EQUIPMENT USED FOR THE EXPERIMENTS
Weighing scale 00176 china
Mortar and pistol
Nigeria
RA -50 Spectrophotometer 22p 6286 Japan
Centrifuge Model DNP
China
Stop watch Model 0024
China
Water bath Model 8414
Japan
Soxhlet Extractor Model
2671 Japan
Rotary evaporator
RE52CS China
Incubator
C.RMDX-280
CHEMICALS /REAGENTS
All the chemicals and reagents used
in this study were of analytical grade.
i. Alkaline phosphatase Reagent (Randox)
(Diethanolamine buffer, MgCl2, P-nitro phenyl phosphate).
ii.
ALT and AST Reagent (Randox) (Phosphate buffer, L-alanine for ALT,
L-aspartate for AST, α- oxaloglutarate, 2,4 –dinitrophenylhydrazine).
iii. Protein Reagent (Randox)
(Sodium hydroxide, Sodium potassium tartarate,
Potassium Iodide, Cupric sulphate).
iv.
Albumin Reagents (Randox)
(Succinate buffer 0.10mol/l, succinic
acid, sodium hydroxide, sodium azide, Bromocresol green solution, distilled
water and 30 % Bovin serum albumin)
V
Malondialdehyde Reagents (Sigma –Aldrich)
(Thiobarbituric acid, 1, 1, 3, 3-tetraethoxy
propane, Trichloroacetic acid and Normal saline).
vi. Reduced Glutathione Reagents (Sigma
–Aldrich)
(EDTA solution, precipitating
reagent, disodium hydrogen phosphate solution and 5,
5-dithio-bis-2-nitrobenzoic acid).
vii. Super oxide Dismutase Reagents (Sigma-Aldrich)
(Sodium pyrophosphate buffer,
phenazine methosulphate, glacial acetic acid, n-butanol and nitroblue
tetrazoline).
viii. Catalase Reagents (Sigma-Aldrich)
(Phosphate buffer and hydrogen
peroxide).
ix. Bilirubin (Total and Direct)
Reagents (Randox)
COLLECTION OF BIOLOGICAL
MATERIALS
The leaves of Aloe barbadensis
(400g) and Allium sativum (200g) were purchased from Abakpa Market in
Abakaliki, Ebonyi State.
32 albino rats were purchased from
the University of Nigeria Nsukka Animal House and were acclimatized for 2 weeks
in the Animal House of Biochemistry Department, Ebonyi State University,
Abakaliki.
METHODS
3.2.1 ETHANOLIC EXTRACTION OF PLANT MATERIALS;
SOXHLET EXTRACTION
200 g of Allium sativum was air dried, grounded into powdered form
and poured into a filter paper. This was then placed into the soxhlet extractor
and coupled to a condenser. 500 ml of ethanol ( 99 %, BDH ) was used as the
extraction solvent. The extract obtained was dried at 50 °C using an oven and
the dried extract stored.
COLD EXTRACTION
400 g of Aloe barbadensis was
collected washed, sliced, mashed gently with mortar and pistol and soaked in
400 ml of Ethanol (99% BDH) for 24 hours which turned out light green in
coloration. Because Aloe barbadensis have thick leaves that can be
difficult to dry, this method of extraction was used (Sofowora, 1982).
The residue was removed from the
extract using filter paper and the extract evaporated at 50 °C using an oven
until a gel like substance was formed.
PREPARATION OF EXTRACTS
0.02g, 0.04g and 0.08g of each of the extracts
were weighed respectively and each diluted in 100 ml of distilled water to form
200 µg/ml, 400 µg/ml and 800 µg/ml of the extracts, respectively.
3.2.3 ADMINSTRATION OF PARACETAMOL OVERDOSE AND
EXTRACTS.
The overdose of paracetamol is an
excellent example of toxication of the liver and Allium sativum and Aloe
barbadensis according to Zimmerman, 1996 is being considered as a liver
protecting plant because of its rich antioxidant activity.
After acclimatization, the rats were
divided into 4 groups. The first group A served as the control group; group B, C
and D were used as test groups, each had 3 rats.Hepatotoxicity was induced by
administering 500 mg of paracetamol per kg rat.
The control (group A) was fed with
rat feed and water for 7 days. Group B was fed with rat feed, water and
paracetamol orally at 500 mg/kg/day without any extract. Group C made up of 12
rats of 3 sub groups received paracetamol overdose plus extract of Allium
sativum at 3 different concentrations of 200 µg/ml, 400 µg/ml and 800 µg/ml for
7 days.
Group D also contained 12 rats of 3
sub groups received overdose of paracetamol plus extract of Aloe barbadensis at
different concentrations of 200 µg/ml, 400 µg/ml and 800 µg/ml.
BIOCHEMICAL ANALYSIS
After the treatment period (7 days), the animals of all
groups were sacrificed. The rats were dissected and 5 ml of blood drawn directly from the heart using
syringe. The blood was poured into test tubes, placed inside the centrifuge and
centrifuged at 5000 rmp for 10 minutes. After centrifugation, the serum was
then separated and used for biochemical assays.
DETERMINATION OF SERUM
ASPARTATE TRANSAMINASE ACTIVITY (AST)
The aspartate transaminase activity was determined
using the method of Ghosh et al, (2007).
PRINCIPLE: The activity of Aspartate
transaminase was measured by monitoring the concentration of oxaloacetate
hydrazone formed when 2, 4-dinitrophenyl hydrazine (colour developer) reacts
with the substrate buffer.
PROCEDURE: The tubes were selected and
pipetted with 0.5 ml of substrate buffer followed by addition of 0.1 ml of
sample. It was mixed and incubated for exactly 30 minutes at 37 ºC. At the end
of the incubation, 0.5 ml of 2, 4 diphenyl hydrazine was added to all the tubes
while 0.1 ml of sample was added to test only and the tubes were allowed to
stand for 20 minutes at 25 ºC. 5.0 ml of NaOH was added, mixed and the
absorbance of the sample against the blank was read after 5 minutes at 550 nm.
DETERMINATION OF SERUM ALANINE
TRANSAMINASE ACTIVITY (ALT)
The Alanine transaminase activity was
determined using the method of Ghosh et al, (2007).
PRINCIPLE: The activity of alanine
transaminase was measured by monitoring the concentration of pyruvate hydrazone
formed when 2,4-dinitrophenyl hydrazine reacts with the substrate buffer in the sample medium.
PROCEDURE: The tubes were selected and labeled
as blank and sample. About 0.5 of substrate buffer was put followed by addition
of 0.1 ml of sample. It was mixed and incubated at 37 ºC for exactly 30
minutes. At the end of the incubation, 0.5 ml of 2,4 dinitrophenyl hydrazine
was added into the tubes and 0.1 ml of sample added to test and allowed to
stand for 20 minutes at 25 ºC. 5.0 ml of NaOH was added, mixed and the
absorbance of the sample against blank read after 5 minutes at 550 nm.
DETERMINATION OF SERUM ALKALINE
PHOSPHATE (ALP) ACTIVITY
ALP Activity was determined using
Ghosh et al method, (2007).
PRINCIPLE: Based on the ability of ALP enzyme
to convert p-nitro phenyl phosphate to p-nitro phenol and phosphate in a
dephosphorylation reaction.
PROCEDURE: The tubes were selected and labeled
as blank, sample and control. 0.01 ml of sample was put into the test tube only
and 0.5 ml of substrate buffer was added to all the tubes and mixed. The
absorbance was read at 405 nm at time intervals of 1, 2 and 3 minutes and the
mean value obtained.
3.3.4 DETERMINATION OF TOTAL PROTEIN
IN SERUM
Total protein was determined using
Tietz method, (2000).
PRINCIPLE: The proteins are made up of amino
acid containing peptide bonds. In aqueous phase, two or more peptide bonds
react with copper sulphate (blue solution of biuret) and result in purple-
violet coloured product. The number of peptide bonds present in a sample
governs the colour intensity of the sample.
PROCEDURE: 1 ml of Biuret reagent was pipetted
into test tubes labeled blank, standard and sample, followed by addition of
0.02 ml of serum to standard and samples respectively. The mixture was
incubated for 15 minutes at room temperature of 25ºC and the absorbance (colour
intensity) read against a biuret reagent blank at 540nm.The absorbance value is
directly proportional to protein concentration in the sample.
DETERMINATION OF SERUM ALBUMIN
Albumin value was determined using
Tietz, 2000 method.
PRINCIPLE: At pH 4.2, using succinate buffer
system, the anionic dye bromocresol green will preferentially bind to the
albumin molecule of plasma to give a blue coloured complex.
PROCEDURE: 1 ml of Bromocresol dye was
pipetted into test tubes labeled blank, control and samples. About 0.01 ml of
standard solution was added to the control tube and 0.01 ml of sample was added
to the test samples. The solutions were mixed and allowed to stand for 10
minutes at room temperature. Absorbance was read at 540 nm.
GLOBULIN VALUE
Globulin values were gotten by
subtracting albumin values from total proteins.i.e
Total protein – Albumin = Globulin.
DETERMINATION OF BILIRUBIN
(TOTAL AND DIRECT)
Bilirubin was assayed using the
method of Malloy and Evelyn, 1937.
PRINCIPLE: Sulphanilic acid is diazotized by
the Nitrous acid produced from the reaction between sodium nitrite and
hydrochloric cid. Bilirubin reacts with the diazotized sulphanilic acid (diazo
reagent) to form azobilirubin.
The pink acid azobilirubin is converted
to blue azobilirubin by an alkaline tartrate reagent and absorbance of blue
green solution read at 580 nm for total bilirubin and 540 nm for direct
bilirubin. Measurement of the azobilirubin in an alkaline medium removes
turbidity and increases specificity.
PROCEDURE: 0.05 ml of the sample (serum) and
Reagent 1 were pipetted into two test tubes set-up. Reagent 2 (0.05 ml) was
taken into a test tube blank and then, 1 ml of Reagent 3 was added to all the
test tubes and shaken. They were then incubated at room temperature for 10
minutes and cooled. 1 ml of Reagent 4, was added to all test tubes and allowed
to stand for 5-30 minutes at room temperature. The absorbance of the sample
against the blank was read at 580 nm for total bilirubin and 540 nm direct
bilirubin.
DETERMINATION OF ANTIOXIDANT
ACTIVITY
The livers were excised, rinsed in normal saline, followed by
Tris-HCL (pH 7.2), blotted dry under air and weighed with weighing balance. A 2
g portion of the liver tissue was sliced and then homogenized with 10 ml of
normal saline. A part of the homogenate after precipitation of protein using 10
% TCA was used for the following estimation
DETERMINATION OF THIOBARBITURIC
ACID REACTIVE SUBSTANCES (TBARS).
TBARS in tissues was estimated by the
method of Fraga et al, (1981).
PRINCIPLE: Thiobarbituric acid reacts in a
protein free solution to form a colored complex read at 532 nm.
PROCEDURE:
To 0.5 ml tissue homogenate, 0.5 ml normal saline and 1 ml of 10 % TCA
were added, mixed well and centrifuged at 3000 rpm for 20 min. To 1.0 ml of the
protein –free supernatant, 0.25 ml of
Thiobarbituric acid (TBA) reagent was added; the contents were mixed well and
boiled for 1 hr at 95ºC. The test tubes were then cooled to room temperature under
running water and absorbance measured at 532 nm.
DETERMINATION OF REDUCED
GLUTATHIONE (GSH)
GSH was determined by the method of
Ellman et al (1958).
PRINCIPLE: This is based on the formation of
relatively stable yellow colour when Ellman's reagent is added to a sulfhydryl
compound (GSH) to form a chromophoric product known as 2-nitro- 5 –thiobenzoic
acid.
PROCEDURE: About 0.2 ml of tissue homogenate
was mixed with 1.8 ml of EDTA solution. To this 3.0 ml precipitating reagent
(1.67 g of meta phosphoric acid, 0.2 g of EDTA ,0.2 g disodium salt, 30 g
sodium chloride in 1 liter of distilled water) was added, mixed thoroughly and
kept for 5 min before centrifugation. To 2.0 ml of the filtrate, 4.0 ml of
disodium hydrogen phosphate solution and 1.0 ml of DTNB (5,
5-dithio-bis-2-nitrobenzoic acid) reagent were added and read at 412 nm.
DETERMINATION OF SUPER OXIDE
DISMUTASE (SOD)
The activity of SOD in tissue was
assayed by the method of Kakkar et al (1984).
PRINCIPLE: The ability of the enzyme SOD to
catalyze the dismutation of super oxide into oxygen and hydrogen peroxide.
PROCEDURE: The assay mixture contained 1.2 ml
sodium pyrophosphate buffer (pH 8.3), 0.1 ml phenazine methosulphate, 0.3 ml
NTB (Nitro blue tetrazoline), 0.2 ml NADH and approximately diluted enzyme
preparation and water in a total volume of 3 ml. After incubation at 30 ºC for
90 sec, the reaction was terminated by the addition of 1.0 ml of glacial acetic
acid. The reaction mixture was stirred vigorously and shaken with 4.0 ml
n-butanol. The color intensity of the chromogen in the butanol layer was
measured at 560 nm against n-butanol.
DETERMINATION OF CATALASE (CAT)
Catalase was
assayed according to the method of Maehly and Chance (1954).
PRINCIPLE: Based on the ability of Catalase
enzyme to catalyze the decomposition of hydrogen peroxide to water and oxygen.
PROCEDURE: The estimation was done
spectrophotometrically at 230 nm. The tissue was homogenized in 2 ml phosphate
buffer (pH 7.0) at 1-4 º C and centrifuged at 5000 rpm. The reaction mixture
contained 0.01M phosphate buffer, 2 ml Hydrogen peroxide and the enzyme
extract. The specific activity of catalase is expressed in terms of (mole of H2O2
consumed/min/mg of protein).
HISTOLOGICAL ANALYSIS OF THE LIVER
Fresh liver specimens were initially
collected in formol acetic acid solution (1:1:18 v/v mixture of formaldehyde,
glacial acetic acid and 70 % alcohol ). The fixed specimens were then rinsed
twice in distilled water and dehydrated with graded series of ethanol (30, 50,
70 and 95 % ethanol) each for 2 hrs and absolute ethanol overnight.
The dehydrated specimens were cleared
by passing through 3:1, 1:1 and 1:3 v/v absolute ethanol-xylene series followed
by pure xylene each for 3 hrs. The specimens were kept in molten anatomical wax
at 50-60 °C for 12-48 hrs to allow infiltration of wax into the tissues.
Embedding of specimens was done by
pouring molten wax into moulds and the specimens were positioned in the moulds.
The wax was then allowed to cool. Glycerol was rubbed on the inside surface of
the moulds for easier removal of wax blocks from the moulds. The wax blocks
containing the specimens were attached plastic blocks which was then attached
to a rocking microtome for sectioning of the specimen (6 µm sections).
The microtometric sections were laid
on slides previously smeared with egg albumin. The specimen sections on slides
were cleared with graded ethanol –xylene solutions (1:3, 1:1 and 3:1 v/v) and
stained with 30 % and 0.5 % AgNo3 (3-10 minutes). The stained
sections were counter – stained lightly with 0.01 % safranin solution to
enhance visibility of the tissues.
Sections were then dehydrated across
increasing graded ethanol series (30, 50, 70 and 95 % ethanol), cleared thought
ethanol- xylene series (3:1, 1:1 and 1:3 v/v ethanol-xylene solution) and
mounted in Canada balsam.
Mounted sections (i.e. prepared slides) were
allowed to dry in an oven ( 60 °C ). Microscopic examination of specimen
sections was done using a light microscope after which a photo microgram was
taken using a computer
STATISTICAL ANALYSIS
Data for antioxidant activity, reduced GSH, SOD, CAT,
AST, ALT,ALP, Total Protein, Albumin, Globulin and Blilrubin were expressed as
mean ± SD and analyzed statistically using One Way Analysis of Variance
(ANOVA).The minimum level of significance was fixed at P<0.05.