Analgesic effects of eucalyptus essential oil in mice

Abstract

Background: The use of aroma oils dates back to at least 3000 B.C., where it was applied to mummify corpses and treat the wounds of soldiers. Since the 1920s, the term "aromatherapy" has been used for fragrance therapy with essential oils. The purpose of this study was to determine whether the essential oil of Eucalyptus (EOE) affects pain pathways in various pain conditions and motor coordination.

Methods: Mice were subjected to inhalation or intraperitoneal injection of EOE, and its analgesic effects were assessed by conducting formalin, thermal plantar, and acetic acid tests; the effects of EOE on motor coordination were evaluated using a rotarod test. To determine the analgesic mechanism, 5'-guanidinonaltrindole (κ-opioid antagonist, 0.3 mg/kg), naltrindole (δ-opioid antagonist, 5 mg/kg), glibenclamide (δ-opioid antagonist, 2 mg/kg), and naloxone (μ-opioid antagonist, 4, 8, 12 mg/kg) were injected intraperitoneally.

Results: EOE showed an analgesic effect against visceral pain caused by acetic acid (EOE, 45 mg/kg); however, no analgesic effect was observed against thermal nociceptive pain. Moreover, it was demonstrated that EOE did not have an effect on motor coordination. In addition, an anti-inflammatory effect was observed during the formalin test.

Conclusions: EOE, which is associated with the μ-opioid pain pathway, showed potential effects against somatic, inflammatory, and visceral pain and could be a potential therapeutic agent for pain.

Keywords: Acetic acid; Analgesics; Aromatherapy; Eucalyptus; Formaldehyde; Glyburide; Intraperitoneal injections; Mice; Naloxone; Naltrindole; Opioid antagonists; Rotarod performance test.

Fig. 1

Intraperitoneal (i.p.) injection of essential oil of Eucalyptus (EOE) reduced licking time in the second phase of the formalin test. Licking time in mice injected with control (0.9% saline, i.p.), vehicle (almond oil, i.p.), essential oil (11.25, 22.5, and 45 mg/kg, i.p.), or morphine (4 mg/kg, i.p.) 30 min prior to the injection of 20 μl of formalin (2% v/v) into the dorsal surface of their left, hind paws. Morphine, a positive control, reduced the licking time in the first phase (0–5 min). Meanwhile, EOE (45 mg/kg) and morphine significantly reduced licking time in the second phase (20–25 min). Each value represents the mean and standard error of mean. *P < 0.05, **P < 0.001 compared to the vehicle group. One-way analysis of variance followed by Tuckey’s post hoc test were performed for statistical analyses. The control group had 12 mice while the others had 8 mice.

Fig. 2

The inhalation of essential oil of Eucalyptus (EOE) reduced licking time in the second phase of the formalin test. Licking time with the inhalation of control (distilled water [DW]) or EOE mixed with the same volume of dextrose water for 1, 2, 4, 8, and during groups before the formalin test. Each value represents the mean and standard error of mean. *P < 0.05, **P < 0.001, compared to the control group. One-way analysis of variance followed by Tukey’s post hoc test was used. The number of animals in the control group was 7 and in the during group was 8.

Fig. 3

The analgesic effects of essential oil of Eucalyptus (EOE) is via the μ-opioid pathway as demonstrated by the formalin test. The relevance of antinociceptive effect of EOE via the opioid pathway was investigated in this experiment. The licking time after pretreatment with an antagonist of the opioid receptor 15 min before the injection of the EOE (45 mg/kg, intraperitoneal) is shown. 5′-guanidinonaltrindole (GNTI, κ-opioid antagonist, 0.3 mg/kg), naltrindole (NTD, δ-opioid antagonist, 5 mg/kg), and naloxone (Nal, μ-opioid antagonist, 4, 8, 12 mg/kg) plus EOE group were injected intraperitoneally. During the second phase, there was a noteworthy variance in licking time between the EOE group and the vehicle group. The naloxone group (8, 12 mg/kg) and the naloxone plus EOE group showed statistically significant differences compared to the EOE treatment group in the second phase. Nal + Mor: naloxone 4 mg/kg + morphine 4 mg/kg. Each value represents the mean and standard error of mean. *P < 0.05, **P < 0.001, compared to the EOE (45 mg/kg) only group. One-way analysis of variance followed by Tuckey’s post hoc test were used to perform the statistical analyses. There were 8 mice in all groups, except there were 7 in the naloxone plus EOE group.

Fig. 4

Effect of essential oil of Eucalyptus (EOE) on writhing induced by acetic acid. Acetic acid-induced writhing test was used to determine whether EOE has an effect on visceral pain caused by an irritant injection. Control (0.9% saline, i.p.), vehicle (almond oil, i.p.), essential oil (11.5, 22.5, and 45 mg/kg, i.p.), or indomethacin (10 mg/kg, i.p.) were injected 30 min prior to administration of a 0.5% acetic acid solution (10 ml/kg). Five minutes after acetic acid injection, the total number of writhing movements were recorded for 30 min. Writhing decreased from indomethacin and EOE (22.5, 45 mg/kg). Each value represents the mean and standard error of mean. *P < 0.05, **P < 0.001 compared to the vehicle group. ^#P < 0.05 compared to the control group. One-way analysis of variance followed by Tuckey’s post hoc test was used. There were 10 mice in the control group and 8 in the other groups. i.p.: intraperitoneal.

Fig. 5

Essential oil of Eucalyptus (EOE) does not have dose-dependent effects against thermal stimuli. To find out whether EOE alters physiological pain associated with thermal stimulus, the thermal plantar test was used. A pretreatment injection of EOE (45, 90, and 180 mg/kg), or a vehicle were administered intraperitoneally. A focused beam of heat was aimed at the plantar surface of the hind paw in mice, 30 min after the pretreatment. Compared to the vehicle group, no remarkable difference in mean withdrawal latencies of the EOE groups (45, 90, and 180 mg/kg) was observed. Each value represents the mean and standard error of mean. One-way analysis of variance was used. There were 9 mice in each group.

Fig. 6

Essential oil of Eucalyptus (EOE) did not affect motor coordination. A rotarod test was conducted to investigate whether the EOE could influence motor coordination. Vehicle (almond oil) or EOE (45, 90, or 180 mg/kg) were used for pretreatment. No significant differences were evident, although animals in the EOE group stayed on the rod for a shorter period than animals of the vehicle group. Each value represents the mean and standard error of mean. One-way analysis of variance was used. There were 10 mice in each group.