Inhibitory action of palatinose and its hydrogenated derivatives on the hydrolysis of α-glucosylsaccharides in the small intestine

Article abstract of DOI:10.1021/jf7035824

This study was conducted to investigate the inhibitory effects of palatinose and Palatinit, which are disaccharides (or disaccharide alcohol) connected through an α-1,6-glucosyl linkage, on the hydrolysis of other carbohydrates, using an enzyme extract from the rat small intestine and a purified sucrase-isomaltase complex. Palatinose and its hydrogenated product, Palatinit, an equimolar mixture of α-O-D-glucopyranosyl-1,6-D-sorbitol (GPS) and α-O-D-glucopyranosyl-1,6-D-mannitol (GPM), inhibited the hydrolysis of sucrose and maltose. Palatinose and Palatinit also inhibited the hydrolysis of dextrin and soluble starch. Kinetic analysis of the enzymatic inhibition by GPS and GPM on sucrose hydrolysis revealed that both GPS and GPM competitively inhibit sucrase catalytic activity. These results suggest that disaccharides with an α-1,6-glucosyl linkage competitively inhibit intestinal α-glucosidases and may reduce the rate of hydrolysis of sucrose and other α-glucosylsaccharides.

Full text of DOI:10.1021/jf7035824

5892 J. Agric. Food Chem. 2008, 56, 5892–5895
Inhibitory Action of Palatinose and Its Hydrogenated
Derivatives on the Hydrolysis of
r-Glucosylsaccharides in the Small Intestine
,
†
†
‡
JUN KASHIMURA,* YUKIE NAGAI, AND TOSHINAO GODA
Research Laboratory, Mitsui Sugar Co., Ltd., 1-2-14, Honson, Chigasaki-shi, Kanagawa,
2
53-0042 Japan, and School of Food and Nutritional Sciences and Global COE Program,
The University of Shizuoka, 52-1, Yada, Suruga-ku, Shizuoka-shi, Shizuoka, 422-8526 Japan
This study was conducted to investigate the inhibitory effects of palatinose and Palatinit, which are
disaccharides (or disaccharide alcohol) connected through an R-1,6-glucosyl linkage, on the hydrolysis
of other carbohydrates, using an enzyme extract from the rat small intestine and a purified
sucrase-isomaltase complex. Palatinose and its hydrogenated product, Palatinit, an equimolar mixture
of R-O-D-glucopyranosyl-1,6-D-sorbitol (GPS) and R-O-D-glucopyranosyl-1,6-D-mannitol (GPM),
inhibited the hydrolysis of sucrose and maltose. Palatinose and Palatinit also inhibited the hydrolysis
of dextrin and soluble starch. Kinetic analysis of the enzymatic inhibition by GPS and GPM on sucrose
hydrolysis revealed that both GPS and GPM competitively inhibit sucrase catalytic activity. These
results suggest that disaccharides with an R-1,6-glucosyl linkage competitively inhibit intestinal
R-glucosidases and may reduce the rate of hydrolysis of sucrose and other R-glucosylsaccharides.
KEYWORDS: Palatinose; r-O-D-glucopyranosyl-l,6-D-sorbitol; r-O-D-glucopyranosyl-1,6-D-mannitol;
sucrase-isomaltase complex
INTRODUCTION
Table 1. Inhibitory Effects of Palatinose and Its Hydrogenated Product,
a
Palatinit, on the Hydrolysis of Sucrose and Maltose
Considerable attention has been paid in recent years to the
quality and quantity of dietary carbohydrates (1, 2). Psomaltulose
6-O-R-D-glucopyranosyl-D-fructose) is a noncariogenic disac-
rate of glucose production
(
µmol glucose produced/tube/min)
(
charide (3), a compound of glucose and fructose connected
through an R-1,6-glucosidic linkage. Isomaltulose is digested
and absorbed more slowly than sucrose (4–6). Palatinit, a hydro-
genated product of palatinose, is an equimolar mixture of R-D-
glucopyranosyl-l,6-D-sorbitol (GPS) and R-D-glucopyranosyl-1,6-
D-mannitol (IUPAC: R-D-glucopyranosyl-1,1-D-mannitol) (GPM).
Palatinit is minimally hydrolyzed by human digestive enzymes (7).
We have previously demonstrated in intestinal studies that when a
mixture of palatinose and sucrose is ingested, the increase in blood
glucose concentration is reduced as compared with the situation
when sucrose is digested alone (8). However, it is unclear whether
the inhibition of sucrose digestion in the presence of palatinose is
due to a specific action of palatinose at the active site of sucrase.
It is also unclear whether the property of palatinose, that is, a
disaccharide with an R-1,6-glucosidic linkage, plays an important
role in this inhibition. In this study, we investigated the inhibitory
effects of palatinose, GPS, and GPM on the intestinal hydrolytic
activities of various R-glucosylsaccharides using a rat intestinal
enzyme preparation and a purified sucrase-isomaltase complex.
28 mM sucrose
0.262 ( 0.002
2.198 ( 0.042
0.049 ( 0.002
0.017 ( 0.002
0.230 ( 0.005**
0.235 ( 0.002**
2
2
2
2
8 mM maltose
8 mM palatinose
8 mM Palatinit
8 mM sucrose + 28 mM palatinose
28 mM sucrose + 28 mM Palatinit
28 mM maltose + 28 mM palatinose
#
#
1.962 ( 0.020
1.892 ( 0.108
#
28 mM maltose + 28 mM Palatinit
a
Data were expressed as means ( SEM for three determinations. Palatinit is
an equimolar mixture of GPS and GPM. ** denotes a significant difference as
#,##
compared with the value obtained for 28 mM sucrose at p < 0.01.
denotes
significant differences as compared with the value obtained for 28 mM maltose at
p < 0.05 and p < 0.01, respectively.
MATERIALS AND METHODS
Enzyme Preparation. Intestinal acetone powder from rats (Sigma-
Aldrich Inc., Montana) was used as a source of R-glucosidase. The
intestinal acetone powder was dissolved in 10 volumes of 0.1 M
phosphate buffer (pH 6.8) and centrifuged at 12000g for 15 min, and
the supernatant was filtrated through a 0.8 µm membrane filter before
use as a crude enzyme preparation. The crude intestinal enzyme
preparation was further used for purification of the sucrase-isomaltase
complex by the method of Kolinska et al. (9), which involves
fractionation with ammonium sulfate, and column chromatographies
using DEAE-cellulose and DEAE-Sephadex A25. The specific activity
of the purified enzyme was 1300 U/mg for sucrase activity, which is
*
To whom correspondence should be addressed. Tel: +81-467-52-
8
882. Fax: +81-467-52-8889. E-mail: jun.kashimura@mitsui-sugar.co.jp.
†
‡
Mitsui Sugar Co., Ltd.
The University of Shizuoka.
1
0.1021/jf7035824 CCC: $40.75  2008 American Chemical Society
Published on Web 06/28/2008

Inhibitory Actions of Palatinose and Palatinit
J. Agric. Food Chem., Vol. 56, No. 14, 2008 5893
Table 2. Inhibitory Effects of Palatinose and Its Hydrogenated Product,
a
Palatinit, on the Hydrolysis of Soluble Starch and Dextrin
Palatinit. The assay mixture contained 0.1 M phosphate buffer (pH
6.8). The concentrations of dextrin and soluble starch were expressed
as glucose equivalents. After incubation for 20 min at 37 °C, the reaction
mixture was boiled for 3 min to stop the reaction. The released glucose
concentration in the mixture was determined using a D-glucose kit (R.
Biopharm GmbH, Darmstadt, Germany).
rate of glucose
production (µmol glucose
produced/tube/min)
b
2
2
2
2
2
2
2
2
8 mM soluble starch
b
1.504 ( 0.097
1.535 ( 0101
0.049 ( 0.001
0.017 ( 0.002
1.360 ( 0.038
1.345 ( 0.079
1.278 ( 0.079*
1.268 ( 0.061*
To study the inhibitory actions of palatinose and Palatinit on the
hydrolysis of sucrose using the purified sucrase-isomaltase complex,
the purified enzyme (0.20 U of sucrase activity) was incubated with
8 mM dextrin
8 mM palatinose
8 mM Palatinit
2
0 mM sucrose in the absence and the presence of either palatinose,
b
8 mM soluble starch + 28 mM palatinose
b
Palatinit, or maltitol at concentrations of 5, 20, and 50 mM for 20 min
at 37 °C. The released glucose and fructose concentrations in the
mixture were determined using a D-glucose kit and D-fructose kit (R.
Biopharm GmbH, Darmstadt, Germany), respectively.
8 mM soluble starch + 28 mM Palatinit
b
8 mM dextrin + 28 mM palatinose
8 mM dextrin + 28 mM Palatinit
b
a
Kinetic Analysis. In the kinetic studies, GPS and GPM were used
as inhibitory substances, because the rate of hydrolysis of GPS and
GPM was considered to be minimal (7). An enzyme preparation
containing 0.25 U of sucrase activity was incubated with 10, 25, or 50
mM sucrose in the absence and the presence of 50 or 100 mM of either
GPS or GPM for 10 min at 37 °C. The amount of released glucose in
the mixture was determined using a D-glucose kit.
Chemicals. Palatinose (trade name of Hitsui Sugar Co., Ltd., another
name is isomaltulose) and Palatinit, an equimolar mixture of GPS and
GPM, were provided by Mitsui Sugar Co. Ltd. (Tokyo, Japan). Other
sugars were purchased as follows: sucrose and GPM from Wako Pure
Chemical Industries Ltd. (Tokyo, Japan), GPS from Carl Roth GmbH
(Karlsruhe, Germany), maltose from Junsei Chemical Co., Ltd. (Tokyo,
Data were expressed as means ( SEM for three determinations. Palatinit is
an equimolar mixture of GPS and GPM. * denotes a significant difference as
b
compared with the value obtained for 28 mM dextrin at p < 0.05. Concentration
of glucose residue equivalent to 28 mM glucose.
9
80 times that of the crude enzyme solution. One unit (U) of
R-glucosidase is defined as the activity of enzyme that can release 1
µmol of glucose from substrate in 1 min.
Assay for Hydrolytic Activities. To study the effect of palatinose
and its sugar alcohol, Palatinit, on the hydrolysis of sucrose and other
R-glucosylsaccharides, the enzyme preparation containing 0.26 U of
sucrase activity was mixed with 28 mM each of sucrose, maltose,
dextrin, or soluble starch in the absence and presence of palatinose or
Figure 1. Lineweaver-Burk plots of sucrose hydrolytic activity in rat small intestinal extract with or without GPS and GPM.
Figure 2. Dixon plots of sucrose hydrolytic activity in rat small intestinal extract with or without GPS and GPM.

5894 J. Agric. Food Chem., Vol. 56, No. 14, 2008
Kashimura et al.
Figure 3. Inhibitory effects of palatinose (a), Palatinit (b), an equimolar mixture of GPS and GPM, and maltitol (c) on the hydrolysis of sucrose using
,
a purified SI complex. Data were expressed as means ( SEM. * ** denote significant differences at p < 0.05 and p < 0.01, respectively.
Japan), dextrin from Sigma-Aldrich Inc. (Montana), and soluble starch
from Merck KGaA (Darmstadt, Germany). The D-glucose kit and
D-fructose kit were purchased from R.Biopharm GmbH (Dannstadt,
Germany).
Data Analysis. Data were analyzed using a Student’s t test or
Bonferroni’s multiple comparison test where appropriate.
the absence of Palatinit or when compared with the theoretical
additive rates (Table 1). The rate of glucose release from
maltose was significantly lower (p < 0.01 and p < 0.05,
respectively) in the presence of palatinose or Palatinit than that
of maltose alone.
The rate of glucose release from dextrin was significantly
lower (p < 0.05) in the presence of palatinose than that in the
absence of palatinose (Table 2). The rate of glucose release
from dextrin was also significantly lower (by 19.3%, p < 0.05)
than the theoretical additive rates of glucose from dextrin and
palatinose. In the presence of Palatinit, the rates were also
significantly lower (p < 0.05) than that in the absence of
Palatinit or when compared with the theoretical additive rates.
The rate of glucose release from soluble starch was lower in
the presence of palatinose or Palatinit than in the absence of
RESULTS
Inhibitory Effects of Palatinose and Palatinit on the Hy-
drolysis of r-Glucosylsaccharides. The rate of glucose release
from sucrose was significantly lower (p < 0.01) in the presence
of palatinose than in the absence of palatinose (Table 1). The
rate of glucose release from sucrose was also significantly lower
(
by 26%, p < 0.01) than the theoretical additive rate of glucose
release from sucrose and palatinose. In the presence of Palatinit,
the rates were also significantly lower (p < 0.01) than that in

Inhibitory Actions of Palatinose and Palatinit
J. Agric. Food Chem., Vol. 56, No. 14, 2008 5895
palatinose or Palatinit, but these differences were not significant.
The results suggested that palatinose and Palatinit inhibit the
hydrolysis of carbohydrates by R-glucosidases, such as maltase
and glucoamylase, as well as sucrase and isomaltase.
that the inhibitory action of palatinose and Palatinit is related to
its common structure, that is, R-1,6 glucosyl linkage.
It was reported that when a mixture of palatinose and sucrose
was orally administered to humans, the increase in plasma
glucose concentration was smaller than with the ingestion of
sucrose only (8). The inhibitory effect of palatinose on R-glu-
cosidases demonstrated herein explains one of the mechanisms
involved in this phenomenon. Because the rates of hydrolysis
of palatinose and Palatinit are low in the small intestine (7), it
is likely that such an inhibitory effect of palatinose and Palatinit
may be reflected by a reduced rate of digestion and absorption
of R-glucosylsaccharides including starch and sucrose.
In conclusion, the results in this study suggest that palatinose
and its hydrogenated derivatives, both with an R-1,6-glucosyl
linkage, competitively inhibit intestinal R-glucosidases and may
reduce the rate of hydrolysis of sucrose and other R-glucosyl-
saccharides.
Kinetic Analysis. The inhibition by GPS and GPM on sucrose
hydrolysis is illustrated in Figures 1 (Lineweaver-Burk plots) and
2
(Dixon plots). It is clear that GPS and GPM are competitive
inhibitors of sucrose hydrolysis, as demonstrated by the inhibition
patterns observed with the Lineweaver-Burk plots (Figure 1).
There was essentially no difference between GPS and GPM in
either the Km or Vmax values. The inhibitor constant (Ki) for sucrose
hydrolysis calculated from the Dixon plots was 72 (GPS) and 74
mM (GPM), respectively (Figure 2).
Comparison of Disaccharide Alcohols with an r-1,6-
Glucosyl Linkage vs Those with an r-1,4-Glucosyl Linkage.
The inhibitory effects of palatinose, Palatinit, and maltitol on
the hydrolysis of sucrose in the presence of the purified SI
complex are shown in Figure 3. The inhibitory effects of
palatinose on the hydrolysis of sucrose by the purified SI enzyme
preparation appeared when 20 mM or more of palatinose was
added to 20 mM sucrose. Similarly, the inhibitory effects of
Palatinit on sucrose hydrolysis were also observed when 20 mM
or more of Palatinit was added to 20 mM sucrose. On the other
hand, maltitol did not inhibit the hydrolysis of sucrose (Fig-
ure 3).
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This study demonstrated that palatinose and its hydroge-
nated derivative, Palatinit, inhibited the hydrolytic activity
of R-glucosidases in the small intestine. The results in this
study have shown that the inhibitory effects of palatinose
and Palatinit on the hydrolysis of sucrose, maltose, dextrin,
and starch are characteristic properties of R-1,6-binding
saccharides. We have investigated the effects of palatinose
and Palatinit on the hydrolysis of trehalose and lactose and
found that both have virtually no effect (unpublished data).
The results of the present study suggest that palatinose, which
has an R-1,6-linkage, inhibits the hydrolysis of carbohydrates
normally hydrolyzable by R-glucosidases that are also present
in the small intestine. Similar effects were recognized in the
case of R-1,6-binding saccharides, such as palatinose and
Palatinit. On the other hand, maltitol, an R-1,4-binding
saccharide, did not inhibit the hydrolysis of sucrose.
(
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In this study, we have shown that both palatinose and Palatinit
inhibit the activity of maltase/ glucoamylase as well as that of
sucrase. Therefore, it is likely that palatinose and Palatinit inhibit
the hydrolysis of sucrose, maltose, dextrin, and starch by
interacting with the catalytic active sites of these R-glucosidases
in the small intestine. Because maltitol, a disaccharide alcohol with
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Received for review December 11, 2007. Revised manuscript received
April 3, 2008. Accepted April 16, 2008.
JF7035824