Enhancement of Pentobarbital-induced Sleep by the Vaporized Essential Oil of Citrus keraji var. kabuchii and its Characteristic Component, y-Terpinene

Kabuchii (Citrus keraji var. kabuchii hort. ex Tanaka, Rutaceae) is a peculiar Okinawan citrus fruit. Local farmers cultivating various Citrus fruits say that the fragrance of Kabuchii is the most relaxing, but, there are few reports on the biological effects of the essential oil of Kabuchii and its chemical components [1]. In this study, the sedative effects of inhalation of the vaporized Kabuchii essential oil in open field, Rotarod, and pentobarbital sleep tests are compared with diazepam, as a positive control. In the open field test, both Kabuchii essential oil and diazepam decreased the spontaneous motor activity dose-dependently. The reduction in spontaneous motor activity in the 0.3 mg/cage (ca. 0.0278 mg/L) Kabuchii essential oil group was greater than that in the 1 mg/kg diazepam group. In the Rotarod test, Kabuchii did not affect the motor performance, even at the highest dosage tested (3 mg/cage), whereas diazepam decreased it dose-dependently. The effects of the major or characteristic components of Kabuchii, d-limonene, γ-terpinene, thymol, and p-cymene, were also evaluated in the open field and Rotarod tests. γ-Terpinene and thymol significantly decreased spontaneous motor activity at a dosage of 0.3 mg/cage, without affecting motor performance. Thus, γ-terpinene was estimated to be the main active component. Reduction in spontaneous motor activity by γ-terpinene in the open field test was not observed in intranasal zinc sulfate irrigation-induced anosmic mice. In the pentobarbital sleep test, both Kabuchii essential oil and diazepam potentiated pentobarbital-induced loss of the righting reflex (LRR). The LRR duration prolonging effects of both treatments were inhibited by pretreatment with flumazenil, a benzodiazepine receptor antagonist. The LRR latency reducing effect of Kabuchii was not affected by flumazenil, while that of diazepam was suppressed by it. γ-Terpinene showed similar potentiating effects on pentobarbital-induced sleep. Thus, vaporized Kabuchii essential oil and its active component, γ-terpinene, have sedative effects comparable with diazepam without inducing motor incoordination, which is a well-known side effect of diazepam.