Evaluation of Acute and Subacute Toxicity of Auraptene in Rat

Objective: Auraptene is a natural bioactive monoterpene coumarin ether. It has anti-inflammatory, anti-carcinogenic, anti-bacterial, neuroprotective, and hepatoprotective activities. The aim of the present study was to assess acute and subacute toxicity of oral Auraptene administration in Rats, by evaluating changes in clinical signs, hematology and biochemical factors and pathological changes. Materials Methods: Auraptene is a crystalize whitish compound. To preparation for gavage, 2-mercaptoethanol as emulsifier and sonication was used to have Auraptene in emulsion with normal saline.Five groups of rats were obtained, each were contained of 5 male Wistar Rats to determine LD50. The LD50 of some coumarin derivative - 500 mg/kg – were chosen as base administration dose. Subsequently, according to ICH and OECD protocols, doses which included 125, 250, 1000 and 2000 mg/kg were selected, prepared and administrated to rats by gavage.In case of evaluation of subacute toxicity Auraptene was administered once daily in the morning by oral gavage for 28 days in doses 125 and 250 mg/kg. The animals were monitored for food and water consumption, clinical and behavioral symptoms such as diarrhea, immobility, neuromuscular problems and mortality during the first day and thereafter for 28 days. After suffocating by CO2 gaz, samples consisted of blood and serum were used to evaluate the hematological and biochemical changes. Organ tissues consist of heart, lung, kidney and liver were used to explore the pathological changes.Immune-toxicity study was performed by two tests; Delayed-type Hypersensitivity test (DTH) was considered as a generalized assessment of cellular immunity. There were two groups to perform DTH test, treatment and control. Auraptene was administrated by oral gavage at 250 mg/kg dose, in treatment group. In these groups, in day 23, sRBC in complete Freund’s adjuvant was administrated intraperitoneally and in day 28, sRBC in incomplete Freund’s adjuvant was administrated intracutaneously in rats left toe. In day 29 and 30 swelling of the toes was measured and compared with the right toes, and the percentages of the swelling were compared with the control group.To evaluate humoral immunity, Hemagglutination test (HA) was performed. Auraptene was administrated by oral gavage at 250 mg/kg dose, in treatment group. In both treatment and control groups, in day 23, sRBC in complete Freund’s adjuvant was administrated intra-peritoneally and one day after last day of oral administration, titration of serum and hemagglutination test was performed.Finally, to complete immune system assessment, comparison between WBC counts of studied groups and exploring the pathological changes in spleen tissue of treatment and control groups were done. Results conclusions: Our experiment results show that the Auraptene in high values up to 2gr/kg has no lethal toxicity. So, any LD50 values were not allocated to this compound.In long time oral administration over 28 days, exploring subacute toxicity, results are included:Administration of Auraptene did not produce any difference in the food consumption of male and female rats of most groups when compared to control groups. But just in group male 125, food consumption and weighting was unexpectedly about a constant amount that was abnormal in comparing aging and in compared by other groups. The average food intake of male rats was nearly 19 g/day/animal and female was 10 g/day/animal. Similarly the water consumption did not alter in treated groups when compared to control animals (Fig. 2).In comparing hematological and biochemical data, among those data have normal distribution, ANOVA test shows that, between PCV, HB, RBC, Plt,, Glucose, Triglyceride and Cholesterol are meaningful differences (p 0.05).Among those data have no normal distribution, we have used Kruskal-Wallis test and in pairwise comparison, Mann withney U test with Bonferroni correction show that there is meaningful differences between serum creatinine (p 0.017). Pathological comparison between groups show up there are no meaningful differences between them (p 0.05).In immune-toxicity assessment part of the study, Mann-Whitney U test shows no meaningful differences between treatment and control groups in DTH (p=0.808) and HA test (p=0.347) (Fig. 3 and Fig. 4). Pathological exploring of spleen tissues, show no toxic change or any meaningful differences.In conclusion, although there were some meaningful differences between treatment and control groups in hematological and biochemical data, but, almost all data were in normal limit ranges. There were no LD50, no pathological effects and no immunotoxicity. So, we can cautiously name Auraptene as a safe compound in oral administration.