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Home , Valerenic acid, Valerian (herb)

Biomedicine & Pharmacotherapy 99 (2018) 913–920

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Biomedicine & Pharmacotherapy

journal homepage: www.elsevier.com/locate/biopha

Valerian/Cascade mixture promotes sleep by increasing non-rapid eye T movement (NREM) in rodent model ⁎ Hyeon-Son Choia, Ki-Bae Hongb, Sung Hee Hanc, Hyung Joo Suhd, a Department of Food Science and Technology, Seoul Women’s University, Seoul, 01797, Republic of Korea b Department of Biological Sciences and Environmental Science Program, Southern Illinois University-Edwardsville, Edwardsville, IL, 62026, United States c BK21Plus, College of Health Science, Korea University, Seoul, 02841, Republic of Korea d Department of Public Health Sciences, Korea University, Seoul, 02841, Republic of Korea

ARTICLE INFO ABSTRACT

Keywords: The aim of this study was to investigate the beneficial effect of /Cascade mixture on sleeping in mammal Sleep-promotion models. In -induced sleep model, Valerian, Cascade, and Valerian/Cascade mixture significantly Valerian reduced the latency time for sleeping, and total sleeping time effectively increased in these sample groups Cascade compared with the control. Valerian/Cascade mixture increased sleep duration by 37%. The mixture sig- Non-rapid eye movement nificantly increased the non-rapid eye movement (NREM) sleep time by 53% compared with the control, while GABA REM sleeping time was decreased by 33% with Valerian/Cascade mixture, in Electroencephalography (EEG) analysis, resulting in the increase of total sleep time and the decrease of awakening. This sleep-promoting effect was obvious in caffeine-induced awakening model; Valerian, Cascade, and the mixture significantly enhanced NREM and total sleep time, which were reduced by caffeine. Caffeine-induced increase of awakening was ef- fectively deceased to the normal level by these three samples. In particular, delta wave responsible for deep sleep in NREM was greatly increased by the mixture in both normal and caffeine-induced awake models. This sleep- promoting effect of Valerian/Cascade mixture was shown to be due to the upregulation of gamma-aminobutyric

acid A receptor (GABAAR). Valerian/Cascade mixture showed 91% binding capacity to GABAA-BZD receptor. Two compounds, and Xanthohumol, were shown to significantly contribute to the binding activity of Valerian/Cascade mixture on the GABA receptor.

1. Introduction make it hard to fall asleep or stay asleep, causing unrefreshing or non- restorative sleep [4]. Sleep is a resting state of mind and body characterized by the re- Chronic can give a serious take a toll on physical and mental duced sensory reactivity to stimuli and altered unconsciousness, dis- health. It has been implicated with the increase of risk of various health tinguished from wakefulness [1]. It plays an important role in sus- problems including cardiovascular disease, diabetes, obesity, and taining healthy life style [2]. Sleep helps most of the body systems to Alzheimer’sdisease[5]. The treatment of insomnia has been performed by restore normal functions. It is necessary to keep mood, and cognitive the use of such as benzodiazepin and [6]. However, performance, and specifically, contribute to restoration of the function these medications present many side effects such as daytime of the endocrine and immune systems [2]. fatigue, cognitive impairments, and the increase of other accidents [6]. Sleep occurs in two repeating periods known as rapid eye movement Many foods and nutritional intakes can affect duration and quality (REM) and non-rapid eye movement (NREM) [3]. These two periods are of sleep. A recent study reported the relationship of dietary supplements classified as clear behavioral states. REM is characterized by the de- and the sleep duration [7], suggesting that ingested nutrients play an synchronized and fast brain wave with intense dream, while NREM is important role in better sleep. In contrary, a stimulant such as caffeine, associated with the deep sleep with slow wave of brain, showing no eye an antagonist, inhibits sleep [8]. Meanwhile, several movement or no muscle paralysis [3]. Sleep disorders adversely affect extracts or resources have shown a positive effect on the improvement health, safety, and well-being. It causes fatigued feeling, with low en- of insomnia conditions [9]. They include Valerian ( offcinalis), ergy in daytime, depressed mood, weakened concentration, reduced (Humulus lupulus L.), (Matricaria chamomilla), and productivity at work. Insomnia is a common sleep disorder that can passion flower (Passiflora) [10–12].

⁎ Corresponding author. E-mail address: [email protected] (H.J. Suh). https://doi.org/10.1016/j.biopha.2018.01.159 Received 1 November 2017; Received in revised form 8 January 2018; Accepted 29 January 2018 0753-3322/ © 2018 Elsevier Masson SAS. All rights reserved. H.-S. Choi et al. Biomedicine & Pharmacotherapy 99 (2018) 913–920

Valerian is one of the most studied herbs for the sleep disorders. Its 2.4. Total sugar, total polyphenol, and total flavonoid analysis from the root extract have been traditionally used for the improvement of sleep extracts disorders in and USA [1,13]. However, its effects have been controversial from several studies [10,14]. Hops including Cascade Total sugar content in the extracts was analyzed using 5% have also been implicated with sleep promotion with effect and sulfuric acid. The extract (0.5 mL) and 5% phenol (0.5 mL) were [15]. Sleep onset is induced by sleep-promoting neurons which are mixed with concentrated sulfuric acid (2.5 mL), and stand for 20 min at believed to project GABA type A receptor, but inhibited by wakefulness- room temperature, after which the absorbance was measured at 490 nm promoting neurons such as cholinergic, histaminergic, by microplate reader (SpectraMax 340PC384, Molecular Devices, LLC., neurons [16]. Gamma-aminobutyric acid (GABA), a sleep-promoting Sunnyvale, CA, USA) with external calibration curve of glucose. For , responds to the GABA receptors and activates sleep total phenol content, the diluted extract was mixed with 50% Folin- promotion neurons, while wakefulness such as ser- Ciocalteu reagent (20 μL) to stand for 3 min at room temperature. otonin, histamine, and acetylcholine, respond to wakefulness-pro- Sodium carbonate solution (2%, 100 μL) was added to the mixture, and moting neurons to decrease sleep onset [16]. allowed to stand for 30 min. The reaction was monitored by measuring Therefore, the regulation of these neurotransmitters determines the the absorbance at 750 nm. For determination of the total flavonoid sleep onset and wakefulness. The activation of GABA receptors is be- content, Sodium nitrite solution (5% NaNO2,30μL) was added to the lieved to lead to sleep onset by inhibiting wakefulness promoting diluted extract sample, after which aluminum chloride solution (2% neurons [16]. These receptors are classified into two types, GABAA and AlCl3,60μL) and sodium hydroxide solution (4% NaOH, 100 μL) were GABAB, according to the degree of response to GABA. Sleeping aids added to the mixture. The reaction mixture was stand for 15 min at have been generally known to respond to GABAA receptors, while sti- room temperature in dark place. The color change was examined at the mulants usually work on acetylcholine systems [10]. The previous absorbance at 510 nm. Gallic acid and (Sigma Co., St. Louis, study showed the mixture of Valerian and Hops promotes sleep beha- MO, USA) were used as a standard marker for total phenol and total viors in fruit fly[17]. However, the mixture of these herbal extracts has flavonoid, respectively. been yet studied on the duration and quality of sleep in mammal models. In this study, the effect of the mixture or individual extract on 2.5. Animals duration and quality of sleep is described with EEG analysis in mammal models, mice and rat. The protocol for animal experiments was approved by the Korea University Animal Care Committee (KUIACUC-20170307-3, Seoul, Korea). All animals (4-week old ICR mice and 8-week old Sprague- 2. Materials and methods Dawley rats) were obtained from Daehan Biolink (Cheongju, Korea). All rodents maintained acrylic cages with a 12-h light/dark cycle and at- 2.1. Materials mospheric humidity of 50–60% at 24 °C. Food and water were freely provided. After an adaptation period (1 week), the mice were employed Valerian root (Valeriana officinalis) was obtained from Frontier Co. for pentobarbital-induced sleep experiment and the rats were used for Ltd. (CA, USA) and Cascade was gifted from Hongcheon Institute of EEG analysis and gene expression experiment. Medicinal Herb (Hongcheon, Korea). All other chemicals and reagents including Valerenic acid and Xanthohumol were purchased from 2.6. Pentobarbital-induced sleep experiment Sigma-Aldrich (St. Louis, MO, U.S.A.). The tests were performed between 1:00 and 5:00 p.m. after all mice were fasted for a day. All tested samples were suspended in 0.9% 2.2. Preparation of extracts physiological saline and orally administrated. The groups were ad- ministered Valerian (160 mg/kg), Cascade (40 mg/kg), and the Valerian roots (40 g) were extracted with 70% (1600 mL) in Valerian/Cascade mixture (160 mg/kg and 40 mg/kg each), and then, room temperature by stirring for 48 h. Cascade (40 g) was extracted 1 h later, pentobarbital (42 mg/kg) was injected into all mice (n = 6 per with 70% ethanol (800 mL) with a Soxhlet apparatus for 3 h, twice. group). (BDZ, 2.5 mg/kg) was used as a positive con- ® Then, all extracts were filtered by filter paper (Whatman qualitative trol. After injection, as long as mice were placed in individual cages, the filter paper, Grade 1) and evaporated at 40 °C using a rotary vacuum sleep latency time and sleep duration were measured. Animals that did evaporator (Buchi Rotavapor R-100). Valerian and Cascade con- not sleep within 15 min after pentobarbital injection were removed centrates were freeze-dried and stored at 4 °C. from the tests. The administration concentrations of the extracts were decided from preliminary experiments.

2.3. HPLC analysis of chemicals from the extracts 2.7. EEG acquisition and analysis

Acetoxyvalerenic acid and Valerenic acid from Valerian were de- Sprague-Dawley (SD) rats were anesthetized with isoflurane termined using Agilent HPLC series 1100 (Agilent, Waldbronn, (Troikaa Pharmaceuti-cals Ltd., Gujarat, India), then placed in a ste- Germany). A linear gradient of 40–80% acetonitrile in aqueous solution reotaxic instrument frame (Stoelting Co. Wood Dale, IL 60191, USA). A of 0.1% H3PO4 was performed over 20 min with a flow rate of 1.5 mL/ top area of the rat’s head was shaved and cleaned with disinfectant min. The analytical column was a Luna C18 150 × 4.6 mm, 5 μm re- under anesthesia. A midline skin incision was made over the skull to be versed phase column maintained at 25 °C (Phenomenex, Torrance, CA, exposed. The periosteum was removed and hemostasis was maintained U.S.A.). Acetoxyvalerenic acid and Valerenic acid were detected at by sterile cotton pellets. Four holes for positioning the electrodes, 218 nm. Xanthohumol from Cascade were analyzed using a Luna C18 equipped with mounting screw and socket, were drilled on the skull column (4.6 × 150 mm, 5 μm, Phenomenex, Torrance). The mobile surface corresponding to the frontal cortex, striatum, and hippocampus, phases consisted in solvent A (0.025% trifluoroacetic acid (TFA) in after which the electrodes were implanted with dental cement. All rats water), and solvent B (0.025% TFA in acetonitrile). The flow rate was were given an antibiotic for better recovery and protection of infection, adjusted to 1 mL/min. Separation was performed using gradient elution and individually housed in a thermos-regulating facility with food and 25% A and 75% B to 25 min, 5% A and 95% B to 35 min, 65% A and water. After recovery for 7 days, rats were divided into control and 35% B to 50 min. Detection was performed at 372 nm. treatment groups. The experiments were conducted between10:00 a.m.

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