The essential oil of nutmeg seeds was isolated with a yield of 6.85% w/w. The major compounds in the oil were sabinene (21.38%), 4-terpineol (13.92%) and myristicin (13.57%). On the other hand, allylbenzene and propylbenzene derivatives (myristicin, safrole, eugenol, and derivatives thereof) were the predominant compounds in nutmeg seeds. The data describing all the compounds extracted from nutmeg seeds are presented in Table 1.

This research showed that the essential oil of nutmeg seeds is of good quality as it has ideal percentages of essential oil and active compounds. The standard of Materia Medica Indonesia (MMI) requires that the essential oil content in nutmeg seeds be 5 to 10%, and it was found to be 6.85% in this study. Additionally, the content of myristicin and safrole measured in this study exceeded MMI requirements of 5 to 10% [8].

To determine the effects of nutmeg seed essential oil on locomotion, we administered it to mice via inhalation. A strong inhibitory effect of the essential oil on locomotion was observed; doses of 0.1, 0.3, and 0.5 mL/cage decreased locomotor activity by 62.81%, 65.33%, and 68.62%, respectively (Table 2). Thus, the effect of nutmeg oil on locomotion was dose-dependent.

In this research, we compared the activity of nutmeg essence to that of lavender oil as positive control, because lavender oil has been proven to decrease locomotion of female and male laboratory animals [9]. As shown in Figure 1, nutmeg oil afforded a greater inhibitory effect than did lavender oil. Sonavanne et al. [6]. had previously shown that oral administration of nutmeg seeds reduces the number of head pokes and potentiates pentobarbital-induced sleep. The preceding observations suggest that nutmeg seeds have sedative effect. Since the mechanism of anxiogenic activity is obscure, it is difficult to infer the mechanism by which nutmeg seeds potentiate pentobarbital-induced sleep, but it is possible that the allybenzene or propylbenzene derivatives in nutmeg contribute to sedation.

The roles of volatile compounds in locomotor activity can differ. However, all the active compounds in nutmeg seeds are contained in the essential oil. The compounds identified in blood plasma with high bioavailability after 30 to 120 minutes of exposure are likely responsible for the observed locomotor inhibition and are thus lead compounds [10].

In this study, myristicin and 4-terpineole were the predominant nutmeg seed essential oil compounds identified in mice made to inhale the extract. It was strange that sabinene, as the highest component in the nutmeg seeds essential oil, was not identified in the blood plasma. Furthermore, the concentrations of both myristicin and 4-terpineole in the blood plasma of mice increased with duration of exposure to nutmeg seed essential oil (Figure 2). Table 3 summarizes the volatile compounds found in blood plasma as a function of duration of inhalation.

On the other hand, some esters such as methyl myristate, methyl palmitate, methyl 10-octadecanate, methyl oleate, and methyl stearate were also identified in the plasma of mice following exposure to nutmeg seed essential oil. Of these esters, the plasma concentration of methyl palmitate exceeded that of the other compounds.

Half an hour after the addition of 1 mL/cage of nutmeg seed oil, the concentration of myristicin identified in the plasma was 3.7 μg/mL. After one and two hours inhalation of 1 mL of nutmeg seed oil, the myristicin levels in plasma increased to 5.2 μg/mL and 7.1 μg/mL, respectively (Table 3). Based on previous studies [6,11], myristicin, safrole, and 4-terpineol were hypothesized as the active compounds responsible for the inhibitory activity on the locomotion studied here. Structurally, safrole has the basic structure of an allylbenzene and of a propylbenzene, both of which contribute to the aromatic properties of plants.

Myristicin is a safrole derivative with methoxy group attached at carbon 4. The methoxy group in myristicin confers a strong sedative effect on the compound, as does a methoxy group in phenylpropanolamine [12]. Moreover, it has been reported that myristicin undergoes metabolism in the body, and its metabolite is known as a sedative 3-methoxy-4,5-methylenedioxyamphetamine (MMDA) [5,11]. This evidence supports the hypothesis that myristicin in nutmeg seed oil contributed to the locomotor inhibition in mice.

In this study, plasma levels of myristicin and safrole in mice correlated with extent of locomotor inhibition. After inhalation of nutmeg seed oil at a dose of 0.5 mL, mouse locomotor activity was inhibited by 68.82%. This effect was dose-dependent and more pronounced compared to that of other essential oils. Greater concentrations of myristicin were detected in plasma following inhalation of larger doses. Along the same lines, the concentration of 4-terpineol in blood plasma after inhalation of 0.1-, 0.3-, and 0.5-mL doses were 1.49, 2.95, and 6.28 μg/mL, respectively (Table 3).

Other authors [13,14] reported that 4-terpineol potentiates the GABA_A receptor-mediated response at a low concentration of GABA as effectively as α-terpineol. In particular, 4-terpineol potentiates the GABA response remarkably. The terpineols potentiate the effect of 5 μM GABA. The potentiation caused by a mixture of 4-terpineol and citral or cineol is greater than that caused by citral, cineol, or butanol, but less than that by 4-terpineol itself. This suggests there might be competitive binding of 4-terpineol and the three other compounds to the potentiating site.

According to the results above, myristicin, safrole, and 4-terpineol verify our prediction that these three compounds play a role in inhibiting locomotor activity in mice. Figure 3 showed that myristicin, safrole, and 4-terpineole have a strong capacity to inhibit locomotor activity in mice. Safrole effects extreme inhibition compared to others as can be seen in Table 4.

Based on these results, we suppose that the volatile compounds detected in blood samples, such as myristicin, 4-terpineole, and safrole, were responsible for the observed inhibition of locomotor activity. It is suggested that locomotor inhibition by nutmeg seed essential oil is due, at least in part, to the direct pharmacological action of one or more of its constituents.

Animals—Male mice weighing 25 to 30 g and 2 to 3 months old were used. The mice were adapted for one week to the laboratory in which locomotor activity experiments were conducted and were selected for wheel rotations of between 150 to 300 before the experiments were started.

Aromatic plant material—The nutmeg seeds (Myristica fragrans Houtt.) used were obtained from Bogor, West Java.

Chemicals—Methanol puriss. p.a. (Merck) was used as eluent for SPE. Heparin tubes (Boehringer) were used for blood collection. Pure lavender (Lavandula officinalis) oils were obtained from Martina Bertho. Alkane standards C₈–C₂₀, C₂₁–C₄₀, 1,4-dichlorobenzene, and myristicin, and safrole were obtained from Sigma.