Loganin exerts sedative and hypnotic effects via

modulation of the serotonergic system and GABAergic

neurons
Jiekun Xu1*, Rui Shi1, Yu Yan2, Haoyi Qiao1, Yan Han1, Jun He2, Congyuan Xia2, Wenwne
Lian2, Tingli Li3

1
Beijing University of Chinese Medicine, China, 2China-Japan Friendship Hospital, China,
3
Heilongjiang University of Chinese Medicine, China
Submitted to Journal:
Frontiers in Pharmacology

Specialty Section:

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Ethnopharmacology

ISSN:
1663-9812

Article type:

sio n a
i
Original Research Article

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Received on:

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23 Jan 2019

P r Accepted on:
01 Apr 2019

Provisional PDF published on:

01 Apr 2019

Frontiers website link:

www.frontiersin.org

Citation:
Xu J, Shi R, Yan Y, Qiao H, Han Y, He J, Xia C, Lian W and Li T(2019) Loganin exerts sedative and
hypnotic effects via modulation of the serotonergic system and GABAergic neurons. Front.
Pharmacol. 10:409. doi:10.3389/fphar.2019.00409

Copyright statement:
© 2019 Xu, Shi, Yan, Qiao, Han, He, Xia, Lian and Li. This is an open-access article distributed under
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This Provisional PDF corresponds to the article as it appeared upon acceptance, after peer-review. Fully formatted PDF
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Frontiers in Pharmacology | www.frontiersin.org

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Dear Prof. Rong-Rong He,
We greatly appreciate for your and the reviewers′ careful and professional comments
on our manuscript entitled “Loganin, an iridoid glycoside isolated from Corni fructus,
exerts sedative and hypnotic effects via modulation of the serotonergic system and
GABAergic neurons” (Manuscript ID: 449827).
In the revised version, changes to our manuscript within the document were all
highlighted by using yellow colored texts. Point-by-point responses to the reviewer’s
comments are enclosed after the letter for your consideration.
About your questions:
1. The information about source, isolation, purification, and structural
determination should be supplied . A critical aspect that should be considered
is how these assays and extraction protocols are linked to local and traditional
uses.

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Answer: We are thankful for your kind advisements.

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Firstly, the information about source, isolation, purification, and structural

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determination of loganin have been supplemented in the revised manuscript.

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Seondly, it is well known that insomnia is due to heart and kidney deficiency to a

P large extent in traditional Chinese medicine theory. Taking the facts, including
Corni fructus has been used as a tonic for the kidney in China for thousands of years
[1]
, and loganin is a major chemical constituents [2] into account, the sedative and
hypnotic effects of loganin on different animal models were investigated in our lab.
Finally, as described in manuscript, extraction of loganin from Corni fructus was
used by hot water in our lab, which is also consistent with the regulation of local
and traditional uses.
[1] Archives of Pharmacal Research, 2009, 32, 677-683.
[2] China Journal of Chinese Materia Medica, 1994, 19(12), 738-763
2. The active dose in mouse is 50 mg/kg, please discuss the possibility of the
relative dose for human.
Answer: As described in manuscript, the active dose of locomotor activity test and
pentobarbital-induced sleep test in mice about loganin are 50 mg/kg, and its active
dose in EEG and EMG recordings of rats are 35 mg/kg in our experiments. When
converting human dose into a mouse or rat dose, the conversion factor is 9.1 or 6.3,
respectively [3]. Therefore, the possibility of the relative dose for human is about 5.5
mg/kg (330 mg for a 60 kg normal person).
It is well known that serious side effects are always accompanied with clinical
chemotherapy drugs (such as benzodiazepines, zolpidem and zopiclone, etc.) for
insomnia, including drug dependence, drug tolerance, rebound insomnia, and
amnesia [4, 5].
Although the dose of loganin for human might be higher than clinical chemotherapy
drugs, our reseach confirmed that loganin had almost no side effects, and even at
the dose of 5000 mg/kg the administration of loganin was safe with no indication
of an adverse physiological effects observed by biochemical measurements and
histopathological examinations. The revelant data will be published soon.

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In addition, aqueous extract of valerian root ( Valeriana officinalis L.) improves

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sleep quality in man at the dose of 400 mg. Several “over-the-counter” sedatives

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containing valerian are sold in Switzerland and the dose at 400 mg was
recommended in the Swiss pharmacopoeia

r
[6].
The dose of loganin for human is

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lower than the commercial preparation containing valerian.
On the other side, the dose of loganin for human is also lower than many bioactive
compounds with sleep-enhancing effects from the Chinese medicines or natural
products. For example,
(1) Tenuifolin, a saponin derived from Radix Polygalae, exhibits sleep-enhancing
effects at the dose of 80 mg/kg in mice [7]. When converting into human dose,
the therapeutical dose of tenuifolin is 8.8 mg/kg (528 mg for a 60 kg normal
person).
(2) Safranal, one bioactive constituent of saffron, enhances sleep in mice at the
[8]
dose of 180 and 360 mg/kg . When converting into human dose, the
therapeutical dose of safranal is 19.8 and 39.6 mg/kg (1188 mg and 2376 mg
for a 60 kg normal person).
(3) Tetrandrine, a bisbenzylisoquinoline alkaloid from Chinese herb Radix, exertes
 the hypnotic effectl through serotonergic system at a dose of 60 mg/kg [9]. When
converting into human dose, the therapeutical dose of tetrandrine is 6.6 mg/kg
(396 mg for a 60 kg normal person).
[3] Journal of Pharmacy and Pharmacology, 2014, 66(6), 823-834.
[4] British Journal of Clinical Pharmacology, 1986, 21, 647-653.
[5] Psychopharmacology, 1999, 143, 373-379.
[6] Pharmncoiogy Biochemistry & Behavior, 1982, 17, 65-71.
[7] Phytomedicine, 2016, 23, 1797-1805.
[8] CNS Neuroscience & Therapeutics, 2012, 18(8), 623-630.
[9] European Journal of Pharmacology, 2004, 506, 101-105.
3. Please discuss how specific is the effect?
Answer: The results of the EEG and EMG recordings of rats showed that loganin
significantly increased NREM sleep and total sleep, and remarkably shortened

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wakefulness. Loganin did not significantly reduce the EEG power of delta, which

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indicated that loganin-induced sleep is compatible with physiological sleep.

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As described in manuscript, the results of our research further indicated that 5-HT

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and GABAergic neurons activation were involved in the mechanism of sleep-

P promoting effect and sleep architecture regulation of loganin, which means

serotonergic system and GABAergic neurons might be the specific targets of
loganin. Further studies are required to identify the role of loganin in the related
signaling pathways in future.
4. Loganin is water soluble. Can it pass the BBB? Please discuss.
Answer: In 2006, Xiao-Na Li, et al. reported that after oral administration of
loganin (20 mg/kg), it could be respectively determined in brain of rats at 15 and
90 min, which proved that loganin could pass through the blood-brain barrier and
reach the brain [10].
In addition, nowadays we have been developing a rapid, sensitive and selective LC-
MS/MS method for determination of loganin in rat tissues, and we detected loganin
in brain after oral administration of loganin (50 mg/kg).
[10] Biomedical Chromatography, 2006, 20(10): 1087-1092.
Thank you very much for your understanding again!
Best regards,

Jie-Kun Xu
School of Life Sciences,
Beijing University of Chinese Medicine,
No. 11, Bei San Huan Dong Lu, Chaoyang District,
Beijing 100029, P. R. China
Phone: +86-10-84205662
Fax: +86-10-84205662
E-mail: xjkbucm@163.com

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Reviewer 1
1. I appreciate the answer by the Authors and the fact they have changed the title,
but, if they have not used commercial loganin and isolated it from the plant,
still all the information about source, isolation, purification, and structural
determination are missing.
Answer: We are thankful for your kind advisements!
Firstly, the information about source, isolation, purification, and structural
determination of loganin have been supplemented in the revised manuscript.
Secondly, The NMR spectra data of loganin are as follows:
1
H-NMR (CD3OD, 400MHz), δ: 5.28 (1H, d, 4.4 Hz, H-1), 7.40 (1H, s, H-3), 3.37
(1H, m, H -5), 1.63 (1H, m, H-6), 1.88 (1H, m, H-6), 3.67 (1H, m, H-7), 2.05 (1H,
m, H-8), 2.25 (1H, m, H-9), 1.11 (3H, d, 7.2 Hz, H-10), 3.70 (3H, s, COOCH3),
4.66 (1H, d, 8.0 Hz, H-1′), 3.10~3.39 (m, H-2′~H -5′), 3.66 ( dd, 1H, 1.2, 11.6 Hz,
H-6′), 3.89 (dd, 1H, 1.2, 11.6 Hz, H-6′);

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13
C-NMR (CD3OD, 100 MHz), δ: 97.7 (C-1), 152.2 (C-3), 114.1 (C-4), 32.2 (C-5),

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42.7 (C-6), 75.1 (C-7), 42.2 (C-8), 46.5 (C-9), 13.5 (C-10), 169.6 (C-11), 51.7 (-

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OCH3), 100.1 (C-1′), 74.8 (C-2′), 78.4 (C-3′), 71.6 (C-4′), 78.1 (C-5′), 62.8 (C-6′).

P The data are identical to those of the reference [11].

[11] Phytomedicine, 2012, 19(3): 317-321.
The above information have been added into from Line 93 to Line 109 in the revised
manuscript.
 Figure 1. 1H-NMR Spectrum of loganin in Methanol-d4

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Figure 2. 13C-NMR Spectrum of loganin in Methanol-d4

2. I suppose you have extracted the fruits of the plant: please specify in the text.
I am very surprised about the high amount of plant material you have used!!
Did you only run NMR? Did you check the purity of ypour fractions by other
methods?
Answer: Thanks for your nice suggestions!
As you said, Corni fructus, the fruit of Cornus officinalis Sieb. et Zucc., were
extracted and we had specified in the text.
In fact, before studies of pharmacological activities of loganin, chemical
constituents of Corni fructus have been investigated in our lab from 2013 until now
[12-18].
To further the search for structurally diverse and biologically significant
compounds from Corni Fructus, the high amount of plant material (63.0 kg) were
collected and extracted. Finally, loganin (200 g) was obtained after repeatly
isolation and recrystallization procedures.
Besides the NMR spectra of loganin, the purity of loganin (99%, tR = 16.7 min) is
detected by HPLC using an ES Sonoma C18 column (4.6×250 mm, 5 μm) with
acetonitrile/water (from 5% to 20% in 20 min) as a mobile phase with DAD detector
(230 nm, 30°C, 1.0 mL/min). The HPLC chromagraphy of loganin is following:

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[12] Jie-Kun Xu, et al. Bioorganic Chemistry. 2019, 82: 1-5
[13] Jie-Kun Xu, et al. Journal of Natural Products. 2017, 80 (12): 3103–3111
[14] Jie-Kun Xu, et al. Fitoterapia. 2017, 120: 136-141
[15] Jie-Kun Xu, et al. China Journal of Chinese Materia Medica
. 2018, 43(21): 4264-4266
[16] Jie-Kun Xu, et al. Chinese Pharmaceutical Journal . 2017,
52 (14): 1212-1216
[17] Jie-Kun Xu, et al. China Journal of Chinese Materia Medica
. 2016, 41(24): 4605-4609
[18] Jie-Kun Xu, et al. A preparation method of loganin. Chinese Invention Patent
. CN 106831910A
 Loganin exerts sedative and hypnotic effects via modulation of the
serotonergic system and GABAergic neurons
1 Rui Shi 1, 2 §, Yan Han 1, 2 §, Yu Yan 2, Hao-Yi Qiao 1, 2, Jun He 2, Wen-Wen Lian 2, Cong-Yuan
2 Xia 2, Ting-Li Li 3, Wei-Ku Zhang 2 *and Jie-Kun Xu 1 *
1
3 School of Life Sciences & School of Chinese Medicine Sciences, Beijing University of Chinese
4 Medicine, Beijing 100029, People’s Republic of China
2
5 Institute of Clinical Medical Sciences & Department of Pharmacy, China-Japan Friendship Hospital,
6 Beijing 100029, People’s Republic of China
3
7 School of Chinese Medicine Sciences, Heilongjiang University of Chinese Medicine, Harbin 150070,
8 People’s Republic of China
9 * Correspondence:
10 W.-K. Zhang, zhangweiku@zryhyy.com.cn

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11 J.-K. Xu, xjkbucm@163.com

12

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Author Contributions
§

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Rui Shi and Yan Han contributed equally to this research.

14

15 ABSTRACT

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Keywords: Loganin1; Sedative2; Hypnotic3; serotonergic system4; GABAergic neurons5.

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17
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Corni fructus, the fruit of Cornus officinalis Sieb. et Zucc., has been used as a tonic for the kidney in
China for thousands of years. Loganin is one of the major constituents derived from Corni fructus. In
this study, we revealed the sedative and hypnotic activity of loganin and investigated its mechanisms
for the first time. Pentobarbital-induced sleep test and insomnia mice models (induced by caffeine and
p-chlorophenylalanine (PCPA)) were used for the assessment of sedative and hypnotic effects of
loganin. It was found that loganin (20-50 mg/kg) exerted sedative effect in normal mice. Loganin
22 exhibited hypnotic effect by increasing sleep onset and sleep duration in pentobarbital-treated mice,
23 recovering PCPA-induced insomnia and exerting synergistic hypnosis effect with 5-HTP. In addition,
24 electroencephalograph (EEG) and electromyography (EMG) recordings of rats showed that loganin
25 (35 mg/kg) prolonged the ratio of non-rapid eye movement (NREM) sleep and shortened wakefulness
26 significantly, further immunohistochemistry showed that loganin (35 mg/kg) increased c-Fos
27 expression in GABAergic neurons of rats in the ventrolateral preoptic nucleus (VLPO). The levels of
28 norepinephrine (NE), dopamine (DA), serotonin (5-HT) and its metabolite were measured in the
29 hippocampus, prefrontal cortex and striatum of mice, 1 h after loganin (35 mg/kg) treatment. 5-HT, 5-
30 HIAA/5-HT, DA and DOPAC were decreased significantly in the prefrontal cortex. In conclusion,
31 these results indicated that loganin produced beneficial sedative and hypnotic activity, which might be
32 mainly mediated by modification of the serotonergic system and GABAergic neurons.

33 INTRODUCTION

34 Insomnia is referred to as continuous difficulty in initiating or maintaining sleep, which can induce
35 extreme medical and psychiatric disorders (Cao et al., 2016). It was reported that twenty-seven percent
 Loganin exerts hypnotic effect

36 of people in the world suffering from insomnia (Doghramji, 2006), and approximately 3 to 10 percent
37 of people would frequently depend on hypnotics to overcome insomnia by 2050 (Chu et al., 2007).
38 Clinically, benzodiazepines are the most extensively used drug for insomnia. Nevertheless, serious side
39 effects are always accompanied with their treatment, including drug dependence, drug tolerance,
40 rebound insomnia, and amnesia (Griffiths et al., 1986; Bocca et al., 1999). Zolpidem and zopiclone, a
41 new type of hypnotic, also show some undesirable side effects. Therefore, novel drug for insomnia
42 with better effect and less side effect deserves more effort, and novel herbal ingredients in traditional
43 Chinese medicine might be a better choice for drug discovery (Griffiths et al., 1986).

44 Corni fructus, the fruit of Cornus officinalis Sieb. et Zucc. (Cornaceae), has long been used to nourish
45 the liver and kidney and treat diabetes and other diseases in China, Korea and Japan (Lee et al., 2009).
46 Loganin (Fig. 1), a major iridoid glycoside isolated from Corni fructus, has been reported to show
47 neuroprotective activity, immunity regulatory activity, and lipid metabolism regulatory activity, and
48 further exhibit preventative and treatment effects in diabetes, tumors, inflammation, and Alzheimer's
49 disease (Hui-Jun et al., 2003; Frédérich et al., 2004; Lee et al., 2009). In addition, loganin has been
50 proved to pass through the blood-brain barrier and reaches the brain (Li et al., 2010), which might be
51 the basis of its neuroprotective activity. However, the sedative and hypnotic effects of loganin have
52 not yet been reported.

53 Monoamine neurotransmitters and their metabolites play a significant role in the nervous system and
54
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56
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have also been associated with insomnia. Mammalian sleep is composed of non-rapid eye movement

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(NREM) and rapid eye movement (REM) sleep alternation (Weber, 2017). The ventrolateral preoptic

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nucleus (VLPO) cluster is a necessary component of sleep circuitry, the discharge rate of which

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57 increases just before or at the transition from wakefulness to NREM sleep and decreases prior to the

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58 transition from NREM or REM sleep to wakefulness (Szymusiak et al., 1998). However, to the best of

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59 our knowledge there is no reported mechanism of sedative and hypnotic effects of loganin is related to

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60 monoamine neurotransmitters and VLPO neurons.

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In this paper, we investigated the sedative activity of loganin using locomotor activity test and then
detected the hypnotic effect of loganin in pentobarbital-induced sleep test and insomnia mice models
(induced by caffeine and p-chlorophenylalanine (PCPA)). In addition, we detected the effects of
loganin on sleep architecture in free-moving rats through electroencephalograph (EEG) and
electromyography (EMG) recording. Furthermore, we evaluated the concentration of monoamine
neurotransmitters and their metabolites using high-performance liquid chromatography with
67 electrochemical detection (HPLC-ECD) and assessed the ratio of c-Fos-positive glutamic acid
68 decarboxylase (GAD) neurons in VLPO, the main brain region triggers NREM sleep (Cao et al., 2016),
69 trying to explain the mechanism of loganin.

70 MATERIALS AND METHODS

71 Animals
72 Adult male ICR mice (20 ± 2 g) and adult male Sprague-Dawley rats (260 - 280 g) were purchased
73 from Vital River Laboratories (Beijing, P. R. China). The animals were housed in acrylic cages (45 ×
74 60 × 25 cm) with water and food available ad libitum under an artificial 12 h light/dark cycle (light
75 from 8:00 am to 8:00 pm) in a sound-proof room, and the temperature (24 ± 2 ºC) the humidity (55 ±
76 15%) were controlled.
77 Experiments were conducted in compliance with the National Institutes of Health Guide for the Care
78 and Use of Laboratory Animals of China and were approved by the Animal Care Committee of China-
79 Japan Friendship Hospital. Every effort was made to minimize the use of animals and any pain or
80 discomfort.

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 Loganin exerts hypnotic effect

81 Drugs
82 Commercially available drugs used in this study were purchased: pentobarbital (Sigma Aldrich),
83 Estazolam tablet (1.0 mg/ 60 mg; Shandong Xinyi Pharmaceutical Co., Ltd, Shandong, P. R. China),
84 anhydrous sodium acetate (Fisher Scientific), citric acid (Coolaber), sodium octane sulfonate
85 (Damasbeta), EDTA-2Na (Beijing Biodee Biotechnology Co., Ltd, Beijing, P. R. China), KCl (Fisher
86 Scientific), perchlorate (Tianjin Damao Chemical Reagent Factory, Tianjin, P. R. China). HPLC-grade
87 water and methanol were obtained from Fisher Scientific (Thermo Fisher). 3,4-Dihydroxybenzylamine
88 hydrobromide (DHBA), norepinephrine (NE), 3,4-dihydroxy-phenyl acetic acid (DOPAC), dopamine
89 (DA), 5-hydroxyindoleacetic acid (5-HIAA), homovanillic acid (HVA), and serotonin (5-HT) were
90 purchased from Sigma Aldrich. PCPA, caffeine, and 5-HTP were obtained from Melonepharma Co.,
91 Ltd. (Dalian, P. R. China).
92 Extraction, isolation, and structural elucidation of loganin
93 Corni fructus (63.0 kg) collected in Foping, Shaanxi province, P.R. China in November 2013 were
94 refluxed twice with hot water (630 L, 100 °C) for two hours. The solvent was concentrated to afford
95 28.9 kg of crude extract, which was separated via a macroporous adsorption resin (D101) column
96 eluting with H2O, 30% ethanol, 55% ethanol and 95% ethanol. The 30% ethanol residue (2.9 kg) was
97 separated on a silica gel column with CHCl3−CH3OH (100:0 to 1:1) to give five fractions (A−E).
98 Fraction B (1.0 kg) was dissolved in CH3OH, and placed at the room temperature for 24 hours. The

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99 sediment (300 g) was repeatedly dissolved in ethanol and ethyl acetate to recrystallize to obtain loganin

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100 (200 g).
101
102
103

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The NMR data of loganin are as follows: 1H-NMR (CD3OD, 400 MHz), δ: 5.28 (1H, d, 4.4 Hz, H-1),
7.40 (1H, s, H-3), 3.37 (1H, m, H -5), 1.63 (1H, m, H-6), 1.88 (1H, m, H-6), 3.67 (1H, m, H-7), 2.05

i
(1H, m, H-8), 2.25 (1H, m, H-9), 1.11 (3H, d, 7.2 Hz, H-10), 3.70 (3H, s, COOCH3), 4.66 (1H, d, 8.0

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104 Hz, H-1′), 3.10~3.39 (m, H-2′~H -5′), 3.66 ( dd, 1H, 1.2, 11.6 Hz, H-6′), 3.89 (dd, 1H, 1.2, 11.6 Hz,

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105 H-6′); 13C-NMR (CD3OD, 100 MHz), δ: 97.7 (C-1), 152.2 (C-3), 114.1 (C-4), 32.2 (C-5), 42.7 (C-6),
106
107
108
109
110
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75.1 (C-7), 42.2 (C-8), 46.5 (C-9), 13.5 (C-10), 169.6 (C-11), 51.7 (-OCH3), 100.1 (C-1′), 74.8 (C-2′),
78.4 (C-3′), 71.6 (C-4′), 78.1 (C-5′), 62.8 (C-6′). The data are identical to those of the reference (Jeong
et al., 2012). The purity of loganin is 99% by HPLC detection.
Treatments
The animals were adapted to laboratory conditions seven days before the behavioral study and were
maintained in the laboratory until the study was completed.
112 For oral administration (0.2 ml/10 g for mouse, 1.0 ml/100 g for rat, volume/body weight, i.g.). Animals
113 were orally administered loganin (5, 20, and 50 mg/kg for mouse, 35 mg/kg for rat) and estazolam
114 (0.15 mg/kg for mouse, 0.1 mg/kg for rat), which were suspended in distilled water and administered
115 25 min before pentobarbital administration (Qiao et al., 2017).
116 In the pentobarbital-induced sleep test, 45 mg/kg pentobarbital (i.p.) was used as the hypnotic dose
117 (sleep onset = 100%), and 23 mg/kg (i.p.) was used as the subhypnotic dose (sleep onset < 10%).
118 Caffeine (7.5 mg/kg, i.p.), PCPA (300 mg/kg, s.c.) and 5-HTP (2.5 mg/kg, i.p.), were suspended in
119 physiological saline. For the PCPA-induced insomnia, PCPA was injected in mice between 08:00 am
120 and 09:00 am, 24 h prior to the pentobarbital injection. For the caffeine-induced insomnia model, mice
121 received an injection of caffeine between 1:00 pm and 1:30 pm, with the caffeine treatment 30 min
122 prior to the pentobarbital injection. 5-HTP was injected 15 min prior to pentobarbital administration
123 (i.p.).
124 All drugs were prepared before the experiment each day.
125 Behavioral tests

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 Loganin exerts hypnotic effect

126 The behavioral tests were conducted in a soundproof room. The experimenters were blind to the
127 treatment.
128 Locomotor activity test
129 Experiments were carried out between 1:00 pm and 5:00 pm. The spontaneous locomotor activity of
130 mice was used to investigate the sedative effects.
131 A mouse was placed in 1 of 4 boxes in an apparatus (ZIL-2, Institute of Materia Medica, Chinese
132 Academy of Medical Sciences, P. R. China). The locomotor activities of the mice were recorded 5 min,
133 30 min, 60 min, 120 min, 180 min, and 240 min after loganin and estazolam injection. Mice were
134 acclimated to the boxes for 2 min, and the locomotor activity of each mouse was monitored for 5 min
135 with activity counts recorded. After each testing session, the enclosures were cleaned with 75% ethanol
136 and water (Xiao-Yu et al., 2014).
137 Pentobarbital-induced sleep test
138 Pentobarbital-induced sleep test in mice were performed, which is a behavioral method to evaluate
139 sedative-hypnotic activity (Masur et al., 1971). Experiments were carried out between 1:00 pm. and
140 5:00 pm. Mice were placed on their back and observed for sleep onset following pentobarbital injection.
141 The criterion for a mouse to fall asleep was that the mouse lost the righting reflex and remained on
142 their back for more than 1 min. The loss of righting reflex was defined as a failure of the mouse to right

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143 itself within 10 s after being placed on its back. The time between the administration of pentobarbital

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144 and the loss of righting reflex was recorded as the time to sleep onset, while the time from the loss of

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145 righting reflex to recovery was recorded as sleep duration. In the subhypnotic pentobarbital test, the

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146 percentage of mice that fell asleep was calculated as follows: Sleep onset (%) = No. falling asleep/Total

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147 No. × 100%.
148
149

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EEG and EMG recordings and analysis
Under deep anesthesia with pentobarbital sodium (45 mg/kg, i.p.), rats were placed in a stereotaxic

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150 apparatus to position their bodies. Briefly, two stainless steel screws attached to an insulated wire were
151 implanted in the skull over the frontal parietal cortex for EEG recordings. One was placed
152 approximately 2 mm anterior and 2 mm to the right of bregma; another was placed approximately 3
153 mm posterior and 2 mm to the left of bregma. A pair of wire electrodes was inserted into the neck
154 muscle to record the EMG. The connecting wires attached to the electrodes were welded to a six-pin
155 connector and attached to the skull, secured with dental cement. After surgery, the rats were given
156 antibiotics for three days, placed in separate cages and allowed to recover for a week. The recordings
157 were carried out immediately after intragastric administration of control drug or loganin (35 mg/kg).
158 The signals were amplified and sampled at 200 Hz by a polygraph (Model MP 150; BIOPAC Systems,
159 Goleta, California, USA). The signals were filtered (EEG, 0.5-30 Hz; EMG, 16-128 Hz), then digitized
160 at a sampling rate of 128 Hz and recorded using AcqKnowledge software (BIOPAC Systems, Inc.,
161 USA). The analysis was based on the standard criteria embedded in SleepSign v.3.0 software (BIOPAC
162 Systems). Each epoch was characterized as follows: wakefulness (low amplitude EEG activity, high-
163 voltage EMG activity), NREMS (high amplitude slow or spindle EEG activity, low-amplitude EMG
164 activities), and REMS (low-voltage EEG featured theta wave and very low EMG activities). Total sleep
165 time was calculated as nonrapid eye movement (NREM) time plus rapid eye movement (REM) time.
166 the details of the data parameters with four frequency ranges as follows: the delta band (0.5–4 Hz),
167 theta band (4–8 Hz), alpha band (8–13 Hz) and beta band (13–30 Hz) (Cui et al., 2012). The data were
168 analyzed by automatic scoring and manually checked for possible errors in the automatic scoring.
169 Recordings were performed during the normal wake period of mice to better observe the hypnotic
170 effect of loganin. The power of each frequency band was averaged and expressed as a percent of the
171 total power within the frequency range of 0.5-30 Hz (Qiao et al., 2017).
172 c-Fos immunohistochemistry analysis

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 Loganin exerts hypnotic effect

173 One hour after loganin (35 mg/kg) administration (12:00 am), the rats were anesthetized with
174 pentobarbital (45 mg/kg, i.p.) and the heart was perfused with saline, followed by ice-cold 4%
175 polyformaldehyde (PFA) in 0.01 M phosphate buffered saline (PBS) (pH 7.4). Then, their brains were
176 removed, post-fixed in 4% PFA for 24 h at 4°C and immersed in 30% sucrose at 4°C until they sank.
177 VLPO was obtained by mapping the brains of mice (Konsman, 2003). After that, the frozen sections
178 were cut into pieces of 30 μm thickness on the coronal plane using a cryotome. Sections were double
179 immunofluorescence labeled using methods previously described with some modification (Sherin et
180 al., 1998). Each section was double-immunostained with nucleus-specific neurotransmitter markers
181 labeled green and c-Fos labeled red. The sections were examined using an Olympus IX71 fluorescence
182 microscope (Olympus Corporation, Tokyo, Japan).
183 The c-Fos positive ratio was calculated as follows:
184 The c-Fos positive ratio = 100 × (No. c-Fos (+)-neurotransmitter synthesizing enzyme (+) cells/No. c-
185 Fos ( )-neurotransmitter synthesizing enzyme (+) cells).
186 The average number of c-Fos (+) cells in the nuclei of both hemispheres was taken for statistical
187 analysis.
188 Monoamine neurotransmitters content analysis by HPLC-ECD
189 The rats were sacrificed 1 h after loganin administration (12:00 am). The process and conditions of

l
190 tissue preparation and monoamine testing were conducted as described with little modification (Zhang
191
192
193
a
et al., 2014). Hippocampus, frontal cortex, and striatum were obtained according to the mouse brain

n
atlas (Konsman, 2003). Brain neurotransmitters and metabolites were analyzed using HPLC-ECD.

sio
As shown in Fig. 2, the peak areas in the sample chromatograms and the reference standards were

i
194 determined by the interior standard method. Reference standards using solutions of NE, 3, 4-

v
195 dihydroxybenzylamine hydrobromide (DHBA, interior label), DA, DOPAC, 5-HT, HVA and 5-HIAA.
196
197
198
199
200
201
P r o
The results are expressed in nanograms per milligram of fresh tissue.
Statistical analysis
The results were expressed as the mean ± S.E.M. For multiple comparisons in the pentobarbital-
induced sleep test, data were analyzed using one-way analysis of variance (ANOVA) was used
followed by the Students-Newman-Keuls (SNK) test. One-way analysis of variance (ANOVA)
followed by the Least Significance Difference test was applied to the results of EEG and EMG
202 recordings. For comparisons between two-group data, the unpaired Student's t-tests was used. For the
203 subhypnotic dose of pentobarbital test, a chi-square test was used to compare the number of mice that
204 fell asleep. Differences were considered statistically significant at *P < 0.05.
205 RESULTS
206 Sedative effect of loganin in normal mice
207 To explore whether the excitability of the central nervous system was affected by loganin, locomotor
208 activity in normal mice was assessed. As shown in Fig. 3, locomotor activity was recorded at 5, 30, 60,
209 120, 180 and 240 min after drug administration. And the locomotor activity of the 5 groups gradually
210 decreased. Compared with the control group, the locomotor activity of loganin (20 and 50 mg/kg)
211 decreased significantly from 5 min (*P < 0.05 at 20 and 50 mg/kg) to 180 min (*P < 0.05 at 20 and 50
212 mg/kg), which demonstrated that the sedative effect of loganin appeared as early as 5 min and last for
213 180 min. Estazolam also showed a similar inhibition on locomotor activity in normal mice (*P < 0.01
214 at 5 and 180 min).
215 Onset and duration of sleep in pentobarbital-induced sleep test
216 To investigate the hypnotic activity of loganin, the effect of loganin on the onset and duration of

5
 Loganin exerts hypnotic effect

217 pentobarbital-induced sleep in mice was evaluated. In mice treated with subhypnotic doses of
218 pentobarbital (23 mg/kg, i.p.), loganin (5, 20 and 50 mg/kg, i.g.) prominently increased sleep onset in
219 mice in a dose-dependent manner (*P < 0.05 at 5, 20 and 50 mg/kg, Tab. 1), compared with the vehicle
220 group. Loganin at 20 and 50 mg/kg increased the sleep onset to 60%, which was slightly lower than
221 the positive control estazolam at 1 mg/kg.
222 In mice with a hypnotic dose of pentobarbital (45 mg/kg, i.p.) treatment, loganin at 50 mg/kg only
223 showed a tendency to reduce sleep latency with no significance (Fig. 4A). In terms of sleep duration,
224 loganin at 20 mg/kg and 50 mg/kg significantly prolonged sleep duration (*P < 0.05 at 20 mg/kg; **P
225 < 0.01 at 50 mg/kg, Fig. 4B) to a slightly lower extent than the positive control estazolam at 0.15 mg/kg.
226 Caffeine-induced sleep disturbance in pentobarbital- induced sleep test
227 Mice treated with caffeine, which have been used as an insomnia model, was used to explore the effect
228 of loganin on sleep disturbances in mice. As shown in Fig. 5, caffeine decreased sleep time significantly
229 (**P < 0.01, Fig. 5B), but did not affect the sleep latency (Fig. 5A). Loganin at 50 mg/kg attenuated
230 sleep latency (Fig. 5A), However it only showed a tendency to decrease sleep latency when combined
231 with caffeine. Loganin at 50 mg/kg increased sleep time (Fig. 5B) compared with the vehicle group,
232 but it had no effect on the decreased sleep time induced by caffeine in pentobarbital-treated mice.
233 PCPA-induced insomnia in pentobarbital-induced sleep test

l
234 As shown in Fig. 6, administration of PCPA could induce absolute insomnia by attenuating sleep time

a
235 (**P < 0.01, Fig. 6B), but it had no effect on sleep latency. Loganin (50 mg/kg) remarkably decreased

n
236 sleep latency (**P < 0.01, Fig. 6A) in the absence or presense of PCPA. Loganin (50 mg/kg) could also

o
237 extend decreased sleep time induced by PCPA (**P < 0.01, Fig. 6B).
238
239
240
i si
Synergistic hypnosis effect of low-dose loganin with 5-HTP in pentobarbital-induced sleep test

v
Furthermore, low-dose loganin and 5-HTP was given individually or together in pentobarbital-induced

r o
sleep test. Neither loganin (5 mg/kg) nor 5-HTP (2.5 mg/kg) individually affected the sleep latency or

P
241 sleep time induced by the hypnotic dose of pentobarbital (Fig. 7A); however, coadministration of
242 loganin (5 mg/kg) and 5-HTP (2.5 mg/kg) exhibited a synergistic effect of shortening sleep latency (*P
243 < 0.05, Fig. 7A) and prolonging sleep time significantly (**P < 0.01, Fig. 7B). The coadministration
244 significantly increased sleep onset (**P < 0.01, Tab. 2) in mice treated with a subhypnotic dose of
245 pentobarbital as well.
246 Sleep architecture in freely moving rats
247 The EEG and EMG recordings in freely moving rats showed that loganin clearly prolonged total sleep
248 time (**P < 0.01, Fig. 8A). Loganin (35 mg/kg, i.g.) prominently increased the ratio of NREM sleep
249 and shortened wakefulness (**P < 0.01, Fig. 8B), meanwhile, and it increased REM sleep (*P < 0.05,
250 Fig. 8B) at 6 h after intragastric administration compared with the vehicle group. As the positive control
251 group, estazolam also significantly increased NREM sleep and REM sleep (Fig. 8B). In addition, the
252 EEG power spectra showed that, similar to estazolam, loganin had almost no effect on the EEG power
253 density of NREM sleep (Fig. 8C and D), which is similar to estazolam.
254 The monoamine neurotransmitter levels in hippocampus, prefrontal cortex and striatum of mice
255 The result in 3.2.3 and 3.2.4 section suggested that, neurotransmitter might be involved in the hypnotic
256 activity of loganin. Therefore, the concentrations of monoamines and their metabolites in the mouse
257 hippocampus, prefrontal cortex and striatum 1 h after loganin (35 mg/kg) treatment were measured. As
258 shown in Tab. 3, in the prefrontal cortex, loganin significantly increased the ratio of 5-HIAA/5-HT,
259 while decreasing the concentration of 5-HT, and simultaneously decreased the concentration of DA
260 and its metabolite DOPAC. No changes in NE and 5-HIAA concentrations in the hippocampus,
261 prefrontal cortex or striatum occurred after loganin treatment.

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 Loganin exerts hypnotic effect

262 The activation of GABAergic neurons in VLPO of rats

263 To study the possible mechanism of the sleep-enhancing effects of loganin in rats, the activity of
264 loganin in the VLPO sleep center was investigated. GAD is a marker for GABAergic cells. c-Fos
265 expression is a cellular marker of neuronal activity. As shown in Fig. 9, at 1 h after administration,
266 loganin at a hypnotic dose of 35 mg/kg observably increased the ratio of c-Fos (+) in glutamic acid
267 decarboxylase (GAD) (+) neurons in the VLPO (**P < 0.01, Fig. 9D). These results suggested that
268 treatment with loganin significantly increased c-Fos expression in the GABAergic neurons of the
269 VLPO (**P < 0.01), as compared with the vehicle control.
270 DISCUSSION
271 Loganin, one of the major bioactive iridoid glycosides from Corni fructus, has been studied in many
272 aspects; however, the sedative and hypnotic effects have not been reported. The present study showed
273 that loganin significantly decreased locomotor activity in normal mice, potentiated the hypnotic effect
274 of sodium pentobarbital. These results indicated that loganin might show central nervous inhibition
275 activity.
276 Caffeine administration provides a simple and effective insomnia model, with difficulty in sleep
277 initiation (Alexeev et al., 2012). In the present study, loganin could not promote the decreased sleep
278 time induced by caffeine.

l
279 Previous studies have proved that 5-HT is the key regulator of sleep-wake. To investigate the role of

a
280 5-HT on the hypnotic activity of loganin, the effects of PCPA and 5-HTP were elevated. PCPA, a

n
281 blocker of tryptophan hydroxylase, has been shown to inhibit more than 95% of 5-HT biosynthesis

o
282 (Khanna et al., 1979). The effect of PCPA has been characterized by an initial sedative phase, which is

si
283 followed by a phase of reduced sleep and a recovery phase (Borbély et al., 1981). 5-HTP, the precursor
284
285
286
i
of 5-hydroxytryptamine (5-HT), prolonged pentobarbital-induced sleep time in a dose-dependent

v
manner (Zhao et al., 2004). In our study, PCPA-induced insomnia could be recovered by loganin (50

r o
mg/kg). Loganin (5 mg/kg) exerted synergistic hypnosis effect with 5-HTP, promoting sleep onset with

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287 a subhypnotic dose of pentobarbital and reducing sleep latency and prolonging sleep time induced by
288 a hypnotic dose of pentobarbital in mice. Taken together, we supposed that serotonergic system might
289 be involved in the mechanism underlying the hypnotic effects of loganin.
290 Neurotransmitters and their metabolites are known to play a significant role in the nervous system for
291 numerous organisms. Changes in the metabolism and levels of monoamine neurotransmitters have also
292 been associated with insomnia (Wei et al., 2014). Thus, we detected the effects of loganin on the levels
293 of 5-HT, DA, NE, and their metabolites 5-HIAA, DOPAC and HVA. Our results showed that loganin
294 decreased the concentration of 5-HT, DA and DOPAC, and increased the 5-HIAA/5-HT ratio in the
295 prefrontal cortex. These results suggested that the serotonergic and dopaminergic neurons might be
296 mainly involved in the mechanism of the hypnotic effects of loganin.
297 Mammalian sleep is composed of NREM and REM sleep alternation (Weber, 2017). Loganin
298 significantly increased NREM sleep and total sleep, and remarkably shortened wakefulness. Similar to
299 estazolam, loganin did not significantly reduce the EEG power of delta, which indicated that the EEG
300 power following administration of loganin was similar to that of natural sleep. This suggests that
301 loganin is compatible with physiological sleep. Therefore, loganin might be used for the treatment of
302 insomnia.
303 Many studies have shown that neurons in the VLPO region show c-Fos activation after sleep and
304 provide GABAergic innervation of the major monoamine arousal systems, which suggests that they
305 may be a necessary part of the brain circuitry (Gallopin et al., 2000). It was reported that the VLPO
306 cluster is a necessary component of sleep circuitry, without which NREM sleep is severely impaired.
307 The discharge rate of VLPO neurons increases just before or at the transition from wakefulness to
308 NREM sleep and decreases prior to the transition from NREM or REM sleep to wakefulness

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 Loganin exerts hypnotic effect

309 (Szymusiak et al., 1998). Results showed that loganin at a hypnotic dose of 35 mg/kg observably
310 increased the ratio of c-Fos (+) in glutamic acid decarboxylase (GAD) (+) neurons in the VLPO, which
311 indicated that the hypnotic effect of loganin may be mediated by activation of the sleep-promoting
312 GABAergic neurons in the VLPO.
313 In conclusion, this is the first study to confirm the sedative and hypnotic effects of loganin. Moreover,
314 5-HT, DA and GAD activation might be involved in the mechanism of sleep-promoting effect and sleep
315 architecture regulation of loganin. Other mechanisms involved in the sedative and hypnotic effects of
316 loganin deserve more attention and further investigation. Taken together, these findings suggest that
317 loganin might be a potential candidate for insomnia.

318 CONFLICT OF INTEREST

319 The authors declare that the research was conducted in the absence of any commercial or financial
320 relationships that could be construed as a potential conflict of interest

321 AUTHOR CONTRIUBUTIONS

322 J-KX and W-KZ designed the research and polished the manuscript. RS, YH, and H-YQ performed
323 the experiments. T-LL provided the EGG and EMG instruments and experimental guidance. RS, YH,
324 and YY drafted the manuscript. JH, W-WL, and C-YX revised the manuscript. All of the authors read
325

326
and approved the final manuscript.

ACKNOWLEDGMENTS

o n al
327
328
329
330
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This work was supported by G20 Engineering Innovation Research Project of Beijing Municipal

v
Science & Technology Commission (Nos. Z171100001717009, Z181100002218029), Chinese

o
Academy of Medical Sciences Basic Research Business Fund Youth Medical Talent Award Project

r
(No. 2018RC350019) and Distinguished Young Talents Project of Beijing University of Chinese
331

332

333
334
335
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Medicine (No. 2018-JYB-XJQ007).

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403

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Fig. 1. Chemical structure of loganin.

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404 Fig. 2. Chromatograms of monoamine neurotransmitter with HPLC-ECD assay. NE (tR= 2.7 min),
405 DHBA (tR= 4.3 min, internal standard), DOPAC (tR = 5.1 min), DA (tR = 6.4 min), 5-HIAA (tR = 7.9
406 min), HVA (tR = 13.5 min), 5-HT (tR = 15.7 min). (A-Blank solution, B-Solution of mixed standard
407 substances, C-Sample solution of rat brain).

408 Fig. 3. Inhibitory effect of loganin on locomotor activity in normal mice. 5-240 min after administration
409 of loganin (5, 20, 50 mg/kg, i.g.) or estazolam (0.15 mg/kg, i.g.), spontaneous locomotor activity was
410 monitored. Data are presented as Means ± S.E.M. (n=12). +P < 0.05, ++P < 0.01, loganin 5 mg/kg, vs.
411 the control group; XP < 0.05, XXP < 0.01, loganin 20 mg/kg, vs. the control group; *P < 0.05, **P <
412 0.01, loganin 50 mg/kg vs. the control group; #P < 0.05, ##P < 0.01, estazolam 0.15 mg/kg, vs. the
413 control group (Oneway ANOVA followed by Student-Newman-Keuls test).

414 Fig. 4. Effect of loganin on the hypnotic response to pentobarbital-induced sleep in mice. The sleep
415 latency (A) and the sleep time (B) were measured. Data are presented as mean ± S.E.M. (n=12). *P <
416 0.05, **P < 0.01, vs. the control group (Oneway ANOVA followed by Student-Newman-Keuls test).

417 Fig.5. Effects of loganin on caffeine-induced insomnia in pentobarbital-treated mice. The sleep latency
418 (A) and sleep time (B) were assessed. Data are presented as mean ± S.E.M. (n=10). *P < 0.05, **P <
419 0.01, vs. vehicle. (Oneway ANOVA followed by Student-Newman-Keuls test).

420 Fig.6. Effects of loganin on PCPA-induced insomnia in pentobarbital-treated mice. The sleep latency
421 (A) and sleep time (B) were assessed. Data are presented as mean ± S.E.M. (n=10). **P < 0.01 vs.
422 vehicle. (Student-Newman-Keuls test); ##P < 0.01, vs. PCPA alone (unpaired Student's t-test).

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 Loganin exerts hypnotic effect

423 Fig.7. Synergic effects of loganin with 5-HTP on sleep in pentobarbital-treated mice. The sleep latency
424 (A) and sleep time (B) were assessed. Data are presented as mean ±S.E.M. (n=12). *P < 0.05, **P <
425 0.01, vs. vehicle (Student-Newman-Keuls test); #P < 0.05, ##P < 0.01, vs. group treated loganin and 5-
426 HTP (unpaired Student's t-test).

427 Fig.8. Effect of loganin on the sleep architecture in freely moving rats. EEG and EMG recordings were
428 immediately applied 6 h after intragastric administration of loganin (35 mg/kg, i.g.) or vehicle, and
429 determined total sleep, sleep latency, wakefulness, REM sleep and NREM sleep. Power density
430 percentage of delta (0.5-4 Hz), theta (4-8 Hz), alpha (8-13 Hz) and beta bands (13-30 Hz) of NREM
431 sleep were examined. Data are presented as mean ± S.E.M. (n=4-5). *P < 0.05, **P < 0.01, vs. vehicle
432 (Oneway ANOVA followed by Least Significant Difference).

433 Fig.9. The effect of Loganin on c-Fos expression in the VLPO. The GABAergic neurons were labeled
434 by green, c-Fos was labeled by red. (A) An example of photomicrographs of the double-immunostained
435 neurons (scale bar=50 μm; original magnification 400×). Fluorescence micrographs of c-Fos and GAD
436 in normal mice (B) and mice treated with loganin 35 mg/kg (C) (scale bar=100 μm; original
437 magnification 200×). (D) Quantification of the number of c-Fos positive GABAergic neurons. Data
438 are represented as mean ± S.E.M. (n=5). **P < 0.01, vs. vehicle (unpaired Student's t-test).

439 Tab. 1. Effect of loganin on sleep onset of mice induced by subhypnotic dosage of pentobarbital. 25
440
441
442 square test).

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min after administration of distilled water, estazolam and loganin; pentobarbital (23 mg/kg, i.p.) was

a
given to mice. The number of mice falling asleep was recorded (n=12), *P < 0.05, vs. vehicle (chi-

443
444
445

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Tab. 2. Effect of loganin combined with 5-HTP on sleep onset of mice treated with hypnotic dosage

v
of pentobarbital (45 mg/kg, i.p.). The number of mice falling asleep was recorded (n=12). **P < 0.01,

o
vs. vehicle (chi-square test). #P < 0.05, vs. 2.5 mg/kg 5-HTP + 2.5 mg/kg loganin (chi-square test).

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P
446 Tab.3. The concentrations of NE, 5-HT, DA and their metabolites in the hippocampus, prefrontal
447 cortex and striatum after loganin treatment. Data are presented as Means ± S.E.M. (n=9). *P < 0.05,
**
448 P < 0.01, vs. control (unpaired Student's t-test); ND: not detectable.

449 Tab. 1.

Groups Dosage (mg/kg, i.g.) No. of falling asleep/total 450

Sleep onset (%)

Vehicle - 1/25 4.0 451

Estazolam 0.15 25/25 100.0*

452
Loganin 5 9/25 36.0*
453
20 15/25 60.0*
454
50 15/25 60.0*

455

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 Loganin exerts hypnotic effect

456 Tab. 2.

Groups Dosage (mg/kg) No. of falling asleep/total 457

Sleep onset (%)

Vehicle - 0/12 0.0 458

459
5-HTP 2.5 (i.p.) 2/12 16.7*
460
Loganin 5 (i.g.) 2/12 16.7*
461
5-HTP + Loganin 2.5 (i.p.) +5 (i.g.) 12/12 91.67** 462

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 Loganin exerts hypnotic effect

463 Tab. 3.

Group NE(ng/g) 5-HT(ng/g) 5-HIAA(ng/g) DA(ng/g) DOPAC(ng/g) 5-HIAA/5-HT DOPAC/DA

Control 673.5 ± 119.1 1115 ± 151.4 337.5 ± 49.21 1031 ± 698.4 417.3±145.4 0.29±0.02 0.58±0.18
PFC
Loganin 469.5 ± 72.33 687.7 ± 91.16* 241.3 ± 40.44 223.8 ± 56.61** 124.7±8.557** 0.35±0.05* 0.63±0.071

Control 263 ± 64.29 466.8 ± 99.63 322.1 ± 73.21 16052 ± 3693 1088 ± 190.4 0.78 ± 0.14 0.09 ± 0.005
STR
Loganin 265.6 ± 26.61 723 ± 80.8 605.2 ± 92.07* 15287 ± 1645 1284 ± 125.5 0.80 ± 0.07 0.09 ± 0.003

Control 478.4 ± 78.22 445.6 ± 61.47 346.8 ± 84.66 66.42 ± 16.19 ND 0.60 ± 0.10 ND
HIP
Loganin 515.9 ± 68.09 499.3 ± 62.03 382.7 ± 98.16 75.44 ± 29.22 ND 0.59 ± 0.11 ND

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