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Evaluation of Neuroactive Effects of Ethanol Extract of Schisandra

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Evaluation of Neuroactive Effects of Ethanol Extract of Schisandra Chinese Journal of Natural Chinese Journal of Natural Medicines 2018, 16(12): 09160925 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, schisan- drin 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): 916925 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): 916925 mean of 36 larvae of each group. Groups of EESC (10 μg·mL−1), same concentrations used in the egg-laying experiment.
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