Anti-Inflammatory and Antimicrobial Effects of Eucalyptus spp. Essential Oils: A Potential Valuable Use for an Industry Byproduct

In Brazil, the use of Eucalyptus is focused on the production of wood or pulp for the paper industry but without any general recovery of waste, with leaves and branches being left on the ground. One possibility is to use these residues as raw materials in the production of industrially relevant and value-added compounds such as essential oil. The aim of the present study was to investigate the chemical composition, yield, anti-inflammatory/antinociceptive activities, and acute toxicity in mice, as well as the antimicrobial effects of essential oils from the leaves of 7 varieties of Eucalyptus and hybrids against Escherichia coli, Staphylococcus aureus, and Candida albicans. The extraction of oils was carried out using hydrodistillation, and they were analyzed by gas chromatography coupled to mass spectrometry. Urocam and Grancam were the plants that obtained the highest oil yield, with yields of 3.32 and 2.30%, respectively. The main chemical components identified in these plants were 1.8 cineole and α-pinene. The antinociceptive effect of the 7 oils (50 mg/kg, p.o.) was initially assessed in the acetic acid-induced writhing test. In this assay, a significant (p < 0.05) antinociceptive/anti-inflammatory effect was observed from 4 tested essential oils (E. benthamii, E. saligna, and the hybrids Urocam and Grancam) when compared to the vehicle-treated group. This effect was then confirmed in the formalin-induced paw licking test. No toxicological effects or alterations were observed in motor coordination after the administration of the studied oils to the animals. In the antimicrobial evaluation, the seven essential oils inhibited the growth of S. aureus, E. coli, and C. albicans at different concentrations. Collectively, these results demonstrate that the essential oil from the leaves and branches of Eucalyptus species and varieties present potential biomedical applications and represent a source of antimicrobial and/or anti-inflammatory compounds.


Introduction
Eucalyptus species (Myrtaceae) are native to Australia, New Zealand, and Tasmania [1] and are well known for their economic importance in wood and paper production (forest industry) [2,3]. Eucalyptus plantations occupy 5.7 million hectares of planted trees in Brazil [4], and the main destination of these plants is the forestry industry, generating economic benefts but, on the other hand, millions of tons of waste, which result in considerable costs for the industry and the environment [3].
In Brazil, the use of Eucalyptus is focused on the production of wood or pulp for the paper industry [5], but without any general recovery of waste, with leaves and branches being left on the ground. One possibility is to use these residues as raw materials in the production of industrially relevant and value-added compounds such as essential oil [6]. In fact, Eucalyptus essential oil is widely used in industry, especially in pharmaceuticals and perfumery. Eucalyptus is a heteroblastic species, and genetic variations have been detected both within and between populations at the juvenile coppice and adult leaf stages, suggesting that the populations may involve quite diferent ontogenetic trajectories [7].
Currently, nonsteroidal anti-infammatory drugs (NSAIDs) and glucocorticoids are the frst-line drugs used to reduce the harmful events associated with infammation [13]. Tese drugs exhibit important side efects, ranging from gastric irritation and ulcers to liver toxicity and chronic renal failure [14]. Te scarce options for safe drugs for the treatment of chronic infammatory diseases, such as arthritis, have led to the discovery of new medicinal agents derived from plants [15]. Tus, the disadvantages of using NSAIDs can be minimized by replacing them with safer and more efcient compounds derived from medicinal plants [15].
Essential oils from Eucalyptus species have been reported to have antimicrobial activity against Gram-negative (Escherichia coli) and Gram-positive (Staphylococcus aureus) bacteria, indicating that the oil can be studied as a possible natural antibiotic for the treatment of various infectious diseases caused by these two bacteria [16]. Such investigations suggest that Eucalyptus major compound, 1,8cineole, may be responsible for this action, highlighting the importance of the discovery of new substances with antimicrobial potential. Microbial resistance has been growing and rendering treatment of infections difcult with the therapeutic options available; therefore, the research and development of new substances with antimicrobial potential are of primary importance [17].
Literature appraisal on the subject demonstrates the importance of preclinical investigation of the possible anti-infammatory, antinociceptive, and antimicrobial action of diferent essential oils from the leaves of Eucalyptus species ( Urocam − E. urophylla × E. camaldulensis), which are abundantly cultivated in Brazil and had never been studied regarding these biological activities [18].
Considering the abovementioned scenario, the exploration of the biological and chemical properties of plant residues from the paper industry could represent a valuable approach for these by-products. Terefore, the aim of this study was to evaluate the antinociceptive, antiinfammatory, and antimicrobial properties of the essential oils extracted from the leaves of seven varieties of Eucalyptus, as well as to determine their yield and chemical composition.  [19]. Te yield % (wt/wt, based on the fresh weight of the mature leaves) of the extracted oil from each sample was determined by the following equation:

Materials and Methods
Yield of essential oil (%) � Weight of oil Weight of Eucalyptus leaves * 100. (1) Chemical composition of the essential oil samples was determined by gas chromatography using an Agilent GC/MS (7890B) coupled to a quadripolar mass spectrometer (5977A) (Agilent Technologies, Palo Alto, CA, USA). A HP-SMS 5% Phenyl Methyl Silox capillary column (30 m × 250 μm × 0.25 μm) connected to a fame ionization detector (FID) was used with mobile phase fow (carrier gas: He) adjusted to 1.0 mL·min −1 . Te GC temperature program was 60°C at 4.0 min, then up to 240°C at a rate of 10°C per min, then to 300°C at a rate of 40°C per min (maintained for 5 min). Te injector temperature was 280°C. Oil samples were diluted with methanol.
For GC/MS detection, an electron ionization system was used with ionization energy set at 70 eV and mass range m/z 40-300. Te chemical components were detected and identifed by comparison of the mass spectra using the NIST 5.01 Mass Spectral Library (Agilent P/N G1033A) and comparing retention times; homologous C8-C30 n-alkane series by determining the linear retention index (LRI) according to Van Den Dool and Kratz [20]. Te relative amounts of individual components were performed by integrating the peak in the FID chromatogram and expressed as percent of area.  [25]. Te protocols were approved by the Ethics Committee of the Community University of Chapecó Region (approval number 005/2020).

Acetic Acid-Induced Abdominal Writhing
Test. In this model, abdominal writhing was induced in mice according to the procedure described by Santos et al. [26] and adapted by our research group [27]. Contractions of the abdominal muscles together with the extension of one of the hind legs occur in response to an intraperitoneal (i.p.) injection of 0.6% acetic acid (10 mL/kg). Te animals received vehicle, indomethacin (10 mg/kg -positive control [28]), or 50 mg/kg of the oils orally (p.o.) 1 h before the exposure to the injection of acetic acid. Before acetic acid administration, the animals remained in the transparent observation chamber for 20 min for recognition and adaptation to the site. Immediately after administration, abdominal contortions were quantifed for 20 min. Te oils that presented better efectiveness for the follow-up of the next behavioral trials were selected.

Formalin-Induced Paw Licking Test.
Tis assay allows evaluating both neurogenic and infammatory pain: direct stimulation of nociceptive fbers happens at frst (1 st phase) and the infammatory reaction (characterized by the release of infammatory mediators) in a second moment (2 nd phase) [29]. Te test was performed as previously described by Santos and Calixto [30] with minor modifcations [27]: infammation induction occurred by intraplantar (i.pl.) administration of 1% formalin (20 μL) in the dorsal region of the right hind paw of mice. Tey were treated orally with vehicle, indomethacin (10 mg/kg -positive control [23]), or the oils under study 1 h before the exposure to formalin. Before formalin administration, the animals remained in the transparent observation chamber for 20 min for recognition and adaptation to the site. Immediately after formalin administration, mice were observed for 30 min: the number of paw elevations was quantifed in the frst 5 min and in the last 15 min. It was considered as paw elevation behavior any movement not associated with locomotion, ranging from a discrete elevation or contraction of the muscles of the animal's thigh to vigorous movement or licking and biting the paw.  [31] in order to verify a possible nonspecifc efect of the oils on the spontaneous exploratory and locomotor activities of the animals, which may infuence the results regarding the antinociceptive/anti-infammatory activity. Mice were treated orally with vehicle or the oils that showed positive results in the nociception/infammation tests. After 60 min, they were placed in the center of a black waterproofed MDF box (40 × 30 × 30 cm) with its bottom divided in 24 equal squares. After 5 min of familiarization, the number of crossings, rearing, and fecal bolus were quantifed for 10 min.

Rotarod Test.
Tis model was carried out to verify the efects of the oils on motor coordination of the animals, as described by Neves et al. [32]. Te rotarod device consisted of a cylinder (6 cm of diameter) rotating at 6.5 rpm. Initially, mice were trained to balance on the device for 5 min, including falls. Twenty-four hours later, the animals were submitted to a baseline responsiveness determination, when only the ones that presented a minimum continuous permanence time of 90 s were considered able to continue the test. Immediately after this session, the selected animals were treated orally with vehicle or the oils. One hour later, the test was performed and the longer permanence time (seconds) and number of falls within the observation period (5 min) were recorded.

Acute Toxicity.
Te acute oral toxicity test was carried out following Guideline 423 from the Organization for Economic Cooperation and Development [33]. Nonpregnant female mice were treated with vehicle (n � 3, control group) or the Eucalyptus essential oils that showed signifcant results in the nociception/infammation assays, at 2000 mg/kg (n � 6). Teir behavior was observed individually with special attention in the frst 4 h and 12 h after the treatment with regard to the following occurrence of: piloerection, palpebral ptosis, abdominal contortions, changes in locomotion, hypothermia, increased muscular tonus, shaking, posterior paws stoppage, salivation, bronchial secretion, convulsions, and deaths. Te animals' body weight, food intake, and deaths were registered every 2 days. On the 15 th day after the treatment, they were euthanized by inhalation of isofurane (dose of 10-12% in inhaled air) and confrmation by cervical displacement [34]; CONCEA [35] and the organs (liver, kidneys, adrenal glands, spleen, lungs, heart, and brain) relative weight (%) and macroscopic aspect were registered.

Yield and Chemical Analyses of Essential Oils.
Te yield and compounds identifed in the essential oils with their respective concentrations are shown in Table 1. Te essential oil yield of the diferent varieties of Eucalyptus was in the range of 0.77-3.32%, in agreement with other works reported in the literature [40]. Knowledge of the essential oil yield is important for both commercial purposes and scientifc research since it can afect the availability and economic viability of Eucalyptus essential oil production [41]. Te plants that showed the greatest potential were Urcan and Grancan varieties, with yields of 3.32 and 2.30%, respectively. It can also be seen from Table 1 that 1,8-cineole (eucalyptol) is present in all species studied except for E. benthamii. Note that the hybrid Urocam presented the highest concentration of this compound (82.18%), which makes this plant a potential source for this compound. Besides being a biologically active compound, it has a high therapeutic index and antiseptic properties [42]. According to Caceres et al. [43], eucalyptol inhibits the arachidonic acid pathway, thus inhibiting infammatory mediators, and also 1,8-cineole and glucocorticoids have a mechanism of inhibiting infammatory mediators in common.
Except for Grancam, α-pinene was also abundant in all Eucalyptus varieties studied. Such a compound has been considered an anti-infammatory agent, mainly in osteoarthritis, besides presenting antioxidant and antiallergic activities and antimicrobial efectiveness against some strains of S. aureus and methicillin-resistant Staphylococcus aureus, among others [44,45].
Another pinene that is part of the chemical composition of Eucalyptus essential oils is β-pinene, which presents biological activities such as anti-infammatory, antidepressant, anxiolytic, hypotensive, antimicrobial, and myorelaxant, among others [46]. A high concentration of p-cymene in Eucalyptus essential oil was reported by Almeida et al. [47], with possible antiseptic activity that inhibited the growth of S. aureus and E. coli.
c-Terpinene has been reported to present signifcant antimicrobial activity [48] and bacteriostatic activity against some microorganisms has been attributed to terpineol [49]. Te Benthamii plant presented globulol as one of the major components (18.01%); this sesquiterpene has antifungal properties against a variety of fungal species as well as bacteria [50] and antimicrobial and antioxidant activities [51].
D-Limonene, a monoterpene found in several essential oils of aromatic species, was observed in 5 of the 7 Eucalyptus varieties studied, with concentrations ranging from 3.23 to 10.56%. D-limonene is a multifunctional compound with a potent therapeutic efect, widely used in the food and pharmaceutical industries [52,53].
Overall, this study provides valuable information about the chemical composition of essential oils from the seven varieties of Eucalyptus cultivated in Brazil, which may explain the results of anti-infammatory and antimicrobial activity obtained in this work and may have important applications in several industries, including pharmaceutical, cosmetic, and food. It is known that 1,8-cineole presents anti-infammatory and antinociceptive properties, while α-pinene shows antiinfammatory and antiallergic activities [45,54]. Results reported in this work agree with other studies that demonstrated that Eucalyptus essential oil reduced the number of abdominal writhing, similarly to the positive control used (indomethacin) [55], which may be related to the presence of 1,8-cineole and α-pinene in the essential oils.

Antinociceptive and Anti-Infammatory
Based on the chemical composition of the oils investigated, the following may be pointed out as the most efcient with regard to pharmacological efect: Urocam

Formalin-Induced Paw Licking Test.
In order to confrm the antinociceptive and/or anti-infammatory activities of Eucalyptus essential oils, the ones that presented the best results on acetic acid-induced abdominal writhing assays were also tested in the formalin-induced paw licking test. Tis assay consists of two phases: the frst one (0-5 min) corresponds to neurogenic pain, and the second one (15-30 min) is related to the infammatory response. Neurogenic pain occurs in response to the direct stimulation of nociceptors by formalin, which results in the release of other mediators that perform local responses, causing vasodilation, activation of synaptic fbers, and leukocyte chemotaxis. In the infammatory response induced by formalin, there is the release of infammatory mediators that end up activating the infammatory cascade and sensitizing nociceptive pathways [56]. Both phases can be inhibited by centralacting drugs, such as opioids, while NSAIDs are more effective in preventing infammatory pain [57]. Figure 2 represents the efects of E. benthamii, E. saligna, Urocam, and Grancam, tested in the formalin test. In the frst phase (Figure 2(a)), the time that the animals spent licking the paw was signifcantly (F (5, 42) � 32.60, P < 0.0001) reduced by the essential oils of the four species when compared to the vehicle-treated group, as well as the animals treated with indomethacin.
Te short nociception time in the frst phase may indicate that the antinociceptive efect had the involvement of the opioid pathway, since it is known that some compounds of the oils, such as β-pinene and p-cymene, present an antinociceptive efect acting as a partial agonist of μ-opioid receptors [58,59]. LR-N-bookstore linear retention index national institute of standards and technology, RT-retention time, and RI-E-experimental retention index.

Evidence-Based Complementary and Alternative Medicine
In the second phase of the test (Figure 2(b)), when the anti-infammatory response of Eucalyptus essential oils tested were evaluated, and there was also a signifcant (F (5, 42) � 24.12, P < 000.1) reduction in paw licking time for all oils tested as well as indomethacin when compared to the vehicle-treated group.
Te results of this test indicate that the essential oils of the leaves of the species E. benthamii, E. saligna, Urocam, and Grancam present antinociceptive action with central and peripheral efects by responding to neurogenic and infammatory pain caused by formalin injection. Tis is probably related to the major compounds of the essential oils studied, 1,8-cineole, p-cymene, and α-pinene, which present anti-infammatory and antinociceptive activities [43,59].
Te compound 1,8-cineole inhibits cyclooxygenase enzymes, resulting in the decrease on the production of TNF-α, leukotriene B4, and thromboxane A2, and it is also a potent inhibitor of cytokines [43]. Te study of Santos and Rao [60] corroborates such facts: in the carrageenan test, which induces paw edema, 1,8-cineole at 400 mg/kg (p.o.) inhibited the edema, and, according to the authors, it indicates a decrease in the production of infammatory mediators. In addition, there was also a reduction in the nociception time in both phases of the formalin test at the same dose of 1,8cineole.
α-Pinene, when investigated through the carrageenaninduced paw edema test, inhibited the edema and also decreased leukocyte migration at 200 and 400 mg/kg [45]. In the study by Lee et al. [55], Eucalyptus essential oil at 45 mg/ kg p.o. signifcantly reduced the nociception time only in the 2 nd phase of the formalin test.

Open Field Test.
Te open feld test was carried out to exclude the possibility that the results of the behavioral assays of nociception and infammation were related to a nonspecifc efect of the essential oils of E. benthamii, E. saligna, Urocam, and Grancam on spontaneous exploratory and locomotor activities of the animals. Figure 3 illustrates the number of crossings of the animals treated with the essential oils. Tere was a signifcant reduction in the number of crossings (Figure 3(a)) of animals treated with all oils studied (F (5, 42) � 18.03, P < 000.1). Also, according to Figure 3(b), mice treated with E. benthamii, Urocam, and Grancam (F (5, 42) � 6.53, P � 0.0004) showed a signifcant reduction in the number of rearing compared to the vehicle-treated group. On the one hand, substances that increase the number of crossings and rearing are considered as stimulants [61]. On the other hand, a reduction in these parameters, as caused by the oils under study, is indicative of central depressant efects [61]. In line with our observations, Santos and Rao [60] demonstrated that 1,8-cineole caused a reduction in the locomotion of the animals in the open feld test, hence suggesting a sedative efect. Furthermore, it is known that the number of crossings can be afected by central-acting drugs or peripheral muscle relaxers [62]; the number of rearing assesses the degree of sedation and anxiety and can be altered by drugs with anxiolytic/anxiogenic activity; and the number of fecal bolus can be altered by anxiolytic, anxiogenic, spasmolytic, or spasmogenic drugs [63].
In Figure 3(c), the number of fecal bolus is depicted: the animals treated with Grancam showed a signifcant (F (5, 42) � 2.767, P � 0.038) reduction in the number of fecal bolus expelled compared to the group treated with vehicle. Te reduction in the number of fecal bolus may be related to the involvement of the opioid receptors in the mechanism of action of this oil since opioid drugs present regularly reported peripheral side efects such as constipation, urinary retention, and pruritus [64].
Te satisfactory antinociceptive activity as well as the constipation and sedative efects are qualities of opioid drugs [65]. Indeed, the chemical composition of the oils tested demonstrated the presence of some compounds that act as opioid agonists, such as β-pinene [58]. Terefore, it can be suggested that the oils tested are very efcient in the pain management process, perhaps with theinvolvement of the opioid pathway. However, their mechanism of antinociceptive action must be elucidated.   Table 2 shows the results of the permanence time and number of falls of the animals treated with the oils in the rotarod test. Tere was no signifcant diference between the parameters evaluated between the groups. Tus, these results indicate that the essential oils of E. benthamii, E. saligna, Urocam, and Grancam did not alter the integrity of the motor coordination of the animals. Sobreira et al. [66] also noted that 1,8-cineole, the major component of Eucalyptus Evidence-Based Complementary and Alternative Medicine essential oil, did not cause changes in the motor coordination of the animals in the rotarod test, therefore in agreement with the results of this study.

Acute Toxicity.
In the acute toxicity assay, the selected essential oils were administered at a single dose of 2000 mg/ kg (p.o.). Figures 4(a) and 4(b) present the relative body weight and food intake of the animals, respectively. A two-way RM ANOVA revealed a signifcant efect of day (F (5, 125) � 56.77 , P < 0.0001])and a signifcant interaction between factors (day x treatment) (F (20, 125) � 7.943, P < 0.0001) on the relative body weight of animals. No efect of treatment was detected on mice relative body weight (F (4, 25) � 0.7158, P � 0.589).
Regarding food intake, a two-way RM ANOVA revealed signifcant efect of day (F (4, 100) � 656.8 , P < 0.0001), treatment (F (4, 25) � 4.94, P � 0.0045) and a signifcant interaction between factors (day × treatment) (F (16, 100) � 49.20, P < 0.0001). Posthoc analysis revealed that on the 3 rd day of the experiment, a signifcant decrease in body weight (P < 0.01 for E. benthamii and P < 0.001 for the other ones) and a signifcant increase in food intake (P < 0.001) were observed in the groups treated with Eucalyptus species when compared to the vehicle-treated group. At the last experimental day, the animals treated with Eucalyptus ingested signifcantly (P < 0.001) less food and their body weight signifcantly increased (P < 0.01 for Urocam and P < 0.001 for the other ones) in comparison with the vehicletreated group.
Tese data suggest that Eucalyptus essential oils present a metabolic side efect. On the 15 th day of observation, when the animals treated with the oils ingested less food and recovered their body weight, it was likely that the efect of Eucalyptus essential oils on metabolism had stopped [66,67]. Some tests on the toxicity of Eucalyptus essential oils in rats showed that their median lethal dose (LD50) was 4,440 mg/ kg, and their major oil compound, 1,8-cineole, had a LD50 of 2,480 mg/kg. Tests on mammals such as koalas demonstrated a certain toxicity of the Eucalyptus oils tested [68]. In addition, there was no diference in both the body weight of animals and organs compared to the vehicle-treated group when isolated 1,8-cineole was tested [69].
Te efect of essential oils on the weight of animals' organs is shown in Figure 5. Te treatments with all studied essential oils signifcantly reduced the relative weight of spleen (F (4, 25) � 7.024, P � 0.0006) (Figure 5(a)) and heart (F (4, 25) � 6.205, P � 0.0013) ( Figure 5(b)). Tere were no efects of the essential oils on the relative weight of mice's adrenals (F (4, 25) � 1.347, P � 0.280) ( Figure 5 Unlike the results found here, there are no reports of splenic and cardiac toxicity related to Eucalyptus essential oils in the literature. Furthermore, the reduction in the proportion of cardiac weight has also not been described for Eucalyptus species and; therefore, there is no conclusive cause of the reason why this efect was observed. Te results of acute toxicity need to be confrmed in repeated dose toxicity studies, with the use of extrapolated working doses, as recommended by the OECD [33]. Anyway, the treatment with the Eucalyptus oils studied did not cause any toxicity sign or death during the study period. Tus, according to Guideline 423 [33], they can be classifed in category 5 of the Globally Harmonized System (LD50 is > 2000 mg/kg-5000 mg/kg for nonvulnerable populations).

Antimicrobial Assay.
Te seven oils demonstrated activity against S. aureus (Gram-positive bacteria) and E. coli (Gram-negative bacteria) (Table 3). Besides, they also presented activity against the fungus C. albicans, as shown in Table 4. Estanislau et al. [70] evaluated the antimicrobial activity of 5 Eucalyptus species and also obtained promising results against S. aureus and E. coli for all oils tested. However, the technique used by them was the disk difusion method; therefore. it is not possible to advance in this discussion.
Additionally, E. dunnii and Grancam had the best performance in MIC and MMC assays for the two bacteria under study. Te 2 species have diferent concentrations of active substances: E. dunnii presents 8.55% α-pinene and 63.01% of 1,8-cineole, while Grancam presents 35.92% of α-pinene and 38.43% of 1,8-cineole. Tese compounds are responsible for the main antimicrobial efectiveness against some strains of S. aureus and have high therapeutic indexes [42,44]. E. dunnii and Grancam present 8.42% and 2.62% of terpineol, respectively, which is responsible for bacteriostatic action [49].
Te compound D-limonene is present in E. dunnii (3.52%) and in Grancam (10.52%) and possesses antimicrobial activity [71]. β-pinene also presents antimicrobial activity and is part of Grancam (2.75%) essential oils [46]. Furthermore, the essential oil of the studied varieties has other components in lower concentrations in its composition. According to Chorianopoulos et al. [72], antimicrobial activity occurs not only through the presence of major compounds but also by the infuence of other components at lower concentrations that can cause synergistic, additive, or antagonistic interactions. Te antifungal activity of the oils was slightly more promising in view of the results of MIC and MMC. Cimanga et al. [73] compared the antimicrobial activity of the essential oils of some Eucalyptus species. Te major compounds (such as 1,8-cineole and α-pinene) were also tested isolated and had less activity than the essential oil, stating that the combination of compounds is important for the antimicrobial activity of Eucalyptus oil [73], thus showing that it is not possible to compare the compounds of the oils isolated.
Te mechanism of inhibitory/microbicidal action of the essential oils may be related to their characteristics, such as hydrophobicity, which allows their interaction with cellular lipid structures, promoting increased permeability and consequent extensive efux of electrolytes, which play an essential role in cell homeostasis [74].
According to Burt [75], the difculty in comparing the results obtained by several authors is often mentioned as a problem faced in the study of the antimicrobial activity of essential oils and plant-derived products, since there are variations in the methods used.      Figure 5: Acute oral toxicity (2000 mg/kg, p.o.) on relative organs weight (%): spleen (a), heart (b), adrenals (c), brain (d), kidneys (e), liver (f ), and thymus (g). Mice were treated with vehicle (0.9% NaCl plus 1% of tween 80, 10 ml/kg, p.o., negative control group, n � 3) or the essential oils of Urocam, Grancam, E. benthamii, or E. saligna (2000 mg/kg, p.o., n � 6). One-way ANOVA followed by Bonferroni test: * P < 0.05, * * P < 0.01, and * * * P < 0.001 compared to the vehicle-treated (negative control) group at the same day. Results expressed as mean ± S.E.M. Results reported in this work reinforce the information that Eucalyptus essential oils have antimicrobial activity, especially antifungal, with the advantage of being a natural product. According to Rahman and Kang [76], the risk that pathogenic microorganisms will develop resistance to essential oils is very low, since they contain a mixture of antimicrobial substances that act through various mechanisms. Tese characteristics represent an advantage of essential oils as antimicrobial drugs, which can be promising for applications in diferent areas.

Conclusion
In this study, the great chemical variability that exists between the essential oils of the various Eucalyptus species was demonstrated, highlighting Urocam, Grancam, and E. dunii, which obtained the highest oil yield with high concentrations of 1.8 cineole and a-pinene. In addition, the essential oils of Urocam, Grancam, E. benthamii, and E. saligna have antinociceptive and anti-infammatory activities and are devoid of acute toxicity. Also, the seven varieties of Eucalyptus studied are active against S. aureus, E. coli, and C. albicans. Finally, the Eucalyptus essential oils represent an important source of antinociceptive, anti-infammatory, and antimicrobial compounds, such as 1,8-cineole and α-pinene. Future studies on the elucidation of the mechanism of the antinociceptive action of the oils are required.

Data Availability
Te data used to support the fndings of this study are available from the corresponding author upon request.

Conflicts of Interest
Te authors declare that they have no conficts of interest.