Studies on absorption and hydrolysis of ethyl α-D-glucoside in rat intestine

Article abstract of DOI:10.1021/jf0508753

Ethyl α-D-glucoside (α-EG) is normally contained in Sake, which has been taken by Japanese people since ancient times. In this study, the intestinal absorption of α-EG was investigated using rat everted intestinal sac. Furthermore, the α-EG hydrolytic activity in rat intestine was compared with disaccharides hydrolytic activities, and the effects of α-EG on disaccharides hydrolysis were examined using crude enzyme preparation from rat intestinal acetone powder. Glucose liberated from α-EG was detected in a serosal solution of everted rat intestinal sac, but it was only less than 4% of absorbed intact α-EG. α-EG absorption into small intestinal tissue was reduced by elimination of sodium ion from the mucosal solution or under the presence of phlorizin. The hydrolytic activity for α-EG was detected in crude enzyme preparation from rat intestinal acetone powder, but it showed a low value as compared to those for disaccharides. α-EG showed mixed type inhibition for maltose and sucrose hydrolysis, but inhibitory concentrations of α-EG required for 50% inhibition for the maltose and sucrose hydrolysis were higher than those of arabinose and acarbose. In conclusion, a small amount of α-EG was hydrolyzed and most of it was absorbed via SGLT1 as an intact form in the rat small intestine, and the inhibitory effect of α-EG on disaccharides hydrolysis was weak.

Full text of DOI:10.1021/jf0508753

J. Agric. Food Chem. 2005, 53, 7257−7261 7257
Studies on Absorption and Hydrolysis of Ethyl
in Rat Intestine
r- -Glucoside
D
TOMOYUKI MISHIMA,*,† KEIKO TANAKA,‡ HARUHITO TSUGE,‡,§ JUN SUGITA,|
†
MICHIO NAKAHARA,| AND TAKASHI HAYAKAWA
The United Graduate School of Agricultural Science and Faculty of Agriculture, Gifu University,
1-1 Yanagido, Gifu 501-1193, Japan, and Basic Research Laboratory, Kanebo Cosmetics Inc.,
Odawara, Kanagawa 250-0002, Japan
Ethyl R-D-glucoside (R-EG) is normally contained in Sake, which has been taken by Japanese people
since ancient times. In this study, the intestinal absorption of R-EG was investigated using rat everted
intestinal sac. Furthermore, the R-EG hydrolytic activity in rat intestine was compared with
disaccharides hydrolytic activities, and the effects of R-EG on disaccharides hydrolysis were examined
using crude enzyme preparation from rat intestinal acetone powder. Glucose liberated from R-EG
was detected in a serosal solution of everted rat intestinal sac, but it was only less than 4% of absorbed
intact R-EG. R-EG absorption into small intestinal tissue was reduced by elimination of sodium ion
from the mucosal solution or under the presence of phlorizin. The hydrolytic activity for R-EG was
detected in crude enzyme preparation from rat intestinal acetone powder, but it showed a low value
as compared to those for disaccharides. R-EG showed mixed type inhibition for maltose and sucrose
hydrolysis, but inhibitory concentrations of R-EG required for 50% inhibition for the maltose and sucrose
hydrolysis were higher than those of arabinose and acarbose. In conclusion, a small amount of R-EG
was hydrolyzed and most of it was absorbed via SGLT1 as an intact form in the rat small intestine,
and the inhibitory effect of R-EG on disaccharides hydrolysis was weak.
KEYWORDS: Ethyl r-D-glucoside; absorption; hydrolysis; glucose transporter; intestine; rat
INTRODUCTION
in Sake, was hydrolyzed by sucrase in the sucrase-isomaltase
complex in rat intestine. Vitamin glucosides (11, 12) and
flavonoid glucosides (13) were completely or incompletely
hydrolyzed in the animal body, and then, they provide glucose
and aglycons. On the other hand, methyl R-D-glucoside (R-MG),
nonmetabolic glucoside, is usable for analyzing the transport
activity of sodium-dependent glucose transporters (SGLTs) (14-
16).
Ethyl R-D-glucoside (R-EG) is normally contained in Sake
(1-4), which has been taken by Japanese people since ancient
times as an alcoholic beverage and used in cooking as a flavor
enhancer. In our previous study, R-EG was detected in plasma
and urine of rats fed an R-EG solution (5). Imanari and Tamura
(2) and Teague et al. (6) showed that R-EG was detected in the
urine of a man who took Sake or rice wine. Thus, R-EG was
not hydrolyzed completely in the animal body. On the other
hand, ultraviolet B (UVB)-induced epidermal barrier disruption
was reduced by oral administration of R-EG or Sake concentrate
containing R-EG on mice (7), which suggested that R-EG
showed physiological effects in the animal body.
Released monosaccharides are absorbed through glucose
transporters [SGLT and facilitative glucose transporters (GLUTs)]
distributed in the small intestinal membrane (17, 18). Brush
border membrane transport of monosaccharides in small intes-
tinal mucosa proceeds via SGLT1 and GLUT5, and basolateral
membrane transport is also mediated by GLUT2 (19, 20).
Landau et al. demonstrated that R-EG was effectively concen-
trated in everted hamster intestinal sacs (21). Ramaswamy et
al. demonstrated that hamster intestinal SGLT1 showed affinity
to â-alkyl glucosides, and the affinity of alkyl glucosides for
SGLT1 increased with the increase in the length of the alkyl
chain (22). Kipp et al. also demonstrated that hog renal SGLTs
showed differences of affinity to alkyl chain length in alkyl
glucosides (23). R-EG might be actively transported via SGLT1
in the small intestine.
In general, disaccharides are hydrolyzed by R-glucosidases
localized in the small intestinal mucosa (8, 9). Some glucosides
were hydrolyzed by enzymes distributed in the small intestine
as well as disaccharides. According to Takenaka and Uchiyama
(10), (2S)-1-O-R-D-glucosylglycerol, which was also contained
* To whom correspondence should be addressed. Tel: +81-58-293-2929.
Fax: +81-58-293-2840. E-mail: h6103007@cc.gifu-u.ac.jp.
† The United Graduate School of Agricultural Science, Gifu University.
‡ Faculty of Agriculture, Gifu University.
§ Present address: Faculty of Food Culture, Kurashiki Sakuyou Univer-
sity, 3515 Tamashima-Nagao, Kurashiki 710-0292, Japan.
| Kanebo Cosmetics Inc..
In the present study, we examined the intestinal absorption
of R-EG by rat everted intestinal sacs. Moreover, we determined
10.1021/jf0508753 CCC: $30.25
© 2005 American Chemical Society
Published on Web 08/09/2005

7258 J. Agric. Food Chem., Vol. 53, No. 18, 2005
Mishima et al.
R-EG and disaccharides hydrolytic activities and effects of R-EG
on disaccharides hydrolysis using crude enzyme preparation
from rat intestinal acetone powder.
MATERIALS AND METHODS
Reagents and Animals. Reagent grade R-EG (purity > 98%) was
prepared by Kanebo Cosmetics Inc. (Kanagawa, Japan). Acarbose,
R-MG, R-glucosidase (EC 3.2.1.20) from Saccharomyces sp., and
Glucose B-Test Wako were purchased from Wako Pure Chemicals Co.
Ltd. (Osaka, Japan). Rat intestinal acetone powder was purchased from
Sigma Chemical Co. (St. Louis, MO). Other reagents were purchased
from Nacalai Tesque Inc. (Kyoto, Japan). Male Wistar ST clean rats
were purchased from Japan SLC Inc. (Hamamatsu, Japan), and care
and use of the rats in the present study followed the guidelines of
governmental legislation in Japan on the proper use of laboratory
animals (1980), and animal experiments were approved by the ethical
committee of the Faculty of Agriculture in Gifu University (Gifu,
Japan).
Figure 1. Absorption of
serosal solution of everted rat intestinal sac was examined. Everted rat
intestinal sacs were incubated for 30 min in the Krebs Ringer buffer
containing 20 mM -EG. -EG absorption was calculated by subtracting
glucose from totally absorbed sugar (glucose plus -EG). Significant
difference was indicated by asterisk (**, P < 0.01). Data showed means
standard errors (n 6).
R-EG and glucose liberated from R-EG into
−
R
R
R
±
)
concentrations with the same buffer. After preincubation of 200 µL of
each sugar solution for 5 min, the reaction was started by adding 100
µL of crude enzyme solution. Incubation was performed at 37 °C for
180 min for R-EG and R-MG or 30 min for disaccharides. To stop the
reaction, 100 µL of 1 N NaOH was added and then neutralized with 1
N HCl. The reaction was performed in triplicate. After centrifugation
at 12000g for 10 min with centrifuge MR-150, and the supernatant
was used for glucose determination by Glucose B-Test Wako. The
hydrolyzing activity for maltose was expressed as half the amount
(µmol) of glucose liberated per mg protein per hour, since 1 mol of
maltose is hydrolyzed to liberate 2 mol of glucose.
Preparation of Everted Rat Intestinal Sacs. Wistar ST rats,
approximately 8-12 weeks old and approximately weighing 200-350
g, were killed by drawing blood under anesthesia, and the small intestine
was excised immediately and rinsed briefly with cold 0.9% KCl. The
preparation of everted rat intestinal sacs was performed by the method
described by Wilson and Wiseman (24). The adherent mesenteric tissue
and fat were trimmed off from the jejunum region. The jejunum was
divided into tubes of similar size (4 cm in length) and then everted.
Both ends of these tubes were bound tightly with silk string after
inclusion of 500 µL of Krebs-Ringer buffer. R-EG absorption and
sugar determination methods were performed as described in a previous
study (5). In brief, the everted intestine was dipped into Krebs-Ringer
buffer containing 20 mM R-EG, which was bubbled with O2/CO2 (95:
5, v/v) gas adequately. After incubation for 30 min, glucose in the
serosal solution was determined by glucose test kit (Glucose B-Test
Wako). The total sugar (R-EG plus glucose) in the serosal solution
was determined by the same kit after hydrolysis by R-glucosidase from
Saccharomyces sp. R-EG absorbed was calculated by subtracting the
glucose from the total sugar. The absorption of R-EG was expressed
as the µmol of R-EG absorbed per 100 mg of small intestine.
Time Course of r-EG Absorption by Everted Rat Intestinal Sacs.
Intestinal sacs were incubated in each experimental solution containing
20 mM R-EG as indicated in parentheses: control (Krebs-Ringer
buffer), Na+-free (Krebs-Ringer buffer replaced sodium ion with
equimolar amount of potassium ion), and phlorizin (Krebs-Ringer
buffer containing 1.55 mM phlorizin). After incubation for 0 (blank),
1, 5, 15, 30, or 45 min at 37 °C, the serosal solution was collected in
a sample tube to determine the absorbed sugars. The same volume (v/
w) of ice-cold Krebs-Ringer buffer was added to the residual intestinal
tissue and then homogenized with a HG30 homogenizer (HITACHI
High-Technologies Co., Tokyo, Japan). After centrifugation at 12000g
for 10 min at 4 °C with centrifuge MR-150 (TOMY SEIKO Co., Ltd.),
the supernatant was collected in a sample tube. Sugars in serosal solution
and supernatant from tissue homogenate were determined by the method
described above.
Preparation of Crude Enzyme Solution from Rat Intestinal
Acetone Powder. The crude enzyme solution was prepared from rat
intestinal acetone powder as described by Takenaka and Uchiyama (10).
The acetone powder was suspended with 10 volumes (v/w) of 0.2 M
sodium phosphate buffer (pH 6.0) and then homogenized with a HG30
homogenizer. The homogenate was sonicated for 15 min using a Sharp
UT-104 Silentsonic (Sharp Manufacturing System Co., Osaka, Japan)
in an ice bath. To remove large particles, the homogenate was
centrifuged at 3000g for 30 min at 4 °C using a refrigerated centrifuge
RS-20IV (TOMY SEIKO Co., Ltd.). The supernatant as a crude enzyme
solution was collected in a sample tube and kept on ice until use. The
protein concentration in each supernatant was determined by the method
of Lowry et al. (25) using bovine serum albumin as the standard.
Measurement of Hydrolyzing Activity for r-EG, r-MG, and
Disaccharides. The crude enzyme solution was diluted with 0.2 M
sodium phosphate buffer (pH 6.0) appropriately. Each substrate solution
(R-EG, R-MG, maltose, sucrose, or lactose) was diluted at various
Assessment of IC50 for Disaccharides Hydrolysis by r-EG and
r-Glucosidase Inhibitors. To start the hydrolyzing reaction, 100 µL
of the crude enzyme solution was added to 200 µL of 50 mM each
disaccharide (maltose, sucrose, or lactose) solution containing various
concentrations of R-EG, arabinose, or acarbose. The hydrolyzing
reaction and glucose analysis were performed by the method described
above.
Determination of Inhibition Type of r-EG for Disaccharides
Hydrolysis. The reaction mixture consisted of 100 µL of the crude
enzyme solution and 200 µL of various concentrations of maltose or
sucrose solution containing various concentrations of R-EG. The
hydrolyzing reaction and glucose analysis were performed by the
method described above.
Statistical Analysis. Statistical significance among means was
estimated at P < 0.05 according to Student’s t-test.
RESULTS
Figure 1 shows the absorption of R-EG by everted rat
intestinal sacs. R-EG and glucose liberated from R-EG were
detected in serosal solution, but absorbed glucose was signifi-
cantly less than absorbed intact R-EG (P < 0.01).
Figure 2 shows the time course of R-EG absorption in small
intestinal tissue (cells) and in serosal solution of everted rat
intestinal sacs. R-EG absorption in the small intestinal tissue
and that in the serosal solution was significantly reduced by
elimination of sodium ion from experimental solution or adding
phlorizin in the mucosal solution (P < 0.05).
R-EG hydrolysis by crude enzyme preparation from rat
intestinal acetone powder was indicated in Figure 3. R-EG was
hydrolyzed by crude enzyme solution prepared from rat
intestinal acetone powder.
Table 1 summarizes Km and Vmax values for R-EG, R-MG,
and disaccharides in this hydrolysis experiment. Vmax values for
the hydrolysis of R-EG, maltose, sucrose, and lactose were 0.09,
13.1, 4.46, and 0.22 µmol/mg protein/h, respectively. The
hydrolyzing activity for R-MG was not detected; therefore, Km
and Vmax values were not determined.

Absorption and Hydrolysis of
R
-EG
J. Agric. Food Chem., Vol. 53, No. 18, 2005 7259
Table 1. Kinetics Constants for the Hydrolysis of Glucose Conjugates
by Crude Enzyme Preparation from Rat Intestinal Acetone Powdera
Km (mM)
Vmax
(µ
mol/mg protein/h)
R
R
-EG
-MG
18.7
ND
14.1
19.9
22.5
0.09
ND
13.1
4.46
0.22
maltose
sucrose
lactose
a Incubations were performed at 37
°C for 180 min for R-EG and R-MG or 30
min for disaccharides. The reaction was performed in triplicate. ND, Km and Vmax
values were not determined.
Figure 2. Time courses of
R-EG absorption into intestinal tissue (a) and
serosal solution (b) on everted rat intestinal sac were indicated. Everted
rat intestinal sacs were incubated for 0 (blank), 1, 5, 15, 30, or 45 min in
the Krebs
Ringer buffer without sodium ion in which potassium ion was substituted
by sodium ion (white triangle), or in the Krebs Ringer buffer with 1.55
mM phlorizin (white square). -EG absorption was calculated by
subtracting glucose from totally absorbed sugar (glucose plus -EG).
Significant difference at each concentration was indicated by asterisk (**,
P < 0.01; and *, P < 0.05). Each point was the mean standard error (n
6).
−Ringer buffer containing sodium ion (white circle), in the Krebs−
−
R
R
±
)
Figure 4. Inhibition of disaccharide hydrolysis by
R-EG was indicated.
Figure 3. Hydrolysis of
intestinal acetone powder was examined. Incubation was performed for
180 min at 37 C. Concentrations of -EG were set at 0, 10, 21, 42, 83,
R-EG by crude enzyme preparation from rat
Concentrations of -EG were set at 0, 21, 42, 83, 167, 333, and 667
R
mM. Incubations were performed for 30 min at 37
performed in triplicate.
°C. Reaction was
°
R
167, and 333 mM. Reaction was performed in triplicate.
was 20% even at 600 mM). Figure 5 shows the Lineweaver-
Burk plot for disaccharides hydrolysis by rat intestinal crude
enzyme with or without R-EG. R-EG showed mixed type
inhibition for the hydrolysis of maltose and sucrose.
Figure 4 shows the effect of R-EG on disaccharides hydroly-
sis by rat intestinal crude enzyme solution. Hydrolyzing
activities for maltose (a) and sucrose (b) were decreased by
adding R-EG to the reaction mixture, but that for lactose (c)
was decreased only slightly in the presence of R-EG at high
concentrations. Table 2 summarizes the concentrations required
for 50% inhibition of disaccharide hydrolysis (IC50). The IC50
of R-EG for maltose and sucrose was 408 and 101 mM,
respectively. The IC50 of acarbose for maltose and sucrose was
2.6 × 10-3 and 2.5 × 10-3 mM, respectively. The IC50 of
arabinose for sucrose hydrolysis was 3.4 mM, whereas that for
maltose was not detected (maximal inhibition rate for maltose
DISCUSSION
Disaccharides and oligosaccharides are absorbed as monosac-
charides into the blood stream by hexose transport systems
distributed in the intestinal membrane after membrane digestion
(19). In our previous investigation, the hydrolyzing activity for
R-EG was detected in a crude enzyme preparation from small
intestinal mucosa of Wistar rats (5). Landau et al. (21) suggested

7260 J. Agric. Food Chem., Vol. 53, No. 18, 2005
Table 2. IC50 Values of -EG, Arabinose, and Acarbose for
Mishima et al.
the mucosal solution or under the presence of phlorizin (Figure
2a), a specific inhibitor of SGLT transporters (27, 28). These
data suggested that R-EG was absorbed into small intestinal
cells via SGLT1 in a brush border membrane. As shown in
Figure 2b, R-EG was detected in serosal solution, which
suggested that absorbed R-EG into intestinal cells was trans-
ported to the serosal side via GLUT2 in basolateral membrane.
Reduction of R-EG transport to serosal solution was associated
with a reduction of R-EG absorption into intestinal cells via
SGLT1. Although the ability of GLUT5 and other transport
systems on R-EG transport in the small intestine remained
unknown, results in this study showed that R-EG was transported
through the small intestinal wall. Therefore, most of the R-EG
ingested was absorbed into the blood stream as the intact form
(5, 26).
R
Disaccharide Hydrolysis by Crude Enzyme Preparation from Rat
Intestinal Acetone Powdera
IC50 values (mM)
R-EG
arabinose
acarbose
-
3
maltose
sucrose
408
101
ND
3.4
2.6
2.5
×
×
10
10
-3
a IC50, inhibitory concentration required for 50% inhibition of the disaccharide
hydrolysis. Incubation was performed at 37 C for 30 min. The reaction was
°
performed in triplicate. ND, IC50 was not determined. Inhibition for maltose hydrolysis
by arabinose was maximum (20%) at 600 mM.
Disaccharides, e.g., maltose and sucrose, are hydrolyzed by
small intestinal enzymes (17, 19, 29), and these enzymes could
hydrolyze various glucose conjugates (11-13, 30). R-EG
showed mixed type inhibition for maltose and sucrose hydrolysis
(Figure 5), showing the possibility that R-EG might be
hydrolyzed by maltase and sucrase in rat intestines. On the other
hand, R-MG was not hydrolyzed by the intestinal crude enzyme
preparation (data not shown). At present, R-MG is considered
as a nonmetabolic glucoside because it was not hydrolyzed by
mammalian enzymes (14-16). Therefore, R-EG hydrolyzing
enzymes in rat small intestine might recognize differences in
carbon length between ethyl and methyl in aglycon. R-Glucosi-
dase in mammalian small intestine has been reported to catalyze
transglucosidation (10, 31-33). However, despite extensive
research into the metabolic fate of ethanol over many years,
glucosidation to ethanol as an acceptor in the mammalian body
has not been described. Therefore, R-EG might not be detected
in the mammalian body unless it was ingested from Sake or
rice wine.
As shown in Table 2, IC50 values of R-EG for maltose and
sucrose hydrolysis were higher than those of arabinose and
acarbose. Arabinose showed uncompetitive inhibition for sucrose
hydrolysis (29); thus, it was expected for the prevention of
increasing plasma glucose levels after sucrose ingestion. Ac-
arbose strongly inhibited disaccharides hydrolysis by intestinal
enzymes; therefore, it is used as a diabetic medicine, a
suppressor of the postprandial increase in blood glucose level
(34, 35). R-EG as an inhibitor for disaccharides hydrolysis was
less effective as compared to arabinose and acarbose.
Figure 5. Lineweaver
in the presence and absence of
performed for 30 min at 37 C. Concentration of
(black circle), 167 (black triangle), or 667 mM (black square) for maltose
hydrolysis and 0 (white circle), 83 (white triangle), or 167 mM (white
square) for sucrose hydrolysis. Reaction was performed in triplicate.
−
Burk plot for maltose (a) and sucrose (b) hydrolysis
-EG were indicated. Incubations were
-EG was set at 0
R
°
R
In conclusion, most of the ingested R-EG was absorbed
through SGLT1 and GLUT2 localized in rat small intestines.
The hydrolyzing activity for R-EG was lower than those for
disaccharides. R-EG was only slowly hydrolyzed by maltase
and sucrase in rat intestines, and effects as an inhibitor on
disaccharides hydrolysis were weak as compared to arabinose
and acarbose.
the presence of R-EG hydrolyzing enzyme in hamster intestines.
As shown in Figure 1, glucose liberated from R-EG was
detected in a serosal solution of everted rat intestinal sacs, but
the glucose absorbed was only less than 4% of absorbed intact
R-EG. R-EG was hydrolyzed in rat intestine (Figure 3), but
the hydrolyzing activity for R-EG showed a low value as
compared to that for maltose and sucrose. Our previous study
reported that more than 60% of R-EG ingested was excreted
into urine (5, 26). These data suggested that R-EG was hardly
hydrolyzed and easily absorbed as an intact form in the small
intestine.
According to Landau et al. (21), R-alkyl glucosides with the
small aglycon, i.e., methyl, ethyl, and isopropyl glucosides, were
extensively concentrated in everted hamster intestines. Further-
more, hamster intestinal SGLT showed affinity to â-alkyl
glucosides, and the affinity of alkyl glucosides for SGLT1
increased with the alkyl chain length (22). In this experiment
using everted rat intestinal sacs, R-EG absorption into the small
intestinal tissue was reduced by elimination of sodium ion from
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Received for review April 16, 2005. Revised manuscript received July
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