JP2004010488A - Intestinal tract permeation inhibitor of allergen - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To obtain a new substance effective for prophylaxis of food allergies and having inhibitory effects on intestinal tract permeation. <P>SOLUTION: An intestinal tract permeation inhibitor of allergens comprises a tryptophan ester, a phenylalanine ester or tyrosine ester as an active ingredient. The inhibitor comprises a peptide composed of 2-5 amino acids and comprising at least the one tryptophan, phenylalanine or tyrosine as the active ingredient. Furthermore, an extract from a spice is contained as the active ingredient. <P>COPYRIGHT: (C)2004,JPO

Description

【００１５】
ロスマリン酸
【化２】

【００１６】
ケルセチン−３−Ｏ−グルクロニドは、以下の化学式で表される化合物である。ケルセチン−３−Ｏ−グルクロニドは、コリアンダーから常法により抽出することができる。抽出法について実施例で具体的に説明する。
【００１７】
ケルセチン−３−Ｏ−グルクロニド
【化３】

【００１８】
ルチンは、以下の化学式で表される化合物である。ルチンは、それぞれコリアンダー及びタラゴンから常法により抽出することができる。抽出法について実施例で具体的に説明する。
【００１９】
ルチン
【化４】

【００２０】
また、ピメントールは、以下の化学式で表される化合物である。ピメントールは、オールスパイスから常法により抽出することができる。抽出法について実施例で具体的に説明する。
【００２１】
ピメントール
【化５】

【００２２】
本発明の腸管透過抑制剤を投与するに際しては、有効成分であるアミノ酸誘導体、オリゴペプチドまたは香辛料抽出物をそのままの状態で用いることもできるが、常法に従って、粉末、顆粒剤、錠剤、カプセル剤、乳化剤、ドリンク剤など製剤化して用いることもできる。さらに、この腸管透過抑制剤を各種栄養剤や食品などに混ぜてアレルギーを予防することも可能である。
【００２３】
例えば、本発明の腸管透過抑制剤は、トリパルミチンからなる担体を用いたマイクロカプセルにすることが、製造の容易さ、腸管での溶出の容易さ、無味化等の観点から好ましい。さらに、前記担体に界面活性剤が含まれていてもよい。界面活性剤としては、造粒方法に応じて油性及び水性界面活性剤を用いることができる。油性界面活性剤としては、例えば、ＰＯ５００（花王社製）、水性界面活性剤としては、ＭＬ７５０（花王社製）等を挙げることができる。
【００２４】
上記マイクロカプセルは、例えば、腸管透過抑制剤、トリパルミチン及び界面活性剤の溶融物、水溶液又は水分散体をスプレイドライすることで製造することができる。また、例えば、腸管透過抑制剤及びトリパルミチンをトリパルミチン飽和ヘキサンに添加し、得られるスラリーをスプレイドライすることでも、上記マイクロカプセルを製造することができる。スプレイドライの条件は、溶融物、水溶液、水分散体又はスラリーに含まれる固形分濃度や腸管透過抑制剤の耐熱性、酸化安定性等を考慮して適宜決定される。具体的には、例えば、スプレイドライの加熱温度を約８０℃とすることができる。
【００２５】
本発明の腸管透過抑制剤の投与量は、年齢、治療効果、病態などにより異なるが、通常、一人当たり一回に体重１ｋｇ当たり約１μｇ〜１００ｍｇの範囲で、一日一回から数回投与すればよい。
【００２６】
なお、本発明の腸管透過抑制剤の効果の確認は、Ｃａｃｏ−２細胞を透過性フィルター上で約１４日間培養したものを用いて行った。詳細は実施例において説明する。
【００２７】
本発明の腸管透過抑制剤は、種々のアレルゲンに対して腸管透過抑制効果を有する。本発明の腸管透過抑制剤は、特に、腸管から吸収される食物由来のアレルゲンに対して効果的であり、食物由来のアレルゲンとしては、例えば、卵白アルブリン、オボムコイド、β−ラクトグロブリン、ＧｌｙＭＢｄ３０Ｋ（大豆アレルゲン）、アミラーゼインヒビター、ペルオキシダーゼ、低分子量グルテニン、等を挙げることができる。但し、これらに限定されるものではない。
【００２８】
【実施例】
以下、本発明を実施例によりさらに説明する。
腸管透過性試験方法
食物アレルゲンとして蛍光標識した卵白アルブミン（ＦＩＴＣ−ＯＶＡ）を、腸管モデルとしてＣａｃｏ−２細胞を透過性フィルター上で約１４日間培養したものを用いた。蛍光標識した卵白アルブミン（ＦＩＴＣ−ＯＶＡ）は、市販卵白アルブミンを常法（微アルカリ性）でフルオレッセンイソチオシアネートと反応させて結合後、流水透析、ＳｅｐｈａｄｅｘＧ−１５カラムクロマトグラフィーで精製して調製した。
【００２９】
Ｃａｃｏ−２細胞の培養は以下の方法で行った。
Ｃａｃｏ−２細胞を　Ａｍｅｒｉｃａｎ　Ｔｙｐｅ　Ｃｕｌｔｕｒｅ　Ｃｏｌｌｅｃｔｉｏｎ　（Ｕ．Ｓ．Ａ．）から入手し、継代数３０〜４０のものを透過試験に用いた。培養液の組成はＤｕｌｂｅｃｃｏ’ｓ　ｍｏｄｉｆｉｅｄ　Ｅａｇｌｅ’ｓ　ｍｅｄｉｕｍ（Ｇｉｂｃｏ，　Ｌｉｆｅ　Ｔｅｃｈｎｏｌｏｇｉｅｓ，　Ｉｎｃ．，　Ｕ．Ｓ．Ａ．）にウシ胎仔血清（２０％，大日本製薬）、非必須アミノ酸溶液（１％，　Ｇｉｂｃｏ，　Ｌｉｆｅ　Ｔｅｃｈｎｏｌｏｇｉｅｓ，　Ｉｎｃ．）、ペニシリン（１００　ＩＵ／ｍｌ，和光純薬）、ストレプトマイシン（１００μｇ　／ｍｌ，和光純薬）およびゲンタマイシン（５０μｇ　／ｍｌ，和光純薬）を加えたものとした。細胞を３７℃、５％ＣＯ_２、加湿条件下で培養し、３，　４日おきに継代を行った。透過試験に際しては、細胞を１２穴トランズウェル内のメンブレンフィルター（Ｎｏ．　３４６０，　Ｃｏｒｎｉｎｇ，　Ｕ．Ｓ．Ａ．）で培養した。培養液をベイサル（ｂａｓａｌ）側には１．５ｍｌ、アピカル（ａｐｉｃａｌ）側には０．５ｍｌを加え約２週間培養した。Ｍｉｌｌｉｃｅｌｌ−ＥＳＲ（Ｍｉｌｌｉｐｏｒｅ　Ｃｏ．，　Ｕ．Ｓ．Ａ．）を用いて経上皮電気抵抗　（ＴＥＥＲ）を測定し、ＴＥＥＲが　３００Ω・ｃｍ^−２以上となり単層が完成したものを透過試験に用いた。
【００３０】
透過試験前夜より試料溶液をアピカル（ａｐｉｃａｌ）側ベイサル（ｂａｓａｌ）側両方に加え、前培養した。培養後、試料共存下でＯＶＡを１ｍｇ／ｍｌの濃度でハンクス平衡塩溶液に溶解したものをアピカル（ａｐｉｃａｌ）側に添加し、３０分間培養した。３０分後ベイサル（ｂａｓａｌ）側の透過液を回収し、透過したＯＶＡ量を蛍光強度により測定した。
蛍光強度測定は、島津製作所社製の分光蛍光光度計を用いて励起波長４９５ｎｍ、蛍光波長５２０ｎｍで行った。
【００３１】
実施例１
トリプトファンエチルエステル、フェニルアラニンエチルエステル及びチロシンエチルエステルをそれぞれ以下の方法により合成した。
アミノ酸（１０ｇ）と１Ｎになるように塩化水素を吹き込んだエタノール（１００ｍｌ）を１時間還流することで製造した。還流の後、塩化水素とエタノールを溜去した残渣をそのまま用いた。
得られた各エステルのＯＶＡ透過抑制作用を、上記腸管透過性試験方法により求めた。その結果、いずれのエステルも１０^−７Ｍの濃度において、ＯＶＡの透過を顕著に抑制した。いずれのエステルも、ＯＶＡの透過を対照に対して約５０％阻害した。
【００３２】
実施例２
アミノ酸オリゴマー
ＷＧは市販品であり、ＷＳＮＳＧ、ＷＳＮ及びＷＳは、固相合成法により調製した。
得られた各アミノ酸オリゴマーのＯＶＡ透過抑制作用を、上記腸管透過性試験方法により求めた。その結果、いずれのアミノ酸オリゴマーも１０^−７Ｍの濃度において、ＯＶＡの透過を顕著に抑制した。いずれのペプチドも、ＯＶＡの透過を対照に対して約５０％阻害した。
【００３３】
実施例３
３３種の食品の５０％メタノール抽出物について上記腸管透過性試験方法によりスクリーニングを行った。その結果、オールスパイス、タイム、コリアンダー、タラゴンに強い活性が認められた。そこで、これらの食品から、以下に示す方法により抽出し、さらに抽出物から非逆相ＨＰＬＣを用いて活性成分を単離した。
【００３４】
ピメントールの抽出
オールスパイス５３７．７ｇを５０％メタノール８００ｍｌに浸漬し、吸引濾過し、濾液を減圧濃縮し、約１ｇの抽出物を得た。この抽出物を１００ｍｌの水に加え、Ｓｅｐ−Ｐａｋに吸着させ、６０％メタノール水溶液で溶出し、減圧濃縮によりメタノールを除去した。得られた溶液を以下の条件の逆相ＨＰＬＣに付し、活性成分を単離した。
【００３５】
ＨＰＬＣ条件
ファーストラン
カラム：Ｓｈｏｄｅｘ　ＲＳ　ｐａｋ　ＲＰ１８−４１５
溶媒：４０％メタノール０．１％ＴＦＡ（トリフルオロ酢酸）水溶液
流速：１ｍｌ／ｍｉｎ．
検出波長：２２０ｎｍ
クロマトグラムを図１に示す。
セカンドラン
カラム：Ｓｈｏｄｅｘ　ＲＳ　ｐａｋ　ＤＥ６１３
溶媒：アセトニトリル／０．１％ＴＦＡ水溶液２５〜３５％（１５分）リニアグラージェント
流速：１ｍｌ／ｍｉｎ．
検出波長：２２０ｎｍ
クロマトグラムを図２に示す。
【００３６】
単離した活性成分は、ＮＭＲおよび質量分析（ＦＡＢ−ＭＳ（Ｎｅｇａｔｉｖｅ）ｍ／ｚ４９３［Ｍ−Ｈ］⁻）の結果、オールスパイスの活性成分はピメントールであった。
【００３７】
^１Ｈ　ＮＭＲ　（５００ＭＨｚ，　ＣＤ_３ＯＤ）：δ３．０１　（２Ｈ，　ｂｒ．ｄｄ，　Ｊ＝５．５，　５．６Ｈｚ，　Ｈ−Ｅ７），　３．４５　（ｍ，Ｈ−Ｇｌｃ４），　３．５０　（ｍ，　Ｈ−Ｇｌｃ３），　３．５１　（ｍ，　Ｈ−Ｇｌｃ２），　３．７２　（ｄｄｄ，　Ｊ＝２．０，　６．８，　９．２Ｈｚ，　Ｈ−Ｇｌｃ５），　３．７８　（３Ｈ，　ｓ，　Ｈ−Ｅ１０（ＯＭｅ）），　４．４４　（ｄｄ，　Ｊ＝６．８，　１１．８Ｈｚ，　Ｈ−Ｇｌｃ６ａ），　４．５８　（ｄｄ，　Ｊ＝２．０，　１１．８Ｈｚ，　Ｈ−Ｇｌｃ６ｂ）　４．７３　（ｄ，　Ｊ＝７．７Ｈｚ，　Ｈ−Ｇｌｃ１），　４．９０　（２Ｈ，ｍ，　Ｈ−Ｅ９），　５．７４　（ｍ，　Ｈ−Ｅ８），　６．４６　（ｄ，　Ｊ＝１．６Ｈｚ，　Ｈ−Ｅ３），　６．５２　（ｄ，　Ｊ＝１．６Ｈｚ，　Ｈ−Ｅ５），　７．０８　（２Ｈ，　ｓ，　Ｈ−Ｇａ２，６）．
^１３Ｃ　ＮＭＲ　（５００ＭＨｚ，　ＣＤ_３ＯＤ）：δ４０．７　（Ｃ−Ｅ７），　５６．７　（Ｃ−Ｅ１０（ＯＭｅ）　），　６４．８　（Ｃ−Ｇｌｃ６），　７１．８　（Ｃ−Ｇｌｃ４），　７４．８　（Ｃ−Ｇｌｃ２），　７５．８　（Ｃ−Ｇｌｃ５），　７７．４　（Ｃ−Ｇｌｃ３），　１０４．１　（Ｃ−Ｇｌｃ１），　１０８．５　（Ｃ−Ｅ３），　１１０．２　（Ｃ−Ｇａ２，　Ｇａ６），　１１５．６　（Ｃ−Ｅ９），　１１１．６　（Ｃ−Ｅ５），　１２１．１　（Ｃ−Ｇａ１），　１３２．６　（Ｃ−Ｅ４），　１３５．６　（Ｃ−Ｅ１），　１３９．０　（Ｃ−Ｅ８），　１４０．２　（Ｃ−Ｇａ４），　１４６．６　（Ｃ−Ｇａ３，　Ｇａ５），　１４６．７　（Ｃ−Ｅ６），　１４９．５　（Ｃ−Ｅ２），　１６８．３　（Ｃ−Ｇａ７）．
【００３８】
ルテオリン−７−Ｏ−グルクロニド及びロスマリン酸の抽出
タイムを原料として上記オールスパイスの場合と同様に抽出溶液を得、以下の条件の逆相ＨＰＬＣに付し、活性成分を単離した。
ＨＰＬＣ条件
ファーストラン
カラム：Ｓｈｏｄｅｘ　ＲＳ　ｐａｋ　ＲＰ１８−４１５
溶媒：メタノール／０．１％ＴＦＡ水溶液３０〜５０％（１５分）リニアグラージェント
流速：１ｍｌ／ｍｉｎ．
検出波長：２２０ｎｍ
クロマトグラムを図３に示す。２つの活性ピークを得た（タイム３及び４）。
タイム３及び４のセカンドラン
カラム：Ｓｈｏｄｅｘ　ＲＳ　ｐａｋ　ＤＥ６１３
溶媒：アセトニトリル／１０ｍＭ酢酸アンモニウム水溶液１０〜３０％（１５分）リニアグラージェント
流速：１ｍｌ／ｍｉｎ．
検出波長：２２０ｎｍ
クロマトグラムを図４に示す。
タイム３のサードラン
カラム：Ｓｈｏｄｅｘ　ＲＳ　ｐａｋ　ＤＥ６１３
溶媒：アセトニトリル／０．１％ＴＦＡ水溶液２５〜５０％（１５分）リニアグラージェント
流速：１ｍｌ／ｍｉｎ．
検出波長：２２０ｎｍ
クロマトグラムを図５に示す。
タイム４のサードラン
カラム：Ｓｈｏｄｅｘ　ＲＳ　ｐａｋ　ＤＥ６１３
溶媒：アセトニトリル／０．１％ＴＦＡ水溶液４０〜６０％（１５分）リニアグラージェント
流速：１ｍｌ／ｍｉｎ．
検出波長：２２０ｎｍ
クロマトグラムを図６に示す。
【００３９】
単離した活性成分は、ＮＭＲおよび質量分析の結果、タイム３がルテオリン−７−Ｏ−グルクロニドであり、タイム４がロスマリン酸であった。
【００４０】
ロスマリン酸（Ｒｏｓｍａｒｉｎｉｃ　ａｃｉｄ）
ＦＡＢ−ＭＳ　（ｎｅｇａｔｉｖｅ）　ｍ／ｚ　４８３　［Ｍ−Ｈ＋Ｎａ−Ｈ］⁻．
^１Ｈ　ＮＭＲ　（５００ＭＨｚ，　ＣＤ_３ＯＤ）δ２．９６　（ｄｄ，　Ｊ　＝　８．９，　１４．３Ｈｚ，　Ｈ−７’）　，　３．０９　（ｄｄ，　Ｊ＝　３．３，　１４．３Ｈｚ，　Ｈ−７’），　５．１４　（ｄｄ，　Ｊ　＝　３．３，　８．９Ｈｚ，　Ｈ−８’），　６．２６　（ｄ，　Ｊ　＝　１６．０Ｈｚ，　Ｈ−８），　６．６１　（ｄｄ，　Ｊ　＝　１．５，　８．１Ｈｚ，　Ｈ−６’），　６．６８　（ｄ，　Ｊ　＝　８．１Ｈｚ，　Ｈ−５’），　６．７５　（ｄ，　Ｊ　＝　１．５Ｈｚ，　Ｈ−２’），　６．７６　（ｄ，　Ｊ　＝　８．１Ｈｚ，　Ｈ−５），　６．９３　（ｄｄ，　Ｊ　＝　２．０，　８．１Ｈｚ，　Ｈ−６），　７．０３　（ｄ，　Ｊ　＝　２．０Ｈｚ，　Ｈ−２），　７．５２　（ｄ，　Ｊ　＝　１６．０Ｈｚ，　Ｈ−７）．
^１３Ｃ　ＮＭＲ　（５００ＭＨｚ，　ＣＤ_３ＯＤ）δ３８．３　（Ｃ−７’），　７６．３　（Ｃ−８’），　１１５．０　（Ｃ−８），　１１５．２　（Ｃ−２），　１１６．３　（Ｃ−５’），　１１６．５　（Ｃ−５），　１１７．６　（Ｃ−２’），　１２１．８　（Ｃ−６’），　１２３．１　（Ｃ−６），　１２７．８　（Ｃ−１），　１３０．１　（Ｃ−１’），　１４５．１　（Ｃ−３’），　１４６．１　（Ｃ−４’），　１４６．８　（Ｃ−３），　１４７．３　（Ｃ−７），　１４９．６　（Ｃ−４），　１６８．８　（Ｃ−９）．
これらのデータは文献値と一致していた。Ｊ．　Ａｇｒｉｃ．　Ｆｏｏｄ　Ｃｈｅｍ．　２００１，　４９，　２−８．
【００４１】
ルテオリン−７−Ｏ−グルクロニド（Ｌｕｔｅｏｌｉｎ−７−Ｏ−ｇｌｕｃｕｒｏｎｉｄｅ）
ＦＡＢ−ＭＳ　（ｎｅｇａｔｉｖｅ）　ｍ／ｚ　４６１　［Ｍ−Ｈ］⁻．
^１Ｈ　ＮＭＲ　（５００ＭＨｚ，　ＣＤ_３ＯＤ）δ３．５４　（３Ｈ，　ｍ，　Ｈ−Ｇｌｃ２，　３　ａｎｄ　４），　３．８５　（ｂｒ．ｄ，　Ｊ　＝　９．１Ｈｚ，　Ｈ−Ｇｌｃ５），　５．０８　（ｄ，　Ｊ　＝　７．３Ｈｚ，　Ｈ−Ｇｌｃ１），　６．４９　（ｄ，　Ｊ　＝　２．２Ｈｚ，　Ｈ−８），６．５９　（ｓ，　Ｈ−３），　６．８２　（ｄ，　Ｊ　＝　２．２Ｈｚ，　Ｈ−６），　６．８９　（ｄ，　Ｊ　＝　８．９Ｈｚ，　Ｈ−５’），　７．３９−７．４１　（２Ｈ，　ｍ，　Ｈ−２’　ａｎｄ　６’）．
これらのデータは文献値と一致していた。Ｐｈｙｔｏｃｈｅｍｉｓｔｒｙ．　２０００，　５５，　２６３−２６７．
【００４２】
ケルセチン−３−Ｏ−グルクロニドの抽出
コリアンダーを５０％メタノール水溶液で抽出した。抽出物をＳｅｐ−Ｐａｃｋに吸着させ、６０％メタノール水溶液で溶出し、溶出画分を分取ＨＰＬＣに供した。
逆相分取ＨＰＬＣの条件
ファーストラン
カラム：Ｓｈｏｄｅｘ　ＲＳ　ｐａｋ　ＲＰ　１８−４１５
溶媒：４０％メタノール０．１％ＴＦＡ水溶液
流速：１ｍｌ／ｍｉｎ．
検出波長２２０ｎｍ
クロマトグラムを図７に示す。
【００４３】
セカンドラン
カラム：Ｓｈｏｄｅｘ　ＲＳ　ｐａｋ　ＲＰ　１８−４１５
溶媒：アセトニトリル／１０ｍＭ酢酸アンモニウム水溶液
アセトニトリル濃度が１０〜３０％（１５分）リニアグラジェント
流速：１ｍｌ／ｍｉｎ．
検出波長２２０ｎｍ
クロマトグラムを図８に示す。
【００４４】
サードラン
カラム：Ｓｈｏｄｅｘ　ＲＳ　ｐａｋ　ＲＰ　１８−４１５
溶媒：アセトニトリル／０．１％ＴＦＡ水溶液
アセトニトリル濃度が０〜３０％（１５分）リニアグラジェント
流速：１ｍｌ／ｍｉｎ．
検出波長２２０ｎｍ
クロマトグラムを図９に示す。
【００４５】
ケルセチン−３−Ｏ−グルクロニド（Ｑｕｅｒｃｅｔｉｎ−３−Ｏ−ｇｌｕｃｕｒｏｎｉｄｅ）
ＦＡＢ−ＭＳ　（ｎｅｇａｔｉｖｅ）　ｍ／ｚ　４７７　［Ｍ−Ｈ］⁻．
^１Ｈ　ＮＭＲ　（５００ＭＨｚ，　ＣＤ_３ＯＤ）δ３．４６−３．６４　（４Ｈ，　ｍ，　Ｈ−Ｇｌｃ２，　３，　４　ａｎｄ　５）　，　５．３０　（ｄ，　Ｊ　＝　７．６Ｈｚ，　Ｈ−Ｇｌｃ１），　６．１９　（ｂｒ．ｓ，　Ｈ−６），　６．３８　（ｂｒ．ｓ，　Ｈ−８），　６．８５　（ｄ，　Ｊ　＝８．４Ｈｚ，　Ｈ−５’），　７．４８　（ｄｄ，　Ｊ　＝　２．１，　８．４Ｈｚ，　Ｈ−６’），　７．９３　（ｂｒ．ｓ，　Ｈ−２’）．
^１３Ｃ　ＮＭＲ　（５００ＭＨｚ，　ＣＤ_３ＯＤ）　δ７３．４，　７５．６，　７７．６　ａｎｄ　７８．１　（Ｃ−Ｇｌｃ２，　３，　４　ｏｒ　５），　９４．３　（Ｃ−８），　１００．０　（Ｃ−６），　１０４．４　（Ｃ−Ｇｌｃ１），　１０３．８　（Ｃ−１０），　１１６．２　（Ｃ−５’），　１１８．１　（Ｃ−２’），　１２２．８　（Ｃ−６’），　１３５．８　（Ｃ−３），　１４５．９，　１４９．９，　１５８．５　（Ｃ−９），　１５９．２（Ｃ−２），　１６３．０　（Ｃ−５），　１６６．１　（Ｃ−７），　１７６．３　（Ｃ−Ｇｌｃ６），　１７９．５　（Ｃ−４）．
これらのデータは文献値と一致していた。Ｐｈｙｔｏｃｈｅｍｉｓｔｒｙ．　１９８５，　２４，　４６５−４６７．
【００４６】
ルチンの抽出
タラゴンの乾燥葉を５０％メタノール水溶液で抽出した。抽出物をＳｅｐ−Ｐａｃｋに吸着させ、６０％メタノール水溶液で溶出し、溶出画分を分取ＨＰＬＣに供した。
逆相分取 ＨＰＬＣ の条件
ファーストラン
カラム：Ｓｈｏｄｅｘ　ＲＳ　ｐａｋ　ＲＰ　１８−４１５
溶媒：メタノール／０．１％ＴＦＡ水溶液
メタノール濃度が３０〜７０％（１５分）リニアグラジェント
流速：１ｍｌ／ｍｉｎ．
検出波長２２０ｎｍ
クロマトグラムを図１０に示す。
【００４７】
タラゴン３は水で結晶化したので少量を取り、水に溶かしてＨＰＬＣでチェックした。
分析条件
カラム：Ｓｈｏｄｅｘ　ＲＳ　ｐａｋ　ＲＰ　１８−４１５
溶媒：アセトニトリル　／１０ｍＭ酢酸アンモニウム水溶液
アセトニトリル濃度が０〜３０％（１５分）リニアグラジェント
流速：１ｍｌ／ｍｉｎ．
検出波長２２０ｎｍ
クロマトグラムを図１１に示す。
【００４８】
ルチン（Ｑｕｅｒｃｅｔｉｎ−３−Ｏ−（６−Ｏ−ｒｈａｍｎｏｓｙｌ）−ｇｌｕｃｏｓｉｄｅ　（ｒｕｔｉｎ））
ＦＡＢ−ＭＳ　（ｎｅｇａｔｉｖｅ）　ｍ／ｚ　４９９　［Ｍ−Ｈ＋Ｎａ−Ｈ］⁻．
^１Ｈ　ＮＭＲ　（５００ＭＨｚ，　ＣＤ_３ＯＤ）δ１．０８　（３Ｈ，　ｄ，　Ｊ　＝　６．２Ｈｚ，　Ｈ−Ｒｈａ６），　３．２２〜３．２６　（２Ｈ，　ｍ，　Ｈ−Ｇｌｃ４，　Ｒｈａ４），　３．３０　（ｂｒ．ｓ，　Ｈ−Ｇｌｃ５），　３．３５　（ｍ，　Ｈ−Ｇｌｃ６），　３．３９〜３．４５　（３Ｈ，　ｍ，　Ｈ−Ｇｌｃ２　ａｎｄ　３，　Ｒｈａ５），　３．５１　（ｂｒ．ｄ，　Ｊ　＝　９．４Ｈｚ，　Ｈ−Ｒｈａ３），　３．６１　（ｂｒ．ｓ，　Ｈ−Ｒｈａ２），　３．７６　（ｂｒ．ｄ，　Ｊ　＝　１０．５Ｈｚ，　Ｈ−Ｇｌｃ６），　４．４８　（ｂｒ．ｓ，　Ｈ−Ｒｈａ１），　５．０６　（ｄ，　Ｊ　＝　７．４Ｈｚ，　Ｈ−Ｇｌｃ１），　６．１４　（ｂｒ．ｓ，　Ｈ−６），　６．３２　（ｂｒ．ｓ，　Ｈ−８），　６．８２　（ｄ，　Ｊ　＝　８．４Ｈｚ，　Ｈ−５’），　７．５８　（ｂｒ．ｄ，　Ｊ　＝　８．４Ｈｚ，　Ｈ−６’），　７．６２　（ｂｒ．ｓ，　Ｈ−２’）．
^１３Ｃ　ＮＭＲ　（５００ＭＨｚ，　ＣＤ_３ＯＤ）δ１７．９　（Ｃ−Ｒｈａ６），　６８．６　（Ｃ−Ｇｌｃ６），　６９．７（Ｃ−Ｒｈａ５），　７１．４　（Ｃ−Ｇｌｃ４），　７２．１　（Ｃ−Ｒｈａ２），　７２．３　（Ｃ−Ｒｈａ３），　７４．０　（Ｃ−Ｒｈａ４），　７５．８　（Ｃ−Ｇｌｃ２），　７７．２　（Ｃ−Ｇｌｃ５），　７８．２　（Ｃ−Ｇｌｃ３），　９４．９　（Ｃ−８），　１００．０　（Ｃ−６），　１０２．４　（Ｃ−Ｒｈａ１），　１０４．８　（Ｃ−Ｇｌｃ１），　１０５．６　（Ｃ−１０），　１１６．１　（Ｃ−５’），　１１７．８　（Ｃ−２’），　１２３．２　（Ｃ−１’），　１２３．６　（Ｃ−６’），　１３５．７　（Ｃ−３），　１４５．８　（Ｃ−３’），　１４９．８　（Ｃ−４’），　１５８．５　（Ｃ−９），　１５９．３　（Ｃ−２），　１６２．９　（Ｃ−５），　１６６．１　（Ｃ−７），　１７９．４（Ｃ−４）．
これらのデータは文献値と一致していた。Ｊ．　Ａｇｒｉｃ．　Ｆｏｏｄ　Ｃｈｅｍ．　１９９９，　４７，　４８８０−４８８２．
【００４９】
得られたピメントール、ルテオリン−７−Ｏ−グルクロニド及びロスマリン酸、ケルセチン−３−Ｏ−グルクロニド、ルチンのＯＶＡ透過抑制作用を、上記腸管透過性試験方法により求めた。その結果、いずれの抽出物も１０^−７Ｍの濃度において、ＯＶＡの透過を顕著に抑制した。抑制結果を表１に示す。
【００５０】
【表１】

【００５１】
参考例１
マイクロカプセルの作製
カプセル材料としてトリパルミチン（ＴＰ）３ｇ及び脂溶性界面活性剤ＰＯ−５００（ＨＬＢ４．９）（ＴＰの１％）の混合物を７０℃で融解し、４２〜４３℃にまで冷却してからシリカゲル上で乾燥したトリプトファンエチルエステル１５０μｇを加え、攪拌しながらＴＰ飽和ヘキサン（３重量）に流し込んでスラリーとし、スプレー乾燥した。マイクロカプセル化した後、１８０μｍの篩を通過する画分は口腔内でのざらつきがなかったので、カプセルはこの篩で分級した。１％ＭＬ−７５０（ＨＬＢ１４．８）で表面処理し、２００メッシュ篩で濾過した後に減圧乾燥して、３．１ｇのマイクロカプセルを得た（トリプトファンエチルエステルの回収率９０％以上）。
【図面の簡単な説明】
【図１】オールスパイス抽出物のＨＰＬＣファーストランのクロマトグラム。
【図２】オールスパイス抽出物のＨＰＬＣセカンドランのクロマトグラム。
【図３】タイム抽出物のＨＰＬＣファーストランのクロマトグラム。
【図４】タイム抽出物のＨＰＬＣセカンドランのクロマトグラム。
【図５】タイム３のＨＰＬＣサードランのクロマトグラム。
【図６】タイム４のＨＰＬＣサードランのクロマトグラム。
【図７】コリアンダー抽出物のＨＰＬＣファーストランのクロマトグラム。
【図８】コリアンダー抽出物のＨＰＬＣセカンドランのクロマトグラム。
【図９】コリアンダー抽出物のＨＰＬＣサードランのクロマトグラム。
【図１０】タラゴン抽出物のＨＰＬＣファーストランのクロマトグラム。
【図１１】タラゴン３のＨＰＬＣクロマトグラム。
【図１２】香辛料抽出物のＯＶＡ透過抑制活性を示す。[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention relates to an intestinal permeation inhibitor for an allergen. More specifically, the present invention relates to an intestinal permeation suppressor for an allergen containing an amino acid derivative, an oligopeptide or an extract from a spice as an active ingredient.
The intestinal permeation suppressant of the present invention suppresses permeation of allergen substances and the like in the intestinal tract, and is therefore useful for prevention and treatment of allergic diseases and the like.
[0002]
[Prior art]
The increasing number of food allergy patients has become a social problem. The symptoms of food allergies vary, including diarrhea, dermatitis, and asthma symptoms. Food allergies are caused by food-derived allergens (allergens) absorbed from the intestinal tract. In fact, the amount of allergen absorbed from the intestinal tract is increased in patients with food allergies, and this is considered to contribute deeply to the onset of allergy (J. Allergy Clin. Immunol. 2000 97 975-990).
[0003]
The permeation of substances in the intestinal tract involves a tight junction existing between epithelial cells of the intestinal tract. The tight junction seals the membrane surface cells without leaking, and surrounds the surface cells in a headband shape. Low molecular substances such as ions pass through the tight junction and are absorbed into the body. Macromolecules such as bacteria, viruses, and proteins that remain undigested may also pass through the intestinal epithelial cells and be absorbed into the body. This absorption is thought to be one of the causes of infections and food allergies.
[0004]
The permeability to tight junction materials is not constant, and the presence of glucose, cytochalasin D, etc., reduces the permeability of tight junctions. Further, it is also known that whey protein such as β-lactoglobulin or a degradation product thereof reduces tight junction permeability (Japanese Patent Application Laid-Open No. 8-73375). Apart from this, no other substances are known which reduce the permeability of high molecular weight compounds. On the other hand, diseases caused by allergies are so many that they are said to be modern diseases. In particular, in order to prevent food allergies, a search for an intestinal permeation-suppressing effect is indispensable.
[0005]
[Problems to be solved by the invention]
Accordingly, an object of the present invention is to provide a substance that is effective for preventing food allergy, is easy to manufacture and process, is highly safe, and has an intestinal permeation suppression effect.
[0006]
The present inventors have conducted intensive studies on substances that suppress substance permeation between intestinal epithelial cells, and found that specific amino acid derivatives, oligopeptides and spice extracts have an effect of suppressing intestinal substance permeation of allergens, The present invention has been completed.
[0007]
[Means for Solving the Problems]
The present invention for achieving the above object is as follows.
The intestinal permeation inhibitor of the allergen of the present invention contains a tryptophan ester, a phenylalanine ester or a tyrosine ester as an active ingredient. In the intestinal permeation suppressor for allergen, the ester is preferably a lower alkyl ester having 1 to 6 carbon atoms.
Further, the intestinal permeation inhibitor of the allergen of the present invention is composed of 2 to 5 amino acids, and contains a peptide containing at least one tryptophan, phenylalanine or tyrosine as an active ingredient. In the agent for suppressing intestinal permeation of an allergen, the peptide is preferably WSNSG, WSN, WS or WG.
[0008]
In addition, in the present invention, the intestinal permeation suppressor for an allergen contains an extract from spice as an active ingredient. In the intestinal permeation suppressor of this allergen, the extract from the spice is luteolin-7-O-glucuronide, rosmarinic acid, quercetin-3-O-glucuronide, rutin or Pimenthol.
The intestinal permeation inhibitor of the present invention has an intestinal permeation-suppressing effect on various allergens, but is particularly effective on food-derived allergens absorbed from the intestinal tract.
[0009]
BEST MODE FOR CARRYING OUT THE INVENTION
The active ingredient of the intestinal permeation inhibitor of the allergen of the present invention, examples of the lower alkyl ester constituting tryptophan ester, phenylalanine ester and tyrosine ester include, for example, methyl, ethyl, propyl, butyl, pentyl, hexyl and the like. it can. However, ethyl ester is preferred from the viewpoint of safety for living organisms, production cost, and intestinal permeation suppression effect. Further, in the case of the same type of ester, tryptophan ester, phenylalanine ester and tyrosine ester have almost the same intestinal permeation suppression effect.
[0010]
The amino acid ester can be produced, for example, by reacting an amino acid (tryptophan, phenylalanine or tyrosine) with an alkanol under acid-catalyzed conditions. Examples of the alkanol include methanol, ethanol, propanol, butanol, pentanol, hexanol and the like. Specifically, the ethyl ester can be produced by refluxing ethanol blown with hydrogen chloride so as to be 1N with the amino acid for 0.5 to 2 hours. After the reflux, the residue obtained by distilling off hydrogen chloride and ethanol may be used as it is. An acid other than hydrogen chloride may be used, and distillation may be omitted. Further, it can be purified by a known purification method such as crystallization with ethanol-hexane.
[0011]
The intestinal permeation inhibitor of an allergen consisting of 2 to 5 amino acids according to the present invention comprises an oligopeptide containing at least one tryptophan, phenylalanine or tyrosine as an active ingredient. The oligopeptide may contain one or more of tryptophan, phenylalanine and tyrosine, and is preferably, for example, WSNSG, WSN, WS, WG, or the like. In addition, W is tryptophan, S is serine, N is asparagine, and G is glycine.
[0012]
The oligopeptide can be produced using a known amino acid synthesis method (solid phase synthesis method). Alternatively, it can also be produced by decomposing a protein containing the above peptide and separating and recovering the fragment by a conventional method.
[0013]
The spices in the intestinal permeation suppressor for allergens containing an extract from spices as an active ingredient of the present invention include, for example, thyme, allspice, coriander, tarragon and the like.
Further, examples of the extract from spices include flavonoids.
Luteolin-7-O-glucuronide and rosmarinic acid, which are specific examples of extracts from spices, are compounds represented by the following chemical formulas, respectively. Luteolin-7-O-glucuronide and rosmarinic acid can be respectively extracted from thyme by a conventional method. The extraction method will be specifically described in Examples.
[0014]
Luteolin-7-O-glucuronide
Embedded image

[0015]
Rosmarinic acid
Embedded image

[0016]
Quercetin-3-O-glucuronide is a compound represented by the following chemical formula. Quercetin-3-O-glucuronide can be extracted from coriander by a conventional method. The extraction method will be specifically described in Examples.
[0017]
Quercetin-3-O-glucuronide
Embedded image

[0018]
Rutin is a compound represented by the following chemical formula. Rutin can be extracted from coriander and tarragon respectively by conventional methods. The extraction method will be specifically described in Examples.
[0019]
Rutin
Embedded image

[0020]
In addition, Pimenthol is a compound represented by the following chemical formula. Pimenthol can be extracted from allspice by a conventional method. The extraction method will be specifically described in Examples.
[0021]
Pimenthol
Embedded image

[0022]
When administering the intestinal permeation suppressant of the present invention, the active ingredient amino acid derivative, oligopeptide or spice extract can be used as it is, but according to a conventional method, powder, granule, tablet, capsule , Emulsifiers, drinks and the like can be formulated and used. Further, the intestinal permeation suppressant can be mixed with various nutrients, foods and the like to prevent allergy.
[0023]
For example, the intestinal permeation suppressant of the present invention is preferably formed into a microcapsule using a carrier made of tripalmitin, from the viewpoints of ease of production, easy dissolution in the intestinal tract, and tastelessness. Further, the carrier may contain a surfactant. As the surfactant, oily and aqueous surfactants can be used depending on the granulation method. Examples of the oily surfactant include PO500 (manufactured by Kao Corporation), and examples of the aqueous surfactant include ML750 (manufactured by Kao Corporation).
[0024]
The microcapsules can be produced, for example, by spray-drying a melt, aqueous solution or aqueous dispersion of an intestinal permeation suppressant, tripalmitin and a surfactant. Further, for example, the microcapsules can also be produced by adding an intestinal permeation inhibitor and tripalmitin to tripalmitin-saturated hexane and spray-drying the obtained slurry. The spray-drying conditions are appropriately determined in consideration of the solid concentration contained in the melt, the aqueous solution, the aqueous dispersion or the slurry, the heat resistance of the intestinal permeation suppressant, the oxidation stability, and the like. Specifically, for example, the heating temperature of spray drying can be set to about 80 ° C.
[0025]
The dosage of the intestinal permeation suppressant of the present invention varies depending on age, therapeutic effect, disease state and the like, but is usually in the range of about 1 μg to 100 mg per kg of body weight per person once, and once to several times a day. Just fine.
[0026]
The effect of the intestinal permeation inhibitor of the present invention was confirmed using Caco-2 cells cultured on a permeable filter for about 14 days. Details will be described in Examples.
[0027]
The intestinal permeation suppressant of the present invention has an intestinal permeation suppressing effect on various allergens. The intestinal permeation inhibitor of the present invention is particularly effective against food-derived allergens absorbed from the intestinal tract. Examples of food-derived allergens include egg white albumin, ovomucoid, β-lactoglobulin, GlyMBd30K (soybean) Allergen), amylase inhibitor, peroxidase, low molecular weight glutenin, and the like. However, it is not limited to these.
[0028]
【Example】
Hereinafter, the present invention will be further described with reference to examples.
Intestinal permeability test method
Ovalbumin (FITC-OVA) fluorescently labeled as a food allergen was used and Caco-2 cells cultured on a permeable filter for about 14 days were used as an intestinal tract model. Fluorescently labeled ovalbumin (FITC-OVA) was prepared by reacting commercially available ovalbumin with fluorescein isothiocyanate by a conventional method (slightly alkaline), binding, and then purifying by running water dialysis and Sephadex G-15 column chromatography.
[0029]
Caco-2 cells were cultured by the following method.
Caco-2 cells were obtained from American Type Culture Collection (U.S.A.) and passages 30-40 were used for permeation studies. The composition of the culture solution was Dulbecco's modified Eagle's medium (Gibco, Life Technologies, Inc., USA), fetal bovine serum (20%, Dainippon Pharmaceutical), non-essential amino acid solution (1%, Gibco, Life Technologies, Inc., penicillin (100 IU / ml, Wako Pure Chemical), streptomycin (100 μg / ml, Wako Pure Chemical) and gentamicin (50 μg / ml, Wako Pure Chemical). Cells at 37 ° C, 5% CO₂The cells were cultured under humidified conditions and subcultured every 3 or 4 days. For the permeation test, the cells were cultured with a membrane filter (No. # 3460, #Corning, #USA) in a 12-well transwell. The culture solution was added to 1.5 ml on the basal side and 0.5 ml on the apical side, and cultured for about 2 weeks. Transepithelial electrical resistance (TEER) was measured using a Millicell-ESR (Millipore Co., USA), and the TEER was measured to be 300 Ω · cm.^-2As described above, a completed single layer was used for the transmission test.
[0030]
From the night before the permeation test, the sample solution was added to both the apical side and the basal side, and pre-cultured. After the culture, a solution prepared by dissolving OVA in a Hanks balanced salt solution at a concentration of 1 mg / ml in the presence of a sample was added to the apical side and cultured for 30 minutes. After 30 minutes, the permeate on the basal side was collected, and the amount of OVA that had passed was measured by fluorescence intensity.
The fluorescence intensity was measured at an excitation wavelength of 495 nm and a fluorescence wavelength of 520 nm using a spectrofluorometer manufactured by Shimadzu Corporation.
[0031]
Example 1
Tryptophan ethyl ester, phenylalanine ethyl ester and tyrosine ethyl ester were synthesized by the following methods, respectively.
It was produced by refluxing an amino acid (10 g) and ethanol (100 ml) into which hydrogen chloride had been blown so as to be 1N for 1 hour. After the reflux, the residue obtained by distilling off hydrogen chloride and ethanol was used as it was.
The OVA permeation inhibitory effect of each of the obtained esters was determined by the intestinal permeability test method described above. As a result, each ester was 10^-7At a concentration of M, transmission of OVA was significantly suppressed. Both esters inhibited OVA transmission by about 50% relative to controls.
[0032]
Example 2
Amino acid oligomer
WG is a commercial product, and WSNSG, WSN and WS were prepared by a solid phase synthesis method.
The OVA permeation inhibitory effect of each of the obtained amino acid oligomers was determined by the intestinal permeability test method described above. As a result, each amino acid oligomer was 10^-7At a concentration of M, transmission of OVA was significantly suppressed. Both peptides inhibited OVA penetration by about 50% relative to controls.
[0033]
Example 3
Screening was performed on 50% methanol extracts of 33 foods by the intestinal permeability test method described above. As a result, strong activity was observed in allspice, thyme, coriander, and tarragon. Therefore, these foods were extracted by the method described below, and the active ingredients were isolated from the extracts using non-reverse phase HPLC.
[0034]
Extraction of Pimenthol
537.7 g of allspice was immersed in 800 ml of 50% methanol, filtered by suction, and the filtrate was concentrated under reduced pressure to obtain about 1 g of an extract. This extract was added to 100 ml of water, adsorbed on Sep-Pak, eluted with a 60% aqueous methanol solution, and methanol was removed by concentration under reduced pressure. The obtained solution was subjected to reverse phase HPLC under the following conditions to isolate the active ingredient.
[0035]
HPLC conditions
First run
Column: Shodex RS RS pak RP18-415
Solvent: 40% methanol 0.1% TFA (trifluoroacetic acid) aqueous solution
Flow rate: 1 ml / min.
Detection wavelength: 220 nm
The chromatogram is shown in FIG.
Second run
Column: Shodex \ RS \ pak \ DE613
Solvent: acetonitrile / 0.1% TFA aqueous solution 25-35% (15 minutes) linear gradient
Flow rate: 1 ml / min.
Detection wavelength: 220 nm
The chromatogram is shown in FIG.
[0036]
The isolated active ingredient was analyzed by NMR and mass spectrometry (FAB-MS (Negative) m / z 493 [MH].⁻As a result, the active ingredient of allspice was Pimenthol.
[0037]
¹H NMR (500 MHz, CD₃OD): δ3.01 (2H, ．br.dd, J = 5.5, 5.6 Hz, H-E7), 3.45 (m, H-Glc4), 3.50 (m, H-Glc3), 3.51 (m, H-Glc2), 3.72 (ddd, J = 2.0, 6.8, 9.2Hz, H-Glc5), 3.78 (3H, s, H-E10 (OMe) ), {4.44} (dd, J = 6.8, 11.8 Hz, H-Glc6a), 4.58 (dd, J = 2.0, 11.8 Hz, H-Glc6b) 4.73 (d, J). = 7.7 Hz, H-Glc1), 4.90 (2H, m, H-E9), 5.74 (m, H-E8), 6.46 (d, J = 1.6 Hz, H-E3) , {6.52} (d, ΔJ = 1.6 Hz H-E5), 7.08 (2H, s, H-Ga2,6).
¹³C NMR (500 MHz, CD₃OD): δ 40.7 {(C-E7), {56.7} (C-E10 (OMe)}), {64.8} (C-Glc6), {71.8} (C-Glc4), {74.8} (C-Glc2) , {75.8} (C-Glc5), {77.4} (C-Glc3), {104.1} (C-Glc1), {108.5} (C-E3), {110.2} (C-Ga2, {Ga6), {115. 6 (C-E9), {111.6} (C-E5), {121.1} (C-Ga1), {132.6} (CE4), {135.6} (CE1), {139.0} (CE8) ), {140.2} (C-Ga4), {146.6} (C-Ga3, {Ga5), {146.7} (CE6), {149.5} (CE2), {168.3} (C-Ga7).
[0038]
Extraction of luteolin-7-O-glucuronide and rosmarinic acid
Using thyme as a raw material, an extraction solution was obtained in the same manner as in the case of the allspice, and subjected to reverse phase HPLC under the following conditions to isolate the active ingredient.
HPLC conditions
First run
Column: Shodex RS RS pak RP18-415
Solvent: methanol / 0.1% TFA aqueous solution 30 to 50% (15 minutes) linear gradient
Flow rate: 1 ml / min.
Detection wavelength: 220 nm
The chromatogram is shown in FIG. Two activity peaks were obtained (times 3 and 4).
Second run of time 3 and 4
Column: Shodex \ RS \ pak \ DE613
Solvent: 10-30% acetonitrile / 10 mM ammonium acetate aqueous solution (15 minutes) Linear gradient
Flow rate: 1 ml / min.
Detection wavelength: 220 nm
The chromatogram is shown in FIG.
Time 3 Third Run
Column: Shodex \ RS \ pak \ DE613
Solvent: acetonitrile / 0.1% TFA aqueous solution 25 to 50% (15 minutes) linear gradient
Flow rate: 1 ml / min.
Detection wavelength: 220 nm
The chromatogram is shown in FIG.
Time 4 Third Run
Column: Shodex \ RS \ pak \ DE613
Solvent: acetonitrile / 0.1% TFA aqueous solution 40-60% (15 minutes) linear gradient
Flow rate: 1 ml / min.
Detection wavelength: 220 nm
The chromatogram is shown in FIG.
[0039]
As a result of NMR and mass spectrometry, Time 3 of the isolated active ingredient was luteolin-7-O-glucuronide, and Time 4 was rosmarinic acid.
[0040]
Rosmarinic acid
FAB-MS (negative) m / z 483 [MH + Na-H]⁻.
¹H NMR (500 MHz, CD₃OD) δ 2.96 {(dd, {J} = {8.9, {14.3 Hz, {H-7 ')}, {3.09} (dd, {J = {3.3, {14.3 Hz, {H-7'), {5.14} ( dd, {J} = {3.3, {8.9 Hz, {H-8 '), {6.26} (d, {J} = {16.0 Hz, {H-8), {6.61} (dd, {J} = {1.5, << 8.1 Hz}. , {H-6 '), {6.68} (d, {J} = {8.1 Hz, {H-5'), {6.75} (d, {J} = {1.5 Hz, {H-2 '), {6.76} (d, {J = {8.1 Hz, {H-5}), {6.93} (dd, {J} = {2.0, {8.1 Hz, {H-6}), {7.03} (d, {J} = {2.0 Hz, {H-2), {7. 52 {(d, {J} = {16.0 Hz, {H-7}).
¹³C NMR (500 MHz, CD₃OD) [delta] 38.3 {(C-7 '), {76.3} (C-8'), {115.0} (C-8), {115.2} (C-2), {116.3} (C-5 '), 116.5 (C-5), {117.6} (C-2 '), {121.8} (C-6'), {123.1} (C-6), {127.8} (C-1), {130.1 (C-1 '), {145.1} (C-3'), {146.1} (C-4 '), {146.8} (C-3), {147.3} (C-7), {149.6} (C -4), {168.8} (C-9).
These data were consistent with literature values. J. {Agric. {Food} Chem. $ 2001, $ 49, $ 2-8.
[0041]
Luteolin-7-O-glucuronide
FAB-MS (negative) m / z 461 [MH]⁻.
¹H NMR (500 MHz, CD₃OD) δ 3.54 (3H, m, H-Glc2, 3 and 4), 3.85 (br.d, J = 9.1 Hz, H-Glc5), 5.08 (d, J = 7.3 Hz, H-Glc1), {6.49} (d, {J} = {2.2 Hz, {H-8), 6.59} (s, {H-3), {6.82} (d, {J} = {2.2 Hz, {H-6), 6.89 {(d, {J} = {8.9 Hz, {H-5 "), {7.39-7.41} (2H, {m, {H-2 '} and \ 6').
These data were consistent with literature values. Phytochemistry. $ 2000, $ 55, $ 263-267.
[0042]
Extraction of quercetin-3-O-glucuronide
Coriander was extracted with a 50% aqueous methanol solution. The extract was adsorbed on Sep-Pack, eluted with a 60% aqueous methanol solution, and the eluted fraction was subjected to preparative HPLC.
Reverse phase preparative HPLC conditions
First run
Column: Shodex RS RS pak RP 18-415
Solvent: 40% methanol 0.1% TFA aqueous solution
Flow rate: 1 ml / min.
Detection wavelength 220nm
The chromatogram is shown in FIG.
[0043]
Second run
Column: Shodex RS RS pak RP 18-415
Solvent: acetonitrile / 10 mM ammonium acetate aqueous solution
Linear gradient with acetonitrile concentration of 10 to 30% (15 minutes)
Flow rate: 1 ml / min.
Detection wavelength 220nm
The chromatogram is shown in FIG.
[0044]
Third run
Column: Shodex RS RS pak RP 18-415
Solvent: acetonitrile / 0.1% TFA aqueous solution
Linear gradient with acetonitrile concentration of 0 to 30% (15 minutes)
Flow rate: 1 ml / min.
Detection wavelength 220nm
The chromatogram is shown in FIG.
[0045]
Quercetin-3-O-glucuronide
FAB-MS (negative) m / z 477 [MH]⁻.
¹H NMR (500 MHz, CD₃OD) δ 3.46-3.64 {(4H, m, H-Glc2, 3, 4 and 5)}, 5.30 (d, J = 7.6 Hz, H-Glc1), 6.19 (br.s, H-6), {6.38} (br.s, {H-8), {6.85} (d, {J} = 8.4 Hz, {H-5 '), {7.48} (dd, {J} = {2.1, {8. 4 Hz, {H-6 '), {7.93} (br.s, {H-2').
¹³C NMR (500 MHz, CD₃OD) 73.4, 75.6, 77.6 and 78.1 (C-Glc2, 3, 4 or 5), 94.3− (C-8), 100.0 (C-6), 104.4. (C-Glc1), {103.8} (C-10), {116.2} (C-5 '), {118.1} (C-2'), {122.8} (C-6 '), {135.8} (C -3), {145.9, {149.9, {158.5} (C-9), {159.2 (C-2), {163.0} (C-5), {166.1} (C-7), {176. 3 {(C-Glc6), {179.5} (C-4).
These data were consistent with literature values. Phytochemistry. $ 1985, $ 24, $ 465-467.
[0046]
Rutin extraction
Dried leaves of tarragon were extracted with a 50% aqueous methanol solution. The extract was adsorbed on Sep-Pack, eluted with a 60% aqueous methanol solution, and the eluted fraction was subjected to preparative HPLC.
Reverse phase fractionation HPLC Condition
First run
Column: Shodex RS RS pak RP 18-415
Solvent: methanol / 0.1% TFA aqueous solution
Linear gradient with methanol concentration of 30-70% (15 minutes)
Flow rate: 1 ml / min.
Detection wavelength 220nm
The chromatogram is shown in FIG.
[0047]
Tarragon 3 crystallized in water, so a small amount was taken, dissolved in water and checked by HPLC.
Analysis conditions
Column: Shodex RS RS pak RP 18-415
Solvent: acetonitrile / 10 mM ammonium acetate aqueous solution
Linear gradient with acetonitrile concentration of 0 to 30% (15 minutes)
Flow rate: 1 ml / min.
Detection wavelength 220nm
The chromatogram is shown in FIG.
[0048]
Rutin (Quercetin-3-O- (6-O-rhamnosyl) -glucoside (rutin))
FAB-MS (negative) m / z 499 [MH + Na-H]⁻.
¹H NMR (500 MHz, CD₃OD) δ1.08 (3H, d, J = 6.2 Hz, H-Rha6), 3.22 to 3.26 (2H, m, H-Glc4, Rha4), 3.30 (br.s, H- Glc5), {3.35} (m, H-Glc6), 3.39 to 3.45 (3H, m, H-Glc2 and 3, Rha5), 3.51 (br.d, J = 9.4 Hz, H). -Rha3), {3.61} (br.s, {H-Rha2), {3.76} (br.d, {J} = {10.5 Hz, {H-Glc6), {4.48} (br.s, {H-Rha1), # 5. 0.06 (d, {J} = {7.4 Hz, {H-Glc1), {6.14} (br.s, {H-6), {6.32} (br.s, {H-8), {6.82} (d, {J} =.4Hz, H-5 '), 7.58 (br.d, J = 8.4Hz, H-6'), 7.62 (br.s, H-2 ').
¹³C NMR (500 MHz, CD₃OD) δ 17.9 (C-Rha6), {68.6} (C-Glc6), {69.7 (C-Rha5), {71.4} (C-Glc4), {72.1} (C-Rha2), {72.3. (C-Rha3), {74.0} (C-Rha4), {75.8} (C-Glc2), {77.2} (C-Glc5), {78.2} (C-Glc3), {94.9} (C-8) , {100.0} (C-6), {102.4} (C-Rha1), {104.8} (C-Glc1), {105.6} (C-10), {116.1} (C-5 '), {117.8. (C-2 '), {123.2} (C-1'), {123.6} (C-6 '), {135.7} (C-3), {145.8} (C-3'), {149.8} ( C-4 '), {158.5} (C-9), 159.3 (C-2), 162.9 (C-5), 166.1 (C-7), 179.4 (C-4).
These data were consistent with literature values. J. {Agric. {Food} Chem. $ 1999, $ 47, $ 4880-4882.
[0049]
The OVA permeation inhibitory effects of the obtained pimenthol, luteolin-7-O-glucuronide, rosmarinic acid, quercetin-3-O-glucuronide and rutin were determined by the intestinal permeability test method described above. As a result, all the extracts were 10^-7At a concentration of M, transmission of OVA was significantly suppressed. Table 1 shows the suppression results.
[0050]
[Table 1]

[0051]
Reference Example 1
Preparation of microcapsules
A mixture of 3 g of tripalmitin (TP) as encapsulant and the fat-soluble surfactant PO-500 (HLB4.9) (1% of TP) is melted at 70 ° C., cooled to 42-43 ° C. and then on silica gel. Was added, and the mixture was poured into TP-saturated hexane (3% by weight) with stirring to form a slurry, followed by spray drying. After microencapsulation, the fraction passing through the 180 μm sieve had no roughness in the oral cavity, so the capsules were classified with this sieve. The surface was treated with 1% ML-750 (HLB 14.8), filtered through a 200-mesh sieve, and dried under reduced pressure to obtain 3.1 g of microcapsules (recovery rate of tryptophan ethyl ester of 90% or more).
[Brief description of the drawings]
FIG. 1. Chromatogram of HPLC first run of allspice extract.
FIG. 2. Chromatogram of HPLC second run of allspice extract.
FIG. 3. Chromatogram of HPLC first run of thyme extract.
FIG. 4: Chromatogram of HPLC second run of thyme extract.
FIG. 5: Chromatogram of a time 3 HPLC third run.
FIG. 6: Chromatogram of the HPLC third run at time 4.
FIG. 7: HPLC first run chromatogram of coriander extract.
FIG. 8: Chromatogram of HPLC second run of coriander extract.
FIG. 9: Chromatogram of HPLC third run of coriander extract.
FIG. 10: Chromatogram of HPLC first run of tarragon extract.
FIG. 11: HPLC chromatogram of tarragon 3.
FIG. 12 shows the OVA permeation inhibitory activity of the spice extract.

Claims (7)

An allergen intestinal permeation inhibitor comprising a tryptophan ester, a phenylalanine ester or a tyrosine ester as an active ingredient. The intestinal permeation suppressant according to claim 1, wherein the ester is a lower alkyl ester having 1 to 6 carbon atoms. An agent for suppressing intestinal permeation of an allergen, comprising a peptide consisting of 2 to 5 amino acids and containing at least one tryptophan, phenylalanine or tyrosine as an active ingredient. The intestinal permeation inhibitor according to claim 3, wherein the peptide is WSNSG, WSN, WS or WG. An intestinal permeation inhibitor for an allergen containing an extract from spices as an active ingredient. The intestinal permeation inhibitor according to claim 5, wherein the extract from the spice is luteolin-7-O-glucuronide, rosmarinic acid, quercetin-3-O-glucuronide, rutin or pimenthol. The intestinal permeation inhibitor according to any one of claims 1 to 6, wherein the allergen is derived from food.

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