Anti-inflammatory activity of linalool and linalyl acetate constituents of essential oils.
Linalool and linalyl acetate are the principal components of many essential oils known to possess several biological activities, attributable to these monoterpene compounds. In this work, we evaluated individually the anti-inflammatory properties of (-) linalool, that is, the natural occurring enantiomer, and its racemate form, present in various amounts in distilled or extracted essential oils. Because in the linalool-containing essential oils, linalyl acetate, is frequently present, we also examined the anti-inflammatory action of this monoterpene ester. Carrageenin-induced edema in rats was used as a model of inflammation.
The experimental data indicate that both the pure enantiomer and its racemate induced, after systemic administration, a reduction of edema. Moreover, the pure enantiomer, at a dose of 25 mg/kg, elicited a delayed and more prolonged effect, while the racemate form induced a significant reduction of the edema only one hour after carrageenin administration. At higher doses, no differences were observed between the (-) enantiomer and the racemate; a further increase in the dose of both forms did not result in an increased effect at any time of observation.
The effects of equi-molar doses of linalyl acetate on local edema were less relevant and more delayed than that of the corresponding alcohol. These finding suggest a typical pro-drug behavior of linalyl acetate.
The results obtained indicate that linalool and the corresponding acetate play a major role in the anti-inflammatory activity displayed by the essential oils containing them, and provide further evidence suggesting that linalool and linalyl acetate-producing species are potentially anti-inflammatory agents.
Key words: (-) Linalool, ([+ or -]) linalool, linalyl acetate, essential oil, anti-inflammatory activity
Linalool and linalyl acetate are monoterpene compounds reported to be major volatile components of the essential oils of several aromatic species. A number of linalool- and linalyl acetate-producing species are used in traditional medicin systems to relieve symptoms and cure a variety of ailments, both acute and chronic. Their pharmacological activities are attributable to the content of alcohols like linalool and its corresponding ester (linalyl acetate) (Peana and Moretti, 2002). Linalool was evaluated recently for its psychopharmacological activity in mice, revealing marked dose-dependent sedative effects on the central nervous system (CNS) (Jirovetz et al. 1991; Buchbauer et al. 1991), including protection against pentylenetetrazol (PTZ), picrotoxin and transcorneal electroshock-induced convulsions, hypnotic and hypothermic properties (Elisabetsky et al. 1995; Elisabetsky et al. 1999). It has also been reported that linalool modulates glutamate activation expression in vitro (competitive antagonism of L-[ [H.sup.3]]glutamate binding) and in vivo (delayed subcutaneous N-methyl-D-aspartate-induced convulsions and blockade of intracerebroventricular quinolinic acid-induced convulsions) (Silva Brum et al. 2001; Brum et al. 2001). Anesthetic activity related to its effects on the nicotinic receptor-ion channel (Ghelardini et al. 1999; Re et al. 2000) and a spasmolytic effect (Lis-Balchin and Hart, 1999) were also reported, as well as antimicrobial activity against several bacteria and fungi (Carson and Riley, 1995; Pattnaik et al. 1997; Peana et al. 1999). Moreover, linalool, as well as some terpenes and terpenoids, could enhance the permeability of a number of drugs through biological tissues like skin or mucus membranes (Kunta et al. 1997; Kommuru et al. 1998; Ceschel et al. 2000).
Among the many papers on linalool, no data about its anti-inflammatory activity have been reported, although some linalool-producing species have been reported to possess good anti-inflammatory activity and a peripheral analgesic action (Moretti et al. 1997; Peana and Moretti, 2002).
The aim of the present study was to test the anti-inflammatory activity of linalool and linalyl acetate. Individual dose curves of (-) linalool, the natural occurring enantiomer, and its racemate form, present in various amounts in distilled or extracted essential oils, were examined. The anti-inflammatory effect of equimolar doses of linalyl acetate, frequently present in linalool-containing essential oils, was also evaluated. Carrageenin-induced edema in rats was used as a model of local inflammation.
Materials and Methods
The present study was carried out in accordance with Italian law, which allows experiments on laboratory animals only after submission of a research project to the competent authorities, and in accordance with the "Principles of laboratory animal care" (NIH publication no. 85-23, revised 1985). According to these principles, with the aim to reduce the number of experimental animals, positive control tests were not performed.
The experiments were performed on male albino Wistar rats weighing 150 170 g each (Harlan, Italy). They were housed in groups of three per cage and maintained under controlled environmental conditions (temperature 22 +/- 2[degrees]C; humidity 60-65%; 12-h Light-dark cycle). All animals were given standard laboratory diet and aqua fontis, available ad libitum.
Drugs and treatments
([+ or -]) Linalool (Sigma) was purified by steam distillation before use; (-) linalool and linalyl acetate (Sigma) were used as received. All compounds were tested on six rats, with six animals as control. Aspirin (ASA) was used as positive control. (-) Linalool was administered at doses of 25,50 and 75 mg/kg body wt., the racemate at doses of 12.5, 25, 50 and 75 mg/kg body wt., while linalyl acetate was administered at doses equivalent to the molar content of (-) linalool (32, 64 and 96 mg/kg body wt.). ASA was utilized at the dose of 150 mg/kg body wt. All compounds tested were administered by abdominal subcutaneous injection after their dissolution in PEG-200 (vehicle). The control groups received only the vehicle, injected in the same site of administration, which did not cause any effect per se. All experiments were performed between 09.00 and 15.00 h.
Anti-inflammatory activity was evaluated on the basis of inhibition of carrageenin-induced hind paw edema (Winter et al. 1962; Dolara et al. 1989). Thirty mm after dosing with the compounds of interest, 0.05 ml of a 1% carrageenin [lambda] suspension in saline (0.9% NaCl) was injected into the plantar surface of the hind paw. Paw volumes were determined using a water plethysmometer (Basile, Italy), at time zero (basal) and at the 1st, 3rd and 5th hour after carrageenin injection. The results are expressed as the difference in mean paw volumes (nil) between basal and treatment values at different times [+ or -] S.E. The activity was evaluated according to edema inhibition at the 1st, 3rd and 5th hour after carrageenin injection, using the following ratio: (Vt-Vo) control - (Vt-Vo) treated x 1 00/(Vt-Vo) control where Vt is the average volume at each time point after carrageenin injection and Vo is the average basal volume.
Each value represents the mean [+ or -] S.E., from six rats. The statistical significance was assessed using analysis of variance (ANOVA), supplemented by F-test for contrast, comparing each group to control groups. (Newman-Keuls test *P < 0.05, **P < 0.01, ***P < 0.005, and ****P < 0.001 were considered as significant differences).
* Results and Discussion
No evident local irritant effects at the injection site were observed during all treatments with active components; this suggests that the monoterpene compounds tested do not possess any marked irritant effect when administered subcutaneously.
Figs. 1, 2, 3 and 4 show the inhibitory effect of (-) linalool, ([+ or -]) linalool, linalyl acetate and ASA, respectively, on carrageenin-induced edema in rats.
(-) Linalool (Fig. 1), at the dose of 25 mg/kg body wt., did not exhibit any activity one hour after administration of carrageenin, but after 3 and 5 hours produced significant inhibition of edema (28%, P = 0.008 and 25%, P = 0.0004, respectively). The administration of higher doses (50 and 75 mg/kg body wt.) resulted in the maximum inhibitory effect against edema, which appeared one hour after carrageenin injection (58%, P = 0.008 and 60%, P = 0.006, respectively). This effect slowed down in the course of the 3rd and 5th hours; the inhibition induced by these two doses after these times did not differ significantly from that induced by (-) linalool at 25 mg/kg body wt., at the corresponding times of observation.
([+ or -]) Linalool (Fig. 2), at the dose of 12.5 mg/kg, failed to exert any effect. At a dose of 25 mg/kg, it showed a significant anti-edematous effect (55%, P = 0.03) only one hour after carrageenin administration, while no effect was present after 3 and 5 hours. By contrast, the racemate compound at 50 and 75 mg/kg body wt. failed to induce any observable effect one hour after carrageenin, but induced a significant effect after 3 (51%, P = 0.03 and 38%, P = 0.02, respectively) and 5 hours (45%, P = 0.01 and 34%, P = 0.04, respectively). The effects of these two doses did not show any statistically significant difference at each time of observation.
The comparison of the data obtained at the dose of 25 mg/kg body wt. shows that the pure enantiomer elicited a delayed and prolonged action with respect to the racemate form, which induced a relevant reduction of the edema only one hour after carrageenin administration.
All in all, statistical analyses showed an higher level of statistical significance for (-) linalool with respect to the racemate form, despite similar percent inhibition values found. This observation pointed to a better reproducibility of experimental data, leading us to hypothesize a more specific action for the pure enantiomer on receptors involved in the inflammation.
Linalyl acetate (Fig. 3) failed to exert any effect at the dose of 32 mg/kg body wt.; at 64mg/kg body wt., it produced an inhibition of inflammation observable only 3 (40%, P = 0.01) and 5 hours (42%, P = 0.004) after carrageenin injection. At the highest dose (96 mg/kg body wt.) the effect was present only after 3 hours (36%, P = 0.03). At this time of observation, the effects of these two last doses (64 and 96 mg/kg body wt.) did not show any statistically significant difference. All in all, the comparison of the data obtained at equi-molar doses evidenced a less pronounced and more delayed effect of linalyl acetate, with respect to the corresponding alcohol.
These differences could be ascribed to in vivo bio-transformation of linalyl acetate into the corresponding alcohol by hydrolysis. These findings could provide a rational basis to confirm an intrinsic ability of linalyl acetate to reduce local edema by a typical pro-drug behavior.
Fig. 4 shows that ASA, at a dose of 150 mg/kg body wt., 1 and 3 hours after administration of carrageenin, produced a significant inhibition of edema (52%, P = 0.02 and 64%, P = 0.002, respectively), but after 5 hours did not exhibit any activity.
The correlation between anti-inflammatory effect and administered doses could suggest a dose-independent effect. This observation is consistent with the possibility of a saturation of the receptors involved in the inflammatory reaction.
The results obtained thus for support the hypothesis that linalool and linalyl acetate play a major role in the anti-inflammatory activity displayed by some essential oils containing them. The present data suggest that all plant species producing a relevant amount of these monoterpene compounds are potential anti-inflammatory agents.
The mechanism by which the anti-inflammatory effect occurs remains to be determined, although several observations suggest a possible involvement of NMDA receptors. Indeed, linalool is a competitive NMDA receptor antagonist (Elisabetisky et al. 1999) and the administration of excitatory amino acid receptor antagonists selectively attenuates carrageenin-induced behavioral hyperalgesia in rats (Ren et al. 1992). In the framework of this hypothesis, linalool might counter the possible effect of glutamate release with the subsequent activation of NMDA receptors, in response to a noxious somatic stimulus.
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Brum LFS, Emanuelli T, Souza DO, Elisabetisky E (2001) Effects of linalool on glutamate release and uptake in mouse cortical synaptosomes. Neurochem Res 26(3): 191-94
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 46 (11-12): 1067-1072
Carson CF, Riley TV (1995) Antimicrobial activity of the major components of the essential oil of Melaleuca alternifolia. J Appl Bacteriol 78:264-269
Ceschel GC, Maffei P, Moretti MD, Demontis S, Peana AT (2000) In vitro permeation through porcine buccal mucosa of Salvia desoleana Atzei & Picci essential oil from topical formulations. Int J Pharm 195: 171-177
Dolara P, Franconi F, Mugelli A (1989) In: Farmacologia e Tossicologia Sperimentale (Pirisino R), pp 191-192, Pitagora. ed, Bologna
Elisabetsky E, Coelho De Souza GP, Dos Santos MAC, Siquieira IR, Amador TA, Nunes DS (1995) Sedative properties of linalool. Fitoterapia LXVI (5): 407-414
Elisabetsky E, Brum LF, Souza DO (1999) Anticonvulsant properties of linalool in glutamate-related seizure models. Phytomedicine 6:107-113
Ghelardini C, Galeotti N, Salvatore G, Mazzanti G (1999) Local anaesthetic activity of the essential oil of Lavandula angustifolia. Planta Med 65: 700-703
Jirovetz L, Jager W, Buchbauer G, Nikiforov A, Raverdino V (1991) Investigations of animal blood samples after fragrance drug inhalation by gas chromatography/mass spectrometry with chemical ionization and selected ion monitoring. Biol Mass Spectrom 20 (12): 801-803
Kommuru TR, Khan MA, Reddy IK (1998) Racemate and enantiomers of ketoprofen: phase diagram, thermodynamic studies, skin permeability, and use of chiral permeation enhancers. J Pharm Sci 87: 833-840
Kunta JR, Goskonda VR, Brotherton HO, Khan MA, Reddy IK (1997) Effects of menthol and related terpenes on the percutaneous absorption of propanolol across excised hairless mouse skin. J Pharm Sci 86: 1369-1373
Lis-Balchin M, Hart S (1999) Studies on the mode of action of the essential oil of lavender (Lavandula angustifolia P. Muller). Phytother Res 23(6): 540-542
Moretti MDL, Peana AT, Satta M (1997) A study on anti-inflammatory and peripheral analgesic action of Salvia sclarea oil and its main components. J Essent Oil Res 9: 199-104
Pattnaik S, Subramanyam VR, Bapaji M, Kole CR (1997) Antibacterial and antifungal activity of aromatic constituents of essential oils. Microbios 89: 39-46
Peana AT, Moretti MDL, Juliano C (1999) Chemical composition and antimicrobial action of the essential oils of Salvia desoleana and S. sclarea. Planta Med 65: 752-754
Peana AT, Moretti MDL (2002) Pharmacological activities and applications of Salvia sclarea and Salvia desoleana essential oils. Studies in Natural Product Chemistry. 26, pp 391-423, Elsevier Science Publishers, UK
Re L, Barocci S, Sonnino S, Mencarelli A, Vivani C, Paolucci G, Scarpantonio A, Rinaldi L, Mosca E (2000) Linalool modifies the nicotinic receptor-ion channel kinetics at the mouse neuromuscular junction. Pharmacol Res 42: 177-182
Ren K, William GM, Hylten JL, Ruta MA, Dubner R (1992) The introthecal administration of excitatory amino acid receptor antagonists selectively attenuated carrageenan-induced behavioral hyperalgesia in rats. Eur J Pharmacol 219(2): 235-243
Silva Brum LF, Elisabetsky E, Souza D (2001) Effects of linalool on [H.sup.3] MK801 and [H.sup.3] muscimol binding in mouse cortical membranes. Phytother Res 15: 422-425
Winter CA, Risley EA, Nuss GW (1962) Carrageenin-induced oedema in hind paw of the rat as an assay for anti-inflammatory drugs. Proc Soc exp Biol Med 111: 544-547
A. T. Peana, Dipartimento di Scienze del Farmaco, Universita degli Studi di Sassari, via Muroni 23/a, 07100 Sassari, Italy
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A. T. Peana, P. S. D'Aquila, F. Panin, G. Serra, P. Pippia (1), and M. D. L. Moretti
Dipartimento di Scienze del Farmaco, Universita degli Studi di Sassari, Sassari, Italy
(1.) Dipartimento di Scienze Fisiologiche Biochimiche e Cellulari, Universita degli Studi di Sassari, Sassari, Italy