Volume 5 Number 2 February 2014 Pages 179–402

Food &
Function
Linking the chemistry and physics of food with health and nutrition
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ISSN 2042-6496

PAPER
Atsushi Kato et al.
Effects of eugenol-reduced clove extract on glycogen phosphorylase b
and the development of diabetes in db/db mice
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Function
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Effects of eugenol-reduced clove extract on

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glycogen phosphorylase b and the development of

Cite this: Food Funct., 2014, 5, 214
diabetes in db/db mice
Fujiko Sanae,a Ogusa Kamiyama,a Kyoko Ikeda-Obatake,a Yasuhiko Higashi,a
Naoki Asano,b Isao Adachic and Atsushi Kato*c

Received 21st October 2013
Accepted 18th November 2013
DOI: 10.1039/c3fo60514k
www.rsc.org/foodfunction
We found that the 50% aqueous EtOH extract of clove (Syzygium aromaticum) had potent dose-dependent
inhibitory activity toward glycogen phosphorylase b and glucagon-stimulated glucose production in
primary rat hepatocytes. Among the components, eugeniin inhibited glycogen phosphorylase b and
glucagon-stimulated glucose production in primary rat hepatocytes, with IC50 values of 0.14 and 4.7 mM,
respectively. In sharp contrast, eugenol showed no significant inhibition toward glycogen phosphorylase
b, even at a concentration of 400 mM. Eugenol-reduced clove extracts (erCE) were prepared and when
fed to a db/db mouse they clearly suppressed the blood glucose and HbA1c levels. Furthermore, plasma
triglyceride and non-esterified fatty acid levels in 5% and 10% erCE-fed db/db mice were significantly
lowered, compared with control db/db mice without erCE supplementation. These results suggested
that dietary supplementation with the erCE could beneficially modify glucose and lipid metabolism and
contribute to the prevention of the progress of hyperglycemia and metabolic syndrome.

There are a number of reports on natural products and

Introduction
Diabetes is now considered as one of the main threats to human
health in the 21st century. Pronounced changes in human
behavior and lifestyle have resulted in a dramatic increase in the
incidence of diabetes worldwide. Noninsulin-dependent dia-
betes (type 2 diabetes) is a heterogeneous disease characterized
by hyperglycemia, which is caused by a disorder of insulin
secretion, insulin resistance in target tissues, and activation of
the hepatic glucose production pathway in the liver.1,2 Current
scientic evidence demonstrates that much of the morbidity and
mortality of diabetes can be eliminated by aggressive treatment
with diet, exercise and pharmacological approaches, to achieve
better control of the blood glucose level. Recently, the possibility
of preventing the onset of diabetes using dietary supplements
and/or herbal medicines has attracted increasing attention. It is
generally recognized that the hepatic glucose output in type 2
diabetes is elevated and thus signicantly contributes to hyper-
glycemia.3,4 A possible way to suppress hepatic glucose produc-
tion in type 2 diabetes may be through glycogen phosphorylase
inhibition.5 Although inhibitors targeting glycogen phosphory-
lase have been developed and studied as potential therapy for
attenuating hyperglycemia associated with type 2 diabetes,6,7
such antidiabetic agents are not yet commercially available.
a
Faculty of Pharmaceutical Sciences, Hokuriku University, Kanazawa 920-1181, Japan
b
BioApply Co., Ltd., 1-95 Tsuchishimizu, Kanazawa 920-0955, Japan
c
Department of Hospital Pharmacy, University of Toyama, 2630 Sugitani, Toyama 930-
0194, Japan. E-mail: kato@med.u-toyama.ac.jp
dietary supplements suppressing hepatic glucose production in
hepatic cells and lowering blood glucose in diabetic rodent
models through hepatic glycogen phosphorylase inhibition. In
enzyme assays, 1,4-dideoxy-1,4-imino-D-arabinitol (DAB), which
was rst isolated from the fruits of the legume Angylocalyx
boutiqueanus,8 and chemically synthesized isofagomine were
found to be potent inhibitors of hepatic glycogen phosphory-
lase.9–11 These iminosugars inhibited hepatic glycogen break-
down in vivo and displayed an accompanying antihyperglycemic
effect, which was most pronounced in obese mice.9 Green tea
and green tea extracts have been demonstrated to benecially
modify glucose metabolism in experimental models of type 2
diabetes.12,13 Wolfram et al. reported that epigallocatechin
gallate (EGCG), which is the most abundant catechin, bene-
cially modied glucose and lipid metabolism in H4IIE rat
hepatoma cells and markedly enhanced glucose tolerance in
diabetic db/db mice.14 We recently reported that the gallated
catechins in green tea inhibited glycogen phosphorylase b, with
IC50 values ranging from 6.3 to 34 mM, and further suppressed
glucose release from rat hepatocytes with IC50 values ranging
from 15 to 46 mM.15
We searched for glycogen phosphorylase b-inhibiting mate-
rials among spices and medicinal herbs and found cloves
(Syzygium aromaticum) as the most potent inhibitory materials
toward glycogen phosphorylase b. In this paper, we report in
vitro inhibition of glycogen phosphorylase b by this extract and
its components, and inhibition of glucagon-stimulated glucose
production in primary rat hepatocytes. We furthermore

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Paper
investigated the suppressive effects of eugenol-reduced CE on
body weights, blood glucose, HbA1c, plasma triglyceride, and
non-esteried fatty acid levels using diabetic db/db mice in vivo.
Materials and methods
Materials
Phosphoglucomutase, glucose 6-phosphate dehydrogenase,
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glucose-1,6-bisphosphate, rabbit glycogen phosphorylase b and
a-D-glucose 1-phosphate dipotassium salt hydrate were
purchased from Sigma-Aldrich Fine Chemicals (St. Louis, MO).
Adenosine 50 -monophosphate and eugenol (1) were purchased
from Wako Pure Chemical Industries (Osaka, Japan). Clove 3
(2), biorin (3), casuarictin (4), eugeniin (5), tellimagrandin I (6)
and 1,3-di-O-galloyl-4,6-O-[S]-hexahydroxydiphenoyl-D-glucose
(7) were purchased from Nagara Science Co. (Gifu, Japan).
Flower buds of S. aromaticum (cloves) produced in Zanzibar
from a crude drug shop Maechu Co. (Nara, Japan). Voucher
specimens of S. aromaticum (HU040120) were deposited at the
Herbarium of the Medicinal Plants Garden in Hokuriku
University, Japan.
Measurement of glycogen phosphorylase b and rat intestinal
a-glucosidase activity
Dried clove was homogenized with 50% aqueous EtOH in a
Waring blender and allowed to stand for 24 h at room
temperature. The mixture was ltered and centrifuged. The
supernatant was concentrated and lyophilized. The extract was
dissolved in 50% aqueous EtOH and used for measurement of
glycogen phosphorylase b and intestinal a-glucosidase inhibi-
tory activity. Glycogen phosphorylase b activity was assayed in
the direction of glycogen breakdown from the rate of NADPH
formation in an assay coupled to phosphoglucomutase, glucose
6-phosphate dehydrogenase according to the literature.16 The
IC50 values were obtained by single determination. Brush
border membranes prepared from rat intestine according to the
method of Kessler et al.17 were used as the source of rat intes-
tinal maltase and sucrase. These activities were determined
using maltose for maltase and sucrose for sucrase as substrates.
The released glucose was determined colorimetrically using the
Glucose CII-test Wako (Wako Pure Chemical Ind.).
Preparation of hepatocytes and glucagon-induced
glycogenolysis
The protocol was approved by the Animal Experiments
Committee of Hokuriku University. Isolation and culturing of
rat hepatocytes and measurement of hepatic glucose produc-
tion were performed according to the methods described in our
previous paper.18
Analysis of clove components
The chromatographic separation was performed using a model
LC-10ATvp pump (Shimadzu, Kyoto, Japan) equipped with a
Rheodyne injection valve (Cotati, CA, U.S.A.) with a 100 mL loop.
The HPLC analysis of clove phenolic components was performed
using a Cosmosil 5C18-MS-II column (150 mm  4.6 mm I.D.;
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Food & Function
Nacalai Tesque, Kyoto, Japan). The mobile phase was acetoni-
trile–H2O–triuoroacetic acid (60 : 440 : 1, v/v, for components 2–
8; 240 : 760 : 1, for component 1). The ow rate was 0.8 mL min1
at room temperature. Components were detected using a Model
SPD-10Avp UV detector (Shimadzu, Kyoto, Japan) at 280 nm.
Preparation of eugenol-reduced clove extract (erCE) and
feeding experiments
The ower bud powder (3 kg) of S. aromaticum (clove) produced
in Zanzibar was extracted three times with ethyl acetate (10 L)
and once with EtOH (4 L) to remove eugenol. The resulting
powder was further extracted three times with 50% aqueous
EtOH (10 L). The extract was concentrated and lyophilized to
give a brown powder (erCE, 450 g).
Animal care and diets
Male diabetic db/db mice (C57BLKS/J db+/db+) and non-diabetic
misty mice (C57BLKS/J +m/+m) were purchased from Nihon SLC
(Hamamatsu, Japan) at an age of ve weeks. Mice were main-
tained on a 12 h light and 12 h dark cycle at a humidity of 55–
60% and a temperature of 22  2  C, fed a powdery commercial
chow (Labo MR Stock) for a week aer arrival and then randomly
divided into three groups. Thereaer, both db/db and misty
mice were fed the diets with or without erCE for four weeks. All
mice consumed diets with free access to water ad libitum.
Animals were treated in accordance with Hokuriku University
Guidelines for the Care and Use of Laboratory Animals.
Blood glucose and glycosylated hemoglobin (HbA1c)
concentrations
The blood glucose concentration was monitored in venous
blood drawn from the tail vein using a glucometer (Nipro,
Japan) every week aer a 15 h fast. The blood HbA1c concen-
tration was measured with an analyzer (DCA-vantage, Siemens,
Japan) at the start and end of the experimental period.
Plasma biomarkers
Plasma triglyceride (TG) and non-esteried fatty acid (NEFA)
concentrations were determined using an enzymatic method
(Triglyceride E-test Wako and NEFA C-test Wako, respectively,
Wako Pure Chemical Ind.).
Results
Inhibition of 50% aqueous EtOH extract of clove against
glycogen phosphorylase b and intestinal a-glucosidases
Cloves are the dried aromatic ower buds of S. aromaticum
(Myrtaceae) and one of the most important spices. They are
renowned for their diverse medicinal benets and high nutri-
tional content. Eugenol (1), which is the dominant ingredient of
clove oil, has been reported to improve diabetic vascular and
nerve function in streptozotocin-induced diabetic rats.19 We
rst compared the ability of clove 50% extract to inhibit a
glycogen phosphorylase b and intestinal maltase and sucrase
activities (Table 1). Miglitol and isofagomine were used as
Food Funct., 2014, 5, 214–219 | 215

Food & Function
positive controls.20–22 Miglitol is well-known as a clinically used
potent a-glucosidase inhibitor for the treatment of type 2 dia-
betes patients. Isofagomine shows potent inhibition of glycogen
phosphorylase b and it has been put forward as a candidate for
a clinical trial for type 2 diabetes. We found that the clove
extract was weakly inhibitory to rat intestinal maltase, iso-
maltase and sucrase, with IC50 values of 190, 250 and 84 mg
mL1, respectively. The activity was much weaker than miglitol
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(IC50 ¼ 0.27, 8.1, and 0.21 mg mL1 against maltase, isomaltase,
and sucrase, respectively). In sharp contrast, the clove 50%
extract showed potent inhibition against glycogen phosphory-
lase b, with an IC50 value of 0.86 mg mL1. It is noteworthy that
the IC50 value was almost the same as that of isofagomine (IC50
¼ 0.85 mg mL1).
Inhibition of glycogen breakdown by the 50% aqueous EtOH
extract of cloves
As described above, the 50% aqueous EtOH extract of cloves
potently inhibited glycogen phosphorylase b, with an IC50 value
of 0.86 mg mL1 (Table 1). We next investigated the effect of this
extract on glucagon-induced glucose production in primary rat
hepatocytes. The extract from cloves suppressed 10 nM
glucagon-stimulated glycogenolysis in primary rat hepatocytes,
with an IC50 value of 8.0 mg mL1. In our previous paper, we
reported that the 50% aqueous EtOH extract of green tea and its
gallated catechins inhibited glycogen phosphorylase b and
glucose release from rat hepatocytes in a dose-dependent
manner.15 Among the gallated catechins, gallocatechin gallate
inhibited glycogen phosphorylase b and glucose release from
rat hepatocytes, with IC50 values of 6.3 and 25 mM, respectively.15
Next, we investigated the effects of the clove components on
glycogen phosphorylase b and glucose release from rat
hepatocytes.
Effects of the clove components on glycogen phosphorylase b
and glucose release from rat hepatocytes
Major secondary components from S. aromaticum belong to three
different chemical classes: essential oils, chromone-C-glucosides
Table 1 Concentration of clove 50% EtOH extracts, miglitol, iso-
fagomine, and DAB giving 50% inhibition of rat intestinal a-glucosi-
dases and rabbit muscle glycogen phosphorylase b activities
IC50 (mg mL1)
Clove 50%
EtOH extracts Miglitol Isofagomine DAB
Rat intestinal
a-glucosidases
Maltase 190 0.27 96 7.3
Isomaltase 250 8.1 43 0.77
Sucrase 84 0.21 78 2.1
Rabbit muscle 0.86 NIa 0.85 0.044
glycogen
phosphorylase b
a
NI: less than 50% inhibition at 1000 mM.
216 | Food Funct., 2014, 5, 214–219
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and tannins. The major component of the essential oil is euginol
(1) and the yield of it from the ower buds is about 15–20%.23,24
This plant also has high levels of polyphenolic compounds such
as chromone-C-glucosides and tannins. Two chromone
C-glucosides, clove 3 (2) and biorin (3), and four ellagitannins,
casuarictin (4), eugeniin (5), tellimagrandin I (6), and 1,3-di-O-
galloyl-4,6-O-[S]-hexahydroxydiphenoyl-b-D-glucose (7), have been
isolated from cloves25,26 (Fig. 1). Thus, we rst investigated their
inhibitory activities toward glycogen phosphorylase b and
glucose production in primary rat hepatocytes. As shown in
Table 2, ellagitannins 4–7 potently inhibited glycogen phos-
phorylase b, with IC50 values ranging from 0.14 to 0.73 mM.
Among them, eugeniin (5) was the most potent inhibitor of
glycogen phosphorylase b and also inhibited glucose production
in primary rat hepatocytes, with an IC50 value of 0.14 and 4.7 mM,
respectively. This study revealed that in vitro inhibition of euge-
niin (5) toward glycogen phosphorylase b was more potent than
that of gallocatechin gallate. We were also intrigued by the fact
that eugenol (1) showed no signicant inhibition toward
glycogen phosphorylase b, even at a concentration of 400 mM
(Table 2).
Preparation of eugenol-reduced clove extract (erCE) for in vivo
experiments and HPLC analysis of components in the extract
Eugenol is widely used as a component of zinc oxide eugenol
cement in dentistry and is also applied in medicine as an anti-
septic and anesthetic agent. It has a distinctive aroma, spicy bitter
taste and undesirable irritant effects on the mucosa.27 In this
study, we found that eugenol showed no signicant inhibition
toward glycogen phosphorylase b despite the strong inhibition of
eugeniin. Considering this result, we envisaged that a further
improvement in activity might be gained by reducing the eugenol
in the extracts. To test this hypothesis, we prepared the eugenol-
reduced clove extract (erCE) by successive extraction with ethyl
acetate, EtOH, and 50% aqueous EtOH. The amounts of compo-
nents 1–7in the erCE were determined with a reversed-phase
HPLC method using a C18 stationary phase (Table 3). The relative
amounts of ellagitannins 4–7, which potently inhibited glycogen
phosphorylase b and glucose release from rat hepatocytes, were
1.3%, 1.5%, 0.71%, and 0.92%, respectively. The erCE thus
prepared was used in the following in vivo experiments.
Fig. 1 Structures of phenolic components in cloves.
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Paper Food & Function

Table 2 Concentration of clove components giving 50% inhibition of

glycogen phosphorylase b and glucagon-stimulated glucose
production in primary rat hepatocytes

IC50 (mM)

Glycogen Glucose
Compound phosphorylase b production

Eugenol (1) NIa —b

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Clove 3 (2) 24 NDc

Biorin (3) 48 ND
Casuarictin (4) 0.33 10.0
Eugeniin (5) 0.14 4.7 Fig. 2 Body weight of db/db (A) and misty mice (B) fed diet supple-
Tellimagrandin (6) 0.38 22.5 mented with eugenol-reduced clove extract (erCE). Mice consumed
1,3-Di-O-galloyl-4,6-O-[S]-hexa- 0.73 13.2 diets supplemented with 5% or 10% erCE for 28 days. Data values are
hydroxydiphenoyl-b-D-glucose (7) mean  S.D. (n ¼ 6–7). Differences among groups were analyzed by
Gallocatechin gallate 6.3d 25.0d Dunnet's multiple comparison test and those at *P < 0.05 were
significantly different from those of control.
a
Less than 50% inhibition at 400 mM. b No effect c
Not determined.
d
Our previous data (Kamiyama et al., 2010).

Table 3 The contents of phenolic components in the eugenol-

reduced clove extract (erCE)

Compound Content (%, w/w)

Eugenol (1) 2.6

Clove 3 (2) 2.0
Biorin (3) 3.6
Casuarictin (4) 1.3
Eugeniin (5) 1.5
Tellimagrandin (6) 0.71
1,3-Di-O-galloyl-4,6-O-[S]-hexa- 0.92
hydroxydiphenoyl-b-D-glucose (7)

Diabetes alleviation in diabetic db/db mice
The db/db mice have a defect in leptin signaling resulting
from a point mutation in the gene for the leptin receptor and
hence are unresponsive to leptin.28,29 Leptin is involved in
eating behavior and energy homeostasis.30 For this reason, the
db/db mice aer birth have unrepressed eating behavior,
become obese, and develop severe insulin resistance associ-
ated with hyperinsulinemia, hyperglycemia, and
hypertriglyceridemia.
Diabetic db/db and non-diabetic misty mice were fed 0, 5,
and the 10% erCE in the diet for 4 weeks. In the db/db mice,
there was no difference in body weight between control (0%
erCE-supplemented) and 5% erCE-fed groups, whereas the body
weight in the 10% erCE-fed group was signicantly lower than
the other groups (Fig. 2). An increase in body weight in the misty
mice was suppressed by the 10% erCE supplementation in
3 weeks.
In non-diabetic misty mice, erCE supplementation did not
affect the blood glucose levels and HbA1c concentrations, while
erCE supplementation in db/db mice suppressed an increase of
blood glucose (Fig. 3). Particularly, in the 10% erCE-fed db/db
group, elevation of the blood glucose levels was not observed
throughout the experimental periods, while the HbA1c
Fig. 3 Blood glucose level (A) and HbA1c level (B) in db/db mice and
misty mice fed diet supplemented with erCE. Mice consumed diets
supplemented with 5% or 10% erCE for 28 days. The blood glucose
concentration was monitored in venous blood drawn from the tail vein
every week after a 15 h fast. The blood HbA1c concentration was
measured at the start and end of the experimental period. Data values
are mean  S.D. (n ¼ 6–7). Differences among groups were analyzed
by Dunnet's multiple comparison test and those at *P < 0.05 were
significantly different from those of control.
concentrations were signicantly lowered by 64.5%, compared
with the control group aer 4 weeks.
Changes in plasma triglyceride (TG) and non-esteried fatty
acid (NEFA) levels aer 4 weeks for erCE-fed db/db and misty
mice are shown in Fig. 4. Plasma concentrations of TG in 5%
and 10% erCE-fed db/db mice were signicantly lowered by
50.0% and 43.5%, respectively, compared with the control
group, while in misty mice, no signicant change in plasma TG
levels were observed. Increased NEFA levels are observed in
obesity and type 2 diabetes, and associated with insulin resis-
tance.31 Plasma NEFA concentrations in 5% and 10% erCE-fed
db/db mice were signicantly lowered by 56.1% and 50.7%,
respectively. By contrast, the NEFA levels in misty mice were
slightly elevated by erCE-supplementation.

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Fig. 4 Plasma triglyceride (TG) level (A) and non-esterified fatty acid
(NEFA) level (B) in db/db mice and misty mice fed diet supplemented
with erCE. Mice consumed diets supplemented with 5% or 10% erCE
for 28 days. The TG and NEFA levels were measured at the end of the
experimental period. Data values are mean  S.D. (n ¼ 6–7). Differ-
ences among groups were analyzed by Dunnet's multiple comparison
test and those at *P < 0.05 were significantly different from those of
control.
Discussion
In the present study, we found that the 50% aqueous EtOH
extract of cloves (CE) and the components, ellagitannins,
exhibit very potent inhibitory activity toward glycogen phos-
phorylase b and also inhibit glucagon-stimulated glucose
production in primary rat hepatocytes. Eugeniin (5) is the most
potent inhibitor of glycogen phosphorylase among natural
products.11 Furthermore, the present study demonstrated that
the dietary eugenol-reduced CE (erCE) supplementation
improved hyperglycemia and hyperlipidemia in db/db mice,
which are obese–diabetic animals with insulin resistance, but
had little effect on non-diabetic misty mice. This study eluci-
dated that CE and its components show potent inhibitory
activities toward glycogen phosphorylase b and glucose release
from rat hepatocytes. However, whether the inhibition of
glycogen phosphorylase b by erCE is the critical role of the
hypoglycemic effects in db/db mice should be further investi-
gated. The hypoglycemic effects may be due to a combination of
glycogen phosphorylase b inhibition and other activities. Pra-
sad et al. reported that the clove extract acts like insulin in
hepatocytes and hepatoma cells by reducing phosphoenolpyr-
uvate carboxykinase and glucose 6-phosphatase gene
expression.32 Recently, it has been reported that Syzygium aro-
maticum-derived oleanoic acid attenuates the activities of gly-
cogenic enzymes with concomitant increases of hepatic and
muscle glycogen concentrations of streptozotocin-induced dia-
betic rats.33 In addition, Kuroda et al. have reported that the
clove EtOH extract signicantly suppresses an increase in blood
glucose level in type 2 diabetic KK-A(y) mice, and that the
components, dehydrodieugenol and dehydrodieugenol B, show
the hypoglycemic effects through proliferator-activated
218 | Food Funct., 2014, 5, 214–219
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receptor-g (PPAR-g) activation.34 Thus, since cloves have
multiple anti-diabetic activities, they could potentially
contribute to nutritional strategies for the prevention and
treatment of type 2 diabetes.
Glycogen phosphorylase catalyzes the breakdown of
glycogen to glucose-1-phosphate in liver and skeletal muscles,
and it exists in two interconvertible forms a and b. The less
active b form is transformed into the active form a by phos-
phorylation. Inhibition of hepatic glycogen phosphorylase is a
promising treatment strategy for attenuating hyperglycemia in
type 2 diabetes.7,35 However, drug development efforts have
failed to discover inhibitors with the specicity for the hepatic
glycogen phosphorylase isoenzyme because of a signicant
homology between hepatic and skeletal muscle glycogen phos-
phorylase isoenzymes.36 Baker et al. have reported that glycogen
phosphorylase inhibition aimed at attenuating hyperglycemia
is unlikely to negatively impact muscle metabolic and func-
tional capacity.37 Such inhibitors have been shown to be more
potent at reducing the hepatic glucose output in the presence of
high glucose concentrations.37,38 Although it has been pointed
out that pharmacological inhibition of hepatic glycogen phos-
phorylase has potential as an effective therapeutic strategy for
the treatment of type 2 diabetes, such inhibitors have not yet
been on the market for the treatment of type 2 diabetes.
Accordingly, further studies aimed at establishing the effects of
glycogen phosphorylase inhibition aer chronic oral dosing are
required to fully clarify the impact that glycogen phosphorylase
inhibitors have on skeletal muscle metabolism and function.
Acknowledgements
The authors wish to thank Dr. Hirotoshi Fushimi and Hayashi
Tamao (Museum of Material Medica, Institute of Natural
Medicine, University of Toyama).
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