Antioxidant Activity of Caffeic Acid and Dihydrocaffeic Acid in Lard and Human Low-Density Lipoprotein

Article abstract of DOI:10.1021/jf9805799

Caffeic acid (CA) has been implied as an important source of natural antioxidants in various agricultural products. We compared the antioxidative activity of CA and dihydrocaffeic acid (HCA) in lard and the low-density lipoprotein (LDL) system to know the role of the 2,3-double bond on the appearance of its antioxidative property. HCA was more effective than CA in enhancing oxidative stability of lard at 60 °C. Inhibition by HCA of copper ion-induced oxidation of human LDL was less effective than that by CA, whereas there was no significant difference between the two compounds in the capacity of 1,1-diphenyl-2-picrylhydrazyl radical scavenging and the activity of lipid peroxyl radical scavenging in solution. It can be concluded that the 2,3-double bond in the structure of CA affects the efficiency of antioxidative activity depending on the environment where the oxidation happens, although it is rarely responsible for its inherent activity.

Full text of DOI:10.1021/jf9805799

5062
J. Agric. Food Chem. 1998, 46, 5062−5065
An tioxid a n t Activity of Ca ffeic Acid a n d Dih yd r oca ffeic Acid in La r d
†
a n d Hu m a n Low -Den sity Lip op r otein
J ae-Hak Moon and J unji Terao*
Department of Nutrition, School of Medicine, The University of Tokushima, Kuramoto-cho 3,
Tokushima 770-0042, J apan
Caffeic acid (CA) has been implied as an important source of natural antioxidants in various
agricultural products. We compared the antioxidative activity of CA and dihydrocaffeic acid (HCA)
in lard and the low-density lipoprotein (LDL) system to know the role of the 2,3-double bond on the
appearance of its antioxidative property. HCA was more effective than CA in enhancing oxidative
stability of lard at 60 °C. Inhibition by HCA of copper ion-induced oxidation of human LDL was
less effective than that by CA, whereas there was no significant difference between the two
compounds in the capacity of 1,1-diphenyl-2-picrylhydrazyl radical scavenging and the activity of
lipid peroxyl radical scavenging in solution. It can be concluded that the 2,3-double bond in the
structure of CA affects the efficiency of antioxidative activity depending on the environment where
the oxidation happens, although it is rarely responsible for its inherent activity.
Keyw or d s: Antioxidants; caffeic acid; dihydrocaffeic acid; free radical-scavenging activity; low-
density lipoprotein
INTRODUCTION
Lipid peroxidation induces oxidative deterioration of
lipid-containing foods. Effective and nontoxic natural
antioxidants are therefore required to prevent this event
during storage and processing. In addition, the meta-
bolic fate of antioxidants should be known because of
not only their safety but also their physiological function
after absorption into the body.
Caffeic acid [3-(3,4-dihydroxyphenyl)-2-propenoic acid,
F igu r e 1. Structures of caffeic acid (CA) and dihydrocaffeic
acid (HCA).
CA], which is a hydroxycinnamic acid derivative (Figure
Thus, this study was undertaken to compare the
1
), is one of the antioxidative compounds in various
activity of HCA with that of CA in edible oil and human
low-density lipoprotein (LDL). We discuss the effective-
ness of HCA and CA as antioxidative additives in edible
oil and potential antioxidants for preventing oxidative
damage in blood circulation.
agricultural products such as coffee beans, potatoes,
grains, and vegetables (Friedman, 1997; Gutfinger,
1
981; Hudson et al., 1980; Kimura et al., 1985; Ky et
al., 1997). CA and some of its related compounds are
absorbed into the body after oral administration, and
specific metabolites were detected in urine and plasma
MATERIALS AND METHODS
(
1
Finkle et al., 1962; Goldstein et al., 1984; Herrman,
956; Horning et al., 1967; J acobson et al., 1983; Nardini
et al., 1995, 1997). In addition, dihydrocaffeic acid
3-(3,4-dihydroxyphenyl)-2-propionic acid, HCA] (Figure
) is formed by intestinal bacteria as a degradation
product of CA (Peppercorn et al., 1971) and eriocitrin
Miyake et al., 1997) which was isolated from lemon
fruit (Citrus limon BURM. f.) as a flavonoid glycoside
eriodictyol 7-glycoside). It should be also noted that
Ch em ica ls. CA was obtained from Nacalai Tesque Inc.
Kyoto, J apan) and purified by HPLC (254 nm, 3.0 mL/min)
(
[
1
using ODS column (YMC-Pack, 6 × 150 mm, 5 µm; YMC,
Kyoto, J apan; water/methanol/acetic acid ) 75:25:1, v/v/v). The
purified CA showed a single peak in the HPLC chromatogram.
HCA was synthesized chemically from CA. Briefly, CA in a
solution of methanol reacted with palladium carbon under
hydrogen gas bubbling. After filtration of palladium carbon
(
(
(
0.45 µm, GL chromatodisk; GL Science, Tokyo, J apan), HCA
HCA was detected as a catechol metabolite of CA from
human plasma (Goldstein et al., 1984). CA may there-
fore act as an in vivo antioxidant after absorption into
the body. However, little is known on the antioxidative
activity of HCA. Furthermore, the role of the 2,3-double
bond in CA on the antioxidant activity is not known.
was separated from the reaction solution using HPLC (col-
umn: YMC-Pack, 6 × 150 mm, 5 µm; YMC, Kyoto, J apan;
water/methanol/acetic acid ) 72:28:1, v/v/v). The HCA fraction
obtained was detected as a single peak in the HPLC chro-
matogram. HCA fraction was identified by GC-MS analysis
after trimethylsilylation using N,O-bis(trimethylsilyl)aceta-
mide (Tokyo Kasei Co., Ltd., Tokyo, J apan). A QP-5000 mass
spectrometer (Shimadzu, Kyoto, J apan) equipped with a fused
silica DB-1 capillary column (0.25-mm i.d. × 30 m, 0.25-mm
film thickness; G & W Scientific, CA) was used with in electron
impact mode (70 eV). The carrier gas was helium at a flow
rate of 1.0 mL/min, and the injection port temperature was
250 °C. The column oven temperature was held at 50 °C for
†
This work was done at the National Food Research
Institute, Tsukuba, J apan, where J .-H.M. worked as a Science
and Technology Agency fellow.
*
Corresponding author (tel, +81-886-31-3111, ext.
511; fax, +81-886-33-7089; e-mail, terao@nutr.med.
tokushima-u.ac.jp).
2
1
0.1021/jf9805799 CCC: $15.00 © 1998 American Chemical Society
Published on Web 11/12/1998

Caffeic and Dihydrocaffeic Acids as Antioxidants
min, before being elevated to 240 °C at 10 °C/min and then
J. Agric. Food Chem., Vol. 46, No. 12, 1998 5063
6
kept constant for 5 min. The compound was identified as 3,4-
bis(trimethylsilyloxy)hydroxycinnamic acid trimethylsilyl ester
+
by the mass spectrum: m/z (rel int) 73 (SiMe
3
, 100%), 179
+
+
+
(C
9
H
7
O
4
, 89%), 219 [(SiMe
3
)
3
, 5%], 267 [(OSiMe
, 8%), 398 (M , 50%). d-R-Tocopherol (R-Toc)
3
)
3
, 38%],
+
+
3
83 (M - CH
3
was kindly supplied by Eisai Co. (Tokyo, J apan). 2,2′-Azobis-
2,4-dimethylvaleronitrile) (AMVN), 1,1-diphenyl-2-picrylhy-
(
drazyl (DPPH), and cupric sulfate were obtained from Kanto
Chemical Co. Inc. (Tokyo, J apan). Methyl linoleate was
purchased from Tokyo Kasei Co. Ltd. (Tokyo, J apan). Methyl
linoleate was further purified by column chromatography
using Florisil (Wako Pure Chemical Co., Osaka, J apan) to
remove contaminant hydroperoxides and impurities (Terao et
al., 1977). Lard without any additives was a kind gift from
Tsukishima Food Industrial Co. Ltd. (Tokyo, J apan). HPLC
analysis in this study was carried out under room temperature.
All other chemicals and solvents were of analytical grade.
F igu r e 2. Effect of CA, HCA, and R-Toc on autoxidation of
lard at 60 °C. Antioxidants (0.4 µmol) were mixed with lard
(1.0 g): (O) no addition; (f) caffeic acid; (4) dihydrocaffeic acid;
(0) R-tocopherol. The data are representative of two experi-
ments.
Mea su r em en t of Oxid a tive Sta bility of La r d . The
procedures for the measurement of oxidative stability of lard
were followed by the method of Koga et al. (1994). Lard (1.0
g) was measured accurately and spread out in a glass dish
(5.0-cm diameter). CA, HCA, or R-Toc (0.4 µmol) dissolved in
a solution of methanol/chloroform (2:8, v/v, 3.0 mL) was added
to the lard and then evaporated under nitrogen and in vacuo.
The mixture was allowed to autoxidize at 60 °C in the dark
with circulating air. At regular intervals, the weight gain was
recorded.
Mea su r em en t of Cop p er Ion -In d u ced LDL Oxid a tion .
LDL particles were isolated from heparinized plasma of
healthy volunteers by differential density-gradient ultracen-
trifugation according to the method described previously (Silva
et al., 1997). Isolated LDL preparation was used immediately
for the experiment or was stored at 4 °C under nitrogen
atmosphere until use for a maximum of 1 week. After the
solution was incubated for 5 min at room temperature, CA or
HCA (each final concentration of 12 µM) dissolved in methanol
was added to the reaction solution such that the final
concentration of methanol in LDL suspension was 0.5% (v/v).
The oxidation reaction was initiated by the addition of cupric
sulfate (final concentration of 5 µM) dissolved in PBS buffer
F igu r e 3. Effect of CA and HCA on copper ion-induced
oxidation of human LDL. LDL (0.4 mg of protein/mL) was
incubated at 37 °C in the absence or presence of CA or HCA.
At the indicated times, aliquots were withdrawn and the levels
of cholesteryl ester hydroperoxides (CE-OOH) were determined
as described in Materials and Methods. Final concentrations
of cupric sulfate and CA/HCA were 5 and 12 µM, respectively,
and initial concentration of R-tocopherol was 12.5 nmol/mg of
protein: (O) no addition; (f) caffeic acid; (4) dihydrocaffeic
acid. The data are representative of two experiments.
(pH 7.4). Cholesteryl ester hydroperoxides (CE-OOH) were
measured by HPLC as an index of oxidation. The procedures
of CE-OOH determination were the same as those described
previously (Silva et al., 1997).
RESULTS
Sca ven gin g of 1,1-Dip h en yl-2-p icr ylh yd r a zyl (DP P H)
Ra d ica l. Free radical-scavenging activity of each antioxidant
was assayed using a stable free radical, DPPH, according to
the method of Blois (1958). The reaction mixture contained
Effect of CA, HCA, a n d r-Toc on th e Oxid a tive
Sta bility of La r d . Lard with or without additives
showed a rapid weight gain after an induction period
when stored at 60 °C (Figure 2). The end of the
induction period was easily recognized by a sharp
weight gain. The induction periods of lard with and
without antioxidants were 5, 11, 28, and 38 days for
control (without additive), R-Toc, CA, and HCA, respec-
tively. Therefore, CA and HCA were more effective than
R-Toc on the improvement of oxidative stability. Inter-
estingly, HCA showed a longer induction period than
CA.
E ffect of CA a n d HCA on t h e Cop p er Ion -
In d u ced LDL Oxid a tion . LDL isolated from human
plasma was oxidized with copper ion at 37 °C under air.
Antioxidant activity was determined by the inhibition
of CE-OOH formation with or without CA and HCA
1
.0 mL of 0.5 mM DPPH and 0.1 mL of an ethanol solution of
the antioxidant at different concentrations (5-350 µM) except
for cysteine (dissolved with 100 mM Tris-HCl buffer, pH 7.4).
Finally, the total volume of the reaction mixture was adjusted
to 2.0 mL with 100 mM Tris-HCl buffer (pH 7.4). After the
reaction was carried out at room temperature for 20 min in
the dark, the free radical-scavenging activity of each antioxi-
dant was quantified by decolorization at 517 nm.
Mea su r em en t of P er oxyl Ra d ica l-Sca ven gin g Activity
in Solu tion . The peroxyl radical-scavenging activities of CA
and HCA were determined by measuring the inhibition of an
AMVN-induced peroxidation of methyl linoleate in a solution
(Terao et al., 1993). The reaction mixture contained 100 mM
methyl linoleate, 80 µM antioxidant, and 10 mM AMVN in a
solution (1.0 mL) of n-hexane/2-propanol/ethanol (8:3:1, v/v/
v) and was incubated in the dark at 37 °C. At specific
intervals, an aliquot of the reaction mixture (10.0 µL) was
withdrawn and injected onto the HPLC column. The HPLC
conditions employed and the calculation of kinetic parameters
of peroxyl radical-scavenging activity for antioxidants have
been described previously (Burton et al., 1981; Nagao et al.,
(
Figure 3). Control (no external addition of antioxidant)
showed some induction period at the initial stage of the
oxidation reaction. It could be derived from the internal
antioxidants present in LDL such as R-Toc (12.5 nmol/
mg of protein LDL). Although the addition of HCA gave
a significant increase in the induction period, it was
shorter than that obtained by CA.
1
990; Terao, 1989; Yamamoto et al., 1982).

5064 J. Agric. Food Chem., Vol. 46, No. 12, 1998
Moon and Terao
-
7
3
reaction during induction periods (Rinh, 0.85 × 10
-
1
Ms ) and the term of induction period (tinh, 14.3 × 10
s) for HCA were not significantly different from those
-
7
-1
3
of CA (Rinh, 1.13 × 10
Ms ; tinh, 18.0 × 10 s).
Consequently, it is concluded that CA and HCA scav-
enge lipid peroxyl radical by a similar mechanism, and
their activities in solution are within the same magni-
tude.
DISCUSSION
Considerable studies have demonstrated that the
o-dihydroxyl structure is essential for free radical-
scavenging and metal-chelating effects in hydroxycin-
namic acid derivatives (Chen et al., 1997; Laranjinha
et al., 1994; Nardini et al., 1995; Terao et al., 1993).
However, the role of the conjugated double bond at the
F igu r e 4. DPPH radical-scavenging effect of antioxidants.
The reaction mixture contained 0.5 mM DPPH (1.0 mL) and
an ethanol solution (0.1 mL) of the antioxidant at different
concentrations (5-350 µM) except for cysteine (dissolved with
1
00 mM Tris-HCl buffer, pH 7.4). Absorbance of each reaction
2
,3-position on its antioxidant activity has not been
mixture was monitored at 517 nm as described in Materials
and Methods after the total volume of the reaction mixture
was finally adjusted to 2.0 mL with 100 mM Tris-HCl buffer
verified as far as we know. Our attention was focused
on the contribution of the 2,3-double bond on its anti-
oxidant activity. We therefore compared the antioxi-
dant activity of CA with that of its derivative containing
no 2,3-double bond, HCA, in lard, and we also examined
their antioxidant capacities on copper ion-induced oxi-
dative modification of human LDL.
(
pH 7.4): (f) caffeic acid; (4) dihydrocaffeic acid; (0) R-toco-
pherol; (b) cysteine. The data are representative of two
experiments.
It is unlikely that the 2,3-double bond of CA is the
radical-targeting site because no difference was ob-
served between their DPPH radical-scavenging capaci-
ties (Figure 4). Furthermore, the inhibitory effects of
CA and HCA on radical chain oxidation of methyl
linoleate in solution were indistinguishable (Figure 5);
that is, the olefinic double bond in CA hardly affects its
inherent chain propagating peroxyl radical-scavenging
ability in solution. Nevertheless, antioxidant activity
in the autoxidation of lard increased in the order of
R-Toc < CA < HCA (Figure 2). This result suggests that
the olefinic double bond in CA is an important factor
for enhancing antioxidant activity in bulk oil autoxida-
tion.
Nardini et al. (1997) demonstrated that CA ac-
cumulated at ca. 1.0 µg/mL concentration in nonfasting
rats (plasma) fed a 0.8% CA-containing diet. In addi-
tion, HCA was found in human plasma as a metabolite
of CA after dietary supplementation and was suggested
to be formed by the intestinal bacteria (Goldstein et al.,
1984). Therefore, both CA and HCA seem to ac-
cumulate in human plasma by the intake of CA-rich
food. Our copper ion-induced LDL oxidation study
(Figure 4) demonstrated that HCA has considerable
antioxidant capacity on oxidative modification of LDL.
Several works (Abu-Amsha et al., 1996; Meyer et al.,
1998; Nardini et al., 1995) have shown that among
hydroxycinnamic acids, CA exerts an effective inhibition
of copper ion-induced LDL in vitro. In addition, this
study first demonstrated that HCA is also a possible
candidate for the antioxidant in blood circulation. o-
Dihydroxyl structure of HCA seems to be responsible
for the radical-scavenging activity as well as the copper
ion-chelating activity, similarly to CA (Nardini et al.,
1995). It is very likely that CA acts as an in vivo
antioxidant after absorption as well as a food antioxi-
dant for the prevention of degradation of food quality.
In addition, HCA, a possible metabolite of CA containing
the o-dihydroxyl structure, may also play a role in the
antioxidant capacity of dietary CA.
F igu r e 5. Effects of CA and HCA on AMVN-initiated oxida-
tion of methyl linoleate in solution. The reaction system
consisted of methyl linoleate (100 mM), AMVN (10 mM), and
antioxidants (80 µM) in a mixture of n-hexane/2-propanol/
ethanol (8:3:1, v/v/v): (O) caffeic acid; (2) hydrocaffeic acid;
(b) no addition. The data are representative of two experi-
ments.
Sca ven gin g E ffect of CA a n d H CA on DP P H
Ra d ica l. Figure 4 shows the relationship between the
concentration of DPPH trapped (indicated by the de-
crease of 517 nm) and the concentration of antioxidants
in the DPPH radical-scavenging assay. The number of
DPPH radicals trapped by antioxidants was calculated
from the assumption that one molecule of cysteine
scavenges one molecule of DPPH radical. The numbers
of CA, HCA, and R-Toc were calculated to be 5.0, 5.0,
and 2.5, respectively. Thus, both CA and HCA can
scavenge five DPPH radicals regardless of the structual
difference in the 2,3-double bond. In addition, the
DPPH radicals to be scavenged by CA and HCA were 2
times more than that by R-Toc.
P er oxyl Radical-Scaven gin g Activity of CA, HCA,
a n d r-Toc in Solu tion . We determined the peroxyl
radical-scavenging activities of CA, HCA, and R-Toc by
measuring the inhibition of free radical oxidation of
methyl linoleate in solution (Figure 5). The induction
period was obtained from the accumulation rate of ML-
OOH when CA, HCA, or R-Toc was added to the reaction
mixture. The values of chain propagation reaction rate
In conclusion, both CA and HCA can be effective
antioxidants for lard and the human plasma LDL
oxidation system. The conjugated olefinic double bond
-
7
-1
for CA (3.36 × 10 Ms ) was quite similar to that of
-
7
-1
HCA (3.82 × 10 Ms ), although the rate of oxidation

Caffeic and Dihydrocaffeic Acids as Antioxidants
J. Agric. Food Chem., Vol. 46, No. 12, 1998 5065
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Received for review J une 1, 1998. Revised manuscript received
September 29, 1998. Accepted October 2, 1998.
J F9805799