Composition for improving blood vessel repair function, composition for improving male function, drug, cosmetic, and food or beverage

Abstract

The present invention comprises: at least one selected from the group consisting of astaxanthin, glucosamine hydrochloride, chondroitin sulfate, tocopherol, resveratrol, hesperidin (vitamin P), beta-carotene, ubiquinone, lipoic acid, zinc, arsine, arginine, citrulline, taurine, vitamin E and mucin; and stem cells.

Description

Compositions for improving vascular repair function, compositions for improving male sexual function, pharmaceuticals, cosmetics, and foods or beverages

[0001] The present invention relates to a composition for improving vascular repair function, a composition for improving male sexual function, a pharmaceutical product, a cosmetic product, and a food or beverage.

[0002] Among lifestyle-related diseases, diabetes, high blood pressure, dyslipidemia, and obesity are known as the death quartet, and people who have all four of these conditions are said to have a mortality risk ten times higher than normal people.

[0003] In these diseases, vascular endothelial cells are constantly under strain and damaged (such as by arteriosclerosis), increasing the risk of blood clots and blood vessel rupture. Therefore, there is a strong need to improve vascular repair functions by increasing the flexibility and elasticity of vascular endothelial cells.

[0004] A straightforward method for evaluating vascular repair function is the wound-healing effect, which measures the speed at which a wound heals. Patent Document 1 describes that, in preventing or treating cartilage tissue-related diseases, it is preferable to use mesenchymal stem cells in the form of spheroids (cell masses; cell aggregates) in order to improve the wound-healing effect of the disease. Patent Document 1 does not mention vascular repair function. However, forming spheroids as described in Patent Document 1 requires the preparation of specific conditions, which is time-consuming and costly.

[0005] Regarding male sexual function, when excitation is transmitted via the autonomic nerve, neurotransmitters are secreted from the ends of the cavernous nerves, causing the smooth muscles of the corpus cavernosum to relax, increasing the amount of blood flowing from the arteries into the corpus cavernosum, causing the corpus cavernosum to expand. Furthermore, as the corpus cavernosum expands, the external pressure increases and the veins within the corpus cavernosum become blocked, so the blood that has flowed into the corpus cavernosum accumulates in the penis without returning to the systemic circulation, resulting in penile erection. Here, blood pressure regulation and control of neuronal excitation through the relaxation of smooth muscle are mediated by the classical NO / cGMP pathway (nitric oxide / cyclic GMP), but when large amounts of cyclic GMP (cGMP) are decomposed by phosphodiesterase (hereinafter sometimes referred to as "PDE"), male sexual dysfunction, for example, occurs.

[0006] Inhibition of PDE is thought to be effective in treating male sexual dysfunction, and PDE inhibitors such as Viagra (sildenafil), Levitra (vardenafil), and Cialis (tadalafil) are commercially available, and compounds with even higher PDE inhibitory activity are also being investigated (see, for example, Patent Document 2). Patent Document 3 also investigates the use of stem cells, but does not mention any connection with PDE, and only presents results based on self-reporting by subjects.

[0007] International Publication No. 2018 / 101481 Japanese Patent Application Laid-Open No. 2017-88571 Japanese Patent Application Laid-Open No. 2021-80255

[0008] The first object of the present invention is to provide a good composition for improving vascular repair function using stem cells. The second object of the present invention is to provide a good composition for improving male sexual function using stem cells. Another object of the present invention is to provide pharmaceuticals, cosmetics, and foods or beverages that use the above-mentioned composition for improving vascular repair function and the composition for improving male sexual function. After extensive research, the inventors discovered that the above-mentioned objects can be achieved by blending certain ingredients, and have thus completed the present invention.

[0009] That is, according to the present invention, there are provided: (1) a composition for improving vascular repair function, comprising at least one selected from the group consisting of astaxanthin, glucosamine hydrochloride, chondroitin sulfate, tocopherol, resveratrol, hesperidin (vitamin P), beta-carotene, ubiquinone, lipoic acid, zinc, allicin, arginine, citrulline, taurine, vitamin E, and mucin, and stem cells; (2) a pharmaceutical containing the composition for improving vascular repair function according to (1); (3) a cosmetic containing the composition for improving vascular repair function according to (1); (4) a food or beverage containing the composition for improving vascular repair function according to (1); (5) a composition for improving male sexual function, comprising at least one selected from the group consisting of zinc, allicin, arginine, citrulline, taurine, vitamin E, and mucin, and stem cells; (6) a pharmaceutical containing the composition for improving male sexual function according to (5); (7) a cosmetic containing the composition for improving male sexual function according to (5); (8) (5) A food or beverage containing the composition for improving male function is provided.

[0010] According to the present invention, a composition for improving vascular repair function using stem cells is provided. The present invention also provides a composition for improving male sexual function using stem cells. The present invention also provides pharmaceuticals, cosmetics, and foods or beverages that use the above-mentioned composition for improving vascular repair function and composition for improving male sexual function.

[0011] 1 is an image showing the results of Example 1-1. 2 is a graph showing the results of Example 1-2. 3 is a graph showing the results of Example 1-3. 4 is an image and a graph showing the results of Example 1-4. 5 is an image and a graph showing the results of Example 1-5. 6 is a graph showing the calibration curve in Example 2-1. 7 is a graph showing the results of Example 2-1. 8 is a graph showing the calibration curve in Example 2-2. 9 is a graph showing the results of Example 2-2. 10 is a graph showing the results of Example 2-3.

[0012] (Composition for Improving Vascular Repair Function) First, a composition for improving vascular repair function according to a first embodiment of the present invention will be described. The composition for improving vascular repair function of the present invention comprises at least one selected from the group consisting of astaxanthin, glucosamine hydrochloride, chondroitin sulfate, tocopherol, resveratrol, hesperidin (vitamin P), beta-carotene, ubiquinone, lipoic acid, zinc, allicin, arginine, citrulline, taurine, vitamin E, and mucin, and stem cells. (Astaxanthin) Astaxanthin may be synthesized or extracted from natural sources. (Glucosamine Hydrochloride) Glucosamine hydrochloride may be, but is not limited to, a depolymerized and acetylated form of shrimp or crab shell. (Chondroitin Sulfate) Chondroitin sulfate may be, but is not limited to, derived from cows, pigs, sharks, salmon, or squid.

[0013] (Tocopherol) Tocopherol is a type of vitamin E, and includes α-tocopherol, β-tocopherol, γ-tocopherol, and δ-tocopherol.

[0014] (Resveratrol) Resveratrol is a type of stilbenoid polyphenol, and is contained in, for example, grape skin, peanuts, cocoa, etc., and although not particularly limited, those derived from these sources can be used.

[0015] (Hesperidin (vitamin P)) Hesperidin (vitamin P) is a type of polyphenol and is found, for example, in the peel and thin skin of Satsuma mandarins, Hassaku oranges, bitter oranges, etc., and although not particularly limited, products derived from these can be used.

[0016] (Beta-carotene) Beta-carotene may be synthesized or extracted from natural products, and examples of natural products include ginseng, palm oil, and Dunaliella. (Ubiquinone) Ubiquinone is also known as coenzyme Q10, and may be produced, for example, by fermentation. (Lipoic acid) As lipoic acid, α-lipoic acid is preferably used, and may be synthesized or extracted from natural products.

[0017] (Zinc) Zinc is present in cells throughout the human body and is an element necessary for maintaining health. It is an essential mineral that must be ingested from food. Zinc is found in meats such as pork liver, seafood such as oysters, beans such as soybeans, nuts, etc., and can be obtained, for example, from at least one of these raw materials. Zinc derived from ores or yeast can also be used, but the manufacturing method and raw materials for zinc are not particularly limited. In addition, in the present invention, zinc may be used in the form of zinc gluconate, zinc sulfate, zinc acetate, etc. (Allicin) Allicin is found in garlic, leeks, onions, chives, etc., and, although not particularly limited, allicin derived from these can be used. (Arginine) Arginine is a naturally occurring amino acid and can be extracted from natural products, for example. (Citrulline) Citrulline is an amino acid and, although not particularly limited, can be obtained from watermelon, corn, etc.

[0018] (Taurine) Taurine may be synthesized or extracted from natural sources, such as squid, octopus, and crustaceans.

[0019] Vitamin E contains four tocopherols and four tocotrienols, including α-tocotrienol, β-tocotrienol, γ-tocotrienol, and δ-tocotrienol. The four tocopherols are as described above.

[0020] (Mucin) Mucin is a component contained in animal mucus, and is not particularly limited, but mucin made from, for example, the stomach mucosa of animals such as pigs, bird's nest, eel, or the like can be used as a raw material.

[0021] (Stem Cells) The composition for improving blood repair function of the present invention contains stem cells. Examples of stem cells that can be used include, but are not limited to, mesenchymal stem cells, hematopoietic stem cells, neural stem cells, epidermal stem cells, embryonic stem (ES) cells, embryonic germ (EG) cells, and all other tissue stem cells. Furthermore, in the case of tissue stem cells, stem cells derived from tissues such as fat, umbilical cord, umbilical cord blood, bone marrow, placenta, dental pulp, amniotic membrane, skeletal muscle, periosteum, and endometrium can be obtained, but are not limited to these. Among these, adipose-derived stem cells are preferred.

[0022] Stem cells may be derived from humans (autologous cells) or from different species (allogeneic cells). Examples of stem cells derived from different species include cows, horses, pigs, dogs, cats, mice, rats, and sheep. When using autologous cells, the cells may be the patient's own or may be cells from another person.

[0023] (Formulation and function of the composition for improving vascular repair function) The composition for improving vascular repair function of the present invention comprises at least one selected from the group consisting of astaxanthin, glucosamine hydrochloride, chondroitin sulfate, tocopherol, resveratrol, hesperidin (vitamin P), beta-carotene, ubiquinone, lipoic acid, zinc, allicin, arginine, citrulline, taurine, vitamin E, and mucin, and stem cells.

[0024] Furthermore, the blending ratios of astaxanthin, glucosamine hydrochloride, chondroitin sulfate, tocopherol, resveratrol, hesperidin (vitamin P), beta-carotene, ubiquinone, lipoic acid, zinc, allicin, arginine, citrulline, taurine, vitamin E, and mucin are not particularly limited as long as they do not impair the effects of the present invention, but it is preferable to blend them at a specific blending ratio.

[0025] Furthermore, the blending ratio of the additional factors described below and the stem cells is not particularly limited as long as it does not impair the effects of the present invention, but it is preferable to blend them at a specific blending ratio.

[0026] The composition for improving vascular repair function of the present invention stimulates undifferentiated stem cells with additional factors and then administers them intravascularly to induce stem cells to repair blood vessels, or to induce stem cells naturally present in the body to direct them to damaged blood vessels and create an environment for repair. The additional factors are at least one selected from the group consisting of astaxanthin, glucosamine hydrochloride, chondroitin sulfate, tocopherol, resveratrol, hesperidin (vitamin P), beta-carotene, ubiquinone, lipoic acid, zinc, allicin, arginine, citrulline, taurine, vitamin E, and mucin.

[0027] The three major aging reactions—glycation, oxidation, and chronic inflammation—cause excessive intracellular production of reactive oxygen species (ROS). ROS production is triggered by glycation induced by advanced glycation end products (AGEs) and oxidation induced by hydrogen peroxide (H2O2). AGEs are produced in vivo by glycation, a non-enzymatic binding reaction between proteins and sugars, and accumulate in vascular endothelial cells. Two distinct types of AGE receptors are known: RAGE and AGE-R2, which activate intracellular signaling pathways to produce ROS; and FEEL-1, FEEL-2, AGE-R1, AGE-R3, and CD36, which are involved in the degradation and digestion of AGEs. Inhibiting the former and promoting the latter may prevent aging in skin and vascular endothelial cells. Furthermore, the inhibitory effects of antioxidants on ROS production resulting from oxidative stress, such as hydrogen peroxide (H2O2) treatment, are being investigated. Suppressing glycation and oxidation and inhibiting ROS production play an important role in vascular repair. Furthermore, it is known that treatment with hydrogen peroxide (H2O2) accelerates cell aging, and delaying this process is effective for vascular repair. In this invention, we used the wound-healing effect, which measures the speed at which a wound heals, as a straightforward method for evaluating vascular repair function.

[0028] (Composition for enhancing male function) Next, a composition for enhancing male function according to a second embodiment of the present invention will be described. The composition for enhancing male function of the present invention comprises at least one selected from the group consisting of zinc, allicin, arginine, citrulline, taurine, vitamin E, and mucin, and stem cells.

[0029] The zinc, allicin, arginine, citrulline, taurine, vitamin E, and mucin are the same as those described in the composition for improving vascular repair function according to the first embodiment. The stem cells are also the same as those described in the composition for improving vascular repair function according to the first embodiment.

[0030] (Formulation and Function of the Composition for Enhancing Male Function) The composition for enhancing male function of the present invention comprises at least one selected from the group consisting of zinc, allicin, arginine, citrulline, taurine, vitamin E, and mucin, and stem cells.

[0031] Furthermore, the blending ratio of zinc, allicin, arginine, citrulline, taurine, vitamin E and mucin is not particularly limited as long as it does not impair the effects of the present invention, but it is preferable to blend them in a specific blending ratio.

[0032] Furthermore, the blending ratio of at least one selected from the group consisting of zinc, allicin, arginine, citrulline, taurine, vitamin E, and mucin to the stem cells is not particularly limited as long as it does not impair the effects of the present invention, but it is preferable to blend them at a specific blending ratio.The composition for improving male function of the present invention can contribute to the improvement of male function by inhibiting PDE.

[0033] (Pharmaceuticals) The composition for improving vascular repair function and / or the composition for improving male sexual function of the present invention can be incorporated into pharmaceuticals for these purposes. Pharmaceuticals can be used as either preventive or therapeutic drugs.

[0034] When compounded into a pharmaceutical product, the component compound may be used alone or may be mixed with generally pharmaceutically acceptable additives to form a formulation. The dosage form may be an oral dosage form such as tablets, granules, capsules, pills, powders, liquids, suspensions, emulsions, syrups, elixirs, or extracts, or a parenteral dosage form such as injections, liquids, suppositories, ointments, patches, poultices, or lotions, but is not particularly limited and can be appropriately selected depending on the purpose of treatment or prevention.

[0035] In the case of tablets, granules, pills, capsules, and powders, additives such as excipients, binders, disintegrants, lubricants, etc. Examples of excipients include starch, carboxymethyl cellulose, sucrose, dextrin, and corn starch.

[0036] Examples of binders include crystalline cellulose, crystalline cellulose-carmellose sodium, methylcellulose, hydroxypropyl cellulose, low-substituted hydroxypropyl cellulose, hydroxypropyl methylcellulose, hydroxypropyl methylcellulose phthalate, hydroxypropyl methylcellulose acetate succinate, carmellose sodium, ethyl cellulose, carboxymethyl ethyl cellulose, hydroxyethyl cellulose, wheat starch, rice starch, corn starch, potato starch, dextrin, pregelatinized starch, partially pregelatinized starch, hydroxypropyl starch, pullulan, polyvinylpyrrolidone, aminoalkyl methacrylate copolymer E, aminoalkyl methacrylate copolymer RS, methacrylic acid copolymer L, methacrylic acid copolymer, polyvinyl acetal diethylaminoacetate, polyvinyl alcohol, gum arabic, powdered gum arabic, agar, gelatin, white shellac, tragacanth, refined sucrose, and macrogol.

[0037] Disintegrants include crystalline cellulose, methylcellulose, low-substituted hydroxypropyl cellulose, carmellose, carmellose calcium, carmellose sodium, croscarmellose sodium, wheat starch, rice starch, corn starch, potato starch, partially pregelatinized starch, hydroxypropyl starch, carboxymethyl starch sodium, and tragacanth.

[0038] Lubricants include wheat starch, rice starch, corn starch, stearic acid, calcium stearate, magnesium stearate, hydrous silicon dioxide, light anhydrous silicic acid, synthetic aluminum silicate, dried aluminum hydroxide gel, talc, magnesium aluminometasilicate, calcium hydrogen phosphate, anhydrous calcium hydrogen phosphate, sucrose fatty acid esters, waxes, hydrogenated vegetable oils, and polyethylene glycol.

[0039] In addition, in the case of liquids, syrups, suspensions, emulsions, and elixirs, in addition to commonly used inactive diluents such as water and vegetable oils, coloring agents, flavoring agents, fragrances, etc. may be contained as additives.

[0040] Injections may contain additives such as suspensions, emulsions, and solubilizers for use. Ointments and suppositories may contain additives such as fats, fatty oils, lanolin, petrolatum, paraffin, wax, resins, plastics, bases, glycols, higher alcohols, water, emulsifiers, and suspending agents. Patches may contain additives such as glycerin, water, water-soluble polymers, and water-absorbent polymers. Lotions may contain additives such as solvents, emulsifiers, and suspending agents.

[0041] (Cosmetics) The composition for improving vascular repair function and / or the composition for improving male sexual function of the present invention can also be incorporated into cosmetics. Examples of cosmetics include lotions, emulsions, facial cleansers, cleansers, serums, creams, foundations, eyebrow products, mascara, eye shadow, eyeliner, lipsticks, glosses, blushers, face powders, and nail polishes. The cosmetics can be in the form of liquids, creams, solids, sticks, powders, and the like.

[0042] (Food or Beverage) The composition for improving vascular repair function and / or the composition for improving male sexual function of the present invention may be incorporated into foods, beverages, etc. Examples of foods include breads, noodles, confectioneries, processed meat products, processed seafood products, frozen foods, jellies, ice cream, dairy products, various seasonings, etc. In addition to general foods, the composition may also be incorporated into foods for specified health uses, quasi-drugs, health foods, and supplements. Examples of beverages include soft drinks, dairy drinks, alcoholic beverages, tea, black tea drinks, coffee, fruit juice drinks, carbonated drinks, mineral waters, fruit and vegetable drinks, etc.

[0043] Furthermore, foods and beverages containing the composition for improving vascular repair function and / or the composition for improving male function of the present invention may be in the same form as oral preparations such as tablets, capsules, syrups, etc.

[0044] Furthermore, when producing foods or beverages containing the composition for improving vascular repair function and / or the composition for improving male sexual function of the present invention, additives such as sweeteners, colorants, preservatives, thickeners, stabilizers, gelling agents, antioxidants, color formers, bleaching agents, emulsifiers, leavening agents, acidulants, glossing agents, and flavorings; solvents; and oils may be added as needed within a range that does not impair the effects of the present invention. These additives may be used alone or in combination of two or more.

[0045] The proportion of the composition for improving vascular repair function and / or the composition for improving male sexual function of the present invention to be blended into the above-mentioned foods and beverages can be adjusted appropriately depending on the intended use, but the proportion of the ingredient blend blended into the above-mentioned foods and beverages is preferably 0.01 to 20% by weight, more preferably 0.01 to 15% by weight, and even more preferably 0.1 to 10% by weight.

[0046] The present invention will be described below with reference to examples, but the present invention is not limited thereto. (Example 1: Vascular repair function) (Composition for improving vascular repair function) Adipose-derived stem cells, specifically, those isolated and cultured from adipose tissue excised from the abdominal cavity of a mouse, were prepared as stem cells. The above-mentioned adipose-derived stem cells were cultured in a Culture Flask at 37°C and 5% CO ２ The cells were cultured in an incubator under the conditions of (1) for 7 days, and then cultured in a culture medium (Mesenchymal Stem Cell Growth Medium 2; C-28009, PromoCell or Opti-MEM I Reduced Serum Medium; 11058-021, Gibco). The culture supernatant was collected when the cells reached approximately 80% confluence.

[0047] The above culture supernatant was mixed with astaxanthin, glucosamine hydrochloride, chondroitin sulfate, tocopherol, resveratrol, hesperidin (vitamin P), beta-carotene, ubiquinone, lipoic acid, zinc, allicin, arginine, citrulline, taurine, vitamin E, and mucin in a predetermined ratio, and ultrapure water or saline was added as needed to achieve a predetermined concentration, thereby obtaining a composition for improving vascular repair function (hereinafter sometimes referred to as STEM CELL+ alpha).

[0048] In addition, we also prepared a mixture of the above culture supernatant alone (hereinafter sometimes referred to as STEM CELL alone), astaxanthin, glucosamine hydrochloride, chondroitin sulfate, tocopherol, resveratrol, hesperidin (vitamin P), beta-carotene, ubiquinone, lipoic acid, zinc, allicin, arginine, citrulline, taurine, vitamin E, and mucin in a specified ratio, with ultrapure water or saline added as needed to achieve a specified concentration (hereinafter sometimes referred to as plus alpha alone).

[0049] (Example 1-1: Wound healing effect) The wound healing effect was evaluated using an established method for verifying the wound healing effect. Specifically, a wound of a certain width was created in cultured vascular endothelial cells, and STEM CELL + alpha was added. The cells were then cultured at 37°C, and the number of days until the wound closed was observed. In other words, whether or not the cell-free area was filled was examined. In addition, instead of STEM CELL + alpha, STEM CELL alone and plus alpha alone were added, and observations were also made. A system (control) without STEM CELL + alpha was also observed. The results are shown in Figure 1.

[0050] As shown in Figure 1, the wound in the control had not yet closed even after 9 days of culture, but the wound closed after 9 days in the STEM CELL alone and STEM CELL plus alpha alone cultures, and after 6 days in the STEM CELL + alpha cultures, demonstrating a wound healing effect in each. The wound healing effect was particularly excellent in the STEM CELL + alpha cultures.

[0051] (Example 1-2: Effect on the mechanism by which cells degrade AGEs produced by glycation reactions - Measurement of AGE receptor mRNA expression) The mRNA level of the receptor that degrades AGEs present on the cell surface within the cell was examined using quantitative RT-PCR. An increase in the mRNA expression level increases the ability to take up and degrade extracellular AGEs, improving vascular endothelial cells and ultimately the vascular wall.

[0052] Specifically, the mRNA levels of various genes (FEEL-1-2, CD36, and AGER-1) were measured by quantitative RT-PCR in cultured vascular endothelial cells treated with both STEM CELL +alpha and AGE, and compared with untreated controls. Cells were incubated in a 37°C incubator and assayed after 4 hours. Measurements were also performed using 1 / 1 volume (final concentration: 10,000 ng / ml), 1 / 10 volume (final concentration: 1,000 ng / ml), and 1 / 100 volume (final concentration: 100 ng / ml) of the culture medium.

[0053] The specific procedure for quantitative RT-PCR was as follows. Total RNA was extracted from STEM CELL +alpha and vascular endothelial cells supplemented with AGE using Trizol reagent (Ambion). The extracted RNA was reverse transcribed to cDNA according to the method of PrimeScript RT Master Mix (Takara), and amplified using SYBR Premix EX Taq II (Takara). The PCR reaction solution consisted of 50 μL (25 μL SYBR Green Mix (2x), 1 μL cDNA, 2 μL primer pair mix (5 pmol / μL each primer), 22 μL H ２ The PCR reaction solution (O) was used, and the reaction was carried out under the conditions of one cycle at 95°C for 30 seconds, followed by 50 cycles of 95°C for 5 seconds and 60°C for 30 seconds.

[0054] Furthermore, instead of STEM CELL + alpha, STEM CELL alone and STEM CELL plus alpha alone were also added and measurements were performed. The results are shown in Figure 2.

[0055] The mRNA expression of AGE receptors; FEEL-1, FEEL-2, CD-36, and AGE-R1 each increased. This indicates that the use of STEM CELL alone, plus alpha alone, and STEM CELL + alpha each increases the ability of AGEs to be taken up into cells and decomposed. Furthermore, the results show that the use of STEM CELL + alpha was the most effective in taking up and decomposing AGEs into cells.

[0056] (Example 1-3: Improving cell survival rate under oxidative stress) When cells are treated with hydrogen peroxide (H2O2) for a certain period of time, they enter a state of oxidative stress, and continuing to culture them in this state leads to cell death. We investigated whether STEM CELL +alpha can prevent this.

[0057] Specifically, endothelial cells were incubated for 2 hours in medium supplemented with 0.2 mM hydrogen peroxide, followed by the addition of a predetermined amount of STEM CELL +alpha. The cells were then incubated for 4 hours, washed with PBS, and replaced with standard medium. After 48 hours, the cells or culture medium were harvested and cell viability was measured. Measurements were also performed on a 10-fold dilution (labeled "10" in Figure 3) and a 100-fold dilution (labeled "100" in Figure 3) of the hydrogen peroxide solution. The system using 0.2 mM hydrogen peroxide is labeled "1" in Figure 3.

[0058] In addition, instead of STEM CELL + alpha, STEM CELL alone and STEM CELL + alpha alone were added and observations were also performed. Observations were also performed on a control system without STEM CELL + alpha. The results are shown in Figure 3.

[0059] As shown in Figure 3, H2O2 treatment reduced cell viability to 62% (38% death) after 48 hours in the control group, but treatment with STEM CELL alone, STEM CELL plus alpha alone, and STEM CELL + alpha protected the cells and improved their viability. In particular, STEM CELL + alpha showed the greatest improvement in viability.

[0060] (Example 1-4: Inhibitory effect on reactive oxygen species (ROS) production) Reactive oxygen species (ROS) accumulate in cells that have been subjected to oxidative stress due to H2O2 treatment, causing the cells to become senescent. We investigated whether STEM CELL +alpha can inhibit this.

[0061] Specifically, endothelial cells were incubated for 2 hours in medium supplemented with 0.2 mM hydrogen peroxide, followed by the addition of a predetermined amount of STEM CELL +alpha and incubation for 4 hours. The cells were then washed with PBS and replaced with standard medium. After 48 hours, the cells or culture medium were harvested. After washing with PBS, 1 M fluorescent probe (CM-H2DCFDA (Molecular Probes Inc., Eugene, OR) was added and incubated at 37°C for 60 minutes. The amount of intracellular reactive oxygen species (ROS) produced was measured using a microplate reader (SYNERGY / HT, BioTek, Japan) and quantified.

[0062] In addition, instead of STEM CELL + alpha, STEM CELL alone and STEM CELL + alpha alone were added and observations were also performed. Observations were also performed on a system (control) without STEM CELL + alpha. The results are shown in Figure 4.

[0063] As shown in Figure 4, H2O2 treatment causes the accumulation of reactive oxygen species (ROS) within cells, but the use of STEM CELL alone, plus alpha alone, and STEM CELL + alpha each showed an inhibitory effect on ROS production. In particular, STEM CELL + alpha was found to have the greatest inhibitory effect on ROS production.

[0064] (Example 1-5: Delaying the appearance of senescent cells) Cells that have been subjected to oxidative stress by H2O2 treatment turn into senescent cells earlier than normal cultured cells. Senescent cells exhibit the property of staining blue with β-Gal. We examined whether STEM CELL +alpha can delay this process.

[0065] Specifically, endothelial cells were incubated for 2 hours in medium containing 0.2 mM hydrogen peroxide, followed by the addition of a predetermined amount of STEM CELL +alpha. The cells were then incubated for 4 hours, washed with PBS, and replaced with standard medium. After 4 weeks, the cells or culture medium were harvested. After washing with PBS, β-Gal was added and observations were performed.

[0066] In addition, instead of STEM CELL + alpha, STEM CELL alone and STEM CELL + alpha alone were added and observations were also performed. Observations were also performed on a control system without STEM CELL + alpha. The results are shown in Figure 5.

[0067] As shown in Figure 5, H2O2 treatment resulted in the early appearance of senescent cells (stained blue with β-Gal). After four weeks, senescent cells appeared at 5000 intensity in the image. Meanwhile, the use of STEM CELL alone, plus alpha alone, and STEM CELL +alpha resulted in fewer blue-stained cells, indicating a delay in cellular senescence. In particular, STEM CELL +alpha was found to have the greatest effect in delaying cellular senescence.

[0068] (Example 2: Male function) Measurements and observations were carried out when human smooth muscle cells were given stem cell stimulation or stem cell plus α stimulation.

[0069] (Composition for improving male sexual function) The same stem cells as those used in the composition for improving vascular repair function were prepared. The culture supernatant obtained from the stem cells was mixed with zinc, allicin, arginine, citrulline, taurine, vitamin E and mucin in a predetermined ratio, and ultrapure water or saline was added as needed to achieve a predetermined concentration, thereby obtaining a composition for improving male sexual function (hereinafter sometimes referred to as STEM CELL+ alpha).

[0070] In addition, we also prepared the above culture supernatant alone (hereinafter sometimes referred to as STEM CELL alone), a mixture of zinc, allicin, arginine, citrulline, taurine, vitamin E, and mucin in a specified ratio, with ultrapure water or saline added as needed to achieve a specified concentration (hereinafter sometimes referred to as plus alpha alone).

[0071] (Example 2-1: PDE inhibitory effect) The PDE inhibitory effect was measured using a PDE1A Assay Kit (manufactured by Bioscience) according to the instructions attached to the kit, measuring the PDE inhibitory activity of STEM CELL alone, plus alpha alone, and STEM CELL + alpha. Measurements were also performed for a control. The calibration curve is shown in Figure 6, and the results are shown in Figure 7.

[0072] Treatment of cells with STEM cells alone, plus alpha alone, or STEM cells plus alpha inhibited PDE activity, suggesting that it may regulate vascular smooth muscle contraction.

[0073] (Example 2-2: Amount of NO (Nitric Oxide) Production) Changes in nitric oxide production in human aortic smooth muscle cells with increasing concentrations (μg / mL) of components in the culture medium of human aortic smooth muscle cells were evaluated. The production of nitric oxide in human aortic smooth muscle cells is mediated by nitrite (NO), an oxidized metabolite of nitric oxide. ２－ The relative fluorescence intensity (RFU) of the fluorescent probe that emits fluorescence upon reacting with Fluorescent Protein (F) was used as an index for evaluation.

[0074] Specifically, human aortic smooth muscle cells (HAoSMCs; PromoCell) were cultured in a dedicated medium (Smooth Muscle Cell Growth Medium 2; PromoCell). The medium was supplemented with 10% bovine serum and antibiotics. 5 × 10 cells were cultured in a 24-well flat-bottom plate. ４ Cells were seeded and cultured until 80% confluent. The medium was then replaced with 0.1, 1, 10, or 100 mg / mL of each component (STEM CELL alone, plus alpha alone, or STEM CELL + alpha) or control medium without any component. After 1 hour, the culture supernatant was collected. Total NO production was measured using the OxiSelect In Vitro Nitric Oxide (Nitrite / Nitrate) Assay Kit (CELLBIOLABS) according to the standard protocol.

[0075] Total RNA was extracted using RNeasy Mini Kit (QIAGEN), and reverse transcription was performed using OneStep RT-PCR Kit (NEB). The NO synthase gene and the constitutively expressed GAPDH gene were then amplified using specific primers.

[0076] The difference in DeltaCt value between each sample and GAPDH (DeltaCt) was calculated by quantitative PCR, and the difference in DeltaCt value between each sample and a control sample containing no components (DeltaDeltaCt value) was also calculated. The calibration curve of the assay kit is shown in Figure 8, and the measurement results are shown in Figure 9, where the mRNA expression level is expressed as a multiple of the mRNA level in the control sample.

[0077] As shown in Figure 9, the amount of NO production increased depending on the concentration of STEM cells alone, plus alpha alone, or STEM cells + alpha. The greatest increase was observed in STEM cells + alpha.

[0078] (Example 2-3: mRNA levels of NO synthase, sGC, and cGMP-dependent protein kinase (PKG)) Measurements were performed according to the following procedures 1 to 8. 1. Human vascular smooth muscle cells were cultured in a 6-well plate at 37°C in a 5% CO2 incubator. 2. Stem Cell alone, plus alpha alone, or Stem Cell + alpha were serially diluted and added to the culture medium, and the culture was continued. 3. After stimulation for a certain period of time, the culture medium was removed, the cell surface was washed with PBS (phosphate buffer), and total RNA was extracted for mRNA measurement. (Details were in accordance with the Trizol reagent protocol.) 4. The amount of total RNA obtained was quantified, and a certain amount was used to synthesize cDNA by reverse transcription (RT). (Details were in accordance with the Takara Primscript Master Mix Kit.) 5. Using the cDNA, a reaction solution was prepared for each sample using a primer pair (synthesized separately) based on the various Hsp-mRNA sequences and a PCR reaction solution (SYBR green system). 6. These were placed in a 96-well PCR plate and loaded into a quantitative PCR device. 7. Using a dedicated program, the original amounts of various Hsp-mRNA were relatively quantified (by the Delta-Delta CT method). 8. The Hsp induction ability of each sample was examined based on the obtained Hsp amounts. The results are shown in Figure 10.

[0079] Figure 10 shows that the expression of various mRNAs involved in NO production increased depending on the concentration of STEM CELL alone, plus alpha alone, and STEM CELL + alpha. PKG expression was particularly elevated. Furthermore, STEM CELL + alpha showed the highest mRNA levels for NO synthase, sGC, and PKG.

Claims

1. A composition for improving vascular repair function comprising stem cells and at least one selected from the group consisting of astaxanthin, glucosamine hydrochloride, chondroitin sulfate, tocopherol, resveratrol, hesperidin (vitamin P), beta-carotene, ubiquinone, lipoic acid, zinc, allicin, arginine, citrulline, taurine, vitamin E, and mucin.

2. A pharmaceutical comprising the composition for improving vascular repair function described in claim 1.

3. A cosmetic product containing the composition for improving vascular repair function described in claim 1.

4. A food or beverage containing the composition for improving vascular repair function described in claim 1.

5. A composition for enhancing male sexual function comprising at least one selected from the group consisting of zinc, allicin, arginine, citrulline, taurine, vitamin E and mucin, and stem cells.

6. A pharmaceutical comprising the composition for enhancing male sexual function described in claim 5.

7. A cosmetic product containing the composition for enhancing male function according to claim 5.

8. A food or beverage containing the composition for enhancing male function according to claim 5.

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