Collection,
identification and processing of the plant.
The
fresh stem bark of Acacia polyacantha were collected from Ahmadu Bello
University main campus
and environment. It was identified in the Department of Biological Sciences
Herbarium of Ahmadu Bello University, Zaria
by Mallam A.U. Gallah. It was identified with a voucher specimen number 1905. The
specimen was then cleaned and air dried at room temperature for 2 weeks and
then made into powder using pestle and mortar. The powdered samples was then
collected and stored in a clean polythene bag until required for extraction.
Method of
extraction
The
dried sample of the stem bark was extracted using 70% methanol and later
macerated using separating funnel plugged with cotton wool, then the filtrate
was concentrated over water bath at about 80⁰c. After which
the extract is scrapped off, to yield a residue of 280 gm of aqueous methanolic
extract and the residue obtained was placed in a well dried and clean air tight
container.
Acute oral
toxicity study
The LD50 was
determined using the method of Lorke (1983). In the initial phase, male Wistar
rats were divided into three groups of three rats each. They were treated with
the aqueous methanolic stem
bark extract of Acacia polyacantha at doses of 10, 100 and 1000 mg/kg
per orally. The Wistar rats were observed for 24 hours for signs of toxicity.
There was no mortality recorded. In the second phase of the toxicity study the
animal were grouped into three groups of one rat each .They were treated with
the aqueous methanolic stem
bark extract of Acacia polyacantha at doses of 1600 mg/kg, 2900 mg/kg,
and 5000 mg/kg per orally. The Wistar rats were observed for 24 hours. The
median lethal dose (LD50) was calculated using the second phase.
Phytochemical Screening
The
preliminary Phytochemical screening of the crude extract of acacia
polyacantha was carried out in order to ascertain the presences of its
constituents by utilizing conventional protocols of Trease and Evans 1983. The
extract was dissolved in methanol and
the following tests was carried out. Alkaloids, Anthraquinones, Cardiac
glycosides, Carbohydrate, Flavonoids, Glycosides, Saponins, Tannins, Steroids
and triterpenes.
Procedure
i.
Test for Alkaloids
The test for
alkaloid was carried out using the three different reagents:
a)
Dragendroff’s
reagent (potassium iodide 16g, Distilled water 40 ml).
1ml of Dragendroff’s reagent was added
to the sample of the plant extract. A rose red precipitate was observed,
indicating the presence alkaloids.
b)
Meyer’s
reagent ( Mercury chloride 1.36g, water 60ml,postassium iodide 5g,water 20ml).
1ml
of Meyer’s reagent was added to the sample of the plant extract. A cream
precipitate was observed, indicating the presence alkaloids.
c)
Wagner’s
reagent( iodine 2g, potassium iodide 3g, water 100ml)
1ml of Wagner’s reagent was added to the
sample of the plant extract. A whitish precipitate was observed indicating the
present of alkaloids (Trease and Evans, 1983).
ii.
Test for Anthraquinones derivative
Free
anthraquinones (Borntrager’s test)- sample of the extract was shaken with 10ml of
benzene and filtered.5ml of 10% of ammonia solution was added to the filtrate
and stirred. A pink-red or violent colour was observed indicating the presence
of free anthraquinones.
Combined
anthracene (Modified Borntrager’s test) - Sample was boiled with 5ml of 10%
hydrochloric acid for 3 mins. (This will hydrolyze the glycosides to yield
aglycones which are soluble in hot water only). The solution was
filtered hot and allowed to cool down and extracted with 5mls of benzene. The
benzene layer was filtered off and shaken gently with half its volume of 10%
ammonia solution. A rose-pink -colour was observed indicating the presence combined
anthracene (Trease and Evans 1983)
iii.
Test for Cardiac glycosides
The following
test were performed respectively on separate portions of the plant extract.
(a)
Kella
– Killiani Test –Extract was dissolved in glacial acetic acid containing traces
of chloride. The test tube was held at an angle of 45 degree, 1ml of
concentrated sulphuric acid was added down the side of the tube, a purple ring
color at the interface was observed indicating the presence cardiac glycosides (Trease
and Evans 1983).
(b)
Kadde
test- 1ml of 2% 3,5-dinitrobenzoic acid in 95 alcohol was added to extract, the
solution was made alkaline with 5% sodium hydroxide. A purple – color was
observed indicating the presence of cardenolides in the ring (Trease
and Evans, 1983).
(c)
Salkowsk Test- to about 0.5g of extract was
dissolved in 2ml of chloroform and few drops of concentrated H2SO4
was added to form a lower layer. A reddish-brown colour at the interface was
observed indicating the presence of a steroidal ring (Trease and Evans 1983).
iv. Test for
Carbohydrates
Molisch’s test-
few drops of molisch reagent was added to a little quantity of extract in a
test tube and small quantity of concentrated
sulphuric acid was allowed to run down the test tube. a purple to violent colored
layer was formed at the interface indicating
the presence of carbohydrates (Trease and Evans 1983).
Fehlings test-
To 2ml of the extract,5ml of a mixture
of Fehling solution A(copper sulphate
69.2g,conc. Sulphuric acid 1ml, water 100ml) and Fehling solution B(potassium sodium tartrate 176g, sodium
hydroxide 77g, water 500ml) in the ratio of 1:1 was added and the mixture boiled
for a few minutes. A brick- red
precipitate was observed indicating the presence of carbohydrate (Trease and
Evans, 1983).
v. Test for
flavonoids
Shinoda test –
About 0.5g of the extract was dissolve in 1-2ml of 50% methanol in the heat.
Metallic magnesium and four-five drops of concentrated. HCl were added. A red
or orange colour was observed indicating the presence of flavonoidaglycones.
Sodium hydroxide
test - few drops of aqueous NaOH was added to 5ml of extract, a yellow
colouration was observed showing the presences of flavonoids (Trease and Evans,
1983).
vi. Test for
Glycosides
5ml of
concentrated sulphuric acids was added to the extract and boiled for 15 mins.
This was then cooled and neutralized with 20% KOH and was divided into two
portions Another part of the extract was dissolved in distilled water, this was
used as a control; no acid hydrolysis.
Fehlings
solution test - 1:1 fehlings solution A (copper sulphate 69.2g, conc. Sulphuric
acid 1ml, water 100ml) and Fehlings solution B (potassium sodium tartrate 176g,
sodium hydroxide 77g, water 500ml) was added to the extract and boiled for few
mins. A brick red- precipitate was observed indicating the glycone portion as a
result of hydrolysis of glycoside.
Ferric chloride
test – 3 drops of ferric chloride solution was added to the extract, a brown to
black precipitate was observed indicating phenol aglycone as a hydrolysis of
glycoside (Trease and Evans, 1983).
vii. Test for
Tannins
Lead sub- acetate test - 3 drops of lead
sub-acetate solution was added to a solution of the extract. A colored precipitate
was observed which indicate the presence
of tannins.
Ferric chloride test- about 0.5ml of extract
was dissolved in 10ml of distilled water, then filtered. Few drops of ferric
chloride solution was added to the filtrate. A blue-black precipitate was
observed which indicates the presence of condensed tannin (Trease and
Evans, 1983).
viii. Test for Saponins
Frothing test-
small quantity of the extract was dissolved in 10ml of distilled water. This was
then shaken vigorously for 30 seconds and allowed to stand for 30 minutes. A honey
comb was formed for more than 30 minutes indicating the presence of saponin (Trease and
Evans, 1983).
ix Test for Steroid and triterpenes (Lieberman-
Burchards test)
Equal volume
of acetic anhydride was added to the sample of the extract. 1ml of concentrated
sulphuric acid was added down side the tube. A Red- pink colour was observed immediately indicating
the presence of triterpenes and later, while blue-green colouration was
observed indicating the presence of steroids (Trease and Evans, 1983).
Experimental Animals
A
total of 50 adult Wistar rats of both sexes were obtained from animal house of
the Department of Pharmacology and Clinical pharmacy in A.B.U. Zaria, after
random selection, two weeks prior to the experiment, The rats were 10-12 weeks
old weighing between 120-200grams. They were housed in metallic cages, fed with
standard rat feed (growers mash containing corn, grounded, cray fish soya beans
and millets) and allowed access to fresh water ad libitum.
Preparation of Dose
The dried aqueous methanolic stem bark extract of Acacia polyacantha was
weighted and then dissolved in 3% propylene glycol and 7% water to make a
solution. The respective doses used for this experiment were prepared (600mg, 300mg,
150mg) per kg body weight of the rats, shortly before administration. This was
carried out as follows. On the administration of the extract per body weight,
6g of the extract was dissolved in 15ml of which 3% is propylene glycol and 7% water
to make a stock solution.
The dose of the extract to be administered to each
rat was calculated as follows:
Volume to be injected = Dose × Wr
Stock solution
Wr = weight of rats.
Stock
solution = original concentration of the plant extract which is used.
Dose = quantity of the plant
extract to be injected.
EXPERIMENTAL
DESIGN
Administration
of the extract
The animals were divided into five
groups of five male rats each. Groups A (control) is given distilled water, B,
C, and D were administered graded doses of 150, 300, and 600 mg/kg respectively
of the aqueous methanolic stem bark extract of Acacia polyacantha orally using cannular for 14 days, group E were
given standard drug sildenafil citrate (viagra) 5mg/kg purchased from Yusuf
pharmaceutical kaduna, manufacture by West-coast pharmaceutical works Ltd Gota,
Ahmedaba -382481 –India with batch number 52651208. 1 hour prior to the
commencement of the experiment.
Mounting frequency
The
female Wistar rats were artificially brought into oestrus (heat) using the
method of (Szechtman et al., 1981) (as female allow mating only during
estrus phase), 48 hours prior to the experiment the female
rats were given ethinyl oestradiol 100µg/kg and progesterone 1mg/kg body weight
subcutaneous 6 hours to the experiment. On the 14th day the male Wistar
rats were paired individually with the receptive female rats in a separate
cage. The male Wistar rat assuming the copulatory position over the female, but
fail to achieve intromission, was considered as a mount (Subramoniam et al.,
1997). The behavior of the animals was noted and recorded for 3
hours.
Sample
Collection
The rats were anaesthetized by placing them in an
anaesthetizing chamber already contain chloroform form for few seconds: after
which they are laid supine on a dissecting board and the limbs fastened to the
board with dissecting pins and dissected exposing the thoracic cavity and blood
sample collected through the apex of heart stored in non- heparinzed EDTA test-
tube and centrifuged, the serum was used for hormonal assay (testosterone). The
testes was then exposure by scrotal incision, removed and transferred into a
Petri dish, the adipose tissues, and blood vessels were removed from the testes
before they were washed with normal saline maintained at 37⁰c, the
testes were weighed in a digital weighing balance, before the epididymis were removed and weighed separately. The testes
were stored in sample bottle containing 10% formal –saline (formalin 100ml,
sodium chloride 8.5g, water 900ml) for histological examination.
Sperm count,
Motility and Morphology
The epididymis was teased into Petri dish and 1ml of
normal saline at temperature of 36⁰c was added to the semen to enhance sperm survival invitro during the
period of the study. The semen mixture was then sucked in to a red blood
pipette to the 0.5 mark, then diluted with warm normal saline which was sucked
up to the 101 mark. The normal saline at the stem of the pipette was discarded
and then the contents of the bulb of the pipette were mixed toughly. A drop of
the semen mixture was placed on the neuber counting chamber which then spread
under the cover- slip by capillary action (charging the chamber). The counting
chamber was then mounted on the slide stage of the microscope and viewed under
the magnification of x40. A grid system divides the counting chamber into 5
major squares using the top and right or left and bottom system of counting
(Sinha et al,.2001). The total number of sperm cells were counted and
expressed in million per mil.
Analysis of sperm motility and sperm morphology was
carried out by placing a drop of the sperm –saline mixture on two separate
slide one for sperm motility (labelled A) and the other for sperm morphology
(labelled B). Slide A was covered with a cover-slip and examined under the
light microscope at a magnification of x40 and the sperm motility was graded
based on progressive forward movement of the sperm cells and were estimated in
%.
A smear was made on slide B by using another slide
(spreader) inclined at an angle of 45⁰C, 95% ethanol was
immediately added for 2 minutes, followed by 1ml of Gimsa stain (Gimsa powder
0.3g, glycerine 25ml, Acetone free alcohol 25ml,), then allowed to stand for 10
minutes, after which it was washed with buffer distilled water, and allow to
air dry. The sperm cells were counted by putting a drop of immersion oil and
placed on a microscope at a magnification of x100. Counting was in a zigzag
pattern. Both normal sperm cells (for rodents having a hook – shaped head) and
abnormal cell (abnormality in head, midpiece or tail) were observed and
counted, and the sperm morphology were estimated in % (Keel, 1990).
Hormonal
assay
The stored blood serum in the non- heparinzed
EDTA-test tubes were analyzed at the Department of Chemical Pathology Ahmadu
Bello Teaching Hospital Zaria, using testosterone kit (Syntron Bioresearch,
Inc. Microwell Testosterone EIA, Reference number 4410 – 96 (96 test kit). The microwell
testosterone EIA is a solid- phase enzyme immunoassay utilizing the competitive
binding principle. Testosterone present in the sample will compete with enzyme-
labelled testosterone for binding with anti- testosterone anti body immobilized
on the microwell surface. The amount of conjugate that binds to the microwell
surface will decrease in proportion to the concentration of testosterone in the
sample.
The unbound sample and conjugate are then removed by
washing and the colour development reagents (substrates = 6.0ml buffer hydrogen
peroxide solution and 6.0 ml buffered 3,3’, 5,5’- Tetramethylbenzidine
solution) are added. Upon exposure to the bound enzyme, a color will take
place. The intensity of the colour reflect the amount of bound enzyme-
testosterone conjugate and is inversely proportional to concentration of
testosterone in the sample within dynamic range of the assay. After stopping
the reaction the resulting colour is measured using a spectrophotometer at 450
nm. The testosterone concentration in the sample and concurrently run controls
were determine from the standard curve thus as follows (Wilkle and Utley, 1987).
·
The average absorbance value (A.450) for
each reference standard, control and test sample were calculated.
·
A standard curve was prepared by plotting the
average absorbance (A.450) versus the corresponding concentration of
the standards on a log-log graph paper.
·
Using the absorbance (A.450) value for
each test sample to determine the corresponding concentration of testosterone
in ng/ml from standard curve.
Histological Analysis
i. Preparation of tissue for histology.
The technique adopted for this process are out lined by (Carleton, 1967).
·
Tissue processing
The tissue obtained after sacrificing the treated and control rats were
trimmed to size and fixed in 10% formal –saline (formalin 100ml, sodium
chloride 8.5g, water 900ml).Using the tissue processor, the tissues were
dehydrated using graded concentrations of ethanol as follows:
i) 70%
alcohol was used to dehydrate the tissue for 1 hr twice.
ii) 90% alcohol was used to dehydrate the tissue
for 1 hr twice.
i)
Absolute alcohol was used to dehydrate the tissue
for 1 hr twice.
They were cleared in xylene by transferring them into equal volumes of
alcohol (Absolute) and xylene for one hour; in order to avoid tissue distortion
due to sharp transition from alcohol to xylene. The tissues were then passed
through two changes of xylene for one hours each.
·
Embedding
The tissues were embedded in paraffin wax at 55⁰c for 2
hours each. This infiltration was carried out in two changes of paraffin wax
for 2 hours each. The tissues were later blocked out using L- shaped metal
molder and subsequently mounted on a wooden block and trimmed to size for
sectioning.\
·
Sectioning
Using rotary microtome, the tissue blocks were cut into ribbons of 5µ
thickness each. The cut sections were picked with horsed – brush onto a slide.
The section on the slide floated in 20% alcohol and then in warm water bath to
allow for proper straightening. The sectioned were mounted on albumenized
slides. They were dried in oven at 37⁰c overnight.
ii.
Staining (Haematoxylin-Eosine
staining method)
Deparaffinizing. The sections were dewaxed using two changed of xylene
for 2 minutes each.
·
Re-hydration
The sections were passed through descending graded of alcohol from
absolute to 70% alcohol ,each for one minute, then washed in water followed by
staining with Haematoxylin for twenty minutes then washed in water, the tissues
were then differentiated with1 % acid alcohol for 5 sec. then washed with
water, blued in ammonical water for 2 minutes, washed in water, counter- stained with 1% aqueous eosin for 2 minutes
and rinsed in water, dehydrated in ascending grades of alcohol from 70%,
90% and absolute alcohol for 1 minutes
each, then cleared in xylene and finally
mounted in DPX(distrene, tricresyl phosphate, xylene).
Statistical Analysis
Result are presented as mean ± SEM. Graphs were
drawn using the excel package for the drawing graphs. Statistical analysis was
done using one way analysis of variance (ANOVA) followed by a post-hoc test of Duncan and
Scheffe. Values of p<0.05 was considered statistically significant (Duncan et
al., 1977).
Limitations of the study
The initial experimental design was
to use cavernous tissue of Wistar male rats, but the equipment to carry out the
test was lacking, so mounting frequency was used instead.