Antibacterial, Acute Toxicity and Histological Study of Dodonaea viscosa, Eucalyptus camaldulencis and Senna siamae: Ethnobotanical Plants for Typhoid Fever in Kano State, Nigeria

Collection and extraction of plant materials

Fresh leaves of Eucalyptus camaldulensis, Senna siamae and Dodonaea viscosa were collected from commercial botanical garden at Tarauni local government area of Kano State. The samples were taken to the herbarium of the department of plant Biology, Bayero University Kano for identification. The selected plant materials were assigned voucher numbers BUKHAN 347 for Eucalyptus camaldulensis. BUKHAN 70 for Senna siamae and BUKHAN 118 for Dodonaea viscosa.

The leaves of the selected plants were properly washed in distilled water and dried under shade for 7 days. Fifty grams (50g) of the pulverized plant materials were macerated in 500ml of ethanol over a period of 72 hours with intermittent shaking. The mixtures were filtered using No.1 Whatman filter paper (15). The filtrates were concentrated using rotary evaporator at reduced temperature and pressure. It was further concentrated using water bath at 50^oC. The crude ethanolic extracts were stored in the air tight container at room ready for the analysis.

Antibacterial activity assay

The antibacterial activity of Eucalyptus camaldulensis, Dodonaea viscosa, and Senna siamae crude ethanolic extracts against Salmonella typhi was assessed using the agar well diffusion method, as previously described (16). Briefly, 0.2 ml of standardized bacterial inoculum (10⁷ CFU/ml) was spread on Mueller-Hinton Agar plates. Wells of 6 mm diameter were filled with extract concentrations of 6.25, 12.5, 25, 50, and 100 mg/ml. Ciprofloxacin served as a positive control. Plates were incubated at 37°C for 24 h, and inhibition zone diameters (IZD) were measured to the nearest millimeter

The minimum inhibitory concentration (MIC) of the extracts was determined using the broth dilution technique. For this quantitative assay, a maximum starting concentration of 50 mg/ml was selected to ensure adequate solubility and to allow reliable visual assessment of bacterial growth in liquid medium. Two-fold serial dilutions were prepared to obtain concentrations of 50, 25, 12.5, 6.25 and 3.125 mg/ml in nutrient broth. A test tube without extract served as the negative control. Each tube was inoculated with 0.5 ml of a 0.5 McFarland standard suspension of S. typhi and incubated at 37 °C for 24 hours. The MIC was defined as the lowest concentration of the extract that showed no visible turbidity.

The minimum bactericidal concentration (MBC) was determined as described by (13). Aliquots (0.1 ml) from MIC tubes that showed no visible growth were sub-cultured onto fresh Mueller–Hinton agar plates and incubated at 37 °C for 24 hours.

Acute toxicity study

The acute toxicity study was conducted using a modified OECD Guideline 420 Fixed-Dose Procedure as described by (17). Female mice (7 weeks old, 19–30 g) were selected for the experimentation due to their suitability for preliminary toxicity screening, lower compound requirements, and reduced cost, while acknowledging that species differences may influence toxicity outcomes compared to rats.

Mice were fasted overnight prior to dosing. Each extract (Eucalyptus camaldulensis, Dodonaea viscosa, and Senna siamae) was administered orally at a single limit dose of 5000 mg/kg to groups of three mice, while the control group received distilled water. Mice were monitored for 5 days for signs of toxicity, including tremors, convulsions, salivation, lethargy, and changes in body weight. Body weights were recorded daily to detect potential extract-induced effects. No mortality within this period indicated an LD₅₀ >5000 mg/kg. Following the observation period, mice were sacrificed for histopathological evaluation of target organs.

Histology study

Histological investigation of the liver, Kidney and Heart were performed according to the method described by (18). After sacrificing the mice, parts of the liver, kidney and heart tissues were collected for histological studies. The tissues were washed in normal saline and fixed in 10% neutral formalin fixative followed by dehydration in ascending grades of alcohol, clearing in xylene and embedded in paraffin wax. Paraffin sections (5μm thickness) were stained with hematoxylin-eosin (H and E) and examined under a light microscope at a magnification of ×400 (Leica Buffalo, N.Y. 14240 U.S.A. Model CME Microscope 220–240 VAC 50/60Hz).

Statistical analysis

Descriptive statistics were expressed as Mean ± SEM. To substantiate comparative claims regarding antibacterial efficacy, one-way Analysis of Variance (ANOVA) was applied to IZD data across all concentrations of the plant extracts. Post hoc comparisons were performed using Tukey’s Honestly Significant Difference (HSD) test to determine statistically significant differences between groups (p < 0.05).

Antibacterial assay

The result of the antibacterial activity of the selected plants crude extracts against Salmonella typhi by agar well diffusion method was depicted in table 1.

Interestingly, all the plants were found to have effect at almost all the concentrations. D. viscosa has the highest activity of 13.0±0.00mm at a concentration of 100mg/ml and lowest activity of 12.5±7.25mm at 12.5mg/ml, no activity was seen at 6.25mg/ml. this is in contrast to the study of (19) where the ethanolic crude extract of D. viscosa did not show activity against S.typhi using agar well diffusion method, the activity was only seen in the ethyle acetate and n-butanol fractions of the extract. This may be due to some differences in the extraction procedures that may prevent the extraction of the compounds that are responsible for the activity in the extract. In a report by (16) a crude methanol extract of D. viscosa exhibit activity against S. typhi.

In S. siamae, activity of 15.0±0.00mm was observed at a concentration of 100mg/ml which is the highest and lowest activity of 7.7±6.66mm was observed at 6.25mg/ml. similar activity of 17.20±0.20mm at 100mg/ml was observed in a research by (13) using the same agar well diffusion method and their research also confirm the antibacterial activity of S. siamae against E.coli, K. pneumoniae and P. aeruginosa. This showed that the antibacterial activity of S. siamae is not limited against S. typhi but also other pathogenic bacteria.

Similarly, for E. camaldulensis all the concentrations were having activity against the tested organism, highest activity of 20.3±2.52mm was observed at 100mg/ml and lowest activity of 13.0±1.00mm was observed at 6.25mg/ml. This is confirmed in a study by (5) where the methanol leaf extract of E.camaldulensis showed antibacterial activity against S.typhi. This is also in accordance to the report by (6) where both the methanol and ethanol extracts of leaf E.camaldulensis exhibit activity against Salmonella typhi, Salmonella paratyphi A and Salmonella paratyphi B at a concentration between 30 mg/ml to 120 mg/ml.

Generally, our study showed that all the three extracts demonstrated dose-dependent activity against S. typhi, with higher concentrations producing larger inhibition zone diameter (IZD) which showed a statistically significant different (p < 0.05) with the control. Although the agar well diffusion assay showed variability—particularly for D. viscosa (12.5 ± 7.25 mm) and S. siamae (7.7 ± 6.66 mm)—this is expected due to the semi-quantitative nature of the method, which is sensitive to diffusion rates, inoculum density, and agar properties. Consequently, MIC and MBC provide a more reliable quantitative assessment of antibacterial efficacy

The MIC and MBC values are summarized in Table 2. lower MIC and MBC values correspond to higher antibacterial activity because smaller concentrations are sufficient to inhibit or kill the test organism (20). E. camaldulensis had the lowest MIC (6.25 mg/ml) and MBC (12.5 mg/ml), indicating the highest antibacterial potency. Both D. viscosa and S. siamae showed MIC values of 12.5 mg/ml and MBC values of 25 mg/ml. These results confirm that E. camaldulensis is the most potent of the three extracts against S. typhi.

Acute toxicity test

Oral administration of a single dose of 5000 mg/kg of each extract resulted in no mortality, indicating an LD₅₀ > 5000 mg/kg. Body weight changes over the 5-day observation period were minimal and comparable to the control group (Table 3), suggesting low acute lethality for all extracts.

This is in line with the reports of previous research (21,22,17) that the LD50 values of ethanol extracts of the investigated plants in rats is greater than >5000 mg/kg b.w. However, research by (23) observed that D. viscosa possesses reproductive toxicity in Zebrafish. Therefore, its use and doses must be carefully monitored in pregnant patients.

Histology test

Histological examination of the liver, kidney, and heart revealed normal features for E. camaldulensis and S. siamae-treated mice, as well as in the control group. In D. viscosa-treated mice, minimal tubular necrosis was observed in the kidney and slight hepatic necrosis in the liver. Necrosis was graded using standardized pathology criteria (minimal = <10% of tissue affected), affecting 1out of 3 mice per group. Heart tissues in all groups showed normal histology (Figure 1–3). The extracts exhibit low acute lethality but possesses target organ toxicity at the limit dose as in the case of D. viscosa. This sub-lethal toxicity is a major safety flag for chronic traditional use and demands detailed risk assessment, similar to how stability issues in long-term drugs require comprehensive investigation.