Essential oils from leaves of Pilocarpus pennatifolius and Cordia verbenacea in silver catfish (Rhamdia quelen): cardiovascular aspects and participation of the GABAergic system in the sedative and anesthetics effects

Carlos Herminio Magalhães Fortes¹, Bernardo Baldisserotto^1,2, Frederico Dimas Fleig³, Valério Valdetar Marques Portela Júnior² and Berta Maria Heinzmann^1,3

PDF: EN    XML: EN | Cite this article

Associate Editor: Renata Moreira

Section Editor: José Birindelli

Editor-in-chief: Carla Pavanelli

Abstract​

---

Introduction​

---

Several synthetic drugs are used in global aquaculture to induce depression of the central nervous system (CNS), especially to lead to stages of sedation and anesthesia. Those commonly used to minimize stressful effects are mainly tricaine methanesulfonate (MS-222) and benzocaine (Mattson, Ripple, 1989; Gomes et al., 2001). However, they may induce undesirable adverse effects, such as hypoxia, loss of mucus, tissue irritation, among others (Sneddon, 2012; Zahl et al., 2012; Barbas et al., 2017). Therefore, agents of natural origin, such as essential oils (EOs), can be an excellent alternative to synthetic drugs, because they have similar or greater efficacy and have lower toxicity rates for fish (Okamoto et al., 2009; Souza et al., 2019). However, some physiological parameters need to be evaluated when using these sedatives and/or anesthetics, as they can be stressful for fish.

Primary stress induces a significant increase in circulating concentrations of catecholamines and corticosteroids, which act to stimulate glycogen hydrolysis. As a result, there is an increase in blood glucose levels and heart rate (hr) (Jerez-Cepa, Ruiz-Jarabo, 2021). Secondary responses occur through an increase in the levels of these hormones (Barton, 2002; Maricchiolo, Genovese, 2011). When the stressor persists, tertiary responses are observed, resulting in severe damage to the fish well-being and health (Maricchiolo, Genovese, 2011; Robinson et al., 2019; Sutherland et al., 2019; Jerez-Cepa; Ruiz-Jarabo, 2021).

It is extremely important to know the mechanism of action of sedative and/or anesthetic agents, especially to predict important aspects regarding sedation and/or anesthesia and also to determine possible antagonists in case of need to reverse the drug effect for some specific reason, such as an administration error. In this context, many anesthetics exert their effects through modulation of the gamma-aminobutyric acid (GABA) receptor complex, the main CNS inhibition neurotransmitter (Sieghart, 2006). Activation for synaptic release of GABA occurs through ionotropic (GABAa and GABAc) and metabotropic (GABAb) receptors (Tanelian et al., 1993; Alexander et al., 2008). Expressions of this system have already been observed in several vertebrates, with functional GABAergic evidence described in the brain of fish (Kim et al., 2004; Delgado, Schmachtenberg, 2008; Garlet et al., 2019a). Benzodiazepines (BDZ) have high affinity for a specific site on the GABAa receptor, and through its binding, the pharmacological action occurs through allosteric modulation (Heldwein et al., 2012). Diazepam (DZP/ BDZ agonist) and flumazenil (FLU/imidazo-benzodiazepine), an antagonist of the central effects of BDZ by competition for the BDZ site (Darragh et al., 1982) are widely used to establish possible positive interaction effects and mechanism of action of EOs, respectively (Heldwein et al., 2012; Silva et al., 2012; dos Santos et al., 2017). Previous work suggested possible GABAergic modulation through GABAa receptors for some EOs, such as those of Ocimum gratissimum and Lippia alba in silver catfish, Rhamdia quelen (Quoy & Gaimard, 1824) (Heldwein et al., 2012; Silva et al., 2012).

The EOs of the plants Cordia verbenacea (CVOL) (Boraginaceae) and Pilocarpus pennatifolius (PPOL) (Rutaceae), native to South America (Bayeux et al., 2002; Carmo et al., 2018), need to have their mechanism of action elucidated, as both showed promising sedative and/or anesthetic effects in Nile tilapia, Oreochromis niloticus (Fortes et al., 2024b). Cordia verbenacea is popularly known as “erva-baleeira” (Michielin et al., 2009; Faria et al., 2023) and is found along the Brazilian coast (Dutra et al., 2016), Atlantic Forest, Amazon, and Pampa (Bayeux et al., 2002; Melo et al., 2021; Faria et al., 2023). It is worth highlighting that CVOL is extracted by Brazilian companies that produce and sell herbal medicines. Pilocarpus pennatifolius is popularly known as “jaborandi” or “canela-de-agouti” (Souza et al., 2003; Santos, Moreno, 2004). It has a latitudinal distribution (Sawaya et al., 2011) and occurs in several Brazilian states (Souza et al., 2003). Therefore, this study aimed to evaluate whether the sedative and anesthetic effects of CVOL and PPOL would involve the benzodiazepine site of the GABAa receptor. To this goal, it was assessed whether there would be a positive interaction when these EOs were associated with DZP and the reversal of the effect with the use of FLU. Blood glucose and hr were also determined in silver catfish exposed to these EOs, aiming to suggest a possible safe use of sedative and/or anesthetic concentrations in this species.

Material and methods

---

Chemicals. The synthetic drugs BDZ agonist diazepam (DZP, injectable solution 5 mg mL^-1) and antagonist flumazenil (FLU, injectable solution 0.1 mg mL^-1), were purchased from Cristália Produtos Químicos e Farmacêuticos LTDA, São Paulo, Brazil.

Plant material, extraction, and chemical analysis of natural anesthetics. Leaves of Pilocarpus pennatifolius were collected in Santa Maria, Rio Grande do Sul State, Brazil (29º11’52”S 53º16.8’56”W). The identification of the species was made by Prof. Dr. Frederico Dimas Fleig, from the Department of Forestry Sciences at the UFSM. Voucher material was deposited in the herbarium of the Department of Biology, under registration number SMDB nº 23007. Fresh leaves of P. pennatifolius were used to obtain the EO (PPOL). The EO of fresh leaves of Cordia verbenacea, CVOL, was purchased commercially from Laszlo Aromatologia Eireli (Brazil). The extraction of PPOL was carried out by hydrodistillation for three hours, in triplicate, in modified Clevenger apparatus (European Pharmacopoeia, 2007).The PPOL was sealed and stored in an amber glass bottle at -4ºC, and both EOs had a qualitative analysis of the composition and percentage of components carried out by gas chromatography in an Agilent 7890A hyphenated system, equipped with 5975C series mass selective detector and by flame ionization detector (FID), respectively. The parameters for analysis were DB5-MS fused silica capillary column (Film thickness 0.25 µm, 5% phenylmethylsiloxane, 30 m x 0.25 mm); Carrier gas 1 mL min^-1, He; Split input injection mode (1:50); Oven heating program (40°C), for 4 min/Ti, 40–320 °C at 4 °C min-1; 250 ºC injector, detector and interface temperature. The constituents were identified by comparing the fragmentation patterns of the mass spectra and the Kovats retention indices [KI], determined through a calibration curve of a homologous series of n-alkanes (C8–C40), with data from literature and the equipment library (NIST, 2008, 2024; Adams et al., 2011; Silva et al., 2015; Garlet et al., 2019b).

Animal model (Rhamdia quelen, voucher number Universidade Federal do Rio Grande do Sul, UFRGS 29744) and maintenance. The fish were purchased commercially from a fish farm near Santa Maria and transported to the Fish Physiology Laboratory at the UFSM. The fish were acclimatized for one week, kept protected from light, at 23 ºC, with constant aeration in 250 L tanks, fed three times a day (8, 12 and 19 h) until satiety with commercial feed (32% CP, Supra Water Line | Alisul Alimentos S.A.® Brazil). Every day, 30 min after feeding, feces and food debris were removed, and 10% of the water in the tanks was replaced. Dissolved oxygen levels (6.64 ± 0.22 mg L^-1) and temperature (23.0 ± 1.15 °C) were measured daily with an YSI55 oximeter (Xylem Inc, EUA), pH (7.49 ± 0.26) with a pH meter (DMPH- 2 – Digimed, SP, Brazil).

Assessment of the participation of the benzodiazepine site in the observed effect

Sedative and/or anesthetic induction. Possible central nervous depression was evaluated in juvenile silver catfish (4.98 ± 0.83 g and 6.79 ± 0.49 cm) exposed to the experimental groups (Tab. 1). The EOs were diluted previously in 95% ethanol (1:10). The anesthetic and/or sedative inducing potential and recovery was evaluated individually in 12 h-fasted juveniles in aquariums (11.5 cm high x 12.5 cm wide x 17.5 cm long) containing 1 L water with aeration according to the stages described by Gomes et al. (2011) with some adaptations (n = 6 juveniles): S2 (Deep sedation, without reaction to external stimuli); S3a (Partial loss of balance, the fish begin to swim sideways); S3b (Total loss of balance, the fish do not swim, but there is a response to pressure in the caudal peduncle and the animals remain at the bottom of the aquarium); S4 (Anesthesia and loss of reflexes, with zero response to stimuli in the caudal peduncle). Thus, when the fish reached the S4 stage or after a maximum time of 30 min, they were transferred to recovery aquaria, which contained water or FLU (5 µM), and oxygenation (Heldwein et al., 2012). Each juvenile was used only once, and the induction and recovery times were measured using a digital stopwatch. The control group, containing only ethanol, did not show effects of CNS depression (Heldwein et al., 2012; dos Santos et al., 2017).

TABLE 1 | Acronyms and concentrations used in the study of ethanol, diazepam (DZP), DZP associated with sedative and anesthetic concentrations of essential oils (EOs) of Cordia verbenacea (CVOL) or Pilocarpus pennatifolius (PPOL) and the sedative or anesthetic concentrations of these EOs in the absence of DZP. (+) Association of synthetic drug and anesthetic and/or sedative agents (n = 6 each group).

Experimental groups	Acronyms
	CVOL	PPOL
Ethanol (3000 µL L-1)	Ethanol
Diazepam (150 µM)	DZP
DZP (150 µM) + Sedative (20 mg L-1)	C1	P1
DZP (150 µM) + Anesthetic (300 mg L-1)	C2	P2
Sedative (20 mg L-1)	C3	P3
Anesthetic (300 mg L-1)	C4	P4

Sedative and/or anesthetic recovery: water versus flumazenil. Based on the evaluations and observations of the induction times of the effect on the CNS, the fish were placed in a recovery aquarium with or in the absence (n = 3) of FLU (5 µM) (Heldwein et al., 2012). This was to determine the possible involvement of the GABAergic system BDZ site in the observed effect (sedation or anesthesia) on the fish, with both aquariums containing water (1 L) and oxygenation. Thus, for both groups, the juveniles’ recovery behavior was scored after 1, 5, 10, 15, and 20 min, adapted from Heldwein et al. (2012): 0 (No sign of recovery); 0.5 (Reaction after stimulation of the caudal peduncle); 1 (No posture, but showing first sign of recovery); 1.5 (Swam irregularly and then stopped); 2 (No reflexes after external stimulus, but normal swimming); 2.5 (No reflexes after external stimulus, remaining still after swimming normally); 3 (Reflexes after external stimuli and normal swimming). At the end of 20 min, the score was added up for each fish, with 0.5 being added to this sum when agitation behavior was observed. In each observation, the fish was subjected to an external stimulus with a glass rod at the bottom of the aquarium, as these tend to remain still when not exposed to external stimuli.

Determination of blood glucose levels. Blood collect was performed in the caudal region (caudal vein) of the juveniles with a sterile hypodermic needle and syringe (1mL/CC, U100, 0.45×13, 26Gx1/2”) 30 min after the start of recovery. Fish were contained with a damp cloth, covering the cranial part of the animal (eyes). Glucose was determined using a digital glucometer (G-TECH Free¹®, South Korea) and analytical strips (G-TECH Free¹®, South Korea). Measurements were carried out on all fish exposed to different concentrations (Tab. 1) and also on six animals that were not subjected to any previously described protocol (basal group).

Determination of heart rate (HR). Fish (421.8 ± 78.3 g and 35.3 ± 3.0 cm) were exposed to 20 mg L^-1 of CVOL (C3), 20 mg L^-1 of PPOL (P3), eugenol (50 mg L^-1, positive control) and another group without any substance immersed in the water (negative control) (n = 8 each group). When sedation (S2) was confirmed, the fish was removed from the aquarium and contained in a damp cloth (cranioventral position), to assess HR using an ultrasound device with color doppler (Mindray® Z6 Vet Gold). Heartbeats were counted with a digital stopwatch for 15 sec and the number was multiplied by four to obtain the individual’s heart rate. After the treatments, the fish were placed in aquariums with 10 L of water and aeration for full recovery.

Statistical analysis. Levene’s test was performed to evaluate the homoscedasticity of the data. The data are presented as mean ± standard deviation, with the significance level considered 95% (p < 0.05). Recovery scores were analyzed by One-Way ANOVA, followed by Tukey’s test. The Kruskal-Wallis test, for non-parametric data (anesthetic induction, hr, glucose), followed by the Dunn test, were used to compare the other data, using the Prism version 9.0® software.

Results​

---

Chemical composition of EOs and extraction yield. The identification of the PPOL composition was 99.8%, with 2-Undecanone (57.2%) and 2-Tridecanone (28.3%) as major components. The average yield of PPOL was 0.1 ± 0.007%. The major compound of CVOL was α-Pinene (34.8%) (Tab. 2).

TABLE 2 | Chemical composition of the essential oils of Cordia verbenacea (CVOL) and Pilocarpus pennatifolius (PPOL). Subtitle: ^aRI = Retention index; ^bExperimental; ^cLiterature Adams (2011) and NIST (2024).

RIa Eb	RIa LC	Compound	%
			CVOL	PPOL
929	939	α-pinene	34.8	–
1027	1028	Limonene	1.3	–
1292	1291	2-Undecanone	–	57.2
1301	1303	2-Undecanol	–	1.5
1388	1392	Elemene	2.7	–
1388	1387	Damascone	–	1.3
1453	1452	a-Humulene	3.8	–
1479	1480	Germacrene D	–	10.4
1494	1494	2-Tridecanone	–	28.3
1504	1504	Undecenol acetate	–	0.5
1574	1571	Spathulenol	2.8	0.6
Identified components			45.4	99.8
Unidentified components			54.6	0.2

Assessment of the participation of the benzodiazepine site in the observed effect

Sedative and/or anesthetic induction. DZP led silver catfish to stages S2, S3a, and S3b, but did not anesthetize the fish at the tested concentration. The times for DZP induce S2 and S3a are significantly higher than those of the association DZP and anesthetic concentration of CVOL (Figs. 1A–C). The fish exposed to 20 mg L^-1 CVOL (C3) reached only S2 and the time to reach this stage was higher than the combination DZP and anesthetic concentration of CVOL and the anesthetic concentration of CVOL alone (Fig 1). The combination DZP and anesthetic concentration of CVOL was faster to induce S3 and S4 than the anesthetic concentration of CVOL alone (Figs. 1C, D).

FIGURE 1| Times to juveniles of Rhamdia quelen reach the stages S2 (A), S3a (B), S3b (C) and S4 (D) in response to diazepam (DZP) 150 µM, DZP (150 µM) associated to the sedative (C1) or anesthetic (C2) concentrations of Cordia verbenacea essential oil (CVOL), and sedative (C3) and anesthetic (C4) concentrations of CVOL in the absence of DZP (n = 6). C1: DZP 150 µM + CVOL 20 mg L-1; C2: DZP 150 µM + CVOL 300 mg L-1; C3: CVOL 20 mg L-1; C4: CVOL 300 mg L-1.

Rhamdia quelen juveniles exposed to 20 mg L^-1 PPOL alone (P3) reached only stage S2, but in combination with DZP (P1) reached up to S3b (Fig. 2). Fish submitted to both concentrations of PPOL with DZP (P1 and P2) induced stage S2 faster than P3, and those exposed to P2 reached stage S3b faster than fish exposed to P1 and P4. Fish exposed to P2 reached S4 faster than P4 (Fig. 2).

FIGURE 2| Times to juveniles of Rhamdia quelen reach the stages S2 (A), S3a (B), S3b (C) and S4 (D) in response to diazepam (DZP) 150 µM, DZP (150 µM) associated to the sedative (P1) or anesthetic (P2) concentration of Pilocarpus pennatifolius essential oil from leaves (PPOL), and sedative (P3) and anesthetic (P4) concentrations of PPOL in the absence of DZP (n = 6). [P1 (DZP 150 µM + PPOL 20 mg L-1); P2 (DZP 150 µM + PPOL 300 mg L-1); P3 (PPOL 20 mg L-1); P4 (PPOL 300 mg L-1)].

Sedative and/or anesthetic recovery: water versus flumazenil. The recovery scores of juveniles exposed to both EOs alone or in association with DZP were higher in those that recovered in FLU than those that recovered in water (Fig. 3). Furthermore, fish exposed to C2 placed to recover in water (100%) reached the S5 stage. After 6 h of completing observations of the recovery of juveniles exposed to C2, no interest in the diet provided was observed. After 24 and 48 h, all juveniles fed, and no additional mortality was observed.

FIGURE 3| Sum of the total recovery scores of fish (n = 6) exposed only to diazepam® (DZP 150 µM) or associated with essential oils (EOs) (C1; C2; P1; P2) and EOs in the absence of DZP at concentrations of 20 and 300 mg L-1 (C3; C4; P3; P4). The fish were exposed to these until they reached S4 or for a maximum time of 30 min and then placed in an aquarium containing only water (n = 3) or the benzodiazepine antagonist drug flumazenil® (n = 3). Refers to (A) CVOL – Cordia verbenacea essential oil from leaves [C1 (DZP 150 µM + CVOL 20 mg L-1); C2 (DZP 150 µM + CVOL 300 mg L-1); C3 (CVOL 20 mg L-1); C4 (CVOL 300 mg L-1)] and (B) PPOL – Pilocarpus pennatifolius essential oil from leaves [P1 (DZP 150 µM + PPOL 20 mg L-1); P2 (DZP 150 µM + PPOL 300 mg L-1); P3 (PPOL 20 mg L-1); P4 (PPOL 300 mg L-1)]. The scores were recorded at 1, 5, 10, 15 and 20 min, with the minimum score (0) being no sign of recovery and the maximum (3) fish swimming normally and showing reflexes after stimuli. Furthermore, according to Heldwein et al. (2012), the sum of the scores was added the value of 0.5 when the fish showed signs of agitation after the 20 min assessment. One-way ANOVA followed by Tukey’s test. Different letters indicate statistical difference between water and flumazenil within each treatment.

Blood glucose levels. Blood glucose levels were higher in fish exposed to C2 than basal group and those exposed to ethanol, while juveniles exposed to C4 presented higher levels than basal group (Fig. 4A). The C4 group showed higher blood glucose levels than the C3 group. In the second experiment, exposure to DZP led to higher blood glucose levels than basal group (Fig. 4B).

FIGURE 4| Blood glucose levels in Rhamdia quelen juveniles exposed to diazepam® (DZP) 150 µM, ethanol (3000 µL L-1), (A) CVOL – Cordia verbenacea essential oil from leaves [C1 (DZP 150 µM + CVOL 20 mg L-1); C2 (DZP 150 µM + CVOL 300 mg L-1); C3 (CVOL 20 mg L-1); C4 (CVOL 300 mg L-1)] and (B) PPOL – Pilocarpus pennatifolius essential oil from leaves [P1 (DZP 150 µM + PPOL 20 mg L-1); P2 (DZP 150 µM + PPOL 300 mg L-1); P3 (PPOL 20 mg L-1); P4 (PPOL 300 mg L-1)]. Measurements were also carried out on fish from the basal group (only exposed to water). Different letters indicate significant difference between treatments (n = 6).

Cardiovascular effects: hr. Sedation (S2) with eugenol took longer than with C3. Exposure to eugenol increased heart rate, while both C3 and P3 did not change it compared to basal group (Fig. 5).

FIGURE 5| Time to reach sedation (A) and (B) heart rate of Rhamdia quelen fish exposed to eugenol (50 mg L-1) and to 20 mg L-1 of the essential oils from the leaves of Cordia verbenacea (C3) and Pilocarpus pennatifolius (P3). The basal group does not appear in figure a because fish were only exposed to water. Different letters indicate significant difference between treatments (n = 8).

Discussion​

---

CVOL. CVOL has a proven anesthetic effect on native and exotic fish, such as juvenile silver catfish (Fortes et al., 2024a) and Nile tilapia (Fortes et al., 2024b). α-pinene (34.8%) was found in CVOL in a percentage similar (29.6%) to that described by Carvalho Jr. et al. (2004). This compound was previously observed in EOs from young (26.8%) and old (26.1%) leaves of Nectandra megapotamica, which induced sedation and anesthesia in Centropomus parallelus. Spathulenol, which was found in a low percentage in CVOL (2.8%), was also found in small amounts (1.2–2.1%) in the EO of A. citriodora and showed a direct relationship with time to induce deep anesthesia in silver catfish (Parodi et al., 2020). The EOs from N. megapotamica leaves did not prevent the stress caused by anesthesia, according to the parameters evaluated (Tondolo et al., 2013). In our study, the highest concentration of CVOL also apparently did not prevent stress, as there was an increase in glucose levels compared to the basal group.

The results indicate the possibility of a positive interaction between CVOL and DZP and participation of the BDZ site of GABAa receptors in the effects observed with CVOL, highlighting the association of the sedative concentration (20 mg L^-1) with DZP (C1), which resulted in S4. Apparently, this is a strong indication of synergism or additive effect between the synthetic and natural agents, as C3 only resulted in S2 and DZP alone did not reach S4, signaling that the association possibly resulted in greater CNS depression. Another fact that could strengthen the suggestion of an additive effect or synergism between the drugs would be the 50% of fish that reached the S5 stage after exposure to C2, possibly due to deepening anesthesia (Garlet et al., 2017). The anesthetic concentrations of CVOL with DZP apparently proved to be more effective in inducing the deeper anesthetic stages (S3b and S4), that is, faster than the isolated use of CVOL, being yet another possible evidence of aditive or synergistic interaction between these compounds. Regarding the possible elucidation of the mechanism of action of CVOL, a significant statistical difference was found in the fish recovered in FLU compared to those that recovered in its absence (Heldwein et al., 2012; Silva et al., 2012), suggesting therefore the involvement of the benzodiazepine site of the GABAergic receptor complex in the effects observed for CVOL.

The data found in the literature reinforces these results, as some studies indicate that α-pinene (Aoshima, Hamamoto, 1999) is a positive modulator of GABAa receptors. Furthermore, CVOL presents spathulenol at 2.8%, a constituent that has already been indicated with possible GABAergic involvement in its central depressant effects in rodents and fish (Oliveira Júnior et al., 2018; Garlet et al., 2019a). However, other possible mechanisms of action and interactions cannot be ruled out, as CVOL has other compounds that have not been identified. A factor to highlight in these EOs is the recovery times, as in studies with tilapia (Fortes et al., 2024b) and silver catfish (Fortes et al., 2024a) they were quite prolonged, that is, it could be an indication of high potency. This is because, when analyzing synthetic drugs, the most potent are those that recover later, for example, fish in an immersion bath with propofol recover later than those in an immersion bath with benzocaine (Gonçalves, Giaquinto, 2020). This reflects the high potency of propofol compared to benzocaine, therefore, the choice of inducing agent will depend on the need for the duration of central depression (Gonçalves, Giaquinto, 2020). The association of EOs with synthetic pharmacological agents, such as DZP, has also been observed in other studies as a strategy to improve anesthetic efficacy (Silva et al., 2012; Heldwein et al., 2014; dos Santos et al., 2017). Furthermore, the faster and more profound effect presented in this study when associating DZP with CVOL demonstrates this trend and can be explained by the interaction of DZP with the benzodiazepine site in the CNS, which apparently facilitates the sedative and anesthetic action of EOs, as observed in this study. The interaction between the compounds seems to be essential for obtaining faster and more effective responses.

The higher blood glucose levels in fish from the C2 group suggests that C2 caused stress to the juveniles. A different result was observed in fish exposed to P2, a sample that corresponds to the association of DZP + PPOL, which did not increase the glucose levels of juveniles compared to the basal group. Thus, the results indicate that CVOL associated with DZP cannot be used for deep anesthesia in juvenile silver catfish, as it apparently leads to strong central depression, causes stress and death when anesthesia is not reversed with a BZP antagonist, such as flumazenil. Therefore, when longer procedures need to be performed for example, transport or something that requires longer-lasting sedation for the fishing industry, the use of CVOL could be suggested. Furthermore, the sedative concentration in silver catfish juveniles did not increase blood glucose levels compared to the basal group and when evaluated in fish of larger size and weight, it caused good deep sedation and did not change heart rate compared to the basal group, unlike what was observed for eugenol. In this sense, the sedative concentration of 20 mg L^-1 can be used to transport juvenile silver catfish and also larger fish. However, more stress parameters must be evaluated, such as plasma cortisol and others.

PPOL. Sedative and/or anesthetic activities have not yet been described for 2-Undecanone (57.2%) and 2-Tridecanone (28.3%), the main compounds found in PPOL. However, germacrene D (10.4%) was identified in EOs from young (9.1%) and old (9.2%) leaves of N. megapotamica (Tondolo et al., 2013) and also in the EO of Hyptis mutabilis (Silva et al., 2013), which presented sedative and anesthetic effects, respectively, on C. parallelus and silver catfish. Previous studies that analyzed the chemical composition of PPOL have reported the predominance of Tridecane (56.8%), Pentadecane (25.5%) and Spathulenol (2.6%) (Santos et al., 2004). The majoritarian composition of PPOL, when considered the biosynthetic origin, is quite similar to that found in our work. However, in PPOL, the simple hydrocarbon derivatives underwent oxygenation producing ketones, which were not described by Santos et al. (2004). Differences in the chemical composition of essential oils from the same species can be explained by genetic differences expressed in different populations, which justify the occurrence of distinct chemotypes, as well as geographical and environmental influences, plant development conditions and processing methods, among other factors (Pant et al., 2021; Etri, Pluhár, 2024). Spathulenol was found in PPOL at 0.6%. This is also a compound indicated for contributing to the anesthetic activities of the EOs of Aloysia gratissima (Benovit et al., 2015) and N. grandiflora, which have anesthetic and sedative activity in silver catfish (Garlet et al., 2019a). However, a possible contribution of sedative or anesthetic activity of 2-Undecanone and 2-Tridecanone cannot be ruled out. Thus, the anesthetic activity of these compounds could be investigated, as promising results can be found, considering that no changes were found in glucose levels and no difference in heart rate of fish exposed to PPOL compared to the basal group could be detected. No adverse effects, side effects, or abnormal clinical and behavioral signs were found with the use of PPOL in silver catfish or tilapia (Fortes et al., 2024b). In this sense, PPOL could be recommended in the future in procedures such as medication and vaccine applications that require sedation and/or anesthesia for faster recovery, as this is within the indications of some researchers regarding induction and recovery time (Keene et al., 1998; Roubach et al., 2001; Obirikorang et al., 2020). In view of the elucidation of the possible mechanism of action of PPOL and possible positive interaction with DZP, it was seen that P3 differed from P1 in the evaluation of stratification in S2, as P1 led to this stage in a shorter time. Furthermore, in S3b P4 differed from P2, that is, when associated with DZP, the time to reach S3b was shorter. Furthermore, to achieve deep anesthesia there was a statistically significant difference between P2 and P4, as S4 was reached more quickly when combined with DZP. In this sense, we offer evidence that suggests a positive interaction of PPOL concentrations with DZP, which was demonstrated in decreasing time to reach the CNS depression stages. Another fact to be highlighted regarding the modulation of the anesthetic effect of PPOL associated with DZP is the evidence that the fish were taken to deeper stages.

The possible mechanism of action of PPOL in view of the evidence from this study seems to involve the benzodiazepine site of the GABAa receptor complex, however no description of the action was found for the two major constituents of PPOL, 2-Undecanone and 2-Tridecanone. A study with the EO of Croton conduplicatus leaves, which presents spathulenol (15.47%) as main compound, suggests the involvement of GABAa receptors in mice as a possible mechanism of action (Oliveira Júnior et al., 2018). Another study, in which spathulenol was present in 3.48% of the EO, sedated silver catfish and it was suggested that its effect was probably the result of GABAa-mediated action (Garlet et al., 2019a). In this context, it may be that this compound present in PPOL has contributed to the possible involvement of the GABAergic receptor, observed in silver catfish juveniles. These findings offered by our study are in agreement with several pharmacological reports of possible interaction of sesquiterpenes and monoterpenes in GABA/BZP mediation (Granger et al., 2005; Rivera et al., 2014; Sousa et al., 2015; Milanos et al., 2017). More specifically in silver catfish, it was also suggested that sesquiterpenes present in the EO of N. grandiflora modulated GABAergic activity (Garlet et al., 2019a). However, other mechanisms of action cannot be ruled out since there is a lack of studies with the two major compounds of PPOL and the participation of spathulenol in possible interactions with muscarinic receptors (M2, M3 and M4) and opioids (Delta and Mu) (Oliveira-Júnior et al., 2018), for example. Thus, given the complex chemical composition of EOs, the mechanism of action may result from the interaction of different components with more than one system, depending on the chemical composition. Another important point of our study is the recovery time, as the results showed that fish recovered in FLU more quickly and efficiently compared to those that recovered in water. The data reinforce the role of benzodiazepine antagonists in reversing sedative and anesthetic effects. Furthermore, the fish recovered satisfactorily, with a return to normal feeding after 24 h, indicating that treatments with EOs did not cause prolonged adverse effects. This is yet another positive indication for the use of these compounds in aquaculture. PPOL showed promising effects in the analysis of glucose levels in all its concentrations, compared to the basal group and in the hr evaluation experiment used in this study, contrary to what was observed for eugenol.

The methodology of evaluating hr with Doppler ultrasound used in our work was recently used to demonstrate that anesthesia with MS-222 (300 mg L^-1) and the EO of Protium heptaphyllum (PHEO) (600 mg L^-1) increased hr in silver catfish (da Silva et al., 2024). While fish anesthetized with CVOL and PPOL did not showed change in hr compared to the control, eugenol increased it. In this sense, eugenol may cause a transient sympathic activation that significantly elevates hr as seen with PHEO. This may be due to the ability of eugenol and PHEO to interact with adrenergic receptors and induce the release of catecholamines such as adrenaline and noradrenaline (Joyce et al., 2023). Therefore, the cardiovascular effects observed in the study are extremely relevant to the safety of using these EOs. This is because the increase in hr with the use of eugenol was observed, however in the groups exposed to CVOL (C3) and PPOL (P3) no significant changes were observed. Thus, these results suggest that eugenol may have a stimulating effect on the cardiovascular system, while CVOL and PPOL do not induce significant changes in hr. Therefore, the use of these EOs can be considered an advantage in terms of safety for the species under study. Ultimately, this device was satisfactory for determining hr and we also suggest its use for other cardiac assessments in fish, such as myocardial contractions in catfish sedated and/or anesthetized with EOs from Brazilian biodiversity and other countries.

The results of the study demonstrate that concentrations of CVOL and PPOL are effective in inducing sedation and anesthesia, but when associated with DZP, they may be more effective in inducing sedation and anesthesia in Rhamdia quelen. The positive interaction between these compounds may allow a faster and more effective induction of deep anesthetic stages, while recovery was accelerated with the use of flumazenil. In this sense, the results suggest the involvement of the benzodiazepine site of the GABAergic receptor complex for both CVOL and PPOL in silver catfish sedation and anesthesia, but further studies regarding the activation of CNS receptors for these EOs need to be carried out. The anesthetic concentration of CVOL increased blood glucose levels compared to the basal group and after deep anesthesia apparently decreased feed consumption, indicating that it did not prevent stress in the fish. The sedative concentration did not change glucose levels and also did not increase heart rate. Apparently CVOL has potential to be used in sedative concentration (20 mg L^-1). In the case of PPOL, the results were promising for the development of a sedative/anesthetic for juvenile silver catfish and larger sizes (around 35 cm) for sedation 20 mg L^-1 and for deep anesthesia 300 mg L^-1, as blood glucose levels and hr did not increase compared to the basal group. Although the effects on blood glucose levels and heart rate require further investigation, the findings suggest that these EOs have potential for use in aquaculture as alternatives to conventional anesthetics. Essentially, as they offer a less invasive and safer approach, no adverse effects, mortality or altered clinical and behavioral signs were found for the concentrations suggested above.

References​

---

Adams TB, Gavin CL, McGowen MM, Waddell WJ, Cohen SM, Feron VJ et al. The FEMA GRAS assessment of aliphatic and aromatic terpene hydrocarbons used as flavor ingredients. Food Chem Toxicol. 2011; 49(10):2471–94. https://doi.org/10.1016/j.fct.2011.06.011

Alexander SP, Mathie A, Peters JA. Guide to receptors and channels (GRAC), 3rd ed. Br J Pharmacol. 2008; 153(2):1–209. https://doi.org/10.1038/sj.bjp.0707746

Aoshima H, Hamamoto K. Potentiation of GABAA receptors expressed in Xenopus oocytes by perfume and phytoncid. Biosci Biotechnol Biochem. 1999; 63(4):743–48. https://doi.org/10.1271/bbb.63.743

Barbas LAL, Hamoy M, Mello VJ, Barbosa RPM, Lima HST, Torres MF et al. Essential oil of citronella modulates electrophysiological responses in tambaqui Colossoma macropomum: a new anaesthetic for use in fish. Aquaculture. 2017; 479:60–68. https://doi.org/10.1016/j.aquaculture.2017.05.027

Barton BA. Stress in fishes: a diversity of responses with particular reference to changes in circulating corticosteroids. Integrative Comp Biol. 2002; 42(3):517–25. https://doi.org/10.1093/icb/42.3.517

Bayeux MC, Fernandes AT, Foglio MA, Carvalho JE. Evaluation of the antiedematogenic activity of artemetin isolated from Cordia curassavica DC. Braz J Med Biol Res. 2002; 35(10):1229–32. https://doi.org/10.1590/S0100-879X2002001000017

Benovit SC, Silva LL, Salbego J, Loro VL, Mallmann CA, Baldisserotto B et al. Anesthetic activity and bio-guided fractionation of the essential oil of Aloysia gratissima (Gillies & Hook.) Tronc. in silver catfish Rhamdia quelen. An Acad Bras Cienc. 2015; 87(3):1675–89. https://doi.org/10.1590/0001-3765201520140223

Carmo G, Fernandes TS, Pedroso M, Ferraz A, Neto AT, Silva UF et al. Phytochemical and antimicrobial study of Pilocarpus pennatifolius Lemaire. Fitoterapia. 2018; 131:1–08. https://doi.org/10.1016/j.fitote.2018.09.009

Carvalho Jr. PM, Rodrigues RFO, Sawaya ACHF, Marques MOM, Shimizu MT. Chemical composition and antimicrobial activity of the essential oil of Cordia verbenacea DC. J ethnopharmacol. 2004; 95(2–3):297–301. https://doi.org/10.1016/j.jep.2004.07.028

Darragh A, Lambe R, Kenny M, Tijolo I, Taaffe W, O’boyle C. RO 15-1788 antagonises the central effects of diazepam in man without altering diazepam biovailability. Br J Clin Pharmacol. 1982; 14(5):677–82. https://doi.org/10.1111/j.1365-2125.1982.tb04956.x

Delgado L, Schmachtenberg O. Immunohistochemical localization of GABA, GAD65, and the receptor subunits GABA_Aα1 and GABA_B1 in the zebrafish cerebellum. Cerebellum. 2008; 7:444–50. https://doi.org/10.1007/s12311-008-0047-7

Dutra RC, Campos MM, Santos AR, Calixto JB. Medicinal plants in Brazil: pharmacological studies, drug discovery, challenges and perspectives. Pharmacol Res. 2016; 112:4–29. https://doi.org/10.1016/j.phrs.2016.01.021

Etri K, Pluhár Z. Exploring chemical variability in the essential oils of the Thymus genus. Plants. 2024; 13(10):1375. https://doi.org/10.3390/plants13101375

European Pharmacopoeia. European Directorate for the Quality of Medicines. 6th ed. Strassbourg; 2007. Available from: https://www.edqm.eu/en/european-pharmacopoeia

Faria RD, Cabral IR, Oliveira TAS, Thiesen LV, Rakes M, Nesi CN et al. Essential oils from Cordia verbenacea and Elionurus latiflorus and their binary mixture: bioactivity against the Mexican bean weevil and an aflatoxin-producing fungal species. Ind Crop Prod. 2023; 206:117674. https://doi.org/10.1016/j.indcrop.2023.117674

Fortes CHM, Baldisserotto B, Fleig FD, Heiznmann BM. Sedative and anesthetic efficacy of the essential oils from the Brazilian native plants Pilocarpus pennatifolius and Cordia verbenacea in Nile Tilapia. An Acad Bras Ciênc. 2024b; 96(3):e20240235. https://doi.org/10.1590/0001-3765202420240235

Fortes CHM, Ferrari FT, Baldisserotto B, Schmidt D, Sutili FJ, Heiznmann BM. Anesthetic potential of essential oils from Brazilian native plants in Rhamdia quelen juveniles (silver catfish). Neotrop Ichthyol. 2024a; 22(3):e240034. https://doi.org/10.1590/1982-0224-2024-0034

Garlet QI, Pires LC, Milanesi LH, Marafiga JR, Baldisserotto B, Mello CF et al. (+)-Dehydrofukinone modulates membrane potential and delas seizure conset by GABAa receptor-mediated mechanism in mice. Toxicol Appl Pharmacol. 2017; 332:52–63. https://doi.org/10.1016/j.taap.2017.07.010

Garlet QI, Rodrigues P, Barbosa LB, Londero AL, Mello CF, Heinzmann BM. Nectandra grandiflora essential oil and its isolated sesquiterpenoids minimize anxiety-related behaviors in mice through GABAergic mechanisms. Toxicol Appl Pharmacol. 2019b; 375:64–80. https://doi.org/10.1016/j.taap.2019.05.003

Garlet QI, Souza CF, Rodrigues P, Descovi SN, Martinez-Rodriguez G, Baldisserotto B et al. GABAa receptor subunits expression. In silver catfish (Rhamdia quelen) brain and its modulation by Nectandra grandiflora Nees essential oil and isolated compounds. Behav Brain Res. 2019a; 376:112178. https://doi.org/10.1016/j.bbr.2019.112178

Gomes DP, Chaves BW, Becker AG, Baldisserotto B. Water parameters affect anaesthesia induced by eugenol in silver catfish, Rhamdia quelen. Aquacult Res. 2011; 42:878–86. https://doi.org/10.1111/j.1365-2109.2011.02864.x

Gomes LC, Chippari-Gomes AR, Lopes NP, Roubach R, Araujo-Lima CARM. Efficacy of benzocaine as an anesthetic in juvenile tambaqui Colossoma macropomum. J World Aquac Soc. 2001; 32(4):426–31. https://doi.org/10.1111/j.1749-7345.2001.tb00470.x

Gonçalves B, Giaquinto PC. Propofol and benzocaine anesthetics responses profiles in Nile tilapia. Int Aquat Res. 2020; 12(3):219–25. https://doi.org/10.22034/IAR.2020.1895923.1026

Granger RE, Campbell EL, Johnston GA. (+)- and (-)-borneol: efficacious positive modulators of GABA action at human recombinant α₁β₂γ_2L GABA_A receptors. Biochem Pharmacol. 2005; 69(7):1101–11. https://doi.org/10.1016/j.bcp.2005.01.002

Heldwein CG, Silva LL, Reckziegel P, Barros FMC, Bürger ME, Baldisserotto B et al. Participation of the GABAergic system in the anesthetic effect of Lippia alba (Mill.) N. E. Brown essential oil. Braz J Med Biol Res. 2012; 45(5):436–43. https://doi.org/10.1590/S0100-879X2012007500052

Jerez-Cepa I, Ruiz-Jarabo I. Physiology: an important tool to assess the welfare of aquatic animals. Biology. 2021; 10(1):61. https://doi.org/10.3390/biology10010061

Joyce W, Warwicker J, Shields HA, Perry SF. Evolution and divergence of teleost adrenergic receptors: why sometimes ‘the drugs don’t work’ in fish. J Exp Biol. 2023; 226(19):1–14. https://doi.org/10.1242/jeb.245859

Keene JI, Noakes DLG, Moccia RD, Soto CG. The efficacy of clove oil as an anaesthetic for rainbow trout, Oncorhynchus mykiss (Walbaum). Aquacult Res. 1998; 29:89–101. https://doi.org/10.1046/j.1365-2109.1998.00927.x

Kim YJ, Nam RH, Yoo YM, Lee CJ. Identification and functional evidence of GABAergic neurons in parts of the brain of adult zebrafish (Danio rerio). Neurosci Lett. 2004; 355(1–2):29–32. https://doi.org/10.1016/j.neulet.2003.10.024

Maricchiolo G, Genovese L. Some contributions to knowledge of stress response in innovative species with particular focus on the use of the anaesthetics. Open Marine Biol J. 2011; 5:24–33. https://doi.org/10.2174/1874450801105010024

Mattson NS, Ripple TH. Metomidate, a better anesthetic for cod (Gadus morhua) in comparison with benzocaine, MS222, chlorobutanol, and phenoxyethanol. Aquaculture. 1989; 83(1–2):89–94. https://doi.org/10.1016/0044-8486(89)90063-X

Melo CPB, Saito P, Vale DL, Rodrigues CC, Pinto IC, Martinez RM et al. Protection against UVB deleterious skin effects in a mouse model: effect of a topical emulsion containing Cordia verbenacea extract. Photochem Photobiol Sci. 2021; 20:1033–51. https://doi.org/10.1007/s43630-021-00079-x

Michielin EMZ, Salvador AA, Riehl CAS, Smânia Jr. A, Smânia EFA, Ferreira SRS. Chemical composition and antibacterial activity of Cordia verbenacea extracts obtained by different methods. Bioresour Technol. 2009; 100(24):6615–23. https://doi.org/10.1016/j.biortech.2009.07.06

Milanos S, Elsharif SA, Janzen D, Buettner A, Villmann C. Metabolic products of linalool and modulation of GABA_A receptors. Front Chem. 2017; 5:46. https://doi.org/10.3389/fchem.2017.00046

National Institute of Standards and Technology (NIST). Mass spectral search for the NIST/EPA/NIH mass spectral library, 2. National Institute of Standards and Technology, Gaithersburg, USA; 2008.

National Institute of Standards and Technology (NIST). National Institute of Standards and Technology. NIST Web chemistry book, SRD 69. 2024. https://doi.org/10.18434/T4D303

Obirikorang KA, Asante-Tuoh DT, Agbo NW, Amponsah AK, Skov PV. Anaesthetic potential of propofol for Nile tilapia (Oreochromis niloticus): effect of anaesthetic concentration and body weight. Scientific African. 2020; 10:e00595. https://doi.org/10.1016/j.sciaf.2020.e00595

Okamoto MH, Tesser MB, Louzada LR, Santos RA, Sampaio LA. Benzocaine and eugenol as anaesthetics for pompano juvenile Trachinotus marginatus. Cienc Rural. 2009; 39(3):866–70. https://doi.org/10.1590/S0103-84782008005000100

Oliveira Júnior RG, Ferraz CAA, Silva JC, Teles RBA, Silva MG, Diniz TC et al. Neuropharmacological effects of essential oil from the leaves of Croton conduplicatus Kunth and possible mechanisms of action involved. J Ethnopharmacol. 2018; 221:65–76. https://doi.org/10.1016/j.jep.2018.04.009

Pant P, Pandey S, Dall’Acqua S. The influence of environmental conditions on secondary metabolites in medicinal plants: a literature review. Chem Biodivers. 2021; 18(11):e2100345. https://doi.org/10.1002/cbdv.202100345

Parodi TV, Gressler LT, Silva LL, Becker AG, Schmidt D, Caron BO et al. Chemical composition of the essential oil of Aloysia triphylla under seasonal influence and its anaesthetic activity in fish. Aquac Res. 2020; 51(6):2515–24. https://doi.org/10.1111/are.14594

Rivera EM, Cid MP, Zunino P, Baiardi G, Salvatierra NA. Central α- and β-thujone: Similar anxiogenic-like effects and differential modulation on GABA_A receptors in neonatal chicks. Brain Res. 2014; 1555:28–35. https://doi.org/10.1016/j.brainres.2014.01.039

Robinson NA, Johnsen H, Moghadam H, Andersen O, Tveiten H. Stress affects subsequent gene expression response to an acute stress in Atlantic salmon: an approach for creating robust fish for aquaculture? G3. 2019; 9(5):1597–611. https://doi.org/10.1534/g3.119.400152

Roubach R, Carvalho GL, Val AL. Safest level of tricaine methanesulphonate (MS-222) to induce anesthesia in juveniles of Matrinxã, Brycon cephalus. Acta Amaz. 2001; 31(1):159–63. https://doi.org/10.1590/1809-43922001311163

Santos AP, Lopes MC, Limberger RP, Apel MA, Henriques AT, Moreno PRH. Analysis of the volatile oil from Pilocarpus pennatifolius Lemmaire (Rutaceae) leaves by GC–MS. Flavour Fragr J. 2004; 19(4):325–26. https://doi.org/10.1002/ffj.1306

Santos AP, Moreno PRH. Pilocarpus spp: a survey of its chemical constituents and biological activities. Rev Bras Cienc Farm. 2004; 40(2):116–37. https://doi.org/10.1590/S1516-93322004000200002

dos Santos AC, Bandeira-Júnior G, Zago DC, Zeppenfeld CC, Silva DT, Heinzmann BM et al. Anesthesia and anesthetic action mechanism of essential oils of Aloysia triphylla and Cymbopogon flexuosus in silver catfish (Rhamdia quelen). Vet Anaesth Analg. 2017; 44(1):106–13. https://doi.org/10.1111/vaa.12386

Sawaya ACHF, Vaz BG, Eberlin MN, Mazzafera P. Screening species of Pilocarpus (Rutaceae) as sources of pilocarpine and other imidazole alkaloids. Genet Resour Crop Ev. 2011; 58:471–80. https://doi.org/10.1007/s10722-011-9660-2

Sieghart W. Structure, pharmacology, and function of GABA_A receptor subtypes. Adv Pharmacol. 2006; 54:231–63. https://doi.org/10.1016/S1054-3589(06)54010-4

Silva LL, Garlet QI, Benovit SC, Dolci G, Mallmann CA, Bürger ME et al. Sedative and anesthetic activities of the essential oils of Hyptis mutabilis (Rich.) Briq. and their isolated components in silver catfish (Rhamdia quelen). Braz J Med Biol Res. 2013; 46(9):771–79. https://doi.org/10.1590/1414-431X20133013

Silva LL, Garlet QI, Koakoski G, Abreu MSD, Mallmann CA, Baldisserotto B et al. Anesthetic activity of the essential oil of Ocimum americanum in Rhamdia quelen and its effects on stress parameters. Neotrop Ichthyol. 2015; 13(4):715–22. https://doi.org/10.1590/1982-0224-20150012

Silva LL, Parodi TV, Reckziegel P, Garcia VO, Bürger ME, Baldisserotto B et al. Essential oil of Ocimum gratissimum L.: anesthetic effects, mechanism of action and tolerance in silver catfish, Rhamdia quelen. Aquaculture. 2012; 350–53:91–97. https://doi.org/10.1016/j.aquaculture.2012.04.012

da Silva HNP, Dornelles JL, Bandeira Junior G, Barany A, Silva LVF, Mourão RHV et al. Protium heptaphyllum essential oil from the fruit as a sedative and anesthetic in Rhamdia quelen: influence in cardiac frequency, biochemical, and oxidative parameters. Fish Physiol Biochem. 2024; 50:2137–47. https://doi.org/10.1007/s10695-024-01380-z

Sneddon LU. Clinical anesthesia and analgesia in fish. J Exot Pet Med. 2012; 21(1):32–43. https://doi.org/10.1053/j.jepm.2011.11.009

Sousa DP, Almeida SHP, Andrade LN, Andreatini R. A systematic review of the anxiolytic-like effects of essential oils in animal models. Molecules. 2015; 20(10):18620–60. https://doi.org/10.3390/molecules201018620

Souza CF, Baldissera MD, Baldisserotto B, Heinzmann BM, Martos-Sitcha JA, Mancera JM. Essential oils as stress reducing agents in fish farming: a review. Front Physiol. 2019; 10:785. https://doi.org/10.3389/fphys.2019.00785

Souza LA, Mourão KSM, Moscheta IS, Rosa SM. Floral morphology and anatomy of Pilocarpus pennatifolius Lem. (Rutaceae). Braz J Bot. 2003; 26(2):175–84. https://doi.org/10.1590/S0100-84042003000200005

Sutherland M, Dowling S, Shaw R, Hickey J, Fraser D, Cameron C et al. Stress-induced immunomodulation in low and high reactive sheep. Animals. 2019; 9(3):1–13. https://doi.org/10.3390/ani9030104

Tanelian DL, Kosek P, Mody I, Maciver MB. The role of the GABAA receptor/chloride channel complex in anesthesia. Anesthesiol. 1993; 78:757–76. https://doi.org/10.1097/00000542-199304000-00020

Tondolo JSM, Amaral LDP, Simões LN, Garlet QI, Schindler B, Oliveira TM et al. Anesthesia and transport of fat snook Centropomus parallelus with the essential oil of Nectandra megapotamica (Spreng.) Mez. Neotrop Ichthyol. 2013; 11(3):667–74. https://doi.org/10.1590/S1679-62252013000300020

Zahl IH, Samuelsen O, Kiessling A. Anaesthesia of farmed fish: implications for welfare. Fish Physiol Biochem. 2012; 38(1):201–18. https://doi.org/10.1007/s10695-011-9565-1

Authors

---

Carlos Herminio Magalhães Fortes¹, Bernardo Baldisserotto^1,2, Frederico Dimas Fleig³, Valério Valdetar Marques Portela Júnior² and Berta Maria Heinzmann^1,3

^[1]    Programa de Pós-Graduação em Farmacologia, Universidade Federal de Santa Maria, Av. Roraima, 1000, 97105-900 Santa Maria, RS, Brazil. (CHMF) medvet.chmf@gmail.com, (BB) bernardo.baldisserotto@ufsm.br, (BMH) berta.heinzmann@gmail.com (corresponding author).

^[2]    Departamento de Fisiologia e Farmacologia, Universidade Federal de Santa Maria, Av. Roraima, 1000, 97105-900 Santa Maria, RS, Brazil. (VVMPJ) valerio.portela@ufsm.br.

^[3]    Departamento de Ciências Florestais, Universidade Federal de Santa Maria, Santa Maria, Av. Roraima, 1000, 97105-900 Santa Maria RS, Brazil. (FDF) fredyfleig@gmail.com.

^[4]    Departamento de Farmácia Industrial, Universidade Federal de Santa Maria, Av. Roraima, 1000, 97105.900 Santa Maria, RS, Brazil.

Authors’ Contribution

---

Carlos Herminio Magalhães Fortes: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Project administration, Validation, Visualization, Writing-original draft, Writing-review and editing.

Bernardo Baldisserotto: Conceptualization, Data curation, Funding acquisition, Investigation, Methodology, Resources, Supervision, Validation, Visualization, Writing-review and editing.

Frederico Dimas Fleig: Resources, Writing-review and editing.

Valério Valdetar Marques Portela Júnior: Resources, Writing-review and editing.

Berta Maria Heinzmann: Data curation, Methodology, Project administration, Resources, Supervision, Validation, Visualization, Writing-review and editing.

Ethical Statement​

---

The methodology was approved by the UFSM Ethics Committee under n^o 9303280623 and the project was registered in SISGEN under n^o AE4FE76.

Competing Interests

---

The author declares no competing interests.

Data availability statement

---

The authors confirm that the data supporting the findings of this study are available within the article.

Funding

---

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Ph.D. Scholarship for CHMF – Financial Code 001 and the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) for the a PQ scholarship for Professor BB, process 301816/2022–0.

How to cite this article

---

Fortes CHM, Baldisserotto B, Fleig FD, Portela Júnior VVM, Heiznmann BM. Essential oils from leaves of Pilocarpus pennatifolius and Cordia verbenacea in silver catfish (Rhamdia quelen): cardiovascular aspects and participation of the GABAergic system in the sedative and anesthetics effects. Neotrop Ichthyol. 2025; 23(3):e250014. https://doi.org/10.1590/1982-0224-2025-0014

Copyright​

---

This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

Distributed under

Creative Commons CC-BY 4.0

© 2025 The Authors.

Diversity and Distributions Published by SBI

Accepted July 3, 2025

Submitted January 30, 2025

Epub November 7, 2025