May 2008 Notes Biol. Pharm. Bull. 31(5) 1017—1020 (2008) 1017
Antinociceptive Activity of ( )-Carvone: Evidence of Association with Decreased Peripheral Nerve Excitability
a a a Juan Carlos Ramos GONÇALVES, Fernando de Sousa OLIVEIRA, Rubens Batista BENEDITO, b ,a Damião Pergentino de SOUSA, Reinaldo Nóbrega de ALMEIDA,* and a Demetrius Antônio Machado de ARAÚJO a Laboratory of Pharmaceutical Technology, Federal University of Paraíba; CP 5009, CEP 58051–970, João Pessoa, Paraíba, Brazil: and b Department of Physiology, Federal University of Sergipe; CEP 49100–000, São Cristóvão, Sergipe, Brazil. Received November 17, 2007; accepted December 26, 2007; published online February 13, 2008
( )-Carvone is a monoterpene ketone that is the main active component of Mentha plant species like Men- tha spicata. This study aimed to investigate the antinociceptive activity of ( )-carvone using different experimen- tal models of pain and to investigate whether such effects might be involved in the nervous excitability elicited by others monoterpenes. In the acetic acid-induced writhing test, we observed that ( )-carvone-treated mice exhib- ited a significant decrease in the number of writhes when 100 and 200 mg/kg was administered. It was also demonstrated that ( )-carvone inhibited the licking response of the injected paw when 100 and 200 mg/kg was administered (i.p.) to mice in the first and second phases of the formalin test. Since naloxone (5 mg/kg, s.c.), an opioid antagonist, showed no influence on the antinociceptive action of ( )-carvone (100 mg/kg), this suggested nonparticipation of the opioid system in the modulation of pain induced by ( )-carvone. Such results were un- likely to be provoked by motor abnormality, since ( )-carvone-treated mice did not exhibit any performance al- teration on the Rota-rod apparatus. Because the antinociceptive effects could be associated with neuronal ex- citability inhibition, we performed the single sucrose gap technique and observed that ( )-carvone (10 mM) was able to reduce the excitability of the isolated sciatic nerve through a diminution of the compound action potential amplitude by about 50% from control recordings. We conclude that ( )-carvone has antinociceptive activity as- sociated with decreased peripheral nerve excitability. Key words ( )-carvone; antinociception; compound action potential
Although a considerable number of analgesic drugs are writhing nociceptive response induced by acetic acid in available for the treatment of pain, the search for develop- mice,10,11) although the mechanisms underlying such proper- ment of new compounds as therapeutic alternatives continues ties remain unclear. since the available analgesic drugs exert a wide range of side A previous study reported a central nervous system (CNS) effects.1) Because of their relatively low cost and easy avail- depressive activity of citronellol, another monoterpene, that ability in several countries, natural active products could be exhibited peripheral activity, reducing nervous excitability used as synthesis models of more selective and powerful through a diminution of the compound action potential drugs. (CAP) amplitude.13) Another study using monoterpenes indi- The essential oils are natural products that exhibit a variety cated a neuroactive property affecting neuronal intracellular of biological properties, such as analgesic,2) anticonvul- signaling which could be provoked through neurotransmitter- sant,3,4) and anxiolytic.5) Those effects are attributed to the gated current modulation induced by these terpenes.14) For monoterpenes, which are the major chemical components of these reasons, it appeared possible that ( )-carvone could these oils. Previous studies showed that some monoterpenes also have analgesic activity involving the central and periph- also possess antinociceptive6) and anesthetic7) activities in an- eral nervous systems. Therefore our objective was to verify imal experiments, demonstrating their potential contribution the antinociceptive effect of ( )-carvone using different ex- to the development of more efficient analgesic drugs. perimental models of pain and to investigate whether such ( )-Carvone (p-mentha-6,8-dien-2-one) is a monoterpene effect might be involved in nervous excitability using the sin- ketone (Fig. 1) found as the main active component of gle sucrose gap technique. spearmint oil (Mentha spicata), a relative of common mint distilled from this plant.8,9) Recently, our group has demon- MATERIALS AND METHODS strated that ( )-carvone derivatives, such as rotundifolone,10) hydroxydihydrocarvone,11) and epoxy-carvone12) appear to Drugs For all in vivo experiments the following drugs have analgesic properties. Such compounds decreased the were used: ( )-carvone (Aldrich, U.S.A.), morphine hy- drochloride (Merck, U.S.A.), naloxone hydrochloride (Re- search Biochemical, U.S.A.) 37% formaldehyde (Vetec, Brazil), diazepam, acetylsalicylic acid (ASA), and acetic acid (Sigma, U.S.A.). Vehicle was 5% Tween 80 (Sigma, U.S.A.) dissolved in 0.9% saline solution and used to dilute the test drugs. The physiologic solution used for the in vitro tests was
composed of (in mM): NaCl 150; KCl 4; CaCl2 2; MgCl2 1; glucose 10; and [N-(2-hydroxyethyl)piperazine-N -2-ethane- Fig. 1. Chemical Structure of ( )-Carvone sulfonic acid] (HEPES) 10, adjusted to pH 7.4 with NaOH.
∗ To whom correspondence should be addressed. e-mail: [email protected] © 2008 Pharmaceutical Society of Japan 1018 Vol. 31, No. 5
Animals Adult (3-month-old) male albino Swiss mice partments electrically. ( )-Carvone (10 mM), diluted in phys- (25—35 g) and Wistar rats (350 g) were randomly housed in iologic solution with 0.5% Tween 80, was introduced into the appropriate cages at 21 1 °C with a 12/12-h light/dark cycle test compartment. The potential difference between the test (light from 06:00 to 18:00), with free access to food (Purina, and the fifth (last) compartment was recorded every 10 min. Brazil) and tap water. All experimental observations were Data were converted to digital form by a microcomputer- conducted between 12:00 and 17:00. The animals were killed based 12-bit A/D converter at a rate of 10.5 kHz and later an- by cervical dislocation, and all procedures were carried out alyzed using a suite of programs (Lynx, Brazil). To quantify in accordance with Ethical Committee guidelines (CEPA/ the effects of ( )-carvone, we used the CAP amplitude as a LTF-UFPB, process number 0208/06). parameter, measured by the potential difference between the Acetic Acid-Induced Writhing Initially the mice were baseline and the maximal voltage of the CAP. divided into five groups (n 8). Subsequently, ( )-carvone Statistical Analysis The in vivo and in vitro experimen- (50, 100, 200 mg/kg), vehicle (control), and morphine tal data obtained were evaluated using oneway analysis of (6 mg/kg) were administered intraperitoneally (i.p.) 30 min variance (ANOVA), followed by Dunnett’s test. Differences before an injection of 0.8% acetic acid (10 ml/kg). Each ani- were considered to be statistically significant when p 0.05. mal was isolated in an individual observation chamber and 10 min after acetic acid injection the cumulative number of RESULTS writhing responses was recorded during 10 min.1,15) Formalin-Induced Nociception This test was per- In the acetic acid-induced writhing test, the antinocicep- formed according to the model described by Vaz et al.,16) tive effect, represented by writhe reduction, elicited by which represents a modification of the original model de- 100 mg/kg of ( )-carvone (2.6 1.1) in mice was similar to scribed by Hunskaar et al.17) The animals were divided into that of morphine 6 mg/kg (3.3 1.4), a standard opioid six groups (n 8) and treated i.p. with vehicle (control), ( )- drug,1) when both groups were compared with control carvone (50, 100, 200 mg/kg), morphine (6 mg/kg), and (14.4 2.7). The nociceptive response was almost extin- 100 mg/kg of ASA. After 30 min, 20 ml of 2.5% formalin so- guished when 200 mg/kg of ( )-carvone was administered lution (0.92% formaldehyde in 0.9% saline) was injected into (0.3 0.1), although a dose of 50 mg/kg did not produce any the subplantar area of the right hindpaw. The duration of paw significant effect (Fig. 2A). licking was measured at 0—5 min (first phase) and 15— ( )-Carvone inhibited the licking response to the inject- 30 min (second phase) after formalin administration. ed paw when 100 mg/kg (63.5 6.4 s; 83.9 39.4 s) and Possible Antagonism of the ( )-Carvone Antinocicep- 200 mg/kg (40.7 8.5 s; 16.8 16.4 s) were administered i.p. tive Effect by Pretreatment with Naloxone Mice were in mice compared with the control group (119.7 9.9 s; subcutaneously (s.c.) pretreated (n 8) with 5 mg/kg of 296.4 31.8 s) in the first and second phases of the formalin naloxone, a nonselective opioid antagonist, 15 min before the i.p. administration of vehicle (control), ( )-carvone (100 mg/kg), or morphine (6 mg/kg). Subsequently, the acetic acid-induced writhing test was performed as described above. Rota-Rod Test Initially, the mice able to remain on the Rota-rod (Ugo Basile, Model 7750, Italy) longer than 180 s (7 rpm) were selected 24 h before the test.18) Then the se- lected animals were divided into five groups (n 8) and treated i.p. with vehicle (control), ( )-carvone (50, 100, 200 ml/kg), and diazepam (4 mg/kg). Thirty minutes later, each animal was tested on the Rota-rod and the time (s) they remained on the bar for up to 180 s was recorded after 30, 60, and 120 min. Electrophysiologic Assays The single sucrose gap tech- nique was used as described in a previous papers.13,19) Briefly, the sciatic nerves from Wistar rats were carefully removed and desheathed. One nerve bundle was positioned across the five compartments of the experimental chamber, which con- tained solid petroleum jelly at the partitions to isolate them electrically. Compartments 1 and 2 at one end of the nerve bundle were used to apply supramaximal stimulation, which consisted of 100-ms isolated rectangular voltage pulses deliv- ered by a stimulator (CF Palmer, Model 8048, U.K.) trig- Fig. 2. Antinociceptive Activity of ( )-Carvone ([ ]-CV) Using Differ- gered manually. These parameters were chosen to stimulate ent Experimental Models of Pain fast-conducting myelinated fibers (Aa) selectively. All com- (A) Effects of ( )-CV in the acetic acid-induced writhing test in mice. Vehicle (con- trol), ( )-CV (50, 100, 200 mg/kg), or morphine (MOR) were administered i.p. 30 min partments were filled with physiologic solution, except for before acetic acid injection. (B) Effects of ( )-carvone (50, 100, 200 mg/kg), MOR the fourth compartment, which was filled with isotonic su- (6 mg/kg), and acetylsalicylic acid (ASA, 100 mg/kg) on formalin-induced nociception in mice. Each column represents mean S.E.M. (n 8). ∗ p 0.01 vs. control; a p 0.01 M crose (280 m in ultrafiltrated water) solution that was con- vs. 1st-phase control, b p 0.01 vs. 2nd-phase control (ANOVA followed by Dunnett’s tinuously renewed to isolate the neighboring recording com- test). May 2008 1019
Fig. 3. Effects of Naloxone (NAL) in ( )-Carvone ([ ]-CV) and Mor- phine (MOR) Antinociceptive Activity The acetic acid-induced writhing test was performed as described in the text. Pre- treatment with NAL (5 mg/kg, s.c.) was performed 15 min before treatment (i.p.) with vehicle (control), ( )-CV (100 mg/kg), or MOR (6 mg/kg). The number of writhes was counted over a period of 10 min. Each column represents mean S.E.M. (n 8). a p 0.01 vs. control, b p 0.01 vs. control when NAL was injected (ANOVA followed by Dunnett’s test).
Table1.Time (s) on the Rota-Rod Observed in Mice after i.p. Treatment with Vehicle (Control), ( )-Carvone (50, 100, 200 mg/kg), or Diazepam (4 mg/kg)
Dose Time (s) on Rota-rod/180 s Treatment (mg/kg, Fig. 4. ( )-Carvone Reduces the CAP Amplitude Recordings in Isolated Period (observation) Rat Nerves i.p.) 30 min 60 min 120 min (A) Representative CAP recordings obtained every 10 min (during 30 min) in the presence of ( )-carvone (10 mM). Control records were acquired when the sciatic nerve Vehicle (control) — 177.0 3.0 180.0 0.0 180.0 0.0 was incubated with physiologic solution alone. (B) CAP amplitude decrease with ( )- ( )-Carvone 50 179.6 0.4 179.5 0.5 166.9 12.2 carvone (10 mM) incubation. Stimulation parameters were 6—8 V/100 ms. Values are 100 161.3 16.7 154.4 16.5 179.4 0.6 expressed as mean S.E.M. (n 4). ∗ p 0.05 vs. control (ANOVA followed by Dun- 200 162.1 17.9 168.4 11.6 180.0 0.0 nett’s test). Diazepam 4 32.0 7.3* 75.3 21.4* 166.9 7.8
The motor response was recorded for the following 180 s after drug treatment. Statis- DISCUSSION tical differences vs. control group were calculated using ANOVA, followed by Dun- nett’s test (n 8). ∗ p 0.01. The present study demonstrated the antinociceptive activ- ity of ( )-carvone through two distinct in vivo models of test, respectively (Fig. 2B). As expected, morphine (6 mg/kg) analgesia, the acetic acid-induced writhing and formalin also reduced the licking time in both phases of this test tests. The latter method is believed to resemble clinical pain (55.8 9.0 s; 52.4 15.3 s), while ASA (100 mg/kg) reduced more closely in comparison with other tests that employ me- it only during the second phase (140.3 34.3 s). ( )-Carvone chanical or thermal stimuli.20,21) (50 mg/kg) did not produce any significant changes in both The writhing behavior in mice produced by an i.p. injec- phases of the formalin test. tion of acetic acid is used to evaluate central and peripheral It was observed that naloxone (5 mg/kg, s.c.) antagonized analgesic activities.1) The acetic acid-induced algesia pro- the antinociceptive response of morphine from 2.4 1.2 motes endogenous substance release and many other effects (with vehicle only) to 23.3 2.5 (with vehicle plus naloxone) stimulating nervous termination of pain.22,23) Based on the writhes in the acetic acid-induced writhing test. However, percentage of writhing inhibition obtained with ( )-carvone naloxone did not reverse the effect of ( )-carvone administration, in the different doses tested, the analgesic ef- (100 mg/kg), and also failed to modify the acid-induced fect was similar to that of morphine, a standard drug (Fig. writhing response when only the vehicle was administered 2A). (Fig. 3). The formalin test is a valuable tool for assessing the anal- In the Rota-rod test, ( )-carvone-treated mice did not gesic properties of drug candidates.24) This test is a model of show any significant motor performance alterations with the nociceptive response in two distinct phases involving differ- doses of 50, 100, or 200 mg/kg (Table 1). As might be ex- ent mechanisms. The first phase (neurogenic pain) results pected, the CNS depressant diazepam (4 mg/kg) reduced the from the direct chemical stimulation of myelinated and un- time of treated animals on the Rota-rod after 30 (from myelinated nociceptive afferent fibers, mainly C fibers, which 177.0 3.0 to 32.0 7.3 s) and 60 (from 180.0 0.0 to can be suppressed by opioid analgesic drugs like morphine.1) 75.3 21.4 s) but not after 120 min of treatment with this The second phase (inflammatory pain) results from the re- standard drug. lease of inflammatory mediators in the peripheral tissues and Figure 4A illustrates ( )-carvone (10 mM) effects on the of functional changes in the neurons of the spinal dorsal horn CAP waveform of the isolated peripheral nerve, acquired by that, in the long term, promote facilitation of synaptic trans- supramaximal stimulation (6—8 V, 100 ms) every 10 min, mission at the spinal level.25,26) This latter phase was reported using the single sucrose gap technique. After 30-min incu- to be sensitive to the action of the majority of nonsteroidal bation, ( )-carvone produced about 50% CAP blockade, antiinflammatory drugs (NSAIDs), including ASA, indo- reducing the CAP amplitude from 50.8 3.4 mV to methacin, and naproxen.20,27) 27.2 7.1 mV (p 0.05) (Fig. 4B). In this model, ( )-carvone inhibited the licking response of mice in both phases of the formalin test (Fig. 2B), sug- 1020 Vol. 31, No. 5 gesting this monoterpene could exert its antinociceptive ef- 3) Almeida R. N., Motta S. C., Leite J. R., Bol. Latinoam. Caribe Plantas fects connected with central or peripheral mechanisms. How- Med. Aromat., 2, 3—6 (2003). 4) Elisabetsky E., de Souza G. P. C., Santos M. A. C., Siqueira I. R., ever, naloxone, an opioid antagonist, showed no influence on Amador T. A., Fitoterapia, 66, 407—414 (1995). the antinociceptive action of ( )-carvone (100 mg/kg, i.p.) in 5) Almeida R. N., Motta S. C., Faturi C. B., Catallani B., Leite J. 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