Therapeutic Effect of Silybum marianum Plant Extract on Tamoxifen-Induced Fatty Liver in Rats
author: Nasrin Ziamajidi, Department of Clinical Biochemistry, School
of Medicine, Hamadan University of Medical Sciences, Hamadan, IR Iran.
Tel: +98-8138380574, Email: email@example.com
(TMX) is a synthetic drug that is widely used for treatment of breast
cancer. Despite the beneficial effects of TMX, the use of this drug has
several side effects such as the development of Non-Alcoholic Fatty
Liver Disease (NAFLD) in the patients. Silybum marianum is the most researched plant in treatment of liver disease.
Objectives: In this study the effect of Silybum marianum Extract (SME) on histological and biochemical parameters in TMX-treated rats was investigated.
Materials and Methods: Adult
female Wistar rats were divided to four groups; 1) normal control
untreated rats, 2) SME-treated rats, that received only SME in a dose of
1.6 g/kg body weight/day, oral gavage for 14 days, 3) TMX-treated rats,
that received TMX in a dose of 1 mg/kg body weight/day, subcutaneously,
for seven days, 4) TMX-SME-treated rats, that received TMX in a dose of
1 mg/kg body weight/day, subcutaneously, for seven days and then SME by
oral gavage for 14 days. The groups were compared in terms of serum
glucose, triglyceride, cholesterol, Alanine Aminotransferase (ALT),
Aspartate Aminotransferase (AST), Alkaline Phosphatase (ALP),
High-Density Lipoprotein-Cholesterol (HDL-C), Low-Density
Lipoprotein-Cholesterol (LDL-C), total protein and liver triglyceride
amount, and histological findings.
of HDL-C and protein decreased and activity of ALT increased in TMX
group compared with the control group. The SME increased protein
concentration and decreased ALT activity in TMX-SME group compared with
the TMX group. Histopathological examination and triglyceride assay in
liver tissues showed lipid accumulation in the TMX-treated rats and
amelioration of lipid accumulation in the liver of TMX-SME-treated rats.
Conclusions: Intake of water extract of Silybum marianum is a useful treatment for liver function in drug-induced fatty liver.
Keywords: Fatty Liver; Tamoxifen; Silybum marianum
2-[4-[(Z)-1,2-diphenylbut-1-enyl phenoxy] - N,N-dimethylethanamine, is a
synthetic non-steroidal anti-estrogen drug that is widely used for
treatment of breast cancer (1). It is also used for infertility treatment due to its stimulatory effect on the secretion of pituitary gonadotropin hormones (2, 3). Despite the beneficial effects of TMX, the use of this drug has several side effects in patients with breast cancer (4).
Hepatic side effects of TMX include elevation in liver function tests,
jaundice, hepatitis, steatohepatitis, cholestasis and massive hepatic
necrosis (5). Non-Alcoholic Fatty Liver Disease (NAFLD) is a well-known adverse effect of TMX (4).
This disease is the accumulation of fat in the liver in patients
without a history of alcohol abuse. Non-Alcoholic Fatty Liver Disease is
classified into simple steatosis and Non-Alcoholic Steatohepatitis
(NASH). Simple steatosis is a benign condition that can be reversed by
some drugs, whereas NASH is an irreversible form of the disease, in
which not only steatosis but also hepatocellular inflammation injury are
present and can often cause progress to fibrosis, cirrhosis and
Hepatocellular Carcinoma (HCC) (6).
Herbal medicines derived from plant extracts due to their low cost,
availability, and fewer side effects, are increasingly being used for
treatment of a wide variety of clinical diseases (7, 8). Among various plants, Silybum marianum (milk thistle) is the most researched plant for the treatment of liver diseases from ancient times until now (9, 10). Several studies showed that the extract of this plant protects the liver against chemical and environmental toxins (11-14).
In the current study we investigated the effect of Silybum marianum on histological and biochemical parameters in TMX-treated rats.
3. Materials and Methods
Tamoxifen was purchased from Sigma, USA. Biochemical assay kits
were purchased from Pars Azmoon, Iran. The other chemicals used were of
3.2. Preparations of Aqueous Silybum Marianum Extract (SME)
Silybum marianum extract was prepared as described previously (11).
Briefly, 5 g seeds of this plant were dissolved in 60 mL of boiling
distilled water and left to brew for 10 to 15 minutes. After filtration,
4 mL of this solution was given to the rats by gavage, daily.
3.3. Induction of Fatty Liver in Rats by Tamoxifen
Tamoxifen dissolved at a concentration of 0.2 mg/mL in sesame
oil, containing 1% benzyl alcohol, was subcutaneously injected into the
rats, 1 mg/kg body weight/day for seven days (15).
3.4. Animals and Treatment
Adult female Wistar rats weighing 190 - 210 g were purchased from
the Pasteur Institute of Iran. They were kept in metal wire cages in a
room with 12-hour light-dark cycles, at a constant temperature of 25 ±
2°C and free access to food and water. The animals were acclimatized for
at least five days under these conditions before the start of the
experiments. The rats were divided to four groups of six animals. 1)
Normal control untreated rats. 2) SME-treated rats; this group received
only SME, at a dose of 1.6 g/kg body weight/day, by oral gavage for 14
days. 3) TMX-treated rats, this group received TMX at a dose of 1 mg/kg
body weight/day, subcutaneously, for seven days. 4) TMX-SME-treated
rats, this group received TMX at a dose of 1 mg/kg body weight/day,
subcutaneously, for seven days and then SME by oral gavage for 14 days.
3.5. Biochemical Assay
Biochemical parameters such as level of glucose, triglycerides,
cholesterol, high density lipoprotein-cholesterol (HDL-C), low density
lipoprotein-cholesterol (LDL-C), and total protein in sera were
determined using Pars Azmoon diagnostic kits. For evaluation of liver
function, activities of Alanine Aminotransferase (ALT), Aspartate
Aminotransferase (AST), Alkaline Phosphatase (ALP) in sera were
determined by Pars Azmoon diagnostic kits. To distinguish triglycerides
accumulation in liver tissues, triglyceride concentration was quantified
by a colorimetric assay after extraction by the method of Folch et al. (16).
Tissues were homogenized in chloroform/methanol (2/1) to a final volume
of 20 times the volume of the tissue sample (1 g in 20 mL of solvent
3.6. Liver Histological Analysis
The liver tissues of different groups were quickly removed at the
end of treatment, cut into small pieces, soaked, and fixed in 10%
aqueous formalin to paraffin embedment. Staining of paraffin sections
with hematoxylin and eosin (H and E) was done for histological
3.7. Statistical Analysis
Statistical analysis was performed with the SPSS software Version
16 (SPSS, Chicago, IL, USA) and Analysis of Variance (ANOVA) was used
to compare means in different groups. A probability (P) value of less
than 0.05 was considered signiﬁcant in all statistical analyses. All
experiments were performed independently at least three times. Data are
expressed as Means ± Standard Deviation (SD).
shows the serum levels of glucose, protein, triglycerides, cholesterol,
HDL-C and LDL-C in control and experimental groups of rats. The levels
of glucose, triglycerides, cholesterol, and LDL-C did not change
significantly, whereas protein and HDL-C concentration were
significantly decreased (P < 0.05) in the TMX group compared with the
control group. Oral administration of SME increased the protein level
significantly (P < 0.05) and the HDL-C concentration
non-significantly, in TMX+SME group compared with the TMX group. Liver
function test results are shown in Table 2.
The activity of ALT was elevated significantly (P < 0.05) in
TMX-treated rats in comparison with the control group. Oral
administration of SME in the TMX-SME group decreased this enzyme
activity significantly (P < 0.05) close to normal levels. Activities
of AST and ALP followed the same pattern, although they were not
significant. Figure 1
depicts the level of triglycerides in the liver tissues of control and
experimental animals. A significant increase (P < 0.05) in
triglyceride level was observed in TMX-treated rats, which changed to
normal values after treatment with SME in the TMX-SME group.
Histological results confirmed biochemical data showed that SME can
improve fatty liver-induced by TMX (Figure 2).
Biochemical Parameters in the Serum of Rats a, b
Liver Enzymes Activity in the Serum of Rats a, b
Triglycerides Concentration in Liver Tissues
Liver injury is one of
the side effects of tamoxifen treatment in women with breast cancer.
Tamoxifen is a toxic drug for liver tissue because of its higher
affinity for hepatocytes compared with the other cells (17).
Among various liver problems, NAFLD is the most common condition due to
activation of fatty acid and triglyceride biosynthesis (18, 19). In these patients, liver enzymes activities in serum and liver sonography are abnormal (4).
In this study tamoxifen was used in order to induce fatty liver in an
animal model. As expected, liver enzyme activities in serum of
TMX-treated rats elevated indicating liver injury. Although the increase
of ALT serum levels was significant, it was not considerable for AST
and ALP. High triglyceride concentration in liver tissues of TMX-treated
rats proved lipid accumulation and fatty liver in these kinds of rats.
Histopathological examination confirmed these biochemical results.
Moreover, significant decrement of total protein concentration in serum
of TMX-treated rats, was the reason for liver dysfunction. In similar
studies with induction of fatty liver by drugs in animal models,
different plant extracts were used for improvement of liver function (1, 7, 20). Madani et al. showed that Silybum marianum and Cichorium intybus had protective effects on rat liver cells treated by thioacetamide (10). Butt et al. demonstrated that Cichorium intybus had a hepatoprotective effect on paracetamol-induced liver damage in rats (21). Ozturk et al. showed that Silybum marianum had a preventive effect on CCl4 induced liver damage (11). In this study Silybum marianum
was used for treatment of lipid accumulation in fatty liver induction
by tamoxifen. Because SME have a strong protection effect against
multiple drugs (10, 22),
applying SME in TMX-treated rats decreased liver enzymes activities
close to normal levels. In addition, the rise of the serum total protein
indicated that liver function was improved in the presence of SME. One
of the most important problems caused by some drugs such as tamoxifen,
is the induction of oxidative stress during their metabolisms in the
Since different plant extracts such as SME have antioxidant components,
it might be that these compounds protect cells from damage caused by
oxidative stress. On the other hand, the effect of SME on ameliorating
of fatty liver is possibly due to inhibition of fatty acid synthesis or
increasing of mitochondria and peroxisomal β-oxidation (18, 19).
Data of this study indicated that SME has therapeutic effects on
tamoxifen induced fatty liver. Further investigations are needed to
determine the involved mechanisms.
El-Beshbishy HA. The effect of dimethyl dimethoxy biphenyl dicarboxylate
(DDB) against tamoxifen-induced liver injury in rats: DDB use is
curative or protective. J Biochem Mol Biol. 2005;38(3):300-6. [PubMed]
Adashi EY, Hsueh AJ, Bambino TH, Yen SS. Disparate effect of clomiphene
and tamoxifen on pituitary gonadotropin release in vitro. Am J Physiol. 1981;240(2):E125-30. [PubMed]
Gill-Sharma MK, Balasinor N, Parte P, Aleem M, Juneja HS. Effects of
tamoxifen metabolites on fertility of male rat. Contraception. 2001;63(2):103-9. [PubMed]
CL, Huang JK, Cheng SP, Chang YC, Lee JJ, Liu TP. Fatty liver and
transaminase changes with adjuvant tamoxifen therapy. Anticancer Drugs. 2006;17(6):709-13. [DOI] [PubMed]
Nishino M, Hayakawa K, Nakamura Y, Morimoto T, Mukaihara S. Effects of
tamoxifen on hepatic fat content and the development of hepatic
steatosis in patients with breast cancer: high frequency of involvement
and rapid reversal after completion of tamoxifen therapy. AJR Am J Roentgenol. 2003;180(1):129-34. [DOI] [PubMed]
Ziamajidi N, Khaghani S, Hassanzadeh G, Vardasbi S, Ahmadian S, Nowrouzi
A, et al. Amelioration by chicory seed extract of diabetes- and oleic
acid-induced non-alcoholic fatty liver disease (NAFLD)/non-alcoholic
steatohepatitis (NASH) via modulation of PPARalpha and SREBP-1. Food Chem Toxicol. 2013;58:198-209. [DOI] [PubMed]
El-Beshbishy HA. Hepatoprotective effect of green tea (Camellia
sinensis) extract against tamoxifen-induced liver injury in rats. J Biochem Mol Biol. 2005;38(5):563-70. [PubMed]
Gilani AH, Rahman AU. Trends in ethnopharmocology. J Ethnopharmacol. 2005;100(1-2):43-9. [PubMed]
Abenavoli L, Capasso R, Milic N, Capasso F. Milk thistle in liver
diseases: past, present, future. Phytother Res. 2010;24(10):1423-32. [DOI] [PubMed]
Madani H, Asgary S, Talebolhosseini M, Naderi GH. Hepatoprotective
Activity of Silybum marianum and Cichorium intybus Against Thioacetamide
in Rat. Pak J Nutr . 2008;7(1):172-6. [DOI]
Ozturk M, Akdogan M, Keskin I, Kisioglu AN, Oztas S, Yildiz K. Effect of
Silybum marianum on acute hepatic damage caused by carbon tetrachloride
in rats. Biomed Res. 2012;23(2):268-74.
KR, Kowdley KV. A review of Silybum marianum (milk thistle) as a
treatment for alcoholic liver disease. J Clin Gastroenterol. 2005;39(6):520-8. [PubMed]
Flora K, Hahn M, Rosen H, Benner K. Milk thistle (Silybum marianum) for
the therapy of liver disease. Am J Gastroenterol. 1998;93(2):139-43. [DOI] [PubMed]
Elitok B. Efficacy of Herbal Remedies in The Treatment of Nonalcholic
Fatty Liver Disease/Hepatic Steatosis in Human and Animals. Kocatepe Vet J. 2012;5(2)
EA, Solheim E, Ueland PM. Distribution of tamoxifen and its metabolites
in rat and human tissues during steady-state treatment. Cancer Res. 1991;51(18):4837-44. [PubMed]
Folch J, Lees M, Sloane Stanley GH. A simple method for the isolation
and purification of total lipides from animal tissues. J Biol Chem. 1957;226(1):497-509. [PubMed]
Desai PB, Nallani SC, Sane RS, Moore LB, Goodwin BJ, Buckley DJ, et al.
Induction of cytochrome P450 3A4 in primary human hepatocytes and
activation of the human pregnane X receptor by tamoxifen and
4-hydroxytamoxifen. Drug Metab Dispos. 2002;30(5):608-12. [PubMed]
LK, Jacobs RL, Vance DE. Tamoxifen induces triacylglycerol accumulation
in the mouse liver by activation of fatty acid synthesis. Hepatology. 2010;52(4):1258-65. [DOI] [PubMed]
Gudbrandsen OA, Rost TH, Berge RK. Causes and prevention of
tamoxifen-induced accumulation of triacylglycerol in rat liver. J Lipid Res. 2006;47(10):2223-32. [DOI] [PubMed]
Al-Jassabi S, Saad A, Al-Omari A. Toxic effects of tamoxifen and the
protective role of silymarin and zizyphus. Middle East J Sci Res. 2011;9:110-4.
K, Yunas S, Sheikh RM. Hepatoprotective Effect of Cichorium intybus on
Paracetamol Induced Liver Damage in Albino Rats. Libyan Agric Res Center J Int. 2012;3(2):60-3.
SH, Cheon HJ, Yun N, Oh ST, Shin E, Shim KS, et al. Protective effect of
a mixture of Aloe vera and Silybum marianum against carbon
tetrachloride-induced acute hepatotoxicity and liver fibrosis. J Pharmacol Sci. 2009;109(1):119-27. [PubMed]
Ferlini C, Scambia G, Marone M, Distefano M, Gaggini C, Ferrandina G, et
al. Tamoxifen induces oxidative stress and apoptosis in oestrogen
receptor-negative human cancer cell lines. Br J Cancer. 1999;79(2):257-63. [DOI] [PubMed]