In phytotherapy, essential oils tend to be used daily for a period of days or weeks, rather than in a single application. However, the literature contains very little information on repeated use of essential oils. In this study, we investigated the effects on behavior and the accumulation in the brain and liver of α-pinene, an essential oil component, when inhaled by mice. Animals were individually housed in cages for 1 week. Mice inhaled α-pinene or water vapor (negative control) for 90 min/day for 1 day, 3 days, or 5 days, and they were then submitted to the elevated plus maze test for 10 min. We used gas chromatography with flame ionization detection to quantify concentrations of α-pinene in the brain and liver. There was significant anxiolytic-like activity, which remained constant for the 5 days’ inhalation of α-pinene. On the other hand, the accumulation of α-pinene in the brain and liver peaked on the third day of inhalation. The existence of stress related to the new environment appears to have affected the change in the accumulation of α-pinene in the internal organs, keeping the anxiolytic-like action constant.

Essential oils are mainly administered by inhalation. Administration by inhalation is considered to occur through two pathways, neurological transfer and pharmacological transfer. However, the relationship between the two routes is not clear. To clarify this relationship, we administered α-pinene, which has an anxiolytic-like effect, to mice. Emotional behavior and accumulation and expression of relevant mRNAs in the brain (brain-derived neurotrophic factor (BDNF); tyrosine hydroxylase (TH)) were examined following inhaled administration of α-pinene (10 μL/L air for 60 or 90min). To evaluate the anxiolytic-like effect, the elevated plus maze (EPM) test was used. Inhalation of α-pinene for 60 min produced a significant increase in the total distance traveled in the EPM test compared with control (water). The concentration of α-pinene in the brain after 60 min of inhalation was significantly increased compared with that after 90 min of inhalation. The expression of BDNF mRNA in the olfactory bulb and in the hippocampus was almost the same after 60 min of inhalation compared to that after 90 min of inhalation. The expression of TH mRNA in the midbrain after 60 min of inhalation was significantly increased compared with that of the control. Thus, an increase in α-pinene in the brain induces an increase in TH mRNA expression and increases locomotor activity. The anxiolytic-like effect may be related to both neurological transfer and pharmacological transfer.

Several reports have described the relationship between the quantity of an essential oil (EO), delivered by injection, and the effects on emotional behaviour. However, few studies have examined the relationship between the duration of EO inhalation and effects on emotional behaviour. It is necessary to clarify this relationship to accomplish effective utilization of an EO. Therefore, we aimed to clarify the relationship between EO inhalation time and anxiolytic-like effects. The anxiolytic-like effect of inhaled EO at various administration times was examined in normal healthy mice using the elevated-plus maze (EPM) test. The effect of a dose of 3.5 mg/l air of Alpinia zerumbet EO was assessed using the EPM test. A time-dependent potentiation in the anxiolytic-like effect was observed when the inhalation time was altered from 0 to 120 min. A significant (P < 0.05 compared to time 0) anxiolytic-like effect was observed to peak at inhalation times of 90 and 120 min. However, the anxiolytic-like effect returned to normal with a 150-min exposure. There was an optimal time of EO inhalation for expressing an anxiolytic-like effect.

Essential oils are composed of multiple components. It is thought that the effect of essential oils is due to specific component ratios, which may differ from the original ratio when the essential oil is absorbed. However, very little detailed research exists in this area. We studied the distribution of essential oil components after inhalation of single and mixed components in mice. This research was done using four main components of Alpinia zerumbet (Pers.) B. L. Burtt. and R. M. Sm.: α-pinene, p-cymene, 1,8-cineole, and limonene. After inhalation of single or mixed components for 90 min, component levels in the brain and liver of mice were measured. The results indicated that the amount of α-pinene in the brain and liver was twofold greater after mixed-component inhalation than that after single-component inhalation. In a comparison of the components of the mixed inhalation, the ratio of α-pinene increased to about three times that of 1,8-cineole. It is thought that the absorption via the nasal mucus greatly influences this phenomenon. The results of this investigation of the bodily distribution of essential oil volatile components may provide clues for elucidating their action.

The anxiolytic-like and stress reduction effects following inhaled administration of essential oil from Chamaecyparis obtusa (EOCO) have been reported. Volatile components are thought to produce these effects of EOCO by neurological transfer and pharmacological transfer. The regions of the brain in which inhaled compounds are found due to pharmacological transfer of EOCO are not known. This research was undertaken to clarify the relationship between the intracerebral distribution of -pinene, which is the main component of EOCO, and emotional behavior. -Pinene was detected as the main component of volatile EOCO. The amount of -pinene in each region of the brain was measured following inhaled administration of EOCO. The amount of -pinene was different in each region of the brain. With inhaled administration of 32 L/L air EOCO, a high concentration of -pinene was observed. However, no significant differences in the concentration of -pinene among brain regions were found. A therapeutic concentration of -pinene (8 L/L air EOCO) in each region of the brain may induce an anxiolytic-like effect, and a high concentration of -pinene (32 L/L air EOCO) in each region of the brain may induce an excitatory-like effect. The increases in the concentration of -pinene from 8 to 32 L/L air EOCO in the striatum and the hippocampus were significantly lower compared with the increases in other brain regions. These results indicate that regions besides the striatum and the hippocampus participated in the increase in locomotor activity due to the high concentration of -pinene in the brain.