To determine acute oral toxicity, the method of acute oral toxicity at fixed doses was used.13 The extract was administered at doses of 5 mg/kg to 100 mg/kg, with animals showing no notable signs of toxicity. The 50% lethal dose was found to be greater than 100 mg/kg,

which is twice the highest dose (50 mg/kg) used for evaluation of a possible diuretic effect. Animals were maintained under standard condition of temperature and humidity and underwent for an adaptation period of three days. The animals were divided into four groups (n = 6). Group 1, as the negative control, received normal saline solution (25 ml/kg oral administration); group 2 received the reference diuretic, furosemide (Lasix, SANOFI-AVENTIS) at 20 mg/kg administered intraperitoneally LY2109761 order 14 and 15; groups 3 and 4 received the ethanolic extract of G. seemannii Peyr. at 25 mg/kg p.o. and 50 mg/kg p.o. respectively, in normal saline solution (25 ml/kg p.o.) and the diuretic activity was carried out based on the method of Lipschitz et al. 16 Immediately after administration

by gavage using an 18 G intragastric cannula, the animals were placed in metabolic cages (1 per cage), especially designed to separate urine and feces, and kept at a controlled temperature of 22–25 °C. At the end of 12 h, the volume of urine collected was measured. During this period, no food and water was available to the animals. During the two-week experimental period, the parameters measured were body weight (before and after the

test period), total urine volume, and concentration INCB024360 ic50 of Na+, K+ and Cl− in the urine. Na+, K+, Cl− concentrations were GPX6 determined by an ion sensitive electrode (Roche Hitachi 917) automatic analyzer. After the experiment, animals were sacrificed by ether anesthesia.17 Results are expressed as the mean ± SEM. Data was analyzed by one-way analysis of variance (ANOVA) followed by Dunnett’s multiple comparison test. A value of p < 0.001 was considered statistically significant. The LD50 was estimated to be greater than 100 mg/kg. The experimental extracts of G. seemannii Peyr. were used in concentrations of 25 mg/kg and 50 mg/kg, with animals showing no signs of acute toxicity. No macroscopic alterations were noted in the viscera of the treated rats. The animals were observed with no signs of dehydration at 12 h intervals. The reference diuretic (furosemide) significantly increased urine output compared to the control (p > 0.001), with a diuretic index of 2.86. Administration of the test drug at 25 and 50 mg/kg also resulted in a significant increase in urine volume, although less than that found with the reference drug. The diuretic index for these two doses was 1.49 and 1.75, respectively, compared to 2.86 found for furosemide ( Table 1). Ethanolic extract of G. seemannii Peyr.