JP6805399B2 - 植物性乳酸菌を含むスキンケア剤 - Google Patents
植物性乳酸菌を含むスキンケア剤 Download PDFInfo
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- JP6805399B2 JP6805399B2 JP2020550194A JP2020550194A JP6805399B2 JP 6805399 B2 JP6805399 B2 JP 6805399B2 JP 2020550194 A JP2020550194 A JP 2020550194A JP 2020550194 A JP2020550194 A JP 2020550194A JP 6805399 B2 JP6805399 B2 JP 6805399B2
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Description
(1)受託番号NITE BP−02707として寄託されたラクトバチルス カゼイ亜種カゼイ(Lactobacillus casei subsp.casei)327株の菌体を有効成分として含むスキンケア剤。
(2)ヒアルロン酸合成促進剤、角層形成促進剤又は皮膚常在菌叢のバランス調整剤である(1)のスキンケア剤。
(3)上記菌体が、死菌体である(1)又は(2)のスキンケア剤。
(4)皮膚外用剤の形態にある、(1)〜(3)いずれかのスキンケア剤。
(5)(1)〜(4)いずれかに記載のスキンケア剤を皮膚に塗布することを特徴とするシワの形成、肌pHの上昇及び/又は肌水分量の低下を予防又は改善するための非治療的方法。
(6)皮膚状態を改善する方法であって、受託番号NITE BP−02707として寄託されたラクトバチルス カゼイ亜種カゼイ(Lactobacillus casei subsp. casei)327株の菌体を有効成分として含む組成物を対象者の皮膚に塗布する工程を含む方法。
(7)皮膚状態の改善が、ヒアルロン酸合成促進、角層形成促進又は皮膚常在菌のバランス調整である(6)に記載の方法。
(8)対象者の皮膚のシワの形成、肌pHの上昇及び/又は肌水分量の低下を予防又は改善するための(6)又(7)に記載の方法。
(9)皮膚におけるヒアルロン酸の合成若しくは角質形成の促進、又は皮膚常在菌のバランスを調整するための組成物を製造するための、受託番号NITE BP−02707として寄託されたラクトバチルス カゼイ亜種カゼイ(Lactobacillus casei subsp. casei)327株菌体の使用。
(10)皮膚のシワの形成、肌pHの上昇及び/又は肌水分量の低下を予防又は改善するための組成物を製造するための、受託番号NITE BP−02707として寄託されたラクトバチルス カゼイ亜種カゼイ(Lactobacillus casei subsp. casei)327株菌体の使用。
(I)植物性乳酸菌
(II)スキンケア剤及びその作用
(III)用途
(IV)美容的方法
本発明に使用される植物性乳酸菌は、味噌、醤油、漬物、糠、牧草、米、麦、麦芽など加工食品を含む植物由来のものから分離され、糖質などを利用して乳酸を産生する乳酸菌である。好ましくは、米を原料とした発酵食品から分離されたラクトバチルス属に属する乳酸菌を用いることができる。例えば、米及び米加工品から分離され、抗変異原性を有する複数の植物性乳酸菌が報告されている(日本食品科学工学会誌、第48巻、第9号、693〜696頁、2001年)。本発明の有効成分として用いられる植物性乳酸菌は、これらの中で、亜種カゼイ327株又はその変異株が最も好ましい。なおここで「変異株」とは、特定の菌株に対し、当業者に周知の方法により当業者がその性質に変化を及ぼさない範囲で変異させたもの、あるいは、それと同等であると当業者が確認できるものを包含する意味である。
本明細書における「スキンケア剤」とは、上記K−1株の菌体を有効成分として含み、スキンケア作用を有する製剤又は組成物を意味する。ここで、「スキンケア」とは、典型的には、塵埃、化学物質、微生物などの環境的影響や、皮膚組織内の水、天然脂肪、電解質などの内因性物質の損失に対する障壁としての皮膚の自然な機能が強化又は再生されることをいう。本発明における、「スキンケア剤」のより具体的な態様には、「ヒアルロン酸合成促進剤」、「角層形成促進剤」及び「皮膚常在菌叢のバランス調整剤」等を含むがこれらに限定されない。また、好ましい実施形態において用いられる死菌体は、上述したスキンケア作用等の効果が期待できるだけでなく、生菌の場合、製品製造以降の配送時や陳列時に形態変化を起こす可能性があるのに対し、それ以上形態変化を起こさない死菌体は好適に使用できる。
(各種組成物)
本発明のスキンケア剤を、例えば皮膚外用剤(外用医薬品、化粧品等)の各種組成物の形態で用いる場合、有効成分としてのK−1株の菌体を、乳酸菌培養の常法に従って培養し、得られた培養物から遠心分離等の集菌手段によって分離されたものをそのまま用いることができる。また、当該培養・発酵液(培養上清)、その培養物の粗精製品あるいは精製品、それらの凍結乾燥品、或いは菌体を酵素や物理的手段を用いて処理した細胞質や細胞壁画分も用いることができる。
本発明のスキンケア剤を化粧品、外用医薬品、医薬部外品等の外用剤組成物とする場合は、本発明の有効成分と共に、製剤学的に許容される適当な製剤担体を用いて、一般的な外用剤組成物の形態に調製されて実用される。かかる製剤担体としては、例えば、グリセリン、ワセリン、尿素、ヒアルロン酸、ヘパリン等の保湿剤;PABA誘導体(パラアミノ安息香酸、エスカロール507等)、桂皮酸誘導体(ネオヘリオパン、パルソールMCX、サンガードB等)、サリチル酸誘導体(オクチルサリチレート等)、ベンゾフェノン誘導体(ASL−24、ASL−24S等)、ジベンゾイルメタン誘導体(パルソールA、パルソールDAM等)、複素環誘導体(チヌビン系等)、酸化チタン等の紫外線吸収剤・散乱剤;エデト酸二ナトリウム、エデト酸三ナトリウム、クエン酸、クエン酸ナトリウム、酒石酸、酒石酸ナトリウム、乳酸、リンゴ酸、ポリリン酸ナトリウム、メタリン酸ナトリウム、グルコン酸等の金属封鎖剤;サリチル酸、イオウ、カフェイン、タンニン等の皮脂抑制剤;塩化ベンザルコニウム、塩化ベンゼトニウム、グルコン酸クロルヘキシジン等の殺菌・消毒剤;塩酸ジフェンヒドラミン、トラネキサム酸、グアイアズレン、アズレン、アラントイン、ヒノキチオール、グリチルリチン酸及びその塩、グリチルリチン酸誘導体、グリチルレチン酸等の抗炎症剤;ビタミンA、ビタミンB群(B1,B2,B6,B12,B15)、葉酸、ニコチン酸類、パントテン酸類、ビオチン、ビタミンC、ビタミンD群(D2,D3)、ビタミンE、ユビキノン類、ビタミンK(K1,K2,K3,K4)等のビタミン類;アスパラギン酸、グルタミン酸、アラニン、リジン、グリシン、グルタミン、セリン、システイン、シスチン、チロシン、プロリン、アルギニン、ピロリドンカルボン酸等のアミノ酸及びその誘導体;レチノール、酢酸トコフェロール、アスコルビン酸リン酸マグネシウム、アスコルビン酸グルコシド、アルブチン、コウジ酸、エラグ酸、胎盤抽出液等の美白剤;ブチルヒドロキシトルエン、ブチルヒドロキシアニソール、没食子酸プロピル等の抗酸化剤;塩化亜鉛、硫酸亜鉛、石炭酸亜鉛、酸化亜鉛、硫酸アルミニウムカリウム等の収斂剤;グルコース、フルクトース、マルトース、ショ糖、トレハロース、エリスリトール、マンニトール、キシリトール、ラクチトール等の糖類;甘草、カミツレ、マロニエ、ユキノシタ、芍薬、カリン、オウゴン、オウバク、オウレン、ジュウヤク、イチョウ葉等の各種植物エキス等の他、油性成分、界面活性剤、増粘剤、アルコール類、粉末成分、色素等が挙げられる。
本発明の他の実施形態では、上述したK−1株の菌体を有効成分として含むスキンケア剤を皮膚に塗布することを含むシワを予防又は改善するための非治療的方法が提供される。この美容方法は、好ましくは皮膚外用剤の形態にあるスキンケア剤を皮膚に塗布することを含み、少なくとも1週間以上連続して塗布することが好ましい。本実施形態にかかる美容方法を用いることにより、本発明のスキンケア剤を使用した際に、より効果的にシワを改善することが可能となる。「連続して塗布する」とは、少なくとも皮膚上及び/又は皮膚内にK−1株の菌体が留まるように塗布することを意味し、1日に1回〜複数回塗布したり、1〜数日置きに塗布したりする方法も包含する。
(試験方法の概要)
分化誘導した表皮角化細胞(NHEK)は、抗生物質(Gibco(商標)Antibiotic−Antimycotic(100X))及びサプリメントS7を添加したEpiLife(Thermo Fisher Scientific)を用いて培養した。K−1株は、MRS培地を用いて37℃で24時間培養した後、4℃冷却下で生理食塩水を用いて遠心洗浄(3000rpm、10分)を4回繰り返した菌体を純水にて10質量%の懸濁液を調製し、オートクレーブで滅菌したものを使用した。上記分化誘導した表皮角化細胞(NHEK)に、K−1株の菌体を所定の濃度となるように混合し、24時間培養後、回収した表皮角化細胞からDNAを抽出した。RT−PCR法により、β−ディフェンシン3、角層因子、及びヒアルロン酸合成酵素の発現量変化を解析した。一方、培養後の細胞上清を回収し、ELISA法を用いて、培養上清中に分泌された、β−ディフェンシン3、及びヒアルロン酸の合成量を定量した。
24穴プレートにて細胞密度がコンフルエントになるまで表皮角化細胞(NHEK細胞)を、CO2インキュベーターにて37℃で培養した。これに最終濃度が1mMになるようにカルシウムを添加し、24時間分化誘導を行った。分化誘導後の細胞に対し乳酸菌K−1の菌体を、質量比でそれぞれ、0.004%、0.02%及び0.1%となるように添加し、さらに24時間培養した。培養後の細胞より、mRNAを精製した。mRNAの精製にはQIAGEN社より販売されているQIAshreder及びRNeasy Mini Kitを用いた。
24穴プレートにて細胞密度がコンフルエントになるまで表皮角化細胞(NHEK細胞)を、CO2インキュベーターにて37℃で培養した。これに最終濃度が1mMになるようにカルシウムを添加し、24時間分化誘導を行った。分化誘導後の細胞に対し乳酸菌K−1を、質量比でそれぞれ、0.004%、0.02%及び0.1%となるように添加し、さらに24時間培養した。培養後の培養上清より、β−ディフェンシン3及びヒアルロン酸を定量した。β−ディフェンシン3の検出にはPEPROTECHより販売されているELISAキット、Human BD−3 Mini ELISA Development Kitを使用し、実験方法は取り扱い説明書に準じて行った。細胞培養上清に放出されたヒアルロン酸の定量については、コスモ・バイオ株式会社より販売されている、Hyaluronan Quantification Kitを使用した。実験方法は取扱説明書に準じて行った。
これらの結果より、K−1株は、β−ディフェンシン3の有する抗菌作用により、皮膚からの病原性微生物の感染防御の役割を果たすとともに、皮膚の常在菌叢のバランスを調整して皮膚のバリア機能を維持する作用を有すると考えられる。また、皮膚におけるヒアルロン酸の産生を促進して肌のみずみずしさ、ハリ、弾力を維持してシワを伸ばす作用を有すると考えられる。
表皮ブドウ球菌(Staphylococcus epidermidis)標準菌(ATCC12228)と、各種濃度に希釈したK−1株の菌体とを混合し、37℃で24時間培養後、その一部を培養培地としてニュートリブイヨンNo.2(日水製薬株式会社)プレートに播種して37℃にて2日間培養した。プレート上に形成されたコロニー数を計数し、表皮ブドウ球菌によるK−1株の資化性を調べた。K−1株の菌体は、実施例1と同様の方法により10質量%の死菌体懸濁液を調製し、終濃度が質量比でそれぞれ0.000%、0.025%、0.050%、0.100%となるように純水で希釈した。表皮ブドウ球菌は、2.67×109CFU/mLであった凍結試料を解凍し、終濃度が3×106CFU/mLとなるように水で希釈して用いた。
まず、表2に示す各K−1株菌体濃度群における表皮ブドウ球菌のコロニー数の平均値(セット1から3でカウントした表皮ブドウ球菌のコロニー数の平均値)を算出した。0.000%K−1株菌体濃度群は0、0.025%K−1株菌体濃度群は9.3個、0.050%K−1株菌体濃度群は24.3個、および0.100%K−1株菌体濃度群は104.7個であった。
X=(2.5×103)×N ・・・・・(式1)
(Xは図7で示す生菌濃度、Nは各K−1株菌体濃度群の表皮ブドウ球菌のコロニー数の平均値)
(試験方法)
27〜60歳の健康な男性20名を被験者として本モニター試験を行った。10名の被験者がK−1塗布群(平均年齢45歳)、10名の被験者がプラセボ塗布群(平均年齢44歳)と設定して、当該試験を行った。試験開始(0日)から4週間の間、合計20名の被験者は、表3に記載の組成の洗顔料を用いて、毎日朝晩の洗顔を行った。毎回の洗顔後、K−1塗布群の被験者は表4記載の組成のジェル0.8gを顔に塗布し、プラセボ塗布群の被験者はK−1未含有の表5記載の組成のジェル0.8gを顔に塗布した。
その結果を、表6と表7に示す。肌pH測定結果を示した表6では、各投与群(各部位:左頬、右頬、額)の0週のpHを0として、4週のpHの値を相対値として示している。なお、0週のpHは5.7〜6.1の範囲内であった。ヒトの健康な皮膚のpHは、通常、弱酸性(約pH4.5〜6.0)と考えられているが、K−1塗布群では4週においても0日と比べpHの変化が起きなかったが、プラセボ塗布群ではpHの上昇が確認された。
Claims (4)
- 受託番号NITE BP−02707として寄託されたラクトバチルス・カゼイ亜種カゼイ(Lactobacillus casei subsp. casei)327株の菌体を有効成分として含む皮膚外用剤。
- ヒアルロン酸合成促進剤、角層形成促進剤又は皮膚常在菌叢のバランス調整剤である請求項1に記載の剤。
- 前記菌体が、死菌体である請求項1又は2に記載の剤。
- シワの形成、肌pHの上昇及び/又は肌水分量の低下を予防又は改善するための請求項1〜3の何れか一項に記載の剤。
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