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.
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