JP6774731B2 - 卵殻膜成分を含む分子シャペロン遺伝子活性化剤ならびにそれを用いた組成物 - Google Patents
卵殻膜成分を含む分子シャペロン遺伝子活性化剤ならびにそれを用いた組成物 Download PDFInfo
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Description
本発明の分子シャペロン遺伝子活性化剤は、卵殻膜成分を有効成分とする。卵殻膜成分としては、卵殻膜そのもの、卵殻膜の加工物、抽出物等のいずれであってもよく、たとえば卵殻膜含有粉末または可溶性卵殻膜成分(加水分解物等)を利用することができる。
本発明に使用される卵殻膜成分は、卵殻膜の可溶性成分、たとえば卵殻膜の分解または抽出物であることができる。卵殻膜加水分解物は、公知の方法、たとえば卵殻膜をアルカリ性含水有機溶媒中で分解後、得られた分解液を中和、濾過することを特徴とする可溶化卵殻膜の製法(特公平6−21047号公報)、卵殻膜をタンパク分解酵素で処理することを特徴とする水溶性卵殻膜の製造法(特公平7−110210号公報)、アルカリ性含水有機溶媒中で加水分解後に陰イオン交換樹脂で処理する方法(特許第5179847号公報)、米国特許第8211477号公報(発明の名称:Solubilized protein composition obtained from eggshell membrane)に記載のアルカリ加水分解法およびそれらの改変法にしたがって製造することができる。
本発明において使用される卵殻膜含有粉末は、少なくとも卵殻膜を含む粉末であれば特に制限はないが、卵殻膜含有微粉末であって、体積平均粒子径が6μm以下であることが好ましい。また、本発明において使用される卵殻膜含有微粉末は、体積最大粒子径が20μm以下であることが好ましい。なお、本願明細書において、粉末または微粉末の「体積平均粒子径」および「体積最大粒子径」は、レーザー回折式粒度分布測定機(LMS−30、株式会社セイシン企業製)を用いて測定した値を意味する。ここで、「体積平均粒子径」は、粒度分布における小粒径側からの累積値が50%における粒子径を意味する。また、卵殻膜含有粉末または微粉末の粒子径の測定に際しては、卵殻膜含有粉末または微粉末を、界面活性剤を用いて水に分散させた測定試料を用いる。なお、「粉末」は粒子のサイズにかかわらずあらゆる粉体を指し、「微粉末」は粉末のうち最大粒子径および/または平均粒子径が概ね100μmより小さいものを指すが、厳密な区別を意図するものではない。
本発明に使用される卵殻膜含有粉末の製造には、剥離された卵殻膜または卵殻に卵殻膜が付着した状態の原料を使用してもよく、当該原料と卵殻膜粉末とを併用することもできる。このような原料を粉末化する方法は公知のいずれのものでもよい。市販の卵殻膜粉末を卵殻膜含有粉末として用いてもよく、市販の卵殻膜粉末としては、たとえば、商品名「EMパウダー300」(キューピー株式会社製)を利用することができる。卵殻膜含有微粉末を製造する場合、市販の卵殻膜粉末、または、市販の卵殻膜粉末および卵殻カルシウムを利用して、これをさらに体積平均粒子径が6μm以下、および/または、体積最大粒子径が20μm以下まで微粉砕してもよい。
本発明の組成物は、本発明の分子シャペロン遺伝子活性化剤とともに、少なくとも一種の賦形剤を含有する。本発明の分子シャペロン遺伝子活性化剤は刺激性がないため、医薬または化粧品等の組成物とする場合、そのような組成物は、剤型に特に制限はなく、経口用または外用のあらゆる組成物とすることができる。点眼薬、点鼻薬、点耳薬、口腔薬(含嗽剤、噴霧剤)、坐薬(坐剤、軟膏剤、浣腸)等の外用組成物は、通常用いられる公知の成分を配合することによって、液剤、固形剤、半固形剤等のその使用目的に応じた各種剤型に調製することができる。好ましい組成物としては、たとえば、ローション、軟膏、ゲル、クリーム、スプレー剤、貼付剤、粉末等を挙げることができる。経口投与または摂取される用途では、錠剤、散剤、顆粒剤、カプセル剤、液剤などの経口用組成物とすることが好ましい。経口用組成物は、舌下薬(錠剤だけでなく、オブラートのようなシート、ペースト)やゼリー、微粉末を懸濁させたドリンク剤でもよい。口腔粘膜からの吸収は、活性成分が毛細血管から内頸静脈を経て直接心臓へ入るため、消化管腔内での分解、代謝、肝での代謝による初回通過効果を回避でき、全身に一気にまわるので好都合である。上記で例示したものを含む種々の剤型の医薬または化粧品等の組成物を製造するための各種成分および製造法は、医薬および化粧品等の製造にかかる分野で公知であり、当業者は必要に応じて適宜選択することができる。なお、ここで「医薬組成物」は、ヒト用に限定されず、ペットや家畜として飼育されている犬や猫などの哺乳動物用の医薬組成物を含む。また、「化粧品組成物」は、化粧品のみでなく、薬事法上の各種医薬部外品、薬用化粧品等を含む。
局所適用剤とするには、その使用目的に応じて、本発明の分子シャペロン遺伝子活性化剤を通常用いられる公知の成分に配合することによって、液剤、固形剤、半固形剤等の各種剤形に調製することが可能である。本発明の外用組成物には、本発明の分子シャペロン遺伝子活性化剤および賦形剤に加え、たとえば、美容上または医薬的な有効成分、芳香成分(香料など)、着色剤などを使用することができる。他の有効成分の例としては、たとえば、消炎剤、抗炎症剤、メラニン産生抑制剤、メラニン還元剤、脱色剤、メラニン排水促進剤、細胞賦活剤、抗酸化剤、酸化防止剤、角質溶解・剥離剤、皮脂抑制剤、保湿剤、エモリエント剤、皮脂分泌抑制・促進剤、紫外線吸収剤、制汗剤、血行促進剤、角質除去・柔軟剤、美白剤、抗アレルギー剤、ステロイドホルモン、免疫抑制剤、抗生物質などが挙げられる。
本発明の分子シャペロン遺伝子活性化剤は、錠剤、散剤、顆粒剤、カプセル剤、液剤などの経口用組成物とすることできる。種々の剤型の経口用組成物を製造するための各種成分および製造法は、医薬および化粧品等の製造にかかる分野で公知であり、当業者は必要に応じて適宜選択することができる。
本発明の分子シャペロン遺伝子活性化剤を、単独で、または他の食品添加物などの生理的に許容される各種成分と組み合わせて、菓子類、健康食品、保存食品、加工食品などの食品に添加するための食品添加物とすることができる。本発明の食品添加物は、分子シャペロン遺伝子活性化を目的として、当該技術分野で公知の方法により各種食品に添加して使用することができる。たとえば、卵殻膜の食品への適用については、粉末状に粉砕した卵殻膜を含む錠剤や菓子類などが提案されている(特許第3862600号、特開2009−165421号公報)。これらに記載された錠剤や菓子類において使用されている卵殻膜粉末として、本発明の分子シャペロン遺伝子活性化剤を含有する食品添加物を使用することができる。
アルカリ加水分解卵殻膜(以下「ASESM」という)としては、キューピー(Kewpie Corporation, Tokyo, Japan)から入手した商品名「EM PROTEIN‐P」を使用した。このASESMについてサイズ排除クロマトグラフィ(ゲルろ過)により測定した相対分子量は、主要な部分が約12〜14kDaであることが見出された(非特許文献1:Ohto-Fujita et al, Cell Tissue Res. 2011 July; 345(1): 177−190)。
動物は、ヘアレスマウス(Hos:HR−1、6週齢、雄)を用いた(コントロール群:n=2、ASESM処理群:n=2)。ASESM処理群には、前記で製造した10%(W/V)ASESM溶液を、外用で(局所的に)背部皮膚に、10日間(40μl/回×2)適用した。コントロール群には、ASESMを含まない上記基剤溶液を同様に適用した。
卵殻膜含有粉末サンプルとして、キューピー株式会社の商品名「EMパウダー300」をジェットミルで粉砕したものを用いた。ジェットミルとしてはシングルトラックジェットミル(株式会社セイシン企業製、FS−4)を用いて、風量:1.2m3/min、動力:11kwにて、体積最大粒子径が800メッシュ(目開きで約20μm)程度となるまで粉砕を実施した。レーザー回折式粒度分布測定機(株式会社セイシン企業製、LMS−30)を用いて粉砕後の粒径を測定したところ、体積最大粒子径は19.6μm、体積平均粒子径は5.8μmであった。
8週齢の雄Hos/HR-1マウスを前日より絶食させたのち、卵殻膜微粉末と卵殻カルシウムのみを有効成分として含有する実験用サプリメント(「8φCR 200mg」、上記で製造した卵殻膜含有微粉末(800メッシュ) 37.50%(75.0mg); 卵殻カルシウム(キューピー株式会社) 11.75%(23.5mg); 乳糖(グランビアフーズ社) 43.75%(87.5mg); トウモロコシ蛋白(小林香料株式会社) 5.00%(10.0mg); 菜種硬化油(川研ファインケミカルズ株式会社) 2.00%(4.0mg))0.5mg、または対照として賦型剤のみを含むコントロール錠剤(「9φCR 250mg」、乳糖 93.00%(232.5mg); トウモロコシ蛋白 5.00%(12.5mg); 菜種硬化油 2.00%(5.0mg)) 0.5mg(錠剤を乳鉢で粉にしたもの)を、動物用薬物投与ゼリー(商品名MediGel Sucralose、日本エスエルシー株式会社)100μLに懸濁し、軽くエーテル麻酔したマウスにゾンデを使って胃に直接全量投与した(n=各3)。8時間後にマウスを解剖し、皮膚組織中の細胞でのHspb5およびHspb2遺伝子発現を、上記と同様にして定量的リアルタイムPCRで評価した。
タンパク質などの含窒素化合物を炭酸リチウムと混合し中性子照射すると、Li6 (n,α)3H反応で生成したトリチウムにより標識される。これを利用して、トリチウム標識された卵殻膜含有粉末をマウスに経口投与した場合の体内動態を以下のようにして調べた。
卵殻膜含有粉末0.32g(「EMパウダー)、キューピー)と炭酸リチウム0.65gを十分に混合し石英管に減圧封入後、日本原子力研究機構原子力科学研究所(JRR4原子炉)で20分間中性子照射した。照射試料を石英管から取り出し、水と混合して未反応の炭酸リチウムを溶解した。卵殻膜粉末は水に不溶であるので濾過して回収し、卵殻膜に未結合のトリチウムを除去するため、濾液の放射能が十分に減少するまで水で洗浄した。
オリエンタル酵母より6週齢で購入したC57BL/6Jマウスを1週間程度の予備飼育(温度23±2℃、相対湿度55±10%、12時間明暗サイクルの環境下)後、7週齢時に実験を行った。マウスをスギヤマゲンの代謝ケージ(メタボリカMM)(86.5cm2×14.5cm、スペース約2000cm3)内で1匹ずつ飼育し、固形飼料(MF、オリエンタル酵母)と水道水を自由摂取させた。
投与前16時間絶食させたマウスに水で懸濁した標識卵殻膜含有粉末を、プラスチック製ディスポーザブルゾンデを用いて胃内に単回強制経口投与した。投与放射能は、約4.5MBq/kg(122mCi/kg)体重とし、投与量は250mg/kg体重とした。
放射能の測定は、調製された放射能測定試料にシンチレーターを加え、液体シンチレーションカウンター(Packard,2200CA)により行った。クエンチングの補正は外部標準線源比法により行った。
標識卵殻膜含有粉末投与後0.25、0.5、1、2、4、6、9、12、24時間ならびに2、3、4、5、6日に尾静脈より血液5mlを採取した。この試料に組織可溶化剤(Soluene−350(Perkin Elmer)/イソプロピルアルコール(1:1)) 1mlを加え、50℃で3時間加温振盪した後、30%過酸化水素水500mlを加えた。この試料にシンチレーター(Hionic fluor, Perkin Elmer)10mlを加え、放射能を測定した。
標識化合物投与後、マウスを代謝ケージ(メタボリカMM、スギヤマゲン)に入れ、投与後1日ごとに6日間、尿・糞を分離して採取した。採取した糞の一部を精秤し、これに組織溶解剤2mlを加え、3〜4時間、50℃で加温し、その後、イソプロパノール1mlを加え、50℃で2時間加温した。この試料に30%過酸化水素水を0.5ml加え、シンチレーター(Hionic Fluor, Perkin Elmer)を10ml加え、放射能を測定した。尿は、各画分の1mlにシンチレーター(ウルチマゴールドLLT)5mlを加え、放射能を測定した。
前記と同様にしてゾンデを用いて胃内に、5,568,000dpmのトリチウム標識卵殻膜を3個体のマウスに投与した。投与後、2時間、6時間および12時間後に、各個体から組織の一部または全部を摘出し、重量を測定した。各組織に2mlの可溶化剤(Soluene-350)を添加し、60℃で3時間インキュベートした。この試料に30%過酸化水素水を0.5ml加え、シンチレーター(Hionic Fluor)を10ml加えて室温で1時間インキュベートした後、液体シンチレーションカウンターにより放射能を測定した。結果を、表4および図3に示す。
(1)打錠用の顆粒の製造
上記で製造した卵殻膜含有微粉末(800メッシュ):20.0 質量部、日食株式会社製「ワキシa」:10.0質量部、松谷化学株式会社製「パインファイバー」:20.0質量部、乳糖(メグレ社製):25.9質量部、卵殻カルシウム(キューピー株式会社製「カルホープ」):10質量部、β−カロチン:5.0質量部、ビタミンB:20.05質量部、ビタミンE:0.05質量部およびナイアシン:2.0質量部を、V型混合機を用いて混合することにより、原料混合物を調製した。次いで、この原料混合物93.0質量部に対して、エチルアルコール15質量部を混合し、これにより得られた混合物を、湿式造粒装置を用いて造粒し、次いで温度50℃で約16時間乾燥して、打錠用の顆粒を製造した。
次に、打錠用の顆粒100質量部に対して、ビタミンCを9質量部およびショ糖脂肪酸エステルを1質量部の割合で混合し、それにより得られた混合物を、打錠装置を使用して、1粒が200mgの裸錠を製造した。
次に、裸錠の表面に、コーティング装置を使用して、岐阜セラック株式会社製「セラック」の水溶液を塗布し、温度40℃ で2時間乾燥して、保護コーティングされた錠剤(保護コーティング錠)を得た。
十分に乾燥させた保護コーティング錠の表面に、糖衣被覆装置を使用して、糖衣用ペーストA(グラニュー糖70質量部、アラビアガム3質量部、ゼラチン4質量部、卵殻カルシウム3質量部および水65質量部を混合したペースト)を被覆した後、温度約40℃で約4 時間乾燥した。その後、糖衣用ペーストAに水を加えて希釈した糖衣用ペーストBを調製した。さらに、糖衣用ペーストAでコーティング処理および乾燥処理された錠剤の表面に、糖衣被覆装置を使用して、糖衣用ペーストBを、被覆した後、温度約40℃で約4 時間乾燥した。これにより糖衣用ペーストでコーティングされた錠剤(糖衣コーティング錠)を得た。
糖衣コーティング錠の表面に、三栄源社製「SRレッドK3」を含む着色液を塗布した後、40〜50℃で4時間乾燥して、赤色に着色した錠剤(着色錠)を製造した。
着色錠の表面に、カルナウバロウを用いて、艶だしを行った。これにより得られた錠剤1個の質量は400mgであり、錠剤1個当たり卵殻膜成分を約40mgの割合で含有していた。
艶出し処理を行った錠剤を選別して不良品を除き、製品検査後に計量し、乾燥剤を同封した二重袋で包装した。なお、錠剤は、十分な硬度および形状保持性を有しており、選別、検査、包装時に変形したり、崩壊したりすることがなく、取り扱い性に優れていた。
Claims (1)
- 体積最大粒子径が800メッシュの卵殻膜含有微粉末を含有し、Hspa1b(Hsp70−1)、Hspd1(Hsp60)、Hsp90aa1、Hsp90ab1、Hsp90b1、Trap1およびHspb2のうちの1以上の遺伝子の発現を増強することを特徴とする、分子シャペロン遺伝子活性化剤。
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US3558771A (en) * | 1968-02-12 | 1971-01-26 | Leslie L Balassa | Process for using eggshell compositions for promoting wound healing |
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