Assessment of antinociceptive, anti-inflammatory and antioxidant properties of Cymbopogon winterianus leaf essential oil

Abstract

The present study investigated the antinociceptive, anti-inflammatory and antioxidant effects of the leaf essential oil (LEO) of Cymbopogon winterianus Jowitt (Poaceae). In the acetic acid-induced writhing and formalin tests, the LEO (50, 100, and 200 mg/kg, p.o.) significantly reduced (p < 0.05) the number of writhings and paw licking times in the first (0-5 min) and second (15-30 min) phases, respectively. In contrast, the LEO did not alter the latency time for mice licking the rear paws in hot-plate test. The LEO inhibited the carrageenan-induced neutrophil migration to the peritoneal cavity in a dose-dependent manner (35.5%, 42.8%, and 66.1% at doses of 50, 100, and 200 mg/kg, respectively, p < 0.001). Moreover, LEO exhibited higher scavenging activity toward 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals with an IC₅₀ (12.66 ± 0.56 μg/mL). Our present results demonstrated that the LEO has antinociceptive, anti-inflammatory, and antioxidant properties.

Keywords::

• Cymbopogon winterianus
• essential oil
• antinociceptive
• anti-inflammatory
• antioxidant

Introduction

The Cymbopogon genus (Poaceae) is composed of more than 100 species found in tropical countries (Lorenzi & Matos, 2003). About 56 species are aromatic and some of them have medicinal, pharmacological and industrial importance (Barbosa-Filho et al., 2005). Two species of Cymbopogon: Cymbopogon nardus (Jamarosa) and C. winterianus (Java citronella) are known to have similar volatile oil scent and medicinal uses, but they show different citronellal content.

Cymbopogon winterianus Jowitt is an important aromatic grass cultivated in India and Brazil that yields essential oil. The essential oil of the leaves, extracted by steam, is used in the perfumery, cosmetics, pharmaceutical, and flavoring industries (Duarte et al., 2005). Leaf essential oil (LEO) of C. winterianus is rich in geraniol (36%) and citronellal (42.7%) and shows repellent, antimycotic and acaricidal activities (Mendonça et al., 2005; Cassel & Vargas, 2006). Blank et al. (2007) reported that the LEO is an air freshener. The main traditional use is as a repellant (Tawatsin et al., 2001). However, folk medicine practitioners in northeastern Brazil use the infusion of the fleshy leaves and/or unguent for the treatment of pain and anxiety (oral communication). There is little published information about the plant. Preliminary behavioral screening realized in our laboratory with the LEO showed depressant activity on the central nervous system (CNS) and anticonvulsant property (Quintans-Júnior et al., 2008). Therefore, this study investigated the antinociceptive, anti-inflammatory, and antioxidant effects of LEO.

Methods

Plant material and essential oil extraction

Leaves were collected in October 2006 from the cultivation of the C. winterianus genotypes established at the Research Station “Campus Rural” of the Federal University of Sergipe (10° 55′ S, 37° 11′ W), Brazil, and a voucher sample has been deposited in the Herbarium of the Department of Biology of the same University. Plants were cut 20 cm above soil level in Spring at 09:00 h and dried at 40°C in a forced air oven (Marconi^®, Brasil) for 5 days. The essential oil (EO) of those leaves were extracted by hydrodistillation for 3 h (Carvalho-Filho et al., 2006), using a Clevenger-type apparatus (British Pharmacopoeia, 1988). The oils were separated from the aqueous phase and kept in the freezer (-20°C) until further use. The oil content was estimated based on dry herbage weight using three samples of 75 g of dry leaves (American Spice Trade Association, 1985). We obtained 3.4% essential oil content. Our oil showed in gas chromatography-mass spectrometry (GC-MS) analysis a mixture of monoterpenes, such as geraniol (40.06%), citronellal (27.44%) and citronellol (10.45%) as the main compounds in the LEO (Quintans-Júnior et al., 2008).

Animals

Male Swiss mice weighing 20-35 g each, maintained under standard environmental conditions, were used distributed in groups of 10 (writhing, formalin and hot-plate tests) and 6 male Wistar rats weighing 150-200 g each (neutrophil migration activity). The animals had free access to pellet diet (Purina chow) and tap water. Federal University of Sergipe Animal Care and Use Committee (CEPA/UFS no. 09/07) approved experimental protocols and procedures.

Acetic acid-induced writhing

The abdominal constrictions were produced according to the procedure described previously by Koster et al. (1959). The number of stretches or writhes (arching of the back, development of tension in the abdominal muscles, elongation of body, and extension of the forelimbs) per animal was counted during a 10-min period starting 5 min after i.p. administration of an 0.85% acetic acid solution. Vehicle (saline + 1 drop of Tween 80, 0.2%) and LEO was administered in doses of 50, 100 and 200 mg/kg (orally, p.o.) 60 min before acetic acid injection. The reference drug, acetylsalicylic acid (aspirin) (300 mg/kg) was administered intraperitoneally to the control group. The animals were observed in experimental cages. Hand-operated counters and stopwatches were used to score the writhing frequency.

Formalin test

The method used was similar to that described previously (Shibata et al., 1989; Vianna et al., 1998). LEO (50, 100, and 200 mg/kg p.o.) and acetylsalicylic acid (300 mg/kg p.o.) were administered 60 min before formalin injection (n = 10, each group). Control animals received the same volume of saline solution orally. An aliquot of 1% Formalin (20 µL) was injected subcutaneously into the right hind paw of the mice. The time (in seconds) spent in licking and biting responses of the injected paw was taken as an indicator of pain response. Responses were measured for 5 min (first phase) and 15-30 min after the formalin injection (second phase).

Hot-plate test

The hot-plate test measured response latencies according to the method described by Eddy and Leimback (1953). Animals were placed on an Insight^® hot-plate (Model EFF-361) maintained at 50° ± 1°C and the time between placement of the animal on the hot-plate and the occurrence of either the licking of the hind paws, shaking or jump off from the surface was recorded as response latency. Mice with baseline latencies of more than 10 s were eliminated from the study 24 h later. The cut-off time for the hot-plate latencies was set at 30 s. Animals were treated with LEO (50, 100 and 200 mg/kg p.o.) and morphine (5mg/kg i.p.) 60 min before the experiments. Control animals received the same treatment as with the abdominal constriction test.

Neutrophil migration to the peritoneal cavity

The leukocyte migration was induced by injection of carrageenan (500 µg/cavity i.p. 500 µL) into the peritoneal cavity of rats 1 h after administration of LEO (50, 100, and 200 mg/kg, p.o.) or dexamethasone (2 mg/kg s.c., n = 6) by modification of the technique previously described by Bastos et al. (2007). The animals were euthanized by cervical dislocation 4 h after carrageenan injection. Shortly after, phosphate buffered saline (PBS) containing EDTA (1 mM i.p., 10 mL) was injected. Immediately a brief massage was done for further fluid collection, which was centrifuged (2000 rpm for 5 min) at room temperature. The supernatant was discarded and 1 mL of PBS was introduced to the precipitate. An aliquot of 10 μL from this suspension was dissolved in 200 μL Turk solution and the total cells were counted in a Neubauer chamber, under optic microscopy. The results were expressed as the number of neutrophils/mL. The percentage of the leukocyte inhibition = (1 – T/C) × 100, where T represents the treated groups leukocyte counts and C represents the control group leukocyte counts.

Free radical scavenging activity

The free radical scavenging activity of the extract was determined based on its ability to scavenge the stable DPPH free radical. The test was based on the modified method of Chi and Jun (1990). Stock solution (10 mg/mL) of the LEO was prepared in EtOH and serial dilutions were carried out to obtain concentrations of 1, 5, 10, 15, 20, 25, 30 and 35 µg/mL. Diluted solutions (2 mL) were added to 2 mL of a 0.004% EtOH solution of DPPH, mixed and allowed to stand for 30 min for reaction to occur. The equation of the concentration × absorbance calibration curve for the DPPH radical was C = 110.547-0.02804A, where C is the concentration of the DPPH radical in medium, A is the absorbance at 515 nm. The correlation coefficient was R = 0.9983. The percentage of remaining DPPH (%DPPH_REM) was calculated according to Brand-Williams et al. (1995), as follows: %DPPH_REM = [DPPH]_T/[DPPH]_T0 × 100, where T is the time when absorbance was determined (1-60 min) and T₀ is the zero time. The amount of antioxidant necessary to decrease the initial concentration of DPPH radical by 50% (IC₅₀) was calculated by plotting the percentage of DPPH_REM at time of 60 min against various concentrations of LEO. The results were expressed as µg antioxidant/ml DPPH ± standard deviation. Butylated-hydroxy-toluene (BHT) was used as the positive control.

Statistical analysis

The data obtained were evaluated by one-way analysis of variance (ANOVA) followed by Dunnett`s test and Tukey’s test for antioxidant activity. Differences were considered to be statistically significant when p <0.05. The percentage of antinociceptive inhibition was determined for each experimental group using the following formula (Reanmongkol et al., 1994):

Inhibition % = 100 × (control-experiment)/control

Results and discussion

The number of writhes during the 15-min test period was statistically reduced in doses of 50, 100, 200 mg/kg of LEO in relation to the control group (Table 1). Moreover, the inhibition (%) by 50, 100 and 200 mg/kg of LEO was lower than that produced by 300 mg/kg acetylsalicylic acid (49.5%, 72.4%, 93.8% and 95.8%, respectively). Acetic acid-induced abdominal writhing causes algesia by liberation of endogenous substances, which excite the pain nerve endings (Taesotikul et al., 2003). Increased levels of PGE₂ and PGF_2α in the peritoneal fluid have been reported to be responsible for pain sensation caused by intraperitoneal administration of acetic acid (Derardt et al., 1980). Based on this result, it can be assumed that the mode of action of this activity might involve a peripheral mechanism.

Table 1.  Effect of LEO or aspirin on writhing induced by acetic acid.

Treatment	Dose (mg/kg)	Number of writhings^a	% Inhibition
Vehicle	-	19.2 ± 3.7	-
LEO	50	9.7 ± 2.1^b	49.5^d
LEO	100	5.3 ± 1.8^b	72.4^d
LEO	200	1.2 ± 0.9^c	93.8^e
Aspirin	300	0.8 ± 0.3^c	95.8^e

n = 10.

^aValues represent mean ± SEM.

^bP <0.01 (one-way ANOVA and Dunnett`s test), significantly different from control.

^cP <0.001 (one-way ANOVA and Dunnett`s test), significantly different from control.

^dP <0.01 (Fisher`s test), significantly different from control.

^eP <0.001 (Fisher`s test), significantly different from control.

In the formalin test (Table 2), LEO decreased the licking time during the first and second phases at doses of 50, 100, and 200 mg/kg. The advantage of using the formalin model of nociception is that it can discriminate pain in its central and peripherical components (Tjolsen et al., 1992). The test consists of two different phases separated in time: the first one is generated in the periphery through the activation of nociceptive neurons by the direct action of formalin, and the second phase occurs through the activation of the ventral horn neurons at the spinal cord level. Morphine, a typical narcotic drug, inhibits nociception in both phases (Shibata et al., 1989), but drugs with peripheric action, such as indomethacin and corticosteroids inhibit only the second phase. Moreover, drugs such as acetylsalicylic acid and paracetamol, which inhibit prostaglandin synthesis, block only the second phase of the formalin test (Hunskaar & Hole, 1987; Rosland et al., 1990).

Table 2.  Effect of LEO or aspirin on formalin-induced pain.

Treatment	Dose (mg/kg)	Number of licks (s)
		0-5 min		15-30 min
		Score of pain^a	% inhibition	Score of pain^a	% inhibition
Vehicle	-	65.9 ± 10.3	-	137.1 ± 29.7	-
LEO	50	35.6 ± 8.2^b	46.0^d	94.3 ± 21.8	31.2^d
LEO	100	5.8 ± 2.9^c	91.2^f	21.1 ± 9.4^b	84.6^e
LEO	200	4.3 ± 3.5^c	93.4^f	8.3 ± 7.1^c	93.9^f
Aspirin	300	22.4 ± 9.1^b	66.0^e	7.8 ± 4.4^c	94.3^f

n = 10.

^aValues represent mean ± SEM.

^bP <0.05 (one-way ANOVA and Dunnett`s test), significantly different from control.

^cP <0.001 (one-way ANOVA and Dunnett`s test), significantly different from control.

^dP <0.05 (Fisher`s test), significantly different from control.

^eP <0.01 (Fisher`s test), significantly different from control.

^fP <0.001 (Fisher`s test), significantly different from control.

Moreover, the hot plate test checked a possible central antinociceptive effect of the LEO since this is considered a specific test for central pain analysis. LEO did not interfere with nociception in this test (Table 3).

Table 3.  Effect of LEO on the hot plate test in mice.

Treatment	Dose (mg/kg)	Reaction time (s)^a
		Basal	0.5h	1h	1.5h	2h
Vehicle	-	8.5 ± 2.9	8.1 ± 2.5	7.2 ± 3.2	9.2 ± 3.7	10.1 ± 5.9
LEO	50	6.3 ± 2.1	7.9 ± 4.3	6.8 ± 4.9	9.2 ± 4.2	11.4 ± 5.2
LEO	100	9.4 ± 3.7	9.8 ± 3.9	8.4 ± 3.7	8.8 ± 5.1	11.2 ± 4.2
LEO	200	7.1 ± 3.1	11.5 ± 4.5	10.3 ± 5.2	11.2 ± 4.6	12.1 ± 4.8
Morphine	5	8.1 ± 2.7	29.2 ± 5.3^b	26.8 ± 4.2^b	28.4 ± 4.6^b	28.9 ± 5.5^b

n = 10.

^aValues represent mean ± SEM.

^bP <0.01 (one-way ANOVA and Dunnett`s test), significantly different from control.

Mild analgesics such as aspirin lack antinociceptive action in thermally motivated tests such as the hot-plate test, but have significant antinociceptive activity in tonic tests (writhing and formalin tests), which are characterized by the direct chemical stimulation of nociceptors. Since it has been reported that thermally motivated and tonic tests elicit the selective stimulation of A-γ fibers and C fibers, respectively (Yeomans et al., 1996), it is tempting to propose that LEO may interfere with the transmission of both fibers or a common pathway.

Carrageenan (500 µg/cavity) induced neutrophil migration to the peritoneal cavity 4 h after stimulus. Figure 1 shows the inhibitory effect of LEO on carrageenan-induced responses in a dose-dependent manner (35.5%, 42.8%, and 66.1% at doses of 50, 100, and 200 mg/kg, respectively, p <0.001). The results obtained with the control group support the effect of LEO since the vehicle presented no activity, and the control drug dexamethasone (2 mg/kg s.c., 1 h beforehand) inhibited (92.2%, p <0.001) the carrageenan-induced neutrophil migration to the peritoneal cavity.

Figure 1.  Effect of LEO on leukocyte migration into the peritoneal cavity induced by carrageenan in rats. Groups of rats were pre-treated with vehicle (C, control group, 10 mL/kg p.o., open column), dexamethasone (Dexa, 2 mg/kg s.c., cross-hatched column), or LEO in concentrations of 50, 100, and 200 mg/kg (p.o., right-hatched columns) 60 min before carrageenan-induced (500 µg/cavity, 500 µL i.p.) peritonitis. Cell counts were performed 4 h after the injection of carrageenan. Each value represents the mean ± SEM. Asterisks denote statistical significance, *p <0.001 related to control group. ANOVA followed by Tukey’s test (n = 6).

Cell recruitment during inflammation depends on the orchestrated release of local mediators that are responsible for local vascular and tissue changes as well as for the recruitment of host defense cells (Luster et al., 2005). The inflammation induced by carrageenan involves cell migration, plasma exudation and production of mediators such as nitric oxide, prostaglandin E₂, interleukin (IL)-1β, IL-6 and tumor necrosis factor (TNF)-α (Salvemini et al., 1996; Loram et al., 2007). Those mediators are able to recruit leukocytes, such as neutrophils, in several experimental models. LEO inhibited leukocyte migration induced by i.p. injection of carrageenan (in the peritonitis model) in a dose-dependent manner. A putative mechanism associated with this activity may be inhibition of the synthesis of many inflammatory mediators whose involvement in the cell migration is well established.

The amount of the DPPH radical, which reacted with the essential oil (30 µg/ml, 30 min) was 99.88% (Table 4). The leaf essential oil presented a response similar to the positive control butylated-hydroxy-toluene (BHT) (30 µg/ml, 30 min, Table 4). According to the IC₅₀ values, the antioxidant concentration needed to decrease by 50% the initial concentration of DPPH radical was not statistically different (p >0.05) of the positive control butylated-hydroxy-toluene (BHT). However, LEO showed a higher inhibition potential before 30 min that was able to reduce over 95% of the DPPH radical after only 1 min of reaction (Figure 2).

Figure 2.  Kinetic behavior of the LEO determined spectrophotometrically at 515 nm by reaction with the methanol solution of DPPH (30 µg/mL).

Table 4.  Radical scavenging activities of LEO determined by the reduction of DPPH free radical.

Samples	IP (%)	IC50 (µg/mL DPPH)
LEO	99.88	12.66 ± 0.56^*
BHT	95.39	16.33 ± 0.96^*

n = 3. Different small letters means significantly different at P <0.05 in the same column.

*IC₅₀ and IP (30 μg/mL) of extracts were calculated at the steady state (30 min).

The DPPH test is a very convenient method for screening small antioxidant molecules because the intensity of reaction can be analyzed by simple spectrophotometric assay (Sánchez-Moreno et al., 1998; Soler-Rivas et al., 2000). The DPPH radical is scavenged by antioxidants through the donation of hydrogen to form the stable reduced DPPH molecule. Thus, the antioxidant radicals are stabilized through the formation of non-radical products (Argolo et al., 2004). Pal Singh et al. (2009) showed that essential oil of Artemisia Scoparia, rich in citronellal (15.2%) and β-citronellol (11%), exhibited strong antioxidant and radical scavenging activity against hydroxyl ions (OH) and hydrogen peroxide (H₂O₂). These monoterpenes may be involved in LEO antioxidant activity.

We concluded that LEO possesses antinociceptive and anti-inflammatory properties, which are probably mediated via inhibition of prostaglandin synthesis, as well as a great antioxidant potential. Further studies will enable us to understand the precise mechanisms.

Declaration of interest

We are grateful to the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Fundação de Amparo à Pesquisa e à Inovação Tecnológica do Estado de Sergipe (FAPITEC-SE) and Fundação e Coordenação de Projetos, Pesquisas e Estudos Tecnológicos (COPPETEC) for fellowship support. Author Bárbara Lima Simeoni Leite has scholarships from Rede Nordeste de Biotecnologia (RENORBIO).