MATERIALS AND METHODS
Materials
Plant Material
The leaves of Senna mimosoides were collected from Ibagwa Roadside, Nsukka in Enugu State of Nigeria, during the months of July and August, 2011. The plant characterisation and identification was carried out by a taxonomist, Mr P.O. Ugwuozor, in the Department of Plant Science and Biotechnology, University of Nigeria, Nsukka and the sample was kept in the Herbarium Voucher of the Department.
Animal
Wistar albino rats and mice of thesame sex weighing between 130-250g and 20-30g respectively were obtained from the Animal House, Faculty of Biological Sciences, University of Nigeria, Nsukka. These animals were given standard feeds (vital) for at least one week after purchase to acclematize them to their new environment before use. Sheep weighing 300 kg was obtained from the Animal Farm, Faculty of Veterinary Medicine, University of Nigeria, Nsukka.
Chemicals and Reagents
Chemicals
All chemicals used in this study were of analytical grade and were obtained from Merck (Germany), May and Baker (England), Sigma Chemical Company (U.S.A.), or BDH Chemicals Limited (Poole, England), Sterile distilled and deionized water were used in the preparation of the chemicals and or reagents.
Agar was supplied by LABM
Adrenalin was supplied by BDH chemicals
Bismuth carbonate was supplied by BDH chemicals
Glutathione was supplied by Qualikems
5,5-dithiobis-(-nitrobenzoic acid) was supplied by BDH chemicals
1-chloro-2,4-dinitrobenzene was supplied by BDH chemicals
Potassium heptaoxodichromate vi was supplied by BDH chemicals
Α-napthol was supplied by BDH chemicals
Petroleum ether was supplied by Aldrich
Phosphoric acidwas supplied by BDH chemicals
Phosphomolybdic acidwas supplied by BDH chemicals
Picric acidwas supplied by BDH chemicals
Sodium Potassium Tartarate was supplied byRiedel-de Haen
Sulfosalicylic acidwas supplied Biolaboratories
Thiobarbituric acid was supplied by BDH chemicals
Trichloroacetic acidwas supplied by JHD chemicals
Triswas supplied by Meck
Tartaric acid was supplied by BDH chemicals
Reagents
Adrenaline solution (0.059%): Adrenaline (0.01g) was dissolved in 17ml of distilled water. Agar suspension (3%): A 3% (w/v) agar suspension in normal saline was prepared by dissolving 3 g of agar in 100 ml of distilled water.
Alkaline iodine solution: NaOH (8 g) and 5 g of KI were dissolved in distilled water and made up to 1 dm3.
Alkaline copper reagent: Anhydrous sodium carbonate (40 g) + 400 ml of distilled water +
7.5 g of tartaric acid + 45 g of CuSO4 are mixed and diluted to 1 dm3 with water.
Alkaline picrate solution: 1 g of picric acid and 5 g of sodium carbonate were dissolved in warm water and made up to 200 ml with distilled water.
Aqueous phosphomolybdic acid: 5 g of phosphomolybdic acid was dissolved in water and made up to 100 ml.
Biuret reagent: KNaC4H4O6 (Na K tartarate) (45 g) + 40 ml of 0.2M NaOH + 15 g of CUSO4.5H2O + 5 g of KI were all dissolved and made up to 1 dm3 with 0.2 M NaOH.
1-chloro-2,4-dinitrobenzene, CDNB, (20 mM): CDNB (3.37 mg) was dissolved in 1ml of absolute ethanol in simple ratio to required volume.
Chloroform: The commercial product was obtained.
Chromagen: FeCl3 5 g was dissolved in 200 ml of phosphoric acid. Then 4 ml of the solution was pipetted and diluted to 24 ml with conc. H2SO4.
Conc. H2SO4: The commercial product was obtained.
Dichromate/acetic acid: This was prepared by mixing 5% solution of K2Cr2O2 with glacial acetic acid in a 1:3 volume ratio.
Dilute ammonia: Equal volume of distilled water and commercial ammonia was mixed. Dilute HCl: It was prepared by adding 200 ml of concentrated HCl to 800 ml of distilled water.
Dilute H2SO4: It was prepared by adding 127 ml of the conc. H2SO4 was added to 500 ml of distilled water and made up to 1 dm3 with distilled water.
Dragendorffs’ reagent: Bismuth carbonate (0.85 g) was dissolved in 100 ml of glacial acetic acid and 40 ml of distilled water to give solution A. Another solution B, was prepared by dissolving 8.0 g of potassium iodide in 20 ml of distilled water. Both solutions were then mixed to give the stock solution.
Ellman’s reagent (5,5’-dithio-2-nitrobenzoic acid): Ellman’s reagent (40 mg) was dissolved in, and made up to 100ml with the prepared buffer.
Ethanol: The commercial product (absolute) was obtained.
Ethylacetate: The commercial product was obtained.
FeCl3: It was prepared by dissolving 27 g of FeCl3 in water and making up to 1dm3. Fehling’s solution A: Fehling’s solution was prepared by dissolving 35 g of hydrated CuSO4 in water and few drops of conc. H2SO4 were added and the solution diluted to 500ml with distilled water.
Fehling’s solution B: 60 g of pure NaOH and 173 g of Rochelle salt (sodium potassium tartarate) were dissolved in 500 ml of distilled water.
Ferric chloride (5%): This was prepared by dissolving 2.5 g of ferric chloride in 50 ml of distilled water.
Formaldehyde (0.5%): This was prepared by adding 0.5 ml of formaldehyde to distilled water and making it up to 100 ml.
HCl (0.1N): HCl (9 ml) was added to 1dm3 of distilled water.
HCl (0.1M): This was prepared by adding 3.65 ml of concentrated HCl to 996.35 ml of water.
H2O2 (0.2M): This was prepared by adding 22.68 ml of 30% H2O2 (8.82 M) to distilled water and making up to 1 dm3.
H2SO4 (20%):It was prepared by dissolving 20 ml of H2SO4 in 80 ml of distilled water. H2SO4 (60%): It was prepared by dissolving 60 ml of H2SO4 in 40 ml of distilled water. KOH (20%): It was prepared by dissolving 20 g of KOH in distilled water and made up to 100 ml
Mayer’s reagent: Mercuric iodide (1.35 g) was dissolved in 50 ml of distilled water. Also 5 g of potassium iodide was dissolved in 20 ml of distilled water. The two solutions were mixed and the volume made up to 100 ml.
Molisch’s reagent: This was prepared by dissolving 0.1 g of α-napthol or (1 naphthol) in 100 ml of ethanol.
NaOH solution: This was prepared by dissolving 30 g of NaOH in distilled water and making up to 1 dm3.
Petroleum ether: The commercial product was obtained.
Picric acid solution: This was prepares by dissolving 10 g of picric acid in distilled water and making up to 1 dm3.
Phosphate buffer, pH 7.0, (0.1M): Na2HPO4.12H2O (3.58 g) and 1.19 g of NaH2PO4.2H2O were dissolved in water and made up to 1 dm3 with distilled water. The pH was adjusted to 7.0.
Phosphate buffer, pH 7.8, (0.05M): This was prepared by dissolving 6.97 g of K2HPO4 and
1.36 g of KH2PO4 in distilled water and making up to 1000ml with distilled water. The pH was adjusted to 7.8.
Phosphate buffer, pH 7.4, (0.1M): This was prepared by dissolving 7.163g of Na2HPO4 and 1.5603 of NaH2PO4.2H2O in distilled water and making up to 300ml. The pH was adjusted to 7.4.
Phosphate buffer, pH 6.5, (0.1M): KH2PO4 (4.96g) were added to distilled water and made up to 100 ml with distilled water. The pH was adjusted to 6.5.
Potassium dichromate, K2Cr2O7, (5%): K2Cr2O7 (5 g) was dissolved in distilled water and made up to 100 ml.
Reduced Glutathione, GSH, (0.1M): This was prepared by dissolving 40 mg of GSH in phosphate buffer and making up to 100 ml with the buffer. This served as the stock solution for the GSH standard calibration curve.
Sulfo-salicyclic acid (4.0%): Sulfosalicyclic acid (4g) was dissolved in distilled water and made up to 100 ml with distilled water too.
Thiobarbituric acid, TBA, (0.75%): Thiobarbituric acid (0.75 g) was dissolved and made up to 100 ml with 0.1 M HCL. The solution was allowed to dissolve properly by shaking in a boiling water bath
Trichloroacetic acid, TCA, (20%): Trichloroacetic acid (20 g) was dissolved in distilled water and made up to 100ml with distilled water too. The solution was stored in the refrigerator.
Tris KCl buffer (0.15 M): This was prepared by dissolving 1.15 g of KCl and making up to 100 ml with distilled water. 2.36 g of Tris-HCl were dissolved and made up to 100 ml with distilled water. The two solutions were then mixed together.
Wagner’s reagent: Iodine crystals (2 g) and 3 g of potassium iodide were dissolved in 100 ml of distilled water to obtain the reagent.
Methods
Aqueous Extraction
A known amount, 2000g, of Senna mimosoides leaves was extracted with 8400ml of distilled water using cold maceration. It was filtered first with calico and subsequently with glass wool and finally Whatmann No.4 filter paper. The filtrate was concentrated using the method of lyophilisation. A brown slurry-like substance was obtained and stored in the refrigerator for further investigation.
Determination of Percentage Yield
The percentage yield of the extract was calculated using the following formular
% yield = ×
Experimental Design
For the animal model experiment, one hundredand thirty (130) albino rats were used. The experimental designs were divided into four (4) phases containing five (5) or six (6) groups of five (5) rats in each group.
Phase 1
A total of twenty five (25) Wistar albino rats of same sex used in in vivo leukocyte mobilization were grouped as follows:
Group A: Rats were treated with agar and normal saline
Group B: Rats were treated with agar and 100 mg/kg body weight of extract Group C: Rats were treated with agar and 250 mg/kg body weight of extract Group D: Rats were treated with agar and 500 mg/kg body weight of extract Group E: Rats were treated with agar and 25 mg/kg body weight of indomethacin
Phase II
A total of fifty (50) Wistar albino rats used in delayed type hypersensitivity reaction and humoural antibody titre (twenty five (25) rats for each parameter) were grouped as follows:
Group A: Rats were treated with SRBC and normal saline and it served as control Group B: Rats were treated with SRBC and 50 mg/kg body weight of extract Group C: Rats were treated with SRBC and 100 mg/kg body weight of extract Group D: Rats were treated with SRBC and 250 mg/kg body weight of extract Group E: Rats were treated with SRBC and 25 mg/kg body weight of levamisol
Phase III
A total of twenty five (25) Wistar albino rats used in cyclophosphamide-induced myellosuppression were grouped as follows:
Group A: Rats were treated with 30 mg/kg of CP and normal saline and it served as control Group B: Rats were treated with 30 mg/kg of CP and 50 mg/kg body weight of extract Group C: Rats were treated with 30 mg/kg of CP and 100 mg/kg body weight of extract Group D: Rats were treated with 30 mg/kg of CP and 250 mg/kg body weight of extract Group E: Rats were treated with 30 mg/kg of CP and 25 mg/kg body weight of levamisol
Phase IV
A total of thirty (30) Wistar albino rats used in hepatotoxicity were grouped as follows: Group A: Rats were treated with CCl4 and normal saline and it served as control
Group B: Rats were treated with CCl4 and 50 mg/kg body weight of extract Group C: Rats were treated with CCl4 and 100 mg/kg body weight of extract Group D: Rats were treated with CCl4 and 250 mg/kg body weight of extract Group E: Rats were treated with CCl4 and 25 mg/kg body weight of levamisol
Phytochemical Analysis
Phytochemical analysis of the organic extract was carried out according to the method of Harborne (1998). Basic phytochemical screening was carried out using simple chemical tests to detect the presence of secondary plant constituents such as alkaloids, tannins, flavonoids, saponins, triterpenes, sterols, phenols, glycosides, reducing sugars and soluble carbohydrates in the sample. The methods used were those outlined by Harborne (1998) except otherwise stated.
Qualitative Phytochemical Analysis
Test for saponins
A 5.0ml aliquot of the extract was diluted with 20ml of deionized water, shaken vigorously and observed. Persistent foaming indicated the presence of saponins.
Test for alkaloids
A known quantity of the extract, 0.1 mg was added to 6ml of dilute hydrochloric acid and boiled, after boiling, it was cooled and filtered. The filtrate was divided into three portions and subjected to the following tests.To the first portion, 2 drops of Dragendorff’s reagent were added. The formation of a red precipitate indicated the presence of alkaloids.To the second portion, 2 drops of Meyer’s reagent were added. A creamy white precipitate indicated the presence of alkaloids.
To the third portion, 2 drops of Wagner’s reagent were added. A reddish-brown precipitate indicated the presence of alkaloids.
Test for tannins
The extract, 1 ml was added to 10 ml of deionised water and then treated with 3 drops of ferric chloride. A greenish-brown precipitate indicated the presence of tannins.
Test for flavonoides
A quantity of the extract was boiled in ethylacetate (10 ml) for 3 minutes, filtered and cooled. Then the filtrate (4 ml) was shaken with 1ml of dilute ammonia solution. An intense yellow colouration indicated the presence of flavonoids.
Test for terpenoids
Nine millilitre (9 ml) of ethanol was added to 1g of the extract and refluxed for a few minutes and filtered. The filtrate was concentrated down to 2.5 ml in a boiling water bath. Hot distilled water (5ml) was added to the concentrated solution; the mixture was allowed to stand for 1 hour and the waxy mater was filtered off. The filtrate was extracted with 2.5 ml of chloroform using a separating funnel. The chloroform extract was evaporated to dryness in a water bath and dissolved in 3 ml of concentrated sulphuric acid and then heated for 10 min in a water bath. A grey colour indicated the presence of terpenoids.
Test for steroids
A known quantity (0.1g) of the test sample was extracted in the chloroform and filtered. The filtrate was mixed with 2 ml of conc. H2SO4 carefully so that the sulphuric acid formed a lower layer. A reddish-brown colour at the interphase indicated the presence of steroidal ring.
Test for phenols
The test sample 0.1 g was added to 10 ml of distilled water. The solution was heated in a boiling water bath for 3 min and filtered. A 2 ml aliquot of the filtrate was placed in each of 3
test tubes. The filtrate in one of the test tubes was diluted with distilled water in the ratio 1:4. A blue or greenish colour indicated the presence of phenols.
Test for glycosides
Dilute sulphuric acid (5 ml) was added to 0.1 g of the extract in a test tube and boiled for 15 min in a water bath, then cooled and neutralized with 20% potassium hydroxide solution. Ten millilitre (10 ml) of a mixture of equal parts of Fehling’s solution A and B was added and boiled for 5 min. A more dense brick red precipitate indicated the presence of glycoside.
Test for reducing sugar
Five millilitre (5 ml) of a mixture of equal parts of Fehling’s solution A and B was added to 5 ml of extract and then heated in a water bath for 5 min. Brick red precipitate showed the presence of reducing sugar.
Test for cyanide
Distilled water (15 ml) was added to 0.1 g of the extract in a test tube. An alkaline picrate paper was suspended over the mixture and held in place by rubber bung. The arrangement was allowed to stand for 18 hr at room temperature. Colour change from yellow to orange indicated the presence of cyanide.
Test for soluble carbohydrate (Molisch test)
The extract (0.1 g) was boiled with 2 ml of distilled water and filtered. To the filtrate, few drops of naphthol solution in ethanol (molisch’s reagent) were added. Concentrated sulphuric acid in a Pasteur pipette was then gently poured down the side of the test tube to form a lower layer. A purple interfacial ring indicated the presence of carbohydrate.
Quantitative Phytochemical Analysis
Quantitative phytochemical screening of the aqueous extract of S. mimosoides leaves using the method of Harborne (1998).
Test for saponins
The extract (1g) was marcarated with 10 ml of petroleum ether and decanted into a beaker. Another 10 ml of the petroleum ether was added into the beaker and the filterate evaporated into dryness. The residue was dissolved in 6 ml of ethanol. The solution (2 ml) was put in a test tube and 2 ml of chromagen solution added into it. It was left to stand for 30 min and the absorbance was read at 550 nm.
Test for alkaloids
The extract (1 g) was marcarated with 20 ml of ethanol and 20% H2SO4 (1:1 v/v). The filterate (1 ml) was added to 5 ml of 60% H2SO4. After 5 min, 5 ml of 0.5% formaldehyde in 60% H2SO4 was mixed with the mixture and allowed to stand for 3 hr. The absorbance was read at 565 nm.
Test for tannins
The extract (1 g) was marcarated with 50 ml of methanol and filtered. To the filterate (5 ml),
0.3 ml of 0.1N ferric chloride in 0.1N HCl and 0.3 ml of 0.0008 M of potassium ferricyanide were added and the absorbance read at 720 nm.
Test for flavonoids
The extract (1 g) was marcarated with 20 ml of ethylacetate for 5 min and filtered. To the filterate (5 ml), 5 ml of dilute ammonia was added and shaken for 5 min. The upper layer was collected and the absorbance read at 490 nm.
Test for terpenoids
The extract (1 g) was marcarated with 50 ml of ethanol and filtered. To the filtrate (2.5 ml),
2.5 ml of 5% aqueous phosphomolybdic acid solution was added and 2.5 ml of concentrated H2SO4 was gradually added and mixed. The mixture was left to stand for 30 min and then made up to 12.5 ml with ethanol. The absorbance was taken at 700 nm.
Test for steroids
The extract (1 g) was marcarated with 20 ml of ethanol and filterd. To the filterate (2 ml), 2 ml of chromagen solution was added and the solution left to stand for 30 min. The absorbance was read at 550 nm.
Test for glycosides
The extract (1 g) was marcarated with 50 ml of distilled water and filtered. To the filterate (1 ml), 4 ml of alkaline pirate solution was added. The mixture was boiled for 5 min and allowed to cool. The absorbance was read at 490 nm.
Test for reducing sugar
The extract (1 g) was marcarated with 20 ml of distilled water and filtered. To 1 ml of the filterate, 1 ml of alkaline copper reagent was added. The mixture was boiled for 5 min and allowed to cool. Then 1 ml of phosphomolybdic acid reagent and 2 ml of distilled water was added and the absorbance read at 420 nm.
Test for soluble carbohydrate
The extract (1 g) was marcarated with 50 ml of distilled water and filtered. To the 1 ml of the filterate, saturated aqueous solution of picric acid was added and absorbance read at 580 nm.
Test for cyanide
The extract (1 g) was marcarated with 50 ml of distilled water and then filtered. To 1 ml of the filterate, 4 ml of alkaline picrate solution was added. The mixture was boiled for 5 min and allowed to cool. The absorbance was measured at 490 nm.
Test for phenols
The extract (1 g) was marcarated with 20 ml of 80% ethanol and then filtered. The filterate (5 ml) was added to 0.5 ml of folin ciocalteus reagent and allowed to stand for 30 min. Then 2 ml of 20% sodium carbonate was added and absorbance measured at 650 nm.
Determination of Biological Activity
Acute Toxicity and Lethality
Investigation on acute toxicity of the extract with estimation of the median lethal dose (LD50) was carried out using the method of Lorke (1983). Thirteen experimental animals (mice with weight range of 20g-30g) were used for the test. In the investigation, three groups of mice containing three mice each were administered 10-, 100- and 1000g/kg respectively of the aqueous extract intraperitoneally (ip). They were observed closely for 24 hr for lethality or any other behavioural response. Based on the result, further increased doses of 1500-, 2000-, 3000- and 5000 mg/kg were administered ip to four other mice respectively. They were also observed for 24 hr for any death or behavioural changes.
Immunomodulatory Activity of Extracts
Preparation of Antigen
Fresh sheep blood was obtained from the animal farm of the Faculty of Veterinary Medicine, University of Nigeria, Nsukka. Sheep red blood cells (SRBCs) were washed three times in continous volume of pyrogen-free sterile normal saline by centrifugation at 3000×g for 10
min on each occasion. The washed SRBCs were adjusted to a concentration of 109cells/ml for immunization and challenge.
Delayed Type Hypersensitivity (DTH) Reaction
Delayed type hypersensitivity (DTH) reaction was investigated using the method of Naved et al. (2005). DTH reaction was induced in rats using SRBCs as antigen. Animals were sensitized by subcutaneous injection of 0.02 ml of 109cells/ml SRBCs (day 0) in the plantar region of right hind foot paw and challenged on day 5 by subcutaneous injection of the same amount of antigen into the left hind paw. The oedema produced by antigenic challenge in the left hind paw was measured as the difference in the paw thickness before challenge; 24 hr and 48 hr after the challenge. The paw thickness was measured with a pocket-sized screw guage (Naved et al., 2005). The extracts were administered three days prior to sensitization and continued daily till the challenge.
Humoural Antibody (HA) Synthesis
Rats were immunized by an intraperitoneal injection (i.p) of 0.1ml of 109 SRBC ml-1 on day 0 and challenged by similar ip injection of the same amount of 0.1 ml on day 5. Primary antibody titre was determined on day 5 (before the challenge) and secondary titre on day 10 (Sharma et al., 1996) by haemagglutination technique (Nelson and Mildenhall, 1967). The aqueous extract (50, 100 and 250 mgKg-1) was administered 3 days prior to immunization and continued daily for 5 days after challenge. Blood samples were obtained by retro-orbital puncture and collected in a test tube and allowed to clot. For each sample, a 25µL serum was obtained after centrifugation and serially diluted two-fold in 96-U well microtitre plates using pyrogen-free sterile normal saline as control. The diluted sera were challenged with 25µL of 1% (v/v) SRBC in the plates and then incubated at 370C for 1hr. The highest dilution giving rise to visible haemagglutination was taken and antibody titre expressed in graded manner, the minimum dilution (1/2) being ranked as 1 (calculated as Log2 of the dilution factor). The mean ranks of different treatment groups were compared for statistical significance.
Cyclophosphamide-Induced Myelosuppression
Cyclophosphamide induced myelosuppression was studied with a slight modification of the method of Manjarekar et al. (2000). Blood samples of rats in each group were collected and haematological parameters determined. Then cyclophosphamide (30mg/kg i.p.) was administered to each of the rats in the groups. Three days later, blood samples of the rats were collected and haematological parameters determined. This was followed by oral
administration of different extract doses and levamisol for seven days. On the thirteenth day (from the day of cyclophosphamide administration), the blood samples of the animals were collected and the haematological parameters determined.
Determination of Haematological Parameter
White blood cell (WBC) count, %PCV, Hb concentration and RBC count of the extract were determined by the method described by Jain (1986).
Determination of WBC Count
WBC fluid (380 µl) was put in a test tube, 20 µl of blood was added and mixed well. The mixture was allowed to stand for 5 min so that the fluid will destroy the RBC and then fixed in a counting chamber. The number counted was multiplied by 50.
Determination of PCV Concentration
It was carried out with capillary tube with blood collcted in EDTA tube and sealed with plastacin at the bottom end. The tube was then placed in a centrifuge and spinned for 5 min at 10,000 to 12,000 Rev/min. The reading was then taken with a centrifuge reader and the value taken is the % of PCV in the blood.
Determination of Hb Concentration
Drabkine solution (5 ml) was put in a test tube and 20 µl of blood was added and mixed very well. This mixture was allowed to stand for 5 min and the absorbance of the mixture was read using colorimeter at 540 nm. The value gotten was converted using the Hb chart in g/dl.
Determination of RBC Count
RBC fuid (4 ml) was put in a test tube and 20 µl of blood was added, mixed and allowed to stand for 5 min for the destruction of WBC. The mixture was then inserted in a counting chamber and the number counted was multiplied by 10.
Erythrocyte indices including mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH) and mean corpuscular haemoglobin concentration (MCHC) were determined from the values obtained from RBC count, Hb concentration and PCV values (Duncan et al., 1994).
Effect of the Extract on in vivo Leukocyte Mobilization
The effect of the extract on in vivo leukocyte migration induced by inflammatory stimulus was investigated using the method of Ribeiro et al., (1991). One hour after oral administration of extract, each rat in the groups received intraperitoneal injections of 0.5ml of 3% (w/v) agar suspension in normal saline. Four hours later, the rats were sacrificed and the peritoneum washed with 5ml of 5% solution of EDTA in phosphate buffered saline (PBS). The peritoneal fluid was recovered and total and differential leukocyte counts (TLC and DLC) were performed on the perfusates.
Determination of the Effect of the Extract on CCl4 Induced Hepatotoxicity Wistar albino rats of either sex weighing 180-220 g were used for this study. Animals were divided into groups of five animals each and treated orally with the extract 7 days. CCl4 was
used to induce hepatotoxicity on day 3 and 5.
Animals were sacrificed under light ether anesthesia 24 hr after the last dose. Blood was collected by cardiac puncture in plain tubes and liver was removed, rinsed in cold saline, blotted with filter paper and weighed. Liver homogenate was prepared in 0.25 M sucrose solution and centrifuged at 7000 rpm for 10 min at 40C. The supernatant was used for various biochemical assays. Total protein was estimated according to the method of Lowry et al. (19) using bovine serum albumin (BSA) as a standard. Serum was separated by centrifugation at 3000 rpm at 40C for 10 min and used for measurement of various biochemical markers.
Assay of Serum Alanine Aminotransferase (ALT) Activity
The activity of alanine aminotransferase was assayed by the method of Reitman and Frankel (1957) as outlined in the Randox kit.
Principle
Alanine aminotransferase assay, according to this method, is based on the principle that pyruvate is formed from the reaction below:
α -Oxoglutarate + L-Aspartate L-Glutamate + Oxaloacetate
ALT
Methodology
Alanine aminotransferase is measured by monitoring the concentration of pyruvate hydrazone formed with 2, 4-dinitrophenylhydrazine.
Kit Reagents
S/N
Content
Initial Concentration of Reagents
1.
2.
Phosphate Buffer L – alanine
Α – Oxoglutarate
2, 4-Dinitrophenylhydrazine
100 mmol/l, pH 7.4
200 mmol/l,
2.0 mmol/l 2.0mmol/l
Procedures
Aliquots, 0.5 ml of the ALT substrate phosphate buffer solutions were pipetted into two sets of test tubes labeled B (sample blank) and T (sample test). The serum sample of 0.1 ml was added to the sample test (T) test tubes only and mixed thoroughly and then, incubated in a water bath at temperature of 370 C for 30 minutes.
A volume, 0.5 ml of 2, 4-dinitrophenylhydrazine was added to each of the test tubes labelled T (sample test) and B (sample blank) immediately after the incubation. Also, 0.1 ml of serum sample was added to sample blank (B) only. The entire medium was mixed thoroughly and allowed to stand at 250 C for exactly 20 minutes. After this, 5.0 ml of sodium hydroxide (NaOH) solution was added to each test tube and mixed thoroughly. The absorbance of sample against the sample blank was read at a wavelength of 550 nm against the sample blank after 5 minutes. The activity of ALT in the serum was obtained from the pre-calibrated table.
Assay of Aspartate Aminotransferase (AST) Activity
The activity of aspartate aminotransferase (AST) was assayed by the method of Reitman and Frankel (1957) as outline in Randox kit.
Principle
Oxaloacetate is formed according to the equation as follows.
α – Oxoglutarate + L-aspartate AST L-glutamate+ Oxaloacetate
Methodology
Aspartate aminotransferase activity was measured by monitoring the following information of oxaloacetate hydrazone with 2, 4-dinitrophenylhydrazine.
Kit Reagents
S/N
Content
Initial Concentration of Reagents
1.
Phosphate Buffer
100 mmol/l, pH 7.4
L – Aspartate
100 mml/l
2.
α – Oxoglutarate
2, 4-Dinitrophenylhydrazine
2 mmol/l
2mmol/l
Measurement against reagent blank
The AST substrate phosphate buffer solutions (0.5 ml each) were pipetted into both the reagent blank (B) and sample test (T) test tubes. The serum sample of 0.1 ml was added to the sample test (T) tubes only and mixed thoroughly. Then, 0.1 ml of distilled water was added to the reagent blank (B). Then, the entire reaction medium was well mixed and incubated in a water bath at 370 C for 30 minutes.
Immediately after incubation, 0.5 ml of 2, 4-dinitrophenylhydrazine was added to the reagent blank (B) and the sample test tubes. It was mixed thoroughly and allowed to stand for exactly 20 minutes at 250 C. Finally, 5.0 ml of sodium hydroxide solution was added to both the blank and the reagent test tubes respectively and mixed thoroughly. The absorbance of sample Asample was read at the wavelength of 550 nm against the reagent blank after 5 minutes.
Measurement against sample blank
The AST substrate phosphate buffer solutions (0.5 ml each) were pipetted into both the reagent blank (B) and sample test (T) test tubes. The serum sample of volume, 0.1 ml was added to the sample test (T) test tubes only and mixed thoroughly. Then, 0.1 ml of distilled water was added to the reagent blank (B). Thereafter, the entire reaction medium was well mixed and incubated at 37o C for 30 minutes in a water bath.
A volume, 0.5 ml of 2, 4-dinitrophenylhydrazine was added to the reagent blank (B) and the sample test tubes immediately after incubation. Also, 0.1 ml of the sample was added to blank (B) only. The medium was mixed and allowed to stand for exactly 20 minutes at 250 C. Finally, 5.0 ml of sodium hydroxide (NaOH) solution was added to both the blank (B) and
sample test (T) test tubes and mixed thoroughly. After 5 minutes, the absorbance of sample A was read at 550 nm against the sample blank after 5 minutes. The activity of AST in mice serum was obtained from the already calibrated table.
Assay of Serum Alkaline Phosphatase (ALP) Activity
The activity of alkaline phosphatase (ALP) was assayed by the method of Reitman and Frankel (1957) as outline in Randox kit.
Principle
p-nitrophenol is formed according to the equation as follows p-nitrophenylphosphate + H2O → Phosphate + p-nitrophenol
Methodology Kit Reagent
S/N
Content
Concentration in the Test
R1a
R1b
Diethanolamine buffer Magnesium chloride
p-nitrophenylphosphate
1 mol/l, pH 9.8
0.5 mmol/l 10 mmol/l
Procedure
Pipetted into cuvette was 0.05 ml of sample, and 3.00 ml reagent 1a (Diethanolamine buffer 1mol/l pH 9.8, MgCl2 0.5 mmol/l) and reagent 1b (p-nitrophenylphosphate 10 mmol/l). They were mixed and read at time 0, 1, 2 and 3 minutes at 405 nm.
The calculation of ALP activity was done thus:
ALP activity = 3300 x ΔA
ΔA is change in absorbance of the sample at 405 nm 3300 = a constant
Determination of Serum Bilirubin Concentration
Total bilirubin concentration was determined using the method of Jendrassik and Grof (1938) as outlined in the Randox kit.
Principle
Direct (conjugated) bilirubin reacts with diazotized sulphanilic acid in alkaline medium to form a blue coloured complex. Total bilirubin is determined in the presence of caffeine, which releases albumin bound bilirubin by reaction with diazotized sulphanilic acid.
Methodology
A volume, 0.2 ml of sulphanilic acid was pipetted into the sample blank tube and sample tube. This was immediately followed by the addition of 0.05 ml of sodium nitrite to the sample tube. Caffeine (10 ml) and 0.2 ml of sample were also pipetted into each of the sample blank tube and sample tube. These mixtures were mixed and incubated for 10 minutes at 20 -250 C. Finally, 1.0 ml of tartrate was pipetted into the sample blank tube and sample tube. These mixtures were once again mixed, incubated at 20 – 250 C for 30 minutes and their absorbances read at 578 nm against the sample blank.
Total bilirubin (µmol/l) = 185 × sample blank (578 nm)
Assay of Catalase Activity
The catalase activity of the supernatant fraction from the liver was determined according to the method of Sinha (1972). The standard curve of H2O2 was prepared. To prepare the curve, different amounts of 0.2 M H2O2 ranging from 10 to 100 micromoles were taken in test tubes. Dichromate/acetic acid (2 ml) was added to each. On adding the dichromate/acetic acid, an unstable blue precipitate of perchromic acid was instantaneously produced. Heating for 10 min in a boiling water bath changed the colour of the solution to stable green due to the formation of chromic acetate. After cooling at room temperature, the volumes of the samples were made to 3 ml with distilled water and the absorbance measured at 570 nm. The curve was gotten by plotting absorbance on the vertical axis against concentration on the horizontal axis.
The supernatant fraction of the liver was properly diluted 50 times. 4ml of 0.2M H2O2 was added to 5ml of 0.1M phosphate buffer pH 7.0. 1.0ml of the properly diluted enzyme preparation (the diluted liver fractions) was added to the H2O2/buffer mixture and the mixture was gently mixed at room temperature. Some portion (1.0 ml) of the reaction mixture was withdrawn and blown into 2 ml dichromate/acetic acid reagent at one minute interval and the steady absorbance reading taken at 570 nm.
The monomolecular velocity constant K; for the decomposition of H2O2 by catalase was determined by using the equation for a first order reaction
Where S0 is the initial H2O2 concentration and S is the concentration of H2O2 at a particular time interval given as t(minutes). The value of K are plotted against t, and the velocity constant of catalase K(0) at 0 minute determined by extrapolation (that is the intercept on the vertical axis). The catalase contents of the samples were expressed in terms of katalase fighigkeit or ‘Kat.Kat.f. = K (0)/mg protein per ml.
Determination of Reduced Glutathione concentration
Hepatic reduced glutathione (GSH) level was determined by the method of Ellman modified by Jollow et al.(1974). GSH calibration curve was made by serial dilutions of the stock GSH solution by using 0.1 M phosphate buffer pH 7.4 for dilution (to a total volume of 0.5 ml). Ellman’s reagent (4.5ml) was added to each solution. The absorbance of the coloured solution developed was read at 412 nm within 5 min of the colour generation. Absorbance was plotted against glutathione concentration.
One ml of 4% Sulfo-salicylic acid was added to a mixture of 0.4 ml homogenate and 0.6 ml of distilled water as protein precipitant. The mixture was centrifuged at 17,000 rpm for 15 min at 20C. The supernatant (0.5ml) was added to 4.5 ml of Ellman’s reagent. A blank was prepared by adding 0.5 ml of 4% Sulfo-salicylic acid to 4.5 ml of Ellman’s reagent. The absorbance was read at 412 nm and the equivalent GSH concentration was read from the curve.
Assay of Superoxide Dismutase (SOD) Activity
SOD activity was assyed using the method of Fridovich (1989). The liver homogenate was diluted 20 times and 0.2 ml of it was added to 2.5 ml of 0.05 M phosphate buffer, pH 7.8. The mixture was equilibrated in the spectrophotometer before adding adrenaline solution. The reaction started with the addition of 0.3 ml of freshly prepared adrenaline solution to the mixture followed by quick mixing by inversion in the cuvette. The reference cuvette therefore contained 2.5 ml buffer, 0.3 ml of adrenaline and 0.2 ml of sample. The increase in absorbance was taken at 480 nm for 150 seconds at 30 seconds interval.
The results were calculated as follows
Where A0 = absorbance after 30 seconds A3 = absorbance after 150 seconds % Inhibition = (Increase in absorbance for substrate) × 100 / (Increase in absorbance for blank)
One unit of SOD activity was given as the amount of SOD necessary to cause 50% inhibition of the oxidation of adrenaline.
Note: A blank was prepared with 0.3 ml of adrenaline in 2.5 ml of buffer.