Lotus Leaf Alkaloid Extract Displays Sedative–Hypnotic and Anxiolytic

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Lotus leaf alkaloid extract displays sedative-hypnotic and anxiolytic effects though GABAA receptor Mingzhu Yan, Qi Chang, Yu Zhong, Bingxin Xiao, Li Feng, Fangrui Cao, Rui-Le Pan, Zesheng Zhang, Yong-Hong Liao, and Xinmin Liu J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.5b04141 • Publication Date (Web): 07 Oct 2015 Downloaded from http://pubs.acs.org on October 16, 2015

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Lotus leaf alkaloid extract displays sedative-hypnotic and anxiolytic effects though GABAA receptor

Ming-Zhu Yan†,‡, Qi Chang*,†, Yu Zhong†,‡, Bing-Xin Xiao†, Li Feng†, Fang-Rui Cao†, † ‡ † Rei-Le Pan , Ze-Sheng Zhang , Yong-Hong Liao , Xin-Min Liu*,†



Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100193, People's Republic of China. ‡

Key Laboratory of Food Nutrition and Safety, Ministry of Education, College of Food Engineering and Biotechnology, Tianjin University of Science & Technology, Tianjin 300457, People's Republic of China.

Corresponding to --------------------------------------------------------------* Professor Qi Chang Institute of Medicinal Plant Development Chinese Academy of Medical sciences & Peking Union Medical College No.151, Malianwa North Road, Haidian District, Beijing 100193, PRC Tel: (86) 10-57833224 Fax: (86) 10-57833224 E-mail: [email protected] * Professor Xin-Min Liu Institute of Medicinal Plant Development Chinese Academy of Medical sciences & Peking Union Medical College No.151, Malianwa North Road, Haidian District, Beijing 100193, PRC Tel: (86) 10-57833224 Fax: (86) 10-57833224 E-mail: [email protected]

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Abstract: Lotus leaves have been used traditionally as both food and herbal medicine in

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Asia. Open-field, sodium pentobarbital-induced sleeping and light/dark box tests were used

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to evaluate sedative-hypnotic and anxiolytic effects of the total alkaloids (TA) extracted

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from the herb, and the neurotransmitter levels in the brain were determined by ultra fast

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liquid chromatography/tandem mass spectrometry. The effects of picrotoxin, flumazenil

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and bicuculline on the hypnotic activity of TA, as well as the influence of TA on Cl− influx

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in cerebellar granule cells were also investigated. TA showed sedative-hypnotic effect by

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increasing the brain level of γ-amino butyric acid (GABA), and the hypnotic effect could be

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blocked by picrotoxin and bicuculline, but couldn’t be antagonized by flumazenil.

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Additionally, TA could increase Cl− influx in cerebellar granule cells. TA at 20 mg/kg

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induced anxiolytic-like effects and significantly increased the concentrations of serotonin

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(5-HT), 5-hydroxyindoleacetic acid (5-HIAA) and dopamine (DA). These data

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demonstrated that TA exerts sedative-hypnotic and anxiolytic effects via binding to GABAA

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receptor and activating the monoaminergic system.

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Key words: Lotus leaves; sedative-hypnotic; anxiolytic; GABAA receptor; monoaminergic

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system

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Introduction

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Insomnia and anxiety are among the most common mental health problems and

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frequently cause significant functional impairment. These mental disorders impact the

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quality of suffers’ daily life appreciably and increase the risk for other medical problems.

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Although adequate symptomatic relief is achieved with the currently available drugs, severe

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side effects, such as anterograde amnesia, cognitive impairment, confusion and ataxia, limit

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their use in clinic. Other major problems include drug resistance, dependence, withdrawal

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syndromes and the potential for drug abuse1. Thus, in recent years, growing attention has

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been focused on herbal medicines, which are more effective and less toxic. Due to their

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multicomponent, multitarget actions, herbal medicines generally modulate several

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neurotransmitter systems, such as γ-aminobutyric acid (GABA), serotonin (5-HT) and

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dopamine (DA) to treat psychiatric disorders2,3.

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Lotus (Nelumbo nucifera Gaertn.) is a perennial aquatic medicinal plant which has

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been widely used for centuries in oriental medicine. Almost all parts of lotus, including

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flowers, leaves, leaf stalks, seeds and rhizomes are utilized and its leaves are used as both

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food and herbal medicine. Recently, leaves of the herb are becoming popular in China as a

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“tea drink” and dietary supplements for losing body weight and reducing blood lipids.

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Lotus leaves are known for diuretic, antipyretic and astringent properties, and are primarily

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used to treat sunstroke, sweating, diarrhea and fever in traditional Chinese medicine4.

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Modern pharmacological studies have demonstrated that lotus leaves exhibit a wide range

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of biological activities, including anti-hyperlipidemia, anti-hypoglycemic, anti-obesity,

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anti-oxidant, anti-HIV, anti-inflammatory, and anti-tumor5-12. Alkaloids are recognized as 3

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the

main

active

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benzylisoquinoline-type alkaloids11. Moreover, many investigations have shown that

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aporphine-

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neuropharmacological activities, such as sedative, anticonvulsant, anti-depression,

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anti-amnesic and neuroprotective effects3,12-14. Thus, lotus leaves may have the potential to

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exert the similar neuropharmacological activities.

and

compounds

in

lotus

benzylisoquinoline-type

leaves,

alkaloids

including

possessed

aporphine-

a

variety

and

of

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Our Previous studies have demonstrated that lotus leaves possess sedative-hypnotic

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effects and its alkaloid fraction was found to be responsible for the effect15. However,

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detailed neuropharmacological studies on the alkaloids and possible mechanisms have not

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yet been reported. The present work was undertaken to evaluate the sedative-hypnotic and

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anxiolytic effects after a single and 7-days multiple oral dose of alkaloids from lotus leaves

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in mice. Additionally, the involvements of the GABAergic and monoaminergic systems for

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these neuropharmacological effects were also investigated to clarify their possible action

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mechanisms.

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Materials and methods

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Plant material

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Nelumbo nucifera leaves were collected in June 2012 in Hunan province of China, and

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identified by Professor Bengang Zhang from the Institute of Medicinal Plant Development

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of the Chinese Academy of Medical Sciences.

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Preparation of alkaloid extract

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The air-dried lotus leaves (500 g) were ground into small pieces and exhaustively

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extracted with 0.1% HCl (10 L) three times. The filtered extract solution was loaded onto a 4

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D001 resin column, which was packed with 95% ethanol and equilibrated with water.

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After exhaustedly washing with water, the absorbed alkaloid compounds were then eluted

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from the resin column by 95% ethanol containing 1% ammonia. The eluent was

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concentrated to dryness under reduced pressure to yield an alkaloid extract (6.70 g), namely

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total alkaloids (TA), which is mainly composed of nuciferine (NF, 27.76%),

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N-nornuciferine (N-NF, 13.23%) and 2-hydroxy-1-methoxyaporphine (HMA, 4.23%)

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determined by HPLC method16 (Fig. 1).

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Animals

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Adult male ICR mice, weighing 18-20 g, were used in all experiments. All animals

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were housed in plastic cages, with six animals per cage, under controlled conditions of

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temperature (23 ± 2 oC), humidity (60 ± 5%) and 12 h light-dark cycle. Animals had free

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access to standard feed and water during the whole experiment. All behavioral evaluations

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were performed during the time of 08:00-17:00. The animal experiments were approved by

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the Animal Ethics Committee at the Institute of Medicinal Plant Development, Chinese

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Academy of Medical Sciences.

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Reagents

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Sodium pentobarbital, picrotoxin, bicuculline (BIC), sodium carboxymethyl cellulose

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(CMC-Na) and all calibration standards used in the brain neurotransmitter determination

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were purchased from Sigma (St. Louis, MO, USA), and diazepam (DZP) and flumazenil

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were purchased from National Institute for Food and Drug Control (Beijing, China). All

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chemicals and reagents used in the cell culture were from Gibco (Gaithersburg, MD, USA).

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N-(ethoxycarbonylmethyl)-6-methoxyquinolinium bromide (MQAE) was obtained from 5

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AAT Bioquest Inc. (Sunnyvale, CA, USA).

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Sodium pentobarbital and picrotoxin were prepared in saline, and bicuculline and

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flumazenil were prepared in saline containing 0.5% Tween 80. DZP and TA were suspended

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in 0.5%CMC-Na and administered intragastricly (i.g.). All the drugs were given to mice in

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a volume of 10 mL/kg.

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Measurement of locomotor activity

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The locomotor activity of mice was measured with an Activity Instrument, which

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consisted of a computer control system and four cages (35 × 35 × 30 cm, length × width ×

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height). TA (10, 20 and 40 mg/kg) and DZP (5 mg/kg) were administered orally 30 min

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before the test. Animals were then individually placed in the cages. After a 3 min

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acclimatization period, 10-min movement of mice in testing cages were monitored by

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cameras using a computer-based image-processing system. Locomotion is defined as a

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mouse removed all four paws from one place to another. 6.5cm/s was selected as the

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appropriate motion threshold17. Distance and time of the moving were recorded to explore

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the sedative effect of TA18.

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Sodium pentobarbital-induced sleeping

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Sub-threshold (25 mg/kg) or hypnotic (50 mg/kg) dose of sodium pentobarbital

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solution was injected intraperitoneally (i.p.) to mice 30 min after oral administration of

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vehicle or drugs. Sleep latency and the duration of the loss of righting reflex was

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recorded19.

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In the antagonistic experiments, picrotoxin (1 and 2 mg/kg), a non-competitive channel

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blocker for the GABAA receptor chloride channels, flumazenil (4 and 8 mg/kg), a specific 6

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antagonist of the benzodiazepine site in the GABAA receptor complex and bicuculline (BIC,

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2 and 4 mg/kg), a light-sensitive competitive antagonist of GABAA receptor, were i.p.

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injected 20 min prior to the administration of TA (40 mg/kg).

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Light/dark box test

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The light/dark box consists of a metal box of dimensions 20 × 12 × 12 cm, divided

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into a dark compartment and an illuminated compartment equally. The division between

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zones contains an opening of 4 × 4 cm. Both compartments possess 40 infrared-emitting

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diodes and an infrared camera to detect the movement of tested animals. The instrument is

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connected to a computer that records the number of transitions, latency to the first transition,

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time and activity in each compartment and total activity in a 5 min session. Animals were

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placed in the center of the light area facing away from the opening. An increase of the

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exploration in the lit area is associated with an anxiolytic effect; as such, two parameters

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were selected as a measure of anxiety: the time spent in the lit compartment and the total

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number of transitions20.

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Open-field test

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After finishing the above light/dark box test, animals were then placed in the

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open-field test cages of Activity Instrument and start the recording of activity immediately.

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Increases of time spent in the central part as well as of the ratio of central/total locomotion

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are indications of anxiolysis. Accordingly, the time spent in central area during 5 min was

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registered as described by Rex et al21.

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Acute toxicity

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Five groups of mice were used, with 10 mice for each group, weighing 20-22 g (half 7

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male, half female). The mice received a single oral dose of TA at the five different doses of

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200, 316, 501, 794 or 1000 mg/kg, respectively. After dosing, the animals were observed at

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15, 30, 60, 120, 240 min and 24 h for signs of toxicity and abnormality in behavior.

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Observations were recorded pertaining to the following symptoms: spontaneous activity;

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writhing; tremors; clonic convulsions; tonic convulsions; catalepsy; ataxia; salivation;

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dacryrrhea; aggressiveness or docility; gasping; anisorhythmia; apastia; diarrhea;

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incontinence; piloerection; cyanosis; pupil size and nostril discharge, according to the

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procedure described by Irwin22. Then, daily observations for mortality were made up to 7

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days. The LD50 value of TA was calculated by the equation: LogLD50 =∑1/2(Xi+Xi+1)(Pi+1−Pi)

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Where, X is the logarithm of doses; P represents the mortality; i is the the number of

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divided groups (i = 1, 2, 3, 4).

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Analysis of brain neurotransmitters

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Neurotransmitters,

including

glutamic

acid

(Glu),

GABA,

5-HT,

DA,

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5-hydroxyindoleacetic acid (5-HIAA), epinephrine (E) and norepinephrine (NE) in the

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cerebral cortex of the brain were determined by using an ultra fast liquid

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chromatography/tandem

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Prominence UFLC connected with Applied Biosystem 5500 Q-Trap mass detector) as

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previously reported method with slight modification23. Briefly, mice used in the open field

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test were sacrificed after finishing the test and the cerebral cortex were rapidly removed,

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weighed and stored at -80 oC until extraction. The frozen tissue samples were homogenized

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in 0.2 mL normal saline containing 0.2% formic acid, followed by mixing with 0.4 mL cold

mass

spectrometry

(UFLC-MS/MS)

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acetonitrile, which contained 0.2% formic acid and 45 µg/mL 3,4-dihydroxybenzylamine

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hydrobromide (internal standard). Then, the samples were restored in a -20 oC freezer

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overnight prior to centrifugation at 20000 × g for 30 min at 4 oC. The supernatant was

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collected and a 5 µL aliquot was injected into the UFLC-MS/MS system for analysis.

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Chromatographic separation was conducted on a C18 column (50 × 2 mm, 5 µm;

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Phenomenex, USA) maintained at 35 oC. The mobile phase consisted of 0.05% formic acid

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in water (solvent A) and acetonitrile (solvent B), at a flow rate of 0.4 mL/min. The linear

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gradient elution program was as follows, 20% B (0-1 min), 20%-80% B (1-2 min),

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80%-20% B (2-3 min) and 20% B (3-5 min). The mass spectrometer with electrospray

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ionization source was operated in positive ion mode and the ionization parameters were set

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as follows: curtain gas, 20 psi; ion source gas 1, 60 psi; ion source gas 2, 60 psi; and ion

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source temperature, 550 oC. The quantification was performed by multiple reaction

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monitoring (MRM) of the molecular ion and the related product ion for each

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neurotransmitters.

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Measurement of Cl− influx in cerebellar granule cells

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Cerebellar granule cells were prepared from 7 day-old SD rats using a procedure

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described by Zhu and Baker with a slight modification24. Cells (5×105 cells/mL) were

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plated on poly-L-lysine coated 96-well fluorescence plate. The cells attached to the bottom

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of the plate in 4-6 h. After that, the DMEM (10% heat-inactivated fetal bovine serum, 1%

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nonessential amino acids, 1% penicillin-streptomycin solution) was replaced with 0.2 mL

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growth medium (Neurobasal-A containing 2% B27, 2 mmol/L glutamine, 25 mmol/L KCl,

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0.5% penicillin-streptomycin solution). 9

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N-(ethoxycarbonylmethyl)-6-methoxyquinolinium bromide (MQAE) is a fluorescent

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indicator for intracellular Cl−. Its fluorescence is collisionally quenched upon interaction

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with Cl−, resulting in an ion concentration-dependent fluorescence decrease. Hence, we

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used MQAE to estimate the Cl− influx according to the method of West and Molly with a

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slight modification25. Cells were incubated in 5 mmol/L MQAE dissolved in Cl−-containing

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buffer [in mmol/L: HEPES (10), NaCl (137), KCl (5), MgCl2 (1), NaHCO3 (4.2) KH2PO4

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(0.44), CaCl2 (1) and D-glucose (10),] and loaded for 45 min at 37 oC. After loading, the

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cells were washed twice in the Cl−-free buffer. Then the buffer was replaced by

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Cl−-containing buffer with or without TA or control, and cells were incubated for 5 min

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before switching to the buffer with 2.5 mmol/L nigericin sodium and 3.5 mmol/L tributyltin

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chloride. Then 10.5 mol/L KSCN and 1.75 mmol/L valinomycin were added to the buffer to

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quench the intracellular MQAE fluorescence. Repeat measurements of fluorescence were

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initiated immediately using a fluorescence microplate reader (excitation, 360 nm; emission,

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460 nm). Resting intracellular Cl− concentrations were calibrated using standard Cl−

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solutions of 0, 10, 20, 30, 40 and 50 mmol/L Cl−. The intracellular concentration of Cl−

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( [Cl−] ) can be calculated by the Stern-Volmer equation:

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F0/FCl=1+KCl[Cl−]

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Where, F0 is the maximum fluorescence that can be quenched; FCl represent the increase in

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fluorescence (Ft−Fmin) (Ft is the fluorescence at time 0 or 5 min and Fmin is the fluorescence

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after quenching); KCl is a constant that can be derived from standard curve.

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Statistical analysis

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Statistical comparisons were performed by two-way ANOVA followed by 10

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Dunnett’s-test using SPSS19.0. The results of potentiation of sodium pentobarbital-induced

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loss of righting reflex at sub-threshold dose were analyzed by Fish’s exact test. P < 0.05

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was considered as significant. Data are expressed as mean ± S.E.M.

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Results

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Locomotor activity

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Locomotor activity results are shown in Fig. 2. The data reveals that single dose (day 1)

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TA induced a dose-dependent reduction of distance (Fig. 2A) and time of moving (Fig. 2B).

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TA at 40 mg/kg (P < 0.001) and diazepam (5 mg/kg, P < 0.001) showed a significant

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decrease in spontaneous activity of mice, in comparison with control group. After given TA

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(10, 20 and 40 mg/kg) and diazepam (5 mg/kg) for 7 days, only TA at 20 mg/kg could

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significantly (P < 0.05) decrease the spontaneous activity. Moreover, TA at 40 mg/kg and

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diazepam (5 mg/kg) showed a significant increase in locomotor activity, compared with

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those by single dose.

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Potentiation of sodium pentobarbital-induced sleeping

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Single dose TA and diazepam (2 mg/kg, P < 0.001, Fisher’s exact tests) increased the

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number of mice falling asleep induced by sub-threshold dosage of sodium pentobarbital (25

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mg/kg), compared with control group (Fig. 3). TA at the doses of 20 and 40 mg/kg showed

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an increase in sleeping rates from 0% to 42% (P < 0.05) and 67% (P < 0.01), respectively.

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However, multiple doses of TA (10, 20 and 40 mg/kg/day, for 7 days) did not significantly

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affect (P > 0.05) this parameter, with sleeping rates of 8%, 25% and 33%, respectively. The

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results also revealed a decrease in the number of mice falling asleep compared with the

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corresponding groups of single dose. Similarly, DZP (2 mg/kg) given by multiple doses 11

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(67%) showed a more significant (P < 0.05) decrease compared with that of single dose

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(100%).

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Two-way ANOVA data analysis showed a significant difference between groups in the

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sleep latency (Fig. 4A) and sleep duration(Fig. 4B). Diazepam at dose 2 mg/kg significantly

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(P < 0.001) decreased sleep latency and prolonged sleep duration already at day 1. Similarly,

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single dose TA (40 mg/kg) decreased the sleep latency by 18.71%, and increased duration

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of loss of righting reflex by 76.51%, compared with control group. 40 mg/kg TA did not

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decrease the sleep latency nor increase the sleep duration at day 7, only DZP showed

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statistically significant (P < 0.001 and P < 0.05, respectively). Despite the result that TA (40

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mg/kg) and DZP (2 mg/kg) given by multiple doses both showed a decrease in sleep

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duration (P < 0.05 and P < 0.001, respectively), decrease induced by DZP (2 mg/kg) was

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more significant.

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The decrease of sleep latency and prolongation of sleep duration of TA induce by

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sodium pentobarbital were reversed by picrotoxin (2 mg/kg) (Fig. 5A) and bicuculline (4

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mg/kg) (Fig. 5B). However, there is no antagonism effect of flumazenil (4 or 8 mg/kg) on

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hypnotic activity of TA, a specific antagonist of the benzodiazepine site in the GABAA

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receptor complex (Fig. 5C).

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Light/dark box test

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The influence of TA on the time spent in the lit compartment and the number of

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transitions between lit and dark compartments reached significance (Fig. 6A). Diazepam at

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2 mg/kg by single dose and multiple doses showed an anxiolytic effect in mice

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characterized by a significant increase in the time spent in lit area and the number of 12

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transitions between lit and dark compartments (P < 0.001). Similarly, TA at dose of 20

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mg/kg significantly (P < 0.01) increased the time spent in the lit compartment, but not the

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number of transitions between the two compartments. In contrast, lower (10 mg/kg) and

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higher doses (40 mg/kg) were ineffective. However, TA (10 and 20 mg/kg) by multiple

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doses caused a significant increase (P < 0.01) in the time spent in lit area compared with

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vehicle. These results demonstrate that TA has anxiolytic effects at lower doses but not at

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higher doses.

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Open-field test

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Results of the open-field test showed that TA (20 mg/kg) and DZP (2 mg/kg) by single

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dose and multiple doses produced a significant increase in the time spent in central area, as

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well as the ratio of central/total locomotion, indicating an anxiolytic-like effect (Fig. 6B).

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Acute toxicity

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The LD50 value of TA was estimated to be 457 mg/kg, indicating that TA is medium

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toxic according to ministry of health P. R. China’s classification.

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The levels of Neurotransmitters in brain

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Single dose of TA (40 mg/kg, P < 0.01) and diazepam (5 mg/kg, P < 0.05)

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significantly increased brain GABA (Table 1). TA (40 mg/kg) given by multiple doses

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showed a significant decrease in brain GABA level compared with that by single dose (P