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Relaxing effect of eugenol and essential oils in Pomacea canaliculata/Efeito relaxante de eugenol e oleos essenciais em Pomacea canaliculata.

Adriane Erbice Bianchini, Jessyka Arruda da Cunha, Isabel Cristina Markowski Brusque Carlos Garrido Pinheiro, Bianca Schindler, Berta Maria Heinzmann, Bernardo Baldisserotto

INTRODUCTION

Many mollusk species have economic importance, such in the production of pearls or in research (AQUILINA & ROBERTS, 2000; WYETH et al., 2009). Anesthetic substances have been used to improve their breeding in captivity (GARR et al., 2012), minimizing unpleasant sensations that these animals are likely to experience in handling and surgeries (COOPER, 2011), facilitating pearl withdrawal in bivalves (AQUILINA & ROBERTS, 2000). Almost all anesthetic substances used in shellfish are synthetic products such as benzocaine, MS-222 and 2-phenoxyethanol, and the minority are plant extractives (GARR et al., 2012). Essential oils (EOs) of Lippia alba and Aloysia triphylla (PARODI et al., 2012), for example, and isolated substances such as eugenol (GOMES et al., 2011; PARODI et al., 2012) and linalool (HELDWEIN et al., 2014) are effective to anesthetize fish and shrimps. Thus, we can assume that EOs may also be effective in mollusks.

Although some shellfish species have economic importance, others, such as Pomacea canaliculata, have become pests in agriculture and aquaculture because they are highly invasive and difficult to eliminate or control biologically (HAYES et al., 2015). In addition, they serve as intermediate hosts of disease-causing human parasites (SONG et al., 2016). Pomacea canaliculata is a gastropod from the family Ampullariidae, popularly known as golden apple snail. The uncontrolled spread of these gastropods has increased the demand for preventive and control measures through products that are non-toxic to other non-target organisms and the environment, as synthetic products used for this purpose have high residual effects (CALUMPANG et al., 1995). Products extracted from plants are promising alternatives because they are biodegradable and from renewable sources; they also have fewer adverse effects on the ecosystem. There are studies reporting molluscicidal activity of some plant extracts against P. canaliculata (DAI et al., 2011; KIJPRAYOON et al., 2014) and other mollusk species (RODRIGUES et al., 2013; HAMED et al., 2015). In this context, the aim of this study was to evaluate possible molluscicidal and/or relaxing effects of eugenol and the EOs of Origanum majorana, Ocimum americanum, Hesperozygis ringens, and Piper gaudichaudianum on P. canaliculata.

MATERIALS AND METHODS

The animals were obtained from earth ponds in a fish culture in Sao Joao do Polesine city (Rio Grande do Sul, Brazil) and housed in the Fish Physiology Laboratory at the Universidade Federal de Santa Maria (UFSM) under the following conditions: temperature 19.02 [+ or -] 0.02[degrees]C, pH 7.85 [+ or -] 0.01, and dissolved oxygen levels 8.59[+ or -]0.25mg [L.sup.-1]. Identification of specimens was carried out by Dr. Carla Bender Kotzian (Department of Biology, UFSM).

Essential oils and isolated substance

Eugenol and the EO of leaves of O. majorana were purchased from Sigma-Aldrich, Brazil, and Vimontti[R] (Agroindustria Sao Caetano Ltda, Brazil), respectively. The other plant species (O. americanum, H. ringens, and P. gaudichaudianum) were collected in Sao Joao do Polesine, Sao Francisco de Assis, and Santa Maria cities, Rio Grande do Sul State, respectively. The EOs were obtained from the aerial parts of the plants by hydrodistillation using a Clevenger apparatus for 2h and subsequently stored in amber glasses at -4[degrees]C in the dark prior to phytochemical analysis (EUROPEAN PHARMACOPOEIA, 2007).

Phytochemical analysis

Characterization of EOs was performed by chromatographic analysis using an Agilent 7890A gas chromatograph coupled to an Agilent 5975C mass selective detector (GC-MS). The identification of constituents was achieved by the comparison of retention indices, obtained by the use of a calibration curve of n-alkanes injected at the conditions mentioned for the samples, and the mass fragmentation patterns with the data of ADAMS (2009), NIST (2010), VIEIRA et al. (2014), and PINHEIRO et al. (2016).

Induction of relaxation and recovery

Animals (11.87 [+ or -] 0.76g) were placed in continuously aerated 1-L aquaria (n=five animals/ aquarium) in duplicate. Compounds and EOs previously dissolved in 95% ethanol (1:10) were tested at concentrations of 100, 250, 500, and 750[micro]L [L.sup.-1]. Control experiments were performed using aquaria containing only ethanol at a concentration equivalent to the highest dilution used for samples. The evaluation of relaxation was based on the experimental protocol described by GARR et al. (2012), with some adjustments. The maximum evaluation time was 40min, with the percentage of relaxed animals registered every 10min. Animals were considered relaxed when they did not show any resistance to the pulling of the operculum with the aid of a forceps. After relaxation, animals were transferred to anesthetic-free aquaria. Recovery time was evaluated at 10min intervals and the animal was considered recovered when it presented resistance to pulling of the operculum. Integrity and mortality were evaluated 24 hours after the experiment.

Molluscicidal effect

An additional experiment was performed with gastropods exposed to 10, 25, or 50[micro]L [L.sup.-1] of the EOs of H. ringens and P. gaudichaudianum, which revealed toxicity in the first experiment. The animals were placed in 1-L aquaria (n = five animals aquarium-1, in duplicate) with each EO previously solubilized in 95% ethanol (1:10). A control group (water only) and another exposed to ethanol (450[micro]L [L.sup.-1]) were also observed. Mortality and integrity of animals were evaluated after 24 hours.

Statistical analysis

Data were submitted to Levene's test to determine homogeneity of variances, after which one-way ANOVA was performed, followed by Tukey's test or Kruskal-Wallis test, when appropriate.

The tests were performed using the software package Statistica (version 11.0). The effective concentration that relaxes 50% of the animals ([EC.sub.50]) was calculated with the SigmaPlot software. The minimum significance level was 95% (P<0.05). Data are reported as means [+ or -] SEM.

RESULTS

Chemical composition of essential oils

The EOs presented as major constituents the following compounds: terpinen-4-ol (29.13%), [gamma]-terpinene (17.99%), and [alpha]-terpinene (9.72%) in the EO of O. majorana; dillapiole (84.32%) in the EO of P. gaudichaudianum; eucalyptol (24.31%), linalool (23.44%), and eugenol (14.86%) in the EO of O. americanum; and pulegone (74.27%) in the EO of H. ringens.

Induction of relaxation and recovery

Ethanol did not show any relaxing effect on animals within the time frame of the analysis. Essential oils, as well as eugenol, did not promote relaxation at 100[micro]L [L.sup.-1], but the EOs of O. americanum and O. majorana and eugenol promoted relaxation at concentrations above 100gL [L.sup.-1]. Use of 250[micro]L [L.sup.-1] eugenol induced relaxation of only 40% of the animals, but most animals relaxed at higher concentrations. The EO of O. americanum had a higher percentage of relaxed animals (70%) at 250[micro]L [L.sup.-1], with the effect decreasing to 50% with increasing concentrations. In contrast, the two lowest concentrations (250 and 500[micro]L [L.sup.-1]) of EO of O. majorana relaxed only 10% of the animals, while at 750[micro]L [L.sup.-1], 80% of the animals relaxed (Figure 1A). Other EOs tested (P gaudichaudianum and H. ringens) did not promote relaxation, but caused a loss of mucus in the animals at all concentrations tested. There was no significant difference between the relaxation time in the concentrations of eugenol and EO of O. americanum. It was not possible to perform statistical analysis of the relaxation time between the different concentrations of the EO of O. majorana because of the small number of animals that relaxed (Figure 1B). The [EC.sub.50] of eugenol and the EO of O. majorana were 269.5 and 662.1[micro]L [L.sup.-1], respectively. The [EC.sub.50] of the EO of O. americanum could not be calculated because no significant relationship concentration-response was reported.

The percentage of recovered animals exposed to eugenol was not related to concentration. Mollusks exposed to the highest concentration of the EO of O. americanum presented a higher percentage of recovery, and the few specimens exposed to all concentrations of the EO of O. majorana that relaxed showed recovery within the observation time (Figure 1C). Recovery time was not significantly affected by eugenol concentration. Low number of animals that recovered from the exposure to the EOs did not allow a statistical analysis of this parameter (Figure 1D).

Eugenol and the EOs of O. americanum and O. majorana did not cause mortality after 24 hours; however, the EOs of H. ringens and P. gaudichaudianum at 750[micro]L [L.sup.-1] provoked 20 and 30% mortality, respectively. Other animals exposed to P gaudichaudianum and H. ringens and remained alive exhibited fragile and brittle shells.

Molluscicidal effect

No mortality was observed in any of the treatments after 24h, but gastropods exposed to 50[micro]L [L.sup.-1] of both EOs tested showed damages in their shells, as reported in the previous experiment.

DISCUSSION

Eugenol and the EOs of O. americanum and O. majorana promoted muscle relaxation in P. canaliculata. This effect was expected, since these compounds have an anesthetic effect in fish (GOMES et al., 2011, SILVA et al., 2015; CUNHA, et al., 2017). Eugenol also presented anesthetic activity in crustaceans (PARODI et al., 2012). In gastropods, clove oil at 350[micro]L [L.sup.-1] showed 100% efficacy in relaxing Pomaceapaludosa within about 20min (GARR et al., 2012), while eugenol, the main constituent of clove oil, was 100% effective in the present study at relaxing P canaliculata only at a higher concentration (750 [micro]L [L.sup.-1]) and with a longer exposure time. Animals required less time to recover from eugenol exposure, but only 40% of the animals that relaxed with 750 [micro]L [L.sup.-1] recovered within the observation period. The intermediate concentration of eugenol tested (500 [micro]L [L.sup.-1]) showed the best result, with 100% animals recovered.

The EO of O. americanum presented as major constituents eucalyptol, linalool, and eugenol, corroborating the results reported by SILVA et al. (2015). The relaxing effect of this oil can be attributed to eugenol, which has been proven to have a relaxing effect in P. canaliculata, and linalool, which also has an anesthetic effect on silver catfish, Rhamdia quelen (HELDWEIN et al., 2014). In contrast, eucalyptol showed no anesthetic activity up to 17mg [L.sup.-1] when tested in this species (HELDWEIN, 2011), but was effective in blocking the excitability of the superior cervical ganglion neurons of in vitro rats (FERREIRA-DA-SILVA et al., 2009). Most likely, the relaxing effect of the EO of O. americanum is due to the synergistic action of its compounds. Regarding efficacy, it was possible to relax 70% of the animals at 250mL [L.sup.-1] within 30min, whereas higher concentrations had lower efficacy; although, there was no difference in relaxation time. Similar results were obtained for Ostrea edulis anesthetized with urethane and menthol; namely, lower concentrations were more effective (CULLOTY & MULCAHY, 1992).

Terpinen-4-ol, [gamma]-terpinene, and [alpha]-terpinene were the main constituents identified in the EO of O. majorana. This same EO presented anesthetic activity in silver catfish at concentrations [greater than or equal to] 100 [micro]L [L.sup.-1] (CUNHA, et al., 2017). In P canaliculata, the relaxing effect was obtained from 250 [micro]L [L.sup.-1] (10% efficacy), but only produced a satisfactory effect at a higher concentration (750 [micro]L [L.sup.-1]). However, all animals that relaxed with EO of O. majorana recovered up to 40min. High concentrations of nembutal (1,000[micro]L [L.sup.-1]) and 2-phenoxyethanol (3,000 [micro]L [L.sup.-1]) were also necessary for the relaxation of Haliotis iris (AQUILINA & ROBERTS, 2000) and Pomacea paludosa (GARR et al., 2012), respectively.

The EOs of H. ringens and P. gaudichaudianum at concentrations of up to 750 [micro]L [L.sup.-1] did not cause muscle relaxation in the golden apple snail. The EO of H. ringens promoted deep anesthesia (111 to 554 [micro]L [L.sup.-1]) without mortality in silver catfish (SILVA et al., 2013), and its effect was attributed to the presence of pulegone (96.66%), an allosteric modulator of GABA (TONG & COATS, 2010). As GABA immunoreactive neurons are reported in the central nervous system of gastropods (HATAKEYAMA & ITO, 2000; GUNARATNE et al., 2014; 2016), this result was unexpected.

Pomacea canaliculata can cause losses in agriculture (COWI, 2002) and public health problems because it is an intermediate host of the nematode Angiostrongylus cantonensis, responsible for causing meningitis in humans (SONG et al., 2016). For this reason, the molluscicidal activity of the EOs of H. ringens and P. gaudichaudianum were tested. Both EOs did not cause mortality within 24h exposure at the lower tested concentrations (10, 25, and 50 [micro]L [L.sup.-1]), but loss of mucus and degradation of the calcareous shell were observed. However, as 750 [micro]L [L.sup.-1] of both EOs provoked 20-30% mortality within 40min, it is likely that the 100-750 [micro]L [L.sup.-1] range would induce 100% mortality. Deleterious effects caused by these EOs may be due to some of their constituents or the synergistic combination of several compounds. These EOs have a larvicidal effect against the Coenagrionidae family and fungitoxic activity in vitro against species of pathogenic fungi and wood decay (SILVA et al., 2014; SCHINDLER, 2015). Dillapiole, the major constituent of the EO of P gaudichaudianum, is present in other species of the genus, and its insecticidal activity has previously been described (VOLPE et al., 2016). Natural products, such as glycosides extracted from fresh leaves of Nerium indicum (DAI et al., 2011) and fractions of the methanolic extract of seed flour Camellia oleifera (KIJPRAYOON et al., 2014), also caused P canaliculata mortality. Molluscicidal effect of the EO Cymbopogon winterianus against Biomphalaria glabrata (RODRIGUES et al., 2013) and the hexane and ethyl acetate extracts from the aerial parts of Atriplex inflata against Galba truncatula (HAMED et al., 2015) have also been described.

CONCLUSION

Based on the results obtained, we can conclude that eugenol, as well as the EOs of O. americanum and O. majorana, have relaxing activity in P. canaliculata and can be used for research purposes in this species. The EOs of H. ringens and P. gaudichaudianum did not promote relaxation, even at high concentrations, and were also not effective as molluscicides at up to 50 [micro]L [L.sup.-1] against this species.

http://dx.doi.org/10.1590/0103-8478cr20170210

ACKNOWLEDGMENTS

B. Baldisserotto and B.M. Heinzmann received Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq) research fellowships. J.A. Cunha, B. Schindler, and C.G. Pinheiro received Coordenacao de Aperfeiqoamento de Pessoal de Nivel Superior (CAPES) scholarships.

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Adriane Erbice Bianchini (1) Jessyka Arruda da Cunha (1) Isabel Cristina Markowski Brusque (1) Carlos Garrido Pinheiro (2) Bianca Schindler (2) Berta Maria Heinzmann (3) Bernardo Baldisserotto (1,4,*)

(1) Departamento de Fisiologia e Farmacologia, Universidade Federal de Santa Maria (UFSM), Santa Maria, RS, Brasil.

(2) Departamento de Engenharia Florestal, Universidade Federal de Santa Maria (UFSM), Santa Maria, RS, Brasil.

(3) Departamento de Farmacia Industrial, Universidade Federal de Santa Maria (UFSM), Santa Maria, RS, Brasil.

(4) Programa de Pos-graduacao em Zootecnia, Universidade Federal de Santa Maria (UFSM), 97105-900, Santa Maria, RS, Brasil. E-mail: bbaldisserotto@hotmail.com. * Corresponding author.

Caption: Figure 1--Relaxation percentage (a), time to induction of relaxation (b), recovery percentage (c), and recovery time (d) of Pomacea canaliculata exposed to eugenol and essential oils of Origanum majorana (EO O. majorana) and Ocimum americanum (EO O. americanum). Different letters indicate a significant difference between concentrations in the same treatment by one-way ANOVA and Tukey's test (P < 0.05). No statistical analysis was performed in "a" and "c" and for the EO of O. majorana (b) and the EOs of O. americanum and O. majorana (d).
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