nutrients
Article
Water Extract of Curcuma longa L. Ameliorates
Non-Alcoholic Fatty Liver Disease
Jeongeun Mun 1 , Shintae Kim 1 , Ho-Geun Yoon 2 , Yanghee You 1 , Ok-Kyung Kim 1 ,
Kyung-Chul Choi 3 , Yoo-Hyun Lee 4 , Jeongmin Lee 5 , Jeongjin Park 1, * and Woojin Jun 1, *
1
2
3
4
5
*
Division of Food and Nutrition, Chonnam National University, Gwangju 61186, Korea;
mjo3214@naver.com (J.M.); rotoman1@naver.com (S.K.); yewha@naver.com (Y.Y.);
20woskxm@chonnam.ac.kr (O.-K.K.)
Department of Biochemistry and Molecular Biology, College of Medicine, Yonsei University,
Seoul 03722, Korea; yhgeun@yuhs.ac.kr
Department of Biomedical Sciences, University of Ulsan College of Medicine, Seoul 05505, Korea;
choikc75@amc.seoul.kr
Department of Food and Nutrition, University of Suwon, Gyeonggido 18323, Korea; creamut@suwon.ac.kr
Department of Medical Nutrition, Kyung Hee University, Gyeonggido 17104, Korea; jlee2007@khu.ac.kr
Correspondence: pjj8425@hanmail.net (J.P.); wjjun@chonnam.ac.kr (W.J.); Tel.: +82-62-530-0344 (J.P.);
+82-62-530-1337 (W.J.)
Received: 30 September 2019; Accepted: 18 October 2019; Published: 21 October 2019
Abstract: Our aim was to investigate whether hot water extract (CLW) of Curcuma longa L. could
prevent non-alcoholic fatty liver disease (NAFLD). HepG2 cells were treated with free fatty acid (FFA)
mixture (oleic acid: palmitic acid, 2:1) for 24 h to stimulate in vitro fatty liver. In addition, C57BL/6
mice were fed 60 kcal% high-fat (HF) diet for eight weeks to induce fatty liver in vivo. Intracellular
reactive oxygen species (ROS) and malondialdehyde (MDA) productions were increased by FFA
and HF-diet, but supplementation with CLW significantly decreased these levels. CLW treatment
ameliorated antioxidant activities that were suppressed by exposure to the FFA and HF-diet. Cluster of
differentiation 36 (CD36) and fatty acid transport proteins (FATP2 and FATP5) were increased in HF-diet
groups, while CLW suppressed their expression levels. Moreover, sterol regulatory element-binding
protein-1c (SREBP-1c), acetyl-coenzyme A carboxylase (ACC), and fatty acid synthase (FAS) expression
levels were down-regulated in the CLW groups compared to HF-diet groups. On the other hand,
50 adenosine monophosphate-activated protein kinase (AMPK), Peroxisome proliferator-activated
receptor alpha (PPAR-α), and carnitine palmitoyltransferase 1 (CPT-1) expressions were up-regulated
in the CLW groups. HF-diet fed mice showed high hepatic triglycerides (TG) content compared to
the normal diet mice. However, the administration of CLW restored the hepatic TG level, indicating
an inhibitory effect against lipid accumulation by CLW. These results suggest that CLW could be a
potentially useful agent for the prevention of NAFLD through modulating fatty acid uptake.
Keywords: Curcuma longa L.; non-alcoholic fatty liver disease; fatty acid uptake; lipid accumulation;
oxidative stress
1. Introduction
The liver is the central organ of lipid metabolism. Dietary fatty acids are transported to the liver and
oxidized to produce as much energy as the hepatocyte needs. The excess fatty acids are converted to
triglycerides (TG) and stored in the hepatocyte [1]. When the amount of TG accounts for more than 5% of
the liver weight, the organ function is impaired, and this condition is known as fatty liver disease. Chronic
alcohol intake has long been considered the major cause of alcoholic fatty liver, while non-alcoholic fatty
liver has been shown to be unrelated to alcohol intake [2]. The causes of non-alcoholic fatty liver are
Nutrients 2019, 11, 2536; doi:10.3390/nu11102536
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Nutrients 2019, 11, 2536
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lifestyle habits, which include excessive dietary intake and lack of energy consumption [3]. Therefore, it is
often accompanied by lifestyle diseases such as obesity, diabetes, and metabolic syndrome. In recent times,
with the growing incidence of these diseases, the attention paid to non-alcoholic fatty liver is gradually
increasing [4]. Non-alcoholic fatty liver disease (NAFLD) shows various stages. Simple hepatic steatosis is
characterized by lipid accumulation in hepatocytes. Non-alcoholic steatohepatitis (NASH) can be further
developed into cirrhosis and hepatocellular carcinoma. Although many factors such as obesity, diabetes,
and inflammation have been implicated in relation to NAFLD in humans [5,6], the underlying mechanism
of the development and progression of NAFLD remains unclear.
When free fatty acid (FFA) is overloaded in the body, FFA uptake factors such as cluster of
differentiation 36 (CD36) and fatty acid transport proteins (FATPs) are significantly up-regulated,
and then excessive FFA is accepted into the liver, gradually accumulating [7]. According to previous
studies, palmitic acid and high-fat (HF) diet markedly increased CD36 expression, which is closely
associated with the development of NAFLD [8,9]. Furthermore, several studies have indicated that
fatty acid overflow by activated CD36 subsequently produced a higher level of reactive oxygen species
(ROS), which could be linked to oxidative stress [10].
Oxidative stress is the imbalance between the production of ROS and the activation of antioxidants
in the body and results in several metabolic disorders [11]. It particularly produces a disturbance in
lipid metabolism, eventually leading to lipid accumulation in hepatocytes. Of the several existing
pathogenic mechanisms of NAFLD, oxidative stress is considered a major contributor. According to a
previous study, uric acid-induced oxidative stress in hepatocytes significantly elevated the expression of
lipogenesis factors such as acetyl-coenzyme A carboxylase (ACC), fatty acid synthase (FAS), and sterol
regulatory element-binding protein-1c (SREBP-1c) [12]. Another study has shown that an HF diet
destroyed the antioxidant defense system by decreasing superoxide dismutase (SOD) and catalase
(CAT) activities, and markedly increased the production of ROS [13]. To prevent metabolic disorders
due to oxidative stress, antioxidative capability that suppresses or eliminates ROS production plays an
important role in the body. Plants contain a variety of phytochemicals that have antioxidant ability to
effectively remove ROS [14].
Curcuma longa L. is a flowering plant of the ginger family (Zingiberaceae). As C. longa possesses
natural antioxidants, which leads to free radical scavenging potential, it has been widely used to
prevent many diseases, including liver injury [15].
The aim of our study was to investigate in vitro and in vivo protective effects of hot water extract
(CLW) from C. longa on the development of fatty liver. Furthermore, the mode of action against lipid
accumulation and oxidative stress in non-alcoholic liver damage was determined.
2. Materials and Methods
2.1. Sample and Chemicals
C. longa, which was supplied by the SDC R&D Center (Damyang, Korea), was extracted with
20 volumes of hot water at 250 ◦ C for 3 h. The extracted solution was filtered and concentrated using an
evaporator under vacuum conditions. The concentrate was lyophilized and stored at -20◦ C until further
use. Minimum essential medium (MEM), fetal bovine serum (FBS), antibiotics, trypsin-ethylenediamine
tetraacetic acid, and Hank’s balanced salt solution were products of Gibco BRL (Grand Island, NY,
USA). Sodium salt of 2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxyanilide inner
salt or XTT, H2 O2 , 4-nitrocatechol, 2’,7’-dichlorofluorescein diacetate (DCF-DA), sodium oleate, sodium
palmitate, and Oil Red O were purchased from Sigma-Aldrich (St. Louis, MO, USA). All other chemicals
were of analytical reagent grade.
2.2. Cell Culture
Human hepatocellular carcinoma cell line HepG2 cells were obtained from the American Type
Culture Collection (Manassas, VA, USA). The cells were cultured in MEM supplemented with 10%
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FBS and 1% penicillin/streptomycin and maintained in 10 cm dishes at 37 ◦ C under a humidified
atmosphere of 5% CO2 .
2.3. FFA-Induced Lipid Overloading in HepG2 Cells
HepG2 cells were seeded at 1 × 105 cells/well in a 24-well culture plate and incubated for 24 h to
induce excessive lipid accumulation in an in vitro hepatic steatosis model. On day 2, when the cells
reached approximately 80% confluency, they were pretreated with CLW solution for 2 h, and then,
1 mM FFA mixture was added into each well. To prepare the FFA mixture, sodium oleate and sodium
palmitate were conjugated with a culture medium containing 1% bovine serum albumin (BSA). The FFA
mixture (2:1 ratio of oleate: palmitate) was diluted in the culture medium to obtain the desired final
concentration. Control cells were treated with 1% BSA only.
2.4. Measurement of Intracellular ROS Formation
Intracellular ROS levels were detected using the fluorescence probe (DCF-DA). After 24 h
incubation of the HepG2 cells that had been seeded in a 24-well plate at a concentration of 1 × 105
cells/well, the cells were treated with 50 and 100 µg/mL CLW and 1 mM FFA mixture, respectively,
for 2 h. The cells were then exposed to 30 µM DCF-DA for 30 min at 37◦ C. Fluorescence intensity of
the cells was measured using a fluorescence microplate reader (BioTek Instruments, Winooski, VT,
USA) with an excitation wavelength of 485 nm and an emission wavelength of 530 nm. Control cells
were treated with 1% BSA only.
2.5. Measurement of Lipid Accumulation (Oil Red O Staining)
To evaluate the accumulation of intracellular neutral lipids levels, HepG2 cells were treated with 50
and 100 µg/mL CLW and 1 mM FFA mixture for 24 h, respectively. Then, the cells were fixed with 10%
formalin for 30 min, followed by staining with Oil Red O solution for 40 min. To remove the remnants
of the Oil Red O reagent, the stained cells were washed 3 times with phosphate-buffered saline (PBS)
and then observed under a microscope (Leica DMi1; Leica Microsystems, Wetzlar, Germany). Lipid
droplets were extracted using 60% isopropanol and assessed colorimetrically at 510 nm. Control cells
were treated with 1% BSA only.
2.6. Animal Experiments
Seven-week-old male C57BL/6 mice were purchased from Orient Bio (Seongnam, Korea).
They were acclimated for 1 week before experiments. The animals were housed in stainless steel
cages under controlled laboratory conditions (20–25 ◦ C, 55%–60% humidity, and 12 h light/dark cycle
of lighting). The Institutional Animal Care and Use Committee of Chonnam National University
approved the protocol for the animal study. Animals were cared for according to the “Guidelines
for Animal Experiments” established by Chonnam National University (CNU IACUC-YB-2018-55).
For the experiments, the mice were randomly divided into 5 groups (n = 10 per group) as follows;
(1) CON: Mice fed a normal diet (AIN-76A diet, Research Diet, Inc., New Brunswick, NJ, USA); (2) HF:
Mice fed a 60 kcal% fat diet (D12492, Research Diet, Inc.); (3) C-LOW: Mice fed high-fat diet plus CLW
300 mg/kg/b.w./day; (4) C-HIGH: Mice fed high-fat diet plus CLW 900 mg/kg/b.w./day; and (5) SILY:
Mice fed high-fat diet plus silymarin 50 mg/kg/b.w./day. The animals were fed ad libitum for 8 weeks.
Blood samples were collected from the postcaval vein of each animal immediately after death and
centrifuged at 3000 rpm for 15 min. The serum was stored at -80 ◦ C until further analysis. Each tissue
was immediately weighed and stored at -80◦ C until further analysis.
2.7. Assays for Serum Marker Enzymes, and Hepatic Triglyceride, and Total Cholesterol
Aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were measured using
assay kits (Asan Pharmaceutical, Seoul, Korea). For analyses of hepatic TG and total cholesterol,
Nutrients 2019, 11, 2536
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the frozen liver tissue was homogenized in a glass-Teflon homogenizer (Daihan, Korea) with PBS and
centrifuged at 13,000 rpm for 30 min. The liver lipids were extracted by the Folch method [16] and
quantified. Hepatic TG and total cholesterol (TC) levels were measured by assay kits (Asan Pharm).
2.8. Hematoxylin and Eosin Staining
For hematoxylin and eosin (H&E) staining, the liver was fixed in 10% formaldehyde, embedded
in paraffin, and cut into 10 µm sections. The sections were stained with H&E and then observed under
a microscope.
2.9. Measurement of Antioxidant Enzyme Activity
For the antioxidant activity assays, the liver was homogenized in PBS. The suspension was
centrifuged at 13,000× g for 15 min at 4 ◦ C, and the supernatant was used for the assay. The amount
of protein was estimated using the Bradford assay. The SOD activity was measured by the
method of Alam [17], and CAT activity was assayed by the method of Garg [18]. The activities
of hepatic glutathione-S-transferase (GST), GPx, and GR were determined by methods of Molina [19],
Al Batran [20], and Calberg and Mannervik [21], respectively. Reduced glutathione (GSH) level
was measured using the method of Akerboom and Sies [22]. To measure the lipid peroxidation,
the concentration of malondialdehyde (MDA) was monitored with thiobarbituric acid reactive
substance formation as described by Draper and Hadley [23], and calculated using a standard curve.
2.10. Total RNA Isolation and Real-Time Polymerase Chain Reaction
Total RNA was extracted from the liver by an easy-BLUETM total RNA extraction kit
(Intron Biotechnology, Sungnam, Korea) according to the manufacturer’s instructions. Nano-drop
measurements of RNA samples were processed to determine the quality and quantity of the RNA.
cDNA was synthesized from the purified total RNA using an iScript™ cDNA synthesis kit (Bio-Rad
Laboratories, Hercules, CA, USA). Real-time polymerase chain reaction (RT-PCR) was performed
using an SYBR green RT-PCR kit and custom-designed primers (Table 1) with the CFX96 TouchTM
real-time PCR detection system (Bio-Rad). The cDNA was amplified for 40 cycles of denaturation
(95 ◦ C for 30 s), annealing (58 ◦ C for 30 s), and extension (72 ◦ C for 45 s).
Table 1. Real-time polymerase chain reaction (RT-PCR) primer sequences.
Gene
Primers
Sequence (50 to 30 )
CYP2E1
Forward
Reverse
50 - CGTGGAAATGGAGAAGGAAA-30
50 - GGTGATGAACCGCTGAATCT-30
AMPK
Forward
Reverse
50 - GGCACCCTCCCATTTGATG-30
50 - ACACCCCCTCGGATCTTCTT-30
ACC
Forward
Reverse
50 - TGCAGATCTTAGCGGACCAA-30
50 - GCCTGCGTTGTACAGAGCAA-30
CPT-1
Forward
Reverse
50 - TGTTGGGTATGCTGTTCATGACA-30
50 - GCGGCCTGGGTAGGAAGA-30
SREBP-1c
Forward
Reverse
50 - CGGAACCATCTTGGCAACA-30
50 - GCCGGTTGATAGGCAGCTT-30
PPAR-α
Forward
Reverse
50 - AACATCCAAGAGATTTCGCAATC-30
50 - CCGTAAAGCCAAAGCTTCCA-30
CD36
Forward
Reverse
50 - TGGAACAGAGGCTGACAACT-30
50 - TTGATTTTTGATAGATATGGG-30
FATP2
Forward
Reverse
50 - CTTTCAGCACATTGCTGATTACCT-30
50 -CAGTGATCTCAATGGTGTCCTGTAT-30
FATP5
Forward
Reverse
50 - AGCTCCTGCGGTACTTGTGT-30
50 - AAGGTCTCCCACACATCAGC-30
β-Actin
Forward
Reverse
50 - ACGGCCAGGTCATCACTATTG-30
50 - CAAGAAGGAAGGCTGGAAAAGA-30
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Reverse
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5′- CAAGAAGGAAGGCTGGAAAAGA-3′
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2.11. Western Blotting
Equal amounts
2.11. Western
Blotting of proteins (100 μg/lane) from the liver were separated by sodium dodecyl
sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes
Equal amounts of proteins (100 µg/lane) from the liver were separated by sodium dodecyl
(Bio-Rad). The membranes were blocked with blocking buffer (10% blocker BSA in PBS, Thermo
sulfate-polyacrylamide
electrophoresis
andwith
transferred
polyvinylidene
difluorideAntibodies
membranes
Fisher Scientific) for 30gel
min
before incubating
the firstto
antibody
at 4 °C overnight.
(Bio-Rad).
The membranes
were blocked
with blocking and
buffer
(10% were
blocker
BSA in PBS,
against AMPK,
phospho-AMPK,
ACC, phospho-ACC,
β-actin
purchased
fromThermo
Cell
◦ C overnight. Antibodies
Fisher
Scientific)
for
30
min
before
incubating
with
the
first
antibody
at
4
Signaling Technology (Danvers, MA, USA). Thereafter, the membranes were incubated with a
against
AMPK,
phospho-AMPK,
phospho-ACC,
and β-actin
were purchased
from
secondary
antibody
(anti-rabbitACC,
immunoglobulin
G (IgG),
Cell Signaling
Technology)
forCell
2 h.Signaling
Bands
Technology
(Danvers,
USA).chemiluminescence
Thereafter, the membranes
were incubated
with ablotting
secondary
antibody
were detected
via MA,
enhanced
(ECL) using
ECL western
detection
(anti-rabbit
immunoglobulin G (IgG), Cell Signaling Technology) for 2 h. Bands were detected via
reagents (Bio-Rad).
enhanced chemiluminescence (ECL) using ECL western blotting detection reagents (Bio-Rad).
2.12. Statistical Analysis
2.12. Statistical Analysis
All data are presented as mean ± S.E. Data were statistically evaluated via one-way analysis of
All data
are presented
as mean
± S.E.procedures
Data werefor
statistically
viaStatistics
one-way22.0,
analysis
variance
(ANOVA)
using SPSS
statistical
Windows evaluated
(SPSS PASW
SPSS of
Inc.
Chicago,
IL,
USA),
and
Duncan’s
multiple
range
test
was
used
to
compare
significant
variance (ANOVA) using SPSS statistical procedures for Windows (SPSS PASW Statistics 22.0, SPSS
differences
(p <0.05). multiple range test was used to compare significant differences
Inc.
Chicago, between
IL, USA),groups
and Duncan’s
between groups (p < 0.05).
3. Results
3. Results
3.1. Effect of CLW on Antioxidant Activity in FFA-Treated Cells
3.1. Effect of CLW on Antioxidant Activity in FFA-Treated Cells
The changes in intracellular ROS levels by CLW in FFA-treated cells are depicted in Figure 1.
Compared
to theincontrol
group,ROS
the intracellular
ROSinlevel
of only cells
the FFA-treated
group
was 1.
The changes
intracellular
levels by CLW
FFA-treated
are depicted
in Figure
significantly
increased.
the the
cellsintracellular
were pretreated
50 of
and
100 the
μg/mL
CLW, intracellular
Compared
to the
controlWhen
group,
ROSwith
level
only
FFA-treated
group was
ROS formation
was When
suppressed
in were
a dose-dependent
compared
to that
in the only
significantly
increased.
the cells
pretreated withmanner
50 and 100
µg/mL CLW,
intracellular
ROS
FFA-treated
group.
formation was suppressed in a dose-dependent manner compared to that in the only FFA-treated group.
Figure
Effect
of water
extract
(CLW)
Curcuma
onformation
the formation
of intracellular
Figure
1. 1.
Effect
of water
extract
(CLW)
fromfrom
Curcuma
longalonga
L. onL.the
of intracellular
reactive
reactive
oxygen
species
(ROS)
in free
fatty acid-treated
HepG2
cells.normal
CON, control
normal control
group;free
oxygen
species
(ROS)
in free
fatty
acid-treated
HepG2 cells.
CON,
group; FFA,
FFA,
free fatty acid-treated
group;and
CLW50
and CLW100,
pretreatment
with50
CLW
and
100 μg/mL
fatty
acid-treated
group; CLW50
CLW100,
pretreatment
with CLW
and50
100
µg/mL
groups,
groups,
respectively.
Data
are
expressed
as
mean
±
S.E.,
and
different
letters
indicate
significant
respectively. Data are expressed as mean ± S.E., and different letters indicate significant differences
as determined
via Duncan’s
range test.
(p differences
< 0.05, a > (p
b><0.05,
c), asa>b>c),
determined
via Duncan’s
multiplemultiple
range test.
3.2.3.2.
Effect
ofofCLW
Effect
CLWononLipid
LipidAccumulation
Accumulationin
in FFA-Treated
FFA-Treated Cells
Cells
Lipid
accumulationhad
hadnoticeably
noticeably increased
increased in
to to
that
in in
thethe
Lipid
accumulation
inthe
theFFA-treated
FFA-treatedgroup
groupcompared
compared
that
control
group,
while
CLW
supplementation
decreased
accumulation
in a dose-dependent
control
group,
while
CLW
supplementation
decreased
lipidlipid
accumulation
in a dose-dependent
manner
manner
(Figure
2A).
Oil Red image
O staining
image
of the FFA-treated
group
showed
in the
(Figure
2A).
Oil Red
O staining
of the
FFA-treated
group showed
more
in the more
cell than
thecell
other
than the
groups.
Onpretreated
the other groups
hand, pretreated
had less
lipid droplets,
groups.
Onother
the other
hand,
with CLW groups
had lesswith
lipidCLW
droplets,
indicating
a decrease
a decrease(Figure
in lipid2B).
accumulation (Figure 2B).
in indicating
lipid accumulation
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2019,11,
11,xxFOR
FORPEER
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Figure
2.2.2.Effect
(CLW)
from
Curcumalonga
longaL.
onlipid
lipid
accumulation
in
free
fatty
Figure
Effect
of
water
extract
accumulation
in
fatty
Figure
Effectof
ofwater
waterextract
extract (CLW)
(CLW) from
from Curcuma
Curcuma
longa
L.L.on
on
lipid
accumulation
in free
free
fatty
acid-treated
HepG2
cell.
(A)
Intracellular
lipid
levels
were
colorimetrically
510
nm.
acid-treated
HepG2
cell.
(A)
Intracellular
lipid
levels
were
colorimetrically
measured
510
nm.
(B)
acid-treated
HepG2
cell.
(A)
Intracellular
lipid
levels
were
colorimetricallymeasured
measuredatatat
510
nm.(B)
(B)The
stained
cells
were
observed
under
a
microscope
(400×).
CON,
normal
control
group;
FFA,
FFA,
free
The
stained
cells
were
observed
under
a
microscope
(400×).
CON,
normal
control
group;
FFA,
FFA,
The stained cells were observed under a microscope (400×). CON, normal control group; FFA, FFA,
fatty
acid-treated
group;
CLW50
and
CLW100,
pretreatment
with
CLW
50
and
100
µg/mL
groups,
free
fatty
acid-treated
group;
CLW50
and
CLW100,
pretreatment
with
CLW
50
and
100
μg/mL
free fatty acid-treated group; CLW50 and CLW100, pretreatment with CLW 50 and 100 μg/mL
groups,
Data
as
mean
±± S.E.,
letters
indicate
respectively.
Data are expressed
as mean ±
and
different
letters
indicate
differences
groups, respectively.
respectively.
Data are
are expressed
expressed
asS.E.,
mean
S.E., and
and different
different
letterssignificant
indicate significant
significant
(p
<0.05),
as
determined
via
Duncan’s
multiple
range
test.
(pdifferences
<
0.05),
as
determined
via
Duncan’s
multiple
range
test.
differences (p <0.05), as determined via Duncan’s multiple range test.
3.3.
Effect
ofofofCLW
in
Mice
3.3.
Effect
CLW
on
HF-Induced
Hepatotoxicity
3.3.
Effect
CLWon
onHF-Induced
HF-InducedHepatotoxicity
Hepatotoxicityin
inMice
Mice
Serum
AST
andand
ALTALT
activities
were useful
indicators
of liver damage
and
hepatotoxicity.
Serum
AST
activities
were
useful
indicators
of
damage
and
Serum
AST
and
ALT
activities
were biological
useful biological
biological
indicators
of liver
liver
damage
and
These
enzyme
activities
were
significantly
elevated
in
mice
administered
an
HF
diet.
However,
CLW
hepatotoxicity.
These
enzyme
activities
were
significantly
elevated
in
mice
administered
an
HF
hepatotoxicity. These enzyme activities were significantly elevated in mice administered an HF
diet.
However,
CLW
co-treatment
for
eight
weeks
remarkably
decreased
the
serum
levels
of
these
co-treatment
for
eight
weeks
remarkably
decreased
the
serum
levels
of
these
indicators
(Figure
diet. However, CLW co-treatment for eight weeks remarkably decreased the serum levels of these 3).
indicators
(Figure
supplementation
with
CLW
at
of
b.w./day
Notably,
supplementation
with CLW
at a dose of 300
mg/kg
recovered
the impaired
liver
indicators
(Figure 3).
3). Notably,
Notably,
supplementation
with
CLWb.w./day
at aa dose
dose
of 300
300 mg/kg
mg/kg
b.w./day
recovered
the
liver
resulting
As
shown
in
4,4,the
functions
resulting
from HF-induced
toxicity.
As from
shown
in Figure 4,toxicity.
the liver
TG
and TC
levels
recovered
theimpaired
impaired
liver functions
functions
resulting
from HF-induced
HF-induced
toxicity.
As
shown
in Figure
Figurein
the
liver
TG
TC
in
HF
group
were
higher
than
in
HF
group
higher
than those
the
control
group,
whereas
co-administration
of
CLWwhereas
markedly
the
liverwere
TG and
and
TC levels
levels
in the
thein
HF
group
were
higher
than those
those
in the
the control
control group,
group,
whereas
co-administration
of
CLW
markedly
reduced
TG
and
TC
levels.
These
results
suggested
that
co-administration
CLW markedly
reduced
TG and that
TC levels.
results suggested
thatCLW
CLW
reduced
TG and TCoflevels.
These results
suggested
CLW These
supplementation
suppressed
lipid
supplementation
suppressed
lipid
accumulation
in
the
liver.
supplementation
lipid accumulation in the liver.
accumulation
in thesuppressed
liver.
Figure
3.3.3.Effect
(CLW)
from
Curcuma
longaL.
onhepatic
hepatic
markers
in
serum
Figure
Effect
of
water
extract
markers
in
serum
of
Figure
Effectof
ofwater
waterextract
extract(CLW)
(CLW)from
fromCurcuma
Curcumalonga
longa
L.L.on
on
hepatic
markers
inthe
thethe
serum
of of
high-fat
diet-fed
mice.
ALT;
alanine
aminotransferase,
AST;
aspartate
aminotransferase.
CON,
high-fat
diet-fed
mice.
ALT;
alanine
aminotransferase,
AST;
aspartate
aminotransferase.
CON,
normal
high-fat diet-fed mice. ALT; alanine aminotransferase, AST; aspartate aminotransferase. CON,
normal
control
diet;
HF,
high-fat
diet;
C-LOW,
60%
high-fat
diet
plus
300
b.w./day
of
control
diet;
HF, 60%
diet;
C-LOW,
60%
high-fat
diet
plus 300
mg/kg
of CLW;
C-HIGH,
normal
control
diet;high-fat
HF, 60%
60%
high-fat
diet;
C-LOW,
60%
high-fat
diet
plusb.w./day
300 mg/kg
mg/kg
b.w./day
of
CLW;
C-HIGH,
60%
high-fat
diet
plus
900
mg/kg
b.w./day
of
CLW;
SILY,
60%
high-fat
diet
plus
50
60%
high-fat
diet 60%
plushigh-fat
900 mg/kg
of CLW;
SILY, 60%
high-fat
50 mg/kg
b.w./day
CLW;
C-HIGH,
diet b.w./day
plus 900 mg/kg
b.w./day
of CLW;
SILY,diet
60%plus
high-fat
diet plus
50
b.w./day
of
Data
are
expressed
as
mean
±±S.E.
(n
different
letters
ofmg/kg
silymarin.
Data
are
expressed
± S.E.
= 10),
and
indicate
significant
mg/kg
b.w./day
ofsilymarin.
silymarin.
Dataas
aremean
expressed
as(n
mean
S.E.
(n=different
=10),
10),and
andletters
different
lettersindicate
indicate
significant
differences
<0.05),
via
multiple
range
differences
< 0.05), as(p(p
determined
via Duncan’s
multiple
test.
significant(p
differences
<0.05),as
asdetermined
determined
viaDuncan’s
Duncan’srange
multiple
rangetest.
test.
Nutrients 2019, 11, 2536
Nutrients
Nutrients 2019,
2019, 11,
11, x
x FOR
FOR PEER
PEER REVIEW
REVIEW
7 of 13
77 of
of 13
13
Figure
4. Effect
of water
extract (CLW)
from Curcuma
longa L. on
on hepatictriglyceride
triglyceride and
total
Figure
Figure 4.
4. Effect
Effect of
of water
water extract
extract (CLW)
(CLW) from
from Curcuma
Curcuma longa
longa L.
L. on hepatic
hepatic triglyceride and
and total
total
cholesterol
in high-fat
diet-fed mice.
(A) TG;
triglyceride, (B)
(B) TC; total
total cholesterol.CON,
CON, normal
cholesterol
cholesterol in
in high-fat
high-fat diet-fed
diet-fed mice.
mice. (A)
(A) TG;
TG; triglyceride,
triglyceride, (B) TC;
TC; total cholesterol.
cholesterol. CON, normal
normal
control
diet;
HF,HF,
60%60%
high-fat
diet;diet;
C-LOW,
60% 60%
high-fat
diet plus
300
mg/kg
b.w./day
of CLW;
C-HIGH,
control
diet;
high-fat
C-LOW,
high-fat
diet
plus
300
mg/kg
b.w./day
control diet; HF, 60% high-fat diet; C-LOW, 60% high-fat diet plus 300 mg/kg b.w./day of
of CLW;
CLW;
60%
high-fat60%
diet
plus 900
mg/kg
b.w./day
of
CLW; SILY,
60%SILY,
high-fat
diet plusdiet
50 plus
mg/kg
b.w./day
C-HIGH,
high-fat
diet
plus
900
mg/kg
b.w./day
of
CLW;
60%
high-fat
50
C-HIGH, 60% high-fat diet plus 900 mg/kg b.w./day of CLW; SILY, 60% high-fat diet plus 50 mg/kg
mg/kg
of b.w./day
silymarin. Data
are expressed
as mean ± S.E.
(n = 10), and
different
letters indicate
significant
b.w./day of
of silymarin.
silymarin. Data
Data are
are expressed
expressed as
as mean
mean ±± S.E.
S.E. (n
(n == 10),
10), and
and different
different letters
letters indicate
indicate
differences
(p
<
0.05),
as
determined
via
Duncan’s
multiple
range
test.
significant
significant differences
differences (p
(p << 0.05),
0.05), as
as determined
determined via
via Duncan’s
Duncan’s multiple
multiple range
range test.
test.
3.4.3.4.
Effect of of
CLW ononHistopathological
Morphology
3.4. Effect
Effect of CLW
CLW on Histopathological
Histopathological Morphology
Morphology
The
livers ofofthe
control mice
revealed no
abnormal appearance
appearance orhistopathological
histopathological change.
The
The livers
livers of the
the control
control mice
mice revealed
revealed no
no abnormal
abnormal appearance or
or histopathological change.
change.
However,
thethe
HF
diet
caused
fatty
liver
in
mice,
as
demonstrated
byoutstandingly
outstandinglynumerous
numerous lipid
However,
HF
diet
caused
fatty
liver
in
mice,
as
demonstrated
by
However, the HF diet caused fatty liver in mice, as demonstrated by outstandingly numerous lipid
lipid
droplets
compared
totothe
control
liver
(Figure
5).
The hepatic
hepaticlipid
lipiddroplets
dropletsinduced
induced
by
the
HF
diet
droplets
the
droplets compared
compared to
the control
control liver
liver (Figure
(Figure 5).
5). The hepatic
lipid droplets
induced by
by the
the HF
HF diet
diet
were
significantly
reduced
administeredCLW
CLWat
dose
900
mg/kg
were
significantly
reduced
by
treatment
with
of
900
mg/kg
were
significantly
reducedby
bytreatment
treatmentwith
with CLW.
CLW. When
When administered
administered
CLW
atataa adose
dose
ofof
900
mg/kg
b.w./day,
liver
conditions
were
ameliorated
to
the
normal
liver
b.w./day,
liver
conditions
were
ameliorated
to
the
normal
liver
tissues.
b.w./day, liver conditions were ameliorated to the normal liver tissues.
Figure
5. Effect
of water
extract
(CLW)
from
Curcuma
L. on
liver histopathological
changes
in
Figure
Effect
waterextract
extract(CLW)
(CLW)from
from Curcuma
Curcuma longa
longa
changes
in in
Figure
5. 5.
Effect
ofof
water
longa L.
L. on
onliver
liverhistopathological
histopathological
changes
high-fat
diet-fed
mice.
Liver
sections
of
mice
fed
a
normal
control
diet
or
a
high-fat
diet
for
8
weeks,
high-fat
diet-fed
mice.
Liver
sections
of
mice
fed
a
normal
control
diet
or
a
high-fat
diet
for
8
weeks,
high-fat diet-fed mice. Liver sections of mice fed a normal control diet or a high-fat diet for 8 weeks,
stained
with
hematoxylin
and
eosin
(H&E;
bars:
μm). CON,
normal control
diet; HF,
60%
high-fat
stained
with
hematoxylinand
andeosin
eosin(H&E;
(H&E; bars:
bars: 50
50
HF,
60%
high-fat
stained
with
hematoxylin
50 μm).
µm). CON,
CON,normal
normalcontrol
controldiet;
diet;
HF,
60%
high-fat
diet;
C-LOW,
60%
high-fat
diet
plus
300
mg/kg
b.w./day
of
CLW;
C-HIGH,
60%
high-fat
diet
plus
900
diet;
C-LOW,60%
60%high-fat
high-fat diet
diet plus
b.w./day
of CLW;
C-HIGH,
60% 60%
high-fat
diet plus
diet;
C-LOW,
plus300
300mg/kg
mg/kg
b.w./day
of CLW;
C-HIGH,
high-fat
diet900
plus
mg/kg
b.w./day
of
CLW;
SILY,
60%
high-fat
diet
plus
50
mg/kg
b.w./day
of
silymarin.
mg/kg
b.w./day
of
CLW;
SILY,
60%
high-fat
diet
plus
50
mg/kg
b.w./day
of
silymarin.
900 mg/kg b.w./day of CLW; SILY, 60% high-fat diet plus 50 mg/kg b.w./day of silymarin.
Changes
Antioxidant
Activities
in
Fatty
Liver
by CLW
3.5.
Changes
inAntioxidant
AntioxidantActivities
Activitiesin
inFatty
Fatty Liver
Liver by
CLW Treatment
Treatment
3.5.3.5.
Changes
inin
Treatment
The
functions
of
liver
antioxidants
in
HF
diet-fed
mice
are summarized
in Figure 6A–F.
In
the
HF
The
functions
liverantioxidants
antioxidantsin
in HF
HF diet-fed
diet-fed mice
6A–F.
In In
thethe
HFHF
The
functions
ofofliver
miceare
aresummarized
summarizedininFigure
Figure
6A–F.
group,
levels
of
CAT,
SOD,
GST,
GPx,
GR,
and
GSH
were
significantly
dropped
compared
to
those
group,
levels
of
CAT,
SOD,
GST,
GPx,
GR,
and
GSH
were
significantly
dropped
compared
to
those
group, levels of CAT, SOD, GST, GPx, GR, and GSH were significantly dropped compared to those
of the
control
group.
However,
co-treatment
with
at aa dose
of 300 mg/kg
b.w./day completely
control
group.However,
However,co-treatment
co-treatment with
with CLW
CLW
completely
of of
thethe
control
group.
CLW at
at adose
doseofof300
300mg/kg
mg/kgb.w./day
b.w./day
completely
prevented
a
decrease
in
liver
antioxidants.
In
this
study,
silymarin,
a
well-known
hepatoprotectant
prevented
a
decrease
in
liver
antioxidants.
In
this
study,
silymarin,
a
well-known
hepatoprotectant
prevented
a
decrease
in
liver
antioxidants.
In
this
study,
silymarin,
a
well-known
hepatoprotectant
obtained
obtained from
from the
the milk
milk thistle,
thistle, was
was used
used as
as aa positive
positive control.
control. The
The antioxidant
antioxidant levels
levels of
of CLW
CLW were
were
obtained
from
theofmilk
thistle,The
was
used asstress
a positive
control.
Thediet
antioxidant
levels of CLW
were
similar
to
those
silymarin.
oxidative
induced
by
an
HF
caused
a
significant
similar to those of silymarin. The oxidative stress induced by an HF diet caused a significant increase
increase
similar
to those of asilymarin.
The oxidative
stress induced
by an HF diet caused
a significant(Figure
increase
in
in MDA
MDA levels,
levels, a key
key biomarker
biomarker of
of oxidative
oxidative stress,
stress, in
in comparison
comparison with
with the
the control
control group
group (Figure
in 6G).
MDAOn
levels,
a
key
biomarker
of
oxidative
stress,
in
comparison
with
the
control
group
(Figure
6G).
6G). On the
the other
other hand,
hand, supplementation
supplementation with
with CLW
CLW partially
partially prevented
prevented the
the elevation
elevation of
of MDA.
MDA.
OnThese
the other
hand,
supplementation
with
CLW
partially
prevented
the
elevation
of
MDA.
These
results
These results
results showed
showed that
that CLW
CLW suppressed
suppressed the
the oxidative
oxidative stress
stress in
in the
the liver.
liver.
showed that CLW suppressed the oxidative stress in the liver.
Nutrients 2019, 11, x FOR PEER REVIEW
Nutrients 2019, 11, 2536
Nutrients 2019, 11, x FOR PEER REVIEW
8 of 13
8 of 13
8 of 13
Figure 6. Effect of water extract (CLW) from Curcuma longa L. on antioxidant status in high-fat
Figure
6. 6.
Effect
of water
extract
(CLW)
from
Curcuma
L. onL.antioxidant
status
in high-fat
diet-fed
diet-fed
mice.
(A)water
Catalase
(CAT)
activity,
(B)longa
superoxide
(SOD)
activity,
(C)
Figure
Effect
of
extract
(CLW)
from
Curcuma
longa
ondismutase
antioxidant
status
in high-fat
mice.
(A)
Catalase
(CAT)
activity,
(B)
superoxide
dismutase
(SOD)
activity,
(C)
glutathione-S-transferase
glutathione-S-transferase
(GST)
activity,
(D)
glutathione
peroxidase
(GPx)
activity,
(E)
glutathione
diet-fed mice. (A) Catalase (CAT) activity, (B) superoxide dismutase (SOD) activity, (C)
(GST)
activity,
(D)activity,
glutathione
peroxidase
activity,
(E)(G)
glutathione
reductase
(GR)level
activity,
reductase
(GR)
(F)
reduced
glutathione
(GSH)
level,
malondialdehyde
(MDA)
in
glutathione-S-transferase
(GST)
activity,
(D)(GPx)
glutathione
peroxidase
(GPx) activity,
(E)
glutathione
the
liversglutathione
of different
groups.
Data(G)
are
expressed(GSH)
as mean
± S.E.
(nlevel
= 10),inand
letters
indicate
(F)reductase
reduced
(GSH)
level,
malondialdehyde
(MDA)
the different
livers of(MDA)
different
groups.
(GR)
activity,
(F) reduced
glutathione
level,
(G)
malondialdehyde
level
in
significant
(p<0.05)
as(ndetermined
viaasDuncan’s
multiple
range
CON,
normal
control
Data
are
expressed
as mean
± S.E.
= 10),
and different
letters
indicate
significant
differences
(p
< 0.05)
the
livers
ofdifferences
different
groups.
Data
are
expressed
mean
± S.E.
(n = 10),
andtest.
different
letters
indicate
diet; HF, 60%
high-fat
diet; multiple
C-LOW,
60%
diet plus
300 mg/kg
CLW;
C-HIGH,
as significant
determined
via
Duncan’s
rangehigh-fat
test.
CON,
normal
control
diet;
HF,of60%
high-fat
diet;
differences
(p<0.05)
as determined
via Duncan’s
multiple
rangeb.w./day
test. CON,
normal
control
60% HF,
high-fat
diet
plus
900
mg/kg
b.w./day
of CLW;
SILY,
60%
diet
plus of
50
mg/kg
b.w./day
C-LOW,
60%
high-fat
dietdiet;
plus
300 mg/kg
of
CLW;
60%
high-fat
diet
plusC-HIGH,
900 mg/kg
diet;
60%
high-fat
C-LOW,
60%b.w./day
high-fat
diet
plusC-HIGH,
300high-fat
mg/kg
b.w./day
CLW;
of silymarin.
b.w./day
of CLW;
high-fat
diet plus
mg/kg
b.w./day
of silymarin.
60%
high-fat
dietSILY,
plus60%
900 mg/kg
b.w./day
of 50
CLW;
SILY,
60% high-fat
diet plus 50 mg/kg b.w./day
of silymarin.
3.6.3.6.
Effect
of CLW
onon
Fatty
Acid
Diet-fedMice
Mice
Effect
of CLW
Fatty
AcidUptake-Related
Uptake-RelatedmRNA
mRNAExpression
Expression in HF Diet-fed
3.6.Expression
Effect
of CLW
on
Fatty
Uptake-Related
Expression
insuch
HF
Mice
Expression
levels
ofAcid
mRNA
related
to mRNA
fatty
acid
uptake,
as CD36,
FATP5,
FATP2,
levels
of mRNA
related
to fatty
acid
uptake,
such
asDiet-fed
CD36,
FATP5,
andand
FATP2,
were
wereExpression
up-regulated
themRNA
HF
group
7).diet
The
HF
dietinduced
actively
induced
inflow
of acids
free
fatty
up-regulated
in thelevels
HFingroup
(Figure
7).(Figure
The
HF
actively
free
fatty
in the
of
related
to fatty
acid
uptake,
such asinflow
CD36,ofFATP5,
and
FATP2,
acids
in theinliver,
resulting
fatty
liver disease.
However,
CLW
groups
significantly
liver,
resulting
fattyin
liver
However,
CLW
groups
showed
significantly
down-regulated
fatty
were
up-regulated
the disease.
HF in
group
(Figure
7). The
HF
diet
actively
inducedshowed
inflow
of
free fatty
down-regulated
fatty
acid
levels,
which
were
comparable
to
those
of
the
SILY
group.
These
results
acids
in the
liver,
resulting
in fatty
liver of
disease.
However,
CLW groups
showed significantly
acid
levels,
which
were
comparable
to those
the SILY
group. These
results indicated
that fatty acid
indicated
that fatty
acid
uptake
was
blocked
CLW supplementation.
down-regulated
fatty
acid
levels,
which
wereby
comparable
to those of the SILY group. These results
uptake
was blocked
by
CLW
supplementation.
indicated that fatty acid uptake was blocked by CLW supplementation.
Figure
7. Effect
of water
extract
(CLW)
fromfrom
Curcuma
longalonga
L. onL.fatty
in high-fat
diet-fed
Figure
7. Effect
of water
extract
(CLW)
Curcuma
on acid
fattyuptake
acid uptake
in high-fat
mice.
mRNA
expression
levels
of
fatty
acid
uptake
factors,
including
a
cluster
of
differentiation
36
diet-fed7. mice.
levelsfrom
of Curcuma
fatty acidlonga
uptake
including
Figure
Effect mRNA
of waterexpression
extract (CLW)
L. onfactors,
fatty acid
uptake aincluster
high-fatof
(CD36),
fatty
acid mRNA
transport
protein
2 (FATP2),
and
FATP5.
CON,
normal
control
diet; HF,
60%control
high-fat
differentiation
(CD36),
fatty acid
transport
2 (FATP2),
FATP5.
CON,
normal
diet-fed
mice. 36
expression
levels
of protein
fatty acid
uptake and
factors,
including
a cluster
of
diet;
C-LOW,
60%
high-fat
diet
plus
300
mg/kg
b.w./day
of
CLW;
C-HIGH,
60%
high-fat
diet
plus
diet;
HF,
60%
high-fat
diet;
C-LOW,
60%
high-fat
diet
plus
300
mg/kg
b.w./day
of
CLW;
C-HIGH,
differentiation 36 (CD36), fatty acid transport protein 2 (FATP2), and FATP5. CON, normal control
900
mg/kg
b.w./day
of
CLW;
SILY,
60%
high-fat
diet
plus
50
mg/kg
b.w./day
of
silymarin.
Data
60%
high-fat
diet
plus
900
mg/kg
b.w./day
of
CLW;
SILY,
60%
high-fat
diet
plus
50
mg/kg
b.w./day
diet; HF, 60% high-fat diet; C-LOW, 60% high-fat diet plus 300 mg/kg b.w./day of CLW; C-HIGH,are
expressed
as mean
± S.E.
= 10),
and different
indicate
significant
(p < 0.05),
60% high-fat
diet plus
900(n
mg/kg
b.w./day
of CLW;letters
SILY, 60%
high-fat
diet plusdifferences
50 mg/kg b.w./day
as determined via Duncan’s multiple range test.
Nutrients 2019, 11, x FOR PEER REVIEW
9 of 13
of silymarin. Data are expressed as mean ± S.E. (n = 10), and different letters indicate significant
9 of 13
differences (p <0.05), as determined via Duncan’s multiple range test.
Nutrients 2019, 11, 2536
Effect
CLW
LipidAccumulation-Related
Accumulation-Related mRNA
mRNA Expression
3.7.3.7.
Effect
of of
CLW
ononLipid
Expression in
inHF
HFDiet-fed
Diet-fedMice
Mice
In comparison with the control mice, HF diet mice showed increased mRNA expression levels
In comparison with the control mice, HF diet mice showed increased mRNA expression levels
related to fatty acid synthesis, such as SREBP-1c, FAS, and ACC by approximately 4.1, 3.3, and
related to fatty acid synthesis, such as SREBP-1c, FAS, and ACC by approximately 4.1, 3.3, and 2.6-fold,
2.6-fold, respectively (Figure 8A). Supplementation with CLW reversed the elevations of mRNA
respectively (Figure 8A). Supplementation with CLW reversed the elevations of mRNA factors involved
factors involved in fatty acid synthesis. Moreover, mice co-treated with CLW restored the
in fatty acid synthesis. Moreover, mice co-treated with CLW restored the β-oxidation factors such as
β-oxidation factors such as 5′ adenosine monophosphate-activated protein kinase (AMPK),
50 peroxisome
adenosine monophosphate-activated
protein kinase (AMPK), peroxisome proliferator-activated
proliferator-activated receptor-α (PPAR-α), and carnitine palmitoyltransferase 1
receptor-α
and carnitine
palmitoyltransferase
1 (CPT-1),
indicating
(CPT-1), (PPAR-α),
indicating CLW
as the accelerant
of lipid consumption
(Figure
8B). CLW as the accelerant of
lipid consumption (Figure 8B).
Figure 8. Effect of water extract (CLW) from Curcuma longa L. on lipid metabolism in high-fat
Figure mice.
8. Effect
water extract
(CLW)levels
from Curcuma
on lipid factors,
metabolism
in high-fat
diet-fed
(A)ofmRNA
expression
of fatty longa
acid L.
synthesis
including
sterol
diet-fed
mice.
(A)
mRNA
expression
levels
of
fatty
acid
synthesis
factors,
including
sterol
regulatory
regulatory element-binding protein-1c (SREBP-1c), fatty acid synthase (FAS), and acetyl-coenzyme
element-binding protein-1c (SREBP-1c), fatty acid synthase (FAS), and acetyl-coenzyme A
A carboxylase (ACC). (B) mRNA expression levels of β-oxidation factors, including 50 adenosine
carboxylase (ACC). (B) mRNA expression levels of β-oxidation factors, including 5′ adenosine
monophosphate-activated protein kinase (AMPK), and peroxisome proliferator-activated receptor
monophosphate-activated protein kinase (AMPK), and peroxisome proliferator-activated receptor
alpha (PPAR- α), carnitine palmitoyltransferase 1 (CPT-1). CON, normal control diet; HF, 60% high-fat
alpha (PPAR- α), carnitine palmitoyltransferase 1 (CPT-1). CON, normal control diet; HF, 60%
diet; C-LOW, 60% high-fat diet plus 300 mg/kg b.w./day of CLW; C-HIGH, 60% high-fat diet plus
high-fat diet; C-LOW, 60% high-fat diet plus 300 mg/kg b.w./day of CLW; C-HIGH, 60% high-fat diet
900 mg/kg b.w./day of CLW; SILY, 60% high-fat diet plus 50 mg/kg b.w./day of silymarin. Data are
plus 900 mg/kg b.w./day of CLW; SILY, 60% high-fat diet plus 50 mg/kg b.w./day of silymarin. Data
expressed as mean ± S.E. (n = 10), and different letters indicate significant differences (p < 0.05),
are expressed as mean ± S.E. (n = 10), and different letters indicate significant differences (p <0.05), as
as determined via Duncan’s multiple range test.
determined via Duncan’s multiple range test.
3.8. Effect of CLW on Activation of AMPK and ACC in HF Diet-fed Mice
3.8. Effect of CLW on Activation of AMPK and ACC in HF Diet-fed Mice
AMPK and ACC activities in the liver were measured by performing a western blot assay. Both
AMPK and ACC activities in the liver were measured by performing a western blot assay.
phosphorylated AMPK to AMPK ratio and the phosphorylated ACC to ACC ratio were significantly
Both phosphorylated AMPK to AMPK ratio and the phosphorylated ACC to ACC ratio were
decreased in HF diet-fed mice compared to the control mice (Figure 9). On the other hand, the CLW
significantly decreased in HF diet-fed mice compared to the control mice (Figure 9). On the other
administration
increased
these ratios
back to
the normal
range.
These
results
suggested
CLW
hand, the CLW
administration
increased
these
ratios back
to the
normal
range.
These that
results
might
decrease
lipid
accumulation
in
the
liver
via
enhancing
AMPK
activation
and
suppressing
suggested that CLW might decrease lipid accumulation in the liver via enhancing AMPK activation
ACC
andactivation.
suppressing ACC activation.
Nutrients 2019, 11, 2536
Nutrients 2019, 11, x FOR PEER REVIEW
10 of 13
10 of 13
Figure 9.
9. Effect
Effectofofwater
water
extract
(CLW)
from
Curcuma
on activation
of AMPK
andinACC
extract
(CLW)
from
Curcuma
longalonga
L. on L.
activation
of AMPK
and ACC
in
high-fat
diet-fed
mice.
50 adenosine
monophosphate-activated
protein
kinase
(AMPK)
high-fat
diet-fed
mice.
(A) (A)
5′ adenosine
monophosphate-activated
protein
kinase
(AMPK)
and and
phosphorylation
ofacetyl-coenzyme
acetyl-coenzymeAA
carboxylase
(ACC)
protein
levels
were
monitored
by Western
phosphorylation of
carboxylase
(ACC)
protein
levels
were
monitored
by Western
blot analysis.
analysis. (B)
density
of of
ACC
andand
AMPK.
CON,
normal
control
diet; diet;
HF, 60%
blot
(B)and
and(C)
(C)Protein
Protein
density
ACC
AMPK.
CON,
normal
control
HF, 60%
plus
300300
mg/kg
b.w./day
of CLW;
C-HIGH,
60%60%
high-fat
diet diet
high-fat diet;
diet;C-LOW,
C-LOW,60%
60%high-fat
high-fatdiet
diet
plus
mg/kg
b.w./day
of CLW;
C-HIGH,
high-fat
plus 900
diet
plus
50 50
mg/kg
b.w./day
of silymarin.
DataData
plus
900 mg/kg
mg/kgb.w./day
b.w./dayofofCLW;
CLW;SILY,
SILY,60%
60%high-fat
high-fat
diet
plus
mg/kg
b.w./day
of silymarin.
are expressed
expressed as
(n (n
= 10),
andand
different
letters
indicate
significant
differences
(p <0.05),
are
asmean
mean± ±S.E.
S.E.
= 10),
different
letters
indicate
significant
differences
(p <as0.05),
determined
via via
Duncan’s
multiple
range
test. test.
as
determined
Duncan’s
multiple
range
4.
4. Discussion
Discussion
NAFLD isis characterized
abundant
TGTG
accumulation
in the
In the present
study, we
NAFLD
characterizedbyby
abundant
accumulation
inliver.
the liver.
In the present
study,
showed
thatthat
CLW
possessed
the the
inhibitory
activity
of lipid
accumulation
in hepatocytes.
Elevated
we
showed
CLW
possessed
inhibitory
activity
of lipid
accumulation
in hepatocytes.
Elevated
FFAs from
from dietary
dietary fat
the
crucial
risk
factor
for for
fatty
liver.
Frequently,
not only
FFAs
fatintake
intakeare
areconsidered
considered
the
crucial
risk
factor
fatty
liver.
Frequently,
not only
patients
with
NAFLD
but
also
those
with
obesity
and
type
2
diabetes
show
higher
FFA
levels
in in
patients with NAFLD but also those with obesity and type 2 diabetes show higher FFA levels
serum
and
in
the
liver
[24].
Therefore,
an
FFA
mixture
to
induce
lipid
accumulation
in
HepG2
cells
serum and in the liver [24]. Therefore, an FFA mixture to induce lipid accumulation in HepG2 cells
was used.
used. In
oleic
acid
and
palmitic
acidacid
led led
to lipid
accumulation
in in
was
In previously
previouslypublished
publishedstudies,
studies,
oleic
acid
and
palmitic
to lipid
accumulation
hepatocytes [25,26].
[25,26]. Our
Ourstudy
studyconfirmed
confirmed
exposure
increased
lipid accumulation.
hepatocytes
thatthat
FFAFFA
exposure
increased
lipid accumulation.
However,
However, cell treatment with CLW and FFA decreased accumulation. These observations were
cell treatment with CLW and FFA decreased accumulation. These observations were further confirmed
further confirmed by an HF diet-fed animal experiment. The HF diet led to the development of fatty
by an HF diet-fed animal experiment. The HF diet led to the development of fatty liver, consistent
liver, consistent with that seen in a previous study [27], whereas CLW administration lowered the
with that seen in a previous study [27], whereas CLW administration lowered the lipid accumulation.
lipid accumulation. Alleviation of lipid accumulation in hepatocytes was more clearly shown by
Alleviation of lipid accumulation in hepatocytes was more clearly shown by H&E staining. According
H&E staining. According to the microscopic examination of the CLW-treated mouse liver cells,
to
the microscopic examination of the CLW-treated mouse liver cells, while being fed the HF diet,
while being fed the HF diet, the production of lipid droplets was significantly prevented.
the production
of lipidfactors
droplets
wasassignificantly
prevented.
Fatty acid uptake
such
CD36, FATP2,
and FATP5 actively function when excessive
Fatty
acid
uptake
factors
such
as
CD36,
FATP2,
andbut
FATP5
actively
when
excessive
fatty acids are present owing to high fat intake or obesity,
not in
normalfunction
conditions.
In our
study, fatty
acids
aregroup
present
owing toup-regulated
high fat intake
obesity, but
not in
normal
conditions.
ouragreement
study, the HF
the HF
remarkably
theorexpression
of FFA
uptake
factors,
which In
is in
group
remarkably
up-regulated
the
expression
of
FFA
uptake
factors,
which
is
in
agreement
with earlier studies [28]. These results provide evidence that the CLW administration disturbs FFAwith
earlier
[28].by
These
results provide
that the CLW administration disturbs FFA uptake
uptakestudies
in the liver
suppressing
fatty acidevidence
uptake factors.
in theDespite
liver bybeing
suppressing
fatty acid
uptakean
factors.
useful energy
sources,
excess of FFA evokes excessive β-oxidation and
eventually
produces
high ROS
levels sources,
[29]. Overproduction
of ROS
result
in the hepatocellular
Despite
being useful
energy
an excess of
FFA could
evokes
excessive
β-oxidation and
membrane produces
being attacked
of the
inactivation
of enzymatic
and
non-enzymatic
antioxidants
eventually
highbecause
ROS levels
[29].
Overproduction
of ROS
could
result in the
hepatocellular
and lipid peroxidation
in the
liver, followed
by liver injuries.
When fatty
uptake is suppressed,
membrane
being attacked
because
of the inactivation
of enzymatic
andacid
non-enzymatic
antioxidants
the overproduction
of ROS
could
befollowed
inhibited.by
Serum
and When
AST levels
indicators
of
and
lipid peroxidation
in the
liver,
liver ALT
injuries.
fattyare
aciduseful
uptake
is suppressed,
liver
damage
caused
by
oxidative
stress
[30].
Significantly
decreased
serum
enzyme
levels
were
the overproduction of ROS could be inhibited. Serum ALT and AST levels are useful indicators of liver
observedcaused
in mice
with Significantly
a CLW and HF
diet. Moreover,
MDA, levels
whichwere
is mainly
damage
byco-supplemented
oxidative stress [30].
decreased
serum enzyme
observed
known
as
a
marker
of
oxidative
stress
[31],
was
markedly
reduced
in
the
CLW
groups.
Furthermore,
in mice co-supplemented with a CLW and HF diet. Moreover, MDA, which is mainly known as a
the CLW supplementation ameliorated the impaired antioxidative defense system in mice, as
marker
of oxidative stress [31], was markedly reduced in the CLW groups. Furthermore, the CLW
supplementation ameliorated the impaired antioxidative defense system in mice, as indicated by the
Nutrients 2019, 11, 2536
11 of 13
restoration of liver antioxidants such as CAT, SOD, GST, GPx, GR, and GSH. These results suggest that
CLW has a hepatoprotective potential against oxidative stress.
Oxidative stress is a common risk factor for lipid accumulation in hepatocytes. Due to oxidative
stress, alteration in several transcription factors related to TG metabolism causes NAFLD [32]. SREBP-1c
is a transcription factor that regulates the synthesis of fatty acids, cholesterol, and triglycerides.
It promotes the expressions of ACC and FAS and eventually leads to fatty acid synthesis. SREBP-1c and
AMPK are negatively correlated as AMPK activity controls SREBP-1c inhibition [33]. Several studies
have shown that FFA-induced HepG2 cells and mice fed an HF-diet suppressed AMPK activity [34,35].
In our study, AMPK expression was remarkably down-regulated by the HF-diet. In addition, as shown
by western blotting, the phosphorylation of the AMPK and AMPK ratio was decreased, indicating the
inactivation of AMPK. However, supplementation with CLW up-regulated AMPK expression and
down-regulated SREBP-1c, FAS, and ACC expressions.
Expression levels of CPT-1 and PPAR-α, well-known regulators of fatty acid β-oxidation, were
significantly increased in the CLW groups. CPT-1 is inhibited by malonyl-CoA, which is produced by
ACC [36]. PPAR-α, the main factor for regulating β-oxidation, can potentially prevent NAFLD [37].
Up-regulations of CPT-1 and PPAR-α by supplementation with CLW suggest that CLW contributes to
decreasing β-oxidation, resulting in the inhibition of lipid accumulation in hepatocytes.
Silymarin, an approved agent for the treatment of liver damage, was used as a positive control in
our study to verify the potential of CLW to produce a comparable hepatoprotective effect. It prevents
oxidative stress as well as lipid accumulation [11,38]. In our study, supplementation with CLW showed
similar potential for the prevention of NAFLD to silymarin.
NAFLD is one of the most common liver diseases. NAFLD is associated with obesity, diabetes,
and dyslipidemia. In general, lipid metabolism and accumulation in hepatocytes leads to hepatic
steatosis, the first step of NAFLD. NAFLD can progress to NASH, advanced hepatic fibrosis,
and cirrhosis [39,40]. The present study dealt with the steatosis stage of NAFLD in an animal
model. We confirmed the potential of CLW to prevent the steatosis. Further study with human subjects
is necessary for safe administration with potent efficacy.
5. Conclusions
In conclusion, our study is the first to report that treatment with water extract of C. longa is
effective in the prevention of NAFLD. It is presumed that the mechanism of hepatoprotection by CLW
supplementation against lipid accumulation and oxidative stress might be due to the modulation of
fatty acid uptake. The knowledge gained from the present study could aid in the development of
effective therapeutics to protect from the formation of fatty liver.
Author Contributions: J.M. performed the experiments and wrote the manuscript. S.K. performed the acquisition
of data and statistical analysis. H-G.Y., K-C.C., J.L. performed analysis and interpretation of the data. Y.Y., O-K.K.,
Y-H.L. contributed to the conception of research and important revision. W.J. and J.P. equally contributed to the
design and supervision of the research. All authors read and approved the final manuscript.
Funding: This research was supported by the Basic Science Research Program through the National Research
Foundation of Korea (NRF) funded by the Ministry of Education (2017R1D1A3B03035177).
Conflicts of Interest: The authors declare no conflict of interest.
References
1.
2.
3.
Purohit, V.; Russo, D.; Coates, P.M. Role of fatty liver, dietary fatty acid supplements, and obesity in the
progression of alcoholic liver disease: Introduction and summary of the symposium. Alcohol 2004, 34, 3–8.
[CrossRef] [PubMed]
Yeh, M.M.; Brunt, E.M. Pathological features of fatty liver disease. Gastroenterology 2014, 147, 754–764.
[CrossRef] [PubMed]
Brunt, E.M. Pathology of fatty liver disease. Mod. Pathol. 2007, 20, 40–48. [CrossRef] [PubMed]
Nutrients 2019, 11, 2536
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
12 of 13
Cohen, J.C.; Horton, J.D.; Hobbs, H.H. Human fatty liver disease: Old questions and new insights. Science
2011, 332, 1519–1523. [CrossRef] [PubMed]
Yasui, K.; Hashimoto, E.; Komorizono, Y.; Koike, K.; Arii, S.; Imai, Y.; Shima, T.; Kanbara, Y.; Saibara, T.;
Mori, T.; et al. Characteristics of patients with nonalcoholic steatohepatitis who develop hepatocellular
carcinoma. Clin. Gastroenterol. Hepatol. 2011, 9, 428–433. [CrossRef]
Rawla, P.; Sunkara, T.; Muralidharan, P.; Raj, J.P. Update in global trends and aetiology of hepatocellular
carcinoma. Contemp. Oncol. 2018, 22, 141–150. [CrossRef]
Koonen, D.P.Y.; Jacobs, R.L.; Febbraio, M.; Young, M.E.; Soltys, C.L.M.; Ong, H.; Vance, D.E.; Dyck, J.R.B.
Increased hepatic CD36 expression contributes to dyslipidemia associated with diet-induced obesity. Diabetes
2007, 56, 2863–2871. [CrossRef]
Ouwens, D.M.; Diamant, M.; Fodor, M.; Habets, D.D.J.; Pelsers, M.M.A.L.; El Hasnaoui, M.; Dang, Z.C.;
van den Brom, C.E.; Vlasblom, R.; Rietdijk, A.; et al. Cardiac contractile dysfunction in insulin-resistant rats
fed a high-fat diet is associated with elevated CD36-mediated fatty acid uptake and esterification. Diabetologia
2007, 50, 1938–1948. [CrossRef]
Vallve, J.C.; Uliaque, K.; Girona, J.; Cabre, A.; Ribalta, J.; Heras, M.; Masana, L. Unsaturated fatty acids and
their oxidation products stimulate CD36 gene expression in human macrophages. Atherosclerosis 2002, 164,
45–56. [CrossRef]
Zhong, S.; Zhao, L.; Wang, Y.; Zhang, C.; Liu, J.; Wang, P.; Zhou, W.; Yang, P.; Varghese, Z.; Moorhead, J.F.;
et al. Cluster of differentiation 36 deficiency aggravates macrophage infiltration and hepatic inflammation
by upregulating monocyte chemotactic protein-1 expression of hepatocytes through histone deacetylase
2-dependent pathway. Antioxid. Redox Signal. 2017, 27, 201–214. [CrossRef]
You, Y.; Min, S.; Lee, Y.-H.; Hwang, K.; Jun, W. Hepatoprotective effect of 10% ethanolic extract from
Curdrania tricuspidata leaves against ethanol-induced oxidative stress through suppression of CYP2E1. Food
Chem. Toxicol. 2017, 108, 298–304. [CrossRef] [PubMed]
Choi, Y.-J.; Shin, H.-S.; Choi, H.S.; Park, J.-W.; Jo, I.; Oh, E.-S.; Lee, K.-Y.; Lee, B.-H.; Johnson, R.J.; Kang, D.-H.
Uric acid induces fat accumulation via generation of endoplasmic reticulum stress and SREBP-1c activation
in hepatocytes. Lab. Investig. 2014, 94, 1114–1125. [CrossRef]
Vijayakumar, R.S.; Surya, D.; Nalini, N. Antioxidant efficacy of black pepper (Piper nigrum L.) and piperine
in rats with high fat diet induced oxidative stress. Redox Rep. 2004, 9, 105–110. [CrossRef] [PubMed]
Song, F.L.; Gan, R.Y.; Zhang, Y.; Xiao, Q.; Kuang, L.; Li, H.-B. Total phenolic contents and antioxidant
capacities of selected chinese medicinal plants. Int. J. Mol. Sci. 2010, 11, 2362–2372. [CrossRef] [PubMed]
Lee, M.; Kim, Y.; Yoon, H.-G.; You, Y.; Park, J.; Lee, Y.-H.; Kim, S.; Hwang, K.; Lee, J.; Jun, W. Prevention
of ethanol-induced hepatotoxicity by fermented Curcuma longa L. in C57BL/6 Mice. Food Sci. Biotechnol.
2014, 23, 925–930. [CrossRef]
Folch, J.; Lees, M.; Stanley, G.H.S. A simple method for the isolation and purification of total lipides from
animal tissues. J. Biol. Chem. 1957, 226, 497–509.
Alam, M.N.; Bristi, N.J.; Rafiquzzaman, M. Review on in vivo and in vitro methods evaluation of antioxidant
activity. Saudi Pharm J. 2013, 21, 143–152. [CrossRef]
Garg, S.; Garg, A.; Shukla, A.; Dev, S.K.; Bishnoi, R.S.; Kumar, M. Review on antioxidant evaluation methods:
And models In-vitro in-vivo. Asian J. Pharm. Pharmacol. 2018, 4, 147–154. [CrossRef]
Molina, M.F.; Sanchez-Reus, I.; Iglesias, I.; Benedi, J. Quercetin, a flavonoid antioxidant, prevents and protects
against ethanol-induced oxidative stress in mouse liver. Biol. Pharm. Bull. 2003, 26, 1398–1402. [CrossRef]
Al Batran, R.; Al-Bayaty, F.; Al-Obaidi, M.M.J.; Abdualkader, A.M.; Hadi, H.A.; Ali, H.M.; Abdulla, M.A.
In vivo antioxidant and antiulcer activity of Parkia speciosa ethanolic leaf extract against ethanol-induced
gastric ulcer in rats. PLoS ONE 2013, 8, e64751. [CrossRef]
Carlberg, I.; Mannervik, B. Purification and characterization of the flavoenzyme glutathione reductase from
rat liver. J. Biol. Chem. 1975, 250, 5475–5480. [PubMed]
Akerboom, T.P.; Sies, H. Assay of glutathione, glutathione disulfide, and glutathione mixed disulfides in
biological samples. Methods Enzymol. 1981, 77, 373–382.
Draper, H.H.; Hadley, M. Malondialdehyde determination as index of lipid peroxidation. Methods Enzymol.
1990, 186, 421–431. [PubMed]
Garcia, M.C.; Amankwa-Sakyi, M.; Flynn, T.J. Cellular glutathione in fatty liver in vitro models. Toxicol. Vitr.
2011, 25, 1501–1506. [CrossRef] [PubMed]
Nutrients 2019, 11, 2536
25.
26.
27.
28.
29.
30.
31.
32.
33.
34.
35.
36.
37.
38.
39.
40.
13 of 13
Dai, L.; Xu, S.; Choi, S.-K.; Ha, C.-M.; Thoudam, T.; Cha, S.-K.; Wiederkehr, A.; Wollheim, C.B.; Lee, I.-K.;
Park, K.-S. Oxidative stress and calcium dysregulation by palmitate in type 2 diabetes. Exp. Mol. Med.
2017, 49, 291.
Gomez-Lechon, M.J.; Donato, M.T.; Martinez-Romero, A.; Jimenez, N.; Castell, J.V.; O’Connor, J.E. A human
hepatocellular in vitro model to investigate steatosis. Chem. Biol. Interact. 2007, 165, 106–116. [CrossRef]
[PubMed]
Matsuzawa-Nagata, N.; Takamura, T.; Ando, H.; Nakamura, S.; Kurita, S.; Misu, H.; Ota, T.; Yokoyama, M.;
Honda, M.; Miyamoto, K.; et al. Increased oxidative stress precedes the onset of high-fat diet-induced insulin
resistance and obesity. Metabolism 2008, 57, 1071–1077. [CrossRef]
Clugston, R.D.; Yuen, J.J.; Hu, Y.; Abumrad, N.A.; Berk, P.D.; Goldberg, I.J.; Blaner, W.S.; Huang, L.-S.
CD36-deficient mice are resistant to alcohol- and high-carbohydrate-induced hepatic steatosis. J. Lipid Res.
2013, 55, 239–246. [CrossRef]
Tanikawa, K.; Torimura, T. Studies on oxidative stress in liver diseases: Important future trends in liver
research. Med. Mol. Morphol. 2006, 39, 22–27. [CrossRef]
Kim, Y.; You, Y.; Yoon, H.G.; Lee, Y.H.; Kim, K.; Lee, J.; Kim, M.S.; Kim, J.C.; Jun, W. Hepatoprotective effects
of fermented Curcuma longa L. on carbon tetrachloride-induced oxidative stress in rats. Food Chem. 2014, 15,
148–153. [CrossRef]
Ayala, A.; Munoz, M.F.; Arguelles, S. Lipid Peroxidation: Production, Metabolism, and Signaling Mechanisms
of Malondialdehyde and 4-Hydroxy-2-Nonenal. Oxid. Med. Cell. Longev. 2014, 2014, 360438. [CrossRef]
[PubMed]
Hassan, W.; Rongyin, G.; Daoud, A.; Ding, L.; Wang, L.; Liu, J.; Shang, J. Reduced Oxidative Stress Contributes
to the Lipid Lowering Effects of Isoquercitrin in Free Fatty Acids Induced Hepatocytes. Oxid. Med. Cell.
Longev. 2014, 2014, 313602. [CrossRef] [PubMed]
Song, C.Y.; Shi, J.; Zeng, X.; Zhang, Y.; Xie, W.F.; Chen, Y.X. Sophocarpine alleviates hepatocyte steatosis
through activating AMPK signaling pathway. Toxicol. In Vitro 2013, 27, 1065–1071. [CrossRef] [PubMed]
Lee, M.R.; Park, K.I.; Ma, J.Y. Leonurus japonicus houtt attenuates nonalcoholic fatty liver disease in free
fatty acid-induced HepG2 cells and mice fed a high-fat diet. Nutrients 2018, 10, 20. [CrossRef]
Lee, J.-H.; Lee, J.-J.; Cho, W.-K.; Yim, N.-H.; Kim, H.-K.; Yun, B.; Ma, J.Y. KBH-1, an herbal composition,
improves hepatic steatosis and leptin resistance in high-fat diet-induced obese rats. BMC Complement.
Altern. Med. 2016, 16, 1–12.
Kim, M.K.; Kim, S.H.; Yu, H.S.; Park, H.G.; Kang, U.G.; Ahn, Y.M.; Kim, Y.S. The effect of clozapine on
the AMPK-ACC-CPT1 pathway in the rat frontal cortex. Int. J. Neuropsychopharmacol. 2012, 15, 907–917.
[CrossRef]
Tailleux, A.; Wouters, K.; Staels, B. Roles of PPARs in NAFLD: Potential therapeutic targets. BBA Mol. Cell
Biol. Lipids 2012, 1821, 809–818. [CrossRef]
Cacciapuoti, F.; Scognamiglio, A.; Palumbo, R.; Forte, R.; Cacciapuoti, F. Silymarin in non alcoholic fatty liver
disease. World J. Hepatol. 2013, 5, 109–113. [CrossRef]
Kim, M.H.; Seong, J.B.; Huh, J.W.; Bae, Y.C.; Lee, H.S.; Lee, D.S. Peroxiredoxin 5 ameliorates obesity-induced
non-alcoholic fatty liver disease through the regulation of oxidative stress and AMP-activated protein kinase
signaling. Redox. Biol. 2020, 3, 101315. [CrossRef]
Saitta, C.; Pollicino, T.; Raimondo, G. Obesity and liver cancer. Ann. Hepatol. 2019, in press. [CrossRef]
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