JP2015157772A - 細胞分化促進剤及び化粧料 - Google Patents
細胞分化促進剤及び化粧料 Download PDFInfo
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- JP2015157772A JP2015157772A JP2014032310A JP2014032310A JP2015157772A JP 2015157772 A JP2015157772 A JP 2015157772A JP 2014032310 A JP2014032310 A JP 2014032310A JP 2014032310 A JP2014032310 A JP 2014032310A JP 2015157772 A JP2015157772 A JP 2015157772A
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- lactic acid
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Abstract
【解決手段】キク科植物のオグルマ属(Inula)旋覆花、シカギク属(Matricaria)カミツレ、キク属(Chrysanthemum)野菊に属する植物から選択される1種又は2種以上の花部を、乳酸菌及び/又は酵母菌により発酵させて得られた発酵物を有効成分とする細胞分化促進剤。
【選択図】図−1
Description
まず、本願発明の概要を説明する。
大きくは次の工程に分けられる。
(第一工程)特定の植物の花部を浸出させた浸出液を得る工程。
(第二工程)第一工程で得られた浸出液に、予め培養しておいた乳酸菌等を植菌し、一定条件下で培養して発酵処理物を得る工程。
(第三工程)第二工程で得られた培養処理物をろ過等により、発酵培養液と残渣に分ける工程。
(第四工程)第三工程で得られた残渣に更に溶媒等を加えて発酵抽出液を得る工程。
本願では、第三工程、第四工程で得られた有効物を利用する。
(1)浸出液とは、本願で用いる植物の花部を精製水、エタノール等の溶媒で浸出させたものを言い、植物の花部をろ過等により取り除く前の段階のものを指す。適宜熱を加えた、所謂植物抽出液も含まれる。
(2)培養前エキスとは、浸出液から植物の花部をろ過等により、取り除いたものを指す。
(3)発酵処理物とは、浸出液に乳酸菌等を植菌し、一定期間培養したものを指す。
(4)発酵培養液とは、発酵処理物から、ろ過等により発酵させた花部を取り除いたものを指す。
(5)発酵抽出液とは、(4)にて取り除いた発酵済みの花部に、更に精製水などの抽出溶媒を加え、一定条件下で抽出した後、ろ過などにより残渣を取り除いたものを指す。
(6)発酵物とは、発酵培養液と発酵抽出液を合わせた総称である。
本願では、どちらの菌で処理したかを明確にする為、乳酸菌で処理した場合は「乳酸菌発酵処理物」、「乳酸菌発酵培養液」等と、酵母菌で処理した場合は「酵母菌発酵処理物」、「酵母菌発酵培養液」等と記載する。酵母菌を酵母と称する場合もあるが同義である。
まず、浸出しようとするキク科植物の花部を前記溶媒に浸漬、または分散させる。この場合、植物体の花部は生のまま用いても、又予め乾燥もしくは半乾燥した上用いてもよい。又、形状としては、採取したものをそのまま用いることもできるが、細断或いは粉砕して微細化すれば、後の発酵工程において発酵効率を上げることができる。
本願発明においては、当該浸出液は、浸出させた花部を取り除かないまま、次工程である発酵工程に供する。乳酸菌等を植菌して発酵させることにより、花部の組織を乳酸菌等により分解させることが出来る。これにより、従来法による抽出では抽出できなかった成分も抽出が可能となり、今までにない効果が得られる。
もっとも、浸出を十分に行っている場合等においては、花部から成分を十分抽出出来ているので、花部を取り除いて使用しても差し支えない。この場合は抽出された成分が乳酸菌等により発酵されて、新たな有効成分に変換される。その意味では、通常の植物エキスに該当する状態のものであっても浸出液として利用することが出来る。
加熱殺菌法としては、キク科植物を加えた発酵液を105〜121℃で10〜20分間加熱するオートクレーブ殺菌法や、キク科植物を加えた発酵液を80〜90℃に60〜120分間保持することを1日1回2〜3日間繰り返す間断殺菌法が一般に用いられる。
もっとも、本殺菌工程は必須工程ではない。次工程の発酵工程において障害にならなければ、本殺菌工程を省いても差し支えない。
市販のヨーグルトや、また自然界からも容易に分離することができる。また、微生物分譲機関においても分譲されているため、当該菌株を購入して利用することが可能である。なお分離の方法は、特開2012−105639に記載の方法等により分離が可能である。
たとえば酵母菌であればCzapek培地、Burkholder培地、YNB(Yeast Nitrogen Base)培地を基本培地とすることが出来る。
発酵日数は、至適温度に於いて一般に3〜50日、好ましくは7〜35日の範囲である。発酵日数が上記の一般的範囲より短くなると発酵が十分に行われず発酵物の有効性が低下する場合がある。一方35日を越えて長くしても有効性のそれ以上の有効性は認められなく好ましくない。
発酵工程は静置で行えば十分であるが、発酵時間の短縮等の為、振とう培養、通気培養を行うことも可能である。上記条件で発酵工程を経た浸出液を発酵処理物と称す。この段階では溶媒と植物体が混在した状態である。以上の発酵処理が終ったならば、発酵を停止させる為、発酵処理物に80〜120℃で15〜120分程度の加熱殺菌処理を施す。
尚、発酵抽出液を得る際の抽出温度、及び抽出時間は目的に応じて適宜調整可能であるが、殺菌処理を兼ねて抽出温度は、80〜120℃、好ましくは90℃〜105℃である。抽出時間は、15〜120分間、好ましくは30〜60分間である。
乳酸菌は(独)製品評価技術基盤機構より分譲を受けたLactobacillus delbrueckii (NBRC 102622)、およびLactobacillus plantarum(NBRC 101975)をもちいた。乳酸菌の培養はMRS培地にて、培地30mlに分譲を受けた菌株の1白金耳を摂取し、30℃にて3日間静置培養を行い、乳酸菌培養液とした。
(MRS培地組成:ペプトン10g、牛肉エキス10g、酵母エキス5g、グルコース20g、Tween80 1g、K2HPO4 2g、酢酸ナトリウム 5g、クエン酸二アンモニウム 2g、MgSO4・7H2O 0.2g、MnSO4・nH2O 0.05g、精製水1L)
酵母菌は(独)製品評価技術基盤機構より分譲を受けたSaccharomyces cereviciae(NBRC 0308)を用いた。酵母菌の培養は、YNB(Yeast Nitrogen Base) with Ammonium Sulfate 合成培地にグルコースを2.0%添加した(以下、本明細書中では、YNB-SG培地と称す)培地50mlに分譲を受けた菌株の1白金耳を摂取し、25℃にて5日間静置培養を行い、酵母菌培養液とした。
<YNB-SG培地組成>
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、グルコース20,000(単位は全てmg/L)。
乾燥旋覆花の花部10gに精製水100mlを加え、室温で浸出させた。その後、105℃、15分間滅菌処理を行った。冷却後前記乳酸菌培養液5ml、又は酵母菌培養液5mlを前記浸出液に加えよく撹拌した。乳酸菌を植え付けた浸出液は30℃、酵母菌を植え付けた浸出液は25℃にてそれぞれ28日間静置培養を行い、乳酸菌発酵処理物又は酵母菌発酵処理物を得た。
カミツレ花部発酵処理物、野菊花部発酵処理物も同様に調製を行った。
得られた旋覆花の花部発酵処理物は、発酵工程を停止させる為、105℃、15分間の滅菌処理を行う。滅菌後、ろ布で培養液を絞り出す。ろ液は6,000rpm×10min.で遠心分離を行い、上清を取り効果試験の旋覆花の花部乳酸菌発酵培養液又は旋覆花の花部酵母菌発酵培養液とした。さらに、ろ布に残った旋覆花の花部残渣1gに、精製水を10mlの割合で加え、105℃、15分間滅菌抽出した。滅菌抽出後、ろ布で抽出液を絞り出し、効果試験の旋覆花の花部乳酸菌発酵抽出液又は旋覆花の花部酵母菌発酵抽出液とした。
カミツレ花部発酵物、野菊花部発酵物も同様に調製を行った。
尚、[表−1]中、「培養前」とは「培養前エキス」を指す。
細胞:Hacat細胞
培地:Humedia KG2
D-MEM (Ca濃度:1.8mM)
固定液:Mildform 10NM(和光純薬工業)
染色:0.05%ナフトールブルーブラック溶液(9%酢酸、0.11M酢酸ナトリウム)
ヒト表皮細胞であるHacat細胞をHumedia KG2(クラボウ)培地で培養した。
細胞を12 well plateに50%コンフルーエント程度に植え付け培養した。翌日各試料を添加した。添加後、72時間培養後に細胞を固定し、ナフトールブルーブラック溶液で染色し、細胞の形態を顕微鏡観察し、分化の程度を判定した。
表皮細胞であるケラチノサイトは分化すると細胞同士が接着し、無定形の形を取る。一方、未分化の細胞は一つ一つの細胞が独立し接着しないことが一般に知られている。ケラチノサイトの分化は培地内のCa濃度により促進される。[図―1]の写真のようにHacat細胞をCa濃度1.8mMのD-MEM培地で培養すると細胞は完全に分化し(評価点5番の写真に該当)、Ca濃度が低いHumedia KG2培地では細胞は未分化の状態で増殖する(評価点1番の写真に該当)。この未分化と分化状態の細胞同士の接着具合を5段階で顕微鏡による目視評価を行い、細胞分化度を評価した。細胞分化度の判定基準は[図―1]に示した基準により判定を行った。
[図-1]の細胞分化度評価の評価基準に基づき、各試料を添加した場合の細胞分化度の結果を[表-1]に示した。なお、細胞への発酵物の添加量は、[0051]にて調製したものを培地に2.5%添加して実験を行った。実施例1として旋覆花、実施例2としてカミツレ、実施例3として野菊の結果を示した。実施例1の旋覆花では、発酵前エキスの細胞分化度評点が2点でほとんど細胞分化促進効果を示さなかったが、酵母を用いて得られた発酵培養液では評点4、発酵抽出液では評点4と非常に高い細胞分化促進効果を示した。さらに乳酸菌を用いて得られた発酵培養液においても評点4、乳酸菌を用いて得られた発酵抽出液でも評点4と非常に高い細胞分化促進効果を示した。同様に、実施例2のカミツレ、実施例3の野菊においても、培養前エキスと比較して、高い細胞分化促進効果を示した。尚、酵母菌を他の種類に代えて調製した発酵物、乳酸菌の種類をL.plantarumに変えて調製した発酵物、酵母菌と乳酸菌を混合して調製した醗酵物においても同様の結果を示した。
ヒト上腕内側皮膚に2.5%SDS水溶液を一日5分間塗布し、これを10日間繰り返すことにより、バリア機能が破壊された皮膚モデルとした。その後、香料・防腐剤・酸化防止剤を除いた処方例6に示す化粧水に対して、処方例6の化粧水から、発酵抽出液(カミツレ・花・酵母)、発酵抽出液(カミツレ・花・乳酸菌)、発酵抽出液(野菊・花・酵母)、発酵培養液(野菊・花・酵母)を除いて精製水と置き換えた化粧水を作成した。選出したパネラー5名にそれぞれの試験品を2週間塗布してもらった後、TEWAメーターTM-210(Courage+Khazaka製)にて皮膚水分蒸散量の測定を行った。
尚、発酵培養液(野菊・花・酵母)とは、野菊の花部の浸出液に酵母を植菌し、発酵処理後得られた発酵培養液を意味する。以下、同趣旨で記載する。
この結果を表2に示す。表3より、SDS塗布により皮膚のバリア機能が破壊され、TEWL値が36まで上昇した。一方、実施例-4の本発明品による化粧水の塗布により、TEWL値が16まで減少した。ここで、本発明による比較例-3の発酵抽出液、発酵培養液を添加していない化粧水を塗布した場合のTEWL値は30であったことより、本発明による発酵抽出液、発酵培養液を添加した化粧水を塗布した群が明らかに皮膚バリア機能の改善が認められた。
従って、本願の細胞分化促進剤は、バリア機能改善剤としても利用できる。
被験者として、20〜60歳の女性10名に1日2回(朝、夜)連続1ヵ月間、試験品と比較品のそれぞれの皮膚外用剤を使用させ、塗布部位の状態を試験前後で比較し、改善効果を調べた。本試験には、試験品として処方例8で示した皮膚外用剤を用い(実施例−5)、比較品には処方例8に示した皮膚外用剤から発酵抽出液(旋覆花・花・酵母)、発酵培養液及び発酵抽出液(カミツレ・花・乳酸菌)、発酵抽出液(野菊・花・酵母)を除き、精製水と置き換えた皮膚外用剤を作成し(比較例−4)、その使用による効果について調べた。評価は[表-3]に示す基準に従って行った。本発明の有効成分を配合した皮膚外用剤を毎日使用しながら肌の状態を塗布開始前及び1ヶ月塗布後におけるアンケートで集計し、効果の確認を行った。結果は表−4に示す。表中の数字は、人数を示している。表−4からも明らかなように、発酵物の入った試験品の皮膚外用剤では、評価点数が71点を示した。一方、試験品から発酵物を抜いた比較品では、評価点数が34点であった。この結果から明らかなように、発酵物を添加することにより高い整肌効果が得られることが明らかとなった。
以下の化粧料の処方例で示す発酵物は、[0046]で示した方法で調製した、各培養液、抽出液を示す。尚、「発酵培養液(旋覆花・花・酵母)」と示した場合は、旋覆花の花部を酵母菌で発酵処理した発酵培養液を意味する。以下同様に示す。
(処方例1)化粧用クリーム(質量%)
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)発酵培養液(旋覆花・花・酵母)・・・5.0
h)発酵抽出液(旋覆花・花・酵母)・・・5.0
i)1,3−ブチレングリコール・・・5.0
j)水酸化カリウム・・・0.3
k)防腐剤・酸化防止剤・・・適量
l)精製水・・・残部
製法
a)〜f)までを加熱溶解し、80℃に保つ。i)〜l)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)、h)を加え、攪拌し均一に溶解する。
(処方例2)化粧用クリーム(質量%)
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)発酵培養液(カミツレ・花・乳酸菌)・・・10.0
h)発酵抽出液(カミツレ・花・乳酸菌)・・・5.0
i)1,3−ブチレングリコール・・・5.0
j)水酸化カリウム・・・0.3
k)防腐剤・酸化防止剤・・・適量
l)精製水・・・残部
製法
a)〜f)までを加熱溶解し、80℃に保つ。i)〜l)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)、h)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)発酵抽出液(野菊・花・酵母)・・・0.1
g)発酵培養液(野菊・花・乳酸菌)・・・1.0
h)1,3−ブチレングリコール・・・7.0
i)カルボキシビニルポリマー・・・0.2
j)水酸化カリウム・・・0.1
k)精製水・・・残部
l)防腐剤・酸化防止剤・・・適量
m)エタノール・・・7.0
製法
a)〜e)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)〜e)に加えて乳化し、50℃まで撹拌しながら冷却する。50℃でf)、g)、m)を添加し、40℃まで攪拌、冷却する。
a)発酵抽出液(旋覆花・花・乳酸菌)・・・5.0
b)発酵培養液(カミツレ・花・酵母)・・・0.001
c)発酵培養液(野菊・花・酵母)・・・0.01
d)発酵抽出液(野菊・花・乳酸菌)・・・0.1
e)グリセリン・・・5.0
f)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
g)エタノール・・・6.0
h)香料・・・適量
i)防腐剤・酸化防止剤・・・適量
j)精製水・・・残部
製法
a)〜j)までを混合し、均一に溶解する。
a)発酵培養液(旋覆花・花・乳酸菌)・・・0.01
b)発酵抽出液(旋覆花・花・乳酸菌)・・・10.0
c)発酵抽出液(カミツレ・花・乳酸菌)・・・1.0
d)発酵抽出液(野菊・花・乳酸菌)・・・0.1
e)グリセリン・・・5.0
f)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
g)エタノール・・・6.0
h)香料・・・適量
i)防腐剤・酸化防止剤・・・適量
j)精製水・・・残部
製法
a)〜j)までを混合し、均一に溶解する。
a)発酵抽出液(カミツレ・花・酵母)・・・10.0
b)発酵抽出液(カミツレ・花・乳酸菌)・・・2.0
c)発酵抽出液(野菊・花・酵母)・・・1.0
d)発酵培養液(野菊・花・酵母)・・・0.1
e)グリセリン・・・5.0
f)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
g)エタノール・・・6.0
h)香料・・・適量
i)防腐剤・酸化防止剤・・・適量
j)精製水・・・残部
製法
a)〜j)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)発酵抽出液(カミツレ・花・酵母)・・・1.0
n)発酵培養液(カミツレ・花・乳酸菌)・・・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)、n)を添加し、40℃まで攪拌、冷却する。
a)発酵抽出液(旋覆花・花・酵母)・・・2.0
b)発酵培養液(カミツレ・花・乳酸菌)・・・2.0
c)発酵抽出液(カミツレ・花・乳酸菌)・・・2.0
d)発酵抽出液(野菊・花・酵母)・・・2.0
e)グリセリン・・・5.0
f)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
g)エタノール・・・6.0
h)香料・・・適量
i)防腐剤・酸化防止剤・・・適量
j)精製水・・・残部
製法
a)〜j)までを混合し、均一に溶解する。
Claims (4)
- キク科植物のオグルマ属(Inula)旋覆花(Inula britannica L. subsp. japonica Kitam)、シカギク属(Matricaria)カミツレ(Matricaria chamomilla L.)、キク属(Chrysanthemum)野菊(Chrysanthemum indcum L.)から選択される1種又は2種以上の花部を乳酸菌または酵母菌により発酵させて得られる発酵物を有効成分とする細胞分化促進剤。
- 乳酸菌がLactobacillus delbrueckii 、及び/又はLactobacillus plantarumである請求項1記載の細胞分化促進剤。
- 酵母菌がSaccharomyces cereviciaeである請求項1記載の細胞分化促進剤。
- 請求項1乃至3のいずれかに記載の細胞分化促進剤を配合したことを特徴とする化粧料。
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