Doi:10.1016/j.jep.2003.09.007

Journal of Ethnopharmacology 89 (2003) 277–283 Analgesic and anti-inflammatory effects of essential oils of Eucalyptus Jeane Silva , Worku Abebe , S.M. Sousa , V.G. Duarte , M.I.L. Machado , F.J.A. Matos a Departamento de Biologia, Centro de Ciˆencias, Universidade Federal do Ceará, Fortaleza, Ceará, Brazil b Department of Oral Biology and Maxillofacial Pathology, School of Dentistry, CB-3710, Medical College Georgia, Augusta, GA 30912-1128, USA c Laboratório de Produtos Naturais, Centro de Ciˆencias, Universidade Federal do Ceará, Fortaleza, Ceará, Brazil Received 14 February 2003; received in revised form 26 August 2003; accepted 1 September 2003 Abstract
Many species of the genus Eucalyptus from the Myrtaceae family are used in Brazilian folk medicine for the treatment of various medical conditions such as cold, flue, fever, and bronchial infections. In the current investigation, we evaluated the analgesic and anti-inflammatoryeffects of essential oil extracts from three species of Eucalyptus employing various standard experimental test models. Using acetic acid-inducedwrithes in mice and hot plate thermal stimulation in rats, it was shown that the essential oils of Eucalyptus citriodora (EC), Eucalyptustereticornis (ET), and Eucalyptus globulus (EG) induced analgesic effects in both models, suggesting peripheral and central actions. Inaddition, essential oil extracts from the three Eucalyptus species produced anti-inflammatory effects, as demonstrated by inhibition of rat pawedema induced by carrageenan and dextran, neutrophil migration into rat peritoneal cavities induced by carrageenan, and vascular permeabilityinduced by carrageenan and histamine. However, no consistent results were observed for some of the parameters evaluated, both in terms ofactivities and dose–response relationships, reflecting the complex nature of the oil extracts and/or the assay systems used. Taken together, thedata suggest that essential oil extracts of EC, ET, and EG possess central and peripheral analgesic effects as well as neutrophil-dependent andindependent anti-inflammatory activities. These initial observations provide support for the reported use of the eucalyptus plant in Brazilianfolk medicine. Further investigation is warranted for possible development of new classes of analgesic and anti-inflammatory drugs fromcomponents of the essential oils of the Eucalyptus species.
2003 Elsevier Ireland Ltd. All rights reserved.
Keywords: Analgesic; Anti-inflammatory; Essential oils; Eucalyptus 1. Introduction
Phytochemical analysis has shownthat the profile of the monoterpenoids varies among the Eu- Many species of the genus Eucalyptus from the Myrtaceae calyptus species, with potential variations in medicinal prop- family are used in Brazilian folk medicine for a variety of erties. Eucalyptus citriodora has been shown to contain 60% medical conditions. For instance, hot water extracts of dried of the monoterpenoid, citronellal, whereas Eucalyptus tereti- leaves of Eucalyptus citriodora (EC) Hook are tradition- cornis (ET) and Eucalyptus globulus (EG) contain 60–90% ally used as analgesic, anti-inflammatory, and antipyretic of eucalyptol (1,8-cineole), another major monoterpenoid remedies for the symptoms of respiratory infections, such as cold, flue, and sinus congestion. Essential oils from Eu- calyptus species are also widely used in modern cosmetics, lyptol has been reported to inhibit the production/synthesis of tumor necrosis factor-␣, interleukin-1␤, leukotriene B4, In this regard, monoterpenoid components of the aro- and thromboxane B2 in inflammatory cells matic constituents of the oils are commercially available for These findings provide support at least for some the treatment of the common cold and other symptoms of of the traditionally accepted medicinal uses of eucalyptus in the Brazilian society, although the relationship may not bethat direct. The aim of the present study was to evaluate theanalgesic and anti-inflammatory effects of the essential oils ∗ Corresponding author. Tel.: +1-706-721-3181; fax: +1-706-721-6252.
of the Eucalyptus species, EC, ET, and EG, using various E-mail address: [email protected] (W. Abebe).
standard experimental test models. To our knowledge, this 1 Present address: Developmental Neurobiology, Institute of Molecular Medicine and Genetics, CA-4054, 1120 15th Street, Medical College of is the first attempt addressing such ethnopharmacological Georgia, Augusta, GA 30912-2640, USA.
properties of eucalyptus in a comprehensive manner.
0378-8741/$ – see front matter 2003 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.jep.2003.09.007 J. Silva et al. / Journal of Ethnopharmacology 89 (2003) 277–283 2. Materials and methods
30 min prior to the injection of acetic acid (0.6%, 10 ml/kg).
Acetylsalicylic acid is a well known peripheral analgesic drug and it was used as a positive control in the presentinvestigation. The mice were then placed in an observation Male Wistar rats (150–250 g), and either male or female box, and the number of writhes was counted for 20 min Swiss albino mice (20–25 g) were used. These animals were obtained from colonies maintained at the Departamentode Biologia, Centro de Ciˆencias, Universidade Federal do Ceará (Fortaleza, Brazil). The animals were housed in The hot plate assay method was employed for the purpose groups of 6–10 under environmentally controlled condi- of preferential assessment of possible centrally mediated tions with free access to water and standard food. Food was analgesic effects of the essential oils ( withheld overnight prior to experiments while water was The central analgesic drug, morphine, was used as still provided ad libitum. The handling and use of animals a positive control substance. For three consecutive days were in accordance to the institutional guidelines.
preceding the experiments, rats (200–250 g) were placedon a plate maintained at room temperature for 15 min each day. Essential oil extracts from EC, EG, or ET were givenby intraperitoneal injection at a dose of 10 or 100 mg/kg.
The following drugs and chemicals were used: morphine Morphine (10 mg/kg), and vehicle were also administered (Laboratório Enila Ltda., RJ, Brazil), acetylsalicylic acid in some animals by the same route. Each animal was then (Bristol-Mayers Squibb, Brazil), carrageenan (BDH, UK), placed gently on to a 55 ◦C hot plate. Latency to exhibit no- dextran 70 (Pharmacia, USA), histamine, prostaglandin I2, ciceptive responses, such as licking paws or jumping off the mepiramine (Sigma Chemical Co., MO, USA), dexametha- hot plate, was determined 15, 30, 45, 60, and 90 min after sone (MSD, Brazil), and Evans blue dye (Aldrich Chemical administration of the test substances or vehicle ( Co., USA). All other chemicals were of analytical grade.
2.3. Plant materials and preparation of essential oil 2.5.1. Inflammatory paw edema in rats Essential oils were extracted from samples of the aerial This assay was determined as described by parts of EC and ET, cultivated in the Medicinal Garden of Paw edema was induced by a single 0.1 ml sub- the Federal University of Ceará (Fortaleza, Brazil). The tax- plantar injection of carrageenan (200 ␮g/paw) or dextran onomic identity of the plants was confirmed by Professor (300 ␮g/paw), containing prostaglandin I2 (PGI2, 200 ng/ G. Fernandes, Department of Biology, Federal University paw), into the right hind paw of conscious rats (150–170 g).
of Ceará and voucher samples were deposited in the Prisco While carrageenan is known to result in at least neutrophil- Bezerra Herbarium No. 21 444 (EC) and No. 24 743 (ET), linked edematous inflammation, the effect of dextrane is as- Department of Biology, Federal University of Ceará, Fort- sociated with mast cell degranulation and is less related to aleza, Ceará, Brazil. In the extraction process, leaves were air-dried and ground into a fine powder. The oils were then ume was measured immediately before the injection of the “irritant” substance and at regular selected time intervals (1, extractor which provided a yield of 1%. The essential oils 2, 3, and 4 h) after injection of each of the Eucalyptus essen- of EG, which were also extracted as above providing a 1% tial oils (10 or 100 mg/kg) or equivalent volume of vehicle, yield, were generously provided by Dr. F.A. Matos of Lab- using a plethysmograph (model 7150, UGO Basile, Milan, oratorio de Produtos Naturais-UFC, Brazil.
Italy). Results were expressed as the increase in paw vol-ume (in ml) calculated after subtraction of basal paw volume 2.4.1. Acetic acid-induced writhing in mice 2.5.2. Neutrophil migration into peritoneal cavity of rats The writhing acetic acid test was performed in mice as Carrageenan diluted in sterile physiological buffer solu- tion (PBS) (400 ␮g/ml) was injected intraperitoneally into was used to preferentially evaluate possible peripheral ef- rats (150–170 g) 1 h after subcutaneous injection of EC, ET, fects of the essential oils as analgesic substances. Groups of or EG essential oils at a dose of 100 mg/kg each ( 10 mice were fasted overnight prior the start of the exper- Three hours later, the animals received an in- iment, while given free access to water. The essential oils jection of Evans blue dye (25 mg/kg in 2.5% PBS) and were of EC, ET, and EG (0.1, 10, and 100 mg/kg), acetylsalicylic immediately sacrificed. The peritoneal cavity was washed acid (250 mg/kg), or equivalent volumes of vehicle (0.9% with 10 ml of PBS containing 5 U/ml heparin. The lavage saline plus 4% Tween-20) were injected subcutaneously fluid was removed, and total and differential cell counts J. Silva et al. / Journal of Ethnopharmacology 89 (2003) 277–283 3. Results
results are expressed as the number of cells per milliliterof peritoneal wash fluid The steroidal 3.1. Analgesic effects of essential oils of Eucalyptus anti-inflammatory drug, dexamethasone (1 mg/kg, subcuta-neously), was used as a positive control for inhibiting neu- Intraperitoneal administration of essential oils of EC, ET, trophil migration. Control animals for basal value determi- and EG (0.1, 10, and 100 mg/kg each) significantly de- nation received equivalent volume of vehicle (4% Tween-20 creased the number of acetic acid-induced writhes in mice compared to the animals that received vehicle only (The writhe inhibitory effects of the oil extracts ranged from 2.5.3. Cutaneous vascular permeability in rats 43 to 73%. By comparison, 250 mg/kg acetylsalicylic acid The dorsal region of a rat was shaved and skin vascular produced nearly complete (i.e. 91% effectiveness) analge- permeability test was initiated by injection of carrageenan sia in this nociception model. While the analgesic effect in- (400 ␮g) or histamine (10 ␮g), together with 200 ng duced by the essential oil of ET was dose-related, the effects prostaglandin I2 in a total volume of 0.2 ml after 30 min produced by the oils of EC and EG were not. At the highest pretreatment with either essential oils (10 or 100 mg/kg), dose administered, EC was the most effective followed by dexamethasone (2 mg/kg), mepiramine (0.01 mg/kg) or ve- hicle (4% Tween-20 in PBS) alone administered per via Using the hot plate test, it was also shown that intraperi- intraperitoneal (In this regard, histamine tonial administration of EC, ET, or EG (10 and 100 mg/kg is different from carrageenan in that it has a more direct each) significantly prolonged the reaction time at several effect of causing vascular permeability. Dexamethasone time points after 30 min treatment, as compared to the cor- and mepiramine were used as positive controls for the in- responding control groups (These effects of es- hibition of the carrageenan and histamine-induced vascular sential oils were dose-independent. Although the oil of ET permeability, respectively. One hour after the injection of appeared to induce higher analgesic activity in most cases, inflammatory stimulus (carrageenan or histamine), each there was no consistent pattern of activity among the three rat received an intravenous injection of Evans blue dye essential oils (As expected, in this analgesic testing (25 mg/kg in 2.5% PBS). The animals were sacrificed 1 h model, morphine significantly prolonged the reaction time after the dye injection, and the skin was carefully dissected of the animals with relatively extended duration of stimula- to reveal the blue spots of the invaded dye. Each spot was tion, confirming centrally mediated activity.
excised, and the Evans blue dye was extracted from thetissue with formamide (2 ml per spot) overnight at room 3.2. Anti-inflammatory effects of essential oils of blue dye was assessed by spectrophotometry at 600 nm.
The results were expressed as Evans blue dye extracted per essential oils of EC, ET, and EG significantly reduced edemaof the rat hind paws induced by carrageenan and dextran1–4 h after administration, relative to control values. While, there was no clear difference in “edema-reducing” activityamong the three types of essential oils in most of the exper- Results are expressed as mean ± S.E.M. Statistical evalu- iments, the oil extracts from ET were more effective against ations were made using ANOVA or Student’s t-test, and val- dextran-induced paw edema compared to the other two ex- ues were considered significantly different when P < 0.05.
Table 1Analgesic effects of essential oils of EC, ET, and EG (0.1, 10, and 100 mg/kg each), and acetylsalicylic acid (250 mg/kg) on acetic acid-induced writhesin mice The data represent the mean ± S.E.M. of the number of animals in parentheses.
∗ P < 0.05 compared to control.
J. Silva et al. / Journal of Ethnopharmacology 89 (2003) 277–283 Table 2Analgesic effects of essential oils of EC, ET, and EG (10 and 100 mg/kg each), and morphine (10 mg/kg) on heat stimulation response in the hot platetest in rat The data represent the mean ± S.E.M. of the number of animals in parentheses.
∗ P < 0.05 compared to corresponding control.
Table 3Effect of essential oils of EC, ET, and EG (10 and 100 mg/kg each) on edema of rat hind paws induced by carrageenan (200 ␮g/paw) plus PGI2 (200 ng/paw) The data represent the mean ± S.E.M. of the number of animals in parentheses.
∗ P < 0.05 compared to corresponding control.
Subcutaneous administration of essential oils of EC, Pretreatment of rats with essential oils of ET and EG ET, or EG (100 mg/kg each) also significantly reduced significantly diminished vascular permeability induced by carrageenan-induced neutrophil migration into peritoneal carrageenan The effect of EG was, however, cavity of rats 3 h after injection Compared to the more pronounced than that of ET. Only the extract of ET effect of the positive control anti-inflammatory drug, dexam- produced dose-dependent inhibition of carrageenan-induced ethasone (1 mg/kg), which produced almost total inhibition vascular permeability (Compared to the effect of of neutophil migration, the essential oils caused inhibition dexamethasone (2 mg/kg), which produced an inhibition of by 70, 80, and 76%, respectively, relative to control.
70%, the vascular permeability inhibitions caused by ET Table 4Effect of essential oils of EC, ET, and EG (10 and 100 mg/kg each) on the edema of rat hind paws induced by dextran (300 ␮g/paw) plus PGI2 (200 ng/paw) The data represent the mean ± S.E.M. of the number of animals in parentheses.
∗ P < 0.05 compared to corresponding control.
J. Silva et al. / Journal of Ethnopharmacology 89 (2003) 277–283 However, the effect of the oil extract of EC was greater than Effect of essential oils of EC, ET, and EG (100 mg/kg each), and dex- that of ET. ET, but not EC, produced a dose-dependent ef- amethasone (1 mg/kg) on carrageenan-induced neutrophil migration into fect. On the other hand, essential oils of EG had no effect on histamine-induced vascular permeability The his- tamine receptor antagonist drug, mepiramine (0.01 mg/kg), which was used as a positive control, elicited marked reduc- tion (i.e. by 61%) of the vascular permeability caused by histamine, and by comparison only 10 mg/kg EC generated The data represent the mean ± S.E.M. of the number of animals inparentheses.
4. Discussion and conclusion
∗ P < 0.05 compared to control.
Previously, it was determined that the lethal dose 50 Effect of essential oils of EC, ET, and EG (10 and 100 mg/kg each), and 50) values of essential oil extracts from EC, ET, and EG dexamethasone (2 mg/kg) on Evans blue dye extravasation induced by were 190 ± 22 mg/kg, 240 ± 34 mg/kg, and 353 ± 64 mg/kg subcutaneous injection of carrageenan (400 ␮g per site) (n = 10), respectively, in mice. Therefore, the dosages of the eucalyptus oils used in the present study (i.e. 0.1, 10, and 100 mg/kg) were far below the LD50 values. Conse- quently, no apparent behavioral side effects were observedin the animals during our experimentation. The high LD values also suggest that the oil extracts were relatively safe Two different analgesic testing methods were employed in the current investigation with the objective of identify- ing possible peripheral and central effects of the test sub- stances. Using both acetic acid-induced writhes and hot plate thermal stimulation, it was observed that the essential The data represent the mean ± S.E.M. of the number of animals in oils of EC, ET, and EG possessed analgesic effects against both models. This observation indicates that these eucalyp- ∗ P < 0.05 compared to control.
tus oils have both peripheral (writhe reduction) and central(thermal reaction time prolongation) effects. Among the oils and EG ranged from 24 to 43% (By contrast, tested, extracts from EC demonstrated the highest peripheral the essential oil of EC was without significant effect on anti-nociceptive activity, while extracts from ET were the carrageenan-induced vascular permeability, although at a most potent central anti-nociceptive substance. On the other relatively high dose it tended to produce some inhibition.
hand, while both of these oil extracts induced moderate ef- ws that essential oils of EC and ET signifi- fects in reverse manners to the above, the essential oils of EG cantly inhibited vascular permeability induced by histamine.
appeared to be the least effective in both animal models ofnociception. Taken together, the data presented demonstrate that the essential oils of eucalyptus induce variable degrees Effect of essential oils of EC, ET, and EG (10 and 100 mg/kg each), and of peripheral and central analgesic effects depending upon mepiramine (0.01 mg/kg) on Evans blue dye extravagation induced by the Eucalyptus species where they are obtained from. These subcutaneous injection of histamine (10 ␮g per site) observations can provide useful information if a choice is desired to be made regarding the species of eucalyptus as a From the present work alone, the mechanism(s) of the analgesic effects of the eucalyptus oils tested is not readily apparent. It can, however, be speculated that it may be linked to processes involved in the prevention of sensitization of the nociceptor, down-regulation of the sensitized nocicep- tor and/or blockade of the nociceptor at peripheral and/orcentral levels (One of the well character- ized signaling systems believed to participate in this mech-anism(s) is the arachidonic acid metabolic pathway. In this The data represent the mean ± S.E.M. of the number of animals in regard, it has been previously shown that at least eucalyp- ∗ P < 0.05 compared to control.
tol, one of the major components of eucalyptus oils, inhibits J. Silva et al. / Journal of Ethnopharmacology 89 (2003) 277–283 the production of prostaglandins and thromboxanes similar factory explanations. However, as suggested by this problem may be related, at least in part, to the The anti-inflammatory properties of the eucalyptus oils presence of different chemical components in the extracts.
were studied using several standard pharmacological meth- It is possible that these components can vary in kind as well ods. The results generally reveal that the essential oils of EC, as concentrations. This in turn may result in the manifes- ET, and EG elicited anti-inflammatory activities of different tation of divergent intrinsic activities, depending upon the intensities, as reflected by inhibition of rat paw edema caused type of active ingredients present, and/or different magni- by either carrageenan or dextran, carrageenan-induced neu- tudes of responses, depending upon the concentrations of trophil migration into rat peritoneal cavity, and vascular the active ingredients. The presence of multiple chemicals in permeability induced by carrageenan or histamine. Of the a given extract can also interfere with the pharmacokinetic three eucalyptus oils, ET clearly demonstrated the highest and pharmacodynamic properties of the individual active in- anti-edematogenic activity against edema induced by dex- tran. These data suggest that the anti-edematogenic effect shading light into this problem of crude extract application of the oils of ET has a strong non-neutrophil-mediated is the determination of the composition of the extracts. Such component as compared to the effects of the other two an effort can facilitate a better assessment whether or not the extracts of eucalyptus oils. On the other hand, the fact effects observed with the oil extracts are related to the pres- that all the essential oil extracts effectively inhibited neu- ence of different components or different concentrations of trophil migration as well as carrageenan-induced edema the same active ingredients. In this regard, while the present with similar efficacies provides evidence for the impor- investigation is meant to serve as a preliminary observation, tant roles that neutrophil-linked mechanisms play in the future studies should consider the determination of the com- anti-inflammatory action of the essential oils from the three position of the individual oil extracts and evaluation of the Eucalyptus species. In addition, while the observed effects pharmacology of each active ingredient. It should also be of the eucalyptus oils on carrageenan and histamine-induced recognized that the use of different experimental models can vascular permeability can provide additional support for also play an important role in influencing the effects of the their anti-inflammatory activities, the lack of effect of ex- tracts of either EC (in carrageenan-induced permeability) In the present study, doses of oil extracts ranging from or EG (in histamine-induced permeability) on these inflam- 0.1 to 100 mg/kg were used. These doses were chosen on matory reaction models is hard to explain. It is, however, the basis of effectiveness and the LD50 values determined possible that this could be a reflection of differences in the previously. Doses lower than the minimum indicated for a mechanisms of vascular inflammatory processes caused by particular type of experiment did not usually produce mea- carrageenan and histamine, which can be differentially af- surable responses and doses higher than 100 mg/kg were not fected by the essential oils. Alternatively, this may indicate desired to be used due to questionable practical significance possible differences in the mechanisms of actions of the and closeness to the LD50 values. Compared to the assumed essential oils and/or other components present in the two doses of essential oil extracts used by patients in traditional therapy (i.e. in the form of hot water infusion), the doses of As with the analgesic effects, the exact mechanism(s) of the extracts we used in the present experiments appear to be the anti-inflammatory properties of the essential oils used higher, although one cannot be sure about it. However, such in the present study is unclear. However, other investigators variations in doses of drugs used in animal experiments and have previously reported that the monoterpene components in humans are not unexpected, in which case the doses are of the oils of eucalyptus, such as eucalyptol, are potent in- usually higher in animal studies. In this respect, animal stud- hibitors of the inflammatory mediators, cytokines ( ies are considered useful in providing clues about what may Furthermore, the production of leukotriene happen in humans even at different doses. Based on the out- B2, prostaglandin E2, and other arachidonic acid metabo- comes from such studies, further investigation can then be lites in human monocytes was also shown to be inhibited pursued. It is believed that the results presented in this com- munication have the potential to lead to this development.
ments indicating that monoterpenes have secretolytic proper- In conclusion, the results reported in this communication ties against several inflammatory mediators demonstrate that the essential oils of EC, ET, and EG inhibit These reports imply that the essential oil extracts peripherally and centrally mediated nociception as well as from the Eucalyptus species we used can be associated with neutrophil-dependent and independent inflammatory reac- anti-inflammatory properties at least due to the presence of tions. The variations in biological activities observed could be related, at least in part, to differences in the types and Although attempts have been made to speculate the pos- amounts of the components of the oils and/or experimental sible mechanisms for the analgesic and anti-inflammatory models used, as documented in the literature. Overall, our effects of the eucalyptus oil extracts based on the literature, data provide support for the popular use of the eucalyptus our observation of inconsistent or multiphasic activities in plant in Brazilian folk medicine for pain, and for respira- some situations is difficult to be provided with any satis- tory and other inflammatory conditions. Thus, the present J. Silva et al. / Journal of Ethnopharmacology 89 (2003) 277–283 study warrants further investigations involving components Flores, C.A., Zappellini, A., Prado-Franceschi, J., 1993. Lipoxygenase- of essential oil extracts from various species of eucalyp- derived mediators may be involved in in vitro neutrophil migrationinduced by Brothrops erythromelas and Brothrops alternatus venoms.
tus for possible development new class of analgesic and Gomes-Carneiro, M.R., Felzenszwalb, I., Paumgartten, F.J., 1998. Mu- tagenicity testing (+/−)-camphor, 1,8-cineole, citral, citronellal, (−)-menthol and terpineol with the Salmonella/microsome assay. Mutation Acknowledgements
Jager, W., Nasel, B., Nasel, C., Binder, R., Stimpfl, T., Vycudilik, W., The authors are grateful to Dr. Ana Maria Sampaio As- Buchbauer, G., 1996. Pharmacokinetic studies of the fragrance com-pound 1,8-cineol in humans during inhalation. Chemical Senses 21, sreuy (Department of Physiology and Pharmacology) Dr.
Vˆania Maria Maciel Melo and Dr. Ana de Fatima Urano Juergens, U.R., Stober, M., Vetter, H., 1998a. Inhibition of cytokine Fonteles (Department of Biology, Federal University of production and arachidonic acid metabolism by eucalyptol (1,8-cineole) Ceará), and Dr. Carlos A. Flores for their advice and con- in human blood monocytes in vitro. European Journal of Medical structive criticism in the preparation of the manuscript. This Juergens, U.R., Stober, M., Schmidt-Schilling, L., Kleuver, T., Vetter, work was supported by Fundação Cearense de Amparo H., 1998b. Antiinflammatory effects of eucalyptol (1,8-cineole) in à Pesquisa (FUCAP) and Universidade Federal do Ceará, bronchial asthma: inhibition of arachidonic acid metabolism in human blood monocytes ex vivo. European Journal of Medical Research 3,407–412.
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