and Environmental Sciences, Prefectural University of Hiroshima, Shobara, Hiroshima 727-0023, Japan
Division of Pharmaceutical Sciences, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama 700-8530, Japan
b
a r t i c l e i n f o
a b s t r a c t
Article history:
Background: Vanillin, a compound widely used in foods, beverages, cosmetics and drugs, has been reported to
exhibit multifunctional effects such as antimutagenic, antiangiogenetic, anti-colitis, anti-sickling, and
antianalgesic effects. However, results of studies on the antioxidant activity of vanillin are not consistent.
Methods: We systematically evaluated the antioxidant activity of vanillin using multiple assay systems. DPPH
Received 17 September 2010
Received in revised form 10 November 2010
Accepted 12 November 2010
Available online 21 November 2010
+
radical-, galvinoxyl radical-, and ABTS• -scavenging assays, ORAC assay and an oxidative hemolysis inhibition
assay (OxHLIA) were used for determining the antioxidant activity.
Results and conclusion: Vanillin showed stronger activity than did ascorbic acid and Trolox in the ABTS• -
Keywords:
Vanillin
Antioxidant
Self-dimerization
ABTS radical cation
ORAC
+
scavenging assay but showed no activity in the DPPH radical- and galvinoxyl radical-scavenging assays.
Vanillin showed much stronger antioxidant activity than did ascorbic acid and Trolox in the ORAC assay and
+
OxHLIA. In the ABTS• -scavenging assay, ORAC assay and OxHLIA, vanillin reacted with radicals via a self-
+
dimerization mechanism. The dimerization contributed to the high reaction stoichiometry against ABTS•
Oxidative hemolysis inhibition assay
and AAPH-derived radicals to result in the strong effect of vanillin. Oral administration of vanillin to mice
increased the vanillin concentration and the antioxidant activity in plasma. These data suggested that
antioxidant activity of vanillin might be more beneficial than has been thought for daily health care.
General significance: Based on the results of the present study, we propose the addition of antioxidant capacity
to the multifunctionality of vanillin.
©
2010 Elsevier B.V. All rights reserved.
1
. Introduction
tion, non-homologous DNA end-joining inhibition, and reactive
oxygen species (ROS) scavenging [8].
Vanillin (4-hydroxy-3-methoxybenzaldehyde) is one of the most
In general, ROS generated in vivo, such as hydroxyl radical (HO•) and
peroxyl radical (ROO•), are highly unstable and reactive. It seems crucial
to quench ROS as fast as possible before they attack biomolecules and
cause harm. There are many different kinds of evaluation reports for the
antioxidant activity of vanillin against ROS and other radicals, though
the ROS-scavenging activity as mentioned above seems to be one of the
factors for the antimutagenic property of vanillin. It has been well
reported that the antioxidant ability of vanillin is very low: for example,
vanillin had the weakest superoxide anion scavenging activity in five
tested phenolic compounds and had almost no antioxidative activity
against lipid peroxidation [9], and vanillin exhibited no or little
antioxidant activity in a 1,1-diphenyl-picrylhydrazyl (DPPH) radical-
scavenging assay [10–12], in a β-carotene decolorization assay [11], and
in linoleic acid and cholesterol oxidation assays [13]. However, it has
also been reported that vanillin inhibited protein oxidation and lipid
peroxidation induced by photosensitization in rat liver mitochondria [1]
and that vanillin exhibited hydroxyl radical [14] and 2,2′-azinobis(3-
popularly used flavoring components extracted from the seedpods of
Vanilla planifolia and is widely used in foods, beverages, cosmetics,
and drugs. Concentrations of vanillin used in food and beverage
products range widely from 0.3 to 33 mM [1]. It is expected that the
high level of vanillin intake from foods and beverages will have some
effects on human health. Over the past two decades, the antimuta-
genic properties of vanillin have been studied by many researchers. In
1
986, Ohta et al. first showed the antimutagenic effect of vanillin on
mutagenesis induced by 4-nitroquinoline 1-oxide, furylfuramide,
captan or methylglyoxal in Escherichia coli [2]. Subsequently, vanillin
was reported to significantly reduce mutations induced by ultraviolet
light or X-ray in mammalian cells both in vitro and in vivo [3–5].
Mitomycin C- and methylmethane sulphonate-induced mutations in
somatic cells of Drosophila melanogaster and mouse bone marrow cells
were also reduced by vanillin [6,7]. It has been suggested that the
antimutagenic property of vanillin is achieved by recA-dependent
recombinational repair enhancement, error-prone SOS repair inhibi-
+
ethylbenzothiazoline-6-sulfonic acid) radical cation (ABTS• ) [15]
scavenging activities. Furthermore, it has been reported that vanillin
was a poor antioxidant and that vanillin radicals promoted oxidation of
glutathione, sulfhydryl groups in ovalbumin, and NADPH [16]. There-
fore, there is no consistency in results of studies on the antioxidant
activity of vanillin.
⁎
Corresponding author. Fax: +81 824 74 1779.
E-mail address: atai@pu-hiroshima.ac.jp (A. Tai).
0304-4165/$ – see front matter © 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.bbagen.2010.11.004

A. Tai et al. / Biochimica et Biophysica Acta 1810 (2011) 170–177
171
We have assessed the antioxidant activities with attention to the
following proposals and points of view: 1) Niki et al. reported that
there are two types of antioxidant that scavenge radicals quickly
and quench many radicals, and they proposed assessing reactivity
based on both reaction rate and stoichiometry [17], 2) the activities
of some antioxidants vary depending on the assay method, and thus
the use of multiple methods is recommended [17–20], and 3)
comparative studies using common antioxidants are essential to
clarify the biological significance of the activities of samples. We found
that 2-O-α-D-glucopyranosyl-L-ascorbic acid (AA-2G), a stable ascor-
bic acid derivative, per se exerted radical-scavenging activity toward
was monitored by measuring the absorbance at 524 nm with a
spectrophotometer (Hitachi U-1900, Tokyo, Japan).
2.3. Galvinoxyl radical-scavenging assay
The galvinoxyl radical-scavenging activities were assessed as
described previously [23]. Briefly, galvinoxyl radical (100 μM) was
mixed with an antioxidant (20 μM) in 60% ethanol/40% citric acid–
sodium citrate buffer (10 mM, pH 6). The reaction was carried out at
room temperature for 2 h. The decrease of galvinoxyl radical
concentration was monitored by measuring the absorbance at
+
unnatural model radicals, such as DPPH radical [21–24] and ABTS•
4
32 nm with a spectrophotometer.
[23,25]. The chemical properties of AA-2G as a radical scavenger were
greatly different from those of ascorbic acid, in that the reaction rate
with these model radicals of AA-2G was much slower than that of
ascorbic acid, but the long-term radical-scavenging ability per
molecule of AA-2G was superior to that of ascorbic acid. We also
found by using a cell-based antioxidant assay system, oxidative
hemolysis inhibition assay (OxHLIA) that the radical-scavenging
activity of AA-2G was biologically relevant [26]. A stereoisomer of
AA-2G, 2-O-β-D-glucopyranosyl-L-ascorbic acid, was also found to
have long-term radical-scavenging ability similar to that of AA-2G
+
2
.4. ABTS• -scavenging assay
+
The ABTS• -scavenging activities were assessed as described
previously [25]. Briefly, ABTS• (100 μM) generated with an ABTS/
H O /HRP system was mixed with an antioxidant (10 or 20 μM) in
2 2
citric acid–sodium citrate buffer (50 mM, pH 6) containing 2% EtOH.
The reaction was carried out at room temperature for 2 h. The
decrease of ABTS• concentration was monitored by measuring the
absorbance at 730 nm with a spectrophotometer. The 50% effective
concentration (EC50) value was also determined as the concentration
of vanillin required to give 50% of the absorbance shown by a blank
+
+
[27]. Recently, we reassessed the antioxidant activity of arbutin using
five in vitro assay systems, though arbutin has been reported to
possess weak antioxidant activity compared to that of its precursor,
hydroquinone [28]. We found that arbutin exerted strong antioxidant
activity comparable or even superior to that of hydroquinone.
However, we have felt that in vivo experiments are necessary for a
complete understanding of these antioxidant activities.
+
test. Vanillin (20, 30 and 40 μM) was reacted with ABTS• (100 μM) at
room temperature for 20 and 120 min.
Hence, in this study, we systematically evaluated the antioxidant
activity of vanillin using multiple assay systems. We first performed
assays using model radicals, DPPH radical, galvinoxyl radical and
+
2
.5. HPLC analysis of the reaction mixture of vanillin with ABTS•
Vanillin (50 μM) was reacted with ABTS•+ (250 μM) generated
O /HRP system in citric acid-sodium citrate buffer
2 2
50 mM, pH 6) containing 5% DMSO. An aliquot of the reaction
mixture was periodically withdrawn and directly subjected to HPLC
analysis. The HPLC analyses were carried out with a system consisting
of a Hitachi L-7100 pump, L-7420 UV–VIS detector, L-7300 column
oven, and D-2500 chromato-integrator. The separation of vanillin and
reaction products was achieved by isocratic elution from an Inertsil
ODS-3 column (4.6 i.d.×250 mm, 5 μm, GL Sciences Inc., Tokyo,
+
ABTS• . We then evaluated its antioxidant activity by ORAC assay and
with an ABTS/H
(
OxHLIA using physiologically relevant peroxyl radicals. Finally, we
confirmed the antioxidant activity of vanillin by oral administration of
vanillin to mice. Furthermore, a key intermediate for the antioxidant
+
activity of vanillin was identified in the reaction with ABTS• and
AAPH.
2
. Materials and methods
Japan) kept at 40 °C with MeOH/H
2
O/acetic acid (40:59:1, v/v) at a
flow rate of 0.7 ml/min. The absorbance at 275 nm was monitored.
2
.1. Chemicals
Vanillin was obtained from Nacalai Tesque (Kyoto, Japan).
2
.6. Purification and structural determination of the reaction product of
Ascorbic acid, sodium fluorescein, 2,2′-azobis(2-methylpropionami-
dine) dihydrochloride (AAPH) and heparin were purchased from
Wako Pure Chemical Industries (Osaka, Japan). Galvinoxyl free radical
and vanillic acid were from Tokyo Chemical Industry (Tokyo, Japan).
,1-Diphenyl-2-picrylhydrazyl (DPPH), trans-ferulic acid, H O (3%)
2 2
and 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Tro-
+
vanillin with ABTS•
Vanillin (1.0 g, 6.57 mmol) was dissolved in 20 ml of EtOH and
+
then mixed with 329 ml of 10 mM ABTS• (3.29 mmol). The solution
was diluted with 0.1 M citric acid-sodium citrate buffer (pH 6) to give
1
1
+
000 ml of a mixture of vanillin and ABTS• . The solution was reacted
lox) were from Aldrich Chemical (Milwaukee, WI). 2,2′-Azino-bis
for 30 min at 30 °C. The reaction mixture was twice extracted with
00 ml of ethyl acetate. The organic layer was dried over anhydrous
Na SO overnight to give a white precipitate. After dissolution of the
(
3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS),
horseradish peroxidase (HRP; type VI-A, essentially a salt-free
310 units/mg solid) and protocatechuic acid were from Sigma
Chemical (St. Louis, MO). Sheep erythrocytes were from Nippon
Bio-Supp. Center (Tokyo, Japan). Reagents were used without further
purification. All water used was Milli-Q grade.
5
2
4
1
precipitate by heating, the organic layer was filtered and concentrated
in vacuo to dryness. The residue was suspended with MeOH and then
filtered to give 6,6′-dihydroxy-5,5′-dimethoxy-(1,1′-biphenyl)-3,3′-
dicarboxaldehyde (86.0 mg) as an MeOH insoluble compound. NMR
spectra were recorded on a Varian INOVA-AS600 instrument. An
electron spray ionization mass spectrum was obtained on an AB SCIEX
API 4000 LC/MS/MS system using direct sample injection. 1H NMR
2
.2. DPPH radical-scavenging assay
The DPPH radical-scavenging activities were assessed as described
(600 MHz, DMSO-d
H-4, 4′), 7.43 (2H, d, J=1.8 Hz, H-2, 2′), 9.79 (2H, s, CHO). C NMR
(150 MHz, DMSO-d ) δ : 56.1 (OCH ), 109.0 (C-4, 4′), 125.2 (C-1, 1′),
6 Η 3
) δ : 3.90 (6H, s, OCH ), 7.39 (2H, d, J=1.8 Hz,
13
previously [23]. Briefly, DPPH radical (100 μM) was mixed with an
antioxidant (20 μM) in 60% ethanol/40% citric acid–sodium citrate
buffer (10 mM, pH 6). The reaction was carried out at room
temperature for 2 h. The decrease of DPPH radical concentration
6
C
3
127.4 (C-3, 3′), 128.5 (C-2, 2′), 148.7 (C-5, 5′), 152.0 br (C-6, 6′), 191.2
−
(CHO). ESI-MS (negative ion mode) m/z: 301 [M−H] .

172
A. Tai et al. / Biochimica et Biophysica Acta 1810 (2011) 170–177
2
.7. ORAC assay
Fig. 1. Ascorbic acid and Trolox showed nearly the same reaction
profiles in the three assays (Fig. 2), i.e., 20 μM of these antioxidants
rapidly quenched ca. 40 μM of DPPH radical, galvinoxyl radical and
ABTS• within 5 min. Thus, their reaction stoichiometries (number of
The ORAC assay was carried out as described previously [27].
Briefly, fluorescein (60 nM), antioxidant (3 μM), and AAPH
+
(
(
4
18.75 mM) were incubated in 200 μl of KH
75 mM, pH 7.4) at 37 °C in a 96-well plate. The fluorescence (Ex:
85 nm, Em: 520 nm) was monitored every 2 min for 70 min by
2
PO
4
–K
2
HPO
4
buffer
radical molecules reduced by one molecule of antioxidant) were
about 2 and this was consistent with our previous report [23]. Vanillin
scavenged little or no DPPH radical and galvinoxyl radical, while
vanillic acid and ferulic acid continuously quenched both radicals for
Varioskan Flash (Thermo Fisher Scientific, Waltham, MA).
+
To determine the reaction product of vanillin with AAPH, vanillin
the whole experimental period (Fig. 2A, B). In the ABTS• assay,
(
(
50 μM) was reacted with AAPH (40 mM) in phosphate-buffered saline
PBS: 150 mM NaCl, 8.1 mM Na HPO , and 1.9 mM NaH PO , pH 7.4)
vanillin as well as vanillic acid and ferulic acid showed significant
2
4
2
4
radical-scavenging activity (Fig. 2C). Interestingly, the reaction
+
containing5% DMSO. An aliquot of the reaction mixture was periodically
withdrawn and directly subjected to HPLC analysis. The HPLC analyses
were carried out under the above-described conditions.
between vanillin and ABTS• slowly and continuously proceeded for
120 min. The 50% effective concentration (EC50) of vanillin against
+
ABTS• was also investigated at 20 min and 120 min. The EC50 of
vanillin markedly decreased from 40.6 μM at 20 min to 19.4 μM at
120 min, because vanillin slowly and continuously reacted with ABTS•+
over a period of 120 min. The result showed that 19.4 μM of vanillin
2
.8. Oxidative hemolysis inhibition assay (OxHLIA)
+
The OxHLIA was carried out as described in previous papers
quenched 50 μM of ABTS• for up to 120 min. Thus, the reaction
+
[27,29]. Briefly, sheep erythrocytes suspended at a concentration of
stoichiometry of vanillin against ABTS• was 2.6 at 120 min. Vanillin
0
.7% (v/v) in PBS were incubated with 40 mM of AAPH in the presence
showed no activity in the DPPH radical- and galvinoxyl radical-
scavenging assays but showed stronger activity than ascorbic acid and
Trolox in the ABTS• -scavenging assay.
of an antioxidant (3.1, 6.3, 12.5, 25 or 50 μM) at 37 °C with shaking.
The degree of hemolysis (%) was monitored every 15 min for 165 min
from the concentration of hemoglobin in the centrifuged supernatant
by measuring the absorbance at 524 nm.
+
+
3.2. Reaction products of vanillin with ABTS•
2
.9. Contents of vanillin and its metabolites and antioxidant capacity in
Vanillin slowly and continuously reacted with ABTS•+ over a
mouse plasma
period of 120 min (Fig. 2C). It is noteworthy that 1 mol of vanillin
scavenged 2.6 mol of ABTS• in 120 min of reaction, although one
+
Male ICR mice, 6 weeks old, were purchased from CLEA Japan
Tokyo, Japan). Mice that had been fed ad libitum on a stock diet of CE-
(CLEA Japan) and water were fasted for 16 h prior to use. All mice
molecule of vanillin has only one of the oxidizable phenolic hydroxyl
groups at the C-4 position (Fig. 1). To investigate characteristics of the
reaction, the reaction mixtures of vanillin with ABTS• were analyzed
(
2
+
were used at 7 weeks of age. They were orally given 100 mg/kg of
vanillin (25 mg/ml) dissolved in 20% EtOH/PBS. At the indicated
times, blood samples were collected by heart puncture in a heparin-
coated syringe under sodium pentobarbital anesthesia. Each blood
sample was centrifuged at 10,000×g for 10 min at 4 °C, and the
resulting plasma was subjected to HPLC analysis and plasma ORAC
assay. For HPLC analysis, to 250 μl of the resulting plasma was added
by HPLC. The HPLC profiles of the reaction mixture between vanillin
(50 μM) and ABTS• (250 μM) in citrate buffer (pH 6) containing 5%
+
DMSO at 1, 5, 15, and 120 min of reaction are shown in Fig. 3. At 1 min,
peaks of reaction products 1 and 2, besides vanillin, were found. The
peaks of vanillin and 2 decreased with time, whereas the peak of 1
slowly increased. These results suggest that vanillin scavenged ABTS•+
to generate reaction product 2 at an early stage of the reaction.
Subsequently, the reaction product 2 reacted with additional ABTS•+ to
give the reaction product 1.
Clarifying the reaction products seemed to show the reason why
the radical-scavenging activity of vanillin was higher than that of
ascorbic acid and Trolox at 120 min of reaction. We tried to isolate
2
50 μl of ethyl acetate. After vortexing, the resulting mixture was
centrifuged at 10,000×g for 5 min at 4 °C, and 150 μl of the ethyl
acetate phase was removed and concentrated to dryness with a
stream of air. The resulting residue was dissolved with a HPLC solvent
and subjected to HPLC analysis for estimating the concentrations of
vanillin, vanillic acid and protocatechuic acid. For plasma ORAC assay,
7
0 μl of the resulting plasma was deproteinized by adding 140 μl of
O
OH
acetone/H O/acetic acid (70:29.5:0.5, v/v). The mixture was centri-
2
fuged at 10,000×g for 10 min at 4 °C, and the resulting supernatant
was diluted with 75 mM phosphate buffer (pH 7.4) to give a final
concentration of 50 times dilution for the ORAC assay. The HPLC
analysis and the ORAC assay were carried out under the above-
described conditions.
O
H
O
OH
OCH3
OCH3
OCH3
3
. Results
OH
OH
OH
+
3
.1. DPPH radical-, galvinoxyl radical- and ABTS• -scavenging activities
Vanillin
Vanillic acid
Ferulic acid
DPPH radical, galvinoxyl radical and ABTS•+ are relatively stable
radicals. Their characteristic colors disappear when they are
quenched, and so the decrease of these radicals can be easily
monitored by a spectrometer [30,31]. We assessed the DPPH
HO
HO
HO
O
O
HO
O
+
radical-, galvinoxyl radical- and ABTS• -scavenging activities of
OH
vanillin in buffered solutions at pH 6 and compared them with
those of vanillic acid, ferulic acid, ascorbic acid and Trolox. Vanillic
acid and ferulic acid were used as known antioxidants with similar
structures. Ascorbic acid and Trolox were used as standard anti-
oxidants. The chemical structures of these compounds are shown in
O
OH
L-Ascorbic acid
Trolox
Fig. 1. Chemical structures of vanillin, vanillic acid, ferulic acid, ascorbic acid and Trolox.

A. Tai et al. / Biochimica et Biophysica Acta 1810 (2011) 170–177
173
A
100
80
60
40
20
0
0
0
0
15
15
15
30
30
30
60
120
Time (min)
B
1
00
8
6
4
2
0
0
0
0
0
Fig. 3. HPLC chromatograms of the products of reaction of vanillin with ABTS•+. Vanillin
(50 μM) and ABTS•+ (250 μM) were incubated in 5% DMSO/citrate buffer (50 mM, pH 6).
At 1 (A), 5 (B), 15 (C), and 120 min (D), aliquots of the reaction mixture were withdrawn
and analyzed by HPLC.
60
120
Time (min)
C
organic layer was filtered and concentrated in vacuo to dryness. The
residue was suspended with MeOH and then filtered to give an MeOH
insoluble compound (86.0 mg) as the reaction product 2. The reaction
product 1 dissolved in MeOH was further purified by a silica gel
column but was not isolated. The reaction product 2 was fully
characterized and identified to be 6,6′-dihydroxy-5,5′-dimethoxy-
1
00
80
60
40
20
0
(
1,1′-biphenyl)-3,3′-dicarboxaldehyde (divanillin) by the mass spec-
1
13
trum and several NMR spectra ( H, C, HSQC, and HMBC) (Fig. 4).
3.3. ORAC assay
Vanillin showed stronger activity than did ascorbic acid and Trolox
+
in the ABTS• -scavenging assay but showed no activity in the DPPH
radical- and galvinoxyl radical-scavenging assays. This discrepancy
led us to assess the antioxidant efficacy of vanillin in more
physiologically relevant assay systems, because DPPH radical, galvi-
60
120
Time (min)
+
Fig. 2. Time courses of DPPH radical (A)-, galvinoxyl radical (B)- and ABTS•+ (C)-scavenging
noxyl radical and ABTS• are unnatural radical species that do not
reactions of vanillin, vanillic acid, ferulic acid, ascorbic acid and Trolox. Vanillin ( ), vanillic
acid (○), ferulic acid ( ), ascorbic acid (□), Trolox ( ) (each 20 μM) or control (△) and
DPPH radical or galvinoxyl radical (100 μM) were incubated at room temperature in 60%
exist in the human body. The ORAC assay utilizes an AAPH-derived
ethanol/40% citrate buffer (10 mM, pH 6). Vanillin (20 μM), vanillic acid (20 μM), ferulic acid
OHC
HO
OCH3
+
(10 μM), ascorbic acid (20 μM), Trolox (20 μM) or control and ABTS• (100 μM) were
3
5
2
6
6' 5'
incubated at room temperature in 5% DMSO/citrate buffer (50 mM, pH 6). Changes in the
remaining radicals were measured at the indicated times. Each value is the mean± SD of
three separate experiments. The absence of an SD bar means that the SD bar is within the
symbol.
1
4
4'
1
'
2' 3'
H CO
OH
CHO
reaction products 1 and 2 from reaction mixture of vanillin
3
(
(
6.57 mmol) and ABTS•+ (3.29 mmol) in 2% ethanol/citrate buffer
6
3
,6'-dihydroxy-5,5'-dimethoxy-(1,1'-biphenyl)-
,3'-dicarboxaldehyde (divanillin)
pH 6). The reaction mixture was extracted with ethyl acetate. The
organic layer was dried over anhydrous Na
2 4
SO overnight to give a
white precipitate. After dissolution of the precipitate by heating, the
Fig. 4. Chemical structure of one of the products of the reaction of vanillin with ABTS•+
.

174
A. Tai et al. / Biochimica et Biophysica Acta 1810 (2011) 170–177
peroxyl radical, which mimics lipid peroxyl radicals involved in lipid
peroxidation chain reaction in vivo. Inhibition of peroxyl radical-
induced oxidations of a fluorescent probe, fluorescein, by antioxidants
is serially monitored [30]. Unexpectedly, vanillin had the strongest
antioxidant effect in the ORAC assay. The order of inhibition was
vanillinNvanillic acid≥ferulic acid≫TroloxNascorbic acid (Fig. 5).
To investigate the characteristics of the reaction for the unexpect-
edly strong effect of vanillin, reaction mixtures of vanillin with AAPH-
derived radicals were analyzed by HPLC. The HPLC profiles of the
reaction mixture of vanillin (50 μM) and AAPH (40 mM) in PBS
containing 5% DMSO at 10, 30, and 60 min of reaction are shown in
Fig. 6. At 5 min, a small amount of divanillin was observed (data not
shown). The peak of vanillin decreased with time, whereas the peak of
divanillin gradually increased over a period of 30 min. At 60 min, the
peaks of vanillin and divanillin had almost disappeared. A peak of 1 as
shown in Fig. 3 was not observed in this reaction. These results
suggested that the formation of divanillin resulted in the unexpect-
edly strong effect of vanillin.
3
.4. Oxidative hemolysis inhibition assay (OxHLIA)
OxHLIA is a cell-based antioxidant assay using the same radical
source as that used for the ORAC assay [26,29]. Oxidation of
erythrocyte membranes by an AAPH-derived peroxyl radical induces
oxidation of lipids and proteins and eventually causes hemolysis, and
this hemolysis was inhibited by each antioxidant (Fig. 7A). The order
of inhibition was ferulic acid (25 μM)Nvanillin (25 μM)≥vanillic acid
(
25 μM)≈Trolox (50 μM)Nascorbic acid (50 μM), which was a little
different from that observed in the ORAC assay (Fig. 5).
Concentration-dependency of vanillin for hemolysis assays is
shown in Fig. 7B. The results are expressed as delayed time of
hemolysis (ΔT), which was calculated by the following equation:
Fig. 6. HPLC chromatograms of the products of reaction of vanillin with AAPH. Vanillin
(50 μM) and AAPH (40 mM) were incubated in 5% DMSO/PBS. At 10 (A), 30 (B), and
60 min (C), aliquots of the reaction mixture were withdrawn and analyzed by HPLC.
ΔT = HT50ðsampleÞ−HT50ðcontrolÞ;
3.5. Contents of vanillin and its metabolites and antioxidant capacity in
mouse plasma
where HT50 is the 50% hemolysis time. The average HT50 (control) was
In the some in vitro experiments, vanillin showed potent
antioxidant activity compared with the activities of ascorbic acid
and Trolox used as positive controls and compared with the activities
of known antioxidants, vanillic acid and ferulic acid. To investigate the
absorption and the antioxidant capacity of vanillin, mice were orally
administrated a single dose (100 mg/kg) of vanillin. After 5, 15, 30 and
9
3± 4 min (n=3). The ΔT value of vanillin at 25 μM was 2.6-times
greater than that at 12.5 μM. The ΔT value of vanillin at 12.5 μM was
3
3
.2-times greater than that at 6.3 μM. The ΔT value of vanillin at
.1 μM was scarcely observed. The inhibitory effects of vanillin on
hemolysis were concentration-dependent.
6
0 min, plasma samples were prepared and subjected to HPLC
analysis and plasma ORAC assay. Fig. 8A shows time-course plots of
the plasma concentrations of vanillin and its metabolites after oral
administration of vanillin. Vanillic acid and protocatechic acid were
detected as metabolites of vanillin. The concentration of vanillin
sharply increased up to 5 min and then rapidly decreased until
1.00
0.80
0.60
0.40
0.20
0
1
5 min. The level of protocatechic acid reached its maximum at 5 min
and then rapidly decreased until 15 min. On the other hand, the level
of vanillic acid reached its maximum at 5 min and then gradually
decreased with time. The maximal values of vanillin, vanillic acid and
protocatechic acid at 5 min were 7.1± 2.1, 0.75± 0.09 and 1.4±
0
.4 μg/ml, respectively. Fig. 8B shows ORAC activities in the plasma at
various times after oral administration of vanillin. The plasma sample
at each time showed high ORAC activity, and the order of activity was
5
minN15 minN30 minN60 minN0 min. The highest activity in the
0
10
20
30
40
50
60
70
plasma ORAC assay was observed at 5 min, when the concentrations
of vanillin, vanillic acid and protocatechic acid were high.
Time (min)
Fig. 5. ORAC assay for vanillin, vanillic acid, ferulic acid, ascorbic acid and Trolox.
Reaction mixtures containing vanillin ( ), vanillic acid (○), ferulic acid ( ), ascorbic
acid (□), Trolox ( ) (each 3.0 μM) or control (△), fluorescein (60 nM) and AAPH
4. Discussion
(
7
18.75 mM) in 200 μl of phosphate buffer (75 mM, pH 7.4) were incubated at 37 °C for
0 min. Changes in fluorescence intensity of fluorescein were monitored. Each value is
As mentioned in the Introduction, vanillin has antimutagenic
properties. Recently, it has also been reported that vanillin suppressed
the metastatic potential of human cancer cells through PI3K inhibition
the mean± SD of triplicate experiments. The absence of an SD bar means that the SD
bar is within the symbol.

A. Tai et al. / Biochimica et Biophysica Acta 1810 (2011) 170–177
175
A100
A
1
0
8
6
4
2
0
80
60
40
20
0
0
60
75
90
105
120
135
150
165
Time (min)
0
5
15
30
60
B 50
Time (min)
B
4
3
2
1
0
0
0
0
0
1
0
0
0
.00
.80
.60
.40
3
.1
6.3
12.5
25
0.20
0
Vanillin (μ M)
0
10
20
30
40
50
60
70
Fig. 7. OxHLIA for vanillin, vanillic acid, ferulic acid, ascorbic acid and Trolox. (A) Sheep
erythrocytes at 0.7% (v/v) suspension in PBS were incubated with 40 mM of AAPH in
the absence (△) and in the presence of vanillin (25 μM, ), vanillic acid (25 μM, ○),
ferulic acid (25 μM, ), ascorbic acid (50 μM, □) or Trolox (50 μM, ) at 37 °C for
Time (min)
Fig. 8. Contents of vanillin and its metabolites and total antioxidant capacity in plasma
after an oral administration of vanillin to mice. Male ICR mice were orally administrated
a single dose of vanillin (100 mg/kg). At the indicated times, plasma samples were
collected and subjected to HPLC analysis and plasma ORAC assay. (A) Time-course
profiles of plasma vanillin ( ), vanillic acid (○) and protocatechuic acid ( ) contents
were investigated by HPLC analysis. Each value is the mean± SEM of five separate
experiments. (B) In the absence (△) and presence of plasma at 0 min ( ), 5 min ( ),
15 min (○), 30 min ( ), and 60 min (□), an ORAC assay was carried out. Changes in
fluorescence intensity of fluorescein were monitored. Each value is the mean± SEM of
five separate experiments. The absence of an SEM bar means that the SEM bar is within
the symbol.
165 min with shaking. Each value is the mean± SD of five separate experiments. The
absence of an SD bar means that the SD bar is within the symbol. (B) Sheep erythrocytes
at 0.7% (v/v) suspension in PBS were incubated with 40 mM of AAPH with the indicated
concentration of vanillin, vanillic acid, ferulic acid, ascorbic acid or Trolox at 37 °C for
1
65 min with shaking. Results are expressed as delayed times of hemolysis (ΔT)
defined in the text. Each value is the mean+SD of three separate experiments.
and inhibited angiogenesis in a chick chorioallantoic membrane assay
[32], that vanillin improved and prevented trinitrobenzene sulfonic
acid-induced colitis in mice [33], that vanillin covalently bound with
sickle hemoglobin and inhibited cell sickling [34], and that vanillin
exhibited analgesic effects in a mouse [35] or a rat [36] model.
Therefore, vanillin with multifunctionality seems to be a beneficial
compound for human health. Based on the results of the present
study, we propose the addition of antioxidant capacity to the multi-
functionality of vanillin.
formation contributed to the total radical-scavenging ability of
protocatechuic esters [39]. Hence, we assumed that vanillin also
reacts with radicals via adduct formation or self-dimerization.
To predict the radical-scavenging mechanism of vanillin, we tried to
isolate reaction products 1 and 2 from reaction mixture of vanillin and
+
ABTS• (Fig. 3). The reaction product 2 was purified and identified to be
divanillin. Divanillin is a natural product found in a commercial vanilla
extract and in cured vanilla beans [40]. The reaction product 1 could not
be purified. The reaction product 1 showed a very broad peak compared
to that of divanillin by HPLC analysis (Fig. 3) and several spots besides
divanillin were observed in the reaction mixture by TLC analysis (data
not shown). These data suggested that the reaction product 1 was
composed of several compounds. The peaks of vanillin (from 33.0±
3.6 μM at 1 min to 5.9± 1.0 μM at 120 min) and divanillin (from 4.0±
0.1 μM at 1 min to 1.6± 0.1 μM at 120 min) decreased with time,
whereas the peak of 1 slowly increased. The reaction stoichiometry of
Vanillin showed stronger activity than did ascorbic acid and Trolox
+
in the ABTS• -scavenging assay but showed no activity in the DPPH
radical- and galvinoxyl radical-scavenging assays (Fig. 2). Vanillin
+
slowly and continuously reacted with ABTS• over a period of
1
20 min (Fig. 2C). From the EC50 value, it is estimated that 1 mol of
+
vanillin scavenged 2.6 mol of ABTS• in 120 min of reaction. It is
thought that the ABTS• -scavenging reaction will occur over a more
+
extended time period, since a small amount of vanillin still remained
in the reaction mixture after 120 min (Fig. 3D). However, one
molecule of vanillin has only one oxidizable phenolic hydroxyl
group. There are several findings that are helpful in predicting the
radical-scavenging mechanism of vanillin. It has been reported that a
+
divanillin against ABTS• was 3.0 at 120 min (data not shown). These
+
results suggested that vanillin scavenged ABTS• to generate divanillin
at an early stage of the reaction and that divanillin subsequently reacted
+
2
-pyrone compound [37,38] or AA-2G [24] with one oxidizable –OH
with additional ABTS• to give several reaction products. If the
+
group scavenged more than one equivalent of DPPH radical via an
formation of divanillin is the main reaction route for the ABTS• -
adduct formation with the radical and that an oxidative dimer
scavenging reaction of vanillin, 2 mol of vanillin will scavenge 2 mol of

176
A. Tai et al. / Biochimica et Biophysica Acta 1810 (2011) 170–177
ABTS•+ to generate 1 mol of divanillin, and 1 mol of divanillin will
further react with 3 mol of ABTS• . Thus, the reaction stoichiometry of
the total antioxidative capacity of plasma appears to be due to the
total activity of a variety of compounds, including ascorbic acid,
tocopherol, polyphenols, and possibly other endogenous components.
In this experiment, antioxidative enzymes were not considered as
endogenous components because of the use of deproteinized plasma
samples. The slight discrepancy between the concentrations of
vanillin and its metabolites in plasma and the antioxidant activity in
plasma seems to result from the reproduction of antioxidants and/or
the induction of low molecular endogenous antioxidants by admin-
istered vanillin. Other metabolites that were not detectable under the
analytical conditions also seem to contribute to the antioxidant
activity. These results suggested that vanillin intake directly and
indirectly raised the antioxidant activity in plasma.
+
vanillin is expected to be 2.5. This value agrees fairly well with the actual
reaction stoichiometry of 2.6. Therefore, these results indicated that an
oxidative self-dimerization contributed to the total radical-scavenging
ability of vanillin.
Vanillin showed much stronger antioxidant activity than did
ascorbic acid and Trolox in the ORAC assay (Fig. 5) and OxHLIA
(Fig. 7). The order of inhibition in the ORAC assay was vanillinNvanillic
acid≥ferulic acid≫TroloxNascorbic acid, and the order of inhibition in
the OxHLIA was ferulic acid (25 μM)Nvanillin (25 μM)≥vanillic acid
(
25 μM)≈Trolox (50 μM)Nascorbic acid (50 μM). The antioxidant
activity of ferulic acid was stronger than that of vanillin and vanillic
acid in the OxHLIA but was weaker in the ORAC assay. The ORAC assay
and OxHLIA use the same radical source, AAPH-derived peroxyl radicals,
and results of these assays are therefore correlated with each other to
some extent. Vanillin, vanillic acid and ferulic acid possess the same
chemical structures for radical-scavenging reactions. However, the
orders of activities of ferulic acid and of vanillin and vanillic acid were
opposite between the assays. The ORAC assay and OxHLIA utilize the
same “hydrophilic” peroxyl radicals but different oxidizable targets, i.e.,
a “hydrophilic” fluorescein or “lipophilic” biomembrane of erythrocytes.
The microlocalization of each antioxidant in OxHLIA may reflect the
result that relatively lipophilic ferulic acid was superior to relatively
hydrophilic vanillin and vanillic acid in protection against free radical-
induced membrane damage.
Vanillin is widely used in foods, beverages, cosmetics, and drugs.
Vanillin has been reported to exhibit multifunctional effects such
as antimutagenic [2–8], antiangiogenetic [32], anti-colitis [33], anti-
sickling [34], and antianalgesic effects [35,36]. However, results of
studies on the antioxidant activity of vanillin are not consistent.
Concentrations of vanillin used in food and beverage products range
widely from 0.3 to 33 mM [1]. The high level of vanillin intake from
foods and beverages appears to exert some effects on human health.
In this study, we systematically evaluated the antioxidant activity of
vanillin using multiple assay systems. Vanillin showed stronger
+
activity than did ascorbic acid and Trolox in the ABTS• -scavenging
assay but showed no activity in the DPPH radical- and galvinoxyl
radical-scavenging assays. Vanillin showed much stronger antioxi-
dant activity than did ascorbic acid and Trolox in the ORAC assay and
Divanillin was found in the reaction mixtures of vanillin with
AAPH-derived radicals (Fig. 6), suggesting that the strong activity of
vanillin in the ORAC assay and OxHLIA was expressed by a mechanism
+
OxHLIA. In the ABTS• -scavenging assay, ORAC assay and OxHLIA,
vanillin reacted with radicals via a self-dimerization mechanism. The
+
similar to that in the ABTS• -scavenging assay. At 37 °C, 40 mM of
dimerization contributed to the high reaction stoichiometry against
+
AAPH produces ROO• at a constant rate of 3.3 μM/min [41], and the
total amount of AAPH-derived radical formed over a period of 30 min
is about 100 μM. In our previous study, 50 μM of ascorbic acid was
consumed over a period of 30 min. Hence, the reaction ratio of
ascorbic acid against ROO• was 1:2, and this agreed well with the
present results showing that ascorbic acid reacts with DPPH radical,
ABTS• and AAPH-derived radicals to result in the strong effect of
vanillin. Oral administration of vanillin to mice increased the vanillin
concentration and the antioxidant activity in plasma. Therefore,
antioxidant activity of vanillin might be more beneficial than has been
thought for daily health care.
+
galvinoxyl radical and ABTS• at a ratio of 1:2. Vanillin (50 μM) was
Acknowledgements
consumed over a period of 60 min in the presence of 40 mM AAPH
(
Fig. 6C). The total amount of AAPH-derived radical formed over a
The authors thank Misses M. Inoue and Y. Hirai for their technical
period of 60 min is about 200 μM. Thus, 1 mol of vanillin was expected
to scavenge about 4 mol of ROO•. The peak of vanillin decreased with
time, whereas the peak of divanillin gradually increased over a period
of 30 min and had almost disappeared at 60 min. These results
indicated that the formation of divanillin contributed to the high
reaction stoichiometry against AAPH-derived radicals to result in the
unexpectedly strong effect of vanillin.
To investigate the absorption and the antioxidant capacity of
vanillin, mice were orally administrated a single dose (100 mg/kg) of
vanillin. After oral administration, vanillin and its metabolites, vanillic
acid and protocatechic acid, were detected in the plasma. The
maximal values of vanillin, vanillic acid and protocatechic acid were
assistance in the in vivo experiments.
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