JP4601891B2 - Cell active substance, method for producing the same, and pharmaceutical, cosmetic and food containing the cell active substance - Google Patents

Description

【００６７】
【表２】

【００６８】
【表３】

【００６９】
【表４】

【００７０】
本発明の細胞活性物質を含有する化粧料としては、さらに以下のものを例示することができる。
そのような化粧料としては、固形石鹸、クレンジングフォーム、クレンジングジェル、クレンジングクリーム、クレンジング乳液、クレンジングマスク、クレンジングオイル、洗顔パウダー、シェービングフォーム等の洗顔料類；
柔軟性化粧水、収れん性化粧水、洗浄化粧水、多層式化粧水などの化粧水類；
保湿、柔軟、血行促進、紫外線防御、化粧下地、メークアップベース、角質柔軟、毛髪保護、脱毛防止、整髪、防臭、ひげそり、ボディ用、ハンド用、マッサージ用などの各用途に用いられるオイル類、ジェル類、乳液類、またはクリーム類；
ピールオフタイプ、拭き取りタイプ、洗い流しタイプ、固化後剥離タイプ、貼付タイプ、含浸タイプなどの各タイプのパック・マスク類；
パウダー、白粉、打粉、ファンデーション、口紅、頬紅、アイライナー、マスカラ、アイブロー、ネールエナメル、アイシャドー、エナメルリムーバー、ネールトリートメントなどのメーキャップ化粧品類；
ヘアフォーム、ヘアムース、ヘアスプレー、ヘアジェル、ウォーターグリース、セットローション、ポマード、チック、ヘアクリーム、トリートメントローション、枝毛コート、ヘアオイル、パーマネントウェーブ用剤、染毛剤、ヘアブリーチなどの染毛用化粧料類；
石鹸、薬用石鹸、液体ボディシャンプー、ハンドソープ、フットソープなどの全身洗浄化粧料類；
サンスクリーン、サンタン化粧品、アフターサンケアなどのサンケア化粧料類；
デオドラントローション、デオドラントスプレー、デオドラントスティック、オーデコロン、香水等の防臭芳香化粧料類；
脱毛、除毛、浴用、インセクトリペラーなどの各用途に用いられる衛生用化粧料類；
歯みがき、洗口、防臭、清涼などの各用途に用いられる口腔用化粧料類；
ウエットティッシュ、おしり拭き用ウエットティッシュ、綿棒等の拭き取り用、塗布用紙綿類含浸薬液類；
エアゾール用薬液類などが挙げられる。
【００７１】
上記に、本発明の細胞活性物質を医薬品・化粧料に用いる場合の好ましい態様の例を挙げたが、本発明の効果が得られる限り、本発明の医薬品・化粧料の剤型、投与方法、使用方法等は制限されることはない。また、上記以外にも、本発明の効果を達成し得る限り、本発明の医薬品および化粧料には、医薬品および化粧料の成分として通常使用される成分を配合することができる。
【００７２】
さらに、本発明の細胞活性物質である繊維芽細胞エネルギー代謝・増殖活性物質は、食品に添加して用いることもできる。具体的には、食品を製造する際に、深層水脱塩濃縮液を使用するものであれば、その添加方法、添加量は特に制限されない。本発明の食品が細胞活性物質を含むことにより、消費者は手軽に繊維芽細胞エネルギー代謝・増殖活性物質を摂取することができ、老化症状を効率的に予防あるいは治療することができる。また、生体細胞を活性化することで、免疫力を強化することもできる。
【００７３】
そのような本発明の食品としては、野菜・果汁飲料、炭酸飲料、乳酸菌飲料、珈琲飲料、加工乳飲料、スポーツ飲料、ミネラルウォーター、日本茶、中国茶、紅茶、ハーブティーなどのノンアルコール飲料；
ビール、発泡酒、日本酒、焼酎、泡盛、ワイン、ジン、ウォッカ、ラム、ブランデー、ウイスキー、シャンパン、中国酒、甘酒などのアルコール飲料；
ソース、ドレッシング、みりん、食酢、トマトケチャップなどの調味料；
味噌、納豆、醤油、漬物、ヨーグルトなどの発酵食品；
ゼリー、寒天、シャーベット、豆乳、豆腐、パン、クッキー、餅、炊飯済みライス、生麺、ベビーフードや介護食品等におけるレトルト食品や、乾燥食品、冷凍食品などの加工食品；
カプセル状や錠剤とされたサプリメント等が挙げられる。
【００７４】
上記に、本発明の細胞活性物質を用いることができる食品の例を挙げたが、本発明の効果が得られる限り、これらに限定されるものではなく、かつ本発明の食品の製造方法等も制限されることはない。また、上記以外にも、本発明の効果を達成し得る限り、本発明の食品には、食品の成分として通常使用される成分を配合することができる。
【００７５】
なお、上記好酸球活性物質以外の免疫細胞活性物質についても、好酸球活性物質と同等の効果が得られ、医薬品、化粧品、食品等の成分として有用である。
【００７６】
【発明の効果】
本発明の細胞活性物質（繊維芽細胞エネルギー代謝・増殖活性物質などの皮膚細胞活性物質および好酸球活性物質などの免疫細胞活性物質を含む）は、海洋深層水に特定の処理をすることにより得られる溶液中に含有される物質であり、ゲル濾過クロマトグラフィーなどの適切な手段で水から分離して得られる一定の分子量を有する高分子化合物であろうと推定される。
【００７７】
従来より、海洋深層水からの分離物を皮膚治療あるいはアレルギー治療に使用することは知られているが、これらは、海洋深層水中に含有される無機成分を用いたものである。即ち、海洋深層水中には塩化ナトリウム、塩化カルシウム、塩化マグネシウムのような塩化物のほかに、種々多様なミネラルが含まれており、これらのミネラルを皮膚治療またはアレルギー治療に利用することが知られていた。従って、皮膚治療およびアレルギー治療に海洋深層水を使用する場合には、海洋深層水を濃縮して析出した析出物（ミネラル分）を海水から分離して使用していた。さらに、海洋深層水中には皮膚治療またはアレルギー治療に有効な有機成分は含有されているとは考えられていなかったことから、海洋深層水中に含有される有機物質を分離して使用することは試みられていなかった。
【００７８】
これに対して本発明者は、海洋深層水中には上記のような無機塩とは別に有機物質が含有されており、この有機物質は、海洋深層水中に含有されるミネラルとは全く異なる機能を有しており、この機能を利用することにより、皮膚を形成する繊維芽細胞のエネルギー代謝・増殖活性などの皮膚細胞活性および好酸球活性などの免疫細胞活性を向上させることができること見出したのである。
【００７９】
本発明の細胞活性物質は、海洋深層水に特定の処理を施すことにより得られ得る有機物質を含有しており、この有機物質は繊維芽細胞のエネルギー代謝・増殖活性および好酸球活性を向上させることができる。
こうした細胞活性物質は、種々の有機物質の複合体であり、個々の成分を特定することが極めて困難であるが、海洋深層水に本発明で規定する処理をすることにより繰り返し得ることができる。
【００８０】
そして、この細胞活性物質は、ゲル濾過クロマトグラフィーを用いて分子量により、複数の画分にピークを有する有機物質の複合体であることがわかった。これらの各画分に分離された成分は、単独では、例えば繊維芽細胞に対するエネルギー代謝・増殖活性あるいは好酸球活性が異なるが、これらの複合体をそのまま使用することにより、特に繊維芽細胞に対して優れたエネルギー代謝・増殖活性および好酸球に対する活性の向上効果を示すことが確認された。
【００８１】
しかも、このような本発明の細胞活性物質（繊維芽細胞エネルギー代謝・増殖活性物質などの皮膚細胞活性物質および好酸球活性物質などの免疫細胞活性物質を含む）は、通常の細胞活性物質と比較して、副作用が著しく少なく、極めて安全性が高く、特に繊維芽細胞に対しては、副作用なしに優れた細胞エネルギー代謝・増殖活性（細胞活性）を示すとの特異的な効果が認められるため、特に皮膚外用剤として有用性が高い。また、同様な理由から、特にアトピー性皮膚炎などのアレルギー疾患の治療薬として非常に有用性が高い。
【００８２】
また、本発明の細胞活性物質（繊維芽細胞エネルギー代謝・増殖活性物質などの皮膚細胞活性物質および好酸球活性物質などの免疫細胞活性物質を含む）は、水に対する親和性がよいので、外用薬、内服薬、注射液など種々の形態で利用することができる。
さらに具体的には、本発明の繊維芽細胞活性物質によれば、上述のように、顕著に繊維芽細胞を活性化し得るので、人の皮膚の細胞のエネルギー代謝活性や増殖活性を改善することができ、ヒト皮膚のしわ、しみ、たるみの発生といった老化現象を予防あるいは治療することができる。
【００８３】
また、たとえば、本発明の好酸球活性物質では、上述のように、好酸球を活性化させることができるため、好酸球が活性化して産生したスーパーオキサイドなどの作用により非常に優れた生体防御機能を有する。また、この物質は、炎症をメディエートするヒスタミンなどを代謝させてアレルギー反応の修復に働くヒスタミラーゼなどを放出する。さらに、この好酸球活性物質を好酸球に作用させることにより、好酸球からスーパーオキサイド（Ｏ₂ ^-）が生成し、このスーパーオキサイド（Ｏ₂ ^-）から酵素的反応あるいは非酵素的反応により反応性の高い各種活性酸素種を生成させることができる。そして、これらの活性酸素種に殺菌作用などの重要な生体防御を発現させることができる。
【００８４】
そして、本発明の好酸球活性物質の有する上記のような機能が複合的に作用すると考えられるので、本発明の好酸球活性物質は、喘息や花粉症、皮膚炎などのアレルギー疾患の治療成分として、特にアトピー性皮膚炎などのアレルギー疾患の治療成分として有用性が高い。
【００８５】
【実施例】
次に、実施例を示して本発明をさらに詳細に説明するが、本発明はこれらによって限定されるものではない。
【００８６】
【実施例１】
［細胞エネルギー代謝・増殖活性物質の調製］
駿河湾沖深度６８７mから海洋深層水２０リットルを採取した。
この海洋深層水２０リットルを目開き０．４５μmのメンブランフィルターを用いて濾過し、この海洋深層水中の浮遊物を除去した。
【００８７】
こうして濾過された海洋深層水を減圧蒸留装置に入れ、減圧度２０Torr の圧力条件で、蒸留温度を４０℃以下の温度に設定して、海洋深層水の量が約３．３リットル（元の海洋深層水の1／６の量）になるまで減圧蒸留した。
こうして減圧蒸留することにより、海洋深層水に溶存していた塩化ナトリウム、塩化カリウム、塩化マグネシウムなどの無機塩が析出した。この析出した無機塩を目開き０．５μmのメンブランフィルターで濾過した。なお、濾別された無機塩は、本発明では使用しないので廃棄した。
【００８８】
次いで、上記工程で得られた濾液をモザイク荷電膜（商品名：ＭＣ−５５Ｂモザイク荷電膜、東ソー（株）製）を用いて低温８℃の条件で脱塩操作を行い、さらにこの脱塩された液を減圧蒸留装置に入れて、２０Torrの減圧下に、４０℃の温度を超えないように温度を管理して、液の容量が２００ml（元の海洋深層水の約1／１００の量）になるまで減圧下に水分を蒸留除去した。
【００８９】
上記のモザイク荷電膜を用いた脱塩および減圧蒸留を繰り返して、５mlの濃縮物を得た。
この濃縮物中には、０．２重量％の有機物質が含有されており、さらにこの濃縮物中における無機塩の含有率は１００ppm以下、無機塩化物の含有率は７０ppm以下であった。
【００９０】
この濃縮物をファルマシヤ社製のカラム（セファクリルＳ-300）を有するゲル濾過クロマトグラフィーを用い、展開溶媒としてリン酸バッファー生理食塩水(PBS-)を用いて含有成分を分析した。
この分析結果を、分子量に従って、
分子量１００万以上の第１画分、
分子量３万〜１００万の第２画分、
分子量５０００〜３万の第３画分、および、
分子量５０００以下の第４画分に分けてそれぞれの画分における波長２８０nmでの吸収ピークの有無を調べた。
【００９１】
結果を図２に示す。
図２に示したように、第４画分に最も大きなピークが見られ、第３画分では、第４画分と裾が重なる形で、第４画分の半分以下の大きさのピークが見られた。また、第１画分にも小さいながらも明確なピークが見られた。しかし、第２画分には、ＵＶのピークは見られなかった。
【００９２】
なお、これらの画分に表れたピークの成分の解析の参考にするために、平均分子量13700のRibonuclease Ａおよび平均分子量44万のFerritinを含有する液を調整し、上記と同様にして分析して、その結果を図２に破線で併せて記載する。
［繊維芽細胞エネルギー代謝・増殖活性の測定］
上記のような構成を有する細胞エネルギー代謝・増殖活性物質（繊維芽細胞物質を含む）を、固形分換算で０．１重量％となるように標準液であるリン酸バッファー生理食塩水(PBS-)に加えて本発明の試料を調整した。なお、各試料の最終塩分は標準液であるＰＢＳ−の塩分にあわせた。
【００９３】
ヒト真皮由来繊維芽細胞の3次元細胞組織モデル（東洋紡(株)製、MATREX-LDM）を用いて、MTT法により細胞のミトコンドリアのエネルギー代謝活性を評価した。
ここでMTT法は、3-(4,5-Dimethyl-2-thiazoly)-2,5-diphenyltertrazolium Bromideを用いた細胞のエネルギー代謝活性を測定する方法である。即ち、MTTは、細胞のミトコンドリアのエネルギー代謝過程で生成したNADH(reduced nicotinamide adenine dinucleotide)あるいはNADPH(reduced nicotinamide adenine dinucleotide phosphate)によって還元され、青色のホルマザンを生成する。
【００９４】
青色のホルマザンを波長５７０nmにおいて吸光測定し比色測定し、比色定量することによって細胞の代謝活性及び細胞の増殖活性を評価する方法である。この場合、青色が濃くなればなるほど活性が高いことを示している。
また、本発明では、コントロールとして、上記細胞エネルギー代謝・増殖活性物質を用いずに標準液(PBS-)のみを使用して、同様にしてMTT法により細胞のミトコンドリアのエネルギー代謝活性を調べた。
【００９５】
さらに、ネガティブコントロールとして、典型的な細胞毒性のあるドデシル硫酸ナトリウム（SDS=Sodium dodecyl sulfate）を、０．１重量％配合してMTT法によりエネルギー代謝・増殖活性を調べた。このネガティブコントロールは、エネルギー代謝・増殖活性を全く示さない場合におけるMTT法の最低基準を示すものである。
【００９６】
上記コントロールにより得られたMTT法の値を１００とすると、ネガティブコントロールの値は３３となる。
そして、同様の試験で測定した本発明の細胞エネルギー代謝・増殖活性物質を０．１重量部で含有する試料のMTT法における値は２４５であり、この細胞エネルギー代謝・増殖活性物質は、細胞エネルギー代謝・増殖活性を示すことが明らかである。
【００９７】
また、この試験においてMTT試薬は、細胞のミトコンドリアの代謝過程で生成したNADHあるいはNADPHによって還元され青色になるのではなく、本発明の細胞エネルギー代謝・増殖活性物質自体に還元され青色になる可能性も考えられるため、ドデシル硫酸ナトリウム（SDS＝Sodium dodecyl sulfate）を、０．１重量％に、本発明の細胞エネルギー代謝・増殖活性物質を固形分換算で０．１重量％加え、同様な試験で調べた。その結果、同様な試験における値は２７であり、ドデシル硫酸ナトリウムが単独の場合の値３３とほぼ同様レベルであるため、本発明の細胞エネルギー代謝・増殖活性物質自体がMTT試薬を還元し青色に変色させることはなく、細胞のミトコンドリアが本発明の細胞エネルギー代謝・増殖活性物質を添加することによって代謝活性が活発になり、生成されたNADHあるいはNADPHがMTT試薬を還元して青色に変色させることがわかった。即ち、本発明の細胞エネルギー代謝・増殖活性物質により代謝活性が向上したことは明らかである。また、別の実験で、MTT試薬に上記の深層水細胞活性物質を添加しても、MTT試薬はまったく変色しないことから、上記の結論はさらに証明された。
【００９８】
また、参考のために各画分の成分を固形分換算で０．１重量％となるように標準液であるリン酸バッファ生理食塩水(PBS-)に加えた試料を調整し最終塩分は標準液であるPBS−の塩分にあわせ、MTT法における細胞エネルギー代謝・増殖活性を調べた。結果を図３に示す。
その結果、
分子量１００万以上の第１画分の成分の活性は、１５６、
分子量３万〜１００万の第２画分の成分の活性は、２５７、
分子量３万〜５０００の第３画分の成分の活性は、１５１、
分子量５０００以下の第４画分の成分の活性は、１５５であった。
【００９９】
なお、上記各画分の成分を用いた活性の評価値は、参考的な値であり、さらに詳細に実験を重ねることにより、また、実験の精度を上げることにより上記値がさらに明確な細胞エネルギー代謝・増殖活性を示す可能性が充分にある。
【０１００】
【実施例２】
好酸球活性物質を、実施例１の細胞活性物質の調製と同様の方法にて調製し、得られた好酸球活性物質について、下記の方法により、活性化度を測定した。
［好酸球活性物質の活性化度測定］
本発明の細胞活性物質（好酸球活性物質を含む）の好酸球を活性化する能力（活性化度）は、好酸球が産生するスーパーオキサイド（Ｏ₂ ^-）から派生した過酸化水素（Ｈ₂Ｏ₂）の量を指標にして測定した。前記好酸球活性物質により好酸球が活性化されて産生するスーパーオキサイド（Ｏ₂ ^-）から派生した過酸化水素（Ｈ₂Ｏ₂）の量の測定は、具体的には、2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA)による蛍光をフローサイトメーターを用いて解析することにより行った。
【０１０１】
５×１０⁵cells／ｍｌの好酸球を、2.5μモルDCFH-DAを含むPhosphate-buffer saline glucose(5mモルglucose)(PBSｇ)０．５ｍｌ中で、３７℃で、１５分間インキュベートし、その後、刺激物（ＰＭＡ50ng／ml）を添加し、３７℃で、３０分間インキュベートを行った。PBSｇで２度細胞を洗浄した後、フローサイトメーターで解析・測定した。
【０１０２】
本実験において使用した好酸球は、好酸球に選択的な増殖分化因子であるIL-５を恒常的に産生するトランスジェニックマウス(C3H/HeN-TgN(IL-5)Imeg)の末梢血から分離した好酸球を用いた。末梢血を終濃度０．２２％のクエン酸を含むＣａ⁺⁺フリーのKrebs Ringerリン酸緩衝液（ＫＲＰ）（０．９％ＮａＣｌ、６ｍモルＫＣｌ、１ｍモルＭｇＣｌ₂、１０ｍモルＮａ−リン酸緩衝液（ｐＨ７．４））中に採取し、赤血球、単球を除去する目的で、Percoll不連続密度勾配遠心法を行った。Percoll原液に対して、１０倍濃度のＫＲＰを１／１０量添加したものを１００％Percoll定義細胞混濁の下に６０、７０、８０％Percollを重層し、３５０ｇ、室温、２０分間遠心した後、７０、８０％層を回収した。回収した細胞集団を分離用緩衝液（５ｍモルＥＤＴＡ、０．５％ＢＳＡ、１０ｍモルモルＮａ−Ｋ−リン酸緩衝液（ｐＨ７．２））に更に懸濁（１×１０⁷cells／５０μｌ）し、抗Ｔｈｙ１．２抗体結合磁気ビーズ（１×１０⁷beads／２５μｌ）と抗Ｂ２２０抗体結合磁気ビーズ（１×１０⁷beads／２５μｌ）に添加し、４℃で２０分間反応させた後、永久磁石を用いネガティブセレクションで採取された細胞集団を好酸球集団として実験に供した。なお、分離後の細胞集団の純度はフローサイトメーター（ＢＥＣＴＯＮ ＤＩＣＫＩＮＳＯＮ社製のＦＡＣＳcan）で解析した。
【０１０３】
測定物質は、サンプル／１．８％ＮａＣｌと精製水で調整し、等張に調整したものを終濃度１００μｇ／ｍｌで添加した。
なお、コントロールとしてリン酸バッファー生理食塩水（PBS）を用い、ポジティブコントロールとしてオプソニン化ザイモザン（OZ, 免疫賦活剤）を用いて同様の実験を行った。
【０１０４】
上記のように測定した各被験物質の好酸球活性化度の値（過酸化水素量生産相当）は、図４に示す。また、例として、図５にコントロール（PBS）を用いた場合の好酸球の過酸化水素産生量と、上記好酸球活性物質を用いた場合の好酸球の過酸化水素産生量のフローサイトメーター解析・測定した結果を示す。
図４に示したように、コントロール（PBS）の好酸球活性化度は３．３５であるのに対し、上記好酸球活性物質の好酸球活性化度は７．０９（コントロールの約２．１倍）であった。
【０１０５】
上記のようにして得られた好酸球活性物質について、好酸球活性を調べたところ、コントロール（PBS）およびポジティブコントロール（OZ：オプソニン化ザイモザン）よりも高い好酸球活性を示した。
また、参考のため、上記細胞エネルギー代謝・増殖活性物質と同様に、GPCにより分割した各画分について、好酸球活性を調べたところ、以下のように、いずれの画分においても、コントロール（PBS）およびポジティブコントロール（OZ：オプソニン化ザイモザン）よりも高い好酸球活性を示した。すなわち、
分子量１００万以上の第１画分の成分の活性は、４．５７、
分子量３万〜１００万の第２画分の成分の活性は、５．４８、
分子量３万〜５０００の第３画分の成分の活性は、４．７３、
分子量５０００以下の第４画分の成分の活性は、８．６３であった。
【０１０６】
なお、図３と図４の比較から、上記のようにして得られた高濃縮脱塩海洋深層水を用いた場合は、高分子量の画分では繊維芽細胞活性が高い傾向にあり、また、逆に、低分子量の画分では好酸球活性が高い傾向にあることが示された。
【図面の簡単な説明】
【図１】図１は、本発明の細胞活性物質を得るための工程の例を示す工程図である。
【図２】図２は、本発明の細胞活性物質を、ゲル濾過クロマトグラフィーを用いて各画分に分けて、各画分に表れるピークの例を示した図である。
【図３】図３は、本発明の細胞活性物質の繊維芽細胞活性化度を示すグラフである。
【図４】図４は、本発明の細胞活性物質の好酸球活性化値を示すグラフである。
【図５】図５は、フローサイトメーターで解析・測定した好酸球の過酸化水素産生能のチャートである。[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention relates to a cellular active substance contained in deep ocean water, a method for producing the same, and a pharmaceutical and a cosmetic containing the cellular active substance. More specifically, the present invention relates to a skin cell active substance (including a fibroblast active substance such as a fibroblast energy metabolizing active substance and a fibroblast proliferating active substance), an immune cell active substance contained in deep ocean water. The present invention relates to cell active substances such as (including eosinophil active substances), methods for producing such cell active substances, and pharmaceuticals, cosmetics and foods containing such cell active substances.
[0002]
TECHNICAL BACKGROUND OF THE INVENTION
In the human skin, aging symptoms such as wrinkles, blemishes and sagging appear due to internal factors such as aging or external factors such as ultraviolet rays. Such aging symptoms of the human skin are considered to be caused mainly by a decrease in the function of the cutaneous dermal fibroblasts, and the insufficient secretion of matrix fibers and collagen accompanying the decrease in the function of these cells. Therefore, it is considered that an effective means for preventing the aging of human skin or improving the function of aging skin is to activate skin dermal fibroblasts and further proliferate these cells.
[0003]
Extracts from seaweed and the like are said to be effective for such aging symptoms of human skin, that is, a decrease in the activity of skin dermal fibroblasts. For example, in JP-A-10-330281, extracts from Yakuzusa, Hondara, Fukurinamiji, Fukuronori, Kombu, Kamensou, Kaigankenori, Otosa, Yanagiri, Sozo, Rattle are extracted from dermal fibers. It is described to activate blast cell metabolism. Japanese Patent Application Laid-Open No. 11-116431 discloses an invention for a topical skin preparation containing a culture supernatant of ketoceros gracilis. As used herein, Chaetoceros gracilis is a kind of algae that lives alone in the waters near the sea. It is described that the culture supernatant of this ketoceros gracilis has an effect of improving the metabolic activity of dermal fibroblasts.
[0004]
In addition to these seaweed-derived components, for example, JP-A-2000-169320 discloses any one of a seaweed extract, a chamomile extract, a mugwort extract, a Tamatsuki-suzuraji extract, and a yucca extract. It is described that the combination is effective as a cell activator.
However, such an algae or plant extract may cause fluctuations in the composition of the components extracted depending on the algae or plant habitat, and the extraction operation from such algae or plants is also complicated. There is.
[0005]
JP-A-2000-159654 discloses at least one treatment method selected from ultrafiltration, reverse osmosis membrane filtration methods, electrodialysis methods, ion exchange membrane methods and distillation methods. An invention of cosmetics characterized by containing concentrated water obtained by treatment is disclosed. This publication describes that the conductivity of this concentrated water is 5 mS / cm to 1 S / cm, preferably 10 mS / cm to 500 mS / cm, and seawater has the above conductivity. Is considered to contain a considerable amount of ionic substances. In this publication, the total abundance ratio of chlorine ions, sodium ions and potassium ions per water-evaporated solid in the concentrated water is 35% or less, and the concentrated water actually used in Example 2 and Comparative Example 2 is used. The total abundance of chlorine ions, sodium ions and potassium ions in B1 is 31.5%, and the total abundance of chlorine ions, sodium ions and potassium ions in concentrated water B2 used in Example 2 and Comparative Example 2 is also 32. It shows a very high value of 0.5%. And in this gazette, it is the concentrated water which has these values and is obtained by the method described in this gazette, Comprising: It is irritating with respect to skin among the components contained in deep sea water. It is understood that the invention of cosmetics using deep ocean water containing a high concentration of minerals excluding chlorine ions, sodium ions and potassium ions is disclosed. When chlorine ions, sodium ions, and potassium ions that are irritating to the skin are removed from inorganic substances contained in deep sea water, phosphate ions, silicate ions, nitrate ions, etc., which are generally called nutrient salts The content ratio is increased, and it is considered that these natural minerals have an effect of improving skin dullness, wrinkles and the like and a slimming effect.
[0006]
As described above, JP-A-2000-159654 discloses a cosmetic using concentrated water from deep sea water mainly containing natural minerals contained in deep sea water by concentrating deep sea water. Therefore, it is understood that the effect exerted by the cosmetic disclosed in this publication is a natural mineral component contained in deep ocean water, that is, an inorganic salt.
[0007]
Apart from skin aging due to internal factors such as aging, patients who have various allergic symptoms such as atopic dermatitis due to changes in the living environment and eating habits and stress in recent years as skin diseases Is increasing and is a problem.
Allergic symptoms such as atopic dermatitis are a type of hypersensitivity in which the inherited constitution plays a major role. Antigens that cause allergic symptoms are thought to be in the blood, and the complex of this antigen and antibody releases histamine or histamine-like substances from tissues, resulting in specific symptoms It is believed that. For example, in atopic dermatitis, peripheral blood eosinophils increase, and infiltration of eosinophils is often seen in histological findings. This also suggests that eosinophils are one of the cells that play a central role in atopic dermatitis.
[0008]
Against this background, various studies have been conducted on eosinophil active substances, and it is already known that the activity of eosinophils is enhanced by deep ocean water. The mechanism for eosinophil activity has not been elucidated yet. At present, the idea that eosinophil activity is derived from minerals contained in the deep ocean water is the majority, and the mineral content obtained by concentrating, desalting and separating deep ocean water is large. The isolates contained were used for allergic drugs and the like.
[0009]
Most of the substances obtained by concentrating, desalting and separating the deep ocean water as described above are occupied by inorganic salts such as calcium sulfate. However, it is known that calcium sulfate has substantially no medicinal effects related to allergy.
[0010]
OBJECT OF THE INVENTION
The present invention removes inorganic salts from deep ocean water as much as possible, while skin cell active substances (fibroblasts), which are cellular active substances composed of highly concentrated desalted ocean deep water in which organic substances contained in the ocean deep water are concentrated. It is intended to provide a fibroblast active substance such as a cellular energy metabolic active substance and a fibroblast proliferation active substance) and an immune cell active substance (including an eosinophil active substance).
[0011]
It is another object of the present invention to provide a method for producing a skin cell active substance and an immune cell active substance, which are cell active substances as described above.
Another object of the present invention is to provide pharmaceuticals, cosmetics and foods using cell active substances such as the above-mentioned skin cell active substances and immune cell active substances.
[0012]
Summary of the Invention
The cellular active substance of the present invention can be obtained by performing at least one operation of further concentrating the desalted deep ocean water that has been subjected to the operation of separating at least a part of the inorganic salt dissolved in the deep ocean water. is there.
The desalted marine water may be concentrated desalted marine water concentrated by vacuum distillation or the like, if necessary.
[0013]
In the present invention, the concentration operation (process) refers to an operation for removing dissolved salts dissolved in ocean water. In this step, ocean water may be concentrated and fresh water may be removed.
The cell active substances in the present invention include skin cell active substances and immune cell active substances. The skin cell active substance includes a fibroblast active substance such as a fibroblast energy metabolism active substance and a fibroblast proliferation active substance, and the immune cell active substance includes an eosinophil active substance. It is.
[0014]
In the present invention, deep sea water collected from the deep sea with a depth of 200 m or more is used.
In addition, the method for producing a cellular active substance of the present invention includes a step of separating at least a part of an inorganic salt dissolved from deep ocean water to obtain (concentrated) demineralized ocean deep water, and (concentrated) demineralized ocean. It is characterized in that highly concentrated desalted deep ocean water containing a high concentration of cellular active substances is obtained through a process of further concentrating the deep water to separate precipitates or inorganic salts at least once.
[0015]
Furthermore, the pharmaceutical product of the present invention can be obtained by performing at least one operation of further concentrating (concentrated) demineralized deep ocean water that has undergone an operation of separating at least a part of the inorganic salt dissolved in the deep ocean water. It is characterized by containing a cell active substance to be obtained.
The cosmetic of the present invention is obtained by performing at least one operation of further concentrating (concentrated) demineralized deep ocean water that has undergone an operation of separating at least a part of the inorganic salt dissolved in the deep ocean water. It is characterized by containing a cell active substance that can be obtained.
[0016]
Still further, the food of the present invention is obtained by performing at least one operation of further concentrating (concentrated) demineralized deep ocean water that has undergone an operation of separating at least a part of the inorganic salt dissolved in the deep ocean water. It is characterized by containing a cell active substance that can be obtained.
The deep ocean water contains inorganic salts and organic substances, and the concentration of the inorganic salt dissolved in the deep ocean water is usually about 3.4% by weight. Of these inorganic salts, about 2.2% by weight or more is sodium chloride, magnesium chloride or calcium sulfate, and these inorganic salts have little action as a fibroblast active substance, and particularly in inorganic salts. It is known that calcium sulfate contained in a large amount has substantially no medicinal effects related to allergy.
[0017]
On the other hand, when a concentrated solution obtained by concentration while removing inorganic salts dissolved in deep sea water was analyzed, it was found that the concentrated solution contained an organic substance having a certain molecular weight or more. The details of the origin and structure of such organic substances are not clear, and it is estimated that they contain a very wide variety of components. However, when such an organic substance is analyzed by selecting a developing solvent and using gel filtration chromatography, in the ultraviolet detection analysis at a wavelength of 280 nm, the first fraction having a molecular weight of 1 million or more and the second fraction having a molecular weight of 30,000 to 1,000,000 are used. It was found that an organic substance having a peak was contained in at least one fraction of the fraction, the third fraction having a molecular weight of 5000 to 30,000, and the fourth fraction having a molecular weight of 5000 or less.
[0018]
Then, the present invention selectively collects organic substances contained in such deep ocean waters and investigates cellular activities such as the behavior of fibroblasts and eosinophil activity. The present invention has been completed with the knowledge that there are cell activities such as fibroblast energy metabolism / proliferation activity and eosinophil activity.
[0019]
That is, conventionally, natural minerals, which are inorganic substances other than chlorides, contained in deep ocean water have attracted attention. By blending such natural minerals with a topical medicine for skin such as cosmetics, rare natural minerals can be made into fibers. It was thought that it would act as an active substance having blast cell activity and eosinophil activity, but for these organic substances contained in trace amounts in deep ocean waters, detailed studies on cellular energy metabolism / proliferation activity etc. It has not been. This is because the content of inorganic salts in the deep ocean water is high, and it is extremely difficult to separate organic substances contained in trace amounts by being inhibited by inorganic salts from the inorganic salts. This is because it was extremely difficult to identify the action effect of a very small amount of organic substance in the shadow of an inorganic salt.
[0020]
However, the present inventor has separated and removed inorganic salts as much as possible while repeatedly concentrating deep ocean water to increase the concentration of the organic substance, and a concentrated solution containing this organic substance at a relatively high concentration, for example, fiber buds. When it was allowed to act on cells and eosinophils, it exhibited an excellent effect as a fibroblast energy metabolism / proliferation active substance and an excellent effect as an eosinophil active substance.
[0021]
DETAILED DESCRIPTION OF THE INVENTION
Hereinafter, a fibroblast active substance is taken as an example of a skin cell active substance, and an eosinophil active substance is taken as an example of an immune cell active substance. Cell active substances, methods for producing them, and pharmaceuticals, cosmetics and foods containing the cell active substances will be specifically described.
[0022]
In the present invention, the skin cell active substance refers to a substance that can activate fibroblasts and the like.
In addition, immune cell active substances include leukocytes, ie, monocytes such as macrophages, lymphocytes such as NK cells, T cells, K cells, and B cells, granulocytes such as eosinophils, neutrophils, and basophils. A substance that can activate the like.
[0023]
FIG. 1 shows an example of a typical operation for obtaining the cell active substance of the present invention. In addition, although the cell active substance (skin cell active substance and immune cell active substance) of the present invention can be obtained by the above-described operation, it should not be construed as limited to substances obtained only by this operation. .
First, the cell active substance of the present invention will be specifically described along the production flow illustrated in FIG.
[0024]
As shown in FIG. 1, the cellular active substance of the present invention is produced using deep ocean water as a raw material.
In the present invention, the cell active substance includes a skin cell active substance and / or an immune cell active substance, and particularly preferably includes a fibroblast energy metabolism / proliferation active substance and / or an eosinophil active substance. That is, the cell active substance of the present invention may have a skin cell activity and an immune cell activity at the same time, or may have only a skin cell activity or an immune cell activity.
[0025]
In the present invention, deep ocean water having a depth of 200 m or more, preferably 300 m or more, at least where sunlight does not reach is used. Although the water depth is less than 200m, deep sea water that rises near the sea surface due to the rise of seawater can also be used.
Generally, the depth at which sunlight can reach in ocean water is about 200 m. Therefore, since sunlight does not reach the deep ocean water that exists in the deep ocean below 200m, the photosynthesis by algae or aquatic plants is not performed in this deep ocean water, and plankton, etc. The general view is that there is almost no breeding. In this way, it is thought that organic substances are hardly contained in the ocean currents in the deep ocean, but on the other hand, the mineral content is higher than normal ocean water (meaning ocean water other than ocean deep water). It is known that these minerals also contain rare minerals that tend to be deficient in the current normal diet. Therefore, in the conventional deep sea water, it was thought that the rare minerals as described above act in a complex manner and have activity against fibroblasts or eosinophils.
[0026]
However, when the inventor analyzed the deep ocean water in detail, it was found that unexpectedly many organic substances were dissolved in the deep ocean water. Therefore, it is highly possible that such dissolved organic substances have cell activity, and if such components can be obtained, high cell activity, specifically, fibroblast energy metabolism, Skin cell active substances such as proliferative active substances (fibroblast active substances) or immune cell active substances such as eosinophil active substances can be obtained. These fibroblast energy metabolism / proliferation active substances are effective in preventing skin aging or improving the function of aging skin, and these eosinophil active substances are very useful as therapeutic agents for allergic diseases such as atopy. High nature.
[0027]
The deep ocean water collected in this way is filtered using a filter having a relatively small opening, such as a membrane filter, in order to remove suspended matter that may be mixed during the collection. In the present invention, it is usually filtered using a filter having an opening of 0.01 to 2 μm, preferably 0.1 to 1 μm, particularly a membrane filter. By using such a filter, fine components that cannot be confirmed with the naked eye can be filtered out. If there are precipitates or inorganic salts that precipitate at this stage, such precipitates may be separated at this stage.
[0028]
In the present invention, since the filtrate filtered as described above is used, the substance (residue) that could not pass through the filter is discarded.
In the present invention, the filtrate obtained as described above is distilled under reduced pressure, and at least a part of the water contained in the filtrate is removed by this reduced-pressure distillation operation. When the first deep sea water sampled is 100 parts by volume, the distillation operation under reduced pressure usually results in 1 to 50 parts by volume, preferably 1 to 30 parts by volume with respect to the first deep sea water sample. Further, the distillation under reduced pressure is carried out so that the amount is more preferably 2 to 25 parts by volume, particularly preferably 5 to 20 parts by volume. Specifically, under a reduced pressure of 1 to 40 torr, preferably 5 to 30 Torr, at a temperature of 50 ° C. or lower, preferably 40 ° C. or lower, 10 minutes to 3.0 hours, preferably 30 minutes to 1.0. Distill under reduced pressure over time. Note that the distillation temperature during this vacuum distillation is preferably as low as the above range in order to prevent decomposition of dissolved substances dissolved in deep sea water. Therefore, during the vacuum distillation used here, The vacuum distillation operation can be carried out at a lower temperature by lowering the pressure of. A part of the water in the deep sea water is separated by this vacuum distillation operation, and the separated water (fresh water) is discarded. Moreover, if the processing capacity of the facility for separating the inorganic salt is sufficient, the above-described concentration operation can be omitted.
[0029]
Moreover, instead of concentrating deep sea water by vacuum distillation and separating inorganic salts as described above, an electrolyte (inorganic salt) in deep sea water can be separated using a charged mosaic membrane. By using a charged mosaic membrane, it is possible to selectively remove inorganic salts in deep ocean water, and it is not necessary to heat the deep ocean water for selective removal of these inorganic salts. Therefore, it is possible to reduce the heat effect on the dissolved organic matter.
[0030]
On the other hand, inorganic salts and the like are deposited in the deep ocean water concentrated by separating water by vacuum distillation as described above (hereinafter referred to as concentrated ocean deep water). This precipitate is mainly composed of an inorganic salt such as sodium chloride, calcium sulfate or magnesium chloride, and preferably 2 to 10 after the distillation residue is sufficiently cooled (usually at room temperature (25 ° C.) or lower). The deposited precipitate is separated by cooling to ° C. A general separation method such as filtration, centrifugation, or decantation can be used to separate the precipitate. Moreover, these separation operations can be combined. The amount of precipitates (mainly inorganic chlorides) deposited by concentration under reduced pressure as described above is relatively large, and most of these precipitates are crystallized inorganic salts. Efficient.
[0031]
Although there is no limitation in particular in the filtration apparatus used for isolation | separation of deposits, such as inorganic salt here, a filter with an opening of 0.01-2 micrometers normally, Preferably it is 0.1-1.0 micrometer is used. By filtering under reduced pressure using the above filter, the precipitate and the filtrate can be separated efficiently. As described above, the precipitates separated here are mainly inorganic chlorides, and inorganic salts such as inorganic chlorides (mainly sodium chloride and magnesium chloride) dissolved in the deep sea water used first by this separation operation. In addition, 50% by weight or more, preferably 70% by weight or more, particularly preferably 90% by weight or more of the calcium sulfate is removed. The precipitate (mainly inorganic chloride) separated as described above is discarded without being used in the present invention.
[0032]
In the present invention, when the precipitate is separated as described above, that is, when separated by filtration as shown in the above example, the above desalted filtrate is used in the next step (hereinafter referred to as (concentration) desalting. This is called salt ocean deep water).
That is, the cellular active substance of the present invention performs at least one step of concentrating the desalted filtrate ((concentrated) desalted deep ocean water) obtained in the above step and separating the precipitate deposited by this concentration. It is contained in high concentration demineralized deep sea water which can be obtained by high concentration.
[0033]
Here, the filtrate is usually concentrated by distillation under reduced pressure.
In the present invention, the concentration operation (process) refers to an operation for removing dissolved salts dissolved in ocean water. In this step, ocean water may be concentrated and fresh water may be removed. The pressure at this time can be normally set within the range of 1 to 40 Torr, preferably 5 to 30 Torr, and the distillation temperature is usually 5 to 50 ° C., preferably 10 to 40 ° C. In one operation under the above conditions, distillation is performed so that the amount of the original filtrate is preferably within the range of 5 to 30 parts by volume, more preferably 5 to 20 parts by volume with respect to 100 parts by volume of the original filtrate. By distillation in this way, components (mainly inorganic salts) dissolved in the filtrate are deposited. Next, in the present invention, the precipitate (mainly inorganic salt) thus precipitated is separated.
[0034]
Separation of this precipitate or inorganic salt, that is, removal of dissolved salt, is performed by separation operation using a semi-permeable membrane, operation for electrically separating residual ionic substances, centrifuge operation, gel filtration operation, charged mosaic membrane. It is preferable to employ any one of the separation operation used and the ultrafiltration operation. That is, the precipitate in this process tends to have a smaller particle size than the precipitate in the previous process, and some of the precipitate may be colloidal or even gelled. Difficult to separate.
[0035]
Therefore, the separation of precipitates or inorganic salts and liquid in this step is a method of separating from the dispersion medium using the difference in osmotic pressure of the semi-permeable membrane, and the electrical properties of the remaining ionic substances by applying voltage. A method of collecting and separating from a dispersion medium using characteristics, a method of collecting a precipitate using an external stress such as centrifugal force and separating it from the dispersion medium, and a filtration membrane having a desired permeability such as an ultrafiltration membrane. The precipitate and the dispersion medium can be separated by using a method of filtering under pressure using a method, a method of separating low molecular weight particles from a dispersion medium using gel filtration, or the like.
[0036]
The cellular active substance of the present invention is a dispersion medium (highly concentrated desalted deep ocean water) obtained by performing the above-described concentration operation and separation operation at least once, preferably twice or more. When the above steps are repeated, the concentration operation and separation operation performed in the previous operation and the subsequent concentration operation and separation operation do not need to be the same operation, and the state of the precipitates, etc. Corresponding operations can be combined as appropriate. The precipitate thus separated is considered to be mainly the remaining inorganic salt, dissolved metal compound, etc., and in the present invention, this precipitate is discarded.
[0037]
A dispersion medium (highly concentrated desalted deep ocean water) obtained by repeating concentration and separation as described above is a liquid containing a high concentration of cellular active substances, and this cellular active substance (ie, fibroblast metabolism) Active substance and eosinophil active substance) in a high concentration is usually from 0.00001 to 50.0 parts by volume, preferably 0.0001 to 100 parts by volume of deep sea water used first. -30.0 volume parts, more preferably 0.001-5.0 volume parts, particularly preferably 0.01-0.5 volume parts.
[0038]
The highly concentrated desalted deep ocean water obtained as described above contains various organic substances, and conversely, the content of inorganic substances is very low, usually 2.0% by weight or less, Preferably it is 0.9% by weight or less, more preferably 0.1% by weight or less, particularly preferably 0.01% by weight or less. The ignition residue of this cellular active substance is usually 1.0% by weight or less, Preferably it is 0.1 weight% or less, Most preferably, it is 0.01 weight% or less. In particular, the content of inorganic chloride in the highly concentrated desalted deep ocean water is extremely low and is usually 1000 ppm or less, preferably 100 ppm or less. Further, the total amount of organic substances contained in this highly concentrated desalted deep ocean water is usually 0.001 to 50% by weight, preferably 0.01 to 50% by weight, particularly preferably 0.1 to 10% by weight. It is in the range of%.
[0039]
The highly concentrated desalted deep ocean water obtained as described above contains, for example, skin cell active substances such as substances that activate human skin fibroblasts and / or eosinophilic activity, for example. Cell-active substances such as immune cell-active substances such as substances that enhance the activity are contained. The details of the skin cell active substance and immune cell active substance contained in this highly concentrated desalted deep sea water are unknown, but at least the highly concentrated desalted obtained by treating the deep sea water as described above. Deep sea water contains a component that imparts energy metabolism / proliferation activity to fibroblasts and / or a component that has eosinophil activity. According to the study of the present inventor, it is estimated that this substance is one of the organic substances contained in the highly concentrated desalted ocean deep water.
[0040]
Therefore, using this highly concentrated desalted deep ocean water as a developing solvent, phosphate buffered saline (PBS-) was used, and the product name Sephacryl S-300 (manufactured by Pharmacia) was used as a column. Analysis was performed by ultraviolet detection at 280 nm, and the analysis results were divided into a first fraction having a molecular weight of 1,000,000 or more, a second fraction having a molecular weight of 30,000 to 1,000,000, a third fraction having a molecular weight of 30,000 to 5,000, and a molecular weight of 5,000. When divided into the following fourth fractions and examined for the presence or absence of peaks in each fraction, the highly concentrated desalted deep ocean water obtained as described above is at least the first to fourth fractions. At least one peak is observed in any fraction. A peak appearing in the fraction partitioned by molecular weight as described above means that this highly concentrated desalted deep ocean water contains an organic compound having a molecular weight corresponding to the above fraction. Incidentally, when Ribonuclease A having a number average molecular weight of 13700 is added as a labeling substance, the portion corresponding to Ribonuclease A appears in the third fraction, and when adding Ferritin having a number average molecular weight of 440000 as the labeling substance, The part corresponding to Ferritin appears in the second fraction.
[0041]
FIG. 2 shows an example of a gel filtration chromatography chart measured for the highly concentrated desalted deep ocean water obtained as described above. In FIG. 2, the dashed lines indicate Ribonuclease A having a number average molecular weight of 13700 and Ferritin peaks having a number average molecular weight of 440000 used as the labeling substances.
In the chart shown in FIG. 2, a peak appears in the third and fourth fractions (molecular weight of 30,000 or less), and a peak appears in the first fraction (molecular weight of 1 million or more). In addition, although no peak appears in the second fraction, since fibroblast activity was confirmed, the substance contained in the second fraction is considered to be an organic component having no UV absorption, for example, a polysaccharide component. It is done. Regarding the structure of the substances that appear as these peaks, the details are not yet elucidated because these peaks do not represent a single substance but are peaks caused by multiple compounds. . However, in comparison with the peak of the labeling substance, there is a high possibility that it is a polymerized biologically relevant substance.
[0042]
The highly concentrated desalted ocean deep water as described above has skin cell activity and / or immune cell activity. The skin cell activity and immune cell activity exhibited by the highly concentrated desalted deep ocean water will be described below with reference to cell energy metabolism / proliferation promoting activity and eosinophil activity for fibroblasts, respectively.
First, the cell energy metabolism / proliferation promoting activity for fibroblasts possessed by cell active substances (fibroblast energy metabolism / proliferation active substances) contained in high concentration in the highly concentrated desalted ocean deep water will be described.
[0043]
For example, when the energy concentration activity of the mitochondrial cell is evaluated by the MTT method using the three-dimensional tissue model of human dermis-derived fibroblasts, the highly concentrated desalted deep ocean water is When the solid content is 0.1% by weight, a clear metabolic activity improving effect on fibroblasts is observed.
Furthermore, when the components are separated for each fraction and the energy metabolism / proliferation activity of the fibroblasts is examined, as shown in FIG. 3, the components of the second fraction tend to exhibit the highest energy metabolism / proliferation activity. . In FIG. 2, since a UV peak does not appear in this fraction, the active ingredient contained in the second fraction is considered to be a polysaccharide component having no UV absorption.
[0044]
As described above, the fibroblast energy metabolism / proliferation active substance of the present invention, that is, the fibroblast active substance is obtained from deep ocean water, and particularly exhibits energy metabolism / proliferation activity against fibroblasts. The cell energy metabolism / proliferation active substance (fibroblast active substance) is preferably added to pharmaceuticals, cosmetics, foods and the like.
[0045]
In pharmaceuticals and cosmetics, it can be used as a skin external preparation. In addition, as long as the effect of the present invention can be achieved, the dosage form and administration method of pharmaceuticals / cosmetics are not limited, and in the present invention, the cellular active substance may be an internal medicine. Oral administration is also possible.
The topical skin preparation of the present invention is obtained by performing at least one operation of further concentrating (concentrated) demineralized deep ocean water that has undergone an operation of separating at least a part of the inorganic salt dissolved in the deep ocean water. It contains a fibroblast energy metabolism / proliferation active substance.
[0046]
The fibroblast energy metabolism / proliferation active substance, which is the cell active substance of the present invention, can be used, for example, as a lotion in an aqueous medium, or used as a cream by adding it to an ointment base. You can also. Furthermore, it can be used like an emulsion by blending into an emulsion, and the cell energy metabolism / proliferation active substance can be blended into an aqueous medium, and a thickener can be blended into the aqueous medium, thereby It can also be used in this way.
[0047]
Ingredients other than the cellular energy metabolism / proliferation active substance that can be incorporated into the external preparation for skin of the present invention include mineral oil, vegetable oil, wax, fatty acid, aliphatic alcohol, ester oil, surfactant, wetting agent, polymer, animal and plant Extracted oils, amino acids, vitamins, steroids, anti-inflammatory agents, antiseptics, UV absorbers, metal chelators, antioxidants, pH adjusters, colorants, fragrances, and other ingredients that are commonly used in external skin preparations Can be blended.
[0048]
The external preparation for skin of the present invention can adjust the content of a cell energy metabolism / proliferation active substance (fibroblast active substance) in accordance with its use. That is, the external preparation for skin of the present invention prevents or treats aging symptoms such as wrinkles, blemishes and sagging on human skin by improving the metabolism and proliferation activity of human skin cells. The cell energy metabolism / proliferation active substance (fibroblast active substance) to be blended can be adjusted according to the method and the like. This cell energy metabolism / proliferation active substance (fibroblast active substance) is usually obtained in a dissolved or dispersed state in water as described above, but it prevents aging symptoms such as wrinkles, spots and sagging in human skin. Alternatively, since the dissolved or dispersed components in this aqueous solution or dispersion act on the treatment, in the present invention, it is preferable to specify the usage amount of the cellular energy metabolism / proliferation active substance as the solid weight. The amount of the cellular energy metabolism / proliferation active substance (fibroblast active substance) in 100 parts by weight of the external preparation for skin (agent) of the present invention is usually 0.00001 to 50.0 parts by weight in terms of solid content. The amount is preferably 0.0001 to 30.0 parts by weight, more preferably 0.001 to 5.0 parts by weight, and particularly preferably 0.01 to 1.0 parts by weight. The cellular energy metabolism / proliferation active substance (fibroblast active substance) of the present invention is not necessarily clear in details such as the structure of the components contained in the above-mentioned fractions by molecular weight, and therefore the components contained in these fractions. It is very difficult to clarify the action and effect of, and it is considered that there is also an improvement in action and effect due to the interaction of components contained in each fraction. For the above reasons, in the present invention, it is preferable to directly use an aqueous solution or dispersion containing the cell energy metabolism / proliferation active substance obtained as described above, which is a complex of components contained in each fraction. In this case, the amount used is expressed as a solid content dissolved or dispersed in an aqueous solution or dispersion.
[0049]
By using the external preparation for skin of the present invention containing the cell energy metabolism / proliferation active substance (fibroblast active substance) in the amount as described above, this cell energy metabolism / proliferation active substance (fibroblast active substance) Activates human skin cells, especially fibroblasts, and promotes secretion of anti-aging components such as collagen, for example, and can effectively prevent pigmentation and decrease in hyaluronic acid production. .
[0050]
Together, these can prevent aging symptoms such as wrinkles, blemishes and sagging in human skin, or treat and improve the skin that has fallen into aging symptoms.
Next, the eosinophil activity possessed by immune cell active substances (including eosinophil active substances) contained at high concentrations in the highly concentrated desalted ocean deep water will be described.
[0051]
When eosinophil activity was examined using the above-mentioned highly concentrated desalted deep ocean water by the method described below, phosphate buffered saline (PBS) used as a control and opsonized zymozan (positive control) ( Eosinophil activity higher than OZ, immunostimulator.
Further, even when eosinophil activity was examined for each of the fractions divided as described above, eosinophil activity higher than that of the control was observed in any fraction. In each fraction, the component of the fourth fraction tends to exhibit the highest high eosinophil activity (see FIG. 4).
[0052]
The relationship between allergy typified by atopy and eosinophil activity will be briefly explained.
In an allergic reaction such as atopic dermatitis, locally migrated eosinophils are Histamylase, allylsulfatase B, phospholipase D.₂Release. These released substances are histamine that mediates inflammation, eosinophil chemotactic factor of anaphylaxis and platelet activating factor of abnormal hypersensitivity reaction to foreign substances such as foreign proteins (PAF)) is known to work to repair allergic reactions. Furthermore, recently, eosinophils have come to be considered to act on tissue damage more than the above-mentioned tissue repair action. Four types of basic proteins (major basic protein (MBP), eosinophil peroxidase (EPO), eosinophil cationic protein ()) exist in special granules of eosinophils that play a role in the involvement of eosinophils in tissue damage. ECP), eosinophil-derived neurotoxin (EDN)) and superoxide produced by eosinophils (O₂ ^-) -Derived various active enzyme molecular species. These four basic proteins are released from activated eosinophils into tissues by degranulation and are thought to be involved in tissue damage. In particular, MBP is thought to activate basophils and EPO activates mast cells to release histamine and increase allergic reactions.
[0053]
Superoxide (O₂ ^-) Is produced by activation of a cell membrane-bound enzyme system called NADPH oxidase by various stimuli such as IgE complex, PAF, opsonized zymosan (OZ), and phorbol myristate acetate. This NADPH oxidase oxidizes NADPH produced by a sudden increase in hexose monophosphate shunt activity, and thereby oxygen is reduced by one electron.₂ ^-Is generated. This O₂ ^-From H, due to enzymatic or non-enzymatic reactions₂O₂, HO^-,¹O₂Various reactive oxygen molecular species having high reactivity are generated. These active oxygen molecular species are responsible for important biological defenses such as sterilization. Therefore, eosinophils are superoxide (O₂ ^-) The ability to promote production is very effective in controlling the causative factors of allergic reactions such as atopic dermatitis.
[0054]
In the present invention, the degree of activation of eosinophils can be measured, for example, by the method described below.
First, eosinophils used for measuring the degree of activation are prepared. Specifically, for example, 15 to 20 weeks old of a transgenic mouse (C3H / HeN-TgN (IL-5) Imeg) that constantly produces IL-5 which is a growth and differentiation factor selective for eosinophils. Spleen eosinophils and spinal eosinophils are prepared from these individuals. That is, the spleen and spinal cord²⁺Eosinophils can be obtained by homogenizing in free Krebs-Ringer-phosphate buffer solution (KRP) (pH value 7.4) and separating the cell suspension by a method such as Percoll discontinuous density gradient centrifugation. it can. The spleen is usually 75% and the spinal cord is usually 80% as the eosinophil fraction. The mice used here are preferably those in which 20 μg of cadmium sulfate was intraperitoneally administered 5 days before the experiment for the purpose of increasing IL-5 production under the control of the metallothionein promoter.
[0055]
In addition to the above, for example, isolated from peripheral blood of a transgenic mouse (C3H / HeN-TgN (IL-5) Imeg) that constantly produces IL-5, which is a growth and differentiation factor selective for eosinophils. Eosinophils are also preferably used.
Eosinophil superoxide (O) by the cell active substance (eosinophil active substance) of the present invention contained in deep ocean water₂ ^-) Production improving ability can be calculated by measuring the amount of cytochrome c (Cyt. C) reduced by hydrogen peroxide produced by eosinophils.
[0056]
This measurement can be performed by the method described in Nakagawa. A., Shibata. K., Takeshige. K., et al: Exp. Cell. Res. 101: 224-234 (1976).
Thus, for example, 20 μmol cytochrome c (Cyt. C), 10 mmol glucose, 1 mM CaCl₂And eosinophils derived from the above in KRP maintained at 37 ° C. (5 × 10 5^Fivecells / ml) is suspended, and the absorbance at 550 nm increases with the reduction of Cyt. c (A_550-540) Is measured over time. O₂ ^-The amount of produced is 21.0 mM of Cyt. C molecular absorbance.^-1·cm^-1Can be obtained from For this measurement, it is desirable to use an apparatus (for example, a dual wavelength spectrophotometer (UV-300) manufactured by Shimadzu Corporation) that can measure the absorbance while stirring the reaction solution.
[0057]
Further, the degree of activation of eosinophils can be further measured by the method described below.
That is, for example, superoxide (O₂ ^-Hydrogen peroxide (H) derived from₂O₂) To measure. This hydrogen peroxide (H₂O₂) Indicates the ability of eosinophils to produce hydrogen peroxide, and the degree of activation of eosinophils can be expressed as an activation index. This measurement can be performed by the method described in Irene, A.M.Vint, John, C. Foreman, et al: Eur. J. Immunol. 24: 1961-1965,1994.
[0058]
That is, superoxide (O) produced by eosinophils being activated by eosinophil active substances in deep sea water.₂ ^-Hydrogen peroxide (H) derived from₂O₂) Can be measured by analyzing fluorescence by 2 ', 7'-dichlorodihydrofluorescein diacetate (DCFH-DA) using a flow cytometer. Specifically, 5 × 10^FiveIncubate cells / ml eosinophils in 0.5 ml of Phosphate-buffer saline glucose (5 mM glucose) (PBSg) containing 2.5 μM DCFH-DA at 37 ° C. for 15 minutes, then add stimulus After incubating at 37 ° C. for 30 minutes, and finally washing the cells several times with PBSg, it can be analyzed and measured with a flow cytometer.
[0059]
In addition, said measuring method is an example of the method of measuring the activation degree of an eosinophil, and the measuring method of the activation degree of the eosinophil active substance of this invention is not limited to said method. .
Since the cell active substance (eosinophil active substance) of the present invention has excellent eosinophil activity, it can be used in pharmaceuticals and cosmetics.
[0060]
Moreover, the pharmaceutical (medicine) containing the cell active substance (eosinophil active substance) of the present invention is highly useful as a component of a pharmaceutical used for allergic diseases, particularly atopic dermatitis. Moreover, the cellular active substance of the present invention can also be used as a cosmetic product that can achieve the same effect.
That is, the cell active substance of the present invention (here, eosinophil active substance) activates eosinophils that have migrated locally, and eosinophils that are abnormally sensitive to foreign substances such as histamine and heterologous proteins in allergic reactions. Histamylase, allylsulfatase B, phospolypase D, which metabolizes eosinophil chemotactic factor of anaphylaxis and platelet activating factor (PAF)₂Release. Furthermore, superoxide (O) by eosinophils activating the cell membrane-bound enzyme system.₂ ^-) Can improve the biological defense reaction.
[0061]
However, as described above, the cellular active substance (eosinophil active substance) of the present invention is not necessarily clear in details such as the structure of the components contained in the above fraction by molecular weight. It is very difficult to clarify the action and effect of the components contained in the mixture, and it is considered that the action and effect are improved by the interaction of the components contained in each fraction. Therefore, in the pharmaceutical / cosmetics of the present invention, it is preferable to directly use an aqueous solution or dispersion containing the cell-active substance obtained as described above, which is a complex of components contained in each fraction.
[0062]
The method of use and the like when the cell active substance of the present invention is used as a eosinophil active substance in pharmaceuticals will be described below.
The cell active substance (eosinophil active substance) of the present invention can also be used as an external preparation dissolved or dispersed in a base material. Thus, when used as an external preparation, the amount of the eosinophil active substance in the external preparation is usually 0.00001 to 50.0% by weight, preferably 0.00001 to 30.0% by weight, particularly preferably. 0.001 to 0.1% by weight. In this case, as a base material for the external medicine, those used as a base material for ordinary external medicines such as petrolatum, ceresin, paraffin, squalane, waxes, ozokelide can be used. The dosage form can be applied to emulsion, oil, wax, lotion and the like.
[0063]
Further, the cell active substance (eosinophil active substance) of the present invention can be used as an injection solution by making it into an aqueous solution. In this case, the concentration of the eosinophil active substance is usually set within the range of 0.000001 to 10.0% by weight, preferably 0.0001 to 0.1% by weight. When used as an injection solution, components to be added to ensure the stability of the injection solution such as a pH adjusting agent and an antioxidant can be added.
[0064]
Furthermore, the cell active substance (eosinophil active substance) of the present invention can also be used as an internal medicine for powders, tablets, granules, capsules, syrups and the like.
When used as an internal medicine or injection using the cell active substance (eosinophil active substance) of the present invention, the amount used can be appropriately set in consideration of symptoms, the condition of the user, etc. The amount of can be generally set within the range of 0.0001 to 10000 mg, preferably 0.1 to 100 mg.
[0065]
Further, as cosmetics containing the cellular active substance of the present invention, lotions, emulsions, creams, gels, whitening essences, UV protection essences, cosmetic oils, packs, shampoos, conditioning shampoos, rinses, hair restorers, hair liquids, Examples include liquid toothpaste, mouthwash, and wet tissue chemicals. Table 1 shows one embodiment of these formulations, but the cosmetic of the present invention is not limited to the following formulations. In the following formulation examples, “%” represents “% by weight”, and the total amount of additives is 100% by weight.
[0066]
[Table 1]

[0067]
[Table 2]

[0068]
[Table 3]

[0069]
[Table 4]

[0070]
Examples of cosmetics containing the cellular active substance of the present invention can further include the following.
Examples of such cosmetics include soaps such as bar soap, cleansing foam, cleansing gel, cleansing cream, cleansing milk, cleansing mask, cleansing oil, facial cleansing powder, and shaving foam;
Lotions such as flexible lotions, astringent lotions, cleansing lotions, and multi-layer lotions;
Oils used for various purposes such as moisturizing, softening, blood circulation promotion, UV protection, makeup base, makeup base, keratin softness, hair protection, hair removal prevention, hair styling, deodorization, shaving, body use, hand use, massage use, Gels, emulsions, or creams;
Packs and masks of each type such as peel-off type, wipe-off type, wash-off type, solidified peeling type, pasting type, impregnation type, etc .;
Makeup cosmetics such as powder, white powder, dusting powder, foundation, lipstick, blusher, eyeliner, mascara, eyebrow, nail enamel, eye shadow, enamel remover, nail treatment;
Hair foam, hair mousse, hair spray, hair gel, water grease, set lotion, pomade, tic, hair cream, treatment lotion, split coat, hair oil, permanent wave agent, hair dye, hair bleach and other cosmetics for hair dyeing Kind;
Whole body cleaning cosmetics such as soap, medicated soap, liquid body shampoo, hand soap, foot soap;
Sunscreen cosmetics such as sunscreen, suntan cosmetics and after-suncare;
Deodorant cosmetics such as deodorant lotion, deodorant spray, deodorant stick, eau de cologne and perfume;
Sanitary cosmetics used for hair removal, hair removal, bathing, insectryper, etc .;
Oral cosmetics used for brushing, mouthwashing, deodorization, refreshing, etc .;
Wet tissue, wet tissue for wiping wipes, swabs, etc., coated paper cotton impregnated chemicals;
Examples include aerosol chemicals.
[0071]
Although the example of the preferable aspect in the case of using the cell active substance of this invention for a pharmaceutical and cosmetics was mentioned above, as long as the effect of this invention is acquired, the dosage form, administration method of the pharmaceutical and cosmetics of this invention, The method of use is not limited. In addition to the above, as long as the effects of the present invention can be achieved, the pharmaceuticals and cosmetics of the present invention can be mixed with components that are usually used as components of pharmaceuticals and cosmetics.
[0072]
Furthermore, the fibroblast energy metabolism / proliferation active substance, which is the cell active substance of the present invention, can be used by adding to food. Specifically, when the food is produced, the addition method and the addition amount are not particularly limited as long as the deep-layer water desalting concentrate is used. When the food of the present invention contains a cellular active substance, the consumer can easily take the fibroblast energy metabolism / proliferation active substance, and can effectively prevent or treat aging symptoms. Moreover, immunity can also be strengthened by activating living cells.
[0073]
Examples of such foods of the present invention include vegetable / fruit juice drinks, carbonated drinks, lactic acid bacteria drinks, strawberries drinks, processed milk drinks, sports drinks, mineral water, Japanese tea, Chinese tea, tea, herbal tea and other non-alcoholic drinks;
Alcoholic beverages such as beer, happoshu, sake, shochu, awamori, wine, gin, vodka, rum, brandy, whiskey, champagne, Chinese liquor, amazake;
Seasonings such as sauce, dressing, mirin, vinegar, tomato ketchup;
Fermented foods such as miso, natto, soy sauce, pickles, yogurt;
Retort foods such as jelly, agar, sorbet, soy milk, tofu, bread, cookies, rice cakes, cooked rice, raw noodles, baby food and nursing foods, processed foods such as dried foods and frozen foods;
Examples include capsules and tablets.
[0074]
Although the example of the foodstuff which can use the cell active substance of this invention was mentioned above, as long as the effect of this invention is acquired, it is not limited to these, Moreover, the manufacturing method of the foodstuff of this invention, etc. There is no limit. In addition to the above, as long as the effects of the present invention can be achieved, the food of the present invention can be blended with components that are normally used as food components.
[0075]
In addition, immune cell active substances other than the above eosinophil active substances can achieve the same effects as eosinophil active substances and are useful as components of pharmaceuticals, cosmetics, foods and the like.
[0076]
【The invention's effect】
The cell active substances of the present invention (including skin cell active substances such as fibroblast energy metabolism / proliferation active substances and immune cell active substances such as eosinophil active substances) can be obtained by subjecting deep sea water to a specific treatment. It is presumed that it is a substance contained in the resulting solution and is a polymer compound having a certain molecular weight obtained by separation from water by an appropriate means such as gel filtration chromatography.
[0077]
Conventionally, it is known to use a separated substance from deep ocean water for skin treatment or allergy treatment, but these use inorganic components contained in deep ocean water. That is, deep sea water contains various minerals in addition to chlorides such as sodium chloride, calcium chloride and magnesium chloride, and these minerals are known to be used for skin treatment or allergy treatment. It was. Therefore, when using deep sea water for skin treatment and allergy treatment, the precipitate (mineral content) deposited by concentrating deep sea water is separated from seawater and used. Furthermore, since it was not thought that organic components effective for skin treatment or allergy treatment were contained in deep sea water, it was attempted to separate and use organic substances contained in deep sea water. It was not done.
[0078]
In contrast, the inventor of the present invention contains an organic substance in addition to the inorganic salt as described above in the deep sea water, and this organic substance has a function completely different from the mineral contained in the deep sea water. It has been found that by utilizing this function, skin cell activity such as energy metabolism and proliferation activity of fibroblasts forming the skin and immune cell activity such as eosinophil activity can be improved. is there.
[0079]
The cellular active substance of the present invention contains an organic substance that can be obtained by subjecting deep sea water to a specific treatment, and this organic substance improves the energy metabolism / proliferation activity and eosinophil activity of fibroblasts. Can be made.
Such a cell active substance is a complex of various organic substances, and it is extremely difficult to specify individual components, but it can be repeatedly obtained by treating the deep sea water according to the present invention.
[0080]
This cell-active substance was found to be a complex of organic substances having peaks in a plurality of fractions according to molecular weight using gel filtration chromatography. The components separated into these fractions alone differ in, for example, energy metabolism / proliferation activity or eosinophil activity for fibroblasts. However, by using these complexes as they are, fibroblasts are particularly useful. On the other hand, it was confirmed that it showed excellent energy metabolism / proliferation activity and activity improvement effect on eosinophils.
[0081]
Moreover, such cell active substances of the present invention (including skin cell active substances such as fibroblast energy metabolism / proliferation active substances and immune cell active substances such as eosinophil active substances) include normal cell active substances and Compared with fibroblasts, the side effects are extremely low, and especially fibroblasts have a specific effect that they exhibit excellent cell energy metabolism / proliferation activity (cell activity) without side effects. Therefore, it is particularly useful as a skin external preparation. For the same reason, it is very useful as a therapeutic agent for allergic diseases such as atopic dermatitis.
[0082]
In addition, since the cell active substances of the present invention (including skin cell active substances such as fibroblast energy metabolism / proliferation active substances and immune cell active substances such as eosinophil active substances) have good affinity for water, they are for external use. It can be used in various forms such as drugs, internal medicines, and injection solutions.
More specifically, according to the fibroblast active substance of the present invention, as described above, fibroblasts can be remarkably activated, so that the energy metabolism activity and proliferation activity of human skin cells are improved. It is possible to prevent or treat aging phenomena such as wrinkles, blemishes and sagging in human skin.
[0083]
In addition, for example, the eosinophil active substance of the present invention can activate eosinophils as described above, and thus is very excellent due to the action of superoxide produced by the activation of eosinophils. Has a biological defense function. In addition, this substance metabolizes histamine that mediates inflammation and releases histamylase that works to repair allergic reactions. Furthermore, by making this eosinophil active substance act on eosinophils, superoxide (O₂ ^-) And this superoxide (O₂ ^-) Can generate various reactive oxygen species with high reactivity by enzymatic reaction or non-enzymatic reaction. These active oxygen species can express important biological defenses such as bactericidal action.
[0084]
Since the above-described functions of the eosinophil active substance of the present invention are considered to act in a complex manner, the eosinophil active substance of the present invention is used to treat allergic diseases such as asthma, hay fever and dermatitis. As a component, it is particularly useful as a therapeutic component for allergic diseases such as atopic dermatitis.
[0085]
【Example】
Next, although an Example is shown and this invention is demonstrated further in detail, this invention is not limited by these.
[0086]
[Example 1]
[Preparation of active substance for cell energy metabolism / proliferation]
20 liters of deep ocean water was collected from a depth of 687m off Suruga Bay.
20 liters of this deep sea water was filtered using a membrane filter having an opening of 0.45 μm to remove suspended matters in this deep sea water.
[0087]
The filtered deep ocean water is put into a vacuum distillation apparatus, the distillation temperature is set to a temperature of 40 ° C. or less under a pressure condition of 20 Torr, and the amount of deep ocean water is about 3.3 liters (the original ocean water) Distilled under reduced pressure until 1/6 of the deep water.
By distillation under reduced pressure in this way, inorganic salts such as sodium chloride, potassium chloride and magnesium chloride which had been dissolved in the deep ocean water were precipitated. The deposited inorganic salt was filtered through a membrane filter having an opening of 0.5 μm. The filtered inorganic salt was discarded because it is not used in the present invention.
[0088]
Next, the filtrate obtained in the above step is subjected to a desalting operation at a low temperature of 8 ° C. using a mosaic charged membrane (trade name: MC-55B mosaic charged membrane, manufactured by Tosoh Corporation). The resulting liquid is put into a vacuum distillation apparatus, and the temperature is controlled so as not to exceed the temperature of 40 ° C. under a reduced pressure of 20 Torr, and the volume of the liquid is 200 ml (approximately 1/100 of the original deep sea water). Water was distilled off under reduced pressure until.
[0089]
The desalting and vacuum distillation using the above mosaic charged membrane was repeated to obtain 5 ml of concentrate.
The concentrate contained 0.2% by weight of an organic substance, and the content of inorganic salt in the concentrate was 100 ppm or less, and the content of inorganic chloride was 70 ppm or less.
[0090]
The concentrate was subjected to gel filtration chromatography having a column (Sephacryl S-300) manufactured by Pharmacia, and the components contained therein were analyzed using phosphate buffer saline (PBS-) as a developing solvent.
The result of this analysis, according to molecular weight,
The first fraction with a molecular weight of over 1 million,
A second fraction having a molecular weight of 30,000 to 1,000,000,
A third fraction having a molecular weight of 5000 to 30,000, and
The presence or absence of an absorption peak at a wavelength of 280 nm in each fraction was examined by dividing into fourth fractions having a molecular weight of 5000 or less.
[0091]
The results are shown in FIG.
As shown in FIG. 2, the largest peak is seen in the fourth fraction, and the third fraction has a peak that is less than half the size of the fourth fraction, with the bottom overlapping with the fourth fraction. It was seen. Moreover, although it was small also in the 1st fraction, the clear peak was seen. However, no UV peak was observed in the second fraction.
[0092]
In order to make reference to the analysis of the peak components in these fractions, a solution containing Ribonuclease A with an average molecular weight of 13700 and Ferritin with an average molecular weight of 440,000 was prepared and analyzed in the same manner as described above. The results are also shown in FIG.
[Measurement of fibroblast energy metabolism and proliferation activity]
Cellular energy metabolism / proliferation active substances (including fibroblast substances) having the above-described configuration are phosphate buffered saline (PBS-) which is a standard solution so that the solid content is 0.1% by weight. The sample of the present invention was prepared. The final salt content of each sample was adjusted to the salt content of PBS- which is a standard solution.
[0093]
Using a three-dimensional tissue model of human dermis-derived fibroblasts (MATREX-LDM, manufactured by Toyobo Co., Ltd.), the mitochondrial energy metabolic activity was evaluated by the MTT method.
Here, the MTT method is a method for measuring the energy metabolic activity of cells using 3- (4,5-Dimethyl-2-thiazoly) -2,5-diphenyltertrazolium bromide. That is, MTT is reduced by NADH (reduced nicotinamide adenine dinucleotide) or NADPH (reduced nicotinamide adenine dinucleotide phosphate) generated in the process of mitochondrial energy metabolism of cells to produce blue formazan.
[0094]
In this method, blue formazan is absorbed at a wavelength of 570 nm, colorimetrically measured, and colorimetrically quantified to evaluate cell metabolic activity and cell growth activity. In this case, the darker the blue, the higher the activity.
Further, in the present invention, as a control, the mitochondrial energy metabolic activity of the cells was examined by the MTT method in the same manner using only the standard solution (PBS-) without using the cell energy metabolism / proliferation active substance.
[0095]
Further, as a negative control, 0.1 wt% of typical cytotoxic sodium dodecyl sulfate (SDS = Sodium dodecyl sulfate) was blended and the energy metabolism / proliferation activity was examined by the MTT method. This negative control shows the minimum standard of the MTT method when no energy metabolism / proliferation activity is shown.
[0096]
When the value of the MTT method obtained by the above control is 100, the value of the negative control is 33.
And the value in the MTT method of the sample containing 0.1 parts by weight of the cellular energy metabolism / proliferation active substance of the present invention measured in the same test is 245, and this cellular energy metabolism / proliferation active substance is cell energy. It is clear that it exhibits metabolic / proliferative activity.
[0097]
In this test, the MTT reagent may not be reduced to blue by NADH or NADPH generated during the mitochondrial metabolic process of cells, but may be reduced to blue by being reduced to the cell energy metabolism / proliferation active substance of the present invention itself. Therefore, sodium dodecyl sulfate (SDS = Sodium dodecyl sulfate) was added to 0.1% by weight and 0.1% by weight of the cell energy metabolism / proliferation active substance of the present invention in terms of solid content. Examined. As a result, the value in a similar test is 27, which is almost the same level as the value 33 when sodium dodecyl sulfate is used alone, so that the cell energy metabolism / proliferation active substance of the present invention itself reduces the MTT reagent and turns blue. The cell mitochondria are activated by the addition of the cell energy metabolism / proliferation active substance of the present invention, and the generated NADH or NADPH reduces the MTT reagent and changes the color to blue without causing discoloration. I understood. That is, it is clear that the metabolic activity is improved by the cell energy metabolism / proliferation active substance of the present invention. Further, in another experiment, the above conclusion was further proved because the MTT reagent was not discolored at all even when the above-mentioned deep water cell active substance was added to the MTT reagent.
[0098]
For reference, the final salt content is adjusted by adjusting the sample added to phosphate buffered saline (PBS-), which is a standard solution, so that the components of each fraction are 0.1% by weight in terms of solid content. The cell energy metabolism / proliferation activity in the MTT method was examined according to the salt content of PBS- as a solution. The results are shown in FIG.
as a result,
The activity of the first fraction component having a molecular weight of 1 million or more is 156,
The activity of the component of the second fraction having a molecular weight of 30,000 to 1,000,000 is 257,
The activity of the component of the third fraction having a molecular weight of 30,000 to 5,000 is 151,
The activity of the component of the fourth fraction having a molecular weight of 5000 or less was 155.
[0099]
Note that the activity evaluation values using the components of each of the above-mentioned fractions are reference values. By repeating experiments in more detail and increasing the accuracy of the experiments, the above values are more clearly defined. There is a good possibility of showing metabolic / proliferative activity.
[0100]
[Example 2]
The eosinophil active substance was prepared by the same method as the preparation of the cell active substance of Example 1, and the degree of activation of the obtained eosinophil active substance was measured by the following method.
[Measurement of activation degree of eosinophil active substance]
The ability (activation degree) of activating eosinophils of the cell active substances (including eosinophil active substances) of the present invention is the superoxide (O₂ ^-Hydrogen peroxide (H) derived from₂O₂) Was used as an index. Superoxide (O) produced by activation of eosinophils by the eosinophil active substance₂ ^-Hydrogen peroxide (H) derived from₂O₂Specifically, the amount of) was measured by analyzing fluorescence by 2 ', 7'-dichlorodihydrofluorescein diacetate (DCFH-DA) using a flow cytometer.
[0101]
5 × 10^FiveCells / ml eosinophils were incubated for 15 minutes at 37 ° C. in 0.5 ml of Phosphate-buffer saline glucose (5 mM glucose) (PBSg) containing 2.5 μM DCFH-DA, after which stimuli ( PMA (50 ng / ml) was added and incubated at 37 ° C. for 30 minutes. The cells were washed twice with PBSg, and then analyzed and measured with a flow cytometer.
[0102]
The eosinophils used in this experiment were peripheral blood of a transgenic mouse (C3H / HeN-TgN (IL-5) Imeg) that constantly produces IL-5, a growth and differentiation factor selective for eosinophils. The eosinophils separated from were used. Peripheral blood is Ca containing 0.22% final concentration of citric acid⁺⁺Free Krebs Ringer phosphate buffer (KRP) (0.9% NaCl, 6 mmol KCl, 1 mmol MgCl₂Percoll discontinuous density gradient centrifugation was performed for the purpose of removing erythrocytes and monocytes after collection in 10 mM Na-phosphate buffer (pH 7.4)). 60%, 70, 80% Percoll overlaid with 100% Percoll-defined cell turbidity with 10% concentrated KRP added to Percoll stock solution, centrifuged at 350 g, room temperature for 20 minutes, 70 and 80% layers were collected. The collected cell population was further suspended in a separation buffer (5 mmol EDTA, 0.5% BSA, 10 mmol Na-K-phosphate buffer (pH 7.2)) (1 × 10⁷cells / 50 μl) and anti-Thy1.2 antibody-bound magnetic beads (1 × 10⁷beads / 25 μl) and anti-B220 antibody-coupled magnetic beads (1 × 10⁷bead / 25 μl) and allowed to react at 4 ° C. for 20 minutes, and a cell population collected by negative selection using a permanent magnet was subjected to an experiment as an eosinophil population. The purity of the cell population after separation was analyzed with a flow cytometer (FACScan manufactured by BECTON DICKINSON).
[0103]
The substance to be measured was adjusted with sample / 1.8% NaCl and purified water and adjusted to be isotonic at a final concentration of 100 μg / ml.
The same experiment was performed using phosphate buffered saline (PBS) as a control and opsonized zymozan (OZ, immunostimulant) as a positive control.
[0104]
The value of the degree of eosinophil activation of each test substance measured as described above (equivalent to production of hydrogen peroxide) is shown in FIG. As an example, FIG. 5 shows a flow of hydrogen peroxide production of eosinophils when using the control (PBS) and flow of hydrogen peroxide production of eosinophils when using the eosinophil active substance. The result of cytometer analysis / measurement is shown.
As shown in FIG. 4, the degree of eosinophil activation of the control (PBS) is 3.35, whereas the degree of eosinophil activation of the eosinophil active substance is 7.09 (about that of the control). 2.1 times).
[0105]
When the eosinophil activity of the eosinophil active substance obtained as described above was examined, it showed higher eosinophil activity than the control (PBS) and the positive control (OZ: opsonized zymosan).
For reference, as in the case of the cellular energy metabolism / proliferation active substance, the eosinophil activity of each fraction divided by GPC was examined. As a result, the control ( PBS) and higher eosinophil activity than the positive control (OZ: opsonized zymosan). That is,
The activity of the component of the first fraction having a molecular weight of 1 million or more is 4.57,
The activity of the component of the second fraction having a molecular weight of 30,000 to 1,000,000 is 5.48,
The activity of the component of the third fraction having a molecular weight of 30,000 to 5,000 is 4.73,
The activity of the component of the fourth fraction having a molecular weight of 5000 or less was 8.63.
[0106]
From the comparison of FIG. 3 and FIG. 4, when the highly concentrated desalted deep ocean water obtained as described above is used, the high molecular weight fraction tends to have high fibroblast activity, On the contrary, it was shown that the eosinophil activity tends to be high in the low molecular weight fraction.
[Brief description of the drawings]
FIG. 1 is a process diagram showing an example of a process for obtaining a cell active substance of the present invention.
FIG. 2 is a diagram showing an example of peaks appearing in each fraction when the cellular active substance of the present invention is divided into each fraction using gel filtration chromatography.
FIG. 3 is a graph showing the degree of fibroblast activation of the cell active substance of the present invention.
FIG. 4 is a graph showing the eosinophil activation value of the cell active substance of the present invention.
FIG. 5 is a chart of hydrogen peroxide producing ability of eosinophils analyzed and measured by a flow cytometer.

Claims (12)

At least one operation of further concentrating the concentrated desalted deep ocean water that has been subjected to the operation of separating 90% by weight or more of the inorganic salt dissolved in the deep ocean water to separate the precipitate or the inorganic salt using a charged mosaic membrane. A cellular active substance contained in highly concentrated desalted deep sea water with a total amount of organic substances of 0.1 to 10% by weight and an inorganic salt content of 100 ppm or less .   2. The cell active substance according to claim 1, wherein the cell active substance is a skin cell active substance and / or an immune cell active substance.   The cell active substance according to claim 1 or 2, wherein the deep sea water is collected from a deep sea having a depth of 200 m or more.   The cell active substance according to any one of claims 1 to 3, which can be obtained by performing the operation of further concentrating the concentrated desalted deep ocean water two or more times.   After the concentrated desalted deep sea water is purified by filtering the deep sea water, the filtered deep sea water is in the range of 1 to 30 parts by volume with respect to 100 parts by volume of the deep sea water first used. The cellular active substance according to any one of claims 1 to 4, wherein the cellular active substance is obtained by concentrating so as to be inside and separating an inorganic salt.   The cellular active substance is contained in highly concentrated desalted deep sea water concentrated in the range of 0.0001 to 30.0 parts by volume when the volume of deep sea water used first is 100 parts by volume. The cell active substance according to any one of claims 1 to 5, wherein the cell active substance is a substance.   The cell active substance is a first fraction having a molecular weight of 1 million or more by UV detection at a wavelength of 280 nm by gel filtration chromatography analysis using phosphate buffered saline (PBS-) as a developing solvent, and a molecular weight of 30,000 to 1,000,000. The first fraction has a peak in at least one fraction comprising the second fraction, the third fraction having a molecular weight of 5000 to 30,000, and the fourth fraction having a molecular weight of 5000 or less. Item 7. The cell active substance according to any one of Items 6 to 6.   The cell according to any one of claims 1 to 7, wherein a high-temperature residue of highly concentrated desalted deep ocean water containing the cellular active substance is 0.1 wt% or less. Active substance. Separating 90% by weight or more of inorganic salt dissolved in deep ocean water to obtain concentrated desalted ocean deep water, and further charging the concentrated desalted ocean deep water to deposit precipitates or inorganic salts Through the step of performing the operation of separating at least once using a membrane, the total amount of organic substances is 0.1 to 10% by weight, and the content of inorganic salts is 100 ppm or less. A method for producing a cell-active substance, characterized in that it contains highly concentrated desalted deep ocean water.   The method for producing a cell active substance according to claim 9, wherein the cell active substance is a skin cell active substance and / or an immune cell active substance.   The method for producing a cell active substance according to claim 9 or 10, wherein the deep sea water is collected from a deep sea having a depth of 200 m or more.   12. The production of a cell active substance according to any one of claims 9 to 11, wherein the operation of further concentrating the concentrated desalted deep ocean water and separating the precipitate is repeated twice or more. Method.

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