Anesthetic activity and bio-guided fractionation of the essential oil of Aloysia gratissima (Gillies & Hook.) Tronc. in silver catfish Rhamdia quelen

SIMONE C. BENOVIT LENISE L. SILVA JOSEÂNIA SALBEGO VANIA L. LORO CARLOS A. MALLMANN BERNARDO BALDISSEROTTO ERICO M.M. FLORES BERTA M. HEINZMANN About the authors

Abstracts

This work aimed to determine the efficacy of the essential oil of A. gratissima as anesthetic for silver catfish, and to perform the bio-guided fractionation of essential oil aiming to isolate compounds responsible for the noted effects. Fish were submitted to anesthesia bath with essential oil, its fractions and isolated compounds to determine time of anesthetic induction and recovery. Eugenol (50 mg L-1) was used as positive control. Essential oil of A. gratissima was effective as an anesthetic at concentrations of 300 to 900 mg L-1. Fish presented involuntary muscle contractions during induction and recovery. The bio-guided fractionation of essential oil furnishedE-(-)-pinocamphone, (-)-caryophyllene oxide, (-)-guaiol and (+)-spathulenol. E-(-)-pinocamphone caused the same side effects observed for essential oil. (-)-Caryophyllene oxide, (-)-guaiol and (+)-spathulenol showed only sedative effects at proportional concentrations to those of the constituents in essential oil. (+)-Spathulenol (51.2 mg L-1) promoted deep anesthesia without side effects. A higher concentration of (+)-spathulenol, and lower or absent amounts ofE-(-)-pinocamphone could contribute to increase the activity and safety of the essential oil of A. gratissima. (+)-Spathulenol showed potent sedative and anesthetic activities in silver catfish, and could be considered as a viable compound for the development of a new anesthetic.

anesthesia; (-)-caryophyllene oxide; E-(-)-pinocamphone; (-)-guaiol, silver catfish; (+)-spathulenol


Este trabalho tem como objetivo determinar a eficácia do óleo essencial deA. gratissima como anestésico para jundiás, bem como realizar o fracionamento bio-guiado do óleo essencial a fim de isolar os compostos responsáveis pelos efeitos observados. Os peixes foram submetidos ao banho-anestésico com óleo essencial, suas frações e compostos isolados para determinar o tempo de indução e recuperação da anestesia. O eugenol (50 mg L-1) foi utilizado como controle positivo. O óleo essencial deA. gratissima promoveu anestesia em concentrações de 300 a 900 mg L-1. Os peixes apresentaram contrações musculares involuntárias durante indução e recuperação. Com o fracionamento bio-guiado do óleo essencial foram obtidos E-(-)-pinocanfona, (-)-óxido de cariofileno, (-)-guaiol e (+)-espatulenol. A E-(-)-pinocanfona promoveu os mesmos efeitos adversos observados com o óleo essencial. (-)-Óxido de cariofileno, (-)-guaiol e (+)-espatulenol demonstraram apenas efeitos sedativos nas concentrações proporcionais ao óleo essencial. O (+)-espatulenol (51.2 mg L-1) promoveu anestesia profunda sem efeitos adversos. Uma maior concentração de (+)-espatulenol e menores quantidades ou ausência deE-(-)-pinocanfona poderiam contribuir para melhorar a atividade anestésica e segurança do óleo essencial de A. gratissima. O (+)-espatulenol apresentou potente atividade sedativa e anestésica em jundiás, e poderia ser considerado um composto viável para o desenvolvimento de um novo anestésico.

anestesia; (-)-óxido de cariofileno; E-(-)-pinocanfona; (-)-guaiol, jundiá; (+)-espatulenol


INTRODUCTION

There are different handling practices in fish farming, such as biometrics and transport. These practices can cause stress, which can be detrimental to animal performance. Anesthetics are used in this context to maintain fish welfare, minimizing stressful situations and facilitating handling (Coyle et al. 2004COYLE SD, DURBOROW RM AND TIDWELL JH. 2004. Anesthetics in aquaculture. SRAC Publication 3900., Zahl et al. 2012ZAHL IH, SAMUELSEN O AND KIESSLING A. 2012. Anaesthesia of farmed fish: implications for welfare. Fish Physiol Biochem 38: 201-218.). Natural products have been evaluated as an alternative to synthetic anesthetics, such as 2-phenoxyethanol, quinaldine sulfate and benzocaine, which may cause some side effects in fish and handlers (Inoue et al. 2003INOUE LAKA, NETO CS AND MORAES G. 2003. Clove oil as anesthetic for juveniles of matrinxã Brycon cephalus (Gunther, 1869). Cienc Rural 33: 943-947., Velisek et al. 2007VELISEK J, WLASOW T, GOMULKA P, SVOBODOVA Z AND NOVOTNY L. 2007. Effects of 2-phenoxyethanol anaesthesia on sheatfish (Silurus glanis L.). Vet Med (Praha) 52: 103-110.). Some compounds isolated from plants, such as eugenol (Cunha et al. 2010aCUNHA MA, ZEPPENFELD CC, GARCIA LO, LORO VL, FONSECA MB, EMANUELLI T, VEECK APL, COPATTI CE AND BALDISSEROTTO B. 2010a. Anesthesia of silver catfish with eugenol: time of induction, cortisol response and sensory analysis of fillet. Cienc Rural 40: 2107-2114., Gomes et al. 2011GOMES DP, CHAVES BW, BECKER AG AND BALDISSEROTTO B. 2011. Water parameters affect anaesthesia induced by eugenol in silver catfish, Rhamdia quelen. Aquac Res 4: 878-886., Becker et al. 2012BECKER AG, PARODI TV, HELDWEIN CG, ZEPPENFELD CC, HEINZMANN BM AND BALDISSEROTTO B. 2012. Transportation of silver catfish, Rhamdia quelen, in water with eugenol and the essential oil of Lippia alba. Fish Physiol Biochem 38: 789-796.) and menthol (Simões and Gomes 2009SIMÕES LN AND GOMES LC. 2009. Menthol efficiency as anesthetic for juveniles Nile tilapia Oreochromis niloticus. Arq Bras Med Vet Zootec 61: 613-620.), and also some essential oils, such as the ones obtained fromEugenia caryophyllata (Inoue et al. 2003INOUE LAKA, NETO CS AND MORAES G. 2003. Clove oil as anesthetic for juveniles of matrinxã Brycon cephalus (Gunther, 1869). Cienc Rural 33: 943-947.), Lippia alba (Cunha et al. 2010bCUNHA MA, BARROS FMC, GARCIA LO, VEECK APL, HEINZMANN BM, LORO VL, EMANUELLI T AND BALDISSEROTTO B. 2010b. Essential oil of Lippia alba: a new anesthetic for silver catfish, Rhamdia quelen. Aquaculture 306: 403-406., 2011CUNHA MA, SILVA BF, DELUNARDO FAC, BENOVIT SC, GOMES LC, HEINZMANN BM AND BALDISSEROTTO B. 2011. Anesthetic induction and recovery of Hippocampus reidi exposed to the essential oil of Lippia alba. Neotrop Ichthyol 9: 683-688.),Ocimum gratissimum (Benovit et al. 2012BENOVIT SC, GRESSLER LT, SILVA LL, GARCIA LO, OKAMOTO MH, PEDRON JS, SAMPAIO LA, RODRIGUES RV, HEINZMANN BM AND BALDISSEROTTO B. 2012. Anesthesia and transport of Brazilian flounder, Paralichthys orbignyanus, with essential oils of Aloysia gratissima and Ocimum gratissimum. J World Aquac Soc 43: 896-900., Silva et al. 2012SILVA LL, PARODI TV, RECKZIEGEL P, GARCIA VO, BÜRGER ME, BALDISSEROTTO B, MALMANN CA, PEREIRA AMS AND HEINZMANN BM. 2012. Essential oil of Ocimum gratissimum L.: anesthetic effects, mechanism of action and tolerance in silver catfish, Rhamdia quelen. Aquaculture 350-353: 91-97.) andMelaleuca alternifolia (Hajek 2011HAJEK GJ. 2011. The anaesthetic-like effect of tea tree oil in common carp Cyprinus carpio L. Aquac Res 42: 296-300.) have shown anesthetic activity on different fish species.

The genus Aloysia (Verbenaceae) is originally from South America, with 34 species cataloged, among which 12 can be found in Brazil (Cordo and Deloach 1995CORDO HA AND DELOACH CJ. 1995. Natural enemies of the rangeland weed whitebrush (Aloysia gratissima: Verbenaceae) in South America: potential for biological control in the United States. Biol Control 5: 218-230.). Certain species ofAloysia are known to exhibit central activity. A. gratissima (Gillies & Hook) Tronc. is known in Brazil aserva-santa (Souza and Wiest 20007SOUZA AA AND WIEST JM. 2007. Antibacterial activity of Aloysia gratissima (Gill et Hook) Tronc. (garupá, herb-saint), used in the traditional medicine in Rio Grande do Sul State - Brazil. Rev Bras Pl Med 9: 23-29.), and has been used in folk medicine due to its sedative properties (Goleniowski et al. 2006GOLENIOWSKI ME, BONGIOVANNI GA, PALACIO L, NUÑEZ CO AND CANTERO JJ. 2006. Medicinal plants from the "Sierra de Comechingones", Argentina. J Ethnopharmacol 107: 324-341.). The essential oil of A. triphylla showed potent anesthetic activity in white shrimp (Litopenaeus vannamei) (Parodi et al. 2012PARODI TV ET AL. 2012. The anesthetic efficacy of eugenol and the essential oils of Lippia alba and Aloysia triphylla in post-larvae and sub-adults of Litopenaeus vannamei (Crustacea, Penaeidae). Comp Biochem Physiol C Toxicol Pharmacol 155: 462-468.) and silver catfish (Rhamdia quelen) (Gressler et al. 2014GRESSLER LT ET AL. 2014. Silver catfish Rhamdia quelen immersion anaesthesia with essential oil of Aloysia triphylla (L'Hérit) Britton or tricaine methanesulfonate: effect on stress response and antioxidant status. Aquac Res 45: 1061-1072.). Benovit et al. (2012)BENOVIT SC, GRESSLER LT, SILVA LL, GARCIA LO, OKAMOTO MH, PEDRON JS, SAMPAIO LA, RODRIGUES RV, HEINZMANN BM AND BALDISSEROTTO B. 2012. Anesthesia and transport of Brazilian flounder, Paralichthys orbignyanus, with essential oils of Aloysia gratissima and Ocimum gratissimum. J World Aquac Soc 43: 896-900. verified anesthetic activity of essential oil of A. gratissima in Brazilian flounder (Paralichthys orbignyanus). However, there are no studies correlating its depressant effect with some chemical constituent.

This work aimed to determine the efficacy of the essential oil obtained from leaves of A. gratissima (EOA) as anesthetic for silver catfish (Rhamdia quelen), and to perform the bio-guided fractionation of EOA aiming to isolate the active compounds responsible for the noted effects. Acetylcholinesterase (AChE) activity under the influence of EOA was also investigated.

MATERIALS AND METHODS

PLANT MATERIAL

Aerial parts of A. gratissima were collected in April 2010 in Santa Maria, Rio Grande do Sul, Brazil. A voucher specimen (SMDB 13.077) was kept in the Biology Department of the Universidade Federal de Santa Maria and identified by Professor Marcos Sobral, UFSJ, and Professor Solon Jonas Longhi, UFSM.

ESSENTIAL OIL EXTRACTION

The essential oil was obtained from fresh leaves of A. gratissima by hydrodistillation with a Clevenger type apparatus for 3h (British Pharmacopoeia 2007). Samples of the essential oil were stored at −4ºC in amber glass bottles prior to the tests or isolation.

GAS CHROMATOGRAPHY MASS SPECTROMETRY (GC-MS) AND NUCLEAR MAGNETIC RESONANCE (NMR) ANALYSIS

GC-MS analysis of EOA and fractions were performed using an Agilent-6890 gas chromatograph coupled with an Agilent 5973 mass selective detector, with an HP5-MS column (5% phenyl - 95% methylsiloxane, 30 m x 0.25 mm i.d. x 0.25 µm) and EI-MS of 70 eV. The isolated compounds were analyzed by a Varian CP-3800 gas chromatograph coupled to a Saturno mass selective detector, using VF-5 MS column (Varian, 30 m x 0.25 mm x 0.25 µm). Conditions of analysis: injector 1177 (MS), 250ºC, split 1:20; injetor 1093 (FID), 250ºC. The operating conditions were: split inlet 1:100; temperature program, 40-320ºC at 4ºC min-1; carrier gas He; flow rate 1.3 mL min-1; injector and detector temperature 250ºC. The constituents were identified by comparing their Kovats retention index and mass spectra with a mass spectral library (NIST 2005) and literature data (Adams 2001).

Optical rotations were obtained on a Perkin Elmer 343 polarimeter. NMR spectra were recorded in CDCl3 on Bruker HPX 300 FT-NMR at 400 MHz for 1H and 100 MHz for 13C with TMS as internal standard.

FRACTIONATION AND ISOLATION OF ESSENTIAL OIL (EOA)

EOA (6.03 g) was first submitted to fractionation by column chromatography (CC, 3.5 x 52 cm) with 300 g silica gel 60 (Macherey-Nagel, 70-230 mesh) as stationary phase and hexane:acetone (99:1 v/v) as mobile phase. The fractions obtained (100 mL each) were gathered in 16 main fractions (Fractions A-P) based on TLC profile (silica gel F254; detection: anisaldehyde H2SO4). The fractions G and J were subsequently fractionated.

Fraction G (910.4 mg) was submitted to CC (1.9 x 61 cm) with 89 g of silica gel 60 and eluted with henane:ethyl ether (99:1 v/v) to obtain fractions of 20 mL, resulting in 23 main fractions (G1-23). The fraction G9 (167.7 mg) was rechromatographed by CC (1 x 31.5 cm) using 18 g of silica gel 60 impregnated with 10% silver nitrate (Williams and Mander 2001) and hexane:acetone (99:1 v/v). Fractions of 10 mL were collected and pooled together to render 10 main fractions (G9A-J). Purification of the main constituent of G9B(99.0 mg) was performed with CC (1 x 28.5 cm) using 15 g silica gel 60 and eluted with chloroform:hexane:ethyl acetate (78:20:2 v/v/v), to obtainE-(-)-pinocamphone [76.1 mg; EI-MS m/z (%): 152 (M+); [α]D 20 = − 24.54º (c 0.22, CHCl3)] Fractions G17 - G20 were gathered (112.7 mg) and chromatographed by CC (1 x 30 cm) using 16.31 g of silica gel 60 impregnated with 10% silver nitrate and hexane:ethyl acetate (97:3 v/v) to provide (-)-caryophyllene oxide [91.1 mg; EI-MS m/z (%): 220 (M+); [α]D 20 = − 62.5º (c 0.24, CHCl3)]. Fraction J (493.3 mg) was submitted to CC (1.6 x 47.5 cm) with 48 g of silica gel 60 impregnated with 10% silver nitrate, using hexane:ethyl ether (92:8 v/v) as eluent, and furnished (-)-guaiol [107.0 mg; EI-MS m/z (%): 222 (M+); [α]D 20 = − 28.125º (c 0.16, CHCl3)] and (+)-spathulenol [91.3 mg; EI-MS m/z (%): 220 (M+); [α]D 20 = + 8.66º (c 0.15, CHCl3)].

E-(-)-pinocamphone: 1H NMR, δ (ppm): 0.84 (s, 3H, H9); 1.05 (d, 3H, H10); 1.12 (d, 1H, H7a); 1.29 (s, 3H, H8); 1.88 (dt, 1H, H1); 2.06 (m, 1H, H5); 2.38* (m, 1H, H4a); 2.41* (m, 1H, H7b); 2.57 (m, 1H; H2); 2.58 (m, 1H, H4b) (* These values can be exchanged). 13C NMR (ppm), δ: 14.96 (C10); 19.70 (C9); 26.28 (C8); 29.04 (C7); 38.24 (C5); 39.36 (C6); 44.29 (C4); 44.39 (C1); 46.41 (C2); 215.34 (C3).

(-)-Caryophyllene oxide: 1H NMR, δ (ppm): 0.9 (m, 1H, H); 0.91 (s, 3H, H13); 0.93 (s, 3H, H12); 1.13 (s, 3H, H14); 1.25 (m, 1H, H6a); 1.35 (m, 1H, H2a); 1.58 (m, 3H, H2b, H10a, H10b); 1.7 (m, 1H, H1); 2.02 (m, 1H, H3b); 2.03 (m, 1H, H7b); 2.17 (m, 1H, H6b); 2.27 (m, 1H, H7a); 2.54 (q, 1H, H9); 2.80 (dd, 1H, H5); 4.89 (s, 1H, H15a); 4.97 (s, 1H, H15b). 13C NMR (ppm), δ: 16.94 (C14); 21.59 (C13); 27.18 (C2); 29.78 (C7); 29.84 (C12); 30.14 (C6); 33.96 (C11); 39.12 (C3); 39.72 (C10); 48.68 (C9); 50.74 (C1); 59.74 (C4); 63.68 (C5); 112.69 (C15); 151.76 (C8).

(-)-Guaiol: 1H NMR, δ (ppm): 0.94 (d, 3H, H14); 0.98 (d, 3H, H15); 1.14 (s, 3H, H12); 1.17 (s, 3H, H13); 1.27, 1.95 (both m, 1H each, H3); 1.52 (m, 1H, H7); 1.70, 1.55 (both m, 1H each, H9); 1.80, 1.44 (both m, 1H each, H8); 2.13, 1.87 (dd, m respectively, 1H each, H6); 2.28 (m, 1H, H10); 2.41, 2.05 (both m, 1H each, H2); 2.52 (dd, 1H, H4). 13C NMR, δ (ppm): 19.69 (C15); 19.88 (C14); 25.93 (C12); 27.26 (C8); 27.31 (C13); 27.80 (C6); 30.91 (C3); 33.64 (C10); 33.73 (C9); 35.34 (C2); 46.27 (C4); 49.59 (C7); 73.48 (C11); 138.69 (C5); 140.07 (C1).

(+)-Spathulenol: 1H NMR, δ (ppm): 0.39 (dd, 1H, H6); 0.64 (m, 1H, H7); 0.92 (m, 1H, H8b); 0.97 (s, 3H, H13); 0.98 (s, 3H, H12); 1.21 (s, 3H, H15); 1.25 (dd, 1H, H5); 1.48 (m, 1H, H3b); 1.55 (m, 1H, H2b); 1.70 (m, 1H, H3a); 1.83 (m, 1H, H2a); 1.91 (m, 1H, H8a); 1.98 (m, 1H, H9a); 2.14 (m, 1H, H1); 2.35 (dd, 1H, H9b); 4.60 (d, 2H, H14a/H14b).13C NMR, δ (ppm): 16.29 (C13); 20.21 (C11); 24.74 (C8); 26.03 (C15); 26.68 (C2); 27.46 (C7); 28.62 (C12); 29.90 (C6); 38.83 (C9); 41.71 (C3); 53.37 (C1); 54.27 (C5); 80.90 (C4); 106.22 (C14); 153.38 (C10).

ANIMALS

Silver catfish were purchased from local producers. Fish were kept in continuously aerated 250 L tanks for seven days for acclimatization under the same conditions of each experiment performed in sequence. A semi-static system was used, where 50% of the water volume was exchanged once a day. In experiment 1, the water parameters were maintained at the following values: temperature, 15.90 ± 0.14ºC; pH, 7.37 ± 0.11; dissolved oxygen, 8.65 ± 0.07 mg L-1; total ammonia, 2.43 ± 0.81 mg L-1 and unionized ammonia, 0.016 mg L-1. In the other experiments, the values were 19.33 ± 0.69ºC; pH, 7.19 ± 0.56; dissolved oxygen, 6.61 ± 0.95; total ammonia, 1.06 ± 0.39 mg L-1 and unionized ammonia, 0.006 mg L-1. Dissolved oxygen and temperature were measured with a YSI oxygen meter (model Y5512). The pH was verified with a DMPH-2 pH meter. Total ammonia levels were determined by the salicylate method (Verdouw et al. 1978). Fish were fed once a day until satiation with commercial feed for juveniles containing 28.0% of crude protein. The juveniles were fasted for 24h prior to the experiments. The experimental methodologies were approved by the Ethical and Animal Welfare Committee of the Universidade Federal de Santa Maria (process No. 46/2010).

EXPERIMENTAL PROCEDURE

Three groups of experiments were performed to evaluate the sedative and anesthetic potential, as well as the recovery of silver catfish exposed to EOA, fractions and isolated compounds. Animals were transferred to aquaria containing 1 L of water and the samples to be tested, these samples were previously diluted in ethanol 95% (1:10), to evaluate the time required to reach stages of anesthesia (Gomes et al. 2011GOMES DP, CHAVES BW, BECKER AG AND BALDISSEROTTO B. 2011. Water parameters affect anaesthesia induced by eugenol in silver catfish, Rhamdia quelen. Aquac Res 4: 878-886.). In this method, each juvenile was used only once in order to observe deep sedation (S2), partial (S3a) and total loss (S3b) of equilibrium, anesthesia (S4) and/or medullar collapse (S5). Anesthesia was determined by loss of reflex activity and no reaction to strong external stimuli (pressure on caudal peduncle). The animals remained in the anesthetic bath until they reached S4 or for 30 min. After anesthesia induction, each fish was measured, weighed and transferred to an anesthetic-free aquarium to recover. The fish were considered to have recovered if their normal posture and behavior were restored, and the maximum time observed for this to occur was 30 min. After that, the animals were transferred to 30 L aquariums, where possible side effects (spasms, contortions, secretion of mucus, seizures) or mortality was examined after 24h of exposition. Control experiments were performed using aquarium containing ethanol at the highest concentration used to dilute the EOA.

In experiment 1, the anesthetic activity of EOA was evaluated. Ten silver catfish juveniles (6.82 ± 2.04 g; 9.10 ± 0.91 cm) were used for each concentration of EOA (90, 300, 450, or 900 mg L-1). Experiment 2 was performed to compare the activity among 450 mg L-1 EOA and the fractions with higher chromatographic yield obtained by column chromatography (B, D, F, G and J). Six juveniles (8.66 ± 2.32 g; 9.52 ± 0.86 cm) were used for testing each fraction, at proportional concentrations to those of the major constituents in the EOA. In experiment 3, the sedative and anesthetic activities of the isolated compounds were evaluated. Six juveniles (6.99 ± 2.24 g; 9.44 ± 1.03 cm) were used for each concentration. The isolated compounds were evaluated at proportional concentrations to those of the constituents in the EOA at concentrations of 450, 675 and 900 mg L-1, which corresponded to: 19.06, 28.60 and 38.14 mg L-1of E-(-)-pinocamphone; 10.00, 15.00, 20.00 mg L-1 of (-)-caryophyllene oxide; 32.65, 48.96 and 65.30 mg L-1 of (-)-guaiol; and 12.80, 19.20 and 25.60 mg L-1 of (+)-spathulenol. Additional concentrations of (-)-caryophyllene oxide (40 mg L-1) and (+)-spathulenol (51.20 mg L-1) were tested. (-)-Guaiol and (+)-spathulenol were also evaluated together (32.65 and 12.80 mg L-1, respectively). Eugenol at concentration of 50 mg L-1 was used as positive control (Cunha et al. 2010aCUNHA MA, ZEPPENFELD CC, GARCIA LO, LORO VL, FONSECA MB, EMANUELLI T, VEECK APL, COPATTI CE AND BALDISSEROTTO B. 2010a. Anesthesia of silver catfish with eugenol: time of induction, cortisol response and sensory analysis of fillet. Cienc Rural 40: 2107-2114.).

LONG EXPOSURE TIME

This experiment evaluated the effects of long exposure time (24h) of silver catfish to EOA and (+)-spathulenol at sedative concentrations. Considering the sedative concentrations determined in the experiments described in the previous sections, four experimental groups were evaluated: EOA at 90 mg L-1, (+)-spathulenol at 2.6 mg L-1 (proportional concentration of the constituent in the EOA at 90 mg L-1), ethanol (at the concentration used to dilute the EOA) and water.

Silver catfish (6.72 ± 2.01 g; 8.75 ± 1.02 cm) were transferred to aquaria containing 1 L of water and the sample to be tested. Six animals were used for each group to determine the time it would take to reach stage S3a. Initially, animal condition was evaluated in the first 30 min of exposure to confirm the occurrence of sedation and then after 24h, aiming to evaluate the toxicity of the substances during this time. After exposure, fish were transferred to anesthetic-free 7 L aquariums, where possible signs of intoxication or mortality were examined for an additional 24h.

ACETYLCHOLINESTERASE ASSAY

Six silver catfish juveniles (20.49 ± 6.71 g; 16.50 ± 1.33 cm) were exposed to each concentration of EOA (90, 300 or 450 mg L-1), firstly diluted in ethanol 95% (1:10). Control experiments were performed using water alone or ethanol at the highest concentration used to dilute the EOA. Fish were euthanized by severing the spinal cord after they reached S4 or after 30 min of exposure (control group and concentrations that did not promote anesthesia). Whole brain and muscle samples were removed, packed in eppendorf tubes and kept at −20ºC for AChE analysis. AChE assay was performed in homogenates of brain and muscle according to the method described by Ellman et al. (1961) and modified by Villescas et al. (1981). Homogenates (50-100 µL) were incubated with 0.8 mM of acetylthiocholine and 1.0 mM of 5,5'-dithio-bis-2-nitrobenzoic acid (DTNB) at 25ºC for 2 min. Protein content was determined according to Bradford (1976). Enzyme activity was expressed as micromoles of acetylthiocholine hydrolyzed per minute per milligram of protein.

STATISTICAL ANALYSIS

Values are presented as mean ± SEM (standard error of the mean). A Levene test was used with all data to verify the homogeneity of variances. The results were analyzed by one-way ANOVA and Tukey test or Kruskal-Wallis and Dunn test, when appropriate. Comparison between spathulenol and eugenol was performed with t-test. The minimum significance level was set at P<0.05. Regression analyses were performed to EOA and isolated compounds.

RESULTS

The yield of EOA was 1.94 ± 0.03% (w/w). EOA showed 73 constituents, and 91.86% of the chemical composition was identified. The major compounds of EOA were 1,8-cineole (18.54%), sabinene (9.5%), guaiol (6.79%) and bicyclogermacrene (5.12%) (Table I).

TABLE I
Chemical composition of essential oil of Aloysia gratissima and fractions of higher yield.

Silver catfish reached anesthesia with EOA at concentrations ranging from 300 (approximately 18 min) to 900 mg L-1 (approximately 12 min). Fish exposed to 90 mg L-1 reached stage 3a after 8 min and they remained in this stage for 30 min of observation. There were no significant differences in the induction times to anesthesia between the concentrations of 450 and 900 mg L-1, but these induction times (approximately 12 min) were significantly faster than those of 300 mg L-1. A significant relationship between the EOA concentration and the time required for anesthesia induction was noted for all stages (Table II), except for recovery time. Time of recovery was significantly faster at 900 mg L-1 (approximately 12 min) when compared to 300 and 450 mg L-1. During this experiment, and 24h after it, no mortality occurred. However, involuntary muscle contractions were observed in the fish submitted to EOA during induction and recovery. The group exposed to ethanol showed light sedation (S2 in approximately 7.9 min), but it was significantly different from all EOA-treated groups.

TABLE II
Time (s) required for anesthesia induction and recovery using different concentrations of EOA in Rhamdia quelen.

AChE activity did not show any significant difference among treatments (Table III).

TABLE III
Brain and muscle specific AChE activity (μmol min-1 mg protein-1) of Rhamdia quelen exposed to EOA.

The fractions B, D, F, G and J showed higher yield from the EOA chromatography process. Similar chemical composition was verified for fractions F and G, while the others showed different major constituents (Table I). No sedative effect was observed for fraction D in silver catfish within 30 min of exposure. Fish exposed to fractions B and F reached only S3a (Table IV). Both fractions G and J resulted in anesthesia (S4) in 50% of fish with no significant differences between them. However, fraction G caused involuntary muscle contractions similar to those verified with the EOA. Fraction J caused no side effects, but it resulted in increased recovery time in relation to the other fractions. Since fractions G and J showed anesthetic or side effects, we chose to isolate their constituents in order to identify the possible active compounds. E-(-)-pinocamphone and (-)-caryophyllene oxide were obtained from fraction G, while purification of fraction J resulted in the isolation of (-)-guaiol and (+)-spathulenol. The isolated constituents were identified by comparing the MS and NMR data with literature reports (Coxon et al. 1984COXON JM, HYDES GJ AND STEEL PJ. 1984. Carbon-13 nuclear magnetic resonance spectra of pinane monoterpenoids. J Chem Soc Perkin Trans 2: 1351-1355., Raharivelomana et al. 1995RAHARIVELOMANA P, BIANCHINI JP, CAMBON A, AZZARO M AND FAURE R. 1995. Two-dimensional NMR of sesquiterpenes. 8-complete assignment of 1H and13C NMR spectra of seven sesquiterpene alcohols from Neocallitropsis pancheri. Magn Reson Chem 33: 233-235., Chaves and Santos 2002CHAVES MCO AND SANTOS BVO. 2002. Constituents from Piper marginatum fruits. Fitoterapia 73: 547-549., Höld et al. 2002HÖLD KM, SIRISOMA NS, SPARKS SE AND CASIDA JE. 2002. Metabolism and mode of action of cis- and trans-3-pinanones (the active ingredients of hyssop oil). Xenobiotica 32: 251-265., Bolzan 2007BOLZAN AA. 2007. Constituents of Senecio platensis Arech.: isolation, structural elucidation and evaluation of the antibacterial activity. MSc. Thesis (Ciências Farmacêuticas), Universidade Federal de Santa Maria, RS, Brazil, 150 p. (Unpublished).).

TABLE IV
Time (s) required for induction and recovery from anesthesia using EOA (at concentration of 450 mg L-1) and their fractions of higher yield (at proportional concentrations to those of the major constituents in the EOA) in Rhamdia quelen.

Juveniles submitted to E-(-)-pinocamphone showed different behavior of sedated or anesthetized fish. In the first minutes of exposure, a period of hyperactivity could be noted. Animals tended to present faster and erratic swimming with partial loss of equilibrium, and sudden jumping towards the water surface. Afterwards, there was a period of total loss of equilibrium, animals tumbled on the bottom of the aquarium and remained immobile even after pressure on caudal peduncle, but they responded to external stimuli made with a glass rod in the bottom of the aquarium. Then, fish went back to swimming erratically, and, at that moment, involuntary muscle contractions were noted. Fish writhed to the right and left, or remained contracted in "C or S-shape". There was a significant relationship between the concentration of E-(-)-pinocamphone and the time required for hyperactivity stage (y = 283.198 − 15.111x + 0.228x2; r2 = 1) and tipping stage (y = − 13.117+18.650x-0.406x2; r2 = 1). Three juveniles were found dead 24h after the experiment.

The other isolated compounds reached stages S2 and S3a at proportional concentrations to EOA. Sedation (S2) obtained with (-)-caryophyllene oxide was not significantly different from EOA, except at the lowest concentration tested, or (+)-spathulenol (Fig. 1a). EOA was the most effective for loss of equilibrium (S3a), except at concentration of 675 mg L-1, where it did not differ from (+)-spathulenol. (-)-Caryophyllene oxide and (-)-guaiol did not present statistical differences to reach stage S3a, except at the highest concentration, in which (-)-caryophyllene oxide was more effective (Fig. 1b). In addition, both compounds showed different induction times from EOA.

Figure 1 -
Time required for the induction and recovery from sedation in silver catfish submitted to EOA and the isolated compounds at proportional concentrations to those of the constituents in EOA: (a): Stage 2; (b): Stage 3a; (c): Recovery. For 450 mg L−1 EOA: (-)-guaiol (32.65 mg L−1), (+)-spathulenol (12.8 mg L−1), (-)-caryophyllene oxide (10 mg L−1) and association of (-)-guaiol and (+)-spathulenol (32.65 and 12.8 mg L−1); For 675 mg L−1 EOA: (-)-guaiol (48.96 mg L−1), (+)-spathulenol (19.2 mg L−1) and (-)-caryophyllene oxide (15 mg L−1); For 900 mg L−1 EOA, (-)-guaiol (65.3 mg L−1), (+)-spathulenol (25.6 mg L−1) and (-)-caryophyllene oxide (20 mg L−1). Fish submitted to 675 mg L−1 EOA did not recover within 30 min, so it was not shown in this figure. Stages are according to Gomes et al. (2011)GOMES DP, CHAVES BW, BECKER AG AND BALDISSEROTTO B. 2011. Water parameters affect anaesthesia induced by eugenol in silver catfish, Rhamdia quelen. Aquac Res 4: 878-886.. Maximum observation time was 30 min. N = 6 for each concentration tested. Data are expressed as mean ± SEM. Different letters indicate a significant difference between the different products at the same stage. Stage S3a and recovery time of 675 mg L−1 EOA and S2 of 900 mg L−1 EOA were submitted to Kruskal-Wallis and Dunn tests (P<0.05). The other stages were submitted to ANOVA and Tukey tests (P<0.05).

(-)-Caryophyllene oxide (ln y= 0.769 + 35.066/ x; r2 = 0.995; N = 6), (-)-guaiol (ln y = 1.18 + 0.039x; r2 = 0.999; N = 6), and (+)-spathulenol (y = 28.28 + 6.333x − 0.377x2 + 0.005x3; r2 = 1; N = 6) showed a significant relationship between the concentrations and the time required to reach stage S2. The regression equations to reach stage S3a were y= −3873.656 + 874.635x - 48.433x2 + 0.725x3; r2 = 1; N = 6 to (-)-caryophyllene oxide, ln y= 3.42 + 17.2/x0.5; r2 = 0.999; N = 6 to (-)-guaiol and y = 453.198 - 11.955x − 0.285x2 + 0.007x3; r2 = 1; N = 6 to (+)-spathulenol. (-)-Caryophyllene oxide (y= 2901.2 − 495.21x + 27.753x2 − 0.411x3; r2 = 1; N = 6) and (-)-guaiol (y = 1676.76 − 59.58x + 0.68x2; r2 = 1; N = 6) also showed significant relationships between the concentrations and recovery times. There was no significant difference in the recovery time of juveniles submitted to concentrations of 12.8 to 25.6 mg L-1 of (+)-spathulenol (data not shown). (-)-Caryophyllene oxide, (-)-guaiol and (+)-spathulenol did not promote mortality during induction and recovery and also 24h after exposition, while four fish exposed to EOA were found dead 24h after the experiment. Fish submitted to (-)-caryophyllene oxide (40 mg L−1) and (-)-guaiol (48.96 and 65.30 mg L−1)showed secretion of mucus during exposition, whereas (-)-caryophyllene oxide (15 - 40 mg L−1) caused light spasms in the animals.

(-)-Caryophyllene oxide did not promoted anesthesia even at the highest concentration tested (40 mg L−1). Stage S2 and S3a were reached at 5.50±0.22 s and 25.50±4.29 s, respectively, with 40 mg L−1 of this compound. Moreover, fish recovered after 1168.00±126.08 s. Fish exposed to 51.2 mg L−1 of (+)-spathulenol reached S2 and S3a faster than with the positive control eugenol (Fig. 2). (+)-Spathulenol promoted deep anesthesia in approximately 22 min, which was a significantly different time, than that obtained with eugenol, which was of approximately 13 min, to reach the same stage. Recovery after eugenol exposure occurred in approximately 15 min, while fish exposed to (+)-spathulenol did not recover within 30 min.

Figure 2 -
Time required for the induction of sedation and anesthesia in silver catfish submitted to (+)-spathulenol (51.20 mg L−1) in comparison with eugenol (50 mg L−1). Stages are according to Gomes et al. (2011)GOMES DP, CHAVES BW, BECKER AG AND BALDISSEROTTO B. 2011. Water parameters affect anaesthesia induced by eugenol in silver catfish, Rhamdia quelen. Aquac Res 4: 878-886.. Maximum observation time was 30 min. N= 6 for each concentration tested. Data are expressed as mean ± SEM. Different letters indicate a significant difference between the different products at the same stage using t-test (P<0.05).

The association of (-)-guaiol and (+)-spathulenol resulted in decrease of induction time until stage S3a, when compared to the isolated compounds (Fig. 1b). However, animals submitted to this association showed mucus secretion and greater recovery time than with the isolated compounds (Fig. 1c). When compared with EOA, the recovery time of the association did not differ statistically. The three isolated compounds did not show statistical differences in the recovery time at all concentrations. Moreover, only one fish submitted to EOA recovered within 30 min at the concentration of 675 mg L-1.

Regarding the long exposure time (24h), fish reached S3a at 241.50±20.28 s with EOA and 1752.50±70.94 s with (+)-spathulenol. After 24h of exposure, no mortality was observed, and the animals submitted to EOA and (+)-spathulenol were not sedated. Fish submitted to (+)-spathulenol did not present signs of intoxication or mortality after 24h in anesthetic-free aquaria. However, 33.33% of mortality was observed with EOA.

DISCUSSION

Previous studies showed quantitative differences in the chemical composition of the EO obtained from leaves of A. gratissima. Dambolena et al. (2010)DAMBOLENA JS, ZUNINO MP, LUCINI EI, ZYGADLO JA, BANCHIO E, BIURRUN F, ROTMAN A AND AHUMADA O. 2010. Aromatic plants of northwest Argentina. Constituents of the essential oils of aerial parts of seven Verbenaceae: Lantana and Aloysia. J Essent Oil Res 22: 289-293. identified 1,8-cineole (45.5%) and sabinene (8.3%) as major constituents. However, the proportion of these compounds found by these authors was different from the corresponding data found in the present study. Similar proportion of guaiol (6.27%) was found by Franco et al. (2007)FRANCO ALP, OLIVEIRA TB, FERRI PH, BARA MTF AND PAULA JR. 2007. Evaluation of the chemical composition and antibacterial activity of essential oils of Aloysia gratissima (Gillies & Hook) Tronc., Ocimum gratissimum L and Curcuma longa L. Rev Eletr Farm 4: 208-220.. Varied amounts of bicyclogermacrene (0 - 12.8%) were described by Ricciardi et al. (2006)RICCIARDI GAL, VAN BAREN CM, LIRA PDL, RICCIARDI AIA, LORENZO D, DELLACASSA E AND BANDONI AL. 2006. Volatile constituents from aerial parts of Aloysia gratissima (Gillies & Hook.) Tronc. var. gratissima growing in Corrientes, Argentina. Flavour Fragr J 21: 698-703., and this variation depended on sites and period of collection.

Anesthesia (S4) is achieved when fish lose reflex activity and do not show a reaction to strong external stimuli (Gomes et al. 2011GOMES DP, CHAVES BW, BECKER AG AND BALDISSEROTTO B. 2011. Water parameters affect anaesthesia induced by eugenol in silver catfish, Rhamdia quelen. Aquac Res 4: 878-886.). Silver catfish exposed to concentrations of 300-900 mg L−1 EOA reached this stage within 12-18 min. The concentration of 900 mg L−1 was the most effective, because it showed lower anesthetic induction and recovery time. Similar EOA concentrations (270-900 mg L−1) also provoked deep anesthesia in Brazilian flounder (Benovit et al. 2012BENOVIT SC, GRESSLER LT, SILVA LL, GARCIA LO, OKAMOTO MH, PEDRON JS, SAMPAIO LA, RODRIGUES RV, HEINZMANN BM AND BALDISSEROTTO B. 2012. Anesthesia and transport of Brazilian flounder, Paralichthys orbignyanus, with essential oils of Aloysia gratissima and Ocimum gratissimum. J World Aquac Soc 43: 896-900.). To reach the same level of anesthesia in a similar period of time (approximately 12 min) in silver catfish, 30 mg L−1 of eugenol and 100 mg L−1 of EO ofLippia alba are made necessary (Cunha et al. 2010aCUNHA MA, ZEPPENFELD CC, GARCIA LO, LORO VL, FONSECA MB, EMANUELLI T, VEECK APL, COPATTI CE AND BALDISSEROTTO B. 2010a. Anesthesia of silver catfish with eugenol: time of induction, cortisol response and sensory analysis of fillet. Cienc Rural 40: 2107-2114., bCUNHA MA, BARROS FMC, GARCIA LO, VEECK APL, HEINZMANN BM, LORO VL, EMANUELLI T AND BALDISSEROTTO B. 2010b. Essential oil of Lippia alba: a new anesthetic for silver catfish, Rhamdia quelen. Aquaculture 306: 403-406.). Moreover, 200 µL L−1 of EO of Melaleuca alternifolia and 250 mg L−1 of menthol lead to S4 in common carp, Cyprinus carpio, and in Nile tilapia, Oreochromis niloticus, respectively (Simões and Gomes 2009SIMÕES LN AND GOMES LC. 2009. Menthol efficiency as anesthetic for juveniles Nile tilapia Oreochromis niloticus. Arq Bras Med Vet Zootec 61: 613-620., Hajek 2011HAJEK GJ. 2011. The anaesthetic-like effect of tea tree oil in common carp Cyprinus carpio L. Aquac Res 42: 296-300.).

The fractionation process showed that compounds present in fraction D did not contribute to the depressor effects of EOA, while constituents of the fractions B and F could be involved in its sedative effect. Previous reports described central activity for some compounds present in fractions B and F, such as limonene, 1,8-cineol, α- and β-pinene (Kasanen et al. 1998KASANEN JP, PASANEN AL, PASANEN P, LIESIVUORI J, KOSMA VM AND ALARIE Y. 1998. Stereospecificity of the sensory irritation receptor for nonreactive chemicals illustrated by pinene enantiomers. Arch Toxicol 72: 514-523., Santos and Rao 2000SANTOS FA AND RAO VSN. 2000. Antiinflammatory and antinociceptive effects of 1,8-cineole, a terpenoid oxide present in many plant essential oils. Phytother Res 14: 240-244., Vale et al. 2002VALE TG, FURTADO EC, SANTOS JR JG AND VIANA GS. 2002. Central effects of citral, myrcene and limonene, constituents of essential oil chemotypes from Lippia alba (Mill.) N.E. Brown. Phytomedicine 9: 709-714.), which need to be confirmed in aquatic animals. On the other hand, fractions G and J promoted anesthesia in 50 % of fish, which indicates that the compounds present in these fractions contribute to the anesthetic effect of EOA.

The involuntary muscle contractions and mortality observed in fish exposed to EOA and/or fraction G may be explained by the presence ofE-(-)-pinocamphone in these samples. Additionally, with this isolated compound the fish showed two distinct stages, hyperactivity and tipping. Similar effects were characterized as seizure in zebrafish, Danio renio, exposed to caffeine, pentylenetetrazole (PTZ) and picrotoxin (Wong et al. 2010WONG K ET AL. 2010. Modeling seizure-related behavioral and endocrine phenotypes in adult zebrafish. Brain Res 1348: 209-215.). E- and Z-pinocamphone are known antagonists of gamma-aminobutyric acid type A (GABAA) receptor (Höld et al. 2002HÖLD KM, SIRISOMA NS, SPARKS SE AND CASIDA JE. 2002. Metabolism and mode of action of cis- and trans-3-pinanones (the active ingredients of hyssop oil). Xenobiotica 32: 251-265.). These compounds are able to promote convulsions in mouse that can be alleviated by diazepam, and their signs of intoxication are similar to picrotoxin (Höld et al. 2002HÖLD KM, SIRISOMA NS, SPARKS SE AND CASIDA JE. 2002. Metabolism and mode of action of cis- and trans-3-pinanones (the active ingredients of hyssop oil). Xenobiotica 32: 251-265.). As fish submitted toE-(-)-pinocamphone showed more intense side effects than EOA, the compounds with sedative/anesthetic activity present in EOA may have reduced their effects.

The results obtained for E-(-)-pinocamphone are not inconsistent with the results of the AChE assay of EOA. Action in AChE was researched because the muscle inhibition of this enzyme leads to increased acetylcholine levels, resulting in excessive muscular stimulation (Kirby et al. 2000KIRBY MF, MORRIS S, HURST M, KIRBY SJ, NEALL P, TYLOR T AND FAGG A. 2000. The use of cholinesterase activity in flounder (Platichthys flesus) muscle tissue as a biomarker of neurotoxic contamination in UK estuaries. Mar Pollut Bull 40: 780-791.). However, AChE activity was not affected by EOA at the tested concentrations and, therefore, the contractures observed on the fish were not due to alterations in the activity of this enzyme. On the other hand, this response could be attributed, as reported earlier, to the GABAergic system, and future studies should be conducted to clarify this issue.

(-)-Caryophyllene oxide and (-)-guaiol promoted sedation at all tested concentrations. Higher concentrations of these compounds were not used because they caused mucus secretion on the animals. Additionally, (-)-caryophyllene oxide showed light spasm in fish. Secretion of mucus has been observed with some synthetic anesthetics for fish, such as 2-phenoxyethanol, quinaldine sulfate and benzocaine (Inoue et al. 2003INOUE LAKA, NETO CS AND MORAES G. 2003. Clove oil as anesthetic for juveniles of matrinxã Brycon cephalus (Gunther, 1869). Cienc Rural 33: 943-947., Velisek et al. 2007VELISEK J, WLASOW T, GOMULKA P, SVOBODOVA Z AND NOVOTNY L. 2007. Effects of 2-phenoxyethanol anaesthesia on sheatfish (Silurus glanis L.). Vet Med (Praha) 52: 103-110.); as well as 2,4-D [(2,4-dichlorophenoxy) acetic acid], a herbicide (Sarikaya and Yilmaz 2003SARIKAYA R AND YILMAZ M. 2003. Investigation of acute toxicity and the effect of 2,4-D (2,4-dichlorophenoxyacetic acid) herbicide on the behavior of the common carp (Cyprinus carpio L., 1758; Pisces, Cyprinidae). Chemosphere 52: 195-201.). Caryophyllene oxide, isolated from the Psidium guajava, showed depressant effects on the central nervous system of mice, through potencialization of the pentobarbital induced-sleeping time (Meckes and Calzada 1996MECKES M AND CALZADA F. 1996. Terpenoids isolated from Psidium guajava hexane extract with depressant activity on central nervous system. Phytother Res 10: 600-603.). However, studies on the central activity of (-)-guaiol were not found.

Sedation induced by (+)-spathulenol is in accordance with those described for the EO obtained from Baccharis uncinella, which showed sedative effects in mice and contains spathulenol as one of the major constituents (Ascari et al. 2012ASCARI J, SENS SL, NUNES DS, WISNIEWSKI JR A, ARBO MD, LINCK VM, LUNARDI P, LEAL MB AND ELISABETSKY E. 2012. Sedative effects of essential oils obtained from Baccharis uncinella. Pharm Biol 50: 113-119.). There were no other studies related to the central activity of (+)-spathulenol. Since this compound did not cause any side effects at sedative concentrations, one higher concentration was tested, which resulted in anesthesia. In this concentration, fish submitted to (+)-spathulenol showed faster sedation, slower anesthesia and recovery, than eugenol. Eugenol is largely used as anesthetic for fish and its activity has been proven in different species (Vidal et al. 2007VIDAL LVO, FURUYA WM, GRACIANO TS, SCHAMBER CR, SANTOS LD AND SOARES CM. 2007. Eugenol concentrations for deep anesthesia and acute toxicity in piavuçu (Leporinus macrocephalus) juveniles. Acta Sci Biol Sci 29: 357-362., Honczaryk and Inoue 2009HONCZARYK A AND INOUE LAKA. 2009. Anesthesia in pirarucu by eugenol sprays in the gills. Cienc Rural 39: 577-579.,Cunha et al. 2010aCUNHA MA, ZEPPENFELD CC, GARCIA LO, LORO VL, FONSECA MB, EMANUELLI T, VEECK APL, COPATTI CE AND BALDISSEROTTO B. 2010a. Anesthesia of silver catfish with eugenol: time of induction, cortisol response and sensory analysis of fillet. Cienc Rural 40: 2107-2114., Gomes et al. 2011GOMES DP, CHAVES BW, BECKER AG AND BALDISSEROTTO B. 2011. Water parameters affect anaesthesia induced by eugenol in silver catfish, Rhamdia quelen. Aquac Res 4: 878-886., Becker et al. 2012BECKER AG, PARODI TV, HELDWEIN CG, ZEPPENFELD CC, HEINZMANN BM AND BALDISSEROTTO B. 2012. Transportation of silver catfish, Rhamdia quelen, in water with eugenol and the essential oil of Lippia alba. Fish Physiol Biochem 38: 789-796.). On the other hand, (+)-spathulenol promoted anesthesia in lower concentrations (51.2 mg L-1) than S-(+)-linalool (153 mg L-1) isolated from the EO of L. alba in silver catfish (Heinzmann et al. 2011HEINZMANN BM, BALDISSEROTTO B, FLORES EMM, SCHMIDT D, CARON BO, SOUZA VQ, HELDWEIN CG, PARODI TV, GAI EZ AND ROMAN C. 2011. Processo de obtenção de composto anestésico de Lippia alba, composto anestésico obtido e uso de composto como anestésico. Brazilian Patent PI1103966-3.). Additionally, with S-(+)-linalool, fish did not recover within 30 min, similar to (+)-spathulenol in the present study.

(+)-Spathulenol at 2.6 mg L-1 was shown to be a safe compound to sedate silver catfish for 24h. An opposite pattern occurred with EOA, because mortality was noted after exposure for the same period of time. Spathulenol at 8 mg L-1for 24h was classified by Ito et al. (2004)ITO H, IWAMORI H, KASAJIMA N, KANEDA M AND YOSHIDA T. 2004. Kunzeanones A, B, and C: novel alkylated phloroglucinol metabolites from Kunzea ambígua. Tetrahedron 60: 9971-9976.as ichthyotoxic in medaka, Oryzias latipes. It is probable that this concentration of spathulenol led to S5 of anesthesia induction (medullar collapse) and thus, caused mortality to this fish. An anesthetic may induce different results due to variations between fish species (Zahl et al. 2012ZAHL IH, SAMUELSEN O AND KIESSLING A. 2012. Anaesthesia of farmed fish: implications for welfare. Fish Physiol Biochem 38: 201-218.).

A relationship between concentration and recovery time was noted in fish exposed to (-)-caryophyllene oxide and (-)-guaiol, while the other tested compounds did not show the same relation. Studies with eugenol and EO of L. alba in silver catfish also showed that the concentration of these substances did not influence in the recovery time (Cunha et al. 2010aCUNHA MA, ZEPPENFELD CC, GARCIA LO, LORO VL, FONSECA MB, EMANUELLI T, VEECK APL, COPATTI CE AND BALDISSEROTTO B. 2010a. Anesthesia of silver catfish with eugenol: time of induction, cortisol response and sensory analysis of fillet. Cienc Rural 40: 2107-2114., bCUNHA MA, BARROS FMC, GARCIA LO, VEECK APL, HEINZMANN BM, LORO VL, EMANUELLI T AND BALDISSEROTTO B. 2010b. Essential oil of Lippia alba: a new anesthetic for silver catfish, Rhamdia quelen. Aquaculture 306: 403-406.).

The fractionation of EOA resulted in a decrease of the anesthetic potential. The isolated compounds did not promote anesthesia at proportional concentrations to those of the constituents in the EOA. Moreover, the association of (-)-guaiol and (+)-spathulenol caused a decrease in the time required to reach stage S3a. These evidences indicate that the anesthetic and sedative effects are due to the synergism of the constituents present in EOA. Similar results were observed regarding the anticonvulsive activity of the EO of Ocimum gratissimum in mice. Moreover, the EO of this plant was more effective, than their major constituents tested alone or in association, eugenol, 1,8-cineole andE-caryophyllene, which were evaluated in the same proportion found for the EO (Galindo et al. 2010GALINDO LA, PULTRINI AM AND COSTA M. 2010. Biological effects of Ocimum gratissimum L. are due to synergic action among multiple compounds present in essential oil. J Nat Med 64: 436-441.).

Conversely, the hypothesis that the minor constituents are responsible for the anesthetic activity of EOA in silver catfish cannot be ruled out, since the fractions of lower yield were not tested. Essential oils contain several substances, which can show different mechanisms of action. These different mechanisms can act in synergy resulting in improved activity (Harris 2002HARRIS R. 2002. Synergism in the essential oil world, Int. J Aromath 12: 179-186.), but antagonism can also occur (Savelev et al. 2003SAVELEV S, OKELLO E, PERRY NSL, WILKINS RM AND PERRY EK. 2003. Synergistic and antagonistic interactions of anticholinesterase terpenoids in Salvia lavandulifolia essential oil. Pharmacol Biochem Behav 75: 661-668.). Pharmacokinetic synergism can also be observed, where absorption, distribution, biotransformation or elimination of a compound can be influenced by other substance, obtaining positive results (Spinella 2002SPINELLA M. 2002. The importance of pharmacological synergy in psychoactive herbal medicines. Altern Med Rev 7: 130-137.).

EOA showed anesthetic properties, however, it should be used carefully in silver catfish due to the observed side effects. Additionally, EOA had a higher induction time than ideal, of 3-5 min (Cunha et al. 2010bCUNHA MA, BARROS FMC, GARCIA LO, VEECK APL, HEINZMANN BM, LORO VL, EMANUELLI T AND BALDISSEROTTO B. 2010b. Essential oil of Lippia alba: a new anesthetic for silver catfish, Rhamdia quelen. Aquaculture 306: 403-406.). The presence of E-(-)-pinocamphone and (-)-caryophyllene oxide contributed to the side effects of EOA, whereas anesthetic activity was partially related to (+)-spathulenol. Then, higher concentration of (+)-spathulenol, and lower or absent amounts of E-(-)-pinocamphone, could contribute to improve the anesthetic activity and safety of the EOA. (+)-Spathulenol was considered a promising anesthetic and sedative for aquatic animals.

ACKNOWLEDGMENTS

This study was supported by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq; process 470964/2009-0) and Fundação de Amparo à Pesquisa do Estado do Rio Grande do Sul - Programa de Apoio a Núcleos de Excelência (FAPERGS - PRONEX; process 10/0016-8). B. Baldisserotto received a research fellowship from CNPq and S.C. Benovit, J. Salbego and L.L. Silva received a graduate fellowships from Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES, Brazil) The authors are grateful to Professor Marcos Sobral (UFSJ, MG) and Professor Solon Jonas Longhi (UFSM, RS) for the identification of A. gratissima.

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Publication Dates

  • Publication in this collection
    28 July 2015
  • Date of issue
    Sept 2015

History

  • Received
    06 June 2014
  • Accepted
    05 Dec 2014
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