JP6345537B2 - Skin external preparation or internal preparation - Google Patents
Skin external preparation or internal preparation Download PDFInfo
- Publication number
- JP6345537B2 JP6345537B2 JP2014171255A JP2014171255A JP6345537B2 JP 6345537 B2 JP6345537 B2 JP 6345537B2 JP 2014171255 A JP2014171255 A JP 2014171255A JP 2014171255 A JP2014171255 A JP 2014171255A JP 6345537 B2 JP6345537 B2 JP 6345537B2
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- Prior art keywords
- production
- collagen
- skin
- preparation
- phenylpropanoid
- Prior art date
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Description
本発明は、新規な老化防止作用に優れた皮膚外用剤、しわ改善剤、コラーゲン産生促進剤、マトリックスメタロプロテアーゼ産生抑制剤及びアクチンフィラメント構築促進剤などに関する。 The present invention relates to a novel external preparation for skin, a wrinkle improving agent, a collagen production promoter, a matrix metalloproteinase production inhibitor, an actin filament construction promoter and the like, which are excellent in antiaging effects.
皮膚は、紫外線、乾燥、寒冷、熱、薬物等のさまざまな物理的及び化学的ストレスに日々さらされている。その結果、皮膚の機能低下が引き起こされ、さまざまな皮膚の老化現象が顕在化する。皮膚の老化現象の一つに、しわがある。しわには、表皮性のしわと、真皮性のしわの二種類が存在することが知られている。表皮性のしわは小じわと呼ばれ、皮膚の乾燥により、表皮角質層中の水分量が低下することによって一時的に生じるしわである。小じわの改善方法としては、保湿効果を有する化粧品の使用が一般的である。一方、真皮性のしわは、太陽光線に含まれる紫外線や加齢によって形成されるしわである。その形成メカニズムとしては、紫外線や加齢による真皮線維芽細胞におけるコラーゲン合成能の低下や、マトリックスメタロプロテアーゼ（ＭＭＰ）の増加によるコラーゲンの分解促進が挙げられる。 Skin is exposed to various physical and chemical stresses such as ultraviolet rays, dryness, coldness, heat, and drugs every day. As a result, the skin function is reduced, and various skin aging phenomena become apparent. One of the skin aging phenomena is wrinkles. It is known that there are two types of wrinkles: epidermal wrinkles and dermal wrinkles. Epidermal wrinkles are called fine wrinkles and are temporary wrinkles caused by a decrease in the amount of water in the epidermal stratum corneum due to dry skin. As a method for improving fine lines, the use of cosmetics having a moisturizing effect is common. On the other hand, dermal wrinkles are wrinkles formed by ultraviolet rays contained in sunlight or aging. The formation mechanism includes a decrease in the ability to synthesize collagen in dermal fibroblasts due to ultraviolet rays and aging, and an acceleration of collagen degradation due to an increase in matrix metalloproteinase (MMP).
乾燥に起因する表皮性のしわと真皮性のしわでは、組織学的形態、発症メカニズム、治療方法が異なり、紫外線や加齢により生じる真皮性のしわは、保湿効果を有する化粧品の使用によって改善させることはできない。したがって、紫外線や加齢により生じる真皮性のしわの改善には、真皮線維芽細胞におけるコラーゲン合成能を高める成分やＭＭＰなどのコラーゲン分解酵素の産生や活性を阻害する成分が用いられてきた。 Histological morphology, onset mechanism, and treatment method differ between epidermal wrinkles and dermal wrinkles due to dryness, and dermal wrinkles caused by ultraviolet rays and aging are improved by the use of cosmetics that have a moisturizing effect. It is not possible. Therefore, components that enhance the ability to synthesize collagen in dermal fibroblasts and components that inhibit the production and activity of collagen-degrading enzymes such as MMP have been used to improve dermal wrinkles caused by ultraviolet rays and aging.
また、コラーゲンの生成促進は、皮膚の真皮性のしわを改善するだけでなく、皮膚の創傷治癒の改善、骨粗鬆症、関節炎、腱鞘炎等の治療、高血圧の防止などにも有効である。 Further, the promotion of collagen production is effective not only in improving dermal creases in the skin, but also in improving wound healing of the skin, treating osteoporosis, arthritis, tendonitis, etc., and preventing hypertension.
ＭＭＰは、線維芽細胞や内皮細胞、ガン細胞等が産生する酵素であり、コラーゲン、ゼラチン、エラスチン（動脈、腱、皮膚など弾性組織の特殊成分をなす構造タンパク質）等の基質を分解する。従って、ＭＭＰに対して阻害活性を有する物質は、ガン組織における血管新生やガンの転移を抑制する効果が期待され、ガン疾患の予防、治療に有用であると考えられる。さらにＭＭＰはガン疾患のみならず、潰瘍形成、慢性関節リウマチ、骨粗鬆症、歯周炎等の種々の病態での細胞外基質の分解に関与していることが報告されている。よって、ＭＭＰの阻害活性を有すれば、ガンの転移、潰瘍形成、慢性関節リウマチ、骨粗鬆症、歯周炎等、ＭＭＰの亢進が原因で起こる各種疾患の治療及び改善に有用である。 MMP is an enzyme produced by fibroblasts, endothelial cells, cancer cells, and the like, and degrades substrates such as collagen, gelatin, and elastin (structural proteins that form special components of elastic tissues such as arteries, tendons, and skin). Therefore, a substance having inhibitory activity against MMP is expected to have an effect of suppressing angiogenesis and cancer metastasis in cancer tissue, and is considered useful for the prevention and treatment of cancer diseases. Furthermore, it has been reported that MMP is involved not only in cancer diseases but also in degradation of extracellular matrix in various pathologies such as ulceration, rheumatoid arthritis, osteoporosis, periodontitis and the like. Thus, having MMP inhibitory activity is useful for the treatment and improvement of various diseases caused by MMP enhancement, such as cancer metastasis, ulceration, rheumatoid arthritis, osteoporosis, and periodontitis.
一方、近年皮膚のハリには、皮膚中の線維芽細胞に存在する張力繊維（アクチンフィラメント）が関与し、アクチンフィラメントの構築による線維芽細胞の形態がコラーゲンの産生やＭＭＰの産生に関与しているメカニズムが明らかにされ、アクチンフィラメントの構築促進物質が皮膚のしわ・たるみの改善に有用であることが示唆されている。 On the other hand, in recent years, the tension of the skin has been associated with the tension fibers (actin filaments) present in the fibroblasts in the skin, and the form of the fibroblasts due to the construction of the actin filaments has been implicated in the production of collagen and MMP. It is suggested that actin filament assembly promoters are useful for improving skin wrinkles and sagging.
これまでに、紫外線によって生じる真皮性のしわを改善することを目的として、加水分解アーモンドを有効成分とする皮膚のしわ形成防止・改善剤（特許文献１）、ジョチョウケイ、テンキシ及びキセンウの抽出物を有効成分とする紫外線照射に起因するしわの改善剤（特許文献２）が報告されている。また、コラーゲンの産生促進剤としては、Ｎ−ベンゾイルグリシルグリシンからなるコラーゲン産生促進剤（特許文献３）、ＭＭＰ活性阻害剤としては、Ｃｒａｎｅｓｂｉｌｌ及びＤｏｇｗｏｏｄ Ｂａｒｋ Ａｍｅｒｉｃａｎから選ばれる一種以上の植物又はその抽出物を有効成分とするマトリックスメタロプロテアーゼ活性阻害剤（特許文献４）が報告されている。アクチンの産生促進剤として、γ−グルタミルトランスペプチダーゼを阻害する化合物を有効成分とすることを特徴とする、皮膚線維芽細胞のα−平滑筋アクチン産生促進剤（特許文献５）が報告されている。 So far, for the purpose of improving dermal wrinkles caused by ultraviolet rays, an extract of wrinkle formation preventing / improving agent (Patent Document 1), jojokei, tenki and ginseng containing hydrolyzed almonds as an active ingredient An agent for improving wrinkles caused by ultraviolet irradiation (Patent Document 2) has been reported. Further, as a collagen production promoter, a collagen production promoter composed of N-benzoylglycylglycine (Patent Document 3), and as an MMP activity inhibitor, one or more plants selected from Cranesbil and Dogwood Bark American or their extraction A matrix metalloprotease activity inhibitor (Patent Document 4) containing a product as an active ingredient has been reported. As an actin production promoter, an α-smooth muscle actin production promoter for skin fibroblasts (Patent Document 5) characterized in that it comprises a compound that inhibits γ-glutamyl transpeptidase as an active ingredient has been reported. .
一方、本発明の特定のフェニルプロパノイド配糖体に抗老化効果、しわ改善効果、コラーゲン産生促進効果、マトリックスメタロプロテアーゼ産生抑制効果、アクチンフィラメント構築促進効果などを有することは全く知られていなかった。 On the other hand, it was not known at all that the specific phenylpropanoid glycoside of the present invention had an anti-aging effect, a wrinkle improving effect, a collagen production promoting effect, a matrix metalloproteinase production inhibiting effect, an actin filament construction promoting effect, etc. .
本発明は、このような状況において為されたものであり、しわ形成の予防又は改善、新規なコラーゲン産生促進、マトリックスメタロプロテアーゼ産生抑制作用を有する成分、アクチンフィラメントの構築を促進する成分、並びに、当該成分を含有する皮膚外用剤や内用剤を提供することを課題とする。 The present invention has been made in such a situation, prevention or improvement of wrinkle formation, a novel collagen production promotion, a component having a matrix metalloprotease production inhibitory action, a component that promotes the construction of actin filaments, and It aims at providing the skin external preparation and internal preparation containing the said component.
本発明者らは、多くの化合物について前記のような美容や医薬に関する各種評価系へのスクリーニングを実施してきた。その過程において、本発明者らは鋭意検討した結果、フェニルプロパノイド配糖体が優れたコラーゲン産生促進作用、マトリックスメタロプロテアーゼ産生抑制作用及びアクチンフィラメント構築促進作用をもち、安定性においても優れていることを見出した。さらにフェニルプロパノイド配糖体を含有する皮膚外用剤が、安全で安定であり、しわ改善作用に優れ、内用剤などの医薬用途においても優れることを見出し、本発明を完成するに至った。 The present inventors have carried out screening of various compounds in various evaluation systems relating to beauty and medicine as described above. In the process, as a result of intensive studies, the present inventors have found that phenylpropanoid glycosides have excellent collagen production promoting action, matrix metalloprotease production inhibiting action and actin filament construction promoting action, and are also excellent in stability. I found out. Furthermore, the present inventors have found that a skin external preparation containing a phenylpropanoid glycoside is safe and stable, has an excellent wrinkle improving action, and is excellent in pharmaceutical use such as an internal preparation, and has completed the present invention.
また、特定の構造で表される、フェニルプロパノイド配糖体が新規物質であることを見出した。 Moreover, it discovered that the phenylpropanoid glycoside represented by a specific structure was a novel substance.
すなわち、本発明の皮膚外用剤または内用剤は下記のとおりである。 That is, the external preparation for skin or internal preparation of the present invention is as follows.
[１] 下記一般式（１）及び（３）で表される、フェニルプロパノイド配糖体を一種又は二種以上含有することを特徴とする皮膚外用剤。（（１）の式中、置換基Ｒ １ 〜Ｒ ４ は水素、Ｒ ５ はアセチル基である。（３）の式中、置換基Ｒ１〜Ｒ５は、水素又はアセチル基である。）
[３] [１]記載の一般式（１）及び（３）で表される、フェニルプロパノイド配糖体を一種又は二種以上含有することを特徴とするコラーゲン産生促進剤。
[４] [１]記載の一般式（１）及び（３）で表される、フェニルプロパノイド配糖体を一種又は二種以上含有することを特徴とするマトリックスメタロプロテアーゼ産生抑制剤。
[1] A skin external preparation characterized by containing one or more phenylpropanoid glycosides represented by the following general formulas (1) and (3). (In the formula (1), the substituents R 1 to R 4 are hydrogen and R 5 is an acetyl group. In the formula (3) , the substituents R 1 to R 5 are hydrogen or an acetyl group.)
[3] A collagen production promoter comprising one or more phenylpropanoid glycosides represented by the general formulas (1) and (3) according to [1].
[4] A matrix metalloprotease production inhibitor comprising one or more phenylpropanoid glycosides represented by the general formulas (1) and (3) according to [1].
本発明のフェニルプロパノイド配糖体は、優れたしわ改善作用、コラーゲン産生促進作用、マトリックスメタロプロテアーゼ産生抑制作用及びアクチンフィラメント構築促進作用を有しており、医薬品、医薬部外品、化粧品、食品の分野において貢献できるものである。 The phenylpropanoid glycoside of the present invention has an excellent wrinkle improving action, collagen production promoting action, matrix metalloprotease production inhibiting action and actin filament construction promoting action, and is used for pharmaceuticals, quasi drugs, cosmetics and foods. Can contribute in the field.
以下に、本発明について詳細に述べる。 The present invention will be described in detail below.
上記の一般式（１）〜（３）において、置換基Ｒ１〜Ｒ５は、水素（−Ｈ）又はアセチル基（−Ａｃ、−ＣＯＣＨ３）である。また、一般式の（１）〜（３）の各置換基による物質の中から、一種又は二種以上を組み合わせて用いてもよい。 In the above general formulas (1) to (3), the substituents R 1 to R 5 are hydrogen (—H) or an acetyl group (—Ac, —COCH 3 ). Moreover, you may use 1 type or in combination of 2 or more types from the substance by each substituent of (1)-(3) of general formula.
一般式（１）〜（３）に示される、フェニルプロパノイド配糖体の例としては、下記の物質などがあげられる。ただし、本発明は、これらの物質に限定されない。
構造（４）又は（５）で表される物質は新規物質である。 The substance represented by the structure (4) or (5) is a novel substance.
本発明に用いるフェニルプロパノイド配糖体の由来については、特に問わないが、一例として、ユリ科ユリ属に属する植物から単離することができ、例えば、ユリ科ユリ属カサブランカより抽出・単離精製できる。 The origin of the phenylpropanoid glycoside used in the present invention is not particularly limited. For example, the phenylpropanoid glycoside can be isolated from a plant belonging to the genus Lily family, for example, extracted and isolated from the lily family, Casablanca. It can be purified.
カサブランカとは、ヤマユリ、カノコユリ、タモトユリ等を原種とするオリエンタルハイブリッドとよばれる園芸品種で、広く販売されている。 Casablanca is a horticultural variety called oriental hybrid, which is based on wild lilies, lily kanoko, lily tamoto, etc., and is widely sold.
抽出部位は特に限定されず、すべての部位を用いることができる。抽出方法は特に限定されず、例えば、加熱抽出したものであっても良いし、常温抽出したものであっても良い。 The extraction site is not particularly limited, and all sites can be used. The extraction method is not particularly limited, and for example, it may be a heat extraction or a room temperature extraction.
抽出する溶媒としては、例えば、水、エタノール、メタノール等の水溶性溶媒の単独あるいは混合液などが挙げられる。 Examples of the solvent to be extracted include water, ethanol, methanol and other water-soluble solvents alone or a mixed solution.
本発明で用いるフェニルプロパノイド配糖体は、ダイヤイオンＨＰ−２０、ＯＤＳ等によるカラムクロマトグラフィーや分取高速液体クロマトグラフィー等を用いることにより、上記抽出物から精製することができる。本発明で用いるフェニルプロパノイド配糖体は、必ずしも精製品である必要はなく粗精製品でもよく、また、溶媒を含む状態でもよい。 The phenylpropanoid glycoside used in the present invention can be purified from the above extract by using column chromatography using Diaion HP-20, ODS, or preparative high performance liquid chromatography. The phenylpropanoid glycoside used in the present invention does not necessarily have to be a purified product, and may be a crude product or may contain a solvent.
本発明の皮膚外用剤または内用剤には、フェニルプロパノイド配糖体をそのまま使用しても良く、その効果を損なわない範囲内で、外用剤や内用剤に用いられる成分である油脂類、ロウ類、炭化水素類、脂肪酸類、アルコール類、エステル類、界面活性剤、金属石鹸、ｐＨ調整剤、防腐剤、香料、保湿剤、粉体、紫外線吸収剤、増粘剤、色素、酸化防止剤、美白剤、キレート剤、賦形剤、皮膜剤、甘味料、酸味料等の成分を配合することができる。 In the skin external preparation or internal preparation of the present invention, phenylpropanoid glycosides may be used as they are, and fats and oils which are components used for external preparations and internal preparations within the range not impairing the effects thereof. , Waxes, hydrocarbons, fatty acids, alcohols, esters, surfactants, metal soaps, pH adjusters, preservatives, fragrances, moisturizers, powders, UV absorbers, thickeners, pigments, oxidation Components such as an inhibitor, a whitening agent, a chelating agent, an excipient, a film agent, a sweetener, and a sour agent can be blended.
本発明のフェニルプロパノイド配糖体は、化粧品、医薬部外品、医薬品、食品のいずれにも用いることができ、その剤型としては、例えば、化粧水、クリ−ム、乳液、ゲル剤、エアゾール剤、エッセンス、パック、洗浄剤、浴用剤、ファンデ−ション、打粉、口紅、軟膏、パップ剤、ペースト剤、プラスター剤、エッセンス、散剤、丸剤、錠剤、注射剤、坐剤、乳剤、カプセル剤、顆粒剤、液剤（チンキ剤、流エキス剤、酒精剤、懸濁剤、リモナーデ剤等を含む）、錠菓、飲料等が挙げられる。 The phenylpropanoid glycoside of the present invention can be used in any of cosmetics, quasi drugs, pharmaceuticals, and foods. Examples of the dosage form include lotions, creams, emulsions, gels, Aerosol, essence, pack, cleaning agent, bath preparation, foundation, dusting, lipstick, ointment, poultice, paste, plaster, essence, powder, pill, tablet, injection, suppository, emulsion, capsule Agents, granules, liquids (including tinctures, fluid extracts, spirits, suspensions, limonades, etc.), tablet confections, beverages and the like.
本発明に用いるフェニルプロパノイド配糖体の含有量は、外用の場合、皮膚外用剤全量に対し、固形物に換算して０．００００１重量％以上、好ましくは０．０００１〜１重量％の含有が良い。０．０００１重量％未満では十分な効果は望みにくい。１０重量％を越えて含有した場合、効果の増強は認められにくく不経済である。一方、内用の場合、投与量は年齢、体重、症状、治療効果、投与方法、処理時間等により異なるが、通常、成人１人当たりの１日の量としては、５ｍｇ以上が好ましく、１０ｍｇ〜１ｇがより好ましい。また、添加の方法については、予め加えておいても、製造途中で添加しても良く、作業性を考えて適宜選択すれば良い。 The content of the phenylpropanoid glycoside used in the present invention is 0.00001% by weight or more, preferably 0.0001 to 1% by weight, in terms of solid matter, based on the total amount of the external preparation for skin when used externally. Is good. If it is less than 0.0001% by weight, a sufficient effect is hardly expected. When the content exceeds 10% by weight, the enhancement of the effect is hardly recognized and it is uneconomical. On the other hand, for internal use, the dose varies depending on age, weight, symptoms, therapeutic effects, administration method, treatment time, etc., but the daily dose per adult is usually preferably 5 mg or more, and 10 mg to 1 g. Is more preferable. In addition, the addition method may be added in advance or during the production, and may be appropriately selected in consideration of workability.
次に本発明を詳細に説明するため、実施例として本発明に用いるフェニルプロパノイド配糖体の製造例、処方例及び実験例を挙げるが、本発明はこれに限定されるものではない。実施例に示す部とは重量部を、％とは容量％を示す。 Next, in order to describe the present invention in detail, production examples, formulation examples, and experimental examples of phenylpropanoid glycosides used in the present invention are given as examples, but the present invention is not limited thereto. In the examples, “parts” refers to parts by weight, and “%” refers to volume%.
製造例１ フェニルプロパノイド配糖体（化合物１）
カサブランカの花３０ｇに４００ｇの水を加え２時間熱水で抽出した。濾過後、得られた抽出液を濃縮乾固し、熱水抽出物を８．７ｇ得た。この抽出物の水可溶画分をカラムクロマトグラフィー（カラム：ダイヤイオンＨＰ−２０ ６ｃｍφ×４０ｃｍ、溶出液：０〜１００％エタノール水溶液）に供した。５０％エタノール溶出画分を集め、エタノールを留去後、分取高速液体クロマトグラフィー（カラム：Ｄｅｖｅｌｏｓｉｌ ＯＤＳ−ＨＧ−５ ２×２５ｃｍ、溶出液：５０％メタノール水溶液）を繰り返し用いて精製した。そして該当画分を集めメタノール留去後凍結乾燥することによって化合物１を１２．３ｍｇ得た。化合物１は、ＭＳスペクトル、１Ｄ−ＮＭＲスペクトルおよび２Ｄ−ＮＭＲスペクトルデータを解析することにより構造を決定した。そのデータを下記に示す。
ＭＳ（ＥＳＩ、ｍ／ｚ）７５９（Ｍ＋Ｎａ）＋、
１Ｈ−ＮＭＲ（ｐｙｒｉｄｉｎｅ−ｄ５、δ） ７．９４（１Ｈ、ｄ、Ｊ＝１６．０Ｈｚ）、７．８５（１Ｈ，ｄ、Ｊ＝１６．０Ｈｚ）、７．３０（１Ｈ，ｄ、Ｊ＝８．６Ｈｚ）、７．２７（１Ｈ、ｄ、Ｊ＝２．０Ｈｚ）、７．１６（１Ｈ、ｄｄ、Ｊ＝８．０、２．０Ｈｚ）、７．１１（１Ｈ、ｄｄ、Ｊ＝８．０、２．０Ｈｚ）、７．０３（１Ｈ、ｄ、Ｊ＝８．０Ｈｚ）、７．０１（１Ｈ、ｄ、Ｊ＝８．０Ｈｚ）、６．７２（１Ｈ、ｄ、Ｊ＝１６．０Ｈｚ）、６．５９（１Ｈ、ｄ、Ｊ＝１６．０Ｈｚ）、６．５５（１Ｈ、ｄ、Ｊ＝８．６Ｈｚ）、６．２１（１Ｈ、ｄ、Ｊ＝２．９Ｈｚ）、５．２６（１Ｈ、ｔ、Ｊ＝８．６Ｈｚ）、５．２５（１Ｈ、ｂｒｄ、Ｊ＝９．２Ｈｚ）、５．１０（１Ｈ、ｄｄ、Ｊ＝１１．５、２．０Ｈｚ）、４．２９（１Ｈ、ｄｄ、Ｊ＝９．２、６．３Ｈｚ）、４．７９（１Ｈ、ｄｄ、Ｊ＝１１．５、２．０Ｈｚ）、４．６６（１Ｈ、ｍ）、４．６２（１Ｈ、ｔ、Ｊ＝９．２Ｈｚ）、４．４５（１Ｈ、ｄｄ、Ｊ＝１２．０、６．３Ｈｚ）、４．３８（１Ｈ、ｄｄ、Ｊ＝１２．０、３．０Ｈｚ）、４．２４（１Ｈ、ｄ、Ｊ＝１１．８Ｈｚ）、４．０４（１Ｈ、ｔ、Ｊ＝９．２Ｈｚ）、３．７１（３Ｈ、ｓ）、３．６６（３Ｈ、ｓ）、１．８４（３Ｈ、ｓ）
Production Example 1 Phenylpropanoid glycoside (Compound 1)
400 g of water was added to 30 g of Casablanca flowers and extracted with hot water for 2 hours. After filtration, the obtained extract was concentrated to dryness to obtain 8.7 g of a hot water extract. The water-soluble fraction of this extract was subjected to column chromatography (column: Diaion HP-20 6 cmφ × 40 cm, eluent: 0-100% aqueous ethanol solution). Fractions eluted with 50% ethanol were collected, and after ethanol was distilled off, purification was performed repeatedly using preparative high performance liquid chromatography (column: Develosil ODS-HG-5 2 × 25 cm, eluent: 50% aqueous methanol solution). The corresponding fractions were collected and methanol was distilled off, followed by freeze-drying to obtain 12.3 mg of Compound 1. The structure of Compound 1 was determined by analyzing MS spectrum, 1D-NMR spectrum and 2D-NMR spectrum data. The data is shown below.
MS (ESI, m / z) 759 (M + Na) + ,
1 H-NMR (pyridine-d 5 , δ) 7.94 (1H, d, J = 16.0 Hz), 7.85 (1H, d, J = 16.0 Hz), 7.30 (1H, d, J = 8.6 Hz), 7.27 (1H, d, J = 2.0 Hz), 7.16 (1H, dd, J = 8.0, 2.0 Hz), 7.11 (1H, dd, J = 8.0, 2.0 Hz), 7.03 (1H, d, J = 8.0 Hz), 7.01 (1H, d, J = 8.0 Hz), 6.72 (1H, d, J = 16.0 Hz), 6.59 (1H, d, J = 16.0 Hz), 6.55 (1H, d, J = 8.6 Hz), 6.21 (1H, d, J = 2.9 Hz), 5.26 (1H, t, J = 8.6 Hz), 5.25 (1H, brd, J = 9.2 Hz), 5.10 (1H, dd, J = 11.5, 2.0 Hz), 4 .29 1H, dd, J = 9.2, 6.3 Hz), 4.79 (1H, dd, J = 11.5, 2.0 Hz), 4.66 (1H, m), 4.62 (1H, t , J = 9.2 Hz), 4.45 (1H, dd, J = 12.0, 6.3 Hz), 4.38 (1H, dd, J = 12.0, 3.0 Hz), 4.24 ( 1H, d, J = 11.8 Hz), 4.04 (1H, t, J = 9.2 Hz), 3.71 (3H, s), 3.66 (3H, s), 1.84 (3H, s)
製造例２ フェニルプロパノイド配糖体（化合物２）
製造例１と同様に化合物２（表１の物質２）を１４．９ｍｇ単離し、ＭＳスペクトル、１Ｄ−ＮＭＲスペクトルおよび２Ｄ−ＮＭＲスペクトルデータを解析することにより構造を決定した。そのデータを下記に示す。
ＭＳ（ＥＳＩ、ｍ／ｚ）７５９（Ｍ＋Ｎａ）＋、
１Ｈ−ＮＭＲ（ｐｙｒｉｄｉｎｅ−ｄ５、δ） ７．９６（１Ｈ、ｄ、Ｊ＝１５．６Ｈｚ）、７．８８（１Ｈ，ｄ、Ｊ＝１５．６Ｈｚ）、７．３３（１Ｈ，ｂｒｄ）、７．２９（１Ｈ、ｂｒｄ）、７．１８（１Ｈ、ｂｒｄ、Ｊ＝８．０Ｈｚ）、７．１３（１Ｈ、ｂｒｄ、Ｊ＝８．０Ｈｚ）、７．０４（１Ｈ、ｄ、Ｊ＝８．０Ｈｚ）、７．０２（１Ｈ、ｄ、Ｊ＝８．０Ｈｚ）、６．７６（１Ｈ、ｄ、Ｊ＝１５．６Ｈｚ）、６．６１（１Ｈ、ｄ、Ｊ＝１５．６Ｈｚ）、６．３９（１Ｈ、ｄ、Ｊ＝８．０Ｈｚ）、６．１５（１Ｈ、ｄ、Ｊ＝３．５Ｈｚ）、６．０９（１Ｈ、ｔ、Ｊ＝９．６Ｈｚ）、５．２５（１Ｈ、ｔ、Ｊ＝８．０Ｈｚ）、５．１２（１Ｈ、ｂｒｄ、Ｊ＝１１．８Ｈｚ）、４．９８（１Ｈ、ｄｄ、Ｊ＝９．６、６．３Ｈｚ）、４．８０（１Ｈ、ｄｄ、Ｊ＝１１．８、６．３Ｈｚ）、４．８０（１Ｈ、ｄｄ、Ｊ＝１１．８、６．３Ｈｚ）、４．６１（１Ｈ、ｍ）、４．４８（１Ｈ、ｄｄ、Ｊ＝１１．９、６．０Ｈｚ）、４．４０（１Ｈ、ｄｄ、Ｊ＝１１．９、２．９Ｈｚ）、４．１２（２Ｈ、ｄ、ｂｒｓ）、４．０８（１Ｈ、ｄｄ、Ｊ＝９．６、３．５Ｈｚ）、４．０７（１Ｈ、ｔ、Ｊ＝９．６Ｈｚ）、
３．７４（３Ｈ、ｓ）、３．６５（３Ｈ、ｓ）、１．７３（３Ｈ、ｓ）
Production Example 2 Phenylpropanoid glycoside (Compound 2)
In the same manner as in Production Example 1, 14.9 mg of Compound 2 (Substance 2 in Table 1) was isolated, and the structure was determined by analyzing the MS spectrum, 1D-NMR spectrum and 2D-NMR spectrum data. The data is shown below.
MS (ESI, m / z) 759 (M + Na) + ,
1 H-NMR (pyridine-d 5 , δ) 7.96 (1H, d, J = 15.6 Hz), 7.88 (1H, d, J = 15.6 Hz), 7.33 (1H, brd) 7.29 (1H, brd), 7.18 (1H, brd, J = 8.0 Hz), 7.13 (1H, brd, J = 8.0 Hz), 7.04 (1H, d, J = 8.0 Hz), 7.02 (1H, d, J = 8.0 Hz), 6.76 (1H, d, J = 15.6 Hz), 6.61 (1H, d, J = 15.6 Hz), 6.39 (1H, d, J = 8.0 Hz), 6.15 (1H, d, J = 3.5 Hz), 6.09 (1H, t, J = 9.6 Hz), 5.25 (1H , T, J = 8.0 Hz), 5.12 (1H, brd, J = 11.8 Hz), 4.98 (1H, dd, J = 9.6, 6.3 Hz), 4.8 (1H, dd, J = 11.8, 6.3 Hz), 4.80 (1H, dd, J = 11.8, 6.3 Hz), 4.61 (1H, m), 4.48 (1H, dd, J = 11.9, 6.0 Hz), 4.40 (1H, dd, J = 11.9, 2.9 Hz), 4.12 (2H, d, brs), 4.08 (1H, dd) , J = 9.6, 3.5 Hz), 4.07 (1H, t, J = 9.6 Hz),
3.74 (3H, s), 3.65 (3H, s), 1.73 (3H, s)
製造例３ フェニルプロパノイド配糖体（化合物３）
製造例１と同様に化合物３（表１の物質３）を２６．２ｍｇ単離し、文献記載のスペクトルと比較して構造を確認した。
Production Example 3 Phenylpropanoid glycoside (Compound 3)
26.2 mg of compound 3 (substance 3 in Table 1) was isolated in the same manner as in Production Example 1, and the structure was confirmed by comparison with the spectrum described in the literature.
比較製造例１ ユリ熱水抽出物
カサブランカの花３０ｇに４００ｇの水を加え２時間熱水で抽出した。濾過後、得られた抽出液を濃縮乾固し、熱水抽出物を８．７ｇ得た。
Comparative Production Example 1 Lily hot water extract 400 g of water was added to 30 g of a Casablanca flower and extracted with hot water for 2 hours. After filtration, the obtained extract was concentrated to dryness to obtain 8.7 g of a hot water extract.
処方例１ 化粧水
処方 部
１．化合物１（製造例１） ０．１
２．１，３−ブチレングリコール ８．０
３．グリセリン ２．０
４．キサンタンガム ０．０２
５．クエン酸 ０．０１
６．クエン酸ナトリウム ０．１
７．エタノール ５．０
８．パラオキシ安息香酸メチル ０．１
９．ポリオキシエチレン硬化ヒマシ油（４０Ｅ．Ｏ．） ０．１
１０．香料 適量
１１．精製水にて全量を１００とする
［製造方法］成分１〜６及び１１と、成分７〜１０をそれぞれ均一に溶解し、両者を混合し濾過して製品とする。
Formulation Example 1 Lotion Prescription Section 1. Compound 1 (Production Example 1) 0.1
2. 1,3-butylene glycol 8.0
3. Glycerin 2.0
4). Xanthan gum 0.02
5. Citric acid 0.01
6). Sodium citrate 0.1
7). Ethanol 5.0
8). Methyl paraoxybenzoate 0.1
9. Polyoxyethylene hydrogenated castor oil (40E.O.) 0.1
10. Perfume proper amount11. [Manufacturing method] Components 1 to 6 and 11 and components 7 to 10 are uniformly dissolved in purified water, and both are mixed and filtered to obtain a product.
比較例１ 従来の化粧水１
処方例１において、化合物１を精製水に置き換えたものを従来の化粧水とした。
Comparative Example 1 Conventional lotion 1
In Formulation Example 1, Compound 1 was replaced with purified water to obtain a conventional lotion.
比較例２ 従来の化粧水２
処方例１において、化合物１をユリ熱水抽出物（比較製造例１）に置き換えたものを従来の化粧水とした。
Comparative Example 2 Conventional lotion 2
In Formulation Example 1, a conventional lotion was obtained by replacing Compound 1 with a lily hot water extract (Comparative Production Example 1).
処方例２ クリーム
処方 部
１．化合物２（製造例２） ０．０５
２．スクワラン ５．５
３．オリーブ油 ３．０
４．ステアリン酸 ２．０
５．ミツロウ ２．０
６．ミリスチン酸オクチルドデシル ３．５
７．ポリオキシエチレンセチルエーテル（２０Ｅ．Ｏ．） ３．０
８．ベヘニルアルコール １．５
９．モノステアリン酸グリセリン ２．５
１０．香料 ０．１
１１．パラオキシ安息香酸メチル ０．２
１２．パラオキシ安息香酸エチル ０．０５
１３．１，３−ブチレングリコール ８．５
１４．精製水にて全量を１００とする
［製造方法］成分２〜９を加熱溶解して混合し、７０℃に保ち油相とする。成分１及び１１〜１４を加熱溶解して混合し、７５℃に保ち水相とする。油相に水相を加えて乳化して、かき混ぜながら冷却し、４５℃で成分１０を加え、更に３０℃まで冷却して製品とする。
Formulation Example 2 Cream Formulation Part 1. Compound 2 (Production Example 2) 0.05
2. Squalane 5.5
3. Olive oil 3.0
4). Stearic acid 2.0
5. Beeswax 2.0
6). Octyldodecyl myristate 3.5
7). Polyoxyethylene cetyl ether (20E.O.) 3.0
8). Behenyl alcohol 1.5
9. Glycerol monostearate2.5
10. Fragrance 0.1
11. Methyl paraoxybenzoate 0.2
12 Ethyl paraoxybenzoate 0.05
13.1,3-Butylene glycol 8.5
14 [Manufacturing method] Components 2 to 9 are heated and dissolved and mixed with purified water to a total amount of 100, and kept at 70 ° C to obtain an oil phase. Ingredients 1 and 11 to 14 are dissolved by heating and mixed, and kept at 75 ° C. to form an aqueous phase. The aqueous phase is added to the oil phase to emulsify, and the mixture is cooled while stirring. The component 10 is added at 45 ° C., and further cooled to 30 ° C. to obtain a product.
比較例３ 従来のクリーム
処方例２において、化合物２を精製水に置き換えたものを従来のクリームとした。
Comparative Example 3 Conventional Cream A conventional cream was prepared by replacing Compound 2 with purified water in Formulation Example 2.
処方例３ 乳液
処方 部
１．化合物１（製造例１） ０．０５
２．スクワラン ５．０
３．オリーブ油 ５．０
４．ホホバ油 ５．０
５．セタノール １．５
６．モノステアリン酸グリセリン ２．０
７．ポリオキシエチレンセチルエーテル（２０Ｅ．Ｏ．） ３．０
８．ポリオキシエチレンソルビタンモノオレエート（２０Ｅ．Ｏ．） ２．０
９．香料 ０．１
１０．プロピレングリコール １．０
１１．グリセリン ２．０
１２．パラオキシ安息香酸メチル ０．２
１３．精製水にて全量を１００とする
［製造方法］成分２〜８を加熱溶解して混合し、７０℃に保ち油相とする。成分１及び１０〜１３を加熱溶解して混合し、７５℃に保ち水相とする。油相に水相を加えて乳化して、かき混ぜながら冷却し、４５℃で成分９を加え、更に３０℃まで冷却して製品とする。
Formulation Example 3 Emulsion Formulation Department 1. Compound 1 (Production Example 1) 0.05
2. Squalane 5.0
3. Olive oil 5.0
4). Jojoba oil 5.0
5. Cetanol 1.5
6). Glycerol monostearate 2.0
7). Polyoxyethylene cetyl ether (20E.O.) 3.0
8). Polyoxyethylene sorbitan monooleate (20E.O.) 2.0
9. Fragrance 0.1
10. Propylene glycol 1.0
11. Glycerin 2.0
12 Methyl paraoxybenzoate 0.2
13. [Manufacturing method] Components 2 to 8 are heated and dissolved and mixed with purified water to a total amount of 100, and kept at 70 ° C to obtain an oil phase. Ingredients 1 and 10-13 are dissolved by heating and mixed, and kept at 75 ° C. to form an aqueous phase. The aqueous phase is added to the oil phase to emulsify, and the mixture is cooled while stirring. The component 9 is added at 45 ° C., and further cooled to 30 ° C. to obtain a product.
処方例４ ゲル剤
処方 部
１．化合物１（製造例１） ０．１
２．エタノール ５．０
３．パラオキシ安息香酸メチル ０．１
４．ポリオキシエチレン硬化ヒマシ油（６０Ｅ．Ｏ．） ０．１
５．香料 適量
６．１，３−ブチレングリコール ５．０
７．グリセリン ５．０
８．キサンタンガム ０．１
９．カルボキシビニルポリマー ０．２
１０．水酸化カリウム ０．２
１１．精製水にて全量を１００とする
［製造方法］成分２〜５と、成分１及び６〜１１をそれぞれ均一に溶解し、両者を混合して製品とする。
Formulation Example 4 Gel Formulation Part 1. Compound 1 (Production Example 1) 0.1
2. Ethanol 5.0
3. Methyl paraoxybenzoate 0.1
4). Polyoxyethylene hydrogenated castor oil (60 EO) 0.1
5. Perfume appropriate amount 6.1,3-butylene glycol 5.0
7). Glycerin 5.0
8). Xanthan gum 0.1
9. Carboxyvinyl polymer 0.2
10. Potassium hydroxide 0.2
11. [Production method] Ingredients 2 to 5 and ingredients 1 and 6 to 11 are uniformly dissolved in purified water, and the two are mixed to obtain a product.
処方例５ パック
処方 部
１．化合物１（製造例１） ０．１
２．化合物２（製造例２） ０．１
３．化合物３（製造例３） ０．１
４．ポリビニルアルコール １２．０
５．エタノール ５．０
６．１，３−ブチレングリコール ８．０
７．パラオキシ安息香酸メチル ０．２
８．ポリオキシエチレン硬化ヒマシ油（２０Ｅ．Ｏ．） ０．５
９．クエン酸 ０．１
１０．クエン酸ナトリウム ０．３
１１．香料 適量
１２．精製水にて全量を１００とする
［製造方法］成分１〜１２を均一に溶解し製品とする。
Formulation Example 5 Pack prescription part 1. Compound 1 (Production Example 1) 0.1
2. Compound 2 (Production Example 2) 0.1
3. Compound 3 (Production Example 3) 0.1
4). Polyvinyl alcohol 12.0
5. Ethanol 5.0
6.1,3-Butylene glycol 8.0
7). Methyl paraoxybenzoate 0.2
8). Polyoxyethylene hydrogenated castor oil (20 EO) 0.5
9. Citric acid 0.1
10. Sodium citrate 0.3
11. Perfume appropriate amount 12. [Production method] Components 1 to 12 are uniformly dissolved in purified water to make a total amount of 100 to obtain a product.
処方例６ ファンデーション
処方 部
１．化合物２（製造例２） ０．００１
２．ステアリン酸 ２．４
３．ポリオキシエチレンソルビタンモノステアレート（２０Ｅ．Ｏ．） １．０
４．ポリオキシエチレンセチルエーテル（２０Ｅ．Ｏ．） ２．０
５．セタノール １．０
６．液状ラノリン ２．０
７．流動パラフィン ３．０
８．ミリスチン酸イソプロピル ６．５
９．カルボキシメチルセルロースナトリウム ０．１
１０．ベントナイト ０．５
１１．プロピレングリコール ４．０
１２．トリエタノールアミン １．１
１３．パラオキシ安息香酸メチル ０．２
１４．二酸化チタン ８．０
１５．タルク ４．０
１６．ベンガラ １．０
１７．黄酸化鉄 ２．０
１８．香料 適量
１９．精製水にて全量を１００とする
［製造方法］成分２〜８を加熱溶解し、８０℃に保ち油相とする。成分１９に成分９をよく膨潤させ、続いて、成分１及び１０〜１３を加えて均一に混合する。これに粉砕機で粉砕混合した成分１４〜１７を加え、ホモミキサーで撹拌し７５℃に保ち水相とする。この油相に水相をかき混ぜながら加え、冷却し、４５℃で成分１８を加え、かき混ぜながら３０℃まで冷却して製品とする。
Formulation Example 6 Foundation Formulation Department 1. Compound 2 (Production Example 2) 0.001
2. Stearic acid 2.4
3. Polyoxyethylene sorbitan monostearate (20E.O.) 1.0
4). Polyoxyethylene cetyl ether (20E.O.) 2.0
5. Cetanol 1.0
6). Liquid lanolin 2.0
7). Liquid paraffin 3.0
8). Isopropyl myristate 6.5
9. Sodium carboxymethylcellulose 0.1
10. Bentonite 0.5
11. Propylene glycol 4.0
12 Triethanolamine 1.1
13. Methyl paraoxybenzoate 0.2
14 Titanium dioxide 8.0
15. Talc 4.0
16. Bengala 1.0
17. Yellow iron oxide 2.0
18. Perfume proper amount19. [Manufacturing method] Components 2 to 8 are heated and dissolved in purified water to a total amount of 100, and kept at 80 ° C to obtain an oil phase. Swell component 9 well in component 19, then add components 1 and 10-13 and mix uniformly. To this, components 14 to 17 pulverized and mixed with a pulverizer are added, and the mixture is stirred with a homomixer and kept at 75 ° C. to obtain an aqueous phase. The water phase is added to this oil phase with stirring, cooled, and component 18 is added at 45 ° C., and cooled to 30 ° C. with stirring to give a product.
処方例７ 浴用剤
処方 部
１．化合物３（製造例３） ０．１
２．炭酸水素ナトリウム ５０．０
３．黄色２０２号（１） 適量
４．香料 適量
５．硫酸ナトリウムにて全量を１００とする
［製造方法］成分１〜５を均一に混合し製品とする。
Formulation Example 7 Bath preparation Formulation 1. Compound 3 (Production Example 3) 0.1
2. Sodium bicarbonate 50.0
3. Yellow No. 202 (1) Appropriate amount 4. Perfume appropriate amount 5. [Manufacturing method] Ingredients 1 to 5 are mixed uniformly with sodium sulfate to make a product.
処方例８ 軟膏
処方 部
１．化合物１（製造例１） １．０
２．ポリオキシエチレンセチルエーテル（３０Ｅ．Ｏ．） ２．０
３．モノステアリン酸グリセリン １０．０
４．流動パラフィン ５．０
５．セタノール ６．０
６．パラオキシ安息香酸メチル ０．１
７．プロピレングリコール １０．０
８．精製水にて全量を１００とする
［製造方法］成分２〜５を加熱溶解して混合し、７０℃に保ち油相とする。成分１及び６〜８を加熱溶解して混合し、７５℃に保ち水相とする。油相に水相を加えて乳化して、かき混ぜながら３０℃まで冷却して製品とする。
Formulation Example 8 Ointment Prescription part 1. Compound 1 (Production Example 1) 1.0
2. Polyoxyethylene cetyl ether (30E.O.) 2.0
3. Glycerol monostearate 10.0
4). Liquid paraffin 5.0
5. Cetanol 6.0
6). Methyl paraoxybenzoate 0.1
7). Propylene glycol 10.0
8). [Manufacturing method] Components 2-5 are heated and dissolved and mixed with purified water to 100, and kept at 70 ° C. to obtain an oil phase. Ingredients 1 and 6 to 8 are dissolved by heating and mixed, and kept at 75 ° C. to obtain an aqueous phase. The aqueous phase is added to the oil phase to emulsify, and the product is cooled to 30 ° C. with stirring to obtain a product.
比較例４ 従来の軟膏
処方例８において、化合物１を精製水に置き換えたものを従来の軟膏とした。
Comparative Example 4 Conventional Ointment In Formulation Example 8, Compound 1 was replaced with purified water to obtain a conventional ointment.
処方例９ 錠剤
処方 部
１．化合物２（製造例２） ３．０
２．乾燥コーンスターチ ２７．０
３．カルボキシメチルセルロースカルシウム ２０．０
４．微結晶セルロース ４０．０
５．ポリビニルピロリドン ７．０
６．タルク ３．０
［製造方法］成分１〜４を混合し、次いで成分５の水溶液を結合剤として加えて顆粒成形する。成形した顆粒に成分６を加えて打錠する。１錠０．５２ｇとする。
Formulation Example 9 Tablet Formulation Department 1. Compound 2 (Production Example 2) 3.0
2. Dried cornstarch 27.0
3. Carboxymethylcellulose calcium 20.0
4). Microcrystalline cellulose 40.0
5. Polyvinylpyrrolidone 7.0
6). Talc 3.0
[Production method] Components 1 to 4 are mixed, and then an aqueous solution of component 5 is added as a binder to form granules. Ingredient 6 is added to the formed granules and compressed. One tablet is 0.52 g.
次に、本発明の効果を詳細に説明するため、実験例を挙げる。 Next, experimental examples will be given to explain the effects of the present invention in detail.
実験例１ 紫外線によるコラーゲン産生低下及び分解促進の抑制試験
正常ヒト真皮線維芽細胞（ＮＢ１ＲＧＢ）を６０ｍｍディッシュに播種し、コンフルエントになるまで培養した。その後、最終濃度が１及び１０μｇ／ｍｌになるように化合物１〜３（製造例１〜３）を加え、２４時間培養した。比較用の試料は、ユリの熱水抽出物（比較製造例１）を用いた。培地を除き、細胞をＰＢＳで２回洗浄した後、ＵＶＢ領域の紫外線３０ｍＪ／ｃｍ２（東芝ＦＬ−２０Ｓ・Ｅランプ）を照射した。通常の培地に戻し、さらに２４時間培養後、ＲＮＡｉｓｏ（ＴＡＫＡＲＡ）にて細胞から総ＲＮＡを抽出し、Ｍａｓｔｅｒ Ｍｉｘ ＳＹＢＲ Ｇｒｅｅｎ（インビトロジェン）を用い、ＲＴ−ＰＣＲ法にてタイプＩコラーゲンおよびＭＭＰ１ ｍＲＮＡ発現量の測定を行った。ＰＣＲ反応は、９５℃にて２分間初期変性を行った後、９５℃：１５秒、６０℃：３１秒を１サイクルとして４０サイクル行った。内部標準としては、β−アクチンを用いた。タイプＩコラーゲンおよびＭＭＰ１ ｍＲＮＡ発現量は、β−アクチン ｍＲＮＡ発現量に対する割合として求めた。なお、タイプＩコラーゲン、ＭＭＰ１及びβ−アクチン ｍＲＮＡ発現量の測定に使用したプライマーは次の通りである。
Experimental Example 1 Inhibition test of decrease in collagen production and promotion of degradation by ultraviolet rays Normal human dermal fibroblasts (NB1RGB) were seeded in a 60 mm dish and cultured until confluent. Thereafter, compounds 1 to 3 (Production Examples 1 to 3) were added so that the final concentrations were 1 and 10 μg / ml, and the cells were cultured for 24 hours. As a sample for comparison, a hot water extract of Lily (Comparative Production Example 1) was used. The medium was removed, and the cells were washed twice with PBS, and then irradiated with ultraviolet light 30 mJ / cm 2 (Toshiba FL-20S · E lamp) in the UVB region. After returning to a normal medium and further culturing for 24 hours, total RNA is extracted from the cells with RNAiso (TAKARA), and expression levels of type I collagen and MMP1 mRNA are detected by RT-PCR using Master Mix SYBR Green (Invitrogen). Was measured. The PCR reaction was initially denatured at 95 ° C. for 2 minutes, followed by 40 cycles of 95 ° C .: 15 seconds and 60 ° C .: 31 seconds. Β-actin was used as an internal standard. Type I collagen and MMP1 mRNA expression levels were determined as a percentage of β-actin mRNA expression levels. In addition, the primers used for the measurement of type I collagen, MMP1 and β-actin mRNA expression levels are as follows.
タイプＩコラーゲン用のプライマーセット
ＡＧＧＡＣＡＡＧＡＧＧＣＡＴＧＴＣＴＧＧＴＴ（配列番号１）
ＴＴＧＣＡＧＴＧＧＴＡＧＧＴＧＡＴＧＴＴＣＴＧ（配列番号２）
ＭＭＰ１用のプライマーセット
ＧＧＧＡＧＡＴＣＡＴＣＧＧＧＡＣＡＡＣＴＣ（配列番号３）
ＴＧＡＧＣＡＴＣＣＣＣＴＣＣＡＡＴＡＣＣ（配列番号４）
β−アクチン用のプライマーセット
ＣＡＣＴＣＴＴＣＣＡＧＣＣＴＴＣＣＴＴＣＣ（配列番号５）
ＧＴＧＴＴＧＧＣＧＴＡＣＡＧＧＴＣＴＴＴＧ（配列番号６）
Primer set AGGACAAGAGGGCATGTCTGGTT for type I collagen (SEQ ID NO: 1)
TTGCAGTGGGTAGGTGATGTCTG (SEQ ID NO: 2)
Primer set GGGAGATCATCGGGAACAACTC for MMP1 (SEQ ID NO: 3)
TGAGCATCCCCCCTCAATAC (SEQ ID NO: 4)
Primer set CACTCTCCCAGCCTCTCTCTC for β-actin (SEQ ID NO: 5)
GGTTTGGCGTACAGGTCTTTG (SEQ ID NO: 6)
実験結果を表２に示した。紫外線照射により線維芽細胞におけるタイプＩコラーゲン ｍＲＮＡ発現は低下した。この低下は、化合物１〜３の添加により顕著に抑制された。ユリの熱水抽出物（比較製造例１）も紫外線照射による線維芽細胞におけるタイプＩコラーゲン ｍＲＮＡ発現低下を抑制したが、その効果は、化合物１〜３の方が顕著に高かった。また、紫外線照射により線維芽細胞におけるＭＭＰ１ ｍＲＮＡ発現は促進された。この促進は、化合物１〜３の添加により顕著に抑制された。ユリの熱水抽出物（比較製造例１）も紫外線照射による線維芽細胞におけるＭＭＰ１ ｍＲＮＡ発現促進を抑制したが、その効果は、フェニルプロパノイド配糖体の方が顕著に高かった。これらの結果は、フェニルプロパノイド配糖体が、紫外線による真皮線維芽細胞におけるコラーゲン産生の低下及び分解促進を抑制することにより、従来技術では認められないような優れた紫外線による皮膚のしわの形成を防止・改善効果を有することを示している。 The experimental results are shown in Table 2. UV irradiation reduced type I collagen mRNA expression in fibroblasts. This decrease was significantly suppressed by the addition of compounds 1 to 3. Lily's hot water extract (Comparative Production Example 1) also suppressed the decrease in type I collagen mRNA expression in fibroblasts due to UV irradiation, but the effects of compounds 1 to 3 were significantly higher. Moreover, MMP1 mRNA expression in fibroblasts was promoted by ultraviolet irradiation. This promotion was significantly suppressed by the addition of compounds 1-3. Lily hot water extract (Comparative Production Example 1) also suppressed the promotion of MMP1 mRNA expression in fibroblasts by UV irradiation, but the effect was significantly higher for phenylpropanoid glycosides. These results indicate that phenylpropanoid glycosides suppress the decrease in collagen production and the promotion of degradation in dermal fibroblasts caused by ultraviolet rays, which makes it possible to form excellent skin wrinkles due to ultraviolet rays that are not recognized in the prior art. It shows that it has the effect of preventing and improving.
実験例２ アクチンフィラメント構築促進剤
コラーゲン溶液（新田ゼラチン、Ｃｅｌｌｍａｔｒｉｘ Ｐ−１）を用いてコラーゲンコートしたカバースリップ上に線維芽細胞を播種し、２４時間培養した。細胞が接着したところで、ＰＢＳ存在下でＯｐｔｉｃａｌ Ｍｏｄｕｌｅｘ（ウシオ電機）にて１０Ｊ／ｃｍ２のＵＶ−Ａを２４時間おきに３回照射した。最後の紫外線照射後に、化合物１〜３（製造例１〜３）を含む無血清培地に交換し、さらに２４時間培養した。比較用の試料は、ユリの熱水抽出物（比較製造例１）を用いた。その後、細胞内のアクチンフィラメントをＴＲＩＴＣ−Ｐａｌｌｏｉｄｉｎ（ＳＩＧＭＡ）を用いて染色し、蛍光顕微鏡を用いて観察した。撮影した蛍光画像をＲＧＢ成分に分解し、Ｒ値（０〜２５５）が３０以上の画素を抽出し、アクチンフィラメントの構築量を、細胞あたりの面積として算出した。
Experimental Example 2 Actin Filament Construction Promoter Fibroblasts were seeded on a collagen-coated coverslip using a collagen solution (Nitta Gelatin, Cellmatrix P-1) and cultured for 24 hours. When the cells adhered, they were irradiated with 10 J / cm 2 of UV-A three times every 24 hours using Optical Modex (USHIO) in the presence of PBS. After the last ultraviolet irradiation, the medium was replaced with a serum-free medium containing compounds 1 to 3 (Production Examples 1 to 3), and further cultured for 24 hours. As a sample for comparison, a hot water extract of Lily (Comparative Production Example 1) was used. Thereafter, intracellular actin filaments were stained with TRITC-Palloidin (SIGMA) and observed with a fluorescence microscope. The captured fluorescent image was decomposed into RGB components, pixels with an R value (0 to 255) of 30 or more were extracted, and the amount of actin filaments constructed was calculated as the area per cell.
実験結果を表３に示した。紫外線照射によって、細胞あたりのアクチンフィラメントに相当する蛍光面積は有意に減少した。ユリの熱水抽出物により、アクチンフィラメントの減少は抑制された。しかし、本発明の化合物１〜３を１および１０μｇ／ｍｌ添加することにより、その減少は顕著に抑制された。以上の結果から、化合物１〜３には、紫外線による細胞のアクチンフィラメント構築の低下を格段に抑制する効果があることが示された。 The experimental results are shown in Table 3. UV irradiation significantly reduced the fluorescence area corresponding to actin filaments per cell. The decrease in actin filaments was suppressed by the hot water extract of lily. However, the decrease was remarkably suppressed by adding 1 and 10 μg / ml of the compounds 1 to 3 of the present invention. From the above results, it was shown that the compounds 1 to 3 have an effect of significantly suppressing a decrease in the actin filament construction of cells due to ultraviolet rays.
実験例３ 使用試験
処方例８及び比較例４の軟膏を調製し、顔面に真皮性しわ（小じわではない）を有する女性被験者（４０−５０代）を対象に１ヶ月間の使用試験を実施した。各々の軟膏をそれぞれ６名の被験者の頬部に朝晩１日２回使用させた。試験終了後のしわの改善度を、以下に示す判定基準にて視覚評価することにより、「しわに対する改善効果」を判定した。
（判定基準）
有効 ：真皮性しわ（小じわではない）がかなり目立たなくなった
やや有効：真皮性しわ（小じわではない）が以前より目立たなくなった
効果なし：変化なし
Experimental Example 3 Use Test The ointment of Formulation Example 8 and Comparative Example 4 was prepared, and a 1 month use test was conducted on female subjects (40-50s) who had dermal wrinkles (not fine wrinkles) on the face. . Each ointment was used twice a day in the morning and evening on the cheeks of 6 subjects. The “wrinkle improvement effect” was determined by visually evaluating the degree of wrinkle improvement after the test according to the following criteria.
(Criteria)
Effective: Dermal wrinkles (not fine wrinkles) are slightly inconspicuous Somewhat effective: Dermal wrinkles (not fine wrinkles) are less noticeable than before: No change: No change
その結果、処方例８及び比較例４の軟膏を比較すると、フェニルプロパノイド配糖体を含有する軟膏に、明らかに真皮性しわの改善効果が認められた。なお、試験期間中、皮膚のトラブルは無く、安全性についても問題はなかった。 As a result, when the ointments of Prescription Example 8 and Comparative Example 4 were compared, the ointment containing phenylpropanoid glycosides clearly showed an effect of improving dermal wrinkles. During the test period, there were no skin problems and no safety problems.
以上のことからフェニルプロパノイド配糖体は、優れたコラーゲン産生促進作用、マトリックスメタロプロテアーゼ産生抑制作用及びアクチンフィラメント構築促進作用を有し、安定性にも優れていた。さらに、フェニルプロパノイド配糖体を含有する皮膚外用剤は、安全で優れたしわ改善作用を示した。よって、本発明のフェニルプロパノイド配糖体を含有する皮膚外用剤は、しわ形成防止・改善作用として特に有効である。 From the above, phenylpropanoid glycosides have excellent collagen production promoting action, matrix metalloprotease production inhibiting action and actin filament construction promoting action, and are also excellent in stability. Furthermore, the external preparation for skin containing phenylpropanoid glycoside showed a safe and excellent wrinkle improving action. Therefore, the skin external preparation containing the phenylpropanoid glycoside of the present invention is particularly effective as a wrinkle formation preventing / ameliorating action.
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