Original article
Characterisation of the hypoglycaemic activity of glycoprotein
purified from the edible brown seaweed, Undaria pinnatifida
S.M. Rafiquzzaman,1
Jong Min Lee,1
Raju Ahmed,1
Jong-Hee Lee,2
Jin-Man Kim3
& In-Soo Kong1
*
1 Department of Biotechnology, Pukyong National University, Busan 608-737, Korea
2 World Institute of Kimchi, Gwangju 503-360, Korea
3 Department of Biotechnology and Chemical Engineering, Chonnam National University, Yeosu 550-749, Korea
(Received 18 May 2014; Accepted in revised form 11 August 2014)
Summary Seaweed has been reported to control postprandial hyperglycaemia in various ways. We recently reported
the characterisation of a glycoprotein from Undaria pinnatifida (UPGP) with antioxidant activities. In this
study, we characterised the hypoglycaemic effect of UPGP through monitoring a-glucosidase inhibition
and glucose transport across yeast cell. Dose-dependent inhibitions of UPGP against yeast and rat intesti-
nal a-glucosidase were observed with IC50 values of 0.11 and 0.29 mg mLÀ1
, respectively. UPGP showed
stable inhibition following incubation at different temperatures and metal ions. Regarding bioaccessibility,
the inhibition was decreased slightly during the gastric phase compared to undigested UPGP, with an
increase during the duodenal phase. Kinetics and membrane dialysis revealed mixed and reversible
inhibition, respectively. Furthermore, UPGP with acarbose showed synergistic inhibition against a-gluco-
sidase, and UPGP increased the rate of glucose transport across the yeast cell. In conclusion, our study
demonstrated that UPGP may be used as bioaccessible food additives for controlling postprandial
hyperglycaemia.
Keywords Glucose transport, glycoprotein, hypoglycaemic, Undaria pinnatifida, yeast cell, a-glucosidase.
Introduction
Therapeutic agents from natural sources are gaining
popularity for the treatment of chronic diseases includ-
ing cancer, heart disease and diabetes due to their low
incidence of side effects and cost (Ventakesh et al.,
2003). Several epidemiological studies suggest that
fruits, vegetables, grains, nuts, seaweeds and less-pro-
cessed staple foods provide the best protection against
the development of chronic diseases (Ness & Powles,
1997). Among chronic diseases, diabetes is now consid-
ered a serious global problem. The global prevalence
of diabetes is estimated to increase from 177 million
people in 2000 to at least 366 million by 2030 (Wild
et al., 2004). Diabetes mellitus is a complex metabolic
disorder categorised into type I and type II, resulting
from either insulin insufficiency or insulin dysfunction,
respectively. Type II diabetes is the more common
form of diabetes, accounting for 90% of the diabetic
population.
In individuals with type II diabetes, nutrient uptake
related to the first-phase insulin response is severely
diminished, resulting in elevated postprandial glucose
levels. Thus, controlling postprandial glucose levels is
critical for the early treatment of diabetes. Hydrolysis
of starch and carbohydrate is the major source of
glucose in the blood. Human a-glucosidase is located
in the brush border of the surface membrane of
intestinal cells and is a key enzyme for the hydrolysis
and absorption of carbohydrate in the human body
(Nichols et al., 2002). It is believed that inhibiting
a-glucosidase can delay the breakdown of carbohy-
drate in the small intestine and ultimately decrease
blood glucose levels (Van de Laar, 2008).
Currently, several antidiabetic drugs, including acar-
bose, miglitol and voglibose, which inhibit a-glucosi-
dase activity are available (Van de Laar, 2008). While
these treatments can control postprandial glucose
levels, their continuous use is often associated with
undesirable side effects, including liver toxicity and
adverse gastrointestinal problems (Hanefield et al.,
1991). Thus, there is a need for natural a-glucosidase
inhibitors with no adverse secondary effects. It has
been reported that seaweeds and their organic extracts
contain a wide array of bioactive substances with
diverse health effects (Rindi et al., 2012). Previously,*Correspondent: E-mail: iskong@pknu.ac.kr
International Journal of Food Science and Technology 2015, 50, 143–150
doi:10.1111/ijfs.12663
© 2014 Institute of Food Science and Technology
143

the majority of studies related to seaweed have focused
on the isolation of polysaccharides and characterisa-
tion of their biofunctional activity. Previous studies
have reported that fucoidan and fucoxanthin from
Undaria pinnatifida elicit a wide range of therapeutic
effects, including anti-inflammatory, antiviral and anti-
coagulant activities (Cumashi et al., 2007). However,
our studies found that glycoprotein from U. pinnatifida
and Saccharina japonica showed potent antioxidant
and DNA protective activities (Kim et al., 2012;
Rafiquzzaman et al., 2013). Moreover, seaweeds have
been shown to exhibit antidiabetic properties by inhib-
iting carbohydrate-hydrolysing enzymes (Jung et al.,
2012). Accordingly, it is known that seaweed extracts
and their fractions can act as functional ingredients in
foods to control hyperglycaemia.
Therefore, the objective of this study was to cha-
racterise the antihyperglycaemic effect of UPGP using
in vitro measurements such as a-glucosidase inhibition
and promotion of glucose transport across the yeast
cell membrane. We also performed further characteri-
sation of UPGP, including determination of tempera-
ture and metal ion stabilities, bioaccessibility using in
vitro digestion models, kinetics, and the nature of
inhibition using membrane dialysis. Furthermore, the
combined effect of UPGP and another a-glucosidase
inhibitor (acarbose) was investigated.
Materials and methods
Materials
Undaria pinnatifida was collected from the local mar-
ket in Busan, Korea, and preserved within a plastic
box under dried conditions for experimental use. Yeast
a-glucosidase (EC 3.2.1.20), rat intestinal acetone
powder, p-nitrophenyl-a-D-glucopyranoside (PNPG),
porcine pepsin, taurodeoxycholate, taurocholate, gly-
codeoxycholate and pancreatin were purchased from
Sigma-Aldrich (Busan, Korea). All other chemicals
used, including solvents, were of analytical grade.
Purification of UPGP
Glycoprotein (MW: ~10 kDa) was purified from
U. pinnatifida and identified using sodium dodecyl sul-
phate–polyacrylamide gel electrophoresis (SDS-PAGE)
followed by Coomassie Brilliant Blue (CBB), silver
and periodic acid-Schiff (PAS) staining, as reported
previously (Rafiquzzaman et al., 2013).
Yeast a-glucosidase inhibitory activity assay
The enzyme inhibitory activity of UPGP was evaluated
spectrophotometrically using the procedure reported
by Li et al. (2005). Briefly, 60 lL of a reaction mixture
containing 20 lL of 100 mM phosphate buffer (pH
6.8), 20 lL of 2.5 mM p-nitrophenyl-a-D-glucopyrano-
side (PNPG) and 20 lL of the sample (test concentra-
tion ranging from 0.1 to 1.3 mg mLÀ1
) dissolved in
distilled water (DW) was added to each well, followed
by the addition of 20 lL of a-glucosidase [0.2 U mLÀ1
in 10 mM phosphate buffer (pH 6.8)]. The plate was
incubated at 37 °C for 15 min, after which 80 lL of
0.2 M sodium carbonate solution was added to stop
the reaction. The absorbance was then recorded at
405 nm using a microplate spectrophotometer (Biotek,
USA, ELx-800). Acarbose dissolved in distilled water
was used as a positive control. The percentage inhibi-
tion (%) was obtained using the following equation:
% inhibition = (Ac–As)/Ac 9100, where Ac is the
absorbance of the control and As is the absorbance of
the sample.
Rat intestinal a-glucosidase inhibition assay
Rat intestinal a-glucosidase was prepared as described
by Jo et al. (2009) with minor modifications. Briefly,
rat intestinal acetone powder (200 mg) was dissolved
in 4 mL of 50 mM ice-cold phosphate buffer and soni-
cated for 15 min at 4 °C. After vigorous vortexing for
20 min, the suspension was centrifuged (10 000 g,
4 °C, 30 min) and the resulting supernatant was used
for the assay. A reaction mixture containing 50 lL of
phosphate buffer (50 mM; pH 6.8), 10 lL of rat intesti-
nal a-glucosidase (1 U mLÀ1
) and 20 lL of UPGP at
varying concentrations was pre-incubated for 5 min at
37 °C, after which 20 lL of 1 mM PNPG was added
to the mixture as substrate. After further incubation at
37 °C for 30 min, the reaction was stopped by adding
50 lL of Na2CO3 (0.1 M). The absorbance was then
recorded at 405 nm using a microplate spectrophotom-
eter (Biotek, ELx-800). The percentage inhibition was
obtained using the same equation as used in the yeast
a-glucosidase assay.
Temperature and metal ion stability of UPGP
The stability of UPGP at various temperatures and in
the presence of different metal ions in terms of rat
intestinal a-glucosidase inhibition was investigated as
described by Oh & Lim (2008) with minor modifica-
tions. Briefly, to investigate temperature stability,
UPGP was incubated at different temperatures
(0–100 °C) for 1 h. Following incubation, UPGP was
used for a-glucosidase inhibition assays as described
above. To investigate the effect of metal ions, 100 lL
of UPGP (1 mg mLÀ1
) were reacted with each buffer
(CaCl2, MnCl2, MgCl2, KCl, NaCl, CoCl2 or ZnCl2)
containing 2 mM of metal ions for 2 h at room tem-
perature (RT). The reaction solutions were then
subjected to a-glucosidase inhibition assays.
© 2014 Institute of Food Science and TechnologyInternational Journal of Food Science and Technology 2015
Hypoglycemic activity of glycoprotein S. M. Rafiquzzaman et al.144

Bioaccessibility of UPGP measured using the in vitro
digestion model
The bioaccessibility of UPGP was measured using the
in vitro digestion model as described previously (Raf-
iquzzaman et al., 2013) with minor modifications.
Briefly, UPGP solutions (1 mg mLÀ1
) were acidified to
pH 2 with 1 mL of porcine pepsin preparation (0.04 g
pepsin in 0.1 mol LÀ1
HCl) and incubated at 37 °C in
a shaking water bath at 95 rpm for 1 h. After gastric
digestion, the pH was increased to 5.3 with 0.9 M
sodium bicarbonate, and then 200 lL of the bile salts
glycodeoxycholate (0.04 g in 1 mL of saline), taur-
odeoxycholate (0.025 g in 1 mL of saline) and tau-
rocholate (0.04 g in 1 mL of saline), as well as 100 lL
of pancreatin (0.04 g in 500 mL saline) were added to
the solution. The pH of each sample was increased to
pH 7.4 with 1 M NaOH, and the samples were incu-
bated at 37 °C in a shaking water bath at 95 rpm for
2.5 h to complete the intestinal phase of the in vitro
digestion process. After the intestinal digestion phase,
2 mL of each sample was extracted and stored at
À20 °C. Samples were analysed within 2 weeks.
Analysis of enzyme kinetics
To determine the kinetic mechanism, the Lineweaver
& Burk (1934) plot method was used. Using a Linewe-
aver–Burk (LB) plot, we determined the enzyme kinet-
ics of rat intestinal a-glucosidase using 1, 2 and 3 mM
PNPG as a substrate in the absence or presence of
1.5 mg mLÀ1
(filled circles), 1.0 mg mLÀ1
(open cir-
cles) or 0.5 mg mLÀ1
(filled inverted triangles) UPGP.
The mode of inhibition of UPGP was determined by
analysing data calculated following Michaelis–Menton
kinetics.
Dialysis for reversibility of UPGP action
Rat intestinal a-Glucosidase (100 U mLÀ1
) was incu-
bated with UPGP (10 mg mLÀ1
) in 0.5 mL of sodium
phosphate buffer (50 mM, pH 6.7) for 2 h at 37 °C
and dialysed against sodium phosphate buffer (5 mM,
pH 6.7) at 4 °C for 24 h, changing the buffer every
12 h. Another premixed enzyme solution (0.5 mL) was
maintained 4 °C for 24 h without dialysis for the con-
trol experiment. Reversibility of UPGP was deter-
mined by comparing the residual enzyme activity after
dialysis with that of a nondialysed control (Lee, 2000).
Combined inhibitory effect of UPGP and acarbose
This assay was performed as described previously
(Adisakwattana et al., 2011). The combined inhibitory
effect was evaluated in solution containing acarbose
alone or in a mixture with UPGP to evaluate the
synergistic effect of UPGP and acarbose on rat intesti-
nal a-glucosidase. The reaction was performed as
described above.
Effect of UPGP on glucose transport by yeast cells
Yeast (Saccharomyces cerevisiae) cells were prepared
according to the method of Cirillo (1962). Briefly, yeast
cells were washed by repeated centrifugation (3000 g;
5 min) in distilled water, and a 10% (v/v) suspension
was prepared in distilled water. Various concentrations
of extracts (1–5 mg) were added to 1 mL of glucose
solution (5–20 mM) and incubated together for 10 min
at 37 °C. The reaction was initiated by adding 100 lL
of yeast suspension, vortexed and further incubated at
37 °C for 60 min. After 60 min, the tubes were centri-
fuged (2500 g; 5 min) and glucose concentrations in
the supernatant were determined using the glucose oxi-
dase method. The percentage increase in glucose
uptake by yeast cells was calculated using the following
formula: Increase in glucose uptake (%) = (Abs. con-
trol–Abs. sample/Abs. control) 9100, where Abs con-
trol is the absorbance of the control reaction
(containing all reagents except the test sample) and
Abs sample is the absorbance of the test sample.
Statistical analysis
All experiments were performed in triplicate, and data
are presented as means Æ standard deviation. Analysis
of variance (ANOVA) was performed, and comparisons
of means were conducted using Turkey’s multiple com-
parison tests using the SPSS program (version 16.0 for
windows, SPSS Inc.). Values were considered to differ
significantly if the P value was <0.05.
Results
a-Glucosidase inhibitory activity assay
a-Glucosidase of S. cerevisiae (EC 3.2.1.20) was used
to investigate the inhibitory activity of purified UPGP.
The inhibitory activity against a-glucosidase was deter-
mined using PNPG as substrate. UPGP significantly
inhibited yeast a-glucosidase in a dose-dependent
manner (Fig. S1a). The highest inhibitory activity
(95.03%) was found at 0.9 mg mLÀ1
UPGP, which
inhibited yeast a-glucosidase with an IC50 value of
0.11 mg mLÀ1
. Acarbose (positive control) showed an
IC50 value of 0.69 mg mLÀ1
(Table 1) under similar
conditions. Similarly, rat intestinal a-glucosidase was
used to evaluate the inhibitory activity of UPGP.
UPGP and acarbose inhibit a-glucosidase with IC50 val-
ues of 0.29 and 0.04 mg mLÀ1
, respectively (Table 1).
UPGP also showed dose-dependent inhibitory activities
against rat intestinal a-glucosidase (Fig. S1b).
© 2014 Institute of Food Science and Technology International Journal of Food Science and Technology 2015
Hypoglycemic activity of glycoprotein S. M. Rafiquzzaman et al. 145

Stability at various temperatures and in the presence of
different metal ions
UPGP showed stable rat intestinal a-glucosidase inhi-
bition activity in the presence of different metal ions.
For example, the activities of UPGP in the presence of
Ca2+,
Mn2+
and Zn2+
were highly stable at 80–90%
inhibition, whereas Co2+
showed the lowest inhibition
activity, at 75% (Fig. 1a). As shown in Fig. 1b, the
inhibition by UPGP of yeast a-glucosidase was stable
at a wide range of temperatures (0–100 °C). The high-
est yeast a-glucosidase inhibition activity occurred at
37 °C and the inhibitory activity varied little from 0 to
100 °C; all temperatures examined showed over 80%
inhibition compared to the control.
Bioaccessibility of UPGP measured using the in vitro
digestion model
The rat intestinal a-glucosidase inhibitory activity of
UPGP was measured before and after the in vitro
digestion model to determine its bioaccessibility. There
was little variation in inhibitory activity between
digested and undigested UPGP. However, the a-gluco-
sidase inhibitory activity of UPGP after the gastric
and duodenal phases of in vitro digestion was 9.24%
and 7.7% lower, respectively, compared to that of
undigested UPGP (Fig. 2).
Enzyme kinetics
To investigate the inhibition mode of UPGP, a LB
plot was generated using data obtained from kinetic
studies, which was calculated using the Michaelis–
Menton equation to confirm the inhibition pattern. In
the LB plot, the designated line of various concentra-
tions intersected the axis on the left side of the zero
point, indicative of its ability to act as a mixed-type
inhibitor (Fig. 3).
Reversibility of UPGP action
The rat intestinal a-glucosidase activity was not com-
pletely recovered after dialysis, as shown by the
enzyme-mixed inhibitor curve (EID) that reached
levels corresponding to 70% compared to the enzyme
control without dialysis (EC) (Fig. S2). Proximal run-
ning of ED as an experimental control along with EC
and EID ensured that dialysis alone did not signifi-
cantly affect enzyme activity. However, the nondialy-
sed mixture of enzyme and inhibitor (EIC) showed
inhibitory activity.
Combined effect
The combined effect of UPGP and acarbose on a-glu-
cosidase inhibition is shown in Fig. 4. The percentage
of a-glucosidase inhibition increased upon addition of
UPGP to the mixture containing acarbose. Thus,
Table 1 Inhibitory effects of UPGP and acarbose against yeast and
rat intestinal a-glucosidase
Enzyme type
IC50 (mg mLÀ1
)*
UPGP Acarbose†
Yeast a-glucosidase 0.11 Æ 0.08 0.69 Æ 0.13
Rat intestinal a-glucosidase 0.29 Æ 0.10 0.04 Æ 0.002
*IC50, concentration of inhibitor to inhibit 50% of its activity.
†
A positive control.
(a) (b)
Figure 1 Effect of metal ions (a) and tem-
perature (b) on the inhibitory activities of
UPGP against rat intestinal a-glucosidase.
UPGP (1 mg mLÀ1
) was incubated at differ-
ent temperature (0–100 °C) and metal ions,
and UPGP was then used for a-glucosidase
inhibition. Results are expressed as
mean Æ SD (n = 3). *P ≤ 0.05 compared
with untreated UPGP.
Figure 2 Bioaccessibility of UPGP on the inhibitory activities
against rat intestinal a-glucosidase measured by in vitro digestion
model. Results are expressed as mean Æ SD (n = 3). Different letters
in superscripts indicate significant differences (P ≤ 0.05) compared
with untreated UPGP.
© 2014 Institute of Food Science and TechnologyInternational Journal of Food Science and Technology 2015
Hypoglycemic activity of glycoprotein S. M. Rafiquzzaman et al.146

UPGP and acarbose had an additive inhibitory effect
on a-glucosidase.
Effect of UPGP on glucose transport by yeast cells
The rate of glucose transport across the cell membrane
was investigated using an in vitro system with S. cere-
visiae suspended in 5–20 mM glucose solutions in the
presence/absence of UPGP (Fig. 5). The amount of
glucose remaining in the medium after a specific time
served as an indicator of glucose uptake by yeast cells.
UPGP moderately increased glucose transport in yeast
cells up to 50%, which was directly proportional to
the sample concentration and inversely proportional to
the glucose concentration.
Discussion
Phenolics, flavonoids, fucoidan and fucoxanthin from
seaweed show diverse biofunctional activities, includ-
ing antidiabetic effects (Rindi et al., 2012; Lordan
et al., 2013). However, no antidiabetic activity has
been reported for glycoprotein purified from seaweed.
In our recent study, we reported the purification and
detection of a glycoprotein from U. pinnatifida, as well
as its chemical characterisation, and determination of
its antioxidant and DNA-protecting activity (Rafiquzz-
aman et al., 2013). As part of our continuous effort in
exploring biofunctional activity, we evaluated the anti-
hyperglycaemic effect of UPGP in terms of its a-gluco-
sidase inhibition activity and effect on glucose uptake
by yeast cells. Yeast a-glucosidase is used to identify
inhibitory activity in the extracts of, or active com-
pounds purified from, medicinal plants or food items
(Hogan et al., 2010). However, a-glucosidase prepared
from rat intestinal acetone powder closely mimics the
mammalian system (Ohta et al., 2002) and may be a
better model to identify, design and develop antihyper-
glycaemic agents, particularly for the management of
postprandial hyperglycaemia in diabetes. Therefore, we
tested both systems. The present study reports the
dose-dependent antihyperglycaemic effect of purified
UPGP, which is mediated by inhibition of a-glucosi-
dase (Fig. S1a,b). The inhibitory activity of UPGP on
a-glucosidase from the rat intestine was lower than
that of acarbose. However, UPGP showed consider-
ably higher (P ≤ 0.05) inhibitory activity against yeast
a-glucosidase compared with acarbose (Table 1). The
differences in inhibitory activities may be due to
structural differences between these two enzymes
(Chiba, 1997). These results indicated that UPGP is a
novel type of inhibitor that inhibits a-glucosidase from
yeast or the rat intestine. We used a common substrate
(PNPG) for both enzymes to ensure that the experi-
mental protocols were identical. Regarding further
characterisation of UPGP such as stability, bioaccessi-
bility, kinetics, reversibility and synergistic inhibition
Figure 4 The synergistic effect of acarbose (AC) and UPGP at dif-
ferent concentrations on rat intestinal a-glucosidase inhibition.
Results are expressed as mean Æ SD (n = 3). *P ≤ 0.05 compared
with acarbose alone.
Figure 3 Lineweaver–Burk plots for the rat intestinal a-glucosidase
inhibition by UPGP. Enzyme was treated with various concentration
of PNPG (0.5, 1.0 and 1.5 mM) in the presence of UPGP
[0.5 mg mLÀ1
(▼); 1.0 mg mLÀ1
(○); 1.5 mg mLÀ1
(●)] and found
mixed type of inhibition. Figure 5 Effect of UPGP on glucose transport by yeast cells at dif-
ferent glucose concentrations. Results are expressed as mean Æ SD
(n = 3). *P ≤ 0.05 compared with UPGP (1 mg mLÀ1
) alone.
© 2014 Institute of Food Science and Technology International Journal of Food Science and Technology 2015
Hypoglycemic activity of glycoprotein S. M. Rafiquzzaman et al. 147

with acarbose, we used rat intestinal a-glucosidase as
it closely mimics the mammalian system (Ohta et al.,
2002). Furthermore, we also used yeast a-glucosidase
for those studies and found little variation in terms of
inhibition (data not shown).
When exploring possible industrial use as a food
additive, we investigated the stability of UPGP at 0–
100 °C and in the presence of various metal ions.
UPGP was highly stable (P ≤ 0.05) at these tempera-
tures and in the presence of various metal ions
(Fig. 1a,b). This result is in agreement with the find-
ings of Kim et al. (2005) and suggested that UPGP
would be easier to handle during processing or manu-
facturing steps. Thus, UPGP may have industrial uses
as a food additive. In other words, we determined the
bioaccessibility of UPGP using an in vitro digestion
model to provide an indication of its biofunctional
activities in a biological system, as the model is
designed to simulate in vivo digestion. The overall
inhibitory activities of UPGP were lower (P ≤ 0.05)
during the gastric phase than in the undigested and
duodenal phases (Fig. 2). The differences between the
gastric and duodenal phases were due to changes in
the pH of the UPGP solution, as pH affects enzyme
activity. The inhibitory activity remained after the gas-
tric phase of digestion, indicating that structural defor-
mation or conformational change might not take
place. This result is similar to previous reports (Raf-
iquzzaman et al., 2013) and suggested that purified
UPGP is stable under simulated digestion conditions.
However, some findings regarding the stability of nat-
ural compounds subjected to changes in pH con-
tradicted previous reports (D’Archivio et al., 2010).
To identify the type of enzymatic inhibition, kinetic
analyses were performed at different substrate and
inhibitor concentrations. LB plots were drawn to con-
firm the inhibition pattern; UPGP showed a mixed
inhibition type (Fig. 3). It is possible that UPGP pos-
sesses more than one a-glucosidase inhibitor, which is
supported by the findings of Kim et al. (2005). This
assumption was further supported by previous reports
that UPGP contained a number of protein and carbo-
hydrate, which is linked with O-glycans (Rafiquzz-
aman et al., 2013). It is also possible that UPGP
attaches to a wide region of a-glucosidase or that it
attaches to a unique region and causes structural mod-
ifications. We next performed a dialysis experiment
and found that the inhibitory activity of UPGP was
almost reversible; that is, the enzyme activity was
recovered after dialysis due to removal of inhibitors
(Fig. S2). A similar result was found by Lee (2000)
who reported that dibutyl phthalate from Streptomyces
melanosporofaciens was an almost reversible inhibitor.
Reversible inhibition is a useful property of a-glucosi-
dase inhibitors because the enzyme remains intact even
after elimination of the inhibitor. In contrast, when
the inhibitor binds irreversibly to the enzyme, it may
suffer from hypoglycaemia due to chronic carbohy-
drate malabsorption (Shihabuddin et al., 2011).
Acarbose has been used clinically to treat type II
diabetes mellitus. The lowest dose of acarbose with a
clinical effect is 150 mg per day (Rodier et al., 1998).
However, recent reports have shown that acarbose
treatment is associated with many adverse effects,
such as flatulence, meteorism and abdominal disten-
sion, which occur in a dose-dependent manner (Hane-
field et al., 1991). In general, acarbose may be used
in combination with other agents such as sulfonylurea
and metformin or as a monotherapy for patients with
diabetes (Adisakwattana et al., 2011). Our present
study shows that the combination of acarbose and
UPGP results in additive inhibition (P ≤ 0.05)
(Fig. 4), suggesting that it may have significant clini-
cal benefits for delaying postprandial hyperglycaemia
and hyperinsulinaemia. This could lead to the devel-
opment of a novel combined therapy for patients
with diabetes. Therefore, the dosage of acarbose can
be reduced by combining with UPGP, which would
likely reduce the adverse effects of acarbose in
patients with diabetes.
Other than the inhibition of a-glucosidase by
UPGP, several other mechanisms for the hypoglycae-
mic effect of phytochemicals have been proposed, such
as manipulation of glucose transporters, b-cell regener-
ation and enhancement of insulin release (Tiwari &
Rao, 2002). Our findings demonstrated that glucose
transport across the yeast cell membrane significantly
increased (P ≤ 0.05) in the presence of UPGP (Fig. 5).
The rate of glucose transport was dependent on the
external glucose concentration as well as the sample
concentration. This finding is in agreement with Ah-
med & Urooj (2010). It is generally known that glu-
cose is transported across the yeast cell membrane by
facilitated diffusion. Facilitated carriers are specific
carriers that transport solutes down the concentration
gradient. Therefore, effective transport is possible only
if intracellular glucose is removed (Teysink et al.,
1998).
We performed GC-MS analysis of UPGP; the
results suggested that the glycoside moiety contributed
to the a-glucosidase inhibitory effect (data not shown).
Glycosides are considered promising natural inhibitors
of a-glucosidase (Akkarachiyastit et al., 2010). The
hydroxyl group of glycosides may interact with the
active site of the enzyme through covalent or nonco-
valent interactions, which could modulate a-glucosi-
dase inhibition (Lo Piparo et al., 2008). In addition, it
has been reported that glycoproteins differ from a
nonglycosylated proteins in terms of their large ter-
tiary structure (Nelson & Cox, 2000). This structural
diversity may result in the hypoglycaemic effect of
UPGP.
© 2014 Institute of Food Science and TechnologyInternational Journal of Food Science and Technology 2015
Hypoglycemic activity of glycoprotein S. M. Rafiquzzaman et al.148

Conclusions
Our results confirm the hypoglycaemic properties of
UPGP based on various in vitro methods. This effect
was mediated by inhibiting a-glucosidase and promot-
ing glucose transport across the yeast cell membrane,
as revealed using an in vitro yeast cell model. Based on
our results, UPGP may be applicable as a nutraceuti-
cal or functional food to control hyperglycaemia.
Acknowledgments
This work was supported by a Research Grant of
Pukyong National University (2014 year).
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Hypoglycemic activity of glycoprotein S. M. Rafiquzzaman et al. 149

Supporting Information
Additional Supporting Information may be found in
the online version of this article:
Figure S1. Inhibition of yeast a-glucosidase (a) and
rat intestinal a-glucosidase (b) as a function of concen-
tration of UPGP and acarbose.
Figure S2. Reversibility of UPGP action against rat
intestinal a-glucosidase. Reversibility of UPGP was
determined by comparing the residual enzyme activity
after dialysis with that of nondialysed one. Results are
expressed as mean Æ SD (n = 3).
© 2014 Institute of Food Science and TechnologyInternational Journal of Food Science and Technology 2015
Hypoglycemic activity of glycoprotein S. M. Rafiquzzaman et al.150