Lotus Leaf Alkaloid Extract Displays SedativeâHypnotic 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

2

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

3

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

4

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

5

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

6

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

7

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

8

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

9

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

11

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

13

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

14

receptor and activating the monoaminergic system.

15

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

16

system

17 18 19 20 21 22 2



23

Introduction

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

25

frequently cause significant functional impairment. These mental disorders impact the

26

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

27

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

28

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

29

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

30

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

31

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

32

multicomponent, multitarget actions, herbal medicines generally modulate several

33

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

34

dopamine (DA) to treat psychiatric disorders2,3.

35

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

36

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

37

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

38

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

39

“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-

48

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

49

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

50

exert the similar neuropharmacological activities.

and

compounds

in

lotus

benzylisoquinoline-type

leaves,

alkaloids

including

possessed

aporphine-

a

variety

and

of

51

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

52

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

53

detailed neuropharmacological studies on the alkaloids and possible mechanisms have not

54

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

58

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.

68

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

69

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

70

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%),

72

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

73

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

77

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

78

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

79

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

80

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

81

Academy of Medical Sciences.

82

Reagents

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

84

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

85

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

86

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

87

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

88

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

91

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

92

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

96

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

97

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

98

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

100

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

107

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

110

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

117

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

118

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

134

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)

142 143

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

144

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,

147

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

148

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

149

chromatography/tandem

150

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

151

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

152

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

153

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

154

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

mass

spectrometry

(UFLC-MS/MS)

8


system

(Shimadzu


155

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

156

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

157

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

158

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

159

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

160

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

161

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

162

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

164

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

165

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

166

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

167

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

168

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

171

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

172

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

173

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

174

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

175

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

176

0.5% penicillin-streptomycin solution). 9


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

178

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

179

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

180

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

181

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

182

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

183

(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

187

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

188

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,

190

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

191

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

192

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

193

F0/FCl=1+KCl[Cl−]

194

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

195

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

196

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

197

Statistical analysis

198

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



199

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

200

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

201

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)

205

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

207

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

208

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

209

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

211

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

214

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

215

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

216

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

217

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

218

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

219

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

220

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

222

(100%).

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

224

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

225

(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

227

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

228

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

229

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

230

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

231

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

234

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

235

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

236

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

237

receptor complex (Fig. 5C).

238

Light/dark box test

239

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

240

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

241

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

242

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



243

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

244

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

245

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

246

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

247

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

248

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

249

higher doses.

250

Open-field test

251

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

252

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

253

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

256

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

257

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)

259

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

260

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

Lotus Leaf Alkaloid Extract Displays SedativeâHypnotic and Anxiolytic

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