Phytochemistry 98 (2014) 27–33

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Phytochemistry
journal homepage: www.elsevier.com/locate/phytochem

Alpha-amylase and alpha-glucosidase inhibition is differentially

modulated by fucoidan obtained from Fucus vesiculosus and
Ascophyllum nodosum
Kyung-Tae Kim, Laurie-Eve Rioux, Sylvie L. Turgeon ⇑
Department of Food Science and Nutrition, Institute of Nutrition and Functional Food, Pavillon Paul-Comtois, Laval University, 2425, rue de l’Agriculture, Quebec City, Qc, G1V
0A6, Canada

a r t i c l e i n f o a b s t r a c t

Article history: Fucoidan is a water-soluble, negatively charged, biologically active polysaccharide found in great
Received 31 October 2011 abundance in brown marine algae. However, the inhibition of a-amylase and a-glucosidase by fucoidan
Received in revised form 30 June 2012 derived from two algal species (Ascophyllum nodosum and Fucus vesiculosus) harvested at different
Available online 30 December 2013
periods (accounting for seasonal and yearly variations) has never been investigated. It was found that
fucoidans inhibited a-glucosidase differently, depending on the algal species from which it was extracted
Keywords: and the algae’s season of harvest. Fucoidan extracted from A. nodosum was a more potent inhibitor of
Brown seaweed
a-glucosidase, with an IC50 ranging from 0.013 to 0.047 mg/mL, than the inhibition by fucoidan extracted
Fucus vesiculosus
Ascophyllum nodosum
from F. vesiculosus (IC50 = 0.049 mg/mL). In contrast, fucoidan extracted from F. vesiculosus did not inhibit
Harvest season a-amylase activity, while fucoidan from A. nodosum decreased a-amylase activity by 7–100% at 5 mg/mL
Fucoidan depending upon the algae harvest period. An IC50 of 0.12–4.64 mg/mL for fucoidan from A. nodosum was
Sulfated fucan found for the a-amylase inhibition. The ability of fucoidan to inhibit a-amylase and a-glucosidase thus
a-Amylase varies according to the algae species and harvest period. A. nodosum is more suitable than F. vesiculosus
a-Glucosidase as a source of fucoidan to inhibit a-amylase and a-glucosidase activities. Their potential benefits towards
Enzyme inhibition Type 2 diabetes management should be further investigated.
Type 2 diabetes
Ó 2013 Elsevier Ltd. All rights reserved.

Introduction is suggested as a safe and complementary treatment for diabetes.

Diabetes is a metabolic disorder characterized by high plasma
glucose levels (Mitrakou et al., 1992; Porte, 2001), and is classified
as either Type 1 or Type 2. Type 1 or insulin-dependent diabetes is
due to a failure of the pancreas to secrete insulin, while Type 2 or
non-insulin-dependent diabetes is the result of insufficient insulin
production. Wild et al. (2004) estimated that 246 million persons
in the world suffered from Type 2 diabetes in 2007 and that this
number will reach at least 380 million in 2025.
Type 2 diabetes receives more attention than Type 1 diabetes
because it is considered to be a preventable disease. Type 2 diabetes
is caused by an imbalance between blood sugar absorption and insu-
lin secretion. Post-prandial hyperglycemia plays an important role
in development of Type 2 diabetes (Baron, 1998). Controlling plasma
glucose levels are essential for delaying or preventing Type 2
diabetes. It is possible to reach this goal by stimulating insulin
secretion through medication and/or dietary supervision (Goldberg
et al., 1998; Porte, 2001; Rabasa-Lhoret and Chiasson, 2003; UK
Prospective Diabetes Study (UKPDS) Group, 1998). Dietary control
⇑ Corresponding author. Tel.: +1 418 656 2131x4970; fax: +1 418 656 3353.
E-mail address: sylvie.turgeon@fsaa.ulaval.ca (S.L. Turgeon).
A diet based on the glycemic index of foods is currently one of the
most recommended nutritional treatments. It has been reported
that dietary therapy can be used simultaneously with other medical
treatments to obtain a synergistic effect (Franz, 2000; Gannon et al.,
2001; Jenkins and Wolever, 1981; Wolever et al., 1994). However,
this has the drawback of limiting the types and quantity of food con-
sumed. Another possible solution is to decrease the rate of blood su-
gar absorption from the small intestine by slowing and interrupting
the digestion of dietary starch, the major dietary source of glucose
(Rabasa-Lhoret and Chiasson, 2003). This approach is considered
more efficient than controlling insulin secretion for economic rea-
sons, convenience and the avoidance of side-effects (Porte, 2001).
The inhibition of enzymes, such as a-amylase and a-glucosidase,
which digest dietary starch into glucose, has been studied as a meth-
od to control blood sugar levels (Ali et al., 2006; Geng and Bai, 2008;
Svensson et al., 2004). a-Amylase catalyzes the hydrolysis of a-
(1,4)-glucosidic linkages and produces maltose and glucose from
starch (Søgaard et al., 1993; Teeri, 1991), while a-glucosidase
releases glucose from maltose and/or sucrose (Mohan and Pinto,
2007; Roth et al., 2003). Both of them are secreted in the small
intestine, while only a-amylase is found within saliva. By inhibiting
these two enzymes, the absorption of glucose into the bloodstream

0031-9422/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.phytochem.2013.12.003
28 K.-T. Kim et al. / Phytochemistry 98 (2014) 27–33

can be delayed and thus ameliorate Type 2 diabetes symptoms such sible during the winter because of ice. The range of fucoidan con-
as hyperglycemia. Attempts have been made to identify a-amylase centrations investigated (0–5 mg/mL) was established by
and a-glucosidase inhibitors that can be used as food or food addi- preliminary tests.
tives. Although Seo et al. (2005) and Kato et al. (2005) demonstrated Interestingly, a-amylase inhibitory activity of fucoidan was
that some sugar-like phenolic compounds have a-glucosidase completely different depending on the seaweed source. The fucoi-
inhibitory activity, most studies on a-amylase and a-glucosidase dans extracted from A. nodosum significantly suppressed a-amy-
inhibitors have focused on the utilization of phenolic compounds lase activity; the level of suppression varied depending on the
(Bhandari et al., 2008; He et al., 2007; Kim et al., 2000; Lee et al., harvest period and the fucoidan concentration. In contrast, none
2008; Song et al., 2005; Zhang et al., 2007). of the fucoidan extracts from F. vesiculosus in the range of concen-
Recently, algae have been considered as a source of enzyme tration studied inhibited a-amylase activity (data not shown). This
inhibitors. Similar to plant extracts, algal extracts may be consid- difference indicates that the source of fucoidan has a strong influ-
ered for this purpose because they contain some polyphenolic ence on its inhibitory capacity. It should be noted that the same
compounds, such as bromophenols (Kurihara et al., 1999; Liu extraction method was used for both seaweed species and that
et al., 2011) and phlorotannins (Nwosu et al., 2011; Zhang et al., they were similar in general composition (data not shown). Also,
2007), which are inhibitors of a-glucosidase. Additionally, the purity and the sulfate content were found to be similar
polysaccharides isolated from algae have become attractive in throughout the harvest period, except for the year 2005 where pur-
the biomedical area because of their numerous bioactivities (Gupta ity was lower for F. vesiculosus (Supplementary Fig. 1). Statistical
and Abu-Ghannam, 2011; Holdt and Kraan, 2011; Li et al., 2008). analysis indicated that the purity of the fucoidan fractions did
Marine algae contain large amounts of non-starch polysaccharides not impact their activity (average purity: 87% for A. nodosum and
that cannot be digested completely by the human digestive system 81% for F. vesiculosus) (Table 1).
and which therefore have potential as new sources of dietary fiber, Several concentrations of fucoidan were tested (0.05–5 mg/mL).
prebiotics or other functional ingredients (Lahaye, 1991; Mabeau At a low dose of fucoidan (0.05 mg/mL), no significant effect was
and Fleurence, 1993). As with plant fiber from other sources, sea- observed on the a-amylase activity (Table 1). As the concentration
weed fiber is interesting because its consumption has been associ- increased (0.1–5 mg/mL), the independent variables (year and/or
ated with a significant reduction of chronic diseases such as month of harvest) and the interaction of those variables explain
diabetes, obesity, blood pressure, and so on (Landin et al., 1992; most of the variation observed. This could be visualized with the
Maki et al., 2006; Ou et al., 2001). Soluble fiber can slow down dose-dependent inhibition of a-amylase activity that is presented
digestion and absorption of nutrients by increasing viscosity and in the Supplementary Fig. 2. When the concentration reaches
might thereby decrease blood sugar and cholesterol (Mabeau and 1 mg/mL, only two fucoidan extracts were able to inhibit more
Fleurence, 1993). In this context, fucoidan, a bioactive polysaccha- than 50% of a-amylase while more were found at 5 mg/mL. a-
ride found in brown algae, appears promising. It is abundant in Amylase activity changes caused by fucoidan at 5 mg/mL from
brown seaweed, such as Fucus vesiculosus and Ascophyllum nodo- each harvest period are shown in Fig. 1. Although no clear trends
sum (Percival and McDowell, 1967). It is a mucilaginous, hygro- was observed for the a-amylase activity, the inhibition is signifi-
scopic and sulfated polysaccharide (Black, 1954) that protects cantly lower in May 2002 and 2005 (20% inhibition), and no inhi-
seaweed from dehydration (Percival and McDowell, 1967). Among bition was observed in June 2003. Fucoidan harvested in the late
the biological effects attributed to fucoidan are anti-coagulant, summer months generally demonstrated significantly higher inhi-
anti-HIV and anti-tumor (anti-angiogenesis) activities (Berteau bition activity (>83% in October 2002 and in August 2005) than
and Mulloy, 2003; Gupta and Abu-Ghannam, 2011). Brown algae spring months. The results indicate that important variation in
contain levels as high as 10% fucoidan, with the quantity varying activity occurs according to the harvest period of the seaweed,
depending on the region, species and season of harvest of the algae with IC50 values ranging from 0.12 to 4.64 mg/mL. According to
(Mabeau et al., 1990). The structural and chemical characteristics
of fucoidan also vary among algal species (Bilan et al., 2006; Chevo-
Table 1
lot et al., 2001; Marais and Joseleau, 2001; Pereira et al., 1999). Analysis of variance for the a-amylase measurements.
The aim of this study was to investigate the inhibition of starch
Degrees of freedom F-value Pr > F
digestive enzymes (a-amylase and a-glucosidase) by fucoidan ex-
tracted from F. vesiculosus and A. nodosum harvested in Eastern Fucoidan concentration: 0.05 mg/mL
Covariate: Purity 1 0.4 0.5269
Canada over several years and across seasons.
Year 2 0.5 0.6326
Months of harvest 6 1.3 0.3245
Results and discussion Year  Months 5 0.4 0.8316
Fucoidan concentration: 0.1 mg/mL
Most studies that look for natural extracts that may contribute Covariate: Purity 1 0.9 0.3624
Year 2 1.4 0.2909
to diabetes prevention have focused on a-glucosidase inhibition
Months of harvest 6 11.3 0.0002
because this enzyme plays a role in glucose release from maltose Year  Months 5 7.7 0.0015
and/or sucrose. Although the inhibition of a-amylase also results Fucoidan concentration: 0.5 mg/mL
in the decrease of glucose release, its complete inhibition is not de- Covariate: Purity 1 0.2 0.6354
Year 2 9.4 0.003
sired because it could provoke intestinal disorders (Cho et al.,
Months of harvest 6 15.7 <.0001
2011) due to the undigested starch being utilized by the gut micro- Year  Months 5 6.0 0.0044
flora for gas production. However, partial inhibition of a-amylase Fucoidan concentration: 1 mg/mL
may help modulate the rate of glucose release from starch. Covariate: Purity 1 0.3 0.5946
Year 2 5.3 0.0208
Months of harvest 6 10.8 0.0002
Inhibition of a-amylase by fucoidan Year  Months 5 2.4 0.0979
Fucoidan concentrations mg/mL
In this study, brown algae harvested from eastern Canada from Covariate: Purity 1 0.3 0.5987
May to November in 2002, 2003 and 2005 were used to determine Year 2 29.7 <.0001
Months of harvest 6 17.4 <.0001
the ability of fucoidan collected at different points throughout the
Year  Months 5 15.6 <.0001
harvest period to inhibit a-amylase. No algae harvesting was pos-
 K.-T. Kim et al. / Phytochemistry 98 (2014) 27–33 29

Table 2
Analysis of variance for the a-glucosidase measurements.

Degrees of A. nodosum F. vesiculosus

freedom
F-value Pr > F F-value Pr > F
Fucoidan concentration: 0.005 mg/mL
Covariate: Purity 1 0.19 0.6719 0.03 0.8582
Year 2 0.59 0.5706 1.37 0.2883
Months of harvest 6 4.39 0.0122 2.37 0.0901
Year  Months 5 0.27 0.9203 0.92 0.4992
Fucoidan concentration: 0.01 mg/mL
Covariate: Purity 1 0.08 0.7852 5.44 0.0364
Year 2 1.09 0.3661 1.58 0.2430
Months of harvest 6 11.76 0.0001 2.37 0.0906
Year  Months 5 0.72 0.6194 0.97 0.4719
Fucoidan concentration: 0.025 mg/mL
Covariate: Purity 1 0.27 0.6118 3.88 0.0706
Year 2 5.75 0.0162 6.77 0.0097
Months of harvest 6 16.61 <.0001 3.52 0.0270
Year  Months 5 6.53 0.0030 0.72 0.6224
Fucoidan concentration: 0.05 mg/mL
Covariate: Purity 1 0.00 0.9977 0.21 0.6543
Year 2 8.60 0.0042 13.46 0.0007
Months of harvest 6 52.63 <.0001 7.52 0.0012
Year  Months 5 31.90 <.0001 2.61 0.0755

activity. This study suggests that a possible relationship may exist

Fig. 1. a-Amylase inhibitory activity induced by fucoidans (5 mg/mL) extracted
from A. nodosum harvested in A-2002, B-2003 and C-2005. Identical letters within
the same panel are not significantly different at p < 0.05.
between the fucoidan’s sulfate content and its a-amylase inhibi-
tion capacity. In studies by Cho et al. (2011), a sulfate content as
P51% was required to inhibit a-amylase activity; native fucoidan
with a 42% sulfate content, was unable to affect the enzyme activ-
ity. However, all fucoidan extracts from A. nodosum, which showed
a-amylase inhibition, were less sulfated (<25% sulfate content)
than U. pinnatifida fucoidan. In addition, the average sulfate con-
tent of fucoidan was not significantly different between F. vesiculo-
sus (20.28%) and A. nodosum (21.53%) (Supplementary Fig. 1). The
results of a-amylase inhibition by fucoidan in this study are thus
not in agreement with the result of Cho et al. (2011). Therefore,
it is possible that other structural features might be involved in
a-amylase inhibition.

Inhibition of a-glucosidase by fucoidan

Black (1954), the ratio of L-fucose found in fucoidan obtained from
F. vesiculosus and A. nodosum varies depending on the harvest
period. The seasonal variation in fucoidan’s chemical structure
(i.e., the relative contributions of sulfate, monosaccharide and
uronic acid), has been demonstrated in other brown seaweeds
(Rioux et al., 2009; Skriptsova et al., 2010), but not in the seaweeds
used in this study. The structural changes may result in the sea-
sonal variation of a-amylase inhibitory activity by fucoidan. Addi-
tionally, the structure of fucoidan varies depending on the alga
source and could impact the enzyme activity. Both fucoidan, from
A. nodosum and F. vesiculosus are mainly composed of a-(1–3)
linked sulfated L-fucose sub-units (Chevolot et al., 2001; Patankar
et al., 1993). A low proportion of a-(1–4) linked fucose (Daniel
et al., 1999; Marais and Joseleau, 2001) or a repeating a-(1–3)
and a-(1–4)-linkage (Chevolot et al., 2001; Daniel et al., 2007)
were reported but only for A. nodosum. The molecular weight of
fucoidan also varies according to algal species. Rioux et al.
(2007)found different molecular weights for fucoidan. For exam-
ple, A. nodosum had molecular weights of 1323 kDa, wherever F.
vesiculosus had molecular weights of 887 kDa. Thus, the structural
difference reported between A. nodosum and F. vesiculosus could
impact their enzyme inhibitory activity.
The ability of fucoidan to inhibit a-amylase has been reported
by Cho et al. (2011) using an extract from Undaria pinnatifida
(wakame). A native fucoidan fraction did not show any inhibition
activity, but an oversulfated fucoidan slightly reduced this enzyme
Several concentrations of fucoidan were tested (0.005–0.05 mg/
mL) to determine levels of a-glucosidase inhibition. A statistical
analysis indicates that the purity of the fucoidan made a significant
difference only for F. vesiculosus at 0.01 mg/mL. At low doses of
fucoidan (0.005 and 0.01 mg/mL), the independent variable does
not completely explain the variation in a-glucosidase activity
(Table 2). As the concentration is increased, most of the variation
is explained by the independent variables (year and month
harvested), which suggests that the a-glucosidase activity is signif-
icantly different for each seaweed harvest period. The dose-depen-
dent inhibitions of a-glucosidase activity are presented in the
Supplementary Figs. 3 and 4. When the concentration reaches
0.025 mg/mL only few fucoidan extracts can inhibit more than
50% of a-glucosidase activity for A. nodosum, while none were
found for F. vesiculosus. Figs. 2 and 3 present the a-glucosidase
activity when inhibited by fucoidan (0.05 mg/mL). Fucoidans from
both F. vesiculosus and A. nodosum inhibited a-glucosidase activity.
Inhibition depended on both the harvesting period and the algal
species, though the inhibition was consistently greater for fucoidan
derived from A. nodosum. For F. vesiculosus, the maximal inhibition
capacity was 51.4% (for seaweed harvested in September 2002),
while for A. nodosum the inhibition was 99.6% (for seaweed har-
vested in October 2002 and 2003). Even at a concentration of
0.025 mg/mL, the fucoidan from A. nodosum extracted in autumn
(October/November) showed remarkable a-glucosidase inhibition
30 K.-T. Kim et al. / Phytochemistry 98 (2014) 27–33
Fig. 2. a-Glucosidase inhibitory activity induced by fucoidans (0.05 mg/mL)
extracted from A. nodosum harvested in A-2002, B-2003 and C-2005. Identical
letters within the same panel are not significantly different at p < 0.05.
(over 80%). The IC50 values for a-glucosidase using fucoidan ob-
tained from A. nodosum are shown in Table 3. The results showed
that the IC50 ranged from 0.013 to 0.047 mg/mL for A. nodosum.
On the other hand, the IC50 value for the fucoidan from F. vesiculo-
sus could only be calculated for September 2002 (IC50 = 0.049 mg/
mL), because it is the only harvest period with the concentrations
tested that reached an inhibition of 50%. Overall, fucoidans from A.
nodosum showed better a-glucosidase inhibition than those from F.
vesiculosus, and the highest inhibitory activity was obtained from
the autumn harvest (October/November) of A. nodosum. As ex-
plained previously, the structural difference found between A.
nodosum and F. vesiculosus could explain the inhibition variation
observed for those two brown seaweeds.
The IC50 of the fucoidan obtained from A. nodosum ranged from
0.013 to 0.047 mg/mL, making it more potent than other a-gluco-
sidase inhibitors, such as acarbose (IC50 = 1 mg/mL), which is used
as a medication for Type 2 diabetes (Table 4) (Schäfer and Högger,
2007). Fucoidan’s IC50 is also lower than the IC50 values for several
plant extracts found in tea and other food products, which have an
IC50 range from 11.1 to 519.8 mg/mL (Matsui et al., 1996). How-
ever, polyphenols extracted from A. nodosum have lower IC50
values; those values range from 0.024 to 0.077 mg/mL (Zhang
et al., 2007). Zhang et al. (2007) also studied the effects of a poly-
phenol fraction and a polysaccharide-enriched fraction in the diet
of diabetic mice (200 mg extract/kg body mass). While the poly-
phenol fraction reduced the mice’s glucose levels after 14 days
Fig. 3. a-Glucosidase inhibitory activity induced by fucoidans (0.05 mg/mL)
extracted from F. vesiculosus harvested in A-2002, B-2003 and C-2005. Identical
letters within the same panel are not significantly different at p < 0.05.
on the diet, the polysaccharide-enriched fraction did not decrease
them significantly. Apostolidis and Lee (2010) reported a strong
a-glucosidase inhibition (with an IC50 at 0.24 lg of polyphenol)
and a mild a-amylase inhibitory effect (IC50 at 1.34 lg of polyphe-
nol) with a water-soluble extract from A. nodosum. However, they
did not mention the fucoidan content in the extract. In the content,
herein fucoidans from F. vesiculosus and A. nodosum harvested in
October 2002 contained approximately 0.1% polyphenols. Never-
theless, it is assumed that a-amylase and a-glucosidase inhibition
by fucoidan is not due to the contamination by phenolic
compounds. Fucoidans from A. nodosum and F. vesiculosus have
potentially inhibitory activity against alpha-glucosidase. The exact
mechanism of action needs to be further determined.
Mechanisms of a-glucosidase and a-amylase inhibition differ
among the various inhibitors reported. Previously published arti-
cles on the mechanism of polyphenol compounds suggest that
the principal factor affecting a-glucosidase activity is hydrogen
scavenging because a-glucosidase provides hydrogen needed to
catalyze the hydrolysis of the a-(1,4)-glucosidic linkage (Borges
de Melo et al., 2006; Braun et al., 1995; Mohan and Pinto, 2007).
The inhibitor acts by intercepting the hydrogen ion freed from
the a-glucosidase catalytic site. Some well-known inhibitors, such
as acarbose, mimic the enzyme substrate (Rabasa-Lhoret and
Chiasson, 2003). For a-amylase, the suggested inhibition mecha-
nisms include (1) complex formation between the inhibitor (ex:
 K.-T. Kim et al. / Phytochemistry 98 (2014) 27–33 31

Table 3 Finally, when comparing the inhibition of the two digestive en-
Variation of the a-glucosidase inhibitory activity of fucoidans extracted from A. zymes studied, a-amylase and a-glucosidase, it should be empha-
nodosum for selected harvests.
sized that at least 100 times more fucoidan is needed (5 mg/ml) to
Harvest month IC50 of fucoidan* (mg/mL) inhibit a-amylase to the same degree as a-glucosidase (0.05 mg/
May 0.047 mL). This difference in inhibition is beneficial because, as reported
June 0.037 by Cho et al. (2011), high a-amylase inhibition could be related to
July 0.015–0.036 intestinal discomfort; therefore, a moderated inhibition of a-amy-
August 0.017–0.046
September 0.026–0.029
lase and a stronger glucosidase inhibition would be preferred. The
October 0.013 level of inhibition obtained with fucoidan should therefore result
November 0.014 in the desired effect by slowing the absorption of glucose from
* the intestine into the bloodstream enough to allow the insulin-
Calculation of the fucoidan concentration required to inhibit 50% of the enzyme
activity (performed with Prism software). mediated transport of blood sugar to the muscles before it can
reach harmful levels in the blood of the diabetic patient. These data
suggest that fucoidan could be useful as a component of new med-
Table 4 ication as a food additive or as a food supplement for an enzyme-
Comparison of the inhibition of a-glucosidase by fucoidans extracted from A. nodosum targeted treatment of Type 2 diabetes. Further studies on fucoidan
and other food products. structural features are required to understand the mechanism by
a-Glucosidase inhibitor IC50 (mg/mL) which fucoidan inhibits a-amylase and a-glucosidase and why
only fucoidan obtained from A. nodosum inhibits a-amylase.
Fucoidan from A. nodosum 0.013–0.047
Polyphenolic extracts of A. nodosuma 0.024–0.077
b
Acarbose (Type 2 diabetes medication) 1 Conclusions
Green teac 11.1
Oolong teac 11.3
Sardine muscle hydrolyzatec 48.7 This study has established a novel function of fucoidan as an
Chicken essencec 471.4 efficient inhibitor of the starch-digesting enzymes a-amylase and
Yogurtc 519.8 a-glucosidase. In summary, the enzyme-inhibiting activity of fuco-
a
Zhang et al. (2007). idan was quite variable and depended on the algal species from
b
Schäfer and Högger (2007). which the fucoidan was extracted, the month and year during
c
Matsui et al. (1996). which the algae were harvested and the targeted enzyme (a-amy-
lase or a-glucosidase). Fucoidan from A. nodosum inhibited both
a-amylase and a-glucosidase, but the quantities required for
acarbose) and a-amylase limiting its activity (Nahoum et al., 2000)
a-amylase inhibition were much higher than those required for
or (2) slowing the diffusion of glucose from the active site, for
a-glucosidase. The best harvesting period for a-amylase inhibition
example, by viscous water-soluble dietary fibers, delaying both
from fucoidan extracted from A. nodosum was summer and au-
carbohydrate digestion and glucose absorption (Landin et al.,
tumn, and the best harvesting period for a-glucosidase inhibition
1992; Maki et al., 2006; Ou et al., 2001). Fucoidan could inhibit en-
from both A. nodosum- and F. vesiculosus-derived fucoidan was au-
zyme through electrostatic interaction between the negatively
tumn. These studies using species harvested in eastern Canada
charged sulfate groups of fucoidan and the enzyme. Additionally,
indicate that A. nodosum has a greater potential for the prevention
fucoidan from F. vesiculosus has already shown an higher viscosity
of Type 2 diabetes than F. vesiculosus.
than that of A. nodosum (Rioux et al., 2007) which could impact its
diffusivity in the solvent and subsequently increasing the time for
fucoidan to reach the enzyme. Most studies conducted with fuco- Experimental
idan showed that the sulfate content is closely related to its biolog-
ical properties. Increasing sulfate groups may enhance the activity Algae and chemicals
of fucoidan. Oversulfated fucoidan has disturbed the interaction
between vascular endothelial growth factor 165 and its receptor The brown marine algae F. vesiculosus and A. nodosum were har-
on the cell surface, suppressing its activity (Koyanagi et al., vested near L’Isle Verte (latitude 47° 48.60 N and longitude 69°
2003). Additionally, negatively charged fucoidan binds to a part 33.00 W) from the St. Lawrence River in Quebec (Eastern Canada)
of the positive charges on antithrombin, through electrostatic from May to November in 2002, 2003 and 2005. Algae were milled
interactions (Tissot et al., 2003). Therefore, sulfate groups found in a Cormitrol Mill fitted with perforated plates of 24.5 and 1 mm,
in fucoidan may be involved in the mechanism of the action of en- freeze-dried and then kept at 20 °C until use. Cornstarch was
zyme inhibition but more work will be necessary to establish their used as a substrate for the a-amylase assay (Novation 9230 organic
role. corn starch). All H2O used in the experimental was distilled from
Because the inhibition of a-amylase and a-glucosidase activity de-ionized H2O and filtered (0.2 lm membranes). All chemicals
by fucoidan from the different seaweed sources varies greatly, it is were purchased from Sigma–Aldrich (On, Canada).
considered that the structure of the fucoidan differs in the two sea-
weed sources. Structural features in fucoidan, such as molecular Fucoidan extraction
weight, and the number of sulfate groups, could severely affect
the enzyme activity. Additionally, the amount and type of branch- Lyophilized algae mass (15 g) was suspended in 1% CaCl2 solu-
ing chain on the polysaccharide can affect its anticomplementary tion (450 mL) and stirred for 4 h at 85 °C using an RZR1 stirrer (Caf-
activity (Clement et al., 2010). More work was completed to ex- ramo Ltd. Canada) set at 455 ± 5 rpm. After centrifugation
plain how those structural characteristics influence the enzyme (16,887g, 12 min), the liquid phase was separated by vacuum fil-
activity; this work is discussed in Kim et al. (2013). The molecular tration using Whatman No. 4 filter paper. The filtrate was mixed
weight and the sulfate content were important towards enzyme with two volumes of EtOH and one volume of 1% NaCl solution.
inhibition, while the monosaccharide composition, the glycosidic The mixture was kept at 20 °C for 48 h. The results, precipitate
linkage, and the position of the sulfate groups, had no impact. was collected by centrifugation (16,887g, 20 min) and completely
resuspended in de-ionized H2O (100 mL). This solution was
32 K.-T. Kim et al. / Phytochemistry 98 (2014) 27–33
dialyzed for 48 h using a 15 kDa cut-off dialysis membrane (Fisher
Sci., USA). The dialyzed fucoidan was freeze-dried and kept at
20 °C in sealed tubes. Two extractions were performed for each
seaweed sample.
The fucoidan purity was assessed by HPLC using HPLC Rezex
RPM Monosaccharide 50  7.8 mm precolumn (Phenomenex,
USA) and Rezex RPM Monosaccharide 300  7.8 mm (Phenome-
nex, USA). The system consisted of Waters 715 Ultra wisp sample
processor (Millipore, USA), LKB Bromma 2150 HPLC pump (LKB,
Sweden) and Water 410 differential Refractometer detector
(Millipore, USA) linked to Agilent interface analogue 3590E
(Agilent technology, USA) with HP Chemstation Rev. A.06.03 soft-
ware. The mobile phase was 0.2 lm filtered HPLC grade H2O and
the flow rate was 0.6 mL/min. Commercial fucoidan (98% purity)
of F. vesiculosus (Sigma, On, Canada) was dissolved in deionized
H2O to make several concentrations (125, 250, 300, 400 and
500 ppm) for standard curve.
a-Amylase inhibition assay
The method of Conforti et al. (2005) was modified to determine
the inhibitory effects of the fucoidan extract on a-amylase activity
(from human saliva, EC 3.2.1.1). A 1% starch solution was prepared
by stirring corn starch (1 g) in 20 mM sodium phosphate buffer
(100 mL) with 6.7 mM sodium chloride at a pH of 6.9. The solution
was heated at 100 °C for 15 min and then cooled to room temper-
ature. The volume was brought to 100 mL with distilled H2O. The
a-amylase (A0521, Sigma, On, Canada) solution was prepared as
1 unit/mL. The colorimetric reagent was prepared as shown in
the method of Conforti et al. (2005). The fucoidan solution
(0.1 mL) was added to 1 mL of starch solutions to reach final con-
centrations of 0, 0.05, 0.1, 0.5, 1.0 and 5 mg/mL (dry weight pow-
der). As a control (0 mg/mL fucoidan), distilled water (0.1 mL)
was added instead of the fucoidan solution. The tubes were pre-
pared in duplicate and split into two groups, which were classified
as the test group (TG) and the control group (CG). All tubes were
incubated at 20 °C for 10 min, and then a-amylase solution
(1 mL) was added. For the TG, the tubes were incubated at exactly
20 °C for 5 min. The colorimetric reagent (1 mL of 3,5-dinitrosali-
cylic solution) was added to all of the tubes of the TG, heated at
100 °C for 15 min and then cooled in an ice bath. Distilled H2O
(9 mL) was added to each tube, and the absorbance at 540 nm
was measured. The same steps were completed for the CG, but
the incubation at 20 °C for 5 min was omitted. The degree of en-
zyme inhibition was calculated using the following equation:
ODTG  ODCG
Enzyme inhibition ð%Þ ¼  100ð%Þ
ODCG
a-Glucosidase inhibition assay
To assess of the inhibitory activity of fucoidan on a-glucosidase
(EC 3.2.1.20), all solutions were prepared according to the method
of Halvorson and Ellias (1958). 67 mM potassium phosphate (5 mL,
pH 6.8 at 37 °C) and 3 mM glutathione solution (0.2 mL) were
added into 10 test tubes. They were split into two groups (test
and control), and into the TG tubes, a-glucosidase (G5003, Sigma)
(0.2 mL of 1 unit/mL) was added, while distilled H2O (0.2 mL) was
added to the CG tubes. Next, the fucoidan solutions (0.1 mL) was
added to all of the tubes to reach final concentrations of 0, 0.005,
0.01, 0.025 and 0.05 mg/mL (dry weight powder) in both TG and
CG tubes in parallel. The tubes without any fucoidan were used
to measure the background. All tubes were kept at 37 °C for
20 min, after which 10 mM p-nitrophenyl a-D-glucopyranoside
(0.5 mL) was added to all of the tubes. The tubes were then incu-
bated at 37 °C for 20 min. 1 mL of the solutions was transferred
to new test tubes containing sodium carbonate (4 mL, 0.1 M),
and the new solutions were mixed. The absorbance was measured
at 400 nm, and the degree of enzyme inhibition was calculated
using the following equation:
ODTG  ODCG
Enzyme inhibition ð%Þ ¼  100ð%Þ
ODCG
Statistical analysis
Enzyme inhibition analyses were performed in duplicate with
two repetitions for each fucoidan sample. The results are presented
as the mean ± standard deviation (SD) for each harvesting period.
Significant differences were defined as when p < 0.05. Statistical
analysis was carried out using Prism version 5.0 (GraphPad Soft-
ware, San Diego, USA) for the IC50 calculation and it was deter-
mined only when the inhibition was higher than 50%. The
enzyme activity data were analyzed using SAS 9.2 software (SAS
Institute Inc., Cary, USA). To determine the effect of fucoidan purity
on the enzyme activity, the purity of each fucoidan sample was
added to the statistical model as a covariate in the mixed model
analysis. Because the year when the algae was harvested and the
fucoidan was extracted was considered significant at p < 0.05, an-
other test for the higher fucoidan concentration from each separate
year was performed to compare the least squares means of the dif-
ferent months of harvest using Tukey adjustment with p < 0.05.
Acknowledgements
The authors would like to thanks the CORPAQ program and the
Ministère des pêcheries et de l’alimentation du Québec (MAPAQ)
for the financial support and the seaweeds harvesting. The authors
also wish to thanks Piotr Bryl and Martin Beaulieu for their useful
discussions about seaweeds and fucoidan bioactivity.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.phytochem.2013.
12.003.
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