Acute yeast-induced pyrexia model in rats was used to

Acute toxicity studies (LD50 determination)The median lethal dose of the extract was determined using Lorke’s method (Lorke, 1983). Rats were divided into three groups (n = 3) and fasted overnight, but provided with water ad libitum. In the first phase, the extract was administered orally with doses level (10 mg/kg, 100 mg/kg and 1000 mg/kg). The treated animals were observed for four hours after administration for signs of toxicity. After 24 h, when no death occurred the second phase was carried out. In the second phase, rats were divided into three groups (n = 3) and were given the extract orally with doses level (1600 mg/kg, 2900 mg/kg and 5000 mg/kg). The animals were then observed for signs of toxicity for the first 4 h and mortality for 24 h. Analgesic studies The hot-plate test method was carried out as described by Eddy and Leimback (Eddy et al., 1953). The rats were divided into six groups (n = 6). The different groups were treated orally with PBME (200 and 400 mg/kg), PBCE (200 and 400 mg/kg), tramadol (10 mg/kg), and vehicle control (0.9% NaCl, 10 ml /kg). After the treatment, rats were individually assessed with the hot-plate test maintaining the metal surface temperature at 55± 0.2°C. The latency (seconds) of rats to display hind paw licking or jumping behaviors was observed at 0, 30, 60, 90 and 120 mins after treatment. Maximal allowable control latency was 20 secs, whereas the maximal test latency was 30 secs to avoid tissue damage. Percentage of analgesia was calculated using the following formula (Shook et al., 1987).Analgesia (%) = “(Test latency – control latency)” /”(Cut -off time – control latency)” “× 100” Antipyretic ActivityA brewer’s yeast-induced pyrexia model in rats was used to test the antipyretic activity of extracts (Teotino et al., 1963).  The rats were divided into six groups (n = 6). The different groups were treated orally with PBME (200 and 400 mg/kg), PBCE (200 and 400 mg/kg), paracetamol (100 mg/kg), and control (0.9% NaCl, 10 ml/kg). The rectal temperature of each rat was recorded using the digital thermometer and then pyrexia was induced in all rats by subcutaneous injection 20% aqueous suspension of Brewer’s yeast (10ml/kg). All groups rats fasted overnight with water ad libitum and after 18h rectal temperature of each rat were recorded. The induction of pyrexia was confirmed bythe rise in temperature more than 0.5°F, whereas the rise in temperature less than 0.5°F were excluded from the experiment. After the treatment, rectal temperature was again recorded periodically at 1, 2, 3, and 4h of drugs administration. The percent reduction in pyrexia was calculated by the following formula (Kang et al., 2008). Relative antipyretic (%) = “(T1 -T2 )” /”T0″ “× 100” T0= Initial rectal temperature T1= Temperature after pyrexia induction, T2 = Temperature after 1,2,3, and 4hAnti-inflammatory activityCarrageenan-induced rat paw edema was used to test the anti-inflammatory activity of extracts The rats were divided into six groups (n = 6). The different groups were treated orally with PBME (200 and 400 mg/kg), PBCE (200 and 400 mg/kg), indomethacin (10 mg/kg), and vehicle control (0.9% NaCl, 5 ml/kg). Leaves extracts and drugs were administration 30 min prior to injection of 0.1 ml of 1% freshly prepared carrageenan suspension in normal saline in the right hind paw subplantar of each rat. The paw volume was measured initially and then at 0, 1, 2, 3 and 4 h after the carrageenan injection by using plethysmometer (Lin et al., 1996). The percentage inhibition of edema for each group was calculated using the following formula: – Inhibition of edema (%) = “(EVc – EVt)” /”EVc” “× 100” Where EV= Difference of paw volume before (carrageenan administration and carrageenan administration) .c = control group at a given time; t = test sample animalsData analysis The experimental results are expressed as mean ± S.E.M., with six animals in each group. Two-way ANOVA followed by Dunnett’s test for multiple comparisons was performed for determining the significance level in models such as hot-plate method, brewer’s yeast induced hyperpyrexia, and carrageenan-induced rat paw-edema. GraphPad Prism for Windows, Version 7 (GraphPad Software, San Diego, CA) was used for all statistical analyses. P value <0.05 was considered significant.

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