Available online at http:www.ifg-dg.org [602061]

August 4

Available online at http://www.ifg-dg.org

Int. J. Biol. Chem. Sci. 9(4): 1918-1928, August 201 5

ISSN 1997-342X (Online), ISSN 1991-8631 (Print)

© 2015 International Formulae Group. All rights res erved.
DOI : http://dx.doi.org/10.4314/ijbcs.v9i4.17
Original Paper http://ajol.info/index.php/ijbcs http://inde xmedicus.afro.who.int

Taraxacum officinale and Silybum marianum alone or combined orchestrate
experimentally induced hepatic steatosis through li pogenecity, glucose
tolerance and oxidant/antioxidant status

Nema A. SOLIMAN* and Samia A. El-DARDIRY

Department of Medical Biochemistry, Faculty of Medi cine, Tanta University, Egypt.
*Corresponding author; E-mail: [anonimizat]

ABSTRACT

Natural products with a long history of safety can modulate obesity. Taraxacum officinale , known as
(dandelion) and Silybum marianum known as (milk thistle) have garnered attention fo r their antioxidant and
antiobesity activities. The present study was condu cted to evaluate the potential role of dandelion an d milk
thistle alone or combined against high fat diet (HF D) induced steatohepatitis. 60 male albino rats whi ch were
equally subdivided into four groups: group I was re ceived only HFD, other groups (II, III, IV) were re ceived
dandelion, milk thistle or dandelion/milk thistle c ombination respectively for 8 weeks alongside HFD. Insulin
resistance, glucose tolerance, lipogenesis and anti oxidant capacity were evaluated in the liver tissue . HFD fed
rats exhibited increased insulin resistance-related biomarkers, H 2O2 level, mRNA expression of sterol
regulatory element-binding protein-1c (SREBP-1c) as well as fatty acid synthase (FAS) acti vity with decreased
reduced glutathione (GSH) level. Herbal supplementa tion improved those results with best results were for
dandelion/milk thistle combination group. Results we re confirmed with histopathological examination. Bo th
dandelion and milk thistle alone or combined improv ed glucose tolerance and insulin sensitivity, decre ased
lipogenesis and increased antioxidant capacity with best results obtained in dandelion/milk thistle co mbination
group, implying a potential application in the trea tment of hepatic steatosis associated obesity.
© 2015 International Formulae Group. All rights res erved

Keywords: High fat diet, obesity, dandelion, milk thistle, li pogenesis, insulin resistance.

INTRODUCTION
The worldwide incidence of obesity has
increased dramatically during recent decades
(Holvoet, 2012). By 2015, approximately 2.3
billion adults will be overweight and more
than 700 million will be obese according to
the World Health Organization projections
(Holvoet, 2012). Obesity is associated with a
high incidence of steatosis, insulin resistance
and chronic inflammation (Ismail, 2011). Obesity-related non-alcoholic fatty liver
disease (NAFLD) has recently been
recognized as one of the major causes of
chronic liver disorders, estimated to affect at
least one-quarter of the general population
(Ismail, 2011). NAFLD is characterized firstly
by excess liver lipid accumulation with insulin
resistance and later hepatic inflammation,
leading to nonalcoholic steatohepatitis
(NASH) with subsequent hepatic fibrosis or

N. A. SOLIMAN.and S. A. El-DARDIRY / Int. J. Biol. Chem. Sci. 9(4): 1918-1928, 2015

1919cirrhosis (Abenavoli, 2015). One of the major
causes of steatohepatitis is the inability of the
liver to regulate the changes in lipogenesis
which may result from either increased
triacylglycerol (TAG) synthesis or decreased
fatty acid oxidation, both leading to increased
TAG content in the liver (Tacer and Rozman,
2011). Fatty acid synthase (FAS) is one of the
key enzymes of lipogenesis (Tacer and
Rozman, 2011). Excess lipogenesis with
subsequent fat accumulation worsening
hepatic insulin resistance via a network of
transcription factors, which regulate hepatic
lipogenesis and fatty acid oxidation, including
sterol regulatory element-binding protein-1c
(SREBP-1c) (Berlanga et al., 2014).
The inflammatory response and
Increased oxidative stress associated obesity
involves many components of the classical
inflammatory response and increases in
inflammatory cytokines with subsequent
insulin resistance, dyslipidemia and elevated
levels of oxidized low density lipoprotein (ox-
LDL) (Iantorno et al., 2014).
Taraxacum officinale , known as
dandelion, has been used in folk medicine in
the treatment of hepatic disorders and several
women’s diseases such as breast and uterine
cancers and has choleretic, diuretic, anti-
rheumatic and anti-inflammatory properties
(Choi et al., 2010). Several flavonoids
including caffeic acid, chlorogenic acid,
luteolin, and luteolin 7-glucoside have been
isolated from the dandelion also it is a rich
source of vitamins A, B complex, C, and D, as
well as minerals such as iron, potassium and
zinc (Choi et al., 2010).
Silybum marianum (Milk thistle) has a
long and important history in herbal medicine,
silymarin is the active extract of milk thistle
(Kazazis et al., 2014). It is the most ancient
and extensively used medicinal plant for its
beneficial effects on liver and other organs; it
was primarily recognized for its capacity to
enhance bile flow and to remove obstructions
from liver and spleen (Kazazis et al., 2014).
The present study was designed to explore the
beneficial effects of dandelion and milk thistle
alone or together on biochemical and molecular changes associated obesity in order
to represent a novel approach for treating
steatohepatitis associated obesity.

MATERIALS AND METHODS
This current work was carried out at
Medical Biochemistry Department, in
accordance to the National Institutes of Health
guide for the care and use of Laboratory
animals (NIH Publications No. 8023, revised
1978) to minimize animal suffering and in
accordance to the guidelines of the Ethical
Committee of Medical Research, Faculty of
Medicine, Tanta University, Egypt.

Herbal supplementation
Both dandelion and milk thistle whole
plant extracts were prepared and supplied in
the form of powder by Egyfarma, Egypt.

Animal modeling and grouping
The present study was carried out on 60
male albino rats, aged 3 months, and weighted
80-120 g. During the study, the animals were
kept in wire mesh cages with ad-libitum
access to water. The room temperature was
about 22-24 °C and the animals were exposed
to 12:12 hours light dark cycles. All animals
were given two weeks to acclimate to
laboratory conditions, during that time they
were maintained on ad-libitum laboratory
chow and water. The animals were then
divided into four equal groups each of 15 rats
as follow: obese group (group I) was fed a
HFD (53.15%, 19.68% and 27.17% of energy
from fat, protein and carbohydrate,
respectively) which served as control group
receiving distilled water using a gavage via
intubation daily, dandelion treated group
(group II) was fed HFD alongside dandelion
(dandelion in a dose of 300 mg/kg/day orally
dissolved in distilled water using a gavage via
intubation) (Nnamdi Chinaka et al., 2012);
milk thistle treated group (group III) was fed
HFD alongside milk thistle (milk thistle in a
dose of 200 mg/kg/day orally dissolved in
distilled water using a gavage via intubation)
(Haddad et al., 2011); dandelion/milk thistle
combination treated group (group IV) was fed
HFD alongside both dandelion and milk

N. A. SOLIMAN.and S. A. El-DARDIRY / Int. J. Biol. Chem. Sci. 9(4): 1918-1928, 2015

1920thistle (Nnamdi Chinaka et al., 2012; Haddad
et al., 2011). Obesity was induced in 8 weeks
(Prieto-Hontoria et al., 2009) in which all
groups received HFD or HFD alongside
treatment. Composition of the HFD (g/kg diet)
was according to the formula of Dourmashkin
et al. (2006).

Experimental procedure
The body weight gain was measured
every one week. By the end of the
experimental period, fasting rats (12 hours)
were anaesthetized by ether, and while the
heart was still beating, blood was collected
and serum was separated. The abdomen was
opened liver was taken, cleared of the
adhering fat, weighed, washed by ice cold
saline, dried by filter paper, and weighed then
divided into specimens for preservation in
10% phosphate buffered formalin solution for
histopathological examination using
hematoxylin and eosin (H.&E.) and the other
remaining liver tissues were divided into
small pieces and underwent homogenization
in phosphate buffer saline (PBS) 50 mM, pH
7.4. Supernatant of liver tissue homogenate,
sera and remaining liver tissues were frozen at
-80 °C for further evaluations.

Biochemical analysis of serum
Serum aspartate transaminase (AST)
and alanine transaminase (ALT) activities
were determined using commercial
diagnostics kits supplied by (Elitech, France).
Fasting Blood Sugar (FBS) was measured by
the oxidase method by supplied commercial
kit (Biodiagnostic., Egypt), total Lipid profile
including total cholesterol (TC), triacylglycrol
(TAG), high density lipoprotein cholesterol
(HDL-C) and low density lipoprotein
cholesterol (LDL-C) levels were measured by
enzymatic-colorimetric methods by supplied
commercial kits (Biodiagnostic., Egypt).
Enzyme linked immunosorbent assay
(ELISA) was used to detect serum levels of
insulin (USCN Life Science Inc, Wuhan,
China) and oxidized-LDL (ox-LDL) (Cell
Biolab, Inc., USA) using ELISA Reader (Star
fax 2001). Insulin resistance was assessed by
the homeostatic model assessment of insulin resistance (HOMA-IR) (Duseja et al., 2007)
calculated as: Fasting blood glucose level
(mg/dl) * fasting insulin level ( µIU/mL)/405.

Biochemical analysis of liver tissue
homogenate
Assay hydrogen peroxide (H 2O2) level
By commercial kits supplied by
Biodiagnostic, Egypt. In the presence of horse
radish peroxidase (HRP), H2O2 reacts with 3,
5-dichloro-2-hydroxybenzenesulfonic
(DHBS) acid and 4-aminophenazone (AAP)
to form a chromophore absorbance of the
sample and absorbance of standard were read
against blank at 510 nm using Biosystem
spectrophotometer (BTS 350 semiautomatic
analyzer) (Fossati et al.,1980; Aebi, 1984)
Assay of reduced glutathione (GSH) level
By commercial kits supplied by
Biodiagnostic, Egypt. The reduction of 2-
nitrobenzoic acid (DTNB) with reduced
glutathione (GSH) produced a yellow
compound, the reduced chromogen is directly
proportional to GSH concentration and its
absorbance can be measured at 405 (Ellman,
1959).
Assay of fatty acid synthase (FAS) activity
Using a protocol as previously
described (Nepokroeff et al., 1975), briefly, an
aliquot of supernatant (50 µl) was pre-
incubated with a buffer containing 200 mM
potassium phosphate, pH 6.6, 1 mM DTT, 1
mM EDTA, 0.24 mM NADPH and 30 µM
acetyl-CoA in a final volume of 0.2 ml and
the reaction was monitored at 340 nm for 3
min to measure background NADPH
oxidation. After the addition of 50 µM of
malonyl-CoA, the reaction was assayed for an
additional 15 min to determine FAS-
dependent oxidation of NADPH. The rate of
optical density (OD) at 340nm change was
corrected for the background rate of NADPH
oxidation.
Assay Protein content
According to Lowry et al. (1951) in
which a standard curve of bovine serum
albumin (from 0 to 1000 µg /ml of PBS)
concentration was plotted, and the protein
content of the unknown samples was
measured at wave length 750 nm.

N. A. SOLIMAN.and S. A. El-DARDIRY / Int. J. Biol. Chem. Sci. 9(4): 1918-1928, 2015

1921Liver TAG content
Excessive accumulation of TAG in the
liver is the hallmark of NAFLD. For hepatic
TAG level, 100 mg of liver tissue was
homogenized for extraction of lipid according
to the method of Folch et al. (1957). Level of
TAG was then quantified according to the
manufacturer's procedures with commercial
assay kit (Biodiagnostic., Egypt).

Estimation of SREBP-1c mRNA level in
liver tissue
The frozen liver samples were
processed for total RNA extraction (MagNA
Pure compact Nucleic Acid isolation kit I,
Roche Diagnostics, GmbH, Mannheim,
Germany) according to the manufacturer's
recommendations. Total RNA was treated
with DNase I to eliminate genomic DNA
contamination, followed by synthesis of the
first strand using Transcriptor First Strand
cDNA Synthesis Kit (Roche Diagnostics,
Mannheim, Germany) according to the
manufacturer's instructions. SREBP-1c
mRNA transcripts were quantified, relative to
the house-keeping gene; glyceraldehyde-3-
phosphate dehydrogenase (GAPDH) using the
Roche LightCycler® FastStart DNA
MasterPLUSYBR Green I kits (Roche
Diagnostics, Mannheim, Germany) following
manufacturer's instructions. Sequence specific
primers were designed by Primer3 software:
(http://bioinfo.ut.ee/primer3/) as follows:
SREBP-1c (No: NM_001276707.1) (sense 5'-
GGA GCC ATG GAT TGC ACA TT -3'),
SREBP-1c (antisense 5'- CCT GTC TCA
CCC CCA GCA TA -3'), GAPDH (No:
NM_017008.4) (sense 5'- GGT GAA GTT
CGG AGT CAA CGG A- -3'), GAPDH
(antisense 5'- GAG GGA TCT CGC TCC
TGG AAG A -3'). PCR reaction was
performed following the cycling protocol of
95 °C for 5 min, followed by 45 PCR cycles
with 95 °C for 5 s, 58 °C for 15 s and 72 °C
for 20 s. The values of the target gene under
investigation and the value of the house-
keeping gene (GAPDH) were calculated for
each sample using standard curve. The final
results were automatically calculated from the
(Cp = crossing point) values of the target and the reference genes by LightCycler® 4.0
Relative Quantification Software.

Histopathlogical study
The liver tissue was visualized by
hematoxylin and eosin (H.&E.). staining. The
sections of liver were fixed in 10% formalin,
dehydrated, embedded in paraffin and stained
with H.&E. then photographed.

Statistical analysis
Statistical presentation and analysis of
the present study was conducted, using the
mean, standard deviation by SPSS version.16.
Analysis of variance (ANOVA) and Tukey’s
test were used to determine the significance
between more than 2 groups: according to the
computer program SPSS for Windows. P
value < 0.05 was considered significant.

RESULTS
Effect of dandelion, milk thistle and
dandelion/milk thistle combination on body
weight gain, total liver weight
The body weight of the HFD control
group (group I) was significantly higher than
that of treated groups, after 8 weeks of feeding
and remained significantly higher during the
whole experiment (P<0.05), group II, group
III and group IV had significantly lower body
weight than group I and remained
significantly lower during the experiment with
significant lower results in group IV (P<0.05),
there was no statistically significant difference
between groups II and III (Table 1). The liver
weight in group I rats was significantly
increased when compared to those of treated
groups (II, III and IV) (P<0.05), there was no
statistically significant difference between
groups II and III (Table 2).

Dandelion, milk thistle and dandelion/milk
thistle combination improved the serum
and hepatic lipid profiles as well as hepatic
enzymes
Rats of group I had significantly higher
TAG, TC, LDL-C and ox-LDL levels than
treated groups while HDL-C showed the
opposite (P<0.05) (Table 2), however,
dandelion/milk thistle combination group

N. A. SOLIMAN.and S. A. El-DARDIRY / Int. J. Biol. Chem. Sci. 9(4): 1918-1928, 2015

1922(group IV) showed best results. The level
TAG content in liver tissues was significantly
lowered by dandelion, milk thistle and
dandelion/milk thistle combination in groups
II, III and IV respectively when compared to
group I (P<0.05) (Table 2). In addition, ALT
and AST activities were increased in the
group I, but this was significantly lowered in
treated groups with best results in group IV
(P<0.05) (Table 2).

Dandelion, milk thistle and dandelion/milk
thistle combination lowered insulin
resistance-related biomarkers
HFD fed rats exhibited increased
fasting blood glucose, serum insulin level and
HOMA-IR index (P<0.05) (Table 3).
However, dandelion, milk thistle and
dandelion/milk thistle combination lowered
the aforementioned parameters which are
more obvious in group IV.

Dandelion, milk thistle and dandelion/milk
thistle combination ameliorated
biomarkers of oxidative stress in liver
tissue
Supplementation with dandelion, milk
thistle and dandelion/milk thistle combination
in groups II, III and IV resulted in significant
reduction in elevated H 2O2 level and significant elevation in the lowered GSH level
in group I (P<0.05) (Table 4).

Dandelion, milk thistle and dandelion/milk
thistle combination reduced the expression
of SREBP-1c genes and FAS activity in the
liver
HFD fed rats had higher mRNA
expression of SREBP-1c as well as higher
FAS activity in their liver tissues than animals
in the groups II, III and IV with best lower
significant results in group IV (P<0.05) (Table
4 ).

Histopathological findings
There were histopathological changes
with structural damage of hepatic lobules in
the liver of group I (Figure 1A), the prominent
lesions were many lipid-droplets vacuoles,
fatty cysts and cytoplasmic shrinkage with
dark and small nuclei as well as many
perivascular inflammatory infiltration. In
groups II and III, hepatocytes had displayed
less fatty and inflammatory infiltration
(Figures 1B&C). The fatty and inflammatory
changes of hepatocytes decreased obviously in
group IV (Figure1D).

Table 1: Comparison of body weight gain (gm) throughout the experimental study between all
studied groups.

Group IVd
n=15 Group IIIc
n=15 Group IIb
n=15 Group I a
n=15 Week/Group
100.2±2.9 abc 109.1±1.2 ad 106.2±5.1 ad 112.6±3.2bcd 1 week
109.2±2.1 abc 120.3±2.9 ad 119±4.3 ad 122± 5.2 bcd 2 weeks
120.9±3.1 abc 130.2±1.9 ad 129.1±2.1 ad 134±3.3 bcd 3 weeks
131.4±4.1 abc 141.4±1.2 ad 139.1±2.2 ad 146±2.4 bcd 4 weeks
145.2±2.2 abc 152.4±1.1 ad 150.9±1.3 ad 159±2.5 bcd 5 weeks
152.3±1.9 abc 162.3±2.1 ad 161.2±3.9 ad 173.2±2.2 bcd 6 weeks
159.1±2.7 abc 174.3±2.3 ad 172.1±3.1 ad 186.2±3.1 bcd 7 weeks
164.4±2.6 abc 179.1±4.1 ad 180.4±3.4 ad 193.1±2.1 bcd 8 weeks
a-d: significant difference between groups at p<0.05* a: significant difference with group I, b: significant difference with group
II. c: significant difference with group III. d: significant difference with group VI . Data are m ean ± standard deviation of 15
rats of each. Statistical analysis was carried out using one way analysis of variance (ANOVA) with Tuk ey's post-hoc test,
SPSS computer program.

N. A. SOLIMAN.and S. A. El-DARDIRY / Int. J. Biol. Chem. Sci. 9(4): 1918-1928, 2015

1923Table 2: Effects of dandelion, milk thistle and dandelion/m ilk thistle combination on liver weight,
transaminases activity and serum/lipid profiles amo ng all studied groups.

Group IVd
n=15 Group IIIc
n=15 Group IIb
n=15 Group I a
n=15 Parameter/Group
3.08±0.69abc 4.01±0.09ad 3.9±0.65ad 6.51±0.25bcd liver weight (grams)
52.3±2.7 abc 64.1± 3.6 ad 65.5± 3.51 ad 75.1 ±1.5 bcd Serum ALT activity (U/L)
54.5±4.4 abc 64.13± 2.3 ad 64.2± 1.9 ad 80.5 ±0.95 bcd Serum AST activity (U/L)
6.3±0.94abc 8.2±0.6abd 8.2±0.89acd 13.4±3.9 bcd Hepatic TAG level (mg/gm tissue)
83.7±5.1abc 96.6±4.9 ad 96.2±5.8 ad 169.1±10.7bcd Serum TAG level (mg/dl)
106.1 ±20.8 abc 191.1± 8.7ad 192.5± 8.7ad 222.2 ±46 bcd Serum TC level (mg/dl)
60.1 ±24.8 abc 132.2±23.9ad 135.4±23.1ad 162.1 ±4.2 bcd Serum LDL-C level (mg/dl)
33.8 ±2.3 abc 30.6± 2.0 ad 30.9± 1.6 ad 26.8 ±1.4 bcd Serum HDL-C level (mg/dl)
28.9±9.9 abc 65.8± 1.8 ad 63.4± 5.7 ad 96.6 ±4.6 bcd Serum ox-LDL level (ng/ml)
a-d: significant difference between groups at p<0.05* a: significant difference with group I, b: significant difference with group
II. c: significant difference with group III. d: significant difference with group VI . Data are m ean ± standard deviation of 15
rats of each. Statistical analysis was carried out using one way analysis of variance (ANOVA) with Tuk ey's post-hoc test,
SPSS computer program. ALT: alanine transaminase. A ST: aspartate transaminase. TAG: triacylglycrol . T C: total
cholesterol. LDL-C: low density lipoprotein cholest erol. HDL-C: high density lipoprotein cholesterol . ox-LDL: oxidized low
density lipoprotein.

Table 3: Effects of dandelion, milk thistle and dandelion/m ilk thistle combination on insulin
resistance related biomarkers among all studied gro ups.

Group IVd
n=15 Group IIIc
n=15 Group IIb
n=15 Group I a
n=15 Parameter/Group
92.9 ±7.5 abc 120.2± 4.7 ad 121.7± 4.5 ad 137.9 ±4.9 bcd FBS level (mg/dl)
5.99 ±2.2 abc 14.6± 1.6 ad 14.3± 1.7 ad 29.8 ±2.6 bcd Fasting insulin level ( µIU/ml)
1.46±1.06 abc 4.33± 0.47 ad 4.31± 0.53 ad 10.1 ±1.0 bcd HOMA.IR index
a-d: significant difference between groups at p<0.05* a: significant difference with group I, b: significant difference with group
II. c: significant difference with group III. d: significant difference with group VI . Data are m ean ± standard deviation of 15
rats of each. Statistical analysis was carried out using one way analysis of variance (ANOVA) with Tuk ey's post-hoc test,
SPSS computer program. FBS: fasting blood glucose. HOMA.IR: homeostatic model assessment of insulin re sistance.

Table 4: Effects of dandelion, milk thistle and dandelion/m ilk thistle combination on biomarkers of
oxidative stress and lipogenesis in hepatic tissue among all studied groups.

Group IVd
n=15 Group IIIc
n=15 Group IIb
n=15 Group I a
n=15 Parameter/Group
0.21±0.25abc 0.55± 0.15ad 0.54± 0.22ad 0.88 ±0.12 bcd Hepatic H 2O2
(nmol/min/gm tissue)
3.06±0.24 abc 2.38± 0.08ad 2.36± 0.13ad 1.34 ±0.14 bcd Hepatic GSH
(mg/gm tissue)
3.98±0.20 abc 4.15±0.1abd 4.43±0.19acd 5.8±0.29 bcd Hepatic FAS activity (nmol /mg
protein / min)
0.45±0.11 abc 0.59±0.07abd 0.59±0.09acd 1.13±0.19 bcd Hepatic relative SREBP-1C mRNA
expression
a-d: significant difference between groups at p<0.05* a: significant difference with group I, b: significant difference with group
II. c: significant difference with group III. d: significant difference with group VI . Data are mean ± standard deviation of 15
rats of each. Statistical analysis was carried out using one way analysis of variance (ANOVA) with Tuk ey's post-hoc test,
SPSS computer program. H 2O2: hydrogen peroxide. GSH: reduced glutathione. FAS: fatty acid synthase . SREBP-1C: sterol
regulatory element-binding protein-1c.

N. A. SOLIMAN.and S. A. El-DARDIRY / Int. J. Biol. Chem. Sci. 9(4): 1918-1928, 2015

1924

Figure 1: The liver tissue stained with hematoxylin and eosi n (H.&E.) (200 folds of amplification)
in rats at the end of 8 week. A: group I with severe steatosis; B: group II with improvement of steatosis; C: group III
with improvement of steatosis; D: group IV with res toration of nearly normal architecture. FI: fatty i nfiltration; I:
inflammatory cell infiltration

DISCUSSION
NAFLD is gaining increasing
recognition as a component of the epidemic of
obesity worldwide and is the most common
cause of liver damage (Cheung and Sanyal,
2010). At present, no pharmacological
treatment has been convincingly efficient
against steatohepatitis.
In fact, slight but consistent weight
loss, healthy eating regimen and exercise
together with a number of therapeutic avenues
remain the center of all strategies to improve
or reverse steatohepatitis (Cheung and Sanyal,
2010). With the rising interest in
complementary and alternative medicine,
several natural products became under
focusing for their potential beneficial effects
in liver diseases. Among such attractive novel
therapeutic possibilities are dandelion and
milk thistle, which are widely consumed
around the world (Xu et al., 2010).
Results herein showed that dandelion
and milk thistle alone or combined reduced
body weight gain and lowered the weights of
liver tissues when compared to rats on HFD as
previously reported (Choi et al., 2010; Jahan
et al., 2015). Other studies have revealed that HFD promote hyperlipidemia and
hyperglycemia so it can be used to generate a
valid rodent model for the analysis of the
pathophysiology of dyslipidemia (Puccinelli
et al., 2015).
Results herein showed that dandelion
and milk thistle could ameliorate the abnormal
blood lipid alteration in obese rats (group I)
when compared to treated groups with best
results obtained in the dandelion/milk thistle
combination group as previously reported
(Choi et al., 2010; Jahan et al., 2015).
Additionally, dandelion, milk thistle and
dandelion/milk thistle combination
significantly reduced the elevated liver
transaminases in group I when compared to
treated groups that may be due to their
antioxidant or free radical-antagonizing
actions that increase stability of cellular
membranes of hepatocytes and thereby
promoting hepatic tissue regeneration that is
more obvious in group IV (Shaker et al.,
2010; You et al., 2010).
Hepatic steatosis arises from imbalance
in TAG acquisition and removal (Puccinelli et
al., 2015). dandelion, milk thistle and
dandelion/milk thistle combination

N. A. SOLIMAN.and S. A. El-DARDIRY / Int. J. Biol. Chem. Sci. 9(4): 1918-1928, 2015

1925significantly reduced the elevated liver TAG
content in group I when compared to treated
groups with best results were obtained in
dandelion/milk thistle combination group as
herbal supplementation can reduce intestinal
absorption level of dietary lipid, thereby
decreasing serum and hepatic TAG (Yao et
al., 2011; Mingarro et al., 2015).
Insulin resistance may play an
important role in the development of NAFLD
(Bugianesi et al., 2005). In this current study,
the FBG, fasting insulin as well as HOMA-IR
were significantly increased in group I when
compared to other treated groups, however,
dandelion and milk thistle alone or combined
improved insulin sensitivity, as demonstrated
by a significant reduction in serum insulin and
glucose levels as well as HOMA-IR specially
in group IV. This can be explained that both
herbs together can activate adenosine
monophosphate (AMP)-activated protein
kinase (AMPK) in liver which is resulted in
significantly suppression of lipid
accumulation in the liver, improve insulin
resistance as previously reported (Hardie,
2011; Yao et al., 2011; Mingarro et al., 2015).
In obesity fat accumulation correlated
with systemic oxidative stress in humans and
rodent as the production of reactive oxygen
species (ROS) increased selectively in adipose
tissue (Holvoet et al., 2008; Iantorno et al.,
2014). In this study, the ox-LDL levels were
significantly increased in group I when
compared to treated groups, however,
dandelion and milk thistle supplementation
improved this which is more obvious in
combination group as reported previously by
Wallace et al. (2008) and Colle et al. (2012)
who reported the protective effects of both
herbs most likely through antioxidant and free
radical scavenging mechanisms.
In the current study, there were marked
increase in the levels of H 2O2 as well as a
distinct diminution in GSH level in liver tissue
in group I when compared with treated groups
which is improved by dandelion and milk
thistle supplementations with best
improvement in group IV, a result in
accordance with results herein Choi et al. (2010) and Yao et al. (2011) who reported the
antioxidant properties of silibinin because of
its capacity to decrease lipid peroxidation, to
reduce the release of O 2•− and to restore
hepatic GSH level while Colle et al. (2012)
who reported that dandelion scavenger
activities against ROS and reactive nitrogen
species are attributed to the content of its
phenolic compounds.
Sterol regulatory element binding
proteins (SREBPs) are master transcription
factors for de novo lipogenesis (Zhao et al.,
2014). The three SREBP isoforms, SREBP-1a,
SREBP-1c and SREBP-2, play different roles
in lipid synthesis. Studies using transgenic and
knockout mice suggest that SREBP-1c plays
an essential role in the regulation of most
lipogenic genes involved in fatty acid and
TAG synthesis (Zhao et al., 2014). The
present study showed significant increase in
SREBP-1c mRNA level as well as FAS
activity in the liver tissues of HFD fed rats
when compared to treated groups, however
dandelion and or milk thistle
supplementations attenuated this increase in
treated groups. A recent study by Xiao et al.
(2013) showed that dandelion and milk thistle
inhibited hepatic lipid accumulation through
AMPK activation, the activated AMPK
further phosphorylates acetyl-CoA
carboxylase, which switches off fatty acid
synthesis and accelerates the transport of
long-chain fatty acyl groups into the
mitochondria to undergo β-oxidation (Hardie,
2011).
Other possible mechanism of the
antiobesity activity of both herbs is that
oxidative stress associated obesity which is
attenuated by herbal supplementation can
increase SREBP-1c mRNA expression,
leading to enhanced transcription of FAS with
subsequent hepatic fat accumulation as
reported by Abd Eldaim et al. (2010).
To our knowledge, no previous studies
studied the hypolipidemic effect of dandelion
or milk thistle on the lipogenic gene
expression.
Abnormal lipid metabolism associated
obesity in the liver leads to fatty liver

N. A. SOLIMAN.and S. A. El-DARDIRY / Int. J. Biol. Chem. Sci. 9(4): 1918-1928, 2015

1926formation. Histopathologically, liver sections
from rats fed HFD showed a clear difference
from those of other treated groups in which fat
accumulates in the liver hepatocytes with
marked degenerative changes, necrosis,
disrupted architecture were observed, these
results are in agreement with previous study
of Samuhasaneeto et al. (2007) who reported
the effects of HFD associated inflammation
and oxidative stress in inducing the early stage
steatohepatitis.
Improvement of the histopathological
results was observed with herbal
supplementation in treated groups II and III
with nearly restoration of normal architecture
to some extent in combination group IV
reflecting their anti-oxidative, anti-fatty
infiltration, anti-inflammatory and liver
regenerating effects as previously reported
(Kim et al., 2012)

Conclusion
HFD induced obesity associated with a
disturbed lipid profile, defective antioxidant
stability, and high values of insulin resistance
biomarkers; this may have implications for the
progress of obesity related steatohepatitis.
Treatment with dandelion, milk thistle and
dandelion/milk thistle combination improved
obesity and its associated steatohepatitis so
they may be of particular benefit to
individuals who are unable or unwilling to
reduce their intake of high-fat foods.
Moreover we can conclude that when herbs
are used in combination it helps the body for
better managing. It is therefore preferable to
use herbal combinations instead of depending
on single herbs. Although further research is
required to confirm that, similar effects occur
in humans.

ACKNOWLEDGMENTS
The authors would like to acknowledge
the technicians in Biochemistry department,
the technical assistance in histopathological
examination and the valuable comments of Dr.
Darin A Ali, lecturer of histopathology,
Faculty of Medicine, Tanta University, Egypt.
COMPETING INTERESTS
The authors have no conflict of interest
to declare.

AUTHORS' CONTRIBUTIONS
NAS participated in animal preparation,
sample collection, biochemical and molecular
assays, and together with SAE-D participated
in the design of the study, performed
statistical analysis, and drafted the manuscript.
All authors read and approved the final
manuscript.

REFERENCES
Abd Eldaim MA, Okamatsu-Ogura Y, Terao
A, Kimura K. 2010. Effects of retinoic
acid and hydrogen peroxide on sterol
regulatory element-binding protein-1a
activation during adipogenic
differentiation of 3T3-L1 cells. Jpn. J.
Vet. Res., 58(3-4):149-154.
Abenavoli L. 2015. Non-alcoholic fatty liver
disease and beneficial effects of dietary
supplements. World J. Hepatol.,
7(12):1723-1724. doi: 10.4254/wjh.v7.
i12.1723.
Aebi H. 1984. Catalase in vitro . Methods
Enzymol., 105: 121-126.
Berlanga A, Guiu-Jurado E, Porras JA,
Auguet T. 2014. Molecular pathways in
non-alcoholic fatty liver disease. Clin.
Exp. Gastroenterol., 7: 221-239. doi:
10.2147/CEG.S62831.
Bugianesi E, McCullough AJ, Marchesini G.
2005. Insulin resistance: a metabolic
pathway to chronic liver disease.
Hepatology, 42: 987-1000.
Cheung O, Sanyal AJ. 2010. Recent advances
in nonalcoholic fatty liver disease. Curr.
Opin. Gastroenterol., 26(3): 202–208.
doi: 10.1097/MOG.0b013e328337b0c4.
Choi UK, Lee OH, Yim JH, Cho CW, Rhee
YK, Lim SI, Kim YC. 2010.
Hypolipidemic and antioxidant effects of
dandelion ( Taraxacum officinale ) root
and leaf on cholesterol-fed rabbits. Int. J.
Mol. Sci., 11(1): 67-78. doi:
10.3390/ijms11010067.

N. A. SOLIMAN.and S. A. El-DARDIRY / Int. J. Biol. Chem. Sci. 9(4): 1918-1928, 2015

1927Colle D, Arantes LP, Gubert P, da Luz SC,
Athayde ML, Teixeira Rocha JB, Soares
FA. 2012. Antioxidant properties of
Taraxacum officinale leaf extract are
involved in the protective effect against
hepatoxicity induced by acetaminophen
in mice. J. Med. Food , 15(6):549-556.
doi: 10.1089/jmf.2011.0282.
Dourmashkin JT, Chang GQ, Hill JO, Gayles
EC, Fried SK, Leibowitz SF. 2006.
Model for predicting and phenotyping at
normal weight the long-term propensity
for obesity in Sprague–Dawley rats.
Physiol. Behav., 87(4): 666–678.
Duseja A, Thumburu KK, Das A, Dhiman
RK, Chawla YK, Bhadada S, Bhansali A.
2007. Insulin tolerance test is comparable
to homeostasis model assessment for
insulin resistance in patients with
nonalcoholic fatty liver disease. Indian J.
Gastroenterol., 26: 170-173.
Ellman GL. 1959. Tissue sulfhydryl groups.
Archives of Biochemistry and Biophysics ,
vol. 82(1): 70–77.
Folch J, Lees M, Sloane Stanley GH. 1957. A
simple method for the isolation and
purification of total lipids from animal
tissues. J. Biol. Chem., 226(1): 497–509.
Fossati P, Prencipe L, Berti G. 1980. Use of
3,5-dichloro-2-hydroxybenzenesulfonic
acid/4- aminophenazone chromogenic
system in direct enzymic assay of uric
acid in serum and urine. Clin. Chem.,
26(2): 227-231.
Haddad Y, Vallerand D, Brault A, Haddad PS.
2011. Antioxidant and hepatoprotective
effects of silibinin in a rat model of
nonalcoholic steatohepatitis. Evid. Based
Complement Alternat. Med., 2011 : 164-
173. doi: 10.1093/ecam/nep164.
Hardie DG. 2011. Sensing of energy and
nutrients by AMP-activated protein
kinase. Am. J. Clin. Nutr., 93: 891-896.
doi: 10.3945/ajcn.110.001925.
Holvoet P, Lee DH, Steffes M, Gross M,
Jacobs DR Jr. 2008. Association between
circulating oxidized low-density
lipoprotein and incidence of the
metabolic syndrome. JAMA, 299(19): 2287–2293. doi: 10.1001/jama.299.
19.2287.
Holvoet P. 2012. Stress in Obesity and
Associated Metabolic and Cardiovascular
Disorders. Scientifica, 2012 : 1-17. doi:
10.6064/2012/205027.
Ismail MH. 2011. Nonalcoholic fatty liver
disease and type 2 diabetes mellitus: the
hidden epidemic. Am. J. Med. Sci.,
341(6):485-492. doi: 10.1097/MAJ.
0b013e3182018598.
Jahan S, Khan M, Imran S, Sair M. 2015. The
hepatoprotective role of Silymarin in
isoniazid induced liver damage of rabbits.
J. Pak. Med. Assoc., 65(6): 620-622.
Kazazis CE, Evangelopoulos AA, Kollas A,
Vallianou NG. 2014. The therapeutic
potential of milk thistle in diabetes. Rev.
Diabet. Stud., 11(2): 167-174. doi:
10.1900/RDS.2014.11.167.
Kim M, Yang SG, Kim JM, Lee JW, Kim YS,
Lee JI. 2012. Silymarin suppresses
hepatic stellate cell activation in a dietary
rat model of non-alcoholic steatohepatitis:
analysis of isolated hepatic stellate cells.
Int. J. Mol. Med., 30(3):473-479. doi:
10.3892/ijmm.2012.1029.
Iantorno M, Campia U, Di Daniele N, Nistico
S, Forleo GB, Cardillo C, Tesauro M.
2014. Obesity, inflammation and
endothelial dysfunction. J. Biol. Regul.
Homeost. Agents, 28(2): 169-76.
Lowry OH, Rosenbrough NG, Farr AL,
Randall RJ. 1951. Protein measurements
with the Folin phenol reagent. J. Biol.
Chem., 193(1): 265–275.
Mingarro DM, Plaza A, Galán A, Vicente JA,
Martínez MP, Acero N. 2015. The effect
of five Taraxacum species on in vitro and
in vivo antioxidant and antiproliferative
activity. Food Funct., 6(8):2787-93. doi:
10.1039/c5fo00645g.
Nepokroeff CM, Lakshmanan MR, Porter JW.
1975. Fatty acid synthase from rat liver.
Methods Enzymol., 35: 37–44.
Nnamdi Chinaka C, Uwakwe AA, Chuku LC.
2012. Effect of aqueous and ethanolic
extracts of dandelion ( Taraxacum
officinale F.H. Wigg.) leaves and roots on

N. A. SOLIMAN.and S. A. El-DARDIRY / Int. J. Biol. Chem. Sci. 9(4): 1918-1928, 2015

1928some hematological parameters of normal
and STZ-induced diabetic Wistar albino
rats. G.J.R.M.I., 1(5): 172–180.
Prieto-Hontoria PL, Pérez-Matute P,
Fernández-Galilea M, Barber A, Martínez
JA, Moreno-Aliaga MJ. 2009. Lipoic acid
prevents body weight gain induced by a
high fat diet in rats: effects on intestinal
sugar transport. J. Physiol. Biochem .,
65(1):43-50.
Puccinelli E, Gervasi PG, Trivella MG,
Vornoli A, Viglione F, Pelosi G, Parodi
O, Sampietro T, Puntoni M. 2015.
Modulation of lipid homeostasis in
response to continuous or intermittent
high-fat diet in pigs. Animal, 9(6):1000-
1007. doi: 10.1017/S1751731114003292.
Samuhasaneeto S, Thong-Ngam D,
Kulaputana O, Patumraj S, Klaikeaw N.
2007. Effects of N-Acetylcysteine on
Oxidative Stress in Rats with
Nonalcoholic Steatohepatitis. J. Med.
Assoc. Thai., 90(4):788-797 .
Shaker E, Mahmoud H, Mnaa S. 2010.
Silymarin the antioxidant component and
Silybum marianum extracts prevent liver
damage. Food Chem. Toxicol., 48: 803-
806. doi: 10.1016/j.fct.2009.12.011.
Tacer K, Rozman D. 2011. Nonalcoholic fatty
liver disease: focus on lipoprotein and
lipid deregulation. J. Lipids, 2011 : 1–14.
doi: 10.1155/2011/783976.
Wallace S, Vaughn K, Stewart BW,
Viswanathan T, Clausen E, Nagarajan S,
Carrier DJ. 2008. Milk thistle extracts
inhibit the oxidation of low-density
lipoprotein (LDL) and subsequent
scavenger receptor-dependent monocyte adhesion. J. Agric. Food Chem., 56(11):
3966-3972. doi: 10.1021/jf703694u.
Xiao J, So KF, Liong EC, Tipoe GL. 2013.
Recent Advances in the Herbal Treatment
of Non-Alcoholic Fatty Liver Disease.
Journal of Traditional and
Complementary System, 3(2): 88-94. doi:
10.4103/2225-4110.110411.
Xu RY, Wan YP, Tang QY, Wu J, Cai W.
2010. Carbohydrate-to-fat ratio affects
food intake and body weight in Wistar
rats. Exp. Biol. Med. (Maywood),
235(7):833-838. doi: 10.1258/ebm.
2010.009276.
Yao J, Zhi M, Minhu C. 2011. Effect of
silybin on high-fat-induced fatty liver in
rats. Braz. J. Med. Biol. Res., 44(7): 652-
659.
You Y, Yoo S, Yoon HG, Park J, Lee YH,
Kim S, Oh KT, Lee J, Cho HY, Jun W.
2010. In vitro and in vivo
hepatoprotective effects of the aqueous
extract from Taraxacum officinale
(dandelion) root against alcohol-induced
oxidative stress . Food Chem. Toxicol.,
48(6):1632-1637. doi: 10.1016/j.fct.2010.
03.037.
Zhao X, Xiaoli, Zong H, Abdulla A, Yang ES,
Wang Q, Ji JY, Pessin JE, Das BC, Yang
F. 2014. Inhibition of SREBP
transcriptional activity by a boron-
containing compound improves lipid
homeostasis in diet-induced obesity.
Diabetes , 63(7): 2464-2473. doi:
10.2337/db13-0835.