Hyaluronic acid production promoter

Abstract

To provide pharmaceuticals, quasi-drugs, cosmetics, foods, or materials blended therein, having a hyaluronic acid production-promoting effect and being useful for increasing hyaluronic acid levels in the body.SOLUTION: A hyaluronic acid production promoter comprises Allium fistulosum L. or a water extract thereof as an active ingredient.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to a hyaluronic acid production promoter that increases the amount of hyaluronic acid produced in vivo.

Background Art

[0002] Hyaluronic acid is a linear high-molecular polysaccharide in which disaccharide units of N-acetylglucosamine and glucuronic acid are linked, and in vivo, it is distributed in various tissues such as joints, vitreous humor of the eye, skin, brain, and blood vessels. Hyaluronic acid has the property of binding to a large amount of water, and in joints and skin, it forms an extracellular matrix together with collagen and the like, playing an extremely important role in maintaining the functions and morphology of tissues and cells, as well as preventing bacterial infection.

[0003] The amount of hyaluronic acid present in the skin, joints, etc. decreases with aging, and it is known that the amount of hyaluronic acid decreases in joint diseases such as osteoarthritis, rheumatoid arthritis, septic arthritis, and gouty arthritis. Hyaluronic acid in the skin contributes to skin moisturization and elasticity as an important component constituting the extracellular matrix. However, due to aging and various stresses, the amount of hyaluronic acid decreases, resulting in skin aging such as dryness, rough skin, formation of wrinkles, sagging of the skin, loss of firmness, and decline in barrier function.

[0004] Therefore, increasing the amount of hyaluronic acid in vivo is considered to be effective in preventing or improving joint diseases and preventing or improving skin aging. For example, Non-Patent Document 1 discloses that intra-articular injection of hyaluronic acid is an effective means for treating osteoarthritis of the knee.

[0005] According to the statistics of vegetable production and shipment in Reiwa year 1, the shipment volume of Welsh onion (Allium fistulosum L.) in Japan is approximately 364,100 t. Welsh onion is divided into a green leaf part and a white leaf sheath part (white leaf sheath). The green leaf part is cut and discarded for boxing at the time of shipment. In order to effectively utilize the discarded green leaf part, it is necessary to investigate the composition and amount of the contained substances, as well as specific effects and efficacy such as ingestion effects.

[0006] Conventionally, as the pharmacological action of green onion, for example, Non-Patent Document 2 discloses that the water extract of the green leaf part of green onion reduces xanthine oxidase activity and suppresses oxidative damage of proteins by eliminating active oxygen species such as superoxide radicals and hydroxyl radicals and active nitrogen species such as nitric oxide. In addition, Non-Patent Document 3 examines the antioxidant activity and antioxidant components of green onion (leaf green onion) and white onion (deep-rooted white onion), and discloses that green onion has higher antioxidant activity than white onion, and the antioxidant components are mainly quercetin and kaempferol, which are flavonoids, but their contents in white onion are low. Further, Non-Patent Document 4 discloses that the protein contained in the leaf mucus of the green leaf part of green onion has the effect of increasing the production amounts of TNF-α, MCP-1, and IL-12, while the extracts of the green leaf part and white leaf sheath part of green onion do not have such an effect. In addition, Patent Document 1 discloses that the ethanol extract of Kujo green onion, known as green onion, has an effect of suppressing melanin production, promoting type I collagen production, promoting hyaluronic acid production, suppressing hydrogen peroxide cell damage, promoting epidermal keratinocyte proliferation, and promoting transglutaminase-1 production.

[0007] On the other hand, although it has been reported that quercetin and quercetin have the effect of promoting hyaluronic acid production (Patent Document 2), it is not known that Welsh onion or its water extract and specific sulfur-containing amino acids have the effect of promoting hyaluronic acid production.

Prior Art Documents

Patent Documents

[0008] [Patent Document 1] Japanese Unexamined Patent Application Publication No. 2012-162487 [Patent Document 2] Japanese Translation of PCT International Publication No. 2010-500282 [Non-Patent Document]

[0009] [Non-Patent Document 1] R.R. Bannuru, N.S. Natov, U.R. Dasi, C.H. Schmid, T.E. McAlindon, Therapeutic trajectory following intra-articular hyaluronic acid injection in knee osteoarthritis - meta-analysis. Osteoarthritis and Cartilage, 2011, 19(6),611-619, [Non-Patent Document 2] Wang B. S et al., Wang B. S, Lin S.S, Hsiao W. C, Fan J., Fuh L. F, Duh P.D. Protective effects of an aqueous extract of Welsh onion green leaves on oxidative damage of reactive oxygen and nitrogen species. Food Chem., 2006, 98, 149-157. [Non-Patent Document 3] Sakiko Aoyama et al., "Comparison of antioxidant activities and antioxidant components of three types of green onions", Abstracts of the 2006 Annual Meeting of the Japanese Society of Cooking Science. [Non-Patent Document 4] Yamazaki T et al., Yamazaki T, Ogawa T, Muramoto K, Nakahigashi J, Takeuchi A, Ueda H. Isolation and Biochemical Characterization of Mucus Proteins in Japanese Bunching Onion (Allium fistulosum) Green Leaves. Food Science and Technology Research, 2016, 22(2), 235 - 243.

Summary of the Invention

Problems to be Solved by the Invention

[0010] The present invention relates to providing a pharmaceutical, quasi - drug, cosmetic, food, or a material to be formulated therein, which has an action of promoting hyaluronic acid production and is useful for increasing the amount of hyaluronic acid in vivo.

Means for Solving the Problems

[0011] In view of the above problems, the present inventors conducted studies and found that Welsh onion or its water extract and specific sulfur - containing amino acids have an excellent action of promoting hyaluronic acid production, and this is useful for increasing the amount of hyaluronic acid in vivo.

[0012] That is, the present invention relates to the following 1) to 12). 1) A hyaluronic acid production promoter containing Welsh onion or its water extract as an active ingredient. 2) An agent for preventing or improving skin aging containing Welsh onion or its water extract as an active ingredient. 3) An agent for preventing or improving joint diseases containing Welsh onion or its water extract as an active ingredient. 4) A food for promoting hyaluronic acid production containing the water extract of Welsh onion as an active ingredient. 5) A food for preventing or improving skin aging containing the water extract of Welsh onion as an active ingredient. 6) A food for preventing or improving joint diseases containing the water extract of Welsh onion as an active ingredient. 7) A hyaluronic acid production promoter containing at least one selected from alliin, isoalliin, and cycloalliin as an active ingredient. 8) A skin aging prevention or improvement agent containing at least one selected from alliin, isoalliin, and cycloalliin as an active ingredient. 9) A joint disease prevention or improvement agent containing at least one selected from alliin, isoalliin, and cycloalliin as an active ingredient. 10) A food for promoting hyaluronic acid production containing at least one selected from alliin, isoalliin, and cycloalliin as an active ingredient. 11) A food for preventing or improving skin aging containing at least one selected from alliin, isoalliin, and cycloalliin as an active ingredient. 12) A food for preventing or improving joint diseases containing at least one selected from alliin, isoalliin, and cycloalliin as an active ingredient.

Advantages of the Invention

[0013] According to the hyaluronic acid production promoter of the present invention, the amount of hyaluronic acid in the living body can be increased. Therefore, according to the present invention, pharmaceuticals, quasi-drugs, cosmetics, foods, or materials to be formulated therein useful for preventing or improving skin aging, preventing or improving joint diseases such as osteoarthritis, rheumatoid arthritis, suppurative arthritis, and gouty arthritis can be provided. At the same time, effective utilization of the discarded green leaves of Welsh onions can be achieved.

Brief Description of the Drawings

[0014]

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BEST MODE FOR CARRYING OUT THE INVENTION

[0015] In the present invention, "Welsh onion" refers to Allium fistulosum of the genus Allium in the family Alliaceae, and is also called white onion and deep-rooted onion. Welsh onion is mainly produced in the eastern region of Japan and is distinguished from strains called leaf onions (also known as green onions) such as Kyuujo onion. Welsh onion is cultivated with the leaf sheath being earthed up as it grows, and has a long stature among onions and is characterized by being less likely to tiller from the base of the stem compared to leaf onions. In the present invention, for example, Welsh onions produced in Hokkaido, Akita Prefecture, Miyagi Prefecture, Yamagata Prefecture, Fukushima Prefecture, Tochigi Prefecture, Gunma Prefecture, Ibaraki Prefecture, Chiba Prefecture, Saitama Prefecture, Shizuoka Prefecture, and Kagoshima Prefecture can be preferably used.

[0016] The leaves of Welsh onion consist of a white leaf sheath part (white leaf sheath part) and a green leaf blade part (green leaf part). The green leaf part of Welsh onion is tubular, thick, pointed at the tip, green with a whitish powder blown on it, and contains mucus. The white leaf sheath part is mainly used for food, and the green leaf part is discarded. In the present invention, Welsh onion may be used either for the white leaf sheath part or the green leaf part, but from the viewpoint of promoting hyaluronic acid production and effectively utilizing the green leaf part, it is preferable to use the green leaf part which is the leaf blade.

[0017] In the present invention, Welsh onion can be preferably used either in a dried state (natural drying, freeze-drying, etc.) or in a raw state. Also, Welsh onion can be used as it is after being appropriately pulverized, but it is preferably subjected to extraction.

[0018] In the present invention, water is used as the solvent for extracting Welsh onion. The extracted water is not particularly limited, and examples thereof include tap water, well water, distilled water, deionized water, ultrapure water (for example, Milli-Q water purified by an ultrapure water device Milli-Q), etc. In this case, the extraction temperature may be from room temperature to 100°C, for example, from 25°C to 70°C, preferably from 25°C to 60°C, and may be hot water of 80°C or higher. Also, under pressure, it may be 100°C or higher, for example, from 100°C to 200°C. In the present invention, the water extract extracted using hot water is also referred to as "hot water extract". Here, hot water means water of 80°C or higher and 100°C or lower, and for example, water of 80°C to 98°C, water of 80°C to 95°C. The Welsh onion extract is preferably a water extract (extraction temperature: 25°C to 70°C) when using the green leaf part, and preferably a hot water extract when using the white leaf sheath part.

[0019] Also, the extraction time can be appropriately set in consideration of the extraction temperature, extraction scale, etc. For example, it can be 30 minutes to 12 hours, preferably 1 hour to 5 hours, more preferably 2 hours to 4 hours. Also, the second extraction can be performed using the residue obtained from the first extraction, and the first extract and the second extract can be combined to obtain the extract used in the present invention. In the extraction, the volume ratio of water or hot water to the mass of the green leaf part of dried Welsh onion is not particularly limited as long as the conditions can extract the active ingredient that can obtain the effect of the present invention. For example, the ratio can be 20 to 60 mL of water, preferably 30 to 50 mL, more preferably 30 to 40 mL, per 1 g of dried Welsh onion.

[0020] The extraction means is not particularly limited. For example, ordinary means such as solid-liquid extraction, liquid-liquid extraction, immersion, decoction, leaching, reflux extraction, pressurized heating extraction, distillation, supercritical extraction, etc. can be used.

[0021] It is preferable to use the above extract after removing inert impurities by known techniques such as liquid-liquid partitioning, solid-liquid partitioning, filtration membranes, activated carbon, adsorption resins, ion exchange resins, etc. Also, the extract may be used after being subjected to treatments such as deodorization and decolorization by known methods if necessary. Also, the extract can be further purified and used using known techniques and methods. The water extract of Welsh onion thus obtained can be used as it is, but it can also be prepared in the form of a powder or paste after diluting, concentrating, or freeze-drying the extract.

[0022] The alliins, isoalliins, and cycloalliins of the present invention refer to the sulfur-containing amino acids shown below.

[0023]

Chemical formula

[0024] Among these, alliin and isoalliin are components contained in Welsh onion or its water extract. Alliin and isoalliin can be obtained by separating and purifying from the water extract of Welsh onion green leaves, and can also be obtained from plants of the Liliaceae family such as garlic. It is also possible to chemically synthesize them by known methods. Cycloalliin is known to be converted from isoalliin by heating or alkali treatment (Ueda, Y., Tsubuku, T. and Miyajima, R., Biosci. Biotech. Biochem., 58, 108-110 (1994)). In addition, it is also possible to use commercially available products of alliin, isoalliin and cycloalliin. It has been reported that alliin and isoalliin have various pharmacological effects including fatigue recovery effect, and cycloalliin is known to have an effect such as preventing thrombosis, but it is not known to have an effect of promoting hyaluronic acid production.

[0025] As shown in the examples described below, the water extract of Welsh onion green leaves, alliin and isoalliin contained therein, and cycloalliin have an effect of promoting the production of hyaluronic acid in human dermal fibroblasts. As described above, increasing the amount of hyaluronic acid in the living body is useful for preventing or improving skin aging such as dryness, rough skin, formation of wrinkles, sagging of the skin, loss of firmness, and decline of barrier function caused by the decrease in the amount of hyaluronic acid. It is also useful for preventing or improving joint diseases such as osteoarthritis, rheumatoid arthritis, septic arthritis, and gouty arthritis accompanied by a decrease in the amount of hyaluronic acid. Therefore, one or more selected from Welsh onion or its water extract, alliin, isoalliin and cycloalliin can be a hyaluronic acid production promoter, a skin aging prevention or improvement agent, and a joint disease prevention or improvement agent.

[0026] The above-mentioned hyaluronic acid production promoter, skin aging prevention or improvement agent, and joint disease prevention or improvement agent can be used as pharmaceuticals, quasi-drugs, cosmetics or foods for promoting hyaluronic acid production, preventing or improving skin aging, and further preventing or improving joint diseases, or as materials or preparations for blending them into these. In addition to general foods and beverages, the above-mentioned foods (also referred to as "foods for promoting hyaluronic acid production", "foods for preventing or improving skin aging", and "foods for preventing or improving joint diseases") include foods labeled as such as necessary, functional foods, foods for patients, foods with specified health uses, foods with functional claims, and supplements.

[0027] Here, "promoting hyaluronic acid production" means that the ability of skin epidermal cells to produce hyaluronic acid is promoted and the amount of hyaluronic acid increases. "Skin aging" refers to a physiological phenomenon caused by the interaction of physiological aging associated with aging and photoaging caused by sunlight exposure (ultraviolet rays). Specifically, it includes skin changes or symptoms such as dryness, rough skin, formation of wrinkles, sagging of the skin, loss of firmness, and reduction of barrier function. "Joint disease" refers to a disease in which abnormalities such as deformation, damage, and inflammation occur in joints. Specifically, for example, osteoarthritis, rheumatoid arthritis, suppurative arthritis, gouty arthritis, etc. are included.

[0028] In addition, in the present invention, "prevention" means preventing or delaying the onset of a disease or symptom in an individual, or reducing the risk of onset of a disease or symptom in an individual. "Improvement" means the improvement of a disease, symptom, or condition, the prevention or delay of the deterioration of a disease, symptom, or condition, or the reversal, prevention, or delay of the progression of a disease or symptom, and is a concept including treatment.

[0029] The above-mentioned pharmaceuticals or quasi-drugs containing welsh onion or its water extract, or one or more of alliine, isoalliine, and cycloalliine can be administered in any dosage form. The administration can be oral or parenteral. Dosage forms for oral administration include, for example, solid dosage forms such as tablets, coated tablets, granules, powders, and capsules, as well as liquid dosage forms such as elixirs, syrups, and suspensions. Dosage forms for parenteral administration include injections, infusions, topical applications, external preparations, transdermal, transmucosal, nasal, enteral, inhalation, suppositories, boluses, patches, etc. Preferably, the pharmaceutical or quasi-drug can be in the form of a topical skin preparation.

[0030] The above-mentioned pharmaceutical or quasi-drug may contain one or more of Welsh onion or its water extract, or alliin, isoalliin, and cycloalliin alone, or may be contained in combination with a pharmaceutically acceptable carrier. Examples of such carriers include, for example, excipients, coating agents, binders, extenders, disintegrants, lubricants, diluents, osmotic pressure regulators, pH regulators, dispersants, emulsifiers, preservatives, stabilizers, antioxidants, colorants, ultraviolet absorbers, humectants, thickeners, lubricants, activity enhancers, anti-inflammatory agents, bactericides, fragrances, flavoring agents, odor correctors, and the like. In addition, the said pharmaceutical or quasi-drug may contain other active ingredients or pharmacological ingredients as long as the pharmacological effects of Welsh onion or its water extract are not lost.

[0031] The cosmetic containing Welsh onion or its water extract, or one or more of alliin, isoalliin, and cycloalliin according to the present invention may contain Welsh onion or its water extract, or one or more of alliin, isoalliin, and cycloalliin alone, or may be contained in combination with a carrier acceptable as a cosmetic. Examples of such carriers include, for example, excipients, coating agents, binders, extenders, disintegrants, lubricants, diluents, osmotic pressure regulators, pH regulators, dispersants, emulsifiers, preservatives, stabilizers, antioxidants, colorants, ultraviolet absorbers, humectants, thickeners, lubricants, activity enhancers, anti-inflammatory agents, bactericides, fragrances, flavoring agents, odor correctors, and the like. In addition, the said cosmetic may contain other active ingredients or cosmetic ingredients, for example, humectants, whitening agents, ultraviolet protectants, cell activators, detergents, keratolytic agents, makeup ingredients (for example, makeup base, foundation, powder, blush, lipstick, eye makeup, eyebrow, mascara, etc.), as long as the pharmacological action of the present invention is not lost. Forms as a cosmetic include any form that can be used in cosmetics, such as creams, emulsions, lotions, suspensions, gels, powders, packs, sheets, patches, sticks, cakes, and the like. Preferably, the above cosmetic can be a cosmetic for skin moisturization or a cosmetic for preventing skin aging.

[0032] In addition, the form of the above food containing Welsh onion of the present invention, its water extract, or one or more of alliine, isoalliine, and cycloalliine includes soft drinks, tea-based beverages, coffee beverages, fruit juice beverages, carbonated beverages, jelly, wafers, biscuits, bread, noodles, sausages, and other food and drink products, various foods such as nutritional foods. Furthermore, there are nutritional supplement compositions in the same form as the above oral administration preparations (solid preparations such as tablets, capsules, troches, etc.). Among these, tablets and capsules are preferred. Foods in various forms can be prepared by using the Welsh onion of the present invention or its water extract alone, or by appropriately combining it with other food materials, solvents, softeners, oils, emulsifiers, preservatives, fragrances, stabilizers, colorants, antioxidants, humectants, thickeners, etc.

[0033] The above pharmaceuticals, quasi-drugs, cosmetics, or foods can be produced by a conventional method by combining Welsh onion or its water extract, or one or more of alliine, isoalliine, and cycloalliine, with the above carrier and / or other active ingredients and pharmacological components as needed. The content of Welsh onion or its water extract in the pharmaceuticals, quasi-drugs, cosmetics, or foods is preferably 0.00001% by mass or more, more preferably 0.001% by mass or more, preferably 10% by mass or less, and more preferably 1% by mass or less in the total mass of the preparation on a dry basis. In addition, the content of alliine, isoalliine, or cycloalliine in the pharmaceuticals, quasi-drugs, cosmetics, or foods is preferably 1% by mass or more, more preferably 5% by mass or more, preferably 10% by mass or less, and more preferably 8% by mass or less in the total mass of the preparation.

[0034] In addition, the dosage of the above-mentioned pharmaceuticals and foods may vary according to the condition, body weight, gender, age, or other factors of the subject. However, the daily dosage per adult is usually, as welsh onion or its water extract (dry product), preferably 0.6 g or more, more preferably 1 g or more, and preferably 12 g or less, more preferably 6 g or less. Also, as alliine, isoalliine, or cycloalliine, it is preferably 30 mg or more, more preferably 50 mg or more, and preferably 600 mg or less, more preferably 300 mg or less. The above-mentioned preparation can be administered according to any administration schedule, but it is preferably administered once to several times a day and continuously for several weeks to several months. Examples of the administration targets include humans who need or desire to prevent or improve skin changes or symptoms such as dryness, rough skin, formation of wrinkles, sagging of the skin, loss of firmness, and reduction of barrier function, and humans who need to improve pain due to deformation, injury, and inflammation.

Examples

[0035] Hereinafter, the present invention will be described in more detail based on examples, but the present invention is not limited thereto. <Production Example> 1. Preparation of welsh onion water extract (1) Preparation of water extract of green leaf part or white leaf sheath part of welsh onion 15 mL of Milli-Q water was added to 0.45 g of dry powder of the green leaf part or white leaf sheath part of welsh onion (deep-rooted welsh onion produced in Saitama Prefecture), and extraction was carried out at room temperature for 1 hour and 30 minutes. After centrifugation, the supernatant was collected, 15 mL of Milli-Q water was added again, and after stirring, centrifugation was carried out. The supernatants collected by these two centrifugations were combined and freeze-dried. 0.29 g of the freeze-dried product after extraction from 0.45 g of the green leaf part and 0.40 g of the freeze-dried product after extraction from 0.45 g of the white leaf sheath part were obtained. The obtained freeze-dried products were respectively used as the water extract of the green leaf part of welsh onion (blue water) and the water extract of the white leaf sheath part of welsh onion (white water).

[0036] (2) Preparation of hot water extract of green leaf part or white leaf sheath part of welsh onion 0.45 g of the dried powder of the green leaf part or the white leaf sheath part of Welsh onion (Welsh onion with deep roots produced in Saitama Prefecture) was added with 30 mL of Milli-Q water, and extraction was carried out at 80 °C for 1 hour and 30 minutes. After centrifugation, the supernatant was collected and freeze-dried. 0.32 g of the freeze-dried product after extraction from 0.45 g of the green leaf part and 0.43 g of the freeze-dried product after extraction from 0.45 g of the white leaf sheath part were obtained. The obtained freeze-dried products were designated as the hot water extract of the green leaf part of Welsh onion (blue hot water) and the hot water extract of the white leaf sheath part of Welsh onion (white hot water), respectively.

[0037] (3) Preparation of ethanol extract of green leaf part or white leaf sheath part of Welsh onion 15 mL of 70% ethanol was added to 0.45 g of the dried powder of the green leaf part or the white leaf sheath part of Welsh onion (Welsh onion with deep roots produced in Saitama Prefecture), and extraction was carried out at room temperature for 1 hour and 30 minutes while applying ultrasonic waves. The supernatant after centrifugation was collected, 15 mL of Milli-Q water was added again, and after stirring, centrifugation was carried out. The supernatants collected by these two centrifugations were combined and evaporated to dryness with an evaporator, and then Milli-Q water was added so as to be the same amount as before performing the evaporator. Freeze-drying was carried out to obtain 0.26 g of the ethanol extract of the green leaf part (blue EtOH) and 0.34 g of the ethanol extract of the white leaf sheath part (white EtOH).

[0038] 2. Component analysis It has been reported that a large number of glycosides of quercetin and kaempferol are contained in leek (Allium porrum L) (Soininen TH et al. Soininen TH, Jukarainen N, Soininen P, Auriola SO, Julkunen-Tiitto R, Oleszek W, Stochmal A, Karjalainen RO, Vepsalainen JJ. Metabolite profiling of leek (Allium porrum L) cultivars by (1)H NMR and HPLC-MS. Phytochem Anal., 2014 May-Jun, 25(3), 220-8.). (1) Reverse phase chromatography With reference to the method of Soininen TH et al. (2014), the components of the water extract and hot water extract of the green leaf part of Welsh onion prepared in Production Example 1 were analyzed by reverse-phase chromatography. Each extract was dissolved in methanol at 5 mg / mL and filtered through a 0.45 μm filter (DISMIC-03CP, manufactured by ADVANTEC). In addition, a quercetin standard (1,2,3,4,6-Penta-O-galloyl-β-D-glucopyranose, manufactured by Wako Pure Chemical Industries, Ltd.) and a kaempferol standard ((3,4´,5,7-Tetrahydroxyflavone, manufactured by Wako Pure Chemical Industries, Ltd.) were dissolved in ethanol at 0.2 mg / mL to prepare a standard solution. The sample solution and the standard solution were analyzed by HPLC LC-8020II (manufactured by Tosoh Corporation). The analysis conditions are as follows. · Mobile phase: (A) 0.5% formic acid, (B) methanol · Gradient conditions: 0 - 2 min 5% B, 2 - 10 min 5 - 20% B, 10 - 18 min 20 - 40% B, 18 - 25 min 40 - 100% A, 25 - 27 min 100 - 5% B, 27 - 32 min 5% B · Column: TSKgel ODS-80TsQA · Measurement temperature: 40 °C · Detection wavelength: 340 nm · Injection volume: 10 μL

[0039] (2) NMR analysis Measurements were carried out on the hot water extract of the green leaf part of Welsh onion, the water extract of the green leaf part of Welsh onion, a quercetin standard (1,2,3,4,6-Penta-O-galloyl-β-D-glucopyranose, manufactured by Wako Pure Chemical Industries, Ltd.) and a kaempferol standard ((3,4´,5,7-Tetrahydroxyflavone, manufactured by Wako Pure Chemical Industries, Ltd.). Methanol-d4 containing 0.5% TMS was used as the heavy solvent for measurement. A nuclear magnetic resonance apparatus (NMR) JNM-ECA600 (600 MHz) (manufactured by JEOL Ltd.) was used to acquire the data.

[0040] (3) Results No peaks of quercetin and kaempferol were observed in the aqueous extract and hot water extract of the green leaf part of Welsh onion, and it was confirmed that quercetin and kaempferol were not contained.

[0041] Test Example 1 Promoting effect of Welsh onion extract on hyaluronic acid production 1. Method (1) Cells and cell culture method The cells used were normal human dermal fibroblasts (HFB, Passage 11). For the growth medium, DMEM medium (Dulbecco’s Modified Eagle’s Medium) (low glucose, manufactured by SIGMA) / 10% fetal bovine serum (FBS, manufactured by SIGMA) / 1% PSN Antibiotic Micture (manufactured by GIBCO) was used. For the addition medium, DMEM medium (Dulbecco‘s Modified Eagle’s Medium) (no phenol, low glucose, manufactured by SIGMA) / 1% Sodium pyruvate solution (manufactured by SIGMA) / 1% L-Glutamine (manufactured by GIBCO) was used.

[0042] (2) Addition of test substances The aqueous extract and hot water extract of the green leaf part of Welsh onion were dissolved in PBS to a concentration of 1 mg / mg and filtered using a 0.20 μm cellulose acetate membrane filter (manufactured by ADVANTEC). The ethanol extract of the green leaf part of Welsh onion was dissolved in 70% ethanol to a concentration of 1 mg / mg and filtered using a 0.20 μm cellulose acetate membrane filter (manufactured by ADVANTEC). This sample solution was mixed with the addition medium. Only the addition medium was used as the control. The cells were cultured at 5% CO2 and 37 °C (CO2 incubator MCO-17AIC, manufactured by SANKYO). After growing the cells to a coverage rate of 80 - 90% for the wells used, the growth medium was removed and the cells were washed once with PBS to remove serum components. Immediately afterwards, the cells were replaced with the addition medium containing the sample and cultured at 5% CO2 and 37 °C for 48 hours. After the culture was completed, the supernatant was collected and stored at -20 °C until use.

[0043] (3) Measurement method of hyaluronic acid (HA) in the culture supernatant To a 96-well ELISA plate (manufactured by IWAKI) for ELISA, add 100 μL / well of Hyaluronic Acid Binding Protein (HABP, manufactured by Funakoshi Co., Ltd.) diluted 2000-fold with 1×PBS and leave it standing overnight at 4°C The next day, wash once with PBS-Tween (0.1% Tween 20) (manufactured by Wako Pure Chemical Industries, Ltd.) and then add 200 μL / well of Block Ace (registered trademark) (manufactured by Snow Brand Milk Products Co., Ltd.) solution diluted to 1% with 1×PBS, and react at 37°C for 2 hours. After washing once with PBS-Tween, add 50 μL / well of HA standard coating solution (HANa 1000 μg / mL (manufactured by Wako Pure Chemical Industries, Ltd.) diluted to 1000 ng / mL with 0.5% Block Ace (registered trademark) solution), and leave it standing at 37°C for 1 hour. Wash 3 times with PBS-Tween, add 50 μL / well of the sample and standard HA solutions (0.5% Block Ace (registered trademark) solutions containing 1000, 250, 62.5, 15.625, 3.90625, 0 ng / mL of HA) diluted 5-fold with 0.5% Block Ace (registered trademark) solution. Then, add 50 μL / well of biotin-conjugated HABP solution (manufactured by Funakoshi Co., Ltd.) diluted to 0.125 mg / mL with 0.5% Block Ace (registered trademark) solution from above and react at room temperature for 1 hour. After washing 3 times with PBS-Tween, add 50 μL / well of HRP-conjugated streptavidin solution (manufactured by Thermo scientific) diluted to 0.05 μg / mL with 0.5% Block Ace (registered trademark) solution and react at 37°C for 30 minutes. After washing 5 times with PBS-Tween, add 50 μL / well of TMB reagent (manufactured by Nacalai Tesque, Inc.), cover with aluminum foil to shield from light, and leave it standing at room temperature for 30 minutes to develop color. Add 50 μL / well of 1N H2SO4 solution to stop the reaction. Immediately measure the absorbance at ABS450nm / 630nm using an infinite M200. All measurements were performed in duplicate. The calculation method was to create a logarithmic curve from the standard absorbance to determine the concentration of the sample. For the diluted sample, the dilution factor was multiplied to obtain the original value.

[0044] 2. Results An increase in the production amount of hyaluronic acid was observed in the water extract and hot water extract of the green leaf part of Welsh onion, and the effect was remarkable in the water extract (Figure 3). In addition, an increase in the production amount of hyaluronic acid was also observed in the water extract and hot water extract of the white leaf sheath part of Welsh onion, but the increase amount was less than that in the water extract of the green leaf part of Welsh onion (Figure 4). No increase in the production amount of hyaluronic acid was observed in the ethanol extract of the green leaf part of Welsh onion and the ethanol extract of the white leaf sheath part of Welsh onion (Figures 3 and 4).

[0045] Test Example 2 Effect of Welsh onion or its extract on the photoaging model 1. Method (1) Photoaging skin model The animals were bred in an animal breeding room with a 12-hour light-dark cycle, a temperature of 23.8 ± 0.8 °C, and a humidity of 57 ± 5.2% using a dehumidifier and a humidifier. A solid feed for general breeding was used as the feed, and the animals were group-housed in one cage per group under free feeding and free drinking. After 3 days of preliminary breeding, the animals were grouped so that the body weight, skin moisture content, and transepidermal water loss (TEWL) were equal, and this test was started 2 days after grouping. The administration sample was adjusted to 1 mg / 100 μL with MilliQ water as the solvent and administered at 100 mg / kg body weight.

[0046] The test groups were the UV(-) control group (n = 7), the UV(+) control group (n = 7), the UV(+) Welsh onion green leaf part water extract administration (blue water) group (n = 6), and the UV(+) Welsh onion green leaf part dry powder administration (blue powder) group (n = 6). The ultraviolet irradiation method was carried out according to the method of Tanaka et al. During the test period, the test substance solution was orally administered once a day using a gastric tube. In parallel, ultraviolet irradiation was performed three times a week. As the UVB lamp for ultraviolet irradiation, lump GL20SE (manufactured by SANKYO DENKI) was used, and three of these were installed in parallel and used as an irradiator. This lamp mainly emits light with a wavelength of 280 to 350 nm, with a maximum at 306 nm. The irradiation intensity was measured using a digital ultraviolet intensity meter UV-340 (manufactured by AS ONE). The irradiation was carried out by placing the mice in individual cages of 10 with a length of 10 cm, a width of 6 cm, and a height of 4 cm. Since the irradiation intensity differed depending on the position of each individual cage, rotation of the position was performed for each group every time to mitigate the difference in irradiation intensity. The irradiation was performed three times a week, and the irradiation time was gradually increased from 1 minute in the first week, with a maximum of 3 minutes and 45 seconds to prevent erythema formation. The irradiation period was 6 weeks, and the total irradiation dose was 1.62 J / cm 2 was. Dissection was performed 30 minutes after oral administration of the test substance. After taking a back skin photograph, under anesthesia with sevoflurane (registered trademark) inhalation anesthetic solution (manufactured by Maruishi Pharmaceutical Co., Ltd.), blood was collected from the inferior vena cava to cause euthanasia, and the back skin was collected.

[0047] (2) The skin moisture content was measured using a Corneometer CM825 (manufactured by Courge+Khazaka electronic GmBH) twice a week at the time of grouping and during the test.

[0048] (3) The trans-epidermal water loss was measured using a Tewameter TM300 (manufactured by COURAGE+KHAZAKA electronic GMBH) for the trans-epidermal water loss (TEWL) at the waist of the mice on the day before dissection.

[0049] (4) The degree of wrinkle formation was determined by taking a photograph of the dorsal skin of the hairless mouse under anesthesia with Sevoflurane (registered trademark) inhalation anesthetic solution (Maruishi Pharmaceutical Co., Ltd.) using an OLYMPUS CAMEDIA DIGITAL CAMERA C-3040 ZOOM before dissection. Visual scoring of wrinkles was based on the visual judgment criteria (Inomata et al., J Invest Dermatol. 2003 Jan;120(1):128 - 34.). This image was randomly shown to 7 non - participating test subjects who were not informed of the grouping, and the wrinkles were evaluated on a 5 - point scale of 0, 2, 4, 6, and 8.

[0050] (5) Skin tissue analysis Under anesthesia with Sevoflurane (registered trademark) inhalation anesthetic solution (Maruishi Pharmaceutical Co., Ltd.), after cardiac blood sampling, skin tissue was collected. A biopsy trephine (8 mm diameter, manufactured by Kai Industries Co., Ltd.) was used to make a hole at one point on the upper part of the dorsal midline, and skin for pathological analysis was collected. It was attached to filter paper (filter paper for Kiriyama funnel, No. 4, 21φm / m, manufactured by Kiriyama Seisakusho) with the dermal side facing down, and immediately fixed by placing it in a bottle filled with a fixing solution (Tissue - Tek Yufix, manufactured by Kagoshima Chemical Co., Ltd.). Subsequently, the skin of the entire back was collected, placed on a cutter board with the stratum corneum side down, and the subcutaneous tissue was removed using the non - blade side of a scalpel (Disposables No. 12, manufactured by AS ONE Corporation). The skin tissue was immediately frozen with liquid nitrogen and stored at - 80°C until analysis. The skin collected and fixed during dissection was paraffin - embedded, sectioned, and thinly sliced according to the conventional method, and subjected to hematoxylin & eosin (HE) staining and immunohistochemical staining to prepare respective tissue specimens.

[0051] <HE staining> Deparaffinization was performed, followed by washing with water. Then, it was stained with Carazzi's hematoxylin solution for 5 minutes. Next, it was gently differentiated with 70% hydrochloric acid alcohol solution. After washing with tap water, it was immersed in warm water for 10 minutes, immersed in eosin staining solution for 5 minutes, dehydrated in several tanks of 100% alcohol, cleared with xylene, and mounted. The entire fields of these specimens were observed, and photographs of representative sites were taken using an OLYMPUS CAMEDIA DIGITAL CAMERA C-3040 ZOOM. Using HE-stained specimens, the distance from the basal layer to the upper granular layer was regarded as the epidermis and measured with the image processing software Image J. Three fields were photographed per individual, and the epidermal thickness was measured at 10 locations for each photograph. The average value of the three photographs was calculated and taken as the epidermal thickness of the individual. <Immunohistochemical staining> For the observation of laminin, a component of the basement membrane of the skin, and filaggrin, a component of the epidermis, immunostaining using the enzyme antibody method was performed. Sections with a thickness of 3 μm were prepared from paraffin blocks. After deparaffinization, antigen activation treatment was performed by digestion with Proteinase K (manufactured by Sigma). Subsequently, endogenous peroxidase inhibition treatment with 3% hydrogen peroxide / methanol and blocking treatment with 1% goat serum were performed. After that, primary antibodies diluted with 1% goat serum (rabbit-derived anti-laminin polyclonal antibody, ab11575, derived from Engelbreth-Holm-Swarm sarcoma, manufactured by Abcam, diluted 100-fold; rabbit-derived anti-filaggrin polyclonal antibody, GTX37695, manufactured by GeneTex, diluted 200-fold) were used and reacted at room temperature for 1 hour. After washing with PBS(-), 4 drops of an HRP-labeled secondary antibody (goat-derived anti-rabbit IgG antibody; Simple stain MAX PO, manufactured by Nichirei Biosciences) were added and reacted at room temperature for 30 minutes. After washing, a chromogenic substrate (ImmPact DAB, manufactured by Bector) was reacted for staining. Counterstaining was performed with Mayer's hematoxylin (manufactured by Mutoh Chemical), and after washing with water, dehydration was carried out in several baths with 100% alcohol, clearing was performed with xylene, and mounting was performed with Marinol.

[0052] 2. Results (1) Skin moisture content: (Figure 5) In the UV(+) long-necked green onion leaf dry powder group (blue powder group) and the UV(+) long-necked green onion leaf water extract group (blue water group) compared with the UV(+) control group, a significant increase in skin moisture content was observed after the 3rd week of the test. No significant difference was found between the UV(+) blue powder group and the UV(+) blue water group. (2) Transepidermal water loss (TEWL): (Figure 6) UV exposure slightly increased the TEWL in the UV(+) control group. In the UV(+)-dried green onion leaf powder group (blue powder group), the TEWL decreased, indicating an improvement in skin barrier function. (3) Wrinkling degree: (Figure 7) Although no statistically significant difference was observed in the UV(+)-aqueous extract group of green onion leaf (blue aqueous group), the wrinkles decreased. Also, a slight decrease in wrinkles was observed in the UV(+)-dried green onion leaf powder group (blue powder group). (4) Skin tissue observation: (Figure 8) The stratification of epithelial cells on the basement membrane observed due to UV exposure was suppressed in the UV(+)-dried green onion leaf powder group (blue powder group) and the UV(+)-aqueous extract group of green onion leaf (blue aqueous group). The epidermal thickening was also suppressed.

[0053] Test Example 3. Identification of Active Ingredients (1) Fractionation by Ultrafiltration The aqueous extract of green onion leaf prepared in Production Example 1 was dissolved in Milli-Q water to a concentration of 3 mg / mL. 500 μL of the extract solution was centrifuged at 14,000×g for 45 minutes using a centrifugal ultrafiltration filter (Amicon® Ultra (3K)) capable of fractionating at a molecular weight of 3,000 kDa, and fractionated into a supernatant that did not pass through the membrane and a permeate that passed through the membrane. When the promotion effect of hyaluronic acid production was confirmed for each in the same manner as in Test Example 1, the promotion activity of hyaluronic acid production was observed in the supernatant that did not pass through the membrane.

[0054] (2) Separation and Detection of Alliin (HPLC Analysis) The aqueous extract of green onion leaf prepared in Production Example 1 was dissolved in Milli-Q water to a concentration of 3 mg / mL. It was pulverized with a homogenizer for 2 minutes and allowed to stand at room temperature for 5 minutes. It was filtered using a 0.45 μm cellulose acetate membrane filter to obtain a sample for HPLC analysis. HPLC analysis was performed using a high-performance liquid chromatograph Chromaster (manufactured by Hitachi High-Tech Corporation). The analysis conditions are as follows. · Column: TSKgel ODS-100V(5) (manufactured by Tosoh Corporation) · Liquid delivery: Flow rate 0.4 mL / min · Mobile phase: 40% methanol · Column temperature: 40 °C · Detection wavelength: 220 nm · Injection volume: 5 μL

[0055] (3) Identification of active ingredient (LC / MS analysis) The supernatant and permeate that did not permeate through the membrane obtained in 1) were each filtered using a 0.20 μm cellulose acetate membrane filter and used as samples for LC / MS analysis. LC / MS analysis was performed using LTQ Orbitrap XL (Thermo Fisher Scientific, MA, USA), a high-resolution and high-mass accuracy LC / MS equipped with an ion source by electrospray ionization (ESI). The separation conditions in the liquid chromatograph are as follows. · Column: CAPCELL CORE C18 (particle size 2.7 μm, inner diameter 2.1 mm, length 75 mm) (manufactured by Osaka Soda Co., Ltd.) · Liquid delivery: Flow rate 0.15 mL / min · Mobile phase: 40% methanol · Injection volume: 1 μL

[0056] (4) Results By subjecting the water extract of the green leaves of Welsh onion to HPLC, among the substances detected at elution times of 5.85 and 7.13, it was confirmed that the peak at 7.13 coincided with the time at which alliine could be detected (Figure 9). Since the polymer of glucose was the main component from the previous analysis of sugars, centrifugal filtration using a filtration membrane with a molecular weight of 3000 was also carried out for the purpose of separating alliine and subjected to HPLC in the same manner. As a result, a substance with an elution time of 5.85 was detected in the membrane permeate, and alliine with an elution time of 7.11 was detected in the supernatant that did not permeate through the membrane (Figure 9). The reason why alliine with a molecular weight of 177 could not be filtered through a membrane with a molecular weight of 3000 is thought to be due to some interaction with sugars. However, centrifugation using a membrane with a molecular weight of 3000 enabled the concentration of alliine that did not permeate. Therefore, the reference substance was alliine or isoalliine, and LC / MS analysis of the water extract of the green leaf part of Welsh onion was performed (Figure 10). As a result, substances with molecular weights of 178 and 219 were detected. Since it has been reported that the molecular weights of alliine and isoalliine are equal and cannot be separated, and both components are contained in the green leaf part of Welsh onion, the peak of 178 was identified as alliine or isoalliine. Also, the peak of 219 was glucose.

[0057] Test Example 3 Promoting effect of sulfur-containing amino acids on hyaluronic acid production 1. Method (1) Cell culture and addition of samples The cells used were normal human dermal fibroblasts (HFB). For the culture medium for HFB growth, DMEM (low glucose) medium containing 10% FBS was used. For the medium for sample addition, DMEM (low glucose) medium without FBS was used. Alliine, isoalliine, and cycloalliine (FUJIFILM Wako Pure Chemical Corporation) were dissolved in PBS to a concentration of 10 mM and filtered using a 0.20 μm cellulose acetate membrane filter. This sample solution was mixed with the medium for sample addition to concentrations of 1, 10, 50, and 100 μM. Only the medium for sample addition was used as the control. 1.6×10 4 cells / well were seeded in a 48-well plate. After 48 hours, the growth medium was removed and the cells were washed once with PBS to remove serum components. Immediately thereafter, 0.4 mL / well of the medium for sample addition containing the sample was added, and the cells were cultured at 5% CO2 and 37 °C for 48 hours. After the culture was completed, the supernatant was collected and stored at -20 °C until use.

[0058] (2) Analysis of the amount of hyaluronic acid contained in the cell supernatant using the ELISA method To a 96-well ELISA plate (manufactured by IWAKI) for ELISA, 100 μL / well of Hyalurnic Acid Binding Protein (HABP, manufactured by Funakoshi Co., Ltd.) diluted 1000-fold with 1×PBS was added and left standing overnight at 4°C. The next day, after washing once with PBS-Tween (0.1% Tween20) (manufactured by Wako Pure Chemical Industries, Ltd.), 200 μL / well of a Block Ace (registered trademark) (manufactured by Snow Brand Milk Products Co., Ltd.) solution diluted to 1% with 1×PBS was added and reacted at 37°C for 2 hours and 30 minutes. After washing once with PBS-Tween, 50 μL / well of an HA standard coating solution (HANa 1000 μg / mL (manufactured by Wako Pure Chemical Industries, Ltd.) diluted to 2000 ng / mL with a 0.5% Block Ace (registered trademark) solution) was added and left standing at 37°C for 1 hour. After washing three times with PBS-Tween, it was diluted 50,000-fold with a 0.5% Block Ace (registered trademark) solution. After adding 50 μL / well of the cell supernatant collected in (1) and the standard HA solution (0.5% Block Ace (registered trademark) solution containing 1000, 250, 62.5, 15.625, 3.90625, 0 ng / mL of HA), 50 μL / well of a biotin-conjugated HABP solution (manufactured by Funakoshi Co., Ltd.) diluted to 0.25 mg / mL with a 0.5% Block Ace (registered trademark) solution was added from above and reacted at room temperature for 1 hour. After washing three times with PBS-Tween, 50 μL / well of an HRP-conjugated streptavidin solution (manufactured by Thermo scientific) diluted to 0.05 μg / mL with a 0.5% Block Ace (registered trademark) solution was added and reacted at 37°C for 30 minutes. After washing five times with PBS-Tween, 50 μL / well of TMB reagent (manufactured by Nacalai Tesque, Inc.) was added, covered with aluminum foil to shield from light, and left standing at room temperature for 30 minutes to develop color. 50 μL / well of 1N H2SO4 solution was added to stop the reaction. Immediately afterwards, the absorbance at ABS450nm / 630nm was measured using an infinite M200. All measurements were performed in duplicate. The calculation method was to create a logarithmic curve from the absorbance of the standard and determine the concentration of the sample. For the diluted sample, the dilution factor was multiplied to obtain the original value.

[0059] 2. Results As shown in Fig. 11, the production amount of hyaluronic acid increased by adding alliin, isoalliin or cycloalliin to HFB.

Claims

1. A hyaluronic acid production promoter containing Welsh onion or its water extract as an active ingredient.

2. An agent for preventing or improving skin aging containing Welsh onion or its water extract as an active ingredient.

3. An agent for preventing or improving joint diseases containing Welsh onion or its water extract as an active ingredient.

4. A food for promoting hyaluronic acid production containing the water extract of Welsh onion as an active ingredient.

5. A food for preventing or improving skin aging containing the water extract of Welsh onion as an active ingredient.

6. A food for preventing or improving joint diseases containing the water extract of Welsh onion as an active ingredient.

7. The agent according to any one of Claims 1 to 3, or the food according to any one of Claims 4 to 6, wherein the Welsh onion is the green leaf part of Welsh onion.

8. A hyaluronic acid production promoter containing at least one selected from alliin, isoalliin, and cycloalliin as an active ingredient.

9. An agent for preventing or improving skin aging containing at least one selected from alliin, isoalliin, and cycloalliin as an active ingredient.

10. An agent for preventing or improving joint diseases containing at least one selected from alliin, isoalliin, and cycloalliin as an active ingredient.

11. A food for promoting hyaluronic acid production containing at least one selected from alliin, isoalliin, and cycloalliin as an active ingredient.

12. A food for preventing or improving skin aging containing at least one selected from alliin, isoalliin, and cycloalliin as an active ingredient.

13. A food for preventing or improving joint diseases containing at least one selected from alliin, isoalliin, and cycloalliin as an active ingredient.

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