Anti-inflammatory activity of linalool and linalyl acetate constituents of essential oils.Summary
a natural insecticidal compound found in oil extracted from citrus peel. Similar in activity to d-limonene. 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 enantiomer /en·an·tio·mer/ (en-an´te-o?mer) one of a pair of compounds having a mirror image relationship. , and its racemate racemate
Mixture of equal quantities of two enantiomers, substances whose molecular structures are mirror images of one another (see isomer). The two enantiomers rotate polarized light through opposite angles, canceling each other out so that the racemic mixture has 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 edema (ĭdē`mə), abnormal accumulation of fluid in the body tissues or in the body cavities causing swelling or distention of the affected parts. 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 car·ra·geen·an also car·ra·geen·in
Any of a group of closely related colloids derived from Irish moss and several other red algae, widely used as a thickening, stabilizing, emulsifying, or suspending agent in industrial, pharmaceutical, and 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 psy·cho·phar·ma·col·o·gy
The branch of pharmacology that deals with the study of the actions, effects, and development of psychoactive drugs.
psy activity in mice, revealing marked dose-dependent sedative effects on the central nervous system (CNS See Continuous net settlement.
See continuous net settlement (CNS). ) (Jirovetz et al. 1991; Buchbauer et al. 1991), including protection against pentylenetetrazol (PTZ PTZ Pan-Tilt-Zoom (camera)
PTZ Photo Zenith Tube
PTZ Poisson Truncated At Zero Model
PTZ Point Zero ), picrotoxin pic·ro·tox·in
A bitter crystalline compound derived from the seed of an East Indian woody vine and used as a stimulant, especially in treating barbiturate poisoning. 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 nicotinic /nic·o·tin·ic/ (nik?o-tin´ik) denoting the effect of nicotine and other drugs in initially stimulating and subsequently, in high doses, inhibiting neural impulses at autonomic ganglia and the neuromuscular junction. receptor-ion channel (Ghelardini et al. 1999; Re et al. 2000) and a spasmolytic spas·mo·lyt·ic
Causing arrest of a spasm; antispasmodic.
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 e·qui·mo·lar
Having an equal number of moles. 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 "Not invented here." See digispeak.
NIH - The United States National Institutes of Health. 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 albino (ălbī`nō) [Port.,=white], animal or plant lacking normal pigmentation. The absence of pigment is observed in the body covering (skin, hair, and feathers) and in the iris of the eye. 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 Asa (ā`sə), in the Bible, king of Judah, son and successor of Abijah. He was a good king, zealous in his extirpation of idols. When Baasha of Israel took Ramah (a few miles N of Jerusalem), Asa bought the help of Benhadad of Damascus and ) 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 anova
see analysis of variance.
ANOVA Analysis of variance, see there ), 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 hyperalgesia /hy·per·al·ge·sia/ (-al-je´ze-ah) abnormally increased pain sense.hyperalge´sic
Extreme sensitivity to pain. 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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1. Characteristic of or resulting from the conversion of heat into other forms of energy.
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Of or relating to the structural characteristic of a molecule that makes it impossible to superimpose it on its mirror image.
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A. T. Peana, Dipartimento di Scienze del Farmaco, Universita degli Studi di Sassari, via Muroni 23/a, 07100 Sassari, Italy
Tel.: ++39-079-228738; Fax: ++39-079-228712; e-mail: email@example.com
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