The toxicological effects of Eryngium foetidum extracts on zebrafish embryos and larvae depend on the type of extract, dose, and exposure time.

Abstract

Eryngium foetidum is a herbaceous plant found in tropical and subtropical regions. In vivo pharmacological parameters show that leaf extracts of this plant have antioxidant, anti-inflammatory, antidiabetic, and antimicrobial activities due to their bioactive compounds such as flavonoids and phenols. Despite the evidence for several bioactivities of E. foetidum, information on its safety and tolerability is limited. The objective of this study was to assess the effect and concentration of different extracts of E. foetidum on the development of zebrafish (Danio rerio) embryos. To study the impact of aqueous (AE), ethanolic (EE), and methanolic (ME) extracts, the embryos were exposed to 0.625, 1.25, 2.5, 5, and 10 mg mL⁻¹ for up to 120-h postfertilization to assess embryonic developmental toxicity and then to 0.039, 0.078, 0.156, 0.312, and 0.625 mg mL⁻¹ to assess the antioxidant responses of the enzymes superoxide dismutase catalase, glutathione S-transferase (GST), and cell apoptosis. The results showed that, depending on the extraction solvent, concentration used, and exposure time, E. foetidum extracts caused mortality, altered the hatching time, and promoted changes in enzymatic activities. Delays in development and increased GST activity were found in all treatments. Apoptosis was not observed in any of the treatments. In conclusion, AE, EE, and ME concentrations above 0.625 mg mL⁻¹ can cause adverse effects on the early stages of zebrafish development.

Introduction

Eryngium foetidum is a herbaceous plant found in tropical and subtropical regions.¹ It is popularly known in Brazil as Chicory is a vegetable Pará, culantro, wild coriander, spiny coriander, long coriander, and Chicory is a vegetable Amazon.² Its main use is as a condiment, but its ethnomedicinal uses are also significant. The whole plant or its parts are often used in traditional medicine as home remedies for the treatment of various diseases, such as hypertension, seizures, asthma, infertility complications, malaria, liver problems, and arthritis.^3–9

To understand the advantages and limitations of bioactive isolates of E. foetidum with the aim to increase their applicability as therapeutics, leaf extracts of this plant were studied in vivo and it was shown they contain phenolics, including flavonoids, and exert antioxidant, anti-inflammatory, antidiabetic, and antimicrobial activities.¹⁰ The antimicrobial activity conferred by terpenoids, glycosides, flavonoids, steroids, and tannins enables their use as antimicrobial agents in drugs for the treatment of infectious diseases caused by pathogens.¹¹Eryngium foetidum also has anticlastogenic activity and cytoprotective effects due to the presence of the active compounds terpenoids, phytosterols, polyphenols, and flavonoids, suggesting that their daily consumption may generate health benefits in the form of protection of cells and their genetic material.^12,13

Taken together, these facts make this plant a suitable candidate for studies on the exploration of new drugs.⁴ Even though they have several bioactive compounds, it is necessary to assess their extraction processes, since the recovery of phytochemicals and the antioxidant capacity of plant extracts is influenced by the type of solvent used in the extraction.^14,15 In addition to assessing which solvent is the most efficient to obtain a greater yield of antioxidants for use in phytotherapeutic products, toxicological assessment of these natural products is also necessary. This is because some of its phytochemicals may be potentially toxic, teratogenic, mutagenic, or carcinogenic as an individual compound or in combination. Studying the teratogenicity or toxic effects of medicinal plants using their potential extracts is essential¹⁶ to ensure that the compounds are safe for humans.¹⁷

Zebrafish (Danio rerio) is a widely accepted vertebrate model for developmental and biomedical studies and for in vivo screening of bioactive molecules of medicinal plants.¹⁸ The zebrafish model enables the assessment of the toxicity of bioactive compounds or crude plant extracts, allowing us to determine the ideal processes for obtaining these plant materials.

The objective of this study was to assess the potential toxicity of different E. foetidum extracts to the development of zebrafish in embryonic and larval stages, as well as the biological activity of these extracts.

Materials and methods

Declaration of ethics

All experimental procedures of this study were conducted in strict accordance with the Ethics Committee on Animal Experimentation of the Federal University of Lavras (UFLA), Lavras, Minas Gerais, Brazil, under protocol number 014/2019. They met the guidelines of the National Control Council of Animal Experimentation (Conselho Nacional de Controle de Experimentação Animal—CONCEA) for the care and use of laboratory animals.

Exsiccates of E. foetidum were prepared and deposited in the PAMG herbarium of the Empresa de Pesquisa Agropecuária de Minas Gerais, under registration PAMG 57810.

Plant material and extract preparation

The plant material (shoot of the plant, including flowers, leaves, and stems) from which we obtained extracts was collected between 7 and 9 a.m. in the olericulture sector located in the Department of Agriculture of UFLA. Obtaining plant extracts followed the methodology described by^11,19 with some changes. Fresh materials were washed 2–3 times with running water and once with sterile water and cut into 1 cm² fragments. Plant extracts at 10% (w/v) were prepared using a reflux system, where the plant fragments were extracted with a solution of distilled water, 70% ethanol and 50% methanol for 20 min, boiling in a closed system, to reduce the possible losses by volatilization. The extracts were vacuum filtered and concentrated in an R-114 type rotary evaporator at 40°C under reduced pressure until drying.

Fish and embryos

Nine-month-old zebrafish adults of the wild-type strain, of short fins and of both sexes, were acquired from the Central Animal Facility, UFLA. The animals were raised and kept in specific racks for the species (Rack Hydrus, model ZEB-40) at a temperature of 28 ± 1°C, fed twice a day with commercial flake feed (Alcon basic, Alcon Pet®, SC, BRA), and fed once with Artemia nauplii. Male and female fish in a 2:1 ratio were placed in breeding tanks and kept in a 14:10 h light:dark cycle, turning the light on at dawn to stimulate spawning. The embryos were collected 30 min after natural mating from at least 3 groups of different matrices to avoid genetic variants. After washing with water, the embryos were randomized and transferred randomly to Petri dishes containing 12 mL of embryo culture medium (5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl₂, 0.33 mM CaCl₂, 0.33 mM MgSO₄, 0.33 mM MgSO₄, and methylene blue).^20–22

Developmental toxicity

The embryotoxicity tests were based on the guidelines of OECD No. 236: Fish Embryo Acute Toxicity Test.²³ To assess the lethal and developmental toxicity, 20 embryos were distributed in 96-well microplates (1 embryo/well), each well containing one extract at 0.625, 1.25, 2.5, 5, or 10 mg mL⁻¹ in a volume of 200 μL, whereas the controls contained only the embryo culture medium. The concentrations selected for the study were based on pre-experimental data. All plates were incubated in an oven at 28 ± 1°C.

Embryonic development was monitored daily at 24, 48, 72, 96, and 120 h postfertilization (hpf) using an optical microscope (Olympus CX22LED model) with a focus on the following parameters: embryo coagulation, eye and body pigmentation, somite formation, presence of heartbeat, detachment of the tail, edema of the yolk sac and pericardium, and malformations of the tail.²³

Determination of enzymatic activities

Preparation of homogenate

To determine the activity of the enzymes catalase (CAT), superoxide dismutase (SOD), and glutathione S-transferase (GST), embryos were initially placed after fertilization in Petri dishes with 10 mL of exposure solution until reaching 120 hpf. The exposure studies were performed in triplicate. The plates were incubated at 28 ± 1°C, and the dead eggs or larvae were removed daily. Forty larvae/group were collected and homogenized in 400 μL of cold phosphate-buffered saline (PBS) with the aid of a glass rod. Then, the homogenates were centrifuged at 4000 × g at 4°C for 20 min to obtain the supernatants.^24–25 The concentration of total proteins for each sample was determined spectrophotometrically at 595 nm, according to the Bradford method, using bovine serum albumin as a standard.²⁶

Superoxide dismutase

SOD activity was assessed as described by²⁷ with some modifications. For the quantifications, 153 μL of the reaction system composed of 100 μL of PBS (100 mM, pH 7.8), 2 μL of EDTA (10 μM), 40 μL of methionine, 11 μL of ultrapure water, and 15 μL of 1 mM nitro blue tetrazolium (NBT) was prepared and added to 96-well microplates, which contained 30 μL of sample (homogenate of the larvae) and 2 μL of riboflavin. To start the reaction, the plates were placed under light (400 lux) for 20 min, and then the SOD activity was read in a spectrophotometer at 560 nm/25°C (TECAN Infinity® M200 PRO, Männedorf, Switzerland). One unit of SOD activity (U) was defined as the amount of enzyme required to lower the NBT photoreduction rate by 50%, and the result is expressed as U/mg protein.^27,28 The readings were performed in triplicate.

CATALASE

CAT activity was determined according to the protocol established by.²⁹ For the measurement, 10 μL of the homogenate supernatant of the larvae diluted in 140 μL of KPBS (0.05 M, pH 7.0) and 150 μL of reaction buffer (37.2 μL of H₂O₂ 30%; 15 mL of KPBS) was added to 96-well microplates. The decrease in absorbance was recorded at 240 nm/25°C for 2 min (every 20 s) in a spectrophotometer (PowerWave XS, Biotek, Winooski, VT, USA). The absorbance readings were performed in triplicate, and the CAT activity level was estimated by the molar extinction coefficient of H₂O₂ (43.6 M cm⁻¹). The results are expressed in μmol H₂O₂ degraded/min/mg protein.

Glutathione-S-transferase

GST activity was determined according to the protocol established by.³⁰ For measurement, 15 μL of the homogenate of the larvae, 50 μL of reduced glutathione (GSH) (25 mM), and 180 μL (1 mM) of 1-chloro-2,4-dinitrobenzene (dissolved in 96% ethanol and diluted in 0.1 M sodium phosphate buffer, pH 7.2) were added to microplates. Differences in GST activity were estimated by the absorbance obtained over 5 min in a spectrophotometer at 340 nm (TECAN Infinity® M200 PRO Männedorf, Switzerland). Quantifications were performed in triplicate, and the results are expressed in U/mg protein.

Identification of apoptotic cells

To study the role of apoptosis in the toxicity of E. foetidum extracts, the acridine orange staining method was performed, as described by.³¹ Briefly, after 96 h of exposure to the extracts, the larvae were washed twice with embryo culture medium and stained with 5 mg/L acridine orange for 20 min in the dark at room temperature. After the incubation time, the larvae were again washed with embryo culture medium, anesthetized with 0.16 mg/mL tricaine (Sigma Aldrich), observed, and photographed under a fluorescence microscope at 4× magnification (Axio Observer Z1, Zeiss, Jena, Germany). The fluorescence intensity of the images was quantified in ImageJ software (National Institutes of Health).^31,32 The results are expressed as the percentage change versus the control medium.

Statistical analysis

The toxicity, antioxidant, and apoptosis data were assessed for normality by the Shapiro–Wilk test, and then the data were subjected to analysis of variance and Tukey’s test to find significant differences between treatments. All tests were run in the statistical software Minitab v.19 (State College, PA, USA) with a significance level of 95% (P < 0.05).

Results

Toxicity in embryonic development

The effects of the aqueous (AE), ethanolic (EE), and methanolic (ME) extracts of E. foetidum on the survival rates of zebrafish embryos exposed at 24, 48, 72, 96, and 120 h are shown in Fig. 1. After the first 24 h of exposure, at the highest concentrations tested (10 and 5 mg mL⁻¹) of all extracts and at a concentration of 2.5 mg mL⁻¹ ME, a 100% mortality rate was observed. In contrast, embryos/larvae exposed to 0.625 mg mL⁻¹ of any extract for any time showed similar responses as embryos of the control group (P > 0.05). For the other concentrations, the survival rate changed over time (Fig. 1).

Fig. 1.

Survival rate (%) evaluated daily from 24 to 120 h of zebrafish (D. rerio) embryos/larvae exposed to concentrations of 0.625; 1.25; 2.5; 5, and 10 mg mL⁻¹ of Eryngium foetidum extracts. A) AE; B) EE; and C) ME.

The hatching rate of embryos exposed to E. foetidum extracts varied over time depending on the extract and concentrations assessed (Fig. 2). In the treatments of 10 and 5 mg mL⁻¹ of all extracts and 2.5 mg mL⁻¹ of ME, no hatching was observed at any of the assessed times as a result of 100% embryo mortality. AE and EE at 2.5 mg mL⁻¹ led to no hatching at 48 hpf. Similar results were also observed in the 1.25 mg mL⁻¹ AE and MS groups, in which hatching was not observed at 48 hpf.

Fig. 2.

Hatch rate (%) of zebrafish (D. rerio) embryos at 48, 72, 96 and 120 hpf after exposure to different concentrations (0.625; 1.25; 2.5; 5, and 10 mg mL-1) of the extracts of E. foetidum. A) AE; B) EE; and C) ME.

Regarding morphological changes, normal development was observed in control embryos, whereas a developmental delay was evident at all extract groups (Fig. 3).

Fig. 3.

Zebrafish embryos/larvae at 24, 48, 72, and 120 hpf. In control: normal development at 24, 48, 72, and 120 hpf. AE 1.25 mg ml⁻¹: growth retardation at 24 and 48 hpf; inhibition of melanin release at 48 hpf with permanence at 72 hpf.

Based on the results of the preliminary studies (embryonic development toxicity), concentrations below 0.625 mg mL⁻¹ were selected for the subsequent experiments (enzymatic activity and cell apoptosis). We did not study embryos/larvae at 1.25, 2.5, 5, or 10 mg mL⁻¹ for subsequent analyses due to the very high lethal toxicity.

Enzymatic activities

To further characterize the harmful effects induced by AE, EE, and ME of E. foetidum during zebrafish embryogenesis, we determined the activities of the enzymes SOD, CAT, and GST. As shown in Fig. 4, SOD activity was significantly higher (P < 0.05) than in the control group only for 0.156, 0.312, and 0.625 mg mL⁻¹ AE. CAT activity increased significantly (P < 0.05) in AE and EE at concentrations of 0.039, 0.078, 0.156, 0.0312, and 0.039, 0.156 mg mL⁻¹. GST activity increased significantly (P < 0.05) after exposure to all E. foetidum extracts.

Fig. 4.

Change in the activity of antioxidant enzymes SOD, CAT, and GST in zebrafish embryos/larvae at 96 hpf between control groups and those treated with E. foetidum extracts at different concentrations. A, D, G) AE; B, E, H) EE; and C, F, I) ME. Bars indicate mean ± SEM of 3 replicates (n = 3). Different superscript letters indicate significant differences (P < 0.05) between groups.

Identification of apoptotic cells

The cell death found in the treatment groups did not differ from that in the control (Fig. 5 AC), indicating that the extracts did not exert any cytotoxic effect on zebrafish embryos/larvae at the tested concentrations (0.039, 0.078, 0.156, 0.312, and 0.625 mg mL⁻¹).

Fig. 5.

Effects of E. foetidum extracts on apoptosis of zebrafish larvae. A) AE; B) EE; and C) ME. Apoptosis levels were measured after acridine orange staining by image analysis and fluorescence microscopy. Data obtained from individual analyzes of fluorescence intensity of zebrafish larvae. Values are expressed as mean ± SEM.

Discussion

Eryngium foetidum has a diversity of bioactive compounds, which are reported to have medical and pharmaceutical bioactivities.³³ However, the understanding of their toxicological profiles is limited, especially with regard to embryonic developmental toxicity. To date, no studies on the toxicological effects of E. foetidum using zebrafish embryos/larvae as a model have been found in the literature.

The results of the present study strongly suggest that AE, EE, and ME of E. foetidum are toxic because they lower the survival of zebrafish embryos/larvae, and their effects depend on the dose and exposure time. The toxicity of E. foetidum has been described by other authors, who revealed the larvicidal potential of this species’ essential oil against the mosquito Aedes albopictus³³ and in mice fed a feed supplemented with lyophilized E. foetidum, with signs of abnormalities such as kidney injury.³⁴

Embryos of zebrafish exposed to high concentrations of any of the extracts (10 and 5 mg mL⁻¹) all died within 24 h of exposure. According to³⁵ the mortality of zebrafish embryos in the first hours of development may occur due to the interference of the test substance with cellular organelles, such as mitochondria. In the presence of such interference, the ATP formation process becomes slow or stops, resulting in the failure of the active membrane of the sodium pump, intracellular accumulation of sodium, and external diffusion of potassium, which may cause cell death.^35,36

At concentrations of 1.25 and 2.5 mg mL⁻¹ of the extracts of E. foetidum, where there was no mortality in the first 24 h, embryonic survival declined from the second and/or third day of incubation on. These results show the toxic effects of the extracts depending on the dose and exposure time because as the exposure time increased, survival decreased. Prolonged exposure to an extract may lead to greater accumulation of the extract in the embryo, reaching a concentration that can induce toxicity,³⁷ and/or this can result from the weakened or damaged protective layer of the embryo (chorion).³⁸ The chorion undergoes continuous changes throughout embryonic development, such as in the chorionic protein profile. These changes result in an increase in the opening or expansion of the chorion pore channel, allowing a greater influx of external solute.³⁸

Our results indicate that ME of E. foetidum has a higher toxicity to zebrafish embryos/larvae than AE and EE. This higher toxicity may be due to the ability of methanol to extract more compounds than other polar.^39,40 Nevertheless, because methanol has a lower boiling point (64.7°C), for its evaporation during the extract preparation process, lower temperatures are necessary, causing less damage to the extract than to ethanol and water, which have higher boiling points.³⁹

Among the assessed extracts, the lowest toxicity was found for AE. The higher lethality of EE and ME in zebrafish embryos and larvae may be due to the greater concentration of tannins in these extracts. Reference⁴¹ reported a higher tannin concentration in the extracts of E. foetidum when using EE and ME solvents and a lower presence in water. The lower amount of tannins in the AE extracts is due to the loss of tannins during the boiling process.^14,42 Tannins are compounds that have antioxidant activity,¹⁴ but negative health effects are also reported, as they interfere with the availability of iron and zinc and some digestive enzymes in the body.⁴³ Tannins are considered toxic and nutritionally undesirable because they precipitate proteins, inhibit digestive enzymes, and affect the use of vitamins and minerals.⁴⁴ In addition, a higher intake of sources rich in tannins is also associated with carcinogenic effects, poor use of proteins, and liver and kidney toxicity.⁴⁵ Our findings are in agreement with⁴¹ who showed that the AE of E. foetidum has a low tannin content and therefore has a low probability of causing some negative effect on health.

Like tannins, flavonoids can cause toxicity to organisms. Flavonoids have antioxidant activity by promoting the excessive removal of free radicals, acting as neutralizing agents.⁴⁶ However, depending on their concentrations, flavonoids may have pro-oxidant properties, promoting the oxidation of other compounds.⁴⁷ These antioxidants are easily extracted from E. foetidum in an alcoholic medium but less so in an AE medium because high temperatures degrade flavonoids^41,48 Thus, the lower survival rate of embryos/larvae exposed to EE and ME in the present study might be attributed to the higher concentrations of flavonoids and tannins in these extracts compared with AE.

The zebrafish larvae usually hatch from the chorion between 48 and 72 hpf. Hatching is regulated by a combination of biochemical and physical mechanisms.⁴⁹ Physical and chemical signals detected by the embryo activate the production of the enzyme related to hatching (chorionase, HE1), which degrades the inner layer of the chorion and allows the spontaneous movements of developing embryos to result in hatching.⁵⁰ Some studies have shown that plant extracts and oil delay the hatching process of zebrafish embryos, such as Trachyspermum ammi (Apiaceae) oil, Carpesii fruit extracts, Piper nigrum (black pepper), and Millettia pachycarpa.^51–54 Some authors believe that this delay in hatching may be due to changes in the release of the enzyme chorionase and/or the reduced movement of the larvae due to behavioral changes caused by components present in these plants.^52,54 The study of⁵⁵ suggested that phenolic compounds can induce or inhibit the activity of the chorionase enzyme, delaying the hatching of embryos. Based on these arguments, we assume that, in the present study, delays in the hatching of embryos exposed to E. foetidum extracts were also caused by these factors.

Another hypothesis about delayed hatching is related to the delay in the development of embryos and their inability to rupture the chorion.^51,56 Developmental delay is frequently observed in embryos exposed to teratogenic agents⁵⁷ and is generally considered a reversible and nonspecific effect. In all E. foetidum treatments, there were delays in embryonic development in the first 24 hpf. We believe that due to the high toxicity of concentrations of 1.25 and 2.5 mg mL⁻¹ of the extracts, the embryos reversed their developmental delay more slowly, resulting in late hatching. In contrast, with 0.625 mg mL⁻¹ EE and ME, because of their low toxicity, the embryos reversed the developmental delay more quickly, resulting in hatching at the expected time.

The activities of antioxidant enzymes such as SOD, CAT, and GST are important indicators of the production of reactive oxygen species derived from the toxicity of a compound.⁵⁸ SOD is a metalloenzymes that is part of the first line of defense against excessive amounts of reactive oxygen species. It converts free radical superoxide anions (O₂⁻•) into H₂O₂. This H₂O₂ will be degraded in water and oxygen by the enzyme CAT, thus freeing the cells from the risk of oxidation by these radicals.⁵⁹

After the exposure of embryos/larvae to E. foetidum extracts, significant differences from the control were found only in SOD activity in the groups with 0.156, 0.312, and 0.625 mg mL⁻¹ AE and in CAT in the groups given AE and EE at concentrations of 0.039, 0.078, 0.156, 0.0312, and 0.039, 0.156. We believe that because the concentrations used for the determination of enzymatic activity were lower than those used in the assessment of developmental toxicity, the concentrations of tannins and flavonoids might have been low. Thus, these bioactive agents could act as antioxidants, eliminating reactive oxygen species produced by larval metabolism directly or by stimulating antioxidant enzymes.

GSTs are phase II biotransformation enzymes that play a specific physiological role in zebrafish detoxification.⁶⁰ They are involved in the detoxification of xenobiotics and in the elimination of reactive oxygen species and lipid peroxidation products, acting by catalyzing the conjugation of GSH to a wide variety of substrates, neutralizing their electrophilic sites, making them less toxic, more soluble in water, and more easily excreted.^61,62 We observed a significant increase in GST activity in zebrafish larvae exposed to all concentrations of extracts compared with the control. This increase may be related to the presence of flavonoids in these extracts. According to,^47,63 flavonoids can induce phase II detoxifying enzymes, such as NAD (P) H: quinone oxidoreductase, UDP-glucuronyl transferase, and GST, which are the main defensive enzymes against electrophilic toxics and oxidative stress. In addition, flavonoids are typical xenobiotics for animals and humans, metabolized as such and quickly removed from the circulation.⁴⁷ Thus, the increase in GST activity in embryos/larvae exposed to E. foetidum extracts may have been due to the effect of flavonoids present in the extracts of eliminating toxic compounds or by their own elimination as a xenobiotic.

Redox regulation and apoptosis play crucial roles in the developmental process because their disorder can cause deformity and death.³² Apoptosis can occur naturally during embryogenesis, regulating many developmental processes, such as morphogenesis, removal of vestigial structures and regulation of cell number, or may be the result of cell damage or stress.^22,53,64 In the present study, the presence of acridine orange in the control embryos confirmed that programmed cell death is a normal physiological process during development. The larvae exposed to the extracts also showed cell death, not differing from the control, which suggests that AE, EE, and ME concentrations below 0.625 mg mL⁻¹ do not induce apoptosis in zebrafish embryos/larvae.

Conclusion

Eryngium foetidum extracts can cause toxicity in the development of zebrafish embryos/larvae. This toxicity depends on the solvent used in the extraction, the concentration used, and the exposure time. It can cause death, developmental delays, hatching delays, and antioxidant enzyme activity. Based on our results, for the qualities of these plants and their extracts to be well validated, a more detailed study aiming to understand the biological mechanisms affected by the extracts, with subsequent study of their antioxidant and anti-inflammatory potentials, should be performed.

Authorship contribution statement

TFDC: Conceptualization, Writing—original draft, Investigation, Writing—review and editing. WFC: Conceptualization, Methodology, data curation, formal analysis, writing—review and editing. TR Conceptualization, Methodology, data curation, formal analysis, writing—review and editing. SLF: Methodology, data curation, formal analysis, writing—review and editing. MRFM: Methodology, data curation, formal analysis, writing—review and editing, Funding acquisition. KKCS: Methodology, data curation, formal analysis, writing—review and editing LVR: Conceptualization, Supervision, Data curation, Writing—review and editing. LDSM: Conceptualization, Supervision, Funding acquisition, Project administration, Writing—review and editing.