Composition for improving sleep

Abstract

The problem to be solved by the present invention is to provide safe formulations, foods and beverages that increase non-REM sleep.SOLUTION: To provide a composition for activating DP1, a composition for improving sleeping, and a composition for promoting non-REM sleeping, containing one or more components selected from the group consisting of Eclipta thermalis, Thymus quinquecostatus, and Stellaria media, and to provide preparations, foods, and beverages containing the same.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to a sleep-improving composition that improves sleep quality. The present invention also relates to a sleep-improving composition that improves sleep quality by increasing non-REM sleep. [Background technology]

[0002] Sleep patterns change with age (Non-Patent Document 1). It is known that, among the sleep duration reductions associated with aging, there is a significant decrease in deep non-REM sleep (Non-Patent Document 2). It has also been reported that a decrease in non-REM sleep can cause delays in the removal of waste products from the brain (Non-Patent Document 3). For these reasons, there is a demand for improving sleep quality by promoting non-REM sleep.

[0003] The invention of Patent Document 1 provides a preparation, food, and beverage that suppresses the urge to urinate during sleep, which can improve symptoms of sleep disturbance caused by the urge to urinate at night. However, the invention of Patent Document 1 indirectly improves sleep by suppressing the urge to urinate during sleep, and does not directly improve sleep quality by promoting sleep, particularly non-REM sleep.

[0004] Non-Patent Document 4 reported that injection of prostaglandin D2 (PGD2) into the lateral ventricles of the brain increased the amount of non-REM sleep in wild-type (WT) mice, but not in mice lacking the PGD receptor (DP1). Non-Patent Document 5 reported that sleep induction in mice by PGD2 was mediated by adenosine A 2A It has been reported that PGD2 is mediated by both R-dependent and -independent systems. Non-patent document 6 reports that PGD2 accumulates during wakefulness, releases adenosine via DP1 receptors, and promotes non-REM sleep. Furthermore, activation of DP1 is known to increase non-REM sleep via an increase in intracellular cAMP.

[0005] Thus, PGD2, a lipid mediator, increases non-REM sleep via its receptor DP1. Therefore, the inventors searched for materials with DP1 activating properties in a plant extract library and found several DP1 activating materials. These materials are expected to improve sleep quality by increasing non-REM sleep.

[0006] To date, no food containing ingredients that increase non-REM sleep by activating DP1 has been put to practical use. The ingredients discovered by the present inventors have been safely used in foods, and the use of these ingredients can provide safe foods that increase non-REM sleep.

[0007] Another advantage is that DP1 is expressed in the leptomeninges and does not necessarily need to cross the blood-brain barrier, eliminating delivery issues.

[0008] Furthermore, activation of DP1 has various physiological activities other than sleep, such as suppressing liver inflammation and skin itching, repairing the barrier, preventing allergies, anti-inflammation in eosinophil-related diseases, neuroprotection, and blood pressure control, so it can also be expected to promote overall health. [Prior art documents] [Patent documents]

[0009] [Patent Document 1] Japanese Patent Application Laid-Open No. 2005-343809 [Non-patent literature]

[0010] [Non-Patent Document 1] Li J, Vitiello MV, Gooneratne NS. Sleep in Normal Aging. Sleep Med Clin. 2018 Mar;13(1):1-11. [Non-patent document 2] Mander BA, Winer JR, Walker MP. Sleep and Human Aging. Neuron. 2017 Apr 5;94(1):19-36. [Non-patent document 3] Mander BA. Local Sleep and Alzheimer's Disease Pathophysiology. Front Neurosci. 2020 Sep 23;14:525970. [Non-patent document 4] Mizoguchi A, Eguchi N, Kimura K, Kiyohara Y, Qu WM, Huang ZL, Mochizuki T, Lazarus M, Kobayashi T, Kaneko T, Narumiya S, Urade Y, Hayaishi O. Dominant localization of prostaglandin D receptors on arachnoid trabecular cells in mouse basal forebrain and their involvement in the regulation of non-rapid eye movement sleep. Proc Natl Acad Sci US A. 2001 Sep 25;98(20):11674-9. [Non-Patent Document 5] Zhang BJ, Huang ZL, Chen JF, Urade Y, Qu WM. Adenosine A2A receptor deficiency attenuates the somnogenic effect of prostaglandin D2 in mice. Acta Pharmacol Sin. 2017 Apr;38(4):469-476. [Non-patent document 6] Yoshihiro Urade, Sleep regulation mechanism and food components that control sleep, Chemistry and Biology, Vol. 51, No. 11, 2013, pp. 754-762 Summary of the Invention [Problem to be solved by the invention]

[0011] The problem that the present invention aims to solve is to provide safe formulations, foods, and beverages that increase non-REM sleep. [Means for solving the problem]

[0012] The formulations, foods, and beverages of the present invention contain one or more ingredients selected from the group consisting of hemp seeds, Takasaburo, Ibuki thyme, tangerine pomace, chickweed, and spike pepper. More specifically, the present invention has the following configuration. [Embodiment 1] A composition for activating DP1, comprising one or more ingredients selected from the group consisting of hemp seeds, Takasaburo, Ibuki thyme, tangerine pomace, chickweed, and spike pepper. [Embodiment 2] A sleep-improving composition containing one or more ingredients selected from the group consisting of hemp seeds, Takasaburo, Ibuki thyme, mandarin orange pomace, chickweed, and spike pepper. [Embodiment 3] A composition for promoting non-REM sleep, comprising one or more ingredients selected from the group consisting of hemp seeds, Takasaburo, Ibuki thyme, mandarin orange pomace, chickweed, and spike pepper. [Effects of the Invention]

[0013] The formulations, foods, and beverages of the present invention improve sleep quality and particularly promote non-REM sleep by activating DP1. [Brief explanation of the drawings]

[0014] [Figure 1] FIG. 1 shows the evaluation of DP1 activation ability by luciferase assay. [Figure 2] FIG. 1 shows a concentration-dependent increase in intracellular cAMP of plant extracts. [Figure 3] FIG. 1 shows the results of evaluation of the ability to suppress inflammation in the liver. DETAILED DESCRIPTION OF THE INVENTION

[0015] The present inventors have identified hemp seeds, Eucalyptus thunb., Thymus vulgare, mandarin orange pomace, chickweed, and spike pepper as DP1 activators that promote sleep, particularly non-REM sleep, from among numerous candidate substances. Therefore, the sleep-improving composition and non-REM sleep-improving composition of the present invention contain one or more components selected from the group consisting of hemp seeds, Eucalyptus thunb., Thymus vulgare, mandarin orange pomace, chickweed, and spike pepper.

[0016] The hemp seeds used in the present invention are commercially available from various spice manufacturers, such as S&B Foods. The hemp seeds used in the present invention can be extracted by known methods, such as organic solvents. In the examples of this application, hemp seeds from Yasma Co., Ltd. were extracted with 70% ethanol. In this specification, the term "hemp seeds" may be interchangeably referred to as "hemp seed extract" or "hemp seed extract." The meaning of each term will be clear to those skilled in the art depending on the context. The Takasaburo used in the present invention is commercially available, for example, as an extract from Ryuei Soken Co., Ltd. The Takasaburo used in the present invention can be extracted by known methods, such as using organic solvents, and in the examples of this application, the plant raw material (dried product) obtained from Shinwa Bussan Co., Ltd. was extracted with 70% ethanol. In this specification, the term "Takasaburo" may be alternatively referred to as "Takasaburo extract" or "Takasaburo extract." The meaning of each term will be clear to those skilled in the art depending on the context. Thymus quinquefolia (Thymus quinquefolia) used in the present invention is widely available commercially as a seedling. Thymus quinquefolia (Thymus quinquefolia) used in the present invention can be extracted by known methods, such as using an organic solvent. In the present examples, the plant raw material (dried product) obtained from Shinwa Bussan Co., Ltd. was extracted with 70% ethanol. In this specification, the term "Thymus quinquefolia" may be alternatively referred to as "Thymus quinquefolia extract" or "Thymus quinquefolia extract." The meaning of each term will be clear to those skilled in the art depending on the context. The mandarin orange pomace used in the present invention may include, for example, the juice residue, seeds, peel, etc. of Satsuma mandarins. The mandarin orange pomace used in the present invention can be extracted by known methods, such as using organic solvents. In the examples of the present application, mandarin orange pomace obtained from Ehime Prefectural Agricultural Cooperative Association was extracted with 99.5% ethanol. In the present specification, the term "mandarin orange pomace" may be alternatively referred to as "mandarin orange pomace extract" or "mandarin orange pomace extract." The meaning of each term will be clear to those skilled in the art depending on the context. The chickweed used in the present invention is a plant of the genus Stellaria in the family Caryophyllaceae. There are approximately 120 closely related species worldwide, with approximately 18 species in Japan. Furthermore, "chickweed" is sold as one of the seven herbs of spring. Furthermore, the chickweed used in the present invention can be extracted by known methods, such as organic solvents. In the examples of this application, the plant material (dried product) obtained from Shinwa Bussan Co., Ltd. was extracted with 99.5% ethanol. In this specification, the term "chickweed" may be interchangeably referred to as "chickweed extract" or "chickweed extract." The meaning of each term will be clear to those skilled in the art depending on the context. The spike pepper (Machiko) used in the present invention is a flowering plant of the Piperaceae family, and its leaves, stems, fruits, etc. can be used. The spike pepper used in the present invention can be extracted by known methods, such as using organic solvents. In the present examples, the fruits obtained from Shinwa Bussan Co., Ltd. were extracted with 99.5% ethanol. In this specification, the term "spike pepper" may be interchangeably referred to as "spike pepper extract" or "spike pepper extract." The meaning of each term will be clear to those skilled in the art depending on the context.

[0017] The composition of the present invention containing one or more components selected from the group consisting of hemp seeds, Eucalyptus thunb., Thymus vulgare, mandarin orange pomace, chickweed, and spike pepper can be formulated into dosage forms commonly used in foods, quasi-drugs, and pharmaceuticals, such as solids, semi-solids, and liquids, depending on the intended use of the composition. Preferred dosage forms are solids and liquids. Specific examples include tablets (including orally disintegrating tablets, chewable tablets, effervescent tablets, lozenges, and jelly drops), granules, fine granules, powders, hard capsules, soft capsules, dry syrups, liquids (including drinks, suspensions, and syrups), gels, liposomes, extracts, tinctures, lemonades, and jellies. Among these, tablets, granules, fine granules, powders, hard capsules, soft capsules, and liquids are preferred.

[0018] In addition, the composition of the present invention containing one or more ingredients selected from the group consisting of hemp seeds, Takasaburo, Ibuki thyme, mandarin orange pomace, chickweed, and spike pepper may be appropriately blended with ingredients normally used in foods, quasi-drugs, and pharmaceuticals depending on the use or dosage form of the composition. Ingredients that can be added include, for example, amino acids, various oils and fats, fatty acids, alcohols, sugars, polymeric compounds such as gums, surfactants, coloring agents, preservatives, antibacterial and disinfecting agents, pH adjusters, chelating agents, enzyme components, essential oil components, plant-derived raw materials, naturally derived raw materials, as well as cell activators, tonics, excipients, thickeners, stabilizers, preservatives, isotonicity agents, binders, dispersants, adsorbents, lubricants, disintegrants, disintegration aids, flavorings, sweeteners, humectants or humidity regulators, moisture-proofing agents, flavorings or fragrances, fragrances, refreshing agents, antioxidants, reducing agents, solubilizers, dissolution aids, foaming agents, thickeners or viscosifiers, solvents, bases, fluidizing agents, emulsifiers, plasticizers, buffers, glossing agents, coating agents, etc. [Example]

[0019] [Example 1]

[0020] (1) Primary screening by luciferase assay To identify DP1 activators, we performed a primary screening using plant extracts (230 samples of 99.5% ethanol extracts and 760 samples of 70% ethanol extracts) as follows. The samples were dissolved in DMSO. Preparation of plant extracts To evaluate the DP1 activation ability of various plants, we prepared plant extracts. First, dried plant material was pulverized using a Wonder Blender grinder (Osaka Chemical Co., Ltd.). The pulverized plant material was then subjected to the following procedure using an ASE-200 high-speed solvent extractor (Nippon Dionex Co., Ltd.). Approximately 10 g of the pulverized plant material was filled to approximately 90% of a 22 mL extraction cell. Extraction was performed using 99.5% or 70% ethanol as the solvent at room temperature and a pressure of 1500 psi. The extract was then dried under reduced pressure using a centrifugal evaporator to obtain a dried extract. The weight of the dried extract thus obtained was measured, redissolved in DMSO, and subjected to the DP1 activation ability evaluation test. HEK293 cells, a human embryonic kidney cell line, were transfected with the vector pcDNA3.1-+N-HA-DP1 (using GenScript's artificial gene synthesis service) to obtain DP1-expressing cells (HEK293 / DP1 cells). The vector pcDNA3.1-+N-HA-DP1 was prepared by inserting the DP1 ORF sequence into the KpnI / BamHI site of the cloning vector pcDNA3.1-+N-HA (GenScript, Cat. No. OHu22852C). These cells were then subjected to monoclonalization by limiting dilution. The resulting DP1-expressing monoclonal cells were then transfected with the vector pGL4.29[luc2P / CRE / Hygro] (Promega). This vector encodes luciferase downstream of the cAMP response element CRE. Upon activation of DP1, the transfected cells exhibit increased luciferase expression due to increased intracellular cAMP levels. These cells were also monoclonalized by limiting dilution to generate monoclonal cells expressing DP1 and luciferase (HEK293 / DP1 / Luc cells), which were used for evaluation. As a control, HEK293 cells were transfected with the vector pGL4.29[luc2P / CRE / Hygro] alone, and then monoclonalized HEK293 / Luc cells were generated. 2 × 10 HEK293 / DP1 / Luc cells or control cells 4 Cells were seeded in a 96-well plate at 90 μL / well in DMEM medium containing 1% FBS. After 24 hours of incubation, the plant extract was added to a final concentration of 50 μg / mL, and prostaglandin D2 (PGD2), an endogenous ligand of DP1, was added as a positive control to a final concentration of 20 nM. The cells were then incubated for 4 hours. Luminescence was then measured using the Bright-Glo Luciferase Assay System (Promega) to assess luciferase expression. Measurements were performed according to the accompanying instructions. The luminescence values ​​of the HEK293 / DP1 / Luc cells were normalized using the luminescence values ​​of the control cells to detect increased luciferase expression via DP1 activation. The results showed that the addition of hemp seeds, Takasaburo, Ibuki thyme, mandarin orange pomace, chickweed, and Machiko (spike pepper) increased luminescence levels compared to the control (Figure 1), suggesting that these plant extracts have the effect of activating DP1.

[0021] (2) Secondary screening by intracellular cAMP evaluation DP1 is a G-type G protein-coupled receptor, and the mode of signal transduction upon DP1 activation is known to be an increase in intracellular cAMP. Therefore, samples that were found to have the potential to activate DP1 in the primary screening were evaluated for their ability to increase intracellular cAMP. The HEK293 / DP1 cells prepared in the above primary screening were used in the following experiments. HEK293 cells were used as control cells. The prepared HEK293 / DP1 cells or control cells were seeded in 384-well plates at 5000 cells / 45 μL / well using DMEM medium containing 1% FBS. After 24 hours of incubation, plant extracts were added to final concentrations of 50, 25, 12.5, 6.3, 3.1, and 1.6 μg / mL, and PGD2 (as a positive control) was added to final concentrations of 16, 3.2, 0.64, 0.13, 0.026, 0.0051, and 0.0010 nM, and the cells were incubated for 15 minutes. Intracellular cAMP was then measured using cAMP-Glo ​​(Promega) according to the accompanying instructions. Because luminescence intensity decreases with increasing intracellular cAMP concentration in this test system, samples that showed a concentration-dependent decrease in luminescence intensity were considered to have increased intracellular cAMP in a concentration-dependent manner. Samples that showed an increase in intracellular cAMP in HEK293 / DP1 cells but did not show an increase in intracellular cAMP in control cells were determined to have the ability to activate DP1. The results showed that the addition of hemp seeds, Eucalyptus thunb., Thymus vulgare, mandarin orange pomace, chickweed, and spike pepper caused a concentration-dependent decrease in luminescence (Figures 2A and 2B), demonstrating that these plant extracts increased intracellular cAMP levels via activation of DP1.

[0022] (3) Third screening using anti-inflammatory activity as an index In recent years, it has been reported that activation of DP1 suppresses inflammation in hepatic stellate cells. For plant extracts confirmed to have DP1 activation ability through primary and secondary screening, the ability to activate DP1 can be more stringently confirmed by assessing whether or not they have the ability to suppress inflammation in hepatic stellate cells. Therefore, we performed an inflammation assessment using hepatic stellate cells as follows. 24-well plates were coated with poly-L-lysine (ScienCell) at 2 μg / cm 2 After coating for 1 hour to achieve a plate thickness of 100 μm, the plate was washed twice with sterile water. Human liver-derived hepatic stellate cells (HHSteC) (ScienCell) were added to the plate at a density of 5 × 10 4Cells were seeded at 500 μL / well in Stellate Cell Medium (ScienCell) containing 2% FBS. After 24 hours of culture, the cells were washed with serum-free medium, replaced with 500 μL / well of serum-free medium, and cultured for an additional 24 hours. The test sample was added to the cells at a final concentration of 10 μg / mL, and the DP1 agonist BW245C (Cayman Chemical) was added as a positive control at 0.5 μM, and the cells were cultured for 5 minutes. TNFα (Sigma) was also added to the cells at a final concentration of 10 ng / mL, and the cells were cultured for an additional hour. RNA from the cells was extracted using the RNeasy Mini Kit (QIAGEN) according to the manufacturer's instructions. cDNA was prepared from 100 ng of the extracted RNA using the PrimeScript RT reagent Kit (TaKaRa Bio) according to the manufacturer's instructions. Real-time PCR using probes was performed to examine the expression levels of TNFα and CCL2 (R&D Systems) using the prepared cDNA. The primers and probe used were TaqMan probe (Thermo Fisher Scientific), and the reverse transcriptase was Premix Ex Taq (Perfect Real Time) (TaKaRa Bio). The reaction mixture was prepared according to the attached protocol. The real-time PCR reaction conditions were Stage 1 initial denaturation (95°C for 30 seconds) once, followed by Stage 2 PCR reaction (95°C for 5 seconds, 60°C for 20 seconds) repeated 40 times. The expression level of 18S rRNA was also measured as an endogenous control, and expression levels were compared using the ΔΔCt method. The addition of TNFα induces acute inflammation in hepatic stellate cells, resulting in increased expression of TNFα and CCL2. This increase in TNFα and CCL2 expression is known to be suppressed by the activation of DP1. Therefore, samples that suppressed the increase in TNFα and CCL2 expression due to the addition of TNFα were considered to have DP1 activating activity. These results indicated that hemp seeds, Eucalyptus thunb., Thymus vulgare, mandarin orange pomace, chickweed, and spike pepper (Machiko) suppressed the elevated expression of TNFα and CCL2 during inflammation (Figures 3A and 3B), further supporting the assertion that these plant extracts have the ability to activate DP1.

[0023] (4) Conclusion As mentioned above, activation of DP1 is known to promote non-REM sleep (Non-Patent Document 6). Hemp seeds, Takasaburo, Ibuki thyme, mandarin orange pomace, chickweed, and spike pepper (Machiko), which were confirmed to have DP1 activating ability through primary, secondary, and tertiary screening, promote sleep, particularly non-REM sleep. [Industrial Applicability]

[0024] By utilizing the composition of the present invention, it is possible to provide preparations, foods, and beverages that promote sleep, particularly non-REM sleep.

Claims

1. A composition for activating DP1, comprising one or more components selected from the group consisting of Takasaburo, Thymus vulgare, and Chickweed.

2. A sleep-improving composition comprising one or more ingredients selected from the group consisting of Eucalyptus globulus, Thymus vulgaris, and Chickweed.

3. A composition for promoting non-REM sleep, comprising one or more ingredients selected from the group consisting of Eucalyptus globulus, Thymus vulgare, and Chickweed.

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