Oxytocin receptor agonist composition

Abstract

To provide an oxytocin receptor-operated composition that is obtained from materials of natural origin, has high safety and can be taken readily.SOLUTION: An oxytocin receptor-operated composition includes R1-barrigenol represented by the following formula and / or an R1-barrigenol derivative, the R1-barrigenol derivative being a glycoside or acylated body of R1-barrigenol.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to an oxytocin receptor agonist composition containing R1-valigenol and / or its derivatives as an active ingredient. [Background technology]

[0002] Oxytocin (hereinafter referred to as OXT) is a cyclic peptide hormone consisting of nine amino acids, and has long been used clinically as a uterine muscle contraction agent. OXT is synthesized in the paraventricular nucleus and supraoptic nucleus, and secreted into the blood from the posterior pituitary gland. The secreted OXT acts as a ligand on the oxytocin receptor (hereinafter referred to as OXTR) localized in the cell membranes of various organs in the body, such as the reproductive organs (uterus, mammary gland, ovary, testis), kidney, and brain. OXTR is a type of GPCR with seven transmembrane domains that couples with Gαq / 11 and Gαi as G proteins. When OXTR is stimulated by a ligand, phospholipase C is activated, which activates the intracellular Ca2+ mobilization system and transmits a signal to the downstream cascade, resulting in physiological functions such as uterine muscle contraction and milk ejaculation. Recent studies have revealed that OXTR is also involved in the control of muscle regeneration (Non-Patent Document 1), bone formation (Non-Patent Document 2), modification of sweetness sensitivity (Non-Patent Document 3), inhibition of enteritis (Non-Patent Document 4), stress relief (Non-Patent Document 5), feeding behavior (Non-Patent Document 6), inhibition of alcohol intake (Non-Patent Document 7), pair formation such as couples (Non-Patent Document 8), maternal behavior (Non-Patent Document 9), empathic behavior (Non-Patent Document 10), social cognitive function (Non-Patent Document 11), object recognition function (Non-Patent Document 12), aggressive behavior (Non-Patent Document 9), and anxiolytic effects (Non-Patent Document 13). Furthermore, effects such as increased trust (Non-Patent Document 14), increased understanding of the heart (Non-Patent Document 15), improved marital dispute conversation (Non-Patent Document 16), improved autistic symptoms (Non-Patent Document 17), and reduced snacking (Non-Patent Document 18) have also been reported by administering OXT. Various agonists that activate OXTR have been reported, and are roughly divided into non-peptide and peptide compounds. Non-peptide agonists include WAY-267474 and LIT-001 (Non-Patent Documents 19 and 20), but both are chemically synthesized and have not been put to practical use due to safety concerns. Peptide agonists include OXT and vasopressin (hereinafter abbreviated as AVP), a hormone related to OXT, but they have problems such as being easily decomposed in the body by digestive enzymes and having a short half-life in the body even after being transferred to the blood. On the other hand, AVP, like OXT, is a neuropeptide hormone consisting of nine amino acids, seven of which are highly homologous to OXT. Three types of AVP receptors (hereinafter abbreviated as AVPR) are known: Vasopressin 1A receptor (hereinafter abbreviated as V1aR), Vasopressin V1b receptor (hereinafter abbreviated as V1bR), and Vasopressin 2 receptor (hereinafter abbreviated as V2R). The structurally similar V1aR and V1bR are mainly expressed in blood vessels, the liver, the anterior pituitary gland, etc., and V2R is mainly expressed in the kidney. OXTR and AVPR are highly homologous, and in humans, AVP is known to bind to AVPR (V1aR, V1bR, V2R) and OXTR with the same affinity (Non-Patent Document 21). Here, the relationship between V1aR and psychiatric disorders among AVPRs is known, and it has been reported that AVP acts on V1aR to increase anxiety and fear. Since OXT and AVP are highly homologous, the affinity between OXTR and AVPR is also high, and the affinity between OXTR and AVPR for AVP is similar, there is a risk that OXTR agonists will cause side effects via AVPR. For example, compound 13 (Non-Patent Document 22) and Val3Pro8-OT (Non-Patent Document 23), which have been reported as OXTR ligands, have been reported to act as agonists to activate V1aR. For these reasons, it is desirable for OXTR agonists to be specific for OXTR and non-peptide with a long half-life in the body, without exerting unintended actions on the receptor of ligands highly homologous to OXT and causing side effects, and furthermore, to be derived from natural edible materials that are commonly consumed and can be ingested safely and easily. R1-barrigenol and its derivatives such as glycosides and acylated derivatives are natural components contained in tea flowers and Pittosporum tobira leaves, etc., and it is known that R1-barrigenol has antibacterial activity, etc. (Non-Patent Document 24), and R1-barrigenol glycosides have intestinal motility enhancing effects, free fatty acid production effects, etc. (Patent Document 1), antihyperlipidemic effects, gastrointestinal protective effects, pancreatic lipase inhibitory effects, and food intake suppression effects, etc. (Non-Patent Document 25). However, it was not previously known that R1-barrigenol and its derivatives have OXTR agonist activity. [Prior art documents] [Patent documents]

[0003] [Patent Document 1] JP 2009-57365 A [Non-patent literature]

[0004] [Non-Patent Document 1] Elabd C et al.Nat Commun 5:4082, 2014 [Non-Patent Document 2] Tamma R et al.Proc Natl Acad Sci USA 106:7149-7154, 2009 [Non-Patent Document 3] Sinclair MS et al. Physiol Behav 141:103-1010, 2014 [Non-Patent Document 4] De Araujo AD et al. Nat Commun 5:3165, 2014 [Non-Patent Document 5] Zheng J et al., Am J Physiol Gastrointest Liver Physiol. 2010;299(4):G946-G953.

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[0005] An object of the present invention is to provide an oxytocin receptor agonist composition which is obtained from materials derived from natural products, is highly safe, and can be easily ingested. [Means for solving the problem]

[0006] The present inventors have searched for oxytocin receptor agonists using in vitro systems for various extracts derived from various edible materials. As a result, they have found that a composition containing a material derived from a natural product that is easily available or edible on a daily basis, i.e., the compound of formula I, R1-valigenol and / or its derivatives, has activity as an oxytocin receptor agonist, leading to the completion of the present invention. That is, the present invention is as follows. [1] An oxytocin receptor agonist composition comprising R1-valigenol and / or an R1-valigenol derivative represented by the following formula I, wherein the R1-valigenol derivative is a glycoside or acylated derivative of R1-valigenol. Formula I TIFF0007597554000001.tif6072. [2] The oxytocin receptor agonist composition according to [1], wherein the glycoside or acylated derivative of R1-valigenol is selected from the following compounds: An oxytocin receptor agonist food comprising the oxytocin receptor agonist composition described in TIFF0007597554000002.tif125170[3][1] or [2]. [4] An oxytocin receptor agonist drug comprising the oxytocin receptor agonist composition according to [1] or [2]. [5] A food product according to [3] for preventing or alleviating diseases via activation of oxytocin receptors. [6] The pharmaceutical composition according to [4] for preventing, alleviating, or treating a disease mediated by activation of the oxytocin receptor. Effect of the Invention

[0007] According to the present invention, the composition containing R1-valigenol and / or R1-valigenol derivatives of the present invention has oxytocin receptor agonist activity and can activate the oxytocin receptor. Furthermore, the composition of the present invention is a composition containing ingredients derived from natural products (food ingredients) that are commonly eaten, and can be taken continuously for a long period of time at low cost. Furthermore, the present invention can be used in foods or medicines used for the treatment, amelioration, suppression, or prevention of diseases or symptoms related to oxytocin receptors, such as locomotive syndrome related to muscles and bones, modification of sweet sensitivity, suppression of enteritis, stress relief, suppression of eating behavior, suppression of alcohol intake, regulation of partner preference, promotion of maternal behavior, promotion of empathic behavior, improvement of communication ability, improvement of cognitive function, suppression of aggressive behavior, and anti-anxiety effects. [Brief description of the drawings]

[0008] [Figure 1] The pGL4.3 vector and the pF9A vector for constructing an OXTR agonist evaluation system are shown. [Diagram 2] The results of confirming the operation of the OXTR agonist evaluation system are shown. [Diagram 3] 1 shows the results of confirming the reactivity of the AVPR agonist evaluation system. [Figure 4] 1 shows the results of evaluation of R1-valigenol using an OXTR agonist evaluation system. [Diagram 5] 1 shows the results of evaluation of R1-valigenol using a V1aR agonist evaluation system. [Figure 6] 1 shows the results of an investigation into the concentration dependency of OXTR reactivity of R1-valigenol. [Figure 7]The pGL4.3 vector and a vector in which the OXTR cDNA was removed from pF9A (pF9A control) are shown. [Figure 8] 1 shows the results of an investigation into the non-OXTR-specific signal of R1-valigenol. [Figure 9] The results of confirming the operation of the calcium imaging evaluation system are shown. [Figure 10] 1 shows the results of evaluation of the OXTR agonist activity of R1-valigenol using a calcium imaging evaluation system. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0009] The oxytocin receptor agonist composition of the present invention comprises R1 valigenol and / or R1-valigenol derivatives of formula I below: Formula I TIFF0007597554000003.tif6072.

[0010] In the present invention, the R1-valigenol derivative is a glycoside or acylated derivative of R1-valigenol. The glycoside or acylated derivative of R1-valigenol is not particularly limited as long as it is contained as a component in a natural product. The glycoside or acylated derivative of R1-valigenol is preferably selected from the following compounds. TIFF0007597554000004.tif125170

[0011] The oxytocin receptor agonist composition of the present invention has oxytocin receptor agonist activity. The activity can be evaluated by known methods, including in vitro reporter assays using oxytocin receptor-expressing cells and intracellular Ca2+ + The oxytocin receptor agonist activity can be evaluated by imaging. Furthermore, the oxytocin receptor agonist activity can also be evaluated by determining whether the activity is inhibited by an oxytocin receptor antagonist.

[0012] Oxytocin has already been shown to have a function related to the oxytocin receptor in vivo, and we have used reporter assays and intracellular Ca2+ binding assays using human oxytocin receptor expressing cells in vitro. 2+ Its activity has been confirmed by imaging. From the above, it is clear that there is a correlation between in vitro oxytocin receptor agonist activity and in vivo oxytocin receptor-related effects. In addition, it is said that glycosides are hydrolyzed by intestinal bacteria before the aglycone is absorbed, and the glycosides of R1-valigenol, compound (1) chakasaponin II, compound (2) chakasaponin III, and compound (3) desacyl-floratheasaponin B, can be expected to have the same effect as R1-valigenol when administered orally. In addition, since the acylated form is hydrolyzed by esterase in the body to produce R1-valigenol, compound (4) 21-O-angeloyl-R1-barrigenol, which is an acylated form of R1-valigenol, can be expected to have the same effect as R1-valigenol when administered orally.

[0013] In the present invention, R1-valigenol and / or R1-valigenol derivatives may be contained in natural products such as tea flowers and Pittosporum tobira leaves in which they are contained, or in the form of dried or crushed products, or may be purified from extracts obtained by extraction from tea flowers and Pittosporum tobira, which are materials derived from natural products. Tea is the tea plant (scientific name: Camellia sinensis) of the genus Camellia in the family Theaceae, and Pittosporum tobira is the plant (scientific name: Pittosporum tobira) of the genus Pittosporum in the family Pittosporaceae. For example, tea flowers and Pittosporum tobira leaves can be used as the extracted sample. In the present invention, the storage state, crushing method, variety, or place of origin of these extracted samples are not particularly limited.

[0014] The extract can be obtained by an extraction method known to those skilled in the art, for example, organic solvent extraction, supercritical fluid extraction, heating and pressurizing extraction, etc. The above extraction methods may be used alone or in combination. In the present invention, a particularly preferred extraction method is solvent extraction. Solvent extraction can be performed using a general method known to those skilled in the art. Specifically, water and an organic solvent are first added to the pulverized or dried crushed material, and extraction is performed at room temperature or with heating. Examples of the extraction solvent include, but are not limited to, ethanol, methanol, ethyl acetate, acetone, water, etc. These solvents may be used alone or in combination of two or more. It is preferable to use ethanol because of its safety when used in food, etc. The amount of the solvent to be added is 1 to 100 times, preferably 5 to 50 times, for example 10 times the weight of the extraction material. The extraction method may be, for example, a method in which tea flowers or Pittosporum tobira leaves are mixed with the extraction solvent, and the mixture is stirred at room temperature for 1 to 5 hours or refluxed at the boiling temperature of the extraction solvent for 1 to 5 hours to perform extraction, and then sample residues are removed from the extract by filtration or centrifugation, and the extract is concentrated by reduced pressure or ultrafiltration. If necessary, hydrolysis is performed with an acid or alkali. Furthermore, the extract can be dried by a general method such as drying, freeze-drying, or spray drying.

[0015] From the extract obtained as described above, R1-valigenol and / or R1-valigenol derivatives can be purified by combining an appropriate separation method and purification method for use. Examples of the purification method include liquid-liquid distribution, organic solvent precipitation, various column chromatography (e.g., HPLC, silica gel chromatography, molecular sieve chromatography, ion exchange chromatography, reverse phase chromatography, etc.), crystallization, etc. The obtained R1-valigenol and / or R1-valigenol derivatives can be directly ingested as oxytocin receptor agonists, or can be incorporated into known carriers or various base materials.

[0016] The dosage or intake of R1-valigenol and / or R1-valigenol derivatives is an amount that can provide a therapeutic effect for a given symptom or method of use, and the amount is appropriately determined by a person skilled in the art by carrying out tests using animals and clinical trials, but the age, sex, and weight of the subject to be administered, the applicable disease and its symptoms, dosage form, administration method, etc. should be taken into consideration. For example, when orally administered by oral administration using an oral agent, the dosage or intake can be calculated as the amount of R1-valigenol, and can be 0.01 to 100 mg / kg body weight / day, preferably 0.01 to 20 mg / kg body weight / day. When contained in food, R1-valigenol and / or R1-valigenol derivatives are contained in an amount of 0.01 to 50% by weight, preferably 1 to 30% by weight, calculated as R1-valigenol, and should be ingested in an amount of 0.5 to 5000 mg, preferably 0.5 to 100 mg, per day per adult (body weight approximately 50 kg).

[0017] The present invention relates to a food containing the above-mentioned oxytocin receptor agonist composition. The oxytocin receptor agonist composition of the present invention may be prepared as a health food, a functional food, a specific health food, a nutritional supplement, a nutritional functional food, or other health functional food, a special purpose food (e.g., a food for sick people), a food labeled with a disease risk reduction label, a dietary supplement, a supplement, or the like. As a supplement, for example, it can be in the form of a tablet, a pill, a capsule (including a hard capsule, a soft capsule, and a microcapsule), a powder, a granule, a fine granule, a lozenge, a liquid (including a syrup, a milk, and a suspension) together with various additives used in the manufacture of general supplements. It may also be in the form of a liquid, semi-liquid, or solid, a paste, or added to general food and drink.

[0018] There are no particular limitations on the foods that can be added, but examples include carbohydrate-containing foods and beverages such as rice, rice cakes, noodles, bread, and pasta; various sweets such as Western sweets such as cookies and cakes, Japanese sweets such as buns and yokan, candies, gum, cold desserts and frozen desserts such as yogurt and pudding; various beverages such as juice, soft drinks, milk drinks, tea drinks, functional drinks, nutritional supplements, and non-alcoholic beer; alcoholic beverages such as beer and sparkling wine; foods and beverages such as soup, miso soup, and clear soup; processed products using eggs, processed products (including delicacies) made from seafood (squid, octopus, shellfish, eel, etc.) and meat (including offal such as liver); dashi, soy sauce, mirin, and other seasonings.

[0019] Furthermore, the food of the present invention may contain, in addition to the oxytocin receptor agonist composition, other ingredients such as nutritional supplements, so long as the effects of the present invention are not impaired. Such ingredients include vitamins (e.g., vitamin A, B vitamins, vitamin E, vitamin C, vitamin D, vitamin K, niacin, pantothenic acid, folic acid, etc.), carotenoids (e.g., β-carotene, lycopene, fucoxanthin, etc.), minerals (e.g., seaweed ingredients, CCM, heme iron, iron salts, whey calcium, fermented lactate calcium, bovine bone calcium, coral calcium, eggshell calcium, etc.), various plants, and their extracts, purified products, and fractions. (For example, plantain, chlorella, spirulina, garlic, ginkgo leaves, gymnema, eucommia leaves, shiso leaves, pearl barley, soy globulin, rutin, green tea extract, theanine, polyphenols, licorice, yucca, soy saponin, caffeine, white berry extract, champignon extract, Garcinia cambogia extract, etc.), microorganisms and their growth factors and microbial products (for example, lactic acid bacteria, yeast, lactic acid bacteria growth factors, etc.), dietary fiber and its enzymatic hydrolysis products (e.g. apple fiber, corn fiber, dietary fiber derived from starch, resistant dextrin, enzymatic hydrolysis of guar gum, sweet potato fiber, soybean fiber, seaweed fiber, mushroom fiber, tea fiber, acidic polysaccharides, plant mucilage, wheat bran, etc.), animal bodies and their extracts, refined products, hydrolyzates and products (e.g. royal jelly, propolis, oyster extract, chitin, chitosan, taurine, collagen, gelatin, etc.), various oligosaccharides (e.g. galactooligosaccharides, xylooligosaccharides, soybean oligosaccharides, fructooligosaccharides, isomaltooligosaccharides, lactose oligosaccharides, etc.), lipids (e.g. unsaturated fatty acids (DHA, EPA, etc.), phospholipids, Salatrim, etc.), various proteins and protein hydrolysates (e.g. corn protein, soybean protein, TMP (total milk protein), lactalbumin, casein, whey, glutathione, soybean peptides, egg white peptides, glutamine peptides, etc.), wheat germ such as defatted germ, etc.

[0020] The present invention relates to a medicine containing the above-mentioned oxytocin receptor agonist composition. The oxytocin receptor agonist composition of the present invention can be formulated into any form together with an auxiliary to produce a medicine that can be administered orally or parenterally. For example, oral preparations include granules, powders, tablets (including sugar-coated tablets), pills, capsules, syrups, emulsions, and suspensions. Parenteral preparations include injections (e.g., subcutaneous injections, intravenous injections, intramuscular injections, and intraperitoneal injections), drip infusions, external preparations (e.g., nasal preparations, transdermal preparations, ointments), and suppositories (e.g., rectal suppositories, vaginal suppositories). These preparations can be formulated using pharma- ceutical acceptable carriers by methods commonly used in the art. Pharmaceutically acceptable carriers include excipients, binders, diluents, additives, flavorings, buffers, thickeners, colorants, stabilizers, emulsifiers, dispersants, suspending agents, preservatives, and the like. For example, magnesium carbonate, magnesium stearate, talc, sugar, lactose, pectin, dextrin, starch, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose, low melting point wax, and cocoa butter can be used as carriers.

[0021] In the case of oral preparations, for example, excipients (e.g., lactose, sucrose, starch, mannitol), disintegrants (e.g., calcium carbonate, carboxymethylcellulose calcium), binders (e.g., pregelatinized starch, gum arabic, carboxymethylcellulose, polyvinylpyrrolidone, hydroxypropylcellulose) or lubricants (e.g., talc, magnesium stearate, polyethylene glycol 6000) are added to the active ingredient, followed by compression molding, and then coating by a method known per se for the purposes of taste masking, enteric properties or sustained release, if necessary. Examples of coating agents that can be used include ethyl cellulose, hydroxymethyl cellulose, polyoxyethylene glycol, cellulose acetate phthalate, hydroxypropyl methylcellulose phthalate and Eudragit (manufactured by Rohm, Germany, methacrylic acid-acrylic acid copolymer).

[0022] In the case of the form of an injection, the active ingredient can be dissolved, suspended or emulsified in an aqueous solvent (e.g., distilled water, physiological saline, Ringer's solution, etc.) or an oily solvent (e.g., vegetable oils such as olive oil, sesame oil, cottonseed oil, corn oil, propylene glycol) together with a dispersant (e.g., Tween 80 (manufactured by Atlas Powder, USA), HCO60 (manufactured by Nikko Chemicals), polyethylene glycol, carboxymethylcellulose, sodium alginate, etc.), a preservative (e.g., methylparaben, propylparaben, benzyl alcohol, chlorobutanol, phenol), an isotonicity agent (e.g., sodium chloride, glycerin, sorbitol, glucose, invert sugar), etc. In this case, additives such as a solubilizing agent (e.g., sodium salicylate, sodium acetate), a stabilizer (e.g., human serum albumin), a soothing agent (e.g., benzalkonium chloride, procaine hydrochloride), etc. may be added as desired.

[0023] When the external preparation is used, it can be produced by making the active ingredient into a solid, semi-solid or liquid composition. For example, the solid composition can be produced by adding and mixing the active ingredient as it is, or with excipients (e.g., lactose, mannitol, starch, microcrystalline cellulose, sucrose), thickeners (e.g., natural gums, cellulose derivatives, acrylic acid polymers), etc. to make it into a powder. The liquid composition can be produced in almost the same manner as the injection. The semi-solid composition is preferably an aqueous or oily gel, or a cartilage-like composition. In addition, any of these compositions may contain a pH regulator (e.g., carbonic acid, phosphoric acid, citric acid, hydrochloric acid, sodium hydroxide), a preservative (e.g., paraoxybenzoic acid esters, chlorobutanol, benzalkonium chloride), etc. Suppositories can be produced by making the active ingredient into an oily or aqueous solid, semi-solid or liquid composition. The oil base used in the composition includes glycerides of higher fatty acids (e.g., cacao butter, Witepsols (Dynamite Nobel)), medium fatty acids (e.g., Miglyols (Dynamite Nobel)), or vegetable oils (e.g., sesame oil, soybean oil, cottonseed oil). The aqueous base includes polyethylene glycols and propylene glycol. The aqueous gel base includes natural gums, cellulose derivatives, vinyl polymers, and acrylic acid polymers.

[0024] Since the food and medicine of the present invention contain active ingredients derived from natural products as described above, there is little concern about side effects, etc., and they can be taken continuously over a long period of time at low cost.

[0025] The food or medicine of the present invention can be used for preventing, alleviating, or treating symptoms or diseases mediated by oxytocin receptor activation by oxytocin receptor agonist composition. Examples of such symptoms or diseases include, but are not limited to, muscle and bone-related locomotive syndrome, modification of sweetness sensitivity, suppression of enteritis, stress relief, suppression of eating behavior, suppression of alcohol intake, regulation of partner preference, promotion of maternal behavior, promotion of empathic behavior, improvement of communication ability, improvement of cognitive function, suppression of aggressive behavior, anti-anxiety effect, etc. EXAMPLES

[0026] EXAMPLES In the following, examples are shown to specifically explain the present invention, but the present invention is not limited to the following examples.

[0027] Experiment 1: Construction of an evaluation system for OXTR agonists and confirmation of their reactivity (1) Development of an evaluation system for OXTR agonists To investigate the reactivity of OXTR-acting agonists, we constructed an evaluation system in which exogenous OXTR was expressed in cultured cells and activation of downstream signals of OXTR was detected by luciferase (a reporter gene). That is, pGL4.3 and pF9A-OXTR were double-transfected into HEK293 cells, which are human embryonic kidney cells, to construct an evaluation system for OXTR-acting agonists (see Figure 1; hereafter, this evaluation system is referred to as the OXTR agonist evaluation system). According to this evaluation system, OXTR is expressed by the CMV promoter in the double-transfected cells, and when an agonist acting on OXTR is added to these cultured cells, OXTR is activated and emits a signal, which is transmitted to and activated by NFAT-RE, which is a response element of the downstream signal of OXTR, and firefly luciferase (Luc-2P), which is encoded downstream of OXTR, is expressed, so that the reactivity of OXTR-acting agonists can be evaluated by measuring the activity of this firefly luciferase. Needless to say, this OXTR agonist evaluation system can also be used to evaluate antagonists that act on OXTR. In addition, pF9A-OXTR is a vector in which human OXTR cDNA has been introduced into pF9A CMV hRluc-neo Flexi Vector (Promega) expressing Renilla luciferase (hRluc). pGL4.3 encodes firefly luciferase downstream of NFAT-RE, which is a response element of the downstream signal of OXTR.

[0028] (2) Confirmation of the operation of the evaluation system for OXTR agonists (2)-1 Preparation of transformed cells Human embryonic kidney (HEK293) cells were cultured at 3 × 10 6 The cells were seeded onto a 10 cm cell culture dish at 10 cells / dish and cultured at 37°C under 5% CO2. DMEM + 10% FBS was used as the culture medium. Two types of vectors, pF9A OXTR and pGL4.3, were mixed at a ratio of pF9A OXTR: pGL4.30 = 1:50, and after 24 hours of culture, the cells were transformed using Fugene HD transfection reagent (Promega) to prepare transformed cells for use in the OXTR agonist evaluation system. 24 hours after transformation, the cells were harvested by trypsin treatment, suspended in DMEM + 10% FBS, and plated on a collagen-coated white-walled 96-well microplate at 5 × 10 4 The cells were seeded at 1 cell / well. (2)-2 Ligand reporter assay After seeding in a 96-well microplate and culturing for 12 hours, the medium was replaced with DMEM + 10% FBS, and in test group 3 (OXT + antagonist), L-371,257 (an antagonist against OXTR) dissolved in DMSO was added at a final concentration of 10 -4 M, and incubated for 1 hour. DMSO was added to test plot 1 (negative control) and test plot 2 (OXT) to the same DMSO concentration as in test plot 3. Each test plot had n=7-8. After incubation, the final concentration of OXT in test areas 2 and 3 was 2 × 10 ‐5 M to OXT 10 ‐2The M aqueous solution was added, and the mixture was further incubated for 4 hours at 37°C under 5% CO2 conditions. Note that the same amount of water was added to test group 1. After incubation, the signal intensity of firefly luciferase relative to Renilla luciferase was measured using Dual-Luciferase (registered trademark) Reporter Assay System (Promega). Test area 1 was used as a reference, and the signal intensity of firefly luciferase in test area 1 was set to 1, and the signal intensities in test areas 2 and 3 were calculated as relative values ​​(induction fold relative to the negative control: Hold induction vs. Negative Control). The results are shown in Figure 2. In test area 2, the addition of OXT significantly increased the firefly luciferase signal. In test area 3, the antagonist for OXTR acted on OXTR, so the increase in the firefly luciferase signal due to the addition of OXT was significantly suppressed. From the above, it was shown that the behavior of OXTR can be evaluated when a ligand and an antagonist are applied to OXTR, and it was found to be valid as an OXTR agonist evaluation system.

[0029] Experiment 2: Construction of an evaluation system for agonists acting on AVPR and confirmation of their reactivity Using pF9A AVPR, which had human V1aR cDNA introduced as human AVPR instead of human OXTR cDNA, an evaluation system for agonists acting on AVPR (hereinafter referred to as the V1aR agonist evaluation system) was constructed, and the construction and reactivity of the V1aR agonist evaluation system were confirmed according to Experiment 1, except that AVP was added instead of OXT as a ligand and SR49059 was added instead of L-371,257 as an antagonist to confirm the operation of the evaluation system. Note that NFAT-RE, like OXTR, is known to be activated in response to a signal emitted by AVPR activation. As a result, similar to experiment 1, the addition of AVP significantly increased the firefly luciferase signal, and the increase in the firefly luciferase signal was significantly suppressed by the AVP antagonist (Figure 3). Therefore, it was found that this is a valid evaluation system for OXTR agonists.

[0030] Experiment 3 R 1 -Evaluation of Valigenol as a specific OXTR agonist Experiment 3-1 R using OXTR agonist evaluation system 1 - Valigenol evaluation Instead of OXT, use R1-valigenol at a final concentration of 2 × 10 ‐5 The reactivity of R1-valigenol to OXTR was examined according to Experiment 1, except that R1-valigenol was added to the transformed cells so that the concentration was M (Figure 4). As a result, the addition of R1-valigenol significantly increased the firefly luciferase signal, and this increase in the signal was significantly suppressed by the OXTR antagonist. Therefore, it was shown that R1-valigenol has activity as an OXTR agonist.

[0031] Experiment 3-2 R using V1aR agonist evaluation system 1 - Valigenol evaluation Instead of AVP, R1-valigenol was used at a final concentration of 2 × 10 ‐5 The reactivity of R1-valigenol to V1aR was examined as described in Experiment 2, except that it was added to the transformed cells so that it became M (Figure 5). As a result, the addition of R1-valigenol did not change the firefly luciferase signal, indicating that R1-valigenol does not have V1aR agonistic activity.

[0032] Experiment 3-3 R 1 -Examination of the concentration dependence of OXTR reactivity with Valigenol R1-valigenol was added at a final concentration of 1 x 10 ‐4 , 1×10 ‐5 , 1×10 ‐6 , 1×10 ‐7 The reactivity of R1-valigenol to OXTR was examined according to Experiment 3-1, except that the concentration of R1-valigenol was increased to 1000 mg / kg (Figure 6). As a result, the signal increased as the concentration of R1-valigenol added increased. This confirmed the concentration-dependence of the activity of R1-valigenol as an OXTR agonist.

[0033] Experiment 4 R 1 -Study on OXTR non-specific signaling by Valigenol The NFAT-RE of pGL4.3 used in experiments 1 and 3 may also react to the activation of GPCRs other than OXTR. Therefore, to confirm that the increase in signal due to the addition of R1-valigenol observed in experiment 3 was an OXTR-dependent signal, the firefly luciferase signal due to the addition of OXT or R1-valigenol was evaluated according to experiment 1, except that the cells were transformed with a vector in which OXTR cDNA had been removed from pGL4.3 and pF9A (hereinafter referred to as pF9A control: Figure 7). As a result, no increase in signal was observed due to the addition of OXT or R1-valigenol (Figure 8). This revealed that R1-valigenol increases the signal only in cells transformed with OXTR, and that R1-valigenol increases the signal via OXTR.

[0034] Experiment 5. Construction of calcium imaging evaluation system Experiment 5-1 Establishment of OXTR stable expressing cell line OXTR is a GPCR of the calcium mobilization system, and its activation can be evaluated by detecting calcium ions (hereinafter referred to as calcium imaging evaluation system). R1-valigenol was added to cells stably expressing OXTR, and the reactivity of R1-valigenol to OXTR was evaluated by detecting calcium ions. The method for stably expressing OXTR in cells is as follows. pF9A-OXTR was transformed into HEK293 cells, and the antibiotic G418 was added to a final concentration of 500 μg / ml for drug selection. A neomycin resistance gene was introduced into pF9A OXTR, and cells into which pF9A-OXTR was introduced could be selected with G418. Since OXTR is expressed by the CMV promoter in the cells into which pF9A-OXTR was introduced, the cells obtained by drug selection were OXTR stably expressing cells.

[0035] Experiment 5-2: Operational confirmation of calcium imaging evaluation system 5×10 in a 96-well microplate 4OXTR-highly expressing cells were seeded at 10 cells / well. After seeding in a 96-well microplate and culturing for 12 hours, L-371,257 (an antagonist of OXTR) dissolved in DMSO was added to the test group 3 (OXT+ L371,257) at a final concentration of 10 ‐4 M, and incubated for 1 hour. DMSO was added to test area 1 (negative control) and test area 2 (OXT) to the same DMSO concentration as in test area 3. Each test area had n=7-8, and luminescence signals were detected using Calcium Kit - Fluo 4 (Dojindo Chemical). After incubation, the final concentration of OXT in test areas 2 and 3 was 2 × 10 ‐5 OXT 2×10 to be M ‐4 M aqueous solution was added, and the luminescence signal for 60 seconds after addition was measured using a plate reader. The same amount of water was added to test area 1, and the signal was measured for 60 seconds in the same way. The maximum signal for 60 seconds after addition was taken as the signal value for each well, and the signal increase rate (signal induction) was calculated relative to the signal value before addition for evaluation. As a result, the signal significantly increased with the addition of OXT, and the signal significantly decreased with the addition of an OXTR antagonist, indicating that this is an appropriate evaluation system for OXTR agonists. (Figure 9)

[0036] Experiment 6. Evaluation of OXTR agonist activity using calcium imaging assay 2×10 instead of OXT ‐4 M R1-valigenol final concentration 2 x 10 ‐5 The reactivity of R1-valigenol to OXTR was examined according to Experiment 5, except that R1-valigenol was added to the cells so that the concentration was M (Figure 10). As a result, the addition of R1-valigenol significantly increased the calcium ion signal, and this increase in the signal was significantly suppressed by the OXTR antagonist. This indicates that R1-valigenol has activity as an OXTR agonist.

Claims

1. R of Formula I below 1 -Valigenol and / or R 1 - Valigenol derivatives, 1 - Valigenol derivatives are R 1 - A composition for activating the oxytocin receptor, which is a glycoside or acylated derivative of valigenol Formula I 。

2. The R 1 The composition for activating the oxytocin receptor according to claim 1, wherein the glycoside or acylated derivative of valigenol is selected from the following compounds: 。

3. An oxytocin receptor activating food comprising the oxytocin receptor activating composition according to claim 1 or 2.

4. An oxytocin receptor agonist drug comprising the oxytocin receptor agonist composition according to claim 1 or 2.

5. The food according to claim 3 for preventing or alleviating a disease mediated by activation of oxytocin receptors.

6. The pharmaceutical composition according to claim 4, for preventing, alleviating, or treating a disease mediated by activation of the oxytocin receptor.

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