Antioxidant effects of isorhamnetin 3,7-di-O-β-D-glucopyranoside isolated from mustard leaf (Brassica juncea) in rats with streptozotocin-induced diabetes

Article abstract of DOI:10.1021/jf0202133

To investigate the effects of isorhamnetin 3,7-di-O-β-D-glucopyranoside (isorhamnetin diglucoside), a major flavonoid compound of mustard leaf, on oxidative stress due to diabetes mellitus, in vivo and in vitro studies were carried out. Oral administration of isorhamnetin diglucoside (10 or 20 mg/kg of body weight/day for 10 days) to rats with streptozotocin-induced diabetes significantly reduced serum levels of glucose and 5-(hydroxymethyl)furfural (5-HMF), which is glycosylated with hemoglobin and is an indicator of oxidative stress. After intraperitoneal administration, isorhamnetin diglucoside did not show these activities. In addition, after oral administration, the thiobarbituric acid-reactive substance levels of serum, and liver and kidney mitochondria declined significantly compared with the control group in a dose-dependent manner, whereas after intraperitoneal administration these levels fell only slightly. On the basis of the oral and intraperitoneal results, it was hypothesized that isorhamnetin diglucoside was converted to its metabolite in vivo, and its conversion to its aglycone, isorhamnetin, by β-glucosidase was confirmed; isorhamnetin acted as an antioxidant. Moreover, it was observed that isorhamnetin diglucoside had no effect on the 1,1-diphenyl-2-picrylhydrazyl radical, whereas isorhamnetin showed a potent antioxidant effect in vitro. In addition, intraperitoneal administration of isorhamnetin reduced serum glucose and 5-HMF levels. Furthermore, lipid peroxidation in blood, liver, and kidney associated with diabetes mellitus declined after the administration of isorhamnetin. These results suggest that isorhamnetin diglucoside is metabolized in vivo by intestinal bacteria to isorhamnetin and that isorhamnetin plays an important role as an antioxidant.

Full text of DOI:10.1021/jf0202133

5490 J. Agric. Food Chem. 2002, 50, 5490−5495
Antioxidant Effects of Isorhamnetin
,7-Di-O-â-D-glucopyranoside Isolated from Mustard Leaf
Brassica juncea) in Rats with Streptozotocin-Induced Diabetes
3
(
,†
†
†
‡
TAKAKO YOKOZAWA,* HYUN YOUNG KIM, EUN JU CHO, JAE SUE CHOI, AND
HAE YOUNG CHUNG§
Institute of Natural Medicine, Toyama Medical and Pharmaceutical University,
Sugitani, Toyama 930-0194, Japan, Faculty of Food Science and Biotechnology, Pukyong National
University, Pusan 608-737, Korea, and College of Pharmacy, Pusan National University,
Pusan 609-735, Korea
To investigate the effects of isorhamnetin 3,7-di-O-â-D-glucopyranoside (isorhamnetin diglucoside),
a major flavonoid compound of mustard leaf, on oxidative stress due to diabetes mellitus, in vivo and
in vitro studies were carried out. Oral administration of isorhamnetin diglucoside (10 or 20 mg/kg of
body weight/day for 10 days) to rats with streptozotocin-induced diabetes significantly reduced serum
levels of glucose and 5-(hydroxymethyl)furfural (5-HMF), which is glycosylated with hemoglobin and
is an indicator of oxidative stress. After intraperitoneal administration, isorhamnetin diglucoside did
not show these activities. In addition, after oral administration, the thiobarbituric acid-reactive substance
levels of serum, and liver and kidney mitochondria declined significantly compared with the control
group in a dose-dependent manner, whereas after intraperitoneal administration these levels fell only
slightly. On the basis of the oral and intraperitoneal results, it was hypothesized that isorhamnetin
diglucoside was converted to its metabolite in vivo, and its conversion to its aglycone, isorhamnetin,
by â-glucosidase was confirmed; isorhamnetin acted as an antioxidant. Moreover, it was observed
that isorhamnetin diglucoside had no effect on the 1,1-diphenyl-2-picrylhydrazyl radical, whereas
isorhamnetin showed a potent antioxidant effect in vitro. In addition, intraperitoneal administration of
isorhamnetin reduced serum glucose and 5-HMF levels. Furthermore, lipid peroxidation in blood,
liver, and kidney associated with diabetes mellitus declined after the administration of isorhamnetin.
These results suggest that isorhamnetin diglucoside is metabolized in vivo by intestinal bacteria to
isorhamnetin and that isorhamnetin plays an important role as an antioxidant.
KEYWORDS: Isorhamnetin 3,7-di-O-â-D-glucopyranoside; streptozotocin; 1,1-diphenyl-2-picrylhydrazyl
radical; thiobarbituric acid-reactive substance; 5-(hydroxymethyl)furfural; diabetes
INTRODUCTION
hyperglycemia and cause many of the secondary complications
of diabetes, such as nephropathy, retinopathy, and neuropathy
Oxidative stress is linked to tissue damage and the develop-
ment of pathophysiology and occurs in most, if not all, human
diseases. In addition, oxidative stress and oxidative damage to
tissues are common end points of chronic diseases, such as
atherosclerosis, diabetes, and rheumatoid arthritis. In particular,
since the early 1980s, a role for reactive oxygen species in
diabetes has been widely discussed, and there is considerable
evidence that oxidative damage is increased in diabetes, although
the mechanisms involved are not clear. Furthermore, there are
reports that oxygen species are generated as a result of
(
1-4). Therefore, a wide range of antioxidants, both natural
and synthetic, have been proposed for use in the treatment of
human disease.
Recently attention has been focused on the antioxidative
activities of many plant phenolics, especially flavonoids, because
they inhibit lipid peroxidation, and the significance of potential
protective properties of flavonoids present in vegetables and
fruits has become an important issue (5, 6). Flavonoids are
diphenylpropanes occurring ubiquitously in food plants and are
common components in the human diet. Several studies have
demonstrated the physiological functions of flavonoids, such
as hypotensive, antiallergic, anti-inflammatory, antiviral, anti-
cancer, and anticarcinogenic properties (7-9). Mustard leaf
(Brassica juncea), a major raw material for kimchi in Korea
*
Author to whom correspondence should be addressed (fax +81-76-
4
34-4656; e-mail yokozawa@ms.toyama-mpu.ac.jp).
†
Toyama Medical and Pharmaceutical University.
Pukyong National University.
‡
§
Pusan National University.
1
0.1021/jf0202133 CCC: $22.00 © 2002 American Chemical Society
Published on Web 08/17/2002

Antioxidant Effects of Isorhamnetin 3,7-Di-O-â-D-glucopyranoside
J. Agric. Food Chem., Vol. 50, No. 19, 2002 5491
and one of the cruciferous vegetables well-known to contain
high levels of flavonoids that have attracted much attention as
antioxidants, is also expected to be an effective antioxidant due
to these flavonoids. Although mustard leaf has been demon-
strated to have the antiatherogenic effect of reducing plasma
cholesterol levels and increasing high-density lipoprotein cho-
lesterol levels (10), its antioxidant activities are unclear to date.
In addition, other active compounds besides flavonoids in
mustard leaf, thioglucosides, have been isolated (11). However,
the major active flavonoid in which mustard leaf is rich and its
metabolism in vivo have not been elucidated yet.
(5) Preparation of Mitochondria and Measurement of TBA-ReactiVe
Substance LeVel. Mitochondria from liver or kidney homogenates was
prepared by differential centrifugation (800g or 12000g) with a
refrigerated centrifuge (4 °C) using the methods of Johnson and Lardy
(15) and Jung and Pergande (16) with slight modifications. Each pellet
was resuspended with prepared medium, and the TBA-reactive
substance level was determined according to the method of Uchiyama
and Mihara (17).
(6) Determination of Protein. Protein content was quantified using
a commercial protein assay kit (A/G B-Test Wako; Wako Pure
Chemical Industries) or by the method of Itzhaki and Gill (18) with
bovine serum albumin as the standard.
In this study, we investigated whether the main compound,
isorhamnetin 3,7-di-O-â-D-glucopyranoside (isorhamnetin di-
glucoside), is useful in the improvement of diabetes caused by
oxidative stress and also examined its metabolic pathway and
the antioxidative activity of its metabolite.
Determination of DPPH Radical Level. A 100 µL aliquot of an
aqueous solution of the sample (control ) 100 µL of distilled water)
was added to microwells followed by an ethanolic solution of DPPH
(final concentration ) 60 µM), according to the method of Hatano et
al. (19). After gentle mixing and 30 min of standing at room
temperature, the DPPH radical level was measured with a microplate
reader, model 3550-UV (Bio-Rad, Tokyo, Japan). The antioxidant
activity was expressed as the IC50 (concentration in µg/mL required to
inhibit DPPH radical formation by 50%) determined from the log-
dose inhibition curve.
MATERIALS AND METHODS
Purification of Isorhamnetin Diglucoside from Mustard Leaf.
As described previously (12), dry leaves (14.8 kg) were refluxed with
methanol (MeOH) for 3 h, and the MeOH extract (1600 g) was
Analysis of Hydrolysates by HPLC. The hydrolysates of isorham-
netin diglucoside using HCl (pH 2) or â-glucosidase from naringinase
were analyzed by HPLC. The HPLC system used consisted of a PX-
suspended in distilled H
EtOAc), n-butanol (n-BuOH), and H
fractions (CH Cl , 304 g; EtOAc, 10 g; n-BuOH, 124 g; H
Then, the BuOH fraction (124 g) was subjected to silica gel column
chromatography. Elution with CH Cl containing increasing amounts
2
O and partitioned with CH
O in sequence, thus yielding four
O, 1140 g).
2 2
Cl , ethyl acetate
(
2
8
020, CCPM-II, and UV-8020 (Tosoh Co., Ltd.). The column used
2
2
2
was an ODS-80 Ts analytical column (4.6 i.d. × 150 mm), the mobile
phase consisted of a 20%/80% (by volume) mixture of 100% MeOH/
2
2
-
1
2
% acetic acid at a flow rate of 1 mL min , and the UV detector was
of MeOH (50, 10-20, and 30%) and then MeOH gave 11 subfractions.
Subfraction 6 (41.2 g) was further purified by Sephadex LH-20 column
chromatography using MeOH as a solvent to give isorhamnetin
diglucoside (1800 mg).
set at 265 nm.
Statistical Analysis. Data are presented as means ( SE. Differences
among groups were analyzed by Dunnett’s test, and those at p < 0.05
were considered to be significant.
Preparation of Isorhamnetin from Isorhamnetin Diglucoside.
Isorhamnetin diglucoside (600 mg) was refluxed with 5% H
MeOH (100 mL) at >80 °C for 3 h. The reaction mixture was cooled,
concentrated to half its original volume, added to iced H O, and the
precipitate was collected by filtration. Isorhamnetin (220 mg) was
obtained after washing and drying the precipitate.
Reagents. Streptozotocin (STZ) and naringinase were purchased
from Sigma Chemical Co. (St. Louis, MO). 1,1-Diphenyl-2-picryl-
hydrazyl (DPPH) was obtained from Wako Pure Chemical Industries
Ltd. (Osaka, Japan).
2 4
SO in
RESULTS
2
Serum Glucose and Glycosylated Hemoglobin Levels. The
STZ-induced diabetic rats had higher glucose and 5-HMF levels
than normal rats, as shown in Table 1. However, the serum
glucose levels of rats given 10 and 20 mg of isorhamnetin
diglucoside orally were 510.1 and 492.4 mg/dL, respectively,
whereas the control level was 574.2 mg/dL. Similarly, oral
administration had a very significant and dose-dependent
inhibitory effect on glycosylated hemoglobin formation by 28
and 43% decrease at the oral doses of 10 and 20 mg,
respectively. In contrast, isorhamnetin diglucoside administered
intraperitoneally did not change the serum glucose and 5-HMF
levels in comparison with the controls.
TBA-Reactive Substance Levels in Serum and Hepatic
and Renal Mitochondria. As shown in Figure 1, the diabetic
rats showed higher lipid peroxidation of their serum, livers, and
kidneys than normal rats. The serum TBA-reactive substance
level of diabetic rats given isorhamnetin diglucoside orally was
significantly lower than that of the diabetic control group, but
intraperitoneal administration did not affect the TBA-reactive
substance level. After an oral dose of 20 mg, the serum TBA-
reactive substance level decreased by 57%. Similarly, the hepatic
and renal mitochondrial levels of oral administration showed
significant decreases compared with the control group levels,
whereas after intraperitoneal administration, these levels fell only
slightly. In the rats fed 20 mg orally, the TBA-reactive substance
level of the hepatic mitochondria declined by 35% and that of
the renal mitochondria fell by 38%, whereas after intraperitoneal
administration, the TBA-reactive substance levels of hepatic and
renal mitochondria showed smaller decreases by 12 and 16%,
respectively.
Animal Experiments. (1) Animal Preparation. Male Wistar rats (5
weeks old, 120-130 g) from Japan SLC Inc. (Hamamatsu, Japan) were
used. They were kept in a wire-bottom cage under a conventional
lighting regimen with a dark night. The room temperature (∼25 °C)
and humidity (∼60%) were controlled automatically. Laboratory pellet
chow (CLEA Japan Inc., Tokyo, Japan, comprising 24.0% protein, 3.5%
lipid, and 60.5% carbohydrate) and water were given ad libitum. After
several days of adaptation, STZ (50 mg/kg of body weight) dissolved
in citrate buffer (pH 4.5) was injected intraperitoneally following
overnight fasting. One week after injection, the glucose level of blood
taken from the tail vein of each rat was determined, and then the animals
were divided into experimental groups. The control group was given
physiological saline (vehicle), whereas the other groups were given
isorhamnetin diglucoside (at a dose of 10 or 20 mg/kg of body weight/
day) or isorhamnetin (at a dose of 10 mg/kg of body weight/day) orally
or intraperitoneally. After 10 consecutive days of administration, the
rats were killed by decapitation, the blood was collected, and the livers
and kidneys were removed, rinsed with cold physiological saline, and
frozen at -80 °C until assayed.
(2) Determination of Serum Glucose LeVel. The serum glucose level
was measured using a commercial kit (Glucose CII-Test Wako; Wako
Pure Chemical Industries).
(3) Determination of Serum Glycosylated Hemoglobin LeVel. Gly-
cosylated hemoglobin level in serum was determined by the thiobar-
bituric acid (TBA) assay reported by Fluckiger and Winterhalter (13)
with modification.
(
4) Determination of Serum TBA-ReactiVe Substance LeVel. The
TBA-reactive substance level in serum was determined using the
Identification of Isorhamnetin from Isorhamnetin Diglu-
coside. Hydrolysis of isorhamnetin diglucoside with HCl (pH
method of Naito and Yamanaka (14).

5492 J. Agric. Food Chem., Vol. 50, No. 19, 2002
Yokozawa et al.
Table 1. Effect of Isorhamnetin Diglucoside on Serum Glucose and 5-(Hydroxymethyl)furfural Levels after Oral or Intraperitoneal Administration
dose
(mg/kg of BW/day)
glucosea
(mg/dL)
5-HMFa
(nmol/mg of protein)
route
group
diabetic rats
oral
control
isorhamnetin diglucoside
isorhamnetin diglucoside
574.2 ± 23.0a
510.1 ± 6.5ab
492.4 ± 14.4ab
21.12 ± 1.88a
15.16 ± 0.75ab
11.99 ± 1.01ab
10
20
intraperitoneal
diabetic rats
control
isorhamnetin diglucoside
isorhamnetin diglucoside
522.0 ± 20.2a
538.5 ± 11.4a
520.7 ± 7.8a
110.3 ± 2.1
17.16 ± 0.27a
18.66 ± 0.31a
18.49 ± 0.76a
3.65 ± 0.03
10
20
normal
a
Statistical significance: a, p < 0.001 vs normal rats; b, p < 0.001 vs diabetic control rats.
Figure 1. Effect of isorhamnetin diglucoside on TBA-reactive substance levels after oral (A) or intraperitoneal (B) administration. Statistical significance:
a, p < 0.05; b, p < 0.001 vs diabetic control rats. The value of normal rats is expressed as the horizontal line.
isorhamnetin, its aglycone. Isorhamnetin inhibited DPPH forma-
tion by 50% at a concentration of 26.7 µg/mL (IC50), whereas
isorhamnetin diglucoside, with a high IC50 of 179.7 µg/mL,
showed weak DPPH radical scavenging activity.
Table 2. DPPH Radical Scavenging Activity
compound
IC50 (µg/mL)
isorhamnetin diglucoside
isorhamnetin
179.7 ± 0.8
26.7 ± 0.9
Effects of Isorhamnetin on Glucose, Glycosylated Protein,
and TBA-Reactive Substance Levels. Figure 3 shows the
effects of isorhamnetin on rats with STZ-induced diabetes. We
evaluated the glucose, glycosylated protein, and TBA-reactive
substance levels of serum and hepatic and renal mitochondria.
Intraperitoneal administration of isorhamnetin at 10 mg/kg of
body weight/day reduced the serum levels of glycosylated
protein as well as glucose. The level of glucose declined from
2
) yielded no chromatographic peaks other than isorhamnetin
diglucoside. However, after enzymatic hydrolysis by â-glucosi-
dase, a new peak appeared in the chromatogram, and comparison
with the retention time (42 min) of an authentic sample proved
it was isorhamnetin, as shown in Figure 2. Although there was
no isorhamnetin peak and nothing but isorhamnetin diglucoside
present before incubation, the conversion of isorhamnetin
diglucoside to isorhamnetin by â-glucosidase increased in a
time-dependent manner (32% change of isorhamnetin digluco-
side after 7 h).
5
2
57.2 to 481.9 mg/dL and that of glycosylated protein fell from
0.58 to 16.41 nmol/mg of protein. Isorhamnetin also showed
protective activity against lipid peroxidation. The TBA-reactive
substance levels of serum and hepatic and renal mitochondria
decreased > 20% compared with those of control diabetic rats.
The TBA-reactive substance levels significantly declined by 23
and 27% in hepatic and renal mitochondria, respectively.
DPPH Radical Scavenging Activities of Isorhamnetin
Diglucoside and Isorhamnetin. Table 2 shows the DPPH
radical scavenging activity of isorhamnetin diglucoside and

Antioxidant Effects of Isorhamnetin 3,7-Di-O-â-D-glucopyranoside
J. Agric. Food Chem., Vol. 50, No. 19, 2002 5493
Figure 2. Time course of conversion of isorhamnetin diglucoside by HCl (pH 2) (A) and â-glucosidase (B).
Figure 3. Effect of isorhamnetin on glucose, glycosylated protein, and TBA-reactive substance levels after intraperitoneal administration. Statistical
significance: a, p < 0.01; b, p < 0.001 vs diabetic control rats. The value of normal rats is expressed as the horizontal line.
study on mustard leaf, we found that the EtOAc and BuOH
fractions had stronger DPPH and hydroxyl radical scavenging
DISCUSSION
Recently, much attention has been focused on antioxidants
in food that are potential compounds for preventing diseases
caused by oxidative stress including diabetes because of their
distinctive biological activity and low toxicity. In our previous
activities than the CH2Cl2 and H2O fractions (20). In view of
these results and the yields by weight from dried mustard leaf
(EtOAc fraction, 0.07%; BuOH fraction, 0.85%), we carried
out in vivo and in vitro experiments with the main compound

5494 J. Agric. Food Chem., Vol. 50, No. 19, 2002
Yokozawa et al.
Figure 4. Transformation of isorhamnetin diglucoside to isorhamnetin in vivo.
of the BuOH fraction, isorhamnetin diglucoside (∼1.5% of this
fraction), using rats with STZ-induced diabetes.
protection against lipid peroxidation, whereas after intraperito-
neal administration, this compound had little effect, if any.
Therefore, we hypothesized that after oral administration,
isorhamnetin diglucoside is converted to an active metabolite
in the stomach or intestine and this metabolite has antioxidant
properties. There is a similar report of the antioxidant mecha-
nism and the function of its metabolites in vivo (27). Thus, it
is important to determine how antioxidants in food are metabo-
lized in vivo and how antioxidant metabolites function in vivo.
We also found differences between in vivo and in vitro results
with isorhamnetin diglucoside. It had little DPPH radical
scavenging activity in vitro, whereas after oral administration,
it had a strong antioxidative effect in a diabetic animal model
in vivo. Hence, to investigate the mechanism responsible for
the function of isorhamnetin diglucoside in vivo after oral
administration, we attempted to hydrolyze isorhamnetin diglu-
coside with HCl at pH 2 (reflecting conditions in the stomach)
or â-glucosidase and compared the antioxidant activity of
isorhamnetin diglucoside with that of its metabolite. From the
results, we assume that isorhamnetin diglucoside, a main
compound of mustard leaf, is metabolized by intestinal bacteria
to isorhamnetin, which plays a central antioxidant role in vivo
Diabetes can be produced in animals by administering STZ,
which is toxic to â-cells and is widely used for the induction
of experimental diabetes mellitus, resulting in the production
of active oxygen species (21). Scavengers of oxygen radicals
are effective in preventing diabetes in these animal models (2,
2
2). Therefore, we employed such a diabetic animal model
system to examine the antioxidant effects of isorhamnetin
diglucoside isolated from mustard leaf on oxidative stress.
Hyperglycemia and glycation of proteins are associated with
the development of diabetic complications, resulting in the
generation of oxygen free radicals (23). In particular, increased
glycation of collagen and plasma proteins in subjects with
diabetes may stimulate the oxidation of lipids, which may in
turn stimulate autoxidative reactions of sugars, enhancing
damage to both lipids and proteins in the circulation and the
vascular wall, continuing and reinforcing the cycle of oxidative
stress and damage (2). Bunn et al. (24) reported that 5-HMF is
involved in the nonenzymatic browning process, and nonenzy-
matically bound glucose in serum was found to be released as
5
-HMF (25). Therefore, we evaluated 5-HMF levels to deter-
(Figure 4).
mine the extent of glycosylation of serum proteins. The results
on glucose and glycosylated protein levels indicate that oral
administration of isorhamnetin diglucoside might prevent the
pathogenesis of diabetic complications caused by impaired
glucose metabolism and glycosylation of serum proteins,
eventually resulting in improvement of the diabetic pathological
conditions.
Isorhamnetin diglucoside is a kind of flavonoid present in
vegetables and fruits. Flavonoids generally occur in foods as
O-glycosides with bound sugars, usually at the C3 position. Until
recently, it has been assumed that the flavonoid glycosides
cannot be easily absorbed from the small intestine and the
cleavage of â-glycoside linkage will not occur until the
compounds reach the microflora in the large intestine (28).
However, the â-glucosidase capable of efficiently hydrolyzing
various naturally occurring flavonoid glycoside is found abun-
dantly in the small intestine as well as the large intestine of
humans (29-31). Although the rate and extent of deglycosy-
lation depend on the structure of the flavonoid and the position/
nature of the sugar substitutions, the flavonoids with glucose
at the 3- and 7-positions are all substrates for the â-glucosidase
(31). On the basis of these studies, isorhamnetin diglucoside
from mustard leaf is considered to be hydrolyzed efficiently to
its aglycone, isorhamnetin.
The deglycosylation of flavonoids by human â-glucosidase
could be an important first step in their uptake metabolism,
excretion, and biological activity. In addition, biological activity
depends on the presence or absence of the glycoside residue
(32). The aglycone is likely to have a greater biological effect
than the glycoside, so deglycosylation via a â-glucosidase
activity would be an important step in metabolism. Cyanidine
rather than cyanidine glucosides acts as an antioxidant in living
systems and, after ingestion, the latter can be hydrolyzed by
â-glucosidase of intestinal bacteria (33). In addition, Miyake
et al. (22) reported that the antioxidant activities of the aglycones
of the flavonoid glycosides in lemon fruit were stronger than
those of the flavonoid glycosides themselves. We could also
Oxidative stress is associated with the peroxidation of cellular
lipids, which is determined by measuring TBA-reactive sub-
stance levels. The concentration of lipid peroxidation products
may also reflect the oxidative stress associated with the diabetic
condition. Baynes (2) and Kakkar et al. (26) reported that tissue
and blood malondialdehyde levels of rats with STZ-induced
diabetes increased due to lipid peroxidation. On the basis of
these results, we evaluated TBA-reactive substance levels to
determine the degree of oxidative damage in diabetic rats and
found out that it was significantly higher in the diabetic control
group compared with the normal group. This result suggests
that the increased TBA-reactive substance levels in diabetic rats
result in increased levels of oxygen free radicals, which attack
the polyunsaturated fatty acids in cell membranes and cause
lipid peroxidation, leading to pathological conditions. In contrast,
orally administered isorhamnetin diglucoside reduced the TBA-
reactive substance levels, indicating that isorhamnetin digluco-
side may attract a lot of attention as a new therapeutic agent
for diabetes in view of its inhibition of the lipid peroxidation
and free radical production caused by STZ.
The effects of orally and intraperitoneally administered
isorhamnetin diglucoside on glucose metabolism and lipid
peroxidation differed. Orally administered isorhamnetin diglu-
coside resulted in the improvement of glucose metabolism and

Antioxidant Effects of Isorhamnetin 3,7-Di-O-â-D-glucopyranoside
J. Agric. Food Chem., Vol. 50, No. 19, 2002 5495
confirm that isorhamnetin diglucoside is metabolized by intes-
tinal bacteria to isorhamnetin. Isorhamnetin exhibited antioxidant
activities in rats with STZ-induced diabetes in vivo as well as
in vitro, reducing the levels of DPPH radical, serum glucose,
and glycosylated proteins and protecting serum and tissue
mitochondria against lipid peroxidation. From these results, we
conclude that the antioxidant activity of the active compound
from mustard leaf is attributable to isorhamnetin not to
isorhamnetin diglucoside itself. In view of the antioxidant
activities observed in vivo as well as in vitro, mustard leaf
containing high levels of isorhamnetin diglucoside seems to be
effective for reducing oxidative stress and is expected to
contribute to the prevention of life style-related diseases.
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Received for review February 15, 2002. Revised manuscript received
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