Hepatoprotective Activity of Silybum marianum and Cichorium intybus Against Thioacetamide in Rat

Abstract

The plant phenolic compounds such as flavonoids have an important role in the treatment of many diseases and some of them have a potent hepatoprotective effect. In this study, we have investigated the protective effects of polyphenolic extracts of Silybum marianum and Cichorium intybus on thioacethamide-induced hepatotoxicity in rat. The extracts were injected to the rats, at a dosis of 25 mg kg^-1 body weight together whit thioacetamide at a dosis of 50 mg kg^-1 body weight. To assess the affectivity of extracts, against thioacetamide, the activity of aminotransferases (SGOT and SGPT), alkalin phosphatase, bilirubin, Na⁺ and K⁺ were measured. Significant decrease in the activity of aminotransferases, alkalin phosphatase and bilirubin was observed in the groups treated with extracts and thioacetamide compared with the group that was treated only with thioacetamide. The level of Na⁺, K⁺ and liver weight between different groups was not significantly altered. This results prove the protective effect of extracts on liver cells. The protective effects of this extracts can be due to the presence of flavonoids compounds and their antioxidant effects.

Full text

Pakistan Journal of Nutrition 7 (1): 172-176, 2008
ISSN 1680-5194
© Asian Network for Scientific Information, 2008
172
Hepatoprotective Activity of Silybum marianum and
Cichorium intybus Against Thioacetamide in Rat
H. Madani , M. Talebolhosseini , S. Asgary and G.H. Naderi
1 1 2 2
Department of Biology, Isfahan University, Isfahan, Iran
1
Department of Basic Science, Isfahan University, Isfahan, Iran
2
Abstract: The plant phenolic compounds such as flavonoids have an important role in the treatment of many
diseases and some of them have a potent hepatoprotective effect. In this study, we have investigated the
protective effects of polyphenolic extracts of Silybum marianum and Cichorium intybus on thioacethamide-
induced hepatotoxicity in rat. The extracts were injected to the rats, at a dosis of 25 mg kgG body weight
1
together whit thioacetamide at a dosis of 50 mg kgG body weight. To assess the affectivity of extracts, against
1
thioacetamide, the activity of aminotransferases (SGOT and SGPT), alkalin phosphatase, bilirubin, Na and
+
K were measured. Significant decrease in the activity of aminotransferases, alkalin phosphatase and
+
bilirubin was observed in the groups treated with extracts and thioacetamide compared with the group that
was treated only with thioacetamide. The level of Na , K and liver weight between different groups was not
+ +
significantly altered. This results prove the protective effect of extracts on liver cells. The protective effects of
this extracts can be due to the presence of flavonoids compounds and their antioxidant effects.
Key words: Antioxidants, hepatoprotective, polyphenolic compounds, thioacetamide, Silybum marianum,
Cichorium intybus
Introduction
The liver disorders are a world problem. Despite its
frequent occurrence, high morbidity and high mortality,
its medical management is currently in adequate, no
therapy has successfully prevented the progression of
hepatic diseases, even though newly developed drugs
have been used to treat chronic liver disorders these
drugs have often side effects. Therefore, that is an
essential research about suitable herbal drugs, that
could replace the chemical ones (Bruck et al., 1996).
Plant extracts have been used by traditional medical
practitioners for the treatment of liver disorders for
centuries (Schuppan et al., 1999). Phenylpropanoids or
polyphenolic compounds are a large group of herbal
chemical compounds with well-known treat mental and
protective effects (Pyo et al., 2004; Marja et al., 1999).
Silybum marianum extracts were used as early as
the 4th century B.C. for liver problems (Pyo et al., 2004).
Its pharmacological profile is well defined and studies
in cell culture and animal models clearly show its
hepatoprotective property against carbon tetrachloride,
paracetamol and Amanita phalloide toxin (Muriel and
Mourelle, 1990; Muriel et al., 1992; Vogel et al., 1984).
Silybum marianum seed extract has been called
silymarin and consist of silybin, silychristin, silydianin
and isosilybin. The flavonoid silybin constitutes 60% to
70% of silymarin (Wagner et al., 1974; Quaglia et al.,
1999). Currently standardized extract from the Silybum
marianum are used in many countries as an effective
treatment for liver diseases (Pyo et al., 2004).
Cichorium intybus is widely used as a treatment for liver
disorders. That is a potent antihepatotoxic plant and is
a major component of herbal drugs (Zafar and Mujahid
Ali, 1998). In some study aqueous and alcoholic extracts
of this plant have shown hepatoprotective activity against
carbon tetrachloride (Gazzani et al., 1999; Papetti et al.,
2002).
In the present study the hepatoprotective activities of
Silybum marianum (as positive control) and Cichorium
intybus extracts against thioacetamid toxicity was
evaluated.
Materials and Methods
Herbal extracts: Silybum marianum seeds and
Chicorium intybus were collected from Isfahan Jahad
Sazandegi Research farms and were identified by the
Department of biology of the Isfahan university. To
prepare these extracts, 100g dried herbs were initially
pulverized and deaped in ethanol (once in 96%,than in
70%). The extracts were filtered and purred then
decanted using chloroform and finally dried at 50 C in
O
an oven. This dried extract was dissolved in 10 mL
normal saline and prepared for injection.
Laboratory animals: Adult wistar male rat (200-250g)
were obtained from the Tehran Pasteur Institute. The
animals were kept at 25°C with enough humidity and
under controlled lighting (12-hours light/12-hours dark)
periods. The rats were fed standard diets and allowed
food and water ad libitum.

Madani et al.: Hepatoprotective Activity of Silybum marianum and Cichorium intybus Against Thioacetamide in Rat
173
Animals treatment: Rats were randomly divided into
four groups of five animal. Each group was kept in a
separate cage. Group I served as normal control and in
each injection received only normal saline.
Thioacetamide (Merck company, Germany) was injected
intraperitonally (50 mg kgG) in 3 consecutive days for the
1
second group (intoxicated control). In the 3rd and 4th
groups, 25 mg kgG of extract, of Silybum marianum and
1
Cichorium intybus were administered respectively with
thioacetamide at dose of 50 mg kgG intraperiotenally
1
(Amad et al., 2002).
Biochemical evaluation: Forty-eight hours after the last
injection, the rats were anesthetized with chloroform and
blood sampling was performed by cardic puncture. The
collected blood samples were allowed to clot for 45min
at room temperature. Serum was separated by
centrifugation at 4000 rpm for 20 min. Serum
aminotransferases activities including SGOT and SGPT
and also alkaline phosphatase (ALP), total bilirubinFig. 1: The activity of SGOT
values were checked using the commercial "Man
company" kits. Serum sodium and potassium values
were checked using the flame photometer.
Histological examination: After blood sampling, the rats
were dissected and their livers were separated. The
livers were fixed with 10% formalin solution. Histolologic
sections were prepared from the livers, stained and
examined under light microscope.
Statistical analysis: The data were analyzed and
compared by using the statistical test including analysis
of variances and tukey test. P value less than 0.05 were
considered as significant.
Results
Administration of thioacetamide let to significant
increase of the activity of SGOT, SGPT and ALP,
compared to the control group (Fig. 1, 2, 3). The level of
total bilirubin was also increased (Fig. 4) but there was
no significant difference in the values of sodium,
potassium and liver weight percent between the groups
(Fig. 5, 6).
Treatment with the polyphenolic extracts of Silybum
marianum and Cichorium intybus, reduced the level of
enzymes activity (SGPT, SGOT and ALP) and the level of
total bilirubin, comparing with thioacetamide group.
Values of sodium, potassium and liver weight shows no
significant difference between these groups.
In the thioacetamide treated group, microscopic
examination of the hepatic section showed that central
veins of the hepatic lobules were dilated and filled with
blood. Liver cells around central veins showed relatively
a high number of necrosis and apoptosis. Some acute
and chronic inflammatory cells also seen around the
necrotic cells (Fig. 7).
Fig. 2: The activity of SGPT
In the groups treated with polyphenolic extracts of
Silybum marianum and Cichorium intybus, central veins
were congested and dilted. Som apoptotic cell were also
seen around the central veins. The number of these
cells were much less in treated group by the Silybum
marianum. In the remaining parts of the lobules,
relatively low number of apoptosis were observed.
Necrotic cells were observed very rarely in the Silybum
marianum group (Fig. 8, 9).
Discussion
One of the major function of the liver is detoxification of
xenobiotics and toxin (Mitra et al., 1998). In many cases,

Madani et al.: Hepatoprotective Activity of Silybum marianum and Cichorium intybus Against Thioacetamide in Rat
174
Fig. 3: The activity of ALP
Fig. 4: The level of bilirubin
reactive oxygen species produce during detoxification
(Jeong et al., 1999). Over dose of toxin and some drugs
such as acetaminophen or long time use of some drugs
could produced large amounts of free radicals that
causes oxidative stress and liver injury (Jeong et al.,
2002). Because liver performs many vital functions in the
human body, damage of liver causes unbearable
problems (Mitra et al., 1998; Chattopadhyay, 2003). Thus
study about hepatoprotective compounds is of
importance.
Thioacetamide is a potent hepatotoxic that is
metabolized by Cytp450 enzymes present in the liver
Fig. 5: The level of Na
+
Fig. 6: The level of Ka+
microsomes and is converted to a toxic reactive
intermediate called thioacetamide S-oxide due to
oxidation process (Kim et al., 2000; Sanz et al., 1998).
Thioacetamide S-oxide induced oxidative stress in the
hepatic cells (Zaragoza et al., 2000; Sun, 2000). It is
responsible for the changes in cell permeability,
increase intracellular concentration of Ca , increase in
++
nuclear volume and enlargement of nucleoli and also
inhibits mithochondrial activity wich leads to cell death
and severly affecting those cells wich are located in the
perivenous acinar region (Amad et al., 2002; Diez-
Fernandez et al., 1996).

Madani et al.: Hepatoprotective Activity of Silybum marianum and Cichorium intybus Against Thioacetamide in Rat
175
Fig. 7: Histology of the liver in the thioacetamideFig. 9: Histology of the liver in the thioacetamide
treated group (Magnificatin: *40)
Fig. 8: Histology of the liver in the thioacetamide
supplemented with Silybum marianum treated
group (Magnificatin: *40)
Damage of liver cell is reflected by an increase in the
levels of hepato specific enzymes, these are cytoplasmic
in lection and are released in to circulation after cellular
damage (Sallie et al., 1991). In this study significant
increase in the total bilirubin content and in the SGOT,
SGPT and ALP activities in the thioacetamide treated
group could be taken as an index of liver damage (Zafar
and Mujahid Ali, 1998). Histopathological examination
also confirms these result. Thioacetamide induced
apoptosis and necrosis in liver cells by producing free
radicals during thioacetamide metabolism (Sun, 2000).
Treatment with polyphenolic extracts of Silybum
marianum and chicorium intybus leads to a
thioacetamide decrease, total bilirubin and SGOT, SGPT
and ALP activities as compared with thioacetamide
treated group. These confirm that polyphenolic extracts
have protective effects against hepatic cell injury induced
by thioacetamide.
supplemented with Cichorium intybus treated
group (Magnificatin: *40)
Herbal polyphenolic compounds in the cell can function
as antioxidant and antiprooxidant by scavenging reactive
oxygen species via enzymatic and non-enzymatic
reactions (Pyo et al., 2004; Marja et al., 1999; Sakihama
et al., 2002). This compounds and particularly silimarin,
have multiple mechanisms of action, that may be
hepatoprotective, including: 1) Antioxidant activity:
Flavonoid in general are good free-radical scavengers.
The best known antioxidants are vitamin A, vitamin C,
vitamin E and the mineral selenium. However, in the liver
silymarin is more that 10 times as potent as vitamin E.
2) Anti lipid prooxidation 3) Induce detoxification systems
4) protection of cell against employed glutation 5)
reduction of lukoterin formation from unsaturated free
acid 6) Enhanced protein synthesis 7) Stabilization of
mastcell 8) Regulation of immuno functions. Also
silymarin inhibit cytP450 detoxification system and
prevent metabolism of toxic compound such as
thioacetamide, tetrachloride and acetaminophen
(Papetti et al., 2002; Amad et al., 2002; Mitra et al., 1998;
Janbaz et al., 2002; Baer-Dubowska et al., 1998; Zix and
Agarwal, 1997).
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