JP6049639B2 - クロロフィルc含有脱顆粒抑制剤 - Google Patents
クロロフィルc含有脱顆粒抑制剤 Download PDFInfo
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- JP6049639B2 JP6049639B2 JP2013551859A JP2013551859A JP6049639B2 JP 6049639 B2 JP6049639 B2 JP 6049639B2 JP 2013551859 A JP2013551859 A JP 2013551859A JP 2013551859 A JP2013551859 A JP 2013551859A JP 6049639 B2 JP6049639 B2 JP 6049639B2
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- Coloring Foods And Improving Nutritive Qualities (AREA)
Description
lia、えんどうこんぶ、Laminaria yendoana、くろしおめ属のくろしおめ、Hedophyllum kuroshioense、にせつるも科にせつるも属のほそつるも、にせつるも、Chordaria nagaii、いしげ目いしげ科いしげ属のいしげ、いろろ、foliacea、ひばまた目ひばまた科ひばまた属のひばまた、evanescens、えぞいしげ属のえぞいしげ、Pelvetia babingtonii、Pelvetia wrightii、ほんだわら科すぎもく属のすぎもく、うがのもく属のねぶともく、Cystophyllum crassipes、えぞもく、Cystophyllum geminatum、うがのもく、Cystophyllum hakodatense、やばねもく属のやばねもく、triquetra、Cystoseira prolifera、じょろもく属のじょろもく、yendoi、かいふもく、Cystophyllum sisymbrioides、ひえもく、ほんだわら属のきればもく、かたわもく、すなびきもく、えちごねじもく、つくしもく、あきよれもく、ほっかいもく、たまえだもく、まじりもく、ほそばもく、vulgare var. linearifolium sensu Yendo、ふしすじもく、あつばもく、べりべりもく、こぶくろもく、とさかもく、とげみもく、ふたえもく、ひめこもく、なんかいもく、しだもく、ほんだわら、enerve、ひじき、Hizikia fusiformis、おおばのこぎりもく、こなふきもく、いそもく、あかもく、ふたえひいらぎもく、しまうらもく、とさもく、しろこもく、ながみもく、のこぎりもく、serratifolium auct. japon.、とげもく、ふしいともく、みやべもく、kjellmanianum、たまははきもく、kjellmanianum f. muticum、ひめはもく、opacum、ならさも、たまなしもく、ひらねじもく、うすいろもく、やつまたもく、まめたわら、からくさもく、こばもく、たまきればもく、おおばもく、やなぎもく、ちゅらしまもく、ねじもく、ふくれみもく、ひゅうがもく、ながしまもく、racemosum Yamada et Segi、まるばのがらも、うすばのこぎりもく、よれもく、tortile、きしゅうもく、へらならさも、うすばもく、うみとらのお、たつくり、いとよれもく、なんきもく、あずまねじもく、よれもくもどき、えんどうもく、えながもく、えぞのねじもく、sagamianum var. yezoense、らっぱもく属のかさもく、らっぱもく、たかつきもく、trialata、などの褐藻類が挙げられる。クロロフィルcの原料として褐藻類のあかもく、かじめ、くろめ及びこんぶが特に好ましい。
これまでに脱顆粒抑制作用が知られていなかったクロロフィル類について、以下に記載するRBL−2H3ラット好塩基球由来細胞株を用いた脱顆粒抑制活性評価試験を行った。
実施例1と同様の濃度に希釈したRBL−2H3ラット好塩基球由来細胞株を96ウェル培養プレートに0.1ml/ウェルずつ分注して37℃、5%CO2、100%湿度条件下で一晩培養した。培養上清を除去した後、1ml/ウェルのPBSで細胞表面を洗浄したのち、実施例1と同様の乾燥重量濃度のクロロフィルc2標品を含むMT Bufferを100μl/ウェルずつ添加し、37℃、5%CO2、100%湿度条件下で40分間インキュベーションした。また、脱顆粒反応の抑制を受けないコントロール群には0.5%のエタノールを含むMT Bufferを添加した。40分のインキュベーション後、WST−1試薬((株)同仁化学研究所製)を10μl/ウェルずつ添加し、37℃、5%CO2、100%湿度条件下で30分間インキュベーションした。インキュベーション終了後、室温にて10分間静置したのち450nmの吸光度を測定することで細胞生存率を評価した。
クロロフィルcは、上述したように渦鞭毛藻門、クリプト藻門、黄金色藻綱、ラフィド藻綱、褐藻綱、珪藻綱、真正眼点藻綱を含む不等毛藻門、ハプト藻門などの藻類に多く含まれていることが知られているため、クロロフィルcの含まれる藻類の中から入手が容易で、かつ食経験を有するあかもくを、塩分や多糖類などのクロロフィルc以外の水溶性画分や水不溶性の不純物を除去することで簡便で安価にクロロフィルcを濃縮する目的で、15倍量の水を用いて3分間撹拌しながら洗浄を行い、同様の洗浄操作を3回繰り返した後、25℃の条件下で一晩乾燥させたのち、粉砕機を用いて粉砕しクロロフィルc以外の不要物除去してクロロフィルcを濃縮した褐藻類あかもくの乾燥粉末を製造した。
これまでに脱顆粒抑制作用が知られていなかったクロロフィルc1またはc3について、実施例1と同様にして脱顆粒抑制活性評価を行った。その結果、図8に示す通り、クロロフィルc1標品(非特許文献12などを参考に褐藻類より精製)またはc3標品(DHI 社製)を添加した群において、実施例1で確認されたクロロフィルc2と同様に脱顆粒抑制活性を示すことが明らかとなった。よって、これまでクロロフィル類において全く知られていなかった脱顆粒抑制活性について、実施例1で確認されたクロロフィルc2だけではなく、クロロフィルc1またはc3にも活性を有することが明らかとなり、いずれかのクロロフィルcを用いることによって、脱顆粒反応が関与している花粉症などのアレルギー症状や変形性関節症の予防や治療に有効であることが確認された。
クロロフィルc1またはc3について実施例2と同様にしてRBL−2H3好塩基球由来細胞株を用いた細胞生存率評価試験を行った。その結果、図9に示す通り、クロロフィルc1標品、c3標品を添加したいずれの細胞群においても細胞生存率の低下は認められず、実施例4で明らかとなったクロロフィルc1またはc3による脱顆粒抑制活性は細胞毒性によるものではないことが確認された。
1週間の馴化を行った5週齢のSD系雄性ラットに、実施例3で製造したクロロフィルcを濃縮した褐藻類あかもくの乾燥粉末を、AIN−76文献配合飼料(オリエンタル酵母工業(株)社製)に3重量%または1重量%混合した飼料を28日間自由摂取させた。あるいは実施例3で製造した褐藻類あかもくの80%含水エタノール抽出物をAIN−76文献配合飼料に1.1重量%混合した飼料を28日間自由摂取させた。あるいはAIN−76文献配合飼料のみを28日間自由摂取させた(コントロール群)。これらの各飼料を28日間自由摂取させたSD系雄性ラットの尾静脈に、エバンスブルー色素を投与した。次に、エバンスブルー色素を投与したSD系雄性ラットの背部皮内へ脱顆粒反応を介して血管透過性を惹起する薬剤であるCompound48/80を注射した。麻酔下で頚椎脱臼した後、背部皮膚を剥離し、剥離した皮膚組織に浸透しているエバンスブルー色素の量を、プレートリーダーを用いて620nmの吸光度を測定することで皮膚組織に透過しているエバンスブルー色素の量を定量した。定量したエバンスブルー色素の量より、コントロール群の皮下組織に透過しているエバンスブルー色素の量を100%とした時の、各試験食を与えた際のSD系雄性ラットの皮下組織に透過しているエバンスブルー色素の割合を血管透過率として算出した。その結果、図10に示す通り、1%または3重量%のクロロフィルcを濃縮した褐藻類あかもくの乾燥粉末を含む飼料、または1.1重量%の褐藻類あかもく抽出物を含む飼料を28日間自由摂取させた群において、コントロール群に比べて血管透過率が抑制された。このことから、生体を用いた試験においても、実施例3で製造したクロロフィルcを濃縮した褐藻類あかもくの乾燥粉末または褐藻類あかもくの80%含水エタノール抽出物を経口摂取することによって、脱顆粒反応に起因する血管透過性を抑制し、血管透過性が関与する花粉症などのアレルギー疾患の予防や治療に効果を示すことが明らかとなった。
1週間の馴化を行った4週齢のC57BL/6雄性マウス(日本チャールズ・リバー(株)社製)に、0.5%のCMC溶液に懸濁した実施例3で製造したクロロフィルcを濃縮した褐藻類あかもくの乾燥粉末を2g/kg、または0.5%のCMC溶液に懸濁した実施例3で製造した褐藻類あかもく乾燥粉末から更にクロロフィルcを濃縮した80%含水エタノール抽出物を0.2g/kgまたは1g/kgを4週間反復強制経口投与した。あるいは0.5%のCMC溶液のみ(コントロール群)、または0.5%のCMC溶液に懸濁した脱顆粒抑制作用により花粉症抑制効果を示す医薬品であるオロパタジン(SIMGA社製)(医薬品投与群)を4週間反復強制経口投与した。4週間の反復強制経口投与後、生理食塩水にて50μg/mlに溶解したAntiDNP-IgE溶液をマウス尻静脈内に投与して受動感作した。感作5日後、生理食塩水にて50mg/mlに溶解したDNP−OVA抗原をマウスの両鼻に点鼻投与して鼻炎症状を惹起させ、1時間辺りの鼻掻き回数を数えることによって発症した花粉症症状を評価した。その結果、図11に示す通り、実施例3で製造したクロロフィルcを濃縮した褐藻類あかもくの乾燥粉末2g/kg、または褐藻類あかもくの乾燥粉末から更にクロロフィルcを濃縮した80%含水エタノール抽出物1g/kgもしくは0.2g/kgを摂取した群において、コントロール群に比べて花粉症症状による鼻掻き回数を抑制することが明らかとなり、またその効果は、医薬品であるオロパタジンと同程度の強力な効果であることが示された。このことから、実施例3で製造したクロロフィルcを濃縮した褐藻類あかもくの乾燥粉末または褐藻類あかもくの80%含水エタノール抽出物を経口摂取することによって、脱顆粒反応を起因として実施例6で確認された血管透過性を抑制することによって花粉症症状を抑制し、花粉症などのアレルギー性鼻炎の予防や治療に効果を示すことが明確となった。
1週間の馴化を行った8週齢のDA/Slc雌性ラット(三協ラボサービス(株)社製)に、実施例3で製造したクロロフィルcを濃縮した褐藻類あかもくの乾燥粉末をCRF−1配合飼料(オリエンタル酵母工業(株)社製)に3重量%配合した飼料、またはグルコサミン(焼津水産化学工業(株)社製)を2重量%CRF−1配合飼料に配合した飼料、または食品用コンドロイチン硫酸(SCP(NB)と表記、(株)マルハニチロ食品社製)を5重量%CRF−1配合飼料に配合した飼料、または実施例3で製造したクロロフィルc濃縮褐藻類あかもくの乾燥粉末3重量%に加えてグルコサミンを2重量%CRF−1配合飼料に配合した飼料、または実施例3で製造したクロロフィルc濃縮褐藻類あかもくの乾燥粉末3重量%に加えてSCP(NB)を5重量%CRF−1配合飼料に配合した飼料、またはCRF−1配合飼料のみ(コントロール群)、を4週間自由摂取させた。4週間自由摂取させた後、腰背部の4箇所に関節症惹起物質であるウシII型コラーゲン溶液(コラーゲン技術研修会製)を0.15mgずつ皮内注射することで変形性関節症を惹起した。惹起後、目視により左右の後肢足蹠の状態を観察し、表1に示す後肢足蹠の状態と照らし合わせて発症した関節症を点数付けして評価した。その結果、図12に示す通り、実施例3で製造したクロロフィルc濃縮褐藻類あかもくの乾燥粉末をCRF−1配合飼料に3重量%配合した飼料を自由摂取した群において、コントロール群に比べて関節炎スコアが抑制されることが明らかとなり、また、その効果は変形性関節症の予防や治療に用いられているグルコサミンやSCP(NB)をCRF−1配合飼料に配合した飼料を自由摂取した群と同程度以上の、非常に強力な効果であることが示された。更に、実施例3で製造したクロロフィルc濃縮褐藻類あかもくの乾燥粉末を、変形性関節症の予防や治療に用いられているグルコサミンまたはSCP(NB)と併用して摂取させることにより、グルコサミンやSCP(NB)のみをCRF−1配合飼料に配合した飼料を自由摂取させるよりも、強く変形性関節症を抑制することが確認された。これらの結果から、実施例3で製造したクロロフィルc濃縮褐藻類あかもくの乾燥粉末を摂取することにより変形性関節症を抑制することが明らかとなった。更に、同じ変形性関節症抑制剤であるグルコサミンやSCP(NB)と、実施例3で製造したクロロフィルc濃縮褐藻類あかもくの乾燥粉末を併用することによって、より強力な変形性関節症抑制作用を示すことが明らかとなった。
Claims (10)
- 有効成分としてクロロフィルcを含有する脱顆粒抑制剤組成物。
- 変形性関節症抑制剤又はアレルギー抑制剤として使用する、請求項1に記載の組成物。
- クロロフィルcが褐藻類から抽出されてなる、請求項1又は2に記載の組成物。
- 褐藻類が、あかもく、かじめ、くろめ及びこんぶからなる群から選択される請求項3に記載の組成物。
- グルコサミン及び/又はコンドロイチン硫酸を更に含有する請求項1〜4のいずれかに記載の組成物。
- 有効成分としてクロロフィルcを含有する脱顆粒抑制用食品組成物。
- 変形性関節症抑制用又はアレルギー抑制用として使用する、請求項6に記載の食品組成物。
- クロロフィルcが褐藻類から抽出されてなる、請求項6又は7に記載の食品組成物。
- 褐藻類が、あかもく、かじめ、くろめ及びこんぶからなる群から選択される請求項8に記載の食品組成物。
- グルコサミン及び/又はコンドロイチン硫酸を更に含有する請求項6〜9のいずれかに記載の食品組成物。
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US5661017A (en) * | 1993-09-14 | 1997-08-26 | Dunahay; Terri Goodman | Method to transform algae, materials therefor, and products produced thereby |
JP4012977B2 (ja) | 1996-02-02 | 2007-11-28 | 株式会社紀文フードケミファ | 抗アレルギー剤および抗炎症剤 |
JPH1072362A (ja) | 1996-08-29 | 1998-03-17 | Kyodo Nyugyo Kk | アレルギ−疾患の治療及び予防剤並びにアレルギ−疾患の治療及び予防法 |
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JP3540951B2 (ja) | 1998-12-17 | 2004-07-07 | 麒麟麦酒株式会社 | 高クロロフィル含有耐塩性クロレラ |
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JP2003321474A (ja) | 2002-04-30 | 2003-11-11 | Kentetsuku:Kk | 包接化合物 |
JP4212838B2 (ja) | 2002-06-26 | 2009-01-21 | カルピス株式会社 | 抗アレルギー剤 |
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US9636326B2 (en) | 2017-05-02 |
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EP2799069A1 (en) | 2014-11-05 |
EP2799069A4 (en) | 2015-12-23 |
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