PHYTOTHERAPY RESEARCH

Phytother. Res. (2012)

Published online in Wiley Online Library
(wileyonlinelibrary.com) DOI: 10.1002/ptr.3724

Preventive Effects of Hesperidin, Glucosyl

Hesperidin and Naringin on Hypertension and
Cerebral Thrombosis in Stroke-prone
Spontaneously Hypertensive Rats

Miyako Ikemura,1 Yasuto Sasaki,1* John C. Giddings2 and Junichiro Yamamoto1

1
Laboratory of Physiology, Faculty of Nutrition, Kobe Gakuin University, Nishi-Ku, Kobe, Japan
2
Formerly, Department of Haematology, Cardiff University, UK

The effects of hesperidin, glucosyl hesperidin (G-hesperidin), a water-soluble derivative of hesperidin, and narin-
gin on blood pressure and cerebral thrombosis were investigated using stroke-prone spontaneously hypertensive
rats (SHRSP). Hesperidin, G-hesperidin and naringin were mixed with diet and fed to the animals for 4 weeks. No
effect was evident on body weight, but the supplements significantly suppressed the age related increase in blood
pressure. Thrombotic tendency, as assessed using a He-Ne laser technique in the cerebral blood vessels, was sig-
nificantly decreased in the treated animals compared with the control animals. Measurements of 8-hydroxy-2′-
deoxyguanosine (8-OHdG) demonstrated that the supplements had strong antioxidant activity. Furthermore,
these supplements significantly increased the production of nitric oxide (NO) metabolites in urine measured with
Griess reagent. Vasodilation induced by acetylcholine-mediated NO production in the endothelium was assessed
using thoracic aortic ring preparations and indicated that endothelial function was significantly improved by the
administration of these supplements.
These findings suggest that the strong antioxidant properties of hesperidin, G-hesperidin and naringin could
modulate the inactivation of NO and protect endothelial function from reactive oxygen species (ROS). In this
manner, the flavonoids could contribute beneficial effects on the mechanisms of hypertension and thrombosis
by increasing the bioavailability of NO. Copyright © 2012 John Wiley & Sons, Ltd.

Keywords: hesperidin; glucosyl hesperidin; naringin; thrombosis; nitric oxide; stroke-prone spontaneously hypertensive rats.

mechanisms (Yamamoto et al., 2008b). Nitric oxide is

INTRODUCTION
Recent, growing interest in flavonoids has centered on
their beneficial properties against disease, and many epi-
demiological studies have suggested that a high flavonoid
intake is associated with a wide range of pharmacological
effects leading to a decreased risk of cardiovascular dis-
eases, inflammation and cancer (Jain and Parmar, 2011;
Lee et al., 2010; Morand et al., 2011).
Hesperidin and naringin are flavanone glycosides with
potent antioxidative and free radical scavenging activities,
and are major polyphenol components in citrus fruits and
peels (Wilmsen et al., 2005; El-Sayed et al., 2008). The water
solubility of hesperidin is less than 0.01%, however, and its
use is limited in pharmacological practice (Yamada et al.,
2006). Consequently, glucosyl hesperidin (G-hesperidin)
was synthesized and demonstrated water solubility about
10000 times greater than that of hesperidin with almost
the same antioxidant and biological activities in vitro as
hesperidin (Yamada et al., 2003).
More recently, hesperidin and G-hesperidin were
reported to depress the temporal increase in blood
pressure usually seen in spontaneously hypertensive rats
(SHR), possibly dependent on nitric oxide (NO)-related
* Correspondence to: Dr Yasuto Sasaki, Laboratory of Physiology,
Faculty of Nutrition, Kobe Gakuin University, 518 Arise Ikawadani-Cho,
Nishi-Ku, Kobe, 651-2180, Japan.
E-mail: sasakiya@nutr.kobegakuin.ac.jp
Copyright © 2012 John Wiley & Sons, Ltd.
believed to contribute to the prevention of pathogenesis
in cardiovascular diseases (Napoli and Ignarro, 2009).
Reactive oxygen species (ROS) decrease the bioavail-
ability of NO, and the subsequent oxidative stress causes
endothelial dysfunction and progressive atherogenesis
and thrombosis. Previous studies indicated that strong
antioxidants such as Ginkgo biloba extract (EGb 761)
and sesamin also suppressed the increase in systolic
blood pressure in stroke-prone spontaneously hyperten-
sive rats (SHRSP), improved endothelial function and
inhibited experimental thrombosis (Sasaki et al., 2002;
Noguchi et al., 2004).
The aim of present study was to clarify the role of
flavanone glycosides in reducing hypertension and endo-
thelial dysfunction and the prevention of cardiovascular
diseases. The study focused on the antithrombotic
effects of hesperidin, glucosyl hesperidin and naringin
in cerebral vessels of SHRSP.
MATERIALS AND METHODS
Animals and diets. Stroke-prone spontaneously hyper-
tensive rats (SHRSP), 6 weeks old, were purchased from
a local animal supplier (Japan SLC (Hamamatsu,
Japan)). All experiments were conducted in accordance
with the ethical principles of animal care of Kobe
Gakuin University and the guiding principles for the
Received 31 August 2011
Revised 06 November 2011
Accepted 08 November 2011
 M. IKEMURA ET AL.
care and use of animals in the field of physiological
sciences published by the Physiological Society of Japan
(Japan-Physiological-Society). The animals were eutha-
nized by an overdose of sodium pentobarbital after
experiments.
The powder diet for SHRSP was purchased from
Funabashi farm (Funabashi SP diet, Funabashi, Chiba, Japan).
Hesperidin, glucosyl hesperidin (G-hesperidin), and narin-
gin were kindly provided by Toyo Sugar Refining Co., Ltd,
Tokyo, Japan. The purity of hesperidin, G-hesperidin and
naringin were 95.5%, 78.8% and 88.9%, respectively.
A control group was given an unmodified powder diet.
Hesperidin and G-hesperidin were mixed with the powder
diet at concentrations of 825 and 250, 500, 1000, 2000 mg/
kg and fed for 4 weeks from 7 to 11 weeks of age.
Hesperidin at 825 mg/kg was determined to be equivalent
to G-hesperidin 1000 mg/kg by reference to glycosylation
and the purity of G-hesperidin. The naringin diet was simi-
larly prepared at concentrations of 250, 500, 1000 mg/kg.
The animals were given each diet and water ad libitum.
Each animal was kept in an individual cage and water
and food were changed at 9:00 am every day. Consumption
was assessed by weighing the diet and water on each
occasion. The daily average doses of G-hesperidin,
hesperidin, calculated from food intake in each group were
4.4  0.3 mg/kg/day (n = 6; G-hesperidin 250 mg/kg diet),
8.4  0.2 mg/kg/day (n = 6; G-hesperidin 500 mg/kg diet),
16.2  0.1 mg/kg/day (n = 6; G-hesperidin 1000 mg/kg diet),
31.6  0.2 mg/kg/day (n = 6; G-hesperidin 2000 mg/kg diet)
and 14.5  0.2 mg/kg/day (n = 6; hesperidin 825 mg/kg diet).
The daily average ingestion of naringin in each group was
4.4  0.3 mg/kg/day (n = 6; 250 mg/kg diet), 9.0  0.2 mg/
kg/day (n = 6; 500 mg/mg/kg diet) and 17.7  0.1 mg/kg/
day (n = 6; 1000 mg/kg diet).
Blood pressures and body weights were measured
once a week using a tail-cuff plethysmograph (LE5001,
Pan. Lab, Barcelona, Spain).
Measurement of thrombotic tendency. Closed cranial
windows were created as described previously by Morii
et al. (1986) and thrombotic tendency was measured as
reported (Sasaki et al., 2002). In brief, after anesthetizing
with sodium pentobarbital (60 mg/kg), the animal was
ventilated artificially. Evans blue was administered
through a femoral vein. A closed cranial window with
5 mm in diameter was formed in the right parietal bone
of the animal and artificial cerebrospinal fluid was infused
continuously within the cranial window to adjust the intra-
cranial pressure to 3–5 mmHg. The animals were placed
in a stereotaxic frame on the stage of an optical micro-
scope and the cerebral vessels on the surface of the brain
were monitored with a CCD camera (Pulnix, Takenaka
System, Kyoto) and recorded on videotape. A He-Ne
laser beam (15 mm in diameter) was focused on the center
of selected blood vessels through the optical path of the
microscope and thrombi were formed by repeated irradi-
ation for 10 s at 20 s intervals at a power of 13 mW for
venules (25–40 mm). The number of laser pulses required
to generate an occlusive thrombus was used as an index of
thrombotic tendency.
Measurement of urinary 8-hydroxy-2′-deoxyguanosine
(8-OHdG) and NO metabolites (nitrite/nitrate). Assays
were carried out before and after ingestion of each diet
for 4 weeks. The animals were kept in metabolic cages
and urine samples were collected for 24 h. The
Copyright © 2012 John Wiley & Sons, Ltd.
samples were stored at 70 C until assayed. 8-
Hydroxy-2′-deoxyguanosine (8-OHdG) was determined
using a competitive enzyme-linked immunosorbent assay
(ELISA; 8-OHdG check, Japan Institute for the Control
of Ageing, Shizuoka, Japan). Urinary nitrite/nitrate (NO2/
NO3) concentrations, metabolites of NO, were deter-
mined using Griess reagent (Assay kit-C; Dojindo La-
boratory, Kumamoto, Japan).
Measurement of vasodilation with acetylcholine. Ring
preparations of thoracic aorta were excised after the
experiments and dissected in ice cold Krebs-Henseleit
solution (KHS; composition in mmol/L: 120 NaCl, 4.76
KCl, 25 NaHCO3, 1.18 NaH2PO4
H2O, 1.25 CaCl2,
1.18 MgSO4
H2O, 5.5 glucose) to remove connective
tissue. Rings (3 mm wide) were cut and three seg-
ments from each animal were denuded of endothelium
by gently rubbing the lumen with a small cotton swab
and fine forceps under a stereomicroscope. Two aortic
segments from the same rat, one endothelium intact
and one endothelium-denuded, were mounted on
stainless steel hooks and suspended in a water-
jacketed 5 mL tissue bath filled with KHS maintained
at 37 C and aerated with 95% O2 and 5% CO2 (Easy
Magnus System U-5A (4-channels), Iwashiya Kishimoto
Medical Instruments, Kyoto, Japan). The aortic rings
were stretched to the equivalent of 1.5 g tension for
90 min before commencement of the experiment. After
stretching, the aortic rings were equilibrated and
contracted with phenylephrine (10-5 mol/L). Relaxation
was measured in the presence or absence of endothe-
lium, by adding acetylcholine (10-5 mol/L). Relaxation
was calculated as a percentage of precontractile vascu-
lar tone. The tissue responses were recorded using
isometric transducers (Easy Magnus System U-5A,
Iwashiya Kishimoto Medical Instruments, Kyoto, Japan)
and recorders (Sekonic SS-250F Recorder, Tokyo, Japan).
Statistical analyses. The results are denoted as the num-
ber of animals (n) and the mean and standard error
(SEM) of each experiment. Statistical evaluation was
performed by one-way analysis of variance (ANOVA)
and by the post hoc test of Dunnett and two-tailed un-
paired t-test using commercially available statistical
packages (Prism 5.0; GraphPad Software, Inc., San
Diego, CA, USA). A value of p < 0.05 was considered
to be statistically significant.
RESULTS
Body weights, systolic blood pressures
The body weights of SHRSP given hesperidin, G-hesperidin
and naringin increased over time up to the age of 11 weeks
as shown in Fig. 1A and 1B. Systolic blood pressures
increased with age, but hesperidin, G-hesperidin and nar-
ingin significantly suppressed this increase after treatment
for 4 weeks (Fig. 1C and 1D).
Effects of hesperidin, G-hesperidin and naringin on
cerebral thrombosis
The effects of hesperidin and G-hesperidin on throm-
botic tendency in pial microvessls, as assessed by a
Phytother. Res. (2012)
 ANTITHROMBOTIC EFFECTS OF HESPERIDIN, G-HESPERIDIN AND NARINGIN

A B

C D

Figure 1. The effects of hesperidin, G-hesperidin and naringin on body weight (A, B) and systolic blood pressure (C, D) in SHRSP. SHRSP
were purchased from a local supplier at 6 weeks old and acclimatized for a week. Hesperidin, G-hesperidin (GH) and naringin were fed from
7 weeks old. Body weights and systolic blood pressures were measured weekly for the 4 week period. Statistical analysis was performed by
one-way analysis of variance (ANOVA) and by post hoc test of Dunnett. *p < 0.05, **p < 0.01 vs control.

He-Ne laser technique, are shown in Fig. 2A. The num-
ber of laser pulses required to generate occlusive
thrombi in pial venules in control and hesperidin-treated
animals at 825 mg/kg were 6.3  0.3 (n = 6) and 7.7  0.2,
respectively. The figures for the G-hesperidin diet at 250,
500, 1000, 2000 mg/kg were 7.4  0.5 (n = 6), 7.6  0.3
(n = 6), 10.0  0.7 (n = 4) and 11.5  0.5 (n = 5), respect-
ively. The number of laser pulses in the hesperidin-
treated group at 825 mg/kg was significantly greater than
in the control animals (p < 0.01). Similarly, significant
differences were evident after the G-hesperidin diet at
doses greater than 500 mg /kg (p < 0.05 and p < 0.01).
The effect of naringin on thrombogenesis in cerebral
vessels is depicted in Fig. 2B. The number of laser pulses
required to generate occlusive thrombi in the control
5.6  0.2 (n = 6) and naringin-fed animals at 250, 500,
1000 mg/kg (6.5  0.4 (n = 6), 7.8  0.6 (n = 6) and
8.0  0.5 (n = 6), respectively), were significantly greater
after the 500 and 1000 mg/kg diet (p < 0.01).
Effects of hesperidin, G-hesperidin and naringin on
oxidative stress
The concentrations of 8-OHdG in urine collected for
24 h before and after the experimental diets, adjusted
for body weight, are illustrated in Fig. 3. There were
no significant differences in urinary 8-OHdG in the
control animals before and after the experimental diet.
The amount of urinary 8-OHdG in the hesperidin and
G-hesperidin groups after ingestion at 500, 1000,
2000 mg/kg diet and hesperidin at 825 mg/kg diet,
however, were significantly lower than before (Fig. 3A,
p < 0.05 and p < 0.01). Significant differences, before
and after treatment were also evident in the animals
given the 250, 500, 1000 mg/kg naringin diets (Fig. 3B,
p < 0.05 and p < 0.01).
Copyright © 2012 John Wiley & Sons, Ltd.
Effects of hesperidin, G-hesperidin and naringin on NO
metabolites (NO2/NO3)
The changes in NO metabolites before and after in-
gestion of hesperidin and G-hesperidin are shown in
Fig. 4. The NO2/NO3 levels in the control animals
were significantly lower after the 4-week experimental
period. Ingestion of either hesperidin at 825 mg/kg or
G-hesperidin at 500, 1000, 2000 mg/kg, however,
significantly increased NO metabolites (NO2/NO3)
(Fig. 4A, p < 0.05 and p < 0.01). Similarly, significant
differences were seen after naringin ingestion at
1000 mg/kg (p < 0.05).
Effects of hesperidin, G-hesperidin and naringin on
vascular relaxation
Ingestion of hesperidin, G-hesperidin and naringin for
4 weeks increased vascular responses. Significant increases
in percentage relaxation using 10-5 mol/L acetylcholine were
detected between the control, G-hesperidin (1000 mg/kg)
and hesperidin (825 mg/kg) groups. The figures were
59.0  3.6% (n = 6), 75.0  4.4% (n = 6; p < 0.05) and
74.5  1.7% (n = 6; p < 0.05), respectively. Similarly,
naringin at 1000 mg/kg day 78.0  6.1% (n = 6; p < 0.05)
increased ACh-dependent vascular relaxation compared
with the control 60.6  2.6% (n = 6).
DISCUSSION
The study determined the effects of a flavanone glycoside,
hesperidin, together with its water soluble derivative, G-
hesperidin and naringin on hypertension and cerebral
thrombogenesis in a stroke-prone spontaneously hyper-
tensive animal model. Yamada and colleagues reported
Phytother. Res. (2012)
 M. IKEMURA ET AL.
A A
B **
Figure 2. The effects of hesperidin, G-hesperidin (A) and naringin
(B) on cerebral thombogenesis in SHRSP. Cerebral thrombogenesis
were assessed after ingestion of hesperidin, G-hesperidin (GH) and
naringin for 4 weeks period using a He-Ne laser technique as
described in Materials and Methods. Statistical analysis was per-
formed by one-way analysis of variance (ANOVA) and by post
hoc test of Dunnett. *p < 0.05, **p < 0.01 vs control.
that hesperidin and G-hesperidin had similar metabolic
profiles in vivo, but that the putative metabolite, hespere-
tin, appeared more rapidly and in higher concentration in
sera and urine after ingestion of the water soluble product
than after oral administration of hesperidin (Yamada
et al., 2006; Yamamoto et al., 2008a). Furthermore,
Yamamoto et al. demonstrated that G-hesperidin and
hesperetin had similar effects on systolic blood pressure
and vascular endothelial function in SHR (Yamamoto
et al., 2008a). They showed that a single oral dose of G-
hesperidin (10 to 50 mg/kg) induced a dose-dependent
reduction in systolic blood pressure in SHR, but had no
effects in control (WKY) animals. Intraperitoneal injec-
tion of hesperetin (50 mg/kg) into SHR also caused a
significant reduction in systolic blood pressure. Moreover,
the moderating effects were significantly inhibited by an
NO synthase inhibitor and hesperetin enhanced endothe-
lium-dependent relaxation induced by acetylcholine, but
had no effect on endothelium-independent relaxation
induced by sodium nitroprusside in isolated SHR aortas.
These data suggested that the hypotensive effect of
hesperetin in SHR was associated with NO-mediated
vasodilation. Liu et al. also reported that naringenin and
hesperetin increased the production of NO from human
Copyright © 2012 John Wiley & Sons, Ltd.
*
**
* *
B *
**
Figure 3. The antioxidant effects of hesperidin, G-hesperidin (A)
and naringin (B). Oxidative stress was assessed by measurements
of urinary 8-OHdG samples collected for 24 h before and after in-
gestion of hesperidin, G-hesperidin (GH) and naringin for the
4 week period. Statistical analysis was performed by two-tailed un-
paired t-test. *p < 0.05, **p < 0.01 vs control.
endothelial cells in vitro (Liu et al., 2008). In addition,
Orallo et al. (2004) suggested that the vasorelaxant prop-
erties of hesperetin were related to inhibition of cyclic
nucleotide phosphodiesterase (PDE), showing especially
that hesperetin inhibited PDE isoforms PDE1 and
PDE4. Inhibition of PDE increases cyclic AMP and cyclic
GMP mediating the vasorelaxant effects on rat aortic
smooth muscle.
The current studies investigated the relatively longer
term cardiovascular effects of hesperidin, G-hesperidin
and naringin. The usual temporal increases in systolic
blood pressures in SHRSP were suppressed significantly
after daily administration of the flavonoids for 4 weeks.
In addition, the dietary supplements improved endothe-
lial function as assessed by acetylcholine-induced relax-
ation of aortic ring preparations. It was also shown that
metabolites of NO (nitrite/nitrate) were increased in
urine samples after ingestion of hesperidin, G-hesperi-
din and naringin, and these data were in keeping with
the earlier short-term studies (Yamamoto et al., 2008a).
Many studies have indicated that SHRSP are exposed
to high oxidative stress (Negishi et al., 1999; Mizutani
et al., 2000; Lee et al., 2004) and that this leads to
internal organ dysfunction (Manning et al., 2003). For
example, ROS contribute to the maintenance of hyper-
tension in SHRSP and are involved in mechanisms of
vascular dysfunction induced by oxidative DNA
damage (Negishi et al., 1999; Mizutani et al., 2000,
2001). Increased ROS concentrations reduce the amount
Phytother. Res. (2012)
 ANTITHROMBOTIC EFFECTS OF HESPERIDIN, G-HESPERIDIN AND NARINGIN
A * * *
* In addition, 8-OHdG levels in urine samples were
*
B Furthermore, Orallo et al. reported that hesperidin
*
**
Figure 4. The effects of hesperidin, G-hesperidin (A) and naringin
(B) on nitric oxide (NO) metabolites (NO2/NO3). NO2/NO3 was
measured in urinary samples collected for 24 h before and after
ingestion of hesperidin, G-hesperidin (GH) and naringin for the
4 week period using Griess reagent. Statistical analysis was per-
formed by two-tailed unpaired t-test. The NO metabolites were
significantly decreased in the control, however, were increased
4 weeks after ingestion of hesperidin, G-hesperidin and naringin.
*p < 0.05, **p < 0.01 vs control.
of bioactive NO by chemical inactivation to form toxic
peroxynitrite. Subsequently, peroxynitrite can ‘un-
couple’ endothelial NO synthase (e-NOS) to become a
dysfunctional superoxide-generating enzyme that contri-
butes to vascular oxidative stress. Hence, oxidative stress
and endothelial dysfunction promotes atherogenesis. As
discussed above, it was demonstrated that daily ingestion
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Acknowledgements
We would like to thank Dr IIda at Toyo Sugar Refining Co. Ltd, Tokyo
Japan for kindly providing the hesperidin, G-hesperidin and naringin.
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