2.1 Source of test animal and management
One hundred and forty (140) experimental rats (Rattus norvegicus) of both sexes and age 60 days with a mean weight of 210 ± 10 g were employed for this study. The rats were kept in metal cages in a well-ventilated animal house with five rats placed in each cage and maintained at room temperature (32 ± 2 °C). The rats had free access to water and rodent pellet feeds from the Vital Feeds Limited, Lagos, Nigeria.
2.2 Plant materials and extraction of bioactive compounds
Fresh leaves of Guiera senegalensis, Cassia occidentalis, and Ziziphus mauritiana were collected around Kalgo, Birnin Kebbi, Nigeria, in August 2018. The plants were identified by a taxonomist in the Department of Plant Science and Biotechnology, Kebbi State University of Science and Technology, Aliero. Samples of the authenticated plants with voucher numbers V.N 48, V.N 71, and V.N 258 for G. senegalensis, C. occidentalis, and Z. mauritiana, respectively, were kept in the herbarium section of the department. The plant materials were washed gently to remove impurities and air-dried under shade to a constant dry weight. The dried leaves were milled into fine powder using a laboratory grinder manufactured by TENCAN (model no: XQM-(20-100). About 100, 50, and 100 g powder of Z. mauritiana, C. occidentalis, and G. senegalensis were soaked in flasks containing 600, 300, and 600 ml, 98% methanol, respectively, and were allowed to stand for 24 h. The extracts thus obtained were filtered with muslin cloth, and the solvent (methanol) was removed using a rotary evaporator at 45 °C. The dried extracts were stored in a desiccator before use.
2.3 Qualitative screening of the extracts
The qualitative screening of the extracts was done using standard protocols as described by Sofowora [17].
2.3.1 Test for glycosides
About 2.5 ml of 50% H2SO4 was added to 5 ml of each extract in a test tube, and the mixture was heated in a boiling bath for 15 min. The mixture was allowed to cool and neutralized with 10% NaOH. About 5 ml of Fehling’s solution was added, and the mixture was boiled. A brick-red precipitate was observed, which indicated the presence of glycosides.
2.3.2 Test for anthraquinones
The extract (0.5 g) of each plant was shaken with 10 ml of benzene. The mixture was filtered after which 5 ml of 10% ammonia solution was added to the filtrate and shaken gently. The presence of a pink, red, or violet color in the ammoniacal (lower) phase indicated the presence of anthraquinones.
2.3.3 Test for saponins
About 0.5 g of each extract was dissolved in 10 ml of distilled water in a test tube. The test tube was covered tightly and shaken vigorously for 30 s, after which it was allowed to stand for 45 min. The appearance of frothing, which persists on warming, indicated the presence of saponins.
2.3.4 Test for phlobatanins
The presence of phlobatanins in the extract was determined using the hydrogen chloride test. The extract (0.5 g) was dissolved in distilled water and filtered after which the filtrate was boiled with 2% HCl solution. A red precipitate revealed the presence of phlobatanins.
2.3.5 Test for flavonoids
To a portion of the dissolved extract, a few drops of 10% ferric chloride solution were added. A green or blue color indicated the presence of flavonoids.
2.3.6 Test for steroids
About 0.5 g of each extract was dissolved in 2 ml acetic anhydride and placed in ice. Sulfuric acid was then carefully added, and a color change from violet to blue and finally green indicated the presence of a steroidal nucleus.
2.3.7 Test for tannins
About 0.5 g of the extract was dissolved in 5 ml of water, followed by a few drops of 10% ferric chloride. A blue-black, green, or blue-green precipitate indicated the presence of tannins.
2.3.8 Test for alkaloids
The presence of alkaloids in the extracts was tested using the Wagner-Dragendorff’s test. About 0.2 g of the extracts was heated with 2% H2SO4 for 2 min. The mixture was filtered, and few drops of Dragendorff’s reagent were added. An orange-red precipitate showed the presence of alkaloids.
2.4 Quantitative screening of the extracts
The levels of the phytochemicals detected in the extracts of the plants during the qualitative screening were estimated using the method of Ajuru et al. [18].
2.4.1 Estimation of alkaloids
Five grams of extract of each plant was put in a 250-ml beaker, and 200 ml of 20% acetic acid in ethanol was added and made to stand for 4 h. The mixture was filtered, and the filtrate was concentrated to one quarter of the original volume using a water bath. Few drops of concentrated ammonium hydroxide were added to the extract until the preparation was complete. The solution was allowed to settle, and the precipitate was collected by filtration and weighed. The weight of the alkaloids obtained was then expressed as a percentage of the raw extract of the plant:
$$ \mathrm{Alkaloids}\ \left(\%\right)=\left[\mathrm{weight}\ \mathrm{of}\ \mathrm{alkaloids}/\mathrm{weight}\ \mathrm{of}\ \mathrm{raw}\ \mathrm{extract}\right]\times 100 $$
2.4.2 Estimation of tannins
Five grams of each extract was weighed into a 100-ml plastic bottle containing 50 ml of distilled water and shaken for 1 h in a mechanical shaker. The filtrate obtained was put in a 50-ml volumetric flask and made up to the mark. Then, 5 ml of the filtrate was pipette into a tube and mixed with 3 ml of 0.1 M FeCl3 in 0.1 N HCl and 0.008 M potassium ferrocyanide. The absorbance was measured in a spectrophotometer at 120 nm wavelength within 10 min. A blank sample was prepared, and the color also developed and read at the same wavelength. A standard was prepared using tannic acid to get 100 ppm and measured. The weight was thereafter expressed as a percentage of the raw extract.
2.4.3 Estimation of flavonoids
One hundred grams of each plant extract was extracted repeatedly with 100 ml of 80% aqueous methanol at room temperature. The whole solution was filtered using Whatman filter paper no. 42 (125 mm). The filtrate was later transferred into a crucible, evaporated to dryness over a water bath, and weighed and expressed as a percentage of the raw extract.
2.4.4 Estimation of saponins
The extracts were ground, and 20 g was transferred into 200 ml of 20% ethanol. The suspension produced was heated over a hot water bath for 4 h with continuous stirring at about 55 °C. The mixture was filtered, and the residue re-extracted with another 200 ml of 20% ethanol. The combined extracts were reduced to 40 ml over a water bath at about 90 °C. The concentrate was transferred into a 250-ml separator funnel, and 20 ml of diethyl ether was added and shaken vigorously. The aqueous layer was recovered while the ether layer was discarded. The purification process was repeated, and 60 ml of n-butanol was added. The combined n-butanol extracts were washed twice with 10 ml of 5% aqueous sodium chloride. The remaining solution was heated in a water bath. After evaporation, the samples were dried in the oven to a constant weight and estimated as a percentage of the raw extract.
2.4.5 Estimation of glycosides
One gram of the extract was macerated in 50 ml of distilled water and filtered. Then, 4 ml of the alkaline picrate solution was added to 1 ml of the filtrate. The mixture was boiled for 5 min, allowed to cool, and weighed and expressed as a percentage of the raw extract.
2.5 Experimental design
The toxicity test of methanol extracts of the three plants was determined in two phases. In the first phase, the “Classical LD50” method described by Trevan [19] was used to establish the range of doses capable of producing toxic effects. Ninety rats were randomly divided into 3 groups containing 30 rats each with a group assigned 1 of the extracts and labeled G. senegalensis, C. occidentalis, and Z. mauritiana group, respectively. The thirty rats in each group were subdivided into 5 containing 6 rats each and administered 500, 1000, 2000, 3000, and 5000 mg kg−1 of the extract, respectively. A control group was equally set up, but rats in this group were not given any extract. The rats were monitored for 72 h, and general toxicities of the extracts were evaluated according to a set of signs determined by Almeida [20].
In the second phase, a new set of 36 rats were distributed equally into 4 groups. Rats in groups 1, 2, and 3 were administered 1000 mg kg−1 extracts of G. senegalensis, C. occidentalis, and Z. mauritiana, respectively, for 60 days. The fourth group was made the control and were not fed any extract. At the end of the treatments, the rats were sacrificed by cervical dislocation after sedating them with chloroform in a tightly covered bell-shaped glass jar. Blood samples were then collected for liver function tests (alanine aminotransferase (ALT), apartatate aminotransferase (AST), alkaline phosphatase (ALP), total protein (TP), and albumin) and kidney function tests (creatinine, urea, Cl−, Na+, and K+). The livers of the rats were also harvested for histological examination.
2.6 Blood sample collection procedures
Blood samples were obtained from the rats based on the method of Hugo and Russel [21]. The rats were sedated with chloroform in a bell-shaped glass jar in the laboratory. Total death was prevented to allow continuous flow of blood for proper blood collection. Each rat was pegged down on a work bench and held firmly with office pins. A surgical blade was used to cut through the chest region of the rats in the dorsal-ventral direction. The blood was then collected from a beating heart using a heparinized capillary tube through capillary action into EDTA bottle. EDTA serves as an anticoagulant.
2.7 Liver and kidney function tests
The biochemical parameters, including the ALT, AST, and ALP, were estimated by ultraviolet, colorimetric, and spectrophotometric methods, respectively, as described by Bergneyer and Bernt [22]. The albumin and protein content were calculated by the biuret method as described by Parasuraman et al. [23].
For the determination of kidney functions, creatinine and urea levels were analyzed using the methods of Tietz [24]. The serum ions, including Cl−, Na+, and K+, levels were determined using the flame photometry method as described by Akhigbe et al. [25].
2.8 Histopathological examination
The liver tissues of the rats were prepared for histopathological examination using the method of Taylor et al. [26]. The tissues were excised and processed using routine hematoxylin and eosin staining techniques. Analysis of the tissues was done using qualitative methods with emphasis on the morphology, architectures, and cytological structures of the tissues.
2.9 Data analysis
Data were expressed as mean ± standard error of mean (SEM). The statistical difference between the control and test groups was analyzed using Student’s t test. The value of p ≤ 0.05 was considered significant.
