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The aim of the study was to evaluate the antioxidant action of the medicinal herb
u on rat liver hepatitis induced by carbone tetrachloride (CCl4), and
the content of selenium in this herb. The results showed that the content of selenium in the
surface part of u   was 0.34 ppm, while in the infusion it was 0.14
O ml and 0.12 O ml in the extract.
The most important antioxidant aspect of selenium is its function in the active site of
selenoenzyme Olutathione peroxidase. Glutathione peroxidase (GSHPx) not only allows the
removal of the toxic radicals but also permits the reOeneration of lipid molecules throuOh
reacylation in the cellular membrane. Thus, GSHPx may prevent the harmful effects of free
radicals and may reduce the formation of the reactive metabolites of carbon tetrachloride. Carbon
tetrachloride is a hepatotoxic aOent which Oenerates haloalkane radicals durinO its
biotransformation in the liver and is widely used to make the experimental model of hepatic
damaOe. Therefore, the aim of the present study is to investiOate the possible protective role of
selenium on the experimental liver cirrhosis and some enzyme activities in blood plasma from
rats.
£
º u  Ñ SeleniumÑ atomic absorption spectrometerÑ Liver
enzymesÑ InjuryÑ Carbon tetrachlorideÑ
ÿ

 
The liver is especially sensitive to carbon tetrachloride, commonly used as an
experimental model for the injury and cirrhosis in the liver of the rats 1,2,3,4. Carbon tetrachloride
is metabolized to haloalkane radicals, these radicals may cause the oxidative damaOe of lipids
1,2,3,4,5,6
lipoproteins and other cellular components, such as enzymes, nucleic acids and proteins .
Oxidative damaOe of these components is beinO identified as an important factor in the etioloOy
of deOenerative human liver diseases. Several antioxidants were scavenOed free radicals7,25,26,27
and these radicals in hepatic cellular structures may increase the cellular deOeneration. This
process may be able to affect the levels of liver enzymes due to damaOe of cellular membrane.
Thus, abnormal levels of the liver enzymes in plasma are usually indicative of the hepatic cellular
injury in experimental animals 1,2,3,4,5,6,9.
Selenium is an essential element, plays an antioxidant role, bindinO active site of Olutation
peroxidase (GSHPx). The most important metabolic roles of selenium in mammalian cell occur
due to its function in the active site of selenoenzyme-GSH-Px. GSH-Px not only protects cells
aOainst damaOes by free radicals but also permits reOeneration of a membrane lipid molecule
throuOh reacylation8. Antioxidants may prevent oxidative damaOe caused by free radicals in
bioloOical structures. Interactive relations between antioxidants and carbon tetrachloride may
chanOe the toxicity of hepatotoxic aOent 7,8,9.
The interrelationships between protective effects of the antioxidants and toxic effects of
carbon tetrachloride have been investiOated and it has been reported that administrations of
3,10,11,12
vitamins A , C10,11,12, E4,13, selenium17, selenium±vitamin E18,19,20 and ȕ-carotene20,22
protected the animals from some harmful effects of carbon tetrachloride. Therefore, practical and
effective methods for prevention of the liver injury are very important. The present study was
desiOned to investiOate the effects of selenium on activities of functional liver enzymes in blood
plasma and on the liver fibrosis and cirrhosis Oenerated by carbon tetrachloride in rats.

 



The fresh leaves of ZyOophyllum Potaninii Maxim were collected personally in
the late summer season in the forest area of Gobi-Altai province of MonOolia and authenticated
by specialists of the Dept. of PharmacoloOy, Health Sciences University and Dept. of Plant
Systematics of Herbarium, Institute of BioloOy, Academy of Science MonOolia. The leaves were
fresh dried, powdered and kept for water extract.
 
 
  
 

The water extract was prepared referrinO to the clinical
use of the herb prior to animal treatment. 1 O of powdered leaves was incubated with 10 ml of
deionized water for 15 min at 100°C for further use in animal treatments.
  
      

  1 O of dried leaves was burned at temperature of
530°C in the furnace for 6-7 hours and made into ashes. Ash was dissolved at a temperature of
150°C in 10 ml of HNO3 with a concentration of 1º1. The selenium concentration was determined
usinO atomic absorption spectrometer (Perkin-Elmer Model 5000 Germany) equipped with
Perkin-Elmer HGA-500 Oraphite furnace. Pyrocoated Oraphite tubes were used. The absorbance
was measured at 196.0 nm with a spectral bandwidth of 0.7 nm. A selenium electrodeless
discharOe lamp was operated at a power settinO of 6 watts. The Oraphite furnace settinOs
consistinO of dryinO, charrinO and atomizinO cycles are listed in Table 1.

J Graphite Furnace SettinOs

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? ? ? ? ?  ?

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? ? ?

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?????????????????????????????????????????????????????????? ? ? ???
?
The purOe Oas was arOon. DurinO the atomization cycle the purOinO Oas stream was
interrupted by proOramminO ³stopped flow´ control to increase the residence time of the free
selenium atoms in the liOht beam and to enhance the sensitivity.
c  
 


This study was carried out on 30 Wistar albino male rats weiOhinO 200±250 O body
weiOht. All rats were fed rodent pellets and drinkinO water ad libitum. The animals were
randomly divided into three equal Oroups containinO 10 rats housed in caOes at room temperature
durinO the study.
The first Oroup was used as control and only placebo (physioloOical saline 0.9%) was
injected subcutaneously. The second Oroup was subcutaneously injected CCl4 (0.8ml kO body
weiOht) dissolved in 0.5 ml heliante oil. The third Oroup was subcutaneously injected CCl4 0.8
ml kO plus 1º10 uwater extract at the dose of 200mO kO. CCl4 was
diluted with heliante oil (1 4 v v) for experimental Oroups and these treatments were
administrated three times in a week for a period of 12 weeks. Mortality rates were amonO 5±20%,
which are similar to those reported by investiOators usinO this model19. After 24 h of the last dose
beinO administrated, the blood samples were taken and the animals were sacrificed under ether
anesthesia. The abdomen was opened and the liver was removed and fixed in formaldehyde (10%
v v) for histopatholoOical examination CCl4 was purchased from Merck AG (Darmstadt,
Germany).
D
  
    
  

The blood of all animals was taken by cardiac puncture 12 h after the last application of
CCl4 and water extractu. Whole blood samples were collected in the
heparinizedtubes (Beckon Dickinson Vacuntiner System Cedex, France) subsequently
centrifuOed at 1500xO for 15 min in a Heraus Inst MeOa FuOe 10 and their plasma was removed
into disposable pipettes. In plasma samples, the activities of aspartate aminotransferase (AST),
alanine aminotransferase (ALT), alkaline phosphatase (ALP) and Oamma-Olutamyle transferase
(GGT) were determined by an autoanalyzer (Technicon RA-XT,New York, USA).


  
 
  
 Liver specimens were embedded in paraffin
for anordinary histoloOical examination and sectioned 3±5 mm serial sections usinO a rotary
microtome. The sections were stained with hematoxylin and eosin (H&E) for histoloOical
observations. The histoloOical analyses were performed unawares from Oroup separations by liOht
microscopy. The hepatic injuries were assessed accordinO to Manna et al. (1996). HistoloOical
OradinO was made accordinO to four severity Oradesº 0 (none) no fibrosis and normal liver
architectureÑ I (mild) fatty deOenerations around portal areas and central veins fibrosis increased
in portal areas and sinusoidal space and reOular liver architectureÑ II (moderate) thin fibrous
septa present connectinO portal areas and pseudolobules seen in frequentlyÑ and III (severe) thick
fibrous septa and collaOen bands accompanied by pseudolobules.
Statistical analysis Data were expressed as mean SD. SiOnificance of difference was evaluated
usinO Student¶s t-test and p<0.05 were taken as siOnificant.
Results
The concentration of selenium in the surface part of u  was
determined to be 0.34 ppm, while in the infusion it was 0.14 O ml and in extract 0.12 O ml.
The levels of AST, ALT, ALP and GGT are shown in Table 2. While the activities of
AST, ALT and ALP were siOnificantly increased (p<0,05Ñ p<0,05 and p<0,01Ñ respectively),
GGT activity was not statistically affected (p>0,05) by CCl4-injection. In addition, the
differences between the activities of AST, ALT and GGT (except for ALP) in CCl4-Oroup and the
levels of AST, ALT and GGT in Oroup of CCl4 plus selenium were not siOnificant but the values
of ALP was increased in both CCl4 and CCl4 plus Se-Oroups (p<0,01 and p<0,01Ñ respectively).
While the levels of ALT and ALP in control-Oroup were lower (p<0,05 and p<0,01Ñ respectively)
than those of that in both CCl4 and CCl4 plus Se-Oroups. There are no statistical difference
between the values of AST, ALT and GGT in control-Oroup and the values of the same
enzymatic parameters in CCl4 plus Se-Oroup (Table 2).
J Enzyme activities (U L) of AST ALT ALP and GGT after 12th week in all
Oroups
Groups AST ALT ALP GGT
Control 47.48 2.99 41.9 2.81 59.3 3.1 7.83 1.64
CCl4 72.86 2.35*** 51.73 1.2** 79.5 4.21** 14.6 2.0***
CCl4+Se 52.25 1.1*** 34.07 1.4*** 863 3.6** 5.1 0.85***
ööööööööööööööööööööööööööööööööööööööööööööööööööööööööööööööööööööööööööööö
       !"##$  !!"##     
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HistopatholoOical examinations showed that CCl4 caused hepatic injury. These
histopatholoOical chanOes siOni.cantly decreased in animals treated with selenium plus CCL4 in
compared with the Oroup injected only CCl4 (Table 3).

J   HistopatholoOical OradinO for hepatic injury in liver of control CCl4 and CCl4
plus selenium Oroups
HistopatholoOical OradinO

ööööööööööööööööööööööööööööööööööööööööööööööööööööööööööööööööööööööööööööööööööööööööööööö

Groups n 0 I II III
_____________________________________________________________________________
Control 19 19 0 0 0
CCl4 16 0 0 5 11
CCl4+selenium 15 0 6 8 4
_____________________________________________________________________________
The liver specimens in control animals showed no evidence of histopatholoOical
alterations. The chronic effect of CCl4 on the rat liver usually showed a typical fibrotic and
cirrhotic appearance. The collaOen bands bridOed between portal areas and portal areas or were
extended from central reOions to portal areas. Broad fibrous septa were often seen surroundinO
the liver lobulus. These biopsies were interpreted as stronO suOOestion of cirrhosis (FiO. 5).
 The liver sections obtained from animals treated with selenium showed a consistent
reduction in necrotic fibrotic and cirrhotic processes of the liver. These specimens showed more
reOular liver architecture in which only thin fibrous bands were seen to connect portal areas (FiO.
19).

X Representative slides of H&E-stained liver tissue from (5) CCl4-injured rats Oroup
shows a typical cirrhotic appearance thick fibrous septa were seen bridOed between from central
reOions and portal areas and prominent intralobular inflammatory reaction consistinO of
Oranulocytes and mononuclear inflammatory cellsÑ (19) CCl4 plus selenium-Oroup fibrous
chanOes were seen in portal areas and low deOree intralobular inflammatory reaction consistinO of
Oranulocytes and mononuclear inflammatory cells.
ü 
In vivo and in vitro experiments demonstrate that the antioxidants play protective roles
6,7,8,9
aOainst free radicals . The liver is an important tarOet orOan for CCl4 and the hepatocytes
may be damaOed by haloalkane free radicals produced durinO biotransformation of CCl4 1,2. CCl4
is metabolized in mixed function oxidase system utilizinO the nicotinamide adenine dinucleotide
phosphate (NADPH)±cytochrome P-450 electron transport chain at the level of the hepatic
smooth endoplasmic reticulum and the hemolytic cleavaOe may occur durinO the formation of the
haloalkane free radicals such as trichloro-methyle (CCl3) radical and trichloromethyleperoxy
(CCL3OO) radical. These radicals cause the oxidative damaOe of lipids, lipoproteins and other
biochemical parameters such as enzymes, nucleic acids and proteins. DamaOe of these
components may be an important factor in the pathoOenesis of different liver diseases. In
addition, haloalkane free radicals may bind to cellular macromolecules and can react with free
amino Oroups on proteins and then the macromolecules may lose their physioloOical functions
1,2,5,6
. Thus, the AST, ALT, ALP and GGT may be mobilized into blood plasma and serum
levels of these enzymes may increase. HiOh levels of these enzymes in serum are usually
indicative for acute hepatitis in experimental animals 2,3,4,6 and humans 22,23,24. Indeed, it has been
reported that haloalkane free radicals were held responsible for CCl4-hepatotoxicity and caused
the oxidative injury of unsaturated lipids in some cellular components of hepatic tissues 1,2.
The antioxidant Se may diminish the hepatotoxic effects of CCl4-metabolites by means of
their interactive relations with intermediary metabolites. The most important antioxidant aspect
of Se is its function in the active site of selenoenzyme Olutathione peroxidase. GSH-Px
containinO Se catalyzes the destruction of hydroOen peroxide and lipid hydroperoxides via
8,25
reduced Olutathione . Also, GSH-Px and the other antioxidants such as superoxide dismutase
and catalase may protect cellular membranes aOainst oxidative damaOe caused by toxic free
radicals and so may partially diminish certain types of the hepatic cellular deOeneration. In
addition, GSHPx not only allows the removal of the toxic ROOH but also permits the
4,8,25
reOeneration of lipid molecules throuOh reacylation in the cellular membrane . Reactive
metabolites are produced durinO biotransformation of CCl4 and these metabolites may cause
cellular death in the liver. AST, ALT and ALP may be released into blood plasma and serum
levels of these enzymes may increase as due to the cellular damaOe in the liver16. Thus, the levels
of ALP in blood plasma may also increase in the early periods of liver damaOe 9. HiOh levels of
AST, ALT and ALP in serum are usually indicative of disease and necrosis in the liver of animals
16,17,18,19 22,23,24
and humans . Indeed, it has been reported that haloalkane metabolites were held
responsible for CCl4 hepatotoxicity and caused the oxidative damaOe of unsaturated lipids in
1,2,4,5,6
some cellular components of hepatic tissues . Selenium may also diminish the harmful
effects and the formation of the reactive intermediary metabolites of CCl4. If selenium deficiency
occurs in tissues of the body the cellular membrane may be damaOed. ThrouOh its antioxidant
functions selenium may also prevent the hepatic cellular injury 8,18,19.
The interrelationships between protective effects of antioxidants and toxic effects of CCl4
have been investiOated in different studies. Administrations of vitamins A 3, C10,11,12, E4,13,14,15,16
and selenium±vitamin E17,19,20,26,27 and ȕ-carotene 20,21
could protect the liver of rats from some
harmful effects of hepatotoxic chemicals. Interactive relations between antioxidants and CCl4
may chanOe the toxicity of this hepatotoxic aOent. These effects of selenium on toxicity of CCl4
may also be attributed to the antioxidant function of GSH-Px containinO selenium. Indeed,
selenium is known as an antioxidant ability to inhibit oxidative processes of lipids and
8,9,25
lipoproteins in cell membranes . The activities of AST, ALT and ALP were siOnificantly
increased (p<0,05Ñ p<0,05 and p<0,01Ñ respectively), but GGT activity was not statistically
affected (p>0,05) by CCl4-injection. The values of ALP were increased in CCl4 and CCl4 plus Se-
Oroups (p<0,01 and p<0,01Ñ respectively). In addition, the levels of ALT and ALP in control-
Oroup were lower (p<0,05 and p<0,01Ñ respectively) than those in both CCl4 and CCl4 plus Se-
Oroups (Table 2). Our results are accordance with the results of the other investiOations
performed usinO antioxidants and CCl4 4,5,13,18,19,20.
There usually was the typical fibrotic and cirrhotic appearances in sections of the liver
tissue in the CCl4- Oroup. Thick fibrous septa were seen bridOed between from the central reOions
and portal areas and prominent intralobular inflammatory reaction consistinO of Oranulocytes and
mononuclear inflammatory cells. These biopsies were interpreted as stronO suOOestion of
cirrhosis (FiO.5). Cell injury coaOulative necrosis inflammatory infiltration Oranulocytes and
mononuclear inflammatory cells were occasionally observed in the liver sections of CCl4 plus Se-
Oroup (FiO. 19). These appearances are Oenerally in aOreement with the results of the
4,13,19
investiOations performed usinO antioxidants and hepatotoxic substance . The necrotic
fibrotic and cirrhotic chanOes in the liver sections were reduced after beinO treated with selenium.
These results of the present study suOOest that selenium can diminish liver injury fibrosis and
cirrhosis induced by CCl4. Indeed, there are antioxidant functions of Se in inhibition of the
oxidative processes of lipids and lipoproteins in cell membranes 8,9,15.
We have been considerinO that the administration of u may
have a therapeutic role on the liver toxicity induced by CCl4. Therefore, such onOoinO studies
investiOate the possible protective role of u   aOainst the effects of
CCl4.
†    
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