Potential antioxidant and anxiolytic effects of (+)-limonene epoxide in mice after marble-burying test

Pharmacol Biochem Behav. 2014 Mar;118:69-78. doi: 10.1016/j.pbb.2014.01.006. Epub 2014 Jan 24.


The present study evaluated anxiolytic activity of (+)-limonene epoxide (EL), through the marble burying test (MBT) assay, and the antioxidant potential in vitro and in vivo in mice hippocampus of adult mice subjected to experimental anxiety protocol. For behavioral studies, and in vivo antioxidant analyses, mice were treated orally with 0.05% Tween 80 dissolved in 0.9% saline solution (vehicle), ascorbic acid 250 mg/kg, diazepam (2 mg/kg) and EL (25, 50 and 75 mg/kg). Results suggest an anxiolytic effect of (+)-limonene epoxide. A reduction in number of buried marbles in groups treated with EL doses of 25, 50 and 75 mg/kg was observed when compared with diazepam and vehicle groups. This reduction was observed after treatments with single and repeated doses, reinforcing the hypothesis of anxiolytic effect. The anxiolytic effect was reversed by pretreatment with flumazenil (25 mg/kg, o.r) in the same way as it was observed with diazepam (2 mg/kg, o.r, positive control), suggesting that these drugs possess a similar mechanism of action. In antioxidant tests in vitro, the concentrations from 0.9 to 7.2 μg/ml were tested. The results of in vitro antioxidant tests demonstrated a 50% inhibitory effective concentration of 0.7342, 1.296 and 1.169 μg/ml against the formation of nitrite ion, hydroxyl radical and reactive substances to thiobarbituric acid, respectively. The treatment with EL reduced the lipid peroxidation level and nitrite content, suggesting an antioxidant role in vivo since it was able to reduce the formation of reactive species derived from oxygen and nitrogen. Furthermore, the EL increased activity of enzymes catalase and superoxide dismutase in mice hippocampus, suggesting that their role may be due to antioxidant upregulation of these enzymes.

Keywords: (+)-Limonene epoxide; Antioxidant; Anxiety; Oxidative stress.

MeSH terms

  • Animals
  • Anti-Anxiety Agents / pharmacology*
  • Antioxidants / pharmacology*
  • Anxiety / drug therapy
  • Anxiety / metabolism
  • Anxiety / psychology
  • Catalase / metabolism
  • Cyclohexane Monoterpenes
  • Diazepam / pharmacology
  • Flumazenil / pharmacology
  • Free Radical Scavengers / pharmacology
  • Hippocampus / drug effects
  • Hippocampus / metabolism
  • Male
  • Mice
  • Monoterpenes / pharmacology*
  • Reactive Nitrogen Species / metabolism
  • Reactive Oxygen Species / metabolism
  • Superoxide Dismutase / metabolism


  • Anti-Anxiety Agents
  • Antioxidants
  • Cyclohexane Monoterpenes
  • Free Radical Scavengers
  • Monoterpenes
  • Reactive Nitrogen Species
  • Reactive Oxygen Species
  • limonene-1,2-epoxide
  • Flumazenil
  • Catalase
  • Superoxide Dismutase
  • Diazepam