JOURNAL OF MEDICINAL FOOD

J Med Food 22 (4) 2019, 1–10

# Mary Ann Liebert, Inc., and Korean Society of Food Science and Nutrition
DOI: 10.1089/jmf.2018.4248

Antihyperlipidemic Activity of Ligularia fischeri Extract in Mice Fed

a High-Carbohydrate Diet
Ju-Hyoung Park,1 Eun-Kyung Ahn,2 Jin-Kyu Kim,2 and Joa Sub Oh1
1
Department of Pharmacy, Dankook University, Cheonan, Chungnam, Korea.
2
Department of Bio-Center, Gyeonggido Business and Science Accelerator, Suwon, Gyeonggi, Korea.

ABSTRACT Ligularia fischeri, indigenous to eastern Asia, has been used as a traditional herbal medicine. Ligularia fischeri
reportedly possesses a number of biological activities such as antimutagenic, antioxidant, antigenotoxic, and anti-inflammation.
This study demonstrated the effects of ethanol extracts of Ligularia fischeri (ELF) on a high-carbohydrate diet (HCD)-induced
hyperlipidemia in C57BL/6 mice. The mice were divided into six groups (n = 7/group) as follows: normal diet, HCD, or
Downloaded by East Carolina University from www.liebertpub.com at 02/25/19. For personal use only.

HCD+ELF (100, 200, 400, and 800 mg/kg/day), which were orally administered daily for 12 weeks. Various lipid parameters
and histological changes in liver and fat tissue were compared among the treatment and control groups. ELF remarkably
reduced body weight gain and attenuated hyperlipidemia by improving the plasma levels of total cholesterol, triglycerides,
high-density lipoprotein cholesterol, low-density lipoprotein cholesterol, atherogenic index, and cardiac risk factor. Moreover,
ELF decreased the HCD-induced hepatic accumulation of lipid droplets and adipocyte hypertrophy. These regulatory effects of
ELF appeared to be mediated through the phosphorylation of AMP-activated protein kinase, acetyl-CoA carboxylase, sterol
regulatory element-binding protein-1c, and expression of fatty acid synthase. Taken together, these findings indicate a func-
tional role for ELF in the regulation of HCD-induced obesity and hyperlipidemia.

KEYWORDS:  AMPK  hyperlipidemia  Ligularia fischeri  lipid metabolism  liver

INTRODUCTION and accumulation, such as acetyl-CoA carboxylase (ACC), a

well-characterized target of AMPK and fatty acid synthase

O besity is one of the major global health problems and

is associated with chronic metabolic syndromes, in-
cluding hyperlipidemia and nonalcoholic fatty liver dis-
ease.1,2 These conditions are mainly caused by obesity,
which is due to changes in environmental factors, dietary
habits, and lifestyle.3 Hyperlipidemia is characterized by an
increase in plasma lipid concentrations, such as cholesterol
and triglycerides (TGs).4,5 This is known to be an important
risk factor, causing atherosclerosis and cardiovascular dis-
eases, which in turn increase the risk of stroke and heart and
vascular diseases.6–8
AMP-activated protein kinase (AMPK) is a key sensor to
regulate cellular energy metabolism, including hepatic lipid
metabolism, by activating fatty acid oxidation pathways and
by inhibiting fatty acid and cholesterol synthesis path-
ways.9–11 AMPK is activated by excess energy intake, and
its increased activation leads to the suppression of sterol
regulatory element-binding protein-1c (SREBP-1c), a criti-
cal transcription factor in lipid synthesis. SREBP-1c regu-
lates the expression of proteins involved in lipid synthesis
Manuscript received 11 May 2018. Revision accepted 13 January 2019.
Address correspondence to: Joa Sub Oh, PhD, Department of Pharmacy, Dankook
University, Dandae-ro 119, Dongnam, Cheonan 31116, Chungnam, Korea, E-mail:-
jsoh@dankook.ac.kr
(FAS), a multienzyme that catalyzes the synthesis of pal-
mitate from acetyl-CoA and malonyl-CoA.12–15
Previous studies have indicated that many chemical
agents may reduce obesity and hyperlipidemia but also
cause serious side effects. Since herbal medicines are
usually safe and do not cause side effects, there has been
much interest in natural products as therapeutic agents.
Ligularia fischeri (L. fischer) is widely grown in eastern
Asia, including Korea, China, and Japan, and is widely
distributed in damp shady areas. L. fischeri has long been
used as a traditional herbal medicine.16–18 The leaves and
roots of L. fischeri are pharmacologically effective for
rheumatoid arthritis, contusion, scarlet fever, lower back
pain, inflammation, and hepatic diseases. Previous studies
have reported several biological activities of L. fischeri,
including anticancer, antimutagenic, antigenotoxic, anti-
oxidant, and anti-inflammation.19–23 Quercetin has been
found in L. fischeri extracts and is reported to show anti-
obesity effects.24 However, the in vivo antihyperlipidemic
and antiobesity effect and the related molecular mecha-
nisms of L. fischeri remain unknown.
This study evaluated the effects of an ethanol extract of
L. fischeri (ELF) on biochemical parameters and liver dys-
function in a high-carbohydrate diet (HCD)-induced hy-
perlipidemia C57BL/6 mouse model.

1
 2 PARK ET AL.

MATERIALS AND METHODS
Plant material
Leaves of L. fischeri were collected at Inje-gun,
Gangwon-do, Republic of Korea, in May 2014 and were
identified by Professor J.S.O. The voucher specimen was
deposited in Bio-Center, Gyeonggido Business and Science
Accelerator, Suwon, Republic of Korea.
Preparation of ELF
To prepare ELF, the leaves of L. fischeri were washed with
water, dried in air, and then pulverized. The dried LF at room
temperature was extracted in 10-fold volume (100 g/L) of
ethanol, three times with 50% aqueous ethanol for 48 h. The
extract was subsequently paper filtered, concentrated by
vacuum evaporation under reduced pressure, and centri-
fuged in a vacuum using a rotary evaporator. The extract
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Bohus, Sweden). The mobile phase consisted of water–
trifluoroacetic acid (99.9:0.1; v/v) (solvent A) and acetonitrile
(solvent B). The elution was completed by the following
gradient: initial 90:10 (A:B v/v); 20 min 90:10 (A:B v/v);
30 min 82:18 (A:B v/v); 70 min 82:18 (A:B v/v); and 80 min
0:100 (A:B v/v). The mobile-phase flow rate was 1.0 mL/min.
The volume of injection was 10 lL and the temperature of
column was set at 30C. The Chemstation for LC 3D systems
software (Agilent Technologies) controlled all operations
such as data analysis and acquisition. In Figure 1, HPLC
spectrum data of the 50% ethanol ELF are shown.
Animals
Male C57BL/6 mice, 5 weeks of age, were purchased
from Orient, Inc. (Seoul, Republic of Korea). The mice were
raised in polycarbonate cages in a specific pathogen-free

was completely freeze-dried to obtain a powder.
Apparatus and chromatographic conditions
High-performance liquid chromatography (HPLC) anal-
ysis using an Agilent 1100 series (Agilent Technologies,
Santa Clara, CA, USA) system, including a G1311A qua-
ternary pump, a G1316A thermostat-controlled column
compartment, and a G1315B diode array detector (DAD),
was performed. Separation was accomplished using a Kro-
masil 100-5-C18 column (5 lm, 250 · 4.6 mm id; AkzoNobel,
environment and acclimated to standard conditions of
20C – 2C, a 12-h light/12-h dark cycle, and relative hu-
midity between 35% and 55%. The mice were then divided
into six groups (n = 7/group) as follows: normal diet (ND),
HCD, HCD+ELF (100, 200, 400, and 800 mg/kg/day). ELF,
dissolved in phosphate-buffered saline (PBS), was admin-
istered orally daily for 12 weeks. At the same time, the HCD
group was administered PBS without ELF. During the
treatment period, the mice had free access to experimental
food and tap water. Body weights and food intake were
measured twice a week using a top-loading balance. The use

FIG. 1. A HPLC-DAD chromatogram (254 nm) of a 50% ethanol ELF is shown. Compounds are identified in the figure by number: 1—
neochlorogenic acid, 2—chlorogenic acid, 3—caffeic acid, 4—hyperoside, 5—fukinolic acid, 6—3,4-dicaffeoyl quinic acid, 7—3,5-dicaffeoyl
quinic acid, 8—methyl 3,5-dicaffeoyl quinate, 9—4,5-dicaffeoyl quinic acid, 10—20 -acetyl hyperoside, 11—caffeic acid methyl ester, 12—ethyl
caffeate, 13—quercetin. ELF, extracts of Ligularia fischeri; HPLC-DAD, high-performance liquid chromatography diode array detector. Color
images are available online.
 ANTIHYPERLIPIDEMIC EFFECT OF LIGULARIA FISCHERI 3

of animals in this study was approved by the Institutional
Animal Care and Ethical Use Committee of Bio-Center,
Gyeonggido Business, and Science Accelerator (Approval
No. 2015-05-0003) and was in compliance with the Guide
for the Care and Use of Laboratory Animals.25
Biochemical analysis of blood
At the end of the experiment, the mice in each group were
anesthetized after fasting with a Zoletil–Rompun mixture
to examine physiological changes. Blood from the abdom-
inal vena cava was collected and immediately centrifuged
(Micro 17TR, Hanil Science, Republic of Korea) at 890 g for
15 min at 4C to separate plasma. The plasma was stored at
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-80C until analysis. Plasma levels of total cholesterol (TC),
TGs, high-density lipoprotein (HDL) cholesterol, and low-
density lipoprotein (LDL) cholesterol were measured using
a Hitachi 7020 automatic analyzer (Tokyo, Japan). The
atherogenic index (AI) and cardiac risk factor (CRF) values
were calculated. AI was calculated as (TC–HDL)/HDL and
CRF was calculated as TC/HDL.
Histological analysis of liver and adipose tissue
The liver and white adipose tissue (WAT) were collected
and weighed. These tissues were fixed in 10% neutral buff-
ered formalin and paraffin embedded. The paraffin-embedded
section was cut into 5 lm sections. Each section was stained

FIG. 2. Effect of ELF on body weight and food intake in HCD-fed C57BL/6 mice for 12 weeks. (A, B) During the treatment period, the mice
had free access to ND and HCD. HCD-fed mice were daily treated with PBS or ELF (100, 200, 400, and 800 mg/kg) by oral administration for 12
weeks. The body weight and food intake were measured two times a week. (C–E) At the end of the experiment, WATs were collected and
immediately weighed. (F) To analyze adipocyte size and the quantified area, WATs were fixed in 10% neutral buffered formalin and embedded in
paraffin wax. The paraffin blocks were cut into 5-lm sections. Each section was stained with H&E and observed under light microscopy at a
200 · magnification. Adipocyte area quantification was performed using ImageJ software. Values are expressed as mean – SD; ##P < .01 compared
with the ND group; *P < .05 and **P < .01 compared with the HCD group. HCD, high-carbohydrate diet; H&E, hematoxylin and eosin; ND,
normal diet; PBS, phosphate-buffered saline; SD, standard deviation; WAT, white adipose tissue. Color images are available online.
 4 PARK ET AL.
Downloaded by East Carolina University from www.liebertpub.com at 02/25/19. For personal use only.
FIG. 2.
with hematoxylin and eosin and observed under light micros-
copy at a 200 · magnification. Adipocyte area was quantified
using ImageJ software (US National Institutes of Health, USA).
Hepatic TC and TG content measurement
Liver tissue was homogenized and lysed in tissue lysis
buffer. The lysate was centrifuged at 1000 g for 5 min at
4C. Lipid was extracted by liver and TC and TG contents
were measured.
Western blot analysis
Liver tissue was homogenized with liquid nitrogen using
a pestle and mortar and lysed in lysis buffer (PROPREP
(Continued)
protein extraction solution; iNtRoN Biotechnology, Seoul,
Republic of Korea). The lysate was centrifuged at 16,600 g
for 15 min at 4C, and the total protein concentration in the
supernatant was measured by the Bradford method. These
protein samples (40 lg) were mixed with a loading dye,
electrophoresed in 8% sodium dodecyl sulfate/polyacrylamide
gel and transferred onto nitrocellulose membrane (Whatman,
Dassel, Germany). The membrane in Tris-buffered saline
with 0.1% Tween 20 (TBST) solution was blocked for 1 h at
room temperature and then incubated with primary anti-
bodies against phospho-AMPK Thr-172 (p-AMPK), AMPK,
phospho-SREBP-1c Ser-372 (p-SREBP-1c), phospho-ACC
Ser-79 (p-ACC), FAS (Cell Signaling Technology, Beverly,
MA, USA), and b-actin (Santa Cruz Biotechnology, Inc.,
 ANTIHYPERLIPIDEMIC EFFECT OF LIGULARIA FISCHERI 5
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FIG. 3. Effect of ELF on plasma lipid levels, AI, and CRF in HCD-fed C57BL/6 mice. At the end of the experiment, blood was collected from
the abdominal vena cava and immediately centrifuged at 890 g for 15 min at 4C to separate plasma. (A–D) The amounts of TC, TGs, HDL, and
LDL in plasma were measured using an analyzer. (E, F) AI and CRF values were also calculated. AI = (TC-HDL)/HDL; CRF = TC/HDL. Values
are expressed as mean – SD; ##P < .01 compared with the ND group; *P < .05 and **P < .01 compared with the HCD group. AI, atherogenic index;
CRF, cardiac risk factor; HDL, high-density lipoprotein; LDL, low-density lipoprotein; TC, total cholesterol; TGs, triglycerides.

Santa Cruz, CA, USA), which were used at a 1:1000 dilution,
with gentle shaking at 4C overnight. Each membrane was
washed with TBST and incubated with horseradish
peroxidase-conjugated secondary antibodies, anti-rabbit IgG
(Santa Cruz Biotechnology, Inc.) at 1:2000 dilutions, with
gentle shaking at room temperature for 1 h. The band of
protein was visualized with an enhanced chemiluminescence
reagent and a ChemiDoc XRS system (Bio-Rad, Richmond,
CA, USA). b-actin as a loading control was used.
Statistical analysis
Statistical differences were determined by one-way anal-
ysis of variance, followed by Student’s t-test using Microsoft
 6 PARK ET AL.
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FIG. 4. Effect of ELF on hepatic function in HCD-fed C57BL/6 mice. (A) At the end of the experiment, liver was collected. To analyze lipid
accumulation, the liver was fixed in 10% neutral buffered formalin and embedded in paraffin wax. The paraffin blocks were cut into 5-lm
sections. Each section was stained with H&E and observed under light microscopy at a 200 · magnification. (B, C) Liver was homogenized and
lysed in tissue lysis buffer. The lysates were centrifuged and lipids were extracted. TC and TG contents in the liver were measured by enzymatic
colorimetric assays according to the manufacturer’s instructions. Values are expressed as mean – SD; ##P < .01 compared with the ND group;
*P < .05 with the HCD group. Color images are available online.

Excel 2007. All data are expressed as mean – standard devi-
ation. P-values <.01 and <.05 were considered statistically
significant.
RESULTS
Effects of ELF on biological parameters and function
Dried leaves of L. fischeri were extracted with 50% EtOH
and then analyzed using HPLC-DAD chromatogram
(254 nm). As presented in Figure 1, ELF contained various
compounds such as neochlorogenic acid, chlorogenic acid,
caffeic acid, hyperoside, fukinolic acid, 3,4-dicaffeoyl qui-
nic acid, 3,5-dicaffeoyl quinic acid, methyl 3,5-dicaffeoyl
quinate, 4,5-dicaffeoyl quinic acid, 2-acetyl hyperoside,
caffeic acid methyl ester, ethyl caffeate, and quercetin.
C57BL/6 mice were orally administered ND, HCD, and
HCD+ELF (100, 200, 400, and 800 mg/kg/day) for 12
weeks. As shown in Figure 2A, the body weight of mice
after 12 weeks in the ELF treatment groups was significantly
less than the mice in the HCD group. However, there were
no remarkable changes in the daily amount of food intake
between the ND and HCD groups (Fig. 2B). To evaluate the
effect of ELF on adipose tissue, we measured the weight
of epididymal, peritoneal, and subcutaneous fat. Treatment
with ELF (800 mg/kg) significantly inhibited HCD-induced
increases in peritoneal and subcutaneous fat weight
(Fig. 2C–E). We also confirmed the antiobesity effect of
ELF on the adipocyte size and the quantified area of sub-
cutaneous fat, which were significantly suppressed by ELF
administration in a dose-dependent manner (Fig. 2F). These
findings directly indicated that ELF contributed to reduced
body weight and fat content gain in C57BL/6 mice induced
by the HCD.
Plasma levels of TC, TGs, LDL, and HDL were much
higher in the HCD group than in the ND group. ELF treat-
ment exerted significantly lower TC, TGs, HDL, and LDL
levels than those in the HCD group. Especially, treatment
with 400 and 800 mg/kg of ELF resulted in the greatest
reduction of all parameters after 12 weeks (Fig. 3A–D). As
expected, AI and CRF were observed to be higher in the
HCD group than in the ND group. ELF effectively sup-
pressed the levels of AI and CRF at concentrations above
200 mg/kg (Fig. 3E, F). These data indicated that ELF
 ANTIHYPERLIPIDEMIC EFFECT OF LIGULARIA FISCHERI
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effectively ameliorated HCD-induced hyperlipidemia
through modulation of these pathological changes.
Effect of ELF on hepatic steatosis
To investigate the effect of ELF on hepatic function, we
determined its histological effects on HCD-induced lipid
7
FIG. 5. Effect of ELF on hepatic
AMPK activation in HCD-fed
C57BL/6 mice. At the end of the ex-
periment, liver was collected. Liver
was homogenized and lysed in ice-cold
lysis buffer. The lysates were centri-
fuged at 16,600 g for 15 min at 4C
and the expression of total protein was
measured. (A) The activities of p-
AMPKa, (B) p-ACC, and (C) p-
SREBP-1c and FAS protein expression
determined by western blotting and
densitometry protocol. Values are ex-
pressed as mean – SD; **P < .01 com-
pared with the HCD group. AMPK,
AMP-activated protein kinase; FAS,
fatty acid synthase; p-ACC, phospho-
acetyl-CoA carboxylase; p-SREBP-1c,
phospho-sterol regulatory element-
binding protein-1c.
formation in the liver. Histological analysis showed nu-
merous fat globules in the liver tissue of the HCD group, but
ELF treatment dose dependently decreased fat globules in
the liver tissue (Fig. 4A).
Moreover, ELF treatment suppressed the levels of TC and
TGs in the liver tissue with similar patterns to those in the
plasma of HCD-fed mice (Fig. 4B, C). These data demonstrate
 8 PARK ET AL.
that the inhibition effect of ELF on the production of lipids
leads to morphological changes in the liver as well as to the
reduction of TC and TG levels.
Effect of ELF on hepatic AMPK activation
To find out whether AMPK might play a role in the effect
of ELF on hepatic steatosis, AMPK activity was conducted
by confirming the phosphorylation of AMPK Thr-172, ACC
Ser 79, and SREBP-1c Ser 372 in liver tissue of HCD-fed
mice. The HCD group showed reduced p-AMPK and p-
ACC in the liver tissue compared with those of the ND
group, but these activities were considerably restored by
treatment with 800 mg/kg of ELF (Fig. 5A, B). Hepatic
phosphorylation of SREBP-1c was also sharply decreased in
HCD-fed mice, but ELF treatment (200, 400, and 800 mg/kg)
significantly increased the levels. Moreover, we found that
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ELF treatment inhibited the protein expression of FAS, which
is partially related to the SREBP protein, in a dose-dependent
manner in liver tissue of HCD-fed mice (Fig. 5C). These
findings suggest that phosphorylation of AMPK, ACC, and
SREBP-1c and inhibition of FAS protein expression by ELF
improved hepatic steatosis in HCD-fed mice.
DISCUSSION
Obesity is determined as an excessive accumulation of fat
tissue due to overeating, and hyperlipidemia is an abnormally
increased level of blood lipids such as cholesterol, cholesterol
esters, phospholipids, and TGs.26–28 L. fischeri exhibits vari-
ous activities, for example, against angiogenesis-related dis-
orders and arthritis and anti-inflammatory effects.29–31 In this
study, we first investigated the constituents of ELF. As shown
in Figure 1, most of the phenolic constituents of ELF were
caffeoyl quinic acids (CQAs), coumaric acid ester, cinnamic
acid, and flavonoids. In our previous study, we reported
that the major phenolic compounds found in the leaves of
L. fischeri were CQAs.32 These compounds have shown
antiobesity, antioxidant, anti-inflammatory, antihepatotoxic,
and other biological effects.33–35
Furthermore, we investigated whether obesity and hy-
perlipidemia were improved by ELF in HCD-induced mice.
ELF treatment reduced body and WAT weights compared
with levels in the HCD group, and there were no remarkable
changes in daily food intake and toxicity (Supplementary
Fig. S1) among the groups (Fig. 2). Obesity and hyperlip-
idemia are among the most important risk factors for car-
diovascular diseases such as coronary artery disease and
progression of atherosclerotic lesions. Increases in lipid
levels contribute to the pathogenesis of cardiac disorders,
which are the major cause of mortality in advanced indus-
trial countries.36–38 The HCD group showed high levels of
TC, TGs, HDL, and LDL as well as AI and CRF values,
whereas treatment of hyperlipidemic mice with ELF sig-
nificantly decreased plasma TC, TG, LDL, and HDL levels,
along with decrease of AI and CRF values (Fig. 3). There
were no effects on insulin and glucose levels (Supplemen-
tary Fig. S2).
Since hyperlipidemia usually affects the liver,39 we also
determined the effects of ELF on liver function by evalu-
ating lipid metabolism, focusing on the phosphorylation of
AMPK, ACC, SREBP-1c, and the expression of FAS. We
confirmed increased lipid accumulation and considerable
TG depots in the liver of HCD-fed mice. Histological
analysis showed that ELF effectively inhibited the accu-
mulation of lipid droplets in liver tissue and reduced adi-
pocyte hypertrophy compared with those in the HCD group
(Fig. 4). It has been reported that p-AMPK activation pre-
vents the development of hepatic steatosis by regulat-
ing p-SREBP-1c, p-ACC, and FAS expression. SREBP-1c
phosphorylation is necessary for p-AMPK activation to in-
hibit the cleavage of SREBP-1c and SREBP-2, which are
isoforms of SREBP.40–42 Previous results demonstrate that
ADD1/SREBP-1c in the liver and adipose tissue manages
the genes related to FAS, and overexpression of SREBPs in
adipocyte cells encourages the expression of FAS.43,44 As
shown in Figure 5, inactivated p-AMPK in the HCD group
modulated lipogenesis to reduce phosphorylation of SREBP-
1c and ACC, and increases expression of FAS. However, after
ELF treatment, SREBP-1c phosphorylation was significantly
increased and FAS expression was decreased dose depen-
dently compared with the HCD group.
In conclusion, this study demonstrated for the first time
that ELF improves HCD-induced hyperlipidemia through
the p-AMPK-dependent upregulation of ACC and SREBP-
1c phosphorylation and downregulation of FAS protein
expression in HCD-fed mice. These results suggest that ELF
might be a potential therapy for the remedy and manage-
ment of hyperlipidemia.
ACKNOWLEDGMENT
This study was supported by the research fund of Dan-
kook University in 2015.
AUTHOR DISCLOSURE STATEMENT
J.H.P., E.K.A., J.K.K., and J.S.O. declare that no com-
peting financial interests exist.
SUPPLEMENTARY MATERIALS
Supplementary Figure S1
Supplementary Figure S2
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