JP5002097B2 - Preventive / therapeutic agent for human or animal disease caused by coccidium and adjuvant agent for immunization of human or animal against mild coccidium infection - Google Patents

Description

【０１２１】
サンプル８の吸光度は０．８６と比較的高かった。その理由として他のサンプルは２画分ずつを一緒にしたものであるのに対し、サンプル８は１４画分を合わせたものである。すなわち、サンプル８はテーリングした成分を集めて濃縮したものである。従って、４２０ｎｍの吸光度は高いが、この画分だけを本発明の効果を有する画分として回収することは効率が悪い。
【０１２２】
糖分含量から、サンプル１〜３及び８が非スクロース分画分に相当し、サンプル４〜７がスクロース分画分に相当することがわかる。
【０１２３】
実施例１
製造例１の甘蔗由来のエキス（凍結乾燥品）を用い、ニワトリの体重の推移とEimeria tenellaオーシスト感染後の病態の推移を検査した。
【０１２４】
２週齢近交系ニワトリのヒナ（遺伝子型、Ｈ．Ｂ１５系統）７羽のそ嚢内に、駒込ピペットを用いて甘蔗由来のエキス（５００ｍｇ／ｋｇ体重）を３日間連日で投与した。さらにその最終投与日と同日に、ニワトリコクシジウム症の病原原虫Eimeria tenella（ＮＩＡＨ株）のオーシスト（２０×１０³個／羽）をそ嚢内に接種した（以下、「エキス投与群」という）。
【０１２５】
また、同齢・同種のニワトリのヒナ５羽のそ嚢内に、甘蔗由来のエキスの代わりに生理食塩水をエキス投与群と同様に投与した。さらにその最終投与日と同日に、オーシスト（２０×１０³個／羽）をそ嚢内に接種した（以下、「対照群」という）。
【０１２６】
オーシスト感染（オーシスト接種）に対する甘蔗由来のエキスの効果は、感染後１週間の症状、斃死数、１日当たりの体重増加、血便の有無及び盲腸病変の軽重などを指標として評価した。
【０１２７】
盲腸病変の軽重は、Johnson and Reidの方法に準じて評価し、平均の病変スコアーとして表示した（Johnson, J. and Reid, W. M. Anticoccidial drugs: lesion scoring techniques in battery and floor-pen experiments with chickens. Exp. Parasitol.、２８巻: 第３０〜３６頁、１９７０年; Conway, D. P. Examination of lesions and lesion scoring. In Poultry coccidiosis, Diagnostic and testing procedures 編集Donal P. Conway、１９７０年、第１７〜３６頁、Pfizer International Inc., New York, N.Y. 10017, U.S.A.）。
【０１２８】
表２にその結果を示す。
【０１２９】
【表２】
表２ Eineria tenella オーシスト感染ニワトリにおける斃死数、１日当たりの平均体重の増加、血便の持続性及び盲腸の平均病変スコアー

【０１３０】
斃死数に関して、対照群ではオーシスト感染後３日目に１例が斃死したが、エキス投与群では斃死例は認められなかった。
【０１３１】
感染後１週間における１日当たりの平均体重増加に関して、対照群では平均７．１ｇ／日であるが、エキス投与群では８．３ｇ／日であった。このことから、エキス投与群における感染後１週間における１日当たりの体重増加は、対照群に比べて高かった。
【０１３２】
血便の持続性に関して、対照群では感染後３〜６日に血便が多量、持続的高頻度（＋＋）に観察されたが、エキス投与群では感染後３〜４日に軽度の一過性の血便（＋）がみられた程度であった。
【０１３３】
また、感染後７日の剖検時に盲腸を肉眼的に観察した。対照群では、盲腸は弾力性を欠き膨張し、壁の菲薄化や暗赤色の出血性病巣が認められ、その病変の平均スコアーは＋３．８であった。一方、エキス投与群では、盲腸は弾力性を有し、出血性病変は殆ど認められず、その病変の平均スコアーは＋２．６であった。
【０１３４】
従って、Eimeria tenellaのオーシスト感染前に甘蔗由来のエキスをそ嚢内に投与することにより、感染後の１日当たりの体重増加の低下を抑制することができ、また血便の排泄及び盲腸病変に関しても軽減又は抑制が観察された。これらのことから、製造例１による甘蔗由来のエキスは、成長促進作用及びEimeria tenellaの増殖抑制効果を有することが示された。
【０１３５】
実施例２
製造例１の甘蔗由来のエキス（凍結乾燥品）を用い、ニワトリの体重の推移とEimeria tenellaオーシスト感染後の病態の推移を検査した。
【０１３６】
２系統の２週齢近交ニワトリのヒナ（遺伝子型、Ｈ．Ｂ１５系統を２４羽及びＨ．Ｂ２系統を１２羽）を用いた。そ嚢内に、駒込ピペットを用いて甘蔗由来のエキス（５００ｍｇ／ｋｇ体重）を１日又は３日間連日投与した。さらにその最終投与日と同日に、ニワトリコクシジウム症の病原原虫Eimeria tennela（ＮＩＡＨ株）のオーシスト（２０×１０³個／羽）をそ嚢内に接種した（以下、１日投与した場合を「エキス１回投与群」、３日投与した場合を「エキス３回投与群」という）。
【０１３７】
また、同齢・同種のニワトリのヒナのそ嚢内に、甘蔗由来のエキスの代わりに生理食塩水をエキス１回投与群及びエキス３回投与群と同様に投与した。さらにその最終日と同日に、オーシスト（２０×１０³個／羽）をそ嚢内に接種した（以下、「対照群」という）。
【０１３８】
さらに、同齢・同種のニワトリのヒナを、甘蔗由来のエキスの投与及びオーシストの接種のどちらも行わず、同様の実験に供した（以下、「無処置対照群」という）。
【０１３９】
オーシスト感染（オーシスト接種）に対する甘蔗由来のエキスの効果は、オーシスト感染後１週間の症状、斃死数、１日当たりの体重増加、血便の有無、糞便中のオーシスト数及び盲腸病変の軽重などを指標として評価した。
【０１４０】
糞便中のオーシスト数は、プランクトン計算板を用いる方法にて算出し、１羽当たりの排泄数として表示した。盲腸病変の軽重は前記したJohonson and Reidの方法に準じて評価し、平均の病変スコアーとして表示した。また、病変の病理組織学的検索は、常法に準じて、盲腸組織のヘマトキシリン・エオジン（ＨＥ）染色標本を作成して行われた。
【０１４１】
表３にその結果を示す。
【０１４２】
【表３】
表３ Eimeria tenellaオーシスト感染ニワトリにおける斃死数、1日当りの平均体重の増加、血便の持続性、オーシスト排泄数及び盲腸の平均病変スコアー

【０１４３】
斃死数に関して、対照群ではＨ．Ｂ２系統のオーシスト感染後1週間内に１例が斃死したが、エキス３回投与群では斃死例は認められなかった。
【０１４４】
１日当りの平均体重増加に関し、Ｈ．Ｂ１５系統及びＨ．Ｂ２系統の無処置対照群では、それぞれ１０．７ｇ及び９．８ｇであったが、対照群では、それぞれ７．６ｇ及び７．９ｇと著しく低下した。一方、Ｈ．１５系統のエキス１回投与群及びエキス３回投与群では、それぞれ８．６ｇ及び８．６ｇであり、Ｈ．Ｂ２系統のエキス３回投与群では９．８ｇであった。このように、いずれのエキス投与群においても対照群に比して高い平均体重増加を示した。
【０１４５】
また、Ｈ．Ｂ１５系統及びＨ．Ｂ２系統の双方の感染群のいずれにおいても、感染後３〜６日に鮮血便が多量、持続性かつ高頻度(＋＋)に観察された。しかし、エキス投与群では、感染後３〜４日に軽度、一過性の血便（＋）がみられた程度であった。
【０１４６】
感染６日及び７日目の1羽当りの糞便中の平均オーシスト排泄数に関して、Ｈ．Ｂ１５系統及びＨ．Ｂ２系統の対照群ではそれぞれ２７〜５８×１０⁶及び２６〜３８×１０⁶であったが、Ｈ．Ｂ１５系統のエキス１回投与群及びエキス３回投与群ではそれぞれ１６〜２３×１０⁶及び１２〜１６×１０⁶であり、Ｈ．Ｂ２系統のエキス３回-III投与群においても９〜１２×１０⁶と対照群に比して著しい低下が観察された。
【０１４７】
さらに、ＨＢ１５の感染後７日の剖検時に盲腸を肉眼的に観察したところ、対照群の盲腸は弾力性を欠き腫脹し、壁の菲薄化や暗赤色の出血性病巣が認められ、その病変の平均スコアーは＋４．０であった。一方、エキス３回投与群の盲腸は弾力性を有し、出血性病変は殆ど認められず、その病変の平均スコアーは＋３．４であった。また、Ｈ．Ｂ１５系統のエキス１回投与群の盲腸病変の平均スコアーは＋４．０と高く、回復期と思われる盲腸の委縮がみられたが、出血性病巣はほとんど認められなかった。
【０１４８】
さらに、盲腸組織のＨＥ染色標本について病理組織学的検索をおこなった。その結果、粘膜上皮層の病巣には多数のシゾントに加え、腔内にはガモントやオーシストも散見された。また、出血性病変並びに上皮細胞の破壊及び粘膜固有層の委縮などの退行性変化が観察された。しかし、その病変は各エキス投与群よりも対照群において顕著であった。
【０１４９】
以上の結果より、Eimeria tenellaのオーシスト感染前に甘蔗由来のエキスをそ嚢内に投与することにより、感染後の１日当りの体重の増加の低下が抑制された。また、血便の持続性、糞便中のオーシスト排泄数及び盲腸病変に関しても軽減又は抑制が観察されたことから、製造例１の甘蔗由来のエキスはEimeria tenella原虫の増殖抑制及びその感染を抑制する効果を有することが示された。
【０１５０】
実施例３
製造例１の甘蔗由来のエキス（凍結乾燥品）を用い、ニワトリを使用したEimeria tenella オーシストの軽度感染及び攻撃感染後の病態の推移、抗体産生応答並びに体重の推移を検査した。
【０１５１】
本実施例には７日齢近交系ニワトリのヒナ（遺伝子型、Ｈ．Ｂ１５系統）３６羽をもちい、１群あたり６羽を供試した。ヒナのそ嚢内に、駒込ピペットを用いて甘蔗由来のエキス(５００ｍｇ／ｋｇ体重)を３日間連日投与した。さらにその最終投与日と同日に、ニワトリコクシジウム症の病原原虫Eimeria tenella（ＮＩＡＨ株）のオーシスト（２×１０³個／羽）をそ嚢内に接種した（以下、「エキス投与＋軽度感染」群という）。さらに、「エキス投与＋軽度感染」群において、２８日齢時にオーシスト（１００×１０³個／羽）をそ嚢に接種することにより、攻撃感染をおこなった（以下、「エキス投与＋軽度感染＋攻撃感染」群という）。
【０１５２】
また、同齢・同種のヒナのそ嚢内に、甘蔗由来のエキスの代りに生理食塩水をエキス投与軽度感染群と同様に投与した。さらにその最終投与日と同日に、オーシスト（２×１０³個／羽）をそ嚢内に接種した（以下、「軽度感染群」という）。さらに、「軽度感染群」群において、２８日齢時にオーシスト（１００×１０³個／羽）をそ嚢に接種することにより、攻撃感染をおこなった（以下、「軽度感染＋攻撃感染」群という）。
【０１５３】
さらに、同齢・同種のヒナを、甘蔗由来のエキスの投与及び感染の処置のどちらも行わず、同様の実験に供した（以下、「無処置対照群」という）。さらに、無処置対照群において、２８日齢時にオーシスト（１００×１０³個／羽）をそ嚢に接種することにより、攻撃感染をおこなった（以下、「無処置対照＋攻撃感染」群という）。
【０１５４】
抗体産生応答の評価は、Ｔ細胞依存性抗原である羊赤血球（ＳＲＢＣ）又はＴ細胞非依存性抗原であるBrucella abortus（ＢＡ）を抗原として用い、１４日及び２１日齢時において、無処置対照群、軽度感染群及び「エキス投与＋軽度感染」群には１０％ ＳＲＢＣ及び１％ ＢＡ菌体の混合抗原０．１ｍｌを翼静脈内に接種し、１週後の両抗原に対する血清凝集素力価を測定（Hirota, Y. ら、The development of unusual B-cell functions in the testosterone-propionate-treated chicken. Immunology、第３９巻、第２９〜３６頁、１９８０年; Hirota, Y.ら、Immunopathology of chickens infected in ovo at hatching with the avian osteopetrosis virus MAV.2-0. Eur. J. Immunol.、第１０巻、第９２９〜９３６頁、
１９８０年）することによりおこなった。
【０１５５】
また、血清凝集素力価の測定では、０．２Ｍの２−メルカプトエタノール（２−ＭＥ）で処理後又は未処理の血清を２倍段階希釈し、完全凝集を示す最終希釈の逆数のｌｏｇ２を算出し、それぞれ２−ＭＥ抵抗性抗体価及び総抗体価として表示した。
【０１５６】
攻撃感染に対するエキスの効果は、攻撃感染後における１週間の症状、斃死数、１日当りの平均体重の増加、血便の持続性、糞便中のオーシスト排泄数及び盲腸病変の軽重等を指標として評価した。
【０１５７】
糞便中のオーシスト数は、プランクトン計算板を用いる方法にて算定し、１羽当りの排泄数として表示した。
【０１５８】
盲腸病変の軽重は、前述したJohnson and Reidの方法に準じて評価し、平均病変スコアーとして表示した。また、常法に準じて盲腸組織のＨＥ染色標本を作成し、病変の病理組織学的検索にて評価した。
【０１５９】
さらに、パーコル（Percoll）密度勾配遠心法にて盲腸から分離したリンパ球中の細胞膜マーカー表面抗原（Ｂ細胞マーカーとしてＢｕ−ｌａ⁺、Ｔ細胞マーカーとしてＣＤ４⁺、ＣＤ８⁺、ＴＣＲ１⁺（γδ型レセプター）、ＴＣＲ２⁺（αβ型レセプター）及びＴＣＲ３⁺）について抗原陽性細胞の相対的比率（％）をフローサイトメーターにて解析した（Vainio, O. ら、B-L antigens (class II) of the chicken major histocompatibility complex control T-B cell interaction. Immunogenetics、第１９巻、第１３１頁、１９８４年; Luhtala, M. ら、Analysis of chicken ＣＤ４ by monoclonal antibodies indicates evolutionary conversion between avian and mammalian species. Hybridoma、第１２巻、第６３３〜６４６頁、１９９３年）。
【０１６０】
表４〜表６及び図１に、その結果を示す。
【０１６１】
【表４】
表４ 甘蔗由来のエキスのそ嚢内投与及びEimeria tenella オーシストの軽度感染がＳＲＢＣ及びＢＡに対する抗体産生応答の推移に及ぼす影響

【０１６２】
表４は、ＳＲＢＣ及びＢＡに対する抗体産生応答の結果を示す。ＳＲＢＣに対する抗体応答において、「エキス投与＋軽度感染」群は、１次応答及び２次応答のいずれにおいても、無処置対照群及び対感染群に比して高い応答がみられた。特に、２−ＭＥ処理後の血清抗体価、すなわちＩｇＧクラスを主体とする２−ＭＥ抵抗性抗体が有意に高かった（ｐ＜０．０５）。
【０１６３】
ＢＡに対する１次抗体応答に関し、無処置対照群及び軽度感染群では、それぞれ１２羽中１羽のみが低力価の応答を示したに過ぎなかった。しかし、「エキス投与＋軽度感染」群では、１２羽中４羽に応答が認められ、その力価は無処置対照群及び軽度感染群に比して有意に高い値を示した（ｐ＜０．０５）。
【０１６４】
また、ＢＡに対する２次抗体応答に関して、エキス投与軽度感染群は、無処置対照群及び軽度感染群に比して有意に高い値を示した（ｐ＜０．０５）。
【０１６５】
【表５】
表５ 甘蔗由来のエキスのそ嚢内投与及びEimeria tenella オーシストの軽度感染が攻撃感染後の病態の推移に及ぼす影響

【０１６６】
攻撃感染後の斃死数に関して、「無処置対照群＋攻撃感染」群では、オーシスト感染後３日目に１例が斃死したが、その他の群では斃死例は認められなかった。
【０１６７】
１日当りの平均体重増加に関して、無処置対照群は１０．６ｇであったが、「無処置対照＋攻撃感染」群は８．５ｇであり、「無処置対照＋攻撃感染」群の１日当りの平均体重増加は無処置対照群と比して著しく低下した。また、軽度感染群及び「軽度感染＋攻撃感染」群の場合の１日当りの平均体重増加は、それぞれ１１．７ｇ及び１１．０ｇであり、また「エキス投与＋軽度感染」群及び「エキス投与＋軽度感染＋攻撃感染」群の場合それぞれ１５．９ｇ及び１３．９ｇであった。このことから、前記エキスを投与した群の１日当りの平均体重増加は、いずれにおいても対応する各攻撃感染群に比して高い体重増加を示した。
【０１６８】
血便の持続性に関し、軽度感染を行った全ての群において軽度感染直後では血便は認められなかったが、「無処置対照＋攻撃感染」群では感染後３〜６日に血便が多量、持続性かつ高頻度(＋＋)に観察された。しかし、オーシストの軽度な軽度感染を受けた場合、すなわち「軽度感染＋攻撃感染」群及び「エキス投与＋軽度感染＋攻撃感染」群ではいずれも感染後３〜４日に一過性の軽度の血便（＋）がみられた程度であった。
【０１６９】
攻撃感染６日の１羽当りの糞便中の平均オーシスト排泄数は、攻撃感染群及び「軽度感染＋攻撃感染」群ではそれぞれ４２×１０⁶及び４０×１０⁶であったが、「エキス投与＋軽度感染＋攻撃感染」群では３．３×１０⁶であった。このことから、「エキス投与＋軽度感染＋攻撃感染」群の平均オーシスト排泄数は、攻撃感染群及び「軽度感染＋攻撃感染」群に比して著しい低下が認められた。
【０１７０】
また、攻撃感染後７日の剖検時に盲腸を肉眼的に観察した。その結果、攻撃感染群の盲腸は、弾力性を欠き腫脹し、壁の菲薄化や暗赤色の出血性病巣が認められ、その病変の平均スコアーは＋４．０であった。一方、「軽度感染＋攻撃感染」群及び「エキス投与＋軽度感染＋攻撃感染」群の盲腸は、軽度の出血性病変がみられたが、その平均スコアーはそれぞれ＋３．０及び＋２．０であった。このことから、「エキス投与＋軽度感染＋攻撃感染」群の盲腸の病変は、「軽度感染＋攻撃感染」群に比して軽度であった。なお、軽度感染群及び「エキス投与＋軽度感染」群の盲腸は、弾力性を有し肉眼的病変は認められず、無処置対照群と同様であった。また、甘蔗由来のエキスを投与したいずれの群においても、盲腸の平均病変スコアーが低く抑えられた。
【０１７１】
【表６】
表６ 甘蔗由来のエキスのそ嚢内投与及びEimeria tenella オーシストの軽度感染が攻撃感染後の盲腸内表面マーカー抗原陽性細胞の相対的比率（％）に及ぼす影響

【０１７２】
表６は、３６日齢ニワトリにおける盲腸のリンパ球中における細胞膜マーカー表面抗原陽性細胞の相対的比率をフローサイトメーターにて解析した結果を示す。攻撃感染後７日の盲腸において、Ｂｕ−ｌａ⁺陽性細胞とＣＤ４⁺陽性細胞の相対的比率が無処置対照群に比して著しく低下するのに対して、ＣＤ８⁺陽性細胞及びＴＣＲ１⁺陽性細胞の比率は増加した。軽度感染群及び「軽度感染＋攻撃感染」群ではＴＣＲ２⁺陽性細胞の比率が増加した。また、「エキス投与＋軽度感染」群及び「エキス投与＋軽度感染＋攻撃感染」群では、ＴＣＲ１⁺陽性細胞の比率が増加する傾向が認められた。
【０１７３】
Eimeria tenellaの感染抵抗性には、遺伝的要因の他にインターフェロンγの産生に関わるＣＤ４⁺陽性Ｔ細胞とＴＣＲ２⁺陽性Ｔ細胞の局所への動員・集簇等が関連していることが報告されている（Yun, C.H. ら、Eimeria tenella infection induces local interferon production and intestinal lymphocyte subpopulation changes、第６８巻３号、第１２８２〜１２８８頁、２０００年）。攻撃感染に随伴するＢｕ−ｌａ⁺陽性細胞とＣＤ４⁺陽性細胞の相対的比率の低下は、エキス投与群及び軽度感染群においてみられたように攻撃感染する前にエキス投与又はオーシスト（２×１０³個／羽）の軽度感染を施すことにより、抑制又は軽減させることができた。したがって、エキス投与又はオーシストの軽度感染による感染抑制及び病態軽減効果の機序は不明であるが、本原虫が増殖する盲腸局所へのＢｕ−ｌａ⁺陽性細胞、ＣＤ４⁺陽性Ｔ細胞やＴＣＲ２⁺陽性Ｔ細胞等の集簇が関連していると考えられる。
【０１７４】
盲腸組織のＨＥ染色標本について病理組織学的検査をおこなった（図１）。その結果、攻撃感染群では、粘膜上皮及び固有層の病巣には多数のシゾントが見られ、腔内にはガモントやオーシストも散見された。また、出血性病変、上皮細胞及び粘膜固有層の破壊並びに委縮などの退行性変化が観察された（図１−Ａ）。「軽度感染＋攻撃感染」群では、シゾント数は攻撃感染群に比して少なかった（図１−Ｂ）。「エキス投与＋軽度感染＋攻撃感染」群では、回復期と思われる盲腸の委縮が観察されたが、増殖した原虫はほとんど観察されず及び原虫の寄生は認められなかった（図１−Ｃ）。
【０１７５】
製造例１の甘蔗由来のエキスをそ嚢内に投与することにより、ＳＲＢＣ及びＢＡに対する抗体産生応答の増強が認められた。特に、２週齢時のＢＡ抗原の通常免疫では、通常ＢＡに対する抗体は産生されないにも関わらず、ＢＡ抗原に対して抗体応答を示すヒナの数の増加やその平均力価の上昇が認められた。このことから、甘蔗由来のエキスは、すでにある免疫システムの活性増強及び免疫システムの成熟促進作用を有していると考えられた。Eimeria tenella のオーシスト（２×１０³個／羽）を用いて軽度感染することにより、その後の攻撃感染（１００×１０³個／羽）に随伴する体重減少及び盲腸病変を軽減又は抑制する。しかし、そのオーシスト（２×１０³個／羽）の軽度感染前に、製造例１の甘蔗由来のエキスをそ嚢内に投与することにより、その後の攻撃感染に随伴する体重減少、盲腸の病態並びに病変形成を抑制する効果を示した。
【０１７６】
以上から、甘蔗由来のエキスはコクシジウムの軽度な感染に対するヒトまたは動物の免疫のためのアジュバント効果を有していることが示された。
【０１７７】
【発明の効果】
本発明によれば、甘蔗由来のエキスを有効成分とする剤を動物に投与（例えば、経口投与）することにより、コクシジウムによって引き起こされるヒトまたは動物の病気を予防治療をすることができる。また、本発明によれば、甘蔗由来のエキスを含む剤を動物に投与（例えば、経口投与）することにより、コクシジウムのワクチン投与を含むコクシジウムの軽感染に対するヒトまたは動物の免疫のためのアジュバント効果を発揮することができる。
本発明による予防治療剤及びアジュバント剤は、天然物であるにも関わらず、コクシジウムのオーシストの排泄抑制効果は高く、またアジュバント効果が高い。しかも、少量で上記効果を発揮するため、産業上非常に有用である。従って、抗菌・抗原虫剤の投与回数を減少させる、またはこれらの投与の代替とすることができる。また、アジュバント効果により、ワクチンの投与回数を減少させることができる。また、自然に起きる軽感染に対するアジュバント効果により、ワクチンの投与を省略することができる。さらに、本発明による予防治療剤及びアジュバント剤は、植物由来であり、かつ古来よりヒトが甘蔗そのもの又は含蜜糖として食してきた天然物であるため、ヒト又はヒトが食用とする家畜若しくは家禽などの産業動物の健康を害することもない。このように、本発明による予防治療剤及びアジュバント剤は、安全でありかつ低コストである。
【図面の簡単な説明】
【図１】 攻撃感染後７日目のニワトリ（３５日齢）の盲腸の病理組織像（ＨＥ染色標本、×１００）
図１−Ａ；攻撃感染群；粘膜上皮及び固有層内の多数のシゾント寄生
図１−Ｂ；「軽度感染＋攻撃感染」群；散在性のシゾント
図１−Ｃ；「エキス投与＋軽度感染＋攻撃感染」群；粘膜上皮及び固有層の委縮
【図２】イオン交換樹脂による２番蜜分画サンプルの分析値
【符号の説明】
図１−Ａ（→）；粘膜固有層内の重度のコクシジウ原虫のシゾント寄生
図１−Ｂ（→）；中程度のコクシジウ原虫のシゾントシゾント寄生[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a prophylactic / therapeutic agent for human or animal diseases caused by coccidium and to a feed containing the prophylactic / therapeutic agent. Furthermore, the present invention relates to an adjuvant for immunization of humans or animals against mild coccidial infection, and to a feed containing the adjuvant.
[0002]
[Prior art]
Coccidium is known to be a protozoan that parasitizes animals such as birds, cows, sheep, dogs and cats, and humans, and causes infections. Here, coccidium means Coccidia (Eucoccidiorida) in a broad sense, and the Eimeriaceae in the Coccidiae Eimeriarina in a narrow sense.
[0003]
Specifically, the order Coccidia (Eucoccidiorida) is subdivided into Eimeriorina, Haemospororina and the like.
[0004]
The Eimeria subfamily includes the Eimeriaceae and Cryptosporidiumaceae. Specifically, chicken coccidium (Eimeria) that infects poultry of Eimeriaidae, infects humans from drinking water through livestock of Cryptosporidiumida There are known Cryptosporidium and Sarcocystis which parasitize mammals including humans and reptiles and cause granulosporosis.
[0005]
The Hemosporina subfamily belongs to the Plasmodiidae family, specifically malaria pathogens (Plasmodium), and Leucocytozoon, which causes leucotetozone disease in chickens. ing.
[0006]
Coccidium infection is a problem in animals that are infected with the coccidium. In particular, care must be taken so that poultry and livestock do not develop coccidiosis. Here, coccidium infection simply refers to a state in which an animal is infected with coccidium, and coccidiosis refers to a case where a pathological symptom due to coccidium infection has occurred. Coccidiosis refers to coccidiosis caused by protozoa of Eimeriaidae in particular.
[0007]
In the case of coccidium that infects poultry, bacterial infections and mixed infections with coccidium infections occur in poultry, poultry deaths due to acute coccidiosis occur frequently, or digestive disorders due to chronic diseases Gain And the effect on egg laying is particularly problematic. Therefore, coccidium infection in poultry is a disease that is problematic from the viewpoint of poultry meat and egg productivity. Looking at the incidence of disease in 1997, the incidence of coccidiosis is about 47 in 1 million cattle and 202 in 10 million pigs. Particularly in the case of chickens, the incidence of coccidiosis is very high compared to other animals, and the incidence of coccidiosis is 1044 per 10 million.
[0008]
Coccidium forms a hard-shelled zygote called oocyst during the sexual reproduction of its life cycle. However, this oocyst shows very strong heat resistance and chemical resistance. Therefore, oocysts cannot be easily removed from the environment under the use of synthetic bactericides and heat sterilization conditions used for ordinary microorganism countermeasures.
[0009]
Coccidia belongs to protozoa, which are eukaryotes. Eukaryotes include animals, plants, mushrooms, and the like (basidiomycetes), generally fungi called molds and yeasts. Thus, since coccidium is a eukaryote, coccidium has different characteristics from bacteria and viruses. Specifically, the cellular structure and cellular constituents of coccidium are different from those of bacteria and viruses.
[0010]
Therefore, for the treatment of coccidiosis, antibiotics effective against prokaryotes, for example, antibiotics that inhibit bacterial cell wall synthesis, antibiotics that inhibit prokaryotic protein synthesis, and prokaryotic mRNA synthesis inhibitory action Antibiotics with can not be used. Therefore, the treatment of coccidiosis is performed using antibiotics or antibacterial or antiprotozoal agents effective against eukaryotes.
[0011]
However, administration of antibiotics or antibacterial or antiprotozoal agents effective against eukaryotes also damages the cells of host animals that are eukaryotes. In addition, the expected effect is often not obtained depending on the timing of administration, and side effects are also a problem (“chicken egg information”, Vol. 29, No. 6, pp. 30-33, March 25, 1999). Nikkan, “Clinical Veterinarian”, 16 (6), 92-94, 1998).
[0012]
Furthermore, administration of a coccidia preventive agent may produce coccidium resistant to the prophylactic agent. In addition, it is known that a coccidia preventive agent has a strong effect on a specific type of coccidium, but may not be effective on other coccidiums other than the specific type (“fowl farming” Tomo ", 439, 48-53, August 1998).
[0013]
Regarding mixed infection with coccidium and bacteria, the following two points are specifically problematic ("chicken egg information", Vol. 29, No. 6, pp. 30-33, March 25, 1999) .
[0014]
First, it is said that poultry infected with coccidium are likely to colonize food poisoning bacteria such as Salmonella, Clostridium perfringens (Clostridial), Campylobacter, Staphylococcus and Escherichia coli.
[0015]
Second, coccidial spores form in poultry when: That is, when clostridium and coccidium are mixed and infected in poultry, when the sulfa drug that is an antiprotozoal agent is given to poultry, the administration timing is incorrect, or when only sulfa drugs are given to poultry without noticing mixed infection is there. In particular, in the latter case, the spores formed in the poultry may proliferate significantly in the intestinal tract after completion of sulfa drug administration, and the formed spores may show high poultry mortality.
[0016]
Therefore, the amount and timing of administration of a combination of antibiotics against Clostridium and sulfa drugs against coccidium must be examined according to the mixed infection status of poultry. In any case, such mixed infections show higher poultry mortality compared to single infections.
[0017]
Most of the preventive / therapeutic agents for coccidiosis reported so far are chemical substances or antibiotics, and plant extracts, attenuated vaccines and the like are known.
[0018]
Known chemicals and antibiotics that are the main prophylactic and therapeutic agents for coccidiosis, such as sulfa drugs, organic arsenic compounds, diaminopyrimidine derivatives, carbanilides, nucleic acid-related substances, and polyether antibiotics. ("Poultry Friend", 437, 45-49, July 1998 issue).
[0019]
However, the following side effects have been reported for chemical substances or antibiotics that are preventive / therapeutic agents for coccidiosis.
[0020]
In the side effect report on animal drugs issued by the FDA Central Animal Pharmacy, 1996 side effect report on birds, monensin (polyether antibiotic), nicarbazine (carbanilide), Four cases of roxarson (organic arsenic compound) are coccidium preventive / therapeutic drugs ("Clinical Veterinarian", Vol. 16, No. 6, 1998).
[0021]
As a plant extract effective against coccidiosis, “a preventive and therapeutic agent for coccidiosis in poultry or livestock obtained by administering tissues or cells of Nichinitika (Asteraceae)” (Japanese Patent Laid-Open No. 50-101510), "Prophylactic and therapeutic agents for protozoan diseases of animals containing hydrangea plants" (JP 59-164726 A) have been reported. In addition, an antiprotozoal agent (Japanese Patent Laid-Open No. 2000-143523) using an alkaloid derived from a plant belonging to the genus Stefania belonging to the Rhododendron family has been reported. However, alkaloids derived from plants belonging to the genus Stefania belonging to the family Rhododendronaceae are targeted only for malaria parasites belonging to the subspecies Hemosporulina of Coccidia, and are not described as being effective against coccidiosis.
[0022]
Furthermore, an infection preventive agent containing an extract derived from sweet potato as an active ingredient has been reported (Japanese Patent Laid-Open No. 2000-297046). This publication describes that the extract derived from sweet potato has an effect on bacterial infections and viral infections. However, there is no description or suggestion that an extract derived from sweet potato is effective against infectious diseases caused by coccidium as a pathogen.
[0023]
Attenuated vaccines are used for the purpose of preventive treatment against coccidium. Regarding immunity of coccidium, chickens that were once infected with coccidium in the early 1900 are known to have some kind of immunity, and in the 1940s, the more infected oocysts the first time, It has been reported that the duration of immunity is long ("Poultry Friend", vol. 401, pp. 47-50, July 1995 issue).
[0024]
However, it is known that some avian viral diseases have a long-lasting ability to prevent infection once they have survived infection, and can maintain long immunity even with very mild infections caused by live vaccines.
[0025]
Comparing the administration effect of the vaccine against this avian viral disease and the administration effect of the vaccine against coccidium, it is known that the administration effect of the vaccine against coccidium is weak or the duration of the effect is short. The reason for this is that protozoa such as coccidium have different cell structures, morphology and infection mechanisms compared to viruses and bacteria, for example, or the immune mechanism between immunity against avian viruses and immunity against coccidium. It is thought to be different. Therefore, in the case of coccidiosis, it is necessary to administer a live vaccine at regular intervals, that is, frequently.
[0026]
Vaccine technology against protozoa such as coccidium has not yet been completed, and a satisfactory effective effect has not been achieved ("Farming Friends", vol. 401, pp. 47-50, 1995, 7). Monthly issue).
[0027]
In addition, when inactivated parasites, dead bodies, or antigens of coccidium are administered to a test animal, the antibody titer of the administered test animal increases, but it is known that it does not show a protective effect against subsequent infection. ing.
[0028]
Therefore, it is unclear whether a vaccine adjuvant that is effective for virus and bacterial vaccines and has an action to increase antibody titer has a similar adjuvant effect on coccidium vaccine.
[0029]
So far, an extract derived from sweet potato has been reported as an adjuvant for a plant-derived vaccine (Japanese Patent Laid-Open No. 2000-297046). In this publication, regarding the vaccine adjuvant effect, the extract derived from sweet potato enhances the action of the virus and bacterial vaccine as an antigen, the effect of increasing the antibody titer against the virus and bacterial vaccine, the effect of administering the virus and bacterial vaccine It has been described that it has the effect of increasing the. However, it is not described or suggested whether the extract derived from sweet potato has a similar vaccine adjuvant effect on the coccidial vaccine.
[0030]
[Problems to be solved by the invention]
As an alternative to antibacterial and antiprotozoal agents, prophylactic / therapeutic agents that prevent and treat coccidiosis and have an effect of suppressing oocyst excretion from infected individuals and are highly effective against human or animal diseases caused by coccidiosis are desired. Yes. In addition, there is a demand for an adjuvant agent for immunization of humans or animals against mild infection of coccidium that maintains the immunity to coccidium that can reduce the number of times of vaccine administration and is highly effective. These agents are preferably natural and safe, and are suitable for consumers.
[0031]
[Means for Solving the Problems]
As a result of intensive studies to solve the above-mentioned problems, the present inventors have found that a sweet potato-derived extract obtained by treating sweet candy that has been used as a food since ancient times prevents human or animal diseases caused by coccidium. It has been found that it exhibits a therapeutic effect and an adjuvant effect for immunization of humans or animals against mild infection with coccidium, and the present invention has been completed.
[0032]
One of the present invention relates to a prophylactic / therapeutic agent for human or animal diseases caused by coccidium, which contains an extract derived from sweet potato as an active ingredient, and also relates to a feed containing the agent. In particular, the preventive / therapeutic agent according to the present invention is effective when the animal is a poultry.
[0033]
Another aspect of the present invention relates to an adjuvant for immunization of humans or animals against coccidial mild infection, characterized by containing an extract derived from sweet potato as an active ingredient, and to a feed containing the agent. In particular, the adjuvant agent according to the present invention is effective when the coccidium infects poultry.
[0034]
The sweet potato-derived extract is preferably a fraction obtained by treating a raw material selected from sweet potato juice, a sweet potato solvent extract and molasses derived from sweet potato by column chromatography using a fixed carrier.
[0035]
In addition, an extract derived from sweet potato is adsorbed by the synthetic adsorbent by passing a raw material selected from sweet potato juice, sweet potato solvent extract and molasses derived from sweet potato through a column filled with a synthetic adsorbent as a fixed carrier. The fraction obtained by eluting the component with a solvent selected from water, methanol, ethanol and mixtures thereof is preferable.
[0036]
In addition, the extract derived from sweet potato is processed by column chromatography using the difference in affinity in a column packed with ion-exchange resin as a fixed carrier using a raw material selected from sweet potato juice, sweet potato solvent extract and molasses derived from sweet potato Among the fractions obtained by separation by the above, a fraction that absorbs light having a wavelength of 420 nm is preferable.
[0037]
The sweet potato-derived extract is preferably an extract obtained by extracting bagasse with an extraction solvent selected from the group consisting of water, ethanol, or a mixture thereof.
[0038]
DETAILED DESCRIPTION OF THE INVENTION
In the present invention, the term “coccidium” refers to the order of the coccidiaceae (Eucoccidiorida). More preferably, in the present invention, “coccidia” refers to coccidium contained in the order of coccidia, and includes, for example, Eimeriarina, Haemospororina. Here, the Eimeria subfamily includes the Eimeriaceae, Cryptosporidium, and Scorpiophyceae, and the Eimeriaidae includes, for example, chicken coccidium (Eimeria), and the Cryptosporidium family includes, for example, Cryptosporidium. , And Scorpiophyceae include, for example, sarcocystis (Sarcocystis) and the like. The Hemosporulina family includes the Plasmodiidae family, which includes, for example, malaria pathogens (Plasmodium) and Leucocytozoon pathogens. Even more preferably, in the present invention, “coccidium” refers to coccidium contained in the narrow sense of the order of the coccidial order Eimeriarina.
[0039]
In the present invention, “animal” refers to animals included in mammals and birds. Thus, for example, poultry, livestock and companion animals are included.
[0040]
In the present invention, “poultry” refers to all birds raised as livestock, and examples thereof include chickens, quails, ducks, turkeys, Aigamo, guinea fowls and ostriches.
[0041]
In the present invention, the “prophylactic and therapeutic effect against human or animal disease caused by coccidiosis” refers to the effect of suppressing the symptoms of human or animal disease caused by coccidiosis. For the effect of suppressing disease symptoms, for example, for humans or animals infected with coccidium, it exerts the effect of suppressing oocyst excretion, the effect of suppressing the progression of lesions, and the effect of suppressing immune decline during coccidium infection, As well as the effect of suppressing a decrease in human or animal weight gain per day after coccidium infection.
[0042]
In the present invention, “a prophylactic / therapeutic agent for human or animal disease caused by coccidium” refers to an agent having a preventive / therapeutic effect on the human or animal disease caused by coccidium (hereinafter referred to as “prevention according to the present invention”). Sometimes referred to as “therapeutic agent”).
[0043]
In the present invention, “coccidial mild infection” means that when coccidium infects humans or animals, the infected individual has little or no pathological symptoms such as bloody stool or weight loss. An infection that shows some symptoms. In the present invention, “mild infection with coccidium” and “mild infection with coccidium” are synonymous and are not particularly distinguished.
[0044]
When this coccidial mild infection occurs artificially in poultry, there are differences depending on the species or strain of coccidium, the storage status of the strain, or the age or strain of the poultry. Eimeria tenella It means infection when 5,000 or less oocysts of (NIAH strain) are inoculated per poultry.
[0045]
In Example 3 to be described later, in the case of a chicken infected with 2,000 oocysts, symptoms such as bloody stool and weight loss observed in a normal attack infection were not observed, and 100,000 / Acquires immunity and becomes resistant to attack infection by feather oocysts. From this, the mild infection of coccidium shows the same usefulness as a vaccine.
[0046]
Meanwhile, 2 to 50,000 coccidium per two-week-old chicken Eimeria tenella Infection with oocysts of A. can induce acute coccidiosis, depending on the strain of coccidium or the age or strain of the poultry. Symptoms at this time are, for example, persistence of bloody stool, excretion of oocysts, and weight loss. When infected with 100,000 oocysts, the symptoms worsen and the mortality rate increases.
[0047]
In the present invention, “adjuvant effect for immunization of human or animal against mild infection of coccidium” means not only to increase the antibody titer against antigen but also immune response of human or animal against minor infection of coccidium and other immunity By promoting or activating the mechanism or stimulating immunity, it includes the effect of preventing the next infection against coccidium. In the present invention, “mild infection with coccidium” includes mild infection with coccidium in addition to infection with administration of coccidium vaccine. Therefore, in the present invention, “adjuvant effect for immunization of human or animal against mild infection of coccidium” includes “adjuvant effect for coccidium vaccine”.
[0048]
In the present invention, the term “adjuvant agent for immunization of human or animal against mild infection of coccidium” refers to an agent having an adjuvant effect for immunization of human or animal against mild infection of coccidium (hereinafter referred to as “the present invention”). Sometimes referred to as “adjuvant agent”).
[0049]
In the present invention, “an extract derived from sweet potato” refers to an extract obtained from sweet potato as a raw material.
[0050]
In one embodiment, the extract derived from sweet potato is processed by column chromatography using a fixed carrier for a raw material selected from sweet potato juice, a sweet potato solvent extract and molasses derived from sweet potato (hereinafter, simply referred to as a raw material). This is the fraction obtained. More preferably, the raw material is passed through a column packed with a synthetic adsorbent as a fixed carrier, and the component adsorbed on the synthetic adsorbent is selected from the group consisting of water, methanol, ethanol and mixtures thereof. Among the fractions obtained by elution with the elution solvent, or the fractions obtained by separating the raw material by column chromatography using the difference in affinity in a column packed with an ion exchange resin as a fixed carrier The fraction that absorbs light having a wavelength of 420 nm.
[0051]
Alternatively, in another embodiment, the sweet potato-derived extract is an extract obtained by extracting bagasse derived from sweet potato with a solution selected from water, a hydrophilic solvent, and a mixture thereof, and more preferably, It is an extract obtained by extracting bagasse, which is a residue obtained by pressing sugar juice from sweet potato, with a solvent selected from water, a hydrophilic solvent and a mixture thereof.
[0052]
In the present invention, “sweet potato soup” refers to a squeezed juice obtained by squeezing sugar cane, a brewed juice obtained by leaching sweet potato with water, or a lime-treated clean juice or concentrated juice in a raw sugar manufacturing factory. Include.
[0053]
In the present invention, the “sweet potato solvent extract” means an extract obtained by concentrating and drying an extract obtained by extracting sweet potato with a general-purpose organic solvent, and then re-dissolving in water. Examples of the organic solvent directly above include alcohols such as methanol and ethanol, and these may be used alone or in combination. Further, these solvents and water may be used in combination.
[0054]
In the present invention, “sugar cane derived from sweet potato” means treacle obtained by centrifuging a mixture of sugar crystals and mother liquor obtained in the crystallization process and separating them from sugar crystals, for example, a raw sugar production plant No. 1 honey, No. 2 honey, sugar-making waste nectar, and sugar-washed nectar, 1-7 th. Moreover, the thing which desugared molasses like the isolation | separation liquid which performed alcoholic fermentation using these molasses as a raw material can be used similarly.
[0055]
Bagasse is typically bagasse discharged during the sugar production process in a raw sugar factory. In addition, the bagasse discharged | emitted in the sugar manufacturing process in a raw sugar factory contains not only the final bagasse which came out of the final press machine but the shredded sweet potato bitten by the subsequent press machine containing a 1st press machine. Preferably, bagasse discharged after squeezing the sugar juice in the raw sugar factory in the pressing step is used. The bagasse discharged after squeezing the sugar juice from the pressing step has different moisture content, sugar content and composition ratio depending on the type of sweet potato, harvest time, etc., but in the present invention, these bagasse Can be used arbitrarily. Moreover, you may use the bagasse which remains after the sweet potato press discharged | emitted, for example in a brown sugar manufacturing factory similarly to a raw sugar factory. Alternatively, in a small-scale implementation at the laboratory level, bagasse after squeezing sugar liquid from sweet potato may be used.
[0056]
Specifically, such “sweet potato-derived extract” can be obtained, for example, as follows.
[0057]
First, a method for obtaining “an extract derived from sweet potato” by column chromatography will be described.
[0058]
The sweet potato extract or molasses derived from sweet potato is passed through a column using a fixed carrier. The sweet potato extract or molasses can be passed through a column packed with a fixed carrier as it is or after adjusting to an arbitrary concentration with water. In order to remove foreign substances, it is desirable to filter the sweet potato extract or the molasses derived from sweet potato before processing with the column. The filtration method is not particularly limited, and means such as screen filtration, diatomaceous earth filtration, microfiltration, and ultrafiltration that are widely used in the food industry can be preferably used.
[0059]
As the fixed carrier, a synthetic adsorbent is preferable. As the synthetic adsorbent, an organic resin can be preferably used. For example, an aromatic resin, an acrylic acid-based methacrylic resin, an acrylonitrile aliphatic resin, or the like can be used. An aromatic resin is more preferable, and an unsubstituted aromatic resin can be used. As the synthetic adsorbent, for example, an aromatic resin such as a styrene-divinylbenzene resin can be used. Examples of the aromatic resin include an aromatic resin having a hydrophobic substituent and an unsubstituted aromatic resin. A porous resin such as an aromatic resin obtained by subjecting an unsubstituted group to a special treatment can be used. More preferably, an aromatic resin obtained by subjecting an unsubstituted type to a special treatment can be used. Such synthetic adsorbents are commercially available, for example, HP-10, HP-20, HP-21, HP-30, HP-40, HP-50 (Diaion (trademark) system) SP-825, SP-800, SP-850, SP-875, SP-70, SP-700 (above, specially for the non-substituent group type) Treated aromatic resins, trade names, manufactured by Mitsubishi Chemical Corporation); SP-900 (aromatic resins, trade names, manufactured by Mitsubishi Chemical Corporation); XAD-2 as Amberlite (trademark) , XAD-4, XAD-16, XAD-2000 (above, aromatic resins, all trade names, manufactured by Organo Corporation); Diaion (trademark) system, SP-205, SP-206, SP-207 (End of hydrophobicity Aromatic resins having substituents, all trade names, manufactured by Mitsubishi Chemical Corporation; HP-2MG, EX-0021 (above, aromatic resins having hydrophobic substituents, both trade names, Mitsubishi Chemical Corporation) Company made); Amberlite (trademark) system, XAD-7, XAD-8 (above, acrylic ester resin, both trade names, manufactured by Organo Corporation); Diaion (trademark) system, HP1MG, HP2MG (The above are acrylic acid-based methacrylic resins, both are trade names, manufactured by Mitsubishi Chemical Corporation); Sephadex (trademark) series is LH20, LH60 (all are derivatives of crosslinked dextran, both are trade names, manufactured by Pharmacia Biotech Co., Ltd.) Etc. Among these, SP-850 is particularly preferable.
[0060]
The amount of the fixed carrier varies depending on the size of the column, the type of solvent, the type of fixed carrier, and the like. A wet volume of 0.01 to 5 times the solid content of the raw material (selected from sweet potato juice, sweet potato solvent extract and molasses) is preferred.
[0061]
By passing the raw material (selected from sweet potato juice, sweet potato solvent extract and molasses derived from sweet potato) through the column, the component having the effect of the present invention in the raw material is adsorbed to the fixed carrier, and sucrose, glucose, fructose and Most of the inorganic salts flow out.
[0062]
The component adsorbed on the fixed carrier is eluted with the elution solvent. Here, in order to efficiently elute the component having the effect of the present invention, it is preferable to sufficiently wash away residual sucrose, glucose, fructose and inorganic salts before washing. Thereby, the component which has the target effect adsorbed | sucked can be collect | recovered more efficiently.
[0063]
The elution solvent is selected from water, methanol, ethanol and a mixture thereof. The elution solvent is preferably a mixed solvent of water and alcohol, particularly an ethanol-water mixed solvent. Further, since the components having the desired effect can be efficiently eluted at room temperature, 50 / 50-60 / 40 (volume / volume) ethanol- A water mixed solvent is preferred. Furthermore, by raising the column temperature, the ethanol mixing ratio of the ethanol-water mixed solvent can be reduced, and the target component having the effect of the present invention can be eluted. In this case, the inside of the column is at normal pressure or under pressure. Thus, the component which has the effect of this invention exists in the fraction eluted with the said solvent.
[0064]
The elution rate is not particularly limited because it varies depending on the size of the column, the type of solvent, the type of fixed carrier, and the like, but SV = 0.1-10 is preferable. In addition, SV (Space Velocity, space velocity) is a unit of how many times the volume of the resin is passed per hour.
[0065]
In the method for obtaining “sweet potato-derived extract” by column chromatography, preferred embodiments in the case of using an ion exchange resin as a fixed carrier are as follows.
[0066]
Ion exchange resins are classified into cation exchange resins and anion exchange resins from the viewpoint of ion exchange properties. In the present invention, cation exchange resins can be preferably used. More preferably, a strongly acidic cation exchange resin can be used. More preferably, a strongly acidic sodium ion type or potassium ion type cation exchange resin can be used. The ion exchange resin is classified into a gel type resin and a porous resin such as a porous type, a microporous type, or a high porous type from the viewpoint of the form of the resin. In the present invention, the gel type ion exchange resin is preferably used. Can be used. More preferably, a strongly acidic sodium ion type or potassium ion type cation exchange resin can be used. Such ion exchange resins are commercially available. For example, as diaion (trademark) series, SK1B, SK104, SK110, SK112, SK116 (above, manufactured by Mitsubishi Chemical Corporation), UBK530, UBK550 (above, manufactured by Mitsubishi Chemical Corporation) ), Amberlite (trademark) series as Amberlite IR120BN, IR124, XT1006, IR118, Amberlist 31, Amberlite CG120 for chromatograph, CG6000 (above, manufactured by Organo Corporation), Dowex (trademark) series as HCR- S, HCR-W2, HGR-W2, Monosphere 650C, Marathon C600, 50W × 2, 50W × 4, 50W × 8 (above, manufactured by Dow Chemical Japan Co., Ltd.), Muromac 50WX (manufactured by Muromachi Chemical Industry Co., Ltd.) , Purolite (quotient ) As a system, C-100E, C-100, C-100 × 10, C-120E, PCR433, PCR563K, PCR822, PCR833, PCR866, PCR883, PCR892, PCR945 (above, made by AMP IONEX Co., Ltd.) and the like can be mentioned. . Of these, the UBK series is particularly preferable.
[0067]
The amount of the fixed carrier varies depending on the column size, the type of the fixed carrier, and the like. The amount of the fixed carrier is preferably 2 to 10,000 times, more preferably 5 to 500 times the wet volume of the solid content of the raw material.
[0068]
The raw material is passed through the column and then column chromatographed using water as the eluent. By extracting a fraction that absorbs light having a wavelength of 420 nm from among the many fractions obtained subsequently, the target sweet potato-derived extract can be obtained. Hereinafter, this method is sometimes referred to as ion chromatography separation.
[0069]
Liquid passing conditions vary depending on the composition of the raw materials and the type of the fixed carrier. In the single tower batch separation method, degassed water is used as an eluent, and the flow rate is SV = 0.3 to 1.0 hr. ^-1 The sample supply amount is preferably 1 to 20% of the resin as the liquid amount, and the temperature is preferably 40 to 70 ° C. Each fraction obtained by this separation method is analyzed for absorption at a wavelength of 420 nm, electrical conductivity (a measure of the amount of salt), sucrose, glucose and fructose concentrations. When the values of the respective fractions are represented in a graph in time series, peaks appear in the order of absorbance peak at a wavelength of 420 nm, electrical conductivity peak, sucrose and reducing sugar. Fractions corresponding to fractions 3 to 14 in FIG. 2 to be described later are collected as fractions that absorb light having a wavelength of 420 nm. Particularly preferred are fractions corresponding to fractions 3-8. In addition, for the fraction corresponding to fractions 18 to 30 after sucrose has come out, although the absorbance at a wavelength of 420 nm measured using the fraction as it is is low, for the sample concentrated together, Like the sample 8 of Production Example 2 described later, the absorbance is high. That is, even if the fraction corresponding to fractions 18 to 30 has a low concentration, the purity of the active ingredient per solid content is relatively high. Therefore, the whole non-sucrose fraction (corresponding to the sum of fractions 1-9 and fractions 18-30) has a lower concentration of active ingredient than the fractions corresponding to fractions 3-8, but the same It can be used as an extract derived from sweet potato referred to in the present invention. In addition, the fraction that absorbs light having a wavelength of 420 nm can be further subjected to electrodialysis treatment to reduce the salt content.
[0070]
In addition, when the column chromatography treatment is a simulated moving bed type continuous separation method, in order to set the raw material liquid supply amount, the eluent flow rate, the flow rate of each fraction extraction according to the composition of the raw material, the type of fixed carrier, the amount of resin, General liquid passage conditions cannot be shown.
[0071]
The composition of this fraction obtained by the simulated moving bed type continuous separation method using the second honey at the raw sugar factory varies depending on the type of raw material and the separation capacity of the ion exchange resin, but the sucrose per solid content is 6%. Hereinafter, the non-sugar content is 90% or more, and the apparent pure sugar ratio is 10%. The apparent pure sugar rate is a percentage of the sugar content (direct optical rotation with respect to the specified amount of pure sucrose measured with a saccharimeter) with respect to the Brix (Bx.) Degree (solid percentage measured with a Brix meter).
[0072]
In addition, the extract derived from sweet potato according to the present invention obtained by the single-column batch separation method has a polyphenol content of about 5 per solid (freeze-dried solid) in the case of a fraction obtained from No. 2 honey. % (In terms of catechin), electric conductivity ash (salt) is about 44.7%, and sugar is about 5%.
[0073]
Furthermore, other embodiments of the sweet potato-derived extract can be obtained by extracting bagasse.
[0074]
By extracting bagasse with a solvent selected from the group consisting of water, hydrophilic solvents and mixtures thereof, one of the sweet potato-derived extracts referred to in the present invention (hereinafter sometimes referred to as “bagasse extract”) Is obtained. As the hydrophilic solvent, for example, lower alcohols such as methanol and ethanol, ketones such as acetone, and acetates such as methyl acetate and ethyl acetate can be used. As the hydrophilic solvent, ethanol is particularly preferable. A preferred solvent for extraction is an ethanol-water mixed solvent containing a specific ethanol ratio of 60/40 volume ratio or less, more preferably 50/50 volume ratio or less. The extraction temperature is preferably 50 to 100 ° C. in order to efficiently extract the target component. The extraction time varies depending on the bagasse raw material, type, state, etc., but is usually 1 to 3 hours. The extraction method can be a general versatile method, for example, a method of extracting bagasse and an extraction solvent in a container, a method of extracting by circulating the extraction solvent, a method of continuous extraction, for example, A Desmet extractor, a Lurgi extractor, and the like can be arbitrarily used. Since the bagasse extract has a high sugar content, the sugar may be removed by subjecting the bagasse extract to a column chromatography similar to that described above.
[0075]
The fractions obtained by the above column chromatography or the bagasse extract are collected and concentrated by conventional means (solvent removal under reduced pressure, freeze-drying, etc.) to obtain the sweet potato-derived extract as used in the present invention. be able to.
[0076]
The shape of the “sweet potato-derived extract” of the present invention is not particularly limited, but may be liquid or powder, or may be a solid preparation or a liquid preparation using a commonly used preparation carrier. These preparation methods are known. The “sweet potato-derived extract” thus obtained can be stored in liquid or powder form. The storage is preferably refrigerated storage, particularly when it is liquid.
[0077]
Examples of the liquid sweet potato-derived extract include the sweet potato-derived extract of Production Example 1 described later. In this case, the extract derived from sweet potato is preferably concentrated to a solid content of 20% or more from the viewpoint of preventing spoilage. The storage is preferably refrigerated storage.
[0078]
Examples of the sweet potato-derived extract that is a powder include a sweet potato-derived extract obtained by converting the liquid sweet potato-derived extract into a powder form by a known method. For example, as a known method, for example, a spray drying method, a freeze drying method, a fluidized bed granulation method, or a powdering method using an excipient can be used.
[0079]
Since the “sweet potato-derived extract” is derived from a natural product, its analytical value differs depending on the raw material of the sweet potato, the production area of the sweet potato, the climate, and the production method of the extract. For example, one analytical value of the sweet potato-derived extract of Production Example 1 described below is as follows.
[0080]
The solid content is 37.4% by weight, Brix (Bx.) Is 40.6, and the sugar content is 9.8% per solid content (breakdown: sucrose 6.8%, glucose 1.4%, fructose 1.6%) The polyphenol content is 5.0% per solid content.
[0081]
In addition, solid content was measured with the atmospheric pressure heating drying method, and Brix was measured with the Brix meter. The sugar content (sucrose, glucose, and fructose content) determined by high performance liquid chromatography (HPLC) analysis was shown as a ratio (%) to the solid content weight of each sample. The polyphenol content was measured by a forin-thiocult method in which a calibration curve was drawn using a catechin aqueous solution as a standard solution and reacted with a phenol reagent to measure the absorbance at a wavelength of 765 nm, and was shown as a value in terms of catechin.
[0082]
By administering the preventive and therapeutic agent according to the present invention to an animal (for example, oral administration), the effect of suppressing the excretion of coccidium oocysts can be exerted, and the progression of lesions can also be suppressed. Moreover, while suppressing the suppression of the body weight increase rate by coccidial infection, the suppression of coccidium growth in the cecum and the suppression and reduction effect of cecal lesion formation can be recognized. Furthermore, since the disease caused by coccidium can be prevented and treated, propagation to other chickens can be suppressed, and infection of coccidium to other chickens can be prevented.
[0083]
The preventive / therapeutic agent according to the present invention is particularly effective when the animal is a poultry.
[0084]
The preventive / therapeutic agent according to the present invention includes “an extract derived from sweet potato”, and when the agent is used as a solid preparation, for example, corn starch or defatted rice bran may be mixed with an extract derived from liquid sweet potato. These preparation methods are known.
[0085]
The administration time of the prophylactic and therapeutic agent according to the present invention is not particularly limited.
[0086]
The dose of the prophylactic / therapeutic agent according to the present invention is not particularly limited because it varies depending on the degree of purification and form of the “sweet potato-derived extract”, the type of animal, the health condition, the degree of growth, and the like. For example, when the sweet potato-derived extract described in Production Example 1 described below is used as an agent containing a sweet potato-derived extract, 1 to 1,000 mg is administered per kg of body weight per kg of body weight as a solid content excluding monosaccharides and oligosaccharides. do it.
[0087]
The administration form of the prophylactic / therapeutic agent according to the present invention is not particularly limited. For example, it can be administered by oral, intravenous, intramuscular, subcutaneous, intraperitoneal, intrarectal, sublingual and transdermal methods.
[0088]
The preventive / therapeutic agent according to the present invention includes ingredients contained in sweet candy and honey-containing sugar that have been eaten by humans since ancient times. is there. In addition, sweet potato-derived extracts are natural because they are derived from plants.
[0089]
The adjuvant agent according to the present invention exerts an effect as an adjuvant agent for immunization of human or animal against mild infection of coccidium or coccidium vaccine administration by administering to human or animal (for example, oral administration).
[0090]
The adjuvant agent according to the present invention is particularly effective when coccidium infects poultry.
[0091]
The adjuvant agent according to the present invention contains “an extract derived from sweet potato”, and when the preparation is used as a solid preparation, for example, corn starch or defatted rice bran may be mixed with an extract derived from liquid sweet potato. These preparation methods are known.
[0092]
The administration timing of the adjuvant according to the present invention is not particularly limited. For example, it may be before the mild infection, on the same day as the mild infection, or after the mild infection. In addition, a more certain effect can be expected by administering for several days before and after infection including the same day as mild infection.
[0093]
The frequency of administration of the adjuvant according to the present invention may be one or more. About 1 day to 1 week before mild infection and about 1 to 2 weeks after mild infection, or before mild infection or mild infection. It is preferable to administer continuously or intermittently in any later period. Further, it may be administered continuously for 1 to 6 months.
[0094]
In addition, natural infection of coccidium to chickens is usually orally transmitted. Therefore, coccidiosis co-infection does not suddenly occur in chickens. However, there is a cycle in which coccidiose oocysts that do not cause disease are present in poultry houses, several chickens are mildly infected, and coccididium oocysts grow in the intestinal tract of chickens and are subsequently excreted together with feces. If it continues for a certain period, a large amount of coccidial oocysts will be present in the poultry house. This condition becomes a pathogen for chickens, resulting in the development of coccidiosis. Therefore, by administering the adjuvant agent according to the present invention to chickens in the mild infection state before the onset of disease, the adjuvant effect on the mild infection that occurs naturally regardless of the vaccine administration is exerted, and the chicken does not cause the disease. There is an effect. In the case of chickens, the coccidium vaccine is administered orally.
[0095]
The dosage of the adjuvant according to the present invention is not particularly limited because it varies depending on the purity, form, type of animal, health condition, degree of growth, etc. of the extract derived from sweet potato. For example, when the sweet potato-derived extract described in Production Example 1 described later is used as the sweet potato-derived extract, 1 to 1,000 mg per day per kg of body weight may be administered as a solid content excluding monosaccharides and oligosaccharides. .
[0096]
The administration form of the adjuvant according to the present invention is not particularly limited, and can be administered by, for example, oral, intravenous, intramuscular, subcutaneous, intraperitoneal, intrarectal, sublingual and transdermal methods. In particular, in the case of poultry, oral administration is preferred, and the adjuvant according to the present invention may be administered together with food.
[0097]
Since the adjuvant according to the present invention contains ingredients contained in sweet potato and honey-containing sugar that have been eaten by humans since ancient times, it is safe with food experience and low cost because it uses by-products in the production process of sucrose. . In addition, sweet potato-derived extracts are natural because they are derived from plants.
[0098]
The present invention is a feed containing a prophylactic / therapeutic agent for human or animal diseases caused by coccidium, characterized by using an extract derived from sweet potato as an active ingredient (hereinafter referred to as “a feed containing a prophylactic / therapeutic agent according to the present invention”). Provide). The present invention also includes a feed containing an adjuvant for immunization of humans or animals against mild coccidiosis, characterized by comprising an extract derived from sweet potato as an active ingredient (hereinafter referred to as “feed containing an adjuvant according to the present invention”). ”).
[0099]
The form of the feed containing the preventive / therapeutic agent according to the present invention or the feed containing the adjuvant according to the present invention may be either solid or liquid. Examples of feed applications include poultry, livestock, and companion animals.
[0100]
In the present invention, “feed” means all animals orally ingested for nutritional purposes. Specifically, when classified from the aspect of nutrient content, it includes all of roughage, concentrated feed, inorganic feed, and special feed, and from the aspect of official standards, it includes all of mixed feed, mixed feed, and simple feed. Include. Moreover, when classifying from the aspect of a feeding method, it includes all feeds that are fed directly, feeds that are mixed with other feeds, or feeds that are added to drinking water to replenish nutrients.
[0101]
【Example】
EXAMPLES Hereinafter, the present invention will be described more specifically with reference to examples. However, the present invention is not limited to the contents described in the examples.
[0102]
In the description relating to the dose of sweet potato-derived extract (hereinafter sometimes simply referred to as “extract”) used in the examples, for example, “500 mg / kg” or “500 mg / kg body weight” is derived from 500 mg of sweet potato per kg body weight. Means that the extract was administered.
[0103]
Production Example 1
In the raw sugar factory, ion exchange column chromatography separation was performed using a simulated moving bed type continuous separation method using a separation column packed with a cation exchange resin. As a raw material, sucrose crystals were collected twice with a crystal can, and second honey (Brix (Bx.) Is about 85), which was a honey removed from the crystals by centrifugation.
[0104]
Second honey was diluted to about 50 Brix. To this diluted second nectar, slaked lime and sodium carbonate were added to agglomerate impurities, followed by diatomaceous earth filtration. The analysis values of the obtained filtrate were Brix 47.3, sugar content (Pol.) 23.6, purity rate (Purity) 49.9, and reducing sugar content 2.5%. Using this filtrate as a raw material, ion exchange chromatography by a simulated moving bed type continuous separation method was performed.
[0105]
In addition, since the process from the preparation of the raw material to the ion exchange column chromatography separation is continuously performed, the solid content concentration and composition of the liquid in each process slightly vary with time. Therefore, the following concentrations and compositions showed measured values in steady operation.
[0106]
UBK530 (Mitsubishi Chemical Corporation) was used as a cation exchange resin. The separation tower filled with the resin is divided into 8 parts, and the amount of resin per tower is 6.5 m. ^Three It is. By supplying the raw material liquid and the eluent (water), and switching the extraction position of the sucrose fraction and the non-sucrose fraction at regular intervals, the supply and extraction were performed continuously. The default value for regular operation is a supply flow rate of 3 m. ^Three / Hour, elution water flow 13.5m ^Three / Hour, non-sucrose fraction extraction flow rate 12.13m ^Three / Hour, sucrose fraction extraction flow rate 4.37m ^Three / Hour, switching time was 267 seconds. By this chromatography treatment, a sucrose fraction and a non-sucrose fraction were separated. These correspond to the fractions 10 to 17 in FIG. 2, which will be described later, and the fractions 1 to 9 and the fractions 18 to 30 combined.
[0107]
The sucrose fraction is about 87% sucrose by solids (by HPLC analysis) and the Brix is about 35. This fraction is mixed with clean juice and returned to the sugarmaking process, and sucrose is recovered again.
[0108]
The obtained non-sucrose fraction had sucrose of about 0.3% per solid (by HPLC analysis) and brix of about 8. The non-sucrose fraction was concentrated with a concentrating can to give Brix 40.0, sugar content (Pol.) 2.3, pure sugar ratio (Purity) 5.8, and reducing sugar content 5.4%. This non-sucrose fraction was subjected to lyophilization treatment overnight for use in subsequent tests (hereinafter sometimes referred to as “an extract derived from sweet potato of Production Example 1”).
[0109]
The obtained lyophilized powder (0.25 g) was diluted to 100 ml with 0.5 mM phosphate buffer (pH 7.5), and the absorbance was measured. Absorbance at a wavelength of 420 nm was 1.11.
[0110]
Production Example 2
Using the second honey treatment liquid obtained at the raw sugar factory as a raw material, ion-exchange column chromatography treatment by a single tower batch separation method was performed. The 2nd honey treatment liquid was used as a raw material.
[0111]
The second honey treatment liquid is obtained by diluting the second honey, followed by a cleaning treatment with sodium carbonate and diatomaceous earth filtration. The analytical values of this raw material solution were Brix 47.4, sugar content (Pol.) 23.2, pure sugar rate (Purity) 48.9, and reducing sugar content 3.2%.
[0112]
As a single-column batch separation method, fraction separation was performed using an FPLC system (manufactured by Pharmacia).
[0113]
A gel type strongly acidic cation exchange resin UBK530 and sodium ion type (trademark, Mitsubishi Chemical Corporation) 500 ml were packed in a column. The column has an inner diameter of 26 mm, a height of 1,000 mm, and a flow adapter. The flow-through uses degassed distilled water as an eluent, and a flow rate SV = 0.5 hr. ^-1 (4.17 ml / min) at a temperature of 60 ° C.
[0114]
About 25 ml of raw material was donated to the column. As for the fractionation conditions, collection of the eluate was started 30 minutes after the supply of the raw material, and 3.6 minutes (about 15 ml / tube) was collected per test tube, and a total of 30 tubes were collected.
[0115]
The obtained 30 fractions were measured for absorbance at a wavelength of 420 nm, electrical conductivity, and sugar concentration, and are shown in FIG. Here, for absorbance measurement, 0.1 ml of each fraction was added to 2 ml of 0.5 mM phosphate buffer (pH 7.5) to prepare a sample. For the measurement of electrical conductivity, each fraction was diluted to 0.5% with distilled water and used as a sample. Sugar concentration was measured by HPLC.
[0116]
In order to analyze each peak, the absorption peak portion at a wavelength of 420 nm was combined into four, the sucrose peak portion into three, and the sucrose peak and subsequent portions into one. That is, fractions 3 and 4 are combined into sample 1, fractions 5 and 6 are combined into sample 2, fractions 7 and 8 are combined into sample 3, fractions 9 and 10 are combined into sample 4, Fractions 11 and 12 are combined into sample 5, fractions 13 and 14 are combined into sample 6, fractions 15 and 16 are combined into sample 7, and fractions 17-30 are combined into sample 8. . Fractions 1 and 2 were discarded because there was almost no eluted component. Each sample was lyophilized overnight to form a powder.
[0117]
The absorbance was measured at a wavelength of 420 nm by dissolving 0.25 g of the obtained lyophilized powder in 0.5 mM phosphate buffer (pH 7.5) to 100 ml. Further, the distribution ratio of freeze-dried solids, conductivity ash (salt), sucrose, glucose and fructose content, and polyphenol content were measured.
[0118]
The conductivity ash was calculated by obtaining a coefficient from a calibration curve of the relationship between electrical conductivity and known sulfate ash. The sucrose, glucose, and fructose contents were determined by high performance liquid chromatography (HPLC) analysis, and the ratio (%) to the solid content weight of each sample was shown. The polyphenol content was measured by a forin-thiocult method in which a calibration curve was drawn using a catechin aqueous solution as a standard solution and reacted with a phenol reagent to measure the absorbance at a wavelength of 765 nm, and was shown as a value in terms of catechin. The lyophilized solids distribution ratio is the ratio (%) of the solids weight of each sample to the total solids weight of all samples.
[0119]
The analysis results of each sample are shown in Table 1.
[0120]
[Table 1]
Table 1 Analytical values of each sample (absorbance, electrical conductivity, sucrose, glucose, fructose)

[0121]
The absorbance of sample 8 was relatively high at 0.86. The reason is that the other sample is a combination of two fractions, while the sample 8 is a combination of 14 fractions. That is, the sample 8 is obtained by collecting and concentrating tailing components. Therefore, although the absorbance at 420 nm is high, it is inefficient to collect only this fraction as a fraction having the effects of the present invention.
[0122]
From the sugar content, it can be seen that Samples 1 to 3 and 8 correspond to the non-sucrose fraction, and Samples 4 to 7 correspond to the sucrose fraction.
[0123]
Example 1
Using the sweet potato-derived extract (freeze-dried product) of Production Example 1, Eimeria tenella The changes in the pathology after oocyst infection were examined.
[0124]
An extract derived from sweet potato (500 mg / kg body weight) was administered every day for 3 days into 7 sac of two-week-old inbred chickens (genotype, H.B15 strain). In addition, on the same day as the final administration date, the pathogenic protozoa of chicken coccidiosis Eimeria tenella (NIAH strain) oocyst (20 × 10) ^Three Individual / feather) was inoculated into the sac (hereinafter referred to as “extract administration group”).
[0125]
In addition, physiological saline was administered into the sac of five chicks of the same age and the same kind in the same manner as the extract administration group instead of the extract derived from sweet potato. Furthermore, on the same day as the final administration date, oocysts (20 × 10 ^Three Pieces / feather) were inoculated into the sac (hereinafter referred to as “control group”).
[0126]
The effect of sweet potato-derived extract on oocyst infection (inoculation with oocysts) was evaluated using as an index the symptoms one week after infection, the number of deaths, weight gain per day, the presence or absence of bloody stool, and the severity of cecal lesions.
[0127]
The severity of cecal lesions was evaluated according to the method of Johnson and Reid and expressed as the average lesion score (Johnson, J. and Reid, WM Anticoccidial drugs: lesion scoring techniques in battery and floor-pen experiments with chickens. Exp Parasitol., 28: 30-36, 1970; Conway, DP Examination of lesions and lesion scoring. In Poultry coccidiosis, Diagnostic and testing procedures, edited by Donal P. Conway, 1970, 17-36, Pfizer International Inc., New York, NY 10017, USA).
[0128]
Table 2 shows the results.
[0129]
[Table 2]
Table 2 Eineria tenella Number of moribund deaths, average weight gain per day, persistence of bloody stool and mean cecal lesion score in oocyst-infected chickens

[0130]
Regarding the number of moribunds, one case was drowned on the third day after the oocyst infection in the control group, but no moribund case was observed in the extract administration group.
[0131]
Regarding the average weight gain per day in one week after infection, the average was 7.1 g / day in the control group, but 8.3 g / day in the extract administration group. From this, the weight gain per day in the week after infection in the extract administration group was higher than that in the control group.
[0132]
Concerning the persistence of bloody stool, the control group observed bloody stool abundantly at 3 to 6 days after infection, and sustained high frequency (++), but the extract administration group had mild transient 3 to 4 days after infection. Only bloody stool (+) was observed.
[0133]
In addition, the cecum was visually observed at the time of necropsy on the 7th day after infection. In the control group, the cecum lacked elasticity and swelled, with thin walls and dark red hemorrhagic lesions, with an average score of +3.8 for the lesions. On the other hand, in the extract-administered group, the cecum had elasticity, almost no bleeding lesions were observed, and the average score of the lesions was +2.6.
[0134]
Therefore, Eimeria tenella Administration of a sweet potato-derived extract into the sac before infection of oocysts in the body can suppress a decrease in weight gain per day after infection, and reduction or suppression of blood excretion and caecal lesions is observed. It was. From these, the sweet potato-derived extract according to Production Example 1 has a growth promoting action and Eimeria tenella It was shown to have a growth inhibitory effect.
[0135]
Example 2
Using the sweet potato-derived extract (freeze-dried product) of Production Example 1, Eimeria tenella The changes in the pathology after oocyst infection were examined.
[0136]
Two lines of 2-week-old inbred chicks (genotype, 24 H.B15 lines and 12 H.B2 lines) were used. In the sac, an extract derived from sweet potato (500 mg / kg body weight) was administered daily for 1 day or 3 days using a Komagome pipette. In addition, on the same day as the final administration date, the pathogenic protozoa of chicken coccidiosis Eimeria tennela (NIAH strain) oocyst (20 × 10) ^Three 1 piece / feather) was inoculated into the sac (hereinafter referred to as “extract administration group” when administered for 1 day and “extract administration group of 3 times” when administered for 3 days).
[0137]
In addition, physiological saline was administered into the sac of chicks of the same age and the same kind in the same manner as in the extract 1 administration group and the extract 3 administration group instead of the sweet potato-derived extract. Furthermore, on the same day as the last day, oocysts (20 × 10 ^Three Pieces / feather) were inoculated into the sac (hereinafter referred to as “control group”).
[0138]
Furthermore, chicks of the same age / same species were subjected to the same experiment without administration of sweet potato-derived extract or oocyst inoculation (hereinafter referred to as “untreated control group”).
[0139]
The effects of sweet potato-derived extract on oocyst infection (inoculation with oocysts) are based on symptoms such as symptom one week after oocyst infection, number of deaths, weight gain per day, presence or absence of bloody stool, number of oocysts in stool, and severity of cecal lesions evaluated.
[0140]
The number of oocysts in the stool was calculated by a method using a plankton calculation board, and displayed as the number of excretion per bird. The severity of cecal lesions was evaluated according to the method of Johonson and Reid described above and displayed as an average lesion score. The histopathological search for the lesion was performed by preparing a hematoxylin-eosin (HE) -stained specimen of the cecal tissue according to a conventional method.
[0141]
Table 3 shows the results.
[0142]
[Table 3]
Table 3 Eimeria tenella Number of moribunds, average weight gain per day, bloody stool persistence, oocyst excretion and cecal mean lesion score in oocyst-infected chickens

[0143]
Regarding the number of moribunds, H. One case moribund within one week after the oocyst infection of the B2 strain, but no moribund case was observed in the three-times extract group.
[0144]
Regarding average weight gain per day, B15 line and H.H. In the untreated control group of the B2 line, it was 10.7 g and 9.8 g, respectively, but in the control group, it was significantly reduced to 7.6 g and 7.9 g, respectively. On the other hand, H. In the 15-line extract 1-dose group and the 3-dose extract group, the weight was 8.6 g and 8.6 g, respectively. It was 9.8 g in the B2 strain extract 3 administration group. Thus, any extract administration group showed a higher average weight gain than the control group.
[0145]
H. B15 line and H.H. In both infected groups of the B2 strain, fresh bloody stool was observed in a large amount, persistently and frequently (++) 3 to 6 days after infection. However, in the extract administration group, mild and transient bloody stool (+) was observed 3 to 4 days after infection.
[0146]
Regarding the average number of oocysts excreted per stool on the 6th and 7th day of infection, B15 line and H.H. In the B2 strain control group, 27-58 × 10 respectively ⁶ And 26-38 × 10 ⁶ H. In the B15 strain 1-time extract group and the 3-times extract group, 16 to 23 × 10 respectively. ⁶ And 12-16 × 10 ⁶ H. 9-12x10 in the B3 strain extract 3 times-III administration group ⁶ A significant decrease was observed compared to the control group.
[0147]
Furthermore, when the cecum was macroscopically observed at autopsy 7 days after infection with HB15, the cecum in the control group lacked elasticity and was swollen, and thinning of the walls and dark red bleeding lesions were observed. The average score was +4.0. On the other hand, the cecum of the group administered with the extract 3 times had elasticity, and there was almost no bleeding lesion, and the average score of the lesion was +3.4. H. The average score of cecal lesions in the B15 strain extract once-administered group was as high as +4.0, and cecal atrophy was considered to be in the recovery period, but there was almost no bleeding lesion.
[0148]
Furthermore, a histopathological search was performed on HE stained specimens of cecal tissue. As a result, many schizonts were found in the lesion of the mucosal epithelial layer, and gamont and oocysts were found in the cavity. In addition, hemorrhagic lesions and degenerative changes such as epithelial cell destruction and lamina propria atrophy were observed. However, the lesion was more prominent in the control group than in each extract administration group.
[0149]
based on the above results, Eimeria tenella By administering an extract derived from sweet potato into the sac before oocyst infection, the decrease in the increase in body weight per day after infection was suppressed. In addition, since reduction or suppression of bloody stool persistence, the number of oocysts excreted in feces and cecal lesions was observed, the extract derived from sweet potato of Production Example 1 was Eimeria tenella It has been shown that it has the effect of suppressing the growth of protozoa and suppressing its infection.
[0150]
Example 3
The extract derived from the sweet potato of Production Example 1 (lyophilized product) was used and the chicken was used. Eimeria tenella Changes in pathological conditions, antibody production responses, and changes in body weight after mild infection and challenge infection of oocysts were examined.
[0151]
In this example, 36 chicks (genotype, H.B15 strain) of 7-day-old inbred chickens were used, and 6 birds per group were used. An extract derived from sweet potato (500 mg / kg body weight) was administered to the chick sac for 3 days using a Komagome pipette. In addition, on the same day as the final administration date, the pathogenic protozoa of chicken coccidiosis Eimeria tenella (NIAH strain) oocyst (2 × 10 ^Three Individuals / feather) was inoculated into the sac (hereinafter referred to as “extract administration + mild infection” group). Furthermore, in the “extract administration + mild infection” group, oocysts (100 × 10 10) at the age of 28 days. ^Three Infection was carried out by inoculating the sac into the sac (hereinafter referred to as “extract administration + mild infection + attack infection” group).
[0152]
In addition, physiological saline was administered into the sac of chicks of the same age / same species in the same manner as in the mildly infected group administered with the extract instead of the sweet potato-derived extract. Furthermore, on the same day as the last administration day, oocysts (2 × 10 ^Three Individuals / feather) were inoculated into the sac (hereinafter referred to as “mildly infected group”). Furthermore, in the “mildly infected group” group, oocysts (100 × 10 6) at the age of 28 days. ^Three Infection was carried out by inoculating the sac into the sac (hereinafter referred to as “mild infection + attack infection” group).
[0153]
Furthermore, chicks of the same age and the same species were subjected to the same experiment without administration of the sweet potato-derived extract and treatment of infection (hereinafter referred to as “untreated control group”). Furthermore, in the untreated control group, oocysts (100 × 10 10) at the age of 28 days. ^Three Infection was performed by inoculating the sac into the sac (hereinafter referred to as “no treatment control + attack infection” group).
[0154]
The antibody production response was evaluated using the T cell-dependent antigen sheep erythrocyte (SRBC) or the T cell-independent antigen Brucella abortus (BA) as the antigen, and the untreated control at 14 and 21 days of age. Group, mildly infected group and “extracted administration + mildly infected” group were inoculated with 0.1 ml of mixed antigen of 10% SRBC and 1% BA cells into wing vein, and serum agglutination strength against both antigens after 1 week (Hirota, Y. et al., The development of unusual B-cell functions in the testosterone-propionate-treated chicken. Immunology, 39, 29-36, 1980; Hirota, Y. et al., Immunopathology of chickens infected in ovo at hatching with the avian osteopetrosis virus MAV.2-0. Eur. J. Immunol., 10, 929-936,
(1980).
[0155]
In the measurement of serum agglutinin titer, the serum after treatment with 0.2M 2-mercaptoethanol (2-ME) or untreated serum is diluted 2-fold, and the log2 of the reciprocal of the final dilution showing complete aggregation is obtained. Calculated and expressed as 2-ME resistant antibody titer and total antibody titer, respectively.
[0156]
The effect of the extract on the attack infection was evaluated by using as an index the symptoms for 1 week after the attack infection, the number of moribunds, the increase in average body weight per day, the persistence of bloody stool, the number of excretion of oocysts in the stool, the severity of cecal lesions, etc. .
[0157]
The number of oocysts in the stool was calculated by a method using a plankton calculation plate and displayed as the number of excretion per bird.
[0158]
The severity of cecal lesions was evaluated according to the method of Johnson and Reid described above and displayed as an average lesion score. In addition, an HE-stained specimen of cecal tissue was prepared according to a conventional method and evaluated by histopathological search for lesions.
[0159]
Furthermore, cell membrane marker surface antigens (Bu-la as B cell markers) in lymphocytes separated from the cecum by Percoll density gradient centrifugation ⁺ CD4 as a T cell marker ⁺ , CD8 ⁺ , TCR1 ⁺ (Γδ receptor), TCR2 ⁺ (Αβ type receptor) and TCR3 ⁺ ) Was analyzed with a flow cytometer (Vainio, O. et al., BL antigens (class II) of the chicken major histocompatibility complex control TB cell interaction. Immunogenetics, Vol. 19, 131, 1984; Luhtala, M. et al., Analysis of chicken CD4 by monoclonal antibodies indicates evolutionary conversion between avian and mammalian species. Hybridoma, Vol. 12, 633-646, 1993).
[0160]
The results are shown in Tables 4 to 6 and FIG.
[0161]
[Table 4]
Table 4 Sweet potato-derived extract in the sac and Eimeria tenella Influence of mild oocyst infection on the transition of antibody production response to SRBC and BA

[0162]
Table 4 shows the results of antibody production responses to SRBC and BA. In the antibody response to SRBC, the “extract administration + mild infection” group showed a higher response in both the primary response and the secondary response than the untreated control group and the counter infection group. In particular, the serum antibody titer after 2-ME treatment, that is, the 2-ME resistant antibody mainly composed of IgG class was significantly high (p <0.05).
[0163]
Regarding the primary antibody response to BA, only 1 of 12 birds in each of the untreated control group and mildly infected group showed a low titer response. However, in the “extract administration + mild infection” group, responses were observed in 4 out of 12 birds, and the titers were significantly higher than those in the untreated control group and the mild infection group (p <0). .05).
[0164]
Moreover, regarding the secondary antibody response to BA, the extract-administered mildly infected group showed a significantly higher value than the untreated control group and the mildly infected group (p <0.05).
[0165]
[Table 5]
Table 5 Sweet potato-derived extract in the sac and Eimeria tenella Effects of mild infection of oocysts on changes in pathological conditions after attack infection

[0166]
Regarding the number of moribunds after challenge infection, in the “untreated control group + attack infection” group, one case died on the third day after oocyst infection, but in the other groups, no moribund cases were observed.
[0167]
Regarding the average weight gain per day, the untreated control group was 10.6 g, whereas the “no treatment control + challenge” group was 8.5 g, and the “no treatment control + challenge” group per day. Average body weight gain was significantly reduced compared to the untreated control group. In addition, the average daily weight gains in the mild infection group and the “mild infection + attack infection” group were 11.7 g and 11.0 g, respectively, and the “extract administration + mild infection” group and the “extract administration +” In the case of the “mild infection + attack infection” group, they were 15.9 g and 13.9 g, respectively. From this, the average weight gain per day of the group to which the extract was administered showed a high weight gain in all cases compared to the corresponding challenge groups.
[0168]
Regarding the persistence of bloody stool, no bloody stool was observed immediately after the mild infection in all the groups that were mildly infected, but in the “no treatment control + aggressive infection” group, bloody stool was abundant 3 to 6 days after the infection. And was observed frequently (++). However, in the case of a mild mild infection of oocysts, that is, in both the “mild infection + attack infection” group and the “extract administration + mild infection + attack infection” group, transient mild mild infection 3 to 4 days after infection Only bloody stool (+) was observed.
[0169]
The average number of oocyst excretion in the stool per bird on the 6th day of the attack infection was 42 × 10 in the attack infection group and the “mild infection + attack infection” group ⁶ And 40 × 10 ⁶ However, in the group of “extract administration + mild infection + attack infection” group, 3.3 × 10 ⁶ Met. From this, the average number of oocyst excretion in the “extract administration + mild infection + attack infection” group was significantly lower than that in the attack infection group and the “mild infection + attack infection” group.
[0170]
In addition, the cecum was visually observed at the time of necropsy on the 7th day after the challenge infection. As a result, the caecum in the attack-infected group lacked elasticity and was swollen, and a thin wall and dark red hemorrhagic lesion were observed. The average score of the lesion was +4.0. On the other hand, the cecum of the “mild infection + attack infection” group and the “extract administration + mild infection + attack infection” group had mild hemorrhagic lesions, with average scores of +3.0 and +2.0, respectively. there were. Therefore, the cecal lesions in the “extract administration + mild infection + attack infection” group were milder than those in the “mild infection + attack infection” group. The cecum of the mildly infected group and the “extract administration + mildly infected” group was elastic and showed no gross lesion, and was the same as the untreated control group. Moreover, the average lesion score of the cecum was suppressed low in any group to which the extract derived from sweet potato was administered.
[0171]
[Table 6]
Table 6 Sugarcane-derived extract in the sac and Eimeria tenella The effect of mild oocyst infection on the relative proportion (%) of cecal surface marker antigen-positive cells after challenge infection

[0172]
Table 6 shows the results of analyzing the relative proportion of cell membrane marker surface antigen-positive cells in cecal lymphocytes in 36-day-old chickens using a flow cytometer. In the cecum 7 days after challenge infection, Bu-la ⁺ Positive cells and CD4 ⁺ While the relative proportion of positive cells is significantly reduced compared to the untreated control group, CD8 ⁺ Positive cells and TCR1 ⁺ The proportion of positive cells increased. TCR2 in the mild infection group and the “mild infection + attack infection” group ⁺ The proportion of positive cells increased. In the “extract administration + mild infection” group and the “extract administration + mild infection + attack infection” group, TCR1 ⁺ There was a tendency for the percentage of positive cells to increase.
[0173]
Eimeria tenella In addition to genetic factors, CD4 involved in the production of interferon γ ⁺ Positive T cells and TCR2 ⁺ It has been reported that local mobilization and aggregation of positive T cells are related (Yun, CH et al., Eimeria tenella infection induces local interferon production and intestinal lymphocyte subpopulation changes, Vol. 68, No. 3, pp. 1282-1288, 2000). Bu-la associated with attack infection ⁺ Positive cells and CD4 ⁺ The decrease in the relative proportion of positive cells is due to the administration of extract or oocyst (2 × 10 2) prior to challenge infection as seen in the extract administration group and the mild infection group. ^Three It was possible to suppress or alleviate the infection by giving a mild infection per person / feather). Therefore, although the mechanism of infection suppression and pathological alleviation effect due to extract administration or mild infection of oocysts is unknown, Bu-la to the cecum where the protozoan grows is known. ⁺ Positive cells, CD4 ⁺ Positive T cells and TCR2 ⁺ Concentration of positive T cells is considered to be related.
[0174]
Histopathological examination was performed on the HE-stained specimen of cecal tissue (FIG. 1). As a result, in the attack infection group, many schizonts were found in the mucosal epithelium and the lesion of the lamina propria, and gamont and oocysts were also found in the cavity. In addition, degenerative changes such as hemorrhagic lesions, destruction of epithelial cells and lamina propria and atrophy were observed (FIG. 1-A). In the “mild infection + attack infection” group, the number of schizonts was smaller than that in the attack infection group (FIG. 1-B). In the “extract administration + mild infection + attack infection” group, cecal atrophy considered to be in the recovery period was observed, but almost no proliferated protozoa were observed and no protozoan infestation was observed (FIG. 1-C). .
[0175]
By administering the extract derived from sweet potato of Production Example 1 into the sac, enhancement of antibody production response to SRBC and BA was observed. In particular, in the normal immunization of BA antigen at the age of 2 weeks, an increase in the number of chicks showing an antibody response to the BA antigen and an increase in the average titer thereof are observed although antibodies against BA are not usually produced. It was. From this, it was considered that the sweet potato-derived extract has an action of enhancing the activity of the existing immune system and promoting the maturation of the immune system. Eimeria tenella Oocyst (2 × 10 ^Three Subsequent infection (100 x 10) ^Three Reducing or suppressing weight loss and cecal lesions associated with individuals / feather). However, the oocyst (2 × 10 ^Three Before the mild infection of the individual, the administration of the sweet potato extract of Production Example 1 into the sac showed the effect of suppressing weight loss, caecal pathology and lesion formation associated with subsequent attack infection. .
[0176]
From the above, it was shown that the extract derived from sweet potato has an adjuvant effect for human or animal immunity against mild infection with coccidium.
[0177]
【Effect of the invention】
According to the present invention, a human or animal disease caused by coccidium can be prevented and treated by administering (for example, oral administration) an agent containing an extract derived from sweet potato as an active ingredient. In addition, according to the present invention, an adjuvant effect for immunization of humans or animals against coccidial mild infection including coccidial vaccine administration by administering an agent containing an extract derived from sweet potato to the animal (for example, oral administration) Can be demonstrated.
Although the prophylactic and therapeutic agents and adjuvants according to the present invention are natural products, they have a high effect of suppressing the excretion of coccidium oocysts and a high adjuvant effect. And since the said effect is exhibited with a small quantity, it is very useful industrially. Accordingly, the number of administrations of the antibacterial / antiprotozoal agent can be reduced or substituted for these administrations. In addition, the number of vaccine administrations can be reduced due to the adjuvant effect. Moreover, the administration of the vaccine can be omitted due to the adjuvant effect against a mild infection that occurs naturally. Furthermore, since the prophylactic and therapeutic agents and adjuvants according to the present invention are natural products that are derived from plants and have been eaten by humans as sweet candy itself or as sugar containing sugar since ancient times, humans or domestic animals or poultry that humans eat There is no harm to the health of industrial animals. Thus, the preventive / therapeutic agent and the adjuvant according to the present invention are safe and low in cost.
[Brief description of the drawings]
1 is a histological image of a cecum of a chicken (35 days of age) 7 days after challenge infection (HE-stained specimen, × 100).
Fig. 1-A; attacking group; multiple schizont parasites in mucosal epithelium and lamina propria
Fig. 1-B; "Mild infection + attack infection"group; sporadic schizonts
Fig. 1-C; "Extract administration + mild infection + aggressive infection"group; mucosal epithelium and lamina propria
[Figure 2] Analytical value of the second honey fraction sample by ion exchange resin
[Explanation of symbols]
Fig. 1-A (→): Schizont parasitism of severe wolfberry in the lamina propria
Fig. 1-B (→): Schizonto schizonto parasitism of moderate body beetle

Claims (14)

A prophylactic / therapeutic agent for human or animal diseases caused by coccidium, characterized by comprising a sweet potato-derived extract as an active ingredient , wherein the sweet potato-derived extract is derived from a sweet potato juice, a sweet potato solvent extract and a sweet potato Absorbs light with a wavelength of 420 nm out of fractions obtained by separating the raw material selected from molasses by column chromatography using a difference in affinity in a column packed with a cation exchange resin as a fixed carrier The agent, which is a fraction . The preventive / therapeutic agent according to claim 1, wherein the animal is a poultry. The prophylactic / therapeutic agent according to claim 1 or 2 , wherein the cation exchange resin is a strongly acidic cation exchange resin. The prophylactic / therapeutic agent according to claim 3 , wherein the strongly acidic cation exchange resin is a sodium ion type or a potassium ion type. The prophylactic / therapeutic agent according to any one of claims 1 to 4 , wherein the cation exchange resin is a gel type. The prophylactic / therapeutic agent according to any one of claims 1 to 5 , wherein the column chromatography is performed by a simulated moving bed continuous separation method. The preventive / therapeutic agent according to any one of claims 1 to 6 , wherein a fraction that absorbs light having a wavelength of 420 nm is further subjected to an electrodialysis treatment to reduce a salt content. An adjuvant for immunization of humans or animals against mild coccidial infection , comprising an extract derived from sweet potato as an active ingredient , wherein the extract derived from sweet potato is a sweet potato juice, a solvent extract of sweet potato Among the fractions obtained by separating a raw material selected from molasses derived from sugarcane and sugarcane by column chromatography using a difference in affinity in a column packed with a cation exchange resin as a fixed carrier, light having a wavelength of 420 nm The said agent which is a fraction which absorbs . The adjuvant according to claim 8 , wherein the coccidium infects poultry. The adjuvant according to claim 8 or 9 , wherein the cation exchange resin is a strongly acidic cation exchange resin. The adjuvant agent according to claim 10 , wherein the strongly acidic cation exchange resin is a sodium ion type or a potassium ion type. The adjuvant according to any one of claims 8 to 11 , wherein the cation exchange resin is a gel type. The adjuvant according to any one of claims 8 to 12 , wherein the column chromatography treatment is carried out by a simulated moving bed continuous separation method. The adjuvant according to any one of claims 8 to 13 , wherein a fraction that absorbs light having a wavelength of 420 nm is further subjected to an electrodialysis treatment to reduce a salt content.

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