- Email: [email protected]
Inhaled linalool-induced sedation in mice
ARTICLE IN PRESS
Phytomedicine 16 (2009) 303–307 www.elsevier.de/phymed
Inhaled linalool-induced sedation in mice Viviane de Moura Lincka,b, Adriana Lourenc¸o da Silvaa,b, Micheli Figueiro´a,c, Aˆngelo Luis Piatoa, Ana Paula Herrmanna, Franciele Dupont Bircka, Elina Bastos Carama˜od, Domingos Sa´vio Nunese, Paulo Roberto H. Morenof, Elaine Elisabetskya,b,c, a
Laborato´rio de Etnofarmacologia, Brazil PPG Cieˆncias Biolo´gicas-Bioquı´mica, Brazil c PPG Neurocieˆncias, ICBS, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil d Instituto de Quı´mica, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil e Departamento de Quı´mica, Universidade Estadual de Ponta Grossa, Ponta Grossa, PR, Brazil f Instituto de Quı´mica, Universidade de Sa˜o Paulo, Sa˜o Paulo, SP, Brazil b
Abstract Linalool is a monoterpene often found as a major component of essential oils obtained from aromatic plant species, many of which are used in traditional medical systems as hypno-sedatives. Psychopharmacological evaluations of linalool (i.p. and i.c.v.) revealed marked sedative and anticonvulsant central effects in various mouse models. Considering this profile and alleged effects of inhaled lavender essential oil, the purpose of this study was to examine the sedative effects of inhaled linalool in mice. Mice were placed in an inhalation chamber during 60 min, in an atmosphere saturated with 1% or 3% linalool. Immediately after inhalation, animals were evaluated regarding locomotion, barbiturate-induced sleeping time, body temperature and motor coordination (rota-rod test). The 1% and 3% linalool increased (po0.01) pentobarbital sleeping time and reduced (po0.01) body temperature. The 3% linalool decreased (po0.01) locomotion. Motor coordination was not affected. Hence, linalool inhaled for 1 h seems to induce sedation without significant impairment in motor abilities, a side effect shared by most psycholeptic drugs. r 2008 Elsevier GmbH. All rights reserved. Keywords: Linalool; Sedation; Inhalation; Essential oils
Introduction Aromatic species and essential oils (EOs) are frequently used as Materia Medica in several traditional medical systems (Buchbauer, 2004). Egyptians are Corresponding author at: PPG Neurocieˆncias, ICBS, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil. Tel.: +55 51 3308 3569; fax: +55 51 3308 3121. E-mail address: [email protected] (E. Elisabetsky).
0944-7113/$ - see front matter r 2008 Elsevier GmbH. All rights reserved. doi:10.1016/j.phymed.2008.08.001
thought to have used perfumes over 5000 years ago, and there are nearly 200 references in the Bible relating aromas to ‘‘mental, spiritual and physical healing’’ (Welsh, 1997; Perry and Perry, 2006). Originating in Germany in the 16th century, aromatherapy aims to use EOs for therapeutic purposes (Perry and Perry, 2006). Aromatherapy is currently used in the management of chronic pain, depression, anxiety, some cognitive disorders, insomnia and stress-related disorders (Perry and Perry, 2006). Beneficial effects of inhaled EOs in
ARTICLE IN PRESS 304
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animals and in humans have been reported (Buchbauer et al., 1991, 1993; Buchbauer, 2004; Buckle, 1993; Diego et al., 1998; Moss et al., 2003; Gedney et al., 2004). Despite the growing use of aromatherapy in the treatment of various ailments including those of central origin (Perry and Perry, 2006), and the alleged effects of incenses and other kinds of ambience aromatizers, experimental data on psychopharmacological properties of inhaled EOs are surprisingly scarce. Additionally, despite enough evidence showing that EOs and their components are significantly absorbed by inhalation (Buchbauer et al., 1993), few of the existing studies control the inhalation flow, making it difficult to estimate the actual inhaled concentration and, ultimately, the pharmacological meaning of observed results. Linalool, a monoterpene, is the major component of the EO produced by several well-known species including Lavandula augustifolia Mill., Melissa officinalis L., Rosmarinus officinals L. and Cymbopogon citratus DC. Interestingly, many linalool-producing species are traditionally used in folk medicine, and some in aromatherapy (Elisabetsky et al., 1995). Psychopharmacological evaluations of linalool administered intraperiotoneally (i.p.) or intracerebrally (i.c.v.) revealed marked sedative and anticonvulsant effects in various mouse models (Elisabetsky et al., 1999). Neurochemical assays reveal that (7)-linalool acts as a competitive antagonist of L-[3H]-glutamate binding (Elisabetsky et al., 1999), and shows a dose-dependent non-competitive inhibition of [3H]-MK801 binding (IC50 ¼ 2.97 mM), indicating antagonism of NMDA glutamate receptors (Brum et al., 2001). (7)-Linalool also decreases the potassium-stimulated (but not basal) glutamate release and uptake in mice cortical synaptosomes (Silva Brum et al., 2001). Buchbauer et al. (1991, 1993) showed the sedative effects of inhaled lavender EO in mice and humans. Furthermore, he demonstrated that linalool reversed the physiological alteration parameters produced by stress in humans (Hoferl et al., 2006). Other studies reported the effects of inhaled lavender oil on pain perception (Gedney et al., 2004), and its relaxant effects in humans (Buckle, 1993; Diego et al., 1998; Moss et al., 2003). Notably, there are three registered patents related with linalool, sedation and inhalation, including perfume and aromatherapeutic composition with sedative and/or sleep induction purposes. Dose-dependent sedative effects from linalool (i.p. and inhaled), including hypnotic and hypothermic effects, increased sleeping time, and decreased spontaneous locomotion has been reported in mice (Buchbauer et al., 1991; Elisabetsky et al., 1995). Recently, anxiolytic properties of inhaled lavender were reported for mice (Shaw et al., 2007) and gerbils (Bradley et al., 2007). The purpose of this paper was to determine the
effects of inhaled linalool on locomotion, barbitalinduced sleeping time, body temperature and rota-rod performance in mice.
Materials and methods Animals Male (CF1) adult albino mice, received from Fundac¸a˜o Estadual de Produc¸a˜o e Pesquisa em Sau´de (FEPPS) at 2 months of age (40–45 g), were used. Mice were maintained in our animal facility, under controlled environment (2271 1C, 12 h-light/dark cycle, free access to food [Nuvilab CR1] and water), for at least 2 weeks before the experiments. The project was approved by the University Ethics Committee (approval #2007706), and procedures carried out in accordance with experimental animals institutional policies.
Drugs (7)-Linalool, diazepam and pentobarbital were acquired from Sigma-Aldrich. Linalool was diluted with 1% Tween 80 (v/v). Inhaled 1% Tween 80 and saline intraperitoneal administrations were the controls groups. Diazepam was dissolved with propylene glycol 10% (v/v) and pentobarbital in a sufficient amount of NaOH 0.1 N.
Inhalation apparatus The inhalation apparatus consisted of a sealed cylindrical (internal diameter 15 cm) glass chamber, which included two vaporizers in its upper part so that a continuous fume flow could be maintained in the chamber. Four flasks were attached to the central chamber where mice were kept with their noses facing the chamber. Gas chromatographic analysis of the headspace in the chamber was conducted for determining the correlation of the real concentration of linalool in the chamber and in the flasks. The chromatographic analysis relied on a GC/MS Shimadzu QP 5050A system, an HP-5 (30 m 0.25 mm 0.25 mm) column and isothermal analysis at 120 1C. These analyses indicated that the system needed 15 min to saturate the atmosphere and stabilize the flow (Fig. 3); pilot experiments indicated 60 min as a suitable inhalation period for the expected effects (zero time was defined as the time when the animals were inserted, which happened after the 15-min stabilization time).
ARTICLE IN PRESS V.M. Linck et al. / Phytomedicine 16 (2009) 303–307
305
Locomotion Number of crossings were automatically recorded in activity cages (45 25 20 cm, Albarsch Electronic Equipment), equipped with three parallel photocells (Creese et al., 1976). Immediately after leaving the inhalation apparatus, mice were individually placed in the activity cages. The crossings were recorded for 15 min, being the first 5 min of exploratory and the 10 final minutes of locomotor activity.
Body temperature Immediately after leaving the inhalation apparatus the body temperature was measured with a digital thermometer (sensor probe 1 cm into the rectum). Controls inhaled 1% Tween 80 or had no treatment at all (Elisabetsky et al., 1995).
Fig. 1. Effect of sampling time on the concentration of linalool in the chamber.
200
Barbital-sleeping time
160 crossings
Immediately after body temperature measurements the animals received sodium pentobarbital (35 mg/kg i.p.), and the sleeping-time (time elapsed between loss and recovery of righting reflex) was recorded. As positive control, Diazepam (1 mg/kg) was injected 30 min before sodium pentobarbital administration.
120 80
**
40 0 Saline
Tween 1%
Rota-rod
Linalool 1%
Linalool 3%
250 200 crossing
Mice were initially trained to remain on the rota-rod apparatus (18 rpm, Hugo Basile, Italy) for 120 s; those that did not remain on the bar for at least two out of three consecutive trials were discarded (Bristow et al., 1996). The following day the animals were placed in the inhalation apparatus for 60 min and the latency to fall from the rota-rod (one 60 s trial) was determined immediately, 15, 30, 45 and 60 min after inhalation.
Diazepam 1.0mg/Kg
150 ** 100 50 0 Saline
Statistics Locomotion, body temperature and sleeping time were analyzed with ANOVA/SNK. Rota-rod performance at different time points were analyzed using a general linear model (GLM) with repeated measures (drug treatments vs. time), with time as the repeated measure, followed by SNK. po0.05 was considered statistically significant.
Results GC analysis (Fig. 1) showed that the linalool concentration available in the flasks during the inhala-
Tween 1%
Diazepam 1.0mg/Kg
Linalool 1%
Linalool 3%
Fig. 2. Effects of linalool on spontaneous locomotor activity: (A) first 5 min and (B) final 10 min. Each column represents the mean7SEM. N ¼ 15–23; **po0.01 compared with controls; ANOVA/SNK.
tion period was 0.74% and 2.55% for 1% and 3% linalool, respectively. As can be seen in Fig. 2, 3% linalool, but not diazepam or 1% linalool, significantly decreased the exploratory activity (F4.88 ¼ 10.1, po0.01) and motor activity (F4.88 ¼ 3.6, po0.01) in spontaneous locomotor activity evaluation. The data in Table 1 show that 1% and 3% linalool potentiated the pentobarbital-induced sleeping time
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Table 1. Effects of linalool on sleeping time and body temperature Treatment
Sleeping time (s)
Body temperature (1C)
Control 1% Tween Diazepam 1.0 mg/kg 1% Linalool 3% Linalool
47.6 (5.3) 41.9 (4.9) 149.1 (21.26)**
34.8 (0.1) 34.3 (0.1) –
143.3 (27.1)** 160.2 (23.5)**
33.8 (0.2)** 31.2 (0.2)**#
Data expressed as mean (SEM). N ¼ 12–23. **po 0.01 compared with controls, #po0.01 compared with 1% linalool, ANOVA/SNK.
70
0min
30min
15min
45min
60min
Latency (seg)
60 50 40 30 20 10 0
Saline
Tween
Linalool 1%
Linalool 3%
Fig. 3. Effects of linalool on rota-rod. Data expressed as mean7SEM. N ¼ 7–8. General linear model (GLM) with repeated measures (drug treatments vs. time), followed by SNK.
(F3.67 ¼ 19.7, po0.01) in a manner similar to diazepam (2.0 mg/kg), and also reduced the body temperature (F3.67 ¼ 96.2, po0.01). Linalool did not induce rota-rod deficits (Fig. 3).
Discussion It was recently shown that inhalation of lavender oil composed of 25% of linalool and 46% of linalyl acetate induced anxiolytic (open field) effects in rats after at least 30 min of inhalation (Shaw et al., 2007). Similar effects (elevated plus maze) were observed with lavender inhalation containing 38.47% of linalool and 43.98% of linalyl acetate in gerbils after 1 or 14 days inhalation (Bradley et al., 2007). The pharmacodynamic basis underlying the effects of fragrances as in natura complex mixtures or even of isolated components is far from clarified (Komiya et al., 2006). The mechanism(s) of action of a complex mixtures is(are) far more complex
than a simple sum of each component’s physiological consequence. Interaction among the various substances of an EO can modify the pharmacodynamic and pharmacokinetic properties of each substance; nevertheless, studying the pharmacodynamic basis of isolated components is helpful for a comprehensive understanding of the basis of EO physiological and psychopharmacological effects. The results of this study show that linalool (1% and 3%) inhaled for 60 min is clearly sedative, inducing hypothermia, reducing locomotion and increasing pentobarbital-induced sleeping time (although not hypnotic per se). Plasma linalool concentrations of 1.0, 2.7 and 3.0 ng/ml were found in mice exposed to linalool for 30, 60 or 90 min, respectively (a cage loaded with 27 mg of linalool), resulting in an exposure-dependent decrease in locomotion (Buchbauer et al., 1991). Our data show that although linalool in its higher concentration (3%) reduces locomotion, it does not significantly affect motor coordination in the rota-rod test. Hence, linalool (1% and 3%) inhaled for 1 h seems to induce sedation without impairment of motor abilities. Psychopharmacological effects of inhaled EO were reported for cedrol (Kagawa et al., 2003), and a volatile mixture from the traditional Chinese medicine SuHeXiang Wan composed of 21.4% borneol, 33.3% isoborneol, 5.9% eugenol and other minor components (Koo et al., 2004). Inhalation (2 g of fragrance/day, 2 3 h/day, for 7, 14 or 30 days at home cages) of EO from Acorus gramineus Solander (Araceae) inhibited the activity of GABA transaminase, thereby significantly increasing GABA levels; a decrease in glutamate levels was also reported in this study (Koo et al., 2003). Psychopharmacological activities for i.p. or i.c.v. administered linalool include protection against ECC, PTZ, quinolinic acid and picrotoxin-induced convulsions, delayed onset of NMDA-induced convulsions, as well as hypnotic, and hypothermic properties (Elisabetsky et al., 1995, 1999). In vitro assays demonstrate that linalool acts as an NMDA receptor antagonist and inhibits the glutamate release (Elisabetsky et al., 1999; Silva Brum et al., 2001), compatible with and relevant to the sedative effects observed for lavender and linalool, whether inhaled or otherwise administered. We suggest that the data reported by Shaw et al. (2007) are likely to be associated with the properties here shown for inhaled linalool. Inducing relaxation without producing motor impairments or marked sedation is of obvious advantage. It is worth noting that (7)-linalool was used in this study; using the isolated isomers may significantly alter inhalation time and linalool active concentrations. Although most of the aromatherapy techniques do not result in concentrations as those used in this study, it is arguable that the synergistic effects of complex aromas and the time-dependent effects of inhalation would eventually induce significant sedation.
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References Bradley, B.F., Starkey, N.J., Brown, S.L., Lea, R.W., 2007. The effects of prolonged rose odor inhalation in two animal models of anxiety. Physiol. Behav. 92, 931–938. Bristow, L.J., Flatman, K.L., Hutson, P.H., Kulagowski, J.J., Leeson, P.D., Young, L., Tricklebank, M.D., 1996. The atypical neuroleptic profile of the glycine/N-methyl-Daspartate receptor antagonist, L-701,324, in rodents. J. Pharmacol. Exp. Ther. 277, 578–585. Brum, L.F., Elisabetsky, E., Souza, D., 2001. Effects of linalool on [(3)H]MK801 and [(3)H] muscimol binding in mouse cortical membranes. Phytother. Res. 15, 422–425. Buchbauer, G., 2004. U¨ber biologische Wirkungen von Duftstoffen und a¨therischen O¨len. Wiener medizinische Wochenschrift 154 (21–22), 539–547. Buchbauer, G., Jirovetz, L., Jager, W., Dietrich, H., Plank, C., 1991. Aromatherapy: evidence for sedative effects of the essential oil of lavender after inhalation. Z. Naturforsch. [C] 46, 1067–1072. Buchbauer, G., Jirovetz, L., Jager, W., Plank, C., Dietrich, H., 1993. Fragrance compounds and essential oils with sedative effects upon inhalation. J. Pharm. Sci. 82, 660–664. Buckle, J., 1993. Aromatherapy. Nurs. Times 89, 32–35. Creese, I., Burt, D.R., Snyder, S.H., 1976. Dopamine receptor binding predicts clinical and pharmacological potencies of antischizophrenic drugs. Science 192, 481–483. Diego, M.A., Jones, N.A., Field, T., Hernandez-Reif, M., Schanberg, S., Kuhn, C., McAdam, V., Galamaga, R., Galamaga, M., 1998. Aromatherapy positively affects mood, EEG patterns of alertness and math computations. Int. J. Neurosci. 96, 217–224. Elisabetsky, E., Coelho de Souza, G.P., Dos Santos, M.A.C., Siqueira, I.R., Amador, T.A., 1995. Sedative properties of linalool. Fitoterapia 5, 407–414. Elisabetsky, E., Brum, L.F., Souza, D.O., 1999. Anticonvulsant properties of linalool in glutamate-related seizure models. Phytomedicine 6, 107–113.
307
Gedney, J.J., Glover, T.L., Fillingim, R.B., 2004. Sensory and affective pain discrimination after inhalation of essential oils. Psychosom. Med. 66, 599–606. Hoferl, M., Krist, S., Buchbauer, G., 2006. Chirality influences the effects of linalool on physiological parameters of stress. Planta Med. 72, 1188–1192. Kagawa, D., Jokura, H., Ochiai, R., Tokimitsu, I., Tsubone, H., 2003. The sedative effects and mechanism of action of cedrol inhalation with behavioral pharmacological evaluation. Planta Med. 69, 637–641. Komiya, M., Takeuchi, T., Harada, E., 2006. Lemon oil vapor causes an anti-stress effect via modulating the 5-HT and DA activities in mice. Behav. Brain Res. 172, 240–249. Koo, B.S., Park, K.S., Ha, J.H., Park, J.H., Lim, J.C., Lee, D.U., 2003. Inhibitory effects of the fragrance inhalation of essential oil from Acorus gramineus on central nervous system. Biol. Pharm. Bull. 26, 978–982. Koo, B.S., Lee, S.I., Ha, J.H., Lee, D.U., 2004. Inhibitory effects of the essential oil from SuHeXiang Wan on the central nervous system after inhalation. Biol. Pharm. Bull. 27, 515–519. Moss, M., Cook, J., Wesner, K., Duckett, P., 2003. Aromas of rosemary and lavender essential oils differentially affect cognition and moos in healthy adults. Int. J. Neurosc. 113, 15–38. Perry, N., Perry, E., 2006. Aromatherapy in the management of psychiatric disorders: clinical and neuropharmacological perspectives. CNS Drugs 20, 257–280. Shaw, D., Annett, J.M., Doherty, B., Leslie, J.C., 2007. Anxiolytic effects of lavender oil inhalation on open-field behaviour in rats. Phytomedicine 14, 613–620. Silva Brum, L.F., Emanuelli, T., Souza, D.O., Elisabetsky, E., 2001. Effects of linalool on glutamate release and uptake in mouse cortical synaptosomes. Neurochem. Res. 26, 191–194. Welsh, C., 1997. Touch with oils: a pertinent part of holistic hospice care. Am. J. Hosp. Palliat. Care 14, 42–44.
Phytomedicine 16 (2009) 303–307 www.elsevier.de/phymed
Inhaled linalool-induced sedation in mice Viviane de Moura Lincka,b, Adriana Lourenc¸o da Silvaa,b, Micheli Figueiro´a,c, Aˆngelo Luis Piatoa, Ana Paula Herrmanna, Franciele Dupont Bircka, Elina Bastos Carama˜od, Domingos Sa´vio Nunese, Paulo Roberto H. Morenof, Elaine Elisabetskya,b,c, a
Laborato´rio de Etnofarmacologia, Brazil PPG Cieˆncias Biolo´gicas-Bioquı´mica, Brazil c PPG Neurocieˆncias, ICBS, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil d Instituto de Quı´mica, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil e Departamento de Quı´mica, Universidade Estadual de Ponta Grossa, Ponta Grossa, PR, Brazil f Instituto de Quı´mica, Universidade de Sa˜o Paulo, Sa˜o Paulo, SP, Brazil b
Abstract Linalool is a monoterpene often found as a major component of essential oils obtained from aromatic plant species, many of which are used in traditional medical systems as hypno-sedatives. Psychopharmacological evaluations of linalool (i.p. and i.c.v.) revealed marked sedative and anticonvulsant central effects in various mouse models. Considering this profile and alleged effects of inhaled lavender essential oil, the purpose of this study was to examine the sedative effects of inhaled linalool in mice. Mice were placed in an inhalation chamber during 60 min, in an atmosphere saturated with 1% or 3% linalool. Immediately after inhalation, animals were evaluated regarding locomotion, barbiturate-induced sleeping time, body temperature and motor coordination (rota-rod test). The 1% and 3% linalool increased (po0.01) pentobarbital sleeping time and reduced (po0.01) body temperature. The 3% linalool decreased (po0.01) locomotion. Motor coordination was not affected. Hence, linalool inhaled for 1 h seems to induce sedation without significant impairment in motor abilities, a side effect shared by most psycholeptic drugs. r 2008 Elsevier GmbH. All rights reserved. Keywords: Linalool; Sedation; Inhalation; Essential oils
Introduction Aromatic species and essential oils (EOs) are frequently used as Materia Medica in several traditional medical systems (Buchbauer, 2004). Egyptians are Corresponding author at: PPG Neurocieˆncias, ICBS, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil. Tel.: +55 51 3308 3569; fax: +55 51 3308 3121. E-mail address: [email protected] (E. Elisabetsky).
0944-7113/$ - see front matter r 2008 Elsevier GmbH. All rights reserved. doi:10.1016/j.phymed.2008.08.001
thought to have used perfumes over 5000 years ago, and there are nearly 200 references in the Bible relating aromas to ‘‘mental, spiritual and physical healing’’ (Welsh, 1997; Perry and Perry, 2006). Originating in Germany in the 16th century, aromatherapy aims to use EOs for therapeutic purposes (Perry and Perry, 2006). Aromatherapy is currently used in the management of chronic pain, depression, anxiety, some cognitive disorders, insomnia and stress-related disorders (Perry and Perry, 2006). Beneficial effects of inhaled EOs in
ARTICLE IN PRESS 304
V.M. Linck et al. / Phytomedicine 16 (2009) 303–307
animals and in humans have been reported (Buchbauer et al., 1991, 1993; Buchbauer, 2004; Buckle, 1993; Diego et al., 1998; Moss et al., 2003; Gedney et al., 2004). Despite the growing use of aromatherapy in the treatment of various ailments including those of central origin (Perry and Perry, 2006), and the alleged effects of incenses and other kinds of ambience aromatizers, experimental data on psychopharmacological properties of inhaled EOs are surprisingly scarce. Additionally, despite enough evidence showing that EOs and their components are significantly absorbed by inhalation (Buchbauer et al., 1993), few of the existing studies control the inhalation flow, making it difficult to estimate the actual inhaled concentration and, ultimately, the pharmacological meaning of observed results. Linalool, a monoterpene, is the major component of the EO produced by several well-known species including Lavandula augustifolia Mill., Melissa officinalis L., Rosmarinus officinals L. and Cymbopogon citratus DC. Interestingly, many linalool-producing species are traditionally used in folk medicine, and some in aromatherapy (Elisabetsky et al., 1995). Psychopharmacological evaluations of linalool administered intraperiotoneally (i.p.) or intracerebrally (i.c.v.) revealed marked sedative and anticonvulsant effects in various mouse models (Elisabetsky et al., 1999). Neurochemical assays reveal that (7)-linalool acts as a competitive antagonist of L-[3H]-glutamate binding (Elisabetsky et al., 1999), and shows a dose-dependent non-competitive inhibition of [3H]-MK801 binding (IC50 ¼ 2.97 mM), indicating antagonism of NMDA glutamate receptors (Brum et al., 2001). (7)-Linalool also decreases the potassium-stimulated (but not basal) glutamate release and uptake in mice cortical synaptosomes (Silva Brum et al., 2001). Buchbauer et al. (1991, 1993) showed the sedative effects of inhaled lavender EO in mice and humans. Furthermore, he demonstrated that linalool reversed the physiological alteration parameters produced by stress in humans (Hoferl et al., 2006). Other studies reported the effects of inhaled lavender oil on pain perception (Gedney et al., 2004), and its relaxant effects in humans (Buckle, 1993; Diego et al., 1998; Moss et al., 2003). Notably, there are three registered patents related with linalool, sedation and inhalation, including perfume and aromatherapeutic composition with sedative and/or sleep induction purposes. Dose-dependent sedative effects from linalool (i.p. and inhaled), including hypnotic and hypothermic effects, increased sleeping time, and decreased spontaneous locomotion has been reported in mice (Buchbauer et al., 1991; Elisabetsky et al., 1995). Recently, anxiolytic properties of inhaled lavender were reported for mice (Shaw et al., 2007) and gerbils (Bradley et al., 2007). The purpose of this paper was to determine the
effects of inhaled linalool on locomotion, barbitalinduced sleeping time, body temperature and rota-rod performance in mice.
Materials and methods Animals Male (CF1) adult albino mice, received from Fundac¸a˜o Estadual de Produc¸a˜o e Pesquisa em Sau´de (FEPPS) at 2 months of age (40–45 g), were used. Mice were maintained in our animal facility, under controlled environment (2271 1C, 12 h-light/dark cycle, free access to food [Nuvilab CR1] and water), for at least 2 weeks before the experiments. The project was approved by the University Ethics Committee (approval #2007706), and procedures carried out in accordance with experimental animals institutional policies.
Drugs (7)-Linalool, diazepam and pentobarbital were acquired from Sigma-Aldrich. Linalool was diluted with 1% Tween 80 (v/v). Inhaled 1% Tween 80 and saline intraperitoneal administrations were the controls groups. Diazepam was dissolved with propylene glycol 10% (v/v) and pentobarbital in a sufficient amount of NaOH 0.1 N.
Inhalation apparatus The inhalation apparatus consisted of a sealed cylindrical (internal diameter 15 cm) glass chamber, which included two vaporizers in its upper part so that a continuous fume flow could be maintained in the chamber. Four flasks were attached to the central chamber where mice were kept with their noses facing the chamber. Gas chromatographic analysis of the headspace in the chamber was conducted for determining the correlation of the real concentration of linalool in the chamber and in the flasks. The chromatographic analysis relied on a GC/MS Shimadzu QP 5050A system, an HP-5 (30 m 0.25 mm 0.25 mm) column and isothermal analysis at 120 1C. These analyses indicated that the system needed 15 min to saturate the atmosphere and stabilize the flow (Fig. 3); pilot experiments indicated 60 min as a suitable inhalation period for the expected effects (zero time was defined as the time when the animals were inserted, which happened after the 15-min stabilization time).
ARTICLE IN PRESS V.M. Linck et al. / Phytomedicine 16 (2009) 303–307
305
Locomotion Number of crossings were automatically recorded in activity cages (45 25 20 cm, Albarsch Electronic Equipment), equipped with three parallel photocells (Creese et al., 1976). Immediately after leaving the inhalation apparatus, mice were individually placed in the activity cages. The crossings were recorded for 15 min, being the first 5 min of exploratory and the 10 final minutes of locomotor activity.
Body temperature Immediately after leaving the inhalation apparatus the body temperature was measured with a digital thermometer (sensor probe 1 cm into the rectum). Controls inhaled 1% Tween 80 or had no treatment at all (Elisabetsky et al., 1995).
Fig. 1. Effect of sampling time on the concentration of linalool in the chamber.
200
Barbital-sleeping time
160 crossings
Immediately after body temperature measurements the animals received sodium pentobarbital (35 mg/kg i.p.), and the sleeping-time (time elapsed between loss and recovery of righting reflex) was recorded. As positive control, Diazepam (1 mg/kg) was injected 30 min before sodium pentobarbital administration.
120 80
**
40 0 Saline
Tween 1%
Rota-rod
Linalool 1%
Linalool 3%
250 200 crossing
Mice were initially trained to remain on the rota-rod apparatus (18 rpm, Hugo Basile, Italy) for 120 s; those that did not remain on the bar for at least two out of three consecutive trials were discarded (Bristow et al., 1996). The following day the animals were placed in the inhalation apparatus for 60 min and the latency to fall from the rota-rod (one 60 s trial) was determined immediately, 15, 30, 45 and 60 min after inhalation.
Diazepam 1.0mg/Kg
150 ** 100 50 0 Saline
Statistics Locomotion, body temperature and sleeping time were analyzed with ANOVA/SNK. Rota-rod performance at different time points were analyzed using a general linear model (GLM) with repeated measures (drug treatments vs. time), with time as the repeated measure, followed by SNK. po0.05 was considered statistically significant.
Results GC analysis (Fig. 1) showed that the linalool concentration available in the flasks during the inhala-
Tween 1%
Diazepam 1.0mg/Kg
Linalool 1%
Linalool 3%
Fig. 2. Effects of linalool on spontaneous locomotor activity: (A) first 5 min and (B) final 10 min. Each column represents the mean7SEM. N ¼ 15–23; **po0.01 compared with controls; ANOVA/SNK.
tion period was 0.74% and 2.55% for 1% and 3% linalool, respectively. As can be seen in Fig. 2, 3% linalool, but not diazepam or 1% linalool, significantly decreased the exploratory activity (F4.88 ¼ 10.1, po0.01) and motor activity (F4.88 ¼ 3.6, po0.01) in spontaneous locomotor activity evaluation. The data in Table 1 show that 1% and 3% linalool potentiated the pentobarbital-induced sleeping time
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Table 1. Effects of linalool on sleeping time and body temperature Treatment
Sleeping time (s)
Body temperature (1C)
Control 1% Tween Diazepam 1.0 mg/kg 1% Linalool 3% Linalool
47.6 (5.3) 41.9 (4.9) 149.1 (21.26)**
34.8 (0.1) 34.3 (0.1) –
143.3 (27.1)** 160.2 (23.5)**
33.8 (0.2)** 31.2 (0.2)**#
Data expressed as mean (SEM). N ¼ 12–23. **po 0.01 compared with controls, #po0.01 compared with 1% linalool, ANOVA/SNK.
70
0min
30min
15min
45min
60min
Latency (seg)
60 50 40 30 20 10 0
Saline
Tween
Linalool 1%
Linalool 3%
Fig. 3. Effects of linalool on rota-rod. Data expressed as mean7SEM. N ¼ 7–8. General linear model (GLM) with repeated measures (drug treatments vs. time), followed by SNK.
(F3.67 ¼ 19.7, po0.01) in a manner similar to diazepam (2.0 mg/kg), and also reduced the body temperature (F3.67 ¼ 96.2, po0.01). Linalool did not induce rota-rod deficits (Fig. 3).
Discussion It was recently shown that inhalation of lavender oil composed of 25% of linalool and 46% of linalyl acetate induced anxiolytic (open field) effects in rats after at least 30 min of inhalation (Shaw et al., 2007). Similar effects (elevated plus maze) were observed with lavender inhalation containing 38.47% of linalool and 43.98% of linalyl acetate in gerbils after 1 or 14 days inhalation (Bradley et al., 2007). The pharmacodynamic basis underlying the effects of fragrances as in natura complex mixtures or even of isolated components is far from clarified (Komiya et al., 2006). The mechanism(s) of action of a complex mixtures is(are) far more complex
than a simple sum of each component’s physiological consequence. Interaction among the various substances of an EO can modify the pharmacodynamic and pharmacokinetic properties of each substance; nevertheless, studying the pharmacodynamic basis of isolated components is helpful for a comprehensive understanding of the basis of EO physiological and psychopharmacological effects. The results of this study show that linalool (1% and 3%) inhaled for 60 min is clearly sedative, inducing hypothermia, reducing locomotion and increasing pentobarbital-induced sleeping time (although not hypnotic per se). Plasma linalool concentrations of 1.0, 2.7 and 3.0 ng/ml were found in mice exposed to linalool for 30, 60 or 90 min, respectively (a cage loaded with 27 mg of linalool), resulting in an exposure-dependent decrease in locomotion (Buchbauer et al., 1991). Our data show that although linalool in its higher concentration (3%) reduces locomotion, it does not significantly affect motor coordination in the rota-rod test. Hence, linalool (1% and 3%) inhaled for 1 h seems to induce sedation without impairment of motor abilities. Psychopharmacological effects of inhaled EO were reported for cedrol (Kagawa et al., 2003), and a volatile mixture from the traditional Chinese medicine SuHeXiang Wan composed of 21.4% borneol, 33.3% isoborneol, 5.9% eugenol and other minor components (Koo et al., 2004). Inhalation (2 g of fragrance/day, 2 3 h/day, for 7, 14 or 30 days at home cages) of EO from Acorus gramineus Solander (Araceae) inhibited the activity of GABA transaminase, thereby significantly increasing GABA levels; a decrease in glutamate levels was also reported in this study (Koo et al., 2003). Psychopharmacological activities for i.p. or i.c.v. administered linalool include protection against ECC, PTZ, quinolinic acid and picrotoxin-induced convulsions, delayed onset of NMDA-induced convulsions, as well as hypnotic, and hypothermic properties (Elisabetsky et al., 1995, 1999). In vitro assays demonstrate that linalool acts as an NMDA receptor antagonist and inhibits the glutamate release (Elisabetsky et al., 1999; Silva Brum et al., 2001), compatible with and relevant to the sedative effects observed for lavender and linalool, whether inhaled or otherwise administered. We suggest that the data reported by Shaw et al. (2007) are likely to be associated with the properties here shown for inhaled linalool. Inducing relaxation without producing motor impairments or marked sedation is of obvious advantage. It is worth noting that (7)-linalool was used in this study; using the isolated isomers may significantly alter inhalation time and linalool active concentrations. Although most of the aromatherapy techniques do not result in concentrations as those used in this study, it is arguable that the synergistic effects of complex aromas and the time-dependent effects of inhalation would eventually induce significant sedation.
ARTICLE IN PRESS V.M. Linck et al. / Phytomedicine 16 (2009) 303–307
References Bradley, B.F., Starkey, N.J., Brown, S.L., Lea, R.W., 2007. The effects of prolonged rose odor inhalation in two animal models of anxiety. Physiol. Behav. 92, 931–938. Bristow, L.J., Flatman, K.L., Hutson, P.H., Kulagowski, J.J., Leeson, P.D., Young, L., Tricklebank, M.D., 1996. The atypical neuroleptic profile of the glycine/N-methyl-Daspartate receptor antagonist, L-701,324, in rodents. J. Pharmacol. Exp. Ther. 277, 578–585. Brum, L.F., Elisabetsky, E., Souza, D., 2001. Effects of linalool on [(3)H]MK801 and [(3)H] muscimol binding in mouse cortical membranes. Phytother. Res. 15, 422–425. Buchbauer, G., 2004. U¨ber biologische Wirkungen von Duftstoffen und a¨therischen O¨len. Wiener medizinische Wochenschrift 154 (21–22), 539–547. Buchbauer, G., Jirovetz, L., Jager, W., Dietrich, H., Plank, C., 1991. Aromatherapy: evidence for sedative effects of the essential oil of lavender after inhalation. Z. Naturforsch. [C] 46, 1067–1072. Buchbauer, G., Jirovetz, L., Jager, W., Plank, C., Dietrich, H., 1993. Fragrance compounds and essential oils with sedative effects upon inhalation. J. Pharm. Sci. 82, 660–664. Buckle, J., 1993. Aromatherapy. Nurs. Times 89, 32–35. Creese, I., Burt, D.R., Snyder, S.H., 1976. Dopamine receptor binding predicts clinical and pharmacological potencies of antischizophrenic drugs. Science 192, 481–483. Diego, M.A., Jones, N.A., Field, T., Hernandez-Reif, M., Schanberg, S., Kuhn, C., McAdam, V., Galamaga, R., Galamaga, M., 1998. Aromatherapy positively affects mood, EEG patterns of alertness and math computations. Int. J. Neurosci. 96, 217–224. Elisabetsky, E., Coelho de Souza, G.P., Dos Santos, M.A.C., Siqueira, I.R., Amador, T.A., 1995. Sedative properties of linalool. Fitoterapia 5, 407–414. Elisabetsky, E., Brum, L.F., Souza, D.O., 1999. Anticonvulsant properties of linalool in glutamate-related seizure models. Phytomedicine 6, 107–113.
307
Gedney, J.J., Glover, T.L., Fillingim, R.B., 2004. Sensory and affective pain discrimination after inhalation of essential oils. Psychosom. Med. 66, 599–606. Hoferl, M., Krist, S., Buchbauer, G., 2006. Chirality influences the effects of linalool on physiological parameters of stress. Planta Med. 72, 1188–1192. Kagawa, D., Jokura, H., Ochiai, R., Tokimitsu, I., Tsubone, H., 2003. The sedative effects and mechanism of action of cedrol inhalation with behavioral pharmacological evaluation. Planta Med. 69, 637–641. Komiya, M., Takeuchi, T., Harada, E., 2006. Lemon oil vapor causes an anti-stress effect via modulating the 5-HT and DA activities in mice. Behav. Brain Res. 172, 240–249. Koo, B.S., Park, K.S., Ha, J.H., Park, J.H., Lim, J.C., Lee, D.U., 2003. Inhibitory effects of the fragrance inhalation of essential oil from Acorus gramineus on central nervous system. Biol. Pharm. Bull. 26, 978–982. Koo, B.S., Lee, S.I., Ha, J.H., Lee, D.U., 2004. Inhibitory effects of the essential oil from SuHeXiang Wan on the central nervous system after inhalation. Biol. Pharm. Bull. 27, 515–519. Moss, M., Cook, J., Wesner, K., Duckett, P., 2003. Aromas of rosemary and lavender essential oils differentially affect cognition and moos in healthy adults. Int. J. Neurosc. 113, 15–38. Perry, N., Perry, E., 2006. Aromatherapy in the management of psychiatric disorders: clinical and neuropharmacological perspectives. CNS Drugs 20, 257–280. Shaw, D., Annett, J.M., Doherty, B., Leslie, J.C., 2007. Anxiolytic effects of lavender oil inhalation on open-field behaviour in rats. Phytomedicine 14, 613–620. Silva Brum, L.F., Emanuelli, T., Souza, D.O., Elisabetsky, E., 2001. Effects of linalool on glutamate release and uptake in mouse cortical synaptosomes. Neurochem. Res. 26, 191–194. Welsh, C., 1997. Touch with oils: a pertinent part of holistic hospice care. Am. J. Hosp. Palliat. Care 14, 42–44.