JP2004210687A - Composition having inhibitory action on crisis of alcoholic hepatopathy and healing action, and method for producing the same - Google Patents
Composition having inhibitory action on crisis of alcoholic hepatopathy and healing action, and method for producing the same Download PDFInfo
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【０００１】
【発明が属する技術分野】
本発明は大麦を原料とする焼酎製造において副生する大麦焼酎蒸留残液を固液分離して液体分を得、該液体分を合成吸着剤を使用する吸着分離処理に付して合成吸着剤非吸着画分を得、該合成吸着剤非吸着画分をイオン交換樹脂を使用するイオン交換処理に付してイオン交換樹脂非吸着画分を得、該イオン交換樹脂非吸着画分を限外濾過膜を使用する限外濾過処理に付して濃縮液を得、該濃縮液に有機溶媒を添加することにより分取した、有機溶媒不溶性画分からなるアルコール性肝障害に対する発症抑制作用及び治癒作用を有する組成物及びその製造方法に関する。
【０００２】
【従来の技術】
近年におけるアルコール飲料の消費量の増加に伴って，アルコール性肝障害者の数は増加傾向にあり、生活習慣病の一つとして認識されるようになってきた。こうしたアルコール性肝障害の具体的症状としては、アルコール性脂肪肝、アルコール性肝炎、アルコール性肝線維症、アルコール性肝硬変、或いはアルコール性脂肪肝によって誘発するアルコール性高脂血症等を挙げることができる。そして、アルコール性脂肪肝が慢性に経過すると肝細胞の周りに線維ができるアルコール性肝線維症に移行することが知られており、該アルコール性肝線維症において肝細胞に入る血液量が減少して肝臓のタンパク質合成能や有毒物質分解能が低下すると、肝臓が徐々に硬く変性しアルコール性肝硬変に至ることが知られている。
【０００３】
ところで、大麦焼酎を製造する際に副成する大麦焼酎蒸留残液の肝障害予防作用については、以下のことが知られている。即ち、該大麦焼酎蒸留残液がオロチン酸投与によるラットの肝臓への脂質の蓄積を抑制することが報告されている［日本栄養・食糧学会総会講演要旨集、Ｖｏｌ．５３， ５３（１９９９）参照］（以下、当該文献を非特許文献１と呼称することとし、後述の
【０００５】に別途記載することとする）。さらに該大麦焼酎蒸留残液が有する上記の脂肪肝抑制作用は、ワイン粕やビール粕に比べて強く、該作用はいも焼酎蒸留残液には全く認められず、米焼酎蒸留残液では極めて小さいことから、大麦焼酎蒸留残液のみに特有のものであることが報告されている［日本醸造協会誌、Ｖｏｌ．９４， Ｎｏ．９， ７６８（１９９９）参照］（以下、当該文献を非特許文献２と呼称することとし、後述の
【０００５】に別途記載することとする）。また、前記大麦焼酎蒸留残液がウイルス性肝障害と同様の症状を呈することが知られているＤ−ガラクトサミン誘発性肝障害に対する発症抑制作用を有し、該発症抑制作用は該大麦焼酎蒸留残液を遠心分離に付すことにより得られる液体分に認められることが報告されている［日本醸造協会誌、Ｖｏｌ．９５， Ｎｏ．９， ７０６（２０００）参照］（以下、当該文献を非特許文献３と呼称することとし、後述の
【０００５】に別途記載することとする）。
特開２００１−１４５４７２号公報には、大麦を原料とする焼酎製造において副成する大麦焼酎蒸留残液を固液分離して液体分を得、該液体分にアルカリを添加してアルカリ可溶性画分を分取し、該アルカリ可溶性画分を酸で中和して中性可溶性画分を得、該中性可溶性画分にエタノールを添加することにより分取した、有機酸、タンパク質、及びヘミセルロースを含有するエタノール不溶性画分からなる組成物が、ラットを使用した実験において、オロチン酸誘発性肝障害に対する発症抑制作用を有することが記載されている（以下、当該公報を特許文献１と呼称することとし、後述の
【０００５】に別途記載することとする）。
【０００４】
特許第３１９１９５６号には、トウモロコシフスマより得られたヘミセルロースの部分分解物を主成分とするアルコール性脂肪肝抑制剤が、エタノール溶液投与によるラットの脂肪肝形成に対し、脂肪肝を抑制する作用を有することが記載されている（以下、当該公報を特許文献２と呼称することとし、後述の
【０００５】に別途記載することとする）。また、特許文献２には、前記トウモロコシフスマから得られたヘミセルロースの部分分解物が「セルエース」の商品名で市販されていることが記載されている。［平成３年３月（社）菓子総合技術センター発行「農林水産省食品流通局委託事業 Ｎｏ．８ 飲食料品用機能性素材有効利用技術シリーズ水溶性コーンファイバー（アラビノキシラン）」３頁］には、「セルエース」の商品名で市販されているトウモロコシフスマから得られたヘミセルロースの部分分解物はアラビノース及びキシロースからなるアラビノキシランを主成分とし、キシロース４０．３２％、アラビノース２７．７６％、ウロン酸１１．８６％、ガラクトース５．９１％、及びグルコース２．３０％の組成を有するものであることが記載されている（以下、当該文献を非特許文献４と呼称することとし、後述の
【０００５】に別途記載することとする）。更に、［Ｎｅｗ Ｆｏｏｄ Ｉｎｄｕｓｔｒｙ Ｖｏｌ．４２ Ｎｏ．９， ６（２０００）］には、「セルエース」の商品名で市販されているトウモロコシフスマから得られたヘミセルロースの部分分解物が、急性アルコール性脂肪肝に対する抑制作用を有することが記載されている（以下、当該文献を非特許文献５と呼称することとし、後述の
【０００５】に別途記載することとする）。
【０００５】
【特許文献１】
特開２００１−１４５４７２号公報
【特許文献２】
特許第３１９１９５６号
【非特許文献１】
日本栄養・食糧学会総会講演要旨集、Ｖｏｌ．５３， ５３（１９９９）
【非特許文献２】
日本醸造協会誌、Ｖｏｌ．９４， Ｎｏ．９， ７６８（１９９９）
【非特許文献３】
日本醸造協会誌、Ｖｏｌ．９５， Ｎｏ．９， ７０６（２０００）
【非特許文献４】
平成３年３月（社）菓子総合技術センター発行 「農林水産省食品流通局委託事業 Ｎｏ．８ 飲食料品用機能性素材有効利用技術シリーズ 水溶性コーンファイバー（アラビノキシラン）」 ３頁
【非特許文献５】
Ｎｅｗ Ｆｏｏｄ Ｉｎｄｕｓｔｒｙ Ｖｏｌ．４２ Ｎｏ．９， ６（２０００）
【０００６】
【発明が解決しようとする課題】
大麦焼酎蒸留残液が有する肝障害予防作用については、上述したように、非特許文献１乃至非特許文献３には、大麦焼酎蒸留残液又は大麦焼酎蒸留残液を固液分離することにより得られる液体分（以下、これらを大麦焼酎蒸留残液の液体分と呼称することとする。）がオロチン酸誘発性肝障害及びＤ−ガラクトサミン誘発性肝障害に対する発症抑制作用を有することが記載されているが、アルコール性肝障害に対する発症抑制作用及び治癒作用を有するか否かについては示唆するところすらない。また、特許文献１にはエタノール不溶性画分がオロチン酸誘発性肝障害に対する発症抑制作用を有することが記載されているが、該エタノール不溶性画分が前記アルコール性肝障害に対する発症抑制作用及び治癒作用を有するか否かについては示唆するところすらない。即ち、該大麦焼酎蒸留残液からアルコール性肝障害に対する発症抑制作用及び治癒作用を有する画分を分取した例はこれまでに全く知られていない。
【０００７】
ところで、オロチン酸誘発性肝障害は、オロチン酸により肝臓での脂肪合成が促進され、さらに肝臓から血中への脂肪の移行が抑制され、それにより脂肪肝を誘発する肝障害であることが知られている。また，Ｄ−ガラクトサミン誘発性肝障害は、Ｄ−ガラクトサミンにより肝細胞の壊死が促進され、それにより肝炎を誘発する肝障害であることが知られている。
【０００８】
一方、本発明においていうアルコール性肝障害は、アルコールの過剰摂取によって誘発されるアルコール性肝炎、アルコール性脂肪肝、及びアルコール性高脂血症を包含して意味する。前記アルコール性脂肪肝は、エタノールにより脂肪組織から肝臓への脂肪酸の移行が促進され、肝臓における脂肪酸や中性脂肪の合成が促進され、更に、肝臓における脂肪酸の分解が抑制されること等によって、肝臓内に中性脂肪が蓄積されることにより誘発される脂肪肝であることが知られている。前記アルコール性肝炎は、エタノールの代謝産物であるアセトアルデヒドや酢酸或いはこれらが産生される際に発生する活性酸素が、肝細胞に対して障害性を示すことにより誘発される肝炎であることが知られている。また、前記アルコール性高脂血症は、上述したように肝臓内に蓄積した過剰の中性脂肪が分泌型の超低比重リポタンパク（ＶＬＤＬ）として大量に血中に放出されることにより発症するものであることが知られている。そして、こうしたアルコール性肝障害においては、風船様腫大や肝細胞壊死等の肝炎の病変、或いは大滴性脂肪滴を含有する肝細胞からなる脂肪肝の所見が肝小葉の終末肝静脈周辺領域を中心として進展していくことが知られている。なお、肝臓は主に肝細胞からなり、小葉間結合組織で区切られた肝小葉を一単位として機能しており、この直径１ｍｍほどの肝小葉が多数集合して肝臓ができている。従って、こうしたそれぞれの肝障害の因果関係からすると、アルコール性肝障害は、オロチン酸誘発性肝障害及びＤ−ガラクトサミン誘発性肝障害とは客観的に区別されるものであり、ある種の成分がオロチン酸誘発性肝障害又はＤ−ガラクトサミン誘発性肝障害に対して発症抑制作用或いは治癒作用を有することが判っていても、該成分がアルコール性肝障害に対しても同様の発症抑制作用或いは治癒作用を有するか否かは、容易に予測できるものでは到底ない。
【０００９】
このような従来技術に鑑みて、本発明は大麦焼酎蒸留残液を固液分離して液体分を得、該液体分を合成吸着剤を使用する吸着分離処理に付して合成吸着剤非吸着画分を得、該合成吸着剤非吸着画分をイオン交換樹脂を使用するイオン交換処理に付してイオン交換樹脂非吸着画分を得、該イオン交換樹脂非吸着画分を限外濾過膜を使用する限外濾過処理に付して濃縮液を得、該濃縮液に有機溶媒を添加することにより分取した、アルコール性肝障害に対する強力な発症抑制作用及び治癒作用を有する有機溶媒不溶性画分からなる組成物を提供することを目的とするものである。
【００１０】
【課題を解決するための手段】
本発明者らは、文献１乃至文献３には大麦焼酎蒸留残液の液体分がＤ−ガラクトサミン誘発性肝障害及びオロチン酸誘発性肝障害に対する発症抑制作用を有することが記載されていることに鑑み、該大麦焼酎蒸留残液の液体分が、アルコール性肝障害に対して発症抑制作用を示すか否かを明らかにすることを目的として、実験を介して鋭意検討を行った。その結果、前記大麦焼酎蒸留残液の液体分は、前記アルコール性肝障害に対する発症抑制作用を僅かに有するものの、アルコール性肝障害の発症を積極的に抑制する目的での薬剤としての実使用を示唆する程のものではないことが判明した。
【００１１】
ところで、本発明者らの内の三者は他の二者と共同で、大麦焼酎蒸留残液の液体分を合成吸着剤を用いる吸着分離処理に付すことにより得られる吸着画分は、オロチン酸誘発性脂肪肝及びＤ−ガラクトサミン誘発性肝障害に対する発症抑制作用を有するが、大麦焼酎蒸留残液の液体分を合成吸着剤を用いる吸着分離処理に付すことにより副成する合成吸着剤非吸着画分は、オロチン酸誘発性脂肪肝及びＤ−ガラクトサミン誘発性肝障害に対する発症抑制作用を有しないことを見出した（特願２００２−５６９２９号として出願済）。こうしたことから、前記合成吸着剤非吸着画分は薬理作用を有しないことから無用なものとみなして廃棄していた。ところが、本発明者らは、このように無用なものとして廃棄していた前記合成吸着剤非吸着画分を用いて、そのアルコール性肝障害に対する発症抑制作用及び治癒作用の有無を実験を介して検討したところ、該合成吸着剤非吸着画分がアルコール性肝障害に対する優れた発症抑制作用を有し且つアルコール性肝障害に対する優れた治癒作用を有することを見出した（特願２００２−２５０９９１号として出願済）。当該発見に引き続いて、該合成吸着剤非吸着画分に含まれる有効成分、即ち、アルコール性肝障害に対する強力な発症抑制作用及び治癒作用を有する成分、を分画精製すべく、鋭意、研究を重ねたところ、該合成吸着剤非吸着画分をイオン交換樹脂を使用するイオン交換処理に付してイオン交換樹脂非吸着画分を得、該イオン交換樹脂非吸着画分を限外濾過膜を使用する限外濾過処理に付して濃縮液を得、該濃縮液に有機溶媒を添加することにより分取した有機溶媒不溶性画分が、アルコール性肝障害に対して際立った発症抑制作用及び治癒作用を有することを見出した。
【００１２】
本発明は、このような発見に基づいて完成に至ったものである。本発明の目的は、大麦焼酎蒸留残液を固液分離して液体分を得、該液体分を合成吸着剤を使用する吸着分離処理に付して合成吸着剤非吸着画分を得、該合成吸着剤非吸着画分をイオン交換樹脂を使用するイオン交換処理に付してイオン交換樹脂非吸着画分を得、該イオン交換樹脂非吸着画分を限外濾過膜を使用する限外濾過処理に付して濃縮液を得、該濃縮液に有機溶媒を添加することにより得られる有機溶媒不溶性画分からなる、アルコール性肝障害に対して際立った発症抑制作用及び治癒作用を有する医薬用組成物を提供することにある。本発明の他の目的は、こうした薬理作用を有する組成物の製造方法を提供することにある。
【００１３】
以下に本発明を完成するにあたり、本発明者らが行った実験について詳述する。本発明はこれらの実験において得られた知見に基づいて完成したものである。本発明者らは、上述したように、非特許文献１乃至非特許文献３には大麦焼酎蒸留残液の液体分がＤ−ガラクトサミン誘発性肝障害及びオロチン酸誘発性脂肪肝に対する発症抑制作用を有することが記載されていることに鑑みて、該大麦焼酎蒸留残液の液体分がアルコール性肝障害に対しても発症抑制作用を示すか否かを明らかにするために、該大麦焼酎蒸留残液の液体分（Ａ）を用いて以下の実験を行い、鋭意検討した。
即ち、３週齢Ｗｉｓｔａｒ系雄性ラット（日本ＳＬＣ）２４匹にエタノール含有率を徐々に上げながら（３％→４％→５％）エタノール含有液体飼料を６日間与えた後、１群１２匹として、対照群及び試験群からなる２群に分けた。その際、前記各群におけるラットの平均体重に係る分散に統計学的有意差が生じないように前記２４匹のラットを振り分けた。対照群のラットに対しては５％エタノール含有液体飼料、試験群のラットに対しては該５％エタノール含有液体飼料に大麦焼酎蒸留残液の液体分（Ａ）の凍結乾燥物粉末（Ａ’）１％を添加した液体飼料をそれぞれ４週間与えて飼育した。対照群及び試験群とは別に前記３週齢Ｗｉｓｔａｒ系雄性ラット１２匹からなる無処置群を設け、該無処置群のラットに対しては他の２群と摂取カロリーを同一にするために５％エタノールの代わりにマルトース−デキストリン等量混合物を添加したエタノール非含有液体飼料を４週間与えて飼育した。但し、上記３群とも、各液体飼料の１日あたりの給餌量（摂取カロリー）を７０ｍｌ（７０ｋｃａｌ）に制限した。実験最終日（試験開始後４週間目）に、飼育したラットのそれぞれの腹部大動脈から採血を行い、肝臓を摘出した。採取した血液は血清分離後、血清総コレステロール、血清ＨＤＬ−コレステロール、血清ＬＤＬ−コレステロール、血清トリグリセリド、血清リン脂質、血清遊離脂肪酸、及び血清ＡＬＴ（ＧＰＴ）を測定した。また、摘出した肝臓については、肝臓重量、肝臓総コレステロール、肝臓トリグリセリド、及び肝臓リン脂質を測定した。得られた結果は平均値±標準誤差（ＳＥＭ）で表し、統計処理は以下の手順で行った。即ち、無処置群と対照群の比較はＳｔｕｄｅｎｔ’ｓ ｔｅｓｔ法を用いて解析し、次いで対照群と試験群の比較はＴｕｋｅｙ−Ｋｒａｍｅｒ法を用いて解析し、それぞれの解析において危険率０．０５％以下を有意として判定した。更に、摘出した前記肝臓から採取した肝細胞をＨＥ染色に付した後、オリンパス光学工業（株）製の生物顕微鏡ＢＸ５１を用いて２００倍の倍率で該肝細胞の形態観察を行った。
【００１４】
その結果、対照群は、無処置群と比較して、血清総コレステロール濃度、血清ＨＤＬ−コレステロール濃度、血清ＬＤＬ−コレステロール濃度、血清トリグリセリド濃度及び血清リン脂質濃度が有意に増加して、アルコール性高脂血症が誘発されていることが判明した。また、対照群は、無処置群と比較して、肝臓トリグリセリド濃度及び肝臓リン脂質濃度が有意に増加して、アルコール性脂肪肝が誘発されていることが判明した。更に、対照群は、無処置群と比較して、血中ＡＬＴ（ＧＰＴ）濃度が有意に増加し、肝細胞の生物顕微鏡観察においては肝小葉の終末肝静脈周辺領域における肝細胞壊死と風船様腫大が顕著に認められ、アルコール性肝炎が誘発されていることが判明した。一方、試験群は、対照群と比較して、血清ＬＤＬ−コレステロール濃度、血清トリグリセリド濃度及び肝臓トリグリセリド濃度の上昇が抑制される傾向を示し、肝細胞の生物顕微鏡観察においても肝小葉の終末肝静脈周辺領域における肝細胞壊死と風船様腫大が僅かに減少する傾向を示した。
以上の結果から、前記大麦焼酎蒸留残液の液体分（Ａ）の凍結乾燥物粉末（Ａ’）は、アルコール性肝障害の発症を積極的に抑制する目的での薬剤としての実使用を示唆する程のものではないことが判明した。
【００１５】
次に、本発明者らは、次に述べる実験を介して、大麦焼酎蒸留残液の液体分から得られるどのような画分がアルコール性肝障害に対する発症抑制作用に寄与しているかを明らかにするために、大麦焼酎蒸留残液の液体分を合成吸着剤を用いる吸着分離処理に付すことにより得られる吸着画分をアルカリを用いて溶出することにより得られる脱着画分（Ｂ）を用いて以下の実験を行い、鋭意検討した。
即ち、３週齢Ｗｉｓｔａｒ系雄性ラット（日本ＳＬＣ）２４匹にエタノール含有率を徐々に上げながら（３％→４％→５％）エタノール含有液体飼料を６日間与えた後、１群１２匹として、対照群及び試験群からなる２群に分けた。その際、前記各群におけるラットの平均体重に係る分散に統計学的有意差が生じないように前記２４匹のラットを振り分けた。対照群のラットに対しては５％エタノール含有液体飼料、試験群のラットに対しては該５％エタノール含有液体飼料に上記脱着画分（Ｂ）の凍結乾燥物粉末（Ｂ’）１％を添加した液体飼料をそれぞれ４週間与えて飼育した。対照群及び試験群とは別に前記３週齢Ｗｉｓｔａｒ系雄性ラット１２匹からなる無処置群を設け、該無処置群のラットに対しては他の２群と摂取カロリーを同一にするために５％エタノールの代わりにマルトース−デキストリン等量混合物を添加したエタノール非含有液体飼料を４週間与えて飼育した。但し、上記３群とも、各液体飼料の１日あたりの給餌量（摂取カロリー）を７０ｍｌ（７０ｋｃａｌ）に制限した。実験最終日（試験開始後４週間目）に、飼育したラットのそれぞれの腹部大動脈から採血を行い、肝臓を摘出した。採取した血液は血清分離後、血清総コレステロール、血清ＨＤＬ−コレステロール、血清ＬＤＬ−コレステロール、血清トリグリセリド、血清リン脂質、血清遊離脂肪酸、及び血清ＡＬＴ（ＧＰＴ）を測定した。また、摘出した肝臓については、肝臓重量、肝臓総コレステロール、肝臓トリグリセリド、及び肝臓リン脂質を測定した。得られた結果は平均値±標準誤差（ＳＥＭ）で表し、統計処理は以下の手順で行った。即ち、無処置群と対照群の比較はＳｔｕｄｅｎｔ’ｓ ｔｅｓｔ法を用いて解析し、次いで対照群と試験群の比較はＴｕｋｅｙ−Ｋｒａｍｅｒ法を用いて解析し、それぞれの解析において危険率０．０５％以下を有意として判定した。更に、摘出した前記肝臓から採取した肝細胞をＨＥ染色に付した後、オリンパス光学工業（株）製の生物顕微鏡ＢＸ５１を用いて２００倍の倍率で該肝細胞の形態観察を行った。
【００１６】
その結果、対照群は、無処置群と比較して、血清総コレステロール濃度、血清ＨＤＬ−コレステロール濃度、血清ＬＤＬ−コレステロール濃度、血清トリグリセリド濃度及び血清リン脂質濃度が有意に増加して、アルコール性高脂血症が誘発されていることが判明した。また、対照群は、無処置群と比較して、肝臓トリグリセリド濃度及び肝臓リン脂質濃度が有意に増加して、アルコール性脂肪肝が誘発されていることが判明した。更に、対照群は、無処置群と比較して、血中ＡＬＴ（ＧＰＴ）濃度が有意に増加し、肝細胞の生物顕微鏡観察においては肝小葉の終末肝静脈周辺領域における肝細胞壊死と風船様腫大が顕著に認められ、アルコール性肝炎が誘発されていることが判明した。一方、試験群は、対照群と比較して、該ラットの血清トリグリセリド濃度の上昇が抑制される傾向を示したが、肝臓トリグリセリド濃度の上昇を全く抑制せず、肝細胞の生物顕微鏡観察においても肝小葉の終末肝静脈周辺領域における肝細胞壊死と風船様腫大が顕著に認められた。即ち、上記脱着画分（Ｂ）の凍結乾燥物粉末（Ｂ’）は、アルコール性高脂血症の誘発を抑制する傾向を僅かに示したが、アルコール性脂肪肝及びアルコール性肝炎の誘発を抑制する傾向は全く示さなかった。
以上の結果から、上記脱着画分（Ｂ）の凍結乾燥物粉末（Ｂ’）は、アルコール性肝障害に対する発症抑制作用を実質的に有さないことが判明した。
【００１７】
そこで、本発明者らは、大麦焼酎蒸留残液の液体分を合成吸着剤を用いる吸着分離処理に付すことにより得られる合成吸着剤非吸着画分が、アルコール性肝障害に対して効果的な発症抑制作用を示すのではないかと推測して、大麦焼酎蒸留残液の液体分を合成吸着剤を用いる吸着分離処理に付すことにより得られる合成吸着剤非吸着画分（Ｃ）を用いて以下の実験を行い、鋭意検討した。
即ち、３週齢Ｗｉｓｔａｒ系雄性ラット（日本ＳＬＣ）２４匹にエタノール含有率を徐々に上げながら（３％→４％→５％）エタノール含有液体飼料を６日間与えた後、１群１２匹として、対照群及び試験群からなる２群に分けた。その際、前記各群におけるラットの平均体重に係る分散に統計学的有意差が生じないように前記２４匹のラットを振り分けた。対照群のラットに対しては５％エタノール含有液体飼料、試験群のラットに対しては該５％エタノール含有液体飼料に上記合成吸着剤非吸着画分（Ｃ）の凍結乾燥物粉末（Ｃ’）１％を添加した液体飼料をそれぞれ４週間与えて飼育した。対照群及び試験群とは別に前記３週齢Ｗｉｓｔａｒ系雄性ラット１２匹からなる無処置群を設け、該無処置群のラットに対しては他の２群と摂取カロリーを同一にするために５％エタノールの代わりにマルトース−デキストリン等量混合物を添加したエタノール非含有液体飼料を４週間与えて飼育した。但し、上記３群とも、各液体飼料の１日あたりの給餌量（摂取カロリー）を７０ｍｌ（７０ｋｃａｌ）に制限した。実験最終日（試験開始後４週間目）に、飼育したラットのそれぞれの腹部大動脈から採血を行い、肝臓を摘出した。採取した血液は血清分離後、血清総コレステロール、血清ＨＤＬ−コレステロール、血清ＬＤＬ−コレステロール、血清トリグリセリド、血清リン脂質、血清遊離脂肪酸、及び血清ＡＬＴ（ＧＰＴ）を測定した。また、摘出した肝臓については、肝臓重量、肝臓総コレステロール、肝臓トリグリセリド、及び肝臓リン脂質を測定した。得られた結果は平均値±標準誤差（ＳＥＭ）で表し、統計処理は以下の手順で行った。即ち、無処置群と対照群の比較はＳｔｕｄｅｎｔ’ｓ ｔｅｓｔ法を用いて解析し、次いで対照群と試験群の比較はＴｕｋｅｙ−Ｋｒａｍｅｒ法を用いて解析し、それぞれの解析において危険率０．０５％以下を有意として判定した。更に、摘出した前記肝臓から採取した肝細胞をＨＥ染色に付した後、オリンパス光学工業（株）製の生物顕微鏡ＢＸ５１を用いて２００倍の倍率で該肝細胞の形態観察を行った。
【００１８】
その結果、対照群は、無処置群と比較して、血清総コレステロール濃度、血清ＨＤＬ−コレステロール濃度、血清ＬＤＬ−コレステロール濃度、血清トリグリセリド濃度及び血清リン脂質濃度が有意に増加して、アルコール性高脂血症が誘発されていることが判明した。また、対照群は、無処置群と比較して、肝臓トリグリセリド濃度及び肝臓リン脂質濃度が有意に増加して、アルコール性脂肪肝が誘発されていることが判明した。更に、対照群は、無処置群と比較して、血中ＡＬＴ（ＧＰＴ）濃度が有意に増加し、肝細胞の生物顕微鏡観察においては肝小葉の終末肝静脈周辺領域における肝細胞壊死と風船様腫大が顕著に認められ、アルコール性肝炎が誘発されていることが判明した。一方、試験群は、対照群と比較して、該ラットの血清ＬＤＬ−コレステロール濃度と血清トリグリセリド濃度、及び肝臓トリグリセリド濃度の上昇を有意に抑制し、肝細胞の生物顕微鏡観察においても肝小葉の終末肝静脈周辺領域における肝細胞壊死と風船様腫大がほとんど認められなかった。以上の実験結果から、上記合成吸着剤非吸着画分（Ｃ）の凍結乾燥物粉末（Ｃ’）は、アルコール性肝障害に対する顕著な発症抑制作用を有することが判った。
【００１９】
こうしたことから、大麦焼酎蒸留残液が有するアルコール性肝障害の発症抑制作用に寄与する成分は、大麦焼酎蒸留残液を固液分離して液体分を得、該液体分を合成吸着剤を使用する吸着分離処理に付すことにより得られる合成吸着剤非吸着画分に分画された状態で存在することが判明した。
【００２０】
そこで、本発明者らは、前記合成吸着剤非吸着画分に含まれるアルコール性肝障害に対する強力な発症抑制作用を有する成分を明らかにすることを目的として、鋭意検討を行った。即ち、本発明者らは、大麦を原料とする焼酎製造において副生する大麦焼酎蒸留残液を固液分離して液体分を得、該液体分を合成吸着剤を使用する吸着分離処理に付すことにより得られる合成吸着剤非吸着画分が、成分の１つとして多糖類を含有していることに鑑み、該合成吸着剤非吸着画分に含まれる多糖類がアルコール性肝障害に対する発症抑制作用に関与しているか否かを明らかにするために鋭意検討を行った。即ち、前記有機溶媒不溶性画分に含まれる多糖類以外の成分である、アミノ酸、ペプチド、タンパク質、有機酸、或いは大麦由来のポリフェノール等を除去することを目的として以下の実験を行った。具体的には、大麦焼酎蒸留残液の液体分に含まれる大麦由来のポリフェノール等を除去することを目的として、大麦焼酎蒸留残液を固液分離することにより得た液体分を合成吸着剤を使用した吸着分離処理に付して合成吸着剤非吸着画分を得、次に、該合成吸着剤非吸着画分に含まれるアミノ酸、ペプチド、タンパク質及び有機酸を除去することを目的として、該合成吸着剤非吸着画分を種々のイオン交換樹脂を使用したイオン交換処理に付すことによって異なる種類のイオン交換樹脂非吸着画分を得、得られたそれぞれのイオン交換樹脂非吸着画分に別々に有機溶媒を添加することにより分取したそれぞれの有機溶媒不溶性画分が、アルコール性肝障害に対してどのような発症抑制作用を有するか否かを明らかにするために以下の実験を行った。
即ち、大麦焼酎蒸留残液を固液分離することにより得た液体分を合成吸着剤を使用する吸着分離処理に付すことにより合成吸着剤非吸着画分を得、得られた合成吸着剤非吸着画分を、各種のイオン交換樹脂、具体的には、陽イオン交換樹脂、陰イオン交換樹脂及び両者の混床型イオン交換樹脂を用いたイオン交換処理に付してイオン交換樹脂非吸着画分を得、それぞれのイオン交換樹脂非吸着画分にエタノールを添加することにより有機溶媒不溶性画分を得た。得られたそれぞれの有機溶媒不溶性画分について、遊離糖類、多糖類、有機酸類、及び粗タンパクを測定した。また、それぞれの有機溶媒不溶性画分がアルコール性肝障害に対して発症抑制作用を示すか否かを明らかにするために、以下の実験を行った。
【００２１】
以下の実験１乃至実験７に供する目的で大麦焼酎の製造を行った。原料としては、大麦（７０％精白）を用いた。
【麹の製造】
大麦を４０重量％吸水させ、４０分間蒸した後、４０℃まで放冷し、大麦トンあたり１ｋｇの種麹（白麹菌）を接種し、３８℃、ＲＨ９５％で２４時間、３２℃、ＲＨ９２％で２０時間保持することにより、大麦麹を製造した。
【蒸麦の製造】
大麦を４０重量％吸水させ、４０分間蒸した後、４０℃まで放冷することにより、蒸麦を製造した。
【００２２】
【大麦焼酎及び大麦焼酎蒸留残液の製造】
１次仕込みでは前述の方法で製造した大麦麹（大麦として３トン）に、水３．６キロリットル及び酵母として焼酎酵母の培養菌体１ｋｇ（湿重量）を加えて１次もろみを得、得られた１次もろみを５日間の発酵（１段目の発酵）に付した。次いで、２次仕込みでは、上記１段目の発酵を終えた１次もろみに、水１１．４キロリットル、前述の方法で製造した蒸麦（大麦として７トン）を加えて１１日間の発酵（２段目の発酵）に付した。発酵温度は１次仕込み、２次仕込みとも２５℃とした。上記２段目の発酵を終えた２次もろみを常法により単式蒸留に付し、大麦焼酎１０キロリットルと大麦焼酎蒸留残液１５キロリットルを得た。得られた大麦焼酎蒸留残液を以下の実験１乃至実験７に用いた。
【００２３】
【実験１】
前記
【００２２】において得た大麦焼酎蒸留残液から以下に示す方法により有機溶媒不溶性画分を分画した。即ち、前記大麦焼酎蒸留残液を８０００ｒｐｍ、１０ｍｉｎの条件で遠心分離して大麦焼酎蒸留残液の液体分を得、得られた液体分をオルガノ（株）製の合成吸着剤アンバーライトＸＡＤ−１６を充填したカラムに通して吸着分離処理に付すことにより、該カラムの合成吸着剤に対して非吸着性を示す素通り液からなる合成吸着剤非吸着画分を分取した。得られた合成吸着剤非吸着画分１Ｌを凍結乾燥に付すことにより該合成吸着剤非吸着画分の凍結乾燥物５７．２ｇを得た。
【００２４】
【実験２】
前記
【００２２】において得た大麦焼酎蒸留残液から以下に示す方法により有機溶媒不溶性画分を分画した。即ち、前記大麦焼酎蒸留残液を８０００ｒｐｍ、１０ｍｉｎの条件で遠心分離して大麦焼酎蒸留残液の液体分を得、得られた液体分をオルガノ（株）製の合成吸着剤アンバーライトＸＡＤ−１６を充填したカラムに通して吸着分離処理に付すことにより、該カラムの合成吸着剤に対して非吸着性を示す素通り液からなる合成吸着剤非吸着画分を分取した。得られた合成吸着剤非吸着画分をＢｒｉｘ１０に調整し、Ｂｒｉｘ１０に調整した該合成吸着剤非吸着画分１０Ｌを、５Ｌ容量のオルガノ（株）製アンバーライトＩＲＣ７６（弱酸性陽イオン交換樹脂）を充填したカラムに通して、イオン交換樹脂非吸着画分を得、得られたイオン交換樹脂非吸着画分をＢｒｉｘ６０に調整後、終濃度７５容量％になるようにエタノールを加え、８０００ｒｐｍ、１０ｍｉｎの条件で遠心分離して有機溶媒不溶性画分を分取し、該有機溶媒不溶性画分を凍結乾燥に付すことにより有機溶媒不溶性画分の凍結乾燥物５３ｇを得た。
【００２５】
【実験３】
前記
【００２２】において得た大麦焼酎蒸留残液から以下に示す方法により有機溶媒不溶性画分を分画した。即ち、前記大麦焼酎蒸留残液を８０００ｒｐｍ、１０ｍｉｎの条件で遠心分離して大麦焼酎蒸留残液の液体分を得、得られた液体分をオルガノ（株）製の合成吸着剤アンバーライトＸＡＤ−１６を充填したカラムに通して吸着分離処理に付すことにより、該カラムの合成吸着剤に対して非吸着性を示す素通り液からなる合成吸着剤非吸着画分を分取した。得られた合成吸着剤非吸着画分をＢｒｉｘ１０に調整し、Ｂｒｉｘ１０に調整した該合成吸着剤非吸着画分１０Ｌを、５Ｌ容量のオルガノ（株）製アンバーライトＩＲＡ６７（弱酸性陰イオン交換樹脂）を充填したカラムに通して、イオン交換樹脂非吸着画分を得、得られたイオン交換樹脂非吸着画分をＢｒｉｘ６０に調整後、終濃度７５容量％になるようにエタノールを加え、８０００ｒｐｍ、１０ｍｉｎの条件で遠心分離して有機溶媒不溶性画分を分取し、該有機溶媒不溶性画分を凍結乾燥に付すことにより有機溶媒不溶性画分の凍結乾燥物５９ｇを得た。
【００２６】
【実験４】
前記
【００２２】において得た大麦焼酎蒸留残液から以下に示す方法により有機溶媒不溶性画分を分画した。即ち、前記大麦焼酎蒸留残液を８０００ｒｐｍ、１０ｍｉｎの条件で遠心分離して大麦焼酎蒸留残液の液体分を得、得られた液体分をオルガノ（株）製の合成吸着剤アンバーライトＸＡＤ−１６を充填したカラムに通して吸着分離処理に付すことにより、該カラムの合成吸着剤に対して非吸着性を示す素通り液からなる合成吸着剤非吸着画分を分取した。得られた合成吸着剤非吸着画分をＢｒｉｘ１０に調整し、Ｂｒｉｘ１０に調整した該合成吸着剤非吸着画分１０Ｌを、５Ｌ容量のオルガノ（株）製アンバーライトＩＲ１２０Ｂ（強酸性陽イオン交換樹脂）を充填したカラムに通して、イオン交換樹脂非吸着画分を得、得られたイオン交換樹脂非吸着画分をＢｒｉｘ６０に調整後、終濃度７５容量％になるようにエタノールを加え、８０００ｒｐｍ、１０ｍｉｎの条件で遠心分離して有機溶媒不溶性画分を分取し、該有機溶媒不溶性画分を凍結乾燥に付すことにより有機溶媒不溶性画分の凍結乾燥物５６ｇを得た。
【００２７】
【実験５】
前記
【００２２】において得た大麦焼酎蒸留残液から以下に示す方法により有機溶媒不溶性画分を分画した。即ち、前記大麦焼酎蒸留残液を８０００ｒｐｍ、１０ｍｉｎの条件で遠心分離して大麦焼酎蒸留残液の液体分を得、得られた液体分をオルガノ（株）製の合成吸着剤アンバーライトＸＡＤ−１６を充填したカラムに通して吸着分離処理に付すことにより、該カラムの合成吸着剤に対して非吸着性を示す素通り液からなる合成吸着剤非吸着画分を分取した。得られた合成吸着剤非吸着画分をＢｒｉｘ１０に調整し、Ｂｒｉｘ１０に調整した該合成吸着剤非吸着画分１０Ｌを、５Ｌ容量のオルガノ（株）製アンバーライト２００ＣＴ（強酸性陽イオン交換樹脂）を充填したカラムに通して、イオン交換樹脂非吸着画分を得、得られたイオン交換樹脂非吸着画分をＢｒｉｘ６０に調整後、終濃度７５容量％になるようにエタノールを加え、８０００ｒｐｍ、１０ｍｉｎの条件で遠心分離して有機溶媒不溶性画分を分取し、該有機溶媒不溶性画分を凍結乾燥に付すことにより有機溶媒不溶性画分の凍結乾燥物４１ｇを得た。
【００２８】
【実験６】
前記
【００２２】において得た大麦焼酎蒸留残液から以下に示す方法により有機溶媒不溶性画分を分画した。即ち、前記大麦焼酎蒸留残液を８０００ｒｐｍ、１０ｍｉｎの条件で遠心分離して大麦焼酎蒸留残液の液体分を得、得られた液体分をオルガノ（株）製の合成吸着剤アンバーライトＸＡＤ−１６を充填したカラムに通して吸着分離処理に付すことにより、該カラムの合成吸着剤に対して非吸着性を示す素通り液からなる合成吸着剤非吸着画分を分取した。得られた合成吸着剤非吸着画分をＢｒｉｘ１０に調整し、Ｂｒｉｘ１０に調整した該合成吸着剤非吸着画分１０Ｌを、５Ｌ容量のオルガノ（株）製アンバーライトＩＲＡ４０２ＢＬ（最強塩基性陰イオン交換樹脂）を充填したカラムに通して、イオン交換樹脂非吸着画分を得、得られたイオン交換樹脂非吸着画分をＢｒｉｘ６０に調整後、終濃度７５容量％になるようにエタノールを加え、８０００ｒｐｍ、１０ｍｉｎの条件で遠心分離して有機溶媒不溶性画分を分取し、該有機溶媒不溶性画分を凍結乾燥に付すことにより有機溶媒不溶性画分の凍結乾燥物２３ｇを得た。
【００２９】
【実験７】
前記
【００２２】において得た大麦焼酎蒸留残液から以下に示す方法により有機溶媒不溶性画分を分画した。即ち、前記大麦焼酎蒸留残液を８０００ｒｐｍ、１０ｍｉｎの条件で遠心分離して大麦焼酎蒸留残液の液体分を得、得られた液体分をオルガノ（株）製の合成吸着剤アンバーライトＸＡＤ−１６を充填したカラムに通して吸着分離処理に付すことにより、該カラムの合成吸着剤に対して非吸着性を示す素通り液からなる合成吸着剤非吸着画分を分取した。得られた合成吸着剤非吸着画分をＢｒｉｘ１０に調整し、Ｂｒｉｘ１０に調整した該合成吸着剤非吸着画分１０Ｌを、３．５Ｌ容量のオルガノ（株）製アンバーライトＩＲＣ７６（弱酸性陽イオン交換樹脂）と１．５Ｌ容量のオルガノ（株）製アンバーライトＩＲＡ６７（弱塩基性陰イオン交換樹脂）を混合することにより得た混床イオン交換樹脂を充填したカラムに通して、混床イオン交換樹脂非吸着画分を得、得られた混床イオン交換樹脂非吸着画分をＢｒｉｘ６０に調整後、終濃度７５容量％になるようにエタノールを加え、８０００ｒｐｍ、１０ｍｉｎの条件で遠心分離して有機溶媒不溶性画分を分取し、該有機溶媒不溶性画分を凍結乾燥に付すことにより有機溶媒不溶性画分の凍結乾燥物１９ｇを得た。
【００３０】
上記実験１乃至実験７で得たそれぞれの凍結乾燥物について、以下の方法によりアルコール性肝障害に対する発症抑制作用を評価した。
即ち、７週齢Ｗｉｓｔａｒ系雄性ラット（日本チャールスリバー）８０匹にエタノール含有率を徐々に上げながら（３％→４％→５％）エタノール含有液体飼料を６日間与えて飼育した後、引き続き５％エタノール含有液体飼料で４週間飼育を行い、該４週間目にそれらのラットのそれぞれについて採血を行い、血漿を分離して血清脂質を測定し、１群１０匹として、対照群、試験群１乃至試験群７からなる８群に分けた。その際、前記各群におけるラットの平均体重に係る分散に統計学的有意差が生じないように前記８０匹のラットを振り分けた。該対照群のラットに対しては前記エタノール含有液体飼料投与群と摂取カロリーを同一にするために前記５％エタノールの代わりにマルトース−デキストリン等量混合物を添加したエタノール非含有液体飼料を２週間与えて飼育した。試験群１乃至試験群７に対しては該エタノール非含有液体飼料に前記実験１乃至実験７で得たそれぞれの凍結乾燥物粉末１％を添加した液体飼料を２週間与えて飼育した。更に対照群、試験群１乃至試験群７とは別に前記７週齢Ｗｉｓｔａｒ系雄性ラット１０匹からなる無処置群を設け、該無処置群のラットに対しては該エタノール非含有液体飼料を６週間与えて飼育した。但し、上記９群とも、各液体飼料の１日あたりの給餌量（摂取カロリー）を７０ｍｌ（７０ｋｃａｌ）に制限した。上記９群の全てについて、実験最終日（実験開始後６週間目）に、飼育したラットのそれぞれの腹部大動脈から採血を行い、肝臓を摘出した。採取した血液は血清分離後、血清総コレステロール、血清ＬＤＬ−コレステロール、血清トリグリセリド、血清リン脂質、血清ＡＬＴ（ＧＰＴ）、及び血清ＡＳＴ（ＧＯＴ）を測定した。また、摘出した肝臓については、肝臓重量、肝臓総コレステロール、肝臓トリグリセリド、及び肝臓リン脂質を測定した。得られた結果は平均値±標準誤差（ＳＥＭ）で表し、統計処理は以下の手順で行った。即ち、無処置群と対照群の比較はＳｔｕｄｅｎｔ’ｓ ｔｅｓｔ法を用いて解析し、次いで対照群に対する試験群１乃至試験群７の比較はＴｕｋｅｙ−Ｋｒａｍｅｒ法を用いて解析し、それぞれの解析において危険率０．０５％以下を有意として判定した。更に、摘出した前記肝臓から採取した肝細胞をＨＥ染色に付した後、オリンパス光学工業（株）製の生物顕微鏡ＢＸ５１を用いて２００倍の倍率で該肝細胞の形態観察を行った。
【００３１】
実験１乃至実験７で得たそれぞれの凍結乾燥物について、多糖類含量、多糖類由来のアラビノース及びキシロースの合計含量、及びアルコール性肝障害に対する発症抑制作用の実験結果から以下の事実が判明した。即ち、前記対照群と比較して、前記実験１乃至実験７で得た凍結乾燥物の全てにおいてアルコール性肝障害に対する発症抑制作用が認められた。この中で、前記実験５で得た凍結乾燥物を添加した液体飼料で飼育した試験群５において、極めて顕著なアルコール性肝障害に対する発症抑制効果が認められた。一方、前記実験１で得た凍結乾燥物を添加した液体飼料で飼育した試験群１においては、アルコール性肝障害に対する発症抑制効果の程度が最も低かった。
【００３２】
【多糖類含量の測定】
（多糖類由来のアラビノース及びキシロースの合計含量の測定）
そこで、上記実験１乃至実験７で得たそれぞれの凍結乾燥物について、多糖類含量、及び多糖類由来のアラビノース及びキシロースの合計含量を測定した。
即ち、前記実験１乃至実験７で得たそれぞれの凍結乾燥物０．０５ｇにイオン交換水１ｍｌを加えて溶解し、これに濃塩酸２００μｌを加えて、９５℃、４時間の条件で加水分解を行い、０．８０μｍのメンブランフィルターでろ過してろ液を得、該ろ液を高速液体クロマトグラフに注入して、前記実験１乃至実験７で得たそれぞれの凍結乾燥物に含まれる多糖類の含量、及び多糖類由来のアラビノース及びキシロースの合計含量を求めた。高速液体クロマトグラフ分析は、Ｗａｔｅｒｓ製Ｗａｔｅｒｓ６００を用い、検出器に昭和電工株式会社製示差屈折計ＲＩ−７１を使用し、カラムはＢｉｏＲａｄ社製Ａｍｉｎｅｘ ＨＰＸ−８７Ｈ（３００ｍｍ×７．８ｍｍ）を使用した。カラム温度は６０℃とし、移動相には５ｍＭ硫酸を用い、流量は０．５ｍｌ／ｍｉｎ、試料注入量は２０μｌとした。
【００３３】
実験１乃至実験７で得たそれぞれの凍結乾燥物の多糖類含量の測定結果、並びに、該多糖類由来のアラビノース及びキシロースの合計含量の測定結果から以下の事実が判明した。即ち、前記実験５で得た凍結乾燥物の多糖類含量、並びに、該多糖類由来のアラビノース及びキシロースの合計含量が、それぞれ最も高い値を示した。一方、前記実験１で得た凍結乾燥物の多糖類含量、並びに、該多糖類由来のアラビノース及びキシロースの合計含量が、それぞれ最も低い値を示した。更に、前記実験１乃至実験７で得た凍結乾燥物のそれぞれが有するアルコール性肝障害に対する発症抑制作用の強さは、該凍結乾燥物に含まれる多糖類由来のアラビノース及びキシロースの合計含量に比例して高まる傾向が認められることが明らかになった。
【００３４】
以上の結果から、前記実験１乃至実験７で得たそれぞれの凍結乾燥物が有するアルコール性肝障害に対する発症抑制作用は、該凍結乾燥物に含まれる、アラビノース及びキシロースを主たる構成要素とする多糖類に由来している可能性が極めて高いことが明らかになった。
【００３５】
【分子量分布の測定】
そこで、上記実験１乃至実験７で得たそれぞれの凍結乾燥物に含まれる、アラビノース及びキシロースを主たる構成要素とする多糖類の分子量を明らかにするために、それぞれの有機溶媒不溶性画分が有する分子量分布を測定した。
即ち、昭和電工株式会社製のＳｈｏｄｅｘ ｓｔａｎｄａｒｄ Ｐ−８２（分子量１３００乃至１６６００００）、及びマルトトリオース（分子量５０４）から成る分子量標準品をそれぞれ別々に０．１ｍｏｌ ／Ｌ硝酸ナトリウム溶液に溶解して０．０５Ｗ／Ｖ％濃度の標準液を得、該標準液を高速液体クロマトグラフに注入して検量線を作成した。次に、前記実験１乃至実験７で得たそれぞれの凍結乾燥物０．０２ｇを用意し、これに０．１ｍｏｌ／Ｌ硝酸ナトリウム溶液１０ｍｌ を加え、室温で一晩放置した後、孔径０．４５μｍのメンブランフィルターでろ過してろ液を得、該ろ液を高速液体クロマトグラフに注入して、システムインスツルメンツ株式会社製４８０データステーションＧＰＣプログラムを用いて分子量分布を求めた。高速液体クロマトグラフ分析は、昭和電工株式会社製ＳｈｏｄｅｘＧＰＣ ＳＹＳＴＥＭ−２１ を用い、検出器に昭和電工株式会社製示差屈折計ＲＩ−７１Ｓを使用し、カラムは東ソー株式会社製ＴＳＫｇｅｌ ＧＭＰＷＸＬ（φ７．８ｍｍ×３００ｍｍ）を２本連結して使用した。カラム温度は４０℃とし、移動相には０．１ｍｏｌ／Ｌ硝酸ナトリウム溶液を用い、流量は１．０ｍｌ／ｍｉｎ、試料注入量は１００μｌとした。
【００３６】
上記方法により実験１乃至実験７で得たそれぞれの凍結乾燥物についてそれぞれの分子量分布を測定した結果、該凍結乾燥物に含まれる多糖類由来のアラビノース及びキシロースの合計含量が高いほど、分子量１０，０００乃至１００，０００からなる画分の割合が高まることが判明した。
【００３７】
このようなことから、本発明者らは、前記凍結乾燥物に含まれる、アラビノース及びキシロースを主たる構成要素とする多糖類は、主として１０，０００乃至１００，０００の分子量を有しているのではないかと推測し、限外濾過を用いて、有機溶媒不溶性画分の分画精製を検討した。
［限外濾過による有機溶媒不溶性画分の分画精製］
実験１乃至実験７で得たそれぞれの凍結乾燥物について、上記方法により分子量分布を測定した結果、実験５で得た凍結乾燥物が、分子量１０，０００乃至１００，０００からなる画分の割合が最も高く、しかも多糖類由来のアラビノース及びキシロースの合計含量も最も高いことが判明した。そこで、以下に示す手順により、限外濾過処理を介した前記凍結乾燥物の分画精製を行った。
即ち、
【００２２】で得た大麦焼酎蒸留残液を、８０００ｒｐｍ、１０ｍｉｎの条件で遠心分離して大麦焼酎蒸留残液の液体分を得、得られた液体分をオルガノ（株）製の合成吸着剤アンバーライトＸＡＤ−１６を充填したカラムに通して吸着分離処理に付すことにより、該カラムの合成吸着剤に対して非吸着性を示す素通り液からなる合成吸着剤非吸着画分を分取した。得られた合成吸着剤非吸着画分をＢｒｉｘ１０に調整し、Ｂｒｉｘ１０に調整した該合成吸着剤非吸着画分１Ｌを、５００ｍｌ容量のオルガノ（株）製アンバーライト２００ＣＴ（強酸性陽イオン交換樹脂）を充填したカラムに通して、イオン交換樹脂非吸着画分を得、得られたイオン交換樹脂非吸着画分をＡ／Ｇ テクノロジー社製の限外濾過膜ＵＦＰ−３０−Ｅ−４ＭＡ（分画分子量３０，０００）による処理に付して濃縮液を得、得られた濃縮液をＢｒｉｘ２０に調整後、終濃度７５容量％になるようにエタノールを加え、８０００ｒｐｍ、１０ｍｉｎの条件で遠心分離して有機溶媒不溶性画分を分取し、該有機溶媒不溶性画分を凍結乾燥に付すことにより有機溶媒不溶性画分の凍結乾燥物１．３ｇを得た。
【００３８】
得られた凍結乾燥物について、
【００３２】に記載の方法により、多糖類由来のアラビノース及びキシロースの合計含量を測定した。その結果、多糖類由来のアラビノース及びキシロースの合計含量は、７８．５重量％にまで高まっていることが判明した。また、得られた凍結乾燥物について、
【００３５】に記載の方法により分子量分布の測定を行った。その結果、該凍結乾燥物の分子量分布は１００，０００以上が１１％、３０，０００乃至１００，０００が２９％、１０，０００乃至３０，０００が２４％、３，０００乃至１０，０００が２１％、１，０００乃至３，０００が６％、５００乃至１，０００が２％、５００以下が７％であることが判明した。そして、得られたクロマトグラムの結果から、該凍結乾燥物のクロマトグラムは、分子量３０，０００乃至１００，０００の範囲に最も高いピークを有することが明らかになった。そこで、
【００３０】に記載の方法により、
【００３７】で得た凍結乾燥物のアルコール性肝障害に対する発症抑制作用を評価した。その結果、該凍結乾燥物が有するアルコール性肝障害に対する発症抑制作用は際立ったものであることが判明した。
【００３９】
以上の実験結果から、大麦焼酎蒸留残液の液体分から得られる前記有機溶媒不溶性画分からなる凍結乾燥物が有するアルコール性肝障害に対する前記発症抑制作用は、該有機溶媒不溶性画分からなる凍結乾燥物に含まれる、アラビノース及びキシロースを主たる構成要素とする多糖類に由来し、こうしたアラビノース及びキシロースを主たる構成要素とする多糖類を著量含有する組成物は、大麦を原料とする焼酎製造において副生する大麦焼酎蒸留残液を固液分離して液体分を得、該液体分を合成吸着剤を使用する吸着分離処理に付して合成吸着剤非吸着画分を得、該合成吸着剤非吸着画分をイオン交換樹脂を使用するイオン交換処理に付してイオン交換樹脂非吸着画分を得、得られたイオン交換樹脂非吸着画分を限外濾過膜を使用する限外濾過処理に付して濃縮液を得、該濃縮液に有機溶媒を添加することにより有機溶媒不溶性画分として分取できることが判明した。そして、該有機溶媒不溶性画分からなる凍結乾燥物は、成分分析の結果、多糖類７８．５重量％、粗タンパク３．３重量％、有機酸０．２重量％、遊離糖類１．５重量％を含有し、該画分は多糖類を主たる成分として含有することが判った。
【００４０】
そこで、本発明者らは、アルコール性肝障害に対して発症抑制作用を有することが判明した前記合成吸着剤非吸着画分の凍結乾燥物粉末、及びアルコール性肝障害に対して際立った発症抑制作用を有することが判明した前記有機溶媒不溶性画分の凍結乾燥物粉末が、既に発症したアルコール性肝障害に対して治癒作用を有するか否かを明らかにするために、以下の実験を行った。
即ち、７週齢Ｗｉｓｔａｒ系雄性ラット（日本チャールスリバー）３０匹にエタノール含有率を徐々に上げながら（３％→４％→５％）エタノール含有液体飼料を６日間与えて飼育した後、引き続き５％エタノール含有液体飼料で４週間飼育を行い、該４週間目にそれらのラットのそれぞれについて採血を行い、血漿を分離して血清脂質を測定し、１群１０匹として、対照群、試験群１、及び試験群２からなる３群に分けた。その際、前記各群におけるラットの平均体重に係る分散に統計学的有意差が生じないように前記３０匹のラットを振り分けた。該対照群のラットに対しては前記エタノール含有液体飼料投与群と摂取カロリーを同一にするために前記５％エタノールの代わりにマルトース−デキストリン等量混合物を添加したエタノール非含有液体飼料を２週間与えて飼育した。該試験群１に対しては該エタノール非含有液体飼料に前記合成吸着剤非吸着画分の凍結乾燥物粉末１％を添加した液体飼料を２週間与えて飼育した。該試験群２に対しては該エタノール非含有液体飼料に前記有機溶媒不溶性画分の凍結乾燥物粉末１％を添加した液体飼料を２週間与えて飼育した。更に対照群、試験群１及び試験群２とは別に前記７週齢Ｗｉｓｔａｒ系雄性ラット１０匹からなる無処置群を設け、該無処置群のラットに対しては該エタノール非含有液体飼料を６週間与えて飼育した。但し、上記４群とも、各液体飼料の１日あたりの給餌量（摂取カロリー）を７０ｍｌ（７０ｋｃａｌ）に制限した。上記４群の全てについて、実験最終日（実験開始後６週間目）に、飼育したラットのそれぞれの腹部大動脈から採血を行い、肝臓を摘出した。採取した血液は血清分離後、血清総コレステロール、血清ＬＤＬ−コレステロール、血清トリグリセリド、血清リン脂質、血清ＡＬＴ（ＧＰＴ）、及び血清ＡＳＴ（ＧＯＴ）を測定した。また、摘出した肝臓については、肝臓重量、肝臓総コレステロール、肝臓トリグリセリド、及び肝臓リン脂質を測定した。得られた結果は平均値±標準誤差（ＳＥＭ）で表し、統計処理は以下の手順で行った。即ち、無処置群と対照群の比較はＳｔｕｄｅｎｔ’ｓ ｔｅｓｔ法を用いて解析し、次いで対照群に対するＡ群及びＢ群の比較はＴｕｋｅｙ−Ｋｒａｍｅｒ法を用いて解析し、それぞれの解析において危険率０．０５％以下を有意として判定した。更に、摘出した前記肝臓から採取した肝細胞をＨＥ染色に付した後、オリンパス光学工業（株）製の生物顕微鏡ＢＸ５１を用いて２００倍の倍率で該肝細胞の形態観察を行った。
【００４１】
その結果、対照群は、エタノール含有液体飼料を与えて飼育することにより上昇した血清総コレステロール濃度、血清ＬＤＬ−コレステロール濃度、及び肝臓トリグリセリド濃度が僅かに低下するのみで、それぞれの正常値に近似する程度が極めて小なるものであった。一方、試験群１及び試験群２の血清総コレステロール濃度、血清ＬＤＬ−コレステロール濃度、血清トリグリセリド濃度、血清リン脂質濃度、血清ＡＬＴ（ＧＰＴ）濃度、血清ＡＳＴ（ＧＯＴ）濃度、及び肝臓総コレステロール濃度のそれぞれの値は、対照群のそれぞれの値と比較して有意に低い値、即ち、それぞれの正常値と実質的に同等の値を示し、特に、試験群２のほうが試験群１よりも更に正常値に近似した値を示した。また、試験群１及び試験群２においては、肝細胞の生物顕微鏡観察においても肝小葉の終末肝静脈周辺領域における肝細胞壊死と風船様腫大がほとんど認められなかった。
即ち、前記有機溶媒不溶性画分の凍結乾燥物粉末が有するアルコール性肝障害に対する治癒作用は、前記合成吸着剤非吸着画分の凍結乾燥物粉末が有する同作用を凌駕するものであることが明らかになった。
【００４２】
以上の実験結果から、大麦を原料とする焼酎の製造において副成する大麦焼酎蒸留残液を固液分離して液体分を得、該液体分を合成吸着剤を使用する吸着分離処理に付して合成吸着剤非吸着画分を得、該合成吸着剤非吸着画分をイオン交換樹脂を使用するイオン交換処理に付してイオン交換樹脂非吸着画分を得、該イオン交換樹脂非吸着画分を限外濾過膜を使用する限外濾過処理に付して濃縮液を得、該濃縮液に有機溶媒を添加することにより得られる有機溶媒不溶性画分が、アルコール性肝障害に対する極めて強力な発症抑制作用及び治癒作用を有することが判明した。当該事実から、大麦を原料とする焼酎の製造において副成する大麦焼酎蒸留残液を固液分離することにより得られる液体分は、アルコール性肝障害の発症抑制及び治癒に寄与する成分を含有し、該成分は、前記液体分を合成吸着剤を使用する吸着分離処理に付して合成吸着剤非吸着画分を得、該合成吸着剤非吸着画分をイオン交換樹脂を使用するイオン交換処理に付してイオン交換樹脂非吸着画分を得、該イオン交換樹脂非吸着画分を限外濾過膜を使用する限外濾過処理に付して濃縮液を得、該濃縮液に有機溶媒を添加することにより得られる有機溶媒不溶性画分の中に分画されて含有されることが判明した。
【００４３】
上述したように本発明者らは、大麦を原料とする焼酎製造において副成する大麦焼酎蒸留残液を固液分離することにより得られる液体分は、アルコール性肝障害の発症抑制及び治癒に寄与する成分を含有し、該成分は、前記液体分を合成吸着剤を使用する吸着分離処理に付して合成吸着剤非吸着画分を得、該合成吸着剤非吸着画分をイオン交換樹脂を使用するイオン交換処理に付してイオン交換樹脂非吸着画分を得、該イオン交換樹脂非吸着画分を限外濾過膜を使用する限外濾過処理に付して濃縮液を得、該濃縮液に有機溶媒を添加することにより得られる有機溶媒不溶性画分からなる組成物が、アルコール性肝障害に対して際立った発症抑制作用及び治癒作用を有することを見出した。大麦焼酎蒸留残液から得られる有機溶媒不溶性画分についてのこの発見は、今までに全く例のない新事実である。このように、本発明は、該有機溶媒不溶性画分を治療目的で医薬として使用できるという該有機溶媒不溶性画分の有用な用途を創出するものである。
【００４４】
本発明者らは、上記課題を達成すべく、実験を介して鋭意研究を重ねた結果、大麦を原料とする焼酎製造において副成する大麦焼酎蒸留残液を固液分離して液体分を得、該液体分を合成吸着剤を使用する吸着分離処理に付して合成吸着剤非吸着画分を得、該合成吸着剤非吸着画分をイオン交換樹脂を使用するイオン交換処理に付してイオン交換樹脂非吸着画分を得、該イオン交換樹脂非吸着画分を限外濾過膜を使用する限外濾過処理に付して濃縮液を得、該濃縮液に有機溶媒を添加することにより有機溶媒不溶性画分を分取し、該有機溶媒不溶性画分を成分分析に付した結果、多糖類７８．５重量％、粗タンパク３．３重量％、有機酸０．２重量％、遊離糖類１．５重量％を含有し、該画分は多糖類を主たる成分として含有することが判った。
更に該有機溶媒不溶性画分を凍結乾燥に付した場合、白色の性状を有することが判明した。そして、こうした特徴を有する該有機溶媒不溶性画分は、アルコール誘発性肝障害に対して際立った発症抑制作用及び治癒作用を有することが判明した。本発明はこれらの判明した事実に基づくものである。
【００４５】
なお、特許文献１に記載の脂肪肝抑制作用を有する組成物は、本発明の組成物と同様に大麦焼酎蒸留残液から分取されるものである。しかし特許文献１に記載の該組成物が有する主たる成分は分子量３０００以下のものであり、該組成物に含まれるヘミセルロースはキシロースを主たる構成要素とすることから、本発明の組成物とは明確に区別される別異のものである。
【００４６】
上述したように、特許文献２に記載のアルコール性脂肪肝抑制剤は、トウモロコシフスマから澱粉質及び蛋白質を除去した残部をアルカリ抽出することにより得られるヘミセルロースをキシラナーゼで処理することにより得られる部分分解物を有効成分とするものである。また、非特許文献４には、前記特許文献２に記載のトウモロコシフスマから得られたヘミセルロースの部分分解物（商品名セルエース）が、
【０００４】に記載したとおり、キシロース、アラビノース、ウロン酸、ガラクトース、及びグルコースを含有するものであることが記載されている。よって、特許文献２と非特許文献４は、同じ「トウモロコシフスマから得られたヘミセルロースの部分分解物（商品名セルエース）」に係るものである。一方、本発明の組成物は、前記トウモロコシフスマとは全く異なる大麦焼酎蒸留残液から得られるものである。即ち、本発明の組成物は、特許文献２に記載のヘミセルロースの部分分解物の製造工程とは全く異なる製造工程を介して得られるものである。そして、本発明の組成物は、アラビノース、キシロース及びグルコースのみを含有し、特許文献２に記載のヘミセルロースの部分分解物が有するウロン酸を実質的に含有していない。よって、本発明の組成物は、特許文献２に記載の「トウモロコシフスマより得られたヘミセルロースの部分分解物」を有効成分とするアルコール性脂肪肝抑制剤から明確に区別される別異のものであることは明白である。
【００４７】
尚、非特許文献４には、「トウモロコシフスマより得られたヘミセルロースの部分分解物（商品名セルエース）」の分子量が約２０万であることが記載されているが、当該分子量を決定づけるに至った実験データは全く記載されていない。そこで、本発明者らは、特許文献２において、ヘミセルロースの部分分解物が商品名「セルエース」（日本食品化工株式会社製）として市販されていることが記載されていることから、「セルエース」を入手し、前記
【００３５】に記載の方法により該セルエースの分子量分布を測定した。その結果、該セルエースの分子量分布は、１００万以上が１％、３０万乃至１００万が９％、１００，０００乃至３０万が３０％、３０，０００乃至１００，０００が３０％、１０，０００乃至３０，０００が１１％、３０００乃至１０，０００が４％、１０００乃至３０００が２％、１０００以下が１３％であり、そのクロマトグラムは分子量１００，０００乃至３０万の範囲にピークトップを有する単一のピークのみからなり、その重量平均分子量（Ｍｗ）は１５０，０００であることが判明した。
一方、上述したように本発明の組成物の分子量分布は、１００，０００以上がわずか１１％に過ぎず、分子量３，０００乃至１００，０００からなる成分が全体の７４％を占めている。しかも、本発明の組成物が示すクロマトグラムは、分子量３０，０００乃至１００，０００の範囲に最も高いピークを有している。
従って、本発明の組成物が有する分子量分布は、「セルエース」、即ち、トウモロコシフスマから得られたヘミセルロースの部分分解物からなる組成物が有する分子量分布とは全く異なる。また上述したように、本発明の組成物は、トウモロコシフスマから得られたヘミセルロースの部分分解物が含有するウロン酸を実質的に含有していない。この点からしても、本発明の組成物が、特許文献２に記載の「ヘミセルロースの部分分解物」からなるアルコール性脂肪肝抑制剤とは明らかに区別される別異のものであることは明白である。
【００４８】
【実施態様例】
本発明は上記目的を達成するものであり、アルコール性肝障害に対して強力な発症抑制作用及び治癒作用を有する組成物及びその製造方法を提供する。即ち、大麦を原料とする焼酎の製造において副成する大麦焼酎蒸留残液を固液分離して液体分を得、該液体分を合成吸着剤を使用する吸着分離処理に付して合成吸着剤非吸着画分を得、該合成吸着剤非吸着画分をイオン交換樹脂を使用するイオン交換処理に付してイオン交換樹脂非吸着画分を得、該イオン交換樹脂非吸着画分を限外濾過膜を使用する限外濾過処理に付して濃縮液を得、該濃縮液に有機溶媒を添加することにより得られる有機溶媒不溶性画分からなる、アルコール性肝障害に対して際立った発症抑制作用及び治癒作用を有する組成物及びその製造方法を提供する。また、本発明は、該組成物からなる医薬を提供する。
【００４９】
以下に、本発明の好ましい態様について述べるが、本発明はこれらに限定されるものではない。
本発明の組成物は以下のようにして製造される。即ち該組成物の製造方法は、大麦を使用する蒸留酒の製造において副成する大麦焼酎蒸留残液を固液分離して液体分を得る第１の工程、該液体分を合成吸着剤を使用する吸着分離処理に付して合成吸着剤非吸着画分を得る第２の工程、該合成吸着剤非吸着画分をイオン交換樹脂を使用するイオン交換処理に付してイオン交換樹脂非吸着画分を得る第３の工程、該イオン交換樹脂非吸着画分を限外濾過膜を使用する限外濾過処理に付して濃縮液を得る第４の工程、及び該濃縮液に有機溶媒を添加することにより有機溶媒不溶性画分を分取する第５の工程からなる。
以下に、本発明の該製造方法を実施する際に使用する、大麦を原料とする焼酎の製造において副成する大麦焼酎蒸留残液、及び各工程について詳述する。
【００５０】
本発明において使用する大麦焼酎蒸留残液は、代表的には、大麦又は精白大麦を原料として大麦麹及び蒸麦を製造し、得られた大麦麹及び蒸麦中に含まれるでんぷんを該大麦麹の麹により糖化し、それらを酵母によるアルコール発酵に付して焼酎熟成もろみを得、得られた焼酎熟成もろみを減圧蒸留または常圧蒸留等の単式蒸留装置を用いて蒸留する際に蒸留残渣として副生するもの、即ち、大麦焼酎の蒸留残液を意味する。
【００５１】
本発明において、大麦焼酎蒸留残液を得るに際して、大麦焼酎の製造に用いる大麦麹は、通常の大麦焼酎製造において行われている製麹条件で製造すればよく、用いる麹菌株としては、一般的に大麦焼酎製造で使用する白麹菌（Ａｓｐｅｒｇｉｌｌｕｓ ｋａｗａｃｈｉｉ）が好ましい。或いは泡盛製造で使用する黒麹菌（Ａｓｐｅｒｇｉｌｌｕｓ ａｗａｍｏｒｉ）及び清酒製造等で使用する黄麹（Ａｓｐｅｒｇｉｌｌｕｓ ｏｒｙｚａｅ）などのＡｓｐｅｒｇｉｌｌｕｓ属の菌株を用いることもできる。また大麦焼酎の製造に用いる酵母は、一般的に焼酎製造の際に使用する各種の焼酎醸造用酵母を使用することができる。
【００５２】
本発明において、大麦焼酎の製造における蒸留工程で得られた大麦焼酎蒸留残液を固液分離して液体分を得る第１の工程は、大麦焼酎蒸留残液から原料大麦、あるいは大麦麹由来の水不溶性の発酵残渣等のＳＳ分を除去することを目的として行うものである。この第１の工程における当該固液分離は、スクリュープレス方式やローラープレス方式の固液分離方法によるか、或いはろ過圧搾式の固液分離機を用いて予備分離を行い、次いで遠心分離機、ケイソウ土ろ過装置、セラミックろ過装置、或いはろ過圧搾機等を用いて本発明により実施できる本固液分離処理を行う。
【００５３】
第１の工程で得られた大麦焼酎蒸留残液の液体分を合成吸着剤を使用した吸着分離処理に付すことにより合成吸着剤非吸着画分を得る第２の工程は、大麦焼酎蒸留残液の液体分に含まれているポリフェノール等の成分を除去することを目的として行うものである。この第２の工程で使用する合成吸着剤としては、芳香族系、芳香族系修飾型、或いはメタクリル系の合成吸着剤を用いることができる。第２の工程で使用する合成吸着剤の好適な具体例としては、オルガノ（株）製のアンバーライトＸＡＤ−４、アンバーライトＸＡＤ−１６、アンバーライトＸＡＤ−１１８０及びアンバーライトＸＡＤ−２０００、三菱化学（株）製のセパビーズＳＰ８５０及びダイヤイオンＨＰ２０等の芳香族系（又はスチレン系とも言う）合成吸着剤、オルガノ（株）製のアンバーライトＸＡＤ−７及び三菱化学（株）製のダイヤイオンＨＰ２ＭＧ等のメタクリル系（又はアクリル系とも言う）合成吸着剤を挙げることができる。これらの他、場合によっては三菱化学（株）製のセパピーズＳＰ２０７等の芳香族系修飾型合成吸着剤を用いることができる。
【００５４】
第２の工程で得られた合成吸着剤非吸着画分をイオン交換樹脂を使用するイオン交換処理に付してイオン交換樹脂非吸着画分を得る第３の工程においては、該合成吸着剤非吸着画分に含まれるアミノ酸、ペプチド、タンパク質、及び有機酸を、イオン交換樹脂を用いて除去することを目的として行うものである。第３の工程で使用するイオン交換樹脂としては、陽イオン交換樹脂、陰イオン交換樹脂、或いは両者を混合した混床イオン交換樹脂を使用することができる。陽イオン交換樹脂の場合、強酸性陽イオン交換樹脂及び弱酸性陽イオン交換樹脂のいずれであっても使用することができ、陰イオン交換樹脂の場合、強塩基性陰イオン交換樹脂及び弱塩基性陰イオン交換樹脂のいずれであっても使用することができる。また混床イオン交換樹脂の場合には、上述した陽イオン交換樹脂と陰イオン交換樹脂を自由に組み合わせて所定の割合で混合して使用することができる。こうしたイオン交換樹脂の好適な具体例としては、オルガノ（株）製のアンバーライト２００ＣＴ及びアンバーライトＩＲ１２０Ｂ等の強酸性陽イオン交換樹脂、アンバーライトＩＲＣ７６等の弱酸性陽イオン交換樹脂、アンバーライトＩＲＡ４０２ＢＬ等の最強塩基性陰イオン交換樹脂、アンバーライトＩＲＡ６７等の弱塩基性陰イオン交換樹脂、或いは、前記弱酸性陽イオン交換樹脂と前記弱塩基性陰イオン交換樹脂の両者を所定の割合で混合することにより得られる混床型イオン交換樹脂等を用いることができる。これらのうち、大麦焼酎蒸留残留液に含まれているアミノ酸やペプチド等を除去する観点においては、前記弱酸性陽イオン交換樹脂アンバーライトＩＲＣ７６と前記弱塩基性陰イオン交換樹脂アンバーライトＩＲＡ６７の両者を所定の割合で混合することにより得られる混床型イオン交換樹脂、或いは、強酸性陽イオン交換樹脂アンバーライト２００ＣＴを使用することが特に好ましい。
【００５５】
第３の工程で得られた該イオン交換樹脂非吸着画分を限外濾過膜を使用する限外濾過処理に付して濃縮液を得る第４の工程においては、ＮＫ細胞賦活化作用に関与する成分である、アラビノース及びキシロースを主たる構成要素とする多糖類を限外濾過膜を用いて濃縮することを目的として行うものである。第４の工程で使用する限外濾過膜としては、いかなる膜材質及び膜モジュール型式のものであっても使用することができ、分画分子量は、好ましくは３０００以上、特に好ましくは１０，０００乃至５０，０００のものを使用することができる。
【００５６】
第４の工程で得られた濃縮液に有機溶媒を添加することにより有機溶媒不溶性画分を分取する第５の工程においては、適宜の有機溶媒を所定の終濃度になるまで添加する。この場合、有機溶媒はエタノールが至適であるが、これに限定されるものではない。有機溶媒の終濃度は成分の生産効率に影響し、該有機溶媒の最適終濃度は、好ましくは５容量％以上、より好ましくは、３０乃７５容量％である。
【００５７】
このようにして得られる本発明の組成物である上記有機溶媒不溶性画分はそのままの状態で、或いはこれを凍結乾燥等に付すことにより乾燥物粉末にして、アルコール性肝障害に対する強力な発症抑制作用及び治癒作用を有する医薬として使用することができる。
【００５８】
【実施例】
以下に実施例を挙げて本発明を具体的に説明するが、本発明はこれらの実施例によって何ら限定されるものではない。
【００５９】
【実施例１】
前記
【００２２】において得た大麦焼酎蒸留残液を、８０００ｒｐｍ、１０ｍｉｎの条件で遠心分離して大麦焼酎蒸留残液の液体分を得、得られた液体分をオルガノ（株）製の合成吸着剤アンバーライトＸＡＤ−１６を充填したカラムに通して吸着分離処理に付すことにより、該カラムの合成吸着剤に対して非吸着性を示す素通り液からなる合成吸着剤非吸着画分を分取した。得られた合成吸着剤非吸着画分をＢｒｉｘ１０に調整後、Ｂｒｉｘ１０に調整した該合成吸着剤非吸着画分１Ｌを、５００ｍｌ容量のオルガノ（株）製アンバーライト２００ＣＴ（強酸性陽イオン交換樹脂）を充填したカラムに通して、イオン交換樹脂非吸着画分を得、得られたイオン交換樹脂非吸着画分をＡ／Ｇ テクノロジー社製の限外濾過膜ＵＦＰ−３０−Ｅ−４ＭＡ（分画分子量３０，０００）による処理に付して濃縮液を得、得られた濃縮液をＢｒｉｘ２０に調整後、終濃度７５容量％になるようにエタノールを加え、８０００ｒｐｍ、１０ｍｉｎの条件で遠心分離して有機溶媒不溶性画分を分取し、該有機溶媒不溶性画分を凍結乾燥に付すことにより有機溶媒不溶性画分の凍結乾燥物１．３ｇを得た。該凍結乾燥物を粉砕し灰白色で無味無臭の組成物を得た。
【００６０】
【比較例１】
前記
【００２２】において得た大麦焼酎蒸留残液を８０００ｒｐｍ、１０ｍｉｎの条件で遠心分離して大麦焼酎蒸留残液の液体分を得、得られた液体分をオルガノ（株）製の合成吸着剤アンバーライトＸＡＤ−１６を充填したカラムに通して吸着分離処理に付すことにより、該カラムの合成吸着剤に対して非吸着性を示す素通り液からなる合成吸着剤非吸着画分を分取した。得られた合成吸着剤非吸着画分１Ｌを凍結乾燥に付すことにより該合成吸着剤非吸着画分の凍結乾燥物５７．２ｇを得た。該凍結乾燥物を粉砕し灰白色で無味無臭の組成物を得た。
【００６１】
実施例１で得た凍結乾燥物粉末（組成物）、及び比較例１で得た凍結乾燥物粉末のそれぞれを以下の試験例１に供し、アルコール性肝障害に対する発症抑制作用を評価した。
【００６２】
【試験例１】
本発明の組成物が有するアルコール性肝障害の発症に対する顕著な抑制作用を明らかにするために以下の試験例１を行った。
即ち、３週齢Ｗｉｓｔａｒ系雄性ラット（日本ＳＬＣ）３６匹にエタノール含有率を徐々に上げながら（３％→４％→５％）エタノール含有液体飼料を６日間与えた後、１群１２匹として、対照群、試験群１、及び試験群２からなる３群に分けた。その際、前記各群におけるラットの平均体重に係る分散に統計学的有意差が生じないように前記３６匹のラットを振り分けた。対照群のラットに対しては５％エタノール含有液体飼料を４週間与えて飼育した。試験群１のラットに対しては５％エタノール含有液体飼料に実施例１で得た凍結乾燥物粉末１％を添加した液体飼料を４週間与えて飼育した。試験群２のラットに対しては５％エタノール含有液体飼料に比較例１で得た凍結乾燥物粉末１％を添加した液体飼料を４週間与えて飼育した。対照群、試験群１、及び試験群２とは別に前記３週齢Ｗｉｓｔａｒ系雄性ラット１２匹からなる無処置群を設け、該無処置群のラットに対しては他の３群と摂取カロリーを同一にするために５％エタノールの代わりにマルトース−デキストリン等量混合物を添加したエタノール非含有液体飼料を４週間与えて飼育した。但し、上記４群とも、各液体飼料の１日あたりの給餌量（摂取カロリー）を７０ｍｌ（７０ｋｃａｌ）に制限した。実験最終日（試験開始後４週間目）に、飼育したラットのそれぞれの腹部大動脈から採血を行い、肝臓を摘出した。採取した血液は血清分離後、血清総コレステロール、血清ＨＤＬ−コレステロール、血清ＬＤＬ−コレステロール、血清トリグリセリド、血清リン脂質、血清遊離脂肪酸、及び血清ＡＬＴを測定した。また、摘出した肝臓については、肝臓重量、肝臓総コレステロール、肝臓トリグリセリド、及び肝臓リン脂質を測定した。得られた結果は平均値±標準誤差（ＳＥＭ）で表し、統計処理は以下の手順で行った。即ち、無処置群と対照群の比較はＳｔｕｄｅｎｔ’ｓ ｔｅｓｔ法を用いて解析し、次いで対照群に対する試験群１及び試験群２の比較はＴｕｋｅｙ−Ｋｒａｍｅｒ法を用いて解析し、それぞれの解析において危険率０．０５％以下を有意として判定した。更に、摘出した前記肝臓から採取した肝細胞をＨＥ染色に付した後、オリンパス光学工業（株）製の生物顕微鏡ＢＸ５１を用いて２００倍の倍率で該肝細胞の形態観察を行った。
【００６３】
【評価１】
上記で得られた血清総コレステロール、血清ＨＤＬ−コレステロール、血清ＬＤＬ−コレステロール、血清トリグリセリド、血清リン脂質、血清遊離脂肪酸、及び血清ＡＬＴの測定結果を表１に示し、肝臓重量、肝臓総コレステロール、肝臓トリグリセリド、及び肝臓リン脂質の測定結果から以下の事実が判明した。
即ち、対照群は、無処置群と比較して、血清総コレステロール濃度、血清ＨＤＬ−コレステロール濃度、血清ＬＤＬ−コレステロール濃度、血清トリグリセリド濃度及び血清リン脂質濃度が有意に増加して、アルコール性高脂血症が誘発されていることが判明した。また、対照群は、無処置群と比較して、肝臓トリグリセリド濃度及び肝臓リン脂質濃度が有意に増加して、アルコール性脂肪肝が誘発されていることが判明した。更に、対照群は、無処置群と比較して、血中ＡＬＴ（ＧＰＴ）濃度が有意に増加し、肝細胞の生物顕微鏡観察においては肝小葉の終末肝静脈周辺領域における肝細胞壊死と風船様腫大が顕著に認められ、アルコール性肝炎が誘発されていることが判明した。一方、試験群１及び試験群２は、血清ＬＤＬ−コレステロール濃度、血清トリグリセリド濃度、及び肝臓トリグリセリド濃度の上昇が有意に抑制され、特に、試験群１のほうが試験群２よりも更に正常値に近似した値を示した。また、試験群１及び試験群２には、肝細胞の生物顕微鏡観察においてもアルコール性肝障害に特異的に認められる肝小葉の終末肝静脈周辺領域における肝細胞壊死と風船様腫大がほとんど認められなかった。
【００６４】
以上の結果から、実施例１で得た本発明の凍結乾燥物粉末は、比較例１で得た凍結乾燥物粉末よりも、アルコール性肝障害の誘発を更に著しく抑制し、アルコール性肝障害に対して卓越した発症抑制作用を示すことが判明した。
即ち、本発明の組成物は、アルコール性肝障害に対して際立った発症抑制作用を有することが明らかとなった。
【００６５】
実施例１で得た凍結乾燥物粉末（組成物）及び比較例１で得た凍結乾燥物粉末のそれぞれを以下の試験例２に供し、アルコール性肝障害に対する治癒作用を評価した。
【００６６】
【試験例２】
本試験例では、エタノール含有液体飼料で４週間飼育することによりアルコール性肝障害を発症したラットについて、実施例１で得た凍結乾燥物粉末（本発明の組成物）及び比較例１で得た凍結乾燥物粉末のそれぞれを用いて飼育することにより、アルコール性肝障害に対する治癒作用を評価した。
即ち、７週齢Ｗｉｓｔａｒ系雄性ラット（日本チャールスリバー）３０匹にエタノール含有率を徐々に上げながら（３％→４％→５％）エタノール含有液体飼料を６日間与えて飼育した後、引き続き５％エタノール含有液体飼料で４週間飼育を行い、該４週間目にそれらのラットのそれぞれについて採血を行い、血漿を分離して血清脂質を測定した。その後該３０匹のラットを１群１０匹として、対照群、試験群１及び試験群２の３群に振り分けた。その際、前記各群におけるラットの平均体重に係る分散に統計学的有意差が生じないように該３０匹のラットを振り分けた。対照群のラットに対しては前記エタノール含有液体飼料投与群と摂取カロリーを同一にするために５％エタノールの代わりにマルトース−デキストリン等量混合物を添加したエタノール非含有液体飼料を２週間与えて飼育した。試験群１のラットに対してはエタノール非含有液体飼料に実施例１で得た凍結乾燥物粉末１％を添加した液体飼料を２週間与えて飼育した。試験群２のラットに対してはエタノール非含有液体飼料に比較例１で得た凍結乾燥物粉末１％を添加した液体飼料を２週間与えて飼育した。更に対照群、試験群１及び試験群２とは別に前記７週齢Ｗｉｓｔａｒ系雄性ラット１０匹からなる無処置群を設け、無処置群のラットに対してはエタノール非含有液体飼料を６週間与えて飼育した。但し、上記４群とも、各液体飼料の１日あたりの給餌量（摂取カロリー）を７０ｍｌ（７０ｋｃａｌ）に制限した。上記４群の全てについて、実験最終日（実験開始後６週間目）に、飼育したラットのそれぞれの腹部大動脈から採血を行い、肝臓を摘出した。採取した血液は血清分離後、血清総コレステロール、血清ＬＤＬ−コレステロール、血清トリグリセリド、血清リン脂質、血清ＡＬＴ（ＧＰＴ）、及び血清ＡＳＴ（ＧＯＴ）を測定した。また、摘出した肝臓については、肝臓重量、肝臓総コレステロール、肝臓トリグリセリド、及び肝臓リン脂質を測定した。得られた結果は平均値±標準誤差（ＳＥＭ）で表し、統計処理は以下の手順で行った。即ち、無処置群と対照群の比較はＳｔｕｄｅｎｔ’ｓ ｔｅｓｔ法を用いて解析し、次いで対照群に対する試験群１及び試験群２の比較はＴｕｋｅｙ−Ｋｒａｍｅｒ法を用いて解析し、それぞれの解析において危険率０．０５％以下を有意として判定した。更に、摘出した前記肝臓から採取した肝細胞をＨＥ染色に付した後、オリンパス光学工業（株）製の生物顕微鏡ＢＸ５１を用いて２００倍の倍率で該肝細胞の形態観察を行った。
【００６７】
【評価２】
上記で得られた血清総コレステロール、血清ＬＤＬ−コレステロール、血清トリグリセリド、血清リン脂質、血清ＡＬＴ、及び血清ＡＳＴの測定結果、並びに、肝臓重量、肝臓総コレステロール、肝臓トリグリセリド、及び肝臓リン脂質の測定結果から以下の事実が判明した。
即ち、対照群は、血清総コレステロール及び血清ＬＤＬ−コレステロールが無処置群よりも有意に高い値を示し、血清トリグリセリド及び血清リン脂質も無処置群よりも高まる傾向が認められ、肝細胞の生物顕微鏡観察においてはアルコール性肝障害に特異的に認められる肝小葉の終末肝静脈周辺領域における肝細胞壊死と風船様腫大が顕著に観察された。一方、試験群１及び試験群２は、血清総コレステロール濃度、血清ＬＤＬ−コレステロール濃度、血清トリグリセリド濃度、血清リン脂質濃度、血清ＡＬＴ濃度、血清ＡＳＴ濃度、肝臓総コレステロール濃度、及び肝臓トリグリセリド濃度が、対照群と比較して有意に低い値を示し、特に、試験群１のほうが試験群２よりも更に正常値に近似した値を示した。また、試験群１及び試験群２は、肝細胞の生物顕微鏡観察においてはアルコール性肝障害に特異的に認められる肝小葉の終末肝静脈周辺領域における肝細胞壊死と風船様腫大が対照群と比較して著しく減少しているのが観察された。
以上の結果から、実施例１で得た本発明の凍結乾燥物粉末は、比較例１で得た凍結乾燥物粉末を凌駕し、アルコール性肝障害を更に著しく治癒し、アルコール性肝障害に対して際立った治癒作用を示すことが判明した。
即ち、本発明の組成物は、アルコール性肝障害に対して卓越した治癒作用を有するものであることが判明した。
【００６８】
実施例１で得た凍結乾燥物粉末（組成物）及び比較例１で得た凍結乾燥物粉末のそれぞれを以下の試験例３に供し、アルコール性肝障害に対する治癒作用を試験例２とは異なる方法で評価した。
【試験例３】
７週齢Ｗｉｓｔａｒ系雄性ラット（日本チャールスリバー）３０匹にエタノール含有率を徐々に上げながら（３％→４％→５％）エタノール含有液体飼料を６日間与えて飼育した後、引き続き５％エタノール含有液体飼料で４週間飼育を行い、該４週間目にそれらのラットのそれぞれについて採血を行い、血漿を分離して血清脂質を測定した。その後、該３０匹のラットを１群１０匹として、対照群、試験群１及び試験群２の３群に振り分けた。その際、前記各群におけるラットの平均体重に係る分散に統計学的有意差が生じないように該３０匹のラットを振り分けた。対照群のラットに対しては５％エタノール含有液体飼料を２週間与えて飼育した。試験群１のラットに対しては５％エタノール含有液体飼料に実施例１で得た凍結乾燥物粉末１％を添加した液体飼料を２週間与えて飼育した。試験群２のラットに対しては５％エタノール含有液体飼料に比較例１で得た凍結乾燥物粉末１％を添加した液体飼料を２週間与えて飼育した。更に対照群、試験群１及び試験群２とは別に７週齢Ｗｉｓｔａｒ系雄性ラット１０匹からなる無処置群を設け、該無処置群のラットに対しては、前記５％エタノール含有液体飼料と摂取カロリーを同一にするために５％エタノールの代わりにマルトース−デキストリン等量混合物を添加したエタノール非含有液体飼料を６週間与えて飼育した。但し、上記４群とも、各液体飼料の１日あたりの給餌量（摂取カロリー）を７０ｍｌ（７０ｋｃａｌ）に制限した。上記４群の全てについて、実験最終日（実験開始後６週間目）に、飼育したラットのそれぞれの腹部大動脈から採血を行い、肝臓を摘出した。採取した血液は血清分離後、血清総コレステロール、血清ＬＤＬ−コレステロール、血清トリグリセリド、血清リン脂質、血清ＡＬＴ、及び血清ＡＳＴを測定した。また、摘出した肝臓については、肝臓重量、肝臓総コレステロール、肝臓トリグリセリド、及び肝臓リン脂質を測定した。得られた結果は平均値±標準誤差（ＳＥＭ）で表し、統計処理は以下の手順で行った。即ち、無処置群と対照群の比較はＳｔｕｄｅｎｔ’ｓ ｔｅｓｔ法を用いて解析し、次いで対照群に対する試験群１及び試験群２の比較はＴｕｋｅｙ−Ｋｒａｍｅｒ法を用いて解析し、それぞれの解析において危険率０．０５％以下を有意として判定した。更に、摘出した前記肝臓から採取した肝細胞をＨＥ染色に付した後、オリンパス光学工業（株）製の生物顕微鏡ＢＸ５１を用いて２００倍の倍率で該肝細胞の形態観察を行った。
【００６９】
【評価３】
上記で得られた血清総コレステロール、血清ＬＤＬ−コレステロール、血清トリグリセリド、血清リン脂質、血清ＡＬＴ、及び血清ＡＳＴの測定結果、並びに、肝臓重量、肝臓総コレステロール、肝臓トリグリセリド、及び肝臓リン脂質の測定結果から以下の事実が判明した。
即ち、対照群は、無処置群と比較して、血清総コレステロール濃度、血清ＬＤＬ−コレステロール濃度、血清トリグリセリド濃度、血清リン脂質濃度、血清ＡＬＴ濃度、肝臓重量、肝臓総コレステロール濃度、及び肝臓トリグリセリド濃度が有意に高い値を示し、肝細胞の生物顕微鏡観察においてはアルコール性肝障害に特異的に認められる肝小葉の終末肝静脈周辺領域における肝細胞壊死と風船様腫大が顕著に認められた。一方、試験群１及び試験群２は、血清トリグリセリド濃度、血清総コレステロール濃度、血清リン脂質濃度、血清ＬＤＬ−コレステロール濃度、及び肝臓総コレステロール濃度及び肝臓トリグリセリド濃度が、対照群と比較して有意に低い値を示し、特に、試験群１のほうが試験群２よりも更に正常値に近似した値を示した。また、試験群１及び試験群２は、肝細胞の生物顕微鏡観察においてはアルコール性肝障害に特異的に認められる肝小葉の終末肝静脈周辺領域における肝細胞壊死と風船様腫大が対照群と比較して著しく減少しているのが観察された。
以上の結果から、実施例１で得た本発明の凍結乾燥物粉末は、比較例１で得た凍結乾燥物粉末を凌駕し、アルコール性肝障害を更に著しく治癒し、アルコール性肝障害に対して際立った治癒作用を示すことが判明した。
即ち、本発明の組成物は、アルコール性肝障害に対して卓越した治癒作用を有するものであることが判明した。
【００７０】
以上、試験例１に述べた結果から明らかなように、本発明の組成物は大麦焼酎蒸留残液の液体分を合成吸着剤を使用する吸着分離処理に付すことにより得られる合成吸着剤非吸着画分を卓越した強力なアルコール性肝障害の発症抑制作用を有し、エタノール投与によるアルコール性肝障害の発症を強力に抑制することが判明した。さらに試験例２及び試験例３に述べた結果から明らかなように、本発明の組成物は、既に発症したアルコール性肝障害を顕著に治癒することが判明した。
【００７１】
【使用例１】
実施例１で得た凍結乾燥物粉末を用い、以下に記す材料配合割合で他の担体材料と混合し、打錠機を用いて医薬用錠剤を作製した。
材料配合割合：実施例１で得た凍結乾燥物粉末３５重量％、シュガーエステル５重量％、オリゴ糖３０重量％、乳糖３０重量％
【００７２】
【発明の効果】
以上、詳述したように本発明の大麦を原料とする焼酎製造において副成する大麦焼酎蒸留残液を固液分離して液体分を得、該液体分を合成吸着剤を使用する吸着分離処理に付して合成吸着剤非吸着画分を得、該合成吸着剤非吸着画分をイオン交換樹脂を使用するイオン交換処理に付してイオン交換樹脂非吸着画分を得、該イオン交換樹脂非吸着画分を限外濾過膜を使用する限外濾過処理に付して濃縮液を得、該濃縮液に有機溶媒を添加することにより得られる有機溶媒不溶性画分からなる組成物は、アルコール性肝障害の発症に対しての著しい抑制作用及び治癒作用を有す。[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention provides a liquid fraction by solid-liquid separation of a residual liquid of barley shochu distilled as a by-product in the production of shochu using barley as a raw material, and subjecting the liquid fraction to an adsorption / separation treatment using a synthetic adsorbent. A non-adsorbed fraction is obtained, and the synthetic adsorbent non-adsorbed fraction is subjected to an ion exchange treatment using an ion-exchange resin to obtain an ion-exchange resin non-adsorbed fraction. A concentrated solution is obtained by ultrafiltration treatment using a filtration membrane, and an onset-suppressing effect and a curative effect on alcoholic liver injury composed of an organic solvent-insoluble fraction separated by adding an organic solvent to the concentrated solution are obtained. And a method for producing the same.
[0002]
[Prior art]
With the increase in the consumption of alcoholic beverages in recent years, the number of people with alcoholic liver disorder has been on an increasing trend and has been recognized as one of lifestyle-related diseases. Specific symptoms of such alcoholic liver disease include alcoholic fatty liver, alcoholic hepatitis, alcoholic liver fibrosis, alcoholic cirrhosis, and alcoholic hyperlipidemia induced by alcoholic fatty liver. it can. It is known that when alcoholic fatty liver chronically progresses, it shifts to alcoholic liver fibrosis, in which fibers around the hepatocytes are formed. It is known that when the protein synthesis ability and toxic substance resolution of the liver decrease, the liver gradually denatures and leads to alcoholic cirrhosis.
[0003]
By the way, the following is known about the liver damage prevention effect of the barley shochu distillation residue produced as a byproduct when producing barley shochu. That is, it has been reported that the barley shochu distillation residue suppresses the accumulation of lipids in the liver of rats by orotic acid administration [Summary of the Japanese Society of Nutrition and Food Science, Vol. 53, 53 (1999)] (Hereinafter, this document will be referred to as Non-Patent Document 1 and described later.
[0005] will be described separately). Further, the above-mentioned fatty liver inhibitory effect of the barley shochu distillation residue has a stronger effect than that of wine lees or beer lees, and the effect is not observed at all in potato shochu distillation residue, and is extremely small in rice shochu distillation residue. Therefore, it has been reported that it is unique to only barley shochu distillation residue [Japanese Brewery Association, Vol. 94, no. 9, 768 (1999)] (hereinafter, this document is referred to as Non-Patent Document 2 and will be described later.
[0005] will be described separately). Further, the barley shochu distillation residue has an inhibitory effect on D-galactosamine-induced liver injury, which is known to exhibit the same symptoms as viral liver injury. It has been reported that it is found in the liquid component obtained by subjecting the liquid to centrifugation [Journal of the Japan Brewing Association, Vol. 95, No. 9, 706 (2000)] (hereinafter, this document is referred to as Non-Patent Document 3 and will be described later.
[0005] will be described separately).
JP-A-2001-145472 discloses that a barley shochu distillation residue, which is a by-product in the production of shochu from barley, is subjected to solid-liquid separation to obtain a liquid component, and an alkali-soluble fraction is obtained by adding an alkali to the liquid component. , The alkali-soluble fraction is neutralized with an acid to obtain a neutral soluble fraction, and the organic acid, protein, and hemicellulose separated by adding ethanol to the neutral soluble fraction. It is described that the composition comprising the ethanol-insoluble fraction contained has an onset-suppressing effect on orotic acid-induced liver injury in an experiment using rats (hereinafter, this publication is referred to as Patent Document 1). , See below
[0005] will be described separately).
[0004]
Patent No. 3191956 discloses that an alcoholic fatty liver inhibitor containing a partially decomposed product of hemicellulose obtained from corn puffer as a main component inhibits fatty liver from forming fatty liver in rats by administration of an ethanol solution. (Hereinafter referred to as Patent Document 2 and described later).
[0005] will be described separately). Further, Patent Document 2 describes that a partially degraded product of hemicellulose obtained from the corn bran is commercially available under the trade name of “Cell Ace”. [March 1991 (confectionary) Confectionery Technology Center, “3rd Consignment Business of Food Distribution Bureau of the Ministry of Agriculture, Forestry and Fisheries No. 8 Effective Use of Functional Materials for Food and Beverage Series Water-soluble Corn Fiber (Arabinoxylan)” page 3] The partially decomposed product of hemicellulose obtained from corn bran marketed under the trade name of "Cel Ace" contains arabinoxylan composed of arabinose and xylose as the main components, 40.32% of xylose, 27.76% of arabinose, and 11 of uronic acid 11 It has a composition of 0.86%, galactose 5.91%, and glucose 2.30% (hereinafter referred to as Non-Patent Document 4 and described below).
[0005] will be described separately). Further, [New Food Industry Vol. 42 No. 9, 6 (2000)] describes that a partially decomposed product of hemicellulose obtained from corn flea marketed under the trade name "Cel Ace" has an inhibitory effect on acute alcoholic fatty liver. (Hereinafter, this document will be referred to as Non-Patent Document 5 and described later.
[0005] will be described separately).
[0005]
[Patent Document 1]
JP 2001-145472 A
[Patent Document 2]
Patent No. 3191956
[Non-patent document 1]
Abstracts of Annual Meeting of the Japanese Society of Nutrition and Food Science, Vol. 53, 53 (1999)
[Non-patent document 2]
Journal of the Japan Brewing Association, Vol. 94, no. 9, 768 (1999)
[Non-Patent Document 3]
Journal of the Japan Brewing Association, Vol. 95, No. 9, 706 (2000)
[Non-patent document 4]
March 1991 (published by) Confectionery Technology Center, "Ministry of Agriculture, Forestry and Fisheries Food Distribution Bureau No.8 Effective Use of Functional Materials for Food and Beverage, Water-soluble Corn Fiber (Arabinoxylan)" Page 3
[Non-Patent Document 5]
New Food Industry Vol. 42 No. 9, 6 (2000)
[0006]
[Problems to be solved by the invention]
As described above, Non-Patent Documents 1 to 3 disclose the barley shochu distillation residue or the barley shochu distillation residue obtained by solid-liquid separation of the barley shochu distillation residue. It is described that the resulting liquid components (hereinafter, these are referred to as liquid components of the residual liquid of barley shochu distillation) have an onset-suppressing effect on orotic acid-induced liver injury and D-galactosamine-induced liver injury. However, there is no indication as to whether it has an onset-suppressing effect and a curative effect on alcoholic liver injury. Patent Document 1 discloses that an ethanol-insoluble fraction has an onset-suppressing effect on orotic acid-induced liver injury, but the ethanol-insoluble fraction has an onset-suppressing effect and a healing effect on the alcoholic liver injury. There is no indication as to whether or not it has. That is, there is no known example of fractionating a fraction having an action of suppressing the onset of alcoholic liver injury and a healing action from the barley shochu distillation residue.
[0007]
By the way, it is known that orotic acid-induced liver injury is a liver injury that promotes fat synthesis in the liver by orotic acid and further suppresses the transfer of fat from the liver to the blood, thereby inducing fatty liver. Have been. It is known that D-galactosamine-induced liver injury is a liver injury in which hepatitis is promoted by D-galactosamine, thereby inducing hepatitis.
[0008]
On the other hand, the alcoholic liver disorder referred to in the present invention includes alcoholic hepatitis, alcoholic fatty liver, and alcoholic hyperlipidemia induced by excessive intake of alcohol. In the alcoholic fatty liver, the transfer of fatty acids from adipose tissue to the liver is promoted by ethanol, the synthesis of fatty acids and neutral fats in the liver is promoted, and further, the degradation of fatty acids in the liver is suppressed, and the like. It is known that fatty liver is induced by the accumulation of neutral fat in the liver. The alcoholic hepatitis is known to be hepatitis induced by acetaldehyde and acetic acid, which are metabolites of ethanol, or active oxygen generated when these are produced, show toxic effects on hepatocytes. ing. In addition, the alcoholic hyperlipidemia is caused by excessive triglyceride accumulated in the liver being released into the blood in a large amount as secretory ultra-low-density lipoprotein (VLDL) as described above. It is known to be something. In such alcoholic hepatic disorders, lesions of hepatitis such as balloon-like enlargement and hepatocellular necrosis, or the findings of fatty liver composed of hepatocytes containing large droplets of fat are found in the area around the terminal hepatic vein of the hepatic lobule. It is known that progress will be centered on It should be noted that the liver is mainly composed of hepatocytes, and functions as a unit, with the hepatic lobules separated by interlobular connective tissue as a unit, and a large number of hepatic lobules having a diameter of about 1 mm are assembled to form the liver. Therefore, alcoholic liver injury is objectively distinguished from orotic acid-induced liver injury and D-galactosamine-induced liver injury in view of the causal relationship of each of these liver injury, and certain components have Even if it is known to have an onset-suppressing or curative effect on orotic acid-induced hepatic injury or D-galactosamine-induced hepatic injury, this component has a similar onset-suppressing effect or curative effect on alcoholic hepatic injury. Whether or not it has an effect is hardly predictable.
[0009]
In view of such prior art, the present invention provides a liquid component by solid-liquid separation of the residual liquid of barley shochu distillation, and subjecting the liquid component to an adsorption separation process using a synthetic adsorbent to remove the synthetic adsorbent. A non-adsorbed fraction obtained by subjecting the non-adsorbed fraction to an ion-exchange treatment using an ion-exchange resin to obtain a non-adsorbed fraction of the ion-exchange resin. A concentrated solution was obtained by ultrafiltration using an organic solvent-insoluble fraction having a strong onset-suppressing action and healing action on alcoholic liver injury, which was separated by adding an organic solvent to the concentrated solution. It is an object of the present invention to provide a composition comprising:
[0010]
[Means for Solving the Problems]
The present inventors describe that Documents 1 to 3 describe that the liquid component of the residual liquid from the distillation of barley shochu has a suppressive action against D-galactosamine-induced liver injury and orotic acid-induced liver injury. In view of the above, the present inventors have conducted intensive studies through experiments with the aim of clarifying whether or not the liquid component of the barley shochu distillation residue has an onset-suppressing effect on alcoholic liver injury. As a result, although the liquid content of the barley shochu distillation residue has a slight effect of suppressing the onset of alcoholic liver injury, its use as a drug for the purpose of positively inhibiting the onset of alcoholic liver injury has been considered. It turned out to be less than suggestive.
[0011]
By the way, three of the present inventors, in collaboration with the other two, adsorbed fraction obtained by subjecting the liquid component of the barley shochu distillation residue to adsorption separation using a synthetic adsorbent, orotic acid A synthetic adsorbent non-adsorbed image which has an inhibitory action on induced fatty liver and D-galactosamine-induced liver injury, but is produced as a by-product by subjecting the liquid component of the barley shochu distillation residue to adsorption separation using a synthetic adsorbent. Found that it did not have an onset-suppressing effect on orotic acid-induced fatty liver and D-galactosamine-induced liver injury (filed as Japanese Patent Application No. 2002-56929). For this reason, the non-adsorbed fraction of the synthetic adsorbent has no pharmacological action and has been discarded as deemed useless. However, the present inventors, through the use of the synthetic adsorbent non-adsorbed fraction was discarded as useless in this way, through experiments to determine the onset suppression effect on alcoholic liver injury and the healing effect. As a result of the investigation, it was found that the non-adsorbed fraction of the synthetic adsorbent has an excellent inhibitory action on alcoholic liver injury and an excellent healing action on alcoholic liver injury (as Japanese Patent Application No. 2002-250991). Filed). Subsequent to the discovery, intensive research was conducted to fractionate and purify the active ingredient contained in the non-adsorbed fraction of the synthetic adsorbent, that is, a component having a strong onset-suppressing action and healing action on alcoholic liver injury. When overlapped, the synthetic adsorbent non-adsorbed fraction was subjected to an ion exchange treatment using an ion exchange resin to obtain an ion exchange resin non-adsorbed fraction, and the ion exchange resin non-adsorbed fraction was subjected to an ultrafiltration membrane. A concentrated solution is obtained by ultrafiltration treatment to be used, and an organic solvent-insoluble fraction obtained by adding an organic solvent to the concentrated solution has a remarkable inhibitory action and healing effect on alcoholic liver injury. It has been found to have an effect.
[0012]
The present invention has been completed based on such findings. An object of the present invention is to obtain a liquid component by solid-liquid separation of the residual liquid of barley shochu distillation, and subject the liquid component to an adsorption separation treatment using a synthetic adsorbent to obtain a synthetic adsorbent non-adsorbed fraction. The synthetic adsorbent non-adsorbed fraction is subjected to an ion exchange treatment using an ion exchange resin to obtain an ion exchange resin non-adsorbed fraction, and the ion exchange resin non-adsorbed fraction is subjected to ultrafiltration using an ultrafiltration membrane. A pharmaceutical composition having a remarkable onset-suppressing action and a curative action against alcoholic liver injury, comprising a concentrated solution obtained by subjecting the concentrate to an organic solvent-insoluble fraction obtained by adding an organic solvent to the concentrated solution. To provide things. Another object of the present invention is to provide a method for producing such a composition having a pharmacological action.
[0013]
Hereinafter, an experiment performed by the present inventors in completing the present invention will be described in detail. The present invention has been completed based on the findings obtained in these experiments. As described above, the present inventors have reported in Non-Patent Documents 1 to 3 that the liquid component of the barley shochu distillation residue has an inhibitory effect on D-galactosamine-induced liver injury and orotic acid-induced fatty liver. In order to clarify whether or not the liquid component of the barley shochu distillation residue has an onset-suppressing effect on alcoholic liver injury in view of the fact that the barley shochu distillation residue is described, the barley shochu distillation residue is The following experiment was carried out using the liquid component (A) of the liquid, and was studied diligently.
That is, 24 3-week-old Wistar male rats (Japan SLC) were fed with an ethanol-containing liquid feed for 6 days while gradually increasing the ethanol content (3% → 4% → 5%). , A control group and a test group. At this time, the 24 rats were sorted so that there was no statistically significant difference in the variance of the average weight of the rats in each group. A lyophilized powder (A ') of the liquid component (A) of the distillation residue of barley shochu was added to the control group of rats in a liquid feed containing 5% ethanol, and to the rats in the test group, in the liquid feed containing 5% ethanol. ) A liquid feed supplemented with 1% was fed for 4 weeks each and bred. An untreated group consisting of the 12 3-week-old Wistar male rats was provided separately from the control group and the test group. The rats in the untreated group had 5 rats in order to obtain the same caloric intake as the other two groups. Ethanol-free liquid feed supplemented with a maltose-dextrin equivalent mixture instead of% ethanol was fed for 4 weeks and bred. However, in each of the three groups, the daily feed amount (calorie intake) of each liquid feed was limited to 70 ml (70 kcal). On the final day of the experiment (four weeks after the start of the test), blood was collected from each abdominal aorta of the bred rats, and the liver was extracted. After serum separation, the collected blood was measured for serum total cholesterol, serum HDL-cholesterol, serum LDL-cholesterol, serum triglyceride, serum phospholipid, serum free fatty acid, and serum ALT (GPT). For the isolated liver, liver weight, liver total cholesterol, liver triglyceride, and liver phospholipid were measured. The obtained results were expressed as an average value ± standard error (SEM), and statistical processing was performed in the following procedure. That is, the comparison between the untreated group and the control group was analyzed using the Student's test method, and then the comparison between the control group and the test group was analyzed using the Tukey-Kramer method. % Or less was determined as significant. Further, the hepatocytes collected from the excised liver were subjected to HE staining, and the morphology of the hepatocytes was observed at a magnification of 200 times using a biological microscope BX51 manufactured by Olympus Optical Co., Ltd.
[0014]
As a result, in the control group, the serum total cholesterol concentration, serum HDL-cholesterol concentration, serum LDL-cholesterol concentration, serum triglyceride concentration and serum phospholipid concentration were significantly increased, and the It was found that lipemia was induced. Further, it was found that the liver triglyceride concentration and the liver phospholipid concentration were significantly increased in the control group and the alcoholic fatty liver was induced as compared with the untreated group. Furthermore, in the control group, the blood ALT (GPT) concentration was significantly increased as compared with the non-treatment group, and the hepatocyte necrosis and balloon-like in the area around the terminal hepatic vein of the hepatic lobule were observed by biomicroscopic observation of hepatocytes. Swelling was remarkably observed, and it was found that alcoholic hepatitis was induced. On the other hand, the test group showed a tendency to suppress the increase in serum LDL-cholesterol concentration, serum triglyceride concentration and liver triglyceride concentration as compared with the control group, and the terminal hepatic vein of the hepatic lobule was observed by biomicroscopic observation of hepatocytes. Hepatocyte necrosis and balloon-like swelling in the peripheral area showed a tendency to decrease slightly.
From the above results, it is suggested that the freeze-dried powder (A ′) of the liquid component (A) of the barley shochu distillation residue is used as a drug for the purpose of positively suppressing the onset of alcoholic liver injury. It turned out not to be enough.
[0015]
Next, the present inventors will clarify what fraction obtained from the liquid component of the distillation residue of barley shochu contributes to the onset suppression effect on alcoholic liver injury through the experiments described below. For this purpose, using the desorbed fraction (B) obtained by eluting the adsorbed fraction obtained by subjecting the liquid component of the barley shochu distillation residue to the adsorption separation treatment using a synthetic adsorbent with an alkali, We conducted an experiment and studied diligently.
That is, 24 3-week-old Wistar male rats (Japan SLC) were fed with an ethanol-containing liquid feed for 6 days while gradually increasing the ethanol content (3% → 4% → 5%). , A control group and a test group. At this time, the 24 rats were sorted so that there was no statistically significant difference in the variance of the average weight of the rats in each group. To the rats of the control group, a liquid feed containing 5% ethanol, and to the rats of the test group, 1% of the lyophilized powder (B ') of the above-mentioned desorbed fraction (B) was added to the liquid feed containing 5% ethanol. Each of the added liquid feeds was fed for 4 weeks and bred. An untreated group consisting of the 12 3-week-old Wistar male rats was provided separately from the control group and the test group. The rats in the untreated group had 5 rats in order to obtain the same caloric intake as the other two groups. Ethanol-free liquid feed supplemented with a maltose-dextrin equivalent mixture instead of% ethanol was fed for 4 weeks and bred. However, in each of the three groups, the daily feed amount (calorie intake) of each liquid feed was limited to 70 ml (70 kcal). On the final day of the experiment (four weeks after the start of the test), blood was collected from each abdominal aorta of the bred rats, and the liver was extracted. After serum separation, the collected blood was measured for serum total cholesterol, serum HDL-cholesterol, serum LDL-cholesterol, serum triglyceride, serum phospholipid, serum free fatty acid, and serum ALT (GPT). For the isolated liver, liver weight, liver total cholesterol, liver triglyceride, and liver phospholipid were measured. The obtained results were expressed as an average value ± standard error (SEM), and statistical processing was performed in the following procedure. That is, the comparison between the untreated group and the control group was analyzed using the Student's test method, and then the comparison between the control group and the test group was analyzed using the Tukey-Kramer method. % Or less was determined as significant. Further, the hepatocytes collected from the excised liver were subjected to HE staining, and the morphology of the hepatocytes was observed at a magnification of 200 times using a biological microscope BX51 manufactured by Olympus Optical Co., Ltd.
[0016]
As a result, in the control group, the serum total cholesterol concentration, serum HDL-cholesterol concentration, serum LDL-cholesterol concentration, serum triglyceride concentration and serum phospholipid concentration were significantly increased, and the It was found that lipemia was induced. Further, it was found that the liver triglyceride concentration and the liver phospholipid concentration were significantly increased in the control group and the alcoholic fatty liver was induced as compared with the untreated group. Furthermore, in the control group, the blood ALT (GPT) concentration was significantly increased as compared with the non-treatment group, and the hepatocyte necrosis and balloon-like in the area around the terminal hepatic vein of the hepatic lobule were observed by biomicroscopic observation of hepatocytes. Swelling was remarkably observed, and it was found that alcoholic hepatitis was induced. On the other hand, the test group showed a tendency that the increase in the serum triglyceride concentration of the rat was suppressed as compared with the control group, but did not suppress the increase in the liver triglyceride concentration at all, and even in the biological microscopic observation of hepatocytes. Hepatic cell necrosis and balloon-like swelling in the area around the terminal hepatic vein of the hepatic lobule were remarkably observed. That is, the lyophilized powder (B ′) of the desorbed fraction (B) slightly showed a tendency to suppress the induction of alcoholic hyperlipidemia, but did not induce the induction of alcoholic fatty liver and alcoholic hepatitis. It showed no tendency to suppress.
From the above results, it was found that the lyophilized powder (B ′) of the desorbed fraction (B) had substantially no action of suppressing the onset of alcoholic liver injury.
[0017]
Therefore, the present inventors, the synthetic adsorbent non-adsorbed fraction obtained by subjecting the liquid component of the barley shochu distillation residue to adsorption separation using a synthetic adsorbent, is effective against alcoholic liver injury Presuming that it may have an onset-suppressing effect, the following procedure was performed using the synthetic adsorbent non-adsorbed fraction (C) obtained by subjecting the liquid portion of the barley shochu distillation residue to adsorption separation using a synthetic adsorbent. We conducted an experiment and studied diligently.
That is, 24 3-week-old Wistar male rats (Japan SLC) were fed with an ethanol-containing liquid feed for 6 days while gradually increasing the ethanol content (3% → 4% → 5%). , A control group and a test group. At this time, the 24 rats were sorted so that there was no statistically significant difference in the variance of the average weight of the rats in each group. A lyophilized powder (C ′) of the non-adsorbed fraction (C) of the synthetic adsorbent was added to a 5% ethanol-containing liquid feed for the control group rats and a 5% ethanol-containing liquid feed for the test group rats. ) A liquid feed supplemented with 1% was fed for 4 weeks each and bred. An untreated group consisting of the 12 3-week-old Wistar male rats was provided separately from the control group and the test group. The rats in the untreated group had 5 rats in order to obtain the same caloric intake as the other two groups. Ethanol-free liquid feed supplemented with a maltose-dextrin equivalent mixture instead of% ethanol was fed for 4 weeks and bred. However, in each of the three groups, the daily feed amount (calorie intake) of each liquid feed was limited to 70 ml (70 kcal). On the final day of the experiment (four weeks after the start of the test), blood was collected from each abdominal aorta of the bred rats, and the liver was extracted. After serum separation, the collected blood was measured for serum total cholesterol, serum HDL-cholesterol, serum LDL-cholesterol, serum triglyceride, serum phospholipid, serum free fatty acid, and serum ALT (GPT). For the isolated liver, liver weight, liver total cholesterol, liver triglyceride, and liver phospholipid were measured. The obtained results were expressed as an average value ± standard error (SEM), and statistical processing was performed in the following procedure. That is, the comparison between the untreated group and the control group was analyzed using the Student's test method, and then the comparison between the control group and the test group was analyzed using the Tukey-Kramer method. % Or less was determined as significant. Further, the hepatocytes collected from the excised liver were subjected to HE staining, and the morphology of the hepatocytes was observed at a magnification of 200 times using a biological microscope BX51 manufactured by Olympus Optical Co., Ltd.
[0018]
As a result, in the control group, the serum total cholesterol concentration, serum HDL-cholesterol concentration, serum LDL-cholesterol concentration, serum triglyceride concentration and serum phospholipid concentration were significantly increased, and the It was found that lipemia was induced. Further, it was found that the liver triglyceride concentration and the liver phospholipid concentration were significantly increased in the control group and the alcoholic fatty liver was induced as compared with the untreated group. Furthermore, in the control group, the blood ALT (GPT) concentration was significantly increased as compared with the non-treatment group, and the hepatocyte necrosis and balloon-like in the area around the terminal hepatic vein of the hepatic lobule were observed by biomicroscopic observation of hepatocytes. Swelling was remarkably observed, and it was found that alcoholic hepatitis was induced. On the other hand, the test group significantly suppressed the increase in serum LDL-cholesterol concentration, serum triglyceride concentration, and liver triglyceride concentration of the rats, and showed the terminal hepatic lobule in biological microscopic observation of hepatocytes. Hepatocyte necrosis and balloon-like enlargement in the area around the hepatic vein were scarcely observed. From the above experimental results, it was found that the lyophilized product powder (C ′) of the non-adsorbed fraction (C) of the synthetic adsorbent has a remarkable inhibitory action on alcoholic liver injury.
[0019]
For this reason, the components contributing to the suppression of the onset of alcoholic liver injury contained in the barley shochu distillation residue are obtained by solid-liquid separation of the barley shochu distillation residue to obtain a liquid component, and the liquid component is synthesized using a synthetic adsorbent. It was found that the compound was present in a fractionated state in a non-adsorbed fraction of the synthetic adsorbent obtained by performing the adsorption separation treatment.
[0020]
Therefore, the present inventors have conducted intensive studies for the purpose of elucidating a component contained in the synthetic adsorbent non-adsorbed fraction and having a strong onset-suppressing action against alcoholic liver injury. That is, the present inventors obtain a liquid by solid-liquid separation of the residual liquid of barley shochu distilled as a by-product in the production of shochu using barley as a raw material, and subject the liquid to an adsorption separation treatment using a synthetic adsorbent. In view of the fact that the non-adsorbed fraction of the synthetic adsorbent obtained thereby contains a polysaccharide as one of the components, the polysaccharide contained in the non-adsorbed fraction of the synthetic adsorbent suppresses the onset of alcoholic liver injury. Intensive studies were conducted to clarify whether or not they are involved in the action. That is, the following experiment was conducted for the purpose of removing amino acids, peptides, proteins, organic acids, and barley-derived polyphenols, which are components other than polysaccharides contained in the organic solvent-insoluble fraction. Specifically, for the purpose of removing barley-derived polyphenols and the like contained in the liquid component of the barley shochu distillation residue, a liquid component obtained by solid-liquid separation of the barley shochu distillation residue is used as a synthetic adsorbent. The synthetic adsorbent non-adsorbed fraction is obtained by performing the used adsorption separation treatment, and then, for the purpose of removing amino acids, peptides, proteins and organic acids contained in the synthetic adsorbent non-adsorbed fraction, By subjecting the synthetic adsorbent non-adsorbed fraction to an ion exchange treatment using various ion exchange resins, different types of ion-exchange resin non-adsorbed fractions are obtained, and each obtained ion-exchange resin non-adsorbed fraction is separately separated. The following experiment was conducted to clarify whether or not each organic solvent-insoluble fraction collected by adding an organic solvent to alcohol has the onset-suppressing effect on alcoholic liver injury. It was.
That is, the liquid component obtained by solid-liquid separation of the residual liquid of barley shochu distillation is subjected to an adsorption separation treatment using a synthetic adsorbent to obtain a synthetic adsorbent non-adsorbed fraction, and the obtained synthetic adsorbent non-adsorbed fraction The fraction is subjected to an ion-exchange treatment using various ion-exchange resins, specifically, a cation-exchange resin, an anion-exchange resin and a mixed-bed type ion-exchange resin, and the ion-exchange resin non-adsorbed fraction And an organic solvent-insoluble fraction was obtained by adding ethanol to each ion-exchange resin non-adsorbed fraction. For each of the obtained organic solvent-insoluble fractions, free sugars, polysaccharides, organic acids, and crude proteins were measured. In addition, the following experiment was performed to clarify whether or not each organic solvent-insoluble fraction exhibited an onset-suppressing effect on alcoholic liver injury.
[0021]
Barley shochu was produced for the purpose of the following experiments 1 to 7. Barley (70% refined) was used as a raw material.
[Manufacture of koji]
Barley is allowed to absorb 40% by weight of water, steamed for 40 minutes, allowed to cool to 40 ° C, inoculated with 1 kg of seed koji (white koji mold) per ton of barley, and incubated at 38 ° C and RH95% for 24 hours at 32 ° C and RH92%. For 20 hours to produce barley koji.
[Manufacture of steamed wheat]
Barley was allowed to absorb 40% by weight of water, steamed for 40 minutes, and allowed to cool to 40 ° C. to produce steamed barley.
[0022]
[Production of barley shochu and barley shochu distillation residue]
In the primary preparation, 3.6 kiloliters of water and 1 kg (wet weight) of cultured cells of shochu yeast as yeast were added to the barley koji (3 tons as barley) produced by the above-mentioned method to obtain a primary moromi. The obtained primary moromi was subjected to 5 days of fermentation (first stage fermentation). Next, in the secondary preparation, 11.4 kiloliters of water and steamed barley (7 tons as barley) produced by the above-mentioned method are added to the primary moromi after the first stage fermentation, and fermentation for 11 days ( (Second stage fermentation). The fermentation temperature was 25 ° C. for both the primary charge and the secondary charge. The secondary mash after the second stage of fermentation was subjected to simple distillation by a conventional method to obtain 10 kiloliters of barley shochu and 15 kiloliters of barley shochu distillation residue. The obtained barley shochu distillation residue was used in the following Experiments 1 to 7.
[0023]
[Experiment 1]
Said
The organic solvent-insoluble fraction was fractionated from the barley shochu distillation residue obtained in the above by the following method. That is, the barley shochu distillation residue was centrifuged at 8000 rpm for 10 minutes to obtain a liquid component of the barley shochu distillation residue, and the obtained liquid component was synthesized by Amberlite XAD-16, a synthetic adsorbent manufactured by Organo Corporation. The mixture was subjected to an adsorption separation treatment by passing through a column filled with, and a synthetic adsorbent non-adsorbed fraction consisting of a flow-through liquid having non-adsorbability to the synthetic adsorbent of the column was collected. By subjecting 1 L of the obtained synthetic adsorbent non-adsorbed fraction to lyophilization, 57.2 g of a freeze-dried product of the synthetic adsorbent non-adsorbed fraction was obtained.
[0024]
[Experiment 2]
Said
The organic solvent-insoluble fraction was fractionated from the barley shochu distillation residue obtained in the above by the following method. That is, the barley shochu distillation residue was centrifuged at 8000 rpm for 10 minutes to obtain a liquid component of the barley shochu distillation residue, and the obtained liquid component was synthesized by Amberlite XAD-16, a synthetic adsorbent manufactured by Organo Corporation. The mixture was subjected to an adsorption separation treatment by passing through a column filled with, and a synthetic adsorbent non-adsorbed fraction consisting of a flow-through liquid having non-adsorbability to the synthetic adsorbent of the column was collected. The obtained synthetic adsorbent non-adsorbed fraction was adjusted to Brix 10, and 10 L of the synthetic adsorbent non-adsorbed fraction adjusted to Brix 10 was used in a 5 L capacity of Amberlite IRC76 (a weak acidic cation exchange resin) manufactured by Organo Corporation. Was passed through a column packed with, and an ion-exchange resin non-adsorbed fraction was obtained. After adjusting the obtained ion-exchange resin non-adsorbed fraction to Brix60, ethanol was added to a final concentration of 75% by volume, and 8000 rpm for 10 minutes. The organic solvent-insoluble fraction was collected by centrifugation under the conditions described above, and the organic solvent-insoluble fraction was subjected to freeze-drying to obtain 53 g of a freeze-dried organic solvent-insoluble fraction.
[0025]
[Experiment 3]
Said
The organic solvent-insoluble fraction was fractionated from the barley shochu distillation residue obtained in the above by the following method. That is, the barley shochu distillation residue was centrifuged at 8000 rpm for 10 minutes to obtain a liquid component of the barley shochu distillation residue, and the obtained liquid component was synthesized by Amberlite XAD-16, a synthetic adsorbent manufactured by Organo Corporation. The mixture was subjected to an adsorption separation treatment by passing through a column filled with, and a synthetic adsorbent non-adsorbed fraction consisting of a flow-through liquid having non-adsorbability to the synthetic adsorbent of the column was collected. The obtained synthetic adsorbent non-adsorbed fraction was adjusted to Brix 10, and 10 L of the synthetic adsorbent non-adsorbed fraction adjusted to Brix 10 was used in a 5 L capacity of Amberlite IRA67 (a weak acidic anion exchange resin) manufactured by Organo Corporation. Was passed through a column packed with, and an ion-exchange resin non-adsorbed fraction was obtained. After adjusting the obtained ion-exchange resin non-adsorbed fraction to Brix60, ethanol was added to a final concentration of 75% by volume, and 8000 rpm for 10 minutes. The organic solvent-insoluble fraction was collected by centrifugation under the conditions described above, and the organic solvent-insoluble fraction was subjected to lyophilization to obtain 59 g of a lyophilized organic solvent-insoluble fraction.
[0026]
[Experiment 4]
Said
The organic solvent-insoluble fraction was fractionated from the barley shochu distillation residue obtained in the above by the following method. That is, the barley shochu distillation residue was centrifuged at 8000 rpm for 10 minutes to obtain a liquid component of the barley shochu distillation residue, and the obtained liquid component was synthesized by Amberlite XAD-16, a synthetic adsorbent manufactured by Organo Corporation. The mixture was subjected to an adsorption separation treatment by passing through a column filled with, and a synthetic adsorbent non-adsorbed fraction consisting of a flow-through liquid having non-adsorbability to the synthetic adsorbent of the column was collected. The obtained synthetic adsorbent non-adsorbed fraction was adjusted to Brix 10, and 10 L of the synthetic adsorbent non-adsorbed fraction adjusted to Brix 10 was used in a 5 L capacity of Amberlite IR120B (manufactured by Organo Co., Ltd.) (strongly acidic cation exchange resin). Was passed through a column packed with, and an ion-exchange resin non-adsorbed fraction was obtained. After adjusting the obtained ion-exchange resin non-adsorbed fraction to Brix60, ethanol was added to a final concentration of 75% by volume, and 8000 rpm for 10 minutes. The organic solvent-insoluble fraction was collected by centrifugation under the conditions described above, and the organic solvent-insoluble fraction was subjected to lyophilization to obtain 56 g of a lyophilized organic solvent-insoluble fraction.
[0027]
[Experiment 5]
Said
The organic solvent-insoluble fraction was fractionated from the barley shochu distillation residue obtained in the above by the following method. That is, the barley shochu distillation residue was centrifuged at 8000 rpm for 10 minutes to obtain a liquid component of the barley shochu distillation residue, and the obtained liquid component was synthesized by Amberlite XAD-16, a synthetic adsorbent manufactured by Organo Corporation. The mixture was subjected to an adsorption separation treatment by passing through a column filled with, and a synthetic adsorbent non-adsorbed fraction consisting of a flow-through liquid having non-adsorbability to the synthetic adsorbent of the column was collected. The obtained synthetic adsorbent non-adsorbed fraction was adjusted to Brix 10, and 10 L of the synthetic adsorbent non-adsorbed fraction adjusted to Brix 10 was used as a 5-L capacity of Amberlite 200CT (a strong acid cation exchange resin) manufactured by Organo Corporation. Was passed through a column packed with, and an ion-exchange resin non-adsorbed fraction was obtained. After adjusting the obtained ion-exchange resin non-adsorbed fraction to Brix60, ethanol was added to a final concentration of 75% by volume, and 8000 rpm for 10 minutes. The organic solvent-insoluble fraction was collected by centrifugation under the conditions described above, and the organic solvent-insoluble fraction was subjected to lyophilization to obtain 41 g of a lyophilized organic solvent-insoluble fraction.
[0028]
[Experiment 6]
Said
The organic solvent-insoluble fraction was fractionated from the barley shochu distillation residue obtained in the above by the following method. That is, the barley shochu distillation residue was centrifuged at 8000 rpm for 10 minutes to obtain a liquid component of the barley shochu distillation residue, and the obtained liquid component was synthesized by Amberlite XAD-16, a synthetic adsorbent manufactured by Organo Corporation. The mixture was subjected to an adsorption separation treatment by passing through a column filled with, and a synthetic adsorbent non-adsorbed fraction consisting of a flow-through liquid having non-adsorbability to the synthetic adsorbent of the column was collected. The resulting synthetic adsorbent non-adsorbed fraction was adjusted to Brix 10, and 10 L of the synthetic adsorbent non-adsorbed fraction adjusted to Brix 10 was used in a 5 L capacity of Amberlite IRA402BL (manufactured by Organo Co., Ltd.) (the strongest basic anion exchange resin). ) Was passed through a column packed with the mixture to obtain an ion-exchange resin non-adsorbed fraction. The obtained ion-exchange resin non-adsorbed fraction was adjusted to Brix60, and ethanol was added to a final concentration of 75% by volume. The organic solvent-insoluble fraction was collected by centrifugation at 10 min, and the organic solvent-insoluble fraction was subjected to freeze-drying to obtain 23 g of a freeze-dried organic solvent-insoluble fraction.
[0029]
[Experiment 7]
Said
The organic solvent-insoluble fraction was fractionated from the barley shochu distillation residue obtained in the above by the following method. That is, the barley shochu distillation residue was centrifuged at 8000 rpm for 10 minutes to obtain a liquid component of the barley shochu distillation residue, and the obtained liquid component was synthesized by Amberlite XAD-16, a synthetic adsorbent manufactured by Organo Corporation. The mixture was subjected to an adsorption separation treatment by passing through a column filled with, and a synthetic adsorbent non-adsorbed fraction consisting of a flow-through liquid having non-adsorbability to the synthetic adsorbent of the column was collected. The obtained synthetic adsorbent non-adsorbed fraction was adjusted to Brix10, and 10 L of the synthetic adsorbent nonadsorbed fraction adjusted to Brix10 was used as a 3.5 L capacity of Amberlite IRC76 (manufactured by Organo Co., Ltd.) (weak acidic cation exchange). Resin) and a 1.5 L capacity of Amberlite IRA67 (weakly basic anion exchange resin) manufactured by Organo Co., Ltd., and passed through a column packed with a mixed bed ion exchange resin. A non-adsorbed fraction was obtained, and the obtained mixed-bed ion-exchange resin non-adsorbed fraction was adjusted to Brix60, ethanol was added to a final concentration of 75% by volume, and the mixture was centrifuged at 8000 rpm for 10 minutes to remove the organic solvent. The insoluble fraction was separated, and the organic solvent insoluble fraction was subjected to lyophilization to obtain 19 g of a lyophilized organic solvent insoluble fraction.
[0030]
Each of the freeze-dried products obtained in Experiments 1 to 7 was evaluated for its onset-suppressing effect on alcoholic liver injury by the following method.
That is, 80 7-week-old male Wistar rats (Charles River Japan) were fed an ethanol-containing liquid feed for 6 days while gradually increasing the ethanol content (3% → 4% → 5%), and then reared. The rats were bred for 4 weeks on a liquid feed containing 5% ethanol, blood was collected from each of the rats on the 4th week, the plasma was separated and the serum lipid was measured. The test group 7 was divided into eight groups. At this time, the 80 rats were sorted so that there was no statistically significant difference in the variance of the average weight of the rats in each group. To the rats of the control group, an ethanol-free liquid feed supplemented with an equivalent mixture of maltose-dextrin instead of the 5% ethanol was given for 2 weeks in order to make the calorie intake the same as that of the ethanol-containing liquid feed administration group. Bred. The test groups 1 to 7 were fed with a liquid feed obtained by adding 1% of each of the freeze-dried powders obtained in Experiments 1 to 7 to the ethanol-free liquid feed for 2 weeks. Further, a non-treatment group consisting of the 10 7-week-old Wistar male rats was provided separately from the control group and the test groups 1 to 7, and the ethanol-free liquid feed was administered to the rats of the non-treatment group by 6 rats. They were fed and kept for a week. However, the daily feeding amount (calorie intake) of each liquid feed was limited to 70 ml (70 kcal) in each of the nine groups. On the last day of the experiment (six weeks after the start of the experiment), blood was collected from the abdominal aorta of each of the reared rats, and the liver was extracted from all of the nine groups. After serum separation, the collected blood was measured for serum total cholesterol, serum LDL-cholesterol, serum triglyceride, serum phospholipid, serum ALT (GPT), and serum AST (GOT). For the isolated liver, liver weight, liver total cholesterol, liver triglyceride, and liver phospholipid were measured. The obtained results were expressed as an average value ± standard error (SEM), and statistical processing was performed in the following procedure. That is, the comparison between the untreated group and the control group was analyzed using the Student's test method, and then the comparison of the test groups 1 to 7 with respect to the control group was analyzed using the Tukey-Kramer method. A risk rate of 0.05% or less was determined as significant. Further, the hepatocytes collected from the excised liver were subjected to HE staining, and the morphology of the hepatocytes was observed at a magnification of 200 times using a biological microscope BX51 manufactured by Olympus Optical Co., Ltd.
[0031]
For each of the freeze-dried products obtained in Experiments 1 to 7, the following facts were found from the experimental results of the polysaccharide content, the total content of polysaccharide-derived arabinose and xylose, and the effect of suppressing the onset of alcoholic liver injury. That is, as compared with the control group, all of the freeze-dried products obtained in Experiments 1 to 7 exhibited an inhibitory effect on the development of alcoholic liver injury. Among them, in Test Group 5 bred with the liquid feed to which the freeze-dried product obtained in Experiment 5 was added, a remarkable remarkable effect of suppressing the onset of alcoholic liver injury was observed. On the other hand, in Test Group 1 bred on the liquid feed to which the freeze-dried product obtained in Experiment 1 was added, the degree of the effect of suppressing the onset of alcoholic liver injury was the lowest.
[0032]
[Measurement of polysaccharide content]
(Measurement of total content of arabinose and xylose derived from polysaccharide)
Thus, for each of the freeze-dried products obtained in Experiments 1 to 7, the polysaccharide content and the total content of polysaccharide-derived arabinose and xylose were measured.
That is, 1 ml of ion-exchanged water was added to and dissolved in 0.05 g of the freeze-dried product obtained in each of Experiments 1 to 7, 200 μl of concentrated hydrochloric acid was added, and hydrolysis was performed at 95 ° C. for 4 hours. Then, the mixture was filtered through a 0.80 μm membrane filter to obtain a filtrate. The filtrate was injected into a high-performance liquid chromatograph, and the content of polysaccharide contained in each of the freeze-dried products obtained in Experiments 1 to 7 above. And the total content of arabinose and xylose derived from polysaccharides. High-performance liquid chromatographic analysis was performed using Waters600 manufactured by Waters, a differential refractometer RI-71 manufactured by Showa Denko KK as a detector, and Aminex HPX-87H (300 mm x 7.8 mm) manufactured by BioRad as a column. . The column temperature was 60 ° C., the mobile phase was 5 mM sulfuric acid, the flow rate was 0.5 ml / min, and the sample injection volume was 20 μl.
[0033]
The following facts were found from the measurement results of the polysaccharide content of each freeze-dried product obtained in Experiments 1 to 7, and the measurement results of the total contents of arabinose and xylose derived from the polysaccharide. That is, the polysaccharide content of the freeze-dried product obtained in Experiment 5 and the total content of arabinose and xylose derived from the polysaccharide showed the highest values, respectively. On the other hand, the polysaccharide content of the freeze-dried product obtained in Experiment 1 and the total content of arabinose and xylose derived from the polysaccharide showed the lowest values, respectively. Further, the strength of each of the freeze-dried products obtained in Experiments 1 to 7 for inhibiting the onset of alcoholic liver injury is proportional to the total content of polysaccharide-derived arabinose and xylose contained in the freeze-dried products. It became clear that the tendency to increase was recognized.
[0034]
From the above results, the lyophilizates obtained in Experiments 1 to 7 had an inhibitory effect on the onset of alcoholic liver injury, which was due to the fact that polysaccharides containing arabinose and xylose as main components contained in the lyophilizates It has been revealed that the possibility of the origin of the disease is extremely high.
[0035]
[Measurement of molecular weight distribution]
Therefore, in order to clarify the molecular weights of the polysaccharides containing arabinose and xylose as main components contained in each of the freeze-dried products obtained in Experiments 1 to 7, the molecular weights of the respective organic solvent-insoluble fractions were determined. The distribution was measured.
That is, Shodex standard P-82 (molecular weight: 1300 to 1660000) and standard molecular weight consisting of maltotriose (molecular weight: 504) manufactured by Showa Denko KK are separately dissolved in a 0.1 mol / L sodium nitrate solution to obtain a solution. A standard solution having a concentration of 0.05 W / V% was obtained, and the standard solution was injected into a high performance liquid chromatograph to prepare a calibration curve. Next, 0.02 g of each of the freeze-dried products obtained in Experiments 1 to 7 was prepared, 10 ml of a 0.1 mol / L sodium nitrate solution was added thereto, and the mixture was allowed to stand at room temperature overnight. A filtrate was obtained by filtration through a membrane filter of No. 1, and the filtrate was injected into a high performance liquid chromatograph, and the molecular weight distribution was determined using a 480 data station GPC program manufactured by System Instruments Co., Ltd. High-performance liquid chromatographic analysis was performed using Showex GPC SYSTEM-21 manufactured by Showa Denko KK, using a differential refractometer RI-71S manufactured by Showa Denko KK as a detector, and using TSKgel GMPWXL (φ7.8 mm ×) manufactured by Tosoh Corporation. (300 mm). The column temperature was 40 ° C., the mobile phase was a 0.1 mol / L sodium nitrate solution, the flow rate was 1.0 ml / min, and the sample injection amount was 100 μl.
[0036]
As a result of measuring the molecular weight distribution of each of the freeze-dried products obtained in Experiments 1 to 7 by the above method, the higher the total content of arabinose and xylose derived from polysaccharides contained in the freeze-dried product, the higher the molecular weight was 10,10. It has been found that the proportion of the fraction consisting of 000 to 100,000 increases.
[0037]
From these facts, the present inventors have concluded that the polysaccharide mainly containing arabinose and xylose contained in the freeze-dried product has a molecular weight of 10,000 to 100,000. It was presumed that there was not, and fractionation purification of the organic solvent-insoluble fraction was examined using ultrafiltration.
[Fractionation and purification of the organic solvent-insoluble fraction by ultrafiltration]
The molecular weight distribution of each of the freeze-dried products obtained in Experiments 1 to 7 was measured by the method described above. As a result, the ratio of the fraction of the freeze-dried product obtained in Experiment 5 having a molecular weight of 10,000 to 100,000 was determined. It was found to be the highest, and also the highest in total content of arabinose and xylose from polysaccharides. Therefore, fractionation and purification of the lyophilized product through ultrafiltration were performed by the following procedure.
That is,
The barley shochu distillation residue obtained in the above is centrifuged at 8000 rpm for 10 minutes to obtain a liquid component of the barley shochu distillation residue, and the obtained liquid component is synthesized by Amber, a synthetic adsorbent manufactured by Organo Corporation. By passing through a column filled with light XAD-16 and subjecting it to adsorption separation treatment, a synthetic adsorbent non-adsorbed fraction consisting of a flow-through liquid having non-adsorbability to the synthetic adsorbent of the column was collected. The obtained synthetic adsorbent non-adsorbed fraction was adjusted to Brix 10, and 1 L of the synthetic adsorbent non-adsorbed fraction adjusted to Brix 10 was mixed with 500 ml of Amberlite 200CT (a strong acid cation exchange resin) manufactured by Organo Corporation. Is passed through a column packed with, and an ion-exchange resin non-adsorbed fraction is obtained. The obtained ion-exchange resin non-adsorbed fraction is separated by an ultrafiltration membrane UFP-30-E-4MA (manufactured by A / G Technology). (Molecular weight 30,000) to obtain a concentrated solution. After adjusting the obtained concentrated solution to Brix20, ethanol was added to a final concentration of 75% by volume, and the mixture was centrifuged at 8000 rpm for 10 minutes. The organic solvent-insoluble fraction was collected, and the organic solvent-insoluble fraction was subjected to lyophilization to obtain 1.3 g of a lyophilized organic solvent-insoluble fraction.
[0038]
About the obtained freeze-dried product,
The total content of arabinose and xylose derived from polysaccharide was measured by the method described in (1). As a result, the total content of polysaccharide-derived arabinose and xylose was found to have increased to 78.5% by weight. In addition, about the obtained freeze-dried product,
The molecular weight distribution was measured according to the method described in (1). As a result, the molecular weight distribution of the lyophilized product was 11% for 100,000 or more, 29% for 30,000 to 100,000, 24% for 10,000 to 30,000, and 21% for 3,000 to 10,000. %, 1,000 to 3,000 was 6%, 500 to 1,000 was 2%, and 500 or less was 7%. And the result of the obtained chromatogram revealed that the chromatogram of the freeze-dried product had the highest peak in the molecular weight range of 30,000 to 100,000. Therefore,
According to the method described in
The lyophilizate obtained above was evaluated for its effect on suppressing the onset of alcoholic liver injury. As a result, it was found that the freeze-dried product had a remarkable effect of suppressing the onset of alcoholic liver injury.
[0039]
From the above experimental results, the onset inhibitory effect on alcoholic liver injury having a lyophilized product comprising the organic solvent-insoluble fraction obtained from the liquid component of the barley shochu distillation residue, the lyophilized product comprising the organic solvent-insoluble fraction Contained, derived from a polysaccharide having arabinose and xylose as a main component, a composition containing a significant amount of such a polysaccharide having arabinose and xylose as a main component is a by-product in the production of shochu from barley. Barley shochu distillation residue is solid-liquid separated to obtain a liquid component, and the liquid component is subjected to an adsorption separation treatment using a synthetic adsorbent to obtain a synthetic adsorbent non-adsorbed fraction, and the synthetic adsorbent non-adsorbed fraction is obtained. The fraction is subjected to an ion exchange treatment using an ion exchange resin to obtain an ion exchange resin non-adsorbed fraction, and the obtained ion exchange resin non-adsorbed fraction is subjected to ultrafiltration using an ultrafiltration membrane. To obtain a concentrated solution is subjected to over-treatment, by adding an organic solvent to the concentrate can be fractionated as an organic solvent-insoluble fraction was found. The freeze-dried product comprising the organic solvent-insoluble fraction was analyzed by component analysis to find that 78.5% by weight of polysaccharide, 3.3% by weight of crude protein, 0.2% by weight of organic acid, 1.5% by weight of free saccharide And the fraction was found to contain polysaccharides as the main component.
[0040]
Therefore, the present inventors have found that the synthetic adsorbent non-adsorbed fraction of the lyophilized powder was found to have an effect of suppressing the onset of alcoholic liver injury, and the remarkable suppression of the onset of alcoholic liver injury The following experiment was performed to determine whether the freeze-dried powder of the organic solvent-insoluble fraction found to have an effect has a healing effect on alcoholic liver injury that has already developed. .
That is, 30 7-week-old Wistar male rats (Charles River Japan) were fed an ethanol-containing liquid feed for 6 days while gradually increasing the ethanol content (3% → 4% → 5%), and then raised. The rats were bred for 4 weeks on a liquid feed containing 5% ethanol, blood was collected from each of the rats on the 4th week, the plasma was separated and the serum lipid was measured. , And test group 2. At that time, the 30 rats were sorted so that there was no statistically significant difference in the variance of the average weight of the rats in each group. To the rats of the control group, an ethanol-free liquid feed supplemented with an equivalent mixture of maltose-dextrin instead of the 5% ethanol was given for 2 weeks in order to make the calorie intake the same as that of the ethanol-containing liquid feed administration group. Bred. The test group 1 was fed a liquid feed obtained by adding 1% of a freeze-dried powder of the non-adsorbed fraction of the synthetic adsorbent to the ethanol-free liquid feed for 2 weeks. The test group 2 was fed with a liquid feed obtained by adding 1% of a freeze-dried powder of the organic solvent-insoluble fraction to the ethanol-free liquid feed for 2 weeks. Furthermore, an untreated group consisting of the 10 7-week-old Wistar male rats was provided separately from the control group, the test group 1 and the test group 2. They were fed and kept for a week. However, in each of the above four groups, the daily feed amount (calorie intake) of each liquid feed was limited to 70 ml (70 kcal). On the last day of the experiment (six weeks after the start of the experiment), blood was collected from the abdominal aorta of each of the bred rats, and the liver was extracted from all four groups. After serum separation, the collected blood was measured for serum total cholesterol, serum LDL-cholesterol, serum triglyceride, serum phospholipid, serum ALT (GPT), and serum AST (GOT). For the isolated liver, liver weight, liver total cholesterol, liver triglyceride, and liver phospholipid were measured. The obtained results were expressed as an average value ± standard error (SEM), and statistical processing was performed in the following procedure. That is, the comparison between the untreated group and the control group was analyzed using the Student's test method, and then the comparison between the control group and the group A was analyzed using the Tukey-Kramer method. 0.05% or less was determined as significant. Further, the hepatocytes collected from the excised liver were subjected to HE staining, and the morphology of the hepatocytes was observed at a magnification of 200 times using a biological microscope BX51 manufactured by Olympus Optical Co., Ltd.
[0041]
As a result, in the control group, the serum total cholesterol concentration, the serum LDL-cholesterol concentration, and the liver triglyceride concentration, which were increased by rearing with the feed liquid containing ethanol, were slightly reduced, and were close to the respective normal values. The degree was extremely small. On the other hand, the serum total cholesterol concentration, serum LDL-cholesterol concentration, serum triglyceride concentration, serum phospholipid concentration, serum ALT (GPT) concentration, serum AST (GOT) concentration, and total liver cholesterol concentration of test group 1 and test group 2 were determined. Each value is significantly lower than each value of the control group, that is, a value substantially equivalent to each normal value. In particular, the test group 2 is more normal than the test group 1. The values close to the values are shown. In Test Group 1 and Test Group 2, hepatocyte necrosis and balloon-like enlargement in the area around the terminal hepatic vein of the terminal hepatic lobule were also hardly observed by biological microscopic observation of hepatocytes.
In other words, it is clear that the healing effect of the freeze-dried product powder of the organic solvent-insoluble fraction on alcoholic liver injury has an effect exceeding that of the freeze-dried product powder of the non-adsorbed fraction of the synthetic adsorbent. Became.
[0042]
From the above experimental results, barley shochu distillation residue produced in the production of shochu using barley as a raw material is subjected to solid-liquid separation to obtain a liquid component, and the liquid component is subjected to an adsorption separation treatment using a synthetic adsorbent. The synthetic adsorbent non-adsorbed fraction is obtained by subjecting the synthetic adsorbent non-adsorbed fraction to an ion exchange treatment using an ion exchange resin to obtain an ion exchange resin non-adsorbed fraction. The fraction is subjected to an ultrafiltration treatment using an ultrafiltration membrane to obtain a concentrated solution, and the organic solvent-insoluble fraction obtained by adding an organic solvent to the concentrated solution is extremely powerful against alcoholic liver injury. It was found to have an onset-suppressing action and a healing action. From this fact, the liquid component obtained by solid-liquid separation of the barley shochu distillation residue by-produced in the production of shochu using barley as a raw material contains components that contribute to the suppression of the onset of alcoholic liver injury and the cure. Subjecting the liquid component to an adsorption separation treatment using a synthetic adsorbent to obtain a synthetic adsorbent non-adsorbed fraction, and subjecting the synthetic adsorbent non-adsorbed fraction to an ion exchange resin using an ion exchange resin. To obtain an ion-exchange resin non-adsorbed fraction, and subject the ion-exchange resin non-adsorbed fraction to ultrafiltration treatment using an ultrafiltration membrane to obtain a concentrated solution, and add an organic solvent to the concentrated solution. It was found that it was fractionated and contained in the organic solvent-insoluble fraction obtained by the addition.
[0043]
As described above, the present inventors have found that the liquid component obtained by solid-liquid separation of the residual liquid of barley shochu distilled as a by-product in the production of shochu using barley as a raw material contributes to the suppression of the onset of alcoholic liver injury and its cure. The liquid component is subjected to an adsorption separation treatment using a synthetic adsorbent to obtain a synthetic adsorbent non-adsorbed fraction, and the synthetic adsorbent non-adsorbed fraction is converted to an ion-exchange resin. The ion-exchange resin non-adsorbed fraction is obtained by using an ion exchange treatment to be used, and the ion-exchange resin non-adsorbed fraction is subjected to an ultrafiltration treatment using an ultrafiltration membrane to obtain a concentrated solution. The present inventors have found that a composition comprising an organic solvent-insoluble fraction obtained by adding an organic solvent to a liquid has a remarkable onset-suppressing action and a healing action for alcoholic liver injury. This finding of the organic solvent-insoluble fraction obtained from the distillation residue of barley shochu is a new fact that has never been seen before. Thus, the present invention creates a useful use of the organic solvent-insoluble fraction in that the organic solvent-insoluble fraction can be used as a medicament for therapeutic purposes.
[0044]
The present inventors have conducted intensive studies through experiments in order to achieve the above object, and as a result, solid-liquid separation of barley shochu distillation residue produced as a by-product in shochu production using barley as a raw material to obtain a liquid component. Subjecting the liquid fraction to an adsorption separation treatment using a synthetic adsorbent to obtain a synthetic adsorbent non-adsorbed fraction, and subjecting the synthetic adsorbent non-adsorbed fraction to an ion exchange treatment using an ion exchange resin. By obtaining an ion-exchange resin non-adsorbed fraction, subjecting the ion-exchange resin non-adsorbed fraction to ultrafiltration treatment using an ultrafiltration membrane to obtain a concentrated solution, and adding an organic solvent to the concentrated solution The organic solvent-insoluble fraction was fractionated, and the organic solvent-insoluble fraction was subjected to component analysis. As a result, polysaccharide 78.5% by weight, crude protein 3.3% by weight, organic acid 0.2% by weight, free saccharide 1.5% by weight, said fraction was found to contain polysaccharides as the main component
Further, it was found that when the organic solvent-insoluble fraction was subjected to freeze-drying, it had a white property. And it turned out that the said organic solvent-insoluble fraction which has such a characteristic has a marked onset-suppressing action and a healing action against alcohol-induced liver injury. The present invention is based on these findings.
[0045]
In addition, the composition having a fatty liver inhibitory effect described in Patent Document 1 is fractionated from the residual liquid of barley shochu distillation similarly to the composition of the present invention. However, the main component of the composition described in Patent Document 1 has a molecular weight of 3000 or less, and hemicellulose contained in the composition is mainly composed of xylose. They are distinct and distinct.
[0046]
As described above, the alcoholic fatty liver inhibitor described in Patent Document 2 is partially degraded by treating hemicellulose obtained by removing starch and protein from corn bran with alkali and treating it with xylanase. A substance is used as an active ingredient. In addition, Non-Patent Document 4 discloses a partially degraded product of hemicellulose (trade name: Cell Ace) obtained from corn fusuma described in Patent Document 2 described above.
[0004] As described above, it is described that the composition contains xylose, arabinose, uronic acid, galactose, and glucose. Therefore, Patent Literature 2 and Non-Patent Literature 4 relate to the same “partially decomposed product of hemicellulose (product name: Cell Ace) obtained from corn bran.” On the other hand, the composition of the present invention is obtained from the residual liquid of barley shochu distillation completely different from the above-mentioned corn bran. That is, the composition of the present invention is obtained through a production process completely different from the production process of the partially decomposed product of hemicellulose described in Patent Document 2. The composition of the present invention contains only arabinose, xylose and glucose, and does not substantially contain uronic acid contained in the partially decomposed product of hemicellulose described in Patent Document 2. Therefore, the composition of the present invention is a different thing which is clearly distinguished from the alcoholic fatty liver inhibitor containing, as an active ingredient, a "partially degraded product of hemicellulose obtained from corn flea" described in Patent Document 2. It is obvious.
[0047]
In addition, Non-Patent Document 4 describes that the molecular weight of "partially decomposed product of hemicellulose (trade name: Cell Ace) obtained from corn fusuma" is about 200,000, but has come to determine the molecular weight. No experimental data is described. Therefore, the present inventors have described in Patent Document 2 that a partially decomposed product of hemicellulose is commercially available under the trade name “Cel Ace” (manufactured by Nippon Shokuhin Kako Co., Ltd.). Obtain and said
The molecular weight distribution of Cellace was measured by the method described in (1). As a result, the molecular weight distribution of the Cell Ace is 1% or more in 1,000,000, 9% in 300,000 to 1,000,000, 30% in 100,000 to 300,000, 30% in 30,000 to 100,000, and 10,000. The chromatogram has a peak top in the molecular weight range of 100,000 to 300,000, with 11% in the range of from 30,000 to 30,000 in the range of 4% in 3000 to 10,000, 2% in the range of 1000 to 3000, and 13% in the range of 1000 or less. It consisted of only a single peak and its weight average molecular weight (Mw) was found to be 150,000.
On the other hand, as described above, the molecular weight distribution of the composition of the present invention is only 11% at 100,000 or more, and components having a molecular weight of 3,000 to 100,000 occupy 74% of the whole. Moreover, the chromatogram of the composition of the present invention has the highest peak in the molecular weight range of 30,000 to 100,000.
Therefore, the molecular weight distribution of the composition of the present invention is completely different from the molecular weight distribution of “Cel Ace”, that is, the composition composed of partially decomposed products of hemicellulose obtained from corn fur. Further, as described above, the composition of the present invention does not substantially contain uronic acid contained in a partially decomposed product of hemicellulose obtained from corn bran. Even in this respect, the composition of the present invention is clearly different from the alcoholic fatty liver inhibitor consisting of “partially degraded hemicellulose” described in Patent Document 2. It is obvious.
[0048]
[Example of embodiment]
The present invention achieves the above object, and provides a composition having a strong onset-suppressing action and a curative action on alcoholic liver injury, and a method for producing the same. That is, barley shochu distillation residue produced as a by-product in the production of shochu using barley as a raw material is subjected to solid-liquid separation to obtain a liquid component, and the liquid component is subjected to an adsorption separation treatment using a synthetic adsorbent to produce a synthetic adsorbent. A non-adsorbed fraction is obtained, and the synthetic adsorbent non-adsorbed fraction is subjected to an ion exchange treatment using an ion-exchange resin to obtain an ion-exchange resin non-adsorbed fraction. A concentrated solution is obtained by ultrafiltration treatment using a filtration membrane, and an organic solvent-insoluble fraction obtained by adding an organic solvent to the concentrated solution, a remarkable inhibitory action against alcoholic liver injury. And a composition having a healing action and a method for producing the same. Further, the present invention provides a medicine comprising the composition.
[0049]
Hereinafter, preferred embodiments of the present invention will be described, but the present invention is not limited thereto.
The composition of the present invention is produced as follows. That is, the method for producing the composition comprises a first step of solid-liquid separation of a residual liquid of barley shochu distilled by-product in the production of distilled liquor using barley to obtain a liquid component, and using the liquid component with a synthetic adsorbent. A second step of obtaining a synthetic adsorbent non-adsorbed fraction by subjecting the synthetic adsorbent non-adsorbed fraction to an ion-exchange treatment using an ion exchange resin. A third step of obtaining a concentrate, a fourth step of subjecting the ion-exchange resin non-adsorbed fraction to an ultrafiltration treatment using an ultrafiltration membrane to obtain a concentrate, and adding an organic solvent to the concentrate. A fifth step of fractionating the organic solvent-insoluble fraction.
Hereinafter, the barley shochu distillation residue and by-produced by-products in the production of shochu using barley as a raw material, which are used when carrying out the production method of the present invention, and each step are described in detail.
[0050]
The barley shochu distillation residue used in the present invention is typically a barley koji and steamed barley produced from barley or refined barley, and the starch contained in the obtained barley koji and steamed barley is converted to the barley koji. Saccharified with koji, and subjected to alcohol fermentation with yeast to obtain shochu-ripened moromi, and as a distillation residue when distilling the obtained shochu-ripened moromi using a simple distillation apparatus such as vacuum distillation or atmospheric distillation. It means a by-product, that is, a distillation residue of barley shochu.
[0051]
In the present invention, when obtaining the residual liquid of barley shochu distillation, the barley koji used in the production of barley shochu may be produced under the koji making conditions used in ordinary barley shochu production. Aspergillus kawachii used in barley shochu production is preferred. Alternatively, Aspergillus strains such as Aspergillus awamori used in awamori production and Aspergillus oryzae used in sake production can be used. As the yeast used for producing barley shochu, various types of yeast for brewing shochu generally used for producing shochu can be used.
[0052]
In the present invention, the first step of solid-liquid separation of the barley shochu distillation residue obtained in the distillation step in the production of barley shochu to obtain a liquid component is a raw barley from barley shochu distillation residue, or a barley koji-derived product. This is performed for the purpose of removing SS components such as water-insoluble fermentation residues. The solid-liquid separation in the first step is performed by a solid-liquid separation method such as a screw press method or a roller press method, or a preliminary separation is performed using a solid-liquid separator of a filtration and pressing type, and then a centrifugal separator and a diatom The solid-liquid separation treatment that can be performed according to the present invention is performed using a soil filtration device, a ceramic filtration device, a filtration press, or the like.
[0053]
The second step of subjecting the liquid component of the barley shochu distillation residue obtained in the first step to an adsorption separation treatment using a synthetic adsorbent to obtain a synthetic adsorbent non-adsorbed fraction is a barley shochu distillation residue. The purpose is to remove components such as polyphenols contained in the liquid component. As the synthetic adsorbent used in the second step, an aromatic, aromatic-modified, or methacrylic synthetic adsorbent can be used. Preferable specific examples of the synthetic adsorbent used in the second step include Amberlite XAD-4, Amberlite XAD-16, Amberlite XAD-1180 and Amberlite XAD-2000 manufactured by Organo Corporation, and Mitsubishi Chemical Corporation. Arobe (or also referred to as styrene) synthetic adsorbent such as Sepabeads SP850 and Diaion HP20 manufactured by Co., Ltd., Amberlite XAD-7 manufactured by Organo Co., Ltd., and Diaion HP2MG manufactured by Mitsubishi Chemical Corporation. Methacrylic (or also acrylic) synthetic adsorbent. In addition to the above, an aromatic-modified synthetic adsorbent such as Sepapise SP207 manufactured by Mitsubishi Chemical Corporation may be used in some cases.
[0054]
In the third step of subjecting the synthetic adsorbent non-adsorbed fraction obtained in the second step to an ion exchange treatment using an ion exchange resin to obtain an ion exchange resin non-adsorbed fraction, The purpose is to remove amino acids, peptides, proteins, and organic acids contained in the adsorption fraction using an ion exchange resin. As the ion exchange resin used in the third step, a cation exchange resin, an anion exchange resin, or a mixed bed ion exchange resin in which both are mixed can be used. In the case of a cation exchange resin, any of a strongly acidic cation exchange resin and a weakly acidic cation exchange resin can be used, and in the case of an anion exchange resin, a strongly basic anion exchange resin and a weakly basic cation exchange resin can be used. Any of the anion exchange resins can be used. In the case of a mixed bed ion exchange resin, the cation exchange resin and the anion exchange resin described above can be freely combined and mixed at a predetermined ratio for use. Preferable specific examples of such an ion exchange resin include strongly acidic cation exchange resins such as Amberlite 200CT and Amberlite IR120B manufactured by Organo Corporation, weakly acidic cation exchange resins such as Amberlite IRC76, and Amberlite IRA402BL. The strongest basic anion exchange resin, a weakly basic anion exchange resin such as Amberlite IRA67, or a mixture of the weakly acidic cation exchange resin and the weakly basic anion exchange resin in a predetermined ratio. , Etc. can be used. Among them, from the viewpoint of removing amino acids and peptides contained in the barley shochu distillation residue, both the weakly acidic cation exchange resin Amberlite IRC76 and the weakly basic anion exchange resin Amberlite IRA67 are used. It is particularly preferable to use a mixed bed type ion exchange resin obtained by mixing at a predetermined ratio, or a strongly acidic cation exchange resin Amberlite 200CT.
[0055]
In the fourth step of subjecting the non-adsorbed fraction of the ion-exchange resin obtained in the third step to ultrafiltration using an ultrafiltration membrane to obtain a concentrated solution, the fourth step involves the action of activating NK cells. The purpose of this method is to concentrate polysaccharides containing arabinose and xylose as main components using an ultrafiltration membrane. As the ultrafiltration membrane used in the fourth step, any membrane material and membrane module type can be used, and the molecular weight cutoff is preferably 3,000 or more, particularly preferably 10,000 to 50,000 can be used.
[0056]
In the fifth step of collecting an organic solvent-insoluble fraction by adding an organic solvent to the concentrate obtained in the fourth step, an appropriate organic solvent is added until a predetermined final concentration is reached. In this case, the organic solvent is optimally ethanol, but is not limited thereto. The final concentration of the organic solvent affects the production efficiency of the components, and the optimal final concentration of the organic solvent is preferably 5% by volume or more, more preferably 30% to 75% by volume.
[0057]
The organic solvent-insoluble fraction thus obtained, which is the composition of the present invention, as it is, or as a dry product powder by subjecting it to freeze-drying or the like, is a strong inhibitory agent against alcoholic liver injury. It can be used as a medicine having an action and a healing action.
[0058]
【Example】
Hereinafter, the present invention will be described specifically with reference to examples, but the present invention is not limited to these examples.
[0059]
Embodiment 1
Said
The barley shochu distillation residue obtained in the above was centrifuged at 8,000 rpm for 10 minutes to obtain a liquid component of the barley shochu distillation residue, and the obtained liquid component was synthesized with a synthetic adsorbent Amber manufactured by Organo Corporation. By passing through a column filled with light XAD-16 and subjecting it to adsorption separation treatment, a synthetic adsorbent non-adsorbed fraction consisting of a flow-through liquid having non-adsorbability to the synthetic adsorbent of the column was collected. After adjusting the obtained synthetic adsorbent non-adsorbed fraction to Brix 10, 1 L of the synthetic adsorbent non-adsorbed fraction adjusted to Brix 10 was used in a 500 ml capacity of Amberlite 200CT (a strong acid cation exchange resin) manufactured by Organo Corporation. Is passed through a column packed with, and an ion-exchange resin non-adsorbed fraction is obtained. The obtained ion-exchange resin non-adsorbed fraction is separated by an ultrafiltration membrane UFP-30-E-4MA (manufactured by A / G Technology). (Molecular weight 30,000) to obtain a concentrated solution. After adjusting the obtained concentrated solution to Brix20, ethanol was added to a final concentration of 75% by volume, and the mixture was centrifuged at 8000 rpm for 10 minutes. The organic solvent-insoluble fraction was collected, and the organic solvent-insoluble fraction was subjected to lyophilization to obtain 1.3 g of a lyophilized organic solvent-insoluble fraction. The freeze-dried product was pulverized to obtain an off-white, tasteless and odorless composition.
[0060]
[Comparative Example 1]
Said
The barley shochu distillation residue obtained in the above was centrifuged at 8000 rpm for 10 minutes to obtain a liquid portion of the barley shochu distillation residue, and the obtained liquid portion was synthesized by Amberlite, a synthetic adsorbent manufactured by Organo Corporation. By passing through a column filled with XAD-16 and subjecting it to adsorption separation treatment, a fraction of a synthetic adsorbent non-adsorbed fraction consisting of a flow-through liquid exhibiting non-adsorbability to the synthetic adsorbent of the column was collected. By subjecting 1 L of the obtained synthetic adsorbent non-adsorbed fraction to lyophilization, 57.2 g of a freeze-dried product of the synthetic adsorbent non-adsorbed fraction was obtained. The freeze-dried product was pulverized to obtain an off-white, tasteless and odorless composition.
[0061]
Each of the freeze-dried product powder (composition) obtained in Example 1 and the freeze-dried product powder obtained in Comparative Example 1 was subjected to the following Test Example 1 to evaluate the inhibitory effect on the development of alcoholic liver injury.
[0062]
[Test Example 1]
The following Test Example 1 was conducted to clarify the remarkable inhibitory effect of the composition of the present invention on the development of alcoholic liver injury.
That is, 36-week-old Wistar male rats (Japan SLC) were fed with an ethanol-containing liquid feed for 6 days while gradually increasing the ethanol content (3% → 4% → 5%). , A control group, a test group 1 and a test group 2. At this time, the 36 rats were sorted so that there was no statistically significant difference in the variance of the average weight of the rats in each group. The rats in the control group were fed a liquid feed containing 5% ethanol for 4 weeks and bred. The rats in Test Group 1 were fed a liquid feed containing 1% of the lyophilized powder obtained in Example 1 to a liquid feed containing 5% ethanol for 4 weeks. The rats in Test Group 2 were fed a liquid feed containing 1% of the lyophilized powder obtained in Comparative Example 1 to a liquid feed containing 5% ethanol for 4 weeks. An untreated group consisting of the 12 3-week-old Wistar male rats was provided separately from the control group, the test group 1 and the test group 2, and the caloric intake of the rats in the untreated group was compared with that of the other three groups. In order to make them the same, an ethanol-free liquid feed supplemented with an equivalent mixture of maltose and dextrin instead of 5% ethanol was fed for 4 weeks. However, in each of the above four groups, the daily feed amount (calorie intake) of each liquid feed was limited to 70 ml (70 kcal). On the final day of the experiment (four weeks after the start of the test), blood was collected from each abdominal aorta of the bred rats, and the liver was extracted. The collected blood was subjected to serum separation, and serum total cholesterol, serum HDL-cholesterol, serum LDL-cholesterol, serum triglyceride, serum phospholipid, serum free fatty acid, and serum ALT were measured. For the isolated liver, liver weight, liver total cholesterol, liver triglyceride, and liver phospholipid were measured. The obtained results were expressed as an average value ± standard error (SEM), and statistical processing was performed in the following procedure. That is, the comparison between the untreated group and the control group was analyzed using the Student's test method, and then the comparison between the test group 1 and the test group 2 with respect to the control group was analyzed using the Tukey-Kramer method. A risk rate of 0.05% or less was determined as significant. Further, the hepatocytes collected from the excised liver were subjected to HE staining, and the morphology of the hepatocytes was observed at a magnification of 200 times using a biological microscope BX51 manufactured by Olympus Optical Co., Ltd.
[0063]
[Evaluation 1]
The measurement results of serum total cholesterol, serum HDL-cholesterol, serum LDL-cholesterol, serum triglyceride, serum phospholipid, serum free fatty acid, and serum ALT obtained above are shown in Table 1, and the liver weight, liver total cholesterol, liver The following facts were found from the measurement results of triglycerides and liver phospholipids.
That is, the control group showed a significant increase in serum total cholesterol concentration, serum HDL-cholesterol concentration, serum LDL-cholesterol concentration, serum triglyceride concentration and serum phospholipid concentration as compared with the untreated group, It was found that blood was induced. Further, it was found that the liver triglyceride concentration and the liver phospholipid concentration were significantly increased in the control group and the alcoholic fatty liver was induced as compared with the untreated group. Furthermore, in the control group, the blood ALT (GPT) concentration was significantly increased as compared with the non-treatment group, and the hepatocyte necrosis and balloon-like in the area around the terminal hepatic vein of the hepatic lobule were observed by biomicroscopic observation of hepatocytes. Swelling was remarkably observed, and it was found that alcoholic hepatitis was induced. On the other hand, in Test Group 1 and Test Group 2, increases in serum LDL-cholesterol concentration, serum triglyceride concentration, and liver triglyceride concentration were significantly suppressed, and in particular, Test Group 1 was closer to normal values than Test Group 2. The indicated values were shown. In Test Group 1 and Test Group 2, hepatocyte necrosis and balloon-like enlargement in the area around the terminal hepatic vein of the terminal hepatic lobule, which is specifically observed in alcoholic liver injury, were also observed in biological microscopic observation of hepatocytes. I couldn't.
[0064]
From the above results, the freeze-dried product powder of the present invention obtained in Example 1 further suppressed the induction of alcoholic liver injury more remarkably than the freeze-dried product powder obtained in Comparative Example 1 and caused alcoholic liver injury. On the other hand, it was found to show an excellent onset-suppressing effect.
That is, it was revealed that the composition of the present invention has a remarkable onset-suppressing effect on alcoholic liver injury.
[0065]
Each of the lyophilized product powder (composition) obtained in Example 1 and the lyophilized product powder obtained in Comparative Example 1 was subjected to the following Test Example 2 to evaluate a healing effect on alcoholic liver injury.
[0066]
[Test Example 2]
In this test example, the lyophilized product powder (composition of the present invention) obtained in Example 1 and the lyophilized product powder obtained in Comparative Example 1 were obtained for rats that developed alcoholic liver injury by breeding on a liquid feed containing ethanol for 4 weeks. Each of the freeze-dried product powders was reared to evaluate a healing effect on alcoholic liver injury.
That is, 30 7-week-old Wistar male rats (Charles River Japan) were fed an ethanol-containing liquid feed for 6 days while gradually increasing the ethanol content (3% → 4% → 5%), and then raised. The rats were bred for 4 weeks on a liquid feed containing ethanol, and blood was collected from each of the rats on the 4 weeks, and the plasma was separated and the serum lipid was measured. Thereafter, the 30 rats were divided into three groups, a control group, a test group 1 and a test group 2, with 10 rats per group. At that time, the 30 rats were sorted so that there was no statistically significant difference in the variance of the average weight of the rats in each group. The rats in the control group were fed an ethanol-free liquid feed supplemented with an equivalent mixture of maltose and dextrin instead of 5% ethanol for 2 weeks in order to make the calorie intake the same as that in the above-mentioned ethanol-containing liquid feed administration group. did. The rats in Test Group 1 were fed a liquid feed in which 1% of the lyophilized powder obtained in Example 1 was added to the ethanol-free liquid feed for 2 weeks. The rats in Test Group 2 were fed a liquid feed containing 1% of the freeze-dried powder obtained in Comparative Example 1 to the ethanol-free liquid feed for 2 weeks. Furthermore, an untreated group consisting of the 10 7-week-old Wistar male rats was provided separately from the control group, the test group 1 and the test group 2, and the rats in the untreated group were fed a liquid diet not containing ethanol for 6 weeks. Bred. However, in each of the above four groups, the daily feed amount (calorie intake) of each liquid feed was limited to 70 ml (70 kcal). On the last day of the experiment (six weeks after the start of the experiment), blood was collected from the abdominal aorta of each of the bred rats, and the liver was extracted from all four groups. After serum separation, the collected blood was measured for serum total cholesterol, serum LDL-cholesterol, serum triglyceride, serum phospholipid, serum ALT (GPT), and serum AST (GOT). For the isolated liver, liver weight, liver total cholesterol, liver triglyceride, and liver phospholipid were measured. The obtained results were expressed as an average value ± standard error (SEM), and statistical processing was performed in the following procedure. That is, the comparison between the untreated group and the control group was analyzed using the Student's test method, and then the comparison between the test group 1 and the test group 2 with respect to the control group was analyzed using the Tukey-Kramer method. A risk rate of 0.05% or less was determined as significant. Further, the hepatocytes collected from the excised liver were subjected to HE staining, and the morphology of the hepatocytes was observed at a magnification of 200 times using a biological microscope BX51 manufactured by Olympus Optical Co., Ltd.
[0067]
[Evaluation 2]
Measurement results of serum total cholesterol, serum LDL-cholesterol, serum triglyceride, serum phospholipid, serum ALT, and serum AST obtained above, and measurement results of liver weight, liver total cholesterol, liver triglyceride, and liver phospholipid Found the following facts.
That is, in the control group, serum total cholesterol and serum LDL-cholesterol showed significantly higher values than in the untreated group, and serum triglycerides and serum phospholipids also tended to be higher than those in the untreated group. Hepatic necrosis and balloon-like swelling around the terminal hepatic vein of the hepatic lobule, which were specifically observed in alcoholic liver injury, were observed. On the other hand, test group 1 and test group 2 have serum total cholesterol concentration, serum LDL-cholesterol concentration, serum triglyceride concentration, serum phospholipid concentration, serum ALT concentration, serum AST concentration, liver total cholesterol concentration, and liver triglyceride concentration, The test group 1 showed a value significantly lower than that of the control group, and in particular, the test group 1 showed a value closer to the normal value than the test group 2. Test group 1 and test group 2 showed that hepatocellular necrosis and balloon-like enlargement in the area around the terminal hepatic vein of the hepatic lobule, which was specifically observed in alcoholic liver injury, were observed in the control group. A significant decrease was observed in comparison.
From the above results, the freeze-dried product powder of the present invention obtained in Example 1 surpasses the freeze-dried product powder obtained in Comparative Example 1, and further remarkably cures alcoholic hepatic injury. It was found to have a marked healing effect.
That is, it was found that the composition of the present invention has an excellent healing effect on alcoholic liver injury.
[0068]
Each of the lyophilized product powder (composition) obtained in Example 1 and the lyophilized product powder obtained in Comparative Example 1 was subjected to the following Test Example 3, and the healing effect on alcoholic liver injury was different from that of Test Example 2. The method was evaluated.
[Test Example 3]
Thirty 7-week-old Wistar male rats (Charles River Japan) were fed an ethanol-containing liquid feed for 6 days while gradually increasing the ethanol content (3% → 4% → 5%), followed by 5% ethanol The rats were bred for 4 weeks on the contained liquid feed, and in the 4 weeks, blood was collected from each of the rats, the plasma was separated, and the serum lipid was measured. Thereafter, the 30 rats were divided into three groups, a control group, a test group 1 and a test group 2, with 10 rats per group. At that time, the 30 rats were sorted so that there was no statistically significant difference in the variance of the average weight of the rats in each group. The rats in the control group were fed a liquid feed containing 5% ethanol for 2 weeks and bred. The rats in Test Group 1 were fed a liquid feed containing 1% of the lyophilized powder obtained in Example 1 to a liquid feed containing 5% ethanol for 2 weeks. The rats in Test Group 2 were fed a liquid feed containing 5% ethanol-containing liquid feed and the freeze-dried product powder 1% obtained in Comparative Example 1 for 2 weeks. Furthermore, an untreated group consisting of 10 7-week-old Wistar male rats was provided separately from the control group, the test group 1 and the test group 2, and the 5% ethanol-containing liquid feed and Ethanol-free liquid feed supplemented with a maltose-dextrin equivalent mixture instead of 5% ethanol was fed for 6 weeks in order to make the calorie intake the same. However, in each of the above four groups, the daily feed amount (calorie intake) of each liquid feed was limited to 70 ml (70 kcal). On the last day of the experiment (six weeks after the start of the experiment), blood was collected from the abdominal aorta of each of the bred rats, and the liver was extracted from all four groups. After the serum was separated from the collected blood, serum total cholesterol, serum LDL-cholesterol, serum triglyceride, serum phospholipid, serum ALT, and serum AST were measured. For the isolated liver, liver weight, liver total cholesterol, liver triglyceride, and liver phospholipid were measured. The obtained results were expressed as an average value ± standard error (SEM), and statistical processing was performed in the following procedure. That is, the comparison between the untreated group and the control group was analyzed using the Student's test method, and then the comparison between the test group 1 and the test group 2 with respect to the control group was analyzed using the Tukey-Kramer method. A risk rate of 0.05% or less was determined as significant. Further, the hepatocytes collected from the excised liver were subjected to HE staining, and the morphology of the hepatocytes was observed at a magnification of 200 times using a biological microscope BX51 manufactured by Olympus Optical Co., Ltd.
[0069]
[Evaluation 3]
Measurement results of serum total cholesterol, serum LDL-cholesterol, serum triglyceride, serum phospholipid, serum ALT, and serum AST obtained above, and measurement results of liver weight, liver total cholesterol, liver triglyceride, and liver phospholipid Found the following facts.
That is, the control group was compared with the untreated group, and the serum total cholesterol concentration, serum LDL-cholesterol concentration, serum triglyceride concentration, serum phospholipid concentration, serum ALT concentration, liver weight, liver total cholesterol concentration, and liver triglyceride concentration Showed a significantly high value, and hepatocyte necrosis and balloon-like swelling in the area around the terminal hepatic vein of the hepatic lobule, which was specifically observed in alcoholic liver injury, were observed remarkably in biological microscopic observation of hepatocytes. On the other hand, in the test group 1 and the test group 2, the serum triglyceride concentration, the serum total cholesterol concentration, the serum phospholipid concentration, the serum LDL-cholesterol concentration, and the liver total cholesterol concentration and the liver triglyceride concentration were significantly higher than those of the control group. The test group 1 showed a lower value, and in particular, the test group 1 showed a value closer to the normal value than the test group 2. Test group 1 and test group 2 showed that hepatocellular necrosis and balloon-like enlargement in the area around the terminal hepatic vein of the hepatic lobule, which was specifically observed in alcoholic liver injury, were observed in the control group. A significant decrease was observed in comparison.
From the above results, the freeze-dried product powder of the present invention obtained in Example 1 surpasses the freeze-dried product powder obtained in Comparative Example 1, and further remarkably cures alcoholic hepatic injury. It was found to have a marked healing effect.
That is, it was found that the composition of the present invention has an excellent healing effect on alcoholic liver injury.
[0070]
As is clear from the results described in Test Example 1, the composition of the present invention does not adsorb the synthetic adsorbent obtained by subjecting the liquid component of the distillation residue of barley shochu to an adsorptive separation treatment using a synthetic adsorbent. It was found that it has a strong effect of suppressing the onset of alcoholic liver injury, which has an excellent fraction, and strongly inhibits the onset of alcoholic liver injury caused by ethanol administration. Furthermore, as is clear from the results described in Test Example 2 and Test Example 3, it was found that the composition of the present invention significantly cured alcoholic liver injury that had already developed.
[0071]
[Example 1]
The lyophilized powder obtained in Example 1 was mixed with other carrier materials at the following compounding ratios, and a pharmaceutical tablet was prepared using a tableting machine.
Material blending ratio: 35% by weight of lyophilized powder obtained in Example 1, 5% by weight of sugar ester, 30% by weight of oligosaccharide, 30% by weight of lactose
[0072]
【The invention's effect】
As described in detail above, barley shochu distillation residue produced as a by-product in the production of shochu from barley of the present invention is solid-liquid separated to obtain a liquid component, and the liquid component is subjected to an adsorption separation treatment using a synthetic adsorbent. To obtain a synthetic adsorbent non-adsorbed fraction, and subject the synthetic adsorbent non-adsorbed fraction to an ion exchange treatment using an ion-exchange resin to obtain an ion-exchange resin non-adsorbed fraction. The non-adsorbed fraction is subjected to an ultrafiltration treatment using an ultrafiltration membrane to obtain a concentrated solution, and a composition comprising an organic solvent-insoluble fraction obtained by adding an organic solvent to the concentrated solution has an alcoholic property. It has a remarkable inhibitory and curative effect on the onset of liver damage.
Claims (10)
１００，０００以上 １１％
３０，０００乃至１００，０００ ２９％
１０，０００乃至 ３０，０００ ２４％
３，０００乃至 １０，０００ ２１％
１，０００乃至 ３，０００ ６％
５００乃至 １，０００ ２％
５００以下 ７％A barley shochu distillation residue produced as a by-product in the production of shochu using barley as a raw material is solid-liquid separated to obtain a liquid component, and the liquid component is subjected to an adsorption separation treatment using a synthetic adsorbent, and the synthetic adsorbent non-adsorbed Fraction, and subjecting the synthetic adsorbent non-adsorbed fraction to an ion-exchange treatment using an ion-exchange resin to obtain an ion-exchange resin non-adsorbed fraction. A concentrate is obtained by subjecting to an ultrafiltration treatment to be used, and is obtained by adding an organic solvent to the concentrate and containing substantially no uronic acid, comprising xylose, arabinose and glucose, and comprising xylose A composition comprising a polysaccharide having an / arabinose ratio of> 2.0 and comprising an organic solvent-insoluble fraction having the following molecular weight distribution and having an onset-suppressing action and a curative action on alcoholic liver injury. Molecular weight distribution:
100% or more 11%
30,000 to 100,000 29%
10,000 to 30,000 24%
3,000 to 10,000 21%
1,000 to 3,000 6%
500 to 1,000 2%
500% or less 7%
分子量分布：
１００，０００以上 １１％
３０，０００乃至１００，０００ ２９％
１０，０００乃至 ３０，０００ ２４％
３，０００乃至 １０，０００ ２１％
１，０００乃至 ３，０００ ６％
５００乃至 １，０００ ２％
５００以下 ７％A step of solid-liquid separation of a residual liquid of barley shochu distilled as a by-product in the production of shochu using barley as a raw material to obtain a liquid component, and subjecting the liquid component to an adsorption separation treatment using a synthetic adsorbent to remove the synthetic adsorbent Obtaining a fraction, subjecting the synthetic adsorbent non-adsorbed fraction to an ion exchange treatment using an ion-exchange resin to obtain an ion-exchange resin non-adsorbed fraction, and excluding the ion-exchange resin non-adsorbed fraction A step of obtaining a concentrate by ultrafiltration using a filtration membrane, by adding an organic solvent to the concentrate, substantially free of uronic acid, consisting of xylose, arabinose and glucose, Including a polysaccharide having a xylose / arabinose ratio of> 2.0 and a step of collecting an organic solvent-insoluble fraction having the following molecular weight distribution; Work Method for producing a composition having.
Molecular weight distribution:
100% or more 11%
30,000 to 100,000 29%
10,000 to 30,000 24%
3,000 to 10,000 21%
1,000 to 3,000 6%
500 to 1,000 2%
500% or less 7%
分子量分布：
１００，０００以上 １１％
３０，０００乃至１００，０００ ２９％
１０，０００乃至 ３０，０００ ２４％
３，０００乃至 １０，０００ ２１％
１，０００乃至 ３，０００ ６％
５００乃至 １，０００ ２％
５００以下 ７％Fermenting barley koji and shochu yeast produced from brown barley or refined barley as raw materials to produce ripened moromi, and subjecting the ripened moromi to distillation to produce barley shochu; and The step (A) comprises a step (B) of treating a barley shochu distillation residue as a by-product as a distillation residue when the barley shochu is produced. In the step (B), the barley shochu distillation residue solid-liquid Separating to obtain a liquid component, subjecting the liquid component to an adsorption separation treatment using a synthetic adsorbent to obtain a synthetic adsorbent non-adsorbed fraction, A step of obtaining an ion-exchange resin non-adsorbed fraction by subjecting the ion-exchange resin to use, a step of subjecting the ion-exchange resin non-adsorbed fraction to ultrafiltration using an ultrafiltration membrane to obtain a concentrated solution, And by adding an organic solvent to the concentrated solution An organic solvent-insoluble fraction comprising xylose, arabinose and glucose, which is substantially free from uronic acid, contains a polysaccharide having a xylose / arabinose ratio of> 2.0, and has the following molecular weight distribution: Wherein the step (A) and the step (B) are successively performed, and the composition comprising the barley shochu and the organic solvent-insoluble fraction is continuously produced.
Molecular weight distribution:
100% or more 11%
30,000 to 100,000 29%
10,000 to 30,000 24%
3,000 to 10,000 21%
1,000 to 3,000 6%
500 to 1,000 2%
500% or less 7%
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| JP2002380685A JP4251866B2 (en) | 2002-12-27 | 2002-12-27 | Composition having inhibitory action and curative action on alcoholic liver injury, and method for producing the composition |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2009090961A1 (en) | 2008-01-15 | 2009-07-23 | Sapporo Breweries Limited | Agent for prevention of alcoholic hepatopathy |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2009090961A1 (en) | 2008-01-15 | 2009-07-23 | Sapporo Breweries Limited | Agent for prevention of alcoholic hepatopathy |
| US8747836B2 (en) | 2008-01-15 | 2014-06-10 | Sapporo Breweries Limited | Agent for prevention of alcoholic hepatopathy |
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