Antibacterial and dermal toxicological profiles of ethyl acetate extract from Crassocephalum bauchiense (Hutch.) Milne-Redh (Asteraceae)

The microorganisms used included five ATCC bacterial strains (Staphylococcus aureus ATCC 25922, Enterococcus faecalis ATCC 10541, Escherichia coli ATCC 11775, Pseudomonas aeruginosa ATCC 27853, Salmonella typhi ATCC 6539) and three clinical isolates (Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus) obtained from "Centre Pasteur du Cameroun, Yaoundé". The strains were maintained at +4°C on agar slants.

The leaves of C. bauchiense were dried at room temperature for ten days and powdered to coarse particles. Seven hundred grams of the powder were macerated with 5 l of ethyl acetate for two days with frequent stirring. After filtration, the solvent was removed under reduced pressure using a rotary evaporator (45°C) to yield a paste of 71.75 g (10.25%). The phytochemical analysis was performed following standard methods [5].

The in vitro antibacterial activity of the extract was performed by determining the minimum inhibitory concentrations using broth microdilution method [6]. Briefly, the stock solution of C. bauchiense extract was dissolved in 5% tween 80 in water. 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 6.25 to 0.04 mg/ml for the extract and from 128 to 0.0625 μg/ml for the reference drugs 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. The plates were incubated at 35°C for 18 h. Growth was monitored colorimetrically using iodotetrazolium chloride (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 bactericidal concentrations were determined by adding 50 μl aliquots of the preparations (without INT), which did not show any visible colour change after incubation during MIC assays, into 150 μl of extract-free Mueller Hinton broth [6]. These preparations were further incubated at 35°C for 48 hrs and bacterial growth was revealed by the addition of INT as above. All extract concentrations at which no colour changes were observed were considered as bactericidal concentrations. The smallest of these concentrations was considered as the MBC [6].

The assays were carried out in triplicate. Gentamycin and ciprofloxacin were used as positive controls, and 5% tween 80 solution was used as negative control.

Furs were clipped from about 10% of the body surface area 24 h before the experiment. Infection was established by inoculating the clipped portion, at two different sites, with 0.1 ml of S. Aureus suspension (2 × 10⁸ CFU/ml) prepared from an overnight culture [7]. Infected mice were divided into six groups of five animals each (three control and three test groups). The first control group was not treated, the second and third received vehicle and gentamycin (2%) respectively. The three other groups were treated with gel-extract at 0.5, 1 and 2% (w/v) respectively. Treatment started 48 h after the establishment of the infection by dermal application of 0.05 g of gel-extract once per day for 5 consecutive days. Body weights were measured before inoculation of bacterial suspension and once daily during the experiment period. At the end of the treatment, animals were anaesthetised with chloroform vapour and the skin sample was excised and homogenized in normal saline and further cultured on Mannitol Salt Agar Medium for bacterial count.

A single dose dermal toxicity was conducted according to the OECD guidelines of toxicity studies [8]. Furs were clipped as described above prior to the application of the test substance. Rats were divided into one control and five treated groups (each group consisted of ten animals including 5 males and 5 females). The control group received vehicle (32 g/kg) while each treated group received the C. bauchiense extract gel at 4, 8, 16 and 32 g/kg bw by dermal application. Clinical signs and mortality were observed 15, 30 min and 1, 2, 3 and 6 h after administration of test substances and twice daily for 14 days. Body weights, water and food consumption were recorded throughout the experimental period.

Furs were clipped from at least 10% of the body surface area of rats 24 h before the experiment [9]. Animals were divided into one control and four treated groups. Each group consisted of ten animals (5 females and 5 males). The control group received vehicle (2400 mg/kg) and each treated group received the C. bauchiense extract-gel (30 mg/kg, 300 mg/kg, 1200 mg/kg and 2400 mg/kg) by dermal application for 28 days (once a day).

Observations were made twice each day for motility and mortality. Clinical examinations were made once prior to the first treatment and once weekly. Changes in skin, fur, eyes, mucous membranes and excretions and autonomic activity were noted [9]. At the end of the 21^st days of exposure, motor activity, grip strength and sensory reactivity to stimuli of different types were assessed. Animal weight, water and food consumptions were recorded throughout the experimental period.

On the 28^th day of experiment, urine was collected from individual metabolic cages containing animals subjected to overnight fasting [10].

Blood samples were collected by cardiac puncture from ketamine-diazepam anaesthetized rats into heparinised and non heparinised tubes. The non heparinised tubes were allowed to clot and were centrifuged at 3000 rpm for 5 min to obtain the serum. Animals were further sacrificed and used for gross pathological examinations including weight of different organs (liver, kidney, lung, heart and spleen) [11]. Fifteen percent homogenate of liver and spleen tissues from experimental animals were prepared in normal saline solution. The homogenates obtained were centrifuged at 3.000 rpm for 30 min and the supernatants were used for protein quantification [12].

The serum was assayed for creatinine, aspartate amino transferase (AST), alanine amino tranferase (ALT), total cholesterol, high density lipoprotein (HDL), triglycerides and total protein using commercial kits (Hospitex diagnostic, Roma, Italia). Urine was assayed for total protein and creatinine using the same commercial kits. The liver and spleen homogenates were also assayed for their protein content.

Malassez chamber was used to quantify the total red blood cells (RBCs) and white blood cells (WBCs). Haematocrit was estimated using standard methods [13].

Immediately after collecting the blood samples, vascular perfusion was performed for hepatic tissue fixation using isotonic saline solution (250 ml) followed by 10% phosphate buffered formalin solution (250 ml). Small pieces of liver were subjected to haematoxylin-eosin staining [14]. Pathological observations were performed on gross and microscopic basis. Histological plates were encrypted for analysis by a histopathologist.

Data were subjected to the one way analysis of variance (ANOVA) and recorded as mean ± SD and where differences exist, means were compared using Waller Duncan test at 0.05 significant level.

Phenols, alkaloids, flavonoids, tannins, triterpenes and sterols were identified in the ethyl acetate extract of C. bauchiense. This plant extract expressed antibacterial activity on all the tested microorganisms (Table 1). However, this activity was interesting only on S. aureus (MIC = 48 μg/ml and MBC = 160 μg/ml). P. aeruginosa and E. coli (clinical isolate) were the most resistant to the extract (MIC = 3125 μg/ml and MBC = 6250 μg/ml). MICs values were four fold less than the MBC values, indicating that the bactericidal effect of the C. bauchiense ethyl acetate extract could be expected.

Animals infected with S. aureus showed visible inflammation 48 hours later, characterised by redness and swelling of the skin at the sites of inoculation. Treatment significantly (P < 0.05) reduced the bacterial concentration at the infection sites, with total suppression being noted with the 1 and 2% doses of C. bauchiense gel-extract as gentamycin in 5 days (Figure 1). Animals in all groups showed weight loss after infection (Figure 2). From day 1 of treatment, body weight increased gradually with time. On the other hand, the weight gain by the animals of the negative control was lower than that of all the other groups. However, the weight gain by the animals that received the extract at 1 and 2% were comparable (p > 0.05) to gentamycin group, and significantly higher (p < 0.05) than the 0.5% extract-treated group.

No significant variation was noted in rat's body weight at the end of 14 days of experiment between groups. Besides, no changes in skin, fur, eyes, mucous membranes, respiratory and behavioral pattern were observed. In addition, no sign of convulsion, salivation, diarrhea, lethargy, sleep were noted. Up to 32 g/kg bw in both sexes, no death was observed, indicating that the lethal dose fifty (LD₅₀) of the C. bauchiense extract is higher than 32 g/kg in rats.