Antioxidant Activity and Anxiolytic Effect of Cnidoscolus quercifolius Pohl Stem Bark Extract in Zebrafish

Abstract

Background: Cnidoscolus quercifolius, popularly known as “favela”, is used in traditional medicine to treat various conditions, such as infections and inflammations. However, its therapeutic potentials remain underexplored in scientific research. The present study aimed to evaluate the anxiolytic effect, toxicity, and antioxidant activity of methanolic (EMCq) and ethyl acetate (EAECq) extracts of C. quercifolius bark, as well as determine their chemical composition by HPLC/DAD and their levels of phenolic compounds and flavonoids. Methods: Anxiolytic effect and acute toxicity tests were conducted using the zebrafish model, while antioxidant activity was assessed using the DPPH^• and ABTS⁺ methods. The chemical composition was obtained by HPLC/DAD, and phenolic compounds and flavonoids were quantified with the Folin–Ciocalteu reagents and the aluminum chloride colorimetric method, respectively. Results: Caffeic acid, p-coumaric acid, cinnamic acid, pinocembrin, and apigenin were identified and quantified. The results indicated that both extracts exhibited low antioxidant activity, possibly due to their low levels of phenols (0.187 and 0.293 mg GAE/g) and flavonoids (0.84 and 0.64 mg QE/g). However, the extracts did not show acute toxicity (>400 mg/kg) and reduced the locomotor activity of zebrafish at all the doses tested (40 to 400 mg/kg), while increasing the time the animals remained in the light zone, indicating an anxiolytic effect. Conclusions: These findings suggest for the first time that C. quercifolius has anxiolytic properties, warranting further investigation into specific bioactive compounds and their mechanisms of action. Future studies may explore molecular analysis techniques to identify the responsible compounds, as well as investigate safety and clinical efficacy in mammalian models.

1. Introduction

During normal metabolic activity, the human body produces various reactive species, such as reactive oxygen species (ROS) and reactive nitrogen species (RNS). Although these molecules play important physiological roles at controlled levels, their excessive generation can lead to oxidative stress, a process that accelerates cellular aging and damages vital components of the body, triggering harmful health effects and the development of pathological conditions, including cardiovascular, chronic, and degenerative diseases [1].

Although the mechanisms and pathways involved are not yet fully understood, studies suggest that oxidative stress also plays a key role in the pathophysiology of neuropsychiatric disorders, such as anxiety disorders [2]. Oxidative stress can affect the function of the nervous system by modulating neurotransmitter formation and altering neural transmission mechanisms, which may contribute to the onset of anxious symptoms [3]. Anxiety is characterized by fear, discomfort, and excessive worry, triggered as an anticipatory response to threatening or unknown events [4]. It is a growing psychiatric disorder, with a significant increase in cases, particularly in contexts of chronic stress and behavioral changes, such as those observed during the SARS-CoV-2 pandemic [5,6].

Currently, approximately 10 to 20% of the Brazilian population suffers from anxiety disorders, with Brazil ranking among the countries with the highest global prevalence [7]. Conventional treatment, which includes anxiolytic medications, is increasingly being avoided by patients due to its high costs, ineffectiveness, or adverse effects [6,8]. In this context, the search for effective therapeutic alternatives with a lower risk of side effects has become an urgent need.

The use of experimental models, such as zebrafish (Danio rerio), has shown promise in investigating neurobehavioral disorders and evaluating biologically active substances for the creation of novel medications. Zebrafish exhibit high genetic and physiological similarity to humans, are a low-cost model with the capacity for high-throughput and large-scale analyses, and are widely used in toxicity studies and pharmacological efficacy assessments [9,10].

Among potential therapeutic alternatives, medicinal plants have been widely investigated due to the presence of bioactive compounds such as phenols, flavonoids, tannins, and saponins, which are known for their pharmacological properties [11]. Plants represent a more accessible alternative to conventional treatments, offering an opportunity for the development of new drugs with therapeutic efficacy and a lower risk of side effects [12].

One such species of interest is Cnidoscolus quercifolius (Euphorbiaceae), commonly known as “favela” or “faveleira” [13]. This xerophytic plant, endemic to the Caatinga region, is geographically distributed across several states in Northeast Brazil (RN, PB, PE, CE, BA, AL, SE, and PI), and is recognized for its use in treating various conditions, such as kidney problems, wounds, infections, and inflammations [14,15]. Furthermore, this plant has demonstrated pharmacological activities such as antinociceptive, anti-inflammatory, antimicrobial, and hypoglycemic effects [16,17,18]. However, the anxiolytic potential of C. quercifolius has yet to be investigated, which justifies the need for this study.

Given this, the current study sought to explore, for the first time, the anxiolytic potential and toxicity of the methanolic and ethyl acetate extracts from the stem bark of C. quercifolius in a zebrafish model, as well as to assess its antioxidant activity and the levels of total phenolic and flavonoid compounds. The results may contribute to the development of new treatments for anxiety disorders based on natural resources and provide an effective alternative to conventional treatments.

2. Materials and Methods

2.1. Plant Material and Obtaining Extracts

The bark of the stem of C. quercifolius was commercially purchased in the central market of the city of Juazeiro do Norte, Ceará, Brazil (7°12′16.6″ South, 39°19′01.0″ West). After being reduced to a dry powder (1.5 kg), the bark was extracted in two distinct processes: first with ethyl acetate and then with methanol, for 4 h, using a Soxhlet extractor. Then, the solvents were removed in a rotary evaporator under reduced pressure at an average temperature of 50 °C, obtaining crude ethyl acetate (EAECq) and methanolic (EMCq) extracts, with yields of 4.5% and 11.0%, respectively, based on the dry weight of the extracts.

2.2. Total Phenols

The total content of phenolic compounds was determined by the Folin–Ciocalteau method, following the procedure by Nonato et al. [19] with modifications. Solutions of each extract were prepared with different concentrations (50–1000 μg/mL) diluted in ethanol. The reaction medium consisted of 200 µL of the extract, 400 µL of freshly prepared Folin–Ciocalteu reagent (10%) diluted in water, and 2.8 mL of the Na₂CO₃ (7.5% aq.), having been protected from light. Subsequently, the homogenized mixtures were incubated for 15 min at 45 °C in a water bath and the absorbance was measured in a spectrophotometer at 735 nm. The test was carried out in triplicate, and the result was expressed in equivalent milligrams of gallic acid per gram of extract (mg GAE/g).

2.3. Total Flavonoid

The overall flavonoid concentration was measured using the aluminum chloride colorimetric technique, following the methodology described by Kosalec [20] with adaptations. Aliquots of the samples (100, 500, and 1000 μg/mL) were added to 1 mL of AlCl₃ (15% aq.) and ethanol (3.9 mL, 3.500 mL, and 3 mL, respectively), making a final volume of 5 mL for each concentration. The samples were incubated at room temperature for 30 min and the absorbance was read on a spectrophotometer at 425 nm. The experiments were carried out in triplicate and quercetin was used as the standard for the calibration curve, with the result expressed in equivalent milligrams of quercetin per gram of extract (mg QE/g).

2.4. HPLC/DAD Analysis

Both extracts were analyzed by High-Performance Liquid Chromatography (HPLC) using an Agilent 1260 HPLC system (Agilent Tech., Waldbronn, Germany), equipped with a UV-Vis Diode Array Detector (DAD). The separation was performed using a gradient method, with a C18 chromatographic column (250 mm × 4.0 mm × 5 µm, Macherey-Nagel, Germany). The standard phenolic solutions and mixtures were injected into the system using an automatic injector. The mobile phase consisted of a solvent mixture with A (ultrapure water) and B (methanol: acetonitrile, 60:40, HPLC grade, Agilent Tech., Germany), both acidified with 0.1% formic acid (Sigma Aldrich, St. Louis, MO, USA), following this elution gradient: 0–15 min—15% B in A; 17 min—40% B in A; 30 min—30% B in A; and 38 min—15% B in A, maintaining this composition until 45 min.

The wavelengths used were 310 nm for caffeic acid, p-coumaric acid, and ferulic acid; 290 nm for cinnamic acid, naringenin, and pinocembrin; and 340 nm for apigenin. The mobile phase flow rate was 0.5 mL/min, and the injection volume was 20 µL. All the mobile phases, solutions, and samples were filtered through a Millipore membrane filter, 13 mm diameter, pore size 0.22 µm (Millipore). The samples were dissolved in HPLC-grade methanol (30 mg/mL). Quantification was performed by integrating the peaks using the external standard method.

The analyses were conducted at room temperature and in triplicate, with peaks confirmed by comparing their retention time with those of the reference standards and by DAD spectra (190 to 400 nm). Compound quantifications were based on the analytical curves of the reference standards. The detection limit (LOD) and quantification limit (LOQ) were calculated based on the standard deviation of the responses and the slope, using three independent analytical curves. LOD and LOQ were calculated as 3.3 and 10 σ/S, respectively, where σ is the standard deviation of the response and S is the slope of the calibration curve.

2.5. Antioxidant Activity

2.5.1. ABTS⁺ Radical Discoloration

Radical capture was measured using the method proposed by Rufino et al. [21]. To form the ABTS⁺ radical, a solution formed by 2,2′-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) (7 mM) and potassium persulfate (2.45 mM) was used, with the solution being kept at room temperature in the dark for 16 h. After this period, the solution containing the radical was adjusted to an absorbance of 0.70 ± 0.05 at 734 nm in a spectrophotometer. The concentrations evaluated ranged from 10 to 1000 μg/mL. For the reaction, 30 µL of each concentration and 3.0 mL of the solution containing the ABTS⁺ radical were used. The assays were performed in triplicates, and the absorbance was measured at 734 nm after 6 min of the mixture reaction. Pure ABTS⁺ radical was used as a negative control, methanol as a blank, and ascorbic acid (10 to 1000 μg/mL) as a positive control. The antioxidant activity was calculated according to Equation (1) and activity was expressed as a percentage (%) of elimination of ABTS⁺.

AA% = 100 − {[(AbsSample − AbsBrank)/AbsNegative Control] × 100}

(1)

where AA% refers to the percentage of antioxidant activity and Abs are absorbances.

2.5.2. DPPH^• Radical Scavenging Capacity

The scavenging potential of the DPPH^• (2,2-diphenyl-1-picrylhydrazyl) radical was determined using the method described by Rufino et al. [22] with some modifications. For each extract, a series of dilutions were carried out in methanol, and the preparation of the DPPH^• solution occurred minutes before carrying out the test, also using methanol. Volumes (20 mL) of samples at concentrations of 10 to 1000 µg/mL were mixed with 280 µL of DPPH^• solution (0.06 mM). After 30 min of incubation, at room temperature and in the absence of light, the absorbance was read using a spectrophotometer at 515 nm. All the tests were performed in triplicate, with methanol used as a blank and ascorbic acid as a positive control at concentrations of 10 to 1000 μg/mL. The results were expressed as a percentage of DPPH^• inhibition (or % antioxidant activity) using Equation (1).

2.6. Zebrafish Experimental Model

2.6.1. Animals

Wild zebrafish (Danio rerio), adults of both sexes, aged 90 to 120 days, with similar size (3.5 ± 0.5 cm) and weight (0.4 ± 0.1 g), were purchased commercially (Fortaleza, CE). The fish were acclimatized for 24 h in a 10 L glass aquarium (30 × 15 × 20 cm) (n = 3/L), at a temperature of 25 ± 2 °C, in light/dark cycles with chlorine-free water. The animals received food ad libitum 24 h before the experiment. After the experiments, the animals were euthanized by immersion in ice-cold water (2–4 °C) until loss of opercular movements (1 min). The experiments followed the Ethical Principles of Animal Experimentation and were approved by the Ethics Committee on the Use of Animals of the State University of Ceará (CEUA-UECE; n° 04983945/2021), in accordance with the Ethical Principles of Animal Experimentation.

2.6.2. Determination of Acute Toxicity 96 h

The animals (n = 6/group) were treated orally (v.o) with 20 µL of the extracts (40; 200 or 400 mg/kg) or 20 µL of the vehicle (3% DMSO) intraperitoneally (i.p), and left at rest to analyze the mortality rate for a period of 96 h, recording every 24 h the number of dead fish in each group [23], with the lethal dose capable of killing 50% of the animals (LD₅₀) determined using the Trimmed Spearman–Karber mathematical method with a 95% confidence interval.

2.6.3. Assessment of Locomotor Activity (Open Field Test)

The open field test evaluated the presence or absence of changes in the animals’ motor system [24], whether due to the anxiolytic effect or muscle relaxation. The animals (n = 6/group) were treated as previously described (treated orally (v.o) with 20 µL of the extracts (40; 200 or 400 mg/kg), including the positive control group (Diazepam; 4 mg/kg, i.p). After 60 min of treatment, the animals were placed individually in Petri dishes containing aquarium water (10 × 15 cm; with quadrants at the bottom of the dish). Locomotor activity was verified according to the number of line crossings (LCs) recorded during 0–5 min.

2.6.4. Analysis of Anxiolytic Activity (Light/Dark Test)

Just like rodents, zebrafish naturally have an aversion to illuminated areas, so the light/dark test was used to determine anxiety behavior in these animals [25]. A glass aquarium (30 cm × 15 cm × 20 cm), divided into a light and a dark area, was filled up to 3 cm with chlorine-free tap water, simulating a shallow environment different from the conventional aquarium, inducing behaviors similar to those of anxiety. The animals were treated as previously described (treated orally (v.o) with 20 µL of the extracts (40; 200 or 400 mg/kg) and, after 60 min, they were placed individually in the light zone of the aquarium, with the anxiolytic effect measured based on the time spent in the light zone within 5 min of analysis [26]. A positive control group (Diazepam; 4 mg/kg, i.p) was included. The experiments were conducted in a controlled room, with a 14:10 h light/dark cycle to minimize variations in fish behavior.

2.7. Statistical Analysis

The zebrafish assay results were expressed as mean values ± standard error for each group of 6 animals. After confirming the normality of distribution and homogeneity of the data, the differences between the groups were subjected to analysis of variance—one-way ANOVA, followed by the Tukey test. All the analyses were performed using the GraphPad Prism v software 8.0.1. (San Diego, CA, USA). The level of statistical significance was set at 5% (p < 0.05). The antioxidant tests were carried out in triplicate and the results were expressed as mean (n = 3) ± standard deviation. The IC₅₀ values were obtained by non-linear regression of the curve transforms. For statistical analysis, one-way ANOVA was applied followed by the Bonferroni test, considering a significance of p ≤ 0.05, using the Graphpad Prism 6.0 software (San Diego, CA, USA).

3. Results and Discussion

Based on the results obtained, it was demonstrated that the concentration of total phenols and flavonoids in both extracts was relatively low. The EMCq presented the total phenol and flavonoid contents of 0.187 mg GAE/g and 0.84 mg QE/g, respectively, while EAECq presented a total phenol content of 0.293 mg GAE/g and 0.64 mg QE/g of flavonoids. The quantification was performed using a calibration curve for quercetin (R² = 0.985) and gallic acid (R² = 0.998).

Oliveira Júnior et al. [27] analyzed the phenolic compound content in the ethanolic extract of dried C. quercifolius leaves and observed significantly higher levels of phenols (95.61 ± 2.78 mg GAE/g), which can be explained by differences in the plant materials analyzed (leaves vs. stem bark) and the extraction methodology used. On the other hand, in the study by Peixoto-Sobrinho et al. [28], the bark of C. quercifolius exhibited the lowest values of phenols with 3.58 ± 0.16 mg TAE/g (mg tannic acid equivalent per g of sample) and flavonoids with 0.60 ± 0.05 mg RE/g (mg rutin equivalent per g of sample) when compared to other parts of the plant, such as the leaves (18.32 ± 0.60 mg TAE/g and 26.51 ± 1.78 mg EAT/g, respectively). This again points to a significant variation between different plant parts and their bioactive compounds.

Although colorimetric assays are useful for a preliminary assessment of the concentration of phenols and flavonoids, these assays do not provide sufficiently accurate information about the chemical compounds. Therefore, to obtain a more comprehensive evaluation of the chemical composition of the extracts, additional analyses using HPLC/DAD were performed to verify the presence of phenolic acids and flavonoids commonly found in medicinal species. Although the colorimetric assays revealed low concentrations of total phenols and flavonoids in both extracts, the chromatographic analysis by HPLC/DAD identified a diversity of compounds in EAECq (0.26%), including the presence of caffeic acid (Rt = 8.2 min), p-coumaric acid (Rt = 9.5 min), cinnamic acid (Rt = 10.6 min), pinocembrin (Rt = 24.4 min), and apigenin (Rt = 26 min), with pinocembrin being its main constituent (1.5553 ± 0.05658 mg/g). In contrast, only apigenin was identified in EMCq (Rt = 36.6 min), suggesting a distinct chemical composition between the two extracts (

Table 1. Phenolic acids and flavonoids quantified by HPLC/DAD in the methanolic extract (EMCq) and ethyl acetate extract (EAECq) of C. quercifolius bark.

Compound	LD (mg/mL)	LQ (mg/mL)	EAECq		EMCq
			mg/g	%	mg/g	%
Caffeic acid	0.0001	0.0003	0.2122 ± 0.05513	0.02122	nd	nd
p-Coumaric acid	0.0007	0.0025	0.0506 ± 0.0153	0.00506	nd	nd
Ferulic acid	0.0048	0.0162	nd	nd	nd	nd
Cinnamic acid	0.0009	0.0032	0.3898 ± 0.05011	0.0398	nd	nd
Naringenin	0.0011	0.0036	nd	nd	nd	nd
Pinocembrin	0.0030	0.0102	1.5553 ± 0.05658	0.15553	nd	nd
Apigenin	0.0027	0.0092	0.3940 ± 0.042811	0.03940	0.1484 ± 0.000057	0.01484
Total			2.6019	0.26019	0.1484	0.01484

The results are expressed as the mean (mg/g of the sample) ± standard deviation (n = 3). nd: not detected.

).

Discrepancies in the values obtained in phenol and flavonoid quantification assays for the same species are not uncommon and can be attributed to a range of biotic and abiotic factors [29]. For example, the time of harvest and the growth stage of the plant can significantly affect the concentration of secondary metabolites such as phenols and flavonoids given that these compounds often vary throughout the life cycle of the plant. Variations in the harvest location can also introduce regional differences in climate, soil, and other environmental factors, which can influence both the quantity and quality of the bioactive compounds present in the plant material [30,31].

In addition, along with the choice of extraction method, the choice of solvent used also plays a crucial role in determining the overall composition of the extracts, since different solvents have varying polarities, which can result in the selective extraction of different types of compounds, leading to variations in yields, chemical composition of the extracts, and biological activities of these compounds [32,33]. This may justify the variation in chemical composition between the extract prepared with ethyl acetate (EAECq) and methanol (EMCq).

The chromatographic analysis of this study corroborates the findings of other studies, such as those by Torres et al. [34] and Oliveira Júnior et al. [27], who also identified the presence of phenolic acids and apigenin in C. quercifolius leaves. Furthermore, compounds like apigenin and caffeic acid have been previously reported in other species of the Cnidoscolus genus, such as Cnidoscolus aconitifolius [35]. The identification of apigenin in both samples, as well as in other studies, suggests that this compound could be a useful chemical marker for species in this genus.

Apigenin is known for its antioxidant, anti-inflammatory, and anticancer properties, and may be useful in the treatment of chronic inflammatory diseases and as an adjunct in cancer therapy [36,37]. Other compounds identified, such as caffeic acid, p-coumaric acid, and cinnamic acid, have shown promising effects in cardiovascular protection, anticancer activity, and diabetes control [38,39,40], while pinocembrin offers, among other benefits, neuroprotective effects [41].

Although the presence of antioxidant compounds like phenolic acids and flavonoids has been confirmed in the extracts, the observed antioxidant activity was relatively low at the tested concentrations, with the maximum reduction percentages being approximately 31% (DPPH^•) and 25% (ABTS⁺) for EAECq, and 24% (DPPH^•) and 27% (ABTS⁺) for EMCq, which are much lower than the maximum percentages obtained for ascorbic acid (96% DPPH^• and 100% ABTS⁺) (Figure 1 and Figure 2).

These data are consistent with those reported by Peixoto-Sobrinho et al. [28], who also found no significant antioxidant activity in C. quercifolius extracts (IC₅₀ of 1867.24 ± 53.66 μg/mL for the DPPH^• method and 299.37 ± 14.60 μg/mL for FRAP). A similar result was observed by Nunes et al. [29], who also reported that the ethanolic extract of C. quercifolius stem bark exhibited low antioxidant capacity in both the DPPH^• and ABTS⁺ assays. According to the authors, these results are related to the low total phenol content of the extract.

This hypothesis was also reported by Ribeiro et al. [42], who observed a correlation between antioxidant activity and the total phenol content of the seed (324.92 mg EAG/100 g) and the press cake of C. quercifolius (398.89 mg EAG/100 g). According to the authors, the press cake may have exhibited more potent antioxidant action than the seed due to its higher phenolic compound content. Thus, although it is not the only factor, these results reinforce the idea that the content of these compounds may be responsible for part of the antioxidant potential of the species.

As previously mentioned, although the chromatographic analysis revealed that EAECq presented known antioxidant compounds, the extract did not demonstrate significant antioxidant potential. Thus, the explanation for this result may be partly related to the low concentration of these compounds in the solutions tested. When the concentrations of antioxidant compounds are very low in the samples, traditional assays, such as DPPH^• and ABTS⁺, may not be sensitive enough to effectively detect their activity. The stability of the compounds may also be an important factor. Antioxidant compounds may degrade or undergo structural changes over time or due to handling, which may reduce their effectiveness during the assays [43].

In addition, the extracts may contain other compounds that interfere with the detection or antioxidant activity, such as the presence of other substances, such as sugars or proteins, which may mask or inhibit the antioxidant action of specific compounds in the assays [44]. More specific studies are needed to identify those responsible for the presence or absence of antioxidant potential in the species.

Regarding the toxicity of C. quercifolius, the scientific literature provides limited information, with this study being the first to report acute toxicity using the zebrafish (Danio rerio) model. As indicated in

Table 2. Results of acute toxicity tests of ethyl acetate (EAECq) and methanolic (EMCq) extracts against adult zebrafish.

Sample	Mortality				96 h LD50 (mg/kg)/IV
	NC	D1	D2	D3
EAECq	0	0	1	0	>400
EMCq	0	0	0	0	>400

NC: negative control group (DMSO 3%); D1: Dose 1 (40 mg/kg); D2: Dose 2 (200 mg/kg); D3: Dose 3 (400 mg/kg); LD₅₀: medium lethal dose; IV: confidence interval.

, the extracts suggest an LD₅₀ higher than 400 mg/kg since none of the doses administered caused significant toxicity in the zebrafish model after 96 h of observation. Additional studies, including prolonged exposures and tests in different biological systems, are necessary for a more comprehensive safety assessment. These results are promising, indicating that C. quercifolius could be a potential source of therapeutic substances with a favorable safety profile.

The evaluation of toxicity is crucial, as it ensures the safe use of a species and identifies the doses at which its pharmacological effects do not result in adverse outcomes. The findings of this study are in agreement with previous research that investigated the toxicity of C. quercifolius in different experimental models. For example, Lira et al. [18] reported that the aqueous extract of the plant’s bark exhibited relatively low oral acute toxicity, with an LD₅₀ greater than 2000 mg/kg, and no deaths occurred during the 14-day observation period. Similar results were obtained with extracts from other parts of the plant, such as the hydroalcoholic extract of the periderm (LD₅₀ > 2000 mg/kg) [45] and the seed oil (LD₅₀ > 5000 mg/kg) [46], which also showed no toxic effects in rodents.

Furthermore, the toxicity of different extracts from the species was also evaluated using the Artemia sp. model. Paredes et al. [47] observed that the root extract exhibited an LD₅₀ of 84.76 µg/mL, indicating significant toxicity, while the extracts from the leaves and bark of the roots had LD₅₀ values of 1079.78 µg/mL⁻¹ and 341.45 µg/mL⁻¹, respectively, suggesting lower toxicity for these extracts. These data indicate that the toxicity of C. quercifolius may vary depending on the plant part used, with the bark and leaves being relatively safer compared to the roots.

When placed individually in a different environment, zebrafish tend to display fear and anxiety behavior, swimming in an exploratory manner near the boundaries of the environment in search of predators. However, when treated with anxiolytic substances, the animals show changes in locomotor behavior, such as reduced exploratory activity, indicating a potential sedative or anxiolytic effect [48]. In this sense, the open field test was conducted as a behavioral analysis parameter to indicate whether the substances act on the central nervous system (CNS) [49], causing symptoms suggestive of anxiety, stress, or possible sedative effects similar to anxiolytic drugs, such as Diazepam (DPZ) and other benzodiazepines (BZDs) [50].

Although no evident toxicity was observed, the results suggest that both EAECq and EMCq altered the locomotion of the zebrafish at all the tested doses (Figure 3), with a significant reduction in the number of line crossings in the aquarium compared to the control group, especially at the higher doses of EAECq (200 and 400 mg/kg). These results were similar to those observed with the positive control (DZP), suggesting that the extracts may induce an effect on the animals’ central nervous system. The lowest dose of EAECq (40 mg/kg), as well as all the doses of EMCq (40 mg/kg, 200 mg/kg, and 400 mg/kg), showed significantly different effects from DZP (** p < 0.0001 vs. CONTROL; ## p < 0.01; #### p < 0.0001 vs. DZP).

The impact on locomotion observed with the C. quercifolius extracts was interpreted as indicative of a possible sedative action, which is generally associated with the effect of anxiolytic drugs, such as benzodiazepines (BDZs). However, the smaller alteration in locomotion caused by the extracts suggests that they have a less pronounced sedative effect compared to the positive control DZP, which is considered a beneficial result. This is because excessive sedative effects, often observed with BDZ use, may be regarded as an undesirable adverse effect, limiting their applicability in prolonged treatments [51]. Thus, in addition to the absence of toxicity, the extracts seem to exhibit a low incidence of this side effect, as they interfere less with the voluntary coordination of the animals’ movements, unlike BDZ.

BDZs are described in the literature as responsible for reducing mobility, causing drowsiness, and sedative effects in the CNS through the positive allosteric modulation of the GABA_A receptor [24,52]. Pharmacological studies indicate that the sedative action of BDZs is related to the α1 subunit of the GABA_A receptor, while the α2 and α3 subunits (α2 GABA_A and α3 GABA_A receptors) exert an anxiolytic effect [53]. Thus, it is believed that the alteration in locomotor activity in the animals treated with the extracts (mainly EAECq) may provide potential evidence that the bark of C. quercifolius can reduce central activity, inducing anxiolytic and/or sedative actions similar to BDZ, possibly through binding to GABA_A receptors.

Apigenin, identified in both the C. quercifolius extracts, is described in the literature as a compound with anxiolytic potential, capable of acting without causing excessive sedation, similar to what was observed for EAECq [54,55]. Therefore, it is believed that the presence of apigenin in the extracts may be related to their anxiolytic effects. However, to validate this hypothesis, further studies are needed to investigate the potential of this compound isolated from C. quercifolius. Additionally, studies are also required to characterize the specific mechanisms of action of the extracts, as anxiolytic effects may also be caused by other compounds and mediated through interactions with different neurotransmission systems, such as serotonin receptors (5-HT), NMDA receptors (N-methyl-D-aspartate), and AMPA receptors (alpha-amino-3-hydroxy-5-methyl-4-isoxazolpropionic acid). Investigating such pathways would be essential for a more comprehensive understanding of the behavioral effects observed in the zebrafish.

The light/dark test, another method used to assess anxiolytic behavior, revealed that the extracts significantly increased the time the zebrafish spent in the light area of the aquarium (Figure 4 and Figure 5) compared to the control group (** p < 0.0001 vs. control; ** p < 0.01). This result suggests that the extracts induced an anxiolytic behavior, characterized by reduced preference of the animals for the dark zone (a “safe” area), typical of a predator-avoidance response [26]. At all the doses of EAECq (40, 200, and 400 mg/kg), the results were similar to the DZP (4 mg/kg), indicating that this extract may possess significant anxiolytic effects.

On the other hand, EMCq exhibited an inversely proportional relationship with concentration, with the lowest dose (40 mg/kg) producing the most pronounced anxiolytic effect. This effect was greater than that observed with the higher doses of EMCq (200 and 400 mg/kg), suggesting that for this specific extract, lower doses may have a more potent anxiolytic efficacy, surpassing the higher concentrations. This type of dose-dependent effect is common in plant extracts and may be related to complex interactions between the compounds present. Furthermore, this result is consistent with the literature on drugs that act at low doses to promote anxiety relief but may exhibit reverse or even undesirable effects at high doses [56].

Although the anxiolytic effects observed in this study are promising, it is important to note that no specific studies on the anxiolytic effect of C. quercifolius were found in the literature. However, similar results were observed by Adebiyi et al. [56] when investigating C. aconitifolius, which demonstrated an anxiolytic effect in the elevated plus-maze test in mice. Similarly to EMCq, only the lowest dose (200 mg/kg) was effective, with a decline in anxiolytic efficacy at higher doses, from 400 to 1600 mg/kg.

This finding suggests that the bark of C. quercifolius may act through mechanisms similar to BDZs, particularly through the activation of the GABAergic system. However, the involvement of the GABA_A system and the potential interaction with other neurotransmission systems still needs to be confirmed. To this end, future studies should focus on the bioactive compounds present in the extracts and on mapping the molecular and pharmacological mechanisms underlying their anxiolytic effects. Additionally, more in-depth studies on the long-term safety and therapeutic efficacy of the extracts, as well as clinical trials in mammalian models, are essential for validating the use of C. quercifolius as an anxiolytic phytomedicine [57,58].

4. Conclusions

Although preliminary, this study provides, for the first time, insights into the anxiolytic effects of C. quercifolius stem bark and its safety based on toxicity evaluation in the zebrafish model. Although compounds recognized in the literature for their biological activities, such as caffeic acid, p-coumaric acid, cinnamic acid, pinocembrin, and apigenin, were identified, the results indicated that both the methanolic and ethyl acetate extracts exhibited low antioxidant activity. This low antioxidant performance may be related to the low levels of these compounds in the species’ bark, suggesting that despite the presence of these bioactive compounds, their insufficient concentration may have negatively impacted the observed antioxidant activity.

However, the extracts altered locomotion and increased the time spent by the zebrafish in the light zone, suggesting a possible anxiolytic and sedative effect, similar to that observed with Diazepam. This effect may be related to the bioactive compounds identified, such as apigenin.

These findings suggest that C. quercifolius may be a safe source for the development of anxiolytic therapies. However, further chemical studies are essential to identify the bioactive compounds responsible for the observed effects and to investigate the underlying molecular mechanisms. In this regard, in vivo pharmacological analyses, combined with in silico approaches, would be crucial for mapping the receptors involved in the anxiolytic effect and evaluating the plant’s potential therapeutic efficacy in more complex models.

To better elucidate the mechanisms underlying these findings, additional pharmacological and biochemical studies are needed. Future research should seek to characterize the specific neuropharmacological targets involved, differentiate between anxiolytic and sedative effects, and evaluate potential toxic effects over longer periods. In this sense, in vivo pharmacological analyses, combined with in silico approaches, would be fundamental to map receptor interactions and investigate the therapeutic relevance of C. quercifolius in more complex models.

Given the preliminary results, there is significant potential for the biotechnological exploration of C. quercifolius, either as an ingredient for drug development or as a resource for traditional medicine.

In addition, future investigations should explore preclinical trials in mammalian models and this will allow a better understanding of the pharmacodynamics and pharmacokinetics of the extracts, bringing the results closer to clinical application. Thus, this study paves the way for the development of new phytotherapeutics based on C. quercifolius, with potential use in traditional medicine and in the pharmaceutical sector.