The effects of aqueous extract of chicory root on steatosis, lipid profile and liver damage enzyme markers in tamoxifen-treated rats

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Molecular and Biochemical Diagnosis (MBD)
Vol 1, No 3, 2014
Original Article
Molecular and Biochemical Diagnosis (MBD).Vol.1, No.3 (2014), 185-194
185
The effects of aqueous extract of chicory root on steatosis, lipid
profile and liver damage enzyme markers in tamoxifen-treated
rats
Abolfazl Nasiri1, Nasrin Ziamajidi1*, Hamid Behrouj1, Roghayeh Abbasalipourkabir1,
Arash Dehghan2
1. Department of Clinical Biochemistry, School of Medicine, Hamadan University of Medical Sciences, Hamadan, Iran
2. Department of Pathology, School of Medicine, Hamadan University of Medical Sciences, Hamadan, Iran
Abstract
Background: Accumulation of triglycerides in the liver i.e. steatosis, is a well-known side-
effect of tamoxifen administration to patients suffering from breast cancer. Cichoriumintybus
(chicory) is a plant used as traditional medicine for curing liver disorders. In this study, the
effects of extract prepared from chicory roots on tamoxifen-induced liver steatosis and related
biochemical factors in animal model using rats has been investigated.
Methods: Female rats of Wistar strain were divided into four groups and treated as follows;
1-Control: received vehicle; 2- Chicory root-extract treated: rats were given by gavage the
aqueous chicory root extract (1 g/kg body weight/day for 14 days).3- Tamoxifen-treated: rats
received tamoxifen (1 mg/kg body weight/day, for 7 days). 4- Tamoxifen+chicory-group:
animals received tamoxifen (1 mg/kg body weight/day for 7 days) followed by chicory extract
given by gavage (1 g/kg body weight/day for 14 days). After treatment, blood was collected
by cardiac puncher, plasma was separated and plasma levels of glucose, total protein,
triglyceride, cholesterol,LDL-C, HDL-C and activities of ALT, AST and ALP were
measured. Liver tissues were homogenized used for measuring tissue triglyceride and
histological examinations.
Results: The data show that tamoxifen treatment caused a significant decrease in the level of
serum cholesterol, HDL-C and total protein. However, serum ALT level was increased in
tamoxifen-treated rats compared to controls. Increased serum ALT in tamoxifen-treated rats
was recovered in rats treated with plant extract (tamoxifen+chicory-group). HDL-C and total
protein levels were unaffected in rats fed chicory extracts. Tamaxifen-treated animals showed
signs of liver steatosis as shown by histological examination and accumulation liver
triglyceride. The steatosis markers such as accumulated triglyceride in liver was significantly
reduced due to the plant extract treatments when compared to tamoxifen-group.
Conclusions: Dietary extract prepared from chicory roots is effective in modulation of
tamoxifen-induced liver damage and steatosis.
Keywords: Cichoriumintybus, Steatosis, Tamoxifen, Rats, liver damage
Introduction1
Steatosis of fatty liver is one of the chronic
*Corresponding auth or. Nasrin Ziamaji di, PhD.
Department of Clinical Biochemistry, School of Medicine,
Hamadan University of Medical Sciences, Hamadan, Iran
Tel:+98-8138380574
Email: n.ziamajidi@umsha.ac.ir
liver diseases which affect 10-24% of total
world population. Initially it appears as benign
steatosis, however the disease can progress to
steatohepatitis, fibrosis, cirrhosis, and
hepatocellular carcinoma.

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186 Molecular and Biochemical Diagnosis (MBD). Vol.1, No.3 (2014), 185-194
Non-alcoholic fatty liver disease (NAFLD) is
the results of accumulation of lipids, especially
triglycerides in the liver cells in patients
without a history of alcohol abuse. Several
factors are causing NAFLD, including obesity,
metabolic syndrome, and consumption of some
drugs (Paschos and Paletas, 2009).
Tamoxifen or Nolvadex is one of the drugs that
may induce fatty liver (Murata et al., 2003; Liu et
al., 2006). Tamoxifen, 2-[4-[(Z)-1,2-diphenylbut-
1-enyl]phenoxy]-N,N-dimethylethanamine, is a
synthetic non-steroidal medication that is
widely used for treatment of patients with
estrogen receptor-positive breast cancer (El-
Beshbishy, 2005). Due to its stimulatory effect
on secretion of pituitary gonadotropin, it is
also used in the treatment of infertility and
bodybuilding supplements (Adashi et al., 1981;
Motrich et al., 2007). Several studies have
shown that approximately one third of breast
cancer patients treated with tamoxifen develop
hepatic steatosis (Nemoto et al., 2002).
CichoriumIntybusL., commonly known as
chicory or Kasni (from the Asteraceae family),
is a perennial plant with blue flowers that are
grow in different regions in Iran. It is one of
the oldest known plants used traditionally for
treatment of liver diseases. The protective and
curative effects of the root, leaves, and seeds
of chicory have been investigated (Street et al.,
2013). The major components of chicory roots
are carbohydrates such as fructooligosaccharides
and inulin and the minor components are
polyphenols (Milala et al., 2009). Inulin has
polyfructan structure which is water soluble
and not hydrolyzed by digestive enzymes and
belongs to a group of dietary fiber. Based on
this it has been suggested that the water extract
of chicory root possess hyperlipidemic
properties (Kim and Shin 1998).
The objective of the current study was to
examine the possible effects of aqueous extract
of chicory root on fatty liver and related
biochemical and histological markers in an
experimental model of steatosis induced by
tamoxifen.
Materials and Methods
Preparation of plant extract
Chicory roots were purchased from the
medicinal herb store in Hamadan, Iran. A
voucher number of specimen was deposited at
the herbarium of Bu-Ali Sina University of
Hamadan.
The aqueous extract was prepared according to
the method described by Kim and Shin, 1998
with modification. Briefly, the plant roots were
ground into a powder using electric mill,
dissolved in distilled water in the ratio of 20%
(W/V), and mixed for 50 min at 70°C with
continuous stirring. The mixture was allowed
to cool at room temperature before passing
through Whatman No. 1 filter. Wherever stated
the animals were treated with the plant extract

Modulation of Steatosis by Chicory Extract
Molecular and Biochemical Diagnosis (MBD). Vol.1, No.3 (2014), 185-194
187
of 1 g/kg body weight by gavage for 14 days.
Preparation of tamoxifen solution
Tamoxifen (Sigma Chemical Co., USA) was
dissolved at a concentration of 0.2 mg/ml in
sesame oil containing 1% benzyl alcohol. For
steatosisinduction in rats, each rat received
subcutaneously 1 mg/kg body weight/day for 7
days according to the method described by
Lien et al., 1991.
Treatments
Twenty-four adult female Wistar rats weighing
200±10 g were purchased from Pasteur
Institute of Iran, IRAN. They were housed in
standard cages in animal house and had free
access to food and water. The room
temperature was maintained at a temperature
of 25±2 °C and 12 h light-dark cycle. The
animals were acclimatized for at least 5 days
under this condition before the start of the
experiments.
The rats were randomly divided into four
groups (n=6) and treated as follows:
Group-1, controls treated with vehicle. Group-
2, rats received tamoxifen (1 mg/kg B.W/day,
subcutaneously for 7 days. Group-3) rats
received tamoxifen+chicory. Tamoxifen at a
concentration of 1 mg/kg B.W/day
subcutaneously, for 7 days followed by
aqueous extract of chicory root extract at a
concentration of 1 g/kg B.W/day, gavage, for
14 days. Group-4 rats treated with aqueous
chicory root extract (1 g/kg B.W/day by
gavagefor 14 days.
Blood was collect from each rat; serum was
separated and used for biochemical assays.
Liver tissue was removed, a portion was fixed
in 10% formalin and send to pathology
department for sectioning and staining by
H&E staining method. One gram of liver tissue
was homogenized in chloroform/methanol
solution (2:1 v/v) and used for determination
of triglycerides.
Biochemical assays
Biochemical assay kits were purchased from
Pars Azmoon company of Iran. Glucose,
cholesterol, triglycerides, low density
lipoprotein-cholesterol (LDL-C), high density
lipoprotein-cholesterol (HDL-C), and total
protein levels were measured in the serum of
rats. Liver damage enzymes namely; serum
alanine aminotransferase (ALT), aspartate
aminotransferase (AST) and alkaline
phosphatase (ALP) activities were also
measured.
Triglyceride level was determined in liver
homogenate using a colorimetric assay
following the procedure of Folch et al., 1957
Briefly.
Histological analysis of liver tissues
A small portion of liver tissue obtained from

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188 Molecular and Biochemical Diagnosis (MBD). Vol.1, No.3 (2014), 185-194
each rat was fixed in 10% aqueous formalin
solution, washed with 70% ethanol, dehydrated
using alcohol series from 70% to 100% alcohol
and embedded in paraffin. The paraffin
sections were stained with hematoxylin and
eosin (H&E) dyes and observed under a light
microscope. Histologic scoring system for
non-alcoholic fatty liver disease was
determined according to the method described
by Kleiner et al., 2005: Score 0=5%, score
1=5-33%, score 2=33-66% and score 3=66%
of affected area.
Statistical analysis
Data were expressed as mean ± standard
deviation (SD) and analyzed with SPSS
software Version 16 (SPSS, Chicago, IL,
USA). Comparison between groups was
conducted with one way analysis of variance
(ANOVA) followed by leasts significant
difference (LSD) post hoc test. Results were
considered statistical significant at the
probability (P) value of 0.05.
Results
The results of this study showed that serum
glucose was unaffected in the treatment groups
compared to controls. However, serum total
protein was decreased significantly (P< 0.05) in
tamoxifen-treated rats compared with control
group. Chicory administration by gavage
tamoxifen+chicory group non-significantly
increased protein concentrations (Table 1).
Although there was no significant change in the
serum activities of AST and ALP between the
different groups, but ALT activity elevated
significantly (P< 0.05) in tamoxifen group
compared with control group and it decreased
significantly (P< 0.05) in tamoxifen+chicory
group compared with tamoxifen group (Table 2).
Table 1 Comparison of glucose and total protein concentrations in the serum of rats in different groups.
Groups
Glucose (mg/dl)
Total Protein (g/dl)
Control
175.6 ± 17.5
8.1 ± 0.7
Tamoxifen
161.6 ± 13.8
7 ± 0.2 *
Tamoxifen+Chicory
170.6 ± 14.6
7.3 ± 0.5
Chicory
170 ± 23.2
7.7 ± 0.7
Values are means ± SD;
*P< 0.05, significantly different from Control group.
Table 2 Comparison of hepatic enzymes activity in the serum of rats in different groups.
Groups
ALT (U/L)
AST (U/L)
ALP (U/L)
Control
56 ± 5.8
91.5 ± 8.7
323 ± 52.6
Tamoxifen
77 ± 15.6 *
118.6 ± 5
386 ± 106.1
Tamoxifen+Chicory
56.1 ± 12.4 †
140 ± 33.1
387.8 ± 199
Chicory
53.3 ± 9.5
144.3 ± 12.1
485.8 ± 275.9
Values are means ± SD;
*P< 0.05, significantly different from Control group;
†P< 0.05, significantly different from Tamoxifen group.

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Molecular and Biochemical Diagnosis (MBD). Vol.1, No.3 (2014), 185-194
189
Figure 1 Effects of tamoxifen and chicory extracts on liver triglyceride levels in rats. Values are means ± SD; *P< 0.05,
significantly different from Control group.
Figure 2 Histopathology of rat liver tissue in different groups. The score of steatosis were, 1 in control group, 3 in tamoxifen
group, 2 in tamoxifen+chicory group and 1 in chicory group.
Comparison of the lipid profile did not show
significant alteration in triglyceride and LDL-
C values in different group but cholesterol and
HDL-C concentrations decreased significantly
(P< 0.05) in tamoxifen group compared with
control group (Table-1)
Data presented in Figure 1, show that the
concentration of triglyceride in the liver tissue
of tamoxifen group was elevated significantly
(P< 0.05) compared to control group. The

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190 Molecular and Biochemical Diagnosis (MBD). Vol.1, No.3 (2014), 185-194
triglyceride level was partly recovered in rats
treated with the plant extract (tamoxifen+
chicory group) when compared to that in
tamoxifen group.
Based on histological observations rat the
steatosis score wasin control group, tamoxifen
group, tamoxifen+chicory group and chicory
group was determined as; 1, 3, 2 and 1,
respectively. The results showed that chicory
extract could reverse liver steatosis score
which was induced by tamoxifen (Figure 2).
Discussion
Tamoxifen is used effectively in the treatment
of breast cancer (Mandlekar et al., 2000). One
of the most predominant side-effects of this
drug, is liver dysfunction especially fatty liver
(Nishino et al., 2003). Previous studies
demonstrated that breast cancer patients taking
tamoxifen suffer from breast cancer also suffer
from liver steatosis (Ogawa et al., 1998).
In the current research using an animal model
of steatosis, we showed that tamoxifen
treatments can cause liver dysfunction. A
significant decrease in serum total protein
along with elevation of serum ALT was the
first signs of liver damage in tamoxifen-treated
animals. Accumulation of triglyceride in
hepatocytes that was demonstrated by
biochemical and histological observations
further attest to this finding. This finding was
in accordance with other reports showing that
tamoxifen- related changes in lipid profile
(Morales et al., 1996).
Tamoxifen treatment was associated with a
significant decrease in cholesterol and HDL-C
compared to control group. The effect of
tamoxifen on total cholesterol and LDL-C was
in accordance with the report by Novoa et al.
(2002) who showed that tamoxifen decreased
total cholesterol and LDL-C in male patients
having pubertal gynecomastia, whereas,
tamoxifen treatment had no effect on
triglyceride and HDL-C levels. It seems that
changes in lipid profile in response to
tamoxifen treatments varies depending on the
dose and mode of treatments.
It has been suggested that the complications
raised by conventional drugs such as tamoxifen
can be prevented by consuming the medicinal
plant preparations (Madani et al., 2008; Ozturk
et al., 2012).In this connection, previously it has
been reported that green tea and the dimethoxy
biphenyl dicarboxylate possess protective and
curative effects on tamoxifen-induced liver
injury (El-Beshbishy, 2005). Likewise, the
protective effects of milk thistle extract on fatty
liver induced by tamoxifen have been reported
(Behrouj et al., 2015).
Chicory is well known for its hepatoprotective,
anti-diabetic and lipid lowering properties
(Pushparaj et al., 2007; Madani et al., 2008;
Ghamarian et al., 2012). According to Kim et
al. (1998) chicory extract can improve lipid

Modulation of Steatosis by Chicory Extract
Molecular and Biochemical Diagnosis (MBD). Vol.1, No.3 (2014), 185-194
191
metabolism. It has also been shown that
chicory extract can ameliorate diabetes-
induced fatty liver in rat models (Ziamajidi et
al., 2013).The present study provides
evidences for hepatoprotective action of
chicory. Based on this experimental study, oral
administration of chicory to tamoxifen-treated
rats was effective in modulation of serum ALT
and liver steatosis induced by tamoxifen.
The modulatory effects of chicory on liver
steatosis is probably due to increased β-
oxidation or decreased biosynthesis of fatty
acids and triglycerides (Gudbrandsen et al.,
2006; Cole et al., 2010; Moya et al., 2010).
Also in current study chicory extract did not
have any effect on the serum lipid profile. This
data is justified by knowing that fatty liver did
not parallel with changes in serum total
triglyceride level (Cole et al., 2010).
In the other words, fatty liver patients may be
did not have any hyperlipidemia. However it
seems that the duration of treatment time are
determining factors in these experimental
setups.
In conclusion, based on this experimental
study it was shown that oral administration of
chicory root extract in tamoxifen-treated rats
modulated tamoxifen-related hepatic damages
as shown by changes in serum ALT and
steatosis in the liver. It seems that the aqueous
extract of this plant has beneficial effects on
liver function in drug-induced liver injury,
although more investigations are needed to
further confirm the related pathways and
mechanisms.
Acknowledgement
The author would like to acknowledge the
supports provided by Hamadan University of
Medical Sciences, Hamadan, Iran.
Conflict of interest
The authors declare that there are no conflicts
of interest.
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