Composition for activating muscarinic acetylcholine receptors

Abstract

To provide a composition for activating a muscarinic acetylcholine receptor in a subject.SOLUTION: A composition for activating a muscarinic acetylcholine receptor contains, as an active ingredient, at least one compound selected from the group consisting of sulforaphane, glucoraphanin, sulforaphene and glucoraphanin.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to a composition for activating muscarinic acetylcholine receptors.

Background Art

[0002] Dementia, one of the neurodegenerative diseases, is a progressive disease, and although several possible causes of onset have been suggested, a fundamental treatment method has not yet been found. In patients with Alzheimer's disease (AD), a typical cognitive impairment, the cholinergic hypothesis of Alzheimer's disease has been proposed, which states that the decline in cognitive function is related to the dysfunction of acetylcholine in the brain (Non-Patent Document 1). In fact, when a muscarinic acetylcholine receptor inhibitor was administered to rats to construct an animal model with impaired acetylcholine function, it was shown that well-trained cognitive behaviors, that is, cognitive behaviors recalled from long-term memory, were inhibited. That is, it was revealed that the activation of muscarinic acetylcholine receptors is important for the recall of long-term memory among cognitive functions (Non-Patent Document 2).

[0003] Sulforaphane (SFN) is a natural compound and is contained as a precursor, glucoraphanin (also known as sulforaphane glucosinolate (SGS)), in broccoli and its sprouts. When humans ingest broccoli, SGS is hydrolyzed by myrosinase, an enzyme possessed by plants and intestinal bacteria, to produce sulforaphane. Patent Document 1 reports that extracts of cruciferous plants such as broccoli and sulforaphane have an effect of promoting sebum production. Patent Document 2 reports that extracts of cruciferous plants such as broccoli and sulforaphane have an effect of suppressing melanin production. In addition, sulforaphane is a component whose safety to the human body has also been confirmed when administered orally or the like.

[0004] When sulforaphane is orally administered to mice, it is widely distributed throughout the body's tissues, and it has been shown that sulforaphane concentrations increase in a dose-dependent manner in tissues other than the prostate (including the brain). In particular, it has been shown that sulforaphane concentrations in the brain correlate with sulforaphane concentrations in the blood (Non-Patent Literature 3). [Prior art documents] [Patent Documents]

[0005] [Patent Document 1] Japanese Patent Publication No. 2009-114152 [Patent Document 2] Japanese Patent Publication No. 2007-031297 [Non-patent literature]

[0006] [Non-Patent Document 1] Bartus RT, et al., Science, Vol. 217, pp. 408-417 (1982) [Non-Patent Document 2] Soma S, et al., Frontiers in Aging Neuroscience, Vol. 6, Article 63 (2014) [Non-Patent Document 3] Clarke JD, et al., Pharm. Res. 28(12) pp. 3171-3179 (2011) [Overview of the Initiative] [Problems that the invention aims to solve]

[0007] From the above, it can be said that enhancing the activity of muscarinic acetylcholine receptors is useful in the prevention or treatment of neurodegenerative diseases, including dementia, particularly in the recall of long-term memories, especially in Alzheimer's disease (AD). On the other hand, with the recent increase in consumer health consciousness, there is a demand to manage health through daily diet, to prevent dementia, or to delay or halt its onset and progression. As supplements and functional foods useful for self-medication are attracting attention, it is desirable that the activity of muscarinic acetylcholine receptors be enhanced by ingredients ingested as food. However, to date, no ingredients used as food that have the effect of enhancing the activity of muscarinic acetylcholine receptors have been reported.

[0008] In view of these circumstances and the above-mentioned problems, the present invention aims to provide a composition for activating a target muscarinic acetylcholine receptor. [Means for solving the problem]

[0009] As a result of diligent research to solve the aforementioned problems, the present inventors have found that, as an example, sulforaphane or its analogues can at least partially solve the above problems, and have completed the present invention which incorporates this as one embodiment.

[0010] The present invention encompasses the following embodiments. [1] A composition for activating a target muscarinic acetylcholine receptor, comprising one or more contributing components selected from the group consisting of sulforaphane, glucoraphanin, sulforaphene, and glucoraphenine. [2] The composition according to [1], wherein the muscarinic acetylcholine receptor is an M1 muscarinic acetylcholine receptor. [3] The composition according to [1] or [2], wherein the activation is an increase in the expression level of the muscarinic acetylcholine receptor gene. [4] The composition according to [3], wherein the activation is an increase in the expression level of the M1 muscarinic acetylcholine receptor gene (chrm1). [5] The composition according to any one of [1] to [4] for enhancing acetylcholine-mediated neurotransmission in a subject. [6] The composition according to any one of [1] to [5] for enhancing the function of recall from long-term memory in a subject. [7] The composition according to [6] for enhancing the function of recall from long-term memory in the hippocampus of a subject. [8] The composition according to any one of [1] to [7] for suppressing the functional decline of nerve cells. [9] A method for activating the muscarinic acetylcholine receptor in a non-human animal, comprising using the composition according to any one of [1] to [8].

[10] A method for activating the muscarinic acetylcholine receptor in a human, comprising using the composition according to any one of [1] to [8].

Effects of the Invention

[0011] According to the present invention, it is possible to provide a composition for activating the muscarinic acetylcholine receptor in a subject.

Brief Description of the Drawings

[0012] [Figure 1] It is a graph showing the mRNA amount ratio of Chrm1 and Gapdh in nerve cells incubated with a culture solution supplemented with sulforaphane (SFN) at each concentration.

Modes for Carrying Out the Invention

[0013] 1. Composition for Activating Muscarinic Acetylcholine Receptor The composition for activating the muscarinic acetylcholine receptor of the present invention (hereinafter, also referred to as "the composition of the present invention") contains one or more contributing components selected from the group consisting of sulforaphane, glucoraphanin, sulforaphene, and glucoraphenin. The composition of the present invention can activate the muscarinic acetylcholine receptor in the subject administered therewith, and thereby can contribute to the maintenance or improvement of cognitive function, particularly the recall function from long-term memory, in a subject having a cognitive disorder such as AD. Further, the composition (or agent) of the present invention can appeal to consumers' health consciousness and health management as a functional food with a health claim or a food for specified health use.

[0014] The composition of the present invention may contain, as a contributing component, one or more compounds selected from the group consisting of sulforaphane or its analogs. Examples of analogs of sulforaphane include, but are not limited to, glucoraphanin, sulforaphene, and glucoraphenin. In certain embodiments, sulforaphane or an equivalent of its known analogs may be used.

[0015] As used herein, "subject" refers to an individual who ingests the composition of the present invention by oral administration or the like, and specifically includes humans or non-human animals. Non-human animals include, but are not limited to, experimental animals such as mice, rats, and guinea pigs, livestock animals such as cows, horses, and sheep, and pet animals such as dogs and cats. In the present specification, the subject is preferably a human.

[0016] The composition of the present invention activates muscarinic acetylcholine receptors, preferably M1 muscarinic acetylcholine receptors. In this specification, "muscarinic acetylcholine receptor" refers to a receptor among acetylcholine receptors that is sensitive to muscarine. It is a type of metabotropic receptor (GPCR) coupled to a GTP-binding protein, and is present as a receptor on organ cells in the postganglionic segment of the parasympathetic nervous system. When the parasympathetic nervous system is stimulated, the release of acetylcholine from peripheral receptors is promoted, resulting in excitation or inhibition of organ cells. There are five subtypes of muscarinic receptors, from M1 to M5. In this specification, "M1 muscarinic acetylcholine receptor" refers to a muscarinic acetylcholine receptor of the M1 subtype, and is a receptor known to be present in the cerebral cortex and hippocampus.

[0017] In this specification, "activation" of muscarinic acetylcholine receptors means that the activity of muscarinic acetylcholine receptors in the subject is enhanced compared to the untreated case. The actual effect may be an increase in the biosynthesis of muscarinic acetylcholine receptors in the subject, an increase in the gene expression level of muscarinic acetylcholine receptors, or an increase in the amount of acetylcholine transmitted via muscarinic acetylcholine receptors. Alternatively, the "activation" in this specification may be the maintenance (suppression of decline) of the aforementioned biosynthesis level, gene expression level, or acetylcholine level, which tended to decline due to impaired nerve cell function in the untreated subject.

[0018] In this context, maintaining (suppressing the decline in) the biosynthesis or gene expression level of muscarinic acetylcholine receptors, or the amount of acetylcholine, means that the decline in the activity of muscarinic acetylcholine receptors (indicated by biosynthesis, gene expression, or acetylcholine amount) due to cognitive impairment, etc., is suppressed. In particular, when the composition of the present invention is administered or ingested to an individual with a disease or characteristic that reduces the activity of muscarinic acetylcholine receptors, it means that the decline in the activity of muscarinic acetylcholine receptors over a certain period is prevented compared to when a control composition without contributing components is administered or ingested. For example, let the activity of the target muscarinic acetylcholine receptor at time 1 be 100%, and the activity of the target muscarinic acetylcholine receptor at time 2 be 100%-Δ% (where Δ>0). The decline in the activity of muscarinic acetylcholine receptors during this period is Δ%. If, compared to the case where a control composition is administered during the same period (Δ%), the decrease in muscarinic acetylcholine receptor activity when the composition of the present invention is administered or ingested by the subject is smaller than the aforementioned Δ%, then it can be said that the decrease in muscarinic acetylcholine receptor activity is prevented.

[0019] In another embodiment, when the composition of the present invention is administered to or ingested by an individual with a disease or condition that reduces the activity of muscarinic acetylcholine receptors, the time required for the activity of the muscarinic acetylcholine receptors in the target individual to decrease by Δ% is longer compared to when a control composition without contributing components is administered to or ingested by the target individual. For example, when the composition of the present invention is administered to or ingested by the target individual, if the time required for the decrease in the activity of muscarinic acetylcholine receptors to reach the aforementioned Δ% is 1.1 times or more, 1.2 times or more, 1.5 times or more, or for example, 2 times or more, than the time required for the control composition, it can be said that the decrease in the activity of muscarinic acetylcholine receptors is prevented.

[0020] The composition of the present invention is preferably a composition for increasing the gene expression level of a target muscarinic acetylcholine receptor. In particular, it is preferably a composition for increasing the gene expression level of the M1 muscarinic acetylcholine receptor, but is not limited thereto. The M1 muscarinic acetylcholine receptor gene refers to a gene that codes for a protein having an amino acid sequence that has 70% or more, 80% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 1, or a gene that codes for a protein having an amino acid sequence that has 70% or more, 80% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 2 (rat M1 muscarinic acetylcholine receptor, NCBI Reference Sequence: NP_542951.1), or 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 2.

[0021] In this specification, "sequence identity" refers to the percentage of identical amino acids relative to the total overlapping amino acid sequences (including the amino acid that serves as the translation initiation site) in the optimal alignment, when two amino acid sequences are aligned with or without introducing a gap, and is calculated by formula (1). Sequence identity can be easily checked using BLAST (Basic Local Alignment Search Tool), a commonly used algorithm in this field. For example, BLAST is available to anyone from websites such as NCBI (National Center for Biotechnology Information) and KEGG (Kyoto Encyclopedia of Genes and Genomes), and sequence identity can be easily checked using default parameters. Sequence identity (%) = Number of matches (ignoring gaps) / Length of the shorter sequence (length excluding gaps) × 100 ... Formula (1)

[0022] The gene expression level of muscarinic acetylcholine receptors can be measured by conventional methods such as quantitative PCR (qPCR). However, it can be difficult to directly measure the activity of muscarinic acetylcholine receptors, i.e., the amount of acetylcholine transmitted through these receptors. Therefore, for convenience, this specification uses the gene expression level of muscarinic acetylcholine receptors, particularly the M1 muscarinic acetylcholine receptor, as an indicator of muscarinic acetylcholine receptor activity.

[0023] Alternatively, the activity of muscarinic acetylcholine receptors can be measured, for example, by the amount of muscarinic acetylcholine receptor biosynthesis in the subject. The amount of muscarinic acetylcholine receptor biosynthesis can be measured using known methods such as immunoassays (EIA, RIA, Western blot, etc.) and mass spectrometry using antibodies that can specifically bind to muscarinic acetylcholine receptors.

[0024] In one embodiment, the composition of the present invention may be a composition that enhances neurotransmission mediated by target acetylcholine by activating muscarinic acetylcholine receptors. As disclosed in Non-Patent Literature 2, muscarinic acetylcholine receptors are receptors involved in the function of recalling long-term memories, and activating them may enhance the function of recalling long-term memories. The hippocampus is the region in the brain involved in recalling long-term memories, and muscarinic acetylcholine receptors, particularly M1 muscarinic acetylcholine receptors, are known to be present in the hippocampus. Therefore, in one embodiment, the composition of the present invention may enhance the function of recalling long-term memories, and this function may be enhanced by the composition of the present invention, particularly in the hippocampus. Herein, "enhancement" includes not only an improvement in the function compared to before administration of the composition of the present invention, but also suppression and maintenance of the decline in the function that was trending downward before administration of the composition of the present invention.

[0025] In one embodiment, the composition of the present invention can suppress the decline in neuronal function by activating muscarinic acetylcholine receptors. In one embodiment, the composition of the present invention can suppress the decline in neuronal function that tends to decline due to cognitive impairment, etc., with respect to at least the function involving muscarinic acetylcholine receptors.

[0026] The compositions of the present invention are not limited to a specific mechanism of action, but in some embodiments, the compositions of the present invention can prevent a decrease in the biosynthesis of muscarinic acetylcholine receptors or maintain the amount of acetylcholine transmitted via muscarinic acetylcholine receptors by increasing, maintaining, or suppressing the decrease in the gene expression level of muscarinic acetylcholine receptors. In some embodiments, this can also be used to delay the onset of dementia, AD, or PD, alleviate their symptoms, reduce their severity, or reduce or halt their progression.

[0027] Incidentally, when glucoraphanin is ingested, it is converted to sulforaphane by myrosinase in the body. Therefore, when the composition of the present invention, which contains cruciferous plants containing glucoraphanin, is administered or ingested, it is thought that glucoraphanin is metabolized in the body, and its metabolite, sulforaphane, exerts its activity. Accordingly, if the composition of the present invention contains, for example, sulforaphane and glucoraphanin, and glucoraphanin is converted to sulforaphane in the body, and it is sulforaphane that exerts its activity, then for convenience, the effective amount may mean the total amount of sulforaphane produced after the conversion.

[0028] Sulforaphane or its analogues may be included in preparations or compositions in a purified state, or in one or more forms selected from Brassicaceae plants, their extracts or fractions thereof, and their pulverized products. In this specification, the term “composition” shall include the above-mentioned extracts.

[0029] Glucorafanin and glucorafenin are types of glucosinolates; glucorafanin is a precursor to sulforaphane, and glucorafenin is a precursor to sulforafen. They are hydrolyzed by the enzyme myrosinase, with glucorafanin becoming sulforaphane and glucorafenin becoming sulforafen. When mammals such as humans ingest glucorafanin or glucorafenin, glucorafanin is converted to sulforaphane and glucorafenin is converted to sulforafen before being absorbed from the intestinal tract. Because glucorafanin and glucorafenin, and sulforaphane and sulforafen, are structurally very similar, glucorafenin is expected to have similar effects and properties to glucorafanin, and sulforafen is expected to have similar effects and properties to sulforaphane.

[0030] Examples of Brassicaceae plants include, but are not limited to, broccoli, kale, cauliflower, cabbage, mustard greens, rapeseed, mustard greens, radish, radish leaves, komatsuna, nozawana, Chinese cabbage, Brussels sprouts, and petit vert (a hybrid of kale and Brussels sprouts). The plant part is not particularly limited and may be the growing body of the plant (buds, leaves, stems, roots or flowers, etc.), sprouts (germinating parts), or seeds. In one embodiment, the Brassicaceae plant may be a member of the Brassica genus, such as broccoli (Brassica oleracea var. italica). In one embodiment, the plant part may be broccoli sprouts or seeds. Broccoli sprouts or seeds have a high content of sulforaphane and / or glucoraphanin. Radishes, radish leaves, and radish seeds also have a high content of sulforaphane and / or glucoraphanin.

[0031] When extracting sulforaphane and / or its analogs from broccoli sprouts, sprouts may be used, for example, 1 to 10 days after germination, preferably 1 to 3 days later. Alternatively, broccoli sprouts containing sulforaphane and / or its analogs in an amount of, for example, 50 to 350 mg / 100g (wet weight), preferably 150 to 330 mg / 100g (wet weight), and more preferably 250 to 300 mg / 100g (wet weight) may be used.

[0032] Extraction of sulforaphane and / or its analogs from Brassicaceae plants can be carried out by conventional methods. For example, glucoraphanin can be obtained by extracting part or all of the plant, either whole or dried and pulverized, with a solvent. Suitable solvents for extraction include water, lower monohydric alcohols (methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, etc.), liquid polyhydric alcohols (glycerin, propylene glycol, 1,3-butylene glycol, etc.), lower esters (ethyl acetate, etc.), hydrocarbons (benzene, hexane, pentane, etc.), ketones (acetone, methyl ethyl ketone, etc.), ethers (diethyl ether, tetrahydrofuran, dipropyl ether, etc.), acetonitrile, and combinations thereof. Examples of extraction methods include extraction with hot water (90-100°C) for 10-30 minutes, or extraction with 0-100% by volume aqueous ethanol at room temperature or heated for 1-10 days. The resulting extract may be used as is, or it may be further fractionated and / or purified. Alternatively, glucoraphanin may be extracted by supercritical fluid extraction.

[0033] One method for obtaining sulforaphane from glucoraphanin is to react glucoraphanin with myrosinase. For example, a method may involve crushing, grinding, pulverizing, shearing, or juicing cruciferous plants containing glucoraphanin without heating to react the glucoraphanin with endogenous myrosinase and metabolize it into sulforaphane, or adding myrosinase to glucoraphanin to metabolize it into sulforaphane. Sulforaphane can be obtained from raw materials containing sulforaphane metabolized from glucoraphanin by these methods, using the same method as for glucoraphanin (for example, the extraction method described above). The method for obtaining sulforaphane from glucoraphanin is the same as the method for obtaining sulforaphane from glucoraphanin described above.

[0034] The concentration of glucoraphanin contained in the composition of the present invention can be measured by known methods. For example, high-performance liquid chromatography (HPLC) can be used, and a specific method can be followed, such as the method of Fahey et al. (Fahey et al., Proc. Natl. Acad. Sci. USA, 94, 10367-10372, 1997). The concentration of glucoraphenin can also be measured by the same method as glucoraphanin.

[0035] The concentration of sulforaphane contained in the composition of the present invention can be measured by known methods. For example, high-performance liquid chromatography (HPLC) can be used, and a specific method can be followed, such as the method of Han et al. (Han et al., Int. J. Mol. Sci., 12, 1854-1861, 2011). The concentration of sulforaphene can also be measured by the same method as for sulforaphane.

[0036] The composition of the present invention can take various forms, including but are not limited to supplements, tablets, coated tablets, granules, powders, solutions, emulsions, capsules, injections or liquids, dry syrups, and syrups. In one embodiment, the composition of the present invention may be for oral administration (oral administration). In another embodiment, the composition of the present invention may be in the form of enteral administration, tube administration, or gastrostomy administration. For example, in subjects with advanced dementia, where oral intake is difficult or compliance issues may arise, gastrostomy administration may be used instead of oral administration. In this case, the gastrostomy administration form may be a liquid or liquid food or nutritional composition.

[0037] In some embodiments, the composition of the present invention may contain other components commonly found in oral, enteral, tube-administered, or gastrostomy compositions, such as excipients, disintegrants, binders, lubricants, colorants, flavoring agents, suspending agents, solubilizers, coating agents, auxiliaries, preservatives, flavoring agents, vitamins, pH adjusters, emulsifiers, thickeners, isotonic agents, antioxidants, chelating agents, sweeteners, flavoring agents, etc., but the additional components are not limited to these. In some embodiments, the composition of the present invention may be a food composition or a feed composition. In some embodiments, the composition of the present invention may be a pharmaceutical composition, a drug, or a quasi-drug. In some embodiments, the composition of the present invention may be for the elderly, for example, for the elderly. In this case, the viscosity and fluidity of the composition may be adjusted considering the chewing and swallowing abilities of the elderly.

[0038] Examples of the above-mentioned food compositions include, but are not limited to, beverages (juice, black tea, tea, coffee, carbonated drinks, sports drinks, soft drinks, etc.), confectionery (gum, caramel, candy, chocolate, cookies, biscuits, snacks, jelly, gummies, tablets, etc.), noodles (soba, udon, ramen, etc.), dairy products (milk, ice cream, yogurt, etc.), seasonings (miso, soy sauce, dressings, etc.), soups, and other general foods and processed foods, as well as health foods (tablets, capsules, etc.) and nutritional supplements (supplements, energy drinks, etc.).

[0039] These food compositions can contain various ingredients depending on their type, and may optionally include food additives such as glucose, fructose, sucrose, maltose, raffinose, sorbitol, stevioside, corn syrup, lactose, citric acid, tartaric acid, malic acid, succinic acid, lactic acid, L-ascorbic acid, dl-α-tocopherol, sodium erythorbate, glycerin, propylene glycol, glycerin fatty acid ester, polyglycerin fatty acid ester, sucrose fatty acid ester, sorbitan fatty acid ester, propylene glycol fatty acid ester, gum arabic, carrageenan, casein, gelatin, pectin, agar, B vitamins, nicotinamide, calcium pantothenate, amino acids, calcium salts, colorants, flavorings, and preservatives.

[0040] A contributing component is a substance whose effect is on the physiological function of the body. In pharmaceuticals and quasi-drugs, the contributing component corresponds to what is commonly known as the "active ingredient." In foods for specified health uses and foods with functional claims, the contributing component corresponds to what is commonly known as the "active ingredient" and "functional active ingredient."

[0041] The compositions of the present invention may contain sulforaphane, glucoraphanin, sulforaphene and / or glucoraphenin in amounts of 0.1 mg / kg body weight or more, 0.2 mg / kg body weight or more, 0.5 mg / kg body weight or more, 1 mg / kg body weight or more, 2 mg / kg body weight or more, 5 mg / kg body weight or more, 10 mg / kg body weight or more, 20 mg / kg body weight or more, 50 mg / kg body weight or more, 100 mg / kg body weight or more, 200 mg / kg body weight or more, 500 mg / kg body weight or more, for example 1000 mg / kg body weight or more, for example 0.1 to 1000 mg / kg body weight or more, 0.1 to 500 mg / kg body weight or more, 0.1 to 100 mg / kg body weight or more, 0.1 to 50 mg / kg body weight or more, 0.1 to 20 mg / kg body weight or more, for example 0.1 to 10 mg / kg body weight or more. In another embodiment, the composition of the present invention may contain sulforaphane, glucoraphanin, sulforaphene and / or glucoraphenin in total amounts of 0.01% or more by weight, 0.05% or more by weight, 0.10% or more by weight, 0.20% or more by weight, 0.5% or more by weight, 1% or more by weight, 2% or more by weight, 2.2% or more by weight, 3% or more by weight, 4% or more by weight, 5% or more by weight, 10% or more by weight, 20% or more by weight, 20% or less by weight, 10% or less by weight, 5% or less by weight, for example, 3% or less by weight.

[0042] The amount of the composition of the present invention to be ingested may vary depending on the age, sex, symptoms, and method of administration when the subject is a human, but sulforaphane, glucoraphanin, sulforaphene, and / or glucoraphenin may typically be 1 mg or more, 5 mg or more, 10 mg or more, 20 mg or more, for example 30 mg or more per day for an adult (weighing about 60 kg). Note that "30 mg or more per day" may vary depending on the form of the composition, but refers to the indicated daily intake amount or the amount contained in one bottle of a single-serving beverage that is usually consumed in one go. The daily amount can be achieved by a single dose or multiple doses (ingestion). The timing of administration or ingestion may be before, after, or between meals. The duration of administration or ingestion is not particularly limited, but in some embodiments, the composition of the present invention may be ingested for a long period or continuously. Long-term or continuous ingestion means ingesting for, for example, one month or more, two months or more, three months or more, four months or more, five months or more, for example six months or more. In one embodiment, the composition of the present invention may be packaged in a form suitable for long-term or continuous intake. Furthermore, those skilled in the art can appropriately determine the intake or dosage of the composition of the present invention.

[0043] In certain embodiments, the food composition of the present invention can be designated as a functional food or a food for specified health uses based on the above-mentioned functionality and beneficial effects on the body.

[0044] Regarding the usefulness and functionality of the composition of the present invention, the following indications may be attached to the product when it is commercialized, but are not limited to these. For example, "functionality that activates muscarinic acetylcholine receptors," "functionality that increases the gene expression level of muscarinic acetylcholine receptors," "functionality that prevents a decrease in the activity of muscarinic acetylcholine receptors," "functionality that prevents a decrease in the gene expression level of muscarinic acetylcholine receptors," and similar indications. These indications may be attached to containers and packaging by known methods, displayed or distributed with the above explanations in advertisements, price lists or trade documents related to the product, or information containing these may be provided by electromagnetic means (such as the Internet).

[0045] 2. Methods for activating muscarinic acetylcholine receptors The present invention provides a method for activating muscarinic acetylcholine receptors (hereinafter also referred to as "the method of the present invention"), which includes using the composition described in section "1. Composition for activating muscarinic acetylcholine receptors" to activate muscarinic acetylcholine receptors in non-human animals or humans. The method of the present invention involves administering a composition containing one or more contributing components selected from the group consisting of sulforaphane, glucoraphanin, sulforaphene, and glucoraphenine to non-human animals or humans by oral ingestion or other means, thereby activating muscarinic acetylcholine receptors in the target, and contributing to the maintenance or improvement of cognitive function, particularly the function of recalling from long-term memory.

[0046] In one embodiment, the method of the present invention is a method for activating muscarinic acetylcholine receptors in non-human animals. Examples of non-human animals include, but are not limited to, experimental animals such as mice, rats, and guinea pigs, domesticated animals such as cattle, horses, and sheep, and pet animals such as dogs and cats.

[0047] This specification discloses, apart from the embodiments described above, a method for activating muscarinic acetylcholine receptors in humans, particularly in humans with cognitive impairments such as AD.

[0048] The components of the composition used in the method of the present invention, the preparation method, the dosage form, the mechanism of action, the target recipients, etc., are as described in section "1. Composition for activating muscarinic acetylcholine receptors," unless otherwise specified. [Examples]

[0049] The following examples are for illustrative purposes only and do not limit the technical scope of the present invention. Unless otherwise specified, materials and reagents are commercially available or obtained or prepared according to methods commonly used in the art or procedures described in prior art. The qPCR primers were purchased from Eurofins Genomics.

[0050] [Example 1] 1. Culture of PC12 cells A cryovial containing frozen cells stored in liquid nitrogen (PC12 cells, rat-derived, ACC No.: 88022401) was rapidly thawed in a 37°C water bath. The cells were then added to a 50 mL centrifuge tube containing 10 mL of medium A (DMEM medium containing 10.0% fetal bovine serum, 50 U / mL penicillin, and 50 μg / mL streptomycin) and mixed. After centrifugation at 200 × g for 5 minutes, the supernatant was removed, and the pellet was resuspended in 5 mL of medium A and cultured in a 25 cm³ container. 2 The seeds were sown in a flask.

[0051] After culturing to 60% confluence, two-thirds of the culture medium is transferred to 75 cm³. 2 The cells were transferred to flasks for subculturing, and the remaining one-third was cryopreserved. Subculturing was performed by detaching the cells in the flask with 0.25% trypsin / EDTA solution, washing with PBS, and then transferring them to a new flask with fresh culture medium. The culture medium was changed two days after the start of subculturing and the culture was continued, and then a new 75 cm³ culture medium was used three days later. 2 The cells were transferred to flasks and subcultured. Two days after the start of subculture, the culture medium was changed, and after confirming confluence every two days, the cells were detached using trypsin solution and collected by centrifugation. The collected cells were resuspended in 4 mL of CELLBANKER® 1plus, dispensed into 1 mL portions into cryotubes, and then frozen and stored in liquid nitrogen. The same process was repeated at least two times to prepare a stock of cells for use.

[0052] 1 mL of frozen cell suspension was thawed in a 37°C water bath and added to 6 mL of medium A in a 15 mL centrifuge tube. After mixing the mediums, the cells were centrifuged at 200 × g for 5 minutes, and the supernatant was removed. After adding 10 mL of medium A, the entire volume was seeded into a 100 mm dish and incubated at 37°C. After confirming 80% confluence, the cell saturation was collected into a 50 mL centrifuge tube, the dish was washed with PBS, and the washing solution was collected into the 50 mL tube from which the cell saturation had been collected. After centrifugation at 200 × g for 5 minutes, the supernatant was removed, and the remaining pellet-like cell mass was resuspended in fresh medium B (DMEM medium containing 5.0% fetal bovine serum, 10.0% equine serum, 50 U / mL penicillin, and 50 μg / mL streptomycin). The cells were seeded into 2-3 100 mm dishes, and cell passage for neuronal cell differentiation was performed.

[0053] 2. Neuronal cell-like differentiation of PC cells and SFN stimulation To prepare a collagen-coated 6-well plate, 1 mL / well of collagen solution (Cellmatrix Type IV, manufactured by Nitta Gelatin Co., Ltd., diluted 20-fold with 1 mM hydrochloric acid) was added and spread evenly. After dissolving the solution, the collagen solution was discarded, the plate was dried in a clean bench for 30 minutes, and then UV sterilized for at least 30 minutes. The collagen-coated 6-well plate was washed twice with culture medium before adding the cell solution.

[0054] A stock solution was prepared by diluting 5 mg of sulforaphane (SFN (obtained from CAYMAN, #10496)) with DMSO (TOCRIS BIOSCIENCE, 3176) to a concentration of 10 mM SFN. The 10 mM SFN stock solution was diluted with medium B to a concentration of 100 μM, and then sterilized using an Advantec DISMIC 25CS020AS to prepare a 100 μM SFN solution. As a control, a 0 μM SFN solution was prepared by diluting DMSO 100-fold with medium B instead of the above SFN / DMSO solution.

[0055] After confirming 80% confluence of cultured PC12 cells, the cell saturation was collected in a 50 mL centrifuge tube. The dish was then washed with PBS, and the washing solution was collected in the same 50 mL tube used to collect the cell saturation. After centrifugation at 200 × g for 5 minutes, the supernatant was removed, and the remaining pellet-like cell aggregate was dispersed by adding trypsin solution and incubating. Then, the trypsin reaction was stopped by adding the same amount of fresh medium B as the trypsin solution. After centrifugation at 200 × g for 5 minutes, the supernatant was removed, and medium B was added to the remaining pellet-like cell aggregate to form a 1 × 10⁶ cell mass. 5 The cells were resuspended to a cell / mL concentration, and 1 mL / well was seeded into a washed collagen-coated 6-well plate. The plate was centrifuged at 200 × g for 5 minutes to settle the cells, and the plate was left to stand overnight. After standing overnight, 10 μL / well of 1 μg / mL NGF (nerve growth factor, SIGMA N2513) solution (PBS solution containing 0.1% bovine serum albumin) was added (final concentration 10 ng / mL). Neuronal cell-like differentiation under SFN stimulation was then induced by adding either SFN 100 μM solution or SFN 0 μM solution to each well. The stimulation conditions were SFN 0, 0.1, 0.5, 2.5, 5.0, and 10.0 μM, and the number of samples (n) for each condition is shown in Table 2.

[0056] After incubating cells under each condition for 72 hours, the cells were detached from the flask using a cell scraper, and the cells and medium were collected in a 15 mL centrifuge tube. The plate was then washed with PBS, and the cells and washing solution were collected in the same 15 mL centrifuge tube. The cells were then centrifuged at 200 × g for 5 minutes to collect them.

[0057] 3. mRNA extraction Nerve cells cultured under each condition (1 × 10⁻¹⁶) 5 RNA extraction was performed on the cells. TRIzol, manufactured by Thermo Fisher Scientific, was used for the extraction. TM Reagent (catalog number 15596018) was used. TRIzol was applied to the harvested cells. TM800 μL of Reagent was added, pipetted, and incubated at room temperature for 5 minutes. 160 μL of chloroform was added, mixed by vortexing, and allowed to stand at room temperature for 3 minutes. Centrifuged at 12000 × g for 15 minutes at 4°C, and approximately 400 μL of the upper layer was collected. 400 μL of 2-propanol was added, mixed by vortexing, and allowed to stand at room temperature for 10 minutes. Centrifuged at 12000 × g for 10 minutes at 4°C, and the supernatant was removed. 800 μL of 75% ethanol was added, mixed by vortexing, and then centrifuged at 7500 × g for 5 minutes at 4°C, and the supernatant was removed. The precipitate was dried at room temperature for 5-10 minutes, and the RNA was dissolved in 40 μL of RNase-free water. The eluted RNA was stored at -80°C until cDNA synthesis.

[0058] 4. Gene expression analysis Gene expression analysis was performed using the extracted RNA by RT-qPCR. Specifically, cDNA was synthesized from the extracted RNA, and qPCR was performed using the synthesized cDNA. 10 μL of the RNA solution prepared above was mixed with 4 μL of 5×PrimeScript® RT Master Mix (Perfect Real Time) (TaKaRa, #RR036A) and 6 μL of RNase-free water to obtain 20 μL of reaction solution. The reaction solution was incubated using a thermal cycler (BIO-RAD, T100 thermal cycler) (37°C for 15 minutes, 85°C for 5 seconds, and cooled to 4°C) to obtain cDNA. The procedure was carried out according to the manufacturer's protocol.

[0059] For qPCR, TB Green® Premix ExTaq® II (Tli RNaseH Plus) (TaKaRa, #RR820S) was used as the reagent. The template cDNA amount was 20 ng, and an Applied Biosystems™ 7500 Fast (Thermo Fisher Scientific, #7500-03) was used as the analysis instrument. The qPCR protocol consisted of a pre-amplification step (95°C for 30 seconds), an amplification step (95°C for 5 seconds, 60°C for 34 seconds x 45 cycles), and a thawing step (95°C for 15 seconds, 60°C for 1 minute, then heating up to 95°C).

[0060] The target gene for mRNA analysis was the Chrm1 gene, which is the M1 muscarinic acetylcholine receptor gene. Additionally, the Gapdh gene, which is the glyceraldehyde 3-phosphate dehydrogenase gene, was analyzed as an internal standard. The primer sequences used for qRT-PCR for each gene are shown in Table 1.

[0061] [Table 1]

[0062] The Chrm1 / Gapdh expression ratio was determined for cells under each condition. The results are shown in Table 2 and Figure 1. The values ​​shown in Table 2 and Figure 1 are relative values ​​with the mean value under the condition of 0 μM SFN concentration set to 1.0. As shown in Table 2 and Figure 1, the Chrm1 / Gapdh expression ratio was clearly increased in the test groups in which 0.1 to 5 μM of SFN was added to the culture medium compared with the test group in which SFN was added. Furthermore, when the value of the Chrm1 / Gapdh expression ratio under each condition was analyzed using the Yonk-heel-Tapstra test, the result was P = 0.0001336, indicating a significant upward trend in the Chrm1 / Gapdh expression ratio in relation to the amount of SFN added to the cell culture medium.

[0063] [Table 2]

[0064] In this embodiment, it was shown that the gene expression level of muscarinic acetylcholine receptors increased in nerve cells upon contact with SFN. This indicates that SFN increases the biosynthesis of muscarinic acetylcholine receptors and enhances the overall activity of muscarinic acetylcholine receptors. In this embodiment, SFN was added to the culture medium of nerve cells, but as shown in Non-Patent Literature 3, SFN taken into the body by oral ingestion is transferred to the brain, so it has been shown that activation of muscarinic acetylcholine receptors can occur in subjects such as humans who orally ingest SFN. As mentioned above, muscarinic acetylcholine receptors are receptors involved in the retrieval of long-term memories, and it has also been shown that oral ingestion of SFN may contribute to maintaining and improving their function.

[0065] This specification references several documents, including academic papers. Although the disclosures of these documents are not considered to be related to the patentability of the present invention, their entirety is incorporated herein by reference. More specifically, all referenced documents are incorporated herein by reference in the same way that each individual document is specifically and individually indicated to be incorporated by reference. [Industrial applicability]

[0066] The composition of the present invention can be used in the food, pharmaceutical, and cosmetic fields as a composition for oral intake or other purposes for the purpose of maintaining and improving cognitive function.

Claims

1. A composition for increasing the gene expression level of a target muscarinic acetylcholine receptor, comprising one or more contributing components selected from the group consisting of sulforaphane, glucoraphanin, sulforaphene, and glucoraphenine.

2. The composition according to claim 1, wherein the muscarinic acetylcholine receptor is an M1 muscarinic acetylcholine receptor.

3. The composition according to claim 1 or 2, wherein the activation is an increase in the expression level of the M1 muscarinic acetylcholine receptor gene (chrm1).

4. A composition according to any one of claims 1 to 3, for enhancing acetylcholine-mediated neurotransmission by increasing the gene expression level of a target muscarinic acetylcholine receptor.

5. A method for activating a muscarinic acetylcholine receptor in a non-human animal, comprising increasing the gene expression level of the muscarinic acetylcholine receptor in the non-human animal using the composition described in any one of claims 1 to 4.

Images