JP4698934B2 - Skin collagen production promoter - Google Patents
Skin collagen production promoter Download PDFInfo
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- JP4698934B2 JP4698934B2 JP2003129509A JP2003129509A JP4698934B2 JP 4698934 B2 JP4698934 B2 JP 4698934B2 JP 2003129509 A JP2003129509 A JP 2003129509A JP 2003129509 A JP2003129509 A JP 2003129509A JP 4698934 B2 JP4698934 B2 JP 4698934B2
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- Prior art keywords
- lactoperoxidase
- collagen production
- skin
- skin collagen
- promoting
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Description
【０００１】
【発明の属する技術分野】
本発明は、皮膚の荒れ、シワ、弾性低下等を防止するのに有用な皮膚コラーゲン産生促進剤、皮膚コラーゲン産生促進用飲食品及び皮膚コラーゲン産生促進用化粧料に関する。さらに詳しくは、本発明は、ラクトパーオキシダーゼ及び／またはラクトパーオキシダーゼをタンパク質分解酵素で分解して得られるラクトパーオキシダーゼ分解物を有効成分とする皮膚コラーゲン産生促進剤に関する。
【０００２】
【従来の技術】
近年、皮膚のメカニズムに関する研究が進められ、皮膚の乾燥感や肌荒れ等の原因としてマクロ的には、加齢による新陳代謝の減衰によるもののほかに、太陽光(紫外線)、乾燥、酸化等の作用が複雑に関与していることが確認されてきた。これらの因子による作用によって、真皮の最も主要なマトリックス成分であるコラーゲン繊維が顕著に減少していることが明らかとなってきた。コラーゲン繊維によって保たれていた皮膚のハリや弾力性といった張力保持機構が紫外線等の作用によって、破壊されると、皮膚はシワやたるみを増した状態になる。また、コラーゲンはその分子中に水分を保持することができ、それにより、皮膚をしっとりとした状態に保つことにも役立っているから、外的因子により、コラーゲンが破壊されると肌は、乾燥し、荒れた状態になる。
以上のことから、真皮層の主要な成分の一つであるコラーゲンの生合成を促進させることにより、皮膚のシワやたるみを防止でき、しかも安全性の点でも問題のない皮膚コラーゲン産生促進剤が望まれていた。
【０００３】
ラクトパーオキシダーゼは、乳中に存在し、ヘム鉄を含む糖タンパク質であるが、その詳細な構造はいまだ明らかにされていない。ラクトパーオキシダーゼの利用については、生体内で過酸化脂質の生成抑制効果があることを見いだし、視力低下、運動能力低下、免疫機能低下等を防ぐ老化防止剤とした例（特許文献１参照。）、肝機能改善剤として利用した例（特許文献２参照。）、低う触作用があることから低う触栄養組成物として利用した例（特許文献３参照。）が開示されている。しかし、ラクトパーオキシダーゼ及びそのタンパク質分解酵素で分解して得られる酵素分解物が皮膚コラーゲン産生促進作用を持ち、皮膚コラーゲン産生促進剤として有用であることについては知られていない。
【０００４】
【先行技術文献】
【特許文献１】
特開平５−１２４９８０号公報
【特許文献２】
特開２００１−２２６２８９号公報
【特許文献３】
特開平９−１０７９１７号公報
【０００５】
【発明が解決しようとする課題】
本発明は、皮膚の乾燥感や肌荒れ、しわやタルミを防止でき、しかも安全性の点でも問題のない、コラーゲンの生合成を促進する物質を有効成分とする皮膚コラーゲン産生促進剤を提供することを課題とする。また、本発明は、そのような物質を配合した皮膚コラーゲン産生促進用飲食品及び皮膚コラーゲン産生促進用化粧料を提供することを課題とする。
【０００６】
【課題を解決するための手段】
本発明者らは、これらの課題を解決するために、広く食品素材に含まれている皮膚コラーゲン産生促進作用を示す物質について、鋭意、探索を進めていたところ、ラクトパーオキシダーゼあるいはそのラクトパーオキシダーゼをペプシンやパンクレアチンなどのタンパク質分解酵素で分解して得られるラクトパーオキシダーゼ分解物が、皮膚のコラーゲン量を増加させることができる、すなわち、コラーゲン産生促進作用があることを見出した。そして、このラクトパーオキシダーゼやラクトパーオキシダーゼ分解物を皮膚コラーゲン産生促進剤、皮膚コラーゲン産生促進用飲食品及び皮膚コラーゲン産生促進用化粧料の有効成分として利用できることを見出し、本発明を完成するに至った。
【０００７】
したがって、本発明は、この皮膚コラーゲン産生促進活性を有するラクトパーオキシダーゼ及び／またはラクトパーオキシダーゼをタンパク質分解酵素で分解して得られるラクトパーオキシダーゼ分解物を有効成分とする皮膚コラーゲン産生促進剤に関する。また、本発明は、これらのラクトパーオキシダーゼ及び／またはラクトパーオキシダーゼ分解物を配合した皮膚コラーゲン産生促進用飲食品や皮膚コラーゲン産生促進用化粧料に関する。
【０００８】
本発明で皮膚コラーゲン産生促進剤とは、経口投与あるいは塗布等により、皮膚コラーゲン産生促進効果を発揮するものを言うものとする。また、本発明では、この皮膚コラーゲン産生促進剤のうち、粉末剤、顆粒剤、錠剤、カプセル剤、ドリンク剤などに製剤化してそのまま、あるいは製剤化した後、栄養剤や飲食品などに配合して経口投与するものを皮膚コラーゲン産生促進用飲食品と言うものとする。また、本発明では、皮膚コラーゲン産生促進剤のうち、軟膏、ゲル、クリーム、スプレー剤、貼付剤、ローション等に製剤化して肌に塗布するものを皮膚コラーゲン産生促進用化粧料と言うものとする。また、本発明では、医薬品であるが、製剤化してそのまま経口投与するものを便宜上、前記皮膚コラーゲン産生促進用飲食品の中に含め、医薬品であるが、製剤化して肌に塗布するものを便宜上、前記皮膚コラーゲン産生促進用化粧料の中に含めるものとする。
【０００９】
【発明の実施の形態】
本発明の皮膚コラーゲン産生促進剤の特徴は、ラクトパーオキシダーゼ及び／またはラクトパーオキシダーゼをタンパク質分解酵素で分解して得られるラクトパーオキシダーゼ分解物を有効成分とすることにある。
【００１０】
ラクトパーオキシダーゼは哺乳動物の乳から調製する。給源としては、ウシ、水牛、ヒト、ブタ、ヒツジ、ヤギ、ウマ等の乳があげられる。ラクトパーオキシダーゼは、公知の物質であって、市販されているものであるが、それを製造するには、公知の方法、例えばスルホン化担体を用いてラクトパーオキシダーゼを精製する方法（特開平3-109400号公報）を工業的に有利に利用することができる。また、本発明では、遺伝子工学的手法により生産されたラクトパーオキシダーゼも使用し得る。
【００１１】
ラクトパーオキシダーゼ分解物は、上記のラクトパーオキシダーゼをトリプシン、パンクレアチン、キモトリプシン、ペプシン、パパイン、カリクレイン、カテプシン、サーモライシン、Ｖ８プロテアーゼ等のタンパク質分解酵素で分子量が10,000以下となるように限定分解したペプチド混合物である。分子量下限は500以上が好ましい。
【００１２】
本発明の皮膚コラーゲン産生促進剤は、経口投与あるいは塗布することにより、皮膚コラーゲン産生促進効果を発揮する。
本発明の皮膚コラーゲン産生促進剤を経口投与するに際しては、有効成分であるラクトパーオキシダーゼあるいはラクトパーオキシダーゼ分解物をそのままの状態で用いることもできるが、常法に従い、粉末剤、顆粒剤、錠剤、カプセル剤、ドリンク剤などに製剤化して用いることもできる。本発明において、粉末剤、顆粒剤、錠剤、カプセル剤等の経口剤は、例えば、澱粉、乳糖、白糖、マンニット、カルボキシメチルセルロース、コーンスターチ、無機塩類等を用いて常法によって製剤化される。この種の製剤には、前記賦形剤の他に、結合剤、崩壊剤、界面活性剤、滑沢剤、流動性促進剤、着色料、香料等を適宜使用することが出来る。より具体的には、結合剤としては、例えば、澱粉、デキストリン、アラビアガム末、ゼラチン、ヒドロキシプロピルスターチ、カルボキシメチルセルロースナトリウム、メチルセルロース、結晶性セルロース、エチルセルロース、ポリビニルピロリドンが挙げられる。また、崩壊剤としては、例えば、澱粉、ヒドロキシプロピルスターチ、カルボキシメチルセルロースナトリウム、架橋カルボキシメチルセルロースナトリウム、結晶性セルロース、カルボキシメチルセルロース等が挙げられる。界面活性剤としては、大豆レシチン、蔗糖脂肪酸エステル等が、滑沢剤としては、タルク、ロウ、蔗糖脂肪酸エステル、水素添加植物油等が、流動性促進剤としては無水ケイ酸、乾燥水酸化アルミニウム、ケイ酸マグネシウムなどが挙げられる。
【００１３】
さらには、これらのラクトパーオキシダーゼやラクトパーオキシダーゼ分解物をそのままあるいは製剤化した後、これを栄養剤や飲食品などに配合することも可能である。また、ビタミンＣ等従来からコラーゲン産生に有効な作用を持つと考えられている成分とともにラクトパーオキシダーゼやラクトパーオキシダーゼ分解物を配合すれば、一層の皮膚コラーゲン産生促進作用が期待できる。なお、ラクトパーオキシダーゼあるいはラクトパーオキシダーゼ分解物は、比較的熱に対して安定であるので、ラクトパーオキシダーゼあるいはラクトパーオキシダーゼ分解物を含む原料を通常行われるような条件で加熱殺菌することも可能である。
【００１４】
本発明の皮膚コラーゲン産生促進剤を塗布するに際しては、その使用目的に応じて、通常用いられる公知の成分に配合することによって、液剤、固形剤、半固形剤等の各種剤形に調製することが可能で、好ましい組成物として軟膏、ゲル、クリーム、スプレー剤、貼付剤、ローション、粉末等が挙げられる。例えば、本発明の皮膚コラーゲン産生促進剤をワセリン等の炭化水素、ステアリルアルコール、ミリスチン酸イソプロピル等の高級脂肪酸低級アルキルエステル、ラノリン等の動物性油脂、グリセリン等の多価アルコール、グリセリン脂肪酸エステル、モノステアリン酸ポリエチレングリコール等の界面活性剤、無機塩、ロウ、樹脂、水及び要すればパラオキシ安息香酸メチル、パラオキシ安息香酸ブチル等の保存料に混合することによって、皮膚コラーゲン産生促進用化粧料や医薬品を製造することができる。
【００１５】
本発明の皮膚コラーゲン産生促進剤の経口投与による有効量は、その製剤形態、投与方法、使用目的、及びこれを適用される患者の年齢、体重、病状により適宜規定され一定でないが、ラットを用いた動物実験の結果によると、皮膚コラーゲン産生促進作用を示すためにはラクトパーオキシダーゼあるいはラクトパーオキシダーゼ分解物をラット体重1kg当たり20mg以上摂取する必要があることが判った。したがって、外挿法によると、通常、成人一人当たり一日20mg以上のラクトパーオキシダーゼあるいはラクトパーオキシダーゼ分解物を摂取すれば効果が期待できるので、この必要量を確保できるよう飲食品に配合するか、あるいは、医薬として投与すれば良い。なお、投与は必要に応じて一日数回に分けて行うことも可能である。
【００１６】
本発明の皮膚コラーゲン産生促進剤の塗布による有効量は、剤形により異なるが、適用する組成物全量を基準として、好ましくは、0.001〜2重量％となるように、ラクトパーオキシダーゼあるいはラクトパーオキシダーゼ分解物を配合すればよい。ただし、入浴剤のように使用時に希釈されるものは、さらに配合量を増やすことができる。
【００１７】
次に、実施例及び試験例を示して本発明を詳細に説明するが、これらは単に本発明の実施態様を例示するのみであり、本発明はこれらによって何ら限定されるものではない。
【００１８】
【実施例１】
陽イオン交換樹脂であるスルホン化キトパール（富士紡績社製）400gを充填したカラム（直径5cm×高さ30cm）を脱イオン水で十分に洗浄した後、このカラムに未殺菌脱脂乳40 l (pH 6.7)を流速25ml/minで通液した。通液後、カラムを脱イオン水で十分洗浄し、 2.0M塩化ナトリウムを含む 0.02M炭酸緩衝液(pH 7.0)で溶出した。そしてラクトパーオキシダーゼを含有する溶出画分をS-Sepharose FFカラム（アマシャムバイオサイエンス社製）に吸着させ、脱イオン水で十分洗浄し、10mMリン酸緩衝液（pH7．0）で平衡化した後、0〜2.0M塩化ナトリウムのリニアグラジエントで吸着した画分を溶出し、ラクトパーオキシダーゼを含む画分を回収した。そしてその画分をHiLoad 16/60 Superdex 75 pg（アマシャムバイオサイエンス社製）を用いたゲル濾過クロマトグラフィーで処理し、ラクトパーオキシダーゼ3.0gを得た。なお、このようにして得られたラクトパーオキシダーゼの純度は94％であり、そのまま皮膚コラーゲン産生促進剤として使用可能である。
【００１９】
【実施例２】
実施例１で得られたラクトパーオキシダーゼ1gを水200mlに溶解し、最終濃度が0.01重量％となるようタンパク質分解酵素であるパンクレアチン（シグマ社製）を加え、37℃で5時間酵素処理した。そして、90℃で5分間加熱処理して酵素を失活させた後、凍結乾燥してラクトパーオキシダーゼ分解物 0.8gを得た。なお、このようにして得られたラクトパーオキシダーゼ分解物の分子量は、10,000以下であり、そのまま皮膚コラーゲン産生促進剤として使用可能である。
【００２０】
【実施例３】
実施例１で得られたラクトパーオキシダーゼ1gを水200mlに溶解し、最終濃度が0.01重量％となるようタンパク質分解酵素であるトリプシン（シグマ社製）を加え、37℃で5時間酵素処理した。そして、90℃で5分間加熱処理して酵素を失活させた後、凍結乾燥してラクトパーオキシダーゼ分解物 0.9gを得た。なお、このようにして得られたラクトパーオキシダーゼ分解物の分子量は、10,000以下であり、そのまま皮膚コラーゲン産生促進剤として使用可能である。
【００２１】
【試験例１】
実施例１で得られたラクトパーオキシダーゼ及び実施例２で得られたラクトパーオキシダーゼ分解物について、ラットを用いた動物実験によりコラーゲン産生促進作用を調べた。7週齢のWistar系雄ラットを、生理食塩水投与群（Ａ群）、実施例1で得られたラクトパーオキシダーゼをラット体重1kg当たり20mg投与する群（Ｂ群）、実施例1で得られたラクトパーオキシダーゼをラット体重1kg当たり200mg投与する群（Ｃ群）、実施例２で得られたラクトパーオキシダーゼ分解物をラット体重1kg当たり20mg投与する群（Ｄ群）、実施例２で得られたラクトパーオキシダーゼ分解物をラット体重1kg当たり200mg投与する群（Ｅ群）の5試験群（n=6）に分け、それぞれを毎日1回ゾンデで投与して10週間飼育した。
皮膚のコラーゲン量については、ラットの真皮をNimniらの方法(Arch. Biochem. Biophys, 292頁, 1967年 参照)に準じて処理した後、可溶性画分に含まれるヒドロキシプロリン量を測定した。ヒドロキシプロリンはコラーゲンのみに含まれる特殊なアミノ酸で、コラーゲンを構成する全アミノ酸の約10％を占めることからコラーゲン量の推定ができる(浅野隆司ら，Bio Industory，12頁, 2001年 参照)。その結果を表１に示す。
【００２２】
【表１】
──────────────────
群 ヒドロキシプロリン量(μg/ml)
──────────────────
Ａ群 0.3±0.1
Ｂ群 0.8±0.1※
Ｃ群 1.0±0.2※
Ｄ群 0.7±0.2※
Ｅ群 1.0±0.1※
──────────────────
数値は、平均値±標準偏差（n=6）を示す。
また、※は対照群であるＡ群と比較して有意差があることを示す（p<0.05）。
【００２３】
これによると、10週間後の可溶性画分中ヒドロキシプロリン量は、Ａ群に比べ、Ｂ群、Ｃ群、Ｄ群及びＥ群で有意に高い値を示した。
このことから、ラクトパーオキシダーゼ及びラクトパーオキシダーゼ分解物には皮膚コラーゲン産生促進作用があることが明らかとなり、皮膚コラーゲン産生促進剤として有用であることが示された。
また、この皮膚コラーゲン産生促進作用はラクトパーオキシダーゼまたはラクトパーオキシダーゼ分解物をラット体重1kg当たり最低20mg投与した場合に認められることが明らかとなった。
【００２４】
【実施例４】
表２に示す配合の皮膚コラーゲン産生促進用飲料を常法により製造した。製造した飲料の風味は良好で、常温1年間保存によっても風味が劣化することはなく、沈殿等の問題もなかった。
【００２５】
【表２】
───────────────────────────
混合異性化糖 15.0（重量％）
果汁 10.0
クエン酸 0.5
ラクトパーオキシダーゼ（実施例１） 0.1
香料 0.1
ミネラル混合 0.1
水 74.2
───────────────────────────
【００２６】
【実施例５】
表３に示す配合のドウを常法により作製し、成形した後、焙焼して皮膚コラーゲン産生促進用ビスケットを製造した。
【００２７】
【表３】
──────────────────────────
小麦粉 50.0（重量％）
砂糖 20.0
食塩 0.5
マーガリン 12.5
卵 12.5
水 2.5
ミネラル混合 0.8
ラクトパーオキシダーゼ（実施例１） 1.2
──────────────────────────
【００２８】
【実施例６】
表４に示す配合の皮膚コラーゲン産生促進剤を製造した。
【００２９】
【表４】
───────────────────────────
含水結晶ぶどう糖 83.5（重量％）
ラクトパーオキシダーゼ（実施例１） 10.0
ミネラル混合 5.0
シュガーエステル 1.0
香料 0.5
───────────────────────────
【００３０】
【試験例２】
市販のラクトパーオキシダーゼ（シグマ社製）及び実施例２と同様の方法で市販のラクトパーオキシダーゼをタンパク質分解酵素で処理して得られたラクトパーオキシダーゼ分解物について、正常ヒト線維芽細胞株〔白人女性の皮膚より採取されたCCD45SK(ATCCRL 1506)〕を用いた実験によりコラーゲン産生促進作用を調べた。10容量％ウシ胎児血清(以下FBSと略記)含有変法イーグル培地（MEM、10‐101、大日本製薬社製）を用いて、正常ヒト線維芽細胞株を4×104個/ウエル/0.4mlとなるように24ウエルプレートに播種して、5％炭酸ガス、飽和水蒸気下、37℃で24時間培養した後、0.6容量％FBS含有変法イーグル培地に置換した。そして、市販のラクトパーオキシダーゼ（シグマ社製）及び実施例２と同様の方法で市販のラクトパーオキシダーゼをタンパク質分解酵素で処理して得られたラクトパーオキシダーゼ分解物を、各ウエルに0.1容量％となるように添加（n=6）して、24時間培養した後、β―アミノプロピオニトリルを50μg/ml、トリチウム-L-プロリンを1μCi/mlとなるように添加して、さらに24時間培養して培養液を得た。このようにして得られた培養液より、Websterらの方法(Analytical Biochemistry,220頁,1979年 参照)に従いコラーゲン画分を分画し、コラーゲン画分に取り込まれた放射能を測定した。なお、対照として、ラクトパーオキシダーゼ及びラクトパーオキシダーゼ分解物を添加しないで同様の試験を行った。その結果を表５に示す。
【００３１】
【表５】
また、※は対照群と比較して有意差があることを示す（p<0.05）。
【００３２】
これによると、ラクトパーオキシダーゼ及びラクトパーオキシダーゼ分解物を添加した群は、ラクトパーオキシダーゼ及びラクトパーオキシダーゼ分解物を添加していない群（対照）に比べて2倍程度のコラーゲン産生促進能を示した。
このことから、ラクトパーオキシダーゼ及びラクトパーオキシダーゼ分解物には、皮膚線維芽細胞に働きかけ、コラーゲン産生促進作用があることが明らかとなり、皮膚コラーゲン産生促進剤として有用であることが示唆された。
【００３３】
【実施例７】
表６に示す配合の化粧水を常法により製造した。
【００３４】
【表６】
【実施例８】
表７に示す配合のクリームを常法により製造した。
【００３６】
【表７】
【試験例３】
実施例７で得られた化粧水及び実施例８で得られたクリームを用いて、実使用テストを行った。比較品としては、ラクトパーオキシダーゼを除いた以外は実施例７及び８と同じ配合のものを用いた。
顔面のたるみや小ジワが認められる乾燥肌を有する成人女子20人を、それぞれ10人ずつ無作為に2群（Ａ、Ｂ群）に、また、手に肌荒れが認められる女子20人を、それぞれ10人ずつ無作為に2群（Ｃ、Ｄ群）に分け、Ａ群の顔面には本発明品の化粧水を、Ｂ群の顔面には比較品の化粧水を、Ｃ群の手指には本発明品のクリームを、Ｄ群の手指には比較品のクリームを、それぞれ1日2回通常の使用状態と同様に10日間塗布してもらった。その結果、本発明品の化粧水は、比較品の化粧水に比べて、乾燥感の改善、肌荒れ等の改善が顕著であり、皮膚コラーゲン産生促進効果に優れていることが実証された。また、本発明品のクリームについても、比較品のクリームに比べて、乾燥感の改善、肌荒れに顕著な改善がみられ、肌荒れなどの自然増悪抑制効果を有することが明らかとなった。
【００３８】
【発明の効果】
本発明によりラクトパーオキシダーゼ及び／またはラクトパーオキシダーゼ分解物を有効成分とする皮膚コラーゲン産生促進剤、皮膚コラーゲン産生促進用飲食品及び皮膚コラーゲン産生促進用化粧料が提供される。
本発明の皮膚コラーゲン産生促進剤、皮膚コラーゲン産生促進用飲食品及び皮膚コラーゲン産生促進用化粧料は、皮膚のコラーゲン産生を促進させる作用を有し、皮膚のシワやたるみ、乾燥感や肌荒れの予防や治療に有用である。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a skin collagen production promoter, a food and drink for promoting skin collagen production, and a cosmetic for promoting skin collagen production, which are useful for preventing rough skin, wrinkles, a decrease in elasticity, and the like. More specifically, the present invention relates to a skin collagen production promoter containing as an active ingredient a lactoperoxidase and / or a lactoperoxidase degradation product obtained by degrading lactoperoxidase with a proteolytic enzyme.
[0002]
[Prior art]
In recent years, research on the mechanism of the skin has been promoted. Macroscopically, the effects of sunlight (ultraviolet rays), drying, oxidation, etc., in addition to the deterioration of metabolism due to aging, can be considered as causes of dryness and rough skin. It has been confirmed that it is involved in a complex manner. It has been clarified that collagen fibers, which are the main matrix components of the dermis, are significantly reduced by the action of these factors. When the tension retention mechanism such as skin elasticity and elasticity held by the collagen fibers is broken by the action of ultraviolet rays, the skin becomes wrinkled and sagging. Collagen can also retain moisture in its molecules, thereby helping to keep the skin moist, and when the collagen is destroyed by external factors, the skin becomes dry. And it becomes rough.
From the above, it is possible to prevent skin wrinkles and sagging by promoting the biosynthesis of collagen, which is one of the main components of the dermis layer, and there is no problem in terms of safety. It was desired.
[0003]
Lactoperoxidase is a glycoprotein that exists in milk and contains heme iron, but its detailed structure has not yet been clarified. Regarding the use of lactoperoxidase, it has been found that it has an inhibitory effect on the formation of lipid peroxides in vivo, and is an example of an anti-aging agent that prevents a decrease in visual acuity, a reduction in motor ability, a decrease in immune function, etc. (see Patent Document 1) An example (see Patent Document 2) used as a liver function improving agent, and an example (see Patent Document 3) used as a low palpation nutrition composition because of its low palatability. However, it is not known that lactoperoxidase and an enzyme degradation product obtained by degradation with proteolytic enzyme have a skin collagen production promoting action and are useful as a skin collagen production promoter.
[0004]
[Prior art documents]
[Patent Document 1]
JP-A-5-124980 [Patent Document 2]
JP 2001-226289 A [Patent Document 3]
Japanese Patent Laid-Open No. 9-107917
[Problems to be solved by the invention]
The present invention provides a skin collagen production promoter comprising as an active ingredient a substance capable of preventing skin dryness, rough skin, wrinkles and talmi and having no problem in terms of safety and promoting collagen biosynthesis. Is an issue. Moreover, this invention makes it a subject to provide the food-drinks for skin collagen production promotion which mix | blended such a substance, and the cosmetics for skin collagen production promotion.
[0006]
[Means for Solving the Problems]
In order to solve these problems, the inventors of the present invention have been diligently searching for substances that promote skin collagen production that are widely included in food materials. It was found that a lactoperoxidase degradation product obtained by degrading the protein with a protease such as pepsin or pancreatin can increase the amount of collagen in the skin, that is, has an effect of promoting collagen production. And it discovered that this lactoperoxidase and a lactoperoxidase degradation product can be used as an active ingredient of skin collagen production promoter, food and drink for promoting skin collagen production, and cosmetics for promoting skin collagen production, and completed the present invention. It was.
[0007]
Therefore, the present invention relates to a skin collagen production promoter comprising, as an active ingredient, lactoperoxidase having a skin collagen production promoting activity and / or a lactoperoxidase degradation product obtained by degrading lactoperoxidase with a proteolytic enzyme. In addition, the present invention relates to a food and drink for promoting skin collagen production and a cosmetic for promoting skin collagen production, in which these lactoperoxidase and / or lactoperoxidase degradation products are blended.
[0008]
In the present invention, the skin collagen production promoter refers to an agent that exhibits an effect of promoting skin collagen production by oral administration or application. Further, in the present invention, among these skin collagen production promoters, they are formulated into powders, granules, tablets, capsules, drinks, etc., and are formulated as they are or after being formulated into nutritional agents, foods and drinks, etc. What is orally administered is called food or drink for promoting skin collagen production. Further, in the present invention, among skin collagen production promoters, those formulated into ointments, gels, creams, sprays, patches, lotions and the like and applied to the skin are referred to as cosmetics for promoting skin collagen production. . Further, in the present invention, although it is a pharmaceutical, it is included in the food and drink for promoting skin collagen production for convenience as it is formulated and administered orally as it is, and as a pharmaceutical, it is convenient for those that are formulated and applied to the skin. , And included in the cosmetic for promoting skin collagen production.
[0009]
DETAILED DESCRIPTION OF THE INVENTION
The skin collagen production promoter of the present invention is characterized in that lactoperoxidase and / or lactoperoxidase degradation product obtained by degrading lactoperoxidase with a proteolytic enzyme is an active ingredient.
[0010]
Lactoperoxidase is prepared from mammalian milk. Examples of the source include milk such as cows, buffalos, humans, pigs, sheep, goats and horses. Lactoperoxidase is a known substance and is commercially available. To produce it, a known method, for example, a method for purifying lactoperoxidase using a sulfonated carrier (Japanese Patent Laid-Open No. 3). -109400) can be advantageously used industrially. In the present invention, lactoperoxidase produced by genetic engineering techniques can also be used.
[0011]
The lactoperoxidase degradation product is a peptide obtained by limited degradation of the above-mentioned lactoperoxidase with a proteolytic enzyme such as trypsin, pancreatin, chymotrypsin, pepsin, papain, kallikrein, cathepsin, thermolysin, and V8 protease so that the molecular weight is 10,000 or less. It is a mixture. The molecular weight lower limit is preferably 500 or more.
[0012]
The skin collagen production promoter of the present invention exhibits an effect of promoting skin collagen production by oral administration or application.
When orally administering the skin collagen production promoter of the present invention, the active ingredient lactoperoxidase or lactoperoxidase degradation product can be used as it is, but according to conventional methods, powders, granules, tablets In addition, it can be formulated into capsules and drinks. In the present invention, oral preparations such as powders, granules, tablets and capsules are formulated by conventional methods using, for example, starch, lactose, sucrose, mannitol, carboxymethylcellulose, corn starch, inorganic salts and the like. In this type of preparation, in addition to the above-mentioned excipients, binders, disintegrants, surfactants, lubricants, fluidity promoters, colorants, fragrances and the like can be appropriately used. More specifically, examples of the binder include starch, dextrin, gum arabic powder, gelatin, hydroxypropyl starch, sodium carboxymethylcellulose, methylcellulose, crystalline cellulose, ethylcellulose, and polyvinylpyrrolidone. Moreover, as a disintegrating agent, starch, hydroxypropyl starch, sodium carboxymethylcellulose, bridge | crosslinking sodium carboxymethylcellulose, crystalline cellulose, carboxymethylcellulose etc. are mentioned, for example. As surfactant, soybean lecithin, sucrose fatty acid ester, etc., as lubricant, talc, wax, sucrose fatty acid ester, hydrogenated vegetable oil, etc., as fluidity promoter, anhydrous silicic acid, dry aluminum hydroxide, Examples include magnesium silicate.
[0013]
Furthermore, these lactoperoxidases and lactoperoxidase degradation products can be blended in nutrients, foods and drinks, etc. as they are or after preparation. In addition, if skin peroxidase or a lactoperoxidase degradation product is blended together with components such as vitamin C that are conventionally considered to have an effective action for collagen production, a further skin collagen production promoting action can be expected. In addition, since lactoperoxidase or lactoperoxidase degradation product is relatively stable to heat, it is possible to heat sterilize the raw material containing lactoperoxidase or lactoperoxidase degradation product under normal conditions. It is.
[0014]
When applying the skin collagen production promoter of the present invention, it is prepared into various dosage forms such as a liquid agent, a solid agent, a semi-solid agent, etc., by blending with commonly used known components according to the purpose of use. Preferred compositions include ointments, gels, creams, sprays, patches, lotions, powders and the like. For example, the skin collagen production promoter of the present invention is a hydrocarbon such as petrolatum, a higher fatty acid lower alkyl ester such as stearyl alcohol, isopropyl myristate, an animal fat such as lanolin, a polyhydric alcohol such as glycerin, a glycerin fatty acid ester, a monoester Cosmetics and pharmaceuticals for promoting skin collagen production by mixing with surfactants such as polyethylene glycol stearate, inorganic salts, waxes, resins, water and, if necessary, preservatives such as methyl paraoxybenzoate and butyl paraoxybenzoate Can be manufactured.
[0015]
The effective amount of the skin collagen production promoter of the present invention by oral administration is appropriately defined by the formulation form, administration method, purpose of use, and age, weight, and medical condition of the patient to which it is applied, but is not constant. According to the results of animal experiments, it was found that lactoperoxidase or lactoperoxidase degradation product should be ingested in an amount of 20 mg or more per 1 kg body weight of the rat in order to show skin collagen production promoting action. Therefore, according to the extrapolation method, the effect can be expected usually by ingesting 20 mg or more of lactoperoxidase or lactoperoxidase degradation product per day per adult. Or it may be administered as a medicine. The administration can be divided into several times a day as necessary.
[0016]
The effective amount of the application of the skin collagen production promoter of the present invention varies depending on the dosage form, but preferably, lactoperoxidase or lactoperoxidase is 0.001 to 2% by weight based on the total amount of the composition to be applied. What is necessary is just to mix | blend a decomposition product. However, what is diluted at the time of use like a bath agent can increase a compounding quantity further.
[0017]
EXAMPLES Next, although an Example and a test example are shown and this invention is demonstrated in detail, these only illustrate the embodiment of this invention, and this invention is not limited at all by these.
[0018]
[Example 1]
A column (diameter 5 cm x height 30 cm) packed with 400 g of a cation exchange resin sulfonated chitopearl (Fuji Boseki Co., Ltd.) was thoroughly washed with deionized water. 6.7) was passed at a flow rate of 25 ml / min. After passing through the column, the column was thoroughly washed with deionized water and eluted with 0.02 M carbonate buffer (pH 7.0) containing 2.0 M sodium chloride. The elution fraction containing lactoperoxidase is adsorbed on an S-Sepharose FF column (Amersham Biosciences), washed thoroughly with deionized water, and equilibrated with 10 mM phosphate buffer (pH 7.0). The fraction adsorbed with a linear gradient of 0 to 2.0 M sodium chloride was eluted, and the fraction containing lactoperoxidase was recovered. Then, the fraction was treated by gel filtration chromatography using HiLoad 16/60 Superdex 75 pg (Amersham Bioscience) to obtain 3.0 g of lactoperoxidase. The lactoperoxidase thus obtained has a purity of 94% and can be used as it is as a skin collagen production promoter.
[0019]
[Example 2]
1 g of lactoperoxidase obtained in Example 1 was dissolved in 200 ml of water, pancreatin (produced by Sigma) as a proteolytic enzyme was added so that the final concentration was 0.01% by weight, and the enzyme treatment was performed at 37 ° C. for 5 hours. . The enzyme was inactivated by heat treatment at 90 ° C. for 5 minutes, and then freeze-dried to obtain 0.8 g of a lactoperoxidase degradation product. In addition, the molecular weight of the lactoperoxidase degradation product thus obtained is 10,000 or less, and can be used as it is as a skin collagen production promoter.
[0020]
[Example 3]
1 g of lactoperoxidase obtained in Example 1 was dissolved in 200 ml of water, trypsin (manufactured by Sigma) as a proteolytic enzyme was added to a final concentration of 0.01% by weight, and the enzyme treatment was performed at 37 ° C. for 5 hours. The enzyme was inactivated by heat treatment at 90 ° C. for 5 minutes, and then freeze-dried to obtain 0.9 g of a lactoperoxidase degradation product. In addition, the molecular weight of the lactoperoxidase degradation product thus obtained is 10,000 or less, and can be used as it is as a skin collagen production promoter.
[0021]
[Test Example 1]
About the lactoperoxidase obtained in Example 1 and the lactoperoxidase degradation product obtained in Example 2, the collagen production promoting action was examined by animal experiments using rats. 7-week old Wistar male rats were obtained in physiological saline administration group (Group A), in the group administered with 20 mg of lactoperoxidase obtained in Example 1 per kg body weight (Group B), in Example 1. Group (group C) administered with 200 mg / kg rat peroxidase body weight, group (D group) administered with 20 mg / kg rat body weight degradation product obtained in Example 2, obtained in Example 2. The lactoperoxidase degradation product was divided into 5 test groups (n = 6) in a group (group E) administered with 200 mg / kg rat body weight, and each was administered once daily with a sonde and reared for 10 weeks.
Regarding the amount of collagen in the skin, the amount of hydroxyproline contained in the soluble fraction was measured after treating the rat dermis according to the method of Nimni et al. (See Arch. Biochem. Biophys, page 292, 1967). Hydroxyproline is a special amino acid contained only in collagen and accounts for about 10% of all amino acids constituting collagen, so that the amount of collagen can be estimated (see Takashi Asano et al., Bio Industry, page 12, 2001). The results are shown in Table 1.
[0022]
[Table 1]
──────────────────
Group Amount of hydroxyproline (μg / ml)
──────────────────
Group A 0.3 ± 0.1
Group B 0.8 ± 0.1 *
Group C 1.0 ± 0.2 *
Group D 0.7 ± 0.2 *
Group E 1.0 ± 0.1 *
──────────────────
A numerical value shows an average value +/- standard deviation (n = 6).
In addition, * indicates that there is a significant difference compared with the control group A (p <0.05).
[0023]
According to this, the amount of hydroxyproline in the soluble fraction after 10 weeks was significantly higher in the B, C, D and E groups than in the A group.
From this, it became clear that lactoperoxidase and lactoperoxidase degradation products have skin collagen production promoting action, and it was shown to be useful as a skin collagen production promoter.
It was also revealed that this skin collagen production promoting action was observed when lactoperoxidase or lactoperoxidase degradation product was administered at a minimum of 20 mg / kg rat body weight.
[0024]
[Example 4]
A beverage for promoting the production of skin collagen having the composition shown in Table 2 was produced by a conventional method. The flavor of the produced beverage was good, and the flavor did not deteriorate even after storage at room temperature for 1 year, and there was no problem such as precipitation.
[0025]
[Table 2]
────────────────────────────
Mixed isomerized sugar 15.0 (wt%)
Fruit juice 10.0
Citric acid 0.5
Lactoperoxidase (Example 1) 0.1
Fragrance 0.1
Mineral mixing 0.1
Water 74.2
────────────────────────────
[0026]
[Example 5]
A dough having the composition shown in Table 3 was prepared by a conventional method, molded, and then baked to produce a biscuit for promoting skin collagen production.
[0027]
[Table 3]
──────────────────────────
Wheat flour 50.0 (wt%)
Sugar 20.0
Salt 0.5
Margarine 12.5
Egg 12.5
Water 2.5
Mineral mixing 0.8
Lactoperoxidase (Example 1) 1.2
──────────────────────────
[0028]
[Example 6]
Skin collagen production promoters having the formulations shown in Table 4 were produced.
[0029]
[Table 4]
────────────────────────────
Water-containing crystalline glucose 83.5 (wt%)
Lactoperoxidase (Example 1) 10.0
Mineral mixing 5.0
Sugar Ester 1.0
Fragrance 0.5
────────────────────────────
[0030]
[Test Example 2]
About a commercially available lactoperoxidase (manufactured by Sigma) and a lactoperoxidase degradation product obtained by treating a commercially available lactoperoxidase with a proteolytic enzyme in the same manner as in Example 2, normal human fibroblast cell line [white] Collagen production-promoting action was examined by experiments using CCD45SK (ATCCRL 1506)] collected from female skin. Using a modified Eagle's medium (MEM, 10-101, manufactured by Dainippon Pharmaceutical Co., Ltd.) containing 10% by volume fetal bovine serum (hereinafter abbreviated as FBS), 4 × 10 4 normal human fibroblast cell lines / well / 0.4 The cells were seeded in a 24-well plate so as to be ml, and cultured at 37 ° C. under 5% carbon dioxide gas and saturated steam for 24 hours, and then replaced with a modified Eagle medium containing 0.6% by volume FBS. And the lactoperoxidase degradation product obtained by processing commercially available lactoperoxidase (manufactured by Sigma) and a commercially available lactoperoxidase with a proteolytic enzyme in the same manner as in Example 2 was added to each well in an amount of 0.1% by volume. After incubating for 24 hours, add β-aminopropionitrile at 50 μg / ml and tritium-L-proline at 1 μCi / ml for another 24 hours. Culture was obtained by culturing. The collagen fraction was fractionated from the culture solution thus obtained according to the method of Webster et al. (Analytical Biochemistry, page 220, 1979), and the radioactivity incorporated into the collagen fraction was measured. As a control, the same test was performed without adding lactoperoxidase and lactoperoxidase degradation product. The results are shown in Table 5.
[0031]
[Table 5]
* Indicates that there is a significant difference compared to the control group (p <0.05).
[0032]
According to this, the group to which lactoperoxidase and lactoperoxidase degradation product were added showed about twice the ability to promote collagen production compared to the group to which lactoperoxidase and lactoperoxidase degradation product were not added (control). It was.
From this, it was clarified that lactoperoxidase and lactoperoxidase degradation product act on skin fibroblasts to promote collagen production, suggesting that they are useful as a skin collagen production promoter.
[0033]
[Example 7]
A lotion having the composition shown in Table 6 was produced by a conventional method.
[0034]
[Table 6]
[Example 8]
A cream having the composition shown in Table 7 was produced by a conventional method.
[0036]
[Table 7]
[Test Example 3]
Using the lotion obtained in Example 7 and the cream obtained in Example 8, an actual use test was conducted. As a comparative product, the same formulation as in Examples 7 and 8 was used except that lactoperoxidase was excluded.
Twenty adult women with dry skin with facial sagging and fine wrinkles, each with 10 people randomly divided into 2 groups (Groups A and B), and 20 girls with rough skin on their hands, Randomly divided into 2 groups (groups C and D) of 10 people. The face lotion of the present invention is applied to the face of the group A, the face lotion of the comparative product is applied to the face of the group B, and the fingers of the group C The cream of the present invention was applied to the fingers of group D, and the comparative cream was applied twice a day for 10 days in the same manner as in normal use. As a result, it was proved that the skin lotion according to the present invention is significantly improved in dry feeling and rough skin as compared with the skin lotion of the comparative product, and has an excellent skin collagen production promoting effect. In addition, the cream of the present invention was also found to have an improvement in dry feeling and a marked improvement in rough skin compared to the comparative cream, and to have a natural exacerbation suppressing effect such as rough skin.
[0038]
【The invention's effect】
According to the present invention, there are provided a skin collagen production promoter, a food product for promoting skin collagen production, and a cosmetic product for promoting skin collagen production, comprising lactoperoxidase and / or a lactoperoxidase degradation product as an active ingredient.
The skin collagen production promoter, skin collagen production promoting food and drink, and skin collagen production promoting cosmetic of the present invention have an action of promoting skin collagen production and prevent skin wrinkles and sagging, dryness and rough skin. Useful for treatment.
Claims (2)
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| JP5118398B2 (en) * | 2006-06-23 | 2013-01-16 | ロート製薬株式会社 | Composition having collagen production promoting ability and / or fibroblast proliferation promoting ability |
| JP5653759B2 (en) | 2008-12-15 | 2015-01-14 | カルピス株式会社 | Skin aging inhibitory peptide |
| JP2013079216A (en) | 2011-10-04 | 2013-05-02 | Snow Brand Milk Products Co Ltd | Sense-improving agent |
| JP5955631B2 (en) * | 2012-05-02 | 2016-07-20 | 雪印メグミルク株式会社 | Hyaluronic acid production promoter |
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| JP2001226289A (en) * | 2000-02-16 | 2001-08-21 | Snow Brand Milk Prod Co Ltd | Hepatic function ameliorative agent |
| JP2002087974A (en) * | 2000-09-12 | 2002-03-27 | Maruzen Pharmaceut Co Ltd | Prophylactic and therapeutic agent for inflammatory disease |
| JP2002145737A (en) * | 2000-11-10 | 2002-05-22 | Yashiro Kogyo Kk | Cosmetic with formulated ancient saltwater |
| JP2003095970A (en) * | 2001-09-27 | 2003-04-03 | Maruzen Pharmaceut Co Ltd | Skin cosmetic, and drink and food |
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2003
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH04334310A (en) * | 1991-05-01 | 1992-11-20 | Ichimaru Pharcos Co Ltd | Agent for suppressing formation of lipid peroxide |
| JPH05124980A (en) * | 1991-10-30 | 1993-05-21 | Snow Brand Milk Prod Co Ltd | Aging preventing agent |
| JPH08151331A (en) * | 1994-09-30 | 1996-06-11 | Snow Brand Milk Prod Co Ltd | Bone-strengthening agent |
| JPH09191856A (en) * | 1996-01-17 | 1997-07-29 | Snow Brand Milk Prod Co Ltd | Nutrient-replenishing composition |
| JP2001226289A (en) * | 2000-02-16 | 2001-08-21 | Snow Brand Milk Prod Co Ltd | Hepatic function ameliorative agent |
| JP2002087974A (en) * | 2000-09-12 | 2002-03-27 | Maruzen Pharmaceut Co Ltd | Prophylactic and therapeutic agent for inflammatory disease |
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| JP2003095970A (en) * | 2001-09-27 | 2003-04-03 | Maruzen Pharmaceut Co Ltd | Skin cosmetic, and drink and food |
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