Composition for histamine H1 receptor antagonism

Abstract

The present invention provides histamine H1 receptor antagonism-based uses for various uses (histamine H1 receptor antagonism uses, uses to improve sleep quality and / or modulate balance of the autonomic nervous system, and uses to improve sleep quality and / or modulate balance of the autonomic nervous system. And a use for preventing the onset of autonomic nervous system dysfunction or a symptom caused thereby or for improving autonomic nervous system dysfunction or a symptom caused thereby). The present invention relates to: a composition comprising, as an active ingredient, at least one substance selected from the group consisting of indole-3-lactic acid, alpha-Asp-Phe, Val-Arg, Asp-Gly-Glu, and salts thereof, or a lactic acid bacteria culture supernatant (not containing lactic acid bacteria cells) of a lactic acid bacteria belonging to any genus selected from the group consisting of the genus Lactobacillus, the genus Lactobacillus, and the genus Lactobacillus; the liquid supernatant contains at least one substance selected from the group consisting of indole-3-lactic acid, alpha-Asp-Phe, Val-Arg, Asp-Gly-Glu, and salts thereof. The liquid supernatant contains at least one substance selected from the group consisting of indole-3-lactic acid, alpha-Asp-Phe, Val-Arg, Asp-Gly-Glu.

Description

Technical Field

[0001] This disclosure relates to compositions for histamine H1 receptor antagonism. This disclosure also relates to compositions based on histamine H1 receptor antagonism for, for example, improving sleep quality and / or regulating the balance of the autonomic nervous system. This disclosure further relates to compositions based on histamine H1 receptor antagonism for preventing the onset of autonomic nervous system dysfunction or symptoms caused thereof, or for improving autonomic nervous system dysfunction or symptoms caused thereof. Background Technology

[0002] G protein-coupled receptors (hereinafter also referred to as "GPCRs") recognize a wide variety of molecules as ligands and participate in the regulation of a broad range of signaling and cellular functions. Histamine receptors are also GPCRs. It has been frequently reported that lactic acid bacteria and intestinal bacterial metabolites induce neural activity via GPCRs.

[0003] For example, it has been reported that lactic acid bacteria belonging to the genus *Lactobacillus* can act on the central and autonomic nervous systems via histamine receptors. Specifically, Non-Patent Literature 1 shows that administration of the lactic acid bacteria *Lactobacillus johnsonii* La1 (LJLa1) to the duodenum of rats resulted in parasympathetic nervous system activity being dominated by histaminergic H3 receptors. Non-Patent Literature 2 also shows that administration of the lactic acid bacteria *Lactobacillus paracasei* ST11 (NCC2461) to the duodenum of rats resulted in sympathetic nervous system activity being dominated by histaminergic H1 receptors. These functions are all based on the histamine receptor agonist activity of lactic acid bacteria.

[0004] Histamine H1 receptor antagonists (antagonists) are known to improve sleep. For example, a sleep-improving drug (drug) containing diphenhydramine hydrochloride, a histamine H1 receptor antagonist, as its active ingredient is commercially available. This drug exerts a hypnotic and sedative effect and induces sleep similar to natural sleep by blocking the binding of histamine (a neurotransmitter in the brain) to histamine receptors. However, this drug has been reported to exhibit side effects such as skin rashes, redness, and itching, as well as gastrointestinal symptoms such as stomach pain, nausea, and vomiting (Non-Patent Literature 3).

[0005] Therefore, there is a need for safer sleep-improving compositions that can also be taken as supplements. As active ingredients for such sleep-improving compositions, examples have been proposed, such as lactic acid bacteria cells or cultures thereof (Patent Documents 1 to 5), and amino acids, peptides, and phenyllactic acid produced by lactic acid bacteria (Patent Documents 6 to 7).

[0006] Patent Document 1 discloses a sleep-promoting composition comprising microorganisms or cultures thereof belonging to the genera *Lactococcus*, *Leuconostoc*, *Pediococcus*, *Weissella*, or *Bifidobacterium*. Paragraph

[0019] of this document discloses that the active ingredient of the composition comprises cells of any of these microorganisms (whether they are living or dead cells), or cytoplasm or cell wall fractions of cells treated by enzymatic or physical methods.

[0007] Patent Document 2 discloses a sleep-promoting composition comprising one or more lactic acid bacteria that produce lactic acid classified as other than L-type based on their optical rotation. Paragraph

[0019] of this document discloses that the active ingredient of the composition comprises cells of any of these lactic acid bacteria (whether they are living or dead cells), or cytoplasm or cell wall fractions of cells treated by enzymatic or physical methods.

[0008] Patent Document 3 discloses a blood tryptophan concentration elevation inhibitor comprising, as an active ingredient, cells of lactic acid bacteria belonging to the genus *Lactobacillus* (preferably *Lactobacillus casei*), their processed form, or fermentation products containing these. Paragraphs

[0010] and

[0020] of this document disclose that the blood tryptophan concentration elevation inhibitor inhibits excessive increases in blood tryptophan levels, improves symptoms of niacin deficiency, and is particularly suitable for stress-induced sleep disorders and loss of appetite. As described above, the active ingredient of the blood tryptophan concentration elevation inhibitor comprises lactic acid bacteria cells, their processed form, or fermentation products containing these. Paragraph

[0014] of this document describes that the cell processed form is obtained by subjecting the lactic acid bacteria to specific treatments (e.g., ultrasonic treatment, enzyme treatment, or heat treatment), and discloses cell decomposition products as an example.

[0009] Patent Document 4 discloses a serotonin deficiency improver after stress load relief, comprising Lactobacillus casei cells, its treatments, or ferments containing these as active ingredients. This improver can improve sleep disorders caused by serotonin deficiency after stress load relief. As described above, the active ingredient of the serotonin deficiency improver comprises Lactobacillus casei cells, its treatments, or ferments containing these. Paragraph

[0014] of this document describes that the cell treatments are obtained by subjecting lactic acid bacteria to specific treatments (e.g., ultrasonic treatment, enzyme treatment, or heat treatment), and discloses cell decomposition products as examples.

[0010] Patent document 5 discloses a sleep improver comprising a combination of three components: lactic acid bacteria containing the Lactobacillus helveticus strain LBH MIKI-020, a ferment produced using the lactic acid bacteria, and cell fragments of the lactic acid bacteria. Paragraph

[0033] of this document discloses that the culture supernatant is discarded during the preparation of the lactic acid bacteria (i.e., when collecting the lactic acid bacteria cells); that is, the culture supernatant cannot serve as an active ingredient in the sleep improver.

[0011] Patent document 6 discloses the production of free amino acids and / or functional peptides such as Xaa-Pro and Xaa-Pro-Pro (Xaa represents any amino acid) by fermenting Lactobacillus helveticus strain CM4 (accession number FERM BP-6060) using milk protein as a substrate, and the fermented milk or its processed products containing these free amino acids and / or functional peptides are used as autonomic nervous system regulators or stress relievers, etc.

[0012] Patent document 7 discloses that the culture supernatant of lactic acid bacteria (Lactobacillus plantarum strain SAM2446, FERM ABP-10438) contains phenyllactic acid (D-phenyllactic acid, L-phenyllactic acid), and based on the effect of phenyllactic acid, the composition containing phenyllactic acid is effective in treating diseases and conditions related to autonomic nervous system activity (e.g., autonomic nervous system dysfunction such as anxiety, loss of appetite, fatigue and insomnia).

[0013] Existing technical documents

[0014] Patent documents

[0015] Patent Document 1: JP2021-123561A

[0016] Patent Document 2: JP2021-123562A

[0017] Patent Document 3: WO2017 / 047776

[0018] Patent Document 4: WO2017 / 047777

[0019] Patent Document 5: JP2016-104802A

[0020] Patent Document 6: WO2012 / 063826

[0021] Patent Document 7: WO2008 / 120713

[0022] Non-patent literature

[0023] Non-patent document 1: Tanida M et al., Effects of intraduodenal injection ofLactobacillus johnsonii La1 on renal sympathetic nerve activity and bloodpressure in urethane-anesthetized rats, Neuroscience Letters.2005: 389(2):109-14

[0024] Non-patent document 2: Tanida M et al., Effects of probiotic strain ofLactobacillus Paracasei St11 (Ncc2461) on autonomic nerve activities, bloodpressure and appetite in rats, Curr Top Nutr Res.2007; 5: 157-64

[0025] Non-patent literature 3: Website of Drewell (sleep-improving drug), SSP Co., Ltd.; https: / / www.ssp.co.jp / drewell /

[0026] Non-patent document 4: Asuka Inoue et al., Shinki GPCR kasseika kensyutu ho woriyou shita orphan juyotai no ligand no dotei [Identification of Ligands for Orphan Receptors Using a Novel GPCR Activation Detection Method], Seikagaku, Journal of Japanese Biochemical Society, Vol. 85, No. 11, pp. 1029-1033,2013, The Japanese Biochemical Society.https: / / www.jbsoc.or.jp / seika / wp-content / uploads / 2014 / 06 / 85-11-13.pdf Summary of the Invention

[0027] The problem the invention aims to solve

[0028] The present disclosure aims to provide a histamine H1 receptor antagonist composition, which can also be used as a food or beverage. Another object of the present disclosure is to provide a composition based on histamine H1 receptor antagonism for, for example, improving sleep quality and / or regulating the balance of the autonomic nervous system. Yet another object of the present disclosure is to provide a composition based on histamine H1 receptor antagonism for preventing the onset of autonomic nervous system dysfunction or symptoms caused therefrom, or for improving autonomic nervous system dysfunction or symptoms caused therefrom.

[0029] Solution for solving the problem

[0030] The inventors conducted extensive research to address the aforementioned problems and discovered that indole-3-lactic acid, α-Asp-Phe, Val-Arg, and Asp-Gly-Glu all exhibit antagonistic activity against histamine H1 receptors. Based on this discovery, the inventors proposed that compositions containing at least one of these as active ingredients are suitable for use as histamine H1 receptor antagonists, as sleep-improving compositions based on histamine H1 receptor antagonistic activity, and / or as compositions for regulating the balance of the autonomic nervous system, and further as compositions for preventing or improving the onset of autonomic nervous system dysfunction or its symptoms. This invention is thus completed.

[0031] This disclosure includes the following implementation schemes.

[0032] (I) Histamine H1 receptor antagonist composition

[0033] (I-1) A composition for histamine H1 receptor antagonism comprising at least one selected from indole-3-lactic acid, α-Asp-Phe, Val-Arg, Asp-Gly-Glu and their salts as an active ingredient.

[0034] (I-2) The histamine H1 receptor antagonist composition according to (I-1) comprises, as an active ingredient, a culture supernatant (containing no lactic acid bacteria cells) of lactic acid bacteria belonging to any genus selected from the group consisting of Lacticaseibacillus, Levilactobacillus and Lactiplantibacillus, said supernatant containing at least one selected from indole-3-lactic acid, Val-Arg, Asp-Gly-Glu and their salts.

[0035] (I-3) The histamine H1 receptor antagonist composition according to (I-2),

[0036] Among them, the lactic acid bacteria belonging to the genus Lactobacillus is Lactobacillus casei;

[0037] The lactic acid bacteria belonging to the genus *Levilactobacillus* is *Levilactobacillus brevis*; and

[0038] Lactic acid bacteria belonging to the genus *Lactiplantibacillus* are selected from at least one of *Lactiplantibacillus pentosus* and *Lactiplantibacillus plantarum*.

[0039] (I-4) Use of at least one of indole-3-lactic acid, α-Asp-Phe, Val-Arg, Asp-Gly-Glu and their salts for the manufacture of a composition for histamine H1 receptor antagonism.

[0040] (I-5) Use of a culture supernatant (containing no lactic acid bacteria cells) of any genera of lactic acid bacteria selected from the group consisting of Lactobacillus, Lactobacillus and Lactobacillus for the manufacture of a histamine H1 receptor antagonist composition, said supernatant containing at least one selected from indole-3-lactic acid, Val-Arg, Asp-Gly-Glu and their salts.

[0041] (I-6) According to the uses described in (I-5),

[0042] Among them, the lactic acid bacteria belonging to the genus Lactobacillus is Lactobacillus casei;

[0043] The lactic acid bacteria belonging to the genus *Lactobacillus* is *Lactobacillus shortsum*; and

[0044] Lactic acid bacteria belonging to the genus *Lactobacillus* are selected from at least one of *Lactobacillus pentosus* and *Lactobacillus plantarum*.

[0045] (I-7) A method for imparting histamine H1 receptor antagonism to at least one composition selected from the group consisting of dietary substances, drugs, and quasi-drugs.

[0046] The method includes blending at least one selected from indole-3-lactic acid, α-Asp-Phe, Val-Arg, Asp-Gly-Glu, and their salts during the manufacture of the composition.

[0047] (I-8) A method for imparting histamine H1 receptor antagonism to at least one composition selected from the group consisting of dietary substances, drugs, and quasi-drugs.

[0048] The method includes, during the manufacture of the composition, mixing a lactic acid bacteria culture supernatant (containing no lactic acid bacteria cells) of any genera of lactic acid bacteria selected from the group consisting of *Lactobacillus*, *Lactobacillus*, and *Lactobacillus*, said supernatant containing at least one selected from indole-3-lactic acid, Val-Arg, Asp-Gly-Glu, and their salts.

[0049] (I-9) According to the method described in (I-8),

[0050] Among them, the lactic acid bacteria belonging to the genus Lactobacillus is Lactobacillus casei;

[0051] The lactic acid bacteria belonging to the genus *Lactobacillus* is *Lactobacillus shortsum*; and

[0052] Lactic acid bacteria belonging to the genus *Lactobacillus* are selected from at least one of *Lactobacillus pentosus* and *Lactobacillus plantarum*.

[0053] (II) Sleep-improving compositions

[0054] (II-1) A sleep-improving composition comprising at least one selected from indole-3-lactic acid, α-Asp-Phe, Val-Arg, Asp-Gly-Glu and their salts as an active ingredient.

[0055] (II-2) A sleep-improving composition comprising at least one selected from α-Asp-Phe, Asp-Gly-Glu and their salts as an active ingredient.

[0056] (II-3) A sleep-improving composition comprising, as an active ingredient, a culture supernatant (containing no lactic acid bacteria cells) of lactic acid bacteria belonging to any genus selected from the group consisting of *Lactobacillus*, *Lactobacillus*, and *Lactobacillus*, said supernatant containing at least one selected from indole-3-lactic acid, Val-Arg, Asp-Gly-Glu, and their salts.

[0057] (II-4) A sleep-improving composition comprising, as an active ingredient, a culture supernatant (containing no lactic acid bacteria cells) of lactic acid bacteria belonging to any genus selected from the group consisting of Lactobacillus, Lactobacillus and Lactobacillus, said supernatant containing at least one selected from Val-Arg, Asp-Gly-Glu and their salts.

[0058] (II-5) The sleep-improving composition according to (II-3) or (II-4),

[0059] Among them, the lactic acid bacteria belonging to the genus Lactobacillus is Lactobacillus casei;

[0060] The lactic acid bacteria belonging to the genus *Lactobacillus* is *Lactobacillus shortsum*; and

[0061] Lactic acid bacteria belonging to the genus *Lactobacillus* are selected from at least one of *Lactobacillus pentosus* and *Lactobacillus plantarum*.

[0062] (II-6) Use of at least one of indole-3-lactic acid, α-Asp-Phe, Val-Arg, Asp-Gly-Glu and their salts (preferably at least one of α-Asp-Phe, Asp-Gly-Glu and their salts) for the manufacture of a composition for improving sleep.

[0063] (II-7) Use of a culture supernatant (containing no lactic acid bacteria cells) of any genera of lactic acid bacteria selected from the group consisting of Lactobacillus, Lactobacillus and Lactobacillus for the manufacture of a composition for improving sleep, said supernatant containing at least one selected from indole-3-lactic acid, Val-Arg, Asp-Gly-Glu and their salts (preferably selected from at least one selected from Val-Arg, Asp-Gly-Glu and their salts).

[0064] (II-8) According to the use described in (II-7),

[0065] Among them, the lactic acid bacteria belonging to the genus Lactobacillus is Lactobacillus casei;

[0066] The lactic acid bacteria belonging to the genus *Lactobacillus* is *Lactobacillus shortsum*; and

[0067] Lactic acid bacteria belonging to the genus *Lactobacillus* are selected from at least one of *Lactobacillus pentosus* and *Lactobacillus plantarum*.

[0068] (II-9) is a compound selected from indole-3-lactic acid, α-Asp-Phe, Val-Arg, Asp-Gly-Glu and their salts (preferably selected from α-Asp-Phe, Asp-Gly-Glu and their salts) or a composition containing said compound, for the purpose of improving sleep.

[0069] (II-10) A culture supernatant (containing no lactic acid bacteria cells) of lactic acid bacteria belonging to any genera selected from the group consisting of Lactobacillus, Lactobacillus and Lactobacillus, or a composition containing said supernatant, for the purpose of improving sleep, said supernatant containing at least one selected from indole-3-lactic acid, Val-Arg, Asp-Gly-Glu and their salts (preferably selected from at least one selected from Val-Arg, Asp-Gly-Glu and their salts).

[0070] (II-11) The culture supernatant or a composition comprising the culture supernatant according to (II-10),

[0071] Among them, the lactic acid bacteria belonging to the genus Lactobacillus is Lactobacillus casei;

[0072] The lactic acid bacteria belonging to the genus *Lactobacillus* is *Lactobacillus shortsum*; and

[0073] Lactic acid bacteria belonging to the genus *Lactobacillus* are selected from at least one of *Lactobacillus pentosus* and *Lactobacillus plantarum*.

[0074] (II-12) The culture supernatant or a composition comprising said supernatant according to any one of (II-9) to (II-11),

[0075] The use of it to improve sleep is a non-therapeutic use.

[0076] (II-13) A method for improving sleep, comprising administering to a subject with a sleep disorder (preferably a subject with a sleep disorder due to abnormal regulation of the autonomic nervous system) at least one compound selected from indole-3-lactic acid, α-Asp-Phe, Val-Arg, Asp-Gly-Glu and their salts (preferably at least one compound selected from α-Asp-Phe, Asp-Gly-Glu and their salts) or a composition comprising said compound.

[0077] (II-14) A method for improving sleep, comprising administering to a subject with a sleep disorder (preferably a subject with a sleep disorder due to abnormal autonomic nervous system regulation) a culture supernatant of lactic acid bacteria belonging to any genera selected from the group consisting of *Lactobacillus*, *Lactobacillus*, and *Lactobacillus* (excluding lactic acid bacteria cells) or a composition comprising said supernatant, said supernatant containing at least one selected from indole-3-lactic acid, Val-Arg, Asp-Gly-Glu, and their salts (preferably at least one selected from Val-Arg, Asp-Gly-Glu, and their salts).

[0078] (II-15) Based on the sleep improvement method described in (II-14),

[0079] Among them, the lactic acid bacteria belonging to the genus Lactobacillus is Lactobacillus casei;

[0080] The lactic acid bacteria belonging to the genus *Lactobacillus* is *Lactobacillus shortsum*; and

[0081] Lactic acid bacteria belonging to the genus *Lactobacillus* are selected from at least one of *Lactobacillus pentosus* and *Lactobacillus plantarum*.

[0082] (II-16) The method according to any one of (II-13) to (II-15), wherein the sleep improvement method is a non-therapeutic method.

[0083] (III) Compositions for regulating the autonomic nervous system

[0084] (III-1) A composition for regulating the autonomic nervous system, comprising at least one selected from indole-3-lactic acid, α-Asp-Phe, Val-Arg, Asp-Gly-Glu and their salts as an active ingredient.

[0085] (III-2) A composition for regulating the autonomic nervous system, comprising at least one selected from α-Asp-Phe, Asp-Gly-Glu and their salts as an active ingredient.

[0086] (III-3) A composition for regulating the autonomic nervous system, comprising as an active ingredient a culture supernatant (containing no lactic acid bacteria cells) of lactic acid bacteria belonging to any genus selected from the group consisting of Lactobacillus, Lactobacillus flocculationii, and Lactobacillus lactis, said supernatant containing at least one selected from indole-3-lactic acid, Val-Arg, Asp-Gly-Glu, and their salts.

[0087] (III-4) A composition for regulating the autonomic nervous system, comprising a culture supernatant (containing no lactic acid bacteria cells) of lactic acid bacteria belonging to any genus selected from the group consisting of Lactobacillus, Lactobacillus and Lactobacillus, as an active ingredient, said supernatant containing at least one selected from Val-Arg, Asp-Gly-Glu and their salts.

[0088] (III-5) The composition for regulating the autonomic nervous system according to (III-3) or (III-4),

[0089] Among them, the lactic acid bacteria belonging to the genus Lactobacillus is Lactobacillus casei;

[0090] The lactic acid bacteria belonging to the genus *Lactobacillus* is *Lactobacillus shortsum*; and

[0091] Lactic acid bacteria belonging to the genus *Lactobacillus* are selected from at least one of *Lactobacillus pentosus* and *Lactobacillus plantarum*.

[0092] (III-6) Use of at least one of indole-3-lactic acid, α-Asp-Phe, Val-Arg, Asp-Gly-Glu and their salts (preferably at least one of α-Asp-Phe, Asp-Gly-Glu and their salts) for the manufacture of compositions for the regulation of the autonomic nervous system.

[0093] (III-7) Use of a culture supernatant (containing no lactic acid bacteria cells) of any genera of lactic acid bacteria selected from the group consisting of *Lactobacillus*, *Lactobacillus* and *Lactobacillus* for the manufacture of a composition for regulating the autonomic nervous system, said supernatant containing at least one selected from indole-3-lactic acid, Val-Arg, Asp-Gly-Glu and their salts (preferably selected from at least one selected from Val-Arg, Asp-Gly-Glu and their salts).

[0094] (III-8) According to the uses described in (III-7),

[0095] Among them, the lactic acid bacteria belonging to the genus Lactobacillus is Lactobacillus casei;

[0096] The lactic acid bacteria belonging to the genus *Lactobacillus* is *Lactobacillus shortsum*; and

[0097] Lactic acid bacteria belonging to the genus *Lactobacillus* are selected from at least one of *Lactobacillus pentosus* and *Lactobacillus plantarum*.

[0098] (III-9) at least one compound selected from indole-3-lactic acid, α-Asp-Phe, Val-Arg, Asp-Gly-Glu and their salts (preferably at least one compound selected from α-Asp-Phe, Asp-Gly-Glu and their salts) or a composition containing said compound, for use in regulating the autonomic nervous system.

[0099] (III-10) A culture supernatant of lactic acid bacteria belonging to any genera selected from the group consisting of Lactobacillus, Lactobacillus and Lactobacillus (excluding lactic acid bacteria cells) or a composition containing said supernatant, for use in regulating the autonomic nervous system, said supernatant containing at least one selected from indole-3-lactic acid, Val-Arg, Asp-Gly-Glu and their salts (preferably selected from at least one selected from Val-Arg, Asp-Gly-Glu and their salts).

[0100] (III-11) The culture supernatant according to (III-10) or a composition containing said supernatant,

[0101] Among them, the lactic acid bacteria belonging to the genus Lactobacillus is Lactobacillus casei;

[0102] The lactic acid bacteria belonging to the genus *Lactobacillus* is *Lactobacillus shortsum*; and

[0103] Lactic acid bacteria belonging to the genus *Lactobacillus* are selected from at least one of *Lactobacillus pentosus* and *Lactobacillus plantarum*.

[0104] (III-12) The culture supernatant or a composition comprising the culture supernatant according to any one of (III-9) to (III-11), wherein the use for regulating the autonomic nervous system is a non-therapeutic use.

[0105] (III-13) A method for regulating the autonomic nervous system, comprising administering to a subject with an autonomic nervous system imbalance at least one compound selected from indole-3-lactic acid, α-Asp-Phe, Val-Arg, Asp-Gly-Glu and their salts (preferably at least one compound selected from α-Asp-Phe, Asp-Gly-Glu and their salts) or a composition comprising said compound.

[0106] (III-14) A method for regulating the autonomic nervous system, comprising administering to a subject with autonomic nervous system imbalance a culture supernatant of lactic acid bacteria belonging to any genera selected from the group consisting of *Lactobacillus*, *Lactobacillus*, and *Lactobacillus* (excluding lactic acid bacteria cells) or a composition comprising said supernatant, said supernatant containing at least one selected from indole-3-lactic acid, Val-Arg, Asp-Gly-Glu, and their salts (preferably selected from at least one selected from Val-Arg, Asp-Gly-Glu, and their salts).

[0107] (III-15) Based on the autonomic nervous system regulation method described in (III-14),

[0108] Among them, the lactic acid bacteria belonging to the genus Lactobacillus is Lactobacillus casei;

[0109] The lactic acid bacteria belonging to the genus *Lactobacillus* is *Lactobacillus shortsum*; and

[0110] Lactic acid bacteria belonging to the genus *Lactobacillus* are selected from at least one of *Lactobacillus pentosus* and *Lactobacillus plantarum*.

[0111] (III-16) The method according to any one of (III-13) to (III-15), wherein the autonomic nervous system modulation method is a non-therapeutic method.

[0112] (IV) Used to prevent the onset of autonomic nervous system dysfunction or symptoms caused by it, or to improve autonomic nervous system function. A composition of systemic dysfunction or symptoms caused therefrom

[0113] (IV-1) A composition for preventing or improving the onset of symptoms caused by or inducing autonomic nervous system dysfunction, comprising at least one selected from indole-3-lactic acid, α-Asp-Phe, Val-Arg, Asp-Gly-Glu and their salts as an active ingredient.

[0114] (IV-2) A composition for preventing or improving the onset of symptoms of autonomic nervous system dysfunction or symptoms thereof, comprising at least one selected from α-Asp-Phe, Asp-Gly-Glu and their salts as an active ingredient.

[0115] (IV-3) A composition for preventing or improving symptoms of autonomic nervous system dysfunction or its causes, comprising as an active ingredient a culture supernatant (containing no lactic acid bacteria cells) of lactic acid bacteria belonging to any genus selected from the group consisting of *Lactobacillus*, *Lactobacillus*, and *Lactobacillus*, said supernatant containing at least one selected from indole-3-lactic acid, Val-Arg, Asp-Gly-Glu, and their salts.

[0116] (IV-4) A composition for preventing or improving symptoms of autonomic nervous system dysfunction or its causes, comprising as an active ingredient a culture supernatant (containing no lactic acid bacteria cells) of a lactic acid bacteria belonging to any genus selected from the group consisting of *Lactobacillus*, *Lactobacillus*, and *Lactobacillus*, said supernatant containing at least one selected from Val-Arg, Asp-Gly-Glu, and their salts.

[0117] (IV-5) The composition according to (IV-3) or (IV-4) for preventing the onset of autonomic nervous system dysfunction or symptoms caused therefrom, or for improving autonomic nervous system dysfunction or symptoms caused therefrom.

[0118] Among them, the lactic acid bacteria belonging to the genus Lactobacillus is Lactobacillus casei;

[0119] The lactic acid bacteria belonging to the genus *Lactobacillus* is *Lactobacillus shortsum*; and

[0120] Lactic acid bacteria belonging to the genus *Lactobacillus* are selected from at least one of *Lactobacillus pentosus* and *Lactobacillus plantarum*.

[0121] (IV-6) is used to manufacture a composition for preventing the onset of symptoms of autonomic dysfunction or caused by it, or for improving autonomic dysfunction or caused by it, by selecting at least one of indole-3-lactic acid, α-Asp-Phe, Val-Arg, Asp-Gly-Glu and salts thereof (preferably selected from α-Asp-Phe, Asp-Gly-Glu and salts thereof).

[0122] (IV-7) Use of a culture supernatant (containing no lactic acid bacteria cells) of any genera of lactic acid bacteria selected from the group consisting of *Lactobacillus*, *Lactobacillus*, and *Lactobacillus* for the manufacture of a composition for the prevention of or improvement of symptoms of or caused by autonomic nervous system dysfunction, said supernatant containing at least one selected from indole-3-lactic acid, Val-Arg, Asp-Gly-Glu, and their salts (preferably selected from at least one selected from Val-Arg, Asp-Gly-Glu, and their salts).

[0123] (IV-8) According to the uses described in (IV-7),

[0124] Among them, the lactic acid bacteria belonging to the genus Lactobacillus is Lactobacillus casei;

[0125] The lactic acid bacteria belonging to the genus *Lactobacillus* is *Lactobacillus brevis*; and

[0126] Lactic acid bacteria belonging to the genus *Lactobacillus* are selected from at least one of *Lactobacillus pentosus* and *Lactobacillus plantarum*.

[0127] (IV-9) is the use of at least one compound selected from indole-3-lactic acid, α-Asp-Phe, Val-Arg, Asp-Gly-Glu and their salts (preferably selected from at least one compound selected from α-Asp-Phe, Asp-Gly-Glu and their salts) or a composition comprising said compound for the prevention of onset of autonomic nervous system dysfunction or symptoms thereof or for the improvement of autonomic nervous system dysfunction or symptoms thereof.

[0128] (IV-10) A culture supernatant (containing no lactic acid bacteria cells) of lactic acid bacteria belonging to any genera selected from the group consisting of *Lactobacillus*, *Lactobacillus*, and *Lactobacillus*, or a composition containing said supernatant, for the purpose of preventing or improving the onset of symptoms of autonomic nervous system dysfunction or caused thereby, said supernatant containing at least one selected from indole-3-lactic acid, Val-Arg, Asp-Gly-Glu, and their salts (preferably selected from at least one selected from Val-Arg, Asp-Gly-Glu, and their salts).

[0129] (IV-11) The culture supernatant according to (IV-10) or a composition containing said supernatant,

[0130] Among them, the lactic acid bacteria belonging to the genus Lactobacillus is Lactobacillus casei;

[0131] The lactic acid bacteria belonging to the genus *Lactobacillus* is *Lactobacillus brevis*; and

[0132] Lactic acid bacteria belonging to the genus *Lactobacillus* are selected from at least one of *Lactobacillus pentosus* and *Lactobacillus plantarum*.

[0133] (IV-12) A method for preventing or improving the onset of autonomic nervous system dysfunction or symptoms thereof, comprising administering to a subject having physical discomfort due to an imbalance of autonomic nervous system activity at least one compound selected from indole-3-lactic acid, α-Asp-Phe, Val-Arg, Asp-Gly-Glu and their salts (preferably at least one compound selected from α-Asp-Phe, Asp-Gly-Glu and their salts) or a composition comprising said compound.

[0134] (IV-13) A method for preventing or improving the onset of autonomic nervous system dysfunction or symptoms thereof, comprising administering to a subject suffering from physical discomfort due to an imbalance of autonomic nervous system activity a culture supernatant of lactic acid bacteria belonging to any genera selected from the group consisting of *Lactobacillus*, *Lactobacillus*, and *Lactobacillus* (excluding lactic acid bacteria cells) or a composition comprising said supernatant, said supernatant containing at least one selected from indole-3-lactic acid, Val-Arg, Asp-Gly-Glu, and their salts (preferably selected from at least one selected from Val-Arg, Asp-Gly-Glu, and their salts).

[0135] (IV-14) The method described in (IV-13) for preventing the onset of autonomic nervous system dysfunction or symptoms caused therefrom, or for improving autonomic nervous system dysfunction or symptoms caused therefrom.

[0136] Among them, the lactic acid bacteria belonging to the genus Lactobacillus is Lactobacillus casei;

[0137] The lactic acid bacteria belonging to the genus *Lactobacillus* is *Lactobacillus shortsum*; and

[0138] Lactic acid bacteria belonging to the genus *Lactobacillus* are selected from at least one of *Lactobacillus pentosus* and *Lactobacillus plantarum*.

[0139] The effects of the invention

[0140] This disclosure provides a histamine H1 receptor antagonist composition, which can also be used as a food or beverage. Furthermore, this disclosure provides compositions based on histamine H1 receptor antagonism for, for example, improving sleep quality and / or regulating the balance of the autonomic nervous system. Additionally, this disclosure provides compositions based on histamine H1 receptor antagonism for preventing the onset of autonomic nervous system dysfunction or symptoms caused therefrom, or for improving autonomic nervous system dysfunction or symptoms caused therefrom. Attached Figure Description

[0141] [ Figure 1 The results of the GPCR activation assay (TGF-α cleavage assay) in Experiment Example 1 are shown. The AP-TGFα cleavage amount (%) was measured using doxepin hydrochloride (a known histamine H1 receptor antagonist reagent), indole-3-lactic acid, α-Asp-Phe, Val-Arg and Asp-Gly-Glu as test samples (from left to right in each bar chart).

[0142] [ Figure 2 The results of the GPCR activation assay (TGF-α cleavage assay) in Experiment 2 are shown. The supernatant from the cultures of various lactic acid bacteria (L. casei, L. brevis, L. pentosus, and L. plantarum) was used as the test sample to determine the cleavage rate (%) of AP-TGFα. YPD medium adjusted to a concentration of 2.5% was used as a control. Detailed Implementation

[0143] (I) Histamine H1 receptor antagonist composition

[0144] The histamine H1 receptor antagonist composition disclosed herein is characterized by comprising at least one selected from indole-3-lactic acid, α-Asp-Phe, Val-Arg, Asp-Gly-Glu and their salts as an active ingredient.

[0145] In the following text, "histamine H1 receptor" may be abbreviated as "H1". Specifically, "histamine H1 receptor antagonist composition" is referred to as "H1 antagonist composition", and "histamine H1 receptor antagonism" is referred to as "H1 antagonism".

[0146] There are no particular restrictions on the salts of the above components, as long as they exert their H1 antagonistic effect in the form of salts. Examples of salts include, but are not limited to, alkali metal salts, such as sodium and potassium salts; alkaline earth metal salts, such as calcium and magnesium salts; mineral acid salts, such as hydrochlorides, nitrates, and sulfates; organic acid salts, such as fumarates and methanesulfonates; ammonium salts, such as ammonium salts and tricyclohexylammonium salts; and alkanolamine salts, such as monoethanolamine salts, diethanolamine salts, triethanolamine salts, monoisopropanolamine salts, diisopropanolamine salts, and triisopropanolamine.

[0147] Indole-3-lactic acid, α-Asp-Phe, Val-Arg, Asp-Gly-Glu, and their salts are all commercially available. These commercially available products can be used. Those manufactured through chemical synthesis or according to biochemical methods can also be used. These components are not limited to pure products and can be crude products, as long as they exhibit H1 antagonistic activity.

[0148] Manufacturing based on biochemical methods includes manufacturing based on microbial culture.

[0149] The microorganisms used may be those that, upon culturing, produce at least one selected from indole-3-lactic acid, α-Asp-Phe, Val-Arg, Asp-Gly-Glu, and their salts in the culture supernatant. Specifically, the microorganisms include those capable of producing indole-3-lactic acid, those capable of producing α-Asp-Phe, those capable of producing Val-Arg, and those capable of producing Asp-Gly-Glu. The phrase "capable of production" means having the ability to produce a predetermined component when cultured under suitable conditions (e.g., the composition of the culture medium and culture conditions, such as temperature, pH, and aeration rate). Whether the conditions are suitable can be evaluated by determining the amount of each component contained in the culture supernatant using conventional analytical methods (e.g., LC, MS, or NMR), and can be appropriately set by those skilled in the art.

[0150] These microorganisms include lactic acid bacteria. Specifically, as described in the examples below, examples include lactic acid bacteria belonging to any genera selected from the group consisting of *Lactobacillus*, *Lactobacillus*, and *Lactobacillus*. Lactic acid bacteria belonging to the *Lactobacillus* genus include *Lactobacillus casei*. Lactic acid bacteria belonging to the *Lactobacillus* genus include *Lactobacillus brevis*. Furthermore, lactic acid bacteria belonging to the *Lactobacillus* genus include *Lactobacillus pentosaccharide* and *Lactobacillus plantarum*.

[0151] When lactic acid bacteria are used as microorganisms, the culture medium can be, for example, agar solid medium and / or liquid medium. A carbon or nitrogen source known to those skilled in the art is appropriately added to the medium at the desired concentration, and, if necessary, trace amounts of nutrients such as inorganic ions and vitamins are further added to the medium. More simply, commercially available culture media, such as MRS medium and YPD medium, can be used directly, or additives can be added for use. Where not limited, it is preferred that the medium be adjusted to a pH in the range of 6 to 7; the pH can be adjusted using appropriate acids or bases as needed. Where not limited, the culture temperature is typically 30 to 45°C, preferably 33 to 40°C, more preferably 35 to 40°C. The cultures described herein can suitably be selected from, for example, cultures using solid media and shaken or static cultures using liquid media.

[0152] Where there are no restrictions, preferably, for example, a method can be used to perform anaerobic culture using MRS medium, inoculate the obtained culture solution into YPD medium, and then perform anaerobic culture.

[0153] After the microorganisms have proliferated to a predetermined concentration using this type of culture, the microorganisms (cells) are removed by centrifugation, and optionally subsequently filtered using a filter or the like to obtain a culture supernatant. The degree of microbial proliferation (culture endpoint) can be based on OD. 660nm The absorbance at a certain point is 0.5 or higher as an indicator.

[0154] By using such a culture, as described in the examples below, a culture supernatant containing at least Val-Arg can be obtained by culturing lactic acid bacteria belonging to the genus *Lactobacillus* (preferably *L. casei*) in a culture medium. Furthermore, a culture supernatant containing at least Asp-Gly-Glu can be obtained by culturing lactic acid bacteria belonging to the genus *Lactobacillus* (preferably *L. brevis*) in a culture medium. Additionally, a culture supernatant containing at least indole-3-lactic acid, Val-Arg, and Asp-Gly-Glu can be obtained by culturing lactic acid bacteria belonging to the genus *Lactobacillus* (preferably *L. pentosus* and / or *L. plantarum*) in a culture medium.

[0155] As shown in Experimental Examples 1 and 2 below, indole-3-lactic acid, α-Asp-Phe, Val-Arg, Asp-Gly-Glu, and their salts, as well as compositions containing at least one of these, exhibit H1 antagonistic activity. Therefore, indole-3-lactic acid, α-Asp-Phe, Val-Arg, Asp-Gly-Glu, or their salts, or compositions containing at least one of these, can be effectively used as the active ingredient in H1 antagonistic compositions. Compositions containing at least one selected from indole-3-lactic acid, Val-Arg, and Asp-Gly-Glu include the above-mentioned lactic acid bacteria culture supernatant.

[0156] The composition may be a drug, a quasi-drug, or a food or beverage, or it may be a material or preparation used by compounding with a drug, quasi-drug, or food or beverage. Drugs or quasi-drugs include not only those for human use but also those for non-human animals. Food or beverages include non-therapeutic food or beverages for human use. Furthermore, food or beverages include not only those for human use but also foods (including bait, feed, and experimental feed) for non-human animals such as pets and livestock.

[0157] According to this disclosure, by mixing at least one of indole-3-lactic acid, α-Asp-Phe, Val-Arg, Asp-Gly-Glu and their salts, or a culture supernatant of the above-mentioned lactic acid bacteria containing at least one of indole-3-lactic acid, Val-Arg and Asp-Gly-Glu, during the manufacture of compositions such as food, pharmaceuticals or quasi-pharmaceuticals, the compositions can be endowed with histamine H1 receptor antagonism.

[0158] The content of indole-3-lactic acid, α-Asp-Phe, Val-Arg, Asp-Gly-Glu, and / or their salts in the H1 antagonist composition may suitably be selected from the range of 0.1 to 100% by mass, provided that the H1 antagonist composition exhibits H1 antagonist activity (H1 antagonistic effect). Whether the H1 antagonist composition exhibits H1 antagonist activity (H1 antagonistic effect) can be determined and evaluated based on the GPCR activation assay (TGF-α cleavage assay) described in the Examples section below.

[0159] (II) Sleep-improving compositions

[0160] As described above, indole-3-lactic acid, α-Asp-Phe, Val-Arg, Asp-Gly-Glu, and their salts all exhibit H1 antagonistic activity (H1 antagonistic effect). Furthermore, compositions containing at least one of these (such as the above-mentioned lactic acid bacteria culture supernatant) also exhibit H1 antagonistic activity (H1 antagonistic effect). Therefore, at least one selected from the group consisting of indole-3-lactic acid, α-Asp-Phe, Val-Arg, Asp-Gly-Glu, and their salts, or compositions containing at least one of these (such as the above-mentioned lactic acid bacteria culture supernatant), exhibit sleep-improving effects based on their H1 antagonistic activity and can be effectively used as an active ingredient in sleep-improving compositions.

[0161] The active ingredients in the sleep-improving composition are preferably selected from α-Asp-Phe, Asp-Gly-Glu, and their salts. Furthermore, the active ingredients in the sleep-improving composition are preferably selected from, for example, culture supernatants containing Val-Arg of lactic acid bacteria belonging to the genus *Lactobacillus* (preferably *L. casei*), culture supernatants containing Asp-Gly-Glu of lactic acid bacteria belonging to the genus *Lactobacillus* (preferably *L. brevis*), and culture supernatants containing both Val-Arg and Asp-Gly-Glu of lactic acid bacteria belonging to the genus *Lactobacillus* (preferably *L. pentosus* and / or *L. plantarum*).

[0162] The "sleep improvement" targeted in this disclosure includes the meaning of improving sleep disorders caused by autonomic nervous system dysregulation (dysfunction) through H1 antagonism. Examples of sleep disorders include difficulty falling asleep, sleep persistence disorders in which sleep is not sustained (such as waking during sleep), and / or sleep pattern disorders (such as sleep depth disorders due to a reduction in non-REM sleep). Improving these sleep disorders can alleviate or mitigate symptoms (insomnia) such as insomnia, difficulty falling asleep, short sleep duration, lack of deep sleep sensation, and / or difficulty waking up.

[0163] The content of indole-3-lactic acid, α-Asp-Phe, Val-Arg, Asp-Gly-Glu, and / or their salts in the sleep-improving composition may suitably be selected from the range of 0.1 to 100% by mass, provided that the sleep-improving composition at least exhibits H1 antagonistic activity (H1 antagonistic effect). Whether the sleep-improving composition exhibits H1 antagonistic activity (H1 antagonistic effect) can be determined and evaluated based on the GPCR activation assay (TGF-α cleavage assay) described in the Examples section below.

[0164] The sleep-improving composition may be a drug, quasi-drug, or food supplement for improving sleep disorders, or it may be a material or preparation used by mixing with a drug, quasi-drug, or food supplement. Drugs or quasi-drugs include not only those for human use but also those for non-human animals. Food supplements include non-therapeutic food supplements for human use. Furthermore, food supplements include not only those for human use but also foods (including bait, feed, and experimental feed) for non-human animals such as pets and livestock.

[0165] The dosage form of the drug or quasi-drug can be oral or parenteral (e.g., enteral or injectable), with oral administration being preferred. For administration, a sleep-improving composition containing the above-described active ingredient and comprising a pharmaceutically acceptable non-toxic carrier and additives (e.g., excipients, binders, stabilizers, disintegrants, lubricants, wetting agents, and tonicity agents) suitable for conventional use in oral or parenteral administration can be administered orally or parenterally as is, as a drug or quasi-drug. Optionally, the composition can be mixed with a pharmaceutically acceptable solid or liquid non-toxic carrier (e.g., physiological saline) suitable for conventional use in oral or parenteral administration to prepare a dosage form for administration.

[0166] Examples of formulation forms of drugs or quasi-drugs include, but are not limited to, solid dosage forms such as tablets, granules, powders and capsules; liquid dosage forms such as solutions, suspensions and emulsions; and freeze-dried dosage forms.

[0167] Food and beverage products can be based on the concept of improving sleep (quality) based on H1 antagonistic activity and include specific health foods or foods with functional claims, which, as required, have labels reflecting this concept. The phrase "improves sleep" includes improvements or enhancements in sleep quality, such as falling asleep more easily (improved sleep onset), sleeping soundly without waking during sleep, achieving a feeling of deep sleep, and / or improving wakefulness quality. Food and beverage products can be provided in the form of general food and beverage products or as supplements in the form of solid dosage forms such as tablets, granules, powders, and capsules. General food and beverage products include, but are not limited to, beverages such as soft drinks, fruit juices, milk drinks, lactic acid bacteria drinks, carbonated drinks, and alcoholic beverages; dairy products such as yogurt, cheese, butter, lactic acid bacteria drinks, and milk drinks; processed wheat flour products; processed grain products such as rice; processed legume products such as soybeans; confectionery; and fermented foods obtained using lactic acid bacteria. General food and beverage products, as well as supplements, fall into the category of non-therapeutic food and beverage products not intended for treatment.

[0168] There are no particular limitations on the dosage or intake of the sleep-improving composition, as long as the sleep-improving effect is achieved. Where there are no limitations, the dosage or intake of the active ingredient (i.e., indole-3-lactic acid, α-Asp-Phe, Val-Arg, Asp-Gly-Glu, and their salts) can be, for example, in the range of 25 to 250 mg, and can be taken before bedtime. The dosage or intake mentioned herein is an example and can be appropriately adjusted according to the subject's condition and weight. The subject is a mammal, including humans, and preferably humans. More preferably, the subject is a human with a sleep disorder due to abnormal regulation of the autonomic nervous system. In this disclosure, humans include those in a healthy state and those in a pre-disease state with physical or mental discomfort but who have not yet developed a clearly defined disease.

[0169] (III) Compositions for regulating the autonomic nervous system

[0170] At least one of indole-3-lactic acid, α-Asp-Phe, Val-Arg, Asp-Gly-Glu and their salts, or a composition containing at least one of these (such as the above-mentioned lactic acid bacteria culture supernatant), exhibits a role in regulating the balance of autonomic nervous system activity based on its H1 antagonistic activity (H1 antagonistic effect), and can be effectively used as an active ingredient in compositions for regulating the autonomic nervous system.

[0171] The active ingredient in the composition for regulating the autonomic nervous system is preferably selected from α-Asp-Phe, Asp-Gly-Glu, and their salts. Furthermore, the active ingredient in the composition for regulating the autonomic nervous system is preferably selected from, for example, culture supernatants containing Val-Arg of lactic acid bacteria belonging to the genus *Lactobacillus* (preferably *L. casei*), culture supernatants containing Asp-Gly-Glu of lactic acid bacteria belonging to the genus *Lactobacillus* (preferably *L. brevis*), and culture supernatants containing Val-Arg and Asp-Gly-Glu of lactic acid bacteria belonging to the genus *Lactobacillus* (preferably *L. pentosus* and / or *L. plantarum*).

[0172] The activity of the autonomic nervous system closely influences various regulatory mechanisms in living organisms, such as blood glucose, blood pressure, body temperature regulation, blood flow, hormone secretion, glucose metabolism, and gastric juice secretion. Therefore, imbalances in autonomic nervous system activity can cause physical discomfort, such as medically unexplained symptoms. The autonomic nervous system regulating composition disclosed herein exhibits its effect of regulating imbalances in autonomic nervous system activity primarily through antagonistic activity against histamine H1 receptors. Such autonomic nervous system regulating effects include inhibition of sympathetic nervous system hyperactivity, inhibition of sympathetic nervous system activity, inhibition of parasympathetic nervous system desaturation, and hyperactivity of parasympathetic nervous system activity. For example, medically unexplained symptoms known to be caused by sympathetic nervous system hyperactivity include sleep disturbances, decreased motility of the digestive tract such as the stomach and intestines, thirst, elevated blood pressure, and excessive sweating. Therefore, it is expected that the autonomic nervous system regulating composition disclosed herein will exhibit effects of improving or alleviating such medically unexplained symptoms.

[0173] The content of indole-3-lactic acid, α-Asp-Phe, Val-Arg, Asp-Gly-Glu, and / or their salts in the composition for regulating the autonomic nervous system can be suitably selected from the range of 0.1 to 100% by mass, provided that the composition for regulating the autonomic nervous system at least exhibits H1 antagonistic activity (H1 antagonistic effect). Whether the composition for regulating the autonomic nervous system exhibits H1 antagonistic activity (H1 antagonistic effect) can be determined and evaluated based on the GPCR activation assay (TGF-α cleavage assay) described in the Examples section below.

[0174] The composition for regulating the autonomic nervous system can be a drug, quasi-drug, or diet for regulating the balance of the autonomic nervous system and improving medically unexplained symptoms, or it can be a material or preparation used by mixing with a drug, quasi-drug, or diet. The form of administration or formulation of the drug, quasi-drug, or diet is the same as that described above for the composition for improving sleep, and can be incorporated by reference to the description in (II) above. As in (II), the drugs, quasi-drugs, and diets include not only those for human use but also those for use in non-human animals. Diets include non-therapeutic diets for human use.

[0175] Food and beverage products can be based on the concept of regulating the balance of autonomic nervous system activity based on H1 antagonistic activity, and include specific health foods or foods with functional claims that, where appropriate, have labels reflecting this concept. The phrase "regulating the balance of autonomic nervous system activity" includes improving or alleviating medically unexplained symptoms caused by imbalances in autonomic nervous system activity.

[0176] There are no limitations on the dosage or intake of the composition for regulating the autonomic nervous system, as long as the effect of regulating the autonomic nervous system is obtained. Where there are no limitations, the dosage or intake of the active ingredient (i.e., indole-3-lactic acid, α-Asp-Phe, Val-Arg, Asp-Gly-Glu, and their salts) can be, for example, in the range of 25 to 250 mg, and can be taken once or multiple times daily. The dosage or intake mentioned herein is an example and can be appropriately adjusted according to the condition and weight of the subject. The subject is a mammal, including humans, and preferably humans. More preferably, the subject is a human with autonomic nervous system imbalance. In this disclosure, humans include those in a healthy state and those in a pre-disease state who have physical or mental discomfort but have not yet developed a clearly defined disease.

[0177] (IV) Used to prevent the onset of autonomic nervous system dysfunction or symptoms caused by it, or to improve autonomic nervous system function. A composition of systemic dysfunction or symptoms caused therefrom

[0178] At least one of indole-3-lactic acid, α-Asp-Phe, Val-Arg, Asp-Gly-Glu and their salts, or a composition containing at least one of these (such as the above-mentioned lactic acid bacteria culture supernatant), exhibit the effect of preventing the onset of autonomic nervous system dysfunction or symptoms caused therefrom or improving autonomic nervous system dysfunction or symptoms caused therefrom based on its H1 antagonistic activity (H1 antagonistic effect), and can be effectively used as an active ingredient in compositions for preventing the onset of autonomic nervous system dysfunction or symptoms caused therefrom or improving autonomic nervous system dysfunction or symptoms caused therefrom (these may be collectively referred to as "autonomic nervous system dysfunction, etc." below).

[0179] The active ingredients of the above composition are preferably selected from α-Asp-Phe, Asp-Gly-Glu, and their salts. Furthermore, the active ingredients of the composition are preferably selected from, for example, culture supernatants containing Val-Arg of lactic acid bacteria belonging to the genus *Lactobacillus* (preferably *L. casei*), culture supernatants containing Asp-Gly-Glu of lactic acid bacteria belonging to the genus *Lactobacillus* (preferably *L. brevis*), and culture supernatants containing Val-Arg and Asp-Gly-Glu of lactic acid bacteria belonging to the genus *Lactobacillus* (preferably *L. pentosus* and / or *L. plantarum*).

[0180] Autonomic nervous system dysfunction is physical discomfort caused by an imbalance in the activity of the autonomic nervous system. The compositions disclosed herein for preventing or improving autonomic nervous system dysfunction exhibit an effect of regulating the imbalance in autonomic nervous system activity primarily through antagonistic activity against histamine H1 receptors. Such autonomic nervous system regulation includes inhibition of sympathetic nervous system hyperactivity, inhibition of sympathetic nervous system activity, inhibition of parasympathetic nervous system desaturation, and hyperactivity of parasympathetic nervous system activity. For example, physical discomfort caused by sympathetic nervous system hyperactivity is known to include medically unexplained symptoms such as sleep disturbances, decreased motility of the digestive tract such as the stomach and intestines, thirst, elevated blood pressure, and excessive sweating. Therefore, it is expected that the compositions disclosed herein for preventing or improving autonomic nervous system dysfunction will exhibit effects in preventing the onset of such medically unexplained symptoms and in improving or alleviating such medically unexplained symptoms.

[0181] The content of indole-3-lactic acid, α-Asp-Phe, Val-Arg, Asp-Gly-Glu, and / or their salts in the composition for preventing or improving autonomic nervous system dysfunction may suitably be selected from the range of 0.1 to 100% by mass, provided that the composition at least exhibits H1 antagonistic activity (H1 antagonistic effect). Whether the composition for preventing or improving autonomic nervous system dysfunction exhibits H1 antagonistic activity (H1 antagonistic effect) can be determined and evaluated based on the GPCR activation assay (TGF-α cleavage assay) described in the Examples section below.

[0182] Compositions for preventing or improving autonomic nervous system dysfunction may be drugs or quasi-drugs for regulating the balance of the autonomic nervous system and preventing or improving autonomic nervous system dysfunction, or they may be materials or formulations used by mixing with drugs or quasi-drugs. The form of administration or formulation of the drug or quasi-drug is the same as that described above for compositions for improving sleep, and may be incorporated herein by reference.

[0183] There are no particular limitations on the dosage of compositions used for the prevention or improvement of autonomic nervous system dysfunction, as long as an autonomic nervous system regulatory effect is achieved. Where there are no limitations, the dosage or intake of the active ingredient (i.e., indole-3-lactic acid, α-Asp-Phe, Val-Arg, Asp-Gly-Glu, and their salts) can be, for example, in the range of 25 to 250 mg, and can be taken once or multiple times daily. The dosage or intake mentioned herein is an example and can be appropriately adjusted according to the subject's condition and weight. The subject is a mammal, including humans, and preferably humans. More preferably, the subject is a human with physical discomfort due to imbalance of autonomic nervous system activity.

[0184] In this specification, the terms “comprising,” “including,” and “containing” cover the meanings of “consisting of” and “substantially consisting of”.

[0185] Example

[0186] To aid in understanding the structure and effects of the present invention, experimental examples are provided for illustration. However, the present invention is not limited in any way to these experimental examples. Unless otherwise stated, the following experiments were conducted at atmospheric pressure and room temperature (25±5°C). Unless otherwise stated, the units "%" and "parts" in the following text refer to mass % and mass parts, respectively.

[0187] The reagents, other materials, and measurement methods used in the following experimental examples are as follows.

[0188] [Reagents and other materials]

[0189] HEK293A cells: A cell line established by transforming human fetal kidney cells with an adenovirus gene; obtained from Tohoku University (Japan).

[0190] Lipofectamine 2000: Transfection reagent; obtained from Thermo Fisher Scientific KK.

[0191] Opti-MEM: Reduced Serum Media (the medium used for the above transfection reagents); obtained from Thermo Fisher Scientific KK.

[0192] Plasmid 1 (for AP-TGFα precursor expression); obtained from Tohoku University (Japan).

[0193] Plasmid 2 (for histamine H1 receptor expression); obtained from Tohoku University (Japan). Plasmids 1 and 2 were simultaneously added to cells and cultured for 24 hours for transfection.

[0194] HEPES-buffered Hanks' equilibrium salt solution: A Hanks' equilibrium salt solution containing a final concentration of 20 mM HEPES. The HEPES solution (1 mol / L, pH: 7.2 to 7.4) was mixed with the Hanks' equilibrium salt solution to a concentration of 2% (v / v). The HEPES solution was obtained from FUJIFILM Wako Pure Chemical Corporation, and the Hanks' equilibrium salt solution was obtained from Life Technologies.

[0195] Doxepin hydrochloride: a known H1 antagonist reagent; obtained from Tokyo Chemical Industry Co., Ltd.

[0196] Indole-3-lactic acid: obtained from Sigma Aldrich.

[0197] α-Asp-Phe: Custom-synthesized by Biologica Co., Ltd.

[0198] Val-Arg: Custom-synthesized by Biologica Co., Ltd.

[0199] Asp-Gly-Glu: Custom-synthesized by Biologica Co., Ltd.

[0200] Lactobacillus casei (hereinafter referred to as "L. casei"): Reference strain number JCM 1134T, obtained from RIKEN.

[0201] Lactiplantibacillus brevis (hereinafter referred to as "L. brevis"): Reference strain number JCM 1059T, obtained from RIKEN.

[0202] Lactobacillus pentosus (hereinafter referred to as "L. pentosus"): Reference strain number NCIMB 8026T, obtained from NCIMB.

[0203] Lactobacillus plantarum (hereinafter referred to as "L. plantarum"): Reference strain number NCIMB 11974T, obtained from NCIMB.

[0204] MRS broth: prepared according to a protocol provided by Becton, Dickinson and Company, pH: 6.0 to 6.5.

[0205] YPD medium: prepared according to the protocol provided by Becton, Dickinson and Company; composition of YPD medium (g / 1000ml): 20g peptone, 10g yeast extract and 20g glucose; pH not adjusted.

[0206] Preparation of the test sample

[0207] Preparation of lactic acid bacteria culture supernatant

[0208] One loopful of frozen lactic acid bacteria cells (L. casei, L. brevis, L. pentosus, and L. plantarum) was inoculated into MRS broth and anaerobically cultured at 37°C for 24 hours. After 24 hours, as a second activation treatment, the cells were anaerobically cultured in MRS broth at 37°C for another 24 hours. Subsequently, two drops of each lactic acid bacteria culture were added to 4 ml of YPD medium using a Pasteurized pipette and incubated statically at 37°C under anaerobic conditions. After 24 and 48 hours of culture, OD... 660nm The absorbance was measured, and when the absorbance reached approximately 0.5 or higher, the culture medium was centrifuged, and the collected supernatant was further filtered through a 0.45 μm membrane filter to remove cells. The prepared YPD culture supernatant was stored at -20°C and thawed before evaluation.

[0209] [Determination Method]

[0210] 1. GPCR activation assay (TGF-α cleavage assay)

[0211] The histamine H1 receptor antagonistic activity of the tested samples was evaluated using the GPCR activation assay (TGF-α cleavage assay). The principle of the GPCR activation assay is described in detail in, for example, Seikagaku, Journal of Japanese Biochemical Society, Vol.85, No. 11, pp. 1029-1033, 2013 (The Japanese Biochemical Society) (Non-Patent Document 4).

[0212] HEK293A cells were seeded in 12-well plates, and after 24 hours, a mixture of Lipofectamine 2000 and plasmids 1 (for AP-TGFα precursor expression) and 2 (for histamine H1 receptor expression) in Opti-MEM was added. After 24 hours, the cells were isolated, suspended in HEPES-buffered Hanks' balanced salt solution, seeded in 96-well plates, and incubated in a CO2 incubator for 30 minutes. Histamine solution (final concentration: 20 nM) was added, and after 5 minutes, the test samples (in Experiment 1, each compound was added to a concentration of 10) were added separately. -5 M or 10 -4 M; In Experiment 2, the culture supernatant was added to a concentration of 2.5% and then incubated for 60 minutes. The culture supernatant was aliquoted into individual 96-well plates, and p-nitrophenyl phosphate (p-NPP) solution was added to each plate. The absorbance of each plate was measured at 405 nm immediately after the addition of p-NPP and immediately after 60 minutes of reaction. The AP-TGFα cleavage percentage was calculated from the ratio of alkaline phosphatase (AP) activity in the culture supernatant to that in the cells.

[0213] Experimental Example 1

[0214] GPCR activation assays (TGF-α cleavage assays) were performed using doxepin hydrochloride (a known histamine H1 receptor antagonist), indole-3-lactic acid, α-Asp-Phe, Val-Arg, and Asp-Gly-Glu as test samples, and the cleavage amount (%) of AP-TGFα was calculated.

[0215] Figure 1 Show the results. For example... Figure 1 As shown, the concentration is 10. -5 M and 10 -4 Indole-3-lactic acid, α-Asp-Phe, Val-Arg, and Asp-Gly-Glu from M all showed reduced AP-TGFα cleavage. This confirms that, similar to doxepin hydrochloride, they exert antagonistic activity against histamine H1 receptors.

[0216] Experiment Example 2

[0217] Culture supernatants (with cells removed) of various lactic acid bacteria (L. casei, L. brevis, L. pentosus, and L. plantarum) were used as test samples for GPCR activation assays (TGF-α cleavage assays), and the AP-TGFα cleavage percentage was calculated. YPD medium adjusted to a concentration of 2.5% was used as a control.

[0218] Figure 2 Show the results. For example... Figure 2 As shown, culture supernatants containing *L. casei*, *L. brevis*, *L. pentosus*, and *L. plantarum* exhibited reduced AP-TGFα cleavage, confirming their antagonistic activity against the histamine H1 receptor. In particular, the culture supernatant of *L. casei* showed high antagonistic activity against the histamine H1 receptor.

[0219] Experimental Example 3

[0220] The following methods were used to analyze the components contained in the YPD culture supernatant of various lactic acid bacteria that were confirmed to have antagonistic activity against histamine H1 receptors in Experimental Example 2.

[0221] A mixed solution of methanol and formic acid (99.85% methanol, 0.15% formic acid) was added to the supernatant of YPD cultures of various lactic acid bacteria (L. casei, L. brevis, L. pentosus, and L. plantarum), and the resulting mixture was allowed to stand on ice for 1 hour. After centrifugation (12000 rpm, 10 min), the supernatant was collected and dried to a solid under a nitrogen stream. ODS dissolving solvent (aqueous solution of 0.1% formic acid: methanol of 0.1% formic acid = 9:1, v / v) was added to the sample used for ODS analysis, and HILIC dissolving solvent (aqueous solution of 50% acetonitrile) was added to the sample used for HILIC analysis. The resulting mixture was then filtered using a filter and subjected to quantitative high-resolution accurate mass spectrometry (HRAM) liquid chromatography-mass spectrometry (LC-MS) (Orbitrap LC-MS / MS, manufactured by Thermo Fisher Scientific KK). Based on the data obtained through assays, compounds contained in the YPD culture supernatant of various lactic acid bacteria were experimentally identified using Compound Discoverer 3.1 software (Thermo Fisher Scientific KK).

[0222] For the experimentally identified compounds (indole-3-lactic acid, α-Asp-Phe, Val-Arg, and Asp-Gly-Glu), the same method was used to analyze the standards for these compounds, and FreeStyle 1.4 software (ThermoFisher Scientific KK) was used to confirm whether MS2 spectra similar to those of the standards were observed in the YPD culture supernatant of various lactic acid bacteria.

[0223] The determination conditions for ODS and HILIC analyses are as follows.

[0224] [ODS Analysis]

[0225] Column: Thermo Scientific Hypersil Gold C18 (150×2.1mm, 1.9μm)

[0226] Mobile phase: A) 0.1% formic acid aqueous solution, B) 0.1% formic acid in methanol

[0227] Temperature: 55℃

[0228] Flow rate: 0.1 to 0.25 mL / min (gradient)

[0229] [HILIC Analysis]

[0230] Column: Merck SeQuant ZIC pHILIC (150×2.1mm, 5μm)

[0231] Mobile phase: A) 10 mM ammonium acetate aqueous solution (pH 9.4), B) acetonitrile

[0232] Temperature: 20℃

[0233] Flow rate: 0.25 mL / min

[0234] Table 1 shows the results. In this table, ○ is displayed when a compound is detected in the supernatant of each lactic acid bacteria culture.

[0235] Table 1

[0236]

[0237] As shown in Table 1, the culture supernatant of *L. casei* was confirmed to contain Val-Arg, the culture supernatant of *L. brevis* contained Asp-Gly-Glu, and the culture supernatants of *L. pentosus* and *L. plantarum* contained indole-3-lactic acid, Val-Arg, and Asp-Gly-Glu. These results suggest that the histamine H1 receptor antagonistic activity of the culture supernatants of various lactic acid bacteria may be due to the histamine H1 receptor antagonistic activity of indole-3-lactic acid, Val-Arg, or Asp-Gly-Glu contained in the culture supernatant.

[0238] Industrial availability

[0239] This disclosure provides a safer and more effective composition that exhibits antagonistic activity against histamine H1 receptors and can also be used as a dietary supplement, thus helping to improve sleep and the balance of the autonomic nervous system.

Claims

1. A composition for histamine H1 receptor antagonism comprising at least one selected from indole-3-lactic acid, α-Asp-Phe, Val-Arg, Asp-Gly-Glu and their salts as an active ingredient.

2. The histamine H1 receptor antagonist composition according to claim 1, comprising as an active ingredient a culture supernatant (containing no lactic acid bacteria cells) of lactic acid bacteria belonging to any genera selected from the group consisting of *Lactobacillus*, *Lactobacillus*, and *Lactobacillus*, said supernatant containing at least one selected from indole-3-lactic acid, Val-Arg, Asp-Gly-Glu, and their salts.

3. The histamine H1 receptor antagonist composition according to claim 2, Among them, the lactic acid bacteria belonging to the genus Lactobacillus is Lactobacillus casei; The lactic acid bacteria belonging to the genus *Lactobacillus* is *Lactobacillus shortsum*; and Lactic acid bacteria belonging to the genus *Lactobacillus* are selected from at least one of *Lactobacillus pentosus* and *Lactobacillus plantarum*.

4. A sleep-improving composition comprising at least one selected from indole-3-lactic acid, α-Asp-Phe, Val-Arg, Asp-Gly-Glu and their salts as an active ingredient.

5. The sleep-improving composition according to claim 4, comprising a lactic acid bacteria culture supernatant (containing no lactic acid bacteria cells) of any genera of lactic acid bacteria selected from the group consisting of *Lactobacillus*, *Lactobacillus*, and *Lactobacillus*, as an active ingredient, said supernatant containing at least one selected from indole-3-lactic acid, Val-Arg, Asp-Gly-Glu, and their salts.

6. The sleep-improving composition according to claim 5, Among them, the lactic acid bacteria belonging to the genus Lactobacillus is Lactobacillus casei; The lactic acid bacteria belonging to the genus *Lactobacillus* is *Lactobacillus shortsum*; and Lactic acid bacteria belonging to the genus *Lactobacillus* are selected from at least one of *Lactobacillus pentosus* and *Lactobacillus plantarum*.

7. A composition for regulating the autonomic nervous system, comprising at least one selected from indole-3-lactic acid, α-Asp-Phe, Val-Arg, Asp-Gly-Glu and their salts as an active ingredient.

8. The composition for regulating the autonomic nervous system according to claim 7, comprising as an active ingredient a culture supernatant (containing no lactic acid bacteria cells) of lactic acid bacteria belonging to any genus selected from the group consisting of *Lactobacillus*, *Lactobacillus*, and *Lactobacillus*, said supernatant containing at least one selected from indole-3-lactic acid, Val-Arg, Asp-Gly-Glu, and their salts.

9. The composition for regulating the autonomic nervous system according to claim 8, Among them, the lactic acid bacteria belonging to the genus Lactobacillus is Lactobacillus casei; The lactic acid bacteria belonging to the genus *Lactobacillus* is *Lactobacillus shortsum*; and Lactic acid bacteria belonging to the genus *Lactobacillus* are selected from at least one of *Lactobacillus pentosus* and *Lactobacillus plantarum*.

10. A composition for preventing or improving symptoms of autonomic nervous system dysfunction or its causes, comprising at least one selected from indole-3-lactic acid, α-Asp-Phe, Val-Arg, Asp-Gly-Glu and their salts as an active ingredient.

11. The composition according to claim 10 for preventing or improving the onset of symptoms caused by or inducing autonomic nervous system dysfunction, comprising as an active ingredient a culture supernatant (containing no lactic acid bacteria cells) of lactic acid bacteria belonging to any genus selected from the group consisting of *Lactobacillus*, *Lactobacillus*, and *Lactobacillus*, said supernatant containing at least one selected from indole-3-lactic acid, Val-Arg, Asp-Gly-Glu, and their salts.

12. The composition according to claim 11 for preventing the onset of autonomic nervous system dysfunction or symptoms thereof, or for improving autonomic nervous system dysfunction or symptoms thereof. Among them, the lactic acid bacteria belonging to the genus Lactobacillus is Lactobacillus casei; The lactic acid bacteria belonging to the genus *Lactobacillus* is *Lactobacillus shortsum*; and Lactic acid bacteria belonging to the genus *Lactobacillus* are selected from at least one of *Lactobacillus pentosus* and *Lactobacillus plantarum*.

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