JP6861134B2 - Hyaluronic acid production promoter and hyaluronic acid production promoter food - Google Patents
Hyaluronic acid production promoter and hyaluronic acid production promoter food Download PDFInfo
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Description
本発明はヒアルロン酸の産生を促進するヒアルロン酸産生促進剤に関する。 The present invention relates to a hyaluronic acid production promoter that promotes the production of hyaluronic acid.
ヒアルロン酸は、Ｄ−Ｎ−アセチルグルコサミンとＤ−グルクロン酸が交互に結合して形成された直鎖状の高分子多糖であり、皮膚、軟骨、関節液、臍帯、眼硝子体、その他の結合組織に存在する。このうち、最も多くヒアルロン酸が含まれているのは皮膚である。
皮膚の真皮及び表皮は、表皮細胞、線維芽細胞、及び真皮細胞外マトリックス等によって構成されているが、ヒアルロン酸は、コラーゲン、エラスチン等と共に真皮細胞外マトリックスを構成し、表皮細胞や線維芽細胞等の細胞の外にあって、細胞の保護、組織水分の保持、柔軟性の維持、潤滑性の保持等において重要な役割を担っている。
Hyaluronic acid is a linear high-molecular-weight polysaccharide formed by the alternating combination of DN-acetylglucosamine and D-glucuronic acid, and is a combination of skin, cartilage, articular fluid, umbilical cord, ocular vitreous body, and other bonds. Present in the organization. Of these, the skin contains the largest amount of hyaluronic acid.
The dermis and epidermis of the skin are composed of epidermal cells, fibroblasts, dermal extracellular matrix and the like, but hyaluronic acid constitutes the dermis extracellular matrix together with collagen, elastin and the like, and epidermal cells and fibroblasts. It plays an important role in protecting cells, retaining tissue water, maintaining flexibility, maintaining lubricity, and the like.
皮膚のヒアルロン酸の産生量は、加齢とともに減少することが知られており、その結果、細胞の保湿力を低下させて乾燥、肌荒れ、シミ、皺等の症状を引き起こす原因となる。また、近年、紫外線等の外的要因も、ヒアルロン酸量の減少を誘導する因子とされており、乾燥肌や肌荒れの要因となると考えられる。したがって、ヒアルロン酸産生の促進は、皮膚の保湿機能を維持又は改善する上で重要である。 It is known that the amount of hyaluronic acid produced in the skin decreases with aging, and as a result, the moisturizing power of cells is reduced, causing symptoms such as dryness, rough skin, age spots, and wrinkles. Further, in recent years, external factors such as ultraviolet rays are also considered to be factors that induce a decrease in the amount of hyaluronic acid, and are considered to be factors of dry skin and rough skin. Therefore, promotion of hyaluronic acid production is important for maintaining or improving the moisturizing function of the skin.
そこで、ヒアルロン酸産生を促進する素材を天然物から探索する試みがなされ、植物の抽出物（特許文献１、特許文献２）やその成分（特許文献３）の他、種々の物質にヒアルロン酸産生促進作用があることがこれまでに報告されている。 Therefore, an attempt was made to search for a material that promotes hyaluronic acid production from natural products, and hyaluronic acid production was produced in various substances in addition to plant extracts (Patent Document 1 and Patent Document 2) and their components (Patent Document 3). It has been reported so far that it has a promoting effect.
一方、ブロッコリー等のアブラナ科の植物には、スルフォラファンの前駆体グルコシノレートであるグルコラファニンが多く含まれ、当該植物の組織中に含まれるグルコラファニンは、組織が破壊されると内在性の酵素ミロシナーゼにより加水分解され、スルフォラファンを生じることが知られている（非特許文献１）。また、ヒトがグルコラファニンを摂取した場合においても、腸内細菌の働きによって、スルフォラファンに変換されて、腸管から吸収されると考えられている。 On the other hand, cruciferous plants such as broccoli contain a large amount of glucosinolate, which is a precursor of sulforaphane, and glucoraphanin contained in the tissue of the plant is endogenous when the tissue is destroyed. It is known that sulforaphane is produced by being hydrolyzed by the enzyme myrosinase (Non-Patent Document 1). It is also believed that when humans ingest glucoraphanin, it is converted to sulforaphane by the action of intestinal bacteria and absorbed from the intestinal tract.
斯かるスルフォラファンやその前駆体グルコシノレートであるグルコラファニンには、体内の代謝過程で生じる発ガン物質の活性化を打消す第二相解毒酵素を強く誘導する作用があること、ガン治療に対して有効であること（特許文献４）等が報告されている。
しかしながら、グルコラファニンやスルフォラファンにヒアルロン酸産生促進作用や皮膚の保湿機能を改善する作用があることはこれまでに知られていない。
Such sulforaphane and its precursor glucosinolate, glucosinolate, have the effect of strongly inducing a phase II detoxifying enzyme that counteracts the activation of carcinogens that occur in the metabolic process in the body. It has been reported that it is effective against this (Patent Document 4).
However, it has not been known so far that glucoraphanin and sulforaphane have an action of promoting hyaluronic acid production and an action of improving the moisturizing function of the skin.
本発明の課題は、安全性が高く、長期に適用可能なヒアルロン酸産生促進剤を提供することにある。 An object of the present invention is to provide a hyaluronic acid production promoter that is highly safe and can be applied for a long period of time.
本発明者は、多くの天然物の中からヒアルロン酸産生促進剤を見出すべく検討した結果、グルコラファニン及びスルフォラファンが線維芽細胞におけるヒアルロン酸産生を促進する作用があることを見出し、本発明を完成した。 As a result of studying to find a hyaluronic acid production promoter among many natural products, the present inventor has found that glucorafanine and sulforaphane have an action of promoting hyaluronic acid production in fibroblasts, and the present invention has been developed. completed.
すなわち、本発明は、以下の１）〜４）に係るものである。
１）グルコラファニン及び／又はスルフォラファン、又はこれを含有する植物若しくはその加工品を有効成分とするヒアルロン酸産生促進剤。
２）グルコラファニン及び／又はスルフォラファン、又はこれを含有する植物若しくはその加工品を有効成分とするヒアルロン酸産生促進用食品。
３）グルコラファニン及び／又はスルフォラファン、又はこれを含有する植物若しくはその加工品を有効成分とする皮膚保湿機能改善剤。
４）グルコラファニン及び／又はスルフォラファン、又はこれを含有する植物若しくはその加工品を有効成分とする皮膚保湿機能改善用食品。
That is, the present invention relates to the following 1) to 4).
1) A hyaluronic acid production promoter containing glucoraphanin and / or sulforaphane, a plant containing the sulforaphane, or a processed product thereof as an active ingredient.
2) A food for promoting hyaluronic acid production containing glucoraphanin and / or sulforaphane, a plant containing the same, or a processed product thereof as an active ingredient.
3) A skin moisturizing function improving agent containing glucoraphanin and / or sulforaphane, a plant containing the same, or a processed product thereof as an active ingredient.
4) A food for improving skin moisturizing function containing glucoraphanin and / or sulforaphane, a plant containing the same, or a processed product thereof as an active ingredient.
グルコラファニン及びスルフォラファンは、ブロッコリーやケールから容易に取得可能であり、長期投与又は摂取可能である。したがって、本発明によれば、安全性の高いヒアルロン酸産生促進剤が提供され、ヒアルロン酸の減少に起因する皮膚保湿機能の低下、それに伴う乾燥肌、肌荒れの予防又は改善効果が奏される。 Glucoraphanin and sulforaphane can be easily obtained from broccoli and kale, and can be administered or ingested for a long period of time. Therefore, according to the present invention, a highly safe hyaluronic acid production promoter is provided, and the effect of preventing or improving the skin moisturizing function due to the decrease of hyaluronic acid and the accompanying dry skin and rough skin is exhibited.
本発明のグルコラファニン（Ｇｌｕｃｏｒａｐｈａｎｉｎ）は、別名：１−チオ−β−Ｄ−グルコピラノース１−［Ｎ−（ヒドロキシスルホニルオキシ）−５−（メチルスルフィニル）ペンタンイミダート］であり、スルフォラファン（Ｓｕｌｆｏｒａｐｈａｎｅ）は４−（メチルスルフィニル）ブチルイソチオシアナートである。
前述したとおり、グルコラファニンは、スルフォラファンの前駆体グルコシノレートであり、アブラナ科植物中で酵素ミロシナーゼにより加水分解されてスルフォラファンを生じる。また、ヒトがグルコラファニンを摂取した場合においても、スルフォラファンに変換されて、腸管から吸収される。
The glucoraphanin of the present invention is also known as 1-thio-β-D-glucopyranose 1- [N- (hydroxysulfonyloxy) -5- (methylsulfinyl) pentane imidate] and sulforaphane. ) Is 4- (methylsulfinyl) butyl isothiocyanate.
As mentioned above, glucoraphanin is a precursor glucosinolate of sulforaphane and is hydrolyzed by the enzyme myrosinase in cruciferous plants to produce sulforaphane. Also, when humans ingest glucoraphanin, it is converted to sulforaphane and absorbed from the intestinal tract.
グルコラファニン及びスルフォラファンは、グルコラファニン及び／又はスルフォラファンを含有する植物から公知の方法で単離精製することにより取得することができる。 Glucoraphanin and sulforaphane can be obtained by isolation and purification from plants containing glucoraphanin and / or sulforaphane by a known method.
グルコラファニン及び／又はスルフォラファンを含有する植物としては、例えばアブラナ科（Ｂｒａｓｓｉｃａｃｅａｅ）植物、好ましくはアブラナ科アブラナ属（Ｂｒａｓｓｉｃａ）の植物が例示される。具体的には、ケール、ブロッコリー、カリフラワー、キャベツ等が挙げられる。これらの植物においては、グルコラファニン又はスルフォラファンの含有量の点で、ケールやキャベツではその葉や茎、ブロッコリーやカリフラワーではその花蕾を使用することが望ましい。ケールの品種は、ハイパール（農林水産省品種登録第２０５５５号）、ハイクロップ、エステ、エキストラ・カールド・スコッチ等が利用できる。また、グルコラファニン又はスルフォラファンの含有量を高めたケールに属する新品種を使用することもできる。さらに、グルコラファニン又はスルフォラファンの含有量を指標とすることで、ヒアルロン酸産生促進作用が高いケールに属する新品種の開発を行うこともできる。 Examples of plants containing glucoraphanin and / or sulforaphane include, for example, Brassicaceae plants, preferably Brassicaceae plants. Specific examples include kale, broccoli, cauliflower, cabbage and the like. In these plants, in terms of glucoraphanin or sulforaphane content, it is desirable to use their leaves and stems for kale and cabbage, and their flower buds for broccoli and cauliflower. As kale varieties, Hyperle (Ministry of Agriculture, Forestry and Fisheries Variety Registration No. 20555), High Crop, Esthetics, Extra Carled Scotch, etc. can be used. It is also possible to use new varieties belonging to kale with an increased content of glucoraphanin or sulforaphane. Furthermore, by using the content of glucoraphanin or sulforaphane as an index, it is possible to develop a new variety belonging to kale having a high hyaluronic acid production promoting action.
また、単離精製されたグルコラファニン及びスルフォラファンは市販されており、本発明においては、当該市販品を使用することもできる。 In addition, isolated and purified glucoraphanin and sulforaphane are commercially available, and the commercially available products can also be used in the present invention.
本発明において、グルコラファニン又はスルフォラファンは、単離されたグルコラファニン、スルフォラファンのみならず、上記のグルコラファニン又はスルフォラファンを含有する植物又はその加工品（例えば、該植物の粉砕物、該植物の凍結乾燥物、該植物の搾汁、該植物の抽出物等）を使用することができる。例えば、ケール搾汁物やその乾燥物等が使用可能である。 In the present invention, the glucoraphanin or sulforaphane is not only the isolated glucoraphanin and sulforaphane, but also a plant containing the above-mentioned glucoraphanin or sulforaphane or a processed product thereof (for example, a pulverized product of the plant, the plant). Sulforaphane, squeezed plant juice, extract of the plant, etc.) can be used. For example, kale soup and dried products thereof can be used.
グルコラファニン及び／又はスルフォラファンを含有する植物の加工品、すなわちその粉砕物、凍結乾燥物、及び搾汁は、当業者によって通常用いられる処理方法で、上記植物を処理することにより得ることができる。また、抽出物は、上記の植物から当業者によって通常用いられる抽出処理方法によって得ることができる。例えば、当該植物を粉砕し、水又は含水溶媒（水−アルコール混合溶液等）で含浸又は抽出する方法が挙げられる。 Processed products of plants containing glucoraphanin and / or sulforaphane, ie, crushed products, lyophilized products, and juices thereof, can be obtained by treating the above plants by a treatment method usually used by those skilled in the art. .. In addition, the extract can be obtained from the above plants by an extraction treatment method usually used by those skilled in the art. For example, a method of crushing the plant and impregnating or extracting it with water or a water-containing solvent (water-alcohol mixed solution, etc.) can be mentioned.
斯くして得られた植物の加工品は、必要に応じて、合成吸着剤による処理、ろ過処理、濃縮処理等の分離・精製工程に供することにより、グルコラファニン及び／又はスルフォラファンの含有割合を高めてもよい。 The processed plant product thus obtained is subjected to a separation / purification step such as treatment with a synthetic adsorbent, filtration treatment, concentration treatment, etc., if necessary, so that the content ratio of glucorafanine and / or sulforaphane can be adjusted. You may increase it.
本発明のグルコラファニン及びスルフォラファンは、後記実施例に示すように、正常ヒト皮膚線維芽細胞におけるヒアルロン酸産生を促進する。したがって、グルコラファニン及び／又はスルフォラファン、又はこれを含有する植物若しくはその加工品は、ヒアルロン酸産生促進剤となり、ヒアルロン酸産生促進のために使用できる。
本発明において、ヒアルロン酸の産生促進とは、ヒト皮膚真皮の繊維芽細胞のヒアルロン酸の産生能を促進または改善し、これにより、ヒト皮膚においてヒアルロン酸量が増大し得ることを意味する。皮膚のヒアルロン酸量を増加させることによって、皮膚保湿機能が維持・改善され、乾燥肌、肌荒れの予防又は改善に効果を奏する。したがって、グルコラファニン及び／又はスルフォラファン、又はこれを含有する植物若しくはその加工品は、皮膚保湿機能改善剤にもなり得る。
The glucoraphanin and sulforaphane of the present invention promote hyaluronic acid production in normal human skin fibroblasts, as shown in Examples below. Therefore, glucoraphanin and / or sulforaphane, or a plant containing the same or a processed product thereof, becomes a hyaluronic acid production promoter and can be used for promoting hyaluronic acid production.
In the present invention, promoting the production of hyaluronic acid means promoting or improving the ability of fibroblasts of human skin dermis to produce hyaluronic acid, thereby increasing the amount of hyaluronic acid in human skin. By increasing the amount of hyaluronic acid in the skin, the skin moisturizing function is maintained and improved, and it is effective in preventing or improving dry skin and rough skin. Therefore, glucoraphanin and / or sulforaphane, or a plant containing it or a processed product thereof, can also be a skin moisturizing function improving agent.
上記ヒアルロン酸産生促進剤は、ヒアルロン酸産生を促進するため、皮膚保湿機能を改善するための医薬品、医薬部外品、化粧品、サプリメント又は食品（ヒアルロン酸産生促進用食品、皮膚保湿機能改善用食品）となり、或いはこれらへ配合するための素材又は製剤となり得る。また、本発明のグルコラファニン及びスルフォラファンは何れも経口摂取した場合に体内に吸収されることから、細胞評価試験の結果と同じ効果を奏すると考えられる。
尚、上記食品には、一般飲食品のほか、ヒアルロン酸産生、皮膚保湿機能改善をコンセプトとし、必要に応じてその旨を表示した食品、機能性表示食品、特定保健用食品等が包含される。
Since the above hyaluronic acid production promoter promotes hyaluronic acid production, pharmaceuticals, non-pharmaceutical products, cosmetics, supplements or foods for improving skin moisturizing function (hyaluronic acid production promoting food, skin moisturizing function improving food) ), Or it can be a material or formulation to be incorporated into these. In addition, since both glucoraphanin and sulforaphane of the present invention are absorbed into the body when ingested orally, it is considered that they have the same effect as the result of the cell evaluation test.
In addition to general foods and drinks, the above foods include foods with the concept of hyaluronic acid production and improvement of skin moisturizing function, and foods labeled to that effect, foods with functional claims, foods for specified health use, etc. as necessary. ..
本発明のグルコラファニン及びスルフォラファンを含む上記医薬品（医薬部外品を含む）は、任意の投与形態で投与され得る。投与に際しては、有効成分を経口、直腸内、注射、外用等の投与方法に適した固体又は液体の医薬用無毒性担体と混合して、慣用の医薬品製剤の形態で投与することができる。 The above-mentioned pharmaceutical products (including quasi-drugs) containing glucoraphanin and sulforaphane of the present invention can be administered in any dosage form. Upon administration, the active ingredient can be mixed with a solid or liquid NOAEL suitable for administration methods such as oral, rectal, injection, and external use, and administered in the form of a conventional pharmaceutical preparation.
例えば、経口投与製剤としては、錠剤、顆粒剤、散剤、カプセル剤等の固形剤、溶液剤、懸濁剤、乳剤等の液剤、凍結乾燥剤等が挙げられる。これらの製剤は製剤上の常套手段により調製することができる。上記の医薬用無毒性担体としては、例えば、グルコース、乳糖、ショ糖、澱粉、マンニトール、デキストリン、脂肪酸グリセリド、ポリエチレングリコール、ヒドロキシエチルデンプン、エチレングリコール、ポリオキシエチレンソルビタン脂肪酸エステル、アミノ酸、ゼラチン、アルブミン、水、生理食塩水等が挙げられる。また、必要に応じて、安定化剤、湿潤剤、乳化剤、結合剤、等張化剤、賦形剤等の慣用の添加剤を適宜添加することもできる。 For example, examples of the orally administered preparation include solid preparations such as tablets, granules, powders and capsules, solutions, suspensions, liquid preparations such as emulsions, lyophilizers and the like. These formulations can be prepared by conventional means of formulation. Examples of the above-mentioned pharmaceutical non-toxic carrier include glucose, lactose, sucrose, starch, mannitol, dextrin, fatty acid glyceride, polyethylene glycol, hydroxyethyl starch, ethylene glycol, polyoxyethylene sorbitan fatty acid ester, amino acid, gelatin and albumin. , Water, physiological saline and the like. Further, if necessary, conventional additives such as stabilizers, wetting agents, emulsifiers, binders, isotonic agents, and excipients can be appropriately added.
また、外用剤（化粧品、医薬品および医薬部外品を含む。）の形態としては、軟膏剤、クリーム剤、外用液剤の他、各種化粧品の形態（例えば、化粧水、乳液、各種クリーム、パック、美容液等の基礎化粧料、シャンプー、リンス、トリートメント、ヘアトニック、ヘアリキッド、ヘアクリーム、ヘアミルク等の頭髪用製品、入浴剤等の浴用化粧品、ファンデーション、口紅、マスカラ、アイシャドウ等のメーキャップ化粧品、日焼け止め等）が挙げられる。 The forms of external preparations (including cosmetics, pharmaceuticals, and quasi-drugs) include ointments, creams, and external liquids, as well as various cosmetic forms (for example, cosmetics, milky lotions, various creams, packs, etc.). Basic cosmetics such as beauty liquids, shampoos, rinses, treatments, hair tonics, hair liquids, hair creams, hair milk and other hair products, bath cosmetics such as bathing agents, foundations, lipsticks, mascara, eye shadows and other makeup cosmetics, (Sunscreen, etc.).
また、上記外用剤には、必須成分であるグルコラファニン及び／又はスルフォラファン、又はこれを含有する植物若しくはその加工品の他に、通常、外用剤に配合される任意成分を配合することができる。このような任意成分としては、界面活性剤、油分、アルコール類、保湿剤、増粘剤、防腐剤、酸化防止剤、キレート剤、ｐＨ調整剤、香料、色素、紫外線吸収・散乱剤、粉体、ビタミン類、アミノ酸類、水溶性高分子、発泡剤、顔料、植物抽出物、動物由来成分、海藻抽出物、各種薬剤、添加剤、水等を挙げることができる。 Further, in addition to the essential components glucorafanine and / or sulforaphane, or a plant containing the same or a processed product thereof, the external preparation may contain any component usually contained in the external preparation. .. Such optional components include surfactants, oils, alcohols, moisturizers, thickeners, preservatives, antioxidants, chelating agents, pH regulators, fragrances, pigments, ultraviolet absorbers / scattering agents, and powders. , Vitamins, amino acids, water-soluble polymers, foaming agents, pigments, plant extracts, animal-derived components, seaweed extracts, various chemicals, additives, water and the like.
また、グルコラファニン及び／又はスルフォラファン、又はこれを含有する植物若しくはその加工品を配合した上記食品の形態は、各種食品組成物の他、上述した経口投与製剤と同様の形態（錠剤、カプセル剤、シロップ等）が挙げられる。
種々の形態の食品を調製するには、本発明のグルコラファニン及び／又はスルフォラファン、又はこれを含有する植物若しくはその加工品を単独で、又は他の食品材料や、溶剤、軟化剤、油、乳化剤、防腐剤、香科、安定剤、着色剤、酸化防止剤、保湿剤、増粘剤等を適宜組み合わせて用いることができる。
In addition, the form of the above-mentioned food containing glucoraphanin and / or sulforaphane, or a plant containing the same or a processed product thereof is the same as the above-mentioned orally-administered preparation (tablets, capsules) in addition to various food compositions. , Syrup, etc.).
To prepare various forms of foods, the glucoraphanin and / or sulforaphane of the present invention, or plants containing them or processed products thereof alone, or other food materials, solvents, softeners, oils, etc. Emulsifiers, preservatives, fragrances, stabilizers, colorants, antioxidants, moisturizers, thickeners and the like can be used in appropriate combinations.
上記の医薬品（医薬部外品を含む）、化粧品及び食品中のグルコラファニン及び／又はスルフォラファン、又はこれを含有する植物若しくはその加工品の含有量は、その使用形態により異なるが、通常、グルコラファニン及び／又はスルフォラファンとして、製剤全質量の０．０１〜１０質量％、好ましくは０．１〜１質量％ある。 The content of glucoraphanin and / or sulforaphane in the above-mentioned pharmaceutical products (including quasi-drugs), cosmetics and foods, or plants containing them or processed products thereof varies depending on the usage pattern, but is usually gluco. As raphanin and / or sulforaphane, it is 0.01 to 10% by mass, preferably 0.1 to 1% by mass, based on the total mass of the preparation.
本発明のグルコラファニン及び／又はスルフォラファン、又はこれを含有する植物若しくはその加工品を医薬品、化粧品又は食品として、或いは医薬品や食品に配合して使用する場合の投与量は、患者の状態、体重、性別、年齢又はその他の要因に従って変動し得るが、経口投与の場合の成人１人当たりの１日の投与量は、通常、グルコラファニン及び／又はスルフォラファンとして、好ましくは０．１ｍｇ〜１００ｇ、より好ましくは１ｍｇ〜１０ｇである。
また、上記製剤は、任意の投与計画に従って投与され得るが、１日１回〜数回に分け、数週間〜数ヶ月間継続して投与することが好ましい。
When the glucoraphanin and / or sulforaphane of the present invention, or a plant containing the same, or a processed product thereof is used as a drug, cosmetics or food, or in combination with a drug or food, the dose is determined by the patient's condition and body weight. The daily dose per adult for oral administration is usually 0.1 mg to 100 g, preferably 0.1 mg to 100 g, as glucoraphanin and / or sulforaphane, although it may vary depending on gender, age or other factors. It is preferably 1 mg to 10 g.
The preparation may be administered according to an arbitrary administration plan, but it is preferably administered once to several times a day and continuously for several weeks to several months.
実施例１．ヒアルロン酸産生促進作用の評価
（１）試験品
・グルコラファニン：Ｇｌｕｃｏｒａｐｈａｎｉｎ、 Ｃａｙｍａｎ Ｃｈeｍｉｃａｌ社
・スルフォラファン：Ｒ，Ｓ−Ｓｕｌｆｏｒａｐｈａｎｅ、ＬＫＴ Ｌａｂｏｒａｔｏｒｉｅｓ社
Example 1. Evaluation of hyaluronic acid production promoting action (1) Test product ・ Glucoraphanin: Glucoraphanin, Cayman Chemical Co., Ltd. ・ Sulforaphane: R, S-Sulforaphane, LKT Laboratories
（２）細胞及び培養条件
細胞株：正常ヒト皮膚線維芽細胞（ＮＨＤＦ、クラボウ）
細胞は、３７℃、５％二酸化炭素の雰囲気下にて培養を行った。培地は、Ｄｕｌｂｅｃｃｏ’ｓ Ｍｏｄｉｆｉｅｄ Ｅａｇｌｅ Ｍｅｄｉｕｍ（Ｌｉｆｅ Ｔｅｃｈｎｏｌｏｇｉｅｓ社）を使用した。
(2) Cell and culture conditions
Cell line: Normal human skin fibroblasts (NHDF, Kurabo Industries)
The cells were cultured in an atmosphere of 37 ° C. and 5% carbon dioxide. As the medium, Dulbecco's Modified Eagle Medium (Life Technologies) was used.
（３）試験品溶液の調製
エタノールにグルコラファニン、スルフォラファンを各々溶解し、以下に示す濃度の試験品溶液を調製した。
・グルコラファニン：１５．６２５、３．９０６μｇ／ｍＬ
・スルフォラファン：０．３９、０．０９７μｇ／ｍＬ
(3) Preparation of test product solution Glucoraphanin and sulforaphane were dissolved in ethanol to prepare a test product solution having the following concentrations.
Glucoraphanin: 15.625, 3.906 μg / mL
-Sulforaphane: 0.39, 0.097 μg / mL
（４）ヒアルロン酸産生試験
ＮＨＤＦを９６ｗｅｌｌ ｍｉｃｒｏ ｐｌａｔｅに１×１０４ｃｅｌｌｓ／１００μＬ／ｗｅｌｌの濃度で播種し、２４時間、前培養した。前培養後、上記で調製した各試験品溶液を各ｗｅｌｌに加え４８時間の培養を行った。培養上清を回収し、ＥＬＩＳＡ法（Ｈｙａｌｕｒｏｎａｎ Ｑｕａｎｔｉｋｉｎｅ ＥＬＩＳＡ Ｋｉｔ（Ｒ＆Ｄ Ｓｙｓｔｅｍｓ社））にてヒアルロン酸量を測定した。試験品未添加群（コントロール）のヒアルロン酸量を１００％として、各試験品添加群のヒアルロン酸量産生率を、全細胞の総タンパク質量を測定しタンパク質量当たりのヒアルロン酸産生量で求めた。試験は、３回実施しその平均値を算出した（ｎ＝３）。
(4) Hyaluronic acid production test
NHDF was seeded on a 96- well microplate at a concentration of 1 × 10 4 cells / 100 μL / well and pre-cultured for 24 hours. After the pre-culture, each test product solution prepared above was added to each well and cultured for 48 hours. The culture supernatant was collected, and the amount of hyaluronic acid was measured by an ELISA method (Hyaluronan Quantikine ELISA Kit (R & D Systems)). The amount of hyaluronic acid produced in each test product-added group was determined by measuring the total protein amount of all cells and calculating the amount of hyaluronic acid produced per protein amount, assuming that the amount of hyaluronic acid in the test product-free group (control) was 100%. .. The test was carried out three times and the average value was calculated (n = 3).
（５）結果
結果を図１及び２に示す。
グルコラファニン及びスルフォラファンには、コントロールと比較して有意なヒアルロン酸産生率の増加が認められた。
(5) Results The results are shown in FIGS. 1 and 2.
Glucoraphane and sulforaphane showed a significant increase in hyaluronic acid production rate compared to the control.
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