Biosci. Biotechnol. Biochem.

, 75 (8), 1472–1480, 2011

Protein Tyrosine Phosphatase 1B and -Glucosidase Inhibitory Phlorotannins

from Edible Brown Algae, Ecklonia stolonifera and Eisenia bicyclis
Hye Eun M OON,1 Md. Nurul ISLAM,1 Bo Ra A HN,1 Sabiha Sultana C HOWDHURY,1
Hee Sook S OHN,2 Hyun Ah J UNG,2; y and Jae Sue C HOI1;3; y
1
Department of Food Science and Nutrition, Pukyong National University, Busan 608-737, Republic of Korea
2
Department of Food Science and Human Nutrition, Chonbuk National University, Jeonju 561-756, Republic of Korea
3
Blue-Bio Industry RIC, Dong-Eui University, Busan 614-714, Republic of Korea

Received February 21, 2011; Accepted April 20, 2011; Online Publication, August 7, 2011
[doi:10.1271/bbb.110137]

The present work investigates protein tyrosine phos-
phatase 1B (PTP1B) and the -glucosidase inhibitory
activities of two edible brown algae, Ecklonia stolonifera
and Eisenia bicyclis, as well as in their isolated
phlorotannins. Since the individual extracts and frac-
tions showed significant inhibitory activities, column
chromatography was performed to isolate six phloro-
tannins, phloroglucinol (1), dioxinodehydroeckol (2),
eckol (3), phlorofurofucoeckol-A (4), dieckol (5), and
7-phloroeckol (6). Phlorotannins 3–6 were potent and
noncompetitive PTP1B inhibitors with IC50 values
ranging from 0.56 to 2.64 M; 4–6 exhibited the most
potent -glucosidase inhibition with IC50 values ranging
from 1.37 to 6.13 M. Interestingly, 4 and 6 were
noncompetitive, while 5 exhibited competitive inhibition
in an -glucosidase assay. E. stolonifera and E. bicyclis
as well as their isolated phlorotannins therefore pos-
sessed marked PTP1B and -glucosidase inhibitory
activities; this could lead to opportunities in the devel-
opment of therapeutic agents to control the postprandial
blood glucose level and thereby prevent diabetic com-
plications.
Key words: Ecklonia stolonifera; Eisenia bicyclis; pro-
tein tyrosine phosphatase 1B;
-glucosi-
dase; phlorotannin
Diabetes mellitus is a chronic metabolic disorder
characterized by insulin resistance, including defects in
both insulin secretion and insulin response, resulting in
hyperglycemia as well as an abnormal metabolism of
carbohydrate, fat, and protein. Diabetes mellitus is
mainly classified into two types: an inherited or acquired
deficiency in insulin production and/or insulin secretion
from pancreatic  cells (type I diabetes, insulin-depend-
ent diabetes mellitus), or a combined resistance to both
insulin action and secretory response (type II diabetes,
non-insulin-dependent diabetes mellitus).1,2) Type II
diabetes is a leading health concern due to its escalating
prevalence throughout the world and serious diabetic
complications. Moreover, the high morbidity and mor-
tality rates associated with chronic diabetic complica-
y
To whom correspondence should be addressed. Jae Sue CHOI, Fax: +82-51-629-5842; E-mail: choijs@pknu.ac.kr; Hyun Ah JUNG,
Fax: +82-63-270-3854; E-mail: jungha@jbnu.ac.kr
1
These authors contributed equally to this work.
Abbreviations: DTT, dithiothreitol; EDTA, ethylenediaminetetraacetic acid; IR, insulin receptors; pNPG, p-nitrophenyl
-D-glucopyranoside;
pNPP, p-nitrophenyl phosphate; PTKs, protein tyrosine kinases; PTP1B, protein tyrosine phosphatase 1B; TC-PTP, T-cell PTP
tions make the disease the third largest killer, only after
cancer and cardiovascular diseases.3) In particular, a
persistently increased blood glucose level in diabetic
patients is considered the primary instigator for the
pathogenesis of long-term diabetic complications,
including retinopathy, cataractogenesis, nephropathy,
and neuropathy.4,5) Postprandial hyperglycemia also
plays a critical role in the development of type II
diabetes and associated cardiovascular complications.6)
Modulating postprandial hyperglycemia may therefore
play a crucial role in the treatment and prevention of
diabetes and its complications.
In addition to insulin therapy to reduce hyperglycemia
and maintain normoglycemia, several therapeutic
approaches have been proposed such as
-glucosidase
inhibitors to delay the digestion and absorption of
carbohydrates,7) and protein tyrosine phosphatase 1B
(PTP1B) inhibitors to modulate insulin receptor phos-
phorylation.8) Mammalian
-glucosidase, which is
located in the brush-border surface membrane of
intestinal cells, is the key enzyme that catalyzes the
final step in the digestive process of carbohydrates.9) In
contrast, PTP1B is localized on the cytoplasmic surface
of the endoplasmic reticulum in classical insulin-
targeted tissues such as the liver, muscle and fat.10) This
enzyme is a negative regulator of insulin signaling and
plays a key role in developing insulin resistance, this
being highly implicated in the metabolic syndrome.11)
The former therefore suppresses the influx of glucose
from the intestinal tract to vessels, while the latter
increases the glucose uptake from vessels into cells,
leading to a decrease in postprandial hyperglycemia
which can alleviate diabetes and its complications. An
effective therapeutic approach to treating type II dia-
betes is therefore to inhibit PTP1B and
-glucosidase,
with a subsequently enhanced insulin action and reduced
plasma glucose level to regulate glucose homeostasis.
Marine plants have been reported to possess numer-
ous biological and phytochemical benefits, and these
natural sources, with their great potential, have thus
become intriguing as nutraceuticals and pharmaceuti-
cals.12,13) Ecklonia stolonifera Okamura and Eisenia
 Anti-Diabetic Effects of Phlorotannins from Brown Algae
bicyclis (Kjellman) Setchell, two species of perennial
brown algae belonging to the family Laminareaceae, are
commonly distributed in the mid-Pacific coast around
Korea and Japan. E. stolonifera usually grows well in
subtidal zones at 2–10 m depth, while E. bicyclis grows
in sha1low water down to 8–10 m in the sublittoral zone.
Both brown algae are ecologically and economically
important, and are commonly consumed as a foodstuff
together with Laminaria japonica and Undaria pinna-
tifida.14–16) The Ecklonia and Eisenia species have also
been used as phlorotannin-rich raw materials.17) Phlor-
otannins are secondary metabolites polymerizing phlor-
oglucinol (1,3,5-trihydroxybenzene) through an ether,
phenyl, or 1,4-dibenzodioxin linkage. These polyphe-
nolic compounds are responsible for a variety of
bioactivities, including anti-diabetic complications,17,18)
and antitumorial,19) hepatoprotective,20) anti-plasmin
inhibiting,21) algicidal,22) tyrosinase inhibiting,23) anti-
inflammatory,24,25) nitrite-scavenging,26) anti-skin ag-
ing,27,28) antioxidative,29–31) anti-cholinesterase,32,33) anti-
Alzheimer,34) anti-hyperlipidemic,35) anti-diabetic,36–39)
anti-allergic,40) and angiotensin converting enzyme-I
inhibitory activities.41)
As part of our continuous search for antidiabetic
agents from natural sources, we investigated the MeOH
extract and solvent-soluble fractions of the two selected
brown algae, E. stolonifera and E. bicyclis, together
with six isolated phlorotannins against
-glucosidase
and PTP1B. Enzymatic kinetic analyses of the six
phlorotannins were also performed by using Dixon plots
in order to confirm the type of enzymatic inhibition and
to propose guidelines for phlorotannins as anti-diabetic
agents in discovering new drugs. This work therefore
attempts to evaluate the potential of E. stolonifera and
E. bicyclis, as well as their phlorotannins, as therapeutic
or preventive agents for diabetes and its complications.
Materials and Methods
General experimental procedures. The 1 H- and 13 C-NMR spectra
were determined with a JNM ECP-400 spectrometer (Jeol, Tokyo,
Japan) at 400 MHz for 1 H and at 100 MHz for 13 C. The 1 H- and
13
C-NMR chemical shifts were referenced to the residual solvent peaks
(H 3.31 and C 49.0 for CD3 OD; H 2.50 and C 39.5 for DMSO-d6 ).
The EI-MS data were collected by a GC-MS QP-5050A spectrometer
(Shimadzu, Kyoto, Japan), the LR FAB-MS data were assessed with a
JMS-HX110/110A spectrometer (Jeol, Tokyo, Japan), and the IR
spectrum (KBr) was recorded on a Shimadzu FT-IR spectrometer
(Tokyo, Japan). Column chromatography was conducted by using
silica gel 60 (70–230 mesh; Merck, Darmstadt, Germany), RP-18
Lichroprep (40–63 mm; Merck, Germany), and Sephadex LH20 (20–
100 mm; Sigma, St. Louis, MO, USA). Thin-layer chromatography
(TLC) was conducted on precoated Kieselgel 60 F254 plates
(20
20 cm, 0.25 mm) and RP-18 F254 plates (5
10 cm; Merck,
Darmstadt, Germany), using 50% H2 SO4 as a spray reagent.
Chemicals and reagents. Yeast
-glucosidase, acarbose, p-nitro-
phenyl phosphate (pNPP), p-nitrophenyl
-D-glucopyranoside (pNPG),
and ethylenediaminetetraacetic acid (EDTA) were purchased from
Sigma-Aldrich (St. Louis, MO, USA). PTP1B (human recombinant)
was purchased from Biomol
International LP (Plymouth Meeting,
PA, USA), and dithiothreitol (DTT) was purchased from Bio-Rad
Laboratories (Hercules, CA, USA). All other chemicals and solvents
used were purchased from E. Merck, Fluka, and Sigma-Aldrich, unless
otherwise stated.
Plant materials. The leafy thalli of E. bicyclis were purchased
from a local telemarketing company in Gangwon Province, Korea
1473
(www.ulleumgdomall.com), and authenticated by Emeritus Professor
C. H. Son of Pukyong National University. The leafy thalli of
E. stolonifera were collected at Gijang-gun in Busan, South Korea, in
February 2000 and authenticated by Prof. H. G. Kim of the Faculty of
Marine Bioscience and Technology at Kangnung National University.
Voucher specimens (no. 20000228) have been deposited in the author’s
laboratory (J. S. Choi).
Isolation of phlorotannins 1–6. The powdered leaf thallus of
E. bicyclis and E. stolonifera (500 g each) was refluxed with MeOH
(3
1 L) for 3 h, and each filtrate was concentrated to dryness in vacuo
at 40  C to render the MeOH extract (175.0 and 116.6 g, respectively).
These MeOH extracts were separately suspended in distilled H2 O and
partitioned in sequence with CH2 Cl2 , EtOAc, n-BuOH, and H2 O, thus
yielding four fractions (Fig. 1). The respective yields (%) of the
CH2 Cl2 (16.8 g), EtOAc (51.5 g), n-BuOH (39.6 g), and H2 O fractions
(62.5 g) of E. bicyclis were 9.6, 29.4, 22.6, and 35.7%. The respective
yields (%) of the CH2 Cl2 (9.1 g), EtOAc (4.2 g), n-BuOH (16.6 g), and
H2 O fractions (86.6 g) of E. stolonifera were 7.8, 3.6, 17.3, and 74.3%.
The active EtOAc fraction (51.5 g) obtained from E. bicyclis was
subjected to chromatography in a silica gel column, using EtOAc–
MeOH (50:1 to 5:1) as the eluent, yielding 10 subfractions (EF01–
EF10). Repeated column chromatography of EF01 (6.8 g) was
conducted with a solvent mixture of n-hexane and EtOAc (50:1 to
0:1), yielding 11 subfractions (EF0101–EF0111). Compound 1 (35 mg)
was purified from EF0104 (0.6 g) in an RP-18 column, eluting with
aqueous MeOH (20% MeOH to 100% MeOH, gradient elution). RP-18
column chromatography of EF0105 (3.8 g), using identical solvent
conditions, led to the isolation of 2 (19 mg) and 3 (160 mg).
Compounds 4 (200 mg), 5 (300 mg), and 6 (8.5 mg) were purified
from EF0106 (2.2 g) in RP-18 (20% MeOH to 100% MeOH, gradient
elution) and Sephadex LH-20 columns (100% MeOH). Respective
isolated compounds 1–6 were identified and characterized as
phloroglucinol,42) dioxinodehydroeckol, eckol, phlorofurofucoeckol-
A, dieckol,30) and 7-phloroeckol17,39) by spectroscopic methods, as well
as by comparing with published data. The EtOAc fraction (4.2 g)
obtained from E. stolonifera was chromatographed in a similar manner
to yield 1 (2.2 mg), 2 (0.9 mg), 3 (25.0 mg), 4 (43.5 mg), 5 (42.6 mg),
and 6 (1.2 mg) (Fig. 2).
Phloroglucinol (1). IR max (KBr) cm1 : 3481, 1617, 1499, 1419;
EI-MS m=z (rel. int., %): 126 [M]þ (100); 1 H-NMR (400 MHz,
CD3 OD) : 5.78 (3H, s, H-2/H-4/H-6); 13 C-NMR (100 MHz, CD3 OD)
: 160.9 (C-1/C-3/C-5), 96.3 (C-2/C-4/C-6).
Dioxinodehydroekol (2). IR max (KBr) cm1 : 3243, 1635, 1518,
1494, 1396, 1281, 1243, 1207, 1154, 1118, 1089, 1012, 810; negative
FAB-MS m=z: 369 ½M  H ; positive FAB-MS m=z: 370 [M]þ ;
1
H-NMR (400 MHz, DMSO-d6 ) : 9.77 (1-OH), 9.64 (9-OH), 9.60
(6-OH), 9.27 (3-OH), 9.26 (11-OH), 6.10 (1H, s, H-7), 6.04 (1H, d,
J ¼ 2:7 Hz, H-2), 6.01 (1H, d, J ¼ 2:7 Hz, H-10), 5.84 (1H, d,
J ¼ 2:7 Hz, H-4), 5.82 (1H, d, J ¼ 2:7 Hz, H-12); 13 C-NMR
(100 MHz, DMSO-d6 ) : 153.3 (C-3), 153.0 (C-11), 146.1 (C-1),
146.0 (C-9), 142.1 (C-4a), 141.7 (C-12a), 140.1 (C-6), 137.2 (C-7a),
131.6 (C-13b), 125.9 (C-5a), 122.7 (C-8a), 122.5 (C-13a), 122.3
(C-14a), 98.8 (C-2/C-10), 97.6 (C-7), 93.9 (C-4/C-12).
Eisenia bicyclis and Ecklonia stolonifera (each 500g)
MeOH ( 95 °C reflux for 3h. 3L × 3 times )
MeOH extract (175.0 g / 116.6 g)
Aqueous layer
Partitioned with CH2Cl2
CH2Cl2 layer Aqueous layer
(16.8 g / 9.1 g) Partitioned with EtOAc
EtOAc layer Aqueous layer
(51.5 g / 4.2 g) Partitioned with n-BuOH
n-BuOH layer Aqueous layer
(39.6 g / 16.6 g) (62.5 g / 86.6 g)
Fig. 1. Extraction and Fractionation of E. stolonifera and E. bicyclis.
The former is corresponding weight for E. stolonifera; the latter is
corresponding weight for E. bicyclis in parenthesis.
1474 H. E. M OON et al.
2
HO 1 OH
3

14a 4
14 O 4a
OH
12 13
13b
1 HO O O5
6 2 11 12a 13a 5a

5 10 8a 7a 6
3

HO OH 9 O 7 OH
4 8

OH
Phloroglucinol (1) Dioxinodehydroeckol (2)
4'
HO OH
3' 5'
2' 6'
4'
HO OH OH 1'
3' 5' 15 O
14 1
2' 6'
14a O 15a OH
13
2
1' 12a
OH 3
O 6
5a O 4a
4
10
9 1 12 O 5
9a O 10a OH 7
8 OH OH
2 11a
8
3
7 5a 4a 9
HO 6
O 4
HO
6''
O 11
5 10
OH 5'' 1'' OH
4'' 2''
3''
Eckol (3)
OH

HO
4'
OH
Phlorofurofucoeckol-A (4)
3' 5'
2' 6'
1'
OH O
10
9 1
9a O 10a OH
8 4'
2 HO OH
3 5' 3'
O 7
6
5a O 4a
4
4''' 5 6' 2'
HO OH OH 1'
5''' 3'''
OH O
6''' 2'''
1''' 9 1
OH 9a O 10a OH
O OH 8
10'' 2
9'' 1'' 5"
8''
9a'' O 10a'' OH 6"
5a 4a 3
2'' HO O 7 O
4" 6 4
1"
3''
HO 7'' 5a'' O 4a'' 3" 2" OH
6'' 4''
5'' OH
OH
Dieckol (5) 7-Phloroeckol (6)

Fig. 2. Chemical Structures of the Phlorotannins from E. stolonifera and E. bicyclis.

Eckol (3). Positive FAB-MS m=z: 372 [M]þ ; 1 H-NMR (400 MHz, 7-Phloroeckol (6). 1 H-NMR (400 MHz, DMSO-d6 ) : 9.61 (1H, s,
CD3 OD) : 6.13 (1H, s, H-3), 5.94 (2H, s, H-6/H-8), 5.93 (3H, s, 9-OH), 9.40 (1H, s, 4-OH), 9.20 (1H, s, 2-OH), 9.12 (4H, d,
H-20 /H-40 /H-60 ); 13 C-NMR (100 MHz, CD3 OD) : 162.4 (C-10 ), 160.7 J ¼ 6:3 Hz, 30 , 50 , 200 , 600 -OH), 9.00 (1H, s, H-400 ), 6.14 (1H, s, H-3),
(C-30 /C-50 ), 150.0 (C-8), 147.7 (C-3), 147.6 (C-6), 144.7 (C-9a), 143.8 6.01 (1H, d, J ¼ 3:1 Hz, H-8), 5.86 (2H, s, H-300 /H-500 ), 5.80 (1H, t,
(C-1), 139.0 (C-4a), 126.1 (C-4), 125.3 (C-5a), 125.1 (C-10a), 100.3 J ¼ 2:0 Hz, H-40 ), 5.79 (1H, d, J ¼ 3:1 Hz, H-6), 5.72 (2H, d,
(C-7), 99.9 (C-2), 98.2 (C-40 ), 96.3 (C-9), 95.9 (C-20 /C-60 ). J ¼ 2:0 Hz, H-20 /H-60 ); 13 C-NMR (100 MHz, DMSO-d6 ) : 160.3
Phlorofucofuroeckol A (4). Positive FAB-MS m=z: 602 [M]þ ; (C-10 ), 158.8 (C-30 /C-50 ), 154.8 (C-400 ), 154.5 (C-7), 151.2 (C-200 /
1
H-NMR (400 MHz, CD3 OD) : 6.63 (1H, s, H-7), 6.40 (1H, s, H-11), C-600 ), 146.0 (C-9), 145.9 (C-2), 142.3 (C-5a), 141.8 (C-4), 137.1
6.26 (1H, s, H-2), 5.97 (2H, d, J ¼ 2:1 Hz, H-200 /H-600 ), 5.94 (1H, t, (C-10a), 123.9 (C-9a), 123.1 (C-4a), 122.5 (C-100 ), 122.2 (C-1), 98.9
J ¼ 1:9 Hz, H-40 ), 5.92 (1H, t, J ¼ 2:0 Hz, H-400 ), 5.88 (2H, d, (C-3), 98.3 (C-8), 96.2 (C-40 ), 94.8 (C-300 /C-500 ), 93.6 (C-20 /C-60 ),
J ¼ 2:1 Hz, H-20 /H-60 ); 13 C-NMR (100 MHz, CD3 OD) : 162.7 93.4 (C-6).
(C-100 ), 162.6 (C-10 ), 161.0 (C-30 /C-50 ), 161.0 (C-300 /C-500 ), 154.0
(C-7a), 152.5 (C-10), 152.0 (C-8a), 149.1 (C-3), 149.0 (C-12), 146.7 Protein tyrosine phosphatase 1B inhibitory assay. The PTP1B
(C-6), 144.7 (C-1), 139.2 (C-4a), 136.2 (C-12c), 128.9 (C-5a), 125.9 inhibitory activities of the plant extracts were evaluated by using
(C-13a), 125.6 (C-4), 123.2 (C-9), 106.2 (C-12a), 106.1 (C-12b), 100.8 pNPP.43) To each of 96 wells (100 mL final volume), 40 mL of the
(C-11), 100.2 (C-2), 98.6 (C-40 ), 98.5 (C-400 ), 97.0 (C-7), 96.2 (C-200 / PTP1B enzyme [0.5 unit diluted with a PTP1B reaction buffer
C-600 ), 96.2 (C-20 /C-60 ). containing 50 mM citrate (pH 6.0), 0.1 M NaCl, 1 mM EDTA, and 1 mM
Dieckol (5). Positive FAB-MS m=z: 742 [M]þ ; 1 H-NMR (400 MHz, DTT] was added with or without a sample in a test concentration
CD3 OD) : 6.15 (1H, s, H-300 ), 6.13 (1H, s, H-3), 6.09 (2H, s, H-2000 / ranging from 0.05 to 50 mg/mL for the extract and fractions, and 0.10
H-6000 ), 6.06 (1H, d, J ¼ 2:9 Hz, H-8), 6.05 (1H, d, J ¼ 2:9 Hz, H-600 ), to 100 mM for the phlorotannins dissolved in 10% DMSO. The plate
5.98 (1H, d, J ¼ 2:8 Hz, H-6), 5.95 (1H, d, J ¼ 2:8 Hz, H-6), 5.92 (3H, was preincubated at 37  C for 10 min, and then 50 mL of 2 mM pNPP in
s, H-20 /H-40 /H-60 ); 13 C-NMR (100 MHz, CD3 OD) : 162.7 (C-10 ), the PTP1B reaction buffer was added. After incubatiing at 37  C for
161.0 (C-30 /H-50 ), 158.6 (C-1000 ), 156.8 (C-7), 155.3 (C-700 ), 153.2 20 min, the reaction was terminated by adding 50 mL of NaOH (10 M).
(C-3000 /C-5000 ), 148.1 (C-200 ), 148.01 (C-2), 147.9 (C-900 ), 147.7 (C-9), The amount of p-nitrophenol produced after enzymatic dephosphory-
145.1 (C-5a00 ), 145.0 (C-5a), 144.2 (C-400 ), 144.1 (C-4000 ), 139.4 lation from pNPP was estimated by measuring the absorbance at
(C-10a), 139.3 (C-10a00 ), 127.3 (C-4000 ), 127.0 (C-9a), 126.5 (C-1), 405 nm with a microplate spectrophotometer (Molecular Devices,
126.4 (C-100 ), 125.7 (C-9a00 ), 125.5 (C-4a00 ), 125.4 (C-4a), 100.7 (C-800 ), Sunnyvale, CA, USA). The non-enzymatic hydrolysis of 2 mM pNPP
100.6 (C-8), 100.3 (C-3), 100.2 (C-300 ), 98.5 (C-40 ), 97.0 (C-2000 , 6000 ), was corrected by measuring the increase in absorbance at 405 nm
96.7 (C-600 ), 96.6 (C-60 ), 96.2 (C-20 /C-60 ). obtained in the absence of the PTP1B enzyme. The percentage
 Anti-Diabetic Effects of Phlorotannins from Brown Algae 1475
inhibition (%) was obtained by the following equation: Table 1. Protein Tyrosine Phosphatase 1B Inhibitory Activity of
% inhibition ¼ ðAc  As Þ=Ac
100, where Ac is the absorbance of MeOH Extracts and Different Solvent-Soluble Fractions of E. stolo-
the control, and As is the absorbance of the sample. Ursolic acid was nifera and E. bicyclis
used as a positive control.
IC50 values (mg/mL)a
Test sample

-Glucosidase inhibitory assay. This enzyme inhibition study was E. stolonifera E. bicyclis
carried out spectrophotometrically according to the procedure reported
by Li et al. (2005).44) A 60 mL amount of a reaction mixture containing MeOH 6:39  0:18 0:81  0:09
20 mL of a 100 mM phosphate buffer at pH 6.8, 20 mL of 2.5 mM pNPG, CH2 Cl2 0:91  0:06 0:93  0:15
and 20 mL of a sample (in a test concentration ranging from 0.5 to EtOAc 0:26  0:06 0:18  0:01
200 mg/mL for the extract and fractions, and 0.5 to 200 mM for the n-BuOH 0:23  0:07 0:24  0:01
phlorotannins) dissolved in 10% DMSO were added to each well, H2 O 3:79  0:44 1:36  0:02
before adding 20 mL of
-glucosidase [0.2 unit/mL in a 10 mM Ursolic acidb 3:37  0:13
phosphate buffer at pH 6.8]. The plate was incubated at 37  C for
a
15 min, and then 80 mL of a 0.2 M sodium carbonate solution was added Test concentrations of the samples were 0:05  50 mg/mL dissolved in
to stop the reaction. The absorbance was then immediately recorded at 10% DMSO. 50% Inhibition concentrations are expressed as the
mean  SEM of triplicates;b A positive control.
405 nm with a microplate spectrophotometer (Molecular Devices,
Sunnyvale, CA, USA). The control contained the same reaction
mixture, except for the same volume of a phosphate buffer being added
instead of the sample solution. Acarbose dissolved in 10% DMSO was Table 2. Alpha-Glucosidase Inhibitory Activity of MeOH Extracts
used as a positive control. The percentage inhibition (%) was obtained and Different Solvent-Soluble Fractions of E. stolonifera and E.
by using the same equation as that in the PTP1B enzymatic assay. bicyclis

Kinetic parameters of phlorotannins for two types of enzymatic
inhibition. In order to determine the inhibition mechanism, each
enzymatic inhibition at various concentrations of phlorotannins was
evaluated by monitoring the effect of different concentrations of the
substrate via Dixon plots (a single reciprocal plot). Dixon plots for the
inhibition of PTP1B by phlorotannins 1–6 were obtained in the
presence of different concentrations of the pNPP substrate: 0.5 mM
( ); 1 mM ( ); and 2 mM ( ). The test concentrations for
phlorotannins in the PTP1B kinetic analysis were as follows:
phloroglucinol (100 and 50 mM); dioxinodehydroeckol (50, 25, and
10 mM); eckol (10 and 5 mM); phlorofurofucoeckol-A (25 and 10 mM);
dieckol (5 and 1 mM); and 7-phloroeckol (10 and 5 mM). Dixon plots for

-glucosidase inhibition by the phlorotannins were obtained in the
presence of different concentrations of the pNPG substrate: 0.625 mM
( ); 1.25 mM ( ); and 2.5 mM ( ). The test concentrations of
phlorotannins used in the
-glucosidase kinetic analysis were as
follows: phloroglucinol (500, 250, 200, and 100 mM); dioxinodehy-
droeckol (90, 50, and 10 mM); eckol (30, 10, and 5 mM); phlorofur-
ofucoeckol-A (3 and 1 mM); dieckol (3 and 2 mM); and 7-phloroeckol
(10, 5, and 2.5 mM). Both enzymatic procedures applied the same
aforementioned PTP1B and
-glucosidase assay methods. The inhib-
ition constants (Ki ) were determined by interpreting the Dixon plots, in
which the value of the x-axis implies  Ki .45,46)
Statistics. Each result is expressed as the mean  SEM of
triplicates. Statistical significance was analyzed by one-way ANOVA
and Student’s t-test (Systat; Evaston, IL, USA) and considered at
p < 0:05.
Results
Antidiabetic activities of the extracts and fractions
from E. stolonifera and E. bicyclis
In order to evaluate the anti-diabetic activities of the
two brown algae, E. stolonifera and E. bicyclis, their
inhibitory effects on PTP1B and
-glucosidase were
evaluated. As shown in Table 1, for both types of brown
alga, their MeOH extracts and individual fractions
showed strong inhibitory activity against the PTP1B
enzyme. The EtOAc and n-BuOH fractions of both algae
were found to be the most active, with respective IC50
values of 0:26  0:06 and 0:23  0:07 mg/mL for
E. stolonifera, and 0:18  0:01 and 0:24  0:01 mg/mL
for E. bicyclis, when compared with the positive control
of ursolic acid with an IC50 value of 3.37 mg/mL. The
CH2 Cl2 and H2 O fractions also exhibited significant
PTP1B inhibitory activities with IC50 values ranging
from 0.91 to 3.79 mg/mL. In addition to the PTP1B
IC50 values (mg/mL)a
Test sample
E. stolonifera E. bicyclis
MeOH 2:82  0:63 2:22  0:05
CH2 Cl2 1:46  0:11 4:02  0:36
EtOAc 1:15  0:03 4:88  0:36
n-BuOH 4:59  0:57 1:13  0:06
H2 O 163:63  5:17 111:37  4:15
Acarboseb 187:83  11:06
a
Test concentrations of the samples were 0:5  200 mg/mL dissolved in
10% DMSO. 50% Inhibition concentrations are expressed as the
mean  SEM of triplicates; b A positive control.
inhibitory activity, the
-glucosidase inhibitory activ-
ities of the methanol extracts and different solvent-
soluble fractions of both E. stolonifera and E. bicyclis
were investigated against yeast
-glucosidase (Table 2).
Excepting the H2 O fraction, the extracts and solvent-
soluble fractions of E. stolonifera and E. bicyclis
showed potential inhibitory activity, with IC50 values
ranging from 1.13 to 4.88 mg/mL, when compared with
the positive control of acarbose with an IC50 value of
187.83 mg/mL. Although the two MeOH extracts ex-
hibited similar degrees of inhibitory potential against
-
glucosidase, the CH2 Cl2 , EtOAc, and n-BuOH fractions
of the two algae showed different inhibition with
respective IC50 values of 1:46  0:11, 1:15  0:03, and
4:59  0:57 mg/mL for E. stolonifera, and 4:02  0:36,
4:88  0:36, and 1:13  0:06 mg/mL for E. bicyclis.
Since the EtOAc fraction exhibited the most predom-
inant PTP1B and
-glucosidase inhibitory activities, and
this active fraction was obtained in the highest yield (%)
from E. bicyclis, column chromatography was per-
formed over Si gel, RP-18, and Sephadex LH20, before
evaluating the two enzyme-inhibitory activities of the
subfractions. The results of the TLC analysis enabled
3 subfractions (EF01, EF04, and EF08) to be chosen
for the two enzyme-inhibitory activities to obtain the
following IC50 values: 0:63  0:06, 5:46  0:16, and
8:13  0:66 mg/mL for PTP1B inhibition; and 1:66 
0:28, 7:70  0:10, and 6:93  0:21 mg/mL for
-gluco-
sidase inhibition. Further fractionation was then per-
formed, since each EF01 showed the most significant
inhibitory activities in both assays. Several active
subfractions were successively chromatographed and
1476 H. E. M OON et al.
Table 3. Protein Tyrosine Phosphatase 1B Inhibitory Activity of Table 4. Alpha-Glucosidase Inhibitory Activity of Phlorotannins
Phlorotannins Isolated from E. stolonifera and E. bicyclis Isolated from E. stolonifera and E. bicyclis

Compound IC50 (mM)a Ki b Inhibition typec Compound IC50 (mM)a Ki b Inhibition typec
Phloroglucinol (1) 55:48  1:85 uncompetitive Phloroglucinol (1) 141:18  13:1 170.2 mixed type
Dioxinodehydroeckol (2) 29:97  4:52 16.76 non-competitive Dioxinodehydroeckol (2) 34:60  1:95 51.80 noncompetitive
Eckol (3) 2:64  0:04 2.86 non-competitive Eckol (3) 22:78  2:15 15.48 competitive
Phlorofucofuroeckol-A (4) 0:56  0:10 1.43 non-competitive Phlorofucofuroeckol-A (4) 1:37  0:05 0.45 noncompetitive
Dieckol (5) 1:18  0:02 1.22 non-competitive Dieckol (5) 1:61  0:08 0.27 competitive
7-Phloroeckol (6) 2:09  0:09 2.90 non-competitive 7-Phloroeckol (6) 6:13  0:30 5.67 noncompetitive
Ursolic acidd 10:82  0:32 Acarbosed 157:25  5:92
a a
Test concentrations of the samples were 0.10 to 100 mM dissolved in 10% Test concentrations of the samples were 0.5 to 200 mM dissolved in 10%
DMSO. 50% Inhibition concentrations were expressed as the mean  SEM DMSO. 50% Inhibition concentrations were expressed as the mean  SEM
of triplicates; b Inhibition constants (Ki ) and c inhibition type were deter- of triplicates; b Inhibition constants (Ki ) and c inhibition type were deter-
mined by interpretation of the Dixon plot; d A positive control. mined by interpretation of the Dixon plot; d A positive control.

100
A 3
B
80

2 60

1/V
1/V

40

1
20

0 0
-40 -20 0 20 40 60 80 100 120 -20 0 20 40 60

Concentration (µM) -20

Concentration (µM)

-1

C 30
D 25

20

20
15
1/V

1/V

10
10

0 0
-5 0 5 10 -10 0 10 20 30

Concentration (µM) -5 Concentration (µM)

12
E F
30
9

20
6
1/V

1/V

10
3

0 0
-2 0 2 4 6 -4 0 4 8 12

Concentration (µM) Concentration (µM)

-3

Fig. 3. Dixon Plots for the Inhibition of PTP1B by Phlorotannins.

The results show the effects of the presence of different concentrations of the substrate (0.5 mM ( ); 1 mM ( ); 2 mM ( )) for phloroglucinol
(A), dioxinodehydroeckol (B), eckol (C), phlorofucofuroeckol-A (D), dieckol (E), and 7-phloroeckol (F).

purified to obtain phloroglucinol (1), dioxinodehy- inhibition. Among them, phlorotannins 4–6 were found
droeckol (2), eckol (3), phlorofurofucoeckol-A (4), to be potent inhibitors in both enzyme inhibitory assays
dieckol (5), and 7-phloroeckol (6) via enzyme activity- (Tables 3 and 4). As shown in Table 3, 3–6 were
guided fractionation. determined as potent PTP1B inhibitors with respective
IC50 values of 2:64  0:04, 0:56  0:10, 1:18  0:02,
Anti-diabetic activities of phlorotannins from E. and 2:09  0:09 mM, when compared with the positive
stolonifera and E. bicyclis control of ursolic acid (IC50 ¼ 10:82 mM). In addition to
The anti-diabetic activities of the isolated phlorotan- PTP1B inhibition, all the isolated phlorotannins showed
nins were evaluated by their PTP1B and
-glucosidase stronger
-glucosidase inhibitory activity than commer-
 Anti-Diabetic Effects of Phlorotannins from Brown Algae 1477

A B 1.2

0.8
1.0

0.6 0.8

1/V
0.6

1/V
0.4

0.4

0.2
0.2

0.0 0.0
-400 -200 0 200 400 600 -60 -40 -20 0 20 40 60 80 100

Concentration (µM) -0.2 Concentration (µM)

-0.2

C 1.0
D 1.2

0.8

0.6 0.8

1/V
1/V

0.4
0.4

0.2

0.0 0.0
-20 0 20 40 -2 -1 0 1 2 3

-0.2 Concentration (µM) Concentration (µM)

-0.4

E 2.5
F
2.0
0.6

1.5
1/V

0.4

1.0
1/V

0.2
0.5

0.0 0.0
-2 -1 0 1 2 3 4 -10 -5 0 5 10 15
Concentration (µM) Concentration (µM)
-0.5
-0.2

Fig. 4. Dixon Plots for the Inhibition of
-Glucosidase by Phlorotannins.

The results show the effects of the presence of different concentrations of the substrate (0.625 mM ( ); 1.25 mM ( ); 2.5 mM ( )) for
phloroglucinol (A), dioxinodehydroeckol (B), eckol (C), phlorofucofuroeckol-A (D), dieckol (E), and 7-phloroeckol (F).

cially available acarbose (Table 4). In particular, 4–6 of 16.70, 2.86, 1.43, 1.22, and 2.90 mM, while the
exhibited the most potent
-glucosidase inhibition with phlorotannins showed different inhibition modes with
respective IC50 values of 1:37  0:05, 1:61  0:08, and
-glucosidase; 4 and 6 showed non-competitive inhib-
6:13  0:30 mM, when compared with the positive ition with respective Ki values of 0.45 and 5.67 mM, and
control of acarbose (IC50 ¼ 157:3 mM). Among the 5 exhibited competitive inhibition with a Ki value of
tested phlorotannins, 4 displayed the highest inhibitory 0.27 mM. Since the Ki value represents the concentration
activity in both enzymatic assays. to form an enzyme-inhibitor complex, this value plays
an important role in the development of preventive and
Kinetic parameters of the phlorotannins therapeutic agents. In comparison with the Lineweaver-
In an attempt to explain the mode of enzymatic Burk plot, the Dixon plot makes it easier to obtain a Ki
inhibition, kinetic analyses were performed at different value which is equal to the x intercept in the case
concentrations of the corresponding substrate (pNPP for of non-competitive inhibition. Lineweaver-Burk plots
PTP1B and pNPG for
-glucosidase) and various (double reciprocal plots) were also drawn to confirm the
inhibitor concentrations. Dixon plots are the graphical inhibition pattern, although the data are not shown.
method [a plot of 1/enzyme velocity (1=V) against
inhibitor concentration (I)] for determining the type of Discussion
enzyme inhibition and dissociation or inhibition constant
(Ki ) for an enzyme-inhibitor complex, and were easily The prevalence of type II diabetes represents one of
determined. In the case of competitive inhibition, the the world’s most serious health concerns due to the
x-axis implies Ki when 1=V equals 1=Vmax , while for epidemic of pathogenesis with increased obesity and
non-competitive inhibition, the x-axis implies Ki when advancing age, especially in developing countries
1=V equals 0.45,46) Figures 3 and 4 demonstrate the including India, China, and Korea. Postprandial hyper-
enzymatic kinetic analysis of each phlorotannin 1–6, glycemia, resulting from abnormal insulin secretion by
represented by A–F. Compounds 2–6 showed non- pancreatic  cells in response to starch-rich meals,
competitive PTP1B inhibition with respective Ki values impairs hepatic glucose production, and a defective
1478 H. E. M OON et al.
glucose uptake by peripheral insulin-sensitive tissues,
particularly the skeletal muscles, is closely associated
with the development of type II diabetes. Therapeutic
strategies focusing on the suppression of postprandial
hyperglycemia and improving insulin signaling would
therefore be valuable in the treatment of both diabetic
patients and those individuals with impaired glucose
tolerance.6,47) We hence investigated the PTP1B and

-glucosidase inhibitory activities of the two marine
brown algae, E. stolonifera and E. bicyclis, and the
phlorotannins they contained.
Insulin resistance is a major pathophysiological factor
in the development of type 2 diabetes, occurring in the
peripheral tissues (muscle and adipose tissues) and the
liver, respectively leading to a reduced glucose uptake
and utilization, and increased glucose production.48,49)
Insulin resistance is not only associated with hyper-
insulinemia and hyperglycemia, but also with such other
disorders as atherosclerosis, hypertension, and abnormal
lipid profiles which are now collectively referred to as
the metabolic syndrome or insulin resistance-associated
disorders.50) The amelioration of insulin resistance in
both the peripheral tissues and liver is therefore
considered to be a reasonable treatment for type 2
diabetes. Insulin signaling pathways, which are highly
implicated in insulin resistance, have been relatively
well-established. Insulin receptors (IR), a subclass of a
large family of protein tyrosine kinases (PTKs), undergo
autophosphorylation by binding to insulin. This insulin-
related event enhances the IR kinase activity and
triggers downstream signal events, this being followed
by most of the metabolic action of insulin, including
glucose transport, glycogen synthesis, and inhibition of
gluconeogenesis. PTPs play a crucial role in the negative
regulation of a series of insulin actions by dephosphory-
lating activated IR.51) PTP1B belongs to PTPs, including
T-cell PTP (TC-PTP), PTP LAR, and SHP-2. PTP1B, a
negative regulator of the insulin signaling pathways, was
initially isolated from human placental tissue and is a
widely expressed 50 kDa enzyme that is localized
mainly in the cytoplasmic surface of the endoplasmic
reticulum in such classical insulin-targeted tissues as the
liver and muscle.10,51,52) Considering the negative role of
PTP1B in insulin signaling pathways, inhibiting PTP1B
may be a potential therapeutic strategy for treating type-
2 diabetes mellitus.
Although there are several reports on the PTP1B
inhibitory activities of seaweeds, especially red algae
and lichens53,54) as well as bromophenol,55) limited
research has been performed on brown algae and
phlorotannins. Both the MeOH extracts and solvent-
soluble fractions of E. stolonifera and E. bicyclis
showed strong inhibitory activity in the PTP1B inhib-
itory assay (Table 1). Considering the potential inhib-
itory activity of the MeOH extracts and fractions of
these two brown algae, we further evaluated their
isolated phlorotannins against PTP1B. The tested phlor-
otannins exhibited strong PTP1B inhibitory activity as
shown in Table 3. Moreover, 3–6 were found to be
potent PTP1B inhibitors with five or ten-fold stronger
inhibition than the positive control, ursolic acid. The
results of the PTP1B enzymatic kinetic analysis
(Fig. 3C–F) show that four potent PTP1B inhibitors
3–6 demonstrated non-competitive inhibition such that
the intersection of each line was on the x-axis. The
inhibition constant (Ki ) values for phlorotannins can be
compared as IC50 values. The Ki value is used to
characterize and compare the effectiveness of an
inhibitor relative to Km , the binding constant for the
substrate. This parameter is especially useful and
important in evaluating the potential therapeutic value
of inhibitors (drugs) of a given enzyme-catalyzed
reaction. The lower the Ki value, the tighter the binding
generally is, and hence the more effective an inhibitor is.
However, phlorofucofuroeckol-A (4) had a three-fold
difference in Ki value, indicated by the presence of the
substrate. There are additional advantages of phlorotan-
nins as inhibitors, in addition to enzymatic kinetics. The
PTP1B enzyme binds tyrosine as a substrate at the active
site (His214-Arg221). In addition to the highly charged
PTP1B catalytic site, the WPD loop (Thr177-Pro185)
and a secondary aryl phosphate-binding site in the
enzyme also collaborate with the active site by binding
the phenyl ring of tyrosine.51,52) We have already
mentioned that phlorotannins are naturally occurring
phloroglucinol polymers mainly isolated from Ecklonia
and Eisenia species. Since phloroglucinol is a trihy-
droxylated phenolic nucleus, phlorotannins as oligomers
and polymers produced by ether, phenyl, and 1,4-
dibenzodioxin linkages harbor many hydroxyl groups
and aromatic rings. Considering the results of the
PTP1B inhibitory assay, a relative large molecular size
and number of hydroxyl groups, as characteristics of the
phlorotannin structure, would be key determinants of
PTP1B inhibition.
Mammalian
-glucosidase (EC3.2.1.20), an exo type
of carbohydrase located in the brush-border surface
membrane of intestinal cells, is the key enzyme that
catalyzes the final step in the digestive process of
carbohydrates.
-Glucosidase inhibitors have therefore
become exciting candidates to retard the liberation of
glucose from oligosaccharides and disaccharides in
dietary complex carbohydrates, thereby slowing down
glucose absorption, which in turn results in decreased
postprandial plasma glucose levels and suppressed
postprandial hyperglycemia.9) Numerous reports have
suggested that the main anti-diabetic effects of maritime
plants are due to intestinal
-glucosidase inhibition,
subsequently decreasing the postprandial hyperglycemia
peak.38,56)
Likewise, both the MeOH extracts and solvent-
soluble fractions of E. stolonifera and E. bicyclis
showed strong
-glucosidase inhibitory activity in the
PTP1B inhibitory assay (Table 2). Since the MeOH
extracts and fractions of these two brown algae
exhibited potent inhibitory activities, with the exception
of the H2 O fraction, we attempted to determine the
compounds responsible for the potential
-glucosidase
inhibitory activity of these brown algae, and further
evaluated six phlorotannins 1–6. Phlorotannins 2–6
tested in the present study exhibited strong PTP1B and

-glucosidase inhibitory activities, with the exception of
phloroglucinol (1) (Table 4). In particular, 4–6 exhibited
the most potent
-glucosidase inhibition, this corre-
sponding to previous work indicating phlorotannins to
be potent
-glucosidase inhibitors.37–39,57) These results
strongly suggest that the molecular size of the phlor-
otannins and number of hydroxyl groups present in the
 Anti-Diabetic Effects of Phlorotannins from Brown Algae 1479

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