The Citrus Flavonoids Hesperidin and Naringin Alleviate Alcohol-Induced

Neurotoxicology and Teratology 73 (2019) 22–30

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Neurotoxicology and Teratology

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The citrus ﬂavonoids hesperidin and naringin alleviate alcohol-induced behavioural alterations and developmental defects in zebraﬁsh larvae T ⁎ Xingyu Liu, Meng Li, Xizeng Feng

State Key Laboratory of Medicinal Chemical Biology, The Key Laboratory of Bioactive Materials, Ministry of Education, College of Life Science, Nankai University, Tianjin 300071, China

ARTICLE INFO ABSTRACT

Keywords: This study aimed to investigate the protective effects of two citrus flavonoids, hesperidin and naringin, on Citrus flavonoids alcohol-induced thigmotaxis behavioural and developmental defects in zebrafish. The results of behavioural Alcohol assays indicated that acute exposure to 1% alcohol could induce thigmotaxis behaviour in zebrafish larvae at 3 fi Zebra sh larvae and 5 days post-fertilization (dpf), whereas hesperidin and naringin could inhibit the thigmotaxis behaviour in Embryonic development zebrafish larvae. Moreover, developmental administration of hesperidin and naringin reduced the levels of Thigmotaxis behaviour morphological defects and apoptosis induced by developmental alcohol exposure. In conclusion, this study suggests that the citrus flavonoids hesperidin and naringin could reduce the effects of alcohol damage on embryonic development and neurobehaviour in zebrafish larvae, which would provide useful knowledge for assessing the underlying mechanism of citrus flavonoids and their protective effects.

1. Introduction Selmi et al., 2017). Zhou et al. (2017) and Lin et al. (2017) found that hesperidin (6.25, 12.5, 25 mg/L) and naringenin (5, 10 mg/L), which is Alcohol is the active ingredient in drinks such as wine, beer, and the aglycon of naringin, could alleviate acute alcoholic liver injury by distilled spirits. Acute administration of alcohol in humans could pro- improving lipid metabolism and reducing apoptosis in zebrafish larvae. duce euphoria and disinhibition. As the dose increases, alcohol may Additionally, Martínez et al. (2009) found that hesperidin decreased the lead to impairment in cognitive, motor, and memory functions, as well exploratory activity of in the holeboard test in mice, which indicated as loss of consciousness and even death in severe cases (Lockwood the central nervous system-mediated effects of hesperidin. Several et al., 2004). Additionally, developmental alcohol exposure could lead studies demonstrated that the two chemicals could exert protective to deficits, such as developmental delays, cranial, facial, and cardiac effects against stress-induced anxiety-like behaviour (Fernandez et al., abnormalities in embryonic organisms (Chudley, 2005; Li et al., 2016). 2009; Kwatra et al., 2016; Viswanatha et al., 2012). Fourteen days of Children with foetal alcohol syndrome exhibit similar properties, in- hesperidin pretreatment (50 and 100mg/kg, p.o.) significantly including abnormal facial features, low body weight, small head size, and hibited stress-induced anxiety-behaviour in the elevated maze plus test behavioural and cognitive impairments (Chudley, 2005). in mice (Viswanatha et al., 2012). For naringin, Fernandez et al. found Citrus flavonoids, which commonly exist in citrus fruits such as that naringin administration with a small dose of 1 mg/kg in mice could oranges, lemons, and grapefruits, are critical natural compounds with reduce anxiety-like behaviour in the elevated maze plus test (Fernandez various biological activities (Assini et al., 2013; Tripoli et al., 2007). et al., 2009). Another study showed that naringin administration (50 Hesperidin and naringin are two essential flavanones extracted from and 100 mg/kg, i.p.) could significantly attenuate anxiety and depres- citrus fruits. Hesperidin, which is a flavanone with a disaccharide ru- sion-like behaviour induced by doxorubicin in male Wistar rats assessed tinose, is tasteless, while naringin consists of a flavanone and a neo- with the plus maze test and modified forced swimming test, in which hesperidose that have a bitter taste. These compounds possess anti- the animals showed a decrease in immobility time and an increase in oxidant and anti-inflammatory activities as well as the capacity to swimming time (Kwatra et al., 2016). prevent atherosclerosis and cancer (Tripoli et al., 2007). Furthermore, it The zebrafish shares a large number of genetic homologues with was demonstrated that hesperidin and naringin could both significantly human beings, and it has become a promising model organism for alleviate gastric damage induced by alcohol in rats (Martin et al., 1994; studies of developmental patterns (Brittijn et al., 2009), locomotor

⁎ Corresponding author. E-mail address: [email protected] (X. Feng). https://doi.org/10.1016/j.ntt.2019.03.001 Received 25 November 2018; Received in revised form 13 March 2019; Accepted 14 March 2019 Available online 15 March 2019 0892-0362/ © 2019 Elsevier Inc. All rights reserved. X. Liu, et al. Neurotoxicology and Teratology 73 (2019) 22–30 behaviour (Bailey et al., 2016; Kung et al., 2015; MacPhail et al., 2009), China). Hesperidin and naringin were initially dissolved in DMSO at a sleep patterns (Chen et al., 2017; Zhdanova, 2011), and learning (Li concentration of 1000 mg/L and stored at 4 °C in darkness. Prior to the et al., 2015). In addition, zebrafish have been widely used for assess- embryo exposure, serial dilutions (6.25, 12.5, 25 mg/L, which were ments of drug toxicity (González et al., 2018; Pieróg et al., 2018; Rosa approximately equivalent to 0.01, 0.02, 0.04 mM) of each flavonoid et al., 2018) and for drug screening (Roper et al., 2018; Wang et al., were prepared in the system water with a maximal concentration of 2014). In our study, a model for observing anxiety-like behaviour in 0.2% DMSO. Then, 1% alcohol was prepared in the system water with larval zebrafish was used to investigate the neurobehaviour alterations 0.2% DMSO. induced by acute alcohol exposure. In addition, we determined whether The exposure procedure was conducted as previously described (Liu the administration of flavonoids, specifically hesperidin and naringin, et al., 2018). Briefly, fertilized embryos were transferred randomly into during development could alleviate the behavioural alterations induced 6-well plates with 40 embryos per well. The standard water was re- by alcohol. Furthermore, morphology and apoptosis assessments were moved from the wells, and 10 mL of the experimental solution for each conducted to assess the protective effects of hesperidin and naringin on group was added to each well. For behavioural tests, the embryos were alcohol-induced developmental malformation. initially incubated with a series of concentrations of hesperidin or naringin (6.25, 12.5, 25 mg/L) without alcohol, respectively, from 0.75 2. Material and methods hpf. At 3 dpf, 30–40 zebrafish larvae per group were moved to a single 10-cm Petri dish containing 30 mL 1% alcohol for 10 min (Fig. 2A). For 2.1. Preparation, UPLC analysis, and Q-TOF-MS analysis of hesperidin and the larval development morphological assay, embryos were exposed to naringin 1% alcohol alone, 1% alcohol with hesperidin (6.25, 12.5, 25 mg/L), and 1% alcohol with naringin (6.25, 12.5, 25 mg/L) from 0.75 hpf to 72 A Waters Acquity UPLC system (Waters, MA, USA) equipped with a hpf. For the control group, 0.2% DMSO was used. Each experiment was photodiode array detector was used. The system was controlled by replicated three times independently. MassLynx V4.1 software (Waters Co.). An Acquity BEH C18 column (2.1 × 100 mm, 1.7 μm, Waters Co.) was applied for the separation. For 2.4. Morphological analyses of zebrafish larvae hesperidin, a gradient elution of 0.1% formic acid and acetonitrile was performed as follows: 1.0% B was obtained from 0 to 6 min, 1 to 50% B To determine the adverse effects of alcohol on larvae development from 6 to 8 min, 50 to 60% B from 8 to 10 min, 60 to 80% B from 10 to and the protective effects of the flavonoids hesperidin and naringin, 13 min, and 80 to 99% B from 10 to 15 min. The flow rate was 0.30 mL/ zebrafish larvae were observed at 72 hpf under a stereomicroscope min, and the column temperature was maintained at 45 °C. Accurate (Olympus ZX-10, Japan) and photographed with a Canon digital mass measurements and MS/MS were performed on a Waters Q-TOF- camera. The surviving larvae were anaesthetized with tricaine Premier with electrospray ionization (ESI) system (Synapt G2-S HDMS, (0.016%) and immobilized on 1% low-density agar gels to obtain Waters MS Technologies, Manchester, UK). The ESI-MS spectra were photographs with a consistent orientation. Body length, head area, and acquired in the negative ion voltage modes. The capillary voltages were eye area were measured accordingly. The experiments were replicated set to 2.5 kV for the negative mode. The sample cone voltage was set to three times independently. 40 V. High-purity nitrogen was used as the nebulization and auxiliary gas. The nebulization gas was set at a flow rate of 800.0 L/h, the cone 2.5. Thigmotaxis behavioural assay gas was set at a flow rate of 50 L/h, and the source temperature was 120 °C. The Q-TOF Premier acquisition rate was 0.2 s with a 0.014-s To examine the thigmotaxis behaviour in zebrafish larvae at 3 dpf, scan delay. The instrument was operated with the first resolving behavioural assessments were conducted following the protocol by quadrupole in a wide pass mode (50 to 2500 Da). Leucine Enkephalin Lundegaard et al. (2015) with slight modifications (Fig. 2A). − (200 pg/mL) was used as the lock mass ([M-H] 554.2615). Lundegaard et al. (2015) found that after 1 h exposure to 15 mM roli- pram, 3 dpf larvae could establish thigmotaxis behaviour rapidly within 2.2. Zebrafish maintenance and husbandry 30 s after disturbing their original positions in an open arena (10 cm), which led them to swim rapidly to the edge of the dish. In our study, Wild-type adult zebrafish (AB strain) were maintained in a standard one hour prior to the experiment at 3 dpf, approximately 30–40 larvae water circulation zebrafish facility (KCl 0.05 g/L, NaHCO3 0.025 g/L, from each group were transferred to a 10-cm Petri dish (Corning, cat. NaCl 3.5 g/L, and CaCl2 0.1 g/L, pH 7.0–7.2) at 28.5 °C with a photo- no. 430167). The hesperidin or naringin solution was carefully re- period cycle of 14:10 h light: dark. The fish were fed twice daily with moved, and then 30 mL of 1% alcohol were added. The plate was placed brine shrimp. All experimental procedures concerning zebrafish were in our customized video tracking system (Guo et al., 2014). The larvae approved by the Committee for Animal Experimentation of the College were incubated for 10 min with acute exposure to alcohol under light of Life Science at Nankai University (no. 2008) and were performed in conditions (80 lx) or darkness (0 lx). Then, the Petri dish was swirled to according to the NIH Guide for the Care and Use of Laboratory Animals disrupt the original positions of the larvae, and images of the newly (no. 8023, revised in 1996). established locations were recorded after 30 s to examine the estab- The night before spawning, one female and one male adult zebrafish lishment of thigmotaxis after position disturbance. Only the larvae that were placed into a breeding tank and separated by a transparent sliding were facing the dish edge perpendicularly with their heads sta- barrier. The next morning, the sliding door was removed, which al- tioned < 2 mm from the edge were counted as thigmotaxis-positive lowed the male fish to pursue the female. The fertilized embryos were larvae, while the larvae with their bodies touching the wall were transferred to a Petri dish after spawning and washed with a standard counted as thigmotaxis-negative larvae (Fig. 2B). Thigmotaxis at 3 dpf system water. The dead embryos were removed, and the normal em- was calculated as the percentage (%) of thigmotaxis-positive larvae in bryos were transferred for the experiment according to the experi- the total number of larval zebrafish per group. mental scheme. Exposure solutions were renewed once per day, and the Further assessments of thigmotaxis at 7 dpf in zebrafish were per- embryos were maintained at 28.5 °C. formed. The thigmotaxis assessments followed the procedures of Schnörr et al. (2012) and Pietri et al. (2013), and these procedures were 2.3. Chemicals and exposure procedures moderately adjusted in our assay (Fig. 2E). The larvae were transferred to 12-well plates (1 larva per well) at 2 dpf to minimize the potential Hesperidin (purity ≥98%) and naringin (purity ≥98%) were pur- damage caused by handling (Schnörr et al., 2012). The larvae were chased from Dalian Meilun Biology Technology Co., Ltd. (Dalian, further incubated without disturbance until automated video-tracking

23 X. Liu, et al. Neurotoxicology and Teratology 73 (2019) 22–30 was conducted at 7 dpf. To provide a sufficiently large swimming arena 271 for naringin, which could be naringenin produced by the loss of a for zebrafish larvae and to distinguish between inner and outer zones, neohesperidose moiety, which is consistent with previous studies (Brito 12-well plates were chosen in this occasion for 7 dpf zebrafish larvae. In et al., 2014; Zhang et al., 2014). These possible metabolites could be our modified protocol, 12-well plates (Corning, cat. no. 3513, diameter detected in the plasma of humans who consumed orange or grapefruit 22.6 mm) were used, and each well was divided into two zones: an juice (Josefsson et al., 1996; Nielsen et al., 2006). inner zone at the centre of the well (diameter 6.8 mm) covering 36% of the total area, and an outer ring zone of 64% of the entire area, which 3.2. Alcohol could induce thigmotaxis behaviour in 3 and 7 dpf zebrafish followed similar spatial proportions to those used by Pietri et al. (2013). larvae At 7 dpf, the hesperidin or naringin solution was carefully removed, and 4 mL 1% alcohol were gently added to each well for alcohol exposure. Multiple findings have shown that alcohol treatment could produce Then, the 12-well plate was placed in the video tracking system we behaviour alterations, including hyperactivity, wall-hugging, and edge customized before. Similar to the time scheme set by Schnörr et al. with preference, in both larval and adult zebrafish (Baggio et al., 2017; slight adjustments, zebrafish larvae were first acclimatized under the Baiamonte et al., 2016; Ramlan et al., 2017). In our study, a phenotypic light (80 lx) for 6 min. At minute 7, the lights were turned off im- observation platform for thigmotaxis behaviour in 3 dpf zebrafish mediately for the dark condition (0 lx) or kept on for the light condition larvae was adopted as previously described (Lundegaard et al., 2015). (80 lx), and data were collected in a time period of 7–12 min. The 12- Our previous work has shown that the larvae exposed to 1% alcohol well plate with one larva per well was monitored by using a camera showed significantly increased thigmotaxis (Li et al., 2016) and set (MV-VS078FM, MicroVision, Japan) with a lens (MP5018, Computer). themselves at the edge of the Petri dish, which is an indicator of an- An infrared LED array was used as a backlight source. Thigmotaxis at 7 xiety-like behaviour. The thigmotaxis assay was performed following dpf was presented as the percentage (%) of time spent in the outer ring the same procedure under separate light and the dark conditions, and region. Larvae that did not show sufficient locomotor activity were the results showed that the elevation of the edge preference was higher excluded to avoid inaccuracy in calculations. under the light than in the dark in 3 dpf zebrafish larvae after alcohol exposure (Fig. 2D). 2.6. Quantification of apoptosis in zebrafish larvae The thigmotaxis behaviour in zebrafish larvae at 7 dpf was further evaluated. The results showed that larvae exposed to 1% alcohol under For determination of the apoptosis level in different treatment light conditions presented significantly increased thigmotaxis, and they groups, acridine orange staining was conducted. At 72 hpf, zebrafish spent more time in the outer zone than the control group, which in- embryos exposed to 1% alcohol alone, 1% alcohol with hesperidin dicated there was increased anxiety-like behaviours. Under the dark (6.25, 12.5, 25 mg/L), and 1% alcohol with naringin (6.25, 12.5, conditions, the behaviour alterations induced by alcohol increased 25 mg/L) were washed with system water twice and incubated in ac- slightly. However, there was no statistical significance between the 1% ridine orange (5 μg/mL) dissolved in system water for 30 min (Ramlan alcohol-exposed group and the control group. This outcome might have et al., 2017). After staining, the embryos were rinsed with system water resulted from the fact that the control group presented a relatively three times to remove excessive stains. The larvae were anaesthetized higher level of thigmotaxis under darkness compared to the light con- by 0.016% tricaine and fixed on 1% low-density agar gels for image ditions. records. Observation images were collected in the green channel of and EVOS inverted imaging system (Olympus ZX-10, Japan). Apoptotic cells 3.3. Increased thigmotaxis behaviour induced by alcohol in zebrafish larvae appeared as bright green fluorescence dots, and the apoptotic cells in was alleviated by hesperidin and naringin the head and the heart area were counted by Image-Pro Plus 6.0 (Media Cybernetics, Inc., USA). The experiments were performed in- As described above, thigmotaxis behaviour increased in zebrafish dependently in triplicate (Verma et al., 2018). larvae after 1% alcohol exposure. To investigate whether the flavonoids hesperidin and naringin could alleviate the alcohol-induced beha- 2.7. Data analyses vioural alteration, zebrafish larvae were pre-treated with hesperidin or naringin (6.25, 12.5, 25 mg/L) from 0.75 hpf, respectively. At 3 dpf, the Shapiro-Wilk's test was adopted to test the data normality. One-way larvae were moved into an open arena, and the alcohol exposure and ANOVA with Fisher's LSD was conducted to evaluate the statistical larvae thigmotaxis assessment were conducted in the same way as significance. A nonparametric Kruskal-Wallis test followed by Dunn's mentioned above. The results showed that pretreatment of the two multiple comparisons tests was applied to data that violated the as- flavonoids could significantly prevent the larvae from establishing sumption of normality. The statistical analysis and bar charts were thigmotaxis under the light conditions (Fig. 3A). The thigmotaxis per- obtained by GraphPad Prism 7. All of the figures were assembled using centage of the hesperidin-pretreated groups went from 44.68% at

Photograph CC 2017. 6.25 mg/L to 34.53% at 25 mg/L (F4,18 = 29.52, p < 0.001), while the percentages for the naringin-pretreated counterparts were 36.54% of

3. Results 6.25 mg/L to 23.97% of 25 mg/L (F4,18 = 26.63, p < 0.001, Fig. 3B–C). However, the low dose of ﬂavonoid pretreatment did not 3.1. UPLC/Q-TOF-MS determination of hesperidin and naringin alleviate the alcohol-induced thigmotaxis behaviour in the dark, while only 25 mg/L of naringin could alleviate the alcohol-induced thigmo-

Ultraperformance liquid chromatography (UPLC) and quadrupole taxis effect (F4,10 = 6.358, p = 0.0082, Fig. 3C). time-of-flight mass spectrometry (Q-TOF-MS) analyses were conducted In addition, the thigmotaxis behaviour in zebrafish larvae was − for hesperidin and naringin. The deprotonated [M-H] ions were ob- tested at 7 dpf. In this experiment, thigmotaxis behaviour was pre- tained with characteristic fragment information. The electrospray io- sented as the percentage (%) of time spent in the outer zone (Figs. 2E, nization-mass spectrometry (ESI-MS) spectra were acquired in the ne- 3D–F). The administration of hesperidin and naringin decreased the gative ion voltage modes for each compound. Here, the results of proportion of distance travelled and time spent in the outer zone in hesperidin and naringin in the negative ion voltage mode are presented zebrafish larvae (Fig. 3D, J–I). The thigmotaxis percentage of the he- in Fig. 1 and Table 1. In the negative mode, the most abundant ions that speridin-pretreated groups went from 79.71% at 6.25 mg/L to 77.82% − were observed were singly charged pseudomolecular ions ([M-H] ) for at 25 mg/L (F4,168 = 11.31, p < 0.0001), while the percentages for the hesperidin (609.1776) and naringin (579.1719). The main fragment ion naringin-pretreated counterparts were 83.01% at 6.25 mg/L to 73.74% for hesperidin is at m/z 301 through the loss of rutinose, and it is at m/z at 25 mg/L (F4,170 = 21.67, p < 0.0001, Fig. 3E–F) under the light

24 X. Liu, et al. Neurotoxicology and Teratology 73 (2019) 22–30

Fig. 1. UPLC-Q-TOF/MS BPI chromatograms of hesperidin (A) and naringin (B) in negative ion mode, and the UPLC-Q-TOF/MS spectra of hesperidin (C) and naringin (D) using ESI. conditions. However, the alleviation of thigmotaxis after flavonoid larval zebrafish. The apoptotic cells were characterized as aggregated pretreatment under dark conditions was not as significant as under light green particles. The 1% alcohol treatment group showed an elevated conditions because only 6.25 mg/L and 25 mg/L of hesperidin alle- level of apoptotic cells compared to the control group in both the head viated the thigmotaxis behaviour with the percentage of time spent in area and heart area (Fig. 5). The results of flavonoid-treated groups the outer zone at 82.73% and 79.51%, respectively (F4,168 = 3.967, suggested that hesperidin and naringin could suppress apoptosis in- p = 0.0042, Fig. 3E). duced by alcohol exposure (F7,112 = 15.76, p < 0.001, Fig. 5F).

3.4. Alcohol-induced developmental defects in larvae could be inhibited by 4. Discussion hesperidin and naringin Previous research has indicated that alcohol exposure could disturb ff We further evaluated the adverse e ects of alcohol exposure during cell movement during gastrulation, produce epiboly and gastrulation ff fl developmental stages. Additionally, the protective e ects of the avo- malformation, along with defects in the skeleton and neurobehaviour noids hesperidin and naringin were assessed. In those experiments, (Carvan et al., 2004; Zhang et al., 2010). It has been reported that acute exposure to 1% alcohol alone and joint exposure to 1% alcohol and alcohol exposure could induce behavioural alterations, including hy- hesperidin or to 1% alcohol and naringin were conducted at 0.75 hpf, peractivity and wall-hugging, in a dose- and time-dependent manner in and the morphologies of the larvae were assessed at 3 dpf. No sig- 7 dpf larval zebrafish (Lockwood et al., 2004). They also demonstrated fi ni cant change in survival rate was observed at 72 hpf (Supplementary that the alcohol-exposed AB strain possessed significantly increased Fig. S1). These results are consistent with previous studies (Bilotta thigmotaxis compared to the control after 10 min of alcohol exposure. et al., 2004; Li et al., 2016; Ramlan et al., 2017). Additionally, 1% al- Ramlan et al. (2017) observed increased anxiety-related behaviour with cohol exposure resulted in alteration of larval morphological char- changes in carbohydrates, lipids, nucleic acids, and proteins in 6 dpf acteristics (Fig. 4A). Body length, head area, and eye area were reduced zebrafish larvae after developmental exposure to 0.75% alcohol. Ad- – after alcohol exposure (Fig. 4B D). In addition, pericardium oedema ditionally, acute 1% alcohol exposure could cause defects in learning with a large transparent bubble was observed in the 1% alcohol-treated and memory in adult zebrafish, which was assessed by colour-enhanced group. However, with the developmental administration of hesperidin conditional place preference tests in adult zebrafish (Li et al., 2015). In fi or naringin, the morphological malformation was signi cantly rescued this study, zebrafish larvae at 3 dpf were used to evaluate thigmotaxis, in all groups pretreated with hesperidin and naringin, including body which is a behaviour that involves rapid seeking for and positioning at length (F7,313 = 21.7, p < 0.0001, Fig. 4B), head area (F7,314 = 30.87, the container edge. This phenotype resembles the act of refraining from p < 0.0001, Fig. 4C), and eye area (F7,296 = 32.74, p < 0.001, movement in open spaces and remaining close to the wall in mouse and Fig. 4D). There were no oedema-like characteristics observed in the rat models of anxiety and depression (Prut and Belzung, 2003; fl alcohol- avonoid joint exposure group. The results indicated that he- Seibenhener and Wooten, 2015). The results of the thigmotaxis assay at ff speridin and naringin might inhibit the adverse e ects of alcohol in 3 dpf (Fig. 2D) showed that alcohol could significantly induce thig- fi zebra sh larvae. motaxis in zebrafish larvae, which correlates with previous findings in 7 dpf larvae under different behavioural experiment schemes (Lockwood 3.5. Hesperidin and naringin reduced apoptosis induced by alcohol in et al., 2004). Additionally, Baggio et al. found that exposure to alcohol zebrafish larvae for 2 h led to disrupted social behaviour and elevated anxiety level in novel tank test four months later in embryonic zebrafish at 24 hpf, Apoptosis plays an essential role in the developmental process. The which suggests that embryonic exposure to alcohol may lead to lasting level of apoptosis was evaluated by acridine orange staining at 72 hpf in abnormal behaviours (Baggio et al., 2017).

Table 1 Identiﬁcation of hesperidin and naringin by UPLC-Q-TOF/MS in negative ion mode.

− Identiﬁcation Retention time (min) Molecular formula Measure mass (Da) Exact mass (Da) Error (10 6) Main fragment ions

− − Hesperidin 4.12 C28H34O15 609.1776[M-H] 609.1825[M-H] −8.04 301.0698 − − Naringin 3.98 C27H32O14 579.1705[M-H] 579.1719[M-H] −2.42 459.1148, 217.0608

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Fig. 2. Alcohol promoted thigmotaxis in zebrafish larvae. (A) Schematic representation of thigmotaxis test in 3 dpf zebrafish larvae. (B) Images and illustrations of thigmotaxis-positive zebrafish larvae in an open arena. (C) Representative images in light conditions and (D) quantification of zebrafish larvae in a 10-cm Petri dish. Thigmotaxis at 3 dpf are presented as the percentage (%) of thigmotaxis-positive larvae in the total larval zebrafish per group. Thigmotaxis-positive larvae are presented as red dots, and thigmotaxis-negative larvae are presented as blue dots. n = 6 with 35–40 larvae per group. (E) Schematic representation of the thigmotaxis test in 7 dpf zebrafish larvae. (D) Thigmotaxis of zebrafish larvae at 7 dpf. Thigmotaxis is presented as the percentage (%) of time spent in the outer zone. n = 27–36. L: light conditions; D: dark conditions. **p < 0.01, ***p < 0.001, ****p < 0.0001 (one-way ANOVA with Fisher's LSD). Error bars represent the standard error of the means (SEM).

Previous studies found that hesperidin exhibited an antianxiety-like to induce robust behaviour in zebrafish larvae and compel the larvae to property through an elevated open-platform test in 6-week-old ICR explore the whole spatial area of the well (Schnörr et al., 2012). In male mice (50 mg/kg, p.o.) (Ito et al., 2013). Additionally, pretreat- addition, since larvae at 7 dpf present swimming behaviour without ment of hesperidin (50 and 100 mg/kg, p.o.) for fourteen days in Swiss stimuli, we also assessed the thigmotaxis behaviour under the light albino mice could significantly alleviate the anxiety-like behaviour in- conditions without a sudden light-dark transition. The administration of duced by acute immobilization stress (Viswanatha et al., 2012). Nar- hesperidin and naringin decreased the proportion of distance travelled ingin (50 and 100 mg/kg, p.o.) administration to male Wistar rats could and time spent in the outer zone in zebrafish larvae under light con- attenuate doxorubicin-induced anxiety behaviour (Kwatra et al., 2016). ditions. However, the alleviation of alcohol-induced thigmotaxis after To understand whether the two flavonoids, hesperidin and naringin, flavonoid pretreatment in the dark conditions was not as significant as could alleviate the alcohol-induced behaviour alterations in zebrafish that under the light conditions. It has been reported that the dark- larvae, a series of concentrations (6.25, 12.5, 25 mg/L) for each flavo- avoidance behaviour in light/dark preference test is also one of the noid were chosen according to the methods used in a previous study anxiety-like behaviours in zebrafish larvae. The zebrafish showing an- (Zhou et al., 2017). It has been reported that hesperidin at these con- xiety-like behaviours tend to spend significantly less time in the dark centrations could exert protective effects against acute alcoholic injury compartment as well as display a high latency to enter it and move in zebrafish larvae. Due to the similar chemical structures of naringin significantly less inside it compared to the white compartment of the and hesperidin, the same concentrations were applied for both flavo- testing apparatus (Steenbergen et al., 2011). We estimated that the noids. The results of the 3 dpf thigmotaxis assay showed that both alcohol-induced thigmotaxis was less significant in the darkness than flavonoids could significantly alleviate the 1% alcohol-induced edge under light conditions because zebrafish larvae displayed a relatively effect under the lights. However, the mitigation effect of the flavonoids higher thigmotaxis in the darkness in the control group, which might in the dark was not that apparent, and significant reductions in alcohol- overcome the eff ect caused by alcohol. It was suggested that the an- induced thigmotaxis were only observed in the 25 mg/L naringin- xiolytic effects of hesperidin against immobilization stress were due to treated groups. Furthermore, we evaluated the thigmotaxis behaviour the attenuation in oxidative stress (Viswanatha et al., 2012), which in zebrafish larvae at 7 dpf. Exposure to sudden darkness was intended could be observed in the adult zebrafish brain after acute exposure to

26 X. Liu, et al. Neurotoxicology and Teratology 73 (2019) 22–30

Fig. 3. Hesperidin and naringin alleviate alcohol-induced thigmotaxis in zebrafish larvae. (A) Representative images in light conditions and (B–C) quantification of the percentage of thigmotaxis behaviour of zebrafish larvae in 10-cm Petri dishes for each treatment group at 3 dpf. Thigmotaxis-positive larvae are presented as red dots, and thigmotaxis-negative larvae are presented as blue dots. n = 6–11 with 35–40 larvae per group. (D) Representative images of swimming trajectories in light conditions and (E-F) quantification of the percentage of thigmotaxis behaviour of zebrafish larvae at 7 dpf for each treatment group. n = 27–36. (G–J) Representative 3D reconstructions of movement trajectories for (G) control larvae, (H) 1% alcohol-treated larvae, (I) hesperidin-treated (25 mg/L) larvae, and (J) naringin-treated (25 mg/L) larvae under light conditions at 7 dpf. Different colours indicate different times (0–300 s). The z-axis indicates the time, and the 3D reconstructions revealed the spatiotemporal behavioural phenotypes of larvae. L: light conditions; D: dark conditions. #p < 0.01, ##p < 0.01, ###p < 0.001, ####p < 0.0001 vs. the control, *p < 0.01, **p < 0.01, ***p < 0.001, ****p < 0.0001 vs. 1% EtOH (one-way ANOVA with Fisher's LSD). Error bars represent the standard error of the means (SEM).

1% alcohol (Rosemberg et al., 2010). Similarly, the possible mechanism In our study, the developmental alcohol exposure resulted in mul- of neuroprotective effects against alcohol-induced alterations in nar- tiple morphological defects in zebrafish larvae, including reduced body ingin could be due to the prevention of oxidative stress. Furthermore, length, a smaller head area, and a smaller eye area, which were con- studies have shown that hesperidin and naringin could exert neuro- sistent with previous findings (Bilotta et al., 2004). Kashyap et al. found protective effects by increasing BDNF expression (Antunes et al., 2016; that alcohol exposure during retinal neurogenesis, from 24 hpf to 48 Rong et al., 2012). However, to get a better understanding of the me- hpf, led to persistent microphthalmia, which was caused by overall chanism through the two flavonoids protect against the alcohol-induced developmental delay, lens malformation, and reduced retinal cell dif- neurobehavioural alterations, further studies are necessary. ferentiation (Kashyap et al., 2007). In addition, slight pericardium

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Fig. 4. Phenotypes of zebrafish larvae treated with different concentrations of hesperidin and naringin. (A) Morphological characteristics of the control and the flavonoid administration groups of zebrafish larvae at 72 hpf. (B–D) Measurements of the body length, head area, and eye area of zebrafish larvae (n = 37–48). Error bars represent the standard error of the means (SEM). ****p < 0.0001 (one-way ANOVA with Fisher's LSD). The boxes represent 75/25 percentiles, and the solid lines within the boxes indicate the medians. Dots represent values outside the 5/95 percentile bars. Scale bar = 1 mm.

Fig. 5. Apoptosis assessment in the whole body and head area of zebrafish embryos. (A) Detection of apoptosis by acridine orange staining in 3 dpf zebrafish embryos. (B–E) The apoptotic levels in the head area of the zebrafish embryos for the (B) the control, (C) the 1% alcohol-exposure, (D) the hesperidin-treated (6.25 mg/L) and (E) the naringin-treated (6.25 mg/L) groups, in which 6.25 mg/L was the lowest effective dose for our experiment. White arrows indicate apoptotic cells. (F) Quantification of apoptosis in the head area of zebrafish larvae. (n = 14–17). *p < 0.05, **p < 0.01 (one-way ANOVA with Fisher's LSD). The boxes represent 75/25 percentiles, and the solid lines within the boxes indicate the medians. The dots represent values outside the 10/90 percentile bars.

oedema was observed in the 1% alcohol group in our study. Previous in zebrafish. The morphological assessment results showed that mul- studies found unrecoverable severe pericardium oedema and dorsal tiple morphological defects could be alleviated. Body length, head area, aorta destruction in zebrafish larvae after 2% alcohol exposure (Li et al., and eye area remained at normal levels in all hesperidin-treated and 2016; Soares et al., 2012). It was also reported that cardiac chamber naringin-treated groups. In addition, slight pericardium oedema was malformation could be induced by alcohol during embryogenesis in not observed in the groups with joint exposure to the flavonoids and zebrafish (Sarmah and Marrs, 2017). These studies further demon- alcohol, which may support the potential cardiovascular disease pre- strated that alcohol is teratogenic. vention effects that have been reported before for flavonoids (Morand To confirm whether these two flavonoids could alleviate the de- et al., 2011). Other studies also found that hesperidin could protect velopmental malformation induced by alcohol, exposure to hesperidin against acute alcoholic injury by reducing endoplasmic reticulum or naringin with 1% alcohol was performed during early development stress, reducing DNA damage, and regulating alcohol and lipid

28 X. Liu, et al. Neurotoxicology and Teratology 73 (2019) 22–30

Fig. 6. Schematic of possible mechanisms underlying the protective effects of hesperidin and naringin in zebrafish larvae exposed to alcohol. metabolism in zebrafish larvae (Zhou et al., 2017). Acknowledgements Apoptosis, which is an essential cellular process, plays a critical role in vertebrate development. Impairments at the cellular level in the This research was funded under the Special Fund for Basic Research developmental process and defects in programmed cell death may on Scientific Instruments from the National Natural Science Foundation eventually cause numerous defects in development and neurobeha- of China (Grant Nos. 61327802, 61127006), and the National Basic viour. Our results suggested that 1% alcohol could promote apoptosis in Research Program of China (2015CB856500) supported this work. zebrafish larvae at 72 hpf, which was consistent with a previous study (Ramlan et al., 2017). Alcohol-induced apoptosis was also found in the References foetal brain of a C57BL/6 mouse after maternal exposure (Sari, 2009). It has reported that hesperidin could alleviate acetaminophen-induced Ahmad, S.T., Arjumand, W., Nafees, S., Seth, A., Ali, N., Rashid, S., Sultana, S., 2012. toxicity by decreased apoptosis (Ahmad et al., 2012). In addition, Zhou Hesperidin alleviates acetaminophen induced toxicity in wistar rats by abrogation of fl – fi oxidative stress, apoptosis and in ammation. Toxicol. Lett. 208 (2), 149 161. et al. (2017) found that hesperidin could reduce apoptosis in zebra sh https://doi.org/10.1016/j.toxlet.2011.10.023. larvae. Our results showed that administration of hesperidin and nar- Antunes, M.S., Jesse, C.R., Ruff, J.R., de Oliveira Espinosa, D., Gomes, N.S., Altvater, ingin could inhibit apoptosis induced by alcohol in zebrafish larvae, E.E.T., ... Boeira, S.P., 2016. Hesperidin reverses cognitive and depressive dis- fl turbances induced by olfactory bulbectomy in mice by modulating hippocampal which suggests that the avonoids might inhibit alcohol-induced mal- neurotrophins and cytokine levels and acetylcholinesterase activity. Eur. J. formation in zebrafish larvae by decreasing apoptosis. Further ex- Pharmacol. 789, 411–420. https://doi.org/10.1016/j.ejphar.2016.07.042. ploration is needed to investigate which pathways are regulated during Assini, J.M., Mulvihill, E.E., Huff, M.W., 2013. Citrus flavonoids and lipid metabolism. – the protection process of the flavonoids. Moreover, these flavonoids and Curr. Opin. Lipidol. 24 (1), 34 40. https://doi.org/10.1097/MOL. 0b013e32835c07fd. their metabolites can be found in the plasma of humans who take or- Baggio, S., Mussulini, B.H., de Oliveira, D.L., Gerlai, R., Rico, E.P., 2017. Embryonic al- ange or grapefruit juice, and further pre-clinical and intervention stu- cohol exposure leading to social avoidance and altered anxiety responses in adult fi dies of humans may contribute to assessing whether hesperidin and zebra sh. Behav. Brain Res. https://doi.org/10.1016/j.bbr.2017.08.039. Baiamonte, M., Parker, M.O., Vinson, G.P., Brennan, C.H., 2016. Sustained effects of naringin can protect against alcohol-induced developmental deforma- developmental exposure to ethanol on zebrafish anxiety-like behaviour. PLoS ONE 11 tion and neurobehavioural defects (Fig. 6). (2), e0148425. https://doi.org/10.1371/journal.pone.0148425. Bailey, J.M., Oliveri, A.N., Karbhari, N., Brooks, R.A.J., De La Rocha, A.J., Janardhan, S., Levin, E.D., 2016. Persistent behavioral effects following early life exposure to re- tinoic acid or valproic acid in zebrafish. NeuroToxicology 52, 23–33. https://doi.org/ 5. Conclusion 10.1016/j.neuro.2015.10.001. Bilotta, J., Barnett, J.A., Hancock, L., Saszik, S., 2004. Ethanol exposure alters zebrafish development: a novel model of fetal alcohol syndrome. Neurotoxicol. Teratol. 26 (6), Exposure to 1% alcohol led to behavioural alterations and multiple 737–743. https://doi.org/10.1016/j.ntt.2004.06.011. deformations in the morphology of zebrafish larvae. Pre-treatment with Brito, A., Ramirez, J., Areche, C., Sepúlveda, B., Simirgiotis, M., 2014. HPLC-UV-MS the flavonoids hesperidin and naringin could alleviate these alterations profiles of phenolic compounds and antioxidant activity of fruits from three Citrus species consumed in Northern Chile. Molecules 19 (11), 17400–17421. https://doi. in behaviour and morphology. Additionally, the level of apoptosis in- org/10.3390/molecules191117400. duced by alcohol exposure was reduced when the zebrafish embryos Brittijn, S.A., Duivesteijn, S.J., Belmamoune, M., Bertens, L.F.M., Bitter, W., Debruijn, were incubated in hesperidin and naringin during their developmental J.D., ... Richardson, M.K., 2009. Zebrafish development and regeneration: new tools – – stages. In conclusion, this study suggests that the citrus flavonoids he- for biomedical research. Int. J. Dev. Biol. 53 (5 6), 835 850. https://doi.org/10. 1387/ijdb.082615sb. speridin and naringin could alleviate alcohol damage effects on em- Carvan, M.J., Loucks, E., Weber, D.N., Williams, F.E., 2004. Ethanol effects on the de- bryonic development and neurobehaviour in zebrafish larvae, which veloping zebrafish: neurobehavior and skeletal morphogenesis. Neurotoxicol. – would provide a useful reference for assessing the underlying me- Teratol. 26 (6), 757 768. https://doi.org/10.1016/j.ntt.2004.06.016. fl ff Chen, S., Reichert, S., Singh, C., Oikonomou, G., Rihel, J., Prober, D.A., 2017. Light- chanism of citrus avonoids and their protective e ects. dependent regulation of sleep and wake states by prokineticin 2 in zebrafish. Neuron Supplementary data to this article can be found online at https:// 95 (1), 153–168.e6. https://doi.org/10.1016/j.neuron.2017.06.001. doi.org/10.1016/j.ntt.2019.03.001. Chudley, A.E., 2005. Fetal alcohol spectrum disorder: Canadian guidelines for diagnosis. Can. Med. Assoc. J. 172 (5_suppl), S1–S21. https://doi.org/10.1503/cmaj.1040302. Fernandez, S.P., Nguyen, M., Yow, T.T., Chu, C., Johnston, G.A.R., Hanrahan, J.R., Chebib, M., 2009. The flavonoid glycosides, myricitrin, gossypin and naringin exert Conflicts of interests anxiolytic action in mice. Neurochem. Res. 34 (10), 1867–1875. https://doi.org/10. 1007/s11064-009-9969-9. González, E.A., Carty, D.R., Tran, F.D., Cole, A.M., Lein, P.J., 2018. Developmental ex- The authors declare that there are no conflicts of interests. posure to silver nanoparticles at environmentally relevant concentrations alters swimming behavior in zebrafish (Danio rerio): developmental toxicity of Ag-NPs in

29 X. Liu, et al. Neurotoxicology and Teratology 73 (2019) 22–30

zebrafish. Environ. Toxicol. Chem. https://doi.org/10.1002/etc.4275. org/10.1016/S0014-2999(03)01272-X. Guo, J., Zhao, S., Shu, M., Yan, Z., Sun, M., Zhao, X., Feng, X., 2014. Trajectory Tracking Ramlan, N.F., Sata, N.S.A.M., Hassan, S.N., Bakar, N.A., Ahmad, S., Zulkifli, S.Z., ... of Spasm-oriented Zebrafish Larvae. IEEE, pp. 6849–6853. https://doi.org/10.1109/ Ibrahim, W.N.W., 2017. Time dependent effect of chronic embryonic exposure to ChiCC.2014.6896128. ethanol on zebrafish: morphology, biochemical and anxiety alterations. Behav. Brain Ito, A., Shin, N., Tsuchida, T., Okubo, T., Norimoto, H., 2013. Antianxiety-like effects of Res. 332, 40–49. https://doi.org/10.1016/j.bbr.2017.05.048. Chimpi (dried citrus peels) in the elevated open-platform test. Molecules 18 (8), Rong, W., Wang, J., Liu, X., Jiang, L., Wei, F., Hu, X., Liu, Z., 2012. Naringin treatment 10014–10023. https://doi.org/10.3390/molecules180810014. improves functional recovery by increasing BDNF and VEGF expression, inhibiting Josefsson, M., Zackrisson, A.-L., Ahlner, J., 1996. Effect of grapefruit juice on the phar- neuronal apoptosis after spinal cord injury. Neurochem. Res. 37 (8), 1615–1623. macokinetics of amlodipine in healthy volunteers. Eur. J. Clin. Pharmacol. 51 (2), https://doi.org/10.1007/s11064-012-0756-7. 189–193. https://doi.org/10.1007/s002280050183. Roper, C., Simonich, S.L.M., Tanguay, R.L., 2018. Development of a high-throughput in Kashyap, B., Frederickson, L.C., Stenkamp, D.L., 2007. Mechanisms for persistent mi- vivo screening platform for particulate matter exposures. Environ. Pollut. 235, crophthalmia following ethanol exposure during retinal neurogenesis in zebrafish 993–1005. https://doi.org/10.1016/j.envpol.2018.01.025. embryos. Vis. Neurosci. 24 (03), 409–421. https://doi.org/10.1017/ Rosa, L.V., Ardais, A.P., Costa, F.V., Fontana, B.D., Quadros, V.A., Porciúncula, L.O., S0952523807070423. Rosemberg, D.B., 2018. Different effects of caffeine on behavioral neurophenotypes Kung, T.S., Richardson, J.R., Cooper, K.R., White, L.A., 2015. Developmental deltame- of two zebrafish populations. Pharmacol. Biochem. Behav. 165, 1–8. https://doi.org/ thrin exposure causes persistent changes in dopaminergic gene expression, neu- 10.1016/j.pbb.2017.12.002. rochemistry, and locomotor activity in zebrafish. Toxicol. Sci. 146 (2), 235–243. Rosemberg, D.B., da Rocha, R.F., Rico, E.P., Zanotto-Filho, A., Dias, R.D., Bogo, M.R., ... https://doi.org/10.1093/toxsci/kfv087. Souza, D.O., 2010. Taurine prevents enhancement of acetylcholinesterase activity Kwatra, M., Jangra, A., Mishra, M., Sharma, Y., Ahmed, S., Ghosh, P., Khanam, R., 2016. induced by acute ethanol exposure and decreases the level of markers of oxidative Naringin and sertraline ameliorate doxorubicin-induced behavioral deficits through stress in zebrafish brain. Neuroscience 171 (3), 683–692. https://doi.org/10.1016/j. modulation of serotonin level and mitochondrial complexes protection pathway in rat neuroscience.2010.09.030. hippocampus. Neurochem. Res. 41 (9), 2352–2366. https://doi.org/10.1007/ Sari, Y., 2009. Activity-dependent neuroprotective protein–derived peptide, NAP, pre- s11064-016-1949-2. venting alcohol-induced apoptosis in fetal brain of C57BL/6 mouse. Neuroscience Li, Xiang, Li, X., Li, Y.-X., Zhang, Y., Chen, D., Sun, M.-Z., ... Feng, X.-Z., 2015. The 158 (4), 1426–1435. https://doi.org/10.1016/j.neuroscience.2008.11.021. difference between anxiolytic and anxiogenic effects induced by acute and chronic Sarmah, Swapnalee, Marrs, James, 2017. Embryonic ethanol exposure affects early- and alcohol exposure and changes in associative learning and memory based on color late-added cardiac precursors and produces long-lasting heart chamber defects in preference and the cause of Parkinson-like behaviors in zebrafish. PLoS ONE 10 (11), zebrafish. Toxics 5 (4), 35. https://doi.org/10.3390/toxics5040035. e0141134. https://doi.org/10.1371/journal.pone.0141134. Schnörr, S.J., Steenbergen, P.J., Richardson, M.K., Champagne, D.L., 2012. Measuring Li, Xu, Gao, A., Wang, Y., Chen, M., Peng, J., Yan, H., Chen, D., 2016. Alcohol exposure thigmotaxis in larval zebrafish. Behav. Brain Res. 228 (2), 367–374. https://doi.org/ leads to unrecoverable cardiovascular defects along with edema and motor function 10.1016/j.bbr.2011.12.016. changes in developing zebrafish larvae. Biol. Open 5 (8), 1128–1133. https://doi. Seibenhener, M.L., Wooten, M.C., 2015. Use of the open field maze to measure locomotor org/10.1242/bio.019497. and anxiety-like behavior in mice. J. Vis. Exp.(96). https://doi.org/10.3791/52434. Lin, H., Zhou, Z., Zhong, W., Huang, P., Ma, N., Zhang, Y., Lv, Z., 2017. Naringenin Selmi, S., Rtibi, K., Grami, D., Sebai, H., Marzouki, L., 2017. Protective effects of orange inhibits alcoholic injury by improving lipid metabolism and reducing apoptosis in (Citrus sinensis L.) peel aqueous extract and hesperidin on oxidative stress and peptic zebrafish larvae. Oncol. Rep. 38 (5), 2877–2884. https://doi.org/10.3892/or.2017. ulcer induced by alcohol in rat. Lipids Health Dis. 16 (1). https://doi.org/10.1186/ 5965. s12944-017-0546-y. Liu, X.-Y., Zhang, Q.-P., Li, S.-B., Mi, P., Chen, D.-Y., Zhao, X., Feng, X.-Z., 2018. Soares, A.R., Pereira, P.M., Ferreira, V., Reverendo, M., Simões, J., Bezerra, A.R., Santos, Developmental toxicity and neurotoxicity of synthetic organic insecticides in zebra- M.A.S., 2012. Ethanol exposure induces upregulation of specific MicroRNAs in zeb- fish (Danio rerio): a comparative study of deltamethrin, acephate, and thiamethoxam. rafish embryos. Toxicol. Sci. 127 (1), 18–28. https://doi.org/10.1093/toxsci/kfs068. Chemosphere 199, 16–25. https://doi.org/10.1016/j.chemosphere.2018.01.176. Steenbergen, P.J., Richardson, M.K., Champagne, D.L., 2011. Patterns of avoidance be- Lockwood, B., Bjerke, S., Kobayashi, K., Guo, S., 2004. Acute effects of alcohol on larval haviours in the light/dark preference test in young juvenile zebrafish: a pharmaco- zebrafish: a genetic system for large-scale screening. Pharmacol. Biochem. Behav. 77 logical study. Behav. Brain Res. 222 (1), 15–25. https://doi.org/10.1016/j.bbr.2011. (3), 647–654. https://doi.org/10.1016/j.pbb.2004.01.003. 03.025. Lundegaard, P.R., Anastasaki, C., Grant, N.J., Sillito, R.R., Zich, J., Zeng, Z., Patton, E.E., Tripoli, E., Guardia, M.L., Giammanco, S., Majo, D.D., Giammanco, M., 2007. Citrus 2015. MEK inhibitors reverse cAMP-mediated anxiety in zebrafish. Chem. Biol. 22 flavonoids: molecular structure, biological activity and nutritional properties: a re- (10), 1335–1346. https://doi.org/10.1016/j.chembiol.2015.08.010. view. Food Chem. 104 (2), 466–479. https://doi.org/10.1016/j.foodchem.2006.11. MacPhail, R.C., Brooks, J., Hunter, D.L., Padnos, B., Irons, T.D., Padilla, S., 2009. 054. Locomotion in larval zebrafish: influence of time of day, lighting and ethanol. Verma, S.K., Jha, E., Panda, P.K., Mishra, A., Thirumurugan, A., Das, B., Suar, M., 2018. NeuroToxicology 30 (1), 52–58. https://doi.org/10.1016/j.neuro.2008.09.011. Rapid novel facile biosynthesized silver nanoparticles from bacterial release induce Martin, M.J., Marhuenda, E., Perez-Guerrero, C., Franco, J.M., 1994. Antiulcer effect of biogenicity and concentration dependent in vivo cytotoxicity with embryonic zeb- naringin on gastric lesions induced by ethanol in rats. Pharmacology 49 (3), 144–150. rafish—a mechanistic insight. Toxicol. Sci. 161 (1), 125–138. https://doi.org/10. https://doi.org/10.1159/000139228. 1093/toxsci/kfx204. Martínez, M.C., Fernandez, S.P., Loscalzo, L.M., Wasowski, C., Paladini, A.C., Marder, M., Viswanatha, G.L., Shylaja, H., Sandeep Rao, K.S., Santhosh Kumar, V.R., Jagadeesh, M., Viola, H., 2009. Hesperidin, a flavonoid glycoside with sedative effect, decreases 2012. Hesperidin ameliorates immobilization-stress-induced behavioral and bio- brain pERK1/2 levels in mice. Pharmacol. Biochem. Behav. 92 (2), 291–296. https:// chemical alterations and mitochondrial dysfunction in mice by modulating nitrergic doi.org/10.1016/j.pbb.2008.12.016. pathway. ISRN Pharmacol. 2012, 1–8. https://doi.org/10.5402/2012/479570. Morand, C., Dubray, C., Milenkovic, D., Lioger, D., Martin, J.F., Scalbert, A., Mazur, A., Wang, Y.-N., Hou, Y.-Y., Sun, M.-Z., Zhang, C.-Y., Bai, G., Zhao, X., Feng, X.-Z., 2014. 2011. Hesperidin contributes to the vascular protective effects of orange juice: a Behavioural screening of zebrafish using neuroactive traditional Chinese medicine randomized crossover study in healthy volunteers. Am. J. Clin. Nutr. 93 (1), 73–80. prescriptions and biological targets. Sci. Rep. 4. https://doi.org/10.1038/srep05311. https://doi.org/10.3945/ajcn.110.004945. Zhang, Y., Shao, M., Wang, L., Liu, Z., Gao, M., Liu, C., Zhang, H., 2010. Ethanol exposure Nielsen, I.L.F., Chee, W.S.S., Poulsen, L., Offord-Cavin, E., Rasmussen, S.E., Frederiksen, affects cell movement during gastrulation and induces split axes in zebrafish em- H., ... Williamson, G., 2006. Bioavailability is improved by enzymatic modification of bryos. Int. J. Dev. Neurosci. 28 (4), 283–288. https://doi.org/10.1016/j.ijdevneu. the citrus flavonoid hesperidin in humans: a randomized, double-blind, crossover 2010.04.001. trial. J. Nutr. 136 (2), 404–408. https://doi.org/10.1093/jn/136.2.404. Zhang, J., Gao, W., Liu, Z., Zhang, Z., Liu, C., 2014. Systematic Analysis of Main Pieróg, M., Guz, L., Doboszewska, U., Poleszak, E., Wlaź, P., 2018. Effects of alprazolam Constituents in Rat Biological Samples after Oral Administration of the Methanol treatment on anxiety-like behavior induced by color stimulation in adult zebrafish. Extract of Fructus Aurantii by HPLC-ESI-MS/MS. pp. 13. Prog. Neuro-Psychopharmacol. Biol. Psychiatry 82, 297–306. https://doi.org/10. Zhdanova, I.V., 2011. Sleep and its regulation in zebrafish. Rev. Neurosci. 22 (1). https:// 1016/j.pnpbp.2017.08.025. doi.org/10.1515/rns.2011.005. Pietri, T., Roman, A.-C., Guyon, N., Romano, S.A., Washbourne, P., Moens, C.B., ... Zhou, Z., Zhong, W., Lin, H., Huang, P., Ma, N., Zhang, Y., Lv, Z., 2017. Hesperidin Sumbre, G., 2013. The first mecp2-null zebrafish model shows altered motor beha- protects against acute alcoholic injury through improving lipid metabolism and cell viors. Front. Neural Circuits 7. https://doi.org/10.3389/fncir.2013.00118. damage in zebrafish larvae. Evid. Based Complement. Alternat. Med. 2017, 1–9. Prut, L., Belzung, C., 2003. The open field as a paradigm to measure the effects of drugs https://doi.org/10.1155/2017/7282653. on anxiety-like behaviors: a review. Eur. J. Pharmacol. 463 (1–3), 3–33. https://doi.