Abstract The anticonvulsive potential of proteins extracted from Orthosiphon stamineus leaves (OSLP) has never been elucidated in zebrafish (Danio rerio). This study thus aims to elucidate the anticonvulsive potential of OSLP in pentylenetetrazol (PTZ)-induced seizure model. Physical changes (seizure score and seizure onset time, behavior, locomotor) and neurotransmitter analysis were elucidated to assess the pharmacological activity. The protective mechanism of OSLP on brain was also studied using mass spectrometry-based label-free proteomic quantification (LFQ) and bioinformatics. OSLP was found to be safe up to 800 µg/kg and pre-treatment with OSLP (800 µg/kg, i.p., 30 min) decreased the frequency of convulsive activities (lower seizure score and prolonged seizure onset time), improved locomotor behaviors (reduced erratic swimming movements and bottom-dwelling habit), and lowered the excitatory neurotransmitter (glutamate). Pre-treatment with OSLP increased protein Complexin 2 (Cplx 2) expression in the zebrafish brain. Cplx2 is an important regulator in the trans-SNARE complex which is required during the vesicle priming phase in the calcium-dependent synaptic vesicle exocytosis. Findings in this study collectively suggests that OSLP could be regulating the release of neurotransmitters via calcium-dependent synaptic vesicle exocytosis mediated by the “Synaptic Vesicle Cycle” pathway. OSLP’s anticonvulsive actions could be acting differently from diazepam (DZP) and with that, it might not produce the similar cognitive insults such as DZP. Keywords: Orthosiphon stamineus, plant-derived proteins, epilepsy, seizures, zebrafish 1. Introduction Epilepsy is a chronic non-communicable disease of the brain that affects around 70 million people of all ages worldwide and accounts for about 1% of the global burden of disease. Epilepsy has a high prevalence and an estimated five million people are diagnosed with epilepsy each year. Epilepsy is characterized by recurrent seizures due to brief disturbances in the electrical functions of the brain. It involves brief episodes of involuntary movement that lead to changes in sensory perception, motor control, behavior, autonomic function, or sometimes loss of consciousness [[32]1]. To date, despite having more than 30 antiepileptic drugs (AEDs) on the market [[33]2,[34]3], there are still difficulties in reaching the goal of treating epilepsy and its associated complications without adverse effects. Globally, epilepsy remains a public health imperative. People with epilepsy often require lifelong treatment. AEDs are the mainstay of treatment. These conventional drugs bring about clinically worthwhile improvements but have tolerability issues due to their side effects. Many AEDs used in current mainstream clinical practice have been reported to elicit undesired neuropsychological consequences such as depression (24% lifetime prevalence), anxiety (22%), and intellectual disability, particularly in children with epilepsy (30%–40%) [[35]1]. More than one-third of epileptic seizures are not well controlled by a single AED and often require treatment with two or more AEDs (add-on therapy) [[36]1,[37]2]. Furthermore, about 40%–60% of epileptic patients, accounting for both children and adults, develop neuropsychological impairments [[38]3]. This drives a significant portion of epileptic patients to seek alternative interventions, particularly in herbal medicine [[39]4]. Current systematic studies are reporting promising anticonvulsive activities in a constellation of medicinal plants [[40]5,[41]6]. Orthosiphon stamineus (OS) or Orthosiphon aristatus var. aristatus (OAA), also commonly known as cat’s whiskers or “misai kucing,” is an important medicinal plant. Choo et al. (2018) has shown that the ethanolic extract of OS, exhibited anticonvulsive activity in zebrafish Choo, Kundap [[42]7] and Coelho et al. (2015) has demonstrated the anticonvulsant potential of rosmarinic acid in mice, which is an active chemical constituent in OS extract Coelho, Vieira [[43]8]. Nonetheless, until now the protective potential of OS primary metabolites has not been studied, let alone its proteins. The proteins extracted from OS leaves (OSLP) may also hold valuable protective potential for central nervous system (CNS) disorders such as epilepsy. In the research of epilepsy and drug discovery, zebrafish (Danio rerio) has been widely recognised as an important and promising vertebrate model. Genetic profile of zebrafish shares approximately 70% similarity with human and about 84% of genes known to human diseases are also expressed in zebrafish [[44]9,[45]10]. This makes the zebrafish model particularly useful as a high-throughput screening system in studying mechanisms of brain functions and dysfunctions [[46]11]. To the best of our knowledge, this is the first study on elucidating the anticonvulsive potential of proteins extracted from OSLP. 2. Experimental Section 2.1. Materials Chemicals and Apparatuses L-Glutamic acid (Glu), Gamma-Aminobutyric acid (γ-aminobutyric acid), Pentylenetetrazol (PTZ), Diazepam (DZP), Benzocaine, complete EDTA-free protease inhibitors, phosphatase inhibitors cocktail 2, dithiothreitol (DTT), trifluoroethanol (TFE), ammonium bicarbonate (ABC), 2,3,5-triphenyltetrazolium chloride (TTC), formic acid (FA), and methanol (MeOH) of HPLC-grade were purchased from Sigma-Aldrich (St. Louis, MO, USA). Pierce^®trypsin protease, Pierce^® Radioimmunoprecipitation assay (RIPA) buffer of mass spec grade and Pierce^®C18 mini spin columns were purchased from Thermo Scientific Pierce (Rockford, IL, USA). Protein LoBind microcentrifuge tube (Eppendorf, Enfield, CT, USA), acetonitrile (ACN), trifluoroacetic acid (TFA), indoleacetic acid (IAA) and CHAPS (Nacailai Tesque, Kyoto, Japan) of mass spec grade were from Sigma-Aldrich (St. Louis, MO, USA), Quick Start™ Bradford Protein Assay Kit from Bio-Rad (Hercules, CA, USA), Dimethylsulfoxide (DMSO) and 37% formaldehyde solution were from Friendemann Schmidt Chemical (Parkwood, Western Australia), Milli-Q ultrapure (MQUP) water from Millipore GmbH (Darmstad, Germany), acetic acid (glacial, 100%) from Merck (Darmstadt, Germany) and Phosphate buffered saline (PBS) tablets from VWR Life Science AMRESCO^® (Radnor, PA, USA). Liquid nitrogen was purchased from Linde Malaysia, Hamilton syringes 25 µL (MICROLITER™ #702) from Hamilton Co. (Reno, NV, USA), 35 gauge needles (PrecisionGlide™) were from Becton, Dickinson and Company (Franklin Lakes, NJ, USA), ultrasonic cell crusher (JY88-II N, Shanghai Xiwen Biotech. Co., Ltd., Shanghai, China), Eyela SpeedVac Vacuum Concentrator (Thermo Scientific Pierce, Rockford, IL, USA), Camry High-Precision Electronic Pocket Scale (Model EHA901, Zhaoqing, China) and Classic pH Pen Tester from Yi Hu Fish Farm Trading Pte. Ltd. (Singapore). The other chemicals of analytical grade were from established suppliers worldwide. 2.2. Software and Equipment For the behavioral study, SMART V3.0.05 tracking software (Panlab Harvard Apparatus, Barcelona, Spain) was used for the automated tracking of zebrafish swimming patterns. The video recorded using the camcorder was analyzed using the software. The water-filled tank was divided into two halves of the same size; the upper-half was marked as the top zone and the lower-half as the bottom zone as described by Kundap et al. 2017 [[47]12]. For the neurotransmitter analysis, the solvent delivery was performed using Agilent Ultra High-Performance Liquid Chromatography (UHPLC) 1290 Series (Agilent Technologies, Santa Clara, CA, USA) consisting of Agilent 1290 Series High-Performance Autosampler, Agilent 1290 Series Binary Pump and Agilent 1290 Series Thermostatted Column Compartment; the separations were performed using Zorbax Eclipse Plus C18 (Rapid Resolution HD, 2.1 × 150.0 mm with 1.8 µM pore size reverse-phase column) (Agilent Technologies, Santa Clara, CA, USA), and coupled with Agilent 6410B Triple Quadrupole (QQQ) mass spectrometer equipped with an electrospray ionization (ESI) (Agilent Technologies, Santa Clara, CA, USA) to detect the targeted neurotransmitters. In the protein expression study, Agilent 1200 series HPLC coupled with Agilent 6550 iFunnel Quadrupole Time of Flight (Q-TOF) LC/MS, C-18 300Ǻ Large Capacity Chip (Agilent Technologies, Santa Clara, CA, USA) and Agilent MassHunter data acquisition software were used to identify the differentially expressed proteins (Agilent Technologies, Santa Clara, CA, USA). In addition, PEAKS^®Studio software (Version 8.0, Bioinformatics Solution, Waterloo, ON, Canada) and UniProtKB (Organism: Danio rerio) database were used for the analysis of mass spectrometry-based label-free proteomic quantification (LFQ). Cytoscape software (Version 3.7.2 plugin BiNGO for Gene Ontology (GO) annotated information, Cytoscape Consortium, San Diego, CA, USA), Zebrafish Information Network (ZFIN) Database Information, KAAS (KEGG Automatic Annotation Server Version 2.1, Kanehisa Lab., Kyoto, Japan) and KEGG PATHWAY Database (Organism: Danio rerio) were used to study the functional annotations, protein-protein interactions, and systemic pathway enrichment analysis. 2.3. Zebrafish Maintenance and Housing Conditions Adult zebrafish (Danio rerio; 3–4 months old) of heterogeneous strain wild-type stock (standard short-fin phenotype) were housed in the Animal Facility of Monash University Malaysia and maintained under standard husbandry conditions as follows: standard zebrafish tanks (length of 36 cm × width of 22 cm × height of 26 cm) equipped with circulating water systems to provide constant aeration, controlled water temperature between 26–28 °C and controlled water pH between 6.8–7.1. They were kept in stress-free and hygienic conditions. The zebrafish aquarium was maintained under a 250-lux light intensity with a cycle of 14-h of light to 10-h of darkness controlled by autotimer (light on at 0800 and light off at 2200). Group housing was practiced (10–12 fish per tank) with the females and males separated. The adult zebrafish were fed ad libitum three times a day (TetraMin^® Tropical Flakes) and were supplemented with live brine shrimps (Artemia) purchased from Bio-Marine (Aquafauna Inc., Hawthorne, CA, USA). The adult zebrafish were allowed to acclimatize for a period of seven days to reduce stress before commencing the experiments. The Monash University Malaysia Animal Ethics Committee approved all the animal experimental procedures on 17 January 2019. 2.4. Experimental Design 2.4.1. OSLP Safety Study in Adult Zebrafish A limit test was first performed based on a modified version of the OECD Guidelines for the Testing of Chemicals No. 203 [[48]11,[49]12] and the protocols of Choo et al. [[50]10,[51]13]. Prior to the experimental procedures, all the adult zebrafish were fasted for 24 h. Meanwhile, OSLP powder was completely dissolved in tank water (26–28 °C) and concentrations ranging from 50–1600 µg/kg of zebrafish body weight were freshly prepared. Three-month-old adult zebrafish with an average weight of 0.45–0.50 g were selected. The zebrafish were then divided into 7 groups ([52]Table 1), with 8 fish per group (n = 8) as follows: Table 1. Experimental groups in OSLP safety study. Group Treatment VC Vehicle control (tank water, i.p.) Treatment Group a OSLP (50 µg/kg, i.p.) Treatment Group b OSLP (100 µg/kg, i.p.) Treatment Group c OSLP (200 µg/kg, i.p.) Treatment Group d OSLP (400 µg/kg, i.p.) Treatment Group e OSLP (800 µg/kg, i.p.) Treatment Group f OSLP (1600 µg/kg, i.p.) [53]Open in a new tab A clean observation tank was first set up and filled with 13 L of tank water (Milli-Q filtered water used for keeping the zebrafish; 26–28 °C). One zebrafish from the vehicle control (VC) group was then placed in the observation tank and its behavior was recorded for 10 min using a digital camera (Sony, Japan). After finishing recording, the zebrafish was transferred into a clean individual 1 L tank filled with the same water. This procedure was then repeated for all the other zebrafish in the VC group. For the OSLP-treated groups (II–VII), different concentrations of OSLP were injected intraperitoneally (i.p.) into the zebrafish. Before each IP injection, a zebrafish was individually immersed in anesthesia solution (30 mg/L of Benzocaine) until the cessation of movement [[54]10,[55]13,[56]14]. Immediately, the zebrafish was extracted out to determine the body weight and to calculate the injection volume. The injection volume was calculated at a volume corresponding to 10 microliters per gram of body weight (modified from 15). After injection, the zebrafish was immediately transferred back to the 13 L observation tank. Then, the same recording and tank transfer procedure was repeated, as performed in the VC group. All 56 zebrafish were then kept for 96 h in their respective 1 L tanks. They were checked on every 15 min for the first two hours of exposure and every half an hour thereafter for the first day. On subsequent days, the zebrafish were checked on the morning, afternoon, and evening (3 times per day). Any zebrafish found to exhibit signs of pain, suffering, or anomaly according to our predefined monitoring sheet at any checkpoint were humanely euthanized via an overdose of benzocaine. This protocol deviates from the OECD guidelines in that it does not use mortality as the criterion to determine toxic effects due to the concerns of the MARP-Australia in using death as an endpoint. 2.4.2. Anticonvulsive Potential of OSLP in Adult Zebrafish The anticonvulsive potential of OSLP was investigated in the pentylenetetrazol (PTZ)-induced seizure model. Seizure score and seizure onset time, were one of the primary evaluation parameters used to examine the anticonvulsive activity. Behavioral changes in the zebrafish were determined by evaluating their swimming patterns, total distance travelled (cm) and time spent in the tank (upper-half versus lower-half, s). Three-month-old adult zebrafish with an average weight of 0.45–0.50 g were selected. Prior to beginning the experiments, the zebrafish were kept in 1 L treatment tanks filled with 1 L of tank water (26–28 °C) normally used to fill the zebrafish tanks. In this study, the zebrafish were divided into 5 groups (n = 10) ([57]Table 2) and procedures of experiment ([58]Figure 1) were as follows: Table 2. Experimental groups in the evaluation of OSLP anti-convulsive potential. Group Treatment VC Vehicle control (tank water, i.p. + tank water) NC Negative control (tank water + PTZ 170 mg/kg, i.p.) PC Positive control (DZP 1.25 mg/kg + PTZ 170 mg/kg, i.p.) TC Treatment control (800 µg/kg + tank water, i.p.) O+P OSLP-treated PTZ (800 µg/kg + PTZ 170 mg/kg, i.p.) [59]Open in a new tab Figure 1. [60]Figure 1 [61]Open in a new tab Shows the procedures of experiment. All the groups were habituated in their treatment tanks for a half hour before the administration of PTZ. Before each i.p. injection, a zebrafish was individually immersed in anesthesia solution (30 mg/L of Benzocaine) until the cessation of movement. When multiple IP injections were required in tandem on the same zebrafish, the injections were given at alternating lateral ends, rather than the midline between the pelvic fins 10, 13, 14. The VC group was injected with tank water twice. The NC group was first pre-treated with tank water and then PTZ (170 mg/kg) whereas the PC group was pre-treated with diazepam (1.25 mg/kg) followed by PTZ (170 mg/kg). The TC group was injected with 800 μg/kg of OSLP and tank water. The O+P group was pre-treated with OSLP (800 μg/kg) followed by PTZ (170 mg/kg). PTZ-induced seizures lasted for approximately 10 min after the PTZ injection [[62]10,[63]13,[64]14]. All the groups were then transferred to a 13 L observation tank filled three quarters of the way with water. Behavioral changes of the zebrafish were then recorded individually (10 min) with a digital camera (Sony, Japan). The PTZ injected zebrafish presented diverse seizure profiles, intensities and latency in reaching the different seizure scores and seizure onset times. In order to determine the seizure score and seizure onset time, the individual video was analyzed using a computer as per the scoring system below ([65]Table 3) [[66]10,[67]13,[68]14,[69]15,[70]16]: Table 3. Seizure scoring system. Score Criteria 1 short swim mainly at the bottom of tank 2 increased swimming activity and high frequency of opercular movements 3 burst swimming, left and right movements as well as the erratic movements 4 circular movements [71]Open in a new tab At the end of the experiment, all the groups were sacrificed. The zebrafish were euthanized with 30 mg/L of Benzocaine until the cessation of movement. The brains were then carefully harvested for neurotransmitter analysis, protein expression study and systemic pathway enrichment analysis. 2.5. Extraction of Brains from Zebrafish At the end of the behavioral studies, the zebrafish brains were carefully harvested from the zebrafish skulls and kept in a sterile Petri dish. Each brain was then immediately transferred into a sterile, pre-chilled 2.0 mL microtube and was flash-frozen in liquid nitrogen (LN[2]) before storing them at −152 °C until further analysis. 2.6. Brain Neurotransmitter Analysis Using Nanoflow Liquid Chromatography Coupled with Tandem Mass Spectrometry (Nanoflow-ESI-LC-MS/MS) The levels of neurotransmitters in the brains, namely gamma-aminobutyric acid (GABA) and glutamate (Glu) were estimated using LC-MS/MS with modifications [[72]13,[73]14,[74]17]. All experiments were performed in 3 independent biological replicates. A mother stock of neurotransmitter standards was prepared by mixing GABA and Glu in methanol, MQUP water and 0.1% formic acid, to make up a final concentration of 1 mg/mL. Next, serial dilution was performed to prepare 8 points of standard calibrations ranging from 6.25–1000 ng/mL. A blank (methanol, MQUP water in 0.1% formic acid) with a final concentration of 1 mg/mL was also prepared. Together with the 8 points of standard calibrations, they were used for quantifying the levels of GABA and Glu in LC-MS/MS study. Firstly, each LN[2] flash-frozen zebrafish brain was homogenized in 1 mL ice-cold methanol/MQUP water (3:1, vol/vol) using an ultrasonic cell crusher (JY88-II N, Shanghai Xiwen Biotech. Co., Ltd., Shanghai, China). The homogenate was then vortex-mixed (2500 rpm, 3 m) and later incubated on an agitating shaker (4 °C, 1 h). The homogenate was then centrifuged (4 °C, 10,000× g, 10 min) and the supernatant was carefully transferred into a sterile 2.0 mL microtube. 100 µL of 0.1% formic acid was slowly added, vortex-mixed (2500 rpm, 3 m) and then centrifuged (4 °C, 10,000× g, 10 min). The supernatant was carefully transferred into a sterile insert and vial. Finally, all the brain samples were subjected to LC-MS/MS analysis. LC-MS/MS was run on an Agilent 1290 Infinity UHPLC coupled with an Agilent 6410B Triple Quad MS/MS equipped with an electrospray ionization (ESI). The separations were performed using Zorbax Eclipse Plus C18 (Rapid Resolution HD, 2.1 × 150.0 mm with 1.8 uM pore size reverse-phase column). The flow rate was 0.3 mL/min with the mobile phase consisting of 0.1% formic acid in water (Solvent A) and acetonitrile (Solvent B). The gradient elution used was: (i) 0 min, 5% Solvent B; (ii) 0–3 min, 50% Solvent B and (iii) 3–5 min, 100% Solvent B, with one-minute post time. The injection volume was 1.0 µL per sample with the column compartment temperature and the autosampler temperature set at 25 °C and 4 °C respectively. The total run time for each injection was 5 min. ESI-MS/MS was used in positive ionization mode with a nitrogen gas temperature of 325 °C, gas flow 9 L/min, nebulizer pressure of 45 psi and the capillary voltage of 4000 V. The MS acquisition was scanned in multiple reaction monitoring (MRM) mode. A calibration range of 1.56–200 ng/mL was used for quantifying the targeted neurotransmitters, with a linear plot where r^2 > 0.99. 2.7. Protein Expression Profiling Using Mass Spectrometry-Based Label-Free Proteomic Quantification (LFQ) Brains of these two groups, namely NC (injected with PTZ 170 mg/kg) and O+P (pre-treated with OSLP 800 µg/kg followed by PTZ 170 mg/kg) were subjected to tissue lysis to extract the proteins for mass spectrometry-based label-free proteomic quantification (LFQ). All experiments were performed in 4 independent biological replicates. 2.7.1. Protein Extraction from Zebrafish Brain The zebrafish brain was lysed with 1 mL of ice-cold lysis buffer (RIPA, protease inhibitor 20% v/v, phosphatase inhibitor 1% v/v) in a sterile ProtLoBind microtube and then incubated on an orbital shaker (4 °C; 90 min). Next, the content was homogenized using an ultrasonic cell crusher, briefly centrifuged (18,000 × g, 4 °C; 10 min) and the supernatant produced was harvested. The supernatant extracted was collected into a new sterile ProtLoBind microtube. Protein concentration was estimated using the Quick Start™ Bradford Protein Assay as instructed by the manufacturer (Bio-Rad, Hercules, CA, USA). After that, the brain lysates were concentrated in a speed-vacuum concentrator (300 rpm; 24 h; 60 °C). 2.7.2. In-Solution Digestion of Proteins In-solution protein digestion was carried out according to the instructions (Agilent Technologies, Santa Clara, CA, USA). Briefly, protein samples were re-suspended, denatured and reduced in 25 μL of ABC, 25 μL of TFE and 1 μL of DTT, followed by being vortex-mixed (2500 rpm, 3 m) and then heated in an oven (60 °C, 60 min). Next, the samples were alkylated in 4 μL of IAA and were incubated in the dark (60 min, r.t.). After that, 1 μL of DTT was again added to quench excessive IAA (60 min, r.t., in the dark). 300 μL of MQUP water and 100 μL of ABC were added to dilute and adjust the pH of the protein solutions (pH 7–9). Following that, 1 μL of trypsin was added and was then incubated in an oven (37 °C, 18 h, in the dark). Upon completion of incubation, 1 μL of formic acid was added to terminate the tryptic digestion. Finally, all the samples were concentrated in a speed-vacuum concentrator (300 rpm; 24 h; 60 °C, Eyela SpeedVac Vacuum Concentrator). The dry pellets were kept at −20 °C. 2.7.3. De-Salting of Proteins De-salting of the protein sample was carried out. Each biological replicate was de-salted independently using a Pierce^®C18 mini spin column as instructed (Thermo Scientific Pierce, Rockford, IL, USA), with modifications. Firstly, each mini spin column was activated in 50% ACN (repeated 3 times, r.t.) and equilibrated in 0.5% of TFA in 5% ACN (repeated 3 times, r.t.). Separately, 90 μL of crude protein was added into 30 μL of sample buffer (2% of TFA in 20%) and briefly vortexed at 2200 rpm to mix well. This step was repeated for all the protein samples. Following that, each of the protein samples was loaded onto a mini spin column and was de-salted (repeated 3 times, r.t.). Subsequently, all the protein samples were washed in 0.5% of TFA in 5% ACN (repeated 3 times, r.t.). Lastly, all the protein samples were eluted in 70% ACN (repeated 3 times, r.t.) and all the flow-through produced was collected, vacuum-concentrated (300 rpm; 24 h; 60 °C) and stored at −20 °C prior to mass spectrometry-based LFQ. 2.7.4. Mass Spectrometry-Based Label-Free Proteomic Quantification (LFQ) Using Nanoflow-ESI-LCMS/MS De-salted peptides were loaded onto an Agilent C-18 300Ǻ Large Capacity Chip. The column was equilibrated by 0.1% formic acid in water (Solution A) and peptides were eluted with an increasing gradient of 90% acetonitrile in 0.1% formic acid (Solution B) by the following gradient, 3%–50% Solution B from 0–30 min, 50%–95% Solution B from 30–32 min, 95% Solution B from 32–39 min and 95%–3% Solution B from 39–47 min. The polarity of Q-TOF was set at positive, capillary voltage at 2050 V, fragmentor voltage at 300 V, drying gas flow 5 L/min and gas temperature of 300 °C. The intact protein was analyzed in auto MS/MS mode from range 110–3000 m/z for MS scan and 50–3000 m/z range for MS/MS scan. The spectrum was analyzed using Agilent MassHunter data acquisition software. 2.7.5. Brain Protein and Peptide Identification by Automated de Novo Sequencing and LFQ Analysis Protein identification by automated de novo sequencing was performed with PEAKS^®Studio Version 8.0. UniProtKB (Organism: Danio rerio) database ([75]http://www.uniprot.org/proteomes/UP000000437, 46,847 proteins, accessed on 14 February 2020) was used for protein identification and homology search by comparing the de novo sequence tag, with the following settings: both parent mass and precursor mass tolerance was set at 0.1 Da, carbamidomethylation was set as fixed modification with maximum missed cleavage was set at 3, maximum variable post-translational modification was set at 3, trypsin cleavage, the minimum ratio count set to 2, mass error tolerance set as 20.0 ppm and other parameters were set as default by Agilent. False discovery rate (FDR) threshold of 1% and protein score of −10lgP > 20 were applied to filter out inaccurate proteins. PEAKS^® indicated that a −10lgP score of greater than 20 is of relatively high in confidence as it targets very few decoy matches above the threshold. For LFQ analysis, the differentially expressed proteins between the NC (injected with PTZ 170 mg/kg) and O+P (pre-treated with OSLP 800 μg/kg followed by PTZ 170 mg/kg) groups were identified with the following settings: FDR threshold ≤ 1%, fold change ≥ 1, unique peptide ≥ 1, and significance score ≥ 20. PEAKSQ indicated that a significance score of greater than 20 is equivalent to significance p value < 0.01. Other parameters were set as default by Agilent. 2.8. Bioinformatics Analysis Bioinformatics analysis (functional annotations, protein-protein interactions and systemic pathway enrichment analysis) of the differentially expressed proteins were analyzed and matched with the databases obtained from GO Consortium, ZFIN ([76]www.zfin.org) and the KEGG PATHWAY Database (Danio rerio) [[77]13]. KAAS provides functional annotation of genes by BLAST or GHOST comparisons against the manually curated KEGG GENES database. The result contains KO (KEGG Orthology) assignments (bi-directional best hit) and automatically generated KEGG pathways. The KEGG pathway maps organism-specific pathways: green boxes are hyperlinked to GENES entries by converting K numbers (KO identifiers) to gene identifiers in the reference pathway, indicating the presence of genes in the genome and also the completeness of the pathway. 2.9. Statistical Analysis For behavioral study and neurotransmitter estimation, statistical analysis was performed using GraphPad Prism version 8.0. All data were expressed as mean ± standard error of the mean (SEM). One-way analysis of variance (ANOVA) followed with Dunnett’s post-hoc test at significance levels of * p < 0.05, ** p < 0.01 and *** p < 0.001 against the negative control group (NC, 170 mg/kg PTZ). PEAKSQ statistical analysis (built-in statistical tool of PEAKS^® software) was used in the analysis of differentially expressed proteins identified by LFQ. A significance score of 20% (equivalent to significance level of 0.01) and FDR ≤ 1% was considered statistically significant. In bioinformatics analysis, hypergeometric test followed with Benjamini and Hochberg FDR correction at p value < 0.05 (BiNGO built-in statistical tool) was used to correlate the association between functional annotation of genes and interacting proteins; the built-in statistical tool of KAAS was used to assess the possible association of interacting proteins and systemic pathways in the KEGG PATHWAY Database. 3. Results 3.1. OSLP Safety Study in Adult Zebrafish 3.1.1. Behavioral Study Swim Path Analysis As seen in the swim paths generated by PANLAB SMART v3.0 software, the VC group ([78]Figure 2a) swam throughout the whole tank without showing apparent preference for any part of the tank. The OSLP-treated groups, 50, 100, 200, and 400 µg respectively, showed slight preferences for