Plant material
The leaves of A. cordifolia were collected from Dschang locality, Menoua division, West region in April 2010. Authentification was carried out by the botanists of the Cameroon National Herbarium, Yaounde, where a voucher specimen (9656/SRF CAM) was deposited.
Test bacteria and culture media
The test microorganism, Escherichia coli, was obtained from the Medical Bacteriology Laboratory of the Pasteur Center, Yaounde, Cameroon. Escherichia coli ATCC 10536 obtained from American Type Culture Collection was used as reference strain. Three types of culture media were used during the work: Mueller Hinton Agar (MHA) for the determination of the minimum bactericidal concentration, Mueller Hinton Broth (MHB) for the determination of the minimum inhibitory concentration and Mac conkey agar (MCA) for E. coli culture.
Experimental animals
In the present study, 60 Swiss albino mice (30 males and 30 females) weighing 18–24 g, and 35 Wistar albino female rats weighing 148–187 g were used. These animals were bred in the animal house of the University of Dschang, Cameroon.
Preparation of the extract
The leaves of A. cordifolia were allowed to dry at room temperature (24 ± 2 °C) and were ground. 100 g of the powder were macerated at room temperature in 1 l of water for 48 h and filtered with no 1 whatman paper. The filtrate was concentrated in a drying oven at 45 °C to obtain12.64 g of crude extract [8].
Antibacterial test
The in vitro antibacterial activity of the extract was performed by determining the minimum inhibitory concentrations using broth microdilution method [8]. Briefly, bacterial suspensions of about 1.5 × 108 CFU/ml (Mc Farland turbidity standard no. 0.5) were prepared. To obtain the inocula, these suspensions were diluted 100 times in Muller Hinton broth to give 1.5 × 106 CFU/ml. The antimicrobial susceptibility tests were performed in 96 wells microplates. A serial two-fold dilution of the plant extract was performed to obtain final concentration range of 6000 to 46.87 μg/ml for the extract and of 20 to 0.157 μg/ml for the ciprofloxacin which served as reference drug (positive control) in a total volume of 200 μl/well. Each well contained the test substances at a particular concentration and the bacterial suspension (100 μl) in Muller Hinton broth. For every experiment, sterility control (5% v/v aqueous DMSO and broth) and negative control made up of 5% v/v aqueous DMSO, broth and inoculum were included. The plate was later covered with sterile cover and incubated at 37 °C for 24 h. Growth was monitored colorimetrically using p-iodonitrotetrazolium violet (INT). Viable bacteria change the yellow dye of p-iodonitrotetrazolium violet to a pink colour. All concentrations at which no visible colour changes were observed were considered as inhibitory concentrations and the lowest of these concentrations was considered as the MIC.
The ridges (zigzag) method was used to determine minimal bactericidal concentrations (MBCs). In fact, the wells which showed no growth were cultivated on already prepared petri dishes of 90 mm containing MHA. After 24 h of incubation at 37 °C, concentrations which presented no bacterial growth were considered as bactericidal and the lowest was the MBC.
Phytochemical screening
The phytochemical screening was performed qualitatively using standard methods [10]. The plant sample was screened for the following classes of compounds: phenols, tannins, terpenoids, flavonoids, steroids, alkaloids, anthraquinones, anthocyanins, saponins, coumarins.
Acute toxicity
In acute toxicity studies, 60 Swiss albino mice (30 males and 30 females) were used. Animal treatment was performed according to the method previously described [11]. 3 h following administration of the test substance, the animals were observed frequently for bihavioural and observable physiological variations. The death animals were counted within the first 48 h and the lethal dose 50 (LD50) was determined [12]. Surviving animals were further observed for 2 weeks, during which their weight, food and water intake were recorded.
Infection and treatment
In vivo antibacterial and toxicity studies were performed according to the method previously described [11] with some modifications. The animals were divided into six groups (5 males and 5 females): one simple control (animals who received distilled water), one negative control (infected but not treated), one positive control (infected animals that received the standard antibiotic) and three extract treated groups (58 mg/kg, 116 mg/kg and 232 mg/kg) based on the tradipratician dose which was 58 mg/kg. All the animals were immunosuppressed by intraperitoneal administration of cyclophosphamid at 30 mg/kg, and the infection was done on the third day by intravaginal administration of 100 μl E. coli (1.2 × 108 CFU/ml). From the fourth day post-infection, animals were daily treated with the A. cordifolia extract. The administration of various doses of extract, antibiotic and distilled water was done by gastric intubations once a day, for two consecutive weeks. The food and water intakes were evaluated every day and the animals were also weighed. The evolution of the bacterial load as a function of time was determined based on the diluted vaginal sample.
Effect of the Alchornea cordifolia extract on haematological, biochemical and histological parameters
At the end of treatment, the animals were anesthetized with chloroform vapour prior to dissection. Blood samples were collected by cardiac puncture into heparinised and non- heparinised centrifuge tubes. The heparinised blood was used to estimate hematocrit values, while the non-heparinised blood was allowed to coagulate, centrifuged and the serum was separated. Serum was assayed for proteins, cholesterol, triglycerides, creatinine, and Transaminases (ALT and AST). Immediately after blood collection, the animals were killed for tissue study. Liver, lungs, heart, kidneys, and spleen were isolated, and weighed. Part of each of these organs was cut and stored at −30 °C for the determination of protein concentration, while pieces of livers were kept for the histological analyses.
Preparation of serum sample
The blood was allowed to clot by standing at room temperature for 1 h and then refrigerated for another 1 h. The resultant liquid part was removed and centrifuged at 3000 xg for 5 min, and then the serum (supernatant) was obtained and stored at −30 °C for analysis.
Preparation of tissue homogenate
The homogenate of each organ was prepared in 0.9% NaCl solution at the concentration of 15% (i.e. 15 g organ in 100 ml of solution). Possible damages to the liver, kidneys, heart, lungs, spleen, and red blood cells of the animals as a result of repeated administration of the Alchornea cordifolia extract was studied using some biochemical parameters of tissue damages. Total protein concentrations of the above-mentioned organs were determined by the Biuret method [13]. Serum cholesterol, triglycerides and creatinine levels were determined by colorimetric method using commercial kits of INMESCO [14]. Serum transaminases (ALT and AST) activities were determined by the kinetic method [15, 16] using commercial kits of INMESCO. The determination of the hematological parameters [17] and the histological study were perfomed [18].
Statistical analysis
Statistical analyses were performed using SPSS for Window software version 12.0. Results were expressed as mean ± standard deviation using the ANOVA and the means of different groups were compared using the Waller-Duncan test where P value less than 0.05 was considered statistically significant.