View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Repositório Institucional da Universidade Federal de Sergipe JOURNAL OF MEDICINAL FOOD J Med Food 14 (6) 2011, 630–639 # Mary Ann Liebert, Inc. and Korean Society of Food Science and Nutrition DOI: 10.1089=jmf.2010.0125 Antinociceptive Action and Redox Properties of Citronellal, an Essential Oil Present in Lemongrass Lucindo Quintans-Ju´nior,1 Ricardo Fagundes da Rocha,2 Fernanda Freitas Caregnato,2 Jose´ Claudio Fonseca Moreira,2 Francilene Amaral da Silva,1 Adriano Antunes de Souza Arau´jo,1 Joa˜o Paulo Almeida dos Santos,1 Moˆnica Santos Melo,1 Damia˜o Pergentino de Sousa,1 Leonardo Rigoldi Bonjardim,1 and Daniel Pens Gelain1,2 1Department of Physiology, Federal University of Sergipe, Aracaju, Sergipe, Brazil. 2Center for Studies in Oxidative Stress, Federal University of Rio Grande do Sul, Porto Alegre, Rio Grande do Sul, Brazil. ABSTRACT Citronellal (CT) is a monoterpenoid and the major constituent of the mixture of terpenoids that give the citronella oil its lemon scent. Citronella oil is widely used around the world for various purposes and is mainly obtained from plants of the Cymbopogon genus, which are known as ‘‘lemongrass.’’ Considering these plants have been used worldwide for various medicinal purposes, the interest of researchers to understand the biological activities of monoterpenoids related to the Cymbopogon genus has been increasing. In the present work, we investigated the antinociceptive action and the redox properties of CT. Our results indicate that intraperitoneal injection of CT was effective in reducing nociceptive face-rubbing behavior in both phases of the formalin test, which was also naloxone-sensitive. CT also evoked antinociceptive response in the capsaicin and glutamate tests. The total radical-trapping antioxidant parameter and total antioxidant reactivity assays indicate that CT at doses of 0.1 and 1 mg=mL exerts a significant antioxidant activity, which is probably related to its ability to scavenge superoxide and nitric oxide, but not H2O2 or hydroxyl radicals, as evaluated separately by specific in vitro tests. These results show for the first time the antinociceptive potential of CT and indicate that the antioxidant properties of this compound may rely on its mechanism of biological actions because CT-containing natural products are used to treat various diseases related to oxidative stress and reactive species. KEY WORDS: antinociception citronellal free radicals oxidative stress INTRODUCTION The essential oils are natural products that exhibit vari- ous biological properties, such as analgesic,7 anticonvul- pidemiological studies have been suggested an in- sant,8 and anxiolytic9 effects. Those effects are attributed to verse correlation between the consumption of certain E the monoterpenes, which are the major chemical compo- natural products and the risk of degenerative diseases, can- nents of these essential oils. Citronellal (CT) is a monoter- cer, cardiovascular diseases, and chronic pain.1–4 The pres- pene product from plant secondary metabolism. It is ence of secondary metabolites with antioxidant activity or typically isolated as a non-racemic mixture of its R and S other redox-related properties is generally associated with enantiomers by steam distillation or solvent extraction from such properties, as many of the conditions in which these the oils of Corymbia citriodora Hill and Johnson (former natural products have been observed to exert a preven- Eucalyptus citriodora Hook), Cymbopogon nardus, Cym- tive=therapeutic action are strongly related to oxidative bopogon citratus, and Cymbopogon winterianus (‘‘Java stress and increased reactive species production.5,6 The in- citronella’’).8,10 It is also found in more than 50 other es- crease in reactive species production may result from mul- sential oils. Plants from the Cymbopogon genus are known tiple endogenous (i.e., aging, mitochondrial metabolism) under various names, the most popular being ‘‘lemongrass’’ and exogenous (smoke, ultraviolet radiation, pollution) and ‘‘citronella.’’11 Cymbopogon leaves are used as infusion factors and induce oxidative damage to biomolecules such as preparations for use as spices, medicinal and recreational DNA, proteins, and lipids. teas, soups, and curries.11 Other CT-containing plants, such as members of the genuses Eucalyptus, Melissa, Mentha, Manuscript received 11 May 2010. Revision accepted 3 August 2010. Allium, and Cinnamomum, among others, also present no- table nutritional value and are important food=spice sources Address correspondence to: Daniel Pens Gelain, Ph.D., Departamento de Bioquı´mica, 12 Universidade Federal do Rio Grande do Sul, Rua Ramiro Barcelos, 2600, anexo, CEP in different parts of the world. In Cymbopogon (lemongrass) 90035-003, Porto Alegre, RS, Brazil, E-mail: [email protected] and Melissa officinalis (balm), CT is considered a major 630 PROPERTIES OF CITRONELLAL 631 constituent of the leaves, contributing to a great extent of the thiol), hydrogen peroxide, adrenaline, catalase, and aromatic scent of these plants. The use of lemongrass in superoxide dismutase (SOD) were purchased from Sigma curries in Asia and in spices in Africa is also related to Chemical Co. (St. Louis, MO, USA). Trolox (6-hydroxy- prevention of food spoilage, and the preservative properties 2,5,7,8-tetramethylchroman-2-carboxylic acid) was pur- of these plants are probably related to their high content of chased from Aldrich Chemical Co. (Milwaukee, WI, USA). essential oils.13–16 Nonetheless, isolated properties of the Water was purified using a Milli-QÒ system from Millipore different essential oil components of these medicinal plants, (Milford, MA, USA). All other reagents used in this study such as CT, have been poorly studied. were of highest analytical or high-performance liquid CT is produced by many plants traditionally used to treat chromatography grade. For all in vivo experiments the fol- different conditions by widespread populations around the lowing agents were used: CT [(RS)-(Æ)-CT, 98% purity, world, such as South America and Asia.17–19 Although CT Dierberger, Barra Bonito, Brazil], morphine hydrochloride has been identified, purified, and chemically well defined, its (Unia˜o Quı´mica, Sa˜o Paulo, Brazil), naloxone hydrochlo- biological and antioxidant properties have been very poorly ride (Neoquı´mica, Anapolis, Brazil), 37% formaldehyde explored. For instance, plants of the Cymbopogon genus, (Vetec, Rio de Janeiro, Brazil), Tween 80 (polyoxyethylene- which are rich in mono-, di-, and triterpenes, are widely used sorbitan monolate), and capsaicin (Sigma). Vehicle for an- to treat different forms of pain.20–24 Holanda Pinto et al.25 imals was 1 drop of Tween 80 (0.2%) dissolved in 0.9% demonstrated the analgesic effect of the triterpenoid a,b- NaCl solution. In those protocols the agents were injected amyrin, a compound closely related to CT. Also, CT and intraperitoneally at a dose volume of 1 mL=10 g. The related monoterpenoids were reported to inhibit cytochrome physiologic solution used for in vivo tests was composed of 26 P-450 CYP2B1 and other dehydrogenases, indicating that 150 mM NaCl, 4 mM KCl, 2 mM CaCl2,1mM MgCl2, such compounds may exert a diverse array of biological 10 mM glucose, and 10 mM HEPES, adjusted to pH 7.4 with actions. NaOH. Pain is a complex, multidimensional experience espe- cially relevant to the orofacial region because the face and Formalin test mouth have a particular biological and psychological Orofacial nociception was induced in mice by subcuta- meaning for each individual. Many of the most common neous injection of 20 mL of 2% formalin into the right upper types of pain occur in the orofacial region, and most diffi- lip (perinasal area), using a 27-gauge needle.27,29 This vol- culties in the management of conditions related to acute and ume and percentage concentration of formalin were selected chronic orofacial pain arise from a lack of understanding of from our pilot studies that showed a pain-related biphasic its mechanisms.27 Free radicals, especially nitric oxide behavioral response (face-rubbing) of great intensity at (NO), are also involved in the control of processes related to periods of 0–5 minutes (first phase) and 15–40 minutes pain because such species evoke cell signaling cascades (second phase). Pain was quantified at those periods by related to modulation of neuropathic and inflammatory measuring the time (in seconds) that the animals spent face- pain.28 In the present work, we investigated the anti- rubbing in the injected area with their fore- or hindpaws.27 nociceptive effect of CT and its redox properties. To assess the effects of test drugs, groups of rats (n ¼ 8 per group) were pretreated systemically with vehicle (1 drop of Tween 80 [0.2%] in distilled water, the solvent for CT) or CT MATERIALS AND METHODS (50, 100, and 200 mg=kg, i.p.), 0.5 hours before the local Animals injection of formalin. Morphine (3 mg=kg, i.p.), administered 30 minutes before the algogen, was included as the positive Male Swiss mice (weighing 30–36 g) and male Wistar control. In separate experiments, the possible involvement of rats (weighing 230–260 g), 2–3 months of age, were used opioid mechanism was assessed in the antinociception pro- throughout this study. The animals were randomly housed in duced by CT or morphine with naloxone (1.5 mg=kg, i.p.), an appropriate cages at 22 Æ 28C on a 12-hour light=dark cycle opioid antagonist, injected simultaneously. (lights on 6:00 a.m. to 6:00 p.m.) with free access to food and water. All experiments were carried out between 9:00 Capsaicin test a.m. and 2:00 p.m. in a quiet room. Experimental protocols were approved by the Animal Care and Use Committee The orofacial pain was induced by capsaicin administra- (CEPA=UFS number 12=08) at the Federal University of tion. Mice (n ¼ 8) were injected with capsaicin (20 mL, Sergipe, Aracaju, Brazil.