Kaempferia parviflora Ethanol Extract, a

Peroxisome Proliferator-Activated Receptor γ

Ligand-binding Agonist, Improves Glucose
Tolerance and Suppresses Fat Accumulation in
Diabetic NSY Mice
Masaru Ochiai , Toshiki Takeuchi, Tsutomu Nozaki, Ken-o Ishihara, and Tatsuhiro Matsuo

Abstract: This study assessed the effect of Kaempferia parviflora, also known as black ginger (BG), and its ethanol extract
(BGE) on peroxisome proliferator-activated receptor (PPAR) γ agonistic activity, glucose tolerance, fat accumulation, and
lipids-induced hypertriglyceridemia in mice. PPARγ ligand-binding capacity in vitro and polymethoxy flavone contents
were highly observed in organic solvent extracts. In an animal experiment A, male diabetic Nagoya-Shibata-Yasuda mice
were divided into five dietary groups and fed each diet for 8 weeks: AIN-93G diet (low-fat [LF] diet), high-fat (HF)
diet, HF diet supplemented with 1% BG, HF diet supplemented with 0.19% BGE, and HF diet supplemented with
pioglitazone (PPARγ agonist, 3 mg/kg/day) as a PPARγ agonistic positive control. As determined from glucose and
insulin tolerance tests, plasma glucose levels were improved in the BG and BGE groups. The BGE extract suppressed
fat accumulation in adipose tissues, liver, and muscles without changing the plasma adiponectin level. In an animal
experiment B, in order to investigate the effect of BG and BGE on lipid-induced hypertriglyceridemia, male ddY
mice were divided into three test groups: control, BG-administered group (500 mg/kg), and BGE-administered group
(100 mg/kg). The plasma triacylglycerol level was not different among the groups during the lipids administration
test. These results conclude that the BGE extract containing several kinds of polymethoxy flavones showed PPARγ
ligand-binding capacity in vitro and prevented obesity and insulin resistance independent of adiponectin secretion in mice.

Keywords: fat accumulation, glucose tolerance, Kaempferia parviflora, methoxy flavone, PPARγ

Practical Application: Kaempferia parviflora, also known as black ginger (BG), is often used as a folk medicine and a
functional food material to prevent metabolic syndrome mainly in Asian regions. Here, we have clarified that ethanol
extract from BG (BGE) contains several kinds of polymethoxy flavones to show PPARγ ligand-binding capacity and is an
active extract for the improvement of obesity and insulin resistance. The BGE is expected to be applied for functional food
materials in health food markets. Also, polymethoxy flavones to show PPARγ ligand-binding capacity can be generally
applied as a physiological active compound of functional food supplements.

Introduction well as adipose fat accumulation (Lara-Castro & Garvey, 2008;

Excess energy consumption causes abnormalities in glucose and
fat metabolism and body fat accumulation leading to the onset of
obesity, insulin resistance, and type-2 diabetes mellitus (T2DM).
According to information reported by the International Diabetes
Federation (IDF, 2017), in 2017, approximately 425 million people
suffered from diabetes, and it is estimated that 693 million people
will have diabetes by 2045. Recently, ectopic fat accumulation in
the liver and skeletal muscles have been identified as contributing
Rasouli, Molavi, Elbein, & Kern, 2007). Thus, identification of
foods that can decrease ectopic fat accumulation and improve
insulin resistance has been developed.
Kaempferia parviflora (commonly known as black ginger [BG])
is a perennial Zingiberaceae plant that is indigenous to Thailand
and Laos. BG has been used as a folk medicine to prevent
diet-induced obesity, improve blood flow, and increase energy
consumption (Akase et al., 2011; Shimada et al., 2011). BG
Health, Nutrition, &

factors in the development of insulin resistance and T2DM as contains several kinds of flavonoids and flavonoid glycosides with
plural methoxy groups. Polymethoxyflavones (PMF) including
5,7-dimethoxyflavone (DMF), which is a major PMF in BG,
Food

JFDS-2018-1272 Submitted 8/15/2018, Accepted 12/15/2018. Author Ochiai
is with School of Veterinary Medicine, Kitasato Univ., 23-35-1 Higashi, Towada,
Aomori, 034–8628, Japan. Authors Takeuchi and Matsuo are with Faculty of Agricul-
ture, Kagawa Univ., 2393 Ikenobe, Miki, Kita, Kagawa, 761–0795, Japan. Authors
Nozaki and Ishihara are with BHN Co., Ltd., 1–16, Kandanishiki, Chiyoda, Tokyo,
101-0054, Japan. Direct inquiries to author Ochiai (E-mail: mochiai@vmas.kitasato-
u.ac.jp).
have pharmacological benefits. DMF has anti-inflammatory,
vasorelaxation, and cardio-protection effects (Sae-Wong et al.,
2011; Tep-Areenan, Sawasdee, & Randall, 2010). Mekjaruskul,
Jay, and Sripanidkulchai (2012) also reported that di-, tri-, and
penta-MF (DMF, TMF, and penta-MF) were highly and rapidly
detected in plasma and various tissues of rats that had received
one oral administration of BG. However, the effects of PMF on

C 2019 Institute of Food Technologists

 R

doi: 10.1111/1750-3841.14437 Vol. 84, Iss. 2, 2019 r Journal of Food Science 339
Further reproduction without permission is prohibited
 Black ginger improves glucose tolerance . . .
the accumulation of lipids in tissues can differ depending on their
chemical structure. Other PMF in BG have previously been found
to induce lipids accumulation in adipocyte cells (Horikawa et al.,
2012; Kobayashi et al., 2016). Thus, regulation of fat metabolism
by BG is supposed to be quite different among PMF and various
extractants. BG ethyl acetate or ethanol extracts and crushed BG
rhizome powder have antiobesity effects through enhancement of
energy expenditure and ATP production in mice and mammalian
cells (Akase et al., 2011; Shimada et al., 2011).
Peroxisome proliferator-activated receptor γ (PPARγ ) acts as
the master regulator of adipogenesis and induces adiponectin ex-
pression that can improve glucose metabolism despite the fact that
adipose fat accumulation is accelerated. Many natural product ago-
nists of PPARγ have been introduced and listed in the review; for
example, kaempferol, quercetin, and so on (Wang et al., 2014). BG
and its ethyl acetate extract prevent body weight (BW) gain and
adipose tissue fat accumulation and improve glucose metabolism
in spontaneously obese and diabetic mice (Akase et al., 2011;
Shimada et al., 2011). However, the effect of BG-derived ex-
tracts on glucose tolerance and ectopic fat accumulation through
PPARγ -related mechanisms have not yet been elucidated. If the
preventive effects of BG-derived extracts on T2DM by the ac-
tivation of PPARγ have been clarified, BG-derived extracts can
be widely used as a food-grade material in food supplements.
BG-derived extracts can prevent the increase in the population
suffering from T2DM in the world.
In this study, we compared differences in the contents of PMF
extracted from BG using various extractants and determined their
PPARγ ligand-binding capacity in a cell-free in vitro assay. We then
investigated effects of BG and ethanol extract from BG (BGE) on
glucose tolerance and ectopic and adipose fat accumulation in mice
with T2DM (animal experiment A). And, we also investigated
effects of BG and BGE on lipid-induced hypertriglyceridemia in
ddY mice (animal experiment B).
Materials and Methods
Preparation of BG extract samples and positive control
ingredients
BG crude extract powder was supplied by BHN Co., Ltd.
(Tokyo, Japan). BG powder produced from the rhizome was
prepared and soaked in hexane, ethyl acetate, acetone, ethanol,
methanol, hot water (90 to 100 °C), or water (10:100 [w/v]
BG:diluent) for 24 hr. The solutions or suspensions were filtered
using quantitative filter paper (Whatman TM No1; GE Health-
care Japan Co., Tokyo, Japan). The supernatant was freeze dried
or evaporated, each extract was weighed, the recovery rate of each
sample was calculated, and then samples were stored at −20 °C
until use. For the estimation and quantitation of the chemical com-
pounds in each BG extract, HPLC analysis was performed using
an HPLC system (Shimadzu Co., Kyoto, Japan), which included
a CAPCELL C18 PAK UG120 column (4.6 mm × 250 mm
Health, Nutrition, &
i.d., 5 μm; Shiseido, Tokyo), with a column temperature of
35 °C in reference to the report (Toda et al., 2016). The mo-
Food
bile phase consisted of 0.1% formic acid in acetonitrile (solvent
A) and acetonitrile (solvent B) for gradient elution at a flow rate
of 1.0 mL/min at 35 °C. The gradient elution program was 0
to 35 min, solvent A/solvent B = 65:35; 35 to 35.1 min, sol-
vent A/solvent B = 65:35; 35.1 to 45 min, solvent A/solvent
B = 10:90; 45 to 45.1 min, solvent A/solvent B = 10:90; and 45.1
to 70 min, solvent A/solvent B = 65:35. The detection wavelength
was 260 nm. Identification and/or quantification of each PMF in
340 Journal of Food Science r Vol. 84, Iss. 2, 2019
BG extract was carried out using each pure PMF reagent (5,7-
DMF, 3,5,7-TMF, 5,7,4-TMF, 3,5,7,4-tetra-MF, and 3,5,7,3,4-
penta-MF, purify more than 98%; Funakoshi Co., Ltd. Tokyo,
Japan).
Pioglitazone (Wako Chemicals Industries, Ltd., Osaka, Japan)
and GW1929 (Cayman Chemical, MI, USA) were prepared as
PPARγ agonists. 5,7-DMF (Indofine Chemical Co., Inc., NJ,
USA) at a reagent grade was purchased from Funakoshi Co., Ltd.
and prepared as a standard compound contained in BG for the
measurement of PPARγ ligand-binding capacity.
Measurement of PPARγ ligand-binding capacity in vitro
PPARγ ligand-binding capacity of each extract was determined
using a nuclear receptor cofactor assay kit (EnBio RCAS for
PPARγ , Fujikura Kasei Co., Ltd., Tokyo, Japan). The final con-
centration of each BG extract was <500 μg/mL.
Animal studies
All procedures involving the mice were approved by the Pres-
ident of Kitasato Univ. through the judgment by Institutional
Animal Care and Use Committee of Kitasato Univ. (Approval
No.16-102).
Effect of dietary BGE on fat accumulation in tissues of
diabetic Nagoya-Shibata-Yasuda mice (animal experiment
A)
Animals. Thirty-four Nagoya-Shibata-Yasuda (NSY) diabetic
mice (male, 4 weeks old) were purchased from Japan SLC
(Shizuoka, Japan). The NSY mouse developed by selective breed-
ing for glucose intolerance from ICR mice is a spontaneous model
of T2DM. Mice were individually housed in each stainless wire
cage at 23 ± 2 °C with lights on between 07:00 and 19:00. Free
access to tap water and a chow diet (CE-2; Japan Clea, Tokyo) was
given to them for 1 week of acclimation. NSY mice were divided
into five dietary groups (six to seven mice per group): mice fed a
low-fat (LF) diet (LF group); mice fed a high-fat (HF) diet (HF
group); mice fed an HF diet and orally administered pioglitazone
(3 mg/kg/day) as a positive control group (P group); mice fed an
HF diet supplemented with 1% BG (BG group); and mice fed
an HF diet supplemented with 0.19% BGE (BGE group). BGE
content in the BGE diet was calculated by the collection rate (re-
covery rate) of BGE from the BG (Table 1). Mice were fed their
respective AIN-93G-based diets (Table 2) ad libitum for 8 weeks.
BW and energy intake were monitored once a week. At the end
of the sixth week, an oral glucose tolerance test (OGTT) was
conducted. After an overnight fast, D-glucose (1 g/kg) was orally
administered to each mouse. Before and 30, 60, and 90 min after
the D-glucose administration, blood (20 μL/each) was collected
from the tail vein and centrifuged (6200 × g, 4 °C, 5 min) to ob-
tain plasma for the measurement of glucose level. At the end of the
seventh week, an intraperitoneal insulin tolerance test (ITT) was
conducted. After an overnight fast, porcine insulin (0.33 IU/kg)
was intraperitoneally administered to each mouse. Before and 30,
60, and 90 min after the insulin administration, plasma was ob-
tained for the measurement of glucose level in the same way as
OGTT. The area under or over the curve (AUC or AOC) of the
plasma glucose levels during the OGTT and ITT were calculated
by the trapezoidal rule. Feces collected during the final 3 days were
freeze dried, weighed, and kept at −20 °C until lipid biochem-
ical analyses. At the end of the 8th week, mice were euthanized
under isoflurane anesthesia after an overnight fast. Blood was col-
lected and centrifuged (6200 × g, 4 °C, 15 min) to obtain plasma.
Black ginger improves glucose tolerance . . .

Table 1–Chemical compounds and their contents in each extract of BG.

Compound Recovery 3,5,7,4 -

name rate 5,7-Dimethoxyflavone 5,7,4 -Trimethoxyflavone 3,5,7 -Trimethoxyflavone Tetramethoxyflavone

Extraction
solvent
Water 92.5% 0.56% 0.51% 0.10% 0.24%
Methanol 23.9% 8.77% 13.46% 1.34% 4.46%
Ethanol 18.7% 10.96% 18.48% 1.58% 5.59%
Acetone 16.7% 16.85% 26.02% 2.59% 8.42%
Hexane 7.0% 21.47% 15.24% 5.03% 10.76%
Ethylacetate 17.7% 17.27% 26.40% 2.66% 8.51%
Crude – 2.63% 4.02% 0.39% 1.27%
original
powder
Contents of 5,7-DMF, 3,5,7-TMF, 5,7,4 -TMF, and 3,5,7,4 -tetra-MF were quantified using a HPLC system as described in “Materials and Methodsˮ section.

Table 2–Experimental diet composition (g/kg diet).

HF
LF HF BG BGE
Ingredients
Casein 200.0 250.0 250.0 250.0
L-Cystine 3.0 3.0 3.0 3.0
Cornstarch 529.5 149.5 139.5 149.5
Sucrose 100.0 200.0 200.0 200.0
Cellulose 50.0 50.0 50.0 50.0
Beef tarrow 0.0 230.0 230.0 230.0
Soybean oil 70.0 70.0 70.0 70.0
AIN93G Vitamin mixturea 10.0 10.0 10.0 10.0
AIN93 Mineral mixtureb 35.0 35.0 35.0 35.0
Choline chloride 2.5 2.5 2.5 2.5
BHT 0.014 0.014 0.014 0.014
BG 0.0 0.0 10.0 0.0
BGE 0.0 0.0 0.0 1.9
5,7-Dimethoxyflavonec 0.26 0.21
5,7,4 -Trimethoxyflavonec 0.40 0.35
3,5,7 -Trimethoxyflavonec 0.039 0.030
3,5,7,4 -Tetramethoxyflavonec 0.13 0.11
Othersc 9.2 9.3
Total 1000 1000 1000 1000
Energy composition (%)c
Carbohydrate 64 28 28 28
Fat 16 52 53 52
Protein 20 20 20 20
Total 100 100 100 100
LF, low fat; HF, high fat; BG, black ginger; BGE, black ginger extract. Carbohydrate, fat, and protein provide 4.0, 9.0, and 4.0 kcal/g, respectively.
a
AIN93G vitamin mixture contains 98.1225% sucrose.
b
AIN93 mineral mixture contains 11.8% sucrose.
c
Methoxyflavones content is calculated according to the result (Table 1).
Health, Nutrition, &

Liver, pancreas, intraabdominal adipose tissues, and gastrocnemius v/v) as described by Folch, Lees, and Sloane Stanley (1957). Con-
muscle were harvested immediately after euthanasia and weighed. tents of TG and CHO in the feces, liver, and gastrocnemius muscle
Plasma and tissues were stored at −80 °C until analyses.
Food

Biochemical analyses. The plasma glucose level during the
OGTT and ITT and the final plasma levels of glucose, triacyl-
glycerol (TG), total cholesterol (CHO), unesterified fatty acids
(NEFA), adiponectin, and insulin were measured using commer-
cial kits (Wako Pure Chemical Industries, Ltd.; Shibayagi Co., Ltd.,
Gunma, Japan). Total lipids in the feces, liver, and gastrocnemius
muscle were extracted in a mixture of methanol:chloroform (1:2,
were measured using the same kits as plasma analyses. Fatty acids
composition (C16:0, C16:1, C18:0, C18:1, C18:2, and C18:3) of
the total lipids in the liver and gastrocnemius muscle was deter-
mined using a gas chromatography system (GC 2014, Shimadzu
Co., Kyoto, Japan) in a 25 m capillary column (ULBON HR-
20 M; Shimadzu Co.) as previously described (Ochiai & Matsuo,
2013; Ochiai, Kuroda, & Matsuo, 2014). The sum of the fatty acid
percentages was estimated to be 100%. Indices of stearoyl-CoA

Vol. 84, Iss. 2, 2019 r Journal of Food Science 341

 Black ginger improves glucose tolerance . . .
desaturase (SCD) activity (ratios of C16:1/C16:0 and hot water did not show high ligand-binding capacity to PPARγ
C18:1/C18:0) were calculated.
Effect of single administration of BGE on lipid-induced
hypertriglyceridemia in ddY mice (animal experiment B)
Animals and biochemical analysis. Twenty-five 7-week-
old male ddY mice were purchased from Japan SLC, Inc. The
ddY mice have been reported to be an appropriate animal model
for investigation of lipid-induced hypertriglyceridemia (Yamazaki,
Kishimoto, & Ezaki, 2012). The mice were acclimated for 4 days
prior to initiating experiment B. After a 12-hr fast, the mice
were placed into three groups: control (n = 9), BG (500 mg/kg;
n = 8), and BGE (100 mg/kg; n = 8). Mice in the BG and
BGE groups were administered the BG and BGE dissolved in
distilled water. Mice in the control group were administered water.
The samples and soybean oil (Nacalai Tesque Inc., Kyoto, Japan)
(5 mL/kg) were orally administered to mice simultaneously. Blood
(20 μL/each) was collected from the tail vein before the sample
administration and at 0, 90, 180, 270, and 360 min after the
oil administration. The blood was centrifuged (6200 × g, 4 °C,
5 min) to obtain plasma. Plasma TG levels were measured using a
commercial kit (Wako Pure Chemical Industries).
Statistical analyses. Data are expressed as the mean ± SE.
In experiment A, statistical analysis of the differences between the
treatment groups and control group was performed using one-way
analysis of variance (ANOVA) and the Dunnett test. In experiment
B, statistical analysis of differences among the three groups was
performed using one-way ANOVA and Fisher’s Protected Least
Significant Difference (PLSD) test. A difference with P < 0.05
was statistically significant. All statistical analyses were performed
using a commercially available statistical package (Excel Statistics
2015; SSRI, Tokyo, Japan).
Results
Collection percentages and chemical compounds in each
BG extract
Collection efficiencies of the samples extracted by each solvent
from the BG were 7.0% by n-hexane, 17.7% by ethyl acetate,
16.7% by acetone, 18.7% by ethanol, 23.9% by methanol, 70.0%
by hot water, and 92.5% using water. HPLC chromatograms of
each extract are shown in Figure 1, and the percentage of chemical
compounds in each BG extract is shown in Table 1. 5,7-DMF,
3,5,7-TMF, and 3,5,7,4-tetra-MF were extracted by n-hexane.
5,7,4-TMF was extracted by acetone and ethyl acetate, which had
the highest extraction level among the solvents. These four PMFs
were extracted by any solvent to some extent, although little was
extracted by water (<0.6%). In particular, ethyl acetate, hexane,
and acetone extracted the four PMFs at a high rate (>50%). Two
sharp peaks occurred in the HPLC chromatogram: one was at
15 min and the other was at 19 min. The peak at 19 min have
Health, Nutrition, &
been estimated to be 3,5,7,3 ,4 -PMF, although its content was
not quantified; the other compound observed at 15 min cannot
Food
be identified.
PPARγ ligand-binding capacity in vitro
Results of PPARγ ligand-binding capacity by BG, its extracts,
and positive-control compounds are shown in Figure 1. BG that
was extracted by methanol, ethanol, and acetone showed high
ligand-binding capacity to PPARγ in a concentration-dependent
manner. On the other hand, BG extracted using either water or
342 Journal of Food Science r Vol. 84, Iss. 2, 2019
even at a high concentration (500 μg/mL).
BW gain and energy intake of mice
BW gain, energy intake, and energy efficiency of mice are
shown in Figure 2. BW gain was significantly lower in the LF,
BG, and BGE groups than in the HF group throughout the treat-
ment period. Energy intake was slightly lower in the BG and BGE
groups than in the HF group. Energy efficiency, which was calcu-
lated by dividing BW gain by total energy consumption through-
out the treatment period, was significantly lower in the LF, BG,
and BGE groups than in the HF group.
Fat accumulation and SCD indices in the liver and
gastrocnemius muscle of mice
TG and CHO accumulation and SCD indices in the liver and
gastrocnemius muscle are shown in Figure 2. TG content in the
liver was significantly lower in the BG and BGE groups than in
the HF group. CHO content in the liver was not different among
groups (data not shown). TG content in the gastrocnemius muscle
was significantly lower in the BGE group, but not the BG group,
than in the HF group. SCD index (C18:1/C18:0) of the liver was
significantly lower in the BGE group than in the HF group. SCD
index (C16:1/C16:0) of the muscle was significantly higher in the
P group than in the HF group (data not shown).
Tissue weights and plasma biochemical components of
mice
Tissue weights and plasma biochemical components are shown
in Table 3 and Figure 2. Intraabdominal adipose tissue weights
were significantly lower in the BGE group, but not in the BG
group, than in the HF group. Plasma adiponectin level was signifi-
cantly higher in the P group than in the HF group. No significant
differences in weights of the other tissues and in levels of the
other plasma biochemical components were observed among the
groups.
Fecal fat excretion of mice
Fecal TG and CHO excretion of mice are shown in Figure 3.
Fecal dry weight during the final 3 days of the experiment was
not different among the HF dietary groups. Dietary fat intake was
not significantly different among the HF dietary groups. Fecal TG
excretion was significantly lower, whereas fecal CHO excretion
was significantly higher in the BGE group than in the HF group.
OGTT and ITT
Plasma glucose levels and their AUC and AOC values during
OGTT and ITT are shown in Figure 4. The plasma glucose level
and AUC value in NSY mice during OGTT were significantly
lower in the LF, P, BG, and BGE groups than in the HF group. The
plasma glucose level in NSY mice during ITT was significantly
lower, and the AOC value was significantly higher in the LF, BG,
and BGE groups than in the HF group.
Lipid-induced hypertriglyceridemia test in ddY mice
Plasma TG levels during the lipid-induced hypertriglyceridemia
test are shown in Figure 5. Plasma TG levels at 3 hours after the
administration of lipids were slightly, but not significantly, lower
in the BGE (100 mg/kg) group than in the control group. No
differences in the plasma TG levels were observed between the
control and BG (500 mg/kg) groups.
Black ginger improves glucose tolerance . . .

PPARγ ligand-binding capacity

1.0
Crude

Water
0.8
Hot water

0.6 MeOH

O.D. 450
EtOH
0.4
Acetone

EtAc
0.2

DMF

0.0 PIO

GW1929
-0.2
0 100 200 300 400 500

Concentration (μg/mL)

Figure 1–HPLC chromatogram of each extract from BG and PPARγ ligand-binding capacity BG powder was each extracted by water, methanol, ethanol,
acetone, ethyl acetate, and hexane. Four PMF contents were calculated using a HPLC system as described in “Materials and Methodsˮ section. (a)
5,7-Dimethoxyflavone; (b) 5,7,4 -trimethoxyflavone; (c) 3,5,7 -trimethoxyflavone; (d) 3,5,7,4 -tetramethoxyflavone. PPARγ ligand-binding capacity
was determined using a nuclear receptor cofactor assay system. The capacity of pioglitazone and GW1929 were measured at final concentrations from
0 to 2.5 μM. The capacity of the BG, BG extracts, and DMF was each measured at final concentrations from 0 to 500 or 0 to 100 μg/mL. Data are
expressed as means (n = 2).

Discussion
BG has been reported to prevent diet-induced obesity and im-
prove glucose and lipid metabolism in mice (Akase et al., 2011;
Hidaka et al., 2017; Shimada et al., 2011). In this study, we
have presented data that various PMF can be extracted using
both water-soluble and water-insoluble solvents. Higher PPARγ
ligand-binding capacity was observed in ethanol, methanol, ace-
tone, and ethyl acetate extracts from BG than in water-soluble
solvents. High PPARγ ligand-binding capacity by BG ethanol
extract was correlated with the prevention of glucose intolerance,
BW gain, and fat accumulation in adipose, liver, and muscles of
NSY mice.
Four PMF (5,7-DMF, 3,5,7-TMF, 5,7,4 -TMF, and 3,5,7,4 -
of PMF (Ninomiya et al., 2016) and that ethyl acetate extract
contains four kinds of PMF (5,7-DMF, 3,5,7,4 -tetra-MF, 5,7,4 -
TMF, and 3,5,7,3 ,4 -penta-MF) and five kinds of hydroxy-PMF
(Shimada et al., 2011). With regard to the structural requirements
of flavonoids for PPARγ activity, most flavonoids with hydroxy
groups have lower activity as compared with flavonoids lack-
ing hydroxy groups (Matsuda, Nakamura, & Yoshikawa, 2014).
Flavonoids with methoxy groups have higher activity than hy-
droxy flavonoids, particularly those with a methoxy group at
the 3-position and a methoxy group of flavanols at the B ring
was important (Matsuda et al., 2014). It is in part consistent
with the present results. From the results in Figure 1, PPARγ
ligand-binding capacity was higher in the acetone, ethyl acetate,
Health, Nutrition, &

tetra-MF) were extracted by ethyl acetate, hexane, and acetone at ethanol, and methanol extracts. In terms of the relationships be-
>50% efficiency (Table 1 and Figure 1). In contrast, water had a tween PPARγ ligand-binding capacity and PMF contents, it is
Food

low extraction rate (<0.6%). These data indicate that these PMF
in addition to 3,5,7,3 ,4 -penta-MF are fat-soluble polyphenols
that can be extracted effectively by several kinds of organic sol-
vents. Obvious differences in the four PMF contents were not
observed between the BG and BGE extracts. Toda et al. (2016)
have reported that ethanol-ethyl acetate can elute the PMF more
effectively than hydroxy-PMF. Some reports also shows that ethyl-
acetate-soluble and methanol-eluted fractions contain 12 kinds
possible that fat-soluble extractants elute PMF at a higher level
than aqueous extractants and/or the fat-soluble extractants extract
PMF that have higher PPARγ ligand-binding capacity than those
extracted by water. The chemical compounds other than PMF
that are estimated to be contained in the water extract did not
have high PPARγ ligand-binding capacity.
PMF have multiple mechanisms that contribute to their antiobe-
sity effect and ability to improve insulin resistance. These include

Vol. 84, Iss. 2, 2019 r Journal of Food Science 343

Health, Nutrition, & Black ginger improves glucose tolerance . . .
Food

Figure 2–BW gain, energy intake, intraabdominal adipose tissues weight, and TG content and SCD indices of the muscle and liver in NSY mice. Each data
are expressed as the mean ± SE (n = 6 to 7). Statistical analysis of the differences between the HF group and other NSY mice groups was performed
using one-way ANOVA and Dunnett test. A difference with P < 0.05 was considered to be statistically significant. ∗ P < 0.05; ∗∗ P < 0.01; ∗∗∗ P < 0.001
compared with HF group. LF, low fat; HF, high fat; P, pioglitazone; BG, black ginger; BGE, ethanol extract from BG.

344 Journal of Food Science r Vol. 84, Iss. 2, 2019

Black ginger improves glucose tolerance . . .

Table 3–Effect of BG and BGE on tissues weight and plasma biochemical components of mice.

NSY mice
LF diet HF diet
LF HF P BG BGE
Tissues weight (mg/g)
Liver 35.06 ± 1.20 36.72 ± 1.08 34.37 ± 0.67 35.91 ± 1.19 38.21 ± 1.14
Pancreas 7.91 ± 0.23 7.75 ± 0.26 7.88 ± 0.28 8.27 ± 0.55 8.46 ± 0.38
Soleus muscle 0.50 ± 0.03 0.41 ± 0.07 0.44 ± 0.03 0.43 ± 0.03 0.47 ± 0.03
Gastrocnemius muscle 0.90 ± 0.03 0.85 ± 0.03 0.79 ± 0.03 0.86 ± 0.03 0.88 ± 0.04
Plantaris muscle 6.75 ± 0.15 6.42 ± 0.12 6.17 ± 0.12 6.79 ± 0.17 6.88 ± 0.14
Plasma components
Glucose (mg/dL) 429.3 ± 61.3 534.5 ± 55.4 471.2 ± 65.2 480.1 ± 46.7 421.9 ± 51.4
Insulin (ng/mL) 0.192 ± 0.058 0.587 ± 0.285 0.688 ± 0.232 0.812 ± 0.606 0.362 ± 0.138
Adiponectin (μg/mL) 10.5 ± 0.9 8.3 ± 0.4 17.0 ± 1.1∗∗∗ 9.4 ± 0.4 7.1 ± 0.3
Triacylglycerol (mg/dL) 68.5 ± 4.2 51.0 ± 4.8 61.5 ± 6.4 61.0 ± 4.0 67.3 ± 6.2
Total cholesterol (mg/dL) 83.1 ± 1.7∗ 99.2 ± 4.7 103.9 ± 5.5 106.0 ± 4.1 91.3 ± 3.4
Nonesterified fatty acid (mEq/L) 0.65 ± 0.1∗ 0.41 ± 0.06 0.49 ± 0.05 0.53 ± 0.05 0.52 ± 0.05
LF, low fat; HF, high fat; P, pioglitazone; BG, black ginger; BGE, ethanol extract from BG.
Each data are expressed as the mean ± SE (n = 6 to 7). Area under the curve (AUC) and area over the curve (AOC) were determined by the trapezoidal rule. Statistical analysis of
the differences between the HF group and other NSY mice groups was performed using one-way ANOVA and Dunnett test. A difference with P < 0.05 was considered to be
statistically significant. ∗ P < 0.05; ∗∗ P < 0.01; ∗∗∗ P < 0.001 compared with HF group.

Figure 3–TAG and CHO excretion in the feces of NSY mice during the final 3 days of the experiment. Each data are expressed as the mean ± SE (n = 6
to 7). Feces were collected during the final 3 days of the experiment. Statistical analysis of the differences between the HF group and other NSY mice
groups was performed using one-way ANOVA and Dunnett test. A difference with P < 0.05 was considered to be statistically significant. ∗ P < 0.05;
Health, Nutrition, &

∗∗ P < 0.01; ∗∗∗ P < 0.001 compared with HF group. LF, low fat; HF, high fat; P, pioglitazone; BG, black ginger; BGE, ethanol extract from BG.
Food

affecting PPARγ ligand-binding capacity and enhancing PPARγ
and CCAAT/enhancer-binding protein (C/EBP) expression. In
contrast to our findings, PMF isolated from BG rhizome does not
affect PPARγ ligand-binding capacity in an in vitro cell-free as-
say rather some PMF enhance the mRNA expression of C/EBP,
which is a transcription factor upstream of PPARγ (Horikawa
et al., 2012). Tetra- and penta-MF also increases the mRNA lev-
els of PPARγ and C/EBP as well as adiponectin, glucose trans-
porter 4 (GLUT4), and aP2 (Horikawa et al., 2012). In other
words, they do not activate PPARγ directly in the nuclear re-
ceptor cofactor assay in contrast to pioglitazone, which activates
PPARγ directly (Horikawa et al., 2012). Some reports suggest that
results from a PPARγ ligand-binding in vitro cell-free assay are not
always positively related to results from experiments measuring

Vol. 84, Iss. 2, 2019 r Journal of Food Science 345

 Black ginger improves glucose tolerance . . .

Figure 4–Plasma glucose level during the OGTT and ITT in NSY mice. Each data are expressed as the mean ± SE (n = 6 to 7). Area under the curve
(AUC) and area over the curve (AOC) were determined by the trapezoidal rule. Statistical analysis of the differences between the HF group and other
NSY mice groups was performed using one-way ANOVA and Dunnett test. A difference with P < 0.05 was considered to be statistically significant.
∗ P < 0.05; ∗∗ P < 0.01; ∗∗∗ P < 0.001 compared with HF group. LF, low fat; HF, high fat; P, pioglitazone; BG, black ginger; BGE, ethanol extract from BG.

PPARγ expression in cell and animal studies (Matsuda et al.,
2014). In fact, PPAR dual agonists that increase both PPARα
and PPARγ activity have developed to increase the suppressive
effects on lipid accumulation by PPARα agonists with the insulin
resistance-improving effects by PPARγ agonists. Some reports in-
dicate that BG rhizome suppresses adipose tissue accumulation,
improves glucose metabolism, and enhances energy metabolism in
cell and animal studies (Akase et al., 2011; Kobayashi et al., 2016;
Toda et al., 2016; Yoshino et al., 2014). These results are in part
considered to be the effect of PPARα activation.
Shimada et al. (2011) have reported that PMF containing ex-
tracts from BG rhizome exerts antiobesity effects by inhibiting
pancreatic lipase activity as one of the possible mechanisms. Sup-
pression of pancreatic lipase activity and intestinal lipids absorp-
tion by dietary polyphenols has been frequently reported in re-
views (Buchholz & Melzig, 2015; de la Garza, Milagro, Boque,
Campión, & Martı́nez, 2011). Some flavonoids strongly inhibit
pancreatic lipase activity and suppress digestion and absorption
of dietary lipids. However, fecal lipids excretion was not in-
Health, Nutrition, &
has not well known that methoxy flavonoids are famous as natural
possible pancreatic lipase inhibitors, in contrast many flavonoids
with hydroxy groups are listed in a review (Buchholz & Melzig,
2015). These findings indicate that the antiobesity effect of BG
and BGE is not only induced by the suppression of pancreatic
lipase and intestinal lipids absorption.
As shown in Figure 4, glucose tolerance and insulin sensitivity
were dramatically improved by dietary BG and BGE and no obvi-
ous differences between the BG and BGE groups were observed.
These results indicate that compounds, such as PMF, contained
in the BGE extract are functional compounds to improve glu-
cose intolerance and insulin resistance because PMF contents are
similar between the BG and BGE extracts (Table 1 and 2). It has
been also reported that glucose intolerance is improved by ad-
ministration of BG rhizome in obese and diabetic mice (Akase
et al., 2011; Shimada et al., 2011). Nobiletin and tangeretin,
both are kinds of PMF, are reported to enhance the secretion
of adiponectin, which improves insulin resistance independent of
PPARγ ligand-binding capacity (Kunimasa et al., 2009; Kim, Hur,

creased in the BG and BGE groups (Figure 3). PMF extracted Kwon, & Hwang, 2012; Miyata et al., 2011). Many reports indi-
from BG rhizome has low pancreatic lipase inhibitory activity in cate that PMF improves glucose intolerance through the follow-
Food

an in vitro assay (Shimada et al., 2011); the IC50 values of pan-
creatic lipase inhibitory activity of 5,7-DMF, 3,5,7-TMF, 5,7,4 -
TMF, and 3,5,7,4 -tetra-MF are relatively weak and >600 μg/mL.
Lipid-induced hyperglyceridemia was also not significantly sup-
pressed by a single administration of BG and BGE in ddY mice
(Figure 5). These results are consistent with the low pancreatic
lipase activity of BG-derived PMF (Shimada et al., 2011). Also, it
ing mechanisms: PMF stimulates adiponectin and AMP-activated
protein kinase (AMPK) signaling pathways; GLUT4 expression
and glucose uptake into muscles; improves glycogen metabolism;
and suppresses inflammation (Kunimasa et al., 2009; Lee et al.,
2010; Miyata et al., 2011; Sundaram, Shanthi, & Sachdanandam,
2014). In particular, it is reported that PMF extracted from BG
and 5,7-DMF enhance energy production in C2C12 myocyte

346 Journal of Food Science r Vol. 84, Iss. 2, 2019

Black ginger improves glucose tolerance . . .
Figure 5–Plasma TG level during the lipid-induced hypertriglyceridemia
test in ddY mice. Each data are expressed as the mean ± SE (n = 8 to 9).
Statistical analysis of the differences between the groups was performed
using one-way ANOVA and Fisher’s PLSD test. A difference with P < 0.05
was considered to be statistically significant. LF, low fat; HF, high fat; P,
pioglitazone; BG, black ginger; BGE, ethanol extract from BG.
cells by upregulating AMPK phosphorylation (Toda et al., 2010).
It is well established that upregulation of AMPK phosphorylation
enhances mRNA expression of GLUT4 and PPARγ coactivator
1-α, which increases fatty acid β-oxidation and glucose utilization
as well as mitochondria functions in muscles (Jäger, Handschin,
St-Pierre, & Spiegelman, 2007). With regard to the metabolism
of PMF, Bei and An (2016) have showed the rapid distribution of
5,7-DMF to several tissues, such as liver, muscles, and adipose tis-
sues, from plasma after the single oral administration of 5,7-DMF
in mice. Metabolism of 5,7-DMF can indicate that 5,7-DMF ac-
tivates some protein expressions in addition to PPARγ in adipose
tissues, muscles, and liver in vivo. These previous data can eluci-
date the activation of AMPK by BG and BGE as one of other
mechanisms that improves glucose intolerance.
BW gain and energy efficiency were significantly suppressed in
mice fed the HF diet supplemented with either BG or BGE, but
the visceral fat pad weight was not changed in the BG group.
Akase et al. (2011) also have reported that BG rhizome signif-
icantly suppresses BW gain, but does not influence visceral fat
pad weight in terms of the relative value of BW in obese and
diabetic TSOD mice. Physiological characteristics between the
TSOD and NSY mice are different. An NSY mouse used in this
experiment is late in onset of type-2 diabetic model with mod-
erately obese or resistant to obese because the mouse has both
impaired insulin response to glucose and insulin resistance (Ueda
et al., 1995). Therefore, the HF diet did not increase visceral fat
pad weight, although BW gain was increased (Figure 2). BG and
PMF (5,7,4-TMF and 3,5,7,3,4-penta-MF) extracted from BG
rhizome enhance lipolysis and suppress TG accumulation with an
increase in mRNA expressions of adiponectin, ATGL, and HSL
independent of PPARγ transcription in mature 3T3-L1 adipocyte
cells (Okabe et al., 2014). In contrast, BG and 5,7-DMF contained
in the ethyl acetate extract from BG rhizome increase TG accu-
mulation dependent on PPARγ transcription in brown adipocyte
cells (Kobayashi et al., 2016). Some PMF also enhance TG accu-
mulation in 3T3-L1 cells by PPARγ activation with the increase
in adiponectin level and glucose uptake into cells (Matsuda et al.,
2014). Previously, we reported that pioglitazone decreased hep-
atic TG accumulation and increased muscular TG contents and
SCD indices with the improvement of glucose intolerance in rats
(Ochiai & Matsuo, 2013). Generally, ectopic fat accumulation in-
duces insulin resistance and T2DM, but intramuscular TG accu-
mulation has been recently observed in humans with high insulin
sensitivity as an “athlete paradox” (van Loon & Goodpaster, 2006).
We had expected that BG and BGE would increase muscular TG
accumulation with the improvement of adiponectin secretion and
glucose intolerance because BG and BGE extracts are PPARγ
ligand-binding agonists. However, BG and BGE extracts, but not
pioglitazone, suppressed both hepatic and muscular TG contents
and SCD indices and did not alter plasma adiponectin level in
mice. No change in the plasma adiponectin level by the BG and
BGE may be correlated with no increases in the adipose tissue
accumulation of mice fed with the BG and BGE. To the present,
it has not yet been reported that PMF extracted from BG rhizome
increases plasma adiponectin level in animal studies. These find-
ings indicate that BG and BGE improve glucose intolerance and
ectopic TG accumulation through multiple mechanisms, such as
activation of both AMPK phosphorylation and PPARγ agonistic
activation.
Several reports suggest that plasma lipid levels have little cor-
relation with hepatic and muscular lipid contents in relation to
the improvement of hyperlipidemia (Shimada et al., 2011). The
present findings indicate that BG and BGE in diet also suppress
hepatic TG contents without influencing plasma lipid levels. As
lipid accumulation into the liver and muscles leads to insulin resis-
tance and T2DM, suppressive effects on the hepatic and muscular
TG accumulation by BG and BGE extracts probably contribute
to the improvement of insulin resistance and its related metabolic
diseases.
Conclusions
BGE can be effective for prevention of ectopic fat accumu-
lation, insulin resistance, and T2DM-independent high PPARγ
ligand-binding capacity. The mechanism by which BGE regulates
the decrease in insulin resistance is in part different from that by
pioglitazone. As presented here, BGE, but not pioglitazone, sup-
pressed both hepatic and muscular TG contents and SCD indices
in diabetic NSY mice. Dual mechanisms in addition to PPARγ
ligand-binding capacity may be related to the improvement of
insulin resistance by the BGE extract.
Acknowledgments
This work was supported by the JSPS KAKENHI (Grant Num-
ber 16K21349) and Mishima Kaiun Memorial Foundation (Japan).
Conflict of Interest
The authors declare no competing financial interest.
Health, Nutrition, &
Author Contributions
Food
M. Ochiai designed research and conducted the study and wrote
manuscript. T. Takeuchi helped with the animal experiments. T.
Nozaki and K. Ishihara provided the main materials (BG) and
analyzed PMF profile in the BG. T. Matsuo edited the manuscript.
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