Antioxidative properties of phenolic antioxidants isolated from corn steep liquor

Article abstract of DOI:10.1021/jf0007885

With the immersion of corn into dilute sulfur oxide during starch-manufacturing processes, corn steep liquor (CSL) remains as leftover material. CSL is often used for fermentation, but its components are not fully understood. To determine the properties of CSL, 12 p-coumaric acid-related compounds were isolated from an ethyl acetate extract of CSL with the guidance of antioxidative activity on the rabbit erythrocyte membrane ghost system. The activity of these compounds was compared against oxidative damages, and it was elucidated that the activity of p-coumaric acid derivatives was mainly affected by their functional groups at the 3-position and less, by the conjugated side chain. Moreover, p-coumaric acid derivatives exhibited inhibitory activity stronger than that of tocopherols and ascorbic acid on peroxynitrite-mediated lipoprotein nitration. These findings that p-coumaric acid derivatives, which might play a beneficial role against oxidative damage, exist in CSL suggest this byproduct might be a useful resource of phenolic antioxidants.

Full text of DOI:10.1021/jf0007885

J. Agric. Food Chem. 2001, 49, 177−182
177
An tioxid a tive P r op er ties of P h en olic An tioxid a n ts Isola ted fr om
Cor n Steep Liqu or
Toshio Niwa,*,† Umeyuki Doi,† Yoji Kato,‡ and Toshihiko Osawa§
Department of Research and Development, San-ei Sucrochemical Company, Ltd., Chita 478-8503, J apan;
School of Humanities for Environmental Policy and Technology, Himeji Institute of Technology,
Himeji 670-0092, J apan; and Laboratory of Food and Biodynamics, Nagoya University Graduate School of
Bioagricultural Sciences, Nagoya 464-8601, J apan
With the immersion of corn into dilute sulfur oxide during starch-manufacturing processes, corn
steep liquor (CSL) remains as leftover material. CSL is often used for fermentation, but its
components are not fully understood. To determine the properties of CSL, 12 p-coumaric acid-related
compounds were isolated from an ethyl acetate extract of CSL with the guidance of antioxidative
activity on the rabbit erythrocyte membrane ghost system. The activity of these compounds was
compared against oxidative damages, and it was elucidated that the activity of p-coumaric acid
derivatives was mainly affected by their functional groups at the 3-position and less by the conjugated
side chain. Moreover, p-coumaric acid derivatives exhibited inhibitory activity stronger than that
of tocopherols and ascorbic acid on peroxynitrite-mediated lipoprotein nitration. These findings that
p-coumaric acid derivatives, which might play a beneficial role against oxidative damage, exist in
CSL suggest this byproduct might be a useful resource of phenolic antioxidants.
Keyw or d s: Phenolic antioxidants; corn steep liquor; low-density lipoprotein; peroxynitrite; xanthine
oxidase
INTRODUCTION
(Huie and Padmaja, 1993; Goldstein and Czapski, 1995),
holds interest as a strong plausible oxidant in vivo, and
its marker, 3-nitrotyrosine, has been detected in several
lesions of some (model) diseases (Beckmann et al., 1994;
Kaur and Halliwell, 1994; Matthews and Beal, 1996;
Leeuwenburgh et al., 1997). Thus, we have also evalu-
ated the activity of isolated compounds in preventing
the protein nitration resulting from this powerful
oxidant.
In this paper, the expression of “trans” with respect
to p-coumaric acid derivatives was omitted. We have
also noted the hydrogenated compounds such as 3-(4-
hydroxyphenyl)propionic acid as p-coumaric acid/H2, for
the structure-activity relationships.
The steeping process is the first step in starch
manufacture. It is very effective for softening the corn
kernel and facilitating the separation of germ, hull,
fiber, and gluten from starch granules. Corn steep liquor
(CSL), which is often used as a nitrogen source of
fermentation, is produced through this process. How-
ever, the chemical identification of components in the
exudate has scarcely been mentioned in previous re-
search. In recent studies, much attention has been
focused on oxidative stress from the viewpoint of its
participation in several diseases such as atherosclerosis
(Palinski et al., 1989), cancer (Ames et al., 1993), and
aging (Carney et al., 1991). Some edible plants contain
certain amounts of antioxidants, and they are expected
to prevent these oxidative stresses (Lin et al., 1996; J ang
et al., 1997; Ohta et al., 1997). Also, new antioxidants
have been isolated from a wide variety of fields (Lo et
al., 1994; Murakami et al., 1996; Abe et al., 1998). We
searched for antioxidants from CSL with the guidance
of the inhibitory activity against the oxidation of rabbit
ghost membrane by tert-butyl hydroperoxide. Finally,
we isolated 12 phenolic compounds, of which 11 anti-
oxidants were p-coumaric acid derivatives, and we then
evaluated some structure-activity relationships of the
functional groups at the 3-position and side chains of
p-coumaric acid derivatives.
MATERIALS AND METHODS
Ch em ica ls. p-Coumaric acid, caffeic acid, and vanillic acid
were purchased from Nacalai Tesque Inc., Ltd. (Kyoto, J apan).
L-Ascorbic acid, cinnamic acid, and butylated hydroxyanisole
(BHA) were purchased from Wako Pure Chemical Industries
Ltd. (Osaka, J apan). Ferulic acid was obtained from Tokyo
Kasei Organic Chemicals Co., Ltd. (Tokyo, J apan). Sinapinic
acid and allopurinol were products of Aldrich Chemical Co.,
Inc. (Milwaukee, WI). dl-R-Tocopherol, d-γ-tocopherol, dl-
Trolox, hypoxanthine, and xanthine oxidase (X-4376) were
obtained from Sigma Chemical Co. (St. Louis, MO). â-Carotene
and tert-butyl hydroperoxide were purchased from Merck
(Darmstadt, Germany). The highest quality agents available
were obtained in all cases.
Peroxynitrite was synthesized (Hughes and Nicklin, 1970)
and quantified prior to use as described previously (Hughes
and Nicklin, 1968). Briefly, an acidic solution (10 mL, 0.6 M
HCl) of H2O2 (0.7 M) was mixed vigorously with NaNO2 (10
mL, 0.6 M) in an ice-cold bath, and the reaction was im-
mediately quenched with ice-cold NaOH (20 mL, 1.5 M). The
solution was then frozen for several hours, and the yellowish
top layer was collected.
Peroxynitrite (ONOO-), generated from nitric oxide
and superoxide anion radical at near-diffusion rate
* Corresponding author [telephone (+81)(562)55-5907; fax
(+81)(562)55-5819; e-mail toshio-niwa@sanei-toka.co.jp].
† San-ei Sucrochemical Co., Ltd.
‡ Himeji Institute of Technology.
§ Nagoya University Graduate School of Bioagricultural
Sciences.
10.1021/jf0007885 CCC: $20.00 © 2001 American Chemical Society
Published on Web 11/30/2000

178 J. Agric. Food Chem., Vol. 49, No. 1, 2001
Niwa et al.
In str u m en ts. Preparative high-performance liquid chro-
matography (HPLC) was done with a Wakosil-II 5C18HG
column (i.d. 20 × 250 mm; Wako Pure Chemical) with a flow
rate of 5.0 mL/min at ambient temperature. 1H NMR spectrum
was obtained on a J NM GSX-270 (J EOL, Tokyo, J apan). All
chemical shifts are reported as δ values (parts per million)
relative to the incomplete signal of the deuterated acetone
(2.05 ppm). EI-MS spectrum was measured on a J MS DX-705L
(J EOL).
was carried out on Merck precoated glass plates (silica gel
PF254; 1.0 mm) eluted with a solvent mixture of n-hexane/
EtOAc (1:1).
Sinapinic Acid/H2 Methyl Ester (11): 1H NMR (270 MHz,
acetone-d6) δ 6.99 (1H, br s), 6.53 (2H, s), 3.80 (6H, s), 3.61
(3H, s), 2.81 (2H, t, J ) 7.8 Hz), 2.58 (2H, t, J ) 7.8 Hz).
Isomerization. Isomerization of p-coumaric acid and ferulic
acid was done with daylight irradiation. In brief, the sample
solution dissolved in MeOH in a round-bottom flask was placed
outside to get daylight for 3 h and then evaporated in vacuo.
A novel peak emerged after a trans-compound on reverse-
phase column chromatography and a corresponding fraction
were isolated by preparative HPLC eluted with a solvent
mixture of H2O/MeOH/TFA (650:350:1).
An tioxid a n t P u r ifica tion fr om CSL. CSL (500 g), ob-
tained from our plant (Aichi, J apan), was diluted with 500 mL
of saturated brine and extracted with 500 mL of ethyl acetate
(EtOAc) by centrifugation (3500 rpm, 15 min) in a Teflon
vessel, three times. The EtOAc solution was re-extracted with
saturated NaHCO3 solution (300 mL × 3), and then the
alkaline aqueous phase was acidified by aqueous HCl and
extracted with EtOAc (500 mL × 3). The organic phase was
washed, dehydrated over anhydrous Na2SO4, and evaporated
in vacuo. This acid fraction (1.9 g) was subjected to a silica
gel column chromatography eluted with n-hexane/EtOAc (1:
2) to get four fractions (A1-A4). Further purification of A2
was accomplished with preparative HPLC equipped with an
UV detector at wavelength 254 nm using a solvent mixture of
H2O/MeOH/trifluoroacetic acid (TFA) (700:300:1) and gave
seven fractions (A21-A27). A23 and A25 were further resolved
by preparative HPLC eluted with H2O/acetonitrile/TFA (800:
200:1) at a wavelength of 210 nm. Using this method, we
obtained caffeic acid (4; 4.3 mg) and vanillic acid (1; 14.6 mg)
from A23 and 3-(4-hydroxyphenyl)propionic acid (p-coumaric
acid/H2) (8; 6.1 mg) and 3-(4-hydroxy-3-methoxyphenyl)-
propionic acid (ferulic acid/H2) (9; 1.4 mg) from A25 as active
compounds. Purification of A27 on preparative HPLC with a
solvent mixture of H2O/MeOH/TFA (500:500:1) gave eight
fractions (A271-A278). A271, A272, and A273 were subjected
to reverse-phase chromatography eluted with H2O/MeOH/TFA
(600:400:1) to get p-coumaric acid (5; 4.1 mg), ferulic acid (6;
11.6 mg), cis-p-coumaric acid (2; 0.6 mg), and cis-ferulic acid
(3; 3.3 mg). A3 was purified by preparative HPLC monitoring
at UV 210 nm, and we obtained 3-(3,5-dimethoxy-4-hydroxy-
phenyl)propionic acid (sinapinic acid/H2) (10; 2.0 mg), sinap-
inic acid (7; 6.5 mg), 3-(3,5-dimethoxy-4-hydroxyphenyl)-
propionic acid methyl ester (sinapinic acid/H2 methyl ester)
(11; 1.4 mg), and 3-(3,5-dimethoxy-4-hydroxyphenyl)propionic
acid ethyl ester (sinapinic acid/H2 ethyl ester) (12; 0.9 mg).
The structures of active compounds were deduced from their
spectral data as well as from synthesis as described below,
and 12 compounds were determined.
1
cis-p-Coumaric Acid (6): H NMR (270 MHz, acetone-d6) δ
8.80 (1H, br s), 7.78 (2H, d, J ) 8.6 Hz), 6.86 (1H, d, J ) 12.7
Hz), 6.82 (2H, d, J ) 8.9 Hz), 5.81 (1H, d, J ) 13.0 Hz)
An tioxid a tive Activity Mea su r em en t. Rabbit Erythro-
cyte Membrane Ghost System. Antioxidative activity was
measured by the thiobarbituric acid reactive substances
(TBARS) method on rabbit erythrocyte membrane ghost
treated with tert-butyl hydroperoxide (Osawa et al., 1987) with
slight modification. In brief, rabbit erythrocyte membrane (0.5
mL) prepared from commercially available rabbit blood, by
washing with isotonic buffer solution and lysing in 10 mM
phosphate buffer (pH 7.4), was treated with 25 mM tert-butyl
hydroperoxide (50 µL) with or without samples dissolved in
MeOH (2.0 mM, 100 µL). These reaction mixtures were
incubated for 20 min at 37 °C, and TBARS of each sample were
determined using UV absorption at 532 nm on a spectropho-
tometer (U-2000, Hitachi, Tokyo, J apan).
Detection of 3-Nitrotyrosine with Enzyme-Linked Imunosor-
bent Assay (ELISA). Fresh low-density lipoprotein (LDL) was
obtained from human volunteers (with informed consent) by
density gradient ultracentrifugation (Sparrow et al., 1989). The
detection of 3-nitrotyrosine in protein treated with ONOO- was
accomplished as previously described (Kato et al., 1997). In
the case of LDL, protein was dissolved in phosphate buffer at
1.0 mg/mL (final amount), and the following procedure was
accomplished in the same manner. In brief, 90 µL of protein
solution was placed in a 1.5-mL microtube, and 10 µL of
sample solution (3.0 mM) dissolved in dimethyl sulfoxide
(DMSO) was added, and the mixture was treated with ONOO-
(final concentration ) 1.0 mM) with vigorous vortex mixing.
This modified protein was treated with antiserum against
3-nitrotyrosine and peroxidase-labeled anti-rabbit IgG goat
antibody, successively. Then, o-phenylenediamine was added
as a substrate to peroxidase, and the amount of 3-nitrotyrosine
was evaluated by UV absorption at 492 nm using a multiplate
reader (Spectra Max 250, Molecular Devices Corp., Sunnyvale,
CA).
Mod ifica tion of p-Cou m a r ic Acid Der iva tives. Catalytic
Hydrogenation. Catalytic hydrogenation of p-coumaric acid
derivatives was proceeded with a catalytic amount of 5% Pd/C
(Wako Pure Chemical) under H2 atmosphere for 3 h. When
HPLC analysis of these reactants was done using a Wakosil-
II 5C18HG column (i.d. 4.6 × 250 mm) on a Shimadzu CLASS-
LC10 series HPLC system equipped with a photodiode array
detector (SPD-M10Avp, Shimadzu), there was no absorption
longer than 300 nm derived from a conjugated double bond.
For p-coumaric acid, in practice, a reaction mixture that
dissolved 100 mg of starting material in MeOH (20 mL) was
vigorously mixed with 5% Pd/C (10 mg) in H2 atmosphere.
After 3 h, the reaction mixture was filtered and concentrated
to dryness under reduced pressure. Then, a purification with
preparative HPLC eluted with a solvent mixture of H2O/
MeOH/TFA (650:350:1) monitoring at 254 nm gave p-coumaric
acid/H2.
Inhibitory Activity against Xanthine Oxidase. Ferulic acid
methyl ester was prepared in a manner similar to that used
with sinapinic acid/H2 methyl ester (11) and purified with
preparative HPLC. Enzymatic reaction was generated using
a method of Watanabe et al. (1997) with slight modification.
Briefly, 20 µL of samples dissolved with DMSO, 140 µL of
phosphate-buffered saline (PBS, pH 7.4), and 20 µL of hypox-
anthine (3.0 mM) dissolved in PBS with a minimum amount
of 1.5 M NaOH was placed on a 96-hole plate. The reaction
was started with the addition of 20 µL of xanthine oxidase
(150 milliunits) and proceeded with gentle shaking at ambient
temperature. After 0.5 h, reaction was terminated with the
addition of 20 µL of HCl solution (1.0 M). The reaction
mixtures were diluted 5-fold with distilled water, and 10 µL
of each sample was analyzed with HPLC using a Wakosil-II
5C18HG column (i.d. 4.6 × 250 mm). An aqueous 50 mM
ammonium dihydrogenphosphate was used as the mobile
phase with a flow rate of 1.0 mL/min at 40 °C, and the
remaining hypoxanthine was evaluated by the peak area
eluted at 6.7 min with 254 nm. Fifteen minutes of washing
with 60% MeOH and 20 min of conditioning followed each 10-
min analysis.
1
p-Coumaric Acid/H2 (8): H NMR (270 MHz, acetone-d6) δ
8.12 (1H, br s), 7.08 (2H, d, J ) 7.3 Hz), 6.75 (2H, d, J ) 7.3
Hz), 2.81 (2H, t, J ) 7.6 Hz), 2.55 (2H, t, J ) 7.6 Hz).
Esterification. Esterification of sinapinic acid/H2 (10; 100
mg) was achieved by using a catalytic amount of sulfuric acid
(150 µL) in corresponding alcohol (20 mL) that was refluxed
for 4 h. The reaction mixture was cooled, and saturated
NaHCO3 was then added for neutralization. Alcohol was
evaporated in vacuo, and the remaining residue was parti-
tioned with EtOAc and saturated NaHCO3. The organic layer
was washed and dried in a conventional manner. Purification

Phenolic Antioxidants Isolated from Corn Steep Liquor
J. Agric. Food Chem., Vol. 49, No. 1, 2001 179
F igu r e 1. Scheme for preparation of antioxidants from CSL.
RESULTS AND DISCUSSION
To facilitate the fractionation of CSL, we first exam-
ined the extractions with some organic solvents (n-
hexane, EtOAc, or dichloromethane). Among these
solvents used, the EtOAc extract showed good results
in terms of yield and antioxidative activity (data not
shown). CSL components were distributed between
EtOAc and saturated brine, and then the organic layer
was further separated into acid and neutral fractions.
Although both fractions had antioxidative activity, we
used the acid fraction for isolation because of its
stronger activity as compared to that of the neutral
fraction at the same weight concentration. Repeated
silica gel and reverse-phase column chromatography of
the EtOAc-soluble acid fraction, using an antioxidative
activity as a guide, led to the 12 antioxidants (Figure
1). Each isolated compound was analyzed by spectro-
scopic methods. By comparing the spectrum and the
HPLC traces with those of authentic samples, com-
pounds 1 and 4-7 were identified as vanillic acid, caffeic
acid, p-coumaric acid, ferulic acid, and sinapinic acid,
respectively. On the other hand, the coupling constant
values of olefinic protons of compounds 2 and 3 were
smaller than those of the trans compounds, although
other aromatic signals were similar to those of p-
coumaric acid and ferulic acid. We concluded that 2 and
3 were cis isomers of p-coumaric acid and ferulic acid,
because each compound had emerged from the corre-
sponding trans form by isomerization. Compounds 8-10
had no absorption longer than 300 nm in a photodiode
array-equipped HPLC analysis, and they had two meth-
ylene signals on 1H NMR spectra. By catalytic hydro-
genation of commercially available chemicals, as de-
scribed under Materials and Methods, we determined
that 8-10 were reduced forms of p-coumaric acid, ferulic
acid, and sinapinic acid, respectively. Compounds 11
F igu r e 2. Structures of antioxidants isolated from CSL.
et al., 1996). Among CSL-derived antioxidants, sinapinic
acid derivatives (7 and 10-12) and caffeic acid (4)
strongly inhibited the hydroperoxide-mediated lipid
peroxidation, whereas p-coumaric acid and ferulic acid
derived compounds (2, 3, 5, 6, 8, and 9) showed <50%
inhibition. However, the activities of CSL-derived an-
tioxidants were no more effective than those of R-toco-
pherol and BHA (Figure 3). This study suggested some
structure-activity relationships of p-coumaric acid de-
rivatives: (1) The methoxyl group(s) adjacent to the
4-hydroxyl group enhance(s) the inhibitory activity, and
(2) the adjacent hydroxyl group (catechol) also enhances
the activity more effectively. On the side chain that
conjugated to the aromatic ring, (3) the loss of the double
bond slightly reduced the activity in p-coumaric acid,
ferulic acid, and sinapinic acid; however, (4) we could
not observe any alteration as a result of the isomeriza-
1
and 12 had H NMR spectra very similar to that of 10
except for the methoxyl or ethoxyl signals, and they
were easily prepared from 10 by acid-catalyzed esteri-
fication. Finally, 11 p-coumaric acid derivatives and
vanillic acid were determined (Figure 2).
p-Coumaric acid derivatives are components of plant
cell wall and contribute to plant growth (Kamisaka et
al., 1990). Recent studies have examined these com-
pounds as antioxidants that might be used to prevent
oxidative stresses (Laranjinha et al., 1994; Castelluccio

180 J. Agric. Food Chem., Vol. 49, No. 1, 2001
Niwa et al.
F igu r e 3. Antioxidative activity of the antioxidants isolated
from CSL. Inhibitory activity was obtained with a sample
concentration at 2.0 mM by comparing the TBARS to results
of MeOH alone with or without tert-butyl hydroperoxide. Data
represent the mean ( SD of three independent measurements.
F igu r e 4. Effect of some antioxidants on protein nitration
by peroxynitrite. Protein solutions that contained 0.3 mM (final
concentration) of each sample were treated with 1.0 mM
peroxynitrite, and the nitration was evaluated by ELISA as
described under Materials and Methods. Control means the
peroxynitrite-exposed LDL in the presence of 10% DMSO
without antioxidants. Data represent the mean ( SD of three
separate color developments.
tion of the side chain in p-coumaric acid or ferulic acid.
In this matter, Belguendouz et al. (1997) also observed
little change in the antioxidative activity of resveratrol;
instead, they described a lowered chelating activity.
However, the antioxidative activity of p-coumaric acid
derivatives against hydroperoxide-induced lipid peroxi-
dation was mainly affected by substituent(s) on the
phenolic ring but not the conjugated side chain.
1984; Lo and Chung, 1999). Xanthine oxidase produces
superoxide anion radical, a constitute of ONOO- with
nitric oxide, and some studies tied it to vascular
dysfunction (Tan et al., 1993; Hamer et al., 1995; White
et al., 1996). We aimed to evaluate the isolated com-
pounds of this enzymatic reaction because some p-
coumaric acid derivatives were described as inhibitors
(Chan et al., 1995). According to our evaluation of the
inhibitory activity on xanthine oxidase with the remain-
ing hypoxanthine, cinnamic acid and n-valeric acid
inhibited the enzymatic reaction as well as p-coumaric
acid and ferulic acid at a range of 1.25-10 mM final
concentrations (data not shown). The inhibitory activity
of simple organic acids, such as n-valeric acid, suggested
a carboxylic acid-mediated inhibition of the enzymatic
reaction. To avoid an acidic effect derived from free
carboxylic acid moiety, we prepared ferulic acid methyl
ester and still observed 47.4 ( 6.4% inhibition at 10.0
mM (final concentration), whereas a typical inhibitor,
allopurinol, inhibited completely even at 1.25 mM. Chan
et al. (1995) previously described the inhibitory activity
of cinnamic acid, but the carboxylic acid-derived effect
was not mentioned. Recently, Gao et al. (1999) described
the superoxide scavenging activity of caffeic acid sugar
ester, but they also did not mention any effect on
xanthine oxidase. Although further study is required,
the inhibitory activity of ferulic acid methyl ester on
xanthine oxidase suggested the activity of other p-
coumaric acid derivatives and that the previously
observed “superoxide” scavenging activity of these com-
pounds was derived, at least in part, from the inhibition
of the enzymatic reaction itself.
LDL accumulation in macrophage and the subsequent
formation of foam cell seem to contribute to atheroscle-
rosis (Palinski et al., 1989). However, native LDL does
not make foam cell, and certain oxidative modification
seems to be involved, even though the precise mecha-
nism is unknown. ONOO- is a plausible oxidant in vivo,
and this oxidative modification enables LDL to ac-
cumulate in macrophage via the scavenger receptor
(Graham et al., 1993). Thus, we examined the inhibitory
activity of CSL-derived antioxidants against ONOO--
mediated nitrotyrosine formation on LDL evaluated by
ELISA, as described under Materials and Methods. The
LDL used in our study had a barely detectable amount
of nitrotyrosine residue. In our examination in vitro,
LDL was nitrated by ONOO- but not “decomposed
ONOO-”, indicating that the nitration was not due to
the contaminated impurities of ONOO- synthesis. Phe-
nolic antioxidants obtained from CSL, as shown Figure
2, were potent inhibitors of tyrosine nitration of LDL,
whereas typical antioxidants such as BHA, tocopherols,
ascorbic acid, and â-carotene had weak protection
against nitrotyrosine formation at this concentration
(Figure 4); some previous reports suggested that these
endogenous antioxidants play a role in protection from
ONOO--mediated damage (Hogg et al., 1993; Yermilov,
V. et al., 1995; Whiteman and Halliwell, 1996). Inter-
estingly, p-coumaric acid derivatives produced different
types of reaction products when treated with ONOO-
(Kato et al., 1997; Kerry and Rice-Evans, 1998; Pannala
et al., 1998, Niwa et al., 1999). Cinnamic acid, which
had no phenolic hydroxyl group of p-coumaric acid,
scarcely exhibited the inhibitory effect, and there was
no implication of carboxylic acid-derived effect in our
results. Nor did DMSO, used as a dissolving agent, have
a remarkable effect, at least under our conditions.
Some p-coumaric acid derivatives exert inhibitory
activities on enzymatic reactions such as 5-lipoxygenase
or arylamine N-acetyltransferase (Koshihara et al.,
Corn is cultivated almost all over the world and used
as a feed ingredient and in the production of food and
industrial products (Mehta and Dias, 1999). The isola-
tion of an antifungal benzoxazolinone compound from
this common cereal was reported a few decades ago
(Beck et al., 1957), and we found the same compound
in CSL (data not shown). However, the elucidation of
corn-derived chemical components is still in progress
(Kuga et al., 1993; Buttery and Ling, 1997). In this
study, we revealed 12 phenolic components in CSL that

Phenolic Antioxidants Isolated from Corn Steep Liquor
J. Agric. Food Chem., Vol. 49, No. 1, 2001 181
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ABBREVIATIONS USED
CSL, corn steep liquor; HPLC, high-performance
liquid chromatography; NMR, nuclear magnetic reso-
nance; EI-MS, electron impact mass spectrometry; UV,
ultraviolet; TFA, trifluoroacetic acid; TBARS, thiobar-
bituric acid reactive substances; ELISA, enzyme-linked
immunosorbent assay; LDL, low-density lipoprotein;
ONOO-, peroxynitrite; DMSO, dimethyl sulfoxide; PBS,
phosphate-buffered saline.
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Received for review J une 27, 2000. Revised manuscript
received October 16, 2000. Accepted October 16, 2000.
J F0007885