Phytomedicine 9: 709–714, 2002 © Urban & Fischer Verlag http://www.urbanfischer.de/journals/phytomed Phytomedicine Central effects of citral, myrcene and limonene, constituents of essential oil chemotypes from Lippia alba (Mill.) N.E. Brown T. Gurgel do Vale, E. Couto Furtado, J. G. Santos Jr., and G. S. B. Viana Department of Physiology and Pharmacology, Faculty of Medicine, Federal University of Ceará, Fortaleza, Brazil Summary Citral, myrcene and limonene (100 and 200 mg/kg body wt., i.p.), constituents of essential oils from Lippia alba chemotypes, decreased not only the number of crossings but also numbers for rearing and grooming, as measured by the open-field test in mice. Although muscle relaxation detected by the rota rod test was seen only at the highest doses of citral (200 mg/kg body wt.) and myrcene (100 and 200 mg/kg body wt.), this effect was observed even at the lowest dose of limonene (50 mg/kg body wt.). Also, citral and myrcene (100 and 200 mg/kg body wt.) increased barbiturate sleeping time as compared to control. Limonene was also effective at the highest dose, and although citral did not increase the onset of sleep, it increased the duration of sleep, which is indicative of a poten- tiation of sleeping time. Citral (100 and 200 mg/kg body wt.) increased 2.3 and 3.5 times, respec- tively, the barbiturate sleeping time in mice. Similar effects were observed for myrcene and limonene at the highest dose (200 mg/kg body wt.) which increased the sleeping time around 2.6 times. In the elevated-plus maze, no effect was detected with citral up to 25 mg/kg body wt., while at a high dose it decreased by 46% the number of entries in the open arms. A smaller but signifi- cant effect was detected with limonene (5 mg/kg body wt.). While myrcene (10 mg/kg body wt.) de- creased only by 22% the number of entries in the open arms, this parameter was decreased by 48% at the highest dose. Our study showed that citral, limonene and myrcene presented sedative as well as motor relaxant effects. Although only at the highest dose, they also produced a potentia- tion of the pentobarbital-induced sleeping time in mice, which was more intense in the presence of citral. In addition, neither of them showed an anxiolytic effect, but rather a slight anxiogenic type of effect at the higher doses. Key words: Lippia alba, citral, β-myrcene, d-limonene, central effects j Introduction Lippia alba (Mill.) N.E. Brown (Verbenaceae) is a herb essential oil chemotypes. The presence of citral and known popularly in Brazil as ‘cidreira’, tea from the myrcene characteriizes chemotype I, while chemotypes II leaves of which is used as a tranquilizer and for gastroin- and III present citral and limonene, or carvone and testinal disorders. The plant presents great morphological limonene, respectively, as their main constituents (Matos, and chemical variety. Gas chromatographic analyses of 1996). Recent work (Vale et al., 1999) showed that all the essential oils (EO) from L. alba revealed at least three three EO chemotypes increased significantly not only the chemotypes with the predominance of monoterpene type number and percentage of entries, but also the time and of compounds in all of them. Citral, β-myrcene and d- percentage of time of permanence in the open arms, as limonene are the major monoterpenes from these three demonstrated by the elevated-plus maze test in mice, sug- 0944-7113/02/09/08-709 $ 15.00/0 710 T. Gurgel do Vale et al. gesting an anxiolytic effect. In the open field test, while Barbiturate-induced sleeping time: In this test, per- EO I decreased only the number of rearing, EOs II and III formed according to the method of Ferrini et al. (1974) decreased both the number of rearing and grooming as mouse sleep was induced by i.p. administration of 40 compared to controls. None of them altered the number mg/kg body wt. of pentobarbital. Latency time of sleep of crossings. Anticonvulsive effects against pentylenete- (time to lose the righting reflex) and sleeping time (du- trazole-induced convulsions in mice have also been ration of loss of the righting reflex) were registered. demonstrated (Viana et al., 2000), and experimental pro- Elevated-plus-maze: The apparatus consisted of two tocol animals treated with citral, myrcene and limonene opposite open arms (30 × 5 cm), crossed with two presented significant increases in the latency of convul- closed arms of the same dimensions with 25-cm high sion and percentage of survival as compared to controls. walls. Arms were c onnected to a 5 × 5 cm central In addition, the association of EOs with diazepam signifi- square. The apparatus was elevated 45 cm above the cantly potentiated their anticonvulsant effects. In the pre- floor in a dimly illuminated room. Mice were placed sent work, we decided to further explore those studies, individually in the center of the maze, facing an en- emphasizing the central effects of citral, myrcene and closed arm, and the number of entries and time spent limonene, and trying to clarify their mechanisms of ac- on the open arms was recorded for the next 5 min. The tion. Additionally, we wanted to determine the involve- percentage of time spent in the open arms was also cal- ment of these monoterpenes with the pharmacological ef- culated (Lister, 1987). fects of the EO chemotypes from Lippia alba. Statistical analysis j Material and Methods Results were expressed as means ± S.E.M. and ana- lyzed using analysis of variance (ANOVA) and the Animals Dunnett test as a post hoc analysis. Male Swiss mice (25 g each) from the Animal House of the Federal University of Ceará were used. All animals were maintained at a controlled temperature (23 ± 1°C) j Results under a 12 h dark/light cycle. Experiments were per- formed according to the Guide for the Care and Use of In the open-field test, which measures locomotor activ- Laboratory Animals, U.S. Department of Health and ity, citral (50, 100 and 200 mg/kg body wt., i.p.), signif- Human Services, 1985. icantly reduced (by 84%) the number of crossings at the highest dose. Rearing was decreased by 53 and Drugs 95% at doses of 100 and 200 mg/kg body wt., respec- Citral (minimum 95%, mixture of cis and trans iso- tively, while the number for grooming was already sig- mers, 3,7-dimethyl-2,6-octadienal) beta-myrcene (ap- nificantly decreased (by 50%) at the dose of 50 mg/kg proximately 90%, 7-methyl-3-methylene-1,6-octadi- body wt., reaching a 97% decrease at the highest dose ene) and limonene (minimum 97%, [R]-4-isopropenyl- 1-methyl-1-cyclohexene) were purchased from Sigma relative to controls (Table 1). Chemical Co., USA. Drugs were emulsified in 0.5% In the case of limonene, a 71% decrease in the num- cremophor before use. Animals were injected intraperi- ber of crossings was observed at 200 mg/kg body wt., toneally, 30 min before the experiment. Controls were while similar decreases in the numbers for rearing and injected with vehicle. Diazepam (Diazepam, Uni˜ao grooming (ranging from 55 to 88%) were detected at Química, Brazil) was used as standard. 100 and 200 mg/kg body wt. Significant decreases in the numbers of crossings (49 and 36%), rearing (70 and Pharmacological Tests 79%) and grooming (62 and 65%) were also observed Open-field test: The open-field arena was made of at the doses of 100 and 200 mg/kg body wt. of acrylic (transparent walls and black floow, 30 × 30 × 15 myrcene. The effects observed with all three com- cm), divided into nine squares of equal area. The open pounds were similar to that for the diazepam-treated field was used to evaluate the exploratory activity of group. the animal (Archer, 1973). The observed parameters Muscle relaxation, as measured by the rota rod test, were: number of squares crossed (with four paws), was seen only at the highest dose of citral (200 mg/kg numbers for grooming and rearing. body wt.), which showed a 74% decrease in the time of Rota rod: For the rota rod test, the animal was placed permanence on the bar. Decreases on the order of 49, with the four paws on a 2.5-cm diameter bar, 25 cm 53 and 76% were detected at 50, 100 and 200 mg/kg above the floor, which war turning at 12 rpm. For each body wt. doses of limonene, and of 35 and 48% at 100 animal the number of falls (up to three falls) and the and 200 mg/kg body wt. doses of myrcene, respective- time of permanence on the bar for 1 min were regis- ly (Table 2). Diazepam at the dose used showed no ef- tered (Dunham and Miya, 1957). fect. Central effects of Citral, Myrcene and Limonene, etc. 711 Citral and myrcene (100 and 200 mg/kg body wt.) at i.p., respectively, while myrcene showed an increase of the two highest doses increased significantly (from 1.6 1.6 and 2.6 times at the same doses. On the other hand, to 3.5 times) barbiturate sleeping time as compared to limonene was effective only at the highest dose (200 controls. On the other hand, a significant increase (2.7 mg/kg body wt.), increasing the sleeping time by 2.7 times) of this parameter was detected only at the high- times (Table 3). Diazepam also potentiated barbiturate est dose of limonene (200 mg/kg body wt.). Although sleeping time in a manner similar to citral.