Evaluation of the Anti-Inflammatory and Antinociceptive Effects of the Essential Oil from Leaves of Xylopia laevigata in Experimental Models

Research Article

Received: 5 November 2013, Revised: 16 January 2014, Accepted: 20 January 2014 Published online in Wiley Online Library: 19 February 2014

(wileyonlinelibrary.com) DOI 10.1002/ffj.3195

184

Evaluation of the anti-inflammatory andantinociceptive effects of myrtenol, a plant-derived monoterpene alcohol, in miceRenan O. Silva,a Mirian S. Salvadori,b Francisca Beatriz M. Sousa,a

Maisa S. Santos,a Nathalia S. Carvalho,a Damião P. Sousa,c Bruno S. Gomes,d

Francisco A. Oliveira,d André Luiz R. Barbosa,a Rivelilson M. Freitas,e

Reinaldo Nóbrega de Almeidab and Jand-Venes R. Medeirosa,d*

ABSTRACT: Inflammation is characterized by vasodilatation, increase of blood flow and vascular permeability, migration ofleucocytes to the inflammatory site, and production of cytokines. The aim of this study was evaluate the anti-inflammatoryand antinociceptive effects of (�)-myrtenol, a plant-derived monoterpene alcohol, in mice and its possible mechanisms.Myrtenol was used in classical models of inflammation (paw oedema induced by different agents, carrageenan-induced peri-tonitis, myeloperoxidase levels and cytokine measurement) and nociception (acetic acid-induced writhing, hot-plate test, andpaw licking induced by formalin, glutamate, and capsaicin). Pretreatment with myrtenol effectively inhibited paw oedemainduced by carrageenan, compound 48/80, histamine, serotonin and prostaglandin E2. Myrtenol also reduced the cell counts,myeloperoxidase activity and cytokine levels (interleukin 1β, but not tumour necrosis factor-α) of the peritoneal cavityinduced by carrageenan. In addition, myrtenol inhibited acetic acid-induced writhing, did not significantly prolong the latencytime in the hot-plate test, decreased licking time caused by an intraplantar injection of formalin (only in the second phase),glutamate and capsaicin. Myrtenol reduces the inflammatory response and nociception in mice due to the inhibition of therelease of inflammatory mediators, cell migration and also to the signalling pathway of receptors involved in the transmissionof pain. Copyright © 2014 John Wiley & Sons, Ltd.

Keywords: myrtenol; monoterpene; essential oil; antinociceptive; cell migration; inflammation

* Correspondence to: Jand-Venes R. Medeiros, BIOTEC/LAFFEX/UFPI, Av. SãoSebastião, nº 2819, CEP 64202-020, Parnaíba, PI, Brazil. E-mail: [email protected]

a Biotechnology and Biodiversity Center Research (BIOTEC), Federal Universityof Piauí, Parnaíba, PI, Brazil

b Post-Graduation Program in Natural and Synthetic Bioactive Resources,Federal University of Paraíba, João Pessoa, PB, Brazil

c Department of Pharmaceutical Sciences, Federal University of Paraíba, JoãoPessoa, PB, Brazil

d Center for Research on Medicinal Plants, Post-Graduation Program inPharmacology, Federal University of Piauí, Teresina, PI, Brazil

e Post-Graduation Program in Pharmaceutical Sciences, Federal University ofPiauí, Teresina, PI, Brazil

IntroductionInflammation is the response of living tissue to noxious stimuli,such as pathogens and irritant agents, which involves changesin blood flow, increased vascular permeability, synthesis of in-flammatory mediators (e.g. histamine, serotonin and bradykinin)and intense leucocytes migration for the inflamed site.[1] Someof the mediators produced act as neuromodulators, generatingsustained activation and sensitization of primary nociceptorsand higher-order neurons involved in the transmission of pain.[2]

Although a considerable number of analgesic and anti-inflammatory drugs are available for the treatment of pain andinflammation, there is a continuous search for new compoundsas therapeutic alternatives, because these drugs exert a widerange of side effects and low efficacy, especially for chronic dis-eases.[3] In this context, natural products have been one of themost successful sources for the discovery of new therapeuticagents to benefit those afflicted by inflammatory diseases.[4]

Essential oils are volatile compounds produced as secondarymetabolites by medicinal plants, and have been reported toexhibit a variety of biological properties; the majority of these ef-fects is attributed to monoterpenes.[5] Myrtenol is a monoterpenealcohol (Figure 1) found in the essential oils of some aromaticplants, such as Tanacetum vulgare[6] and Aralia cachemirica,[7]

and can also be obtained through the oxidation of the α-pinene.[8]

Although it is used for its flavouring properties, several studies

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have shown that myrtenol has sedative properties,[9] and hypoten-sive[10] and inhibitory effects against the growth of harmful intes-tinal bacteria.[11]

Considering that many studies have been developed withmonoterpenes, and also based on the fact that these moleculesshowed important pharmacological activities and great thera-peutic potential, the aim of the present study was evaluate theanti-inflammatory and antinociceptive effects of myrtenol, aplant-derived monoterpene alcohol, in mice.

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Figure 1. Chemical structure of myrtenol

Anti-inflammatory and antinociceptive effects of myrtenol

Experimental

Drugs and Reagents

λ-Carrageenan, compound 48/80, serotonin, histamine, prostaglandin E2(PGE2), indomethacin, dimethyl sulfoxide (DMSO), glutamate, capsaicin,MK-801 and (�)-myrtenol (98% purity) were purchased from SigmaChemical (St. Louis, MO, USA). Heparin and morphine were provided byMerck (São Paulo, Brazil). All drugs were dissolved in sterile 0.9% (w/v) NaCl(saline). The myrtenol was dissolved in 2% DMSO. All other chemicals wereof analytical grade and obtained from standard commercial suppliers.

Animals

Male Swiss mice weighing 25–30 g were randomly housed in appropri-ate cages at 23 ± 2°C under a 12/12-h light/dark cycle with free accessto food (Purina®; São Paulo, Brazil) and water. Experimental protocolswere approved by the Ethics Committee in Research of the FederalUniversity of Piauí (protocol no. 0066/10) and handling procedures werein accordance with the Guide for Care and Use of Laboratory Animals(National Institutes of Health, Bethesda, MD, USA). Different groups ofanimals were used for each experimental protocol.

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Anti-inflammatory Activity

Effect of myrtenol on carrageenan-induced paw oedema

Initially, the animals were randomly divided into six groups (n=5) andacute hind paw oedema was produced by injecting of 50 μl of a suspen-sion of carrageenan (500 μg/paw, prepared in 0.9% sterile saline) into theright hind paw (group I). In the other groups, mice were pretreated intra-peritoneally (i.p.) with 2% DMSO (group II, untreated control), indometh-acin 10 mg/kg (group III, standard drug) or myrtenol 25, 50 or 75 mg/kg(groups IV, V and VI, respectively). Right paw volume was measured by thedislocation of the water column of a plethysmometer (Panlab, Barcelona,Spain) before (V0; time zero) at 1, 2, 3 and 4 h after carrageenan treatment(Vt) as described previously.[12] The effect of pretreatment was calculatedas the % inhibition of oedema relative to the paw volume of the DMSO-treated controls using the equation:

% inhibition ¼ Vt � V0ð Þcontrol � Vt � V0ð ÞtreatedVt � V0ð Þcontrol

� 100

Effect of myrtenol on paw oedema induced by different agents

To induce oedema with different agents, the animals received 50 μl in-jections of compound 48/80 (12 μg/paw), serotonin (5-HT; 1% w/v), his-tamine (HIST; 100 μg/paw) or PGE2 (3 nmol/paw) into the right hind paw.The control group, untreated, was injected with only 50 μl of 2.0%DMSO. Myrtenol 75 mg/kg or indomethacin (10 mg/kg, reference con-trol) was injected i.p. 30 min before intraplantar injections of phlogisticagents. Right paw volume was measured by the dislocation of the watercolumn of a plethysmometer (Panlab) before (V0; time zero) at 30, 60, 90and 120 min after (Vt) the administration of phlogistic agents.

Evaluation of neutrophil migration

Mice were injected intraperitoneally with 2.0%DMSO, myrtenol 75mg/kgor indomethacin 10 mg/kg. Thirty minutes later, the mice were injected


with 250 μl of carrageenan (500 μg/cavity). After 4 h, mice were sacrificedand the peritoneal cavity was washed with 1.5 ml of heparinized phos-phate-buffered saline. Total cell counts were performed in a Neubauerchamber and differential cell counts (100 cells total) were carried out oncytocentrifuge slides stained with haematoxylin and eosin. The resultsare presented as the number of neutrophils per millilitre of peritonealexudate. Aliquots of the peritoneal exudates were stored at �70°C forlater analysis of cytokine content and myeloperoxidase (MPO) activity.

Myeloperoxidase activity

The peritoneal exudate was centrifuged at 4500 rpm for 15 min at 4°C. Inthe following section, 10 μl of the exudate was used for the assay ofMPO activity by measuring the change in absorbance at 450 nm usingo-dianisidine dihydrochloride and 1% hydrogen peroxide. MPO activitywas reported as units/ml of exudate. A unit of MPO activity was definedas the amount that converted 1 μmol of hydrogen peroxide to water in1 min at 22°C.[13]

Cytokine measurements

The levels of interleukin 1β (IL-1β) and tumour necrosis factor-α (TNF-α)were evaluated using sandwich ELISA as described previously.[14] Briefly,microlitre plates were coated overnight at 4°C with antibodies againstmouse IL-1β or TNF-α (2 μg/ml). After blocking the plates, the test sam-ples and each standard at various dilutions were added in duplicateand incubated at 4°C for 24 h. The plates were washed three times withbuffer and then incubated with biotinylated sheep polyclonal anti-IL-1βor anti-TNF-α (diluted 1:1000 with assay buffer 1% bovine serum albu-min). After further incubation at room temperature for 1 h, the plateswere washed and 50 μl of streptavidin–horseradish peroxidase (diluted1:5000) was added. The reagent o-phenylenediamine dihydrochloride(50 μl) was added 15 min later, and the plates were incubated in the darkat 37°C for 15–20 min. The reaction was stopped with the addition ofsulfuric acid (1 M) and absorbance was measured at 490 nm. The resultsare expressed as pg/mg protein and reported as mean± SEM.

Antinociceptive Activity

Writhing test

Abdominal constrictions induced by acetic acid (0.6%; i.p.) were accom-plished according to procedures described previously.[15] The animalswere treated with 2% DMSO, myrtenol (75 mg/kg, i.p.) or morphine(5 mg/kg, s.c., reference control), 30 min before the injection of aceticacid. After waiting for 10 min, the number of constrictions, includingabdominal muscle contractions and hind paw extension, was recordedover 20 min.

Hot-plate test

Each mouse was dropped twice on a heated plate (55 ± 1°C), separatedby a 30 min interval. The first trial familiarized the animal with the testprocedure and the second served as the control reaction time (lickinga paw or jumping), recorded as the response latency on a hot-plate(model EFF-361; Insight, Ribeirão Preto, Brazil). Animals with baselinelatencies of more than 20 s were excluded from the study. The mice weretreated with myrtenol (75 mg/kg, i.p.) or morphine (5 mg/kg, s.c.; refer-ence drug) 30 min before the test and the control group received thesame volume of 2% DMSO. Measurements were performed before(zero time) and 30, 60 and 90 min after treatment, with a cut-off timeof 45 s to prevent development of paw lesions.[16]

Formalin test

Nociceptive behaviour was induced by injecting 2.5% formalin (20 μl) in theregion sub-plantar of the right hind paw according to themethod describedpreviously.[17] Mice were given 2% DMSO, myrtenol (75 mg/kg, i.p.) ormorphine (5 mg/kg, s.c.; reference control) 30 min before the formalininjection. Licking time was recorded from 0 to 5 min (phase 1, directchemical stimulation of nociceptors) and 20–25 min after the formalininjection (phase 2, release of inflammatory mediators).

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Glutamate test

The animals were treated with vehicle (2% DMSO, i.p.), myrtenol(75 mg/kg, i.p.) or MK-801 [0.03 mg/kg, i.p.; an N-methyl-D-aspartate(NMDA) receptor antagonist], 30 min later the mice received 20 μl ofglutamate solution (30 μmol/paw) in the right hind paw. The nociceptiveresponse (time that the animal remained licking or biting the injected paw)was timed over a period of 15 min after glutamate administration.[18]

Capsaicin test

The animals were treated with vehicle (2% DMSO, i.p.), myrtenol(75 mg/kg, i.p.) or morphine (5 mg/kg, s.c.; reference control), 30 min laterthe mice received 20 μl of capsaicin solution (3 μg/paw) in the right hindpaw. The nociceptive response (time that the animal remained licking orbiting the injected paw) was timed over a period of 5 min after capsaicinadministration.[19]

Evaluation of Motor Activity

Initially, the animals were evaluated to select those that had shown abil-ity in walking on the revolving bar under the same conditions used inthe test and these were divided into three groups (n=10). On the dayof the test, animals were treated with 2% DMSO (vehicle), myrtenol(25, 50 or 75 mg/kg; i.p.) or diazepam (2 mg/kg; positive control). After60 min, the animals were placed with all four feet onto a bar of 2.5 cmdiameter, 25 cm above the floor, in a rotation of 17 rpm for a period of3 min. The duration of permanence in the swivel bar, in seconds (s),and the number of falls, with three renewals at maximum wasrecorded.[20]

Statistical Analysis

Results are expressed as mean± SEM from at least five animals per groupand statistical analysis was performed using one-way analysis of variance(ANOVA) followed by the Newman–Keuls post hoc test, when appropri-ate. Statistical significance was set at p< 0.05.

Results

Carrageenan-induced Paw Oedema

Table 1 shows that the carrageenan (500 μg/paw) administrationproduced an intense paw oedema which was maintained duringthe first 4 h. This gradual increase in paw volume was observedfrom 1 h (0.058 ± 0.019 ml), with a maximum value observed atthe third hour (0.107 ± 0.016 ml). On the other hand, the admin-istration of myrtenol caused a dose-dependent inhibitory effect,

Table 1. Effect of myrtenol on carrageenan-induced paw oedem

Treatment Dose (mg/kg) Pa

1 h 2

DMSO — 0.002 ± 0.002 0.002 ±Control (Cg) — 0.058 ± 0.019 0.060 ±Indomethacin 10 0.012 ± 0.007* (79.3) 0.014 ±Myrtenol 25 0.034 ± 0.005 (41.3) 0.028 ±

50 0.024 ± 0.005 (58.6) 0.016 ±75 0.035 ± 0.008 (39.6) 0.016 ±

Values of paw oedema are expressed in mean± SEM of five to six% Inhibition of paw edema is indicated in parenthesis.*p< 0.05 versus control group.Cg, carrageenan; DMSO, dimethyl sulfoxide.

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at all doses tested, with maximal effect at dose of 75 mg/kg(0.020 ± 0.008 ml), inhibiting oedema in about 81.3% (p< 0.05).Similarly, indomethacin (10 mg/kg, i.p.), a standard drug, showeda clear inhibition of the inflammation in all study points withmaximum inhibition in about 81.3% at the third hour after carra-geenan injection (Table 1).

Paw Oedema Induced by Different Phlogistic Agents

The oedematogenic response induced by the intraplantar injec-tion of compound 48/80, serotonin, histamine or PGE2 was im-mediately evident and reached the maximum point at 30 minafter administration of the phlogistic agent (0.108 ± 0.011 ml;Figure 2A, 0.084 ± 0.008 ml; Figure 2B, 0.082 ± 0.003 ml; Figure 2Cand 0.071 ± 0.002 ml; Figure 2D, respectively). However,pretreatment with myrtenol (75 mg/kg, i.p.) significantly reduced(p< 0.05) the paw oedema elicited by compound 48/80(0.064 ± 0,007 ml), serotonin (0.030 ± 0.005 ml), histamine(0.028 ± 0.003 ml) or PGE2 (0.018 ± 0.008 ml). In the same way,indomethacin (10 mg/kg, i.p.) also reduced the paw oedemainduced by different phlogistic agents (Figure 2).

Carrageenan-induced Peritonitis

As shown in Figure 3, carrageenan (500 μg/cavity) produced anintense cellular infiltrate 4 h after the stimulus, with a totalleucocyte count (13.22 ± 2.610 leucocytes� 103/ml) and neutro-phils (11.41 ± 2.392 neutrophils� 103/ml) significantly increased(p< 0.05), when compared to animals treated only with 2%DMSO. Pretreatment with myrtenol (75 mg/kg, i.p.) produced adecrease in leucocyte count (1.26 ± 0.22 leucocytes� 103/ml;Figure 3A) and neutrophils (0.67 ± 0.22 neutrophils� 103/ml;Figure 3B) in the peritoneal cavity. Similarly, indomethacin(10 mg/kg, i.p.) decreased the cell migration induced by inflam-matory stimuli.

Myeloperoxidase Activity

Figure 4 shows that myrtenol at 75 mg/kg significantly reducedMPO activity to 1.8 ± 0.1 U/mg of exudate (76.6% inhibition), ascompared to the carrageenan group (7.7 ± 0.8 U/ml of exudate).Similarly, indomethacin, the reference drug, also significantlyreduced the MPO activity (3.2 ± 1.2 U/mg of tissue).

a in mice

w oedema at various times (ml)

h 3 h 4 h

0.002 0.002 ± 0.002 0.002 ± 0.0020.014 0.107 ± 0.016 0.066 ± 0.0060.011* (76.7) 0.020 ± 0.015* (81.3) 0.014 ± 0.011* (78.8)0.004 (53.3) 0.028 ± 0.006* (73.8) 0.038 ± 0.009 (42.40)0.006* (73.3) 0.034 ± 0.006* (68.2) 0.032 ± 0.003 (51.5)0.004* (73.3) 0.020 ± 0.008* (81.3) 0.027 ± 0.009* (59.1)

animals per group.

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Figure 2. Effect of themyrtenol on paw inflammation induced by different agents. Oedemawas induced by: (A), compound 48/80; (B), histamine (Hist); (C),serotonin (5-HT); and (D), Prostaglandin E2 (PGE2). Animals were pretreated with 2% DMSO, myrtenol (Myrt; 75 mg/kg i.p.) or indomethacin (Indo; 10 mg/kg,i.p.). Each column represents mean±SEM of five to six animals per group. *p< 0.05 vs. phlogistic agent. ANOVA and Newman–Keuls test

Figure 3. Anti-inflammatory effect of myrtenol on neutrophil migration inmice. Mice were treated with 2% DMSO, myrtenol (75 mg/kg, i.p.) orindomethacin (Indo; 10 mg/kg, i.p.) and 30 min later was injected 250 μlof carrageenan (500 μg/cavity). Neutrophil migration was evaluated 4 hlater. (A) Total counts and (B) differential counts. Each column representsmeans±SEM of five to six animals per group. *p< 0.05; #p< 0.05 vs. carra-geenan group. ANOVA and Newman–Keuls test

Figure 4. Effect of myrtenol on paw tissue myeloperoxidase activityinduced by carrageenan. Mice were treated with 2% DMSO, myrtenol(75 mg/kg, i.p.) or indomethacin (Indo; 10 mg/kg, i.p.) and 30 min laterwas injected 250 μl of carrageenan (500 μg/cavity). The myeloperoxidase(MPO) activity of peritoneal exudates was determined after 4 h. Resultsare expressed as the mean± SEM of five to six animals per group.*p< 0.05; #p< 0.05 vs. carrageenan group. ANOVA andNewman–Keuls test



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TNF-α and IL-1β Levels in Carrageenan-induced Peritonitis

Figure 5 shows that the intraperitoneal administration of carra-geenan induced a marked increase in IL-1β (1995.0±13.19 pg/ml;Figure 5A) and TNF-α (191.6±18.7 pg/ml; Figure 5B) and concentra-tions in peritoneal exudates fluid. Pretreatment with myrtenol(75 mg/kg, i.p.) significantly reduced IL-1β (1044.0± 221.5 pg/ml)levels, but not TNF-α (194.0 ± 64.09 pg/ml), as compared to thecarrageenan control group (p< 0.05).

Acetic Acid-induced Writhing Test

Figure 6 shows that myrtenol (75 mg/kg, i.p.) produced a signif-icant (p< 0.05) inhibition (69.3%) on the writhing responses,


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Figure 5. Effect ofmyrtenol on carrageenan-induced cytokine productionin peritonitis. Levels of TNF-α (A) and IL-1β (B) in the peritoneal cavities weremeasured 4 h after carrageenan injection. Mice were administered 2%DMSO, myrtenol (75 mg/kg, i.p.) or indomethacin (10 mg/kg, i.p.) and30 min later 250 μl of carrageenan (500 μg/cavity). Each column repre-sents mean± SEM of five to six animals per group. *p< 0.05; #p< 0.05vs. carrageenan group. ANOVA and Newman–Keuls test

Figure 6. Effect of the myrtenol on the acetic acid-induced writhing testin mice. DMSO (control), myrtenol (75 mg/kg, i.p.) or morphine (5 mg/kg,s.c.), were administered 30 min before 0.6% acetic acid (250 μl/cavity).Each column represents mean± SEM of five to six animals per group.*p< 0.05; #p< 0.05 vs. control group. ANOVA and Newman–Keuls test

Figure 7. Effect of the myrtenol on reaction times to thermal stimuli(hot-plate). Mice received 2% DMSO, myrtenol (75 mg/kg, i.p.) or mor-phine (5 mg/kg, s.c.). Each column represents mean± SEM of five to sixanimals per group. *p< 0.05 vs. time zero. ANOVA and Newman–Keulstest.

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when compared to the control group (acetic acid). As expected,morphine (5 mg/kg, s.c.), a standard drug used as a positive con-trol, also produced a significant antinociceptive effect (95.7%).

Hot-plate Test

Figure 7 shows that myrtenol (75 mg/kg, i.p.) did not significantly(p< 0.05) prolong the latency time in the hot-plate test in time


intervals used as compared to time zero. On the other hand,the morphine (5 mg/kg, s.c., positive control) was able to signifi-cantly increase the latency time in the hot-plate test at time inter-vals of 30, 60 and 90 as compared to time 0 (untreated).

Formalin Test

Myrtenol (75 mg/kg, i.p.) showed a significant antinociceptiveeffect, reducing the formalin-induced paw licking time, thoughit did not reduce significantly (p< 0.05) only the second phase(inflammatory) of the test with a reduction of 97.6%, as com-pared to the control group (Figure 8B). As expected, morphine(5 mg/kg, s.c.), the drug used as a positive control, significantlyreduced the licking time in both phases of the test (50.9% inthe first phase to 73.9% in the second phase) (Figure 8).

Glutamate Test

Figure 9 shows that the administration of myrtenol (75 mg/kg,i.p.) (30.90 ± 6.78 s; 44.9%) significantly reduced (p< 0.05) thelicking time induced by the administration of glutamate(56.05 ± 11.21 s). Likewise, MK 801 (0.03 mg/kg, i.p.), the drugused as a positive control, also showed a decrease in the re-sponse (19.08 ± 4.42 s; 66.0%).

Capsaicin Test

The results shown in Figure 10 indicate that myrtenol (75 mg/kg,i.p.) (20.90 ± 2.65 s; 36.8%) presented a significant reduction(p< 0.05) of the capsaicin-induced nociception (33.08± 1.27 s).As expected, morphine (5 mg/kg, s.c.), the drug used as a positivecontrol, caused a reduction in the response (4.19± 1.81 s; 87.3%).

Rota-rod Test

The mice pretreated with myrtenol in doses of 25, 50 or 75 mg/kgshowed no significant motor performance alterations in therota-rod test (data not shown). As might be expected, diazepam(2 mg/kg), a standard drug, significantly reduced (p< 0.05) thetime spent by treated animals on the rota-rod test, comparedwith the control group.


Figure 8. Effect of the myrtenol on the formalin test in mice. The time spent licking was determined during the first 0–5 min. (first phase; left panel)and during 20–25 min. (second phase; right panel) after injection 2.5% formalin. DMSO (control), myrtenol (75 mg/kg, i.p.) or morphine (5 mg/kg, s.c.;reference control) were administered 30 min before formalin injection. Each column represents mean± SEM of five to six animals for each group.*p< 0.05; #p< 0.05 vs. control group. ANOVA and Newman–Keuls test

Figure 9. Effect of the myrtenol on the glutamate test in mice. Animalswere preteated with 2% DMSO (control), myrtenol (75 mg/kg; i.p.) orMK-801 (0.03 mg/kg, positive control; i.p.) 30 min before the injectionof glutamate (30 μmol/paw). The time spent licking was determinedduring the first 0–15 min after injection of glutamate. Each column repre-sents mean± SEM of five to six animals per group. *p< 0.05; #p< 0.05 vs.control group. ANOVA and Newman–Keuls test

Figure 10. Effect of the myrtenol on the capsaicin test in mice. Animalswere preteated with 2% DMSO (control), myrtenol (75 mg/kg; i.p.) ormorphine (5 mg/kg, positive control; s.c.) 30 min before the injectionof capsaicin (3 μg/paw). The time spent licking was determined duringthe first 0–5 min. after injection of capsaicin. Each column representsmean± SEM of five to six animals per group. *p< 0.05; #p< 0.05 vs. con-trol group. ANOVA and Newman–Keuls test


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DiscussionPlants are widely used in many countries to treat different inflam-matory conditions; however, many of their active principles re-main unknown. Thus, experimental pharmacological studies for


the purpose of identifying active principles are needed.[21] Inthe present study, we demonstrated that myrtenol, a plant-derived monoterpene alcohol, exhibits a pronounced anti-inflammatory and antinociceptive effect in different animalmodels.Initially, we conducted the carrageenan-induced paw oedema,

a model which has been widely employed for the screening ofplant-derived anti-inflammatory compounds. This oedema ischaracterized as a biphasic event that involves the release ofdiverse mediators, in which the initial phase (first hour) is mainlymediated by the release of histamine, serotonin and bradyki-nin.[22] The inflammation reaches its maximum approximately 3h post-treatment (second phase), which is sustained by theover-production of prostaglandin followed by the release ofpro-inflammatory cytokines and cellular infiltration.[23] The re-sults of the present study showed that the myrtenol has inhibi-tory effect both in the early and in the late phases of the test.These data suggested allow this molecule to bear a significantanti-inflammatory activity that involves the inhibition of one ormore intracellular signalling pathways involved in the inflamma-tory response.In order to confirm this effect, we performed paw oedema in-

duced by different inflammatory mediators. Paw oedema in-duced by the compound 48/80 is due to the activation andconsequent degranulation of mast cells, culminating in the re-lease of vasoactive amines, including histamine and seroto-nin.[24] Studies have shown that the inhibition of degranulationmast cells serves as a valuable index for the prediction of the ef-ficacy of anti-inflammatory drugs.[25,26] In the present study,myrtenol promoted a reduction of the paw oedema caused bycompound 48/80, what suggests that the prevention of mastcell degranulation is a factor that may contribute to its anti-oedematogenic effect in the early stage of the inflammatoryprocess. These results are in accord with the reduction in theinhibitory effect exercised by myrtenol on oedema vascularityprovoked by the intraplantar administration of histamine andserotonin, suggesting a capacity of this substance to interferein the initial establishment of the inflammatory process.Prostaglandins are important inflammatory mediators involved

in cell migration and development of paw oedema immediatelyafter its administration.[27] Prostaglandin E2 (PGE2), in particular,has been reported to a play a critical role in the inflammatory re-sponse.[28] The intraplantar injection of PGE2 provokes oedemacharacterized by an increase of vascular permeability and pro-duction of several chemo-attractants.[29] The findings of this


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model showed that myrtenol reduced PGE2-induced paw oe-dema, suggesting that the anti-inflammatory effect of myrtenol,a plant-derived monoterpene alcohol, in the delayed phase ofoedema is mediated, at least in parts, by the inhibition of PGE2.

Based on the anti-oedematogenic effect of myrtenol in thelate phase of carrageenan-induced oedema, it is possible to pos-tulate that this molecule could also be interfering in cellularinflammatory events. Carrageenan induces inflammatory responsesaccompanied by neutrophil infiltration, release of neutrophil-derivedmediators and production of neutrophil-derived free radicals.[30]

Thus, to confirm the role of myrtenol on leucocyte migration,we carried the experiments of the carrageenan-induced peritoni-tis model and MPO levels.

Carrageenan-induced peritonitis has been used to inves-tigate the mechanisms involved in acute inflammation andalso to assess the effectiveness of anti-inflammatory drugs.The results showed that the administration of myrtenol wasable to induce a pronounced anti-inflammatory effect throughthe reduction in the number of inflammatory cells into theperitoneal cavity. This observation is also supported by severalstudies showing similar anti-inflammatory effect of the mono-terpenes, a result of the reduction in the neutrophil migrationin the peritonitis model.[31,32]

In sequence, we investigated the effect of myrtenol onmyeloperoxidase activity. MPO is an enzyme found primarily inthe azurophilic granules of neutrophils, released after its activa-tion, commonly used as a quantitative marker of activatedleucocytes and also implicated in exudation and cell migra-tion.[33] Thus, it has been demonstrated that the inhibition ofMPO is an important indicator of anti-inflammatory activity.[34]

The results of this model showed that myrtenol was capable ofreducing the increase of MPO activity in the peritoneal fluidcollected after carrageenan-induced peritonitis.

Considering that activated neutrophils produce and releaseproinflammatory cytokines, which play a relevant role in theinflammatory process,[35] we measured the concentrations ofTNF-α and IL-1β in peritoneal fluid after intraperitoneal injectionof carrageenan. The data demonstrated that myrtenol reducedthe levels of IL-1β, but not TNF-α, corroborating the results ofthe measurement of oedema and leucocyte count, indicatingthat this molecule has anti-inflammatory activities by regulatingthe immune system.

Based on these results, the anti-inflammatory effects of myrtenolwere well evidenced; however, it is worth mentioning that the dataobtained in this study showed that this effect is dependent onthe interference in the release/action of various inflammatorymediators (histamine, serotonin and PGE2) and processes relatedto migration of inflammatory cells. Thus, considering the closerelationship between inflammation and the development of apainful sensation, we decided to investigate the antinociceptivepotential of myrtenol through the following models of acuteinflammation: the acetic acid-induced writhing test, the hot-platetest, and paw licking induced by the administration of eitherformalin, glutamate or capsaicin.

The acetic acid-induced abdominal constriction test is a modelin which this substance directly activates visceral and somaticnociceptors innervating the peritoneum.[36] This test is mediatedby the injection of acetic acid, which causes irritation in the peri-toneal cavity that produces episodes of characteristic stretching(writhing) movements.[37] In general, acetic acid causes painthrough the release of endogenous substances such as seroto-nin, histamine, prostaglandins and proinflammatory cytokines


such as IL-1β, TNF-α via modulation of macrophages and mastcells located in the peritoneal cavity.[38] The results of this modelshowed that myrtenol was able to reduce the number of con-strictions in mice, showing a potent antinociceptive effect. Theprobable mechanism for this effect is due to the reduction of syn-thesis/liberation of inflammatory mediators involved, in additionto the stabilized mast cell membranes, evidenced by the inhibi-tion of paw oedema induced by phlogistic different agents andmeasurement of the cytokine IL-1β.

However, this model of writhing is a non-specific model, sincemany drugs which are not analgesics (such as tricyclic antidepres-sants, anticholinergic and antihistaminic) show antinociceptiveactivity.[39] Thus, to confirm the antinociceptive effect, we usedhot-plate test, also verifying a possible involvement in the centralin analgesic effect of the myrtenol.

This test is widely used in studies of nociception operant re-sponse to a thermal stimulus mediated by central integration.[40]

This model reflects the activity of temperature-sensitive afferentfibres like A-δ and C-fibres, with the participation of substanceP acting as a transmitter of the afferent pathways in the spinalcord.[41] Myrtenol did not significantly increase the latency timein response to thermal stimulus in this test. Therefore, wediscarded a possible central involvement in the antinociceptiveeffects of myrtenol.

To verify the effectiveness of the analgesic effect of myrtenol,we used the formalin test. This typical model of pain occurs in abiphasic pattern; there is an early phase (0–5 min), characterizedby intense neurogenic pain that results from the direct chemicalstimulation on the nociceptive afferent fibres, principally theC-fibres and the release of substance P;[42] there is then thelatephase (20–25 min), which corresponds to inflammatory pain,caused by a release of serotonin, histamine, bradykinin and pros-taglandins.[43] Drugs that act primarily as central analgesicsinhibit both phases while peripherally acting drugs inhibit onlythe second phase.[44] In this test, myrtenol showed a significantantinociceptive effect, reducing the formalin-induced lickingtime only in the second phase. Thus, it is possible to suggest thatthe antinociceptive effect of myrtenol is due to a peripheralmechanism. These findings confirm the data obtained frompaw oedema induced by inflammatory different agents andparameters involved in cell migration described above. Inaddition, these results confirm the data obtained in the hot-platetest, in which myrtenol-treated animals showed similar behav-iour to those treated with vehicle.

Studies of molecules with antinociceptive action, bothperipheral and central, demonstrated an interaction withmechanisms dependent on glutamate[45] and Transient ReceptorPotential (TRP) receptors.[46] It is well established that variousforms of glutamatergic neurotransmission contribute to thedevelopment and/or maintenance of hyperalgesia. Particularly,a mechanism for synaptic potentiation involves NMDA recep-tors located in peripheral structures, spinal cord and abovethese, when activated, increase synaptic transmission betweennociceptive afferent fibres and dorsal horn neurons.[18]

Additionally, activation of these receptors can stimulate theproduction of a variety of intracellular second messengers suchas nitric oxide and pro-inflammatory cytokines, which actsynergistically in the excitation of neurons.[47] In the presentstudy, myrtenol produced an inhibition of the nociceptioninduced by glutamate, suggesting that the inhibition of thissignalling pathway is involved in the antinociceptive effect ofthis compound.



Capsaicin has the ability to activate C or A-δ fibres in afferentneurons through the stimulation of TRP receptors, allowing theinflux of Ca2+ and Na+ and leading to neurogenic pain.[48]

Interestingly, studies have shown that the activation of thesereceptors is involved in mechanisms of various animal modelsof nociception, as in the acetic acid-induced writhing model[49]

and the late phase of the formalin test.[50] The resultsshowed that myrtenol had a significant inhibitory effect. Thus,we can infer a possible antagonism of peripheral vanilloid(TRP) receptors or interference in the cascade mediation initi-ated by the activation of this receptor, culminating in the inhibi-tion of nociception in experimental models mentionedpreviously. A number of monoterpenes have also been de-scribed as agonist[51] or antagonist[46] of different members ofthe TRP channel family.

To investigate whether the treatment with myrtenol could in-fluence the motor activity of the animals and consequently im-pair the assessment of the nociceptive behaviour in theexperimental models, we evaluated with a rota-rod test. Therota-rod apparatus has potential value for testing pharmacolog-ical agents that produce skeletal muscle relaxation, convulsionand depression of the central nervous system.[52] The resultsshowed that myrtenol did not significantly change motor perfor-mance alterations in rota-rod test. These results confirm that theantinociceptive effect exerted by myrtenol is not related tochanges in the motor coordination of animals.

The antinociceptive and anti-inflammatory activity of mono-terpenes alcohols has been reported. Some studies show thatthe chirality influence the pharmacological activity in stereoiso-meric monoterpene alcohols.[53,54] Myrtenol is also a chiralalcohol, which has two stereogenic centres. The structural char-acteristics of this monoterpene alcohol distinguish it from simplealcohols, such as methanol and butanol, which are consideredtoxic. The results for the tests conducted in animals treated with(�)-myrtenol in this study are better than those obtained whenother monoterpenes have been evaluated in models of painand inflammation. (�)-Myrtenol presents a different carbon skel-eton to most of the alcohols described in antinociceptive tests.Furthermore, it is a chiral compound having two asymmetric car-bons and (�)-myrtenol 1R,5S that may confer differences in othercompound as monoterpenes found in essential oils of herbs asthe antinociceptive and anti-inflammatory properties and othersaction mechanisms. The chirality can influence pharmacologicalproperties, causing downtime for example of (+)-mentholcompared with its bioactive enantiomer (�)-menthol.[55]

ConclusionIn the present studywe have demonstrated the efficacy ofmyrtenol,a plant-derivedmonoterpene alcohol, in different anti-inflammatoryand antinociceptive models. Thus, myrtenol was shown to bepromising for treating inflammatory diseases as an effectiveimmunomodulator of the release of inflammatory mediators,inhibition of cell migration and also signalling pathway of recep-tors involved in the transmission of pain.

19

Acknowledgements

The authors gratefully acknowledge the financial support from Na-tional Counsel of Technological and Scientific Development – CNPq(Brazil) and Research Foundation for the State of Piauí – FAPEPI.


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Evaluation of the Anti-Inflammatory and Antinociceptive Effects of the Essential Oil from Leaves of Xylopia laevigata in Experimental Models

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