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transported into 1 L Erlenmeyer flasks, and then 800 mL to give 20% homogenate (w/v). The homogenate was
[21]
of 80% methanol (80:20, methanol:water, v/v) were added centrifuged at 3000 g for 20 min. The supernatant was
to the samples. Extraction was carried out using an orbital separated and stored at -70 °C until the determination of
shaker at a room temperature for 8 h, they were filtrated the levels of malondialdehyde (MDA), total antioxidant
through filter paper (Whatman No. 1), the residue was capacity (TAC), and reduced GSH content. Furthermore, the
re-extracted twice for complete extraction, and then, the relative liver weight of each animal was then calculated as
combined extracts of every sample were evaporated at 45 °C, follows: Relative organ weight = (absolute organ weight [g]
using a rotary vacuum evaporator (Rotavapor R-114 BÜCHI, × 100)/(body weight of rat on sacrifice day [g]).
Switzerland) and stored at -4 °C until use. [15]
Analytical determinations
Animal grouping Colorimetric determinations of serum AST and ALT
Sixty rats were divided into six groups (10 rats each) based on activities were carried out using UV-160 1PC UV-visible
their body weight and treated daily for 28 consecutive days as spectrophotometer (Shimadzu, Japan) for reading the
follows. Group 1: rats were administered with normal saline absorbance. The assay was performed according to the
by gastric intubation and served as control group; group 2: instruction manual of RANDOX reagent kits manufactured by
rats received CFME at a dose of 200 mg/kg (dissolved in RANDOX Laboratories Ltd. (Admore, Diamond road, Crumlin,
normal saline) by gastric intubation; group 3: rats received Co., Antrim, UK BT29 4QY). Colorimetric determination
[18]
FSME at a dose of 200 mg/kg (dissolved in normal saline) by of serum ALP activity was performed according to the
[19]
gastric intubation; group 4: rats were orally administrated instruction manual of Reactivos GPL kits manufactured by
with AZA at a dose of 25 mg/kg (dissolved in normal saline) by Reactivos GPL (Barcelona, Espana, Spain). Serum total- and
gastric intubation; group 5: rats were orally administrated direct- bilirubin levels, triglycerides, and total cholesterol
[20]
with CFME at a dose of 200 mg/kg followed by AZA, after levels were determined colorimetrically using UV-160 1PC
15 min, at a dose of 25 mg/kg by gastric intubation; and UV-visible spectrophotometer for reading the absorbance.
group 6: rats were orally administrated with FSME at a dose The assays were performed according to the instruction
of 200 mg/kg followed by AZA, after 15 min, at a dose of manual of Reactivos GPL kits manufactured by Reactivos
25 mg/kg. GPL (Barcelona, Espana, Spain). MDA as an indirect index
for lipid peroxidation was determined in liver, based on its
During the experiment, the animals were weighed twice every reaction with thiobarbituric acid which forms a pink complex
week. Body weight gain (BWG) of each control and respective that can be measured photometrically. [22] Colorimetric
treated rats was calculated with reference to the initial body determination of hepatic GSH content and TAC were carried
weight recorded at the beginning of the experiment and the out using UV-160 1PC UV-visible spectrophotometer for
final body weight at the end of the experiment. reading the absorbance using Kits produced by Biodiagnostic
Co., Egypt.
Collection of blood samples
At the end of the experimental period, animals were fasted Histopathological examination
overnight. They were slightly anesthetized with diethyl Pieces of the livers from rats of control and treated groups
ether. Blood samples were withdrawn from the retro-orbital were fixed in 10% formalin saline for 24 h. More washing in
venous plexus into serum tubes and left to clot and then tap water overnight was followed by dehydration in graded
centrifuged at 3000 g for 15 min at 4 °C where the clear alcohol, clearing in xylene for 20 min, and embedding in
sera were separated for the determination of alanine paraffin wax. Transverse serial sections were then cut at
[23]
aminotransferase (ALT), aspartate aminotransferase (AST), 5 μm thickness and mounted on albuminized slide. Sections
alkaline phosphatase (ALP) activities, and total cholesterol, were stained with hematoxylin and eosin and investigated
triglycerides, and total- and direct-bilirubin levels. by light microscopy.

Tissue sampling Statistical analysis
At the end of blood collection, each animal was rapidly The obtained data were subjected to one-way analysis
sacrificed, and the liver was dissected out and weighed of variance. The analysis was performed using Statistical
then apart from its left lobe was immediately kept in 10% Analysis System (SAS) program software; copyright (c) 1998
buffered formalin-saline solution for a later histopathological by SAS Institute Inc., Cary, NC, USA. Tukey test was used to
examination. Another part from the same lobe of the liver evaluate the significance between the individual groups at
was washed with saline, dried, weighed, and homogenized P < 0.05. The values in this study were expressed as a
[24]
in 50 mmol/L phosphate buffer (ice-cold) solution (pH 7.4) mean ± standard error.

Hepatoma Research | Volume 1 | Issue 3 | October 15, 2015 127