Quercetin-4'-glucoside is more potent than quercetin-3-glucoside in protection of rat intestinal mucosa homogenates against iron ion-induced lipid peroxidation.

Article abstract of DOI:10.1021/jf035151a

Quercetin is a typical antioxidative flavonoid found in vegetables, which is more commonly present as its glucosides, quercetin-3-glucoside (Q3G) and quercetin-4'-glucoside (Q4'G). The main aim of this study was to estimate the antioxidant activity of Q3G and Q4'G on iron ion-driven lipid peroxidation of the gastrointestinal mucosa. Q4'G markedly suppressed the lipid peroxidation when rat gastrointestinal mucosa homogenates were incubated with Fe(NO3)3 and ascorbic acid. Its effectiveness was greater as compared to that of Q3G and comparable to that of quercetin aglycone. Furthermore, Q4'G yielded higher amounts of quercetin aglycone than Q3G on incubation with the homogenates. However, Q4'G showed a lower chelating activity in comparison to Q3G. These results indicate that Q4'G, even though it has a low chelating activity, because of its efficient conversion to antioxidative aglycone on exposure to the mucosa, can act as a powerful antioxidant on iron ion driven lipid peroxidation in the intestinal mucosa. Thus, vegetables rich in Q4'G, such as onion, are likely to serve as favorable antioxidant sources for suppressing iron-induced oxidative stress in the intestinal tract.

Full text of DOI:10.1021/jf035151a

J. Agric. Food Chem. 2004, 52, 1907−1912 1907
Quercetin-4′-glucoside Is More Potent than
Quercetin-3-glucoside in Protection of Rat Intestinal Mucosa
Homogenates against Iron Ion-Induced Lipid Peroxidation
KAEKO MUROTA,† YUKI MITSUKUNI,† MAMI ICHIKAWA,† TOJIRO TSUSHIDA,‡
SAYURI MIYAMOTO,†,§ AND JUNJI TERAO*,†
Department of Nutrition, School of Medicine, University of Tokushima, Kuramoto-cho 3,
Tokushima 770-8503, Japan, and National Food Research Institute, Kannondai 2-1-2,
Tsukuba 305-8642, Japan
Quercetin is a typical antioxidative flavonoid found in vegetables, which is more commonly present
as its glucosides, quercetin-3-glucoside (Q3G) and quercetin-4′-glucoside (Q4′G). The main aim of
this study was to estimate the antioxidant activity of Q3G and Q4′G on iron ion-driven lipid peroxidation
of the gastrointestinal mucosa. Q4′G markedly suppressed the lipid peroxidation when rat gas-
trointestinal mucosa homogenates were incubated with Fe(NO3)3 and ascorbic acid. Its effectiveness
was greater as compared to that of Q3G and comparable to that of quercetin aglycone. Furthermore,
Q4′G yielded higher amounts of quercetin aglycone than Q3G on incubation with the homogenates.
However, Q4′G showed a lower chelating activity in comparison to Q3G. These results indicate that
Q4′G, even though it has a low chelating activity, because of its efficient conversion to antioxidative
aglycone on exposure to the mucosa, can act as a powerful antioxidant on iron ion driven lipid
peroxidation in the intestinal mucosa. Thus, vegetables rich in Q4′G, such as onion, are likely to
serve as favorable antioxidant sources for suppressing iron-induced oxidative stress in the intestinal
tract.
KEYWORDS: Quercetin glucoside; intestinal mucosa; lipid peroxidation; chelating activity; rat
INTRODUCTION
they interact with other nutrients and nonnutrients present in
the intestinal lumen. Thus, quercetin not only has a beneficial
effect when absorbed into the intestine but also in its unabsorbed
form in the gastrointestinal lumen (7).
Quercetin, 3,3′,4′,5,7-pentahydroxyflavone, is a typical fla-
vonoid widely distributed in vegetables. In recent years, potent
antioxidant activity of quercetin and other flavonoids has
attracted much attention due to the beneficial effect of vegetables
on health (1, 2). Quercetin is well-known as a powerful free
radical scavenger and as a chelating agent that inactivates metal
iron responsible for the generation of reactive oxygen species.
However, dietary quercetin is mostly present as its glycoside,
an “inactive” form as an antioxidant in which one or more sugar
groups are bound to its phenolic group by a glycosidic linkage
(3). A variety of flavonoid glycosides are absorbed into the body
after deglycosylation by enterobacteria (4). Recent studies on
intestinal quercetin glucosides metabolism indicate that the
enterocyte itself has the metabolic ability to absorb quercetin
glucosides, which includes transport and hydrolysis (5). Nev-
ertheless, a large part of dietary quercetin is not absorbed by
the intestine and is excreted into the feces (6), during which
The gastrointestinal mucosa are constantly exposed to a
variety of prooxidants derived from ingested foods. In addition,
the mastication and digestion of lipid-containing foods in the
gastrointestinal fluids can induce lipid peroxidation, due to the
exposure of unsaturated lipids to catalytic heme and/or nonheme
irons (8, 9). Carbonyl compounds, byproducts of lipid peroxi-
dation such as 4-HNE, are potentially toxic compounds (10-
12), and their accumulation in the body can trigger deleterious
effects associated with mucosal inflammation and development
of cancer (13-15). The gastrointestinal mucosa are undoubtedly
a physiological target for the antioxidant activity of flavonoids,
as the concentration of dietary quercetin is much higher in the
lumen of gastrointestinal tract than in plasma and other tissues
in the body (6). Therefore, it is of much interest to study the
preventive action of quercetin glycosides on diet-derived lipid
peroxidation occurring in the gastrointestinal mucosa.
* To whom correspondence should be addressed. Tel: 81-88-633-7087.
Fax: 81-88-633-7089. E-mail: terao@nutr.med.tokushima-u.ac.jp.
† University of Tokushima.
For this study, we selected Q3G and Q4′G (Figure 1), which
are the typical monoglucosides in vegetables, and studied their
antioxidant effectiveness on ferrous ion-induced lipid peroxi-
dation of the gastrointestinal mucosa in comparison to its
aglycone. Q3G is commonly distributed in a wide variety of
‡ National Food Research Institute.
§ Present address: Departamento de Bioquimica, Instituto de Quimica,
Universidade de Sao Paulo, Caxia Postal 26077, CEP 05513-970 Sao Paulo,
Brazil.
10.1021/jf035151a CCC: $27.50 © 2004 American Chemical Society
Published on Web 03/12/2004

1908 J. Agric. Food Chem., Vol. 52, No. 7, 2004
Murota et al.
preparation were performed on ice, and the homogenates were stored
at -30 °C until use.
Mucosa Homogenate Oxidation and Measurement of Lipid
Peroxidation Level. An aliquot (0.6 mL) of each mucosa homogenate
was first diluted with distilled water (2.1 mL), to which quercetin or
its glucosides was added (15 µL of 5 mM quercetin or its glucoside in
methanol solution with a final concentration of 25 µM). For the control,
an equivalent volume of methanol (15 µL) was added to the homoge-
nate. Oxidation was started by the addition of 0.3 mL of the mixed
solution of Fe(NO3)3 and ascorbic acid (final concentrations; 100 µM
and 1 mM, respectively) at 37 °C with continuous shaking. After 4 h,
the peroxidation level was determined with the TBA-fluorometric assay
(21). Hexanal and 4-HNE were measured as additional lipid peroxi-
dation markers (22). In brief, an aliquot of the mixture (0.13 mL) was
mixed with 70 µL of freshly prepared dinitrophenylhydrazine reagent
(0.35 mg/mL in 1 M KCl) and incubated in the dark for 2 h at room
temperature. The reaction mixture was extracted with chloroform and
methanol (1:2, v/v) and subjected to centrifugation. The chloroform
layer was collected and evaporated in vacuo. The residue was then
dissolved in methanol and injected into a Shimadzu HPLC LC-10AS
equipped with an ODS-80Ts column (4.5 mm × 150 mm, Tosoh).
Methanol and water (31:9, v/v) were used as the eluting solvent with
a flow rate of 1.0 mL/min. Eluate was detected at the absorption
wavelength of 378 nm. Peak assignment of 4-HNE and hexanal and
their quantification were done in comparison with their respective
standard compounds.
Quantification of Quercetin and Its Glucosides in the Reaction
Mixture. Quercetin and its glucosides in the reaction mixture were
quantified after incubating the mucosa homogenates with or without
Fe(NO3)3 by reversed phase HPLC as reported previously (23). Ascorbic
acid (the final concentration, 1 mM) was added to the mucosa either
with or without Fe(NO3)3 in order to prevent the autoxidative
decomposition of quercetin and its glucosides during incubation. Briefly,
they were extracted from the reaction mixture with methanol and
injected into HPLC with a C18 column after the concentration by
evaporation. The mobile phase of methanol/water/acetic acid (46:52:
2, v/v/v) was used. The eluate was monitored with a UV detector at
365 nm. Kaempferol was used as an internal standard for calculating
their concentrations.
Interaction of Quercetin and Its Glucosides with Ferrous Ion.
Solutions of quercetin or its glucosides (10 mM) in methanol were
prepared and diluted to 25 µM with Tris-HCl buffer (0.1 M, pH 7.4)
containing 0.135 M KCl. Their absorption spectra were recorded
between 300 and 550 nm (24). Additional scans were taken at 30 and
180 min after the addition of Fe(NO3)3 and ascorbic acid (final
concentrations, 25 and 250 µM) to the diluted 25 µM quercetin or its
glucosides. Furthermore, the effect of deferoxamine salt on the
absorption spectra was measured by adding 250 µM deferoxamine salt
to the 25 µM quercetin or its glucoside solution 10 s after the addition
of Fe(NO3)3 and ascorbic acid.
Data Analysis. Data were represented as the means ( SD of three
independent experiments. Results in Figure 2B,C were shown as the
means of two independent experiments. Statistical analysis was
evaluated by one way analysis of variance or the Kruskal-Wallis
nonparametric test when appropriate, followed by the Student-
Newman-Keuls or Bonferroni/Dunn posthoc multiple comparison.
Differences with p < 0.05 were considered significant.
Figure 1. Structures of Q3G and Q4′G.
vegetables, whereas Q4′G is uniquely present in onion (16).
The results indicate that Q4′G is superior to Q3G in the
suppression of mucosal lipid peroxidation, although free radical
scavenging activity of Q4′G is known to be much lower than
that of Q3G (17).
MATERIALS AND METHODS
Materials. Quercetin, hexanal, 4-HNE, and deferoxamine mesylate
salt were obtained from Sigma Chemical Company (St. Louis, MO).
Q3G and Q4′G were chemically synthesized from quercetin and 2,3,4,6-
tetra-O-acetyl-R-glucopyranosyl bromide using the Koenigs-Knorr
reaction (18); detailed procedures were published elsewhere (16). In
brief, quercetin and tetraacetyl-R-1-bromoglucose were dissolved in
dimethyl sulfoxide and then stirred overnight after the addition of
potassium carbonate. The resulting mixture was then adjusted to an
acidic condition by adding a few drops of formic acid. The precipitate
in the acidic solution was separated by centrifugation. The precipitate
was then washed and concentrated in vacuo. Sodium methylate solution
was added to the residue and kept at room temperature for 20 min.
The solution was then neutralized and filtered. The filtrate was then
chromatographed on a column of Toyopearl HW 40 (Tosoh, Tokyo,
Japan). The quercetin glucoside fractions were further purified by
preparative high-performance liquid chromatography (HPLC) on an
ODS column. Each quercetin glucoside peak was collected and
identified by SIMS and NMR analyses. The purity of the each
preparation was more than 98%. All other chemicals and solvents were
of analytical grade and used without purification.
Mucosa Homogenate Preparation. Male Wistar rats (8 weeks old)
weighing about 200 g were purchased from Japan SLC Co. (Hamamat-
su, Japan). Rats were deprived of food for approximately 20 h and
sacrificed by decapitation under anesthesia. The stomach, small intestine
(jejunum and ileum), and large intestine were removed and flushed
with cold saline to remove intestinal contents. Mucosa were scraped
with a glass plate and washed with cold saline (19). The mucosa
obtained from one or two rats were homogenized with 0.1 M Tris-HCl
buffer, pH 7.4, containing 0.135 M KCl (1.0 mL per 0.5 g of the
mucosa) using Potter-Elvehjem tissue homogenizer (3000 rpm, 2 min)
followed by ultrasonication for 1 min (Branson ultrasonifier). The
protein concentration of each homogenate was adjusted to 5.0 mg/mL
by diluting with 0.1 M Tris-HCl buffer, pH 7.4. Protein concentration
was determined using the Bradford assay, and bovine serum albumin
was used as a standard (20). All procedures of the homogenate
RESULTS
Effect of Quercetin Glucosides on Lipid Peroxidation of
Mucosa Homogenates. TBARS and its aldehydes, hexanal and
4-HNE, were measured as indices of lipid peroxidation level
in the gastrointestinal mucosa. As seen in Figure 2A-C,
addition of Fe(NO3)3 and ascorbic acid extensively elevated the
degree of lipid peroxidation, which increased in the order of
stomach < large intestine < small intestine. Thus, the small
intestinal mucosa were most susceptible to Fe(NO3)3 and
ascorbic acid-induced lipid peroxidation under the experimental
conditions used in this study. Furthermore, quercetin aglycone

Iron Ion-Induced Lipid Peroxidation
J. Agric. Food Chem., Vol. 52, No. 7, 2004 1909
Figure 2. Effect of quercetin glucosides on Fe(NO ) and ascorbic acid induced lipid peroxidation of gastrointestinal mucosa. (A) TBARS, (B) hexanal,
3 3
and (C) 4-HNE. Each mucosal homogenate (5 mg/mL) was diluted (5-fold) with distilled water, and then, lipid peroxidation was started by addition of
Fe(NO ) and ascorbic acid (final concentrations: 100 µM and 1 mM, respectively) in the presence or absence of 25 µM quercetin aglycone/25 µM
3 3
Q3G/25 µM Q4′G at 37 °C with continuous shaking. Fe, Fe(NO ) ; AsA, ascorbic acid; and Q, quercetin aglycone. Results in panel A are the means
3 3
± SD of three independent experiments. Means with different letters for each mucosa are significantly different. Results in panels B and C are the means
of two independent experiments.
and its glucosides, Q3G and Q4′G, significantly suppressed the
accumulation of TBARS (Figure 2A). A similar suppressive
effect of quercetin aglycone and its glucosides on accumulation
of hexanal (Figure 2B) and 4-HNE (Figure 2C) was observed.
The order of the effectiveness of lipid peroxidation suppression
increased in the order of Q3G < Q4′G e quercetin aglycone.
Recovery of Quercetin Aglycone and Its Glucosides after
Incubation with Mucosal Homogenates. Recovery of quercetin
and its glucosides after incubation with mucosa homogenate in
the presence and absence of Fe(NO3)3 is shown in Figure 3.
The levels of quercetin and its glucosides, Q3G and Q4′G, were
adjusted to 25 nmol/mg protein prior to incubation for each
experiment. The recovery of quercetin aglycone on addition of
Fe(NO3)3 was significantly reduced in the stomach, small
intestine, and large intestine mucosa, indicating that the aglycone
was being used as an antioxidant during the incubation with
Fe(NO3)3.
recovery of Q4′G in the presence of Fe(NO3)3 decreased
significantly as compared to that in the absence of Fe(NO3)3 in
all of the mucosa homogenates, whereas total Q3G recovery
showed a significant decrease only in the small intestinal mucosa
homogenate. A large proportion of Q4′G was converted into
its aglycone in the small intestinal mucosa homogenate as
compared to those in the gastric and large intestinal mucosa
homogenates (97.9 ( 1.8, 53.0 ( 10.0, and 79.9 ( 5.6%,
respectively, p < 0.05). Furthermore, quercetin aglycone derived
from Q4′G in the presence of Fe(NO3)3 was significantly
decreased as compared with that in the absence of Fe(NO3)3 in
all of the mucosa homogenates. In contrast, glucoside forms
incubated with Fe(NO3)3 did not decrease in either mucosa
homogenate.
Chelating Activity of Quercetin Aglycone and Its Gluco-
sides. Figure 4 exhibits the absorption spectra of quercetin
aglycone (A), Q3G (B), and Q4′G (C) in the absence or presence
of Fe(NO3)3 and ascorbic acid. The maximum wavelength at
nearly 380 nm is derived from the inherent structure of the
B-ring of quercetin, whereas that in the range from 420 to 450
Both Q3G and Q4′G yielded quercetin aglycone in the
absence of Fe(NO3)3. Interestingly, aglycone was formed at a
significantly higher ratio from Q4′G than from Q3G. Total

1910 J. Agric. Food Chem., Vol. 52, No. 7, 2004
Murota et al.
maximum wavelength was faster than that of quercetin after
the 30 min incubation with deferoxamine salt. In contrast, the
maximum wavelength of Q4′G was completely returned to the
original maximum wavelength after 30 min of incubation with
deferoxamine salt. Therefore, it is apparent that the chelating
activities of quercetin aglycone and its glucosides were different
and increased in the order of Q4′G < Q3G < quercetin
aglycone.
DISCUSSION
We previously examined the antioxidant activity of quercetin
and quercetin glucosides on azo radical-induced lipid peroxi-
dation of liposomal membranes and found that they decreased
the lipid hydroperoxide formation in the order of Q4′G < Q3G
< quercetin aglycone (26). Furthermore, Q4′G in comparison
with Q3G or quercetin aglycone showed very little inhibition
of either azo radical- or ferrous ion-induced lipid peroxidation
of human plasma LDL (27). The o-dihydroxyl group in the
B-ring structure of quercetin is responsible for a greater
proportion of its free radical scavenging activity. The decreased
inhibition of azo radical-induced lipid peroxidation by Q4′G is
because of the loss of the o-dihydroxyl group in the B-ring due
to the introduction of a sugar group (28, 29). While comparing
quercetin with kaempferol, Brown et al. (25) observed that the
o-dihydroxyl structure mainly takes part in chelating copper ion
during the prevention of copper ion-induced LDL oxidation by
quercetin. In this study, we compared the chelating spectrum
of quercetin aglycone, Q3G, and Q4′G and found that the iron
ion chelating activity of Q4′G was much lower than that of Q3G.
Fe(NO3)3- and ascorbic acid-induced lipid peroxidation are
initiated by the generation of ferrous ion from the reduction of
ferric ion by ascorbic acid, followed by the peroxyl radical-
driven free radical chain reaction. Both chelating and radical
scavenging activities are responsible for the inhibition of this
kind of peroxidation. Thus, Q4′G being a poor ferric ion chelator
should have a lesser lipid peroxidation inhibitory effect when
compared to Q3G. However, Q4′G was more effective than Q3G
in the inhibition of Fe(NO3)3 and ascorbic acid-induced lipid
peroxidation of the gastrointestinal mucosa.
Q4′G yielded more amount of quercetin aglycone than Q3G
on incubation with the gastrointestinal mucosa. â-Glucosidase
activity that released quercetin aglycone from its monoglucoside
was observed in the rat intestinal mucosa by Ioku et al. (30)
and later seen in human intestinal epithelial cells (31). Thus,
quercetin glucosides are likely to be converted into quercetin
aglycone before transport into the body. Interestingly, it was
reported that â-glucosidase activity of small intestinal mucosa
from both rat and human prefers the sugar group at the
4′-position to that at the 3-position (30, 31). It is therefore
rational that Q4′G is hydrolyzed to its aglycone more efficiently
than Q3G in rat small intestine. In addition, we found that large
intestinal mucosa prefer Q4′G to Q3G. The predominance of
Q4′G in hydrolysis can explain its higher antioxidant activity
in intestinal mucosa despite its lower free radical scavenging
and chelating activity. Furthermore, lactose phlorizin hydrolase
(LPH; EC 3.2.1.62), a membrane-bound enzyme known to
primarily hydrolyze lactase, was rediscovered as a membrane-
bound â-glucosidase, which is capable of hydrolyzing quercetin
glucosides effectively (32). The Q4′G mucosal hydrolysis was
10 times greater than Q3G hydrolysis in a rat-everted jejunum
sac model (33), suggesting that Q4′G was a better substrate for
LPH in the rat intestine when compared to Q3G. Thus, it is
reasonable to assume that Q4′G is hydrolyzed to its aglycone
before absorption and acts as a strong antioxidant against ferrous
ion-induced oxidation in the intestinal lumen.
Figure 3. Recovery of quercetin and its glucosides after incubation with
gastrointestinal mucosa homogenate. (A) Stomach, (B) small intestine,
and (C) large intestine. Bars represent quercetin aglycone (filled bar) and
its glucosides (open bars). Incubation conditions were the same as those
in Figure 2. None, absence of Fe(NO ) ; Fe2+, presence of Fe(NO ) . In
both cases, ascorbic acid was added to the incubation mixture for
preventing the oxidative decomposition of quercetin or quercetin glucosides.
Results are the means ± SD of three independent experiments. Means
with different letters (a−d) for quercetin aglycone and symbols (*, ‡, #)
for quercetin glucosides are significantly different (p < 0.05).
3 3
3 3
nm is from the chelation of ferrous ion by its B-ring structure
(24, 25). In all compounds, the maximum wavelength at nearly
380 nm was found to be shifted to the higher region by the
addition of Fe(NO3)3 and ascorbic acid. Absorption spectra were
obtained after 30 min and 3 h of incubation with deferoxamine
salt, a strong cheating agent. As aglycone, Q3G, and Q4′G
compete with deferoxamine salt in chelating ferrous ion during
the incubation, the rate of the change in the maximum
wavelength is dependent on the strength of its chelation. For
quercetin aglycone, after addition of deferoxamine salt, the
maximum wavelength stayed shifted to the higher region for
the 30 min incubation, whereas after 3 h, it was shifted to its
original maximum wavelength [seen prior to the addition of
Fe(NO3)3]. In the case of Q3G, the recovery of the original

Iron Ion-Induced Lipid Peroxidation
J. Agric. Food Chem., Vol. 52, No. 7, 2004 1911
Lately, a number of studies have suggested the involvement
of free iron ions in the development of gastrointestinal diseases,
such as inflammation and colon cancer (34-37). Dietary iron
remains substantially unabsorbed in the intestine and, therefore,
could be involved in the generation of hydroxyl radical by the
Fenton type reaction in conjugation with colonic microflora.
Previously, we have demonstrated that phytic acid hydrolysis
products (38) and phosphatidylserine (39) exert a unique
protective effect on iron ion-induced lipid peroxidation. Iron
ion and ascorbic acid used for the initiators of lipid peroxidation
in this study are common food factors present in the digestive
tract. Thus, the protective effect of quercetin glucosides against
lipid peroxidation of the intestinal mucosa indicates the
importance of quercetin glucoside-containing foods for protec-
tion of intestinal mucosa against oxidative stress originating from
dietary prooxidants.
In conclusion, Q4′G is superior to Q3G in protection of the
rat intestinal mucosa from iron ion-induced lipid peroxidation,
despite its lower free radical and iron ion-chelating activity. This
is corroborated by the effective hydrolysis of Q4′G to its active
aglycone in the intestinal mucosa. Thus, vegetables rich in Q4′G
such as onion may be beneficial in preventing oxidative damage
to the intestinal tract. The physiological role of quercetin
glucoside-rich vegetables on the digestive tract needs to be
further explored.
ABBREVIATIONS USED
4-HNE, 4-hydroxynonenal; Q3G, quercetin 3-O-â-glucoside;
Q4′G, quercetin 4′-O-â-glucoside; TBA, thiobarbituric acid;
TBARS, TBA reacting substances; LDL, low-density lipopro-
tein.
ACKNOWLEDGMENT
We thank Dr. S. R. Cheruku, Department of Nutritional
Sciences, Cook College, Rutgers University, for her help in
preparing the manuscript.
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Received for review October 8, 2003. Revised manuscript received
February 2, 2004. Accepted February 3, 2004. This work was partly
supported by the Ministry of Agriculture, Forestry, and Fishery Food
Project, Japan.
JF035151A