Evaluation of Neuroactive Effects of Ethanol Extract of Schisandra

Chinese Journal of

Natural Chinese Journal of Natural Medicines 2018, 16(12): 09160925 Medicines

Evaluation of neuroactive effects of ethanol extract of Schisandra chinensis, Schisandrin, and Schisandrin B and determination of underlying mechanisms by zebrafish behavioral profiling

WANG Jia-Wei1Δ, LIANG Feng-Yin2Δ, OUYANG Xiang-Shuo1, LI Pei-Bo1, PEI Zhong2*, SU Wei-Wei 1*

1 Guangdong Engineering and Technology Research Centre for Quality and Efficacy Re-evaluation of Post-marketed TCM, Guangdong Key Laboratory of Plant Resources, School of Life Sciences, Sun Yat-sen University, Guangzhou 510275, China; 2 Department of Neurology, National Key Clinical Department and Key Discipline of Neurology, Guangdong Provincial Key Laboratory for Diagnosis and Treatment of Major Neurological Diseases, The First Affiliated Hospital, Sun Yat-sen University, Guangzhou 510080, China

Available online 20 Dec., 2018

[ABSTRACT] Schisandra chinensis, a traditional Chinese medicine (TCM), has been used to treat sleep disorders. Zebrafish sleep/wake behavioral profiling provides a high-throughput platform to screen chemicals, but has never been used to study extracts and components from TCM. In the present study, the ethanol extract of Schisandra chinensis and its two main lignin components, schisandrin and schisandrin B, were studied in zebrafish. We found that the ethanol extract had bidirectional improvement in rest and activity in zebrafish. Schisandrin and schisandrin B were both sedative and active components. We predicted that schisandrin was related to serotonin pathway and the enthanol extract of Schisandra chinensis was related to seoronin and domapine pathways using a database of zebrafish behaviors. These predictions were confirmed in experiments using Caenorhabditis elegans. In conclusion, zebrafish behavior profiling could be used as a high-throughput platform to screen neuroactive effects and predict molecular pathways of extracts and components from TCM.

[KEY WORDS] Traditional Chinese medicine; Schisandra chinensis; Zebrafish; Behavior profiling; Neurotransmitter [CLC Number] R965 [Document code] A [Article ID] 2095-6975(2018)12-0916-10

Introduction has been employed to treat insomnia for centuries [4]. The popularity of TCM therapy for insomnia is increasing world- Sleep disorder is a major health issue worldwide and can wide, because of its stable curative effect and minimal ad- have a great negative impact on individuals [1-2]. Pharmacol- verse reactions [5]. ogical agents are the most popular treatments for insomnia, Schisandra chinensis is widely used in many TCM pre- because of easy accessibility and time-saving. Western hypnotic scriptions to cure various neurobehavioral disorders, including drugs have many side effects, including addiction or mental sleep disorder [6]. The ethanol extract of Schisandra chinensis disorders [3]. In contrast, traditional Chinese medicine (TCM) (EESC) is commonly used in pharmacological research of this

plant [7-8]. Lignins are active components of Schisandra [Received on] 1-Oct.-2018 [9-10] [Research funding] This work was supported by Guangzhou Clinical chinensis . Schisandrin and schisandrin B are the major Research and Translational Centre for Major Neurological Diseases lignins of Schisandra chinensis, accounting for up to (No. 201604020010) and Natural Science Foundation of Guangdong 28%-50% of EESC [11]. Thus, we used EESC, schisandrin and Province (No. 2015A030310255). schisandrin B to evaluate the effects of Schisandra Chinen- [*Corresponding authors] Tel: 86-020-84112398; E-mail: lssswwhk@ sis.and explore the underlying mechanisms of action. gmail.com (SU Wei-Wei); Tel: 86-020-87755766-8282; E-mail: Considering the complexity of brain activity, using ap- [email protected]. edu.cn (PEI Zhong) Δ propriate in vivo model for screening novel neuroactive drugs, These authors contributed equally to this work. These authors have no conflict of interest to declare. instead of experiments in vitro, is a rational approach. Com- Published by Elsevier B.V. All rights reserved pared with the high-cost but low-throughput screening in

– 916 – WANG Jia-Wei, et al. / Chin J Nat Med, 2018, 16(12): 916925 mice and rats, zebrafish are small, easy to breed, and inex- methyl sulfoxide (DMSO) group, containing 3‰ DMSO. The pensive to maintain and grow rapidly. Zebrafish larvae and second row contained 10 μg·mL−1 of fluoxetine. The other adults have anatomical, behavioral, pharmacological and rows contained a range of final concentrations of drugs (40, [12-13] molecular correlates of mammalian sleep . Particularly, 20, 10, 5, 1, and 0.2 μg·mL−1). EESC and lignins were dis- drugs that modulate sleep/wake in mammals, including mela- solved in DMSO to obtain stock solutions of 100 mg·mL−1. tonin, barbiturates, and benzodiazepines, have similar effects Zebrafish quantization in zebrafish larvae [14-16]. Furthermore, a large-scale zebrafish The 96-well plate was placed in a plastic groove contain- larvae behavioral profiling database has been established [17]. ing homothermal flowing water at 28.5 °C. The water was circu- It includes the behavior characteristics of nearly 4000 small lated between the groove and water-flow machine (ViewPoint, molecules of known structures and functions. However, the Nice, France). The behaviors of zebrafish larvae were ob- behavior profiling system has not been applied to studying served by a video tracking system (VideoTrack, ViewPoint, neuromodulative effects or mechanism of actions of extracts France). The program parameters for detection were set as or small molecules from TCM. follows: detection sensitivity, 15; detection threshold, 40; burst, In the present study, Caenorhabditis elegans (C. elegans) 25; freeze, 4; and bin size, 60s. The light/dark parameters were was used to confirm the results obtained in zebrafish. It is set as follows: light between 09: 00 and 23: 00; light intensity, considered as an ideal model of neurological disability [18], 50%; and light changing duration, 10s. because the neural pathways of nematodes and mammals are Zebrafish behavior data analysis highly conserved, such as dopamine and serotonin pathways [19]. The raw data for each zebrafish larva were time series Its well-characterized behavioral phenotypes, including egg recording the amount of time it moved in each minute during laying and pharyngeal pumping, and diverse strains are ad- 24 h. Six parameters were calculated for each larva as re- vantageous for studying mechanistic pathways [20-22]. ported previously [17]. One minute with less than 0.1 second of Materials and Methods movement was defined as a “rest minute”. A continuous string of rest minutes was defined as a “rest bout”. The amount of Drug preparation moving time in each minute was defined as “activity”. The six Fructus Schisandrae was purchased from Guangzhou parameters were defined as follows: Daxiang herb shop and identified according to the guidelines Rest total: the total amount of rest minutes; of the Chinese Pharmacopoeia (2015) by Prof. LIAO Wen-Bo, Rest bout: the number of rest bouts; and conserved in Sun Yer-Sen herbarium of type specimens Rest bout length: the average length of rest bouts; (187013). The raw material was extracted with 95% aqueous Rest latency: the duration between a light transition and EtOH thrice by reflux. The combined extracts were concentrated the first rest bout; under reduced pressure to dryness. Schisandrin (CAS#61281- Activity total: average amount of detected activity in 38-7), schisandrin B (CAS#61281-37-6), fluoxetine (CAS#54910- seconds, including all rest minutes; and 89-3) and serotonin (CAS#50-67-9) standard substances were Waking activity: the total amount of detected activity, obtained from Sigma–Aldrich (Saint Louis, MO, USA). excluding all one-minute periods of rest. Zebrafish preparation These parameters were generated automatically by R Parental zebrafish (AB strain, Zebrafish Model Animal software (The Comprehensive R Archive Network, TUAN Team, Facility, Institute of Clinical and Translational Research, Sun Tsinghua University). The program was written in advance. Yat-Sen University) were cultivated in tanks containing cir- The parameters of each group for each experimental day culating water at a constant temperature of 28.5 °C. The and night were the mean of three independent experiments light/dark cycle of the room was 14 h/10 h per day. Two hours (36 larvae), and expressed as means ± standard deviation after fertilization, the eggs were collected and transferred into (SD) from the control group for fingerprints. Color grada- −1 −1 a flat dish with Holt buffer (KCl 0.05 g·L ; NaHCO3 0.025 g·L ; tion was undertaken to present the degrees of standard de- −1 −1 −1 NaCl 3.5 g·L ; CaCl2 0.1 g·L ; 1 mg·L of methylene blue, viation. Positive values were shown in yellow, and negative PH 7.0). Dead embryos were removed and Holt buffer re- value in blue. The standard deviation approaching zero was placed daily. All experimental protocols and procedures in- shown in black. volving zebrafish were undertaken in accordance with the Histogram Guide For The Care And Use Of Laboratory Animals (number Histograms were created to reflect the change of mean 8023, revised in 1996; National Institutes of Health, Bethesda, rest total and waking activity for each concentration (0.2, 1, MD, USA). 5, 10, 20, and 40 μg·mL−1), and separated to day and night. Exposure to drug solutions Each value represents the average of 36 larvae. Bars are the At 4 days post-fertilization (4 dpf), the zebrafish larvae means ± SEM (n = 36). Statistical analysis were done using were transferred into 96-well plates with 300 μL Holt buffer one way ANOVA. separately. Then, 60 µL of mother solution (six times concen- Time series graph tration) was added to each well. Each row of the 96-well plate Rest and waking activity were averaged in 10-min intervals was a group containing 12 larvae. The first row was the di- and then normalized to line graphs. Each line represents the

– 917 – WANG Jia-Wei, et al. / Chin J Nat Med, 2018, 16(12): 916925 mean of 36 larvae of each group. Groups of EESC (10 μg·mL−1), same concentrations used in the egg-laying experiment. schisandrin (20 μg·mL−1) and schisandrin B (20 μg·mL−1) Thirty L4-stage larvae were removed and added to new plates were selected to compare with the respective controls groups. containing drugs. About 20 h later, C. elegans were prepared Instead of a time axis, white and black bars were placed on for use for observation of pharyngeal pumping [25]. The latter the horizontal axis to represent the day and night measure- was recorded using a stereo microscope (Nikon, Tokyo, Japan) ments, respectively. Differences in each 10-min interval be- and videos were generated by a software (UV 200; Nikon). tween treatments and controls were calculated by one-way Ten worms were selected randomly from 30 worms of each ANOVA. Significantly different time series > 1 h are shown by group. The rate of pharyngeal pumping was counted by three a horizontal line. assistants by replaying the videos at one-third of the original Dose correlation speed. The experiments were repeated thrice independently. All correlation analysis was undertaken using SPSS v19.0 (IBM, Armonk, NY, USA). Uncentered Pearson’s correlation Results coefficients were calculated for different concentration pairs Establishment of screening of zebrafish-larvae behavior according to mean ± SD values from the control group. Cor- High-throughput screening based on larval locomotor ac- relation coefficients were also calculated using color grada- tivity was utilized to explore the neural activity of EESC, tion with positive values shown in red. schisandrin, and schisandrin B. The 96-well plates were pre- Target correlation pared by adding 4-dpf larvae to treatment solution and 12 [17] Rihel et al. have reported a high-through put method larvae in each row formed a group. The first row was the about zebrafish larvae and applied to predict the target of control group (containing 3‰ DMSO). The positive control hundreds of compounds. We generated the database from group (containing 10 μg·mL−1 of fluoxetine) was employed [23] fingerprints as reported previously . Representative chemi- for each plate to compare the change in larvae activity be- cals were sorted by neural pathways, including the agonist or tween generations for different plates. Other rows were treat- antagonist of adrenaline, serotonin, dopamine, gamma-amino- ment groups (six concentrations of drugs) (Fig. 1A). The ef- butyric acid (GABA), histamine, adenosine, melatonin, and fects of different treatments were assessed using the total glutamate receptors. A random chemical was selected for each amount of rest, the number and length of rest bouts, the rest target to form a brief database. Based on this database, the latency, and activity (Fig. 1B). correlation coefficients between our treatments and represen- Changes in behavior of zebrafish larvae induced by different tative chemicals were calculated using color gradation. treatments C. elegans preparation Fingerprinting was applied to show the behavioral chara- N2 and tph-1 mutation strains of C. elegans were gener- cters induced by treatments. We found that 40 and 20 μg·mL−1 ated from the Department of Neurology, First Affiliated Hos- of EESC caused massive mortality of larvae and difficult to pital, Sun Yat-Sen University, Guangdong, China). Adult worms be calculated, leaving four concentrations of EESC to be ana- who had not suffered starvation in past three generation were lyzed. Fingerprinting revealed that different concentrations of used. They were fed E. coli OP 50 on nematode growth medium EESC increased the rest parameters (rest total, rest bout, and (NGM) agar plates. rest bout length) slightly and waking activity during the ob- Egg laying servation. Activity total was decreased slightly. Rest latency was increased during daytime and decreased at night. Inter- Egg laying behavior was evaluated following a well-es- [24] estingly, schisandrin presented similar effects to EESC with tablished assay protocol . Thirty L4-stage larvae were re- regard to rest parameters. Rest total and rest bout length were moved and placed on fresh plates seeded with bacteria for improved during day or night, and rest bout length was each group. About 20 h later, the larvae were transferred onto changed remarkably. Rest latency changed in opposite ways a 96-well plate containing 50 μL of solution of a particular during light or dark measurements. Schisandrin had few effects drug at 20 °C. Ten worms from each group were selected on activity total or waking activity. In contrast, schisandrin B randomly and placed into wells. The concentrations of drugs −1 enhanced activity in the daytime but had few effects on rest. in M9 buffer (μg·mL ) were 10 and 100 for EESC, 10 and Quantitative analysis of rest total and waking activity induced 100 for schisandrin, 100 for 5-hydroxytryptamine (5-HT), and by treatments 100 for fluoxetine, respectively. In the control group, 3% DMSO was added in M9 buffer. The plate was left for 60 min The rest total and waking activity were used for quantitative at room temperature. Egg-laying during this period was counted analysis as representative parameters for rest/wake behavior [17, 23] −1 using a dissecting microscope with a 12× objective lens. in larvae . EESC (1−10 μg·mL ) showed a neuroactive −1 Pharyngeal pumping effect during day and night. EESC (< 5 μg·mL ) acted in a After synchronization, the eggs were cultured on NGM dose-dependent manner (Fig. 3A). Schisandrin increased rest agar plates containing different content and placed into an total in light and dark measurement, and the efficacy was −1 incubator with constant temperature (20 °C) for 2 days. The dose-dependent between 1−20 μg·mL . Waking activity was −1 drugs were added to E. coli OP 50 bacterial solution at the changed slightly by schisandrin (except for 40 μg·mL ) (Fig.

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3B). Schisandrin B (2−20 μg·mL−1) improved waking activity during the daytime, but had no effect on rest total (Fig. 3C).

Fig. 1 Quantification of the behavior of zebrafish larvae. (A) Each well of a 96-well plate contained a single 4-dpf larva and a content of treatment. Twelve larvae were prepared for each group and parallel experiments were undertaken for at least thrice. The first group was control with 3‰ DMSO, and the second group was positive control with 10 μg·mL−1 fluoxetine. The concentrations of treatments (EESC, Schisandrin and Schisandrin B) were 40, 20, 10, 5, 1 and 0.2 μg·mL−1. (B) Locomotor activity of a represent larvae. The rest and wake behavior were recorded, including the number of inactivity minutes (rest total), a continuous minute of rest (rest bout), the duration between a light transition and the first rest bout (rest latency), average active time per minute (activity total), and the average activity excluding rest periods (waking activity)

Fig. 2 Drug-induced characteristics of zebrafish larvae behavior. Each row represents a unique concentration of a certain treatment for 36 larvae. Six parameters (rest total, rest bout, rest bout length, rest latency, activity total and waking activity) were calculate to describe behavior characters of zebrafish larvae behavior for each group. White and black bars under the parameter indicate day and night measurements, respectively. The standard deviations of parameters between the control group and each group were calculated and presented by color gradation. Yellow: this parameter is higher than controls. Blue: this parameter is higher than controls

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Fig.3 Quantitative analysis of rest total and waking activity. (A-C)Each value represents the mean of 36 larvae. Error bars represent the standard error of the means (SEM). *P < 0.05 and **P < 0.01 vs control by one way ANOVA. The white and black bars represent the day and night measurements respectively

Changes in behavior characteristics in time series drin was related to a serotonin pathway. Schisandrin B did not We selected representative concentrations of treatments show a correlation to any of the targets in the database. in fingerprint and created graphs of rest and waking activity Confirmation of neuroactive action on C. elegans time series to compare behavior during the day and night To test our prediction, egg laying and pharyngeal pump- more intuitionally. The significant effects (P < 0.05) on rest ing assays were conducted. Pharyngeal pumping is a sero- and activity of EESC (5 μg·mL−1) were separated to several tonin dependent behavior [26]. Egg laying is modulated by periods (Fig. 4A). Schisandrin had significant effect (P < 0.05) serotonin and dopamine system [27]. A serotonin synthesis on rest total at all times. Schisandrin B advanced wakefulness defective tph-1 mutant strain was introduced to study behav- during most of the daytime (Figs. 4B and C). ior in the absence of serotonin [28]. Serotonin and fluoxetine, a Prediction of the neural signaling pathways typical medicine of selective serotonin reuptake inhibitor Correlation matrices of different concentrations were es- (SSRI), were chosen as positive controls [29]. tablished first to exclude uncorrelated concentrations for tar- EESC, schisandrin, serotonin, and fluoxetine improved get prediction. Schisandrin (40 and 0.2 μg·mL−1), and schi- the rate of pharyngeal pumping and egg laying on the N2 sandrin B (0.2 μg·mL−1) were uncorrelated concentrations strain comparing with the control (Figs. 6A and B). Schisan- (Fig. 5A). If the correlation coefficient between concentra- drin (100 μg·mL−1) induced a higher egg laying rate than tions and known chemicals was > 0.6, the treatment was con- serotonin or fluoxetine. The pharyngeal pumping rate (Figs. sidered to be related to the target corresponding to the chemi- 6B and D) was decreased in the serotonin synthesis defective cals (Fig. 5B). Heat maps revealed that, EESC was related to tph-1 mutant strain than in N2 strain (tph-1 vs N2: 30-40/10s different targets, including serotonin and dopamine. Schisan- vs 50-60 /10s). The egg laying rate (Figs. 6A and C) was also

– 920 – WANG Jia-Wei, et al. / Chin J Nat Med, 2018, 16(12): 916925 inhibited on tph-1 mutant (tph-1 vs N2: 0.58/h vs 0.83/h). laying and pharyngeal pumping of schisandrin and fluoxetine Serotonin addition induced a significant improvement in the in the N2 strain disappeared in the tph-1 mutant. Moreover, pharyngeal pumping and egg laying rates in the tph-1 strain 10 and 100 μg·mL−1. EESC also promote the egg laying rate (Figs. 6C and D). Interestingly, the improvement effect on egg in the tph-1 mutant.

Fig. 4 Normalized time series of behavioral profiles. (A−C) Rest and waking activity were averaged in 10-minute intervals of 36 larvae, then normalized to line graphs. The solid line indicates the average of controls. The dotted lines indicate the average of 10 μg·mL−1 of EESC, 20 μg·mL−1 schisandrin and 20 μg·mL−1 schisandrin B groups, respectively. The white and black bars on horizontal axis represent the day and night measurements respectively. *P < 0.05 and **P < 0.01 vs control significantly different by one way ANOVA. Significant different time series > 1 h was shown by a horizontal line

tive effects of treatments. Rest bout is another important cri- Discussion terion for behavior defined sleep-like states, and the duration In the present study, we showed for the first time that EESC of rest bouts reflects the circadian rhythms in zebrafish [30]. had hypnotic and neuroactive effects in zebrafish, probably Rihel et al. have shown that hypnotic drugs, including mela- through serotonin and dopamine pathways. Also schisandrin was tonin and GABA-A agonists, promote the two parameters [17]. one of the components responsible for the sedative effect of In our study, EESC and schisandrin improved rest length and Schisandra chinensis and this effect was serotonin- dependent. rest bout length, indicating a hypnotic effect on zebrafish. In contrast, schisandrin B was an active component. The present Moreover, EESC and schisandrin had considerable similar study indicated the efficacy and mechanism of Schisandra sedative effects, suggesting that schisandrin is one of the chinensis, and the serotonin-like action of schisandrin components responsible for the sedative effects of Schisandra could be used to alleviate sleep disorders and depression. chinensis. Under the maximum non-toxic concentrations, Rest total is the most direct parameter showing the seda- schisandrin induced significant promotion of rest total com-

– 921 – WANG Jia-Wei, et al. / Chin J Nat Med, 2018, 16(12): 916925 pare with EESC, suggesting its potential application for alle- improved on waking activity, suggesting that schisandrin B viating sleep disorders. Conversely, EESC and schisandrin B wasa potential active component of EESC.

Fig. 5 Target correlation analysis. This analysis based on the parameters of each group of 36 larvae, and correlation coefficient was calculated in uncentred Pearson’s correlation coefficient. (A) Each color square of matrix represents the correlation coefficient of different concentrations. Red indicates high correlation and blue indicates a low correlation. (B) Each color square of matrix represents the correlation coefficient between treatments and known chemicals. Neuropathways were separated in the first row, including agonist or antagonist of adrenaline, serotonin, dopamine, GABA, histamine, melatonin, adenosine and the glutamate receptor. A representative chemical for each target was selected randomly. Red indicates high correlation and blue indicates a low correlation

Although EESC had neuromodulative actions, they were Neuroactive molecular pathways were predicted based on relatively weak both in zebrafish or C. elegans because Schi- correlations with known chemicals. This analysis suggested sandra chinensis is a mutiple-function herb with several that EESC was related to a serotonin and dopamine system. non-neuromodulative active sites. Identification and evalua- These two pathways induced improvement on rest and activity in tion of its neuro modulatory active sites could improve spe- zebrafish larvae [17]. Nevertheless, the behavioral characteristics cific and intensive effects [31]. In the present study, schisandrin of these two pathways were distinguishable in the database, was identified as an active chemical of the sedative effect of suggesting that the correlations of EESC differed. Schisandrin EESC. In contrast, schisandrin B had no significantly rest was related to a serotonin system. Slight behavior changes of change, suggesting that it could be removed in sedative site schisandrin B did not match the matrix, because quantitative acquisition of EESC. values from the known compounds in database are large.

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Fig. 6 Drug-induced behaviors in C. elegans. The control group was treated by 3‰ DMSO (grey), positive control groups were treated by 100 μg·mL−1 serotonin and fluoxetine respectively. White/ black bars represent 10 μg·mL−1 and 100 μg·mL−1 treatments. Bars represent mean ± SEM, n = 30, *P < 0.05 and **P < 0.01 vs control, #P < 0.05 vs fluoxetine, calculated by one way ANOVA. (A-B) Different effects of treatments on pharyngeal pumping and egg laying in the N2 strain. (C-D) TPH-1 mutant was used to reduce the production of 5-HT remarkably. The graphs represent pumping and egg laying in an absence of 5-HT

This prediction was confirmed by experiments in C. ele- and wakefulness [39]. These hypotheses suggest the sedative gans. The pharyngeal pumping rate is a serotonin-reliant be- effect of EESC and schisandrin. Given that schisandrin can havior. Mutations bearing the tph-1 deletion do not synthesize pass through the blood-brain barrier [40], identification of the serotonin [28]. Thus, the pharyngeal pumping rate decreased in serotonin-like effects of EESC and schisandrin may provide the tph-1 mutant in our study. EESC and schisandrin increased information to help alleviate depression, anxiety and sleep the pharyngeal pumping rate significantly in the N2 strain, but disorders [41-42]. Nevertheless, the biological activities of other this promotion disappeared in the absence of serotonin. These major chemicals in EESC, the relations between active results demonstrated that EESC and schisandrin were related chemicals, as well as a more accurate target verification of to the serotonin system. The egg-laying rate is modulated by a schisandrin to SSRIs, will be studied further by our research serotonin and dopamine system. The increase in the egg-laying team in the future. rate induced by schisandrin disappeared in the tph-1 strain. In In conclusion, we demonstrated that EESC had sedative contrast, EESC could promote the egg-laying rate in both and neuroactive effects on zebrafish larvae. Schisandrin and strains. Hence, EESC not only induced behaviors through a schisandrin B were two of the sedative and active components serotonin system, but also had effects on a dopamine system. of Schisandrin chinensis. We also predicted that schisandrin Schisandrin has serotonin-dependent effects similar to selective was related to serotonin pathway and EESC was related to serotonin reuptake inhibitors (SSRIs). Some scholars have serotonin and dopamine pathways by using a database of reported that schisandrin is an active component related to zebrafish behavior. These predictions were also confirmed serotonin, and that EESC has a dopaminergic effect [32-33]. Our with experiments of C.elegans. We further demonstrated that research suggested that the molecular pathways of extracts or zebrafish behavior profiling could be used as a high-through components from Schisandra Chinensis could be found effi- platform to screen efficacy and predict neuroactive molecular ciently using profiling of zebrafish behavior. pathways of extracts and components of TCM. Serotonin controls synchronization of the biological clock. It has been proposed to be sedative [34-36] and related to Acknowledgements sleep problems under pathological conditions [37]. Some hyp- We thank the Zebrafish Model Animal Facility, Institute notic medicines elicit their effects through a serotonin path- of Clinical and Translational Research, Sun Yat-Sen Univer- [38] way . Dopamine also participates in the control of sleep sity for providing AB strain zebrafish eggs.

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Cite this article as: WANG Jia-Wei, LIANG Feng-Yin, OUYANG Xiang-Shuo, LI Pei-Bo, PEI Zhong, SU Wei-Wei. Evaluation of neuroactive effects of ethanol extract of Schisandra Chinensis, Schisandrin, and Schisandrin B and determination of underlying mechanisms by zebrafish behavioral profiling [J]. Chin J Nat Med, 2018, 16(12): 916-925.

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