JP5612888B2 - 抗酸化剤、紫外線傷害抑制剤及び抗光老化化粧料 - Google Patents
抗酸化剤、紫外線傷害抑制剤及び抗光老化化粧料 Download PDFInfo
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- JP5612888B2 JP5612888B2 JP2010083765A JP2010083765A JP5612888B2 JP 5612888 B2 JP5612888 B2 JP 5612888B2 JP 2010083765 A JP2010083765 A JP 2010083765A JP 2010083765 A JP2010083765 A JP 2010083765A JP 5612888 B2 JP5612888 B2 JP 5612888B2
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Description
さらに、特開2006−109768(特許文献4)には、サトウキビから砂糖を分離した糖蜜および/または柑橘果汁窄汁残渣より回収する果汁蜜で酵母を培養し、ラジカル消去活性の高い酵母含有組成物を得る方法の記載があり、特開2006−94800(特許文献5)には廃糖蜜を乳酸菌、乳酸菌と酵母、乳酸菌と枯草菌、又は乳酸菌と酵母と枯草菌を用いて発酵させることを特徴とする抗酸化作用が増強された組成物の記載がなされている。
上記に記載したように培地等にいろいろなものを添加し、また各種の微生物を用いて培養した組成物が記載されている。しかしながら、紫サツマイモの抽出液を培地に添加し、Saccharomyces cerevisiae で培養し、培養後の菌体抽出液を利用した抗酸化剤、紫外線傷害抑制剤の記載はなされていない。
本発明で用いるSaccharomyces cerevisiae は市販されているパン酵母、または分譲されているSaccharomyces cerevisiae
に属する酵母であれば特に制限なく使用できる。また、酵母は糖類が存在する自然界からも容易に分離することができるため、植物や土などから分離した酵母であっても、Saccharomyces cerevisiae に属する酵母であれば使用できる。
代表的な品種としては、アヤムラサキ、ムラサキマサリ、パープルスイートロード、種子島紫いも(出願中商標)、紫宝(登録商標)、魚豚紫1号などの品種が知られているが、いずれの紫サツマイモでも本発明に使用できる。
の接種量は107〜108個/mLが適量である。接種量が上記の範囲より多くなっても培養の進行時間は殆ど変わらず、一方上記の範囲より少なくなると培養完了までに長時間を要することとなって好ましくない。
市販されているパン酵母(日清スパーカメリア・ドライイースト:日清フーズ(株))を購入し、120℃で15分間滅菌したYM培地100mlを添加した三角フラスコに0.1g懸濁し、28℃、150rpmで3日間回転培養を行った。懸濁液を1白金耳取り、YM寒天培地上で引き伸ばして単独コロニーを得、これを以下の実験に使用した。なお、YM培地の組成はペプトン5g/L、酵母エキス3g/L、麦芽エキス3g/L、グルコース10g/Lであり、YM寒天培地はYM培地に寒天20g/Lを添加し、加熱溶解して作成した。
紫サツマイモは市販されている紫サツマイモ(種子島紫いも)を購入し、1cm角に細切した。粉砕物100gに精製水150gを加え、80℃で2時間加熱抽出した。抽出液は遠心分離器で上澄み液を取り、培養液に添加した。
培地はYNB(Yeast Nitrogen Base) with Ammonium Sulfate 合成培地にグルコースを2.0%添加した。培地50mlに前記抽出液の調製方法で調製した紫サツマイモ抽出液5mlを加え、120℃、15分間オートクレーブで滅菌処理を行った。培地を冷却後、1白金耳の酵母菌を植え付け、28℃、150rpm、で4日間培養を行った。培養後、2,000rpm、5分間の遠心分離により、菌体と培養液を分離した。分離した菌体に50%エタノール水溶液3mlを加え、室温で1昼夜放置した。その後3000rpmで5分間遠心分離を行い、上清を取り菌体抽出液とした。
YNB(Yeast Nitrogen Base) with Ammonium Sulfate 合成培地の組成は、硫酸アンモニウム5,000、リン酸二水素カリウム1,000、硫酸マグネシウム500、塩化ナトリウム100、塩化カルシウム100、ビオチン0.002、パントテン酸カルシウム0.4、葉酸0.002、イノシトール2.0、ナイアシン0.4、パラアミノ安息香酸0.2、塩酸ピリドキシン0.4、リボフラビン0.2、塩酸チアミン0.4、ホウ酸0.5、硫酸銅0.04、ヨウ化カリウム0.1、塩化第二鉄0.2、硫酸マンガン0.4、モリブデン酸ナトリウム0.2、硫酸亜鉛0.4(単位は全てmg/L)からなる。
DPPH溶液の調製方法
(A)MES(2-(N-モルホリノ)エタンスルホン酸)5.52gを水100mlに溶かし、1N−NaOHでPH6.1に調製する。
(B)DPPH(1,1−ジフェニルー2−ピクリルヒドラジル)15.7mgをエタノール100mlに溶解する。
(C)精製水
DPPH溶液は(A):(B):(C)=1:4:3容量の割合で混合し調製する。
(D)Trolox溶液の調製
Trolox
25mgをDMSO(ジメチルスルホキシド)10mlに溶解し、10mM溶液を作成した。
(E)DPPH試験
測定は試験溶液10μlを96well plateの1well中に加え、次いでDPPH溶液 190μlを迅速に加え混合する。10分後、各wellの吸光度を540nmで測定する。試験溶液のTrolox当量は、0.1mM、0.3mM、0.5mM、1.0mM、3.0mM、5.0mM、10mMのTrolox溶液10μlをとり、DPPH溶液190μlを加え混合し、10分後の540nmの吸光度を測定し、Trolox濃度と540nmの吸光度値の検量線を作成する。試験溶液の540nmの吸光度値から、検量線によりTrolox当量を算出した。標準物質として使用するTroloxは、トコフェロールと類似した構造を有する物質である。Troloxを1とした場合、α-、γ-、δ-トコフェロールは、それぞれ0.50、0.74、1.36を示すといわれております。
表−1には紫サツマイモ抽出液のほかにサツマイモ抽出液を培地に添加して培養した場合の菌体抽出液の結果も示した。サツマイモ抽出液は市販されているサツマイモ(鳴門金時)を用い、前記紫サツマイモ抽出液の調製方法で示した方法と同じ方法により抽出液を得、試験に供した。
表−1の結果より、紫サツマイモ抽出物を10%添加した培養前の培地のDPPH消去活性が0.215mM Trolox当量を示した。0.215mM Trolox当量は、この培養液がTrolox 0.215mMのDPPH消去活性の強さを示すことを示しており、この値が高いほど抗酸化活性が高いことを示すものである。
このDPPH消去活性が培養後では、培養液で0.510mM、菌体抽出液で6.81mM Trolox当量を示した。一方、紫サツマイモ抽出液を添加しないYNB培地のみでパン酵母を培養した培養液および菌体抽出液のDPPH消去活性は0.153mM、−0.07mM Trolox当量であり、明らかに紫サツマイモ抽出液を添加して培養したことにより、菌体抽出液の抗酸化活性が高くなったことが明らかである。
尚、紫サツマイモ、サツマイモをエタノール、エタノール水混液、1,3-ブチレングリコール、1,3−ブチレングリコール水混液等で抽出した抽出液を培地に添加した場合においても、同様の傾向が確認された。
〔細胞の調製〕
ヒト表皮細胞であるHacat細胞を5%FBSを添加したD−MEM培地に分散し24well plateに5×104/wellになるよう細胞を播種し1日間、37℃で培養を行ない50%程度のコンフルーエント状態にした。翌日、各菌体抽出液を細胞培養液500μlに対して25μl添加し2時間、37℃にてインキュベートを行った。その後、培地を捨て、PBS(−)溶液で細胞を洗浄した。洗浄した細胞に15mJ/cm2量のUV−Bを照射した後、5%FBSを添加したD−MEM培地500μlを添加して、2日間培養した。培養終了後、培養液を捨てPBS(−)で洗浄後、10%中性ホルマリン溶液を30分間添加し細胞を固定した。10%中性ホルマリン溶液を除去し、精製水で細胞を洗浄後、0.05%−ナフトールブルーブラック溶液(9%酢酸、0.1M酢酸ナトリウム)で30分間染色した。染色後、細胞を精製水で洗浄し、0.05N−NaOH溶液500μlでナフトールブルーブラック色素を抽出し、595nmの吸光度を測定することによって各well中の細胞数の比較を行った。なお、細胞数の比較はコントロール区の吸光度値を100とした場合の割合を求めて算出した。
紫サツマイモ抽出液を10%培地に添加して培養した酵母菌体抽出液をUV−B照射前に添加しておいた群では、細胞増殖度が90%を示した。また対照としてサツマイモ抽出液を添加して培養した酵母菌体抽出液をUV−B照射前に添加した群では、細胞増殖が71%となり、UV−B傷害による細胞死の抑制効果が少ないことが示された。またYNB培地のみで培養した酵母菌体抽出液添加群の細胞増殖は、57%となり、UV−B傷害による細胞死を抑制できないことが示された。
尚、紫サツマイモ、サツマイモをエタノール、エタノール水混液、1,3-ブチレングリコール、1,3−ブチレングリコール水混液等で抽出した抽出液を培地に添加した場合においても、同様の傾向が確認された。
化粧料の処方例
a)ミツロウ・・・2.0
b)ステアリルアルコール・・・5.0
c)ステアリン酸・・・8.0
d)スクワラン・・・10.0
e)自己乳化型グリセリルモノステアレート・・・3.0
f)ポリオキシエチレンセチルエーテル(20E.O.)・・・1.0
g)紫サツマイモ抽出液添加培養後培養液・・・2.0
h)1,3−ブチレングリコール・・・5.0
i)水酸化カリウム・・・0.3
j)防腐剤・酸化防止剤・・・適量
k)精製水・・・残部
製法
a)〜f)までを加熱溶解し、80℃に保つ。h)〜k)までを加熱溶解し、80℃に保ち、a)〜f)に加えて乳化し、40℃まで撹拌しながら冷却する。その後、g)を加え、攪拌し均一に溶解する。
a)ミツロウ・・・0.5
b)ワセリン・・・2.0
c)スクワラン・・・8.0
d)ソルビタンセスキオレエート・・・0.8
e)ポリオキシエチレンオレイルエーテル(20E.O.)・・・1.2
f)紫サツマイモ抽出液添加培養菌体抽出液・・・5.0
g)1,3− ブチレングリコール・・・7.0
h)カルボキシビニルポリマー・・・0.2
i)水酸化カリウム・・・0.1
j)精製水・・・残部
k)防腐剤・酸化防止剤・・・適量
l)エタノール・・・7.0
製法
a)〜e)までを加熱溶解し、80℃に保つ。g)〜k)までを加熱溶解し、80℃に保ち、a)〜e)に加えて乳化し、50℃まで撹拌しながら冷却する。50℃でf)、l)を添加し、40℃まで攪拌、冷却する。
a)紫サツマイモ抽出液添加培養菌体抽出液・・・10.0
b)グリセリン・・・5.0
c)ポリオキシエチレンソルビタンモノラウレート(20E.O.)・・・1.0
d)エタノール・・・6.0
e)香料・・・適量
f)防腐剤・酸化防止剤・・・適量
g)精製水・・・残部
製法
a)〜g)までを混合し、均一に溶解する。
a)ステアリン酸・・・12 .0
b)ミリスチン酸・・・14 .0
c)ラウリン酸・・・5.0
d)ホホバ油・・・3.0
e)グリセリン・・・10.0
f)ソルビトール・・・15.0
g)1,3−ブチレングリコール・・・10.0
h)POE(20)グリセロールモノステアリン酸・・・2.0
i)水酸化カリウム・・・5.0
j)水・・・残部
k)キレート剤・・・適量
l)香料・・・適量
m)紫サツマイモ抽出液添加培養後培養液・・・1.0
製法
a)〜h)までを加熱溶解し70℃に保つ。j)にi)を溶解後a)〜h)に加えケン化する。その後k)、l)を入れ攪拌しながら冷却する。50℃でm)を添加し、40℃まで攪拌、冷却する。
a)酸化チタン・・・13.0
b)セリサイト・・・25.0
c)タルク・・・残部
d)ベンガラ・・・0.8
e)黄酸化鉄・・・2.5
f)黒酸化鉄・・・0.1
g)紫サツマイモ抽出液添加培養後培養液・・・0.05
h)スクワラン・・・10.0
i)セスキオレイン酸ソルビタン・・・3.5
j)防腐剤・・・適量
製法
顔料a)〜f)を混合し、粉砕機にかけて粉砕する。これを高速ブレンダーに移し、g)〜j)を混合する。これを粉砕機で処理し、圧縮成形する。
本発明の化粧料の抗シワ改善効果につき、使用テストにより効果試験を行った。使用テストは、それぞれ30〜60才の10名の女性をパネラーとし、毎日朝と夜の2回、1ケ月にわたり洗顔後に試験化粧料を顔面に塗布することにより行った。試験化粧料は、段落0050に記載の化粧料を用いた。対照品としては上記の化粧料から、紫サツマイモ抽出液添加培養菌体抽出液の変わりに表中の抽出物に置き換えたものを使用した。結果を表−3に示す。なお、評価基準は下記の基準により評価した。
<抗シワ効果評価基準>
・有効‥‥‥‥肌のシワが改善された。
・やや有効‥‥肌のシワがやや改善された。
・無効‥‥‥‥かわらない。
尚、紫サツマイモ、サツマイモをエタノール、エタノール水混液、1,3-ブチレングリコール、1,3−ブチレングリコール水混液等で抽出した抽出液を培地に添加した場合においても、同様の傾向が確認された。
Claims (4)
- 紫サツマイモ抽出液を配合した培地で培養したSaccharomyces cerevisiae の菌体抽出液からなる抗酸化剤。
- 紫サツマイモ抽出液を配合した培地で培養したSaccharomyces cerevisiae の菌体抽出液からなる紫外線傷害抑制剤。
- 請求項1および/または請求項2記載の各剤を配合したことを特徴とする化粧料。
- 請求項1および/または請求項2記載の各剤を配合したことを特徴とする抗光老化化粧料。
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