JP7811094B2 - Brain function improvers - Google Patents

Description

The present invention relates to a brain function improver that is useful for improving higher brain functions such as memory and learning.

Higher brain functions such as memory and learning are important brain functions directly linked to maintaining life in all animals, including humans, such as food intake and danger avoidance. Humans store information they obtain in their brains on a daily basis and use that information to reason about new problems and make decisions. This process relies on higher brain functions, and properly maintaining higher brain functions is important for maintaining and improving the quality of daily life. Meanwhile, brain-related disorders such as Alzheimer's disease have become a global problem, and overcoming them is an urgent issue in Japan, which is becoming an aging society. Brain dysfunction, such as depression and emotional instability, can also occur in people other than the elderly, for example, due to stress. Therefore, there is a strong demand for technologies that can improve brain function and effectively prevent and treat brain dysfunction.

Various synthetic drugs, such as the cholinesterase inhibitor donepezil hydrochloride (Aricept®), have been developed, approved, and put into practical use as brain function improvers. However, there is a demand for technologies that can maintain and improve brain function through daily dietary intake rather than the regular use of such drugs, and technologies that can meet this demand have been proposed. For example, docosahexaenoic acid (DHA), which is found in large amounts in fish oils such as herring and sardines, has been known to be effective in improving brain function. Patent Document 1 discloses a brain function-improving composition containing a mixture of DHA and phospholipids as an active ingredient. Patent Document 2 discloses that hexanal, hexenal, hexanol, and hexenol, which are relatively abundant in plant leaves, vegetables, and fruits, are effective in improving brain function. Patent Document 3 discloses that Bifidobacterium breve, a species of bacteria belonging to the genus Bifidobacterium that commonly resides in the large intestine of infants, is effective in improving brain function.

In addition, various molecules and signaling pathways have been known to be involved in memory or learning in the central nervous system, including the neuron-specifically expressed RIT2 (Ras-like without CAAX 2) gene, the Ras pathway that uses it as a GTPase, CREB (cAMP-responsive element binding protein) activated by the Ras pathway, and the neurotrophic factor BDNF (brain-derived neurotrophic factor) activated by CREB (Non-Patent Documents 1-7).

Japanese Patent Application Publication No. 7-17855 Japanese Patent Application Laid-Open No. 2007-308435 International Publication No. 2017/209156

Toshihiro Ichiki. Role of cAMP Response Element Binding Protein in Cardiovascular Remodeling. Arteriosclerosis, Thrombosis, and Vascular Biology. Volume 26, Issue 3, 1 March 2006, Pages 449-455. Aislinn J. Williams, Hisashi Umemori. The best-laid plans go oft awry: synaptogenic growth factor signaling in neuropsychiatric disease. Front. Synaptic Neurosci., 18 March 2014. Hiramatsu M, Takiguchi O, Nishiyama A and Mori H. Cilostazol prevents amyloid beta peptide 25-35-induced memory impairment and oxidative stress in mice. Br J Pharmacol. 161, 1899-1912 (2010). J. R. Cardinaux, J. C. Notis, Q. Zhang, N. Vo, J. C. Craig, D. M. Fass, R. G. Brenna and R. H. Goodman. Recruitment of CREB Binding Protein is sufficient for CREB-mediated gene activation. Mol. Cell. Biol., 20, 1546 (2000) Xu-Qiao Chen, Mariko Sawa and William C Mobley. Dysregulation of Neurotrophin Signaling in the Pathogenesis of Alzheimer Disease and of Alzheimer Disease in Down Syndrome. Free Radic Biol Med. 2018 Jan; 114: 52-61. Louis F Reichardt. Neurotrophin-regulated signaling pathways. Philos Trans R Soc Lond B Biol Sci. 2006 Sep 29; 361(1473): 1545-1564. Exploring the involvement of neurotrophin-3 in hippocampal function regulation and antidepressant-like effects, Abstracts of the 94th Annual Meeting of the Japanese Pharmacological Society, https://www.jstage.jst.go.jp/article/jpssuppl/94/0/94_3-O-E3-2/_article/-char/ja/, retrieved July 22, 2021

While conventional brain function improvers are effective in preventing declines in memory and learning ability, there is a demand for more effective agents. Furthermore, these types of agents have side effects, so a high level of safety is required. No brain function improver that can solve these issues has yet been provided.

The objective of the present invention is to provide a brain function improver that is highly effective in improving brain function, has no harmful side effects, and is highly safe.

The present invention is a brain function improving agent containing, as an active ingredient, an alkylresorcinol represented by the following general formula (I):
The present invention also relates to a food or drink or animal feed for improving brain function, which contains the brain function improver of the present invention.

The brain function improver, brain function-improving food and drink, and animal feed of the present invention are highly effective in improving brain function and are useful, for example, for preventing and/or treating symptoms or diseases caused by a decline in learning and memory ability. Furthermore, the brain function improver, brain function-improving food and drink, and animal feed of the present invention typically contain alkylresorcinol, an active ingredient, derived from plants such as grasses, which are widely consumed as food. Therefore, compared to pharmaceuticals or novel functional ingredients whose active ingredients are chemically synthesized, they have the advantages of being highly safe and free of harmful side effects, and can be taken in relatively large amounts daily, making it easier to obtain the desired effects.

FIG. 1 is a graph showing the rate of spontaneous alternation behavior in mice in a Y-maze test.

The present invention encompasses brain function improvers and brain function improving foods, beverages, or animal feeds containing the same. The present invention is used to improve brain function in humans and non-human animals. The "non-human animals" referred to here are not particularly limited and include, for example, dogs, cats, mice, rats, rabbits, cows, horses, monkeys, etc. The present invention is applicable not only to humans, but also to pets (companions), livestock, etc., and can be applied to animals in general for both medical and non-medical purposes.

The present invention can be used to improve higher brain dysfunction. The term "higher brain dysfunction" as used herein is synonymous with cognitive dysfunction and includes brain dysfunction resulting from the death of brain neurons. Specific examples of higher brain dysfunction to which the present invention can be applied include mental disorders such as schizophrenia, bipolar disorder, depression (senile depression, juvenile depression), phobias, sleep disorders, and drug addiction; pervasive developmental disorders such as autism, Asperger's syndrome, mental retardation, hyperactivity disorder, and tic disorder; neurodegenerative diseases such as Alzheimer's disease (also known as "Alzheimer's dementia," senile or juvenile), Parkinson's disease, frontotemporal dementia such as Pick's disease, and Huntington's disease; memory impairment, attention disorder, executive dysfunction, and social behavior disorder.
As used herein, "amelioration" includes "prevention" and "treatment" of symptoms or diseases in subjects (animals including humans). Furthermore, "prevention" as used herein includes preventing, delaying, or reducing the risk of onset of symptoms or diseases in subjects.
Specific examples of the brain function improving effect achieved by the present invention include improvement of decreased perceptual ability, decreased memory and learning ability, decreased thinking ability, decreased concentration, decreased attention, decreased judgment ability, depressive symptoms, decreased cognitive function, and decreased athletic performance resulting from these.
Furthermore, the brain function improving effect achieved by the present invention can be evaluated by cognitive function tests and test items (evaluation items) that are commonly used in human intervention studies.
Examples of the cognitive function tests include Cognitrax, RBANS, and MMSE-J.
Test items (evaluation items) include overall memory, visual memory, verbal memory, cognitive function speed, overall attention, processing speed, executive function, cognitive flexibility, working memory, sustained attention, simple attention, reaction speed (reaction time), motor speed, social cognition, neurocognitive index, logical thinking, delayed recall (delayed memory), orientation, immediate memory, visual-spatial/constructional, language, and attention.

Unlike anti-aging agents that activate sirtuins, the brain dysfunction improving agent of the present invention can be used to improve brain dysfunction not caused by aging, and can be used to treat, for example, psychiatric disorders such as schizophrenia, bipolar disorder, early-onset depression, phobias, sleep disorders, and drug addiction; pervasive developmental disorders such as autism, Asperger's syndrome, mental retardation, hyperactivity disorder, and tic disorder; neurodegenerative diseases such as early-onset Alzheimer's disease and frontotemporal dementia such as Pick's disease; early-onset memory impairment, early-onset attention disorder, early-onset executive dysfunction, and early-onset social behavior disorder. "Early-onset" typically refers to onset before age 65, but can also occur before age 60 or even before age 50. Known causes of brain dysfunction not caused by aging include lack of sleep, diabetes, and stress.

In light of the results of the examples described below, the brain function improver of the present invention is considered to be particularly useful for preventing and/or treating symptoms or diseases caused by a decline in memory and learning ability. Symptoms or diseases caused by a decline in memory and learning ability include dementia and short-term memory impairment.

The brain function improving agent of the present invention contains, as an active ingredient, one or more alkylresorcinols represented by the general formula (I) (hereinafter also referred to as "specific alkylresorcinols").
The saturated or unsaturated alkyl group represented by _R1 in general formula (I) is not limited by the number of carbon atoms, but preferably has 15 to 27 carbon atoms, and more preferably has 15 to 25 carbon atoms. The unsaturated alkyl group represented by _R1 in general formula (I) means an alkenyl group. In this specification, the term "alkylresorcinol" includes cases where the alkyl group represented by _R1 is an unsaturated alkyl group, i.e., an alkenyl group.

Typical examples of saturated alkyl groups having 15 to 27 carbon atoms include linear ones such as n-pentadecyl, n-heptadecyl, n-nonadecyl, n-heneicosyl, n-tricosyl, n-pentacosyl, and n-heptacosyl, and branched or cyclic ones are also acceptable. Among these, saturated alkyl groups having 15 to 25 carbon atoms are preferred, and linear saturated alkyl groups having 15 to 25 carbon atoms are more preferred.
Examples of the unsaturated alkyl group (alkenyl group) having 15 to 27 carbon atoms include those corresponding to the above-mentioned saturated alkyl group having 15 to 27 carbon atoms. There are no particular restrictions on the number and positions of unsaturated bonds contained in the unsaturated alkyl group (alkenyl group).

In addition, _R2 in the general formula (I) is preferably a hydrogen atom, and _R1 is preferably bonded to _R2 at the para position.

Preferred specific examples of the specific alkylresorcinol include the following seven types: The brain function improving agent of the present invention may contain one or more of the following seven types.
1,3-dihydroxy-5-n-pentadecylbenzene (C15:0)
1,3-dihydroxy-5-n-heptadecylbenzene (C17:0)
1,3-dihydroxy-5-n-nonadecylbenzene (C19:0)
1,3-dihydroxy-5-n-heneicosylbenzene (C21:0)
1,3-dihydroxy-5-n-tricosylbenzene (C23:0)
1,3-dihydroxy-5-n-pentacosylbenzene (C25:0)
1,3-dihydroxy-5-n-heptacosylbenzene (C27:0)

As the specific alkylresorcinol, those in which R ₁ is a saturated alkyl group having 15 to 25 carbon atoms and R ₂ is a hydrogen atom are particularly preferred, and the following six types of alkylresorcinols are particularly preferred.
1) Alkylresorcinol (hereinafter also referred to as AR15) in which _R1 in the general formula (I) is a saturated or unsaturated alkyl group having 15 carbon atoms.
2) Alkylresorcinol (hereinafter also referred to as AR17) in which _R1 in the general formula (I) is a saturated or unsaturated alkyl group having 17 carbon atoms.
3) Alkylresorcinol (hereinafter also referred to as AR19) in which _R1 in the general formula (I) is a saturated or unsaturated alkyl group having 19 carbon atoms.
4) Alkylresorcinol (hereinafter also referred to as AR21) in which R ₁ in the general formula (I) is a saturated or unsaturated alkyl group having 21 carbon atoms.
5) Alkylresorcinol (hereinafter also referred to as AR23) in which R ₁ in the general formula (I) is a saturated or unsaturated alkyl group having 23 carbon atoms.
6) Alkylresorcinol (hereinafter also referred to as AR25) in which R ₁ in the general formula (I) is a saturated or unsaturated alkyl group having 25 carbon atoms.

Particularly preferred AR15 is one in which _R1 is a saturated alkyl group having 15 carbon atoms and _R2 is a hydrogen atom, and a specific example thereof is 1,3-dihydroxy-5-n-pentadecylbenzene (C15:0).
Particularly preferred examples of AR17 include those in which R ₁ is a saturated alkyl group having 17 carbon atoms and R ₂ is a hydrogen atom, and specific examples thereof include 1,3-dihydroxy-5-n-heptadecylbenzene (C17:0).
Particularly preferred examples of AR19 include those in which _R1 is a saturated alkyl group having 19 carbon atoms and _R2 is a hydrogen atom, and specific examples thereof include 1,3-dihydroxy-5-n-nonadecylbenzene (C19:0).
Particularly preferred examples of AR21 include those in which _R1 is a saturated alkyl group having 21 carbon atoms and _R2 is a hydrogen atom, and specific examples thereof include 1,3-dihydroxy-5-n-heneicosylbenzene (C21:0).
Particularly preferred examples of AR23 include those in which R ₁ is a saturated alkyl group having 23 carbon atoms and R ₂ is a hydrogen atom, and specific examples thereof include 1,3-dihydroxy-5-n-tricosylbenzene (C23:0).
Particularly preferred AR25 is one in which R ₁ is a saturated alkyl group having 25 carbon atoms and R ₂ is a hydrogen atom, and a specific example thereof is 1,3-dihydroxy-5-n-pentacosylbenzene (C25:0).

As the specific alkylresorcinol, the amount of alkylresorcinol (AR25) in which _R1 in the general formula (I) is a saturated or unsaturated alkyl group having 25 carbon atoms may be 0, but from the viewpoint of the effect of improving brain function, it is preferable that it contains AR25, and in particular, it is preferable that AR25 is contained in an amount of 0.05% by mass or more and 2.0% by mass or less in the alkylresorcinol in formula (I), and it is particularly preferable that AR25 is contained in an amount of 0.05% by mass or more and 2.0% by mass or less in the alkylresorcinol in formula (I) and AR15 is contained in an amount of 0.1% by mass or more in the alkylresorcinol in formula (I).

In the brain function improver of the present invention, the contents of AR15, AR17, AR19, AR21, AR23, and AR25 are preferably each within the following ranges, from the viewpoint of further improving the action and effect of the brain function improver: The brain function improver of the present invention may contain some or all of AR15, AR17, AR19, AR21, AR23, and AR25 so that the total content of these is 100% by mass or less.
The content of AR15 in the brain function improving agent of the present invention is preferably 0.1 to 10.0% by mass, more preferably 0.1 to 5.0% by mass, and particularly preferably 0.5 to 1.5% by mass.
The content of AR17 in the brain function improving agent of the present invention is preferably 1.0 to 20.0% by mass, more preferably 5.0 to 15.0% by mass, and particularly preferably 8.0 to 12.0% by mass.
The content of AR19 in the brain function improving agent of the present invention is preferably 25.0 to 40.0% by mass, more preferably 27.5 to 37.5% by mass, and particularly preferably 30.0 to 35.0% by mass.
The content of AR21 in the brain function improving agent of the present invention is preferably 40.0 to 55.0 mass %, more preferably 42.5 to 52.5 mass %, and particularly preferably 45.0 to 50.0 mass %.
The content of AR23 in the brain function improving agent of the present invention is preferably 1.0 to 15.0% by mass, more preferably 2.5 to 12.5% by mass, and particularly preferably 5.0 to 10.0% by mass.
In terms of the brain function improving effect, the content of AR25 in the brain function improving agent of the present invention is preferably 0.02 to 5.0 mass %, more preferably 0.05 to 2.0 mass %, and particularly preferably 0.09 to 1.5 mass %.

The specific alkylresorcinol may be a chemically synthesized product, a natural product obtained by extraction from a plant, or a commercially available product. Alkylresorcinols, including the specific alkylresorcinol, are known to be contained in various plants as resorcinol lipids, which are natural non-isoterpenoid phenolic amphiphilic compounds. In the present invention, from the viewpoint of making the brain function improving agent free from harmful side effects and highly safe, the specific alkylresorcinol extracted from a plant is preferably used.
Examples of plants that can be sources of specific alkylresorcinols include grains and nuts (nuts and seeds). Examples of grains include grasses, Anacardiaceae, Ginkgoceae, Proteaceae, Ardisiaceae, Primulaceae, Myristicae, Iridaceae, Araceae, Artemisia of the Asteraceae family, and Fabaceae. Examples of nuts include cashew nuts. Among these plants, grasses are suitable as sources of specific alkylresorcinols because they have been widely consumed and are highly safe for the human body.

Examples of grass plants used as a source of specific alkylresorcinols include wheat, durum wheat, rye, triticale, barley, oats, adlay, corn, rice, barnyard millet, foxtail millet, and millet, and these can be used alone or in combination of two or more. Among these grass plants, wheat and rye are preferred because they have a high alkylresorcinol content, and wheat is preferred because it is a composition that can provide high activity in improving brain function.
When a grass family plant is used as a source of the specific alkylresorcinol, grass family plant seeds are usually used. The form of the grass family plant seeds is not particularly limited, and for example, grass family plant seeds (preferably seed husks; bran) themselves; cut, crushed, or powdered grass family plant seeds; dried grass family plant seeds; dried and then crushed or powdered grass family plant seeds can be used. Suitable examples of grass family plant seed husks include bran, flour, rice husks, rice bran, etc., as well as seeds with husks.
It is preferable not to use, as the specific alkylresorcinol, barley grains or their ground products or extracts in which the expression level and/or activity of starch synthase II has been suppressed.

Methods for extracting specific alkylresorcinols from plants, including grasses, include alcohol extraction using alcohol as the extraction solvent. The conditions for alcohol extraction are not particularly limited and may be set appropriately depending on the type of plant used as the source. For example, when using the various forms of grass seeds or nuts as the source, methods include immersing, stirring, or refluxing the source in alcohol, and supercritical fluid extraction. In the former case, the extraction temperature (liquid temperature of the alcohol) is preferably 2 to 100°C, the extraction time is preferably 0.5 to 72 hours, and the amount of alcohol used is preferably 50 to 2,000 parts by mass per 100 parts by mass of the source.

Examples of alcohols used for extraction include monohydric lower alcohols (preferably those with 1 to 4 carbon atoms) such as methanol, ethanol, n-propanol, isopropanol, and n-butanol, and polyhydric alcohols such as 1,3-butylene glycol, propylene glycol, and glycerin, which are liquid at room temperature (25°C). Among these alcohols, ethanol is preferred from the standpoints of ease of use and environmental friendliness. However, hydrous alcohol, which contains aqueous components other than alcohol (water, pure water, distilled water, tap water, acidic water, alkaline water, neutral water, etc.), can also be used for extraction. The alcohol content in hydrous alcohol is typically 70% by volume or more, preferably 80% by volume or more, and more preferably 90% by volume or more.

In the present invention, the alcohol extract of the plant may be used as an active ingredient in the brain function improver either directly or after concentration and drying, and may be purified by known methods, such as partition chromatography. Any type of partition chromatography may be used as long as it can produce the specific alkylresorcinol. However, normal phase chromatography using a non-aqueous solvent as the mobile phase is preferred, and known methods such as open column, medium pressure column, and high performance liquid chromatography can be appropriately selected.

Examples of the mobile phase in partition chromatography include alcohols that are liquid at room temperature (25°C), such as monohydric lower alcohols (preferably those having 1 to 4 carbon atoms), such as methanol, ethanol, n-propanol, isopropanol, and n-butanol, and polyhydric alcohols such as 1,3-butylene glycol, propylene glycol, and glycerin; ethers such as diethyl ether and propyl ether; esters such as butyl acetate and ethyl acetate; ketones such as acetone and ethyl methyl ketone; hexane; methylene chloride; acetonitrile; and chloroform. These solvents can be used alone or in combination of two or more. When multiple solvents are combined to form the mobile phase, partition chromatography (during purification of plant alcohol extracts) can be performed in an isocratic mode, in which the mixing ratio of the multiple solvents is constant, or in a gradient mode, in which the mixing ratio is varied.
Any carrier can be used in partition chromatography as long as it can support and release the desired active ingredient, but typical examples include silica gel, polyacrylamide gel, dextran gel, etc.
The detection wavelength in partition chromatography of the alcohol extract of the plant may be 170 to 320 nm, preferably 190 to 280 nm.

Examples of partition chromatography suitable for purifying alcohol extracts of plants include the following partition chromatography A and B. The specific alkylresorcinol can be obtained as a peak component in the following partition chromatography A or B.
Partition chromatography A: A medium-pressure column method (medium-pressure chromatography) using silica gel as the carrier and a hexane-ethyl acetate mixed solvent as the mobile phase is used, and during the partition chromatography, the mobile phase is changed from a "hexane-ethyl acetate mixed solvent with a relatively high hexane content" to a "hexane-ethyl acetate mixed solvent with a relatively low hexane content" (i.e., a gradient mode of "large to small hexane"), and the peak component at a detection wavelength of 254 nm is separated.
Partition chromatography B: High performance liquid chromatography (HPLC) is used, using silica gel as the carrier and methanol as the mobile phase, and the peak component at a detection wavelength of 215 nm is separated.

A preferred embodiment of the brain function improver of the present invention is one that contains a peak component in partition chromatography (more preferably, partition chromatography A or B) of an alcohol extract (more preferably, an ethanol extract) of grass seed. The peak component contains a specific alkylresorcinol.

The form of the brain function improver of the present invention at room temperature and normal pressure (specifically, an ambient temperature of 25°C and 1 atmosphere) is not particularly limited, and may be, for example, a liquid (liquid or semi-liquid) containing an alcohol extract of a plant, a dried product or powder thereof obtained by evaporating and drying the liquid medium (alcohol or hydroalcohol) in the liquid, or an alcohol solution obtained by dissolving the dried product or powder thereof in an alcohol such as ethanol.

The brain function improver of the present invention can be used as a pharmaceutical, quasi-drug, or food or beverage for animals, including humans, or for producing them. The brain function improver of the present invention can be administered or ingested directly by animals, including humans, as a pharmaceutical, quasi-drug, or food or beverage, or can be added or blended into food, beverage, or animal feed such as pet food to be used as a brain function improving food, beverage, or animal feed. In the latter case, the method of adding or blending the specific alkylresorcinol into the food, beverage, or animal feed is not particularly limited. For example, the specific alkylresorcinol can be directly blended into the raw materials or ingredients before the production of the food, beverage, or animal feed, added during the production process of the food, beverage, or animal feed, or added to the produced food, beverage, or animal feed.
The term "food and beverage" refers to items that can be ingested by humans, and includes general foods and beverages including so-called health foods, as well as health functional foods such as foods for specified health uses and foods with nutrient functions as defined by the Ministry of Health, Labor and Welfare's Health Functional Food System, and supplements.
The "animal feed" refers to a substance given as food to non-human animals (animals kept by humans), such as livestock, poultry, and farmed fish, and examples thereof include livestock feed and pet food.

When the brain function improving agent of the present invention is used as a pharmaceutical or quasi-drug, it may contain the active ingredient, the specific alkylresorcinol, alone, or may further contain a pharmaceutically acceptable carrier. Alternatively, it may further contain other active ingredients or pharmacological ingredients to the extent that the brain function improving effect of the specific alkylresorcinol is not impaired. Examples of such carriers include excipients, coating agents, binders, bulking agents, disintegrants, surfactants, lubricants, diluents, dispersants, buffers, osmotic pressure adjusters, pH adjusters, emulsifiers, preservatives, stabilizers, antioxidants, colorants, UV absorbers, moisturizers, thickeners, activity enhancers, anti-inflammatory agents, disinfectants, flavorings, and odor enhancers.

When the brain function improving agent of the present invention is used as a pharmaceutical or quasi-drug, it can be administered in any dosage form. The dosage form may be oral or parenteral. For example, oral dosage forms include solid dosage forms such as tablets, coated tablets, granules, powders, and capsules, as well as liquid dosage forms such as elixirs, syrups, and suspensions. Parenteral dosage forms include injection, infusion, transdermal, transmucosal, nasal, enteral, inhalation, suppository, bolus, and patch. Of these, oral dosage forms are preferred.

When the brain function improver of the present invention is used as a food or beverage, it may contain the active ingredient, the specific alkylresorcinol, alone, or may further contain various additives used in the manufacture of pharmaceuticals, quasi-drugs, and food or beverage products, provided that the brain function improving effect of the specific alkylresorcinol is not impaired. Examples of such additives include various oils and fats, herbal medicines, amino acids, polyhydric alcohols, natural polymers, vitamins, dietary fiber, surfactants, purified water, excipients, stabilizers, pH adjusters, antioxidants, sweeteners, taste-imparting components, acidulants such as organic acids, stabilizers, flavors, coloring agents, fragrances, etc.

When the brain function improver of the present invention is used as a food or drink, its form is not particularly limited, and examples of the form of the drink include tea drinks, coffee drinks, milk drinks, fruit juice drinks, carbonated drinks, alcoholic drinks, and soft drinks. Furthermore, the form of the food or drink other than a drink may be solid, semi-solid, or liquid, such as tablets, pills, capsules, liquids, syrups, powders, and granules. Specific forms of the food or drink include bread, noodles, jelly foods, various snacks, baked goods, cakes, chocolate, gum, candy, tablets, capsules, soups, dairy products, frozen foods, instant foods, supplements, other processed foods, seasonings, and their ingredients.
When the brain function improver of the present invention is used as a pet food, which is a type of animal feed, it may be of any of the dry type, semi-dry/semi-moist type, and moist type.

The content of the specific alkylresorcinol, which is the active ingredient in the brain function improver of the present invention, is not particularly limited and can be appropriately adjusted depending on the dosage form, symptoms, age and sex of the target (animal), etc. Typically, the content is preferably 70% by mass or more, more preferably 75% by mass or more, relative to the total mass of the brain function improver of the present invention. In other words, the brain function improver of the present invention may be composed of only the specific alkylresorcinol (active ingredient).
For example, when the subject of application of the brain function improving agent of the present invention is humans, the dosage or intake of the active ingredient is preferably 10 to 4500 mg, more preferably 42 to 4200 mg per day for an adult weighing 60 kg.
For example, when the agent for improving brain function of the present invention is applied to pet animals such as dogs and cats, the dosage or intake of the active ingredient is preferably 0.05 to 150 mg, more preferably 0.13 to 130 mg, per kg of body weight of the pet. The content of the specific alkylresorcinol, which is the active ingredient in the agent for improving brain function of the present invention, can be adjusted to fall within the range of the dosage or intake described above.

The brain function improver of the present invention contains a specific alkylresorcinol as an active ingredient and can be used as a pharmaceutical, quasi-drug, or food or beverage product for improving brain function, labeled to that effect, with the intention of improving brain function. The present invention also includes a food or beverage product or animal feed for improving brain function, containing a specific alkylresorcinol as an active ingredient and labeled to that effect, with the intention of improving brain function.

The present invention includes a package for improving brain function, which includes a package and the above-mentioned brain function improving agent of the present invention or a food or drink or animal feed for improving brain function containing the brain function improving agent contained in the package.
The shape and material of the package are not particularly limited as long as it can contain a brain function improving agent, food or drink, or animal feed and can print ingredient labels, etc. Examples of the shape of the package include a box shape, a bag shape, etc. Examples of the material of the package include paper, plastic, paper, woven fabric, metal, etc.
The package clearly indicates various information such as the content of the specific alkylresorcinol in the brain function improver, food, drink, or animal feed contained in the package. The method of presenting the information on such a package is not particularly limited, and for example, the information may be 1) printed on the outer or inner surface of the package, 2) printed on a printing medium such as printing paper contained inside the package together with the brain function improver, food, drink, or animal feed, or 3) an internet URL may be written on the package or on a printing medium contained therein, and the information may be displayed by accessing the URL.

The present invention will be explained in more detail below using examples, but the present invention is not limited to these examples. The animal experiments described below were conducted in fiscal year 2018 (from November 26, 2018 to March 8, 2019).

Example 1
The peak component of the wheat ethanol extract containing specific alkylresorcinols was obtained by partition chromatography using the following extraction and purification method. The composition of the obtained peak component is as follows:
1,3-dihydroxy-5-n-pentadecylbenzene (C15:0) 1.2% by mass, 1,3-dihydroxy-5-n-heptadecylbenzene (C17:0) 10.9% by mass.
33.9% by mass of 1,3-dihydroxy-5-n-nonadecylbenzene (C19:0) 46.4% by mass of 1,3-dihydroxy-5-n-heneicosylbenzene (C21:0).
1,3-dihydroxy-5-n-tricosylbenzene (C23:0) 7.5% by mass
0.1% by mass of 1,3-dihydroxy-5-n-pentacosylbenzene (C25:0).

<Extraction and purification method>
Ethanol was added to wheat bran in an amount five times its mass, and the mixture was stirred at 600 rpm at room temperature for 16 hours. The extract was filtered to remove impurities, and the ethanol extract was recovered. The ethanol was then distilled off to obtain an ethanolic wheat extract.
The wheat ethanol extract was then purified by medium-pressure chromatography under the following medium-pressure chromatography conditions: The peak components appearing 31 to 36 minutes after the start of elution were collected and the solvent was distilled off to obtain the peak components of the wheat ethanol extract partition chromatography.
(Conditions for medium pressure chromatography)
Column: Silica gel (Inject column 3 L, Hi-Flash column 5 L, 60 Å, 40 μm, manufactured by Yamazen Corporation)
Mobile phase: hexane/ethyl acetate mixed solvent (volume ratio) = 90/10 for 9 minutes, 80/20 for 15 minutes, 60/40 for 16 minutes Detection wavelength: 254 nm

The purification of the wheat ethanol extract in the above-mentioned "extraction and purification method" can also be carried out by HPLC instead of medium-pressure chromatography. In this case, methanol is added to the wheat ethanol extract to prepare a methanol-added solution with an ethanol extract concentration of 200 μg/ml, and the methanol-added solution is passed through a filter with a pore size of 0.45 μm, and the passed-through portion is used as the HPLC sample. The HPLC conditions are as follows:
(HPLC conditions)
Column: Silica gel (ODS-80A, 5 μm, 4.6 × 250 mm, manufactured by GL Sciences Inc.)
Guard column: ODS-80A, 5 μm, 4.6 × 50 mm,
Column temperature: 30°C
Mobile phase: 100% methanol
Detection wavelength: 215 nm

(Evaluation test)
To investigate the brain function improving effect of the peak components (hereinafter referred to as "ARs") in the partition chromatography of the wheat ethanol extract of Example 1, the following Y-maze test (spontaneous alternation behavior test) was carried out. The results are shown in Table 1 and Figure 1.

<Y-maze test>
This test uses a Y-maze with three running paths called arms radiating from the center, and evaluates spontaneous alternation behavior observed when mice are allowed to explore the Y-maze as a measure of short-term memory. Specifically, a mouse is placed in one of the arms of the Y-maze and allowed to freely explore the maze for a specified measurement time (8 minutes). The order of the arms entered by the mouse during exploration is recorded, and the number of times the mouse entered each arm (total arm entries) and the number of combinations in which the mouse entered three consecutive arms (number of alternations) are counted. The spontaneous alternation rate (spontaneous percent alternation) is calculated using the following formula:
Spontaneous alternation rate (%) = {number of spontaneous alternations/(total number of arm entries - 2)} x 100
For example, if the three arms of a Y-maze are designated A, B, and C, and a mouse enters each arm in the order ACBABACBAB within a predetermined measurement time, the total number of arm entries will be 10, the number of spontaneous alternations will be 5 (ACB, CBA, BAC, ACB, CBA), and the spontaneous alternation rate according to the above formula will be 62.5% [= {5/(10-2)} × 100]. A higher spontaneous alternation rate indicates better short-term memory, and is therefore evaluated highly.

The Y-maze used in the Y-maze test was a plastic product manufactured by Unicom Co., Ltd., with a running path length of 39.5 cm, running path width of 4.5 cm, and running path height of 12 cm for each arm. During the test, lighting was adjusted so that the illuminance of the Y-maze running path was 10-40 lx. Five-week-old male SPF mice (Slc:ddy, weighing 23-28 g at time of acquisition) were used as subjects. After a five-day quarantine period and a one-day acclimation period, the mice were subjected to the test.
Mice were divided into a β-amyloid injection model group in which β-amyloid was injected to induce short-term memory impairment, and a control group (control example) in which β-amyloid was not injected. The β-amyloid injection model group was further divided into 1) a vehicle administration group (Comparative Example 1) in which the solvent (soybean oil) used to dissolve the sample to be evaluated was administered, 2) a donepezil administration group (Comparative Example 2) in which donepezil (a therapeutic agent for Alzheimer's disease and Alzheimer's dementia) was administered as a sample, 3) an ARs administration group (Example 1) in which ARs was administered as a sample, and 4) a DHA administration group (Comparative Example 3) in which DHA was administered as a sample. Each group contained 10 mice, and the average weights of the mice were adjusted to be approximately equal within each group.
The samples were orally administered to the mice once a day between 9:00 AM and 12:00 PM, a total of 14 times. The sample dose was 10 mL/kg based on the body weight of the mice on the day of administration. On the 8th day of administration, β-amyloid was injected intracerebroventricularly into the mice, followed by oral administration of the samples. On the 14th day of administration, a Y-maze test was performed 1 hour after sample administration.

As shown in Table 1 and Figure 1, among the beta-amyloid injection model groups in which short-term memory impairment was manifested by beta-amyloid injection, the spontaneous alternation behavior rate in the vehicle-administered group (Comparative Example 1), which received no sample, was 56%, whereas the spontaneous alternation behavior rates in the other groups administered some sample (Comparative Examples 2 and 3, Example 1) were all around 70%, similar to the control group (control example) in which short-term memory impairment was not manifested. This indicates that all of the samples used in these other groups were effective in improving short-term memory impairment. Furthermore, the ARs-administered group (Example 1) showed an effect similar to that of the pharmaceutical drug donepezil at a dose about one-tenth that of the DHA-administered group (Comparative Example 3). Therefore, ARs can be said to be a highly safe brain function-improving agent with excellent effects in improving brain function and no harmful side effects.

<Gene expression analysis>
Of the mice in the β-amyloid injection model group subjected to the Y-maze test, hippocampi were collected from the brains of the mice in the vehicle group (Comparative Example 1) and the ARs administration group (Example 1). Hippocampi were collected from four mice in each group.

(Hippocampal fracture)
500 μL of chilled Trizol was added to hippocampi frozen at -80°C, and the mixture was homogenized using a homogenizer (PT1300D, Central Chemical Trading Co., Ltd.) at 30,000 rpm for 5 seconds, 3 times. After leaving the mixture on ice for 5 minutes, it was transferred to a 1.5 mL tube. 600 μL of Trizol was added, and the mixture was centrifuged at 12,000 g for 10 minutes at 4°C. The supernatant was then transferred to a 1.5 mL tube.

(RNA extraction)
RNeasy Mini Kit (QIAGEN) was used. RNA concentration was measured using BioDrop μLITE+ (BioDrop). RNA degradation was confirmed using Agilent 2100 Bioanalyzer and Agilent RNA 6000 Nano Kit (both Agilent).

(Gene expression analysis)
The extracted RNA was subjected to microarray analysis using GeneChip WT PLUS Reagent Kit (Affymetrix). The GeneChip used was a Clariom S Mouse. The procedure followed the standard protocol.

The data obtained from the GeneChip DNA microarray was analyzed using the statistical analysis software R (ver. 3.5.0 + Bioconductor ver. 3.7). The data obtained as fluorescence intensity was normalized using q.farm. A comparison between the vehicle group (Comparative Example 1) and the ARs-administered group (Example 1) was performed using the RankProduct method, and genes with significantly changed expression levels were extracted (FDR < 0.05).
Next, using the analysis software Ingenuity Pathway Analysis (IPA) (manufactured by QIAGEN), pathways contributing to the improvement of brain function were predicted for genes whose expression levels had significantly changed, based on the expression level data and existing information.

(result)
The number of genes whose expression levels changed significantly between the vehicle group (Comparative Example 1) and the ARs-administered group (Example 1) was 348. Of these, the number of genes whose expression levels increased significantly in the ARs-administered group (Example 1) was 199, and the number of genes whose expression levels decreased significantly was 149.
Next, IPA analysis was performed on these genes to predict the pathways contributing to the improvement of brain function. As a result, "CREB Signaling in Neurons" and "Neurotrophin/TRK Signaling" were found to be pathways involved in the improvement of brain function by ARs administration. Genes with altered expression related to the above pathways are shown in Table 2. Note that "increased expression" refers to genes whose expression is increased in Example 1 compared to Comparative Example 1, and "decreased expression" refers to genes whose expression is decreased in Example 1 compared to Comparative Example 1.

(Estimated mechanism of action)
CREB (cAMP-responsive element binding protein) is a transcription factor activated by the MAPK (Mitogen-activated Protein Kinase) pathway, Ras pathway, Akt signaling pathway, etc. (Non-Patent Documents 1 and 2), and activated CREB is known to regulate the expression of memory-related genes such as the neurotrophic factor BDNF and the early response gene c-fos, which regulates learning and memory (Non-Patent Document 3). It has also been reported that mice lacking CREB function due to the introduction of a mutation into the CREB gene exhibit reduced memory (Non-Patent Document 3), and that transgenic mice with consistently elevated CREB activity exhibit improved memory and increased BDNF expression (Non-Patent Document 4).

Neurtrophins are neurotrophic factors secreted by glial cells, and four types (BDNF, NGF, NT-3, and NT-4) have been identified (Non-Patent Document 5). These neurotrophic factors bind to their corresponding tropomyosin receptor kinase (Trk) receptors and activate the transcription factor CREB through activation of the Ras pathway and other pathways (Non-Patent Document 6). It is also known that administration of antidepressants reduces NT-3 expression while increasing BDNF expression (Non-Patent Document 7).

From the results of the above IPA analysis, it was speculated that the brain function improving effect of ARs administration is due to activation of the transcription factor CREB via the Ras pathway, which is caused by activation of the neurotrophin/Trk signaling pathway by ARs intake, and the MAPK pathway, and that activated CREB then induces the expression of memory-regulating genes such as BDNF and c-fos. Furthermore, among the 199 genes whose expression levels were significantly increased in the ARs administration group (Example 1), was RIT2 (Ras-like without CAAX 2), a GTPase in the Ras pathway, supporting the above mechanism of action.

Claims (4)

A brain function improver containing an alkylresorcinol represented by the following general formula (I) as an active ingredient , the alkylresorcinol including a compound in which R ₁ is a saturated alkyl group having 19, 21 or 23 carbon atoms:
( _R1 represents a saturated alkyl group having 15 to 27 carbon atoms, _R2 represents a hydrogen atom, and _R1 is bonded to _R2 at the para position.) The brain function improving agent according to claim 1, wherein the improvement of brain function is the prevention and/or treatment of symptoms or diseases caused by a decline in memory or learning ability. The brain function improver according to claim 1 or 2, wherein the alkylresorcinol is derived from the seeds of a grass family plant. A food, drink, or animal feed for improving brain function, containing the brain function improver described in any one of claims 1 to 3.