Antinociceptive effect of citronellal in mice

Abstract

Citronellal is a monoterpene reported to be a major component of the essential oils in various aromatic species of plants. The present study evaluated the central nervous system depressant and antinociceptive properties of citronellal through behavioral experimental models. Following intraperitoneal injection, citronellal induced the reduction of spontaneous activity, ataxia, analgesia, and sedation. In pentobarbital-induced hypnosis, CTL (citronellal) at 50, 100, and 200 mg/kg (i.p.) significantly increased sleeping time (88.0 ± 11.4, 100.2 ± 16.4, and 119.5 ± 20.9 min) when compared to vehicle solution injections (43.0 ± 6.1). Citronellal (100 and 200 mg/kg, i.p.) significantly reduced the number of writhes (66.4 and 81.9%) in a writhing test and the number of paw licks during phase 1 (47.0 and 66.8%) and phase 2 (71.1 and 79.2%) of a formalin test when compared to control group animals. In addition, the results of a hot plate test showed central analgesic properties for citronellal (p < 0.05). These results indicate depressant, hypnotic, and antinociceptive properties of this monoterpene.

Keywords::

• Antinociception
• central nervous system
• citronellal
• monoterpenes

Introduction

Pain is a sensorial modality, which in many cases represents the only symptom for the diagnosis of several diseases, and often has a protective function. Throughout history man has used many different forms of therapy for the relief of pain, and medicinal herbs are highlighted due to their wide popular use. An example is Papaver somniferum L. (Papaveraceae), from which morphine has been isolated, and is regarded as the prototype of opiate analgesic drugs (Almeida et al., 2001). This fact has stimulated a considerable amount of research into a new drug for pain treatment, and consequently numerous herbal medicines have been recognized as active on the central nervous system (CNS) (Phillipson, 2001). In addition, they have been described to have a hypothetical potential to affect chronic conditions such as anxiety, depression, headaches, pain, or epilepsy, which do not respond well to conventional treatments (Carlini, 2003).

Monoterpenes are the main chemical constituents of the essential oils of these medicinal herbs, and are mixtures of odoriferous components obtained by steam distillation or solvent extraction from a large variety of aromatic plants. They are found in edible as well as medicinal plants with a therapeutic property (Carneiro et al., 1997). Additionally, monoterpenes and their derivative compounds exhibit several types of pharmacological properties, such as anxiolytic (Silva et al., 2007), antinociceptive (De Sousa et al., 2007b), sedative (Elisabetsky et al., 1995; De Sousa et al., 2006a, 2007a), antidepressant (De Sousa et al., 2006b), and anticonvulsant (Viana et al., 2000; De Sousa et al., 2006c, 2007c). In this regard, citronellal (3,7-dimethyl-6-octen-1-al) as a monoterpene is a major component of the essential oils in various aromatic species, such as Cymbopogon winterianus Jowitt (Java citronella) (Quintans-Júnior et al., 2008), Corymbia citriodora (Hook.) K.D.Hill, and C. nardus L. (Lenardão et al., 2007). This monoterpene presents many applications in popular medicine and aromatherapy. Citronellal is used in the perfumery, cosmetic, and soap industries (Lawless, 2002). Along with citral, geranial, linalool, and citronellol, citronellal (CTL) is one of the most important terpenes.

The purpose of the present study was to evaluate the effect of citronellal on the central nervous system and its antinociceptive activity using acetic acid-induced writhing, formalin, and hot plate tests on mice.

Materials and methods

Chemicals

Pentobarbital, acetic acid, and polyoxyethylene-sorbitan monolate (Tween 80) were purchased from Sigma (USA). Morphine, naloxone, and acetylsalicylic acid were purchased from União Química (Brazil). (RS)-(±)-Citronellal (98% purity) (Figure 1) was purchased from Dierberger (Brazil).

Figure 1.  Chemical structure of citronellal.

Animals

Male Swiss mice (30–35 g), 2–3 months of age, were used throughout this study. The animals were randomly housed in appropriate cages at 25 ± 2°C on a 12 h light/dark cycle (lights on 06:00–18:00 h) with free access to food (Purina) and water. They were used in groups of 10 animals each. All experiments were carried out between 09:00 and 16:00 h in a quiet room. Experimental protocols and procedures were approved by the Animal Care and Use Committee (CEPA/UFS N° 12/08) at the Universidade Federal de Sergipe.

Behavioral effects

Behavioral screening of the mice was performed following parameters described by Almeida et al. (1999), and animals were observed at 0.5, 1.0, and 2.0 h after intraperitoneal (i.p.) administration of citronellal (50, 100, and 200 mg/kg, i.p.).

Pentobarbital-induced hypnosis

Sodium pentobarbital, at a hypnotic dose of 50 mg/ kg (i.p.), was injected into four groups (n = 10) of mice 30 min after pretreatment with saline/Tween 80 0.2% (vehicle) and CTL (50, 100, and 200 mg/kg, i.p.), respectively. The latency (interval between injection of sodium pentobarbital and loss of the righting reflex) and duration of sleeping time (interval between loss and recovery of the righting reflex) were recorded (Elisabetsky et al., 1995).

Antinociceptive activity

Acetic acid-induced writhing

This test was done using the method described by Koster et al. (1959) and Broadbear et al. (1994). Muscular contractions were induced by i.p. injection of a 0.85% solution of acetic acid (0.25 mL/animal) to a group of 10 mice. The number of muscular contractions was counted for 15 min after injection and the data represent the average of the total number of writhes observed. CTL was administered in doses of 50, 100, and 200 mg/kg (i.p.). The reference drug, acetylsalicylic acid (ASA), was solubilized in saline plus one drop of Tween 80 0.2% (vehicle) (200 mg/kg) and was administered intraperitoneally to different groups of mice 0.5 h before acetic acid injection.

Formalin test

The observation chamber was a glass box of 30 cm diameter on a transparent acrylic plate floor. Beneath the floor, a mirror was mounted at a 45° angle to allow clear observation of the paws of the animals. The animals were treated with vehicle (saline plus one drop of Tween 80 0.2%), CTL (50, 100, and 200 mg/kg, i.p.), or the reference drug (ASA 200 mg/kg, i.p.) 0.5 h before the formalin injection. Each mouse was placed in the chamber more than 5 min before treatment in order to allow acclimatization to the new environment. The formalin test was carried out as described by Hunskaar and Hole (1987). Twenty microliters of a 2.5% formalin solution (0.92% formaldehyde) in phosphate buffer (pH 7.2) were injected into the dorsal surface of the left hind paw using a microsyringe with a 26-gauge needle. Each animal was then returned to the chamber and the amount of time that the animal spent licking the injected paw was considered to be indicative of pain. Two distinct phases of intensive licking activity were identified: an early acute phase and a late or tonic phase (0–5 and 15–30 min after formalin injection, respectively).

Hot plate test

The hot plate test described by Jacob et al. (1974) and by Jacob and Ramabadran (1978) was used. The animals were placed on an aluminum plate that was adapted to a water bath at 55 ± 0.5°C. The reaction time was noted by observing the licking of the hind paws at basal, 0.5, 1.0, 1.5, and 2.0 h after i.p. administration of 50, 100, and 200 mg/kg of CTL or vehicle (saline plus one drop of Tween 80 0.2%) to different groups of 10 mice. Morphine, 5 mg/kg (i.p.), was used as the reference drug. The effect of pretreatment with naloxone (0.5 mg/kg, i.p.) on the antinociception produced by CTL (100 and 200 mg/kg) and morphine (5 mg/kg, i.p.) was determined.

Statistical analysis

The obtained data were evaluated by one-way analysis of variance (ANOVA) followed by Dunnett’s or Fisher’s tests. In all cases, differences were considered significant if p < 0.05. The percent of inhibition by an antinociceptive agent was determined for the acetic acid-induced writhing and formalin tests using the following formula (Reanmongkol et al., 1994):

Results

Behavioral effects

CTL at doses of 50, 100, and 200 mg/kg (i.p.) showed depressant activity on the CNS based on the following behavioral alterations in animals after 30, 60, and 120 min of elapsed time after treatment: decrease of spontaneous activity, palpebral ptosis, ataxia, analgesia, and sedation.

Pentobarbital-induced hypnosis

As shown in Figure 2A, CTL at doses of 100 and 200 mg/kg (i.p.) did not affect the latency of pentobarbital-induced hypnosis. CTL at 50, 100, and 200 mg/kg (i.p.) significantly (p < 0.05) increased the sleeping time compared to control group animals (Figure 2B).

Figure 2.  Effect of citronellal (CTL) on pentobarbital-induced hypnosis in mice. The parameters evaluated were the onset of sleeping (A) and duration of sleeping (B). Values are mean ± SEM for 10 mice, *p < 0.05; **p < 0.01, as compared to vehicle (control), one-way ANOVA.

Acetic acid-induced writhing

Table 1 shows that CTL significantly (p < 0.01) reduced, in a dose-dependent manner, the number of writhing movements induced by the i.p. administration of acetic acid solution. Although these results are significant, the inhibition (%) of nociceptive behavior by the 50, 100 and 200 mg/kg (61.4, 66.4, and 81.9%) doses was inferior to that produced by the reference drug, aspirin 200 mg/kg (87.8%).

Table 1.  Effect of citronellal (CTL) or aspirin on writhing induced by acetic acid.

Treatment	Dose (mg/kg)	Number of writhings^a	% Inhibition
Vehicle	–	32.1 ± 9.3	–
CTL	50	12.4 ± 6.2^b	61.4^d
CTL	100	10.8 ± 5.9^b	66.4^d
CTL	200	5.8 ± 4.7^c	81.9^e
Aspirin	200	3.9 ± 2.5^c	87.8^e

n = 10.

^aValues represent mean ± SEM.

^bp < 0.05 (one-way ANOVA and Dunnett’s test), ^cp < 0.01 (one-way ANOVA and Dunnett’s test), ^dp < 0.01 (Fisher’s test), ^ep < 0.001 (Fisher’s test), significantly different from control.

Formalin test

In the first and second phases, CTL significantly reduced the licking time compared with the control group (Table 2). This decrease occurred in a dose-dependent manner.

Table 2.  Effect of citronellal (CTL) or indomethacin on formalin-induced pain.

Treatment	Dose (mg/kg)	Licking time (s)
		0–5 min		15–30 min
		Score of pain^a	% Inhibition	Score of pain^a	% Inhibition
Vehicle	–	82.9 ± 19.5	–	74.3 ± 21.1	–
CTL	50	47.3 ± 14.7^b	42.9^d	22.8 ± 10.7^c	69.5^d
CTL	100	43.9 ± 16.8^b	47.0^d	21.5 ± 11.3^c	71.1^d
CTL	200	27.5 ± 17.2^c	66.8^e	15.4 ± 7.3^c	79.2^d
Aspirin	200	35.4 ± 9.8^c	57.3^e	5.9 ± 5.6^c	92.1^e

n = 10.

^aValues represent mean ± SEM.

^bp < 0.05 (one-way ANOVA and Dunnett’s test), ^cp < 0.01 (one-way ANOVA and Dunnett’s test), ^dp < 0.01 (Fisher’s test), ^ep < 0.001 (Fisher’s test), significantly different from control.

Hot plate test

Table 3 shows the results of the hot plate test. Two doses of CTL (100 and 200 mg/kg, i.p.) increased the latency time, in a dose-dependent manner, to the thermal stimulus in both the licking and jumping parameters evaluated. The reaction time parameter was only significantly increased in higher doses of CTL, which induced a nociceptive inhibition similar to the inhibition exerted by morphine (5 mg/kg, i.p.). Naloxone (0.5 mg/kg, i.p.) partially reversed the antinociceptive effect of the monoterpene.

Table 3.  Antinociceptive effect of citronellal (CTL) in the hot plate test in the absence and presence of naloxone in mice.

Treatment	Dose (mg/kg)	Reaction time (licking of hind paws) (s)^a
		Basal	0.5 h	1 h	1.5 h	2 h
Vehicle	–	6.7 ± 2.4	8.1 ± 2.5	7.2 ± 3.2	9.2 ± 3.7	10.1 ± 5.9
CTL	50	7.1 ± 3.2	10.9 ± 3.3	12.4 ± 3.5	13.9 ± 4.4	12.7 ± 5.5
CTL	100	8.0 ± 2.2	15.8 ± 4.7	19.3 ± 4.3^b	18.5 ± 4.9^b	15.0 ± 4.4
CTL	200	7.7 ± 2.9	17.7 ± 3.9^b	21.2 ± 5.1^b	20.3 ± 5.1^b	13.8 ± 5.9
CTL + naloxone	100 + 0.5	8.1 ± 3.1	11.9 ± 3.7	14.1 ± 4.9	12.4 ± 5.8	10.0 ± 3.9
CTL + naloxone	200 + 0.5	6.1 ± 3.4	14.4 ± 4.0	12.6 ± 3.8	15.1 ± 4.3	13.2 ± 4.2
Morphine	5	7.1 ± 2.8	26.5 ± 4.5^c	29.3 ± 3.5^c	28.7 ± 4.1^c	29.1 ± 5.9^c
Morphine + naloxone	5 + 0.5	5.9 ± 4.1	11.4 ± 4.4	12.7 ± 5.8	19.5 ± 4.9^b	13.7 ± 6.2

n = 10.

^aValues represent mean ± SEM.

^bp< 0.05 (one-way ANOVA and Dunnett’s test), ^cp< 0.01 (one-way ANOVA and Dunnett’s test), significantly different from control.

Discussion

Citronellal (CTL) is a monoterpene reported to be a major component of the essential oils in various aromatic plant species (Lenardão et al., 2007; Quintans-Júnior et al., 2008). Recently, various studies have shown that monoterpenes and their derivative compounds also exhibit several types of pharmacological properties, such as antinociception and sedation (Santos & Rao, 2000; Galeotti et al., 2002; De Sousa et al., 2006a, 2007b). In this regard, the aim of the present study was to evaluate the CNS depressant properties and antinociceptive potential of CTL in mice.

Mice treated with CTL (50, 100, or 200 mg/kg, i.p.) presented behavioral alterations, such as decrease of spontaneous activity, palpebral ptosis, ataxia, analgesia, and sedation. These effects were dose-dependent and showed possible evidence that the effects on the CNS are similar to those of drugs that reduce central activity (Morais et al., 2004; De Sousa et al., 2007a).

The potentiation of barbiturate action was investigated, and Figure 2 shows that CTL significantly altered (p < 0.05) the sleeping time. It is established that the potentiation of sleep time induced by pentobarbital must be a sedative or hypnotic action that is attributed to the involvement of central mechanisms in the regulation of sleep (N’Gouemo et al., 1994; Auzi et al., 2007) and involves potentiation of the γ-aminobutyric acid (GABA)ergic system (Steinbach & Akk, 2001; Sivam et al., 2004).

Additionally, our results show that CTL produced dose-dependent inhibition of the inflammatory pain in mice, as determined by a significant reduction in acetic acid-induced abdominal writhing. Acetic acid-induced abdominal constriction is a standard, simple, and sensitive test for measuring analgesia induced by both opioids and peripherally acting analgesics (Hayes et al., 1987; Hunskaar & Hole, 1987). This test, besides being the most appropriate antinociceptive model for opioids (Hayes et al., 1987; Shaw et al., 1988), is also commonly employed as a visceral inflammatory pain model (Barber & Gottschlich, 1992). In acetic acid-induced abdominal writhing, pain is elicited by the injection of an irritant such as acetic acid into the peritoneal cavity, which produces episodes of characteristic stretching (writhing) movements, and inhibition of the number of episodes by analgesics is easily quantifiable. Additionally, these results support the hypothesis of CTL participation in the inhibition of prostaglandin synthesis, as the nociceptive mechanism involves the process or release of arachidonic acid metabolites via cyclooxygenase (COX) and prostaglandin biosynthesis (Duarte et al., 1988) during abdominal writhing induced by acetic acid.

The formalin test involves two phases: a neurogenic phase with release of substance P, and an inflammatory phase with release of serotonin, histamine, bradykinin, and prostaglandins (Murray et al., 1988; Tjølsen et al., 1992). This is of interest, considering that both phases are sensitive to centrally acting drugs, such as opioids (Shibata et al., 1989). However, the second phase is also sensitive to NSAIDs (non-steroidal anti-inflammatory drugs) and corticosteroids (Hunskaar & Hole, 1987). In addition, it is generally agreed that N-methyl-d-aspartate (NMDA) receptors contribute to the persistent chemical stimulus during the late-phase central sensitization of dorsal horn neurons (wind-up; Hunter & Singh, 1994). Most drugs capable of reducing the excitability of spinal cord neurons, including opioids, NSAIDs, and glutamate antagonists, can also reduce or even abolish wind-up (Herrero et al., 2000). Table 2 shows that CTL (50, 100, and 200 mg/kg, i.p.) significantly inhibited (p < 0.05 and p < 0.01) the two phases.

The analgesic action presented by CTL involves supraspinal as well as spinal components, as demonstrated by utilization of the hot plate test (Yaksh & Rubi, 1976). The results suggest that CTL (100 and 200 mg/kg, i.p.) has a central analgesic effect, as evidenced by the prolonged delay in response time when mice were subjected to a nociceptive stimulus during a hot plate test. This central analgesic action was confirmed by the blocking effect of naloxone, a specific antagonist of morphinomimetic receptors (Belvisi et al., 1998; Mundey et al., 2000).

It is concluded that CTL possesses CNS depressant and hypnotic properties. Additionally, CTL was effective as an anti-inflammatory and analgesic compound in various pain models, probably mediated via inhibition of prostaglandin synthesis as well as central inhibitory mechanisms (opioid system). Further studies currently in progress will enable us to understand the precise action mechanisms.

Declaration of interest

We would like to thank the National Council of Technological and Scientific Development (Conselho Nacional de Desenvolvimento Científico e Tecnológico/CNPq/Brazil) and the Research Supporting Foundation of State of Sergipe (Fundação de Amparo à Pesquisa do Estado de Sergipe/FAPITEC-SE) for financial support. Two of the authors (Mônica S. Melo and Lais C. S. Sena) have scholarships from CNPq-UFS.