Previous Article in Journal
A 28-Day Oral Toxicity Study in Wistar Rats for a Highly Bioavailable Curcumin Preparation, CAVACURMIN®
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
Nutraceuticals
Volume 6
Issue 1
10.3390/nutraceuticals6010003
nutraceuticals-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

Toxicological Assessment and Potential Protective Effects of Brassica Macrocarpa Guss Leaf Extract Against Copper Sulphate-Induced Oxidative Stress in Zebrafish Embryos

by
Adele Cicio
1,
Luís M. Félix
2,3,*,
Sandra Mariza Monteiro
2,3,
Maurizio Bruno
1,4,
Maria Grazia Zizzo
1,4,5,* and
Rosa Serio
1
1
Department of Biological, Chemical and Pharmaceutical Sciences and Technologies (STEBICEF), University of Palermo, Viale delle Scienze, 90128 Palermo, Italy
2
Centre for the Research and Technology of Agroenvironmental and Biological Sciences (CITAB), Inov4Agro, University of Trás-os-Montes and Alto Douro (UTAD), Quinta de Prados, 5000-801 Vila Real, Portugal
3
Department of Biology and Environment, School of Life and Environmental Sciences, University of Trás-os-Montes and Alto Douro (UTAD), Quinta de Prados, 5000-801 Vila Real, Portugal
4
NBFC-National Biodiversity Future Center, University of Palermo, 90133 Palermo, Italy
5
ATeN (Advanced Technologies Network) Center, Viale delle Scienze, University of Palermo, 90128 Palermo, Italy
*
Authors to whom correspondence should be addressed.
Nutraceuticals 2026, 6(1), 3; https://doi.org/10.3390/nutraceuticals6010003
Submission received: 5 November 2025 / Revised: 15 December 2025 / Accepted: 16 December 2025 / Published: 23 December 2025
Browse Figures
Versions Notes

Abstract

Background: Oxidative stress is a key contributor to many chronic diseases. Natural biocompounds with antioxidant activity are of growing therapeutic interest. Brassica macrocarpa, a plant from the Brassicaceae family, has shown in vitro safety and antioxidant potential due to its rich content of glucosinolates and phenolics. However, in vivo, its effects remain poorly characterized. This study aimed to evaluate the in vivo safety and biological effects of Brassica macrocarpa leaf extract in zebrafish embryos and to assess its potential to counteract copper sulphate (CuSO4)-induced oxidative stress. Methods: Zebrafish embryos were exposed to Brassica macrocarpa extract at concentrations from 125 to 2000 µg/mL. Embryonic mortality and malformations were monitored daily to determine sub-lethal concentrations (125–500 µg/mL) for further behavioural and biochemical analysis. Antioxidant properties were tested in a CuSO4-induced oxidative stress model. Results: No teratogenic effects were observed over 96 h. Larvae showed normal swimming and no behavioural changes. Pre-treatment with the extract significantly reduced CuSO4-induced ROS and NO production, modulated antioxidant enzyme (SOD, CAT) activity, and lowered lipid peroxidation and protein oxidation, slightly affecting DNA damage. Conclusions: Brassica macrocarpa extract in vivo appears safe at sub-lethal doses and shows promising antioxidant effects, suggesting its potential role in managing oxidative stress-related conditions.

1. Introduction

Inflammation is a key biological immune response that protects the body during infections and tissue injuries, ensuring survival through tissue homeostasis [1]. The molecular mechanisms behind inflammation are complex, and they involve specific molecular patterns orchestrated by key regulators [2]. Recent evidence suggests that redox reactions inducing cellular oxidative stress play a crucial role in the pathophysiology of inflammation [3,4,5]. The accumulation of reactive oxygen species (ROS) and free radicals can damage cellular components like lipids, proteins, and DNA, often leading to the onset and progression of various diseases, including diabetes, cancer, and neurodegenerative disorders such as Alzheimer’s and Parkinson’s diseases [6].
Several synthetic drugs have been produced recently for treating inflammatory diseases and for counteracting oxidative stress. However, the prolonged use of these synthetic agents can lead to drug resistance and induce toxicological side effects [7,8,9]. Consequently, research has increasingly focused on natural compounds, such as polyphenols, flavonoids, and other plant-derived substances, for their potential to mitigate these adverse effects and to improve health outcomes. Plants are a prolific source of secondary metabolites with unique biological properties, including antioxidant and anti-inflammatory effects [10,11,12]. The Brassicaceae family, also known as the cruciferous family, is renowned in both traditional and modern medicine, largely due to its abundance in bioactive compounds such as glucosinolates and phenols, and several plants of this genus have already been shown to have beneficial effects in in vitro and in vivo models of inflammation and oxidative stress [13,14,15]. A recent study, conducted by our research group, characterized the composition of the leaf extract of Sicilian wild Brassica macrocarpa Guss (B. macrocarpa), revealing a rich phytochemical profile, particularly abundant in glucosinolates and flavonoids [16]. The extract was demonstrated to be safe in vitro, tested on the RAW 264.7 murine macrophage cell line. Furthermore, it exhibited protective effects under lipopolysaccharides (LPS)-induced oxidative stress by effectively reducing reactive oxygen species (ROS) and nitrite levels [16]. However, a comprehensive evaluation of the toxicity and beneficial properties of B. macrocarpa extract on an in vivo model is essential to ensure its safety in potential human use.
Over the years, the zebrafish (Danio rerio) has become a widely used model to evaluate different biological and toxicological responses [17,18,19,20]. The small size of the zebrafish and its rapid embryonic development allow for its convenient and easy use in research [21]. Furthermore, significant homologous genes and similarities in physiological response (such as immune response) between humans and zebrafish [22] make this model useful for understanding the mechanisms of toxicity and inflammatory responses.
Moreover, copper sulphate (CuSO4) is widely used in zebrafish models as a pro-oxidant agent due to its well-documented ability to induce oxidative stress by increasing the production of reactive oxygen species (ROS) and disrupting antioxidant defence mechanisms. CuSO4 exposure leads to oxidative damage in various tissues, which in turn leaded to oxidative damage to cellular macromolecules, disrupting normal embryonic development and causing various morphological and physiological defects [23].
Therefore, the aim of this study was at first to evaluate the safety in vivo of B. macrocarpa leaf by assessing its effects on zebrafish embryo development, locomotor behavior, and biochemical parameters. Subsequently, we evaluated whether the extract could have a beneficial effect on CuSO4-induced oxidative stress.

2. Materials and Methods

2.1. Plant Materials and Preparation of Plant Extract

The aerial parts of B. macrocarpa were collected in February 2022 in Favignana Island, Sicily, Italy. The specimen, identified by Professor Vincenzo Ilardi, was deposited in the STEBICEF Department, University of Palermo, Palermo, Italy (voucher no. 109761). The collected aerial parts of B. macrocarpa were dried, and the methanolic extract was prepared as previously described and analyzed by HPLC-MS/MS [16]. The composition published in our previous paper revealed a large amount of glucosinolates and different phenolic compounds [16]. The plant name was verified by “The Plant List” (http://www.theplantlist.org, accessed on 23 December 2012).

2.2. Fish Embryo Acute Toxicity (FET) Test

The AB wild-type zebrafish strain was maintained at the University of Trás-os-Montes and Alto Douro (Vila Real, Portugal) in a constant temperature–light cycle (28 °C and 14:10 h light–dark cycle) as previous reported [24]. The breeding process was facilitated by pairing zebrafish overnight (male-to-female ratio of 2:1) and spawning triggered by the morning light. Fertilized eggs were collected after approximately 1 h, washed one time in Chloramine-T solution at 0.5% w/v, and washed twice with E3 medium (5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl2, and 0.33 mM MgCl2, pH 7.2) [25]. The eggs with normal morphology were selected randomly and distributed in 6-well plates and maintained in E3 medium at 28.5 °C. The solution was refreshed each day, and larvae exhibiting abnormality or mortality were separated. All the experiments were terminated at 96 h post-fertilization (hpf); therefore, no animal test authorization was requested in accordance with European legislation (EU Directive, 2010/63/EU). Embryos at 2 hpf, in 6-well culture plates (20 embryos in 5 mL solution/well), were exposed to different concentrations of B. macrocarpa extract from a stock solution of 50 mg/mL in E3 medium and then diluted to obtain the final concentrations of 125–250–500–1000–2000 μg/mL. Untreated embryos were used as the control. The concentrations of the extract were based on preliminary studies on cellular models [16]. The solutions were replaced daily to maintain the appropriate concentration of the test compounds. Mortalities were recorded at 24, 48, and 96 h. According to the standard of the Organization for Economic Cooperation and Development (OECD), observations of embryo coagulation, failure of somite formation, failure of tail detachment, and lack of heartbeat were used to determine lethality. Quadrupled exposures were performed, and the LC50 value was obtained through probit analysis [26].
To evaluate zebrafish development, animals from each group were analyzed under an inverted microscope (Nikon SMZ-800 stereomicroscope, Nikon, Tokyo, Japan). The following parameters were evaluated to assess toxicity as reported in previous studies [27] at 24 hpf, somite formation, undetached head and tail, and spontaneous movements; at 48 hpf, eye and otolith development, pigmentation, and heart rate; at 72 hpf, hatching rate. At 96 hpf, larvae were immobilized in 3% methylcellulose and assessed for malformation, body length, area of the yolk sac, area of the heart, area of the eye, area of the head, and body angle. The analysis of digital images acquired using the inverted microscope and measurements were performed using Digimizer software (version 5.3.4, MedCalc Software Ltd., Acacialaan 22, 8400 Ostend, Belgium). Embryos were further collected for subsequent biochemical analysis.

2.3. Behavioural Analysis

Six larvae from each replicate were used for behavioural analysis through X, Y coordinate analysis in a video tracking system (TheRealFishTracker) [28]. Locomotor behaviour (open-field exploratory test), avoidance patterns (bouncing ball stimulus), and anxiety-like behaviours (black and white test) were analyzed in a dark, air-conditioned room. Briefly, 12-well agarose-coated plates containing 1 randomly selected larva per well were placed on top of a 15.6″ laptop LCD screen (1366 × 768-pixel resolution) displaying a white Microsoft PowerPoint presentation (Microsoft Corp., Washington, DC, USA). After 5 min of acclimation, an HD digital video camera was used to record the larvae’s exploratory behaviour for 10 min (average speed, total distance travelled, average distance from the centre of the well, average absolute turn angle, and percentage of active time). After an analysis of the exploratory behaviour, the anxious behaviour of the embryo was monitored under an equal division of the well in a white and black area provided by the Microsoft PowerPoint presentation (Microsoft Corp., Redmond, WA, USA). Additionally, avoidance response was measured by the ability of the embryo to respond to a visual stimulus (a bouncing red ball in the top half of the well moving from left to right) for 10 min, using the same plate setup and Microsoft PowerPoint presentation.

2.4. CuSO4 Treatment on Zebrafish Embryo

The experiments were performed on 72 hpf zebrafish larvae when most organs are well developed and zebrafish enter the free-swimming larval stage. A concentration of 20 μM of CuSO4 is the dose required to induce oxidative stress in zebrafish embryos [29]. Preliminarily, zebrafish were treated with CuSO4 (CuSO4·5H2O, CAS 7758-99-8 from Merck, S.A, Lisbon, Portugal) for different times (1 h or 5 h) to determine the effective incubation time at which ROS and NO were released without affecting viability. Then, according to the results obtained and presented below, larvae were treated for 5 h with CuSO4 (20 μM).
Zebrafish larvae were selected and randomly divided into eight groups (n = 30/well in triplicate, containing 5 mL of E3 medium). Specifically, the experimental groups included: a control group (E3 medium), a group exposed to CuSO4 (20 μM for 5 h), and groups treated with increasing sub-lethal concentrations of B. macrocarpa extract (125, 250, and 500 μg/mL) for 26 h, either alone or in combination with CuSO4 exposure (20 μM during the final 5 h) (Figure 1). At the end of exposure, the possible antioxidant effect was investigated using the biochemical analyses reported on below.

2.5. Antioxidant Activity Evaluation

Larvae were collected in 400 μL of cold buffer (0.32 mM of sucrose, 20 mM of HEPES, 1 mM of MgCl2, and 0.5 mM of phenylmethyl sulfonylfluoride (PMSF), pH 7.4) [30] and homogenized in a Tissuelyser II (30 Hz for 30 s—Qiagen, Hilden, Germany). The supernatant was collected at 10,000× g at 4 °C for 10 min (Sigma 3K30, Sigma-Aldrich, Osterode, Germany), and total protein concentration was measured at 280 nm using a Take3 Multi-Volume plate (Take 3 plate, BioTek Instruments, Winooski, VT, USA). The biomarker assessment was conducted in duplicate using a PowerWave XS2 microplate scanning spectrophotometer or a Cary Eclipse fluorescence spectrophotometer at 30 °C as already described [31,32]. Total reactive oxygen species (ROS) levels were determined using the H2DCF-DA fluorescent probe at 485 nm excitation and 530 nm emission wavelengths, based on a DCF standard curve. The level of nitric oxide (NO) was determined using the Griess method, with some modifications. Briefly, samples were mixed with the Griess reagent in a 1:1 ratio and incubated for 15 min at room temperature. Then, the absorbance was read at 540 nm. Sodium nitrate was used to construct a standard curve (0–1 µM). Superoxide dismutase (SOD) activity was assessed using the NBT method at 560 nm, while catalase (CAT) activity was measured based on hydrogen peroxide degradation at 240 nm. Lipid peroxidation was assessed through the determination of thiobarbituric acid reactive substances (TBARS) at 535 nm excitation and 630 nm emission wavelengths. Protein carbonylation was determined at 450 nm based on the reaction between protein carbonyls and 2,4-dinitrophenylhydrazine (DNPH), while DNA damage was determined via the binding of the fluorescent Hoechst 33,258 to DNA at 360 nm excitation and 450 nm emission wavelengths. All data were normalized for protein content and expressed as a percentage of control.

2.6. Statistical Analysis

All data are shown as the mean ± Standard Deviation (SD). The Kolmogorov-Smirnov test was used to evaluate the normality of distribution. A statistical analysis of the data was performed using GraphPad Prism software version 6 (GraphPad Software, San Diego, CA, USA) and a one- or two-way analysis of variance (ANOVA), followed by Dunnett’s post hoc test. p < 0.05 was considered to indicate a statistically significant difference.

3. Results

3.1. Effect of B. macrocarpa Extract on Embryo Survival

The survival rate of the larvae exposed to various concentrations of B. macrocarpa extract was assessed every 24 h over the 96 h treatment period. The survival rate of the larvae at concentrations ranging from 125 μg/mL to 500 μg/mL revealed no significant difference compared to the control at all four time points. In contrast, larvae treated with concentrations of 1000 μg/mL and 2000 μg/mL began to exhibit mortality as early as 48 h post-treatment (Figure 2). At 96 h, LC50 was 762.1 ± 20.6 μg/mL.

3.2. Fish Embryo Acute Toxicity (FET) Test on Zebrafish

The FET test on zebrafish was conducted to assess potential teratogenic effects on embryo development. At 24 hpf, somite formation, as well as head and tail detachment, appeared normal across all treated groups compared to the control group. However, the number of spontaneous movements significantly increased in larvae exposed to the two highest concentrations when compared to the control group (p < 0.05). A tendency toward increased activity was also observed at 250 and 500 µg/mL; however, the trends shown in Figure 3A do not reach statistical significance (Figure 3A). At 48 hpf, animals exposed to the sub-lethal concentrations of 250 μg/mL and 500 μg/mL showed a significant reduction in heartbeat rate (p < 0.05) (Figure 3B).
At 72 hpf, the hatching rate was also assessed, revealing no significant differences between the treated and untreated groups (Figure 3C). At 96 hpf, a morphological evaluation of zebrafish exposed to sub-lethal concentrations showed development comparable to the control group, with only minor malformations observed. The overall morphology, including the backbone angle, body shape (size, eye, head, yolk, pericardial area), and general edema parameters, appeared normal and within the physiological range (Table 1, Figure 4). Based on these findings, further experiments were conducted using sub-lethal concentrations such as 125–250–500 μg/mL.

3.3. Locomotor Behaviour

The effects of B. macrocarpa exposure from 2 hpf to 96 hpf at sub-lethal concentrations on zebrafish behaviour are shown in Table 2. In the open tank test, no significant differences were observed in the mean distance travelled, speed, immobility, absolute turn angle, or distance from the centre (p > 0.05). Similarly, in the white/dark test, no differences were detected between treated embryos and the control group (p > 0.05). Furthermore, in the aversion test, although larvae exposed to the extract spent more time in the stimulus area, this increase was not statistically significant compared to the control (p > 0.05).

3.4. Effect of 26 h Exposure to B. macrocarpa Extract on Oxidative Stress in Zebrafish Larvae

The treatment of zebrafish larvae at 72 hpf with sub-lethal concentrations of B. macrocarpa extract for 24 h did not affect the oxidative stress status (p > 0.05), as shown in Figure 5. Exposure to sub-lethal concentrations of the extract did not result in a statistically significant alteration in the levels of the oxidation products as ROS and NO or the activities of the antioxidant enzymes SOD and CAT. Moreover, no statistically significant increase in lipid, protein, or DNA oxidation products was observed.

3.5. Antioxidant Effect of B. macrocarpa Extract on CuSO4-Induced Oxidative Stress in Zebrafish Larvae

As reported in the methods section, preliminary studies were conducted to determine the optimal exposure time to CuSO4 able to induce oxidative stress in zebrafish larvae without causing mortality. Larvae at 72 hpf were exposed to 20 μM CuSO4, for 1 h or 5 h, and the levels of oxidative mediators were assessed, particularly ROS and NO levels, which are key stress indicators.
The results, expressed as a percentage relative to the control (larvae exposed only to E3 medium = time 0 h), revealed a significant increase in ROS and NO levels, compared to the control, after 5 h of exposure to 20 μM CuSO4 (Figure 6). Additionally, 5 h of exposure to CuSO4 resulted in increased activity of the antioxidant enzymes SOD and CAT, elevated MDA levels, enhanced protein oxidation, and DNA damage (Figure 7). Treatment with the extract significantly reduced the ROS and NO levels, restored SOD and CAT enzyme activity, alleviated the increase in MDA levels, and decreased the protein carbonyl levels. The latter two effects were statistically significant at B. macrocarpa extract concentrations of 250 μg/mL and 500 μg/mL. However, the extract did not significantly reduce the DNA damage caused by CuSO4 exposure.

4. Discussion

Our study aimed to investigate, for the first time, the safety and antioxidant proprieties of a leaf extract of Brassica macrocarpa, a wild species endemic to Favignana Island (Sicily, Italy), in zebrafish larvae, a widely used in vivo model. The results of the present study indicated that in zebrafish larvae, B. macrocarpa extract was non-toxic and displayed a protective effect against CuSO4-induced oxidative stress. The latter was achieved by reducing ROS and NO levels and modulating the activities of antioxidant enzymes.
As above reported, our previous studies revealed a rich phytochemical profile in the leaf extract of Sicilian wild B. macrocarpa, particularly the presence of glucosinolates and flavonoids. In addition, we showed that the B. macrocarpa extract was safe on the RAW 264.7 murine macrophage cell line and exhibited beneficial properties under LPS-induced oxidative stress conditions by effectively reducing the levels of ROS and nitrites [16]. Evidence suggests the beneficial effects of glucosinolates in the in vivo model, and in particular, their supplementation seems to exert positive effects during or after ethanol exposure in zebrafish embryos [33].
In our study, zebrafish embryos at approximately 2 hpf were used to evaluate the toxicity of the B. macrocarpa extract. In fact, due to the transparency and extra-uterine development of the embryos, it was clearly possible to observe phenotypic changes during embryonic development, allowing for the evaluation of the teratogenic and embryotoxic effects of the extract and the determination of safe concentrations.
As previously observed in cellular models, our Brassica extract was also found to be safe in vivo, since a lack of mortality was observed at lower concentrations (125–500 μg/mL), with no malformations observed up to 96 hpf. The LC50 value (762.1 ± 20.6 μg/mL) was higher than the OECD values, suggesting that, at this stage, the methanolic extract is non-toxic and safe for consumption, at least for concentrations lower than its LC50 value. At higher concentrations, as 1000 μg/mL and 2000 μg/mL, the extract induced the death of all zebrafish animals between 48 h and 72 h. The exact toxicity mechanisms of the higher doses of Brassica macrocarpa extract remain to be elucidated. A possible explanation could be based on the known bioactive compounds present in the Brassicaceae family. Indeed, glucosinolates and their hydrolysis products (e.g., isothiocyanates and nitriles) are known to exhibit both protective and potentially cytotoxic effects depending on concentration, duration of exposure, and cellular context [34,35]. Another possible explanation could be due to the visible deposition of the extract components on the chorion surface, which can be interpreted as forming a thin layer or particulate around the embryos, which may cause the obstruction of chorion pores, with a resulting blockade of oxygen transport from the medium into the embryo, as previously demonstrated, for instance, for graphene oxide and iron oxide nanoparticles [36,37].
Moreover, the observation that at 24 hpf, a trend toward hyperactivity emerges, reaching statistical significance at 1000 and 2000 μg/mL, suggests that the extract may begin to disrupt the neural circuits underlying the development of locomotor function. However, it is important to stress that the mortality rate alone is not the definitive criterion for assessing the safety of a plant extract. The impact of the extract on the overall development of the organism must also be taken into consideration [38]. Recently, Deng et al. (2023) reported that a 72 h exposure to 500 μg/mL of a methanolic extract from broccoli (Brassica oleracea) significantly reduced embryo survival and induced teratogenic effects [39]. In contrast, our extract appeared to be safe at the same concentration and exposure duration.
At 96 hpf, morphological aspects in zebrafish larvae exposed to sub-lethal concentrations of the B. macrocarpa extract revealed no effects on their development, suggesting the potential safety of these concentrations. The extract at the concentration of 250–500 μg/mL at 48 hpf induced the inhibition of the heart rate of embryos. However, this decrease did not compromise the delivery of essential nutrients and oxygen required for normal embryonic development [40]; this observation may suggest a mild sedative or anxiolytic effect of the extract. Although overall behavioural outcomes were comparable across treatments, a slight reduction in locomotor activity was observed at 500 μg/mL.
Moreover, the extract did not influence hatching outcomes [41]. In fact, at 72 hpf, the hatching rate showed no significant difference between treated and untreated embryos.
In zebrafish, behaviour analysis is an index of nervous system development, thus allowing us to study the eventual neurotoxic effects of various drugs. The behavioural assessments included tests for locomotor activity, response to aversive stimuli, and anxiolytic-like behaviour. Our data revealed that throughout the observation period, zebrafish from all the experimental groups displayed typical swimming behaviours and appropriate responses to stimuli, suggesting that the extract did not induce any neurotoxic or detrimental effects.
These data establish a baseline for the selection of non-lethal concentrations suitable for subsequent experiments on the biological effects of B. macrocarpa.
Numerous in vitro assays, such as the 2,2-Diphenyl-1-Picrylhydrazyl (DPPH) radical scavenging capacity assay, demonstrated that extracts from various components of the Brassicaceae family also have high antioxidant power [10]. In cellular models, Brassicaceae has been reported to inhibit iNOS expression and function and thus NO production [10].
Oxidative stress can contribute to the development of various diseases [42,43]. Both reactive oxygen species (ROS) and reactive nitrogen species (RNS) can have detrimental effects on critical biological molecules such as DNA, lipids, and proteins [44,45].
Our data suggest that the extract is safe at doses up to 500 μg/mL, as it does not induce oxidative stress under normal physiological conditions. Specifically, it did not affect ROS or NO levels, nor promote lipid peroxidation, protein carbonylation, or DNA damage. In addition, our previous data indicated its ability in vitro both in non-biological and biological systems to counteract oxidative stress [16].
The exposure of zebrafish embryos to CuSO4 was reported to induce an inflammatory status, which is related to exacerbated damage and oxidative stress, and the endogenous signalling molecule adenosine was shown to be involved [46] resulting in a suitable model for studying the effects of plant extracts on the antioxidant system. Our results indicate that CuSO4 (20 μM for 5 h) successfully induced oxidative stress, increasing the levels of ROS, NO, and oxidative damage markers without changes in the viability of the zebrafish larvae at 72 hpf.
B. macrocarpa extract pre-treatment was able to decrease, in a concentration-dependent manner, oxidative stress parameters. In detail, B. macrocarpa extract (250–500 μg/mL) significantly exhibits antioxidant potential, as observed in vitro [11]. Indeed, by using the ROS fluorescent probe H2DCFDA, we observed that B. macrocarpa extract could effectively reduce ROS production stimulated by CuSO4 in vivo in a dose-dependent manner, with the dose of 500 μg/mL being the most effective. These findings agree with those previously reported for other plant extracts displaying a protective effect against stress in vivo by reducing the copper sulphate-induced generation of reactive oxygen species [29,47,48]. Our extract seems to also modulate NO levels and SOD and CAT activities, confirming in vivo its antioxidant effects.
As previously reported, various Brassica extracts, such as those from Brassica oleracea, Brassica juncea, and Brassica rapa, have demonstrated antioxidant properties, including ROS scavenging, the inhibition of ROS production, and the activation of antioxidant enzymes in vivo [10]. The novelty of our study lies in the investigation of Brassica macrocarpa, an endemic and less-studied Sicilian species, particularly in the context of in vivo zebrafish models, where its effects have not been previously characterized.
Based on the known bioactive components of Brassicaceae, such as glucosinolates and their derivatives, it is plausible that the extract modulates antioxidant enzyme activity through redox-sensitive signalling pathways, such as the Nrf2/Keap1 axis. Glucosinolates have been widely recognized for their role in chronic disease prevention; for example, sulforaphane, a well-studied isothiocyanate derived from broccoli, has been shown to activate the Nrf2 pathway, resulting in the upregulation of antioxidant enzymes and attenuation of inflammatory responses [49,50]. However, this hypothesis requires further investigation through specific molecular analyses.
Moreover, a partial reduction in lipid peroxidation and protein oxidation was observed, while no significant decrease in DNA damage was detected. Although polyphenols, flavonoids, and other bioactive compounds are well known for their ability to reduce lipid peroxidation, protein carbonyl formation, and DNA damage [51,52,53], it is possible that the antioxidant components of the extract are more effective in neutralizing ROS at the membrane or cytoplasmic level while offering limited protection at the nuclear level. Additionally, the absence of effect on DNA damage may reflect concentration-dependent limitations or a need for longer exposure times to observe nuclear protection. This aspect represents an important direction for future studies.
Intriguingly, 5 h of CuSO4 exposure increased the expression of SOD and CAT, antioxidant enzymes, which are considered the primary line of defence towards the production of ROS under stress conditions [54]. The observed changes in antioxidant enzyme expression, such as that of SOD and CAT, may reflect a compensatory cellular response rather than a direct induction by the Brassica extract. The phytochemicals in the extract could be able to modulate oxidative stress through indirect mechanisms, including the activation of endogenous antioxidant defences. Then, while the extract itself possesses antioxidant properties, the modulation of SOD and CAT activity may result from a feedback mechanism aimed at maintaining redox homeostasis. Other signalling pathways may be involved, and further studies are needed to clarify the precise molecular mechanisms.
Rodriguez et al. (2004) suggested that the activity of certain antioxidant enzymes may increase under moderate oxidative stress [55]. Alternatively, it is possible to hypothesize that the larvae, to prevent oxidative stress and maintain cellular redox balance, utilize endogenous antioxidant enzymes; in this scenario, the embryos may upregulate enzymes as superoxide dismutase, catalase, or glutathione-related systems to neutralize reactive oxygen species and maintain cellular redox homeostasis. Such an adaptive response has been documented in other metal-induced toxicological contexts; for example, G. Cai et al. (2012) [56] demonstrated that exposure to cobalt induces a similar enhancement in antioxidant defences during embryotoxicity. This observation suggests that zebrafish larvae may employ comparable protective mechanisms when challenged with CuSO4 [56]. Likely, the pre-treatment with the extract, preventing the onset of oxidative stress induced by CuSO4 exposure, did not induce any increase in enzymatic activity levels, remaining comparable to the control group [29,47,48].
Further studies monitoring glutathione (GSH), which directly scavenges free radicals and serves as a substrate for glutathione peroxidase thereby detoxifying peroxides and maintaining redox homeostasis could help to better characterize the antioxidant capacity of Brassica macrocarpa. In addition to the changes in SOD and CAT activity, a reduction in GSH levels may indicate a compromised antioxidant response and increased susceptibility to oxidative damage. Therefore, evaluating the possible modulation of GSH by B. macrocarpa could strengthen and reinforce our current findings.
Thus, these findings revealed the in vivo safety in vivo and antioxidant potential of the B. macrocarpa extract in a model of oxidative stress, likely acting by directly scavenging free radicals and modulating the cellular antioxidant defence system.
Finally, although our previous work initiated the qualitative and quantitative phytochemical profiling of the crude extract [16], further standardization is needed. A detailed identification and quantification of the active compounds responsible for the observed biological effects is essential to ensure reproducibility and enhance its translational relevance.

5. Conclusions

The present study demonstrated that B. macrocarpa leaf extract showed biosafety in toxicity assays conducted in zebrafish and was able to protect this animal model against oxidative stress induced by copper sulphate by reducing ROS and NO levels and modulating the activities of antioxidant enzymes. These data further support the antioxidant capacity of this extract already observed in vitro. As indicated above, one limitation of the present study is the use of a crude extract without the precise identification of its active components. Although our preliminary phytochemical analyses suggest the presence of bioactive compounds such as glucosinolates and flavonoids [16], further investigations are needed to identify the specific constituents responsible for the observed effects. Moreover, the standardization of the Brassica macrocarpa extract is essential to ensure reproducibility and enhance its translational relevance.
Our results support the need for further investigation, including the following: (1) dose–response and long-term studies to confirm the observed effects and evaluate their potential clinical or commercial relevance and (2) mechanistic studies to clarify the underlying mechanisms of the B. macrocarpa extract and to better understand its antioxidant activity, especially in the context of oxidative stress-related diseases.

Author Contributions

A.C.: Investigation; Data analysis; Preparation of figures; Writing—original draft. L.M.F. and M.G.Z.: Conceptualization; Investigation; Data analysis and interpretation; Preparation of figures; Writing—original draft and editing. M.B. Writing—reviewing and editing. S.M.M. and R.S.: Supervision, Conceptualization; Data interpretation; Writing—reviewing and editing. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by a grant from Ministero dell’Università e della Ricerca Scientifica (MIUR) (FFR2024). This work was also supported by National Funds by FCT –Portuguese Foundation for Science and Technology, under the projects UID/04033/2025: Centre for the Research and Technology of Agro-Environmental and Biological Sciences and LA/P/0126/2020 (https://doi.org/10.54499/LA/P/0126/2020).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Data will be made available from the corresponding author on reasonable request.

Conflicts of Interest

The authors declare no conflicts of interest.

Abbreviations

CATCatalase
CuSO4Copper Sulphate
HpfHours Post-Fertilization
LPSLipopolysaccharide
OECDOrganization For Economic Cooperation and Development
NONitric Oxide
ROSReactive Oxygen Species
SDStandard Deviation
SODSuperoxide Dismutase

References

  1. Ahmed, A.U. An Overview of Inflammation: Mechanism and Consequences. Front. Biol. 2011, 6, 274–281. [Google Scholar] [CrossRef]
  2. Medzhitov, R. Origin and Physiological Roles of Inflammation. Nature 2008, 454, 428–435. [Google Scholar] [CrossRef] [PubMed]
  3. Lugrin, J.; Rosenblatt-Velin, N.; Parapanov, R.; Liaudet, L. The Role of Oxidative Stress during Inflammatory Processes. Biol. Chem. 2014, 395, 203–230. [Google Scholar] [CrossRef] [PubMed]
  4. Hussain, T.; Tan, B.; Yin, Y.; Blachier, F.; Tossou, M.C.B.; Rahu, N. Oxidative Stress and Inflammation: What Polyphenols Can Do for Us? Oxidative Med. Cell. Longev. 2016, 2016, 7432797. [Google Scholar] [CrossRef]
  5. Chatterjee, S. Oxidative Stress, Inflammation, and Disease; Elsevier Inc.: Amsterdam, The Netherlands, 2016; ISBN 9780128032701. [Google Scholar]
  6. Hajam, Y.A.; Rani, R.; Ganie, S.Y.; Sheikh, T.A.; Javaid, D.; Qadri, S.S.; Pramodh, S.; Alsulimani, A.; Alkhanani, M.F.; Harakeh, S.; et al. Oxidative Stress in Human Pathology and Aging: Molecular Mechanisms and Perspectives. Cells 2022, 11, 552. [Google Scholar] [CrossRef]
  7. Zhou, N.; Zhao, Y.; Yao, Y.; Wu, N.; Xu, M.; Du, H.; Wu, J.; Tu, Y. Antioxidant Stress and Anti-Inflammatory Activities of Egg White Proteins and Their Derived Peptides: A Review. J. Agric. Food Chem. 2022, 70, 5–20. [Google Scholar] [CrossRef]
  8. Bindu, S.; Mazumder, S.; Bandyopadhyay, U. Non-Steroidal Anti-Inflammatory Drugs (NSAIDs) and Organ Damage: A Current Perspective. Biochem. Pharmacol. 2020, 180, 114147. [Google Scholar] [CrossRef]
  9. Biasi, F.; Astegiano, M.; Maina, M.; Leonarduzzi, G.; Poli, G. Polyphenol Supplementation as a Complementary Medicinal Approach to Treating Inflammatory Bowel Disease. Curr. Med. Chem. 2011, 18, 4851–4865. [Google Scholar] [CrossRef]
  10. Cicio, A.; Sut, S.; Dall’Acqua, S.; Bruno, M.; Luparello, C.; Serio, R.; Zizzo, M.G. Chemical Characterization and Cytotoxic and Antioxidant Activity Evaluation of the Ethanol Extract from the Bulbs of Pancratium maritimun Collected in Sicily. Molecules 2023, 28, 3986. [Google Scholar] [CrossRef]
  11. Cojocariu, R.O.; Balmus, I.M.; Lefter, R.; Hritcu, L.; Ababei, D.C.; Ciobica, A.; Copaci, S.; Mot, S.E.L.; Copolovici, L.; Copolovici, D.M.; et al. Camelina Sativa Methanolic and Ethanolic Extract Potential in Alleviating Oxidative Stress, Memory Deficits, and Affective Impairments in Stress Exposure-Based Irritable Bowel Syndrome Mouse Models. Oxidative Med. Cell. Longev. 2020, 2020, 9510305. [Google Scholar] [CrossRef]
  12. Mattosinhos, P.d.S.; Sarandy, M.M.; Novaes, R.D.; Esposito, D.; Gonçalves, R.V. Anti-Inflammatory, Antioxidant, and Skin Regenerative Potential of Secondary Metabolites from Plants of the Brassicaceae Family: A Systematic Review of In vitro and In vivo Preclinical Evidence (Biological Activities Brassicaceae Skin Diseases). Antioxidants 2022, 11, 1346. [Google Scholar] [CrossRef]
  13. Shankar, S.; Segaran, G.; Sundar, R.D.V.; Settu, S.; Sathiavelu, M. Brassicaceae—A Classical Review on Its Pharmacological Activities. Int. J. Pharm. Sci. Rev. Res. 2019, 55, 107–113. [Google Scholar]
  14. Cicio, A.; Serio, R.; Zizzo, M.G. Anti-Inflammatory Potential of Brassicaceae-Derived Phytochemicals: In vitro and In vivo Evidence for a Putative Role in the Prevention and Treatment of IBD. Nutrients 2023, 15, 31. [Google Scholar] [CrossRef] [PubMed]
  15. Rokayya, S.; Li, C.J.; Zhao, Y.; Li, Y.; Sun, C.H. Cabbage (Brassica oleracea L. var. Capitata) Phytochemicals with Antioxidant and Anti-Inflammatory Potential. Asian Pac. J. Cancer Prev. 2013, 14, 6657–6662. [Google Scholar] [CrossRef] [PubMed]
  16. Cicio, A.; Aloi, N.; Sut, S.; Longo, V.; Terracina, F.; Dall’Acqua, S.; Zizzo, M.G.; Bruno, M.; Ilardi, V.; Colombo, P.; et al. Chemical Characterization, Free Radical Scavenging, and Cellular Antioxidant Properties of the Egadi Island Endemic Brassica macrocarpa Guss Leaf Extract. Biomolecules 2024, 14, 636. [Google Scholar] [CrossRef]
  17. MacRae, C.A.; Peterson, R.T. Zebrafish as Tools for Drug Discovery. Nat. Rev. Drug Discov. 2015, 14, 721–731. [Google Scholar] [CrossRef]
  18. Zon, L.I.; Peterson, R.T. In vivo Drug Discovery in the Zebrafish. Nat. Rev. Drug Discov. 2005, 4, 35–44. [Google Scholar] [CrossRef]
  19. Belo, M.A.A.; Oliveira, M.F.; Oliveira, S.L.; Aracati, M.F.; Rodrigues, L.F.; Costa, C.C.; Conde, G.; Gomes, J.M.M.; Prata, M.N.L.; Barra, A.; et al. Zebrafish as a Model to Study Inflammation: A Tool for Drug Discovery. Biomed. Pharmacother. 2021, 144, 112310. [Google Scholar] [CrossRef]
  20. Arteaga, C.; Boix, N.; Teixido, E.; Marizande, F.; Cadena, S.; Bustillos, A. The Zebrafish Embryo as a Model to Test Protective Effects of Food Antioxidant Compounds. Molecules 2021, 26, 5786. [Google Scholar] [CrossRef]
  21. Jayasinghe, C.D.; Jayawardena, U.A. Toxicity Assessment of Herbal Medicine Using Zebrafish Embryos: A Systematic Review. Evid.-Based Complement. Altern. Med. 2019, 2019, 7272808. [Google Scholar] [CrossRef]
  22. Forn-Cuní, G.; Varela, M.; Pereiro, P.; Novoa, B.; Figueras, A. Conserved Gene Regulation during Acute Inflammation between Zebrafish and Mammals. Sci. Rep. 2017, 7, 41905. [Google Scholar] [CrossRef] [PubMed]
  23. Wang, Y.; Tian, J.; Shi, F.; Li, X.; Hu, Z.; Chu, J. Protective Effect of Surfactin on Copper Sulfate-Induced Inflammation, Oxidative Stress, and Hepatic Injury in Zebrafish. Microbiol. Immunol. 2021, 65, 410–421. [Google Scholar] [CrossRef] [PubMed]
  24. Lanzarin, G.A.B.; Félix, L.M.; Monteiro, S.M.; Ferreira, J.M.; Oliveira, P.A.; Venâncio, C. Anti-Inflammatory, Anti-Oxidative and Anti-Apoptotic Effects of Thymol and 24-Epibrassinolide in Zebrafish Larvae. Antioxidants 2023, 12, 11297. [Google Scholar] [CrossRef]
  25. Meyers, J.R. Zebrafish: Development of a Vertebrate Model Organism. Curr. Protoc. Essent. Lab. Tech. 2018, 16, 19. [Google Scholar] [CrossRef]
  26. Finney, D.J. Probit Analysis: A Statistical Treatment of the Sigmoid Response Curve, 2nd ed.; Cambridge University Press: Cambridge, UK, 1952. [Google Scholar]
  27. Vieira, R.; Venâncio, C.A.S.; Félix, L.M. Toxic Effects of a Mancozeb-Containing Commercial Formulation at Environmental Relevant Concentrations on Zebrafish Embryonic Development. Environ. Sci. Pollut. Res. 2020, 27, 21174–21187. [Google Scholar] [CrossRef]
  28. Félix, L.M.; Antunes, L.M.; Coimbra, A.M.; Valentim, A.M. Behavioral Alterations of Zebrafish Larvae after Early Embryonic Exposure to Ketamine. Psychopharmacology 2017, 234, 549–558. [Google Scholar] [CrossRef]
  29. Nguyen, T.H.; Le, H.D.; Nguyen Thi Kim, T.; Pham The, H.; Nguyen, T.M.; Cornet, V.; Lambert, J.; Kestemont, P. Anti–Inflammatory and Antioxidant Properties of the Ethanol Extract of Clerodendrum Cyrtophyllum Turcz in Copper Sulfate-induced Inflammation in Zebrafish. Antioxidants 2020, 9, 192. [Google Scholar] [CrossRef]
  30. Deng, J.; Yu, L.; Liu, C.; Yu, K.; Shi, X.; Yeung, L.W.Y.; Lam, P.K.S.; Wu, R.S.S.; Zhou, B. Hexabromocyclododecane-Induced Developmental Toxicity and Apoptosis in Zebrafish Embryos. Aquat. Toxicol. 2009, 93, 29–36. [Google Scholar] [CrossRef]
  31. Félix, L.M.; Vidal, A.M.; Serafim, C.; Valentim, A.M.; Antunes, L.M.; Monteiro, S.M.; Matos, M.; Coimbra, A.M. Ketamine Induction of P53-Dependent Apoptosis and Oxidative Stress in Zebrafish (Danio rerio) Embryos. Chemosphere 2018, 201, 730–739. [Google Scholar] [CrossRef]
  32. Félix, L.M.; Vidal, A.M.; Serafim, C.; Valentim, A.M.; Antunes, L.M.; Campos, S.; Matos, M.; Monteiro, S.M.; Coimbra, A.M. Ketamine-Induced Oxidative Stress at Different Developmental Stages of Zebrafish (Danio rerio) Embryos. RSC Adv. 2016, 6, 61254–61266. [Google Scholar] [CrossRef]
  33. Wu, Z.; Chen, S.-Y.; Zheng, L. Sulforaphane Attenuates Ethanol-Induced Teratogenesis and Dysangiogenesis in Zebrafish Embryos. Int. J. Mol. Sci. 2024, 25, 11529. [Google Scholar] [CrossRef]
  34. Wu, X.; Kassie, F.; Mersch-Sundermann, V. Induction of Apoptosis in Tumor Cells by Naturally Occurring Sulfur-Containing Compounds. Mutat. Res./Rev. Mutat. Res. 2005, 589, 81–102. [Google Scholar] [CrossRef] [PubMed]
  35. Kadir, N.H.A.; David, R.; Rossiter, J.T.; Gooderham, N.J. The Selective Cytotoxicity of the Alkenyl Glucosinolate Hydrolysis Products and Their Presence in Brassica Vegetables. Toxicology 2015, 334, 59–71. [Google Scholar] [CrossRef] [PubMed]
  36. Chen, Y.; Hu, X.; Sun, J.; Zhou, Q. Specific Nanotoxicity of Graphene Oxide during Zebrafish Embryogenesis. Nanotoxicology 2016, 10, 42–52. [Google Scholar] [CrossRef] [PubMed]
  37. Rizzo, L.Y.; Golombek, S.K.; Mertens, M.E.; Pan, Y.; Laaf, D.; Broda, J.; Jayapaul, J.; Möckel, D.; Subr, V.; Hennink, W.E. In vivo Nanotoxicity Testing Using the Zebrafish Embryo Assay. J. Mater. Chem. B 2013, 1, 3918–3925. [Google Scholar] [CrossRef]
  38. Sobanska, M.; Scholz, S.; Nyman, A.M.; Cesnaitis, R.; Gutierrez Alonso, S.; Klüver, N.; Kühne, R.; Tyle, H.; de Knecht, J.; Dang, Z.; et al. Applicability of the Fish Embryo Acute Toxicity (FET) Test (OECD 236) in the Regulatory Context of Registration, Evaluation, Authorisation, and Restriction of Chemicals (REACH). Environ. Toxicol. Chem. 2018, 37, 657–670. [Google Scholar] [CrossRef]
  39. Deng, W.W.; Zang, C.R.; Li, Q.C.; Sun, B.; Mei, X.P.; Bai, L.; Shang, X.M.; Deng, Y.; Xiao, Y.Q.; Ghiladi, R.A.; et al. Hydrothermally Derived Green Carbon Dots from Broccoli Water Extracts: Decreased Toxicity, Enhanced Free-Radical Scavenging, and Anti-Inflammatory Performance. ACS Biomater. Sci. Eng. 2023, 9, 1307–1319. [Google Scholar] [CrossRef]
  40. Kopp, R.; Pelster, B.; Schwerte, T. How Does Blood Cell Concentration Modulate Cardiovascular Parameters in Developing Zebrafish (Danio rerio)? Comp. Biochem. Physiol. Part A Mol. Integr. Physiol. 2007, 146, 400–407. [Google Scholar] [CrossRef]
  41. McGrath, P.; Li, C.Q. Zebrafish: A Predictive Model for Assessing Drug-Induced Toxicity. Drug Discov. Today 2008, 13, 394–401. [Google Scholar] [CrossRef]
  42. Bhattacharyya, A.; Chattopadhyay, R.; Mitra, S.; Crowe, S.E. Oxidative Stress: An Essential Factor in the Pathogenesis of Gastrointestinal Mucosal Diseases. Physiol. Rev. 2014, 94, 329–354. [Google Scholar] [CrossRef]
  43. Zuo, L.; Prather, E.R.; Stetskiv, M.; Garrison, D.E.; Meade, J.R.; Peace, T.I.; Zhou, T. Inflammaging and Oxidative Stress in Human Diseases: From Molecular Mechanisms to Novel Treatments. Int. J. Mol. Sci. 2019, 20, 4472. [Google Scholar] [CrossRef] [PubMed]
  44. Juan, C.A.; Pérez de la Lastra, J.M.; Plou, F.J.; Pérez-Lebeña, E. The Chemistry of Reactive Oxygen Species (ROS) Revisited: Outlining Their Role in Biological Macromolecules (DNA, Lipids and Proteins) and Induced Pathologies. Int. J. Mol. Sci. 2021, 22, 4642. [Google Scholar] [CrossRef] [PubMed]
  45. Weidinger, A.; Kozlov, A.V. Biological Activities of Reactive Oxygen and Nitrogen Species: Oxidative Stress versus Signal Transduction. Biomolecules 2015, 5, 472–484. [Google Scholar] [CrossRef] [PubMed]
  46. Singh, M.; Guru, A.; Sudhakaran, G.; Pachaiappan, R.; Mahboob, S.; Al-ghanim, K.A.; Al-misned, F.; Juliet, A.; Gobi, M.; Arokiaraj, J. Copper Sulfate Induced Toxicological Impact on In-Vivo Zebrafish Larval Model Protected Due to Acacetin via Anti-Inflammatory and Glutathione Redox Mechanism. Comp. Biochem. Physiol. Part C 2022, 262, 109463. [Google Scholar] [CrossRef]
  47. da Silva Cordeiro, M.L.; de Queiroz Aquino-Martins, V.G.; da Silva, A.P.; de Souza Paiva, W.; Silva, M.M.C.L.; Luchiari, A.C.; Rocha, H.A.O.; Scortecci, K.C. Bioactivity of Talisia Esculenta Extracts: Antioxidant and Anti-Inflammatory Action on RAW 264.7 Macrophages and Protective Potential on the Zebrafish Exposed to Oxidative Stress Inducers. J. Ethnopharmacol. 2025, 337, 118932. [Google Scholar] [CrossRef]
  48. Liu, N.; Zhang, P.; Xue, M.; Zhang, M.; Xiao, Z.; Xu, C.; Fan, Y.; Liu, W.; Wu, Y.; Wu, M.; et al. Anti-Inflammatory and Antioxidant Properties of Rice Bran Oil Extract in Copper Sulfate-Induced Inflammation in Zebrafish (Danio rerio). Fish Shellfish Immunol. 2023, 136, 108740. [Google Scholar] [CrossRef]
  49. Baldelli, S.; Lombardo, M.; D’Amato, A.; Karav, S.; Tripodi, G.; Aiello, G.; Baldelli, S.; Lombardo, M.; D’Amato, A.; Karav, S.; et al. Glucosinolates in Human Health: Metabolic Pathways, Bioavailability, and Potential in Chronic Disease Prevention. Foods 2025, 14, 912. [Google Scholar] [CrossRef]
  50. Chen, M.; Huang, L.; Lv, Y.; Li, L.; Dong, Q. Sulforaphane Protects against Oxidative Stress-induced Apoptosis via Activating SIRT1 in Mouse Osteoarthritis. Mol. Med. Rep. 2021, 24, 612. [Google Scholar] [CrossRef]
  51. Abbate, F.; Maugeri, A.; Laurà, R.; Levanti, M.; Navarra, M.; Cirmi, S.; Germanà, A. Zebrafish as a Useful Model to Study Oxidative Stress-Linked Disorders: Focus on Flavonoids. Antioxidants 2021, 10, 668. [Google Scholar] [CrossRef]
  52. Ortuno, A.; Benavente-Garcia, O.; Castillo, J.; Alcaraz, M.; Vicente, V.; Del Rio, J.A. Beneficial Action of Citrus Flavonoids on Multiple Cancer-Related Biological Pathways. Curr. Cancer Drug Targets 2007, 7, 795–809. [Google Scholar] [CrossRef]
  53. Bravo, L. Polyphenols: Chemistry, Dietary Sources, Metabolism, and Nutritional Significance. Nutr. Rev. 1998, 56, 317–333. [Google Scholar] [CrossRef]
  54. Lushchak, V.I. Contaminant-Induced Oxidative Stress in Fish: A Mechanistic Approach. Fish Physiol. Biochem. 2016, 42, 711–747. [Google Scholar] [CrossRef]
  55. Rodriguez, C.; Mayo, J.C.; Sainz, R.M.; Antolín, I.; Herrera, F.; Martín, V.; Reiter, R.J. Regulation of Antioxidant Enzymes: A Significant Role for Melatonin. J. Pineal Res. 2004, 36, 1–9. [Google Scholar] [CrossRef]
  56. Cai, G.; Zhu, J.; Shen, C.; Cui, Y.; Du, J.; Chen, X. The Effects of Cobalt on the Development, Oxidative Stress, and Apoptosis in Zebrafish Embryos. Biol. Trace Elem. Res. 2012, 150, 200–207. [Google Scholar] [CrossRef]
Nutraceuticals 06 00003 g001
Figure 1. Scheme of division of treatment groups.
Figure 1. Scheme of division of treatment groups.
Nutraceuticals 06 00003 g001
Nutraceuticals 06 00003 g002
Figure 2. Mortality rate of zebrafish exposed to increasing concentrations of B. macrocarpa extract (125–2000 μg/mL) for 96 h. Data are presented as mean ± SD of four independent experiments. Two-way ANOVA followed by Dunnett’s multiple comparison test was performed. * p < 0.05 vs. control (untreated group).
Figure 2. Mortality rate of zebrafish exposed to increasing concentrations of B. macrocarpa extract (125–2000 μg/mL) for 96 h. Data are presented as mean ± SD of four independent experiments. Two-way ANOVA followed by Dunnett’s multiple comparison test was performed. * p < 0.05 vs. control (untreated group).
Nutraceuticals 06 00003 g002
Nutraceuticals 06 00003 g003
Figure 3. Effects of increasing concentrations of B. macrocarpa extract exposures on early-stage zebrafish development. (A) Spontaneous movements measured in 60″ at 24 hpf and (B) heart rate measured in 60″ at 48 hpf; (C) hatching rate measured at 72 hpf. Data are mean ± SD of 4 replicates. * means significantly different according to one-way ANOVA (p < 0.05).
Figure 3. Effects of increasing concentrations of B. macrocarpa extract exposures on early-stage zebrafish development. (A) Spontaneous movements measured in 60″ at 24 hpf and (B) heart rate measured in 60″ at 48 hpf; (C) hatching rate measured at 72 hpf. Data are mean ± SD of 4 replicates. * means significantly different according to one-way ANOVA (p < 0.05).
Nutraceuticals 06 00003 g003
Nutraceuticals 06 00003 g004
Figure 4. Representative images of zebrafish (Danio rerio) embryos and larvae exposed to Brassica macrocarpa extract. (A) An embryo at 48 h post-fertilization (hpf) exposed to 1000 μg/mL of the extract, showing a thin layer of or particulate surrounding the chorion. (B–E) Larvae at 96 hpf, illustrating normal morphological development. (B) The control group. (C–E) Larvae exposed to sub-lethal concentrations of 125 (C), 250 (D), and 500 (E) μg/mL of B. macrocarpa extract, with no observable malformations when compared to controls. Scale bar: 500 μm.
Figure 4. Representative images of zebrafish (Danio rerio) embryos and larvae exposed to Brassica macrocarpa extract. (A) An embryo at 48 h post-fertilization (hpf) exposed to 1000 μg/mL of the extract, showing a thin layer of or particulate surrounding the chorion. (B–E) Larvae at 96 hpf, illustrating normal morphological development. (B) The control group. (C–E) Larvae exposed to sub-lethal concentrations of 125 (C), 250 (D), and 500 (E) μg/mL of B. macrocarpa extract, with no observable malformations when compared to controls. Scale bar: 500 μm.
Nutraceuticals 06 00003 g004
Nutraceuticals 06 00003 g005
Figure 5. Effect of 26 h treatment with sub-lethal concentrations of B. macrocarpa extract (125–500 μg/ML, grey bars) on healthy zebrafish larvae at 72 hpf on oxidative stress markers: (A) ROS; (B) NO; (C) SOD; (D) CAT; (E) Lipid peroxidation; (F) Protein carbonylation; (G) DNA damage. Data represent mean ± SD of 3 independent experiments conducted in technical duplicate, expressed as percentage of control group (white bar). Mean of control: ROS =198.2 ± 7.5 nmol/mg protein; NO = 16.3 ± 8.1 nmol/mg protein; SOD = 11.9 ± 0.59 U/mg protein; CAT = 2.55 ± 0.40 U/mg protein; Lipid peroxidation: 12.2 ± 1.8 μmol/mg protein; Protein carbonylation: 8.3 ± 0.7 nmol/mg protein; DNA damage: 1.8 ± 0.8 μmol/mg protein.
Figure 5. Effect of 26 h treatment with sub-lethal concentrations of B. macrocarpa extract (125–500 μg/ML, grey bars) on healthy zebrafish larvae at 72 hpf on oxidative stress markers: (A) ROS; (B) NO; (C) SOD; (D) CAT; (E) Lipid peroxidation; (F) Protein carbonylation; (G) DNA damage. Data represent mean ± SD of 3 independent experiments conducted in technical duplicate, expressed as percentage of control group (white bar). Mean of control: ROS =198.2 ± 7.5 nmol/mg protein; NO = 16.3 ± 8.1 nmol/mg protein; SOD = 11.9 ± 0.59 U/mg protein; CAT = 2.55 ± 0.40 U/mg protein; Lipid peroxidation: 12.2 ± 1.8 μmol/mg protein; Protein carbonylation: 8.3 ± 0.7 nmol/mg protein; DNA damage: 1.8 ± 0.8 μmol/mg protein.
Nutraceuticals 06 00003 g005
Nutraceuticals 06 00003 g006
Figure 6. Effect of 20 μM CuSO4-exposed zebrafish larvae at 72 hpf on ROS (A) and NO (B) production at different time points. Data are mean ± SD (n = 3) expressed in percentage relative to control group. One-way ANOVA test was performed. * p < 0.05 vs. control (untreated larvae). Mean of control: ROS = 202.1 ± 9.6 nmol/mg protein; NO = 20.3 ± 10.0 nmol/mg protein.
Figure 6. Effect of 20 μM CuSO4-exposed zebrafish larvae at 72 hpf on ROS (A) and NO (B) production at different time points. Data are mean ± SD (n = 3) expressed in percentage relative to control group. One-way ANOVA test was performed. * p < 0.05 vs. control (untreated larvae). Mean of control: ROS = 202.1 ± 9.6 nmol/mg protein; NO = 20.3 ± 10.0 nmol/mg protein.
Nutraceuticals 06 00003 g006
Nutraceuticals 06 00003 g007
Figure 7. Effect of increasing concentrations of B. macrocarpa extract (125–250–500 μg/mL, grey bars) on CuSO4-induced oxidative stress (A) ROS; (B) NO; (C) SOD; (D) CAT; (E) Lipid peroxidation; (F) Protein carbonylation; (G) DNA damage in zebrafish larvae at 72 hpf. Data are presented as mean ± SD from 3 independent experiments, each conducted in technical duplicate, and expressed as percentage of control group (white bar). Mean of control: ROS = 204.4 ± 12.3 nmol/mg protein; NO = 23.4 ± 11.3 nmol/mg protein; SOD = 11.9 ± 0.59 U/mg protein; CAT = 2.25 ± 0.37 U/mg protein; Lipid peroxidation: 15.3 ± 2.2 μmol/mg protein; Protein carbonylation: 9.2 ± 0.4 nmol/mg protein; DNA damage: 1.5 ± 0.1 μmol/mg protein). Statistical significance calculated by one-way ANOVA: * p < 0.05 vs. control, # p < 0.05 vs. CuSO4 alone.
Figure 7. Effect of increasing concentrations of B. macrocarpa extract (125–250–500 μg/mL, grey bars) on CuSO4-induced oxidative stress (A) ROS; (B) NO; (C) SOD; (D) CAT; (E) Lipid peroxidation; (F) Protein carbonylation; (G) DNA damage in zebrafish larvae at 72 hpf. Data are presented as mean ± SD from 3 independent experiments, each conducted in technical duplicate, and expressed as percentage of control group (white bar). Mean of control: ROS = 204.4 ± 12.3 nmol/mg protein; NO = 23.4 ± 11.3 nmol/mg protein; SOD = 11.9 ± 0.59 U/mg protein; CAT = 2.25 ± 0.37 U/mg protein; Lipid peroxidation: 15.3 ± 2.2 μmol/mg protein; Protein carbonylation: 9.2 ± 0.4 nmol/mg protein; DNA damage: 1.5 ± 0.1 μmol/mg protein). Statistical significance calculated by one-way ANOVA: * p < 0.05 vs. control, # p < 0.05 vs. CuSO4 alone.
Nutraceuticals 06 00003 g007
Table 1. Developmental effects of 96 h B. macrocarpa extract exposure on zebrafish larvae, analyzed by Digimizer software.
Table 1. Developmental effects of 96 h B. macrocarpa extract exposure on zebrafish larvae, analyzed by Digimizer software.
Development
Parameters		0 μg/mL.125 μg/mL.250 μg/mL.500 μg/mL.
Malformations (%)10.00 ± 4.08 15. 00 ± 6.4410.00 ± 5.7710.00 ± 5.7
Edema (%)10.28 ± 4.09 10.90 ± 4.12 15.27 ± 6.0614.75 ± 2.64
Size (mm)3.76 ± 0.053.78 ± 0.053.86 ± 0.063.74 ± 0.06
Angle (°)157.17 ± 0.17 161.80 ± 1.17160.32 ± 1.29156.24 ± 1.11
Head (mm2)0.29 ± 0.010.27 ± 0.010.27 ± 0.010.25 ± 0.03
Eye (mm2)0.10 ± 0.01 0.09 ± 0.010.10 ± 0.010.09 ± 0.01
Pericardium (mm2)0.11 ±0.010.12 ± 0.010.11 ± 0.010.09 ± 0.01
Yolk (mm2)0.44 ± 0.01 0.45 ± 0.010.45 ± 0.010.45 ± 0.01
Data are the mean ± SD of 10 larvae/group in 4 replicates.
Table 2. Larval behavioural analysis after 96 h of B. macrocarpa extract exposure at sub-lethal concentration.
Table 2. Larval behavioural analysis after 96 h of B. macrocarpa extract exposure at sub-lethal concentration.
Behavioural
Parameters		0 μg/mL.125 μg/mL.250 μg/mL.500 μg/mL.
Distance (cm)27.28 ± 945 28.51 ± 7.95 28.32 ± 10.92 17.52 ± 8.75
Speed (mm/s)1.14 ± 0.361.13 ± 0.42 0.74 ± 0.150.71 ± 0.17
Immobility
time (%)		46.56 ± 5.61 45.06 ± 4.9240.51 ± 8.0452.00 ± 8.89
Centre
distance (mm)		7.84 ± 0.21 7.50 ± 0.176.64 ± 0.677.32 ± 0.28
Absolute
turn angle (°)		4.23 ± 0.714.69 ± 0.463.93 ± 0.384.42 ± 0.63
Time in
the lit side (%)		55.43 ± 4.7966.24 ± 5.2463.17 ± 5.5350.13 ± 9.47
Time in the non-stimulus side (%)66.77 ± 6.0472.35 ± 8.8253.40 ± 10.9450.65 ± 5.64
Data are mean ± SD of four independent experiments. Video recordings of larvae subjected to behavioural tests were analyzed with Real Fish Tracker software v0.4.0.
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

Share and Cite

MDPI and ACS Style

Cicio, A.; Félix, L.M.; Monteiro, S.M.; Bruno, M.; Zizzo, M.G.; Serio, R. Toxicological Assessment and Potential Protective Effects of Brassica Macrocarpa Guss Leaf Extract Against Copper Sulphate-Induced Oxidative Stress in Zebrafish Embryos. Nutraceuticals 2026, 6, 3. https://doi.org/10.3390/nutraceuticals6010003

AMA Style

Cicio A, Félix LM, Monteiro SM, Bruno M, Zizzo MG, Serio R. Toxicological Assessment and Potential Protective Effects of Brassica Macrocarpa Guss Leaf Extract Against Copper Sulphate-Induced Oxidative Stress in Zebrafish Embryos. Nutraceuticals. 2026; 6(1):3. https://doi.org/10.3390/nutraceuticals6010003

Chicago/Turabian Style

Cicio, Adele, Luís M. Félix, Sandra Mariza Monteiro, Maurizio Bruno, Maria Grazia Zizzo, and Rosa Serio. 2026. "Toxicological Assessment and Potential Protective Effects of Brassica Macrocarpa Guss Leaf Extract Against Copper Sulphate-Induced Oxidative Stress in Zebrafish Embryos" Nutraceuticals 6, no. 1: 3. https://doi.org/10.3390/nutraceuticals6010003

APA Style

Cicio, A., Félix, L. M., Monteiro, S. M., Bruno, M., Zizzo, M. G., & Serio, R. (2026). Toxicological Assessment and Potential Protective Effects of Brassica Macrocarpa Guss Leaf Extract Against Copper Sulphate-Induced Oxidative Stress in Zebrafish Embryos. Nutraceuticals, 6(1), 3. https://doi.org/10.3390/nutraceuticals6010003

Article Metrics

Article metric data becomes available approximately 24 hours after publication online.
Back to TopTop