Journal of Functional Foods 21 (2016) 249262

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Mulberry leaf polyphenol extract improves

obesity by inducing adipocyte apoptosis and
inhibiting preadipocyte differentiation and
hepatic lipogenesis

Yun-Ching Chang a,b,1, Mon-Yuan Yang a,1, Shu-Chun Chen a, Chau-Jong Wang a,b,*
a
Institute of Biochemistry, Microbiology and Immunology, Medical College, Chung Shan Medical University, Taichung, Taiwan
b
Department of Medical Research, Chung Shan Medical University Hospital, Taichung, Taiwan

A R T I C L E I N F O A B S T R A C T

Article history: This study investigated the effects of mulberry leaf extract (MLE) and mulberry leaf poly-
Received 5 June 2015 phenol extract (MLPE) on lipid accumulation in 3T3-L1 cells and obesity in mice fed a high-
Received in revised form 12 fat diet (HFD). MLPE was extracted from mulberry leaves using ethanol, and those polyphenolic
November 2015 compounds that can be analysed by HPLC. Both MLE and MLPE efficiently suppressed the
Accepted 12 November 2015 expression of SREBP-1c and PPAR- proteins and the target genes A-FABP and FAS, whereas
Available online both of these compounds increased phosphorylation of AMPK in vivo and in vitro. Treat-
ment of quercetin, caffeic acid, hydroxyflavin and hesperetin, the main ingredients of MLPE,
Keywords: also inhibited the differentiation of 3T3-L1 preadipocytes. In addition, orally administering
Morus alba L. leaf extract MLE significantly reduced body weight gain and lipid accumulation in the liver and serum/
Polyphenol hepatic triglyceride and total cholesterol levels compared with those in the HFD group.Therefore,
3T3-L1 adipocyte the mulberry leaf may be used as a dietary supplement in patients with certain diseases
High-fat diet with obesity involvement.
Lipid metabolism 2015 Elsevier Ltd. All rights reserved.
Anti-obesity effect

a major risk factor for metabolic syndrome, which is charac-

1. Introduction
The prevalence of obesity has rapidly increased in the past
several decades because of changes in lifestyle factors such
as diet (Kopelman, 2000). A high-fat diet (HFD) is considered
terized by obesity, hyperlipidaemia, hypertension, cardiovascular
disease, diabetes, renal disease, respiratory complications, os-
teoarthritis, nonalcoholic fatty liver disease (NAFLD), and certain
types of cancer (Harvey, Lashinger, & Hursting, 2011). Obesity
is caused by excessive lipid accumulation in the body, which

* Corresponding author. Institute of Biochemistry, Microbiology and Immunology, Chung Shan Medical University, No. 110, Sec. 1, Jianguo
N. Road, Taichung 402, Taiwan. Tel.: +886 4 24730022 ext 11670; fax: +886 4 23248167.
E-mail address: wcj@csmu.edu.tw (C.-J. Wang).
1
These authors contributed equally to this work and share first authorship.
Abbreviations: MLE, mulberry leaf extract; MLPE, mulberry leaf polyphenolic extract; HFD, high-fat diet; HPLC, high-performance liquid
chromatography; NAFLD, non-alcoholic fatty liver disease; FAS, fatty acid synthase; SREBPs, sterol regulatory element binding proteins;
PPAR-, peroxisome proliferator activated receptors-gamma; A-FABP, adipocyte-specific fatty acid binding protein; FAS, fatty acid syn-
thase; AMPK, adenosine monophosphate-activated protein kinase; ACC, acetyl-Coenzyme A carboxylase; HMGCR, 3-hydroxy-3-
methylglutaryl-Coenzyme A reductase; LDL-c, low-density lipoprotein cholesterol; TG, triglycerides; TC, total cholesterol; GLU, glucose;
BUN, blood urea nitrogen; CRE, creatinine; GOT, glutamic oxaloacetic transaminase; GPT, glutamic pyruvic transaminase
http://dx.doi.org/10.1016/j.jff.2015.11.033
1756-4646/ 2015 Elsevier Ltd. All rights reserved.
250 Journal of Functional Foods 21 (2016) 249262
results from increased adipose tissue mass and dysregulation
of lipid metabolism. Adipocytes are the major cellular com-
ponent of adipose tissue. Many studies have reported treating
obesity by reducing the differentiation of fibroblastic
preadipocytes to mature adipocytes (adipogenesis), inhibit-
ing lipogenesis, increasing lipolysis, and inducing the apoptosis
of adipocytes (Prins & ORahilly, 1997). Obesity has become a
major public health problem worldwide and is a crucial concern
in the field of preventive medicine.
Identifying the molecular basis for controlling adipogenesis
is a potential strategy for obesity prevention and treatment,
because adipocyte differentiation plays a key role in the growth
of adipose tissue mass (Wang et al., 2014). Adipocyte differen-
tiation is a multistep process that is regulated by a network of
transcription factors and adipogenesis-related genes. Repre-
sentatives of two transcription factor families, namely, the nuclear
hormone receptor peroxisome proliferator-activated receptors
(PPARs) and sterol regulatory element-binding proteins (SREBPs),
have been implicated in this process. During adipogenesis, PPAR
activates the expression of lipid-metabolizing enzymes, such as
adipocyte-specific fatty acid-binding protein (A-FABP; also des-
ignated aP2 or FABP4) and fatty acid synthetase (FAS), leading
to morphological changes and lipid accumulation in cells (Farmer,
2006). Two SREBP isoforms, SREBP-1c and -2, are expressed in
the liver. SREBP-1c preferentially regulates fatty acid synthesis
by activating the expression of genes such as acetyl-coenzyme
A carboxylase 1 (ACC1) and FAS. SREBP-2 regulates the tran-
scription of cholesterol-related genes, including 3-hydroxy-3-
methylglutaryl-coenzyme A reductase (HMGCR), which encodes
the rate-limiting enzyme of cholesterol biosynthesis. In addi-
tion, adenosine monophosphate-activated protein kinase (AMPK)
is also a potential molecular candidate involved in the control
of adipocyte differentiation (Lin, Ribar, & Means, 2011). AMPK
regulates the expression of SREBPs and their target genes (e.g.,
A-FABP and FAS) (Ceddia, 2013).
Mulberry (Morus alba L.; Moraceae family) is cultivated in
Asian countries (e.g., China, Korea, Japan, and Taiwan), and its
leaves contain various phytochemical constituents, such as fla-
vonoid and polyphenol compounds. These leaves possess
medicinal benefits and have antibacterial, antihypertensive,
antihypoglycaemic, and antiatherosclerotic effects and con-
sequently have been used as a remedy for several decades (Yang,
Tan, Chen, & Kang, 2014c). In Taiwan, mulberry leaf prepara-
tions are commonly used in commercial beverages (mulberry
tea) and health foods. Water extracts of mulberry leaves have
been shown to exhibit a variety of biological functions,
including antidiabetic, antiobesity, anti-inflammatory,
antioxidative, antiatherosclerotic, antimicrobial, antitumor, and
antihypertensive actions (Lee et al., 2011; Lim, Lee, Kim, Yang,
& Lim, 2013; Yang, Wang, & Li, 2014a; Zhang et al., 2014). Con-
sistently, our previous studies have indicated that mulberry
leaves have not only anticholesteremic effects (Chan et al., 2013)
but also cardiovascular and hepatoprotective properties (Chan
et al., 2010; Yang, Lee, Ou, Chang, & Wang, 2012). Furthermore,
recent scientific evidence has demonstrated that the dried
powder, water extract, and ethanol extract of mulberry leaves
show several entirely distinct, new functions such as antiplatelet
(D. S. Kim et al., 2014), antiosteoporotic (Sungkamanee,
Wattanathorn, Muchimapura, & Thukham-mee, 2014), antiaging
(Zheng et al., 2014), neuroprotective (Bauomy, 2014), and
anti-cancer stem cell (S. Park, Kim, & Kim, 2012) properties. Al-
together, increasing evidence suggests a beneficial role of
mulberry leaves in preventing obesity, although the underly-
ing mechanisms must be elucidated. Hence, in this study, we
investigated protein expression in the livers of hyperlipidaemic
mice (in vivo) and 3T3-L1 cells (in vitro) treated with mulberry
leaf extracts to elucidate the mechanisms underlying the lipid-
lowering effects of mulberry leaves.Two mulberry leaf extracts,
mulberry leaf extract (MLE) and mulberry leaf polyphenol extract
(MLPE), had multiple antiadipogenic effects in these model
systems. Adding quercetin, caffeic acid, hydroxyflavin and hes-
peretin, the main ingredients of MLPE, also inhibited adipocyte
differentiation and induced apoptosis in 3T3-L1 preadipocytes.
2. Materials and methods
2.1. Preparation of MLE and MLPE
Fresh mulberry leaves (200 g) were harvested and immedi-
ately dried at 50 C. The dried leaves were heated in 3000 mL
of deionized water. After filtration, we removed the residue.
The suspension was stored at 80 C overnight and then evapo-
rated with a freeze-dryer. The dried powder remaining was an
aqueous fraction of mulberry leaves (MLE). According to the
previous paper, ethanol is reported to be a better solvent to
extract phenolic compounds from mulberry leaves compared
with other solvents (Jeszka-Skowron et al., 2014; Kim, Chung,
Jung, Wee, & Kwon, 2013; Park, Lee, Lee, & Kim, 2013; Yang,
Park, & Lim, 2014b). For preparation of the polyphenol-rich
extract of mulberry leaves (MLPE), 100 g dried powder of mul-
berry leaves was merged in 300 mL of ethanol and heated at
50 C for 3 h. The extract was filtered and thereafter concen-
trated by evaporation under reduced pressure at room
temperature. The powder was then resuspended in 500 mL of
distilled water, followed by extraction with 180 mL of ethyl
acetate three times, redissolved in 250 mL of distilled water,
stored at 80 C overnight, and lyophilized. MLE and MLPE were
filtrated by 0.22 m filter before use in cell culture.
2.2. HPLC analysis
HPLC was performed with a Hitachi HPLC system (Hitachi,
Danbury, CT, USA) which consisted of a pump (L-6200A), an ul-
traviolet detector (L-4250) and the Hitachi D-7000 HPLC System
Manager program. A reported procedure was used for analysing
the phenolic acids, which contained column, Mightysil RP-18
GP 250 (Kanto, Tokyo, Japan); mobile phase solvent A, acetic acid/
water (2:98, v/v), and solvent B, 0.5% acetic acid in water/
acetonitrile (50:50, v/v). The flow rate was 1 mL/min. The
gradient for the separation was 100% solvent A at 0 min, 70%
solvent A and 30% solvent B at 5 min, 65% solvent A and 35%
solvent B at 50 min, 60% solvent A and 40% solvent B at 55 min,
0% solvent A and 100% solvent B at 60 min, followed by a 5 min
post-run with HPLC grade water. Phenolic acids were de-
tected at 260 nm.
2.3. Cell culture
Mouse embryo 3T3-L1 cells (BCRC 60159) were obtained from
the Bioresource Collection and Research Center (BCRC, Food
 Journal of Functional Foods 21 (2016) 249262
Industry Research and Development Institute, Hsinchu, Taiwan).
The culture medium included DMEM, 10% calf serum, 1.5 g/L
sodium bicarbonate, and 100 U/mL penicillin-streptomycin. The
cell culture condition was 37 C in humidified 5% CO2 incubator.
2.4. Adipocyte maturation assay
Mature adipocyte were seeded in a 6-well plate (3 106 cells)
and treated with indicated concentration of MLE, MLPE or major
polyphenolic constituents (quercetin, caffeic acid, hydroxyflavin
and hesperetin) for 14 days. After being washed twice with
phosphate buffered saline (PBS), the cells were fixed with 4%
formaldehyde for 30 min and then stained with 0.05 g/mL Oil
Red O or 1 g/mL Nile red for 30 min at room temperature. After
staining, the distribution and quantification of lipid in cells was
immediately analysed by flow cytometer (Becton Dickinson,
Mountain View, CA, USA). The red lipid droplets in Oil Red O
were visualized by visible light microscope. Lipid-bounded Nile
red fluorescence was detected using inverted fluorescence
microscope.
2.5. Annexin V-FITC/PI double staining analysis
Annexin V-FITC/propidium iodide (PI) double staining of the
cells was determined using an Annexin V-FITC kit (Vybrant
Apoptosis Assay, V-13242, Molecular Probes, Eugene, OR, USA).
To detect early apoptosis, late apoptosis, and necrosis induced
by MLE, MLPE or major polyphenolic constituents (quercetin,
caffeic acid, hydroxyflavin and hesperetin), mature 3T3-L1
adipocytes (1 106 cells/dish) were added to each well of a 6-cm
dish and treated for 72 h at 37 C in 1 mL culture medium con-
taining testing agents at suitable concentrations to give
indicated concentration of MLE, MLPE or major polyphenolic
constituents (quercetin, caffeic acid, hydroxyflavin and hes-
peretin). Approximately 1 105 cells were then stained for
10 min at room temperature with Annexin V-FITC and PI in a
Ca2+-enriched binding buffer (Annexin V-FITC kit) and analysed
by FACScan flow cytometry. Annexin V-FITC and PI emissions
were detected in the FL1 and FL2 channels of a FACScan flow
cytometry, using emission filters of 525 and 575 nm, respec-
tively. Approximately 1 104 counts were made for each sample.
The percentages of distribution of normal (Annexin V-FITC/PI),
early apoptotic (Annexin V-FITC+/PI), late apoptotic (Annexin
V-FITC+/PI+), and necrotic cells (Annexin V-FITC/PI+) were cal-
culated by CELL Quest software.
2.6. Western blot analysis
Equal amounts of protein samples (50 g) were subjected to
SDS-polyacrylamide gel electrophoresis and electrotransferred
to nitrocellulose membranes (Millipore, Bedford, MA, USA).
Membranes were blocked with 5% nonfat milk powder with
0.05% Tween 20 in PBS and then incubated with the first an-
tibody at 4 C overnight. Thereafter, membranes were washed
three times with 0.05% Tween 20 in PBS and incubated with
the anti-mouse secondary antibody conjugated to horserad-
ish peroxidase (GE Healthcare, Little Chalfont, Buckinghamshire,
UK). Bands were detected and revealed by enhanced chemi-
luminescence using ECL Western blotting detection reagents
and exposed ECL hyper film in FUJFILM Las-3000 (Tokyo, Japan).
251
Protein quantitation was determined by densitometry using
the FUJFILM MultiGauge V2.2 software.
2.7. Animals and diets
A total of 40 C57BL/6 male mice, aged 34 weeks and weigh-
ing 1920 g, were kept at a constant temperature of 24 C and
illuminated for 12 h daily (lights on from 6:00 a.m.to 6:00 p.m.).
After 1 week maintenance for adaptation to the environ-
ment, the mice were randomly grouped by body weight into
four groups; each group was fed a unique diet for 6 weeks and
weighed every 2 weeks. The 4 groups and their correspond-
ing meals were (1) control, normal meals; (2) HFD, normal meals
containing 2% cholesterol and 20% lard oil; (3) HFD + 0.5% NLE,
HFD supplemented with 0.5% NLE as described; (4) HFD + 1.5%
NLE, HFD supplemented with 1.5% NLE as described. After a
6-week application of different diets, whole blood and livers
were collected from mice that had been fasted for 1214 h and
then were sacrificed. The blood was collected by EDTA tubes
and centrifuged at 3000 rpm or 10 min at 4 C. The superna-
tant plasma was transferred into new tubes for determination
of triglycerides, total cholesterol, low-density lipoprotein-
cholesterol (LDL-C), high-density lipoprotein-cholesterol (HDL-
C), GOT, GPT, blood urea nitrogen (BUN) and plasma creatinine.
After the hamsters had been sacrificed, the livers were quickly
frozen in liquid nitrogen for the extraction of liver lipids or
freshly cut into pieces for fixation, embedding, and
haematoxylin and eosin (H&E) staining.
2.8. Extraction of liver lipids
Liver lipids were extracted as described previously (Folch, Lees,
& Sloane Stanley, 1957). Briefly, liver (1.25 g) was homog-
enized with chloroform/methanol (1:2, 3.75 mL), and then
chloroform (1.25 mL) and distilled water (1.25 mL) were added
to the homogenate and mixed well. After centrifugation (1500 g
for 10 min), the lower clear organic phase solution was trans-
ferred into a new glass tube and then lyophilized. The
lyophilized powder was dissolved in chloroform/methanol (1:2)
as the liver lipid extract and stored at 20 C for <3 days. The
lipid extracts were used for quantitation of liver triglycerides
and liver cholesterol.
2.9. Statistics
Data were analysed using an unpaired t test and represented
as means (standard deviation (SD)). Statistical differences were
evaluated using the unpaired t test and considered signifi-
cant at the level p < 0.05. All data collected were analysed using
an unpaired t test after one-way ANOVA testing showed a sig-
nificant difference among all the groups (p < 0.001).
3. Results
3.1. Identification of active components in MLPE by using
high performance liquid chromatography
A high performance liquid chromatography (HPLC) chromato-
gram showed four major peaks and six minor peaks. These
252 Journal of Functional Foods 21 (2016) 249262

Fig. 1 The HPLC chromatogram of MLPE. (A) HPLC chromatogram of fifteen kinds of standard polyphenols. Peaks: 1, gallic
acid (GA); 2, protocatechuic acid (PCA); 3, catechin (C); 4, gallocatechin gallate (GCG); 5, caffeic acid (CA); 6, epicatechin (EC);
7, p-coumaric acid (p-CA); 8, rutin (R); 9, ferulic acid (FA); 10, gossypin (G); 11, hesperetin (H); 12, resveratrol (RV); 13,
quercetin (Q); 14, naringenin (N); 15, hydroxyflavin (FlOH). (B) HPLC chromatogram of free polyphenols from MLPE.

peaks were identified by comparing the retention times of
various standards. The purification procedures have been
applied to the isolation of four major flavonoids from the
ethanolic extract of mulberry leaves: caffeic acid, hesperetin,
quercetin, and hydroxyflavin. The minor active components
were protocatechuic acid, gallocatechin gallate, epicatechin,
rutin, gossypin, and naringenin, as well as unknown compo-
nents without standards. Detailed chemical information on the
compounds is presented in Fig. 1. The contents of quercetin,
caffeic acid, hydroxyflavin and hesperetin in MLPE were esti-
mated to be 1.251 0.81, 1.076 0.69, 0.922 0.59, and
0.899 0.59 mg per 10 mg, respectively.
 Journal of Functional Foods 21 (2016) 249262 253

3.2. MLE reduced HFD-induced body weight and fat/body

weight ratio increase

To test whether MLE prevents HFD-induced body weight gain,

dynamic change in body weight was measured during a 6-week
feeding period. Body weight gain was determined by calculat-
ing the average change during HFD treatment. After 6 weeks,
all mice on a HFD were 40% heavier compared with control
animals (C) (Fig. 2A). By contrast, mice fed a HFD supple-
mented with 0.5% and 1.5% MLE were respectively 7% and 10%
lighter than mice fed only a HFD. In addition, the fat/body
weight ratio was markedly decreased in the HFD + MLE group
compared with the HFD group (Fig. 2B). Thus, MLE effectively
inhibited HFD-induced body weight gain and reduced adipose
tissue mass in mice. These results show that MLE might have
antiobesity effects.

3.3. MLE improved serum lipid profile

To examine the dyslipidaemia-preventing effect of MLE in HFD-

fed mice, serum lipid levels were analysed. Table 1 shows the
serum lipid profile in mice after 6 weeks of treatment with MLE.
The HFD group showed significantly elevated fasting serum
glucose (GLU), total cholesterol (TC), triglyceride (TG), low-
density lipoprotein cholesterol (LDL-c), and FFA concentrations
(P < 0.05) compared with the vehicle control group. By con-
trast, GLU, TC, TG, LDL-c, and FFA concentrations were
markedly reduced to 70.62 8.12 mg/dL, 81.37 8.11 mg/dL,
32.75 5.75 mg/dL, 43.25 4.50 mg/dL, and 5.57 0.92 nmol, re-
spectively, in the HFD + 1.5% MLE group compared with the HFD
group. To determine whether MLE treatment could mitigate
HFD-induced liver injury, the activities (U/L) of serum glu-
tamic oxaloacetic transaminase (GOT) and glutamic pyruvic
transaminase (GPT) were examined. Serum GOT and GPT con-
centrations were significantly higher in the HFD-fed group
(279.28 116.61 and 60.66 12.45, respectively) than in the
control group (16.22 3.07 and 10.33 1.12, respectively)
(P < 0.05). Administering 1.5% MLE significantly prevented HFD- Fig. 2 MLE attenuated body weight and total fat tissue in
induced elevation in serum GOT and GPT concentrations HFD-fed C57BL/6 mice. HFD group were maintained on a
(24.12 6.38 and 13.50 4.00, respectively) in all three groups high-fat diet containing 20% lard oil and 2% cholesterol for
of HFD-fed mice (P < 0.001 versus HFD group). No significant 6 weeks (n = 10). (A) Body weight change of C57BL/6 mice
difference was observed in the levels of serum BUN, CRE, and fed basal laboratory diet or high fat diet during the 6 weeks
ketone bodies among the four groups. These changes can be feeding period. (B) Total fat tissue content epididymal fat,
inhibited or reduced by MLE, suggesting that it has a perirenal fat, inguinal fat and subcutaneous fat tissue.
hypolipidaemic effect. These results indicate that MLE may be C, normal group; HFD, high fat diet without MLE powder;
useful in preventing HFD-induced dyslipidaemia. MLE-0.5%, C57BL/6 mice fed high fat diet with 0.5% MLE
powder; MLE-1.5%, C57BL/6 mice fed high fat diet with
3.4. Effects of MLE on the expression of proteins involved 1.5% MLE powder. Results were statistically analysed with
in hepatic lipid metabolism in HFD-induced obese mice ANOVA. #, P < 0.05 compared with the control group.
*, P < 0.05 compared with the HFD group.
Because obesity can trigger hepatic steatosis, which is asso-
ciated with dyslipidaemia, we measured lipid levels in the liver
to determine whether MLE suppresses lipid accumulation in mice. By contrast, large hepatic lipid droplets were diffusely
the liver. Hepatic TG and TC levels were significantly in- present in the livers of HFD mice. Fatty metamorphosis of the
creased in HFD-fed mice compared with those in normal control liver, which results from injury (most often obesity, alcohol-
group mice, whereas MLE treatment significantly reduced both ism, or diabetes mellitus) leading to lipid accumulation in the
TG and TC levels in the liver (Fig. 3A). In addition, Fig. 3B depicts cytoplasm of hepatocytes, was also observed in the livers of
microphotographs of haematoxylin and eosin staining of hepatic HFD mice. However, the HFD + 0.5% MLE group had signifi-
tissues from experimental mice. Normal hepatocytes were found cantly fewer vacuoles, and the HFD + 1.5% MLE group exhibited
in the sections obtained from the hepatic tissues of normal a normal hepatocellular morphology similar to that of the control
254 Journal of Functional Foods 21 (2016) 249262

Table 1 Biochemical characteristics of C57BL/6 mice after 6 weeks on experimental diets.

C HFD HFD + MLE 0.5% HFD + MLE 1.5%
GOT (U/L) 16.22 3.07 279.28 116.61a
28.55 12.94b
24.12 6.38b
GPT (U/L) 10.33 1.12 60.66 12.45 14.33 7.45b 13.50 4.00b
BUN (mg/dL) 30.98 7.39 29.70 2.04 29.94 3.41 21.63 5.00
CRE (mg/dL) 0.24 0.04 0.54 0.12 0.33 0.08 0.30 0.12
GLU (mg/dL) 70.00 8.5 155.22 29.23a 91.00 6.12b 70.62 8.12b
TCHO (mg/dL) 71.66 7.43 157.33 38.58a 101.11 9.75b 81.37 8.11b
TG (mg/dL) 29.00 5.61 125.77 34.95a 42.11 8.84b 32.75 5.75b
HDL-c (mg/dL) 16.67 2.35 38.88 6.03a 33.11 3.06b 32.00 3.35b
LDL-c (mg/dL) 45.89 4.04 79.33 12.70a 68.67 7.76b 43.25 4.50b
Ketone body (mmol/L) 0.51 0.37 0.77 0.27 0.77 0.29 0.68 0.18
Free fatty acid (nmol) 6.42 0.51 6.89 0.44 6.52 0.17 5.57 0.92
C, normal control group; HFD, mice were fed high fat diet-induced group; HFD + MLE 0.5%, mice were fed HFD with 0.5% MLE group; HFD + MLE
1.5%, mice were fed HFD with 1.5% MLE group. Each value is expressed as the mean SD (n = 10/group). Results were statistically analysed
with ANOVA. Means designated by the same letter within columns are significantly different according to Duncans multiple range test. a, P < 0.05
compared with the C group. b, P < 0.05 compared with the HFD group.

group. Both of these findings suggest that MLE effectively at-
tenuates lipid accumulation in the liver of HFD-fed mice.
To understand the molecular mechanisms underlying the pro-
tective role of MLE in the liver, we assessed changes in the
expression of proteins controlling hepatic lipogenesis and fatty
acid uptake in the HFD-induced model. Compared with the find-
ings of the control group, the expression of SREBP-1, FAS, ACC,
and A-FABP involved in lipid metabolism was higher in the HFD
group; however, a high MLE dose (1.5%) significantly reduced the
expression of SREBP-1 by approximately 28% and that of its target
proteins A-FABP, ACC, and FAS by 3460% (Fig. 3C). Similarly, the
expression of two proteins involved in cholesterol synthesis,
namely, SREBP-2 and HMGCR, was reduced by approximately
15% and 25%, respectively, in the liver of MLE-treated mice (Fig. 3C).
In addition, AMPK is a vital regulator of preadipocyte differen-
tiation and adipogenesis. In this study, administering 1.5% MLE
increased AMPK phosphorylation (pAMPK) and, thus, the acti-
vation of proteins such as HMGCR and ACC; however, it had no
effect on total protein expression (Fig. 3D). These results indi-
cate that MLE administration inhibited lipogenesis through the
regulation of FAS expression by suppressing the expression of
adipogenic transcription factors in the liver.
3.5. Effects of MLE and MLPE on adipogenesis and
adipocyte apoptosis
Cell viability was examined using the MTT assay to test whether
MLE and MLPE had cytotoxic effects in 3T3-L1 adipocytes. Com-
pared with controls, cell viability was not significantly affected
following treatment with MLE (1 and 2 mg/mL) or MLPE (0.25 and
0.5 mg/mL) for 14 days (data not shown). Therefore, for further
investigation, 3T3-L1 cells were treated with the mentioned four
concentrations for 14 days to determine the effects of MLE or
MLPE on the adipogenic differentiation of these cells. In the dif-
ferentiation of 3T3-L1 preadipocytes into adipocytes, preadipocytes
underwent morphological changes such as transformation from
a spindle-like shape to a spherical shape and accumulation of
lipids in the cytoplasm. Microscopic observation of Oil-Red O
staining revealed that the highest MLE and MLPE concentra-
tions (2 and 0.5 mg/mL) suppressed oil droplet accumulation and
reduced the size of the droplets (Fig. 4A, upper panel). Previous
studies have used Nile Red flow cytometry to assess adipocyte
numbers (Smyth & Wharton, 1992). Microscopic observation of
Nile Red staining revealed a reduction in the amount of lipid
droplets with increasing concentrations of MLE and MLPE (Fig. 4A,
middle panel). Nile Red staining and flow cytometry were used
to calculate the percentage of positively stained cells to iden-
tify the proportion of mature adipocytes (Fig. 4B and C). Fig. 4C
shows the quantification of flow cytometry from Fig. 4B. Treat-
ment with 1 and 2 mg/mL MLE reduced the number of 3T3-L1
adipocytes by 15.6% and 3.77%, respectively. In addition, treat-
ment with 0.25 and 0.5 mg/mL MLPE reduced the percentage of
adipocytes to 13.32% and 5.89%, respectively (Fig. 4C). Accord-
ing to the analysis, the different methods exhibited similar effects
on lipid accumulation.These results indicate that MLE and MLPE
may efficiently inhibit adipocyte differentiation and have an-
tiobesity effects in 3T3-L1 cells.
To examine the direct effect of MLE- or MLPE-induced apop-
tosis in 3T3-L1 adipocytes by using annexin V and propidium
iodide double staining assay, cells were treated with various
concentrations of MLE (1 and 2 mg/mL) or MLPE (0.25 and
0.5 mg/mL) for 14 days. Although no significant increase was
observed in the apoptosis of mature adipocytes, and only a
slight increase was observed at 1 mg/mL MLE and 0.25 mg/mL
MLPE, a significant proportion of mature adipocytes under-
went apoptosis at 2 mg/mL MLE and 0.5 mg/mL MLPE (highest
concentrations) (Fig. 4D). MLE at 2 mg/mL or MLPE at 0.5 mg/mL
have increased apoptosis by 6.13% and 7.25%, respectively, in
mature adipocytes relative to preadipocytes (P < 0.01) (Fig. 4E).
The results illustrate MLE- or MLPE-induced cell apoptosis. Thus,
adipocyte number decreases (adipocyte apoptosis) as a result
of decreased proliferation.
3.6. Effects of major polyphenolic constituents (quercetin,
caffeic acid, hydroxyflavin and hesperetin) on adipogenesis
and adipocyte apoptosis
The main polyphenols of MLPE are quercetin, caffeic acid,
hydroxyflavin and hesperetin. To determine whether these four
constituent treatments also affect cell differentiation and sur-
vival in 3T3-L1 preadipocytes, we performed a Nile red stain
and annexin V/PI staining assay. During 3T3-L1 preadipocyte
differentiation into adipocytes, 100 M quercetin, caffeic acid,
hydroxyflavin and hesperetin were treated, and lipid contents
 Journal of Functional Foods 21 (2016) 249262 255

Fig. 3 MLE suppressed lipogenic protein expression in the liver of HFD-fed C57BL/6 mice. The HFD group was maintained
on a high-fat diet containing 20% lard oil and 2% cholesterol for 6 weeks (n = 10). (A) Relative hepatic triglyceride and
cholesterol levels in C57BL/6 mice. Lipids were extracted from liver tissue obtained from the four groups of mice and
analysed using spectrometry. (B) Histologic features of lipid accumulation in the livers of C57BL/6 mice. Paraffin-embedded
sections of the liver from C57BL/6 mice were stained with haematoxylin and eosin. Representative photomicrographs are
shown. The arrow indicates fatty metamorphosis of the liver (100). CV, central vein. (C) The proteins isolated from liver
tissue were analysed using Western blot with anti-SREBP-1/2, anti-A-FABP, anti-HMGCR, anti-FAS, anti-ACC and (D) anti-
phospho-AMPK and anti-AMPK antibodies. C, normal group; HFD, high-fat diet without the MLE powder; MLE-0.5%,
C57BL/6 mice fed a high-fat diet with 0.5% MLE powder; MLE-1.5%, C57BL/6 mice fed a high-fat diet with 1.5% MLE powder.
The data are presented as the mean SD of three replicates per treatment. #P < 0.05 compared with the control group.
*P < 0.05 compared with the HFD group.

were stained with Nile Red and quantified by flow cytometry.
The cells treated with quercetin, caffeic acid, hydroxyflavin and
hesperetin showed that a significantly decreased number of
cells underwent maturation by 11.78, 17.32, 13.53, 14.39%, re-
spectively (Fig. 5A and B). Here, we demonstrated that these
four compounds indeed blocked 3T3-L1 adipogenesis. We next
examined the cells on flow cytometer after annexin V-PI double
staining, which reflects number of apoptotic cells on the basis
of cell membrane phosphatidyl serine (PS) externalization. Treat-
ment with quercetin, hydroxyflavin, hesperetin and caffeic acid
increased apoptosis by 39.44, 20.63, 19.01% (P < 0.01) and 11.45%
(P < 0.05) more than the control after 72-h incubation periods,
respectively (Fig. 5C and D). Taken above, they showed that MLE,
MLPE, and major polyphenolic constituents (quercetin, caffeic
acid, hydroxyflavin and hesperetin) have the ability to reduce
the differentiation and induce apoptosis in 3T3-L1 cells. These
data confirmed our findings treating MLPE (Fig. 4).
3.7. MLE and MLPE reduced expression of adipogenic
transcription factors and lipogenic proteins in 3T3-L1
preadipocytes
We explored whether the effects of MLE observed in vivo could
also be observed in vitro. After culturing, 3T3-L1 preadipocytes
were matured in the presence or absence of MLE or MLPE. The
differentiation of preadipocytes into adipocytes is tightly
256 Journal of Functional Foods 21 (2016) 249262

Fig. 4 MLE and MLPE inhibited lipid accumulation and induced apoptosis in mature adipocytes. Postconfluent 3T3-L1
mature adipocytes were treated with MLE or MLPE. 3T3-L1 cells were stained with Oil-Red O (top, A) and Nile Red (bottom,
A) and analysed using flow cytometry (B). Fluorescence was quantified using flow cytometry (C). 3T3-L1 cells were stained
with annexin V/PI and then analysed using flow cytometry (D). The lower-right phase represents early apoptotic cells.
Fluorescence was quantified using flow cytometry (E). PI: propidium iodide. The data are presented as the mean SD of
three replicates per treatment. *P < 0.05 compared with the mature group. **P < 0.01 compared with the mature group.
 Journal of Functional Foods 21 (2016) 249262 257

Fig. 5 Quercetin, caffeic acid, hydroxyflavin and hesperetin inhibited lipid accumulation and induced apoptosis in mature
adipocytes. Post-confluent 3T3-L1 mature adipocytes were treated with 100 M Quercetin, caffeic acid, hydroxyflavin and
hesperetin for 72 hr. 3T3-L1 cells were stained with Nile Red and analysed using flow cytometry (A). Cellular lipid content
was quantified using flow cytometry (B). 3T3-L1 cells were stained with annexin V/PI and then analysed using flow
cytometry (C). The lower-right phase represents early apoptotic cells. Total apoptotic cell were quantified using flow
cytometry (D). PI: propidium iodide. The data are presented as the mean SD of three replicates per treatment. #, P < 0.05
compared with the pre-adipocyte group. *, P < 0.05 compared with the mature group. **, P < 0.01 compared with the mature
group.

regulated by the sequential activation of several transcrip-
tional factors, including SREBP-1 and PPAR. Hence, we
examined the expression of SREBP-1 and PPAR in mature
adipocytes for 14 days by using Western blot analysis. As shown
in Fig. 6, SREBP-1 and PPAR levels were decreased in MLE- or
MLPE-treated cells during 14 days of culture, in comparison with
the basal levels of these proteins in vehicle-treated control cells.
Because adipogenic transcription factors were downregulated
by MLE or MLPE, we determined the expression and activa-
tion of their downstream protein targets, including ACC and
FAS, which are crucial adipogenic proteins involved in fatty acid
biosynthesis. MLE or MLPE inactivated ACC and reduced the
258 Journal of Functional Foods 21 (2016) 249262

Fig. 6 MLE and MLPE inhibited mature adipocyte differentiation. MLE and MLPE reduced the expression of adipocyte
differentiation markers SREBP-1, PPAR, FAS, and ACC. Postconfluent 3T3-L1 mature adipocytes were treated with MLE or
MLPE. (A) The proteins isolated from cells were analysed using Western blot with anti-SREBP-1, anti-PPAR, anti-FAS, and
anti-ACC antibodies. The detailed method is described in Experimental Procedures. The arrow on the right side
represents the indicated protein. (B) Schematic model of the MLPE ameliorate obesity. MLPE were shown to be capable of
inhibiting hepatic lipogenesis through the mechanism, attenuation of SREBP1/FASN/triglyceride pathway and SREBP2/HMG
CoA reductase/total cholesterol pathway. MLPE also reduced peripheral lipid accumulation by inducing mature adipocyte
apoptosis and suppressing adipocyte differentiation. These results suggested that MLPE can be beneficial for the
suppression of high-fat-diet-induced dyslipidaemia, hepatosteatosis and obesity. All data are expressed as a fold change
relative to control untreated cells. The data are presented as the means SD of three replicates per treatment.
 Journal of Functional Foods 21 (2016) 249262
expression of FAS at concentrations of 2 mg/mL and 0.5 mg/mL,
respectively (Fig. 6A). Altogether, these data indicated that MLE
or MLPE effectively inhibited the expression of key adipogenesis-
related proteins, thereby confirming the antiadipogenic
properties of the extracts.
4. Discussion
Obesity is an increasingly severe health problem worldwide and
in Taiwan. Chiou reported that the rate of obesity is higher in
Taiwan (19.2% in males and 16.6% in females) than in Singapore,
Japan, Malaysia, South Korea, Thailand, and China (Chiou, 2012).
Obesity is abnormal or excessive fat accumulation in the body
and is associated with several chronic diseases. Various strat-
egies have been adopted to address this major public health
problem. However, alternative strategies for developing future
effective, safe, natural antiobesity drugs that can improve energy
imbalance and lipid metabolism are urgently required
(Mohameda, Ibrahimb, Elkhayata, & Dinec, 2014). This study
investigated the effect of mulberry leaves on metabolic disor-
ders in experimental mice and preadipocytes. The results
obtained showed that administering MLE effectively pre-
vented HFD-induced body weight gain and body fat
accumulation and reduced plasma TG and cholesterol levels
in mice on a HFD (Fig. 2 and Table 1). Moreover, we tested the
effects of MLE on serum BUN, creatinine, sodium, and potas-
sium levels and found no difference among the four groups
(Table 1; data not shown). Hence, MLE at the tested concen-
trations did not impair renal function in this study. To control
the progression of hyperlipidaemia, it is essential to under-
stand the regulatory mechanisms underlying lipid accumulation
in the liver. The molecules in these pathways are considered
potential targets for treating obesity and metabolic syn-
drome. Supplementing the diet with MLE (particularly the high
dose of 1.5%) suppressed adipogenesis by attenuating the ex-
pression of SREBP-1/2 and subsequently downregulating the
expression of adipogenic-specific proteins, such as A-FABP and
FAS, compared with the HFD groups. In addition, the findings
of the present study suggest that AMPK is a crucial target of
adipogenesis (Ceddia, 2013). AMPK, in turn, phosphorylates and
inactivates HMGCR and ACC and reduces lipogenesis in the liver
(Fig. 3D). These results indicate that MLE had ameliorating
effects on hyperlipidaemia and hepatic lipid accumulation in
HFD-induced obese mice. In addition, 3T3-L1 preadipocytes were
treated with the water extract of mulberry leaves, MLE, and the
polyphenol extract, MLPE, for 14 days. Fig. 4 shows that MLE
and MLPE have antiadipogenic properties, which were indi-
cated by decreased Oil-Red O staining on differentiation day
14 (Fig. 4A, upper panel). This result is consistent with those
of studies that investigated adipogenesis by using Nile Red
staining and flow cytometry (Fig. 4A, bottom panel and 4B) and
was achieved at a concentration that did not affect cell viabil-
ity according to the MTT assay (data not shown). The two agents
exhibited a potent lipid-lowering effect that was not related
to cytotoxicity. To elucidate the mechanisms underlying the sig-
nalling pathway affected by MLE and MLPE, we determined the
expression of SREBP-1, FAS, and ACC in vitro, which is consistent
with our in vivo results. Our results demonstrate that MLE and
259
MLPE exerted an antiadipogenic effect by inhibiting the ex-
pression of the transcription factors PPAR and SREBP-1 and
their downstream lipogenic targets FAS and ACC (Fig. 6). More-
over, apoptosis in adipose tissue has been recently suggested
as a contributor to body fat loss (Sorisky, Magun, & Gagnon,
2000), and both MLE and MLPE increased apoptosis during
adipocyte differentiation in vitro (Fig. 4D and E). Meanwhile,
in this study, we used quercetin, caffeic acid, hydroxyflavin and
hesperetin, which are main ingredients of MLPE, to show the
effectiveness of reducing the differentiation in cells (Fig. 5). This
is consistent with the results in Fig. 4 that show reduced dif-
ferentiation in 3T3-L1 preadipocytes. Hence, MLE and MLPE
effectively inhibited the differentiation of 3T3-L1 preadipocytes.
In summary, our study demonstrated, for the first time, that
mulberry leaves may be a promising dietary supplement or de-
veloped as a functional food to reduce the risk of obesity and
obesity-related metabolic disorders.
Our previous results have shown that administering mul-
berry leaves alleviates hypercholesteremia (Chan et al., 2013;
Huang, Ou, & Wang, 2013). Similarly, other studies have indi-
cated that mulberry leaves reduce cholesterol (Enkhmaa et al.,
2005; Yang et al., 2014b). In particular, MLEs possess
antihyperlipidaemic effects and the ability to regulate lipid me-
tabolism, as indicated in animal experiments (Chan et al., 2013;
Lim et al., 2013). However, their benefits still must be further
investigated in clinical studies. Two research groups have evalu-
ated the effect of mulberry leaf extract in patients with mild
hyperlipidaemia or diabetes (Andallu, Suryakantham, Lakshmi
Srikanthi, & Reddy, 2001; Aramwit, Petcharat, & Supasyndh,
2011; Aramwit, Supasyndh, Siritienthong, & Bang, 2013). For
the first group, in a clinical study in Thailand by Aramwit et al.,
approximately 20 dyslipidaemic participants received three 280-
mg tablets of mulberry leaf powder three times a day before
meals for 12 weeks (Aramwit et al., 2011, 2013). It was con-
cluded that the mulberry leaf powder could reduce serum TC,
TG, and LDL levels in patients with mild dyslipidaemia without
causing severe adverse reactions and minor side effects. For
the second group, 24 type 2 diabetic patients from India took
six capsules (500 mg/capsule) of mulberry leaf powder three
times a day, amounting to a dosage of 3 g/day for 30 days by
Andallu et al. (2001). They found that mulberry exhibits po-
tential for not only hypoglycaemic but also hypolipidaemic
effects in diabetic patients. These results indicate that mul-
berry leaf tablet therapy could be more effective and are
consistent with those of three studies. To date, although these
beneficial effects have been demonstrated in cultured cells and
animal models (Chan et al., 2013; Enkhmaa et al., 2005; Lim
et al., 2013; Yang et al., 2014b), very few studies have demon-
strated these effects in humans. Thus, human studies should
be performed before these molecules can be considered truly
useful tools for preventing obesity.
Because the currently available drugs for treating obesity
cause serious side effects, a growing demand exists for thera-
peutically potent but less cytotoxic antiobesity drugs. Plants
are valuable natural sources for the detection of new chemi-
cal compounds leading to the development of new drugs. In
addition, natural products have been used worldwide as tra-
ditional herbal medicines to treat different diseases, including
obesity-related metabolic disorders (Marimoutou et al., 2015).
Many medicinal plants and their extracts have therefore been
260 Journal of Functional Foods 21 (2016) 249262

used as dietary supplements because of their beneficial effects adipogenesis in vitro and in vivo (Enkhmaa et al., 2005; Moon,
on health. In particular, polyphenols, a major group of plant Do, Kim, & Shin, 2013). In addition to quercetin, hesperetin
metabolites widely found in cereals, red wine, soy, veg- was proven to reduce HFD-induced body weight gain and
etables, and fruits, have been reported to exhibit many hepatic Cyp2b9 expression involved in lipid homeostasis
biochemical activities. Using HPLC, we demonstrated that (Hoek-van den Hil et al., 2015). It was also noted that hesper-
the phenolic compounds of MLPE contain several major com- etin and its metabolites reduce the risks of atherosclerosis
pounds, such as quercetin, caffeic acid, hydroxyflavin, and by lowering the plasma total cholesterol and lipid peroxide
hesperetin (Fig. 1). Furthermore, Fig. 5 also shows that these levels in hamsters (H. J. Kim et al., 2010). In 2014, Sikder et al.
four major compounds reduced the differentiation and induced pointed out that rutin exerts hypolipidaemic effect by pre-
apoptosis in 3T3-L1 preadipocytes. It was demonstrated that venting the gains in body weight, lipid level, liver function
the main activation components of MLPE are quercetin, caffeic enzymes and lipid peroxidation level (Sikder, Kesh, Das, Manna,
acid, hydroxyflavin and hesperetin. On the other hand, these & Dey, 2014). Other studies supported the fact that mulberry
constituents were illustrated to be effective in reducing obesity leaf extract and its bioactive ingredients, such as protocat-
and its related illnesses (Table 2). Previously, caffeic acid, one echuic acid (Hogan, Canning, Sun, Sun, & Zhou, 2010; Liu,
of the major constituents of MLE, was reported to have anti- Lin, Wang, Mong, & Yin, 2010), flavane and chalcone (Yang
obesity effects (Cho et al., 2010; Liao, Ou, Huang, & Wang, et al., 2014d), exhibit hypolipidaemic effects, implying its po-
2014; Liao, Ou, Wu, & Wang, 2013). Yang et al. discovered that tential for preventing obesity. As mentioned, crude plant extract
caffeic acid and hesperetin can prevent fatty liver and reduce contributing to the antiobesity effect would be due to the
adipose tissue fat by inhibiting lipogenic enzymes and stimu- multiple bioactive compounds. Consequently, the beneficial
lating lipolysis via upregulating AMPK in high-fat fed rat (Yang effects of MLE on obesity should be attributed primarily to its
et al., 2015). An in vivo experiment showed that quercetin polyphenol content.
derivatives can directly regulate gene expression relative to Over the past 40 years, medicinal plants have played a crucial
hepatic lipid metabolism (e.g., Ehhadh, FAS, and GPAT) via role in health care; that is, they have traditionally been used
blocking oxidative stress marker NOX2 (gp91 phox) and in- for treating various diseases, and they are being widely ex-
ducing PPAR-regulated fatty acid -oxidative genes (Sun, perimentally studied throughout the world. Medicinal plants
Yamasaki, Katsube, & Shiwaku, 2015). The previous study include different types of plants used in herbology (also known
also showed that quercetin may have antiobesity effects by as herbalism or herbal/botanical medicine). Herbal teas such
suppressing preadipocyte differentiation and inhibiting as M. alba L. are gaining increased importance because of their
beneficial effects on health. Mulberry (M. alba L.) leaves have
been shown to inhibit adipogenesis, in vitro and in vivo, and
have been used to identify phytochemicals with antiadipogenic
Table 2 Functional ingredients of MLPE on reducing potential. Moreover, combining multiple natural products that
obesity and obesity-related disorder. have synergistic activity resulting both from increases in
Ingredient Effect Reference bioavailability and actions on multiple molecular targets may
Caffeic acid (CA) Decrease body weight Cho et al., 2010; offer advantages over treatments with single compounds.
Yang et al., 2015 Several phytochemicals alone or in combination have been
Attenuate fatty liver Yang et al., 2015 shown to promote adipocyte apoptosis, inhibit lipid accumu-
Promote hepatic Liao et al., 2014; lation, and promote osteogenesis (Sungkamanee et al., 2014).
lipolysis Yang et al., 2015 In particular, Lims group demonstrated that combined mul-
Regulate obesity-related Cho et al., 2010
berry leaf and fruit extract rich in polyphenols showed
hormone
antiobesity and anti-inflammatory effects, including a benefi-
Regulate lipid Cho et al., 2010
metabolism cial effect on dyslipidaemia and hypercholesterolemia in HFD-
Quercetin (Q) Promote hepatic Sun et al., 2015 induced obese mice (Lim et al., 2013; Lim, Yang, Kim, Lee, &
lipolysis Lim, 2013; Valacchi, Belmonte, Miracco, Eo, & Lim, 2014). Based
Inhibit adipocyte Moon et al., 2013 on the previous work, the water extracts of mulberry fruit
differentiation (MWEs) given for 12 weeks was explored to possess the anti-
Suppress adipocytes Moon et al., 2013
obese effect in male Syrian golden hamsters (Peng et al., 2011).
adipogenesis
Decrease body weight Moon et al., 2013
It was shown that MWEs have potential in body weight re-
Improve serum lipid Enkhmaa et al., 2005 duction (approximately 30%) accompanied with lowering TG
profiles and cholesterol of the liver. Furthermore, MWEs significantly
Hesperetin (H) Decrease body weight Hoek-van den Hil decreased the hepatic expression of FAS and HMGCR by ap-
et al., 2015 proximately 80% and 30%, respectively. This result indicated
Regulate lipid Hoek-van den Hil
the similar hypolipidaemic effects between MLE and MWEs but
metabolism et al., 2015
to differing concentrations (1.5% versus 2%) and feeding time
Improve serum lipid Kim et al., 2010
profiles (6 weeks versus 12 weeks). Compared with MWE, mice ap-
Rutin (R) Decrease body weight Sikder et al., 2014 proach the same efficacy as those fed with MLE at lower dosage
Improve serum lipid Sikder et al., 2014 and in shorter time interval. In brief, the identified combina-
profiles tions of natural products with other phytochemicals may
Regulate lipid Sikder et al., 2014 contribute to their potential as a novel therapy for prevent-
metabolism
ing obesity and obesity-related disease.
 Journal of Functional Foods 21 (2016) 249262
5. Conclusions
The present study indicated that MLPE treatment reduced
hyperlipidaemia in mice with HFD-induced obesity. We
showed that administering MLPE to C57BL/6 mice led to (1)
hypertriglyceridemia due to the upregulated expression of pro-
teins involved in lipid biosynthesis, including SREBP-1 and FAS
and (2) hypercholesterolemia due to impaired cholesterol uptake
because of the reduced expression of proteins such as SREBP-2
and HMGCR (Fig. 6B). Moreover, MLPE treatment significantly
attenuated peripheral lipid accumulation by inducing the apop-
tosis of mature adipocytes and suppressing the differentiation
of 3T3-L1 adipocytes. Therefore, mulberry leaves could be a new
therapeutic candidate for preventing and treating obesity.
Conflicts of interest
No potential conflicts of interest were disclosed.
Authors: All research done by the authors.
Acknowledgement
This work was supported by a grant from the National Science
Council (NSC 102-2313-B-040-003-MY3), Taiwan.
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