3.1. Extraction of Shallomin
About 300 g of white shallot bulbs (collected in the spring from the Zagros Mountains; 50 km from Dezful, a city in Southern Iran) was washed thoroughly in water and cut into small pieces by using a kitchen mixer. The pieces were then soaked in 300 mL of distilled water and stirred using a magnetic stirrer for 5 h. Then, the suspension was filtered through Whatman No. 1 filter paper, and the aqueous extract was mixed with ethyl acetate in a 50:50 ratio and stirred for 10 min. The upper organic layer was separated using a separating funnel and centrifuged at 5,000 rpm for 10 min. The ethyl acetate layer was then removed and transferred into a clean flask. This process was repeated 3 times, and the extracts were pooled and dried in a rota-evaporator (Heidolph, Germany) at 50°C. The yield from the extract was weighed and dissolved in ethanol at various concentrations.
3.2. Animal Grouping
A total of 16 groups (n = 10 per group) of male BALB/c mice (body weight, 20 ± 5 g) were used. The groups were housed in separate cages maintained at room temperature, ranging from 25°C to 30°C, with free access to standard laboratory food and tap water. The mice were used in 2 separate series of experiments: in the first series of in vivo experiments, 5 groups (n = 10 per group) were used for the assessment of hematotoxicological, hepatotoxicological, and renal toxicological effects with 3 increasing anticipated doses for the treatment of infections. In the second series designed for assessment of acute toxicity, 11 groups of healthy male mice were used.
3.3. Evaluation of Hematotoxicity, Hepatotoxicity, and Renal Toxicity Effects of Shallomin
In this series of experiments, the first 3 groups (n = 10 per group) were used as the test groups, in which shallomin was administered to the mice at varying doses of 10, 20, or 30 µg/g body weight. The fourth group was used as the placebo group, in which 0.1 mL of ethanol (the solvent) was administered daily via intraperitoneal (IP) injection for 7 consecutive days. The fifth group was used as the non-treated group.
Parameters such as complete blood count (CBC), serum alkaline phosphatase, aspartate aminotranferase (AST), alanine transferase (ALT), creatinine, total bilirubin, direct bilirubin, and blood urea nitrogen (BUN) were measured after 7 days. In addition, the mice were killed, and their lung, liver, kidney, and heart were removed and evaluated for any pathological changes. The dose range for measurement of these parameters ranged from 5 µg/mL to 20 µg/mL (adjusted to body weight in g) and was selected on the basis of previous in vitro concentrations observed to be effective against both fungi and bacteria (2).
3.4. Estimation of Lethal Dose Low of Shallomin in Mice
Seven groups of mice (n = 10 per group; average weight, 20 ± 5 g) were administered IP injection of shallomin dissolved in 0.1 mL of 96% ethanol daily for 7 days (10, 40, 80, 120, 160, 200, or 240 µg/g body weight). The mortality rate in each group was assessed daily for 1 week, and the lowest dose that produced mortality during this period was recorded.
3.5. Statistical Analysis
The results were expressed as mean ± standard error values. The significance of the differences between the mean values was assessed using analysis of variance, followed by Duncan’s test for multiple comparisons among the different groups. A P value <0.05 was considered significant.
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