Bioavailability of glucosyl hesperidin in rats

Article abstract of DOI:10.1271/bbb.50657

Glucosyl hesperidin (G-hesperidin) is a water-soluble derivative of hesperidin. We compared the absorption and metabolism of G-hesperidin with those of hesperidin in rats. After oral administration of G-hesperidin or hesperidin to rats, hesperetin was detected in sera hydrolyzed with β-glucuronidase, but it was not detectable in unhydrolyzed sera. Serum hesperetin was found more rapidly in rats administered G-hesperidin than in those administered hesperidin. The area under the concentration-time curve for hesperetin in the sera of rats administered G-hesperidin was approximately 3.7-fold greater than that of rats administered hesperidin. In the urine of both administration groups, hesperetin and its glucuronide were found. Urinary excretion of metabolites was higher in rats administered G-hesperidin than in those administered hesperidin. These results indicate that G-hesperidin presents the same metabolic profile as hesperidin. Moreover, it was concluded that G-hesperidin is absorbed more rapidly and efficiently than hesperidin, because of its high water solubility.

Full text of DOI:10.1271/bbb.50657

Biosci. Biotechnol. Biochem., 70 (6), 1386–1394, 2006
Bioavailability of Glucosyl Hesperidin in Rats
Mika YAMADA,y Fujimi TANABE, Norie ARAI, Hitoshi MITSUZUMI, Yoshikatsu MIWA,
Michio KUBOTA, Hiroto CHAEN, and Masayoshi KIBATA
Hayashibara Biochemical Laboratories, Inc., 1-2-3 Shimoishii, Okayama 700-0907, Japan
Received December 7, 2005; Accepted February 4, 2006; Online Publication, June 23, 2006
[doi:10.1271/bbb.50657]
Glucosyl hesperidin (G-hesperidin) is a water-soluble
derivative of hesperidin. We compared the absorption
and metabolism of G-hesperidin with those of hesper-
idin in rats. After oral administration of G-hesperidin
or hesperidin to rats, hesperetin was detected in sera
hydrolyzed with ꢀ-glucuronidase, but it was not detect-
able in unhydrolyzed sera. Serum hesperetin was found
more rapidly in rats administered G-hesperidin than
in those administered hesperidin. The area under the
concentration-time curve for hesperetin in the sera
of rats administered G-hesperidin was approximately
3.7-fold greater than that of rats administered hesper-
idin. In the urine of both administration groups,
hesperetin and its glucuronide were found. Urinary
excretion of metabolites was higher in rats administered
G-hesperidin than in those administered hesperidin.
These results indicate that G-hesperidin presents the
same metabolic profile as hesperidin. Moreover, it was
concluded that G-hesperidin is absorbed more rapidly
and efficiently than hesperidin, because of its high water
solubility.
G-Hesperidin becomes markedly more soluble. Its water
solubility is about 10,000 times greater than that of
hesperidin. G-Hesperidin has been reported to possess
almost the same antioxidant properties as hesperidin in
vitro.6) Moreover, it has been confirmed that the bio-
logical activities of G-hesperidin are the same or more
than those of hesperidin.7–9) Nishizaki et al. reported that
G-hesperidin and hesperidin dose-dependently inhibited
passive cutaneous anaphylaxis in rats, and that the effect
of G-hesperidin was more continuous than that of
hesperidin.7) Furthermore, Chiba et al. showed that G-
hesperidin and hesperidin significantly prevented bone
loss in the femur in ovariectomized mice.8) G-Hesper-
idin is susceptible to enzymatic hydrolysis by ꢀ-
glucosidases and this results in release of hesperidin
(Fig. 1).10) Hence, it is assumed that this compound
could behave as hesperidin in vivo. Several studies have
shown that orally administered hesperidin is hydrolyzed
by ꢁ-glucosidases to hesperetin aglycon before absorp-
tion (Fig. 1).11,12) Then absorbed hesperetin is immedi-
ately metabolized to glucuronide and sulfate conjugates
in the intestinal epithelium and liver.11,12) Matsumoto
et al. have determined that the conjugated metabolites in
rat plasma after oral administration of hesperidin are
comprised of hesperetin-7-O-ꢁ-D-glucuronide and hes-
peretin-30-O-ꢁ-D-glucuronide.12) In addition, homoerio-
dictyol conjugates as well as hesperetin conjugates have
been observed in rat plasma.12)
Key words: hesperidin; glucosyl hesperidin; hesperetin
conjugates; intestinal bacteria; bioavailabil-
ity
Chinpi, which is made of Satsuma mandarin peel
(Citrus unshiu Marc.), has traditionally been prescribed
as a natural medicine for inflammation, allergy, and
hepatopathy. It is well-known that hesperidin is one of
the primary constituents of Chinpi. Hesperidin is a
flavanone glycoside consisting of hesperetin aglycon
and disaccharide rutinose. It has multiple biological
activities such as reduction of capillary fragility,
associated with scurvy, and antilipemic activities.1–4)
But the difficulty with hesperidin is low water solubility,
less than 0.01%.
Flavonoids have been reported to function as their
metabolites, including conjugated aglycon, in the body.
For instance, it has been shown that quercetin conjugates
are detected in rat plasma after oral administration of
quercetin and that they exhibit antioxidative activi-
ties.13,14) Recently we found that G-hesperidin is
effective on hyperlipidemic subjects, suggesting that
this derivative functions as hesperetin conjugates in the
body.15)
Hijiya et al. have synthesized a water-soluble deriv-
ative of hesperidin, glucosyl hesperidin (G-hesperidin),
by regioselective transglycosylation with cyclodextrin
glucanotransferase from Bacillus stearothermophilus.5)
Many flavonoids are hardly soluble in water. There-
fore in the oral administration test, these flavonoids are
usually dissolved in vehicles such as carboxymethyl
cellulose sodium and propylene glycol to elevate their
y
To whom correspondence should be addressed. Fax: +81-86-221-6405; E-mail: kaihatsu@hayashibara.co.jp
Abbreviations: G-hesperidin, glucosyl hesperidin; AUC, area under the concentration-time curve; SGLT1, sodium-dependent glucose transporter 1

Bioavailability of Glucosyl Hesperidin
1387
OH
Hayashibara Biochemical Laboratories (Okayama,
Japan). Other chemicals were of analytical or HPLC
grade.
OCH
3
O
CH
OH
O
O
O
2
O
O
HO
CH
3
OH
Animals and diets. Male Sprague-Dawley rats (6
weeks old, Charles River Japan, Atsugi, Japan) were
housed in an air-conditioned room (23 ꢀ 2 ꢁC) under a
12 h light-dark cycle, with free access to tap water and
MF diet (Oriental Yeast, Tokyo). The present study was
approved by the Animal Use Committee of Hayashibara
Biochemical Laboratories, Inc., and the animals were
handled according to the Guidelines for the Care and
Use of Laboratory Animals. Rats were fasted overnight,
and 1 mmol/kg of G-hesperidin or hesperidin in 2 ml of
distilled water was administered orally by gastric
intubation. We have found that G-hesperidin is effective
on hyperlipidemic mice at this dose level per day.6)
Before and at 0.25, 0.5, 1, 3, 6, 9, 12, and 27 h after
administration, blood was withdrawn from the tail vein
into a MICROTAINERꢀ (Becton Dickinson, Franklin
Lakes, NJ). Serum was prepared by centrifugation at
10;000 ꢂ g for 2 min. Urine was collected in metabolic
cages the day before, and for 27 h after administration
of G-hesperidin or hesperidin, in five different fractions:
0–2, 2–4, 4–8, 8–11, and 11–27 h. These serum and
urine samples were stored at ꢃ20 ꢁC. The rats were not
allowed to feed until the last blood sampling.
OH OH
CH OH
OH
2
O
OH
O
HO
OH
G-Hesperidin
α-Glucosidase
OH
OCH
3
O
CH
OH
O
O
O
2
O
O
HO
CH
3
OH
HO
OH OH
OH
Hesperidin
β-Glucosidase
OH
OCH
3
HO
O
O
OH
HPLC analysis. Individual serum and urine samples
were divided into two aliquots. One aliquot of serum and
urine samples was used for the quantification of
metabolites released by hydrolysis with ꢁ-glucuroni-
dase, a deconjugation enzyme. For hydrolysis by ꢁ-
glucuronidase, serum and urine samples (50 ml) were
incubated with 500 units/ml ꢁ-glucuronidase, which
was prepared in 0.1 mM sodium acetate buffer (pH 5.0)
at 37 ꢁC for 2 h in the presence of 0.5 mM castanosper-
mine, an ꢀ-glucosidase inhibitor. Then these mixtures
were acidified with 0.1 ml of 0.01 M oxalic acid. The
other portions of serum and urine samples remained
without hydrolysis by ꢁ-glucuronidase and were used
for direct quantification of non-conjugated metabolites.
These samples were also acidified with 0.1 ml of 0.01 M
oxalic acid. After acidification, both hydrolyzed and
unhydrolyzed samples were applied to a Sep-Pak C18
cartridge (Waters, Milford, MA). The cartridge was
washed with 0.01 M oxalic acid, distilled water, and 25%
methanol, and then 1 ml of methanol eluate was
obtained. The eluate was evaporated to dryness, and
the residue was dissolved in 100 ml of methanol/water/
acetic acid (60:39:1, v/v/v). After filtration (0.45 mm
filter unit), the filtrate (10 ml) was injected onto an
Inertsil ODS-3 column (250 ꢂ 4:6 mm i.d., GL Sci-
ences, Tokyo) and analyzed by HPLC. The mobile phase
contained the following: solvent A, methanol/acetic
acid (100:1, v/v); solvent B, water/acetic acid (100:1,
v/v). The gradient program was as follows: the initial
composition was A/B = 35/65 for 0–5 min, followed
Hesperetin
Fig. 1. Enzymatic Hydrolysis of G-Hesperidin and Hesperidin.
absorption in the body. Quercetin dissolved in propylene
glycol has been found to be absorbed more efficiently
than that suspended in water.16) This means that the
extent of flavonoid absorption depends on solubility in
the vehicle used for administration. That is to say,
elevation of solubility is thought to increase the
bioavailability of supplements. Since G-hesperidin has
high water solubility, there is a possibility that this
derivative undergoes an increase in bioavailability.
The purpose of this study was to compare the
absorption and urinary excretion of G-hesperidin with
those of hesperidin in rats. Additionally, in order to
understand the metabolic fate of G-hesperidin further,
we examined the hydrolysis of this derivative by small
intestine homogenate and cecal content homogenate
from rats.
Materials and Methods
Chemicals. Hesperetin, ꢁ-glucuronidase type VII-A
(EC 3.2.1.31, from Escherichia coli), castanospermine,
p-nitrophenyl-ꢀ-glucoside, and p-nitrophenyl-ꢁ-gluco-
side were purchased from Sigma (St. Louis, MO).
Hesperidin was purchased from Wako Pure Chemical
(Osaka, Japan). G-Hesperidin was obtained from

1388
M. YAMADA et al.
by a liner gradient of 35/65–80/20 for 5–55 min. The
column temperature was kept at 40 ꢁC, and the flow rate
was 0.5 ml/min. The elution was monitored with a
photodiode array detector (Waters) at 285 nm. The
compounds existing in the samples were identified by
comparing their HPLC retention times (tR) with those of
standard G-hesperidin, hesperidin, and hesperetin, and
quantified by measuring the peak areas based on
calibration plots of the peak area of the standards.
Recoveries of these compounds from serum and urine by
this method were >95%. The detection limits for these
compounds were 0.05 mM.
1 ml of enzyme solution. After incubation for the
indicated times at 37 ꢁC, the hesperidin and hesperetin
released from G-hesperidin were determined by HPLC
analysis, as described above.
Data analysis. Values are given as the means ꢀ SD.
The significant difference between the means of two
groups was statistically analyzed by Mann-Whitney’s U
test. The significance level was set at p < 0:05 for all
tests. Statistical analysis was performed with Stat View
version 5.0 for Windows (SAS Institute, Cary, NC).
Results
Preparation of small intestine and cecal content
homogenates. Rats were sacrificed by diethyl ether
anesthesia. The small intestine was excised, rinsed with
cold Dulbecco’s phosphate-buffered saline (Ca2þ and
Mg2þ free), and homogenized in 4 volumes of 100 mM
potassium phosphate buffer (pH 7.0) with a glass-Teflon
homogenizer. The homogenate was centrifuged at
3;000 ꢂ g for 15 min, and the supernatant was used as
an enzyme source. The cecum was removed, and its
contents were drained by finger pressure into a tube.
The cecal contents thus obtained were suspended in 4
volumes of 100 mM potassium phosphate buffer (pH 7.0)
and sonicated for 5 min. After centrifugation at 400 ꢂ g
for 5 min, the supernatant was used as an enzyme source.
Quantification of the metabolites derived from G-
hesperidin and hesperidin in rat serum
The metabolites of flavonoids have frequently been
reported to be present in blood circulation in the form of
glucuronide, sulfate, and methylate conjugates.12,19) To
examine the absorption and metabolism of G-hesperidin
and hesperidin in vivo, each of these compounds was
orally administered to rats. In the case of hesperidin
administration, the conjugates circulating in the blood
were reported to be mainly glucuronides, and, although
minor in quantity, sulfate/glucuronide conjugates were
also detected.12) Therefore in this study, the sera of rats
were subjected to hydrolysis with ꢁ-glucuronidase only,
and this method did not allow us to distinguish the
number and position of conjugated moieties.
Figure 2 shows representative chromatograms of
standards and ꢁ-glucuronidase hydrolysate of rat serum
taken after oral administration of G-hesperidin or
hesperidin. In both administration groups, hesperetin
(tR ¼ 48:4 min) was detected in the hydrolysate (Fig. 2B
and C). A peak (tR ¼ 46:7 min) was assumed to be
homoeriodictyol based on the study of Matsumoto et
al.12) A peak corresponding to hesperetin was not
obtained in the hydrolyate of rat serum before oral
administration of G-hesperidin or hesperidin (data not
shown).
Assay for glucosidase activities. The ꢀ-glucosidase
activity of small intestine and cecal content homoge-
nates was estimated by measuring the rate of formation
of p-nitrophenol from p-nitrophenyl-ꢀ-glucoside.17) Re-
action mixtures (2 ml) consisted of 50 mM potassium
phosphate buffer (pH 7.0), 5 mM p-nitrophenyl-ꢀ-gluco-
side, and a sample of enzyme solution, and were
incubated at 37 ꢁC for 15 min. The p-nitrophenol
liberated was determined by measuring the absorbance
at 400 nm in the presence of 100 mM Na2CO3. One unit
of ꢀ-glucosidase activity was defined as the amount of
enzyme that generates 1 mmol of p-nitrophenol per min
under the conditions mentioned above.
The serum kinetics of hesperetin–glucuronide is
shown in Fig. 3. After dosing with hesperidin, hesper-
etin–glucuronide was detected at 6 h, and reached a
maximum (about 1.3 mM) at 9–12 h. On the other hand,
in the administration of G-hesperidin, hesperetin–glu-
curonide was detected as early as 15 min after admin-
istration, and reached a maximum (6.3 mM) at 6 h. The
serum concentration of hesperetin conjugate observed
after administration of G-hesperidin was higher than that
of hesperidin throughout the experimental period, except
at 27 h. In particular, the differences in the serum
concentrations of the conjugate between the adminis-
tration of G-hesperidin and hesperidin were significant
until 9 h. The area under the concentration-time curve
(AUC) for hesperetin–glucuronide in the sera of rats
administered G-hesperidin was approximately 3.7-fold
higher than that of rats administered hesperidin
(Table 1). After hydrolysis with ꢁ-glucuronidase, a high
The ꢁ-glucosidase activity of small intestine and cecal
content homogenates was determined using p-nitro-
phenyl-ꢁ-glucoside as a substrate.18) Reaction mixtures
(2 ml) consisted of 50 mM sodium acetate buffer
(pH 5.0), 5 mM p-nitrophenyl-ꢁ-glucoside, and a sample
of enzyme solution, and were incubated at 37 ꢁC for
15 min. The p-nitrophenol liberated was determined as
described above. One unit of ꢁ-glucosidase activity was
defined as the amount of enzyme that produces 1 mmol
of p-nitrophenol per min under the assay conditions.
Hydrolysis of G-hesperidin by small intestine and
cecal content homogenates. The hydrolytic ability of
small intestine and cecal content homogenates toward
G-hesperidin was examined by measuring the amounts
of hesperidin and hesperetin released. Reaction mixtures
were composed of 1 ml of 2 mg/ml G-hesperidin and

Bioavailability of Glucosyl Hesperidin
1389
0.015
0.010
0.005
A
B
C
0.000
0.015
0.010
0.005
0.000
0.015
0.010
0.005
0.000
25
30
35
40
45
50
55
Retention time (min)
Fig. 2. Representative HPLC Chromatograms of Standard G-Hesperidin, Hesperidin, and Hesperetin (A), and ꢁ-Glucuronidase Hydrolysate of Rat
Serum Taken at 6 h after Oral Administration of 1 mmol/kg Hesperidin (B) or G-Hesperidin (C).
Hesperetin-Glucuronide
10
G-Hesperidin
Hesperidin
**
**
5
**
**
**
**
0
0
3
6
9
12
27
Time after administration (h)
Fig. 3. Changes in the Serum Concentration of Hesperetin-Glucuronide in Rats Given G-Hesperidin or Hesperidin.
G-Hesperidin ( ) or hesperidin ( ) was given to rats by gastric intubation (1 mmol/kg). Serum samples were treated with ꢁ-glucuronidase.
ꢄꢄ
Values are the means ꢀ SD (n ¼ 5). Significantly different from the value of hesperidin administration at p < 0:01.

1390
M. YAMADA et al.
Table 1. Total Area under the Serum Concentration-Time Curve of
Hesperetin-Glucuronide in Rats Administered G-Hesperidin or Hes-
peridin
(about 586 mM) between 8 and 11 h. A similar tendency
was observed in the administration of G-hesperidin:
hesperetin–glucuronide was detected between 0 and 2 h,
and reached a maximum (about 829 mM) between 8 and
11 h. The urine concentration of hesperetin conjugate
was higher in rats administered G-hesperidin than in
those administered hesperidin throughout the experi-
mental period. The differences in urinary excretion of
the conjugate between the administration of G-hesper-
idin and hesperidin were especially significant until 8 h.
In addition, hesperetin was detected in the unhydrolyzed
urine of rats administered G-hesperidin or hesperidin.
The changes in cumulative urinary excretion of non-
conjugated hesperetin are shown in Fig. 5B. After
supplementation with hesperidin, free hesperetin was
detected between 0 and 2 h, and culminated (at about
115 mM) between 8 and 11 h. Similarly, G-hesperidin
administration also led to detection of free hesperetin
between 0 and 2 h, and its urine level culminated (at
about 661 mM) between 8 and 11 h. The urine concen-
tration of non-conjugated hesperetin was higher in rats
administered G-hesperidin than in those administered
hesperidin throughout the experimental period (p < 0:05
in urine collected between 2 and 4 h, and p < 0:01 in
urine collected between 8 and 11 h).
Supplement
AUC 0 ! 27 h (mM)
G-Hesperidin
Hesperidin
67:6 ꢀ 12:0ꢄꢄ
18:2 ꢀ 7:3
Values are the means ꢀ SD (n ¼ 5). ꢄꢄ Significantly different from the value
of hesperidin administration at p < 0:01.
level of hesperidin was detected in the serum of one out
of five rats administered G-hesperidin (data not shown).
In contrast, no free hesperetin, hesperidin, or G-
hesperidin was detected in the unhydrolyzed sera of
rats administered G-hesperidin or hesperidin.
Quantification of the metabolites derived from G-
hesperidin and hesperidin in rat urine
In order to determine further the metabolic fate and
bioavailability of G-hesperidin and hesperidin, we
examined the excretion of metabolites in rat urine after
administration of both compounds. Figure 4 shows
representative chromatograms of ꢁ-glucuronidase hy-
drolysate of rat urine taken after oral administration of
G-hesperidin or hesperidin. Hesperetin (tR ¼ 48:4 min)
and homoeriodictyol (tR ¼ 46:7 min) were detected in
the hydrolyzed urine (Fig. 4A and B). The peak
corresponding to hesperetin was not detected in the
hydrolysate of rat urine before oral administration of
G-hesperidin or hesperidin (data not shown).
Moreover, glucuronized hesperidin and non-conjugat-
ed hesperidin were found in the urine until 4 h after
administration of G-hesperidin, but they were not found
in the case of hesperidin administration (Fig. 5C and D).
In the urine of rats administered G-hesperidin, the
excretion of hesperidin conjugate and free hesperidin
was earlier than that of hesperetin conjugate and free
hesperetin respectively. Furthermore, the levels of
hesperidin conjugate and free hesperidin were in lower
The changes in cumulative urinary excretion of
hesperetin–glucuronide are shown in Fig. 5A. After
dosing with hesperidin, hesperetin–glucuronide was
detected between 0 and 2 h, and reached a maximum
1.00
A
0.75
0.50
0.25
0.00
1.00
B
0.75
0.50
0.25
0.00
30
35
40
45
50
Retention time (min)
Fig. 4. Representative HPLC Chromatograms of ꢁ-Glucuronidase Hydrolysate of Rat Urine Taken between 4 and 8 h after Oral Administration of
1 mmol/kg Hesperidin (A) or G-Hesperidin (B).

Bioavailability of Glucosyl Hesperidin
1391
B
A
2,000
Hesperetin-Glucuronide
G-Hesperidin
Hesperidin
Hesperetin
2,000
1,500
1,000
500
1,500
1,000
500
0
**
**
**
**
*
0
0
2
4
8
11
27
before
0
2
4
8
11
27
before
C
D
60
Hesperidin
Hesperidin-Glucuronide
60
**
**
40
20
0
40
20
0
**
*
0
2
4
8
11
Time after administration (h)
Fig. 5. Changes in the Urinary Excretion of Hesperetin and Hesperidin in Rats Given G-Hesperidin or Hesperidin.
27
0
2
4
8
11
27
before
before
Time after administration (h)
G-Hesperidin ( ) or hesperidin ( ) was given to rats by gastric intubation (1 mmol/kg). The respective panels represent the concentrations of
hesperetin–glucuronide (A), non-conjugated hesperetin (B), hesperidin–glucuronide (C), and non-conjugated hesperidin (D) in the urine. Values
are the means ꢀ SD (n ¼ 11). ꢄ and ꢄꢄ Significantly different from the value of hesperidin administration at p < 0:05 and p < 0:01 respectively.
Table 2. Glucosidase Activities of Small Intestine and Cecal Content
concentrations than the respective forms of hesperetin in
the urine of rats administered G-hesperidin.
Homogenates from Rats
Enzyme Activity (units/ml)
Hydrolysis of G-hesperidin by small intestine and
cecal content homogenates
ꢀ-Glucosidase
ꢁ-Glucosidase
Small Intestine
Cecal Contents
0.104
0.110
0.039
0.201
It has been reported that G-hesperidin is hydrolyzed to
hesperidin by ꢀ-glucosidases (Fig. 1).10) Furthermore, it
has been explained that orally administered hesperidin is
hydrolyzed to hesperetin by ꢁ-glucosidases from intes-
tinal bacteria and then released hesperetin is absorbed
into blood (Fig. 1).1,11,12,20) We observed that G-hesper-
idin administration as well as hesperidin administration
led to detection of hesperetin conjugate in rat serum
(Fig. 3). Therefore, G-hesperidin was considered to be
finally hydrolyzed to hesperetin via hesperidin in the
intestinal tract. In order to clarify this further, small
intestine and cecal content homogenates from rats were
examined for their hydrolytic ability toward G-hesper-
idin.
One unit of ꢀ- or ꢁ-glucosidase activity was defined as the amount of
enzyme that produces 1 mmol of p-nitrophenol per min under the assay
conditions.
G-hesperidin at 37 ꢁC. As a result, the small intestine
homogenate completely hydrolyzed G-hesperidin to
hesperidin at 1 h (Fig. 6). Moreover, the cecal content
homogenate generated not only hesperidin but also
hesperetin for 4 h (Fig. 6).
Discussion
As shown in Table 2, high activity of ꢀ-glucosidase
was detected in small intestine homogenate, but ꢁ-
glucosidase activity was extremely low in this homoge-
nate. On the other hand, cecal content homogenate
showed high activities of ꢀ- and ꢁ-glucosidases, which
probably derived from intestinal bacteria. These homo-
genates were used as enzyme sources and incubated with
G-Hesperidin, a water-soluble derivative of hesper-
idin, has been shown to exhibit the same biological
activities as hesperidin in vivo,7–9) but the behavior of
this derivative in the body remains to be fully clarified.
In this study, we compared the metabolic fate and the
bioavailability of G-hesperidin with those of hesperidin
in rats.

1392
M. YAMADA et al.
A
B
2,000
1,600
1,200
800
2,000
G-Hesperidin
Hesperidin
Hesperetin
1,600
1,200
800
400
0
400
0
0
1
0
1
4
Incubation time (h)
Incubation time (h)
Fig. 6. Hydrolysis of G-Hesperidin by Small Intestine and Cecal Content Homogenates from Rats.
Small intestine (A) and cecal content (B) homogenates from rats were used as enzyme sources. These enzyme samples were incubated with
G-hesperidin (1 mg/ml) at 37 ꢁC for the indicated times, and the reaction products were determined by HPLC.
After dosing with G-hesperidin or hesperidin, hesper-
etin–glucuronide was found in the sera of both admin-
istration groups (Fig. 3). Moreover, hesperetin–glucu-
ronide (Fig. 5A) and non-conjugated hesperetin
(Fig. 5B) were excreted in the urine of both adminis-
tration groups. These results indicate that G-hesperidin
presents the same metabolic profile as hesperidin. It has
been reported that orally administered hesperidin is
absorbed as hesperetin after hydrolysis by ꢁ-glucosi-
dases from intestinal microflora, and that absorbed
hesperetin is mainly metabolized to glucuronide con-
jugate in the intestinal tissue and liver.12) It appears
likely that G-Hesperidin is converted to hesperidin in the
intestinal tract, thereby causing the metabolism men-
tioned above.
Serum hesperetin–glucuronide was found sooner in
rats administered G-hesperidin than in those adminis-
tered hesperidin (Fig. 3), and furthermore, AUC for this
conjugate was more pronounced in the administration
of G-hesperidin (Table 1). We also observed that the
urinary excretion of metabolites was higher in rats
administered G-hesperidin than in those administered
hesperidin (Fig. 5). These observations indicate that
G-hesperidin is absorbed more efficiently than hesper-
idin. It has been reported that the extent of flavonoid
absorption is affected by its solubility in the digestion
process.16,21) According to the report, quercetin, which is
hardly soluble in water, is elevated in absorption after
administration to rats when it is dissolved in propylene
glycol.16) Therefore, it is thought that the elevated
absorption efficiency of G-hesperidin is due to its high
water solubility.
and cecal content homogenates from rats. It was
observed that G-hesperidin was hydrolyzed to hesper-
idin by small intestine homogenate containing abundant
ꢀ-glucosidases (Table 2 and Fig. 6). On the other hand,
this derivative was hydrolyzed to hesperidin and
hesperetin in an ordered way by cecal content homoge-
nate containing both ꢀ- and ꢁ-glucosidases (Table 2 and
Fig. 6). ꢀ-Glucosidases have been demonstrated to exist
in the brush border of the small intestine.22) Moreover, it
has been shown that cecal contents have an abundance
of intestinal bacteria and that this microflora produces ꢁ-
glucosidases as well as ꢀ-glucosidases.23,24) Further-
more, Kometani et al. have reported that G-hesperidin is
hydrolyzed to hesperidin by ꢀ-glucosidases, but that it
can not undergo the action of ꢁ-glucosidases.10) On the
basis of these findings, our observations suggest that
orally administered G-hesperidin is initially hydrolyzed
to hesperidin by small intestinal ꢀ-glucosidases, and
that released hesperidin is subsequently hydrolyzed to
hesperetin by ꢁ-glucosidases from intestinal bacteria.
In Fig. 6, G-hesperidin released hesperidin just after
the addition of homogenates (0 h). As Kometani et al.
have explained, G-hesperidin has a high susceptibility
to ꢀ-glucosidases.10) Hence it was assumed that this
derivative experienced rapid hydrolysis in the presence
of the enzymes. Moreover, the initial concentration of
G-hesperidin in the reaction with cecal content homo-
genate was slightly lower than that in the reaction with
small intestine homogenate (Fig. 6). This difference
suggests that G-hesperidin partially binds to cecal
contents.
The reason G-hesperidin is efficiently absorbed
remains unclear, but there is a possibility that an active
transport system of glucose by sodium-dependent
Additionally, the profile of G-hesperidin hydrolysis in
the intestinal tract was investigated using small intestine

Bioavailability of Glucosyl Hesperidin
1393
glucose transporter 1 (SGLT1) is involved in the high
absorption efficiency of G-hesperidin. Quercetin glyco-
sides, which have been well investigated as to their
absorption and metabolism, have been verified to be
absorbed rapidly in preference to quercetin agly-
con.25–27) As for the mechanism of this phenomenon,
Hollman et al. have suggested that the aglycon moiety of
quercetin glycosides, namely the quercetin group, might
be drawn into the enterocytes by its glucose moiety
through SGLT1.25) In addition, Day et al. have explained
that lactase phlorizin hydrolase, a ꢁ-glucosidase existing
on the brush border of the small intestine, might
contribute to the rapid absorption of quercetin glyco-
sides in association with SGLT1.27) Similar mechanisms
might lie in the absorption process of G-hesperidin,
because this derivative possesses one glucose molecule
introduced by transglycosylation and efficiently releases
hesperidin, a substrate for ꢁ-glucosidases, by hydrolysis
in the small intestine.
Recently, Kim et al. reported that G-hesperidin
permeated across the Caco-2 cell monolayer, a model
of the small intestinal epithelium, via the paracellular
pathway, but hesperidin did not.28) In this case, the
difference in permeability between G-hesperidin and
hesperidin was considered to be due to the water
solubility of these compounds.28) Such a passive trans-
port system might explain the high absorbability of G-
hesperidin after administration to rats. Nevertheless, the
administration of G-hesperidin did not lead to the
detection of its intact form in rat serum (Fig. 3).
Permeated G-hesperidin might be hydrolyzed to hesper-
etin by ꢀ- and ꢁ-glucosidases derived from enterocytes
during its transepithelial transport in vivo.
In the present study, not only hesperetin and its
conjugate but also hesperidin and its conjugate were
detected in the urine of rats administered G-hesperidin
(Fig. 5). This result suggests the possibility that hesper-
idin released from G-hesperidin is directly absorbed
across the brush border. As mentioned above, it is
thought that orally administered G-hesperidin is hydro-
lyzed to hesperidin and glucose by ꢀ-glucosidases on the
brush border of the small intestine. It appears likely that
a portion of released hesperidin is deglycosylated to
hesperetin by ꢁ-glucosidases from intestinal bacteria or
epitheliocytes and is subsequently absorbed across the
intestinal epithelium. In contrast, another part of
released hesperidin is probably transported into the
enterocytes without deglycosylation. Furthermore, it is
probable that the transported hesperidin, which is
partially conjugated, is immediately removed from
blood circulation and excreted into the urine without
renal tubular reabsorption. We also observed that
hesperidin conjugate was present in the serum of one
out of five rats administered G-hesperidin, supporting
the conclusion that released hesperidin is partially
absorbed in the intact form across the brush border.
It has been reported that hesperidin is not toxic in
high-dose and long-term administration studies.1,29,30)
On the basis of these findings, hesperidin has been
regarded as an extremely safe compound. The safety of
G-hesperidin has also been confirmed in long-term
administration studies using mice and rats.6,31) In a
human study, no clinical changes related to 24-week
administration of G-hesperidin were observed in sub-
jects.32) Hence the safety of G-hesperidin is considered
to be comparable to that of hesperidin. Additionally, we
observed that the metabolites, such as hesperetin,
hesperidin, and their conjugates, were rapidly excreted
in rat urine after administration of G-hesperidin (Fig. 5).
This observation confirms the safety of G-hesperidin.
Acknowledgments
We would like to thank Mr. Osamu Sano and Mr.
Kanso Iwaki for their excellent technical assistance in
this study.
This work was supported in part by the Technical
Development Program for Agribusiness by Utilizing
Concentrated Know-how from the Private Sector of
the Ministry of Agriculture, Forestry, and Fisheries of
Japan.
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