2- Materials and Methods


Rats and Diet:

Male albino rats of Sprague Dawley strain
weighing 170±10g were purchased from the Laboratory Animal Colony, Ministry of
Health and Population, Helwan, Egypt. Basal diet constituents were obtained
from El- Gomhorya Company, Cairo, Egypt.

Chemicals and fed ingredient:

N-Nitrosodiethyamine (NDEA) liver
function kits were purchased from Sigma- Aldrich Co. (St. Louis, Missouri, USA).
Carbon tetrachloride (CCl4) was obtained from El-Gomhorya Company, Cairo,
Egypt.Ginger was
purchased from local Market. Then ,the ginger dried and powdered.   


2-1 Preparation of basal diet:

The basal diet (AIN-93M) was prepared according to Reeves et
al.(1993). Diet was formulated to meet the recommended nutrients levels for

Induction of Hepatocarcinoma

Inducing hepatocarcinoma was
done according to (Sarkar
et al., 1997; Dakshayani
et al., 2005; Singha et al., 2009).
One gram of NDEA was dissolved in 25 mL physiologic saline solution (0.9% NaCl)
and injected intraperitoneally (with least distress to both rat and
investigator) to each rat in a single dose of 200 mg/kg body weight. Two weeks
later, animals received subcutaneous injections of CCl4 dissolved in olive oil at a dose of
3 mL/kg/rat body weight for 6 consecutive weeks to promote the hepatocarcinoma
in rats.

2-3 Experimental Design:

Forty male albino rats were
fed on the basal diet and water was provided ad libi tum. Animals were
maintained under standard conditions of humidity (50-  60%), temperature (20-25°C)
and light (12-h light: 12- h dark cycle) for one week
before starting the experimental for
acclimatization. Rats were divided into
four groups of ten animals each as follows:


1: (N = 7) fed on Ain-93M and used as a negative control (Negative control).

Group 2: Fed on Ain-93M, injected with NDEA and CCl4 according to
the above protocol and used as
Hepatocarcinogenesis control.

Group 3:  Fed on
Ain-93M, injected with NDEA and CCl4 as above and administered daily 0.5 % ginger powder, for 12 weeks.

Group 4: Fed on Ain-93M, injected with NDEA and CCl4 as above and administered daily 0.1 % ginger
powder, for 12 weeks..

Group 5: Fed on Ain-93M, injected with NDEA and CCl4 as above and administered daily 0.2 % ginger
powder, for 12 weeks.

In the first six weeks of the
experiment, animals of groups (2), (3), (4) and (5) were followed the above
protocol to induce hepatocarcinoma in rats. 
At the end of experiment after ~12 weeks of the
experiment, rats were sacrificed after overnight fasting. Blood samples were collected
as practical as possible in clean and dry tubes from the portal vein and left
to clot at room temperature (26-27 ºC). Blood samples were then centrifuged at
3000 rpm for 15 min, serum was carefully separated and kept at -20°C until

2-4 Biochemical Analyses

Aspartate and Alanine Transferases (AST/ALT)

AST and ALT were done
according to (Murray, 1984a) and (Murray, 1984b),
respectively. The principal of AST assay was based on AST catalyses the
reversible transfer of an amino group from aspartate to ?-ketoglutarate forming
glutamate and oxaloacetate. The oxaloacetate produced is reduced to malate by
malate dehydrogenase (MDH) and NADH. Similarly, ALT catalyses the reversible
transfer of an amino group from alanine to ?-ketoglutarate forming glutamate
and pyruvate. The pyruvate produced is reduced to lactate by lactate
dehydrogenase (LDH) and NADH. Reagent kits were used (Spinreact Co., Barcelona, Spain),
and determined spectrophotometrically at 340nm by using
spectrophotometer (BT-260 Plus, Shanghai,
China). The
concentration of the samples were calculated and the results were expressed as (U/L).


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