Menopausal symptom relief

Abstract

To provide a menopausal symptom improving agent useful for improving menopausal symptoms.SOLUTION: A menopausal symptom improving agent comprises delphinidin-3-lucinoside, cyanidin-3-lucinoside, or a combination thereof as an active ingredient.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to an agent for alleviating menopausal symptoms. [Background technology]

[0002] During menopause, women experience a variety of physical complaints, including general fatigue, dizziness, headache, insomnia, hot flashes, sweating, palpitations, nausea, diarrhea, stiff shoulders, lower back pain, and frequent urination. Among these, hot flashes and sweating are characteristic symptoms of menopause and are seen in many women.

[0003] Menopausal symptoms are thought to be caused by a decline in the secretion of female hormones (estrogen) with aging. Therefore, the most common treatment for menopausal symptoms is hormone replacement therapy (HRT), which involves administering estrogen or estrogen and progesterone. However, it is said that menopausal symptoms are not just caused by a decrease in estrogen, but also involve a complex mix of social and psychological factors stemming from work, home environment, etc. Furthermore, it has been pointed out that HRT has negative aspects, such as increasing the incidence of breast cancer and uterine cancer, and there is a need for safer ways to alleviate menopausal symptoms.

[0004] On the other hand, blackcurrant (scientific name: Ribes nigrum), also known as black currant or black gooseberry, is a plant classified in the Ribes genus of the Saxifragaceae family and contains a large amount of four types of anthocyanins. Among the cassis anthocyanins, the rutinosides delphinidin-3-rutinoside and cyanidin-3-rutinoside are characteristic components of cassis anthocyanins. It has been reported that cassis anthocyanins improve blood flow, relieve stiff shoulders, general fatigue, etc. (Patent Document 1), and have estrogenic effects (Non-Patent Document 1). However, it is unclear whether blackcurrant anthocyanins have any effect on improving menopausal symptoms. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] International Publication No. 2001 / 01798 [Non-patent literature]

[0006] [Non-Patent Document 1] Molecules, 23(1), 74, 2018 Summary of the Invention [Problem to be solved by the invention]

[0007] The present invention relates to providing an agent for improving menopausal symptoms, which is useful for improving menopausal symptoms. [Means for solving the problem]

[0008] The present inventors have discovered that delphinidin-3-rutinoside and cyanidin-3-rutinoside are effective in improving menopausal symptoms such as hot flashes and sweating.

[0009] The present invention relates to the following 1) to 7). 1) An agent for improving menopausal symptoms, the active ingredient of which is delphinidin-3-rutinoside, cyanidin-3-rutinoside or a combination thereof. 2) A food for improving menopausal symptoms, containing delphinidin-3-rutinoside, cyanidin-3-rutinoside or a combination thereof as an active ingredient. 3) Use of delphinidin-3-rutinoside, cyanidin-3-rutinoside or a combination thereof for producing an agent for improving menopausal symptoms. 4) Use of delphinidin-3-rutinoside, cyanidin-3-rutinoside or a combination thereof for producing a food for improving menopausal symptoms. 5) Delphinidin-3-rutinoside, cyanidin-3-rutinoside or a combination thereof for use in improving menopausal symptoms. 6) Non-therapeutic use of delphinidin-3-rutinoside, cyanidin-3-rutinoside or a combination thereof to improve menopausal symptoms. 7) A method for improving menopausal symptoms, comprising administering or ingesting an effective amount of delphinidin-3-rutinoside, cyanidin-3-rutinoside, or a combination thereof to a subject in need thereof. [Effects of the Invention]

[0010] The menopausal symptom alleviating agent of the present invention can alleviate menopausal symptoms such as hot flashes (hot flushes, flushing) and sweating. [Brief explanation of the drawings]

[0011] [Figure 1] Graph showing the process of cassis anthocyanin purification. [Figure 2] Preparative HPLC chromatogram of blackcurrant anthocyanins. [Figure 3] 1H, 13C-NMR spectra of Fr.E-3 to -6. DETAILED DESCRIPTION OF THE INVENTION

[0012] In the present invention, delphinidin-3-rutinoside is a glycoside in which rhamnosyl-(α1→6)-glucose is bound to the hydroxyl group at the 3-position of delphinidin, and has the following structure:

[0013] [ka]

[0014] In the present invention, cyanidin-3-rutinoside is a glycoside in which rhamnosyl-(α1→6)-glucose is bound to the hydroxyl group at the 3-position of cyanidin, and has the following structure:

[0015] [ka]

[0016] Delphinidin-3-rutinoside and cyanidin-3-rutinoside may be in the form of a salt or a solvate, and both are included. These may be used alone or in combination of two or more. Among these, delphinidin-3-rutinoside is preferably used because it is easier to obtain the desired effects. Delphinidin-3-rutinoside and cyanidin-3-rutinoside can be obtained by chemical synthesis or by extraction and purification from natural products containing them, particularly plants. Commercially available reagents can also be used. Because delphinidin-3-rutinoside and cyanidin-3-rutinoside are found in black currant (scientific name: Ribes nigrum), it is preferable to use those derived from black currant extract. Black currant extract may also be used as delphinidin-3-rutinoside and cyanidin-3-rutinoside.

[0017] The part of black currant used is not particularly limited, and may be the whole plant, leaves, stems, flowers, buds, peel, fruit, roots, rhizomes, seeds, or a combination thereof. However, it is preferable to use the fruit or peel because they are rich in black currant anthocyanins. Black currant can be used fresh, dried, or processed. Examples of processing include cutting, crushing, grinding, and pulverization. The extraction method can be appropriately selected, and may be any of immersion, decoction, percolation, reflux extraction, supercritical extraction, ultrasonic extraction, microwave extraction, and the like.

[0018] Both polar and nonpolar solvents can be used as the solvent for extraction. Specific examples of solvents include water; monohydric, dihydric, or polyhydric alcohols; ketones such as acetone and methyl ethyl ketone; esters such as methyl acetate and ethyl acetate; linear or cyclic ethers such as diethyl ether and tetrahydrofuran; polyethers such as polyethylene glycol; saturated or unsaturated hydrocarbons such as hexane; aromatic hydrocarbons such as benzene and toluene; halogenated hydrocarbons such as dichloromethane, chloroform, dichloroethane, and carbon tetrachloride; pyridines; dimethyl sulfoxide; acetonitrile; carbon dioxide; supercritical carbon dioxide; fats and oils, waxes, and other oils; and mixtures thereof. Alcohols and aqueous solutions thereof are preferred. Examples of alcohols include methanol, ethanol, 1,3-butylene glycol, n-propanol, isopropanol, n-butanol, isobutanol, sec-butanol, and t-butanol. Ethanol and aqueous ethanol solutions are preferred. The amount of solvent used in the extraction and the extraction conditions are not particularly limited as long as they allow sufficient extraction, and can be adjusted appropriately.

[0019] The blackcurrant extract may be a crude product as long as it meets food and pharmaceutical acceptable standards, contains blackcurrant anthocyanins, and exhibits the effects of the present invention. If necessary, it may be subjected to treatments such as removal of inactive impurities, deodorization, and decolorization using known techniques such as liquid-liquid partitioning, solid-liquid partitioning, membrane filtration, activated carbon, synthetic adsorption resins, ion exchange resins, and sedimentation. Furthermore, the purity of these compounds may be increased by appropriately combining known separation and purification methods, such as organic solvent precipitation, centrifugation, ultrafiltration membrane, high performance liquid chromatography, and column chromatography.

[0020] Blackcurrant extract can be used as is, diluted with an appropriate solvent, or prepared as a concentrated extract, dried powder, or paste. It can also be lyophilized and diluted with a solvent commonly used in extractions, such as water, ethanol, or a water-ethanol mixture, before use. It can also be encapsulated in vesicles such as liposomes or microcapsules.

[0021] The black currant extract preferably contains delphinidin-3-rutinoside and cyanidin-3-rutinoside in a total amount of 0.1% by mass or more, and more preferably 10% by mass or more.

[0022] As shown in the Examples below, oral ingestion of delphinidin-3-rutinoside and cyanidin-3-rutinoside improves menopausal symptoms such as hot flashes and sweating. Blackcurrant anthocyanins have been reported to have estrogenic activity (Molecules, 23(1), 74, 2018). However, the estrogenic activity of blackcurrant anthocyanins is approximately one-thousandth that of estradiol (ibid.). Therefore, the blackcurrant anthocyanins used in this human efficacy study are thought to have approximately one-tenth the estrogenic activity of hormone replacement therapy (HRT). Therefore, delphinidin-3-rutinoside and cyanidin-3-rutinoside are thought to improve menopausal symptoms without estrogen activation. On the other hand, when delphinidin-3-rutinoside and cyanidin-3-rutinoside were contacted with transformed cells in which TRPV4 was functionally expressed by introducing the human TRPV4 gene (TRPV4-expressing cells) in the presence of a TRPV4 agonist, they did not inhibit the increase in intracellular cations (Ca 2+ It was confirmed that the compound has the effect of suppressing the activity of TRPV4, that is, suppressing the influx of ATP (Reference Example 1). TRPV4 (Transient receptor potential cation channel subfamily V member 4) is one of the proteins that make up the thermosensitive TRP channel. TRPV4 is expressed in a wide range of tissues, including the kidney, lung, bladder, heart, skin, brain, and gastrointestinal tract, and is thought to function as a sensor molecule that detects various physicochemical stimuli, such as changes in osmotic pressure, temperature, shear stress caused by blood flow, and changes in organ volume, and transmits these signals to cells. Chlorogenic acid, caffeic acid, and ferulic acid, which are known to inhibit TRPV4 activity, have been reported to improve autonomic nervous function and generalized symptoms (Patent Nos. 6075830, 5931633, and 4077149). Therefore, it is believed that inhibiting TRPV4 activity can improve generalized symptoms in menopausal women. Therefore, delphinidin-3-rutinoside and cyanidin-3-rutinoside are speculated to improve menopausal symptoms through the inhibition of TRPV4 activity.

[0023] Therefore, delphinidin-3-rutinoside, cyanidin-3-rutinoside, or a combination thereof can be an agent for alleviating menopausal symptoms, can be used for alleviating menopausal symptoms, and can also be used to produce an agent for alleviating menopausal symptoms. Here, "use" refers to use in humans or non-human animals, and may be therapeutic or non-therapeutic use. "Non-therapeutic" is a concept that does not include medical procedures, i.e., methods of surgery, therapy, or diagnosis on humans, and more specifically, methods of surgery, therapy, or diagnosis on humans by physicians or those under the direction of physicians.

[0024] The Japan Society of Obstetrics and Gynecology defines menopausal symptoms and disorders as follows: "The five years before and after menopause are called menopause. Of the wide variety of symptoms that appear during this period, those that are not caused by organic changes are called menopausal symptoms, and among these symptoms, pathological conditions that interfere with daily life are called menopausal disorders" (Women's Medical Guidebook, Menopausal Care Edition, 2014 Edition, Japanese Society of Women's Medicine Edition). Examples of menopausal symptoms include dizziness, headache, insomnia, hot flashes, flushing, sweating, palpitations, nausea, constipation, diarrhea, stiff shoulders, lower back pain, joint pain, frequent urination, dry skin or mucous membranes, eczema, etc. In this specification, "amelioration of menopausal symptoms" means improvement of menopausal symptoms, preferably improvement of hot flashes and sweating. As used herein, the term "hot flash" refers to a sudden feeling of heat (fluster, flushing) in the head, chest, or whole body that occurs during menopause. "Amelioration" refers to the improvement of a symptom or condition, the prevention or slowing of the worsening of a symptom or condition, or the reversal, prevention, or slowing of the progression of a symptom or condition.

[0025] The menopausal symptom alleviating agent of the present invention can itself be a pharmaceutical product, quasi-drug product, or food product for humans or animals that exhibits the effect of alleviating menopausal symptoms, or can be a material or preparation to be incorporated into such pharmaceutical products, quasi-drugs, or foods. These foods include functional foods, foods for specified health uses, supplements, etc. that are based on the concept of improving menopausal symptoms and, where necessary, label the product as such. These foods are permitted to make functional claims, so they can be distinguished from general foods.

[0026] The pharmaceuticals (including quasi-drugs) contain delphinidin-3-rutinoside, cyanidin-3-rutinoside, or a combination thereof as active ingredients for improving menopausal symptoms. Furthermore, the pharmaceuticals may contain pharmaceutically acceptable carriers, other active ingredients, pharmacological ingredients, etc., as needed, as long as the functions of the active ingredients are not impaired.

[0027] Examples of dosage forms of the pharmaceuticals (including quasi-drugs) include oral solid preparations such as tablets (including chewable tablets), capsules, granules, powders, and lozenges; oral liquid preparations such as oral liquids and syrups; and parenteral preparations such as injections, suppositories, inhalants, transdermal agents, and topical agents. The preferred administration form is oral administration. These formulations can be prepared according to standard methods by appropriately combining blackcurrant anthocyanins with pharmaceutically acceptable carriers (e.g., excipients, binders, bulking agents, disintegrants, surfactants, lubricants, dispersants, buffers, preservatives, flavoring agents, fragrances, coating agents, diluents, etc.), other medicinal ingredients, etc.

[0028] The food product contains delphinidin-3-rutinoside, cyanidin-3-rutinoside, or a combination thereof as an active ingredient for improving menopausal symptoms. The food product may be in the form of a solid, semi-solid, or liquid (e.g., a beverage). Examples of the food product include beverages such as soft drinks, tea drinks, coffee drinks, fruit juice drinks, carbonated drinks, jelly drinks, and near-water, as well as various foods such as jellies, wafers, biscuits, bread, noodles, and sausages, as well as nutritional foods, and their ingredients. Alternatively, the food product may be a supplement in the form of an oral preparation such as a tablet, capsule, granule, powder, liquid, or syrup.

[0029] The food can be prepared according to a standard method by appropriately combining delphinidin-3-rutinoside, cyanidin-3-rutinoside, or a combination thereof with any food material, or other active ingredient, or additive acceptable for food (e.g., solvents, softeners, oils, emulsifiers, preservatives, acidulants, sweeteners, bittering agents, pH adjusters, stabilizers, colorants, UV absorbers, antioxidants, humectants, thickeners, adhesives, dispersants, flow improvers, wetting agents, aromatics, seasonings, flavor adjusters), etc.

[0030] The content of delphinidin-3-rutinoside, cyanidin-3-rutinoside, or a combination thereof in the above-mentioned drugs, quasi-drugs, or foods may vary depending on their dosage form or shape. For example, in the case of foods or oral preparations that are solid, such as tablets, granules, pills, powders, or gummies, the total amount of delphinidin-3-rutinoside and cyanidin-3-rutinoside is preferably 0.1% by mass or more, more preferably 0.5% by mass or more, even more preferably 1% by mass or more, still more preferably 5% by mass or more, and even more preferably 10% by mass or more. In the case of foods or oral preparations that are liquids such as solutions, syrups, suspensions, emulsions, elixirs, and jellies, the total amount of delphinidin-3-rutinoside and cyanidin-3-rutinoside is preferably 0.001% by mass or more, more preferably 0.005% by mass or more, even more preferably 0.01% by mass or more, and even more preferably 0.05% by mass or more.

[0031] The dosage and administration schedule of delphinidin-3-rutinoside, cyanidin-3-rutinoside, or a combination thereof can be appropriately determined by those skilled in the art depending on the species, weight, sex, age, condition, or other factors of the subject. Hormone replacement therapy for menopausal symptoms has a complex relationship between risks and benefits. While it is suitable for symptom management in some women, it has been suggested that it may increase the risk of serious illnesses (JAMA, 288(3), 321-333, 2002; JAMA, 310(13), 1353, 2013). Therefore, it is important to alleviate menopausal symptoms without exerting hormone-like effects. Therefore, the dosage of delphinidin-3-rutinoside, cyanidin-3-rutinoside, or a combination thereof is, for example, the total amount of delphinidin-3-rutinoside and cyanidin-3-rutinoside per day for an adult (60 kg), preferably 1 mg or more, more preferably 10 mg or more, even more preferably 25 mg or more, and preferably 150 mg or less, more preferably 100 mg or less, even more preferably 50 mg or less, from the viewpoint of an intake concentration that does not exert hormone-like effects. The dosage of delphinidin-3-rutinoside, cyanidin-3-rutinoside or a combination thereof is 1 to 150 mg, more preferably 10 to 100 mg, and even more preferably 25 to 50 mg, per day for an adult (60 kg), as the total amount of delphinidin-3-rutinoside and cyanidin-3-rutinoside.

[0032] In the present invention, the above-mentioned dose is preferably administered or taken, for example, once, twice, or three or more times a day. The administration or intake period is not particularly limited, but is preferably continuous, more preferably one week or more, and even more preferably two weeks or more. The timing of administration or ingestion is not particularly limited, but is preferably after breakfast and / or after dinner until bedtime, and within the period after dinner until bedtime, it is more preferable to administer the drug 30 minutes to 3 hours before the desired time of sleep, and more preferably 1 to 2 hours before.

[0033] The subject to be administered or ingested with the agent for alleviating menopausal symptoms of the present invention is preferably a human or non-human animal who needs or desires alleviation of menopausal symptoms. More preferably, the agent is effectively administered to a human or non-human animal who experiences hot flashes and sweating as menopausal symptoms. The human is preferably a menopausal human, more preferably a menopausal woman.

[0034] In relation to the above-described embodiment, the present invention further discloses the following aspects.

[0035] <1> An agent for improving menopausal symptoms, comprising delphinidin-3-rutinoside, cyanidin-3-rutinoside or a combination thereof as an active ingredient. <2> A food for improving menopausal symptoms, containing delphinidin-3-rutinoside, cyanidin-3-rutinoside or a combination thereof as an active ingredient.

[0036] <3> Use of delphinidin-3-rutinoside, cyanidin-3-rutinoside or a combination thereof for producing an agent for alleviating menopausal symptoms. <4> Use of delphinidin-3-rutinoside, cyanidin-3-rutinoside or a combination thereof for producing a food for improving menopausal symptoms.

[0037] <5> Delphinidin-3-rutinoside, cyanidin-3-rutinoside or a combination thereof for use in improving menopausal symptoms. <6> Non-therapeutic use of delphinidin-3-rutinoside, cyanidin-3-rutinoside or a combination thereof to improve menopausal symptoms.

[0038] <7> A method for improving menopausal symptoms, comprising administering or ingesting an effective amount of delphinidin-3-rutinoside, cyanidin-3-rutinoside, or a combination thereof to a subject in need thereof.

[0039] <8> <1> ~ <7> In the above, the menopausal symptoms are preferably dizziness, headache, insomnia, hot flashes, flushing, sweating, palpitations, nausea, constipation, diarrhea, stiff shoulders, lower back pain, joint pain, frequent urination, dry skin or mucous membranes, or eczema, and more preferably hot flashes or sweating. <9> <1> ~ <8> In the present invention, delphinidin-3-rutinoside and cyanidin-3-rutinoside are preferably derived from black currant extract. <10> <1> ~ <9> In the above, the dosage of delphinidin-3-rutinoside, cyanidin-3-rutinoside or a combination thereof is, in terms of the total amount of delphinidin-3-rutinoside and cyanidin-3-rutinoside, preferably 1 mg or more, more preferably 10 mg or more, and even more preferably 25 mg or more per adult (60 kg) per day, and is preferably 150 mg or less, more preferably 100 mg or less, and even more preferably 50 mg or less, and is preferably 1 to 150 mg, more preferably 10 to 100 mg, and even more preferably 25 to 50 mg. [Example]

[0040] Example 1: Purification of blackcurrant anthocyanins (1) Crude fractionation of blackcurrant extract We fractionated a commercially available blackcurrant extract (Blackcurrant Polyphenol AC10, total anthocyanin content 11.8 wt%, Meiji Food Materials Co., Ltd.) into anthocyanins and other compounds. Based on a literature review (Polyphenols: Functional Components in Medicinal Plants and Foods, CMC Publishing, 250-251, 2012), we performed fractionation using a synthetic adsorption resin. The resulting fraction was divided into an anthocyanin-containing fraction (EtOH-eluted fraction, determined to correspond to four peaks in HPLC analysis, 42.2 wt%) and a more polar fraction (HO-eluted fraction, 60.0 wt%) (see Figure 1).

[0041] (2) Purification of anthocyanins from the EtOH fraction Anthocyanins were purified from the EtOH fraction (anthocyanin-containing fraction). Fractionation was performed by ODS preparative HPLC, based on the analytical HPLC conditions. As a result, four anthocyanin-purified fractions and other fractions were separated into eight fractions (Fr. E-1: 14.9 wt%, Fr. E-2: 1.91 wt%, Fr. E-3: 1.91 wt%, Fr. E-4: 6.41 wt%, Fr. E-5: 1.10 wt%, Fr. E-6: 4.65 wt%, Fr. E-7: 0.44 wt%, Fr. E-8: 6.27 wt%) (see Figure 2).

[0042] (3) Identification of purified anthocyanins The four main peaks were separated and purified by preparative HPLC, and then their identification was attempted. Comparing the results with the HPLC analysis chromatograms in the literature (J Agric Food Chem, 50(11), 3228-3231, 2002), it was highly likely that Fr. E-3 was delphinidin-3-glucoside (1, D3G), Fr. E-4 was delphinidin-3-rutinoside (2, D3R), Fr. E-5 was cyanidin-3-glucoside (3, C3G), and Fr. E-6 was cyanidin-3-rutinoside (4, C3R). Therefore, we performed the following analyses to identify each of the four main peaks. 1 H, 13C-NMR was measured, and the assignment of each signal was compared with that in the literature (J Sep Sci, 40(17), 3506-3512, 2017), confirming that the results were as predicted (see Figure 3).

[0043] Example 2: Efficacy test of black currant extract on menopausal symptoms in humans (1) Eligible persons The subjects were 17 healthy women in their 40s and 50s, and the average age of the subjects at the start of the study was 51.5±1.4 years.

[0044] (2) Preparation of test specimens A commercially available blackcurrant extract containing D3R and C3R (blackcurrant polyphenol AC10, total blackcurrant anthocyanin content 11.8 wt%, D3R 5.4 wt%, C3R 3.8 wt%, manufactured by Meiji Food Materials Co., Ltd.) was made into sugar-coated granules, and each sugar-coated granule contained 100 mg of blackcurrant extract.

[0045] (3) How to take sugar-coated granules The sugar-coated granules (2 granules / dose) were taken with 150 mL of mineral water twice a day (within 30 minutes after breakfast and 60 to 30 minutes before bedtime) for 28 days.

[0046] (4) Evaluation method for menopausal symptoms Before and after taking the sugar-coated granules containing black currant extract, the severity of menopausal symptoms was evaluated using the Kupperman Menopausal Disorder Index. The frequency of hot flashes and sweating was also evaluated using a daily diary. During the test, the following points were taken into consideration: (Note) (i) Try to maintain your normal lifestyle. (ii) Maintain a normal diet. (iii) Not changing exercise habits. (iv) Avoid overeating and extreme dieting. (v) Do not start taking any new supplements.

[0047] (5) Statistical testing The results obtained are shown as mean values ​​± standard error, and Wilcoxon signed rank test was used to compare the results before and after ingestion of sugar-coated granules containing black currant extract. After ingestion of sugar-coated granules containing blackcurrant extract, the total score of the Kupperman Menopausal Index significantly decreased from 23.7±1.9 to 17.6±2.0, and the severity rating of the Kupperman Menopausal Index also significantly decreased from 2.8±0.2 to 1.9±0.2 (P<0.05). In addition, the scores for the vasomotor symptoms of the Kupperman Menopausal Index, "feeling hot in the face" and "easy sweating," significantly decreased from 6.8±0.8 to 4.5±0.7 and from 6.8±0.8 to 4.0±0.9, respectively (P<0.05). After taking sugar-coated granules containing blackcurrant extract, the number of hot flashes per day significantly decreased from 1.2±0.3 to 0.5±0.3, and the number of sweat episodes per day also significantly decreased from 1.2±0.4 to 0.6±0.3 (P<0.05). These results demonstrate that black currant extract has the effect of alleviating menopausal symptoms, particularly hot flashes and sweating, and that ingesting it can improve menopausal symptoms, particularly hot flashes and sweating.

[0048] <Reference Example 1: Evaluation of TRPV4 activity inhibitory effect> (1) Construction of a human TRPV4 gene expression vector Using cDNA obtained by reverse transcription of total RNA extracted from human duodenal cells (Hutu-80 cells, purchased from the American Type Culture Collection) as a template, polymerase chain reaction (PCR) was performed under the following conditions using a primer set consisting of oligonucleotides represented by the base sequence shown below, which were synthesized based on the published human TRPV4 gene sequence.

[0049] <Primer set> Forward primer: 5'-CACCATGGCGGATTCCAGCGAAGGCCC-3' (SEQ ID NO: 1) Reverse primer; 5’-CTAGAGCGGGGCGTCATCAGTCC-3’ (SEQ ID NO: 2)

[0050] <PCR conditions> a) PCR solution composition cDNA (Template) 15 μL 5x PrimeStar GXL Buffer 10 μL dNTPs mixture (2.5 mM) 4 μL PrimeStar GXL DNA Polymerase (Takara Bio) 1 μL Forward Primer (10 μM) 1 μL Reverse Primer (10 μM) 1 μL Water 18 μL b) Temperature and cycle conditions 95°C 2 min ↓ 98°C 10 sec 33 cycles 70°C 2 min

[0051] The obtained PCR product was purified using the High Pure PCR Product Purification Kit (manufactured by Roche). Using the purified PCR product and the pcDNA3.1 Directional TOPO Expression Kit (manufactured by Invitrogen), a human TRPV4 gene expression vector was prepared. <000026⑧> (2) Preparation of human TRPV4-expressing cells Using DMEM / F12 medium (manufactured by Invitrogen) containing 10% fetal bovine serum, a human cervical cancer-derived cell line (HeLa cells, purchased from American Type Culture Collection) was cultured. HeLa cells were seeded at 2×10 5After 2 days of culture, the cells were transfected with the human TRPV4 gene expression vector (7.7 μg) prepared in (1) above using the TransIT-HeLaMONSTER Transfection Kit (Mirus) and cultured for 1 day. The cells were detached using Detachin (Genlantis) and plated onto a 96-well optical bottom plate (Nunc) at 2.0 × 10 4 The cells were seeded at a cell density of 90 μL / well and cultured for another day.

[0053] (3) Measurement of intracellular calcium ion influx activity Intracellular calcium ion influx activity was measured using the Calcium Kit II-fluo 4 (product name, DOJINDO). 90 μL / well of loading buffer containing Fluo4-AM was added to the human TRPV4-expressing cells prepared in (2) above and incubated at 37°C for 1 hour. Fluorescence intensity (excitation wavelength: 488 nm, emission wavelength: 524 nm) was then measured every 2 seconds using a fluorescence plate reader (FDSS / μCELL Drug Discovery Screening Support System C13299, Hamamatsu Photonics) at 37°C. Thirty seconds after the start of measurement, 20 μL / well of the TRPV4 agonist GSK1016790a (Sigma) and the black currant extract prepared in the above Preparation Example, or D3R, C3R, D3G, and C3G were diluted with the dilution buffer provided with the assay kit as samples. The mixture (mixed immediately before addition) was then added. The change in fluorescence intensity was measured every 2 seconds for up to 300 seconds. GSK1016790a was added to a final concentration of 10 nM, and the evaluated concentration of each anthocyanin was set based on its content in the black currant extract. The TRPV4 activity of the sample was calculated using the following formula, assuming the calcium ion influx rate due to treatment with the TRPV4 agonist GSK1016790a as 100%.

[0054] (Formula) Calcium ion influx rate (%) = [(F300 / F0) - (F300C2 / F0C2)] / [(F300C1 / F0C1) - (F300C2 / F0C2)] x 100 F300: Fluorescence intensity of the wells to which GSK1016790a and sample were added 300 seconds after the start of measurement F300C1: Fluorescence intensity of wells to which GSK1016790a and solvent were added 300 seconds after the start of measurement F300C2: Fluorescence intensity of the well to which only solvent was added 300 seconds after the start of measurement F0: Fluorescence intensity of the same well as F300 immediately after the start of measurement F0C1: Fluorescence intensity of the same well as F300C1 immediately after starting measurement F0C2: Fluorescence intensity of the well to which only solvent was added immediately after starting measurement

[0055] The calcium ion influx rate after the addition of test substance was compared with that after the addition of GSK1016790a and solvent using Dunnett's test. The results for the addition of black currant extract, D3R, C3R, D3G, and C3G are shown in the table as mean ± standard error. (In the table, GSK1016790a, black currant extract, black currant extract ethanol fraction, black currant extract water fraction, and a mixture of D3R, C3R, D3G, and C3G (D3R: 45.6 wt%, C3R: 33.0 wt%, D3G: 13.6 wt%, C3G: 7.8 wt%) are abbreviated as GSK, CaE, EtOH, HO, and MIX, respectively.)

[0056] [Table 1]

[0057] As shown in Table 1, black currant extract and the ethanol fraction of black currant extract significantly reduced calcium ion influx. In addition, a mixture (MIX) of the four main components of the ethanol fraction, D3R, C3R, D3G, and C3G, significantly reduced calcium ion influx. D3R in particular significantly reduced calcium ion influx, and C3R tended to reduce calcium ion influx.

[0058] These results indicate that application of black currant extract, D3R, or C3R suppresses TRPV4 activity, indicating that black currant extract, D3R, or C3R is effective in suppressing TRPV4 activity. Furthermore, it has been shown that black currant extract, D3R, and C3R, which have the effect of suppressing TRPV4 activity, are effective in improving menopausal symptoms.

Claims

1. A menopausal symptom improving agent containing delphinidin-3-rutinoside, cyanidin-3-rutinoside, or a combination thereof as an active ingredient, Menopausal symptoms include hot flashes and sweating, Delphinidin-3-rutinoside, cyanidin-3-rutinoside or a combination thereof is administered or ingested in an amount that provides a total amount of delphinidin-3-rutinoside and cyanidin-3-rutinoside of 1 to 150 mg per day for an adult (60 kg), A menopausal symptom improving agent that improves menopausal symptoms without estrogen activation.

2. A menopausal symptom improving agent as described in claim 1, which improves menopausal symptoms by suppressing the activity of TRPV4.

3. 3. The agent for improving menopausal symptoms according to claim 1, wherein delphinidin-3-rutinoside and cyanidin-3-rutinoside are derived from a black currant extract.

4. The agent for improving menopausal symptoms according to any one of claims 1 to 3, wherein delphinidin-3-rutinoside, cyanidin-3-rutinoside, or a combination thereof is administered or ingested in an amount such that the total amount of delphinidin-3-rutinoside and cyanidin-3-rutinoside is 25 to 50 mg per day per adult (60 kg).

5. A food for improving menopausal symptoms, comprising delphinidin-3-rutinoside, cyanidin-3-rutinoside, or a combination thereof as an active ingredient, Menopausal symptoms include hot flashes and sweating, Delphinidin-3-rutinoside, cyanidin-3-rutinoside or a combination thereof is administered or ingested in an amount that provides a total amount of delphinidin-3-rutinoside and cyanidin-3-rutinoside of 1 to 150 mg per day for an adult (60 kg), A food for improving menopausal symptoms that improves menopausal symptoms without activating estrogen.

6. A food for improving menopausal symptoms as described in claim 5, which improves menopausal symptoms by suppressing the activity of TRPV4.

7. 7. The food for improving menopausal symptoms according to claim 5 or 6, wherein delphinidin-3-rutinoside and cyanidin-3-rutinoside are derived from black currant extract.

8. The food for improving menopausal symptoms according to any one of claims 5 to 7, wherein delphinidin-3-rutinoside, cyanidin-3-rutinoside, or a combination thereof is administered or ingested in an amount such that the total amount of delphinidin-3-rutinoside and cyanidin-3-rutinoside is 25 to 50 mg per day per adult (60 kg).

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