JP5714956B2 - Collagen production promoter - Google Patents
Collagen production promoter Download PDFInfo
- Publication number
- JP5714956B2 JP5714956B2 JP2011072313A JP2011072313A JP5714956B2 JP 5714956 B2 JP5714956 B2 JP 5714956B2 JP 2011072313 A JP2011072313 A JP 2011072313A JP 2011072313 A JP2011072313 A JP 2011072313A JP 5714956 B2 JP5714956 B2 JP 5714956B2
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- JP
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- Prior art keywords
- collagen
- extract
- gel
- quince
- added
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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- 239000008158 vegetable oil Substances 0.000 description 1
- 235000019165 vitamin E Nutrition 0.000 description 1
- 229940046009 vitamin E Drugs 0.000 description 1
- 239000011709 vitamin E Substances 0.000 description 1
- 229920003170 water-soluble synthetic polymer Polymers 0.000 description 1
- 230000002087 whitening effect Effects 0.000 description 1
Description
本発明は、コラーゲン産生促進剤、コラーゲンゲル収縮剤及びそれを含有するしわ改善剤に関する。 The present invention relates to a collagen production promoter, a collagen gel contracting agent, and a wrinkle improving agent containing the same.
シワは、加齢や太陽光線による皮膚の老化（光老化）などにより発生する。皮膚老化は環境因子による障害の蓄積が大きく作用していると考えられる。とりわけ紫外線は、皮膚加齢やシワ形成に関与する最大の環境因子と考えられ、紫外線により産生される各種フリーラジカル（特にスーパーオキシド、ハイドロキシラジカル、一重項酸素等の活性酸素）は、日焼け等の急性炎症の原因となるのみならず、その産生が慢性的に繰り返されることにより光老化を誘発することが知られている。活性酸素は、真皮成分のＤＮＡ−蛋白クロスリンク（架橋結合）、コラーゲンやエラスチンの蛋白クロスリンクの障害又は変性、ＳＯＤ等の抗酸化酵素の不活化、細胞成分の膜脂質過酸化とその結果としての細胞機能の劣化などを惹起し、その結果、皮膚の老化やシワの形成を引き起こすと考えられている（非特許文献１）。 Wrinkles occur due to aging and skin aging (photoaging) due to sunlight. Skin aging is thought to be largely due to the accumulation of damage caused by environmental factors. In particular, ultraviolet rays are considered to be the largest environmental factor involved in skin aging and wrinkle formation, and various free radicals produced by ultraviolet rays (especially active oxygen such as superoxide, hydroxy radical, singlet oxygen) It is known not only to cause acute inflammation but also to induce photoaging by its repeated production. Active oxygen is the result of DNA-protein cross-linking (cross-linking) of the dermal component, disorder or denaturation of protein cross-linking of collagen and elastin, inactivation of antioxidant enzymes such as SOD, membrane lipid peroxidation of cell components It is thought that it causes deterioration of the cell function of the skin and, as a result, causes aging of the skin and formation of wrinkles (Non-patent Document 1).
皮膚老化やシワの形成を予防し又は治療するために、ビタミンＥのような抗酸化剤の利用（特許文献１）、シリマリンなど植物エキスの肌への供給（特許文献２）、ヒアルロン酸等細胞外マトリクスの肌への補給（特許文献３）、レチノイン酸やα−ヒドロキシ酸の利用などが提案されている。近年、健康で美しい肌状態を実現することが老若男女を問わず重大な関心事になっている。
一方紫外線によるシワの形成モデルが確立され、これを用いてシワ形成を抑制する成分に探索が行われるようになった。たとえばヒノキ科の植物のアスナロ抽出物がマウスに紫外線を照射することにより形成するシワを抑制する効果を有することが見出されたシワ防止剤として提案されている。（特許文献４：特許第３９１９２５０号公報）。さらに最近ではシリビンとコラーゲントリペプチドを含有する製剤の投与により皮膚の基底層の細胞の増殖を促進させてシワを改善する技術が提案されている（特許文献５）。しかし満足できる物質はあまり発見されていない。
Use of antioxidants such as vitamin E (Patent Document 1), supply of plant extracts such as silymarin to the skin (Patent Document 2), cells such as hyaluronic acid to prevent or treat skin aging and wrinkle formation Replenishment of the outer matrix to the skin (Patent Document 3), use of retinoic acid and α-hydroxy acid, and the like have been proposed. In recent years, achieving healthy and beautiful skin conditions has become a major concern for both young and old.
On the other hand, a wrinkle formation model by ultraviolet rays has been established, and a search for a component that suppresses wrinkle formation has been conducted using this model. For example, it has been proposed as an anti-wrinkle agent that has been found to have the effect of suppressing wrinkles formed by irradiating mice with ultraviolet rays as an asunalo extract of a cypress plant. (Patent Document 4: Japanese Patent No. 3919250). More recently, a technique for improving wrinkles by promoting the proliferation of cells in the basal layer of the skin by administration of a preparation containing silybin and collagen tripeptide has been proposed (Patent Document 5). However, few satisfactory substances have been found.
本発明はヒフ細胞のコラーゲン産生促進剤、コラーゲンゲル収縮剤およびこれを含有するシワ改善剤を提供することを課題とする。 This invention makes it a subject to provide the collagen production promoter of a hif cell, a collagen gel contraction agent, and the wrinkle improving agent containing this.
本発明者らは、マルメロ種子のアルコール抽出物に強いコラーゲン産生促進作用を見出したので、本発明を提案する。
本発明は以下の構成である。
（１）マルメロ種子の、７０％以上の濃度のエタノールによる抽出物を有効成分とするコラーゲン産生促進剤。
（２）マルメロ種子の、７０％以上の濃度のエタノールによる抽出物を有効成分とするコラーゲンゲル収縮剤。
（３）（１）又は（２）に記載の剤を含有するシワ改善剤。
Since the present inventors have found a strong collagen production promoting action on the alcoholic extract of quince seeds, the present invention is proposed.
The present invention has the following configuration.
(1) A collagen production promoter comprising, as an active ingredient, an extract of quince seeds with ethanol having a concentration of 70% or more .
(2) A collagen gel shrinking agent comprising an extract of quince seeds with ethanol having a concentration of 70% or more as an active ingredient.
(3) A wrinkle improving agent containing the agent according to (1) or (2) .
本発明によりコラーゲン産生促進剤ならびにコラーゲンゲル収縮剤が提供される。またコラーゲン産生促進剤及び／又はコラーゲンゲル収縮剤を含有するシワ改善剤が提供される。 The present invention provides a collagen production promoter and a collagen gel contractor. Moreover, the wrinkle improving agent containing a collagen production promoter and / or a collagen gel contraction agent is provided.
本発明に用いるマルメロ（Ｃｙｄｏｎｉａ ｏｂｌｏｎｇａ）は、ヨーロッパやアジア南部（おもにイラン）を原産とするバラ科の植物である。マルメロの種子の水抽出物は化粧品の増粘剤として汎用されている（特許文献６、特許文献７）。またマルメロ果実のアルコール抽出物はチロシナーゼインヒビター、α−アミラーゼインヒビターとしての効果が知られている（特許文献８）。更にまたマルメロ全草の水蒸気蒸留水の香料としての使用（特許文献９）、マルメロ果実エタノール抽出物を保湿、美肌、肌荒れ改善効果を有する化粧料に配合すること、マルメロ種子アルコール抽出物を油性クレンジングに配合すること（特許文献１０）、マルメロ種子の１，３−ブタンジオール抽出物をフォーム状洗浄料に配合すること（特許文献１１）が知られている。しかし本発明のコラーゲン産生促進作用やコラーゲン収縮作用、シワ改善作用は知られていない。 The quince (Cydonia oblonga) used in the present invention is a plant of the Rosaceae originating from Europe and southern Asia (mainly Iran). Quince seed water extract is widely used as a thickener for cosmetics (Patent Document 6, Patent Document 7). Moreover, the alcohol extract of a quince fruit is known for the effect as a tyrosinase inhibitor and an α-amylase inhibitor (Patent Document 8). Furthermore, the use of whole quince as a fragrance of steam distilled water (Patent Document 9), the incorporation of quince fruit ethanol extract into a cosmetic having a moisturizing, beautiful skin, and rough skin improving effect, and the quince seed alcohol extract is oil-based cleansing (Patent Document 10), and 1,3-butanediol extract of quince seeds in a foam-like cleaning material (Patent Document 11) are known. However, the collagen production promoting action, collagen contracting action and wrinkle improving action of the present invention are not known.
本発明に用いるマルメロの抽出部位は種子、果実、花、葉、枝、根のいずれでも良く、種子が特に好ましい。本発明に用いる有機溶媒として、アルコール類、アセトン、アセトニトリル等が挙げられ、親油性有機溶媒としてヘキサン、クロロホルム、酢酸エチル、ジエチルエーテル、トルエン等が挙げられる。マルメロの有機溶媒抽出物の調製に用いる抽出溶媒としては、親水性有機溶媒が好ましい。親水性有機溶媒としてアルコールが特に好ましい。アルコールとしてはエタノール、メタノール、プロパノール、イソプロパノール、ブタノール、１，３−ブチレングリコール等が挙げられる。エタノールが特に好ましい。また、親水性有機溶媒を用いる際は、本発明の効果を損なわない範囲で水と混合して用いることができる。マルメロの抽出溶媒として、親水性有機溶媒と水の混合物を用いる場合は、親水性有機溶媒と水の質量比が５０：５０〜１００：０が好ましく、７０：３０〜１００：０が特に好ましい。使用する有機溶媒は抽出するマルメロの状態により異なるが、抽出対象物の重量の１０倍乃至１００倍量の有機溶媒を用いる。 The extraction part of quince used in the present invention may be any of seeds, fruits, flowers, leaves, branches and roots, and seeds are particularly preferred. Examples of the organic solvent used in the present invention include alcohols, acetone, acetonitrile, and the like. Examples of the lipophilic organic solvent include hexane, chloroform, ethyl acetate, diethyl ether, toluene, and the like. As the extraction solvent used for preparing the organic solvent extract of quince, a hydrophilic organic solvent is preferable. Alcohol is particularly preferred as the hydrophilic organic solvent. Examples of the alcohol include ethanol, methanol, propanol, isopropanol, butanol, 1,3-butylene glycol. Ethanol is particularly preferred. Moreover, when using a hydrophilic organic solvent, it can mix and use with water in the range which does not impair the effect of this invention. When a mixture of a hydrophilic organic solvent and water is used as a quince extraction solvent, the mass ratio of the hydrophilic organic solvent to water is preferably 50:50 to 100: 0, and particularly preferably 70:30 to 100: 0. The organic solvent to be used varies depending on the state of the quince to be extracted, but the organic solvent is used in an amount 10 to 100 times the weight of the extraction object.
抽出にあたっては、新鮮な果実や種子、枝、葉から有機溶媒で直接抽出することもできるが、マルメロの乾燥物を粉砕したものから有機溶媒で抽出する方法が効率的に抽出できるので好ましい。抽出する際には、還流冷却器を付した加熱還流抽出方法が好ましいが、可能な限り低温で抽出する。抽出時間は５〜２４時間循環抽出することで高活性の抽出液を回収することができる。抽出後は、ロータリーエバポレーターなどを用いて低温で溶媒を留去する。 In the extraction, it is possible to directly extract from fresh fruits, seeds, branches, and leaves with an organic solvent. However, a method of extracting a dried quince from a pulverized product with an organic solvent is preferable because it can be efficiently extracted. When extracting, a heating reflux extraction method with a reflux condenser is preferred, but extraction is performed at the lowest possible temperature. Extraction time can collect high activity extract by circulating extraction for 5 to 24 hours. After extraction, the solvent is distilled off at a low temperature using a rotary evaporator or the like.
溶媒を除去した抽出物は、直接又は水や生理食塩水などに希釈してコラーゲン産生促進剤、コラーゲンゲル収縮剤として用いることができる。また賦型剤や安定剤、等張剤など製剤化に必要な成分を添加して製剤化することができる。又凍結乾燥など乾燥操作を行って粉末化することもできる。かくして得られたコラーゲン産生促進剤及び／又はコラーゲンゲル収縮剤を医薬品、化粧品として用いる場合は、直接目的の剤として使用するか、あるいは油剤などに分散・溶解させて配合することができる。化粧品に配合することによりしわ改善剤とすることができる。この化粧品としてのしわ改善剤には、通常の化粧品に用いられる植物油、脂肪酸類、高級アルコール、シリコーン類、界面活性成分、水溶性合成高分子、増粘成分、粉体成分、保湿成分、紫外線吸収剤、紫外線遮蔽物、香料、金属キレート剤、ｐＨ調製剤などの公知の化粧料用成分を含有させることができる。さらには、抗炎症成分、活性酸素消去成分、血行促進成分、美白成分あるいは、その他の公知の添加剤である有効成分を配合することもできる。 The extract from which the solvent has been removed can be used directly or diluted with water or physiological saline to be used as a collagen production promoter or collagen gel contractor. In addition, it can be formulated by adding ingredients necessary for formulation such as excipients, stabilizers, and isotonic agents. It can also be pulverized by a drying operation such as freeze-drying. When the collagen production promoter and / or collagen gel shrinking agent thus obtained is used as a pharmaceutical or cosmetic, it can be used directly as a target agent, or can be blended by being dispersed and dissolved in an oil. It can be used as a wrinkle improving agent by blending into cosmetics. These cosmetic wrinkle improving agents include vegetable oils, fatty acids, higher alcohols, silicones, surface active ingredients, water-soluble synthetic polymers, thickening ingredients, powder ingredients, moisturizing ingredients, UV absorbing agents used in ordinary cosmetics. Well-known cosmetic ingredients such as agents, ultraviolet shielding materials, fragrances, metal chelating agents, and pH adjusting agents can be contained. Furthermore, an active ingredient which is an anti-inflammatory component, an active oxygen scavenging component, a blood circulation promoting component, a whitening component, or other known additives can be blended.
化粧料や医薬部外品あるいは外用剤をしわ改善剤とする場合は、有機溶媒抽出物を乾燥重量換算で化粧料全質量の０．０００１〜５質量％、好ましくは０．００１〜１質量％配合することが適切である。食品や内服剤をしわ改善に用いる場合は一日あたり１００ｍｇ〜１０ｇを経口摂取する。
以下に実施例を示し、本発明を具体的に説明する。
When cosmetics, quasi-drugs, or external preparations are used as wrinkle improving agents, the organic solvent extract is 0.0001-5% by mass, preferably 0.001-1% by mass, based on the total mass of the cosmetics in terms of dry weight. It is appropriate to blend. When foods and internal medicines are used for wrinkle improvement, 100 mg to 10 g per day is taken orally.
Hereinafter, the present invention will be specifically described with reference to examples.
[マルメロ種子のエタノール抽出物の調製]
マルメロの種子の乾燥物を高速ブレンダ―で粉状に粉砕し、粉砕物１０ｇを５００ｍｌの１００％エタノール（含水率０．５％以下）中で常法に従って還流冷却器を付して３日間還流抽出し、抽出物を回収した。抽出物は２．７０ｇで、抽出率は２７％であった。
[Preparation of quince seed ethanol extract]
The dried material of quince seeds was pulverized into powder with a high-speed blender, and 10 g of the pulverized product was refluxed in 500 ml of 100% ethanol (water content of 0.5% or less) with a reflux condenser according to a conventional method for 3 days. Extract and collect the extract. The extract was 2.70 g, and the extraction rate was 27%.
[マルメロ種子のエタノール抽出物のコラーゲン産生促進効果の測定]
（Ａ）測定方法
上記抽出物の添加時と非添加時の線維芽細胞のコラーゲン産生量を比較することによりコラーゲンの産生促進作用を評価した。
１．サンプル
試験区はマルメロ種子エタノール抽出物を添加したサンプル群、アスコルビン酸２グルコシドを添加した陽性対照群、マルメロ種子エタノール抽出物、アスコルビン酸２グルコシドのいずれも添加していないコントロール群を設定し、各サンプルを任意の濃度、１％ペニシリンストレプトマイシン添加ＤＭＥＭ培地（invitrogen 11995-073）に溶解し、終濃度０．５％ＤＭＳＯを添加したものを培養溶液とした。マルメロ種子エタノール抽出物を添加したサンプル群およびアスコルビン酸２グルコシドを添加した陽性対照群は、終濃度６２．５、１２５、２５０、５００μｇ／ｍｌの各４段階の濃度とした。
[Measurement of collagen production promotion effect of ethanol extract of quince seed]
(A) Measurement method Collagen production promoting action was evaluated by comparing the amount of collagen produced by fibroblasts when the extract was added and when it was not added.
1. In the sample test group, a sample group to which quince seed ethanol extract was added, a positive control group to which ascorbic acid 2-glucoside was added, a control group to which none of quince seed ethanol extract and ascorbic acid 2-glucoside was added, Samples were dissolved in DMEM medium (invitrogen 11995-073) supplemented with an arbitrary concentration of 1% penicillin streptomycin, and a culture solution was added with a final concentration of 0.5% DMSO. The sample group to which the quince seed ethanol extract was added and the positive control group to which ascorbic acid 2-glucoside was added had concentrations of 4 stages of final concentrations of 62.5, 125, 250, and 500 μg / ml.
２．培養方法
新生児ヒト***由来線維芽細胞（ＰＤＬ：１８）を用いて実験を行った。９６ウェルプレートに細胞を１×１０４個／ウェルの密度で播種し、１０％の牛胎児血清と１％ペニシリンストレプトマイシンを加えたＤＭＥＭ培地（invitrogen 11995-073）２００μｌで９０％コンフレントになるまで７２時間、３７℃５％ＣＯ２下で前培養を行った。
前培養後、細胞は、ＰＢＳ-で洗浄後、それぞれ調整していたサンプル群、陽性対照群、コントロール群の培地を２００μｌ添加し、３７℃５％ＣＯ2下で４８時間培養を行った。
2. Culture Method Experiments were performed using neonatal human foreskin-derived fibroblasts (PDL: 18). Cells are seeded at a density of 1 × 10 4 cells / well in a 96-well plate, and 200 μl of DMEM medium (invitrogen 11995-073) supplemented with 10% fetal bovine serum and 1% penicillin streptomycin until 72% until it becomes 90% confluent. Pre-culture was performed at 37 ° C. under 5% CO 2 for a period of time.
After pre-culture, cells, PBS - washed, each adjusted to have a sample group, positive control group, the medium of the control group was added 200μl with were cultured for 48 hours under 37 ℃ 5% CO 2.
３．コラーゲン産生量測定
培養終了後、ポリプロピレン製の９６ウェルプレート（nunc 267245）に培地を全量回収し、-３０℃で保管した。培地を取り除いた細胞はＭＴＴアッセイに供した。
線維芽細胞が産生したコラーゲン量は、培地中の１型コラーゲンＣ末ペプチド量に比例することが知られている。そこで、培地中に溶解した１型プロコラーゲンＣ末ペプチドの量を測定し、コラーゲン産生量を算出した。１型コラーゲンＣ末ペプチドの量はPIPEIAキット（タカラバイオ MK101）ELISA法で測定した。測定の際、保存していた培養上清はＰＢＳ-により１０倍に希釈した。コントロール群の培地上清の１型コラーゲンの量をＡ、サンプル群および陽性対照群の各濃度下における培地上清の１型コラーゲン量をＡｓとして、コントロール群の培地上清の１型コラーゲンの量を１００％としたときのコラーゲン産生量の比率を下記の式より算出した。
コラーゲン産生量の比率（％ of control）＝Ａｓ／Ａ×１００
3. Measurement of collagen production After completion of the culture, the entire medium was collected in a 96-well plate (nunc 267245) made of polypropylene and stored at -30 ° C. Cells from which the medium was removed were subjected to the MTT assay.
It is known that the amount of collagen produced by fibroblasts is proportional to the amount of type 1 collagen C-terminal peptide in the medium. Therefore, the amount of type 1 procollagen C-terminal peptide dissolved in the medium was measured, and the amount of collagen produced was calculated. The amount of type 1 collagen C-terminal peptide was measured by the PIPEIA kit (Takara Bio MK101) ELISA method. At the time of measurement, the stored culture supernatant was diluted 10-fold with PBS − . The amount of type 1 collagen in the culture supernatant of the control group, where A is the amount of type 1 collagen in the culture supernatant of the control group, and As is the amount of type 1 collagen in the culture supernatant at each concentration of the sample group and positive control group The ratio of the amount of collagen production was calculated from the following formula when the ratio was 100%.
Collagen production ratio (% of control) = As / A × 100
４．ＭＴＴアッセイ
マルメロ抽出物の毒性評価を細胞生存率で評価した。細胞生存率はＭＴＴアッセイにより求めた。培地を取り除いたプレートにＭＴＴ０．５ｍｇ／ｍｌを含んだＤＭＥＭ培地１００μｌを添加し、３７℃で３時間インキュベートした。インキュベート後、培地を取り除き、イソプロパノールを１００μl添加し１５分撹拌しながらホルマザンを溶解した。マイクロプレートリーダーにて溶解液の５７０nmと６３０nmの波長における吸光度を測定し、その吸光度の差をＭＴＴ定量値とした。コントロール群のＭＴＴ定量値をＢ、サンプル群および陽性対照群の各濃度下のＭＴＴ定量値をＢｓとして、細胞生存率を下記式より算出した。
細胞生存率（％）＝Ｂｓ／Ｂ×１００
4). MTT assay Toxicity evaluation of quince extract was evaluated by cell viability. Cell viability was determined by MTT assay. 100 μl of DMEM medium containing 0.5 mg / ml of MTT was added to the plate from which the medium had been removed, and incubated at 37 ° C. for 3 hours. After incubation, the medium was removed, 100 μl of isopropanol was added, and formazan was dissolved while stirring for 15 minutes. The absorbance of the lysate at wavelengths of 570 nm and 630 nm was measured with a microplate reader, and the difference in absorbance was used as the MTT quantitative value. The cell viability was calculated from the following equation, where B was the MTT quantitative value of the control group and Bs was the MTT quantitative value under each concentration of the sample group and the positive control group.
Cell viability (%) = Bs / B × 100
５．統計処理
サンプル、陽性対照はすべて３点ずつ測定した（ｎ＝３）。コラーゲン産生量の比率、細胞生存率ともに分散分析によって統計処理し、コントロール群の値を対照としてDunnettの検定により５％有意水準でサンプル添加時の値を比較した。
5. Statistical processing All samples and positive controls were measured in triplicate (n = 3). Both the ratio of collagen production and cell viability were statistically processed by analysis of variance, and the values at the time of sample addition were compared at 5% significance level by Dunnett's test using the control group values as controls.
６．結果
コラーゲン産生能の測定結果、細胞毒性の評価結果を図１に示す。
アスコルビン酸２グルコシド、マルメロ種子エタノール抽出物のコラーゲン産生量の比率はともに濃度依存的に増加し、マルメロ種子エタノール抽出物では、５００μg／ｍｌ添加時に有意差が認められた。
6). Results Measurement results of collagen production ability and cytotoxicity evaluation results are shown in FIG.
Ascorbic acid 2-glucoside and the ratio of collagen production of quince seed ethanol extract increased in a concentration-dependent manner, and in quince seed ethanol extract, a significant difference was observed when 500 μg / ml was added.
細胞生存率は、アスコルビン酸２グルコシドでは０−５００μｇ／ｍｌの濃度下では差は認められなかった。マルメロ種子エタノール抽出物では、１２５μｇ／ｍｌおよび２５０μg／ｍｌ添加時に若干の低下が認められるものの、５００μｇ／ｍｌではサンプル無添加（コントロール）と差が認められないことからマルメロ種子エタノール抽出物による毒性はないと判断した。 As for cell viability, no difference was observed in the concentration of 0-500 μg / ml for ascorbic acid 2-glucoside. In the quince seed ethanol extract, although a slight decrease was observed when 125 μg / ml and 250 μg / ml were added, there was no difference from the sample-free addition (control) at 500 μg / ml. Judged not.
以上の結果から、マルメロ種子エタノール抽出物は、細胞の生存率に大きな影響を与えることなくコラーゲン産生量を増加することから、安全なコラーゲン産生促進剤として利用可能であることが明らかとなった。 From the above results, it was clarified that the quince seed ethanol extract can be used as a safe collagen production promoter because it increases the amount of collagen production without greatly affecting the cell viability.
[コラーゲンゲル法による、マルメロ種子エタノール抽出物のコラーゲンゲル収縮作用の評価]
真皮の状態を再現した皮膚モデルとして、線維芽細胞を1型コラーゲンゲルに包埋したコラーゲンゲルが知られている。コラーゲンゲルはゲル内の線維芽細胞の産生するコラーゲンとそれにより形成されるコラーゲン繊維によって、真皮に類似した状態となる。このコラーゲンゲルは、アクチンやインテグリンなどの作用によりゲルに張力がかかり収縮する。生体内においても、同様のメカニズムによって、皮膚のハリが維持される。そこで、コラーゲンゲルを用いてしわ、ハリを改善する指標とすることができる（非特許文献２）。この評価系を用いてシワの改善を評価した。
[Evaluation of collagen gel contraction of quince seed ethanol extract by collagen gel method]
As a skin model that reproduces the state of the dermis, a collagen gel in which fibroblasts are embedded in type 1 collagen gel is known. Collagen gel becomes a state similar to the dermis by collagen produced by fibroblasts in the gel and collagen fibers formed thereby. This collagen gel contracts due to the tension of the gel due to the action of actin, integrin and the like. In the living body, the firmness of the skin is maintained by a similar mechanism. Therefore, it can be used as an index for improving wrinkles and firmness using a collagen gel (Non-patent Document 2). Using this evaluation system, wrinkle improvement was evaluated.
１．コラーゲンゲル収縮作用の評価方法
線維芽細胞（ＰＤＬ２０）を包埋したコラーゲンゲルの収縮率を本発明の抽出物の添加時と非添加時で比較し、コラーゲンゲル収縮作用を評価した。
1. Collagen gel contraction action evaluation method Collagen gel contraction action was evaluated by comparing the contraction rate of collagen gel in which fibroblasts (PDL20) were embedded with and without addition of the extract of the present invention.
１．１ 細胞包埋コラーゲンゲルの作成並びに培養方法
細胞を包埋したコラーゲンゲルは、コラーゲンゲルキット（新田ゼラチン）によって作成した。氷冷下で、CellmatrixTipe1-A(Ａ液)（新田ゼラチン）を終濃度２ｍｇ／ｍｌ（添加量１６ｍｌ）、１０倍濃度ＭＥＭハンクス（Ｂ液）（新田ゼラチン）を終濃度１倍（添加量２ｍｌ）、１倍濃度ＭＥＭハンクスで調整した新生児ヒト***由来線維芽細胞（ＰＤＬ：２０）の細胞懸濁液４．２×１０4ｃｅｌｌｓ／ｍｌ(終濃度)を泡を立てないように混合した後、２ｍｌの再構成用緩衝液（Ｃ液）を混合し、細胞包埋コラーゲンゲルの溶液を作成した。温度が上昇しないようにしながら、作成した細胞包埋コラーゲンゲルの溶液を１２ウェルマルチプレート（住友ベークライト）に８００μｌ添加した。添加終了後、３７℃５％ＣＯ２条件下で２時間インキュベートし、細胞包埋コラーゲンゲルの溶液を完全にゲル化した。
メスおよびスパチュラを用いて、ゲル化したコラーゲンゲルの縁と底面をウェルプレートより剥離した。剥離時のゲル面積をサンプル添加前のゲル面積とし、この時のゲル面積はウェル底面積と同じ３．８ｃｍ2であった。剥離したゲルを入れたウェルプレートにジメチルスルホキシドに各種の濃度で溶解したサンプル（マルメロ種子エタノール抽出物）を１ｍｌ、１％ペニシリンストレプトマイシン添加ＤＭＥＭ培地（invitrogen11995-073）を１ｍｌ添加し、コラーゲンゲルを５日間浮遊させて培養した。この時、培地中に含まれるＤＭＳＯの濃度は０．５％とし、サンプル濃度は０、２０、１００、５００μｇ／ｍｌの４段階を設定した。
浮遊培養後、コラーゲンゲルを、常温で１０％ホルマリン中に２４時間浸漬し、固定した。
コラーゲンゲルの写真を撮影し、Image Jによって画像解析してサンプル添加培養後のコラーゲンゲルの面積を測定した。
次式により、コラーゲンゲルの収縮率を求めた。
ゲル収縮率（％）＝１００-Ａｓ／Ａ×100
Ａ：サンプル添加前のコラーゲンゲルの面積 （３．８ｃｍ２）
Ａｓ：サンプルを添加し、５日間浮遊培養後のコラーゲンゲルの面積
ゲルの収縮状態を撮影した画像を図２に示す。
1.1 Preparation of cell-embedded collagen gel and culture method Collagen gel in which cells were embedded was prepared using a collagen gel kit (Nitta Gelatin). Under ice-cooling, CellmatrixTipe1-A (A solution) (Nitta gelatin) final concentration 2 mg / ml (added amount 16 ml), 10-fold MEM Hanks (B solution) (Nitta gelatin) final concentration 1 time (added) (2 ml in volume) Cell suspension of neonatal human foreskin-derived fibroblasts (PDL: 20) adjusted with 1-fold concentration MEM Hanks 4.2 × 10 4 cells / ml (final concentration) mixed without foaming After that, 2 ml of a reconstitution buffer (solution C) was mixed to prepare a cell-embedded collagen gel solution. While preventing the temperature from rising, 800 μl of the prepared cell-embedded collagen gel solution was added to a 12-well multiplate (Sumitomo Bakelite). After completion of the addition, the cells were incubated at 37 ° C. under 5% CO 2 for 2 hours to completely gel the cell-embedded collagen gel solution.
Using a scalpel and a spatula, the edges and bottom of the gelled collagen gel were peeled from the well plate. The gel area at the time of peeling was defined as the gel area before adding the sample, and the gel area at this time was 3.8 cm 2 which was the same as the well bottom area. To the well plate containing the exfoliated gel, 1 ml of a sample dissolved in dimethyl sulfoxide at various concentrations (malmelloseed ethanol extract), 1 ml of 1% penicillin streptomycin-added DMEM medium (invitrogen 11995-073), and 5 collagen gels were added. The culture was suspended for days. At this time, the concentration of DMSO contained in the medium was set to 0.5%, and the sample concentrations were set in four stages of 0, 20, 100, and 500 μg / ml.
After suspension culture, the collagen gel was fixed by being immersed in 10% formalin for 24 hours at room temperature.
A photograph of the collagen gel was taken, image analysis was performed using Image J, and the area of the collagen gel after the sample addition culture was measured.
The shrinkage ratio of the collagen gel was determined by the following formula.
Gel shrinkage (%) = 100−As / A × 100
A: Area of collagen gel before sample addition (3.8 cm 2 )
As: Area of collagen gel after addition of sample and suspension culture for 5 days An image obtained by photographing the contraction state of the gel is shown in FIG.
１．３ 統計処理
サンプルはすべて３点ずつ測定した（ｎ＝３）。分散分析によって統計処理し、サンプル濃度０μｇ／ｍｌのときのゲル収縮率を対照として、Dunnettの検定により５％有意水準でサンプル濃度２０、１００、５００μｇ／ｍｌのときのゲル収縮率を比較した。ゲルの収縮率を評価した結果を図３に示す。
1.3 Statistical processing All samples were measured in triplicate (n = 3). Statistical analysis was performed by analysis of variance, and the gel contraction rate at a sample concentration of 0 μg / ml was used as a control, and the gel contraction rate at a sample concentration of 20, 100, 500 μg / ml was compared at a 5% significance level by Dunnett's test. The result of evaluating the shrinkage rate of the gel is shown in FIG.
２．結果
培養期間中、ゲル中で正常な細胞増殖が認められた。
サンプル濃度０μｇ／ｍｌを添加したときのコラーゲンゲルの収縮率は３３．４±１．５％であった。
サンプル濃度２０、１００、５００μｇ／ｍｌでは、抽出物の濃度依存的にコラーゲンゲルは収縮した。１００μｇ／ｍｌ添加した場合の収縮率は４１．４±１．９％であり、サンプル濃度０μｇ／ｍｌのときと比べて有意水準５％の差が認められた。５００μｇ／ｍｌ添加した場合の収縮率は４７．４±３．２％であり、サンプル濃度０μｇ／ｍｌのときと比べて有意水準１％の差が認められた。
マルメロ種子エタノール抽出物は、高濃度で添加しても細胞毒性を示さず、コラーゲンゲル収縮能を有することが明らかとなった。したがって、マルメロ種子エタノール抽出物は皮膚のたるみやしわを予防し、改善すると考えられる。
2. Results Normal cell growth was observed in the gel during the culture period.
The contraction rate of the collagen gel when the sample concentration was 0 μg / ml was 33.4 ± 1.5%.
At sample concentrations of 20, 100, and 500 μg / ml, the collagen gel contracted depending on the concentration of the extract. When 100 μg / ml was added, the contraction rate was 41.4 ± 1.9%, and a significant level difference of 5% was observed compared to the sample concentration of 0 μg / ml. When 500 μg / ml was added, the contraction rate was 47.4 ± 3.2%, and a significant level difference of 1% was observed compared to the sample concentration of 0 μg / ml.
It was revealed that quince seed ethanol extract did not show cytotoxicity even when added at a high concentration, and had collagen gel contractility. Therefore, quince seed ethanol extract is considered to prevent and improve skin sagging and wrinkles.
以下にマルメロ抽出物を配合したしわ改善剤の処方例を示す。
［処方例１］クリーム
下記の処方（単位は重量％）により、クリームを製造した。
（１） ステアリルアルコール ６．０
（２） ステアリン酸 ２．０
（３） 水添ラノリン ４．０
（４） スクワラン ９．０
（５） オクチルドデカノール １０．０
（６） ＰＯＥ（２５）セチルアルコールエーテル ３．０
（７） モノステアリン酸グリセリン ２．０
（８） マルメロ種子エタノール抽出物 ０．１
（９） 防腐剤 適量
（１０）香料 適量
（１１）１，３ブチレングリコール ６．０
（１２）ＰＥＧ１５００ ４．０
（１３）精製水 残余
〔製法〕上記成分（１）〜（１０）を８０℃に加熱溶解し油相とする。成分（１１）〜（１３）を７０℃に加熱溶解し水相とする。油相に水相を徐々に加え乳化し、攪拌しながら４０℃まで冷却し、さらに３０℃まで攪拌冷却してクリームを得た。
A prescription example of a wrinkle improving agent containing quince extract is shown below.
[Prescription Example 1] Cream A cream was produced according to the following formulation (unit:% by weight).
(1) Stearyl alcohol 6.0
(2) Stearic acid 2.0
(3) Hydrogenated lanolin 4.0
(4) Squalane 9.0
(5) Octyldodecanol 10.0
(6) POE (25) cetyl alcohol ether 3.0
(7) Glycerin monostearate 2.0
(8) Quince seed ethanol extract 0.1
(9) Preservative appropriate amount (10) perfume appropriate amount (11) 1,3 butylene glycol 6.0
(12) PEG 1500 4.0
(13) Purified water Residue [Production method] The above components (1) to (10) are dissolved by heating at 80 ° C. to obtain an oil phase. Ingredients (11) to (13) are heated and dissolved at 70 ° C. to obtain an aqueous phase. The aqueous phase was gradually added to the oil phase to emulsify, cooled to 40 ° C. with stirring, and further cooled to 30 ° C. with stirring to obtain a cream.
［実施例２］錠剤
下記の処方（単位は重量％）により、錠剤を製造した。
（１）マルメロ種子エタノール抽出物 ２０．０
（２）乳糖 ６５．０
（３）コーンスターチ １４．０
（４）グアーガム １．０
[Example 2] Tablets Tablets were produced according to the following formulation (unit: wt%).
(1) Quince seed ethanol extract 20.0
(2) Lactose 65.0
(3) Cornstarch 14.0
(4) Guar gum 1.0
［処方例３］乳液
下記の処方（単位は重量％）により、乳液を製造した。
（１）ジプロピレングリコール ９．０００
（２）マルメロ種子エタノール抽出物 １．０００
（３）（ヒドロキシエチルアクリル酸／アクリルジメチルタウリンＮａ）
コポリマー ０．１８８
（４）スクワラン ０．１２７
（５）ポリソルベート６０ ０．０２８
（６）ラウロイルグルタミン酸ジ（フィトステリル／オクチルドデシル） １．０００
（７）グリセリン ５．０００
（８）ジメチコン ３．０００
（９）精製水 ７４．７４２
（１０）カルボマー ０．２００
（１１）ベタイン ２．０００
（１２）エタノール ３．０００
（１３）水酸化カリウム ０．０６５
（１４）精製水 ０．６５０
〔製法〕上記成分（１）に（２）を加え８０℃に加熱溶解する。成分（３）〜（８）を加え、８０℃に加熱溶解し油相とする。成分（９）〜（１１）を７０℃に加熱溶解し水相とする。油相に水相を徐々に加え乳化し、攪拌しながら３０℃まで冷却する。成分（１２）および（１３）を（１４）に攪拌溶解したものを加え、攪拌冷却して乳液を得た。
[Prescription Example 3] Emulsion An emulsion was prepared according to the following formulation (unit: wt%).
(1) Dipropylene glycol 9.000
(2) Quince seed ethanol extract 1.000
(3) (Hydroxyethyl acrylic acid / acryl dimethyl taurine Na)
Copolymer 0.188
(4) Squalane 0.127
(5) Polysorbate 60 0.028
(6) Lauroyl glutamate di (phytosteryl / octyldodecyl) 1.000
(7) Glycerin 5.000
(8) Dimethicone 3000
(9) Purified water 74.742
(10) Carbomer 0.200
(11) Betaine 2.000
(12) Ethanol 3.000
(13) Potassium hydroxide 0.065
(14) Purified water 0.650
[Production Method] Add (2) to the above component (1) and dissolve by heating at 80 ° C. Components (3) to (8) are added and heated and dissolved at 80 ° C. to obtain an oil phase. Components (9) to (11) are heated and dissolved at 70 ° C. to obtain an aqueous phase. The aqueous phase is gradually added to the oil phase to emulsify, and cooled to 30 ° C. with stirring. Components (12) and (13) dissolved in (14) with stirring were added and stirred and cooled to obtain an emulsion.
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