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

Abstract

To provide a composition for improving blood vessel repair function using cells.SOLUTION: This composition for improving blood vessel repair function 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.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] The present invention relates to a composition for improving vascular repair function, a composition for improving male function, pharmaceuticals, cosmetics, and foods or beverages.

Background Art

[0002] Among lifestyle-related diseases, especially diabetes, hypertension, dyslipidemia, and obesity are collectively called the "deadly quartet". It is said that people with all of these have a death risk that is 10 to several times higher than that of normal people.

[0003] In these diseases, the vascular endothelial cells are routinely burdened and damaged (such as arteriosclerosis), and there is a high risk of blood clots and the possibility of blood vessels rupturing. Therefore, there is a strong demand to enhance the vascular repair function that can increase the flexibility and elasticity of vascular endothelial cells.

[0004] As a straightforward evaluation method for vascular repair function, there is the wound-healing effect, which measures the speed of wound healing. Here, Patent Document 1 describes that in the prevention or treatment of cartilage tissue-related diseases, from the perspective of improving the wound-healing effect of the disease, it is preferable to use mesenchymal stem cells in the form of spheroids (cell aggregates). Note that Patent Document 1 does not mention the vascular repair function. However, in order to form spheroids as in Patent Document 1, it is necessary to prepare predetermined conditions, which is time-consuming and costly.

[0005] Regarding male function, when excitement is transmitted through the autonomic nerves, neurotransmitters are secreted from the ends of the cavernous nerves, which relax the smooth muscles of the corpus cavernosum, and the amount of blood flowing into the corpus cavernosum from the arteries increases, causing the corpus cavernosum to expand. When the corpus cavernosum expands, the external pressure increases and the veins in the corpus cavernosum become blocked, so the blood that has flowed into the corpus cavernosum does not return to the systemic circulation and accumulates in the penis, resulting in penile erection. Here, blood pressure regulation and control of nerve cell excitement through the relaxation of smooth muscles are due to the classical NO / cGMP pathway (nitric oxide / cyclic GMP), but when a large amount of cyclic GMP (cGMP) is decomposed by phosphodiesterase (hereinafter sometimes referred to as "PDE"), for example, male sexual dysfunction occurs.

[0006] Inhibiting PDE is thought to be effective against male sexual dysfunction, and PDE inhibitors such as Viagra (sildenafil), Levitra (vardenafil), and Cialis (tadalafil) are commercially available, and compounds with 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 the relationship with PDE, and only shows results based on the subjects' self-reports. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] International Publication No. 2018 / 101481 [Patent Document 2] JP 2017-88571 A [Patent Document 3] Patent Publication No. 2021-80255 Summary of the Invention [Problem to be solved by the invention]

[0008] The first object of the present invention is to provide a composition for improving good vascular repair function using stem cells. Further, the second object of the present invention is to provide a composition for improving good male function using stem cells. Also, it is an object to provide pharmaceuticals, cosmetics, and foods or beverages using the above composition for improving vascular repair function and composition for improving male function. As a result of intensive studies, the present inventors have found that the above object can be achieved by blending predetermined components, and have completed the present invention.

[0009] That is, according to the present invention, (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, coenzyme Q10, 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 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 function according to (5), (7) A cosmetic containing the composition for improving male function according to (5), (8) A food or beverage containing the composition for improving male function according to (5) are provided.

Effects of the Invention

[0010] According to the present invention, there is provided a composition for improving good vascular repair function using stem cells. Further, according to the present invention, there is provided a composition for improving good male function using stem cells. Further, according to the present invention, there are provided pharmaceuticals, cosmetics, and foods or beverages using the above composition for improving vascular repair function and the composition for improving male function.

Brief Description of the Drawings

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Modes for Carrying Out the Invention

[0012] (Composition for improving vascular repair function) First, the composition for improving vascular repair function according to the 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 synthetic or extracted from natural products. (Glucosamine hydrochloride) Glucosamine hydrochloride is not particularly limited. For example, those obtained by reducing the molecular weight and acetylating the shells of shrimps and crabs can be used. (Chondroitin sulfate) Chondroitin sulfate is not particularly limited. For example, those derived from cows, pigs, sharks, salmon, and squids can be used.

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

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

[0015] (Hesperidin (vitamin P)) Hesperidin (vitamin P) is a type of polyphenol. For example, it is contained in the peels and thin skins of Satsuma mandarins, hassaku oranges, daidai oranges, etc. It is not particularly limited, and those derived from these can be used. (Beta-carotene)

[0016] Beta-carotene may be synthetic or extracted from natural products. Natural products include ginseng, palm oil, Dunaliella, etc. (Ubiquinone) Ubiquinone is also called coenzyme Q10, etc. For example, those produced by fermentation methods can be used. (Lipoic acid) As lipoic acid, α-lipoic acid can be preferably used, and a synthesized one may be used, or one extracted from natural products may be used.

[0017] (Zinc) Zinc is present in the cells of the whole body of humans and is an element necessary for maintaining health. It is a kind of essential mineral that needs to be ingested from foods, etc. Zinc is contained in meats such as pork liver, seafood such as oysters, beans such as soybeans, nuts, etc. For example, it can be obtained using at least one of these as a raw material, and ores and yeast-derived ones can also be used, but the manufacturing method and raw materials of zinc are not particularly limited. Also, in the present invention, it may be used in the form of zinc gluconate, zinc sulfate, zinc acetate, etc. (Allicin) Allicin is contained in garlic, green onions, onions, and Chinese chives, etc., and although not particularly limited, those derived from these can be used. (Arginine) Arginine is a kind of naturally occurring amino acid and can be extracted from natural products, for example. (Citrulline) Citrulline is a kind of amino acid and although not particularly limited, those using watermelon, corn, etc. as raw materials can be used.

[0018] (Taurine) For taurine, a synthesized one may be used, or one extracted from natural products may be used. Examples of natural products include squid, octopus, crustaceans, etc.

[0019] (Vitamin E) Vitamin E contains four kinds of tocopherols and four kinds of tocotrienols. Examples of tocotrienols include α-tocotrienol, β-tocotrienol, γ-tocotrienol, and δ-tocotrienol. The four kinds of tocopherols are as described above.

[0020] (Mucin) Mucin is a component contained in the mucus of animals. Although not particularly limited, for example, those made from the gastric mucosa of animals such as pigs, bird's nests, eels, etc. can be used as raw materials.

[0021] (Stem cells) The composition for improving blood repair function of the present invention contains stem cells. Although not particularly limited as stem cells, 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, etc. can be used. Also, in the case of tissue stem cells, although not particularly limited, stem cells derived from tissues such as fat, umbilical cord, umbilical cord blood, bone marrow, placenta, dental pulp, amniotic membrane, skeletal muscle, periosteum, endometrium, etc. can be obtained. Among these, adipose-derived stem cells are preferred.

[0022] In addition, the stem cells may be of human origin (autologous cells) or of heterologous origin (allogeneic cells). Examples of the species of heterologous-derived stem cells include cows, horses, pigs, dogs, cats, mice, rats, sheep, etc. Also, when using autologous cells, one's own cells or the cells of others may be used.

[0023] (Composition and function for improving blood vessel repair function) The composition for improving blood vessel 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] Also, the blending ratio 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 is not particularly limited as long as the effects of the present invention are not impaired, but it is preferably blended at a specific blending ratio.

[0025] In addition, regarding the ratio of the positive alpha factor described later and the stem cells, it is not particularly limited as long as the effects of the present invention are not impaired, but it is preferable to mix them at a specific mixing ratio.

[0026] The composition for improving vascular repair function of the present invention is to direct stem cells to vascular repair or create an environment that acts on stem cells originally present in the body and directs them to repair damaged blood vessels by administering undifferentiated stem cells into the blood vessel in a state stimulated by a positive alpha factor. Here, the positive alpha factor is 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] Here, the production of excessive reactive oxygen species (ROS) in cells is caused by the glycation reaction, oxidation reaction, and chronic inflammation reaction, which are regarded as the three major aging reactions. The production of ROS is triggered by glycation stimulation by advanced glycation end products (AGE) and oxidation stimulation by hydrogen peroxide (H2O2). AGE is generated by the non-enzymatic binding of proteins and sugars in the living body through the glycation reaction and also accumulates in vascular endothelial cells. There are two different types of AGE receptors, namely, RAGE, AGE-R2, etc. that are involved in ROS production by activating intracellular signal pathways, and FEEL-1, FEEL-2, AGE-R1, AGE-R3, CD36, etc. that are involved in the degradation and digestion of AGE. It is expected to prevent the aging of the skin and vascular endothelial cells by suppressing the former and promoting the latter. In addition, studies have also been conducted on the inhibitory effect represented by the antioxidant action against the production of ROS caused by oxidative stress such as hydrogen peroxide (H2O2) treatment. Suppressing the glycation reaction and oxidation reaction and inhibiting ROS production play an important role in vascular repair. Furthermore, it is also known that cells age faster by hydrogen peroxide (H2O2) treatment, and delaying it is effective for vascular repair. In the present invention, as a straightforward method for evaluating the blood vessel repair function, the wound-healing effect, which measures the speed of wound healing, was used.

[0028] (Composition for improving male sexual function) Next, a composition for improving male sexual function according to the second embodiment of the present invention will be described. The composition for improving male sexual 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] Zinc, allicin, arginine, citrulline, taurine, vitamin E, and mucin are the same as those described in the composition for improving blood vessel repair function according to the first embodiment above. Also, the stem cells are the same as those described in the composition for improving blood vessel repair function according to the first embodiment above.

[0030] (Formulation and function of the composition for improving male sexual function) The composition for improving male sexual 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] Also, the blending ratio of zinc, allicin, arginine, citrulline, taurine, vitamin E, and mucin is not particularly limited as long as the effects of the present invention are not impaired, but it is preferably blended at a specific blending ratio.

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

[0033] (Pharmaceutical) In addition, the composition for improving vascular repair function and / or the composition for improving male function of the present invention can also be incorporated into pharmaceuticals intended for these purposes. As pharmaceuticals, they can be used as either preventive drugs or therapeutic drugs.

[0034] When incorporated into pharmaceuticals, the component mixture can be used alone, or it can be mixed with additives generally acceptable in formulations and formulated. Also, as dosage forms, dosage forms using oral preparations such as tablets, granules, capsules, pills, powders, liquids, suspensions, emulsions, syrups, elixirs, extracts, etc., or dosage forms using parenteral preparations such as injections, liquids, suppositories, ointments, patches, poultices, lotions, etc. can be mentioned, but there are no particular restrictions, and they can be appropriately selected according to the purpose of treatment or prevention, etc.

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

[0036] Examples of binders include crystalline cellulose, crystalline cellulose - sodium carboxymethylcellulose, methylcellulose, hydroxypropylcellulose, low - substituted hydroxypropylcellulose, hydroxypropylmethylcellulose, hydroxypropylmethylcellulose phthalate, hydroxypropylmethylcellulose acetate succinate, sodium carboxymethylcellulose, ethylcellulose, carboxymethylethylcellulose, hydroxyethylcellulose, wheat starch, rice starch, corn starch, potato starch, dextrin, α - starch, partially α - 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 sugar, macrogol.

[0037] Examples of disintegrants include crystalline cellulose, methylcellulose, low - substituted hydroxypropylcellulose, carboxymethylcellulose, calcium carboxymethylcellulose, sodium carboxymethylcellulose, croscarmellose sodium, wheat starch, rice starch, corn starch, potato starch, partially α - starch, hydroxypropyl starch, sodium carboxymethyl starch, tragacanth.

[0038] Examples of lubricants include wheat starch, rice starch, corn starch, stearic acid, calcium stearate, magnesium stearate, hydrated 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 ester, waxes, hydrogenated vegetable oil, polyethylene glycol.

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

[0040] In the case of injections, additives such as suspensions, emulsions, and solvents for dissolution at the time of use can be included. In the case of ointments and suppositories, fats, fatty oils, lanolin, petrolatum, paraffin, waxes, resins, plastics, bases, glycols, higher alcohols, water, emulsifiers, suspending agents, etc. can be included as additives. In the case of patches, glycerin, water, water-soluble polymers, water-absorbing polymers, etc. can be included as additives. In the case of lotions, solvents, emulsifiers, suspending agents, etc. can be included as additives.

[0041] (Cosmetics) In addition, the composition for improving vascular repair function and / or the composition for improving male function of the present invention can also be formulated into cosmetics. Examples of cosmetics include lotions, milky lotions, facial washes, cleansers, beauty essences, creams, foundations, eyebrow pencils, mascaras, eyeshadows, eyeliners, lipsticks, glosses, blushes, face powders, nail polishes, etc. Also, as the form of cosmetics, forms such as liquids, creams, solids, sticks, and powders can be adopted.

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

[0043] In addition, 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 administration preparations such as tablets, capsules, and syrups.

[0044] In addition, when producing foods and beverages containing the composition for improving vascular repair function and / or the composition for improving male function of the present invention, sweeteners, colorants, preservatives, thickeners, stabilizers, gelling agents, antioxidants, color developers, bleaching agents, emulsifiers, swelling agents, acidulants, brighteners, flavors and other additives; solvents; oils may be added as necessary within a range that does not interfere with the effects of the present invention. These additives may be used individually or in combination of two or more.

[0045] The ratio of the composition for improving vascular repair function and / or the composition for improving male function of the present invention incorporated in the above-mentioned foods and beverages can be appropriately adjusted according to the purpose of use. However, the ratio of the component blend incorporated in the above-mentioned foods and beverages is preferably 0.01 to 20% by weight, more preferably 0.01 to 15% by weight, and still more preferably 0.1 to 10% by weight.

Examples

[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) As stem cells, adipose-derived stem cells, specifically, those isolated and cultured from adipose tissue excised from the abdominal cavity of mice were prepared. The above adipose-derived stem cells were cultured in an incubator at 37°C and 5% CO 2 for 7 days under the conditions, and cultured in a culture medium (Mesenchymal Stem Cell Growth Medium 2; C-28009, PromoCell or Opti-MEM I Reduced Serum Medium; 11058-021, gibco), and the culture supernatant in a state of being about 80% confluent was collected.

[0047] The above-mentioned 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 at a predetermined ratio, and ultrapure water or physiological saline was added as necessary to obtain a predetermined concentration, thereby obtaining a composition for improving vascular repair function (hereinafter sometimes referred to as STEM CELL+ alpha).

[0048] In addition, only the above-mentioned culture supernatant (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 were mixed at a predetermined ratio, and ultrapure water or physiological saline was added as necessary to obtain a predetermined concentration (hereinafter sometimes referred to as plus alpha alone), and preparations were also made for this.

[0049] (Example 1-1: Wound healing effect) For the wound healing effect, a verified method established as the Wound-healing effect was used. That is, a wound of a certain width was created in cultured cells of vascular endothelial cells, STEM CELL+ alpha was added, and the culture was continued at 37°C, and the number of days until the wound was closed was observed. That is, it was verified whether the part without cells was filled. In addition, instead of STEM CELL +alpha, STEM CELL alone and plus alpha alone were each added and observed. In addition, the system without adding STEM CELL +alpha (control) was also observed. The results are shown in Figure 1.

[0050] As shown in Fig. 1, in the control group, the wound was not yet closed even after 9 days of culture. However, in the STEM CELL alone and plus alpha alone groups, the wound closed after 9 days, and in the STEM CELL + alpha group, it closed after 6 days. A wound healing effect was observed in each group. In particular, the STEM CELL + alpha group was found to have an excellent wound healing effect.

[0051] (Example 1-2: Action in the mechanism by which cells decompose AGEs generated by the glycation reaction - Measurement of AGEs receptor - mRNA expression~) The mRNA levels of the receptors for decomposing AGEs present on the cell surface intracellularly were examined by quantitative RT-PCR. Since the ability to take up and decompose extracellular AGEs increases as the mRNA expression level rises, blood vessel endothelium cells, and thus the blood vessel wall, are improved.

[0052] Specifically, for the cultured vascular endothelial cells, the mRNA levels of various types (FEEL-1 -2, CD36, AGER-1) when both STEM CELL + alpha and AGE were added were measured by the quantitative RT-PCR method and compared with the untreated control. They were kept in an incubator at 37°C and measured after 4 hours had passed. Also, the measurements were made for those with 1 / 1 volume (final concentration; 10000 ng / ml), 1 / 10 volume (final concentration; 1000 ng / ml), and 1 / 100 volume (final concentration; 100 ng / ml) in the culture medium.

[0053] Specifically, the quantitative RT-PCR procedure was performed as follows. Total RNA was extracted from vascular endothelial cells supplemented with STEM CELL +alpha and AGE using Trizol reagent (ambion). The extracted RNA was reverse transcribed into cDNA according to the method of PrimeScript RT Master Mix (Takara), and amplification was performed using SYBR Premix EX Taq II (Takara). The PCR reaction solution was 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 O). The reaction was carried out under the conditions of 1 cycle of 30 seconds at 95°C, followed by 50 cycles of 5 seconds at 95°C and 30 seconds at 60°C.

[0054] In addition, instead of STEM CELL +alpha, STEM CELL alone and plus alpha alone were each added and measured. The results are shown in Figure 2.

[0055] The expression of mRNAs of AGEs receptors; FEEL-1, FEEL-2, CD-36, AGE-R1 was each increased. From this, it was shown that by using STEM CELL alone, plus alpha alone, and STEM CELL +alpha respectively, AGEs are taken up into cells and the ability to degrade them is enhanced. Also, among these, the result showed that when STEM CELL +alpha was used, the most AGEs were taken up into cells and degraded.

[0056] (Example 1-3: Improvement of cell viability under oxidative stress) When cells were treated with hydrogen peroxide (H2O2) for a certain period of time, the cells were in an oxidative stress state, and if the culture was continued as it was, it would lead to cell death. It was verified whether STEM CELL +alpha could prevent this.

[0057] Specifically, after incubating vascular endothelial cells in a medium supplemented with 0.2 mM hydrogen peroxide solution for 2 hours, a predetermined amount of STEM CELL +alpha was added, followed by incubation for 4 hours. The cells were then washed and removed with PBS and transferred to a normal medium for continued culture. After 48 hours, the cells or the culture medium was collected, and the cell viability was measured. The same measurements were also performed on the 10-fold diluted (denoted as "10" in Fig. 3) and 100-fold diluted (denoted as "100" in Fig. 3) hydrogen peroxide solutions. The system using 0.2 mM hydrogen peroxide solution was denoted as "1" in Fig. 3.

[0058] In addition, instead of STEM CELL +alpha, STEM CELL alone and plus alpha alone were each added and observed. Also, an observation was made on the system without adding STEM CELL +alpha (control). The results are shown in Fig. 3.

[0059] As shown in Fig. 3, the cell viability decreased to 62% after 48 hours in the control due to H2O2 treatment (38% cell death), but the cells were protected and the viability was improved by treatment with STEM CELL alone, plus alpha alone, and STEM CELL +alpha respectively. In particular, it was confirmed that the viability was most improved in STEM CELL +alpha.

[0060] (Example 1 - 4: Inhibitory effect on reactive oxygen species (ROS) production) Reactive oxygen species (ROS) accumulate in cells under oxidative stress caused by H2O2 treatment, leading to senescent cells. It was verified whether STEM CELL +alpha could suppress this.

[0061] Specifically, after incubating vascular endothelial cells in a medium supplemented with 0.2 mM hydrogen peroxide solution for 2 hours, a predetermined amount of STEM CELL +alpha was added, followed by incubation for 4 hours. The cells were then washed and removed with PBS and transferred to a normal medium for continued culture. After 48 hours, the cells or the culture medium was collected. 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 production amount of reactive oxygen species (ROS) in the cells was measured using a micro plate reader (SYNERGY / HT, BioTek, Japan) to quantify the fluorescence intensity.

[0062] In addition, instead of STEM CELL +alpha, STEM CELL alone and plus alpha alone were each added and observed. Also, an observation was made on a system without the addition of STEM CELL +alpha (control). The results are shown in Figure 4.

[0063] As shown in Figure 4, reactive oxygen species, ROS, accumulate in cells treated with H2O2. However, the use of STEM CELL alone, plus alpha alone, and STEM CELL +alpha each showed an inhibitory effect on ROS production. In particular, it was confirmed that the most significant inhibitory effect on ROS production was obtained with STEM CELL +alpha.

[0064] (Examples 1-5: Delaying effect on the appearance of senescent cells) Cells subjected to oxidative stress by H2O2 treatment change into senescent cells earlier than normal cultured cells. Senescent cells exhibit the property of staining blue with β-Gal. It was verified whether STEM CELL +alpha could delay this process.

[0065] Specifically, after incubating vascular endothelial cells in a medium supplemented with 0.2 mM hydrogen peroxide solution for 2 hours, a predetermined amount of STEM CELL + alpha was added, followed by incubation for 4 hours. The cells were then washed and removed with PBS and transferred to a normal medium for continued culture. After 4 weeks, the cells or the culture medium were collected. After washing with PBS, β-Gal was added for observation.

[0066] In addition, instead of STEM CELL + alpha, STEM CELL alone and plus alpha alone were each added for observation. Also, an observation was made on a system without the addition of STEM CELL + alpha (control). The results are shown in Figure 5.

[0067] As shown in Figure 5, senescent cells (stained blue with β-Gal) appeared early due to H2O2 treatment. After 4 weeks, senescent cells with an intensity of 5000 appeared in the image. On the other hand, when using STEM CELL alone, plus alpha alone, and STEM CELL + alpha, the number of cells stained blue was small, indicating that cell senescence was delayed. In particular, it was confirmed that the most significant delay in cell senescence was obtained with STEM CELL + alpha.

[0068] (Example 2: Male function) Measurements and observations were made on human smooth muscle cells when stimulated with stem cells or stem cells + α.

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

[0070] Also, only the above-mentioned culture supernatant (hereinafter sometimes referred to as STEM CELL alone), zinc, allicin, arginine, citrulline, taurine, vitamin E, and mucin were mixed at a predetermined ratio, and ultrapure water or physiological saline was added as necessary to obtain a predetermined concentration (hereinafter sometimes referred to as plus alpha alone).

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

[0072] The activity of PDE could be inhibited by treating the cells with STEM CELL alone, plus alpha alone, and STEM CELL + alpha. This suggests that it can regulate vascular smooth muscle contraction.

[0073] (Example 2-2: Production amount of NO (nitric oxide)) The change in nitric oxide production in human aortic smooth muscle cells was evaluated with the increase in the component concentration (μg / mL) in the medium of human aortic smooth muscle cells. The production of nitric oxide in human aortic smooth muscle cells was evaluated using the relative fluorescence intensity (RFU) of a fluorescent probe that emits fluorescence by reacting with nitrite (NO 2 - ) as an index.

[0074] Specifically, human aortic smooth muscle cells (HumanAorticSmoothMuscleCells (HAoSMC); PromoCell) were cultured using a dedicated medium (Smooth Muscle Cell Growth Medium 2; PromoCell). 10% fetal bovine serum and antibiotics were added to the medium. 5×104 Cells were seeded and cultured until 80% confluent. Then, the medium was replaced with a medium containing each component (STEM CELL alone, plus alpha alone, or STEM CELL + alpha) at 0.1, 1, 10, 100 mg / mL or a control medium without components. After 1 hour, the culture supernatant was collected. The total NO production was measured according to the standard protocol of the OxiSelect In Vitro Nitric Oxide (Nitrite / Nitrate) Assay Kit (CELLBIOLABS).

[0075] Total RNA was extracted using the RNeasy Mini Kit (QUIAGEN). After performing a reverse transcription reaction using the OneStep RT-PCR Kit (NEB), amplification of the NO synthase gene and the constitutively expressed GAPDH gene was performed using predetermined primers.

[0076] The difference (DeltaCt) from GAPDH for each sample was calculated by quantitative PCR, and further the difference (DeltaDeltaCt value) from the DeltaCt value of the control sample without components was determined. The calibration curve of the measurement kit is shown in Figure 8, and the measurement results are shown in Figure 9, expressed as the fold of the mRNA amount of the control sample as the mRNA expression level.

[0077] As shown in Figure 9, the production amount of NO increased depending on the concentration of each of STEM CELL alone, plus alpha alone, or STEM CELL + alpha. In particular, the most significant increase was observed in STEM CELL + alpha.

[0078] (Example 2 - 3: Each mRNA level of NO synthase, sGC, cGMP - dependent protein kinase (PKG)) The measurement was performed according to the procedures shown in 1 - 8 below. 1. Human vascular smooth muscle cells were cultured in a 6 - well plate in a 37°C, 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 TotalRNA was extracted to measure mRNA. (Details were in accordance with the protocol of Trizol reagent.) 4. After quantifying the amount of the obtained TotalRNA, a certain amount was used to synthesize cDNA by reverse transcription reaction (RT reaction). (Details were in accordance with Takara Primscript master mix Kit.) 5. Using the cDNA, a reaction solution was prepared for each specimen using a primer pair (synthesized separately) according to various Hsp-mRNA sequences and a PCR reaction solution (SYBR green type). 6. These were set in a 96-well PCR plate and loaded into a quantitative PCR apparatus. 7. Using a dedicated program, the original amounts of various Hsp-mRNAs were relatively quantified (by the delta-delta CT method). 8. The Hsp induction ability of various specimens was examined from the obtained Hsp amounts. The results are shown in Fig. 10.

[0079] From Fig. 10, it was shown that the expression of various mRNAs involved in NO production increased depending on the respective concentrations of STEM CELL alone, plus alpha alone, and STEM CELL + alpha. In particular, the increase in PKG was remarkable. Also, the highest mRNA level was shown in STEM CELL + alpha for all of NO synthase, sGC, and PKG.

Claims

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, coenzyme Q10, 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 Claim 1.

3. A cosmetic containing the composition for improving vascular repair function according to Claim 1.

4. A food or beverage containing the composition for improving vascular repair function according to Claim 1.

5. A composition for improving male 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 function according to Claim 5.

7. A cosmetic containing the composition for improving male function according to Claim 5.

8. A food or beverage containing the composition for improving male function according to Claim 5.

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