Antidiarrheal activity of Limonene and Citrus aurantium L. on an experimental model of castor oil-induced diarrhea in mice

Diarrhea is defined as the passage of three or more loose or liquid stools per day (or more frequent passage than is normal for the individual). Diarrhea is usually a symptom of an infection in the intestinal tract, which can be caused by a variety of bacterial, viral and others parasitic organisms. Diarrheal disease is the second leading cause of death in children under five years old and is responsible for killing around 525.000 children every year. Diarrhea can last several days and can leave the body without the water and electrolytes necessary for survival 1. Limonene is a monoterpene commonly used as a flavoring agent in foods and drinks. It is also the major compound in the essential oil of Citrus aurantium L. (Rutaceae), popularly used in traditional medicine for the treatment of gastrointestinal tract disorders. This study investigated the antidiarrheal effect of Citrus aurantium essential oil (CAEO) and its limonene (LIM) major compound in male Swiss mice (n=7-9, 7-8 weeks). The CAEO (50, 100 and 200 mg/kg) and LIM (49, 98 and 196 mg/kg) was given orally. Morphine (5 mg/kg subcutaneous route) or loperamide (10 mg/kg) were used as a positive control and Tween 80™ 8% as vehicle. Diarrhea was induced experimentally by oral administration of castor oil (0.2 mL/animal)2,3. Activated charcoal (10%) was used to evaluate intestinal motility4 and to determinate the intestinal fluid accumulation induced by castor oil5, 6. To evaluate nitric oxide (NO) involvement in the accumulation of intestinal fluid, Nω-nitro-l-arginine methyl ester hydrochloride (L-NAME) a NO synthase inhibitor (100µg/kg) was injected intraperitoneally (i.p.) and after 30 minutes L-arginine was administrated (600 mg/kg i.p.)7. All procedures were approved by the institution's ethics committee. The results are expressed as mean ± S.E.M. and statistical significance was determined by ANOVA followed by Dunnett's or Kruskal-Wallis’ test (p<0.05). After administration of castor oil, treater’s mice with CAEO (200 mg/kg) and LIM (196 mg/kg) had the onset of liquid feces significantly delayed compared to the vehicle-treated group (173.3 ± 21.2 vs. 91.75 ± 10.5 minutes and 194.4 ± 14.61 vs. 91.75 ± 10.5 minutes, respectively). It also in this model, administration of CAEO and LIM reduced the evacuation of liquid feces (1.71 ± 0.68 and 1.75 ± 0.49 number of stools, respective) when compared to the vehicle group (5.43 ± 0.65). CAEO (200 mg/kg) also inhibited intestinal motility significantly when compared to the vehicle group (0.79 ± 0.03 g vs 0.99 ± 0.01 g, respectively). The minimum dose the CAEO and LIM, in the castor oil-induced enteropooling model, inhibited the volume of intestinal contents by 29.65% and 28.52%, respective. However, the effect of LIM (49 mg/kg) is independent of NO in the intestinal accumulation fluid, if compared to the vehicle (1.45 ± 0.04 vs 1.51 ± 0.08 g, respectively). These results show that the antidiarrheal effect of the essential oil of Citrus aurantium may be mediated by the major compound limonene, however further studies are needed to elucidate its mechanism of action.