Central Effects of Citral, Myrcene and Limonene, Constituents of Essential Oil Chemotypes from Lippia Alba (Mill.) N.E

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 potentiation of sleeping time. Citral (100 and 200 mg/kg body wt.) increased 2.3 and 3.5 times, respectively, 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 significant effect was detected with limonene (5 mg/kg body wt.). While myrcene (10 mg/kg body wt.) decreased 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 potentiation 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

᭿ 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 ᭿ 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) ᭿ 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. citral did not increase the onset of sleep in the barbitu- In the elevated-plus maze test in mice, indicative of rate sleeping time test, it significantly increased the du- anxiolytic activity, no effect was seen after citral ad- ration of sleep, indicative of a potentiation of the sleep- ministration at doses of 5, 10 and 25 mg/kg body wt., ing time. The effect was dose-dependent, and citral was i.p. A small but significant effect was, however, ob- the most effective among the three compounds, in- served with limonene (5, 10 and 25 mg/kg body wt., creasing the barbiturate-induced sleeping time by 2.3 i.p.), which decreased the number of entries in the open and 3.5 times at doses of 100 and 200 mg/kg body wt., arms at the smallest dose (30% decrease) without caus-

Table 1. Effects of citral, limonene and myrcene in the open-field test in mice.

Group n Number of squares crossed Rearing Grooming

Control 14 87.1 ± 10.3 41.5 ± 3.1 2.6 ± 0.6 Diazepam 2 10 96.1 ± 9.9 18.6 ± 3.0** 1.0 ± 0.0* Citral 50 9 80.3 ± 5.5 35.2 ± 4.9 1.7 ± 0.3 Citral 100 10 69.4 ± 7.3 19.3 ± 3.0** 1.5 ± 0.5* Citral 200 11 13.8 ± 3.8** 0.2 ± 0.1** 0.09 ± 0.09** Limonene 50 10 70.6 ± 10.3 33.0 ± 4.0 3.2 ± 0.5 Limonene 100 10 79.8 ± 13.6 18.2 ± 5.3** 1.4 ± 0.2* Limonene 200 10 25.8 ± 8.6** 5.0 ± 3.6** 0.4 ± 0.3** Myrcene 50 10 87.2 ± 7.9 39.8 ± 4.1 1.5 ± 0.3 Myrcene 100 10 44.3 ± 11.5* 12.3 ± 4.7** 1.0 ± 0.3* Myrcene 200 9 56.1 ± 17.5 8.9 ± 5.2** 0.9 ± 0.4**

Values are means ± S.E.M. Citral, limonene or myrcene (50, 100 or 200 mg/kg body wt., i.p.) were injected 30 min before the test. *P < 0.05, **P < 0.01 (ANOVAand Dunnet as the post-hoc test).

Table 2. Effects of citral, limonene and myrcene in the rota Table 3. Effects of citral, limonene and myrcene in the pen- rod test in mice. tobarbital-induced sleeping time test in mice.

Group n Number Time of Group n Latency of sleeping Sleeping time of falls permanence (s) time (s) (min)

Control 14 2 (0–3) 50.6 ± 3.3 Control 14 231.2 ± 29.5 40.9 ± 5.1 Diazepam 2 10 2 (0–3) 48.3 ± 4.3 Diazepam 2 10 192.0 ± 25.0 57.0 ± 4.5* Citral 50 9 0 (0–2) 59.0 ± 0.8 Citral 50 9 210.1 ± 16.5 52.7 ± 3.8 Citral 100 10 0 (0–3) 55.7 ± 4.0 Citral 100 10 146.0 ± 11.4 93.1 ± 8.0** Citral 200 11 3 (3–3) 12.9 ± 2.8** Citral 200 11 190.8 ± 16.0 143.3 ± 16.2** Limonene 50 10 3 (3–3) 24.8 ± 3.8 Limonene 50 10 288.0 ± 30.7 41.0 ± 6.5 Limonene 100 10 3 (1–3) 22.9 ± 6.0** Limonene 100 10 270.0 ± 53.1 55.4 ± 4.1 Limonene 200 10 3 (3–3) 11.9 ± 1.6** Limonene 200 10 186.0 ± 18.9 109.2 ± 15.9** Myrcene 50 10 3 (0–3) 43.9 ± 7.0 Myrcene 50 10 225.0 ± 27.2 45.0 ± 4.4 Myrcene 100 10 3 (1–3) 32.9 ± 6.1* Myrcene 100 10 186.0 ± 16.6 64.0 ± 10.2* Myrcene 200 10 3 (2–3) 26.1 ± 6.1** Myrcene 200 10 153.3 ± 24.7 106.6 ± 14.1**

Values are means ± S.E.M. Citral, limonene or myrcene (50, Values are means ± S.E.M. Citral, limonene or myrcene (50, 100 or 200 mg/kg body wt., i.p.) were injected 30 min before 100 or 200 mg/kg body wt., i.p.) were injected 30 min before the test. *P < 0.05, **P < 0.01 (ANOVA and Dunnet as the the test. *P < 0.05, **P < 0.01 (ANOVA and Dunnet as the post-hoc test). post-hoc test). 712 T. Gurgel do Vale et al.

Table 4. Effects of citral, limonene and myrcene in the elevated plus maze test in mice.

Group n NEOA TPOA PTOA

Control 15 10.5 ± 1.3 92.5 ± 10.1 0.40 ± 0.03 Diazepam 2 8 17.6 ± 2.1** 200.9 ± 8.6** 0.74 ± 0.03** Citral 5 11 9.7 ± 0.9 89.3 ± 7.1 0.40 ± 0.03 Citral 10 12 8.0 ± 0.9 87.2 ± 11.0 0.40 ± 0.04 Citral 25 12 10.3 ± 0.7 91.3 ± 7.3 0.40 ± 0.03 Citral 50 10 5.7 ± 0.8* 63.6 ± 8.7 0.30 ± 0.04** Limonene 5 12 8.0 ± 1.0* 91.8 ± 9.8 0.37 ± 0.04 Limonene 10 12 9.2 ± 1.0 89.5 ± 10.4 0.40 ± 0.04 Limonene 25 10 12.8 ± 0.5 131.3 ± 9.5 0.57 ± 0.04 Limonene 50 11 8.2 ± 0.9 98.6 ± 8.3 0.46 ± 0.03 Myrcene 5 10 12.1 ± 1.2 108.3 ± 9.6 0.48 ± 0.04 Myrcene 10 10 8.9 ± 1.3 83.1 • 14.3 0.37 • 0.06 Myrcene 25 10 5.9 ± 0.7* 64.3 ± 10.6 0.32 ± 0.05 Myrcene 50 9 12.3 ± 1.3 98.7 ± 10.6 0.44 ± 0.04 DZP 0.75 8 15.3 ± 1.8 152.4 ± 13.8 0.60 ± 0.05**

Values represent means ± S.E.M. of: NEOA, number of entries in the open arms; TPOA, time of permanence in the open arms; PTOA, percentage of time in the open arms. Citral, limonene or myrcene (5, 10, 25 or 50 mg/kg body wt., i.p.) were injected 30 min before the test. DZP = diazepam (0.75 mg/kg body wt., i.p.) *P < 0.05, **P < 0.01 (ANOVA and Dunnet as the post-hoc test). ing any changes in the time of permanence in the open (Wolffgramm et al., 1994). These authors suggest that arms. A similar but dose-dependent effect was ob- the compound is a partial agonist of central BDZ re- served with myrcene at the same dose range, and this ceptors. Several types of terpene compounds are effect was significant at the doses of 10 and 25 mg/kg known to present analgesic and anti-inflammatory ac- body wt., i.p., which is characteristic of anxiolytic tivities (Nakamura et al., 2001; Zidorn et al., 1999; drugs of the benzodiazepine type. On the other hand, Trentin et al., 1999; Miranda et al., 2000; Matsuda et diazepam used a standard significantly increased by al., 1998), but only a few showed central effects 1.5 and 1.6 times the number of entries in the open (Osuna et al., 1999). arms and the time of permanence in the open arms, re- Although there are literature data presenting the ef- spectively (Table 4). fects of some of these compounds, such as citral on hepatic enzymes and lipid metabolism, as well as de- scribing their toxic effects (Roffey et al., 1990; Diliber- ᭿ Discussion to et al., 1988; Nogueira et al., 1995), there are only few reports on their central effects. Citral is a naturally In the present work, we have shown that citral, occurring aliphatic aldehyde of the terpene series and is limonene and myrcene, the main chemical con- an isomeric mixture of geranial and neral. It is the main stituents of the three chemotypes of L. alba component of several medicinal plants including Lip- widespread in Northeast Brazil, present potent central pia alba, in which it is present in the essential oils from effects. Recently, we showed (Viana et al., 2000) that leaves of chemotypes I and II. Several studies have the essential oils from the three chemotypes of L. alba shown that citral possesses a hemolytic effect through present anticonvulsive activity in the pentylenetetra- its involvement with the formation of free radicals zole-induced convulsions model in mice. Their associ- (Ranner et al., 1996; Segal and Milo-Goldzweig, ation with diazepam significantly potentiated this ef- 1985). It also shows bactericidal and anti-fungal activi- fect, suggesting a similar mechanism of action and ties (Pattnaik et al., 1997). It has been demonstrated also indicating that citral, myrcene and limonene are that citral decreases locomotor activity and potentiates probably the active compounds. Some naturally occur- imipramine effects in the behavioral despair test in ring flavonoids, such as 5,7-dihydroxyflavone, are mice (Komori et al., 1995). Thus, the lemon odor known to displace (3H)-flunitrazepam binding from which presents citral as its main constituent shows an- central benzodiazepine receptors. Such results showed tidepressant effects in the forced swimming test in rats that this flavone derivative possesses an anxiolytic ac- and also decreases locomotor activity in the open-field tion without inducing sedation and muscle relaxation test (Komori et al., 1995). Central effects of Citral, Myrcene and Limonene, etc. 713 Beta-myrcene is an acyclic monoterpene present in the open-field test in mice. Numbers for rearing and chemotype I and also found in essential oils from sever- grooming were also decreased significantly and dose- al aromatic species. It shows a central as well as a pe- dependently with all three compounds. These effects ripheral analgesic effect (Rao et al., 1990). While its are indicative of a dopaminergic-type action. Although central effect was antagonized by the previous adminis- all three constituents showed muscle relaxation at tration of naloxone and yoimbine, the peripheral effects higher doses, as detected by the rota rod test in mice, were blocked by inhibitors of nitric oxide synthesis limonene seemed to be more active. We also showed a (Duarte et al., 1992). Other studies revealed no effect of potentiation of barbiturate sleeping time in mice, in- beta-myrcene in the elevated-plus maze, and condition- dicative of a depressant effect on the CNS, which was al avoidance did not show evidence of central effects dose-dependent and more prominent with citral. The when administered up to 1 g/kg body wt., p.o. Similarly, potentiation of the barbiturate sleeping time observed no protection was observed against pentylenetetrazole- with acute citral administration was also seen after induced convulsions (Silva et al., 1991). chronic treatment for 15 days at a dose of 100 mg/kg It has been shown (Lorenzetti et al., 1991) that the body wt. i.p. (data not shown). oral administration to rats of fresh leaf infusion of In conclusion, we showed that the three main con- lemongrass, which contains myrcene, produced a dose- stituents of the essential oils from L. alba chemotypes dependent analgesia in the carrageenan- or prosta- present sedative as well as motor relaxant effects. glandin E2-induced paw edema, but did not affect that However, this sedative effect was observed only at the induced by a cAMP derivative. These results indicate a highest dose (200 mg/kg body wt.) with all com- peripheral site of action similar to those observed for pounds. They also caused a potentiation of the pento- the plant essential oil. Myrcene also increased the pen- barbital-induced sleeping time in mice that was more tobarbital-induced sleeping time (Freitas et al., 1993), intense in the presence of citral. In addition, none of indicating that it interferes with the in vivo barbiturate them showed an anxiolytic effect, presenting instead an metabolism, and also supporting the view that myrcene anxiogenic type of action which was manifested by all induces the phenobarbital-inducible cytochrome P-450 three essential oil constituents. enzymes in rats. The presence of an anxiolytic effect in mice treated A chemopreventive effect of d-limonene against N- with EOs (Vale et al., 1999) and the absence of this ef- nitrosodiethylamine alone, and along with phenobarbi- fect in those treated with their major constituents (cit- tal-induced hepatocarcinogenesis in mice, has also ral, myrcene and limonene) suggests that the anxio- been demonstrated. That investigation (Giri et al., genic effect observed in the present work might possi- 1999) explains the anti-tumor effect of d-limonene for bly be due to other components present in the EOs. Ad- the first time on the level of oncogene expression in ditionally, the anxiolytic activity observed in mice with those models. Other work (Uedo et al., 1999) suggest all three chemotypes and the anxiogenic activity ob- that limonene attenuates the gastric carcinogenesis en- served in rats (data not shown) indicate that the anxi- hanced by sodium chloride via increased apoptosis and olytic effect is species-dependent, and not due to citral, decreased ornithine decarboxylase activity of gastric myrcene or limonene. Our results also indicated that cancers in rats. A recent work (Larsen et al., 2000) citral, myrcene and limonene are probably responsible showed that limonene enantiomers have some effect on for most of the central effects of the EO chemotypes the respiratory tract in mice, including a mild bron- from Lippia alba. The participation of other compo- choconstrictive effect. nents in these and other actions, such as the anxiolytic Although L. alba, as a popular Brazilian medicinal one, should not, however, be discarded. plant, is also used largely as a tranquilizer, some actions of its essential oil constituents were unexpected. This is the case with the anxiogenic-type effect detect- ᭿ References ed with all three essential oil constituents of L. alba chemotypes. Thus, citral, limonene and myrcene de- Archer J (1973) Tests for emotionality in rats and mice: A re- creased the number of entries in the open arms of the view. Animal Behav 21: 205–235 elevated-plus maze test for mice. We have shown pre- Diliberto JJ, Usha G, Birnbaum LS (1988) Disposition of cit- viously that the aqueous and hydroalcoholic extracts of ral in male Fischer rats. Drug Metabol Disp 16: 721–727 these chemotypes present a potent peripheral analgesic Duarte LD, dos Santos IR, Lorenzetti BB, Ferreira SH (1992) Analgesia by direct antagonism of nociceptor sensitization effect, which was also observed with their essential oils involves the arginine-nitric oxide-cGMP pathway. Eur J (Viana et al., 1998). Pharmacol 217: 225–227 In the present work, we found a sedative effect, not Dunham NW, Miya TS (1957) A note on a simple apparatus only with citral but also with myrcene and limonene, for detecting neurological deficits in rats and mice. J Am which was significant at higher doses as determined by Pharm Assoc 46: 208 714 T. Gurgel do Vale et al.

Ferrini R, Miragoli G, Taccardi B (1974) Neuropharmaco- zymes of microsomal cytochrome P450 and mechanism- logical studies on SB 5833, a new psychotherapeutic agent based inhibition of retinoid oxidation by citral. Mol Phar- of the benzodiazepine class. Arzneim-Forsch 24: 2029 macol 49: 515–522 Freitas JC, Presgave OA, Fingola FF, Menezes MA, Paum- Rao VS, Menezes AM, Viana GSB (1990) Effects of gartten FJ (1993) Effect of beta-myrcene on pentobarbical myrcene on nociception in mice. J Pharm Pharmacol 42: sleeping time. Braz J Med Biol Res 26: 519–523 877–878 Giri RK, Parija T, Das BR (1999) D-limonene chemopreven- Roffey SJ, Walker R, Gibson GG (1990) Hepatic peroxiso- tion of hepatocarcinogenesis in AKR mice: inhibition of c- mal and microsomal enzyme induction by citral and jun and c-myc. Oncol Reprod 6: 1123–1127 linalool in rats. Food Chem Toxicol 28: 403–408 Komori T, Fujiwara R, Tanida M, Nomura J (1995) Potential Segal R, Milo-Goldzweig I (1985) The hemolytic activity of antidepressant effects of lemon odor in rats. Eur Neuropsy- citral – II. Glutathione depletion in citral treated erythro- chopharmacol 5: 477–480 cytes. Biochem Pharmacol 34: 4117–4119 Larsen ST, Hougaard KS, Hammer M, Alarie Y, Wolkoff P, Trentin AP, Santos AR, Guedes A, Pizzolatti MG, Yunes RA, Clausen PA, Wilkins CK, Nielsen GD (2000) Effects of R- Calixto JB (1999) Antinociception caused by the extract of (+)- and S-(–)-limonene on the respiratory tract in mice. Hedyosmum brasiliense and its active principle, the Hum Exp Toxicol 19: 457–466 sesquiterpene lactone 13-hydroxy-8,9-dehydroshizukano- Lister RG (1987) The use of a plus-maze to measure anxiety lide. Planta Med 65: 517–521 in the mouse. Psychopharmacol 92: 180–185 Uedo N, Tatsuta M, Iishi H, Baba M, Sakai N, Yano H, Otani Lorenzetti BB, Souza GE, Sarti SJ, Santos Filho D, Ferreira T (1999) Inhibition of d-limonene of gastric carcinogene- SH (1991) Myrcene mimics the perypheral analgesic ac- sis induced by N-methyl-N′-nitro-N-nitrosoguanidine in tivity of lemongrass tea. J Ethnopharmacol 34: 43–48 Wistar rats. Cancer Lett 137: 131–136 Matos FJA (1996) As ervas cidreiras do Nordeste do Brasil: Vale TG, Matos FJA, Lima TC, Viana GSB (1999) Behav- estudo de três quimiotipos de Lippia alba (Mill.) N. E. ioral effects of essential oils from Lippia alba (Mill.) N.E. Brown (Verbenaceae). Parte II – Farmacoquímica. Rev Brown chemotypes. J Ethnopharmacol 67: 127–133 Bras FArm 77: 137–141 Viana GSB, Vale TG, Matos FJA (2000) Anticonvulsant ac- Matsuda H, Yoshikawa M, Iinuma M, Kubo M (1998) tivity of essential oils and active principles from chemo- Antinociceptive and anti-inflammabory activities of types of Lippia alba (Mill.) N.E.Brown. Biol Pharm Bull. limonin isolated from the fruits of Evodia rutaecarpa var. 23: 1314–1317 bodinieri. Planta Med 64: 339–342 Viana GSB, Vale TG, Rao VSN, Matos FJA (1998) Anal- Miranda AL, Silva JR, Rezende CM, Neves JS, Parrini SC, gesic and anti-inflammatory effects of two chemotypes of Pinheiro ML, Cordeiro MC, Tamborini E, Pinto AC (2000) Lippia alba: a comparative study. Pharm Biol 36: 347–351 Anti-inflammatory and analgesic activities of the latex Wolffgramm J, Mikolaicz KC, Coper H (1994) Acute and containing triterpenes from Himatanthus suuba. Planta sub-chronic benzodiazepine-barbiturate interactions on Med 66: 284–286 behavior and physiological response of the mouse. Nakamura M, Chi YM, Yan WM, Yonezawa A, Nakasugi Y, Naumyn-Schmiedebergs-Arch Pharmacol 349: 279–286 Yoshizawa T, Hashimoto F, Kinjo J, Nohara T, Sakurada S Zidorn C, Dirsch VM, Rungeler P, Sosa S, Della Loggia R, (2001) Structure-antinociceptive activity studies of incar- Merfort I, Pahl HL, Vollmar AM, Stuppner H (1999) Anti- villateine, a monoterpene alkaloid from Incarvillea sinen- inflammatory activities of hypocretenolides from Leon- sis. Planta Med 67: 114–117 todon hispidus. Planta Med 65: 704–708 Nogueira AC, Carvalho RR, Souza CA, Chahoud I, Paum- gartten FJ (1995) Study on the embryofeto-toxicity of citral in the rat. Toxicol 96: 105–113 Osuna L, Pereda-Miranda R, Tortoriello J, Villareal ML (1999) Production of the sedative triterpene galphimine B ᭿ in Galphimia glauca tissue. Planta Med 65: 149–152 Address Pattnaik S, Subramanyam VR, Bapaji M, Kole CR (1997) Production and anti-fungal activity of aromatic con- G. S. B. Viana, Department of Physiology and Pharma- stituents of essential oils. Microbiol 89: 39–46 cology, Faculty of Medicine, Federal University of Ranner GM, Vaz AD, Coon MJ (1996) Metabolism of all- Ceará, Rua Col. Nunes de Melo, 1127 Fortaleza trans, 9-cis, and 13-cis isomer of retinal by purified isoen- 60.430-270, Brazil