Inhibition of Seizure-Like Paroxysms and Toxicity Effects of Securidaca longepedunculata Extracts and Constituents in Zebrafish Danio rerio

Plants used in traditional medicine in the management of epilepsy could potentially yield novel drug compounds with antiepileptic properties. The medicinal plant Securidaca longepedunculata is widely used in traditional medicine in the African continent, and epilepsy is among several indications. Limited knowledge is available on its toxicity and medicinal effects, such as anticonvulsant activities. This study explores the potential in vivo inhibition of seizure-like paroxysms and toxicity effects of dichloromethane (DCM) and ethanol (EtOH) extracts, as well as isolated xanthones and benzoates of S. longepedunculata. Ten phenolic compounds were isolated from the DCM extract. All of the substances were identified by nuclear magnetic resonance spectroscopy. Assays for toxicity and inhibition of pentylenetetrazole (PTZ)-induced seizure-like paroxysms were performed in zebrafish larvae. Among the compounds assessed in the assay for maximum tolerated concentration (MTC), benzyl-2-hydroxy-6-methoxy-benzoate (MTC 12.5 μM), 4,8-dihydroxy-1,2,3,5,6-pentamethoxyxanthone (MTC 25 μM), and 1,7-dihydroxy-4-methoxyxanthone (MTC 6.25 μM) were the most toxic. The DCM extract, 1,7-dihydroxy-4-methoxyxanthone and 2-hydroxy-1,7-dimethoxyxanthone displayed the most significant inhibition of paroxysms by altering the locomotor behavior in GABAA receptor antagonist, PTZ, which induced seizures in larval zebrafish. The EtOH extract, benzyl benzoate, and benzyl-2-hydroxy-6-methoxy-benzoate unexpectedly increased locomotor activity in treated larval zebrafish and decreased locomotor activity in nontreated larval zebrafish, seemingly due to paradoxical excitation. The results reveal promising medicinal activities of this plant, contributing to our understanding of its use as an antiepileptic drug. It also shows us the presence of potentially new lead compounds for future drug development.


INTRODUCTION
Epilepsy is a neurological brain disease that involves predisposition to generate spontaneous recurring seizures, affecting more than 70 million people worldwide. 1Today's pharmacotherapy does not manage to treat approximately onethird of epilepsy patients, despite the availability of numerous antiseizure medications (ASMs) in the western medical market. 2Furthermore, modern ASMs often have side effects. 3,4or this reason, the development of new and better ASMs is a subject of intensive research. 5The use of herbal medicines for the treatment of epilepsy is widespread throughout the world. 6n traditional African medicine, numerous plants are used in the treatment of epilepsy.The violet tree, Securidaca longepedunculata Fresen (Polygalaceae) 7 (sometimes spelled S. longipedunculata), a small tree with pale gray stem bark and sweetly scented pink to purple flowers blooming in bouquets on a peduncle, is an important medicinal plant and known as the "mother of all medicines". 8This tree grows mainly in seasonally dry tropical areas and has been used extensively in African traditional medicine in the treatment of a range of diseases and conditions such as malaria, rheumatism, tuberculosis, and constipation. 9,10Interestingly, S. longepedunculata has been reported to be used against epilepsy in Nigeria, 11,12 Ethiopia, 13 Cameroon, 14 Zimbabwe, 15 and Burkina Faso. 16A crude aqueous extract of the roots was reported to have anticonvulsant activity. 14,17The constituents of the plant responsible for the putative antiepileptic effects appear, however, to be unknown.
A wide range of constituents is reported in S. longepedunculata, including saponins, phenolic acids, benzophenones, benzoates, flavonoids, and sterols (reviewed by Mongalo et al. 9 ).Of note, there is a large number of xanthones, with more than 40 reported so far.To date, no research reports are available on the effects of the xanthones found in S. longepedunculata on the central nervous system (CNS), although other biological effects of these compounds have been reported, e.g., cytotoxic activity against human cancer cells, 18−20 activity against erectile dysfunction through relaxation of rabbit corpus cavernosum smooth muscle, 21,22 and antimicrobial 23 and antiarthritic activities. 24However, other xanthones have been reported as anticonvulsants and antiepileptics. 25,26A nonxanthone constituent, benzyl benzoate, is an acaricide and is used clinically against scabies. 27he knowledge of the toxicological profile of S. longepedunculata is limited.In ritual suicide, a decoction from the stem bark is taken orally in South Africa. 9It is a widespread suicide poison used by women in Zambia, Angola and the Democratic Republic of the Congo, where peeled root or root pulp is introduced into the vagina.As a hunting poison, the allegedly high toxicity of bark and roots comes into play as an arrow and fishing poison used in Nigeria, Zambia, Zimbabwe, the Democratic Republic of the Congo, Senegal, and Angola.S. longepedunculata is also known as murder and trial-by-order poison used in Nigeria, Cameroon, Angola and Central African Republic. 28To our knowledge, no investigations on the toxicity profile of Securidaca spp. in zebrafish have been published.Among the constituents, benzyl benzoate 29 and benzyl 2-hydroxy-6-methoxybenzoate 30 are reported to be toxic to brine shrimps.Additional research is necessary to balance the discrepancy between knowledge on traditional use and the potential bioactivities of compounds of S. longepedunculata.
The present study aims at phytochemically screening extracts through isolation and structural identification of major xanthones and related substances and exploring the toxicological activities and inhibition of antiseizure-like paroxysms of both extracts and compounds of S. longepedunculata bark using locomotor behavior as a readout for convulsions in a zebrafish larvae-based pentylenetetrazole (PTZ) assay for the discovery of new bioactive agents in the treatment of epileptic seizures with a good safety profile.

RESULTS AND DISCUSSION
The present study investigates the toxicity profile and the inhibitory effects on seizure-like paroxysms induced by acute PTZ treatment of extracts and isolated compounds from the African medicinal plant S. longepedunculata, a plant well-known for its use against CNS disesases. 9,10Despite its popularity, how this plant can affect targets in the brain has not been studied.By combining phytochemistry research with pharmacological assays using the locomotor behavior as a readout for seizure-like paroxysms in a zebrafish larvae-based PTZ assay, we were able to explore the possible neurological effects of the main constituents of the lipophilic extract, the benzoates and the xanthones.Additionally, the study demonstrates zebrafish toxicity phenotypes after exposure to the test compounds.By using the zebrafish model for epilepsy and toxicity evaluation, we have obtained new knowledge about the active chemical components of S. longepedunculata which contribute to the understanding of its ethnopharmacological use against epilepsy and the identification of potentially new lead compounds for drug discovery.

Toxicological Evaluation in Larval Zebrafish.
In this in vivo assay, the maximum tolerated concentration (MTC) of the dichloromethane (DCM) and ethanol (EtOH) extracts and compounds 1, 2, 4, 7 and 8 from S. longepedunculata was evaluated and determined by assessing their toxicological effects on wild-type (AB) zebrafish larvae (see results in Table 1).Representative and observable toxicity malformations of treated zebrafish larvae were photographically documented using a microscope camera, as highlighted images shown in Figure 2. Overview of the toxicological traits of each sample at the different time points (3, 24, and 48 h) is also displayed in Table 1.
The knowledge on the toxicological profile of compounds isolated from S. longepedunculata is limited, while there exist in vivo toxicity studies on the aqueous, ethanolic, and methanolic root extract showing various toxic effects of all extracts. 9The EtOH and DCM extracts were highly toxic against the zebrafish larvae with MTC values of 15.7 and 11.8 μg/mL, respectively, compared to the pure compounds tested in this study.The EtOH extract at 31.3 μg/mL caused impaired motility and muscle necrosis of the zebrafish larvae at all time points, in addition to loss of posture after 48 h.An additional toxic abnormality, pericardial edema, occurred after 3 h of treatment when larvae were exposed to a concentration of 47 μg/mL of the EtOH extract, and after 24 h, all larvae were dead.The zebrafish larvae exposed to the DCM extract with a concentration of 15.7 μg/mL showed a consistent toxicological profile at all three time points observed as hypoactivity, impaired movement, and loss of posture, except for cases of death after 24 and 48 h.This is the first time the toxic effects of the DCM extract of S. longepedunculata have been assessed in a larval zebrafish assay.
Compound 1 was well tolerated by the zebrafish larvae with an MTC value of 75 μM.At higher concentrations tested (≥100 μM), the most common abnormalities observed for the treated zebrafish larvae were loss of posture, reduced touch response, and reduced swim bladder or death after 24 and/or 48 h after exposure to compound 1.Previously, one toxicity study on zebrafish larvae (AB strain) has been performed on compound 1 showing toxic effects at higher concentrations, in accordance with our results. 43−46 The MTC value of compound 2 was determined to be 12.5 μM.Compound 2 was not well tolerated by treated larvae, displaying a trend of loss of posture, reduced touch response, and hypoactivity, across all three time points above a concentration of ≥18.75 μM.Pericardial edema, reduced swim bladder, and slow heart rate of the zebrafish larvae were observed at concentrations above 25 μM, whereas muscle necrosis was developing after 24 h of treatment with 50 μM of compound 2. Compound 2 has been reported to have cytotoxic effects in vitro. 30,47In an in vivo assay, compound 2 has also been reported as toxic to brine shrimp larvae. 30,47One in vitro study on benzyl 2-hydroxy-6-methoxybenzoate (compound 2) and methyl 2-hydroxy-6-methoxybenzoate has revealed inactivity against various bacterial and fungal species. 19Zebrafish larvae treated with compound 4 revealed a diverse array of abnormal phenotypes with an MTC value of 25 μM.Treatment of the larvae with a concentration of 37.5 μM resulted in loss of posture, hyperpigmentation, reduced touch response, and reduced swim bladder when examined after 24 and 48 h.At higher concentrations (50 μM), the zebrafish larvae were strongly affected by compound 4, being subject to additional pericardial edema and slow heart rate, showing darkened liver and brain tissues, suggestive of cell death, until 100% death occurred after 48 h, see Figure 2. Interestingly, larvae treated with compound 4 (50 μM) were first hypoactive within the first 3 h and then hyperactive when examined after 24 h, before occurrence of death after 48 h.Compound 4 has been reported only once as a natural product.It was without cytotoxicity on human pancreatic cancer cells. 18Compound 7 was found to be the most toxic compound tested in this study, with an MTC value of 6.25 μM.Curved spine, abnormal urogenital opening, pericardial edema, muscle necrosis, and reduced swim bladder were found as distinct aberrant phenotypes in larvae exposed to 9.4 μM of compound 7, see Figure 2. Compound 7 has been reported to have cytotoxic effects in vitro. 20,36,48As one of the least toxic compounds in this study, compound 8 demonstrated few visible phenotypical changes in larvae exposed to concentrations above the MTC-value (75 μM).Slow heart rate and reduced touch response appeared to be the most common toxic effects observed in larvae when exposed to higher concentrations, 100 and 200 μM.Compound 8 has previously shown moderate cytotoxic activity and no antiviral activity toward influenza virus. 49In another study, compound 8 did not exhibit potent cytotoxic activity against the growth of different human tumor cell lines. 20tudies on benzoates and xanthones isolated from S. longepedunculata are sparse.2,3-Dimethoxy-4-hydroxy-benzophenone (compound 3) has showed low cytotoxicity against pancreatic cancer PANC-1 cells, 18 and in another study, 3 exhibited antiplasmodial activity. 50To date, no in vivo studies have been performed on 1,6-dihydroxy-2,7,8-trimethoxyxanthone (compound 5), 2,7-dihydroxy-1,8-dimethoxyxanthone (compound 9), and 3,7-dihydroxy-1,2,8-trimethoxyxanthone (compound 10).1,6,8-Trihydroxy-2,3,4,5-pentamethoxyxanthone (compound 6) has displayed cytotoxic activity against human pancreatic cancer PANC-1 cells under nutrientdeprived conditions. 18Furthermore, other xanthones previously isolated from S. longepedunculata, 1,6-dihydroxy-2,3,4,5,8-pentamethoxyxanthone, 18 and muchimangin B have induced apoptotic-like cell death of human pancreatic cancer PANC-1 cell line. 19,51A xanthone extract from Garcinia mangostana has shown toxicity to zebrafish embryos at concentrations of 62.5 μg/mL and higher. 52The substances in our experiments were, however, not tested.−57 It was not possible to determine the structure−activity relationship (SAR) of either the benzoates or xanthones tested in this toxicological assay.More data are needed to enable the determination of the chemical group accountable for the toxic effects in larval zebrafish.
To our knowledge, this is the first report on the MTCs of compounds 1, 2, 4, 7, and 8 and extracts isolated from S. longepedunculata on zebrafish larvae.

Inhibition of PTZ-Induced
Seizure-Like Paroxysms in Larval Zebrafish.Anticonvulsant, anxiolytic, and sedative effects of an aqueous root extract of S. longepedunculata on mice have been reported. 9One in vitro study has revealed the ability of xanthones, such as garcinone, γmangosteen, and gartanin, isolated from G. mangostana, to penetrate the blood brain barrier (BBB).In addition, these three xanthones are suggested as anti-Alzheimer agents since they cause antiamyloid plaque formation through inhibitory effects against Aβ42 in Escherichia coli cells, toward Aβ42 selfinduced aggregation in a tube and against BACE1 (β-site amyloid precursor protein-cleaving enzyme 1). 58One newly patented compound, 6-hydroxy-1,2,3,7-tetramethoxyxanthone, has been reported to improve depression by increasing the number of hippocampal neural stem cells. 591,7-Dimethoxy-2hydroxy-xanthone and 1,3,6,8-tetrahydroxy-2,5-dimethoxyxanthone have exhibited activity against erectile dysfunction through relaxation of rabbit corpus carvenosal smooth muscle, 21,22 and some other xanthones have showed moderate antimicrobial activity. 23xtracts and compounds 1, 2, 4, 7, and 8 isolated from S. longepedunculata were tested for their ability to reduce PTZinduced convulsive-like movements in zebrafish larvae at 5 dpf.The MTC of compounds and extracts of S. longepedunculata was determined before the evaluation of anticonvulsant activity through the inhibition of seizure-like paroxysms induced by acute PTZ treatment in larval zebrafish.This behavioral analysis was based on the tracking of PTZ-induced convulsionlike activities of chronically pretreated and nontreated zebrafish larvae detected by video recordings. 60,61The larval locomotor behavior was tracked and divided into four categories: inactivity (<4 mm/s), small movements (4−20 mm/s), large movements (>20 mm/s), and total movements (see Supporting Information with examples of the different movements).The rationale for measuring the basic motor patterns, large and small movements, was to make quantitative descriptions of larval behavior contributing to a better understanding of possible antiseizure effects of S. longepedunculata.Large movements have been associated with tonicclonic like seizures observed as bursts of swimming.Small movements have been described as myoclonic-like behavior characterized as increased orofacial movements and fin fluttering. 62The total movements was defined as equal to the average of the sum of both small and large movements expressed in millimeters per second.As shown in Figure 3, all types of movements are shown in mm within a period of 30 min as averages (summary of 5 min time bins).
As shown in Figure 3a, the DCM extract was the most active in terms of significantly reducing all types of PTZ-induced locomotor activity, both large and small movements, in zebrafish larvae after 20−22 h of incubation at determined MTC (11.8 μg/mL).The major reduction of all types of movements with regard to the DCM extract might reveal synergistic effects and/or the existence of other potent compounds in the extract not tested in this assay.Pretreatment with compounds 7 and 8 isolated from the DCM extract exhibited strong reduction (30−35%) in PTZ-induced large movements of larval zebrafish, tested at their respective MTCvalues at 6.25 and 75 μM.Regarding the molecular structure of the compounds, fewer hydroxyl or methoxyl substituents on the xanthone backbone might appear to be favorable for anticonvulsant activity in treated larvae.Substance 4 seemed to be less active in this respect, although a significant difference was not observed.Substances 7 and 8 have a lower degree of substitution than compound 4.
As shown in Figure 3b, displaying treatment without PTZ, both extracts and compounds of S. longepedunculata were able to reduce both large and small movements of larval zebrafish after 20−22 h of incubation relative to the DMSO control, showing the activity of S. longepedunculata itself.Treatment with compound 1 resulted in hypoactivity, with the highest drop of both large (65% reduction) and small (45% reduction) movements.In contrast, the results for compound 1 corresponded inversely with PTZ-treated zebrafish larvae regarding small movements, indicating the inability to reduce small movements of PTZ-induced larval locomotor behavior, see Figure 3a.
The EtOH extract contributed significantly to increased larval activity of both large and small locomotor activities in the PTZ-treated larvae compared to the PTZ-control (Figure 3a).The increased movements in PTZ-treated larval zebrafish related to compounds 1 and 2 (small movements) and the EtOH extract (large and small movements) might be triggering the γ-amino butyric acid (GABA) paradox, leading to hyperexcitation.To determine this in the future, additional tests such as acoustic startle response with extra control would need to be performed.One comprehensive study has shown that some CNS depressants, such as benzodiazepines, which are related to sedation, also cause paradoxical excitation in zebrafish. 63Paradoxical excitation is defined as decreased neuronal activity but with paradoxically increased activity in the caudal hindbrain, causing escalated motor activity.Substances that cause paradoxical excitation are agonists or positive allosteric modulators (PAMs) of GABA receptors.It must be noted, however, that the behavioral assays performed in the study by McCarroll 63 used the acoustic startle response as the primary readout and is therefore different from seizurelike paroxysms elicited by PTZ.Thus, additional follow up studies comparing the larval response to PAMS or GABA agonists with the responses to compounds 1, 2 and the EtOH extracts are required in order to confirm activation of the GABA paradox.Increased neuronal activity in the brain can be shown with GCaMP imaging and whole brain imaging of pERK, which are potentially useful studies for broadening the understanding of seizuregenic phenotypes beyond simple locomotor assays. 64,65There exists ASMs such as carbamazepine, levetiracetam, and zonisamide with no proven efficacy in pharmacological antiseizure response observed in PTZ-treated zebrafish, meaning that compounds 1 and 2 and the EtOH extract are still interesting as anticonvulsants as they may act via mechanism(s) other than decreasing PTZ-induced seizures in zebrafish.However, the drugs, zonisamide and levetiracetam, are reported with proven efficacy in PTZ-induced seizures in rodents.Also, valproate, diazepam, tiagabine, and ethosuximibe are effective as ASMs in both PTZ-treated zebrafish and rodents. 66The ideal result of a compound acting as an antiepileptic drug in this assay would possibly show a reduction of movements in PTZ-treated fish and no reduction in non-PTZ-treated fish, such as for the DCM extract (small movements).Some reduction in non-PTZ treated fish is also of interest, as this could indicate sedative activity or locomotor impairment.If there is a wide active concentration range for a compound, then ideally, a concentration where there is still an anticonvulsant activity of the compound and no or minimum "sedative" activity is ideal.
PTZ-induced locomotor assays, which include larval zebrafish, are widely used in both pharmacological and genetic screens primarily for the discovery of novel natural compounds and small molecules acting as antiseizure agents. 66−68 After 20−22 h of incubation of the samples of S. longepedunculata, 5 days postfertilization (dpf) zebrafish larvae were exposed to an acute dose of PTZ (20 mM).Shortly after the addition of PTZ in the fish water, behavioral changes ascribed to acute seizures are observed in adult zebrafish. 66Behavioral responses of zebrafish larvae treated acutely with PTZ are elicited and displayed as spasms and hyperactivity along the periphery of the well (stage I), followed by rapid circular swimming often called "whirlpool-like" swimming or corkscrew swimming due to their helical paths of swimming (stage II).At higher PTZ concentrations, seizure-like behavior is observed as sudden jerky movements and brief pauses switching between them, as well as periods of loss of posture and body-stiffening named freezing (stage III). 66,69,70As a proconvulsant drug, PTZ acts as a GABA A receptor blocker. 69Although the mechanism of action of PTZ is not fully defined, one previous study on zebrafish larvae has shown that PTZ causes epileptic seizures resembling clonus-type convulsions in mammals as well as a dose-dependent series of stereotypical behaviors. 70Since the distance traveled by larval zebrafish does not cover all seizure behaviors, secondary bioassays such as brain activity recordings (local field potential recordings in larval zebrafish brains) and studies on mutant zebrafish lines would be highly relevant in future studies.Isolation of further components from both extracts in enough amounts would be relevant for the investigation of new bioactive agents in the treatment of epileptic seizures.Additional behavioral assays, such as thigmotaxis and acoustic startle response, would also be of interest to be performed in future studies.

CONCLUSIONS
In this study, two benzoates (1, 2), one benzophenone (3) and seven xanthones (4−10), were isolated from the DCM extract from S. longepedunculata, an African medicinal plant used against epilepsy.Compounds 2, 4, and 7 displayed the most toxic properties in zebrafish larvae, with MTC values of 12.5, 25, and 9.4 μM, respectively.The highest degree of inhibition of seizure-like paroxysms induced by acute PTZ was found in the DCM extract (small and large movements) and compounds 7 and 8 (large movements).The more highly substituted compound 4 was less active with regard to the reduction of large movements in the zebrafish locomotor activity assay.These findings suggest that the DCM extract as well as compounds 7 and 8 exert promising anticonvulsant-like activity and that they may have a potential to be utilized as a medicinal plant resource for the treatment of epilepsy.The dual effect in the reduction of larval locomotor activity without PTZ treatment and increased locomotor activity in PTZ treated larvae sheds light on the bioactivity of the EtOH extract (small and large movements) and compounds 1 and 2 (small movements) to affect sedation and cause paradoxical excitation in vivo in zebrafish, characterizing them as interesting candidates of GABA A receptor ligands and modulators.

Plant Material.
The bark of S. longepedunculata was collected in Bamako, Mali (coordinates 12°38′21″N 8°0′10″W) in 2017.The plant material was identified by botanists at the Department of Traditional Medicine (DMT), Bamako, Mali.A herbarium specimen (no.2220) is deposited at the DMT.A voucher sample of the bark is kept (no.122) in the Department of Pharmacy, University of Oslo, Norway.The bark was cut into small pieces and air-dried.
4.2.4.2.General Phytochemistry Methods.One-and twodimensional NMR spectra were recorded in CDCl 3 on a Bruker AVIII400 or a Bruker AVII600 instrument (Bruker, Rheinstetten, Germany).Tetramethylsilane (TMS) (Sigma-Aldrich, St. Louis, MO, USA) was used as a reference.Flash chromatography was performed on a Biotage Select Flash instrument equipped with Biotage Sfar silica (Si) gel columns (Biotage, Uppsala, Sweden) or on a VersaFlash system (Supelco, Bellefonte, PA, USA) with Versapak normal phase Si gel columns.Open column chromatography was performed on Sephadex LH20 (Pharmacia, Uppsala, Sweden) or MCI gel CHP20P (Supelco).Fractions were combined as indicated by their UV absorbance or by analytical TLC.
Analytical TLC was carried out on normal phase silica gel 60F 254 , 0.2 mm thick layers (Merck, Darmstadt, Germany).Spots were visualized by irradiation with short-wave (254 nm) and long-wave (366 nm) UV rays (UVGL-58 instrument, Ultra-Violet Products, Upland, CA, USA) and spraying the TLC-plates with a 1% solution of Ce(SO 4 ) 2 in 10% aqueous H 2 SO 4 , followed by heating (105 °C, 5 min).Preparative TLC was done on Si gel 60F 254 plates, 0.5 mm thickness (Merck), with visualization by UV irradiation.Some fractions were purified by centrifugally accelerated TLC (CA-TLC) on a Chromatotron model 7924T (Harrison Research, Palo Alto, CA, USA) on gypsum-containing Si gel (Merck). 71All chemicals and solvents were of the highest quality grade.

Extraction of Plant Material.
The dried bark was pulverized using a blender (RAW Pro X1500) to a fine powder (1 mm).The powdered material (595 g) was mixed with diatomaceous earth (Thermo Scientific, Waltham, MA, USA) (4:1 v/v), loaded in 100 mL steel cartridges, and extracted on an accelerated solvent extraction system (ASE 350; Dionex, Sunnyvale, CA, USA) with dichloromethane (DCM) followed by 80% ethanol (EtOH).The extraction process consisted of a preheating period of 5−7 min and 5 min of static extraction per cycle at 60 °C under a pressure of 1600 PSI.The extraction was performed three times.Solvents (DCM or 80% EtOH) were evaporated on a rotavapor, followed by an oil pump (Edwards, Crawley, UK).A dark brown sticky mass (11 g, 1.79%) was obtained from the DCM extract, and a light brown friable mass (74 g, 12.4%) was obtained from the 80% EtOH extract.
4.5.Animals.Wildtype adult zebrafish (genetic strain AB, Centre for Molecular Medicine Norway (NCMM), Oslo Science Park, Norway) were housed and maintained under controlled environmental conditions at temperatures of 28 °C under 14 h light and 10 h dark cycle (standard aquaculture conditions) as previously described. 72,73Three times a day, adult zebrafish were fed, twice daily with commercial dry feed (Gemma Micro 300, Skretting, Norway) and once daily with live brine shrimp (Artemia).After natural spawning, zebrafish eggs were collected from static tanks and transferred to Petri dishes for further handling and development for the purpose of the experiments.Only fertilized embryos were selected and reared in an incubator at 28 °C.Both embryos and larval zebrafish were raised in artificial fish water (E3 medium) containing 1.5 mmol/L HEPES, 17.4 mmol/L NaCl, 0.21 mmol/L KCl, 0.12 mmol/L MgSO 4 , and 0.18 mmol/L Ca[NO 3 ] 2 at pH 7.6 in the incubator. 74Zebrafish larvae were washed off with embryo medium from the first day of selection of eggs until the experiment day (4 or 5 dpf).
4.6.Toxicological Evaluation in Larval Zebrafish.The MTCs of S. longepedunculata extracts and compounds 1, 2, 4, 7, and 8 (dissolved in DMSO, 0.5% final concentration) were investigated by using zebrafish larvae of 4, 5, and 6 days postfertilization (dpf).The addition of test substance to the larval zebrafish started at 4 dpf, as this is the latest possible time point before the larvae are considered as animals and subject to regulations for animal experimentation.Moreover, the BBB in zebrafish is open from 3 dpf to 10 dpf with no permeability from 11 dpf; 75 thus, the permeability of S. longepedunculata extracts and compounds across the BBB is possible at 4 dpf.At this early stage of the BBB's development, there exist some degree of exclusion of substances from this barrier; these are however mostly identified as larger compounds and peptides. 76ithin the larval stage of 4 dpf, major brain subdivisions have been formed, and differentiated neural subtypes (such as Schwann cells, astrocytes, and oligodendrocytes) have developed. 77The zebrafish larvae were incubated for 3, 24, and 48 h at 28.5 °C at constant light prior to toxicological evaluation.When raised under a static dark− light cycle (14 h light and 10 h dark cycle), the larvae showed the most consistent basal activity levels as previously described. 78Larval fish were arranged in a 24-well plate.Five zebrafish larvae and 5 μL of the test sample were added in 995 μL of the embryo medium to each well.The MTC was estimated to be the highest concentration of a sample in the embryo medium that showed no signs of phenotypical or locomotor abnormalities nor death after 3, 24, or 48 h incubation periods.Each of the larvae was examined for signs of toxicity as well as acute (3 h) and chronic (24 and 48 h) locomotor impairment under a microscope (Leica MDG41, Singapore).The following phenotypes of larvae were scored by binary notations as toxicological characterizations: body deformities (i.e., changes in pigmentation, jaw defects, and body axis), exophthalmos (bulging eyes), hypoactivity (i.e., decreased or no touch response), organ dysfunction (slow heartbeat, deflated swim bladder, abnormal blood flow, pericardial edema, and yolk sac edema), hemorrhaging (i.e., pericardial edema), loss of posture, darkened liver and brain tissues (suggestive of cell death), and death. 79Darkening of mentioned tissues, which are normally clear, were scored with reference to the study of He et al. 2013, showing the darker brown or gray coloration and texture of liver tissues becoming amorphous, suggesting degeneration and/or necrosis. 80Normal visible phenotypes of zebrafish larvae are shown in Figure 2. In order to evaluate the effects of the samples on the larval muscle function and performance, their escape responsiveness was determined. 81Escape behavior registered as "no visible response" was defined as an absent visible covered distance or movement upon light touch stimulus of the zebrafish tail tip with a plastic needle.Spontaneous movements constituting twice the body length of the larva were considered as normal behavior.Shorter movement distances or delayed responses observed were defined as decreased or impaired touch response.The assay was conducted in a minimum of three independent experiments with duplicates for each sample per experiment.A series of various concentrations (1:1 dilutions) per sample were tested on the larvae.At least 30 larvae were used in total per sample to determine the MTC value.Two control groups, embryo medium (negative control) and 0.5% DMSO (vehicle control), and triplicates of these were applied per 24-well plate.Data from the experiments were not recorded if 1 or more larva(s) of the controls were abnormal or dead.4.7.Inhibition of Seizure-Like Paroxysms in a Locomotor Tracking System in Larval Zebrafish.Inhibition of seizure-like paroxysms of S. longepedunculata extracts and compounds was assessed by measuring the level of reduction of larval movements induced by PTZ, as previously described, 79 with minor modifications.Prior to the behavioral analysis, 4 dpf larvae were treated with samples in a total volume of 1000 μL of fish water preincubated for 20−22 h in the dark.Five larvae were added to each well of a 24-well plate in three replicates, meaning that 15 larvae were utilized per concentration of a sample tested.The respective MTC values for each sample used in this assay originated from the toxicological evaluation assay, as previously described.To a 48-well plate was transferred one 5 dpf treated larva to each well filled with 200 μL of fish water.Twelve larvae were used per test sample (extract or compound) per experiment, and eight larvae were used for each control (fish water and 0.5% DMSO).The plate with treated larvae was placed in an experimentally sound-attenuated room.The larvae were allowed to habituate for 15 min in order to acclimate to the environmental conditions of the recording chamber and the experiment room.The chemoconvulsant, PTZ, was added to each well (sample wells and PTZ-control wells) obtaining a final concentration of 20 mM. 79,82,83By bathing the larval zebrafish in a solution of PTZ and fish water, seizures are induced after absorption of the chemoconvulsant through the skin, gills, and gastrointestinal tract from the surrounding medium, eventually reaching and affecting the brain. 79After 5 min of habituation, an automated video tracking system registered the larval activity utilizing Zebrabox hardware and Zebralab software (Viewpoint, Lyon, France) to analyze their behavior for 30 min with 5 min time bins. 79,83The distance traveled by the larva was recorded and quantified in millimeters (mm).All experiments were conducted in the time period between 10:00− 17:00.The Zebrabox is a noise-canceling chamber, providing a highresolution infrared digital video camera that allows automated observation and tracking of larval movements.The Zebralab software is used to analyze the video-tracked larval locomotor activity. 60racking data from each experiment was normalized against the control values (20 mM PTZ or 0.5% DMSO) and set at 1. Results from the tracking assays were assembled from three to six replicates of independent experiments.4.8.Statistical Analysis.After exporting and processing the data from the locomotor movement tracking assay into an Excel format, statistical analyses were performed with GraphPad Prism 9 software.Data from the MTC assay were registered and processed in Excel.Data from each experiment of the locomotor movement tracking assay were normalized against the values from control samples; vehicle (DMSO) + PTZ controls or DMSO controls.The control samples were used as references for each sample tested.Subsequently, the normalized data from all replicate runs were pooled together.Analysis of the electrographic data and the average total movement within 30 min were performed using two-way ANOVA.For comparison of multiple samples, Dunnett's test was applied.

■ ASSOCIATED CONTENT
Public Health, Bamako, Mali, for contributing to the botanical identification.

Figure 1 .
Figure 1.Structures of compounds 1−10 isolated from the DCM extract of S. longepedunculata bark.

Figure 2 .
Figure 2. Examples of toxicological signs from morphological assessments of compounds isolated from S. longepedunculata on zebrafish larvae within the first 3, 24, and 48 h after treatment.(a) Larva exposed to 0.5% DMSO (control) is showing normal urogenital opening (in the dashed box) and normal heart (solid box) at 4 dpf 3 h postexposure, relative to (b) treated larva (compound 7, 9.4 μM) displaying abnormal urogenital opening (dashed box) and abnormal edema of the heart (solid box) at 4 dpf 3 h postexposure.(c) Control larva treated with 0.5% DMSO shows normal body axis (notochord) (arrow), somites [precursors to muscle tissue (solid box)], and swim bladder (solid box) at 5 dpf 24 h postexposure, relative to figure (d) where the treated larva (compound 7, 9.4 μM) displays abnormally curved body axis (notochord) (arrow), darkened tissues, suggestive of cell death (dashed box) and reduced swim bladder (solid box) at 5 dpf 24 h postexposure.(e) Larva as control (0.5% DMSO) shows no signs of darkened brain tissues, suggestive of cell death (dotted arrow), normal liver (dashed box), and normal yolk sac (solid box) at 6 dpf 48 h postexposure, compared to (f) treated larva (compound 4, 43.8 μM) showing darkened brain (dotted arrow), darkened liver (dashed box) indicating cell death, and edema of the yolk sac at 6 dpf 48 h postexposure.All scale bars are presented as 100 μm.

Table 1 .
MTCs and Toxicity Phenotypes of Extracts and Compounds Isolated from S. longepedunculata above MTC after 3, 24, and 48 h in Zebrafish Larvae