JP4380951B2 - Garlic fermentation composition - Google Patents

Description

【００２３】
実施例１ 麹菌を用いたニンニク発酵組成物の製造及びその抗酸化活性（発酵組成物（エキス）の調製）
ニンニクを水洗した後、等量の水を加え、３０分間煮沸した後、破砕し、破砕汁を得た。破砕汁に等量の水、及びグルコース２％を添加し、オートクレーブで１２１℃、２０分間滅菌した（ニンニク培地）。別に、麹菌（Aspergillus oryzae）を、サブロー培地（ペプトン１％、グルコース４％）で２７℃、１６０rpm振盪の条件下で３日間前培養し、ニンニク培地にこの培養液を１％添加し２７℃、１６０rpm 振盪の条件下で２日〜５週間培養し、３００rpm１０分間遠心分離した上清（エキス）を以下の試験に用いた。一方、米、大豆は、乾燥固形物としてニンニク培地と等しくなるように調製した。
【００２４】
（スーパーオキシドアニオン消去能の測定法）
65mM KH₂PO₄−ほう酸緩衝液（pH８．２）２００μl、５mMキサンチン水溶液２００μl、１０mMヒドロキシルアミン水溶液１００μl、水２００μlの混合液にサンプル１００μl及び、キサンチンオキシダーゼ２００μlを加え、３７℃で１５分間インキュベートした。インキュベート終了時に、反応停止及び発色の目的で、３０μＭ Ｎ−ナフチルエチレンジアミン−３mMスルファニル酸−２５％氷酢酸２mLを加えて室温で４５分間放置し、５５０nmで吸光度を測定した。本測定は、２回行い、サンプル添加による吸光度の減少から活性酸素消去活性（％）を求めた。なお、キサンチンオキシダーゼ（キサンチンオキシダーゼ懸濁液：和光純薬）は、吸光度の変化が０．０２０〜０．０２５／分になるように調製した（１５〜２０mU／mL）。
結果を図１及び２に示す。
【００２５】
（ＤＰＰＨラジカル消去能の測定法）
サンプルを０．５Ｍ酢酸緩衝液（pH５．５）で希釈し、その０．５mLにエタノール０．５mLを加え、更に０．５mM ＤＰＰＨ（１，１−ジフェニル−１−２−ピクリルヒドラジル：和光純薬）エタノール溶液０．２５mLを加えてよく混合し、５２０nmにおける吸光度を経時的に測定し、サンプル無添加の対照に対する％を求めた。結果を図３〜５に示す。
【００２６】
（結果）
（１）ニンニク培地及び、ニンニク発酵エキスのスーパーオキシドアニオン消去能
ニンニク培地は、弱い消去活性を示したが、ニンニク発酵エキスは、発酵期間に応じて消去活性が増加した。また、グルコースの添加によって、ニンニク培地の活性は僅かに増加したが、発酵エキスは、ほとんど差が認められなかった（図１）。
ニンニク発酵エキスを希釈して、同様にスーパーオキシドアニオン消去能を測定した。ニンニク発酵エキスは、４週間発酵のサンプルで、１００倍希釈したものと、ニンニク培地の活性が等しくなり、発酵によってスーパーオキシドアニオン消去活性は、１００倍に増強された（図２）。
【００２７】
（２）ＤＰＰＨラジカル消去活性
１００倍希釈液を用いてＤＰＰＨラジカル消去活性を測定した。ニンニク培地（ＢＧ）は、殆ど活性は認められなかったが、ニンニク発酵エキスは、何れの麹菌でも、発酵期間に応じてＤＰＰＨラジカル消去活性が増加した。特にＡ−１、Ａ−６に強い活性が認められた（図３）。
【００２８】
（３） A-1 、 A-6 のＤＰＰＨラジカル消去活性物質産生に及ぼす培地の影響
米培地、大豆培地は殆どＤＰＰＨラジカル消去活性を示さなかった。Ａ−１は、米培地で若干のＤＰＰＨラジカル消去活性が認められ、大豆培地では更に弱いものであったが、ニンニク培地の発酵エキスでは、非常に強い活性が認められた。（図４）Ａ−６では、ニンニク培地での特異性が、更に顕著に認められた。（図５）
【００２９】
実施例２ 紅麹菌を用いたニンニク発酵エキスの抗酸化活性
（発酵エキスの調製法）
ニンニクを水洗した後、等量の水を加え、３０分間煮沸した後、破砕し、破砕汁を得た。破砕汁に等量の水、及びグルコース２％を添加し、オートクレーブで１２１℃、２０分間滅菌した（ニンニク培地）。別に、紅麹菌（Monascus属）１白金耳を、グリセリン培地（グリセリン７％、グルコース３％、Soybean meal３％、ペプトン０．８％、硫酸マグネシウム０．１％、塩化ナトリウム０．２％）で培養し、ニンニク培地にこの培養液を１％添加し２５℃、１６０rpm 振盪の条件下で２〜５週間培養し、３００rpm １０分間遠心分離した上清を以下の試験に用いた。ニンニク培地の他、大豆を乾燥固形物としてニンニク培地と等しくなるように調製した大豆培地、ニンニクと大豆を混合した培地などを用いた。
【００３０】
（ＤＰＰＨラジカル消去能の測定法）
サンプルを０．５Ｍ酢酸緩衝液（pH５．５）で希釈し、その０．５mLにエタノール０．５mLを加え、更に０．５mM ＤＰＰＨ（１，１−ジフェニル−１−２−ピクリルヒドラジル：和光純薬）エタノール溶液０．２５mLを加えてよく混合し、５２０nmにおける吸光度を経時的に測定し、サンプル無添加の対照に対する％を求めた。結果を図６及び７に示す。
【００３１】
（結果）
（１）紅麹培溶液のＤＰＰＨラジカル消去能
ニンニク：大豆（２：２）の混合培地は、弱い消去活性を示したが、紅麹菌で発酵した、ニンニク発酵エキスは、発酵期間に応じて消去活性が増加した。特に、Ａ−２４、Ａ−２６に強い活性が認められた（図６）。
Ａ−２４株を用いて、ニンニクと大豆の混合比を変えて検討した。 大豆のみの培地（０：４）では、ラジカル消去活性は、弱いものであったが、ニンニクの配合比が高くなるにしたがって、活性の増加が認められた。特に、ニンニクを２倍の濃度にした培地（８：０）で最も強い活性が認められた（図７）。
【００３２】
実施例３ ニンニク発酵組成物の抗腫瘍効果及び免疫増強作用
（ニンニク発酵組成物の調製法）
ニンニクを水洗した後、等量の水を加え、３０分間煮沸した後、破砕し、破砕汁を得た。破砕汁に、等量の水及びグルコース２％を添加し、オートクレーブで１２１℃、２０分間滅菌した（ニンニク培地）。別に、麹菌（Aspergillus oryzae）を、サブロー培地（ペプトン１％、グルコース４％）で２７℃、１６０rpm振盪の条件下で３日間前培養し、ニンニク培地にこの培養液を１％添加し２７℃、１６０rpm 振盪の条件下で２週間培養した。得られた発酵物は、１００℃１０分間加熱した後、凍結乾燥し、粉砕した。各種試験に際しては、ニンニク発酵組成物を水に懸濁して経口投与した。
【００３３】
（実験方法）
ＩＣＲ系雄性マウス（７週令:日本クレア）に、sarcoma-180 10⁶cell/mouseを皮下移植し、翌日より被験物質を隔日で１０回経口投与し、最終投与の翌日に、腫瘍の大きさ（３／４×短径×短径×長径mm³）を測定した。その後、無菌的に脾臓を摘出し、常法に従って細胞浮遊液を調製し、赤血球除去用トリス緩衝液（１７mMトリスヒドロキシメチルアミノメタン０．７４７％、NH₄Cl,pH７．６５）で処理後、ＲＰＭＩ １６４０で洗浄し、１０％ＦＣＳを含むＲＰＭＩ１６４０に懸濁した。
【００３４】
ＹＡＣ−１細胞、 Salcoma １８０細胞の標識
１０％ＦＣＳを含むＲＰＭＩ １６４０で２×１０⁶cell／０．１mLに調製したＹＡＣ−１細胞又は、salcom−１８０細胞に、Na₂ ⁵¹CrO₄１００μl（アマシャム、１μCi／μl in sterile０．９％NaCl）を加え、CO₂インキュベータで２時間培養した。その後、１０％ＦＣＳを含むＲＰＭＩ １６４０で４回洗浄し、３×１０⁵cell／mLに調製した。
【００３５】
ＮＫ活性、キラー活性
脾臓細胞を３×１０⁷cell／mL（ＹＡＣ−１細胞、Salcoma−１８０細胞の２００倍）に調製し、ＮＵＮＣ９６ウェルＵ底multi dishに１００μl（３×１０⁶cell）を入れ、３×１０⁵cell／mLに調製した⁵¹Cr標識ＹＡＣ−１細胞又は、Salcoma−１８０細胞５０μl（１．５×１０⁴cell）を加えた（Exp）。これ以外に、最大遊離用として、⁵¹Cr標識ＹＡＣ−１細胞又は、Salcoma−１８０細胞５０μl（１．５×１０⁴cell）に１N HCl １００μlを加えたもの（Tmax）、自然遊離用として、⁵¹Cr標識ＹＡＣ−１細胞又は、Salcoma−１８０細胞５０μl（１．５×１０⁴cell）に１０％ＦＣＳを含むＲＰＭＩ １６４０１００μlを加えたもの（Tspon）を用意した。このmulti dishをＣＯ₂インキュベーターで２４時間培養後、遠心分離した上清１００μlをＲＩＡチューブにとり、ガンマーカウンターで遊離した⁵¹Crを測定した。次の式より、細胞毒性（％）を算出しＮＫ活性、キラー活性とした。
【００３６】
細胞毒性（％）＝（(Exp-Tspon)/(Tmax-Tspon)）×100
【００３７】
統計処理
Ｆ検定の後、Student's-T testもしくは、Aspin-Welch法で検定し危険率５％未満を有意とし＊印で示した。結果を以下に示す。
【００３８】
（結果）
（１）抗腫瘍効果
ガン細胞移植３週間後の腫瘍体積は、コントロール群の３９３mm³に対し、Ａ−１、Ａ−６投与群では統計的に有意な増殖抑制作用が認められた。クレスチンでも同様に有意な作用が認められた。
【００３９】
【表２】

【００４０】
（２）ＮＫ活性
ガン細胞移植３週間後の脾臓細胞を用いて、ＮＫ活性を測定した。ＮＫ活性は、コントロール群の４．６％に対し、Ａ−１、Ａ−６投与群では、１７．８％、１４．１％となり統計的に有意な、ＮＫ活性の増強作用が認められた。クレスチン投与群でも同様に有意な増強作用が認められた。
【００４１】
【表３】

【００４２】
（３）キラー活性
ガン細胞移植３週間後の脾臓細胞を用いて、キラー活性を測定した。キラー活性は、ＮＫ活性と同様に、Ａ−１、Ａ−６投与群では、顕著かつ、統計的に有意な、活性の増強作用が認められた。
【００４３】
【表４】

【００４４】
実施例４ アセトアミノフェン肝障害防護効果
（実験方法）
実施例３と同じニンニク発酵組成物を用いて検討した。６週令のｄｄＹ系雄性マウス（日本ＳＬＣ）を一夜絶食した。アセトアミノフェン投与の２時間前と３０分前の２回被験物質を経口投与し、アセトアミノフェン（４００mg／kg）を腹腔内投与した。６時間後に、腹部大静脈より、ヘパリン処理したシリンジを用いて採血し、血漿ＧＰＴ活性を測定し、肝障害の指標とした。アセトアミノフェン（和光純薬）は、リン酸三カリウム水溶液（１００mg／mL）でpH１１に調製した生理食塩水に沸騰水浴中で溶解した。血漿中ＧＰＴ活性は、ＧＰＴ−ＵＶテストワコー（和光純薬）を用いて測定した。結果を以下に示す。
【００４５】
（結果）
アセトアミノフェンの投与により、コントロール群の血漿中ＧＰＴ活性は、４６８ＩＵ／ｌに増加し激しい肝障害が惹起された。一方、Ａ−１、Ａ−６投与群のＧＰＴ活性は、１４６、６８ＩＵ／ｌの増加に留まり、顕著な肝障害防護作用が認められた。
【００４６】
【表５】

【００４７】
実施例５ 抗糖尿病作用
（実験方法）
実施例３と同じニンニク発酵組成物を用いて検討した。６週令のｄｄＹ系雄性マウス（日本ＳＬＣ）を一夜絶食した。キヤピラリを用いて眼窩静脈叢より２０μl採血した。被験物質４ｇ／kgを経口投与し、１時間後にアロキサン（和光純薬）５０mg／kgを静脈内投与し、１時間後及び６時間後に同様に採血した。絶食は、アロキサン投与直後に解除し、被験物質は、アロキサン投与の前日から、３日後まで１日１回経口投与し、採血は、１、２、４日後に同様に行った。採血後、直ちにヘマトクリット用遠心機で血漿を分離し、グルコースＣII−テスト ワコー（和光純薬）を用いて血糖値を測定した。
統計処理は実施例３と同様に行った。結果を図８に示す。
【００４８】
【表６】

【００４９】
（結果）
ノーマルで示した、正常状態のマウスの血糖値は２００mg／dl程度であるが、アロキサンの投与により、コントロール群の血糖値は、１時間後で５４０mg／dlに激増し、その後も増加を続け、４８時間後では、６５０mg／dlに達した。一方、ニンニク発酵組成物Ａ−６投与群では、アロキサン投与１時間後の血糖値は、３８０mg／dlに留まり、コントロール群との間に、統計的に有意な血糖上昇の抑制作用が認められた。その後も、有意な低値が観察された。Ａ−１も同様に有意な血糖上昇の抑制作用が認められた。しかし、発酵の原料として用いた、ボイルニンニク（ニンニク培地：ＢＧ）では、アロキサン投与による血糖の上昇の抑制作用は認められなかった（図８）。
【００５０】
実施例６ 血糖上昇抑制作用
（実験方法）
実施例３と同じニンニク発酵組成物を用いて検討した。８週令のｄｄＹ系雄性マウス（日本ＳＬＣ）を一夜絶食した後、キヤピラリを用いて眼窩静脈叢より２０μl採血し、以後実験終了時まで絶水とした。被験物質及びスターチを同時に経口投与し、３０、６０、１２０分後に同様に採血した。採血後、直ちにヘマトクリット用遠心機で血漿を分離し、グルコースＣII−テスト ワコー（和光純薬）を用いて血糖値を測定した。統計処理は実施例３と同様に行った。結果を図９に示す。
【００５１】
【表７】

【００５２】
（結果）
スターチを経口投与したコントロール群の血糖値は、３０分後で１５０mg／dl上昇したが、Ａ−１投与群では、１００mg／dlの上昇に留まり、コントロール群に対し有意な血糖上昇の抑制作用が認められた。その後の血糖値は、有意差は無いがコントロール群よりやや高値を推移し、血糖上昇の遅延が観察された。Ａ−６投与群でも、Ａ−１とほぼ同様の結果が得られた。一方、ボイルニンニク（ニンニク培地：ＢＧ）投与群は、コントロールと同様の血糖推移を示し、ニンニク発酵組成物とは明らかに異なる結果が得られた（図９）。
【００５３】
実施例７ 紅麹菌を用いたニンニク発酵エキスのコレステロール合成阻害活性
実施例２と同じニンニク発酵エキスを用いて検討した。
（酵素標本の調製法）
Wistar系雄性ラット（６．５週令）を明時１７：００〜５：００の逆転照明下で、粉末のＥＣ−２（日本クレア製）で６日間の馴致飼育の後、２％コレスチラミン８％コーン油を配合したＣＥ−２で５日間飼育した。深夜に相当する、１０：００にラットを脱血致死後肝臓を摘出し、氷冷下で２倍量のリン酸バッファーを加えて、テフロンホモジナイザーで、ホモジネートを作成した。以下、次の様にミクロゾーム画分を調製しタンパク量１０mg／mLに調製後、−８０℃で保存した。
【００５４】
ラット肝ホモジネート
↓遠心分離（700g、５分）
遠心上清
↓遠心分離（12,000ｇ、30分）
遠心上清
↓遠心分離（105,000g、60分）
沈渣（洗浄）
↓遠心分離（105,000g、60分）
沈渣（ミクロゾーム画分）
リン酸バッファーに懸濁（タンパク量10mg／mL）
【００５５】
（HMG-CoA リダクターゼ阻害活性の測定法）
酵素反応液５０μl中、次の試薬を含むように調製し、３７℃、３０分反応させた。反応の開始は酵素液の添加、反応の停止は２Ｎ塩酸２０μlの添加により行い、塩酸添加後、更に３７℃、１５分間インキュベートした。次に、陽イオン交換樹脂（Bio Rex ５：Bio Rad 社製）の懸濁液（１ｇ／１０mL）４５０μlを加え、１時間振盪後、上清４００μlを液体シンチレーター（ACS II：Amersham社製）１０mLに加えＣｏＡより乖離した¹⁴Ｃをカウントした。
被験物質（実施例２に記載の方法により得た発酵エキス）は、反応液５０μl中に１０μl添加とし、無添加時の酵素反応に対し、５０％阻害を挟む３点以上のデータからLitchfild-Wilcoxon法によりＩＣ₅₀値を算出した。
【００５６】
酵素反応液の組成
０．１１mM dl［３−¹⁴Ｃ］ＨＭＧ−ＣｏＡ（2.25Ci/mol）
１００mM リン酸カリウムバッファー（pH７．４）
１０mM ＥＤＴＡ
１０mM ジチオスレイトール
５mM ＮＡＤＰＨ
３０〜４０μｇ ミクロゾーム蛋白（反応液５０μl中）
【００５７】
（結果）
図１０に各培養液のＨＭＧ−ＣｏＡリダクターゼ阻害活性（ＩＣ₅₀の逆数で表示）を示した。紅麹菌（Ａ−２４株）を大豆培地、ニンニク：大豆（１：１）の混合培地、ニンニク培地を用いて１３〜３４日間培養した。大豆培地では検討期間中殆ど阻害活性は認められなかったが、ニンニク：大豆の混合培地ではＩＣ₅₀値０．０３〜０．０２mL／mLの阻害活性が認められた。更にニンニク培地では培養期間に依存した強い阻害活性（ＩＣ₅₀値０．０２〜０．００７mL／mL）が認められた（図１０）。
【００５８】
この結果から、酵素失活処理ニンニクは、紅麹菌においてHMG-CoA reductase阻害活性物質を効率よく産生することが確認された。
【００５９】
実施例８ 製剤例／錠剤
実施例３で調製したニンニク発酵組成物に乳糖、コーンスターチ、結晶セルロース、カルメロースカルシウム及びステアリン酸マグネシウムを下記の処方量で加え、ボーレコンテナミキサー（コトブキ技研工業製）で１０分間混合した。この混合末を打錠機（コレクト１９Ｋ、菊水製作所製）で圧縮成形し、径８mm、重量１８０mgの錠剤を製した。本錠剤は硬度及び胃内崩壊性に優れていた。
【００６０】
【表８】

【００６１】
実施例９ 製剤例／顆粒剤
実施例３で調製したニンニク発酵組成物にコーンスターチ、結晶セルロース、ヒドロキシプロピルセルロース及びカルメロースカルシウムを下記の処方量で加え混合した後、水を加えて練合した。この練合物を押し出し造粒機（DOME GRAN、不二パウダル製）で径０．８mmの柱状造粒物とし、整粒、乾燥、篩過した後、顆粒剤を製した。本顆粒は胃内崩壊性に優れ、分包等の包装形態で提供することができる。
【００６２】
【表９】

【００６３】
実施例１０ 製剤例／ドリンク剤
実施例２で得られたニンニク発酵組成物に下記の処方で甘味剤、酸味剤、香料、水を加えて滅菌した後、ガラス瓶に充填してドリンク剤を製した。
【００６４】
【表１０】

【００６５】
【発明の効果】
本発明の組成物は臭気がなく、糖尿病、肝臓病、癌、免疫疾患、高脂血症等の予防・治療剤として有用である。
【図面の簡単な説明】
【図１】活性酸素消去活性を示す図である。
【図２】活性酸素消去活性を示す図である。
【図３】ＤＰＰＨラジカル消去活性を示す図である。
【図４】ＤＰＰＨラジカル消去活性を示す図である。
【図５】ＤＰＰＨラジカル消去活性を示す図である。
【図６】ＤＰＰＨラジカル消去活性を示す図である。
【図７】ＤＰＰＨラジカル消去活性を示す図である。
【図８】抗アロキサン糖尿病作用を示す図である。
【図９】耐糖能を示す図である。
【図１０】ＨＭＧ−ＣｏＡリダクターゼ阻害活性を示す図である。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a medicament comprising a garlic fermentation composition as an active ingredient.
[0002]
[Prior art]
Garlic has been used as a seasoning and spice since ancient times, but in recent years, it has become clear that various physiologically active ingredients are contained therein, and is widely used as health foods and pharmaceuticals.
However, garlic contains precursors of odorous components such as allicin and diallyl disulfide, and is easily converted into these, so many people do not like their preferences and refrain. For this reason, methods have been developed in which heat treatment is performed to eliminate bromide (Japanese Patent Laid-Open Publication Nos. 59-216565 and 4-12644), but problems still remain, such as the occurrence of garlic odor after taking the medicine. .
In addition, it is important to determine the processing method of garlic because garlic has different components and physiological activities due to differences in processing conditions (Emiko Mochizuki: FOODS & FOOD INGREDIENTS JOURNAL OF JAPAN)164, 36-45, 1995).
[0003]
On the other hand, in addition to garlic, there is a conventional fermentation method using koji mold and / or koji mold, which has been used for producing miso, soy sauce, sake and the like. Both these koji molds and koji molds have already been proven to be safe from many years of dietary experience and have been reported to have various physiological activities (for example, antioxidant activity by koji molds (Naoko Yamaguchi: Japan Food Industry) Journal26, 71-75, 1979), Angiotensin I-converting enzyme inhibitory activity (Sayuri Teranaka et al .: Journal of Japanese Society for Agricultural Chemistry)69, 1163-1169, 1995) or cholesterol-lowering activity, antihypertensive activity, etc. due to red yeast.
[0004]
Therefore, the present inventor considered that if garlic was fermented with koji mold and / or koji mold, a composition having no garlic odor and useful as a medicine or food could be obtained. Since it has an effect | action, it was not able to ferment with a koji mold and / or a koji mold. Examples of adding a small amount of garlic in the process of producing wine (Japanese Patent Laid-Open No. 48-52995) and examples of adding a large amount of sugar to a small amount of garlic and fermenting (Japanese Patent Laid-Open No. 53-26361) There is no example of fermentation using garlic as the main raw material.
[0005]
[Problems to be solved by the invention]
Accordingly, an object of the present invention is to eliminate odors peculiar to garlic by fermenting garlic with koji molds and to obtain a composition useful as a medicine or food.
[Means for Solving the Problems]
[0006]
In view of such a situation, the present inventors have conducted intensive research and found that if garlic is previously subjected to enzyme inactivation treatment, it can be fermented with gonococcus alone without adding nutrient sources such as glucose and starch, and further obtained. The fermented composition has no garlic odor, has an antioxidant activity about 100 times stronger than ordinary garlic, has anti-diabetic action, liver damage protection action, anti-cancer action, immune enhancement action, cholesterol lowering action, etc. It was also found useful for the treatment or prevention of liver disease, cancer, immune disease, hyperlipidemia, etc., and the present invention was completed.
[0007]
DETAILED DESCRIPTION OF THE INVENTION
That is, the present invention is a pharmaceutical composition comprising an enzyme-deactivated garlic fermented with koji mold, or a composition obtained by adding beans and / or cereals to enzyme-deactivated garlic and fermented with koji mold. Is to provide.
[0008]
Furthermore, the present invention provides a composition in which enzyme-deactivated garlic is fermented with koji mold, or a composition obtained by adding beans and / or cereals to enzyme-deactivated garlic and fermented with koji mold as an active ingredient The present invention provides a preventive / therapeutic agent for a disease selected from liver disease, cancer, immune disease and hyperlipidemia.
[0009]
In the present invention, garlic means Allium sativum L. belonging to the genus Liliaceae and Allium.
The portion of garlic used for fermentation is particularly preferably a bulb portion, and the enzyme-deactivated bulb can be sliced as it is or in an arbitrary size, or crushed into a crushed juice can be used for fermentation. Moreover, you may add beans and / or cereals, such as rice, soybeans, wheat, and barley, as needed.
[0010]
In order to produce the garlic fermentation composition of the present invention, first, garlic enzyme inactivation treatment is performed. The enzyme deactivation treatment can be performed by treating garlic with heat, microwave, high pressure treatment, enzyme, acid or alcohol, and among these, heat treatment is simple and preferable. The temperature for the heat treatment is preferably 50 to 200 ° C. and 1 to 60 minutes, particularly preferably 90 to 121 ° C. and 10 to 30 minutes.
[0011]
Enzyme-inactivated garlic is, for example, Aspergillus oryzae, Aspergillus sojae, Aspergillus kawachi, Aspergillus awamori and other gonococci, Monascus pilosus, Monascus anka, Monascus paxii, Monascus pubigerus, Monascus rupur, Monascus If fermented with one or more bacteria selected from Aspergillus, the desired garlic fermentation composition can be obtained.
As a specific method for preparing the garlic fermentation composition, for example, a method performed according to the following steps may be mentioned.
[0012]
1) Preparation of garlic medium / garlic-containing medium
After washing the garlic with water, add an equal amount of water and boil for 20 to 30 minutes. If necessary, process chopping, crushing, etc. (If crushing, only crushing juice can be used) . If necessary, beans and / or cereals such as rice, soybeans, wheat and pearl barley are added as necessary as nutrients, supplemented with sugar, and sterilized (121 ° C., 20 minutes) with an autoclave or the like. In addition, when adding beans and / or cereals or performing sugar supplementation, these should not exceed 90% by weight (hereinafter simply referred to as “%”).
[0013]
2) Pre-culture of fermenting bacteria
Fermentative bacteria include Aspergillus (Aspergillus oryzae, Aspergillus sojae, Aspergillus kawachi, Aspergillus awamori, etc.) and Aspergillus (Monascus spp .: M.pilosus, M.anka, M.paxii, M.pubigerus, M.purpures, M. .ruber, M.vitreus, M.major, etc.) are preferably used.
The strain is subcultured on a potato dextrose agar medium supplemented with 2% yeast extract, inoculated into the following medium, and precultured at 10 to 50 ° C. for 2 to 14 days, preferably at 20 to 40 ° C. for 2 to 4 days.
The medium for pre-culture is Sabouraud medium (1% peptone, 4% glucose), potato dextrose medium (potato ginseng (200 g / l) glucose 2%), glycerin medium (glycerin 7%, glucose 3%, soybean meal 3 %, Peptone 0.8%, magnesium sulfate 0.1%, sodium chloride 0.2%) and any fungal medium can be used, but edible substances are preferred as the medium composition.
[0014]
3) Fermentation of garlic
0.1 to 50%, preferably 0.5 to 10% of the precultured bacterial solution prepared in Step 2 is added to the garlic medium prepared in Step 1, and more preferably 7 to 60 days at 10 to 50 ° C. The garlic fermentation composition can be prepared by performing shaking culture or stationary culture at 20 to 40 ° C. for 7 to 35 days in the case of Aspergillus or 14 to 42 days in the case of Aspergillus. Moreover, the water | moisture content of a culture medium may be reduced to 40 to 60%, and static culture may be performed in the form of a solid culture.
[0015]
4) Treatment of garlic fermentation composition
At the stage where the fermentation in step 3 is completed, the fermented product is heat sterilized and then used directly or separated from the culture filtrate and used separately. Moreover, only an active ingredient can be extracted and used.
[0016]
The composition of the present invention thus obtained can be made into a food by a conventional method, or it can be made into various dosage forms with a pharmaceutically acceptable carrier.
Among these, when preparing an oral solid preparation, after adding an excipient, a binder, a disintegrant, a lubricant, a coloring agent, a corrigent, a corrigent, etc. to the garlic fermentation composition, Tablets, coated tablets, granules, powders, capsules and the like can be produced by the method. Such additives may be those commonly used in the art. For example, excipients include lactose, sucrose, sodium chloride, glucose, starch, calcium carbonate, kaolin, microcrystalline cellulose, silicic acid As a binder, water, ethanol, propanol, simple syrup, glucose solution, starch solution, gelatin solution, carboxymethylcellulose, hydroxypropylcellulose, hydroxypropyl starch, methylcellulose, ethylcellulose, shellac, calcium phosphate, polyvinylpyrrolidone, etc. As disintegrants, dried starch, carmellose calcium, sodium alginate, agar powder, sodium bicarbonate, calcium carbonate, sodium lauryl sulfate, stearic acid monoglyceride, lactose, etc. are purified as lubricants. Torque, stearate, borax, polyethylene glycol, sucrose as a flavoring agent, orange peel, citric acid, can be exemplified tartaric acid.
[0017]
When preparing an oral liquid preparation, a liquid preparation, a syrup, an elixir, etc. can be manufactured by a conventional method by adding a flavoring agent, a buffer, a stabilizer, a flavoring agent and the like to the garlic fermentation composition. In this case, the flavoring agents may be those listed above, examples of the buffer include sodium citrate, and examples of the stabilizer include tragacanth, gum arabic, and gelatin.
[0018]
The food or medicament of the present invention can be used for the purpose of treatment or prevention of diabetes, liver disease, cancer, immune disease, hyperlipidemia and the like.
The dosage and administration method can be appropriately determined depending on the age, body weight, symptoms, etc. Usually, 0.5 to 2 g of the composition of the present invention can be administered once or several times per day for an adult. preferable.
[0019]
In the composition of the present invention, garlic, koji mold or koji mold, which are main components, are usually used for food, and generally have low toxicity. Moreover, although the composition of the present invention was given to young rats with a 3% diet for 3 weeks, no abnormalities were observed in the growth process and general behavior, and it was confirmed that the composition was a highly safe substance.
[0020]
The garlic fermentation composition of the present invention has a strong antioxidant activity, an anticancer effect, an immune enhancing action such as NK activity, a protective action against acetaminophen liver injury, a protective action against an alloxan diabetes model, and an enhanced glucose tolerance action The anti-diabetic action, cholesterol lowering action, etc. are recognized, and it is extremely useful for the treatment or prevention of adult diseases prevalent in modern society.
In addition, the composition of the present invention is highly safe and has almost no garlic odor (unlike odorless garlic obtained by deactivating allinase, garlic odor is not generated in the mouth). And can be widely used as pharmaceuticals.
[0021]
【Example】
Next, the present invention will be described in more detail with reference to examples. In addition, this invention is not limited by this. Table 1 shows the koji mold and the koji mold used in this example.
[0022]
[Table 1]

[0023]
Example 1 Production of garlic fermentation composition using Aspergillus and its antioxidant activity (Preparation of fermentation composition (extract))
After garlic was washed with water, an equal amount of water was added, boiled for 30 minutes, and then crushed to obtain a crushed juice. An equal amount of water and 2% glucose were added to the crushed juice, and sterilized in an autoclave at 121 ° C. for 20 minutes (garlic medium). Separately, Aspergillus oryzae was pre-cultured in Sabouraud medium (1% peptone, 4% glucose) at 27 ° C. under shaking at 160 rpm for 3 days, and 1% of this culture solution was added to garlic medium at 27 ° C. The supernatant (extract) cultured for 2 days to 5 weeks under the condition of shaking at 160 rpm and centrifuged at 300 rpm for 10 minutes was used for the following test. On the other hand, rice and soybeans were prepared to be equal to the garlic medium as dry solids.
[0024]
(Measurement of superoxide anion scavenging ability)
65mM KH₂PO_Four-Borate buffer (pH 8.2) 200 μl, 5 mM xanthine aqueous solution 200 μl, 10 mM hydroxylamine aqueous solution 100 μl, water 200 μl, 100 μl sample and 200 μl xanthine oxidase were added and incubated at 37 ° C. for 15 minutes. At the end of the incubation, for the purpose of stopping the reaction and coloring, 2 mL of 30 μM N-naphthylethylenediamine-3 mM sulfanilic acid-25% glacial acetic acid was added and left at room temperature for 45 minutes, and the absorbance was measured at 550 nm. This measurement was performed twice, and the active oxygen scavenging activity (%) was determined from the decrease in absorbance due to the addition of the sample. Xanthine oxidase (xanthine oxidase suspension: Wako Pure Chemical Industries, Ltd.) was prepared so that the change in absorbance was 0.020 to 0.025 / min (15 to 20 mU / mL).
The results are shown in FIGS.
[0025]
(Measurement method of DPPH radical scavenging ability)
The sample is diluted with 0.5 M acetate buffer (pH 5.5), 0.5 mL of ethanol is added to 0.5 mL thereof, and 0.5 mM DPPH (1,1-diphenyl-1-picrylhydrazyl: Wako Pure Chemical Industries, Ltd.) 0.25 mL of ethanol solution was added and mixed well, and the absorbance at 520 nm was measured over time, and the percentage relative to the control with no sample added was determined. The results are shown in FIGS.
[0026]
(result)
(1) Superoxide anion scavenging ability of garlic medium and garlic fermentation extract
The garlic medium showed weak scavenging activity, but the garlic fermented extract increased scavenging activity depending on the fermentation period. Moreover, although the activity of the garlic medium increased slightly by addition of glucose, the fermentation extract showed almost no difference (FIG. 1).
The garlic fermentation extract was diluted and the superoxide anion scavenging ability was measured in the same manner. The garlic fermented extract was a 4-week fermentation sample, which was 100-fold diluted, and the activity of the garlic medium became equal. Fermentation enhanced the superoxide anion scavenging activity 100-fold (FIG. 2).
[0027]
(2) DPPH radical scavenging activity
DPPH radical scavenging activity was measured using a 100-fold diluted solution. The garlic medium (BG) showed almost no activity, but the garlic fermentation extract increased DPPH radical scavenging activity depending on the fermentation period in any koji mold. In particular, strong activity was observed in A-1 and A-6 (FIG. 3).
[0028]
(3) A-1 , A-6 Of Medium on Production of DPPH Radical Scavenging Active Substance in Rice
The rice medium and soybean medium hardly showed DPPH radical scavenging activity. A-1 showed some DPPH radical scavenging activity in the rice medium and was weaker in the soybean medium, but very strong activity was observed in the fermented extract of the garlic medium. (FIG. 4) In A-6, the specificity in the garlic medium was more remarkably recognized. (Fig. 5)
[0029]
Example 2 Antioxidant activity of fermented garlic extract using red koji mold
(Method for preparing fermented extract)
After garlic was washed with water, an equal amount of water was added, boiled for 30 minutes, and then crushed to obtain a crushed juice. An equal amount of water and 2% glucose were added to the crushed juice, and sterilized in an autoclave at 121 ° C. for 20 minutes (garlic medium). Separately, 1 platinum ear of Monascus was cultured in glycerol medium (glycerol 7%, glucose 3%, soybean meal 3%, peptone 0.8%, magnesium sulfate 0.1%, sodium chloride 0.2%). Then, 1% of this culture solution was added to garlic medium, cultured for 2 to 5 weeks under conditions of shaking at 25 ° C. and 160 rpm, and the supernatant obtained by centrifugation at 300 rpm for 10 minutes was used for the following tests. In addition to the garlic medium, a soybean medium prepared by using soybean as a dry solid so as to be equal to the garlic medium, a medium in which garlic and soybean were mixed, and the like were used.
[0030]
(Measurement method of DPPH radical scavenging ability)
The sample is diluted with 0.5 M acetate buffer (pH 5.5), 0.5 mL of ethanol is added to 0.5 mL thereof, and 0.5 mM DPPH (1,1-diphenyl-1-picrylhydrazyl: Wako Pure Chemical Industries, Ltd.) 0.25 mL of ethanol solution was added and mixed well, and the absorbance at 520 nm was measured over time, and the percentage relative to the control with no sample added was determined. The results are shown in FIGS.
[0031]
(result)
(1) DPPH radical scavenging ability of red koji culture solution
The mixed medium of garlic: soybean (2: 2) showed a weak erasing activity, but the garlic fermented extract fermented with koji mold increased the erasing activity according to the fermentation period. In particular, strong activity was observed in A-24 and A-26 (FIG. 6).
Using the A-24 strain, the mixing ratio of garlic and soybean was changed and examined. In the soybean-only medium (0: 4), the radical scavenging activity was weak, but as the garlic compounding ratio increased, the activity increased. In particular, the strongest activity was observed in a medium (8: 0) in which garlic was doubled (FIG. 7).
[0032]
Example 3 Antitumor effect and immune enhancement effect of garlic fermentation composition
(Preparation method of garlic fermentation composition)
After garlic was washed with water, an equal amount of water was added, boiled for 30 minutes, and then crushed to obtain a crushed juice. An equal amount of water and 2% glucose were added to the crushed juice, and sterilized in an autoclave at 121 ° C. for 20 minutes (garlic medium). Separately, Aspergillus oryzae was pre-cultured in Sabouraud medium (1% peptone, 4% glucose) at 27 ° C. under shaking at 160 rpm for 3 days, and 1% of this culture solution was added to garlic medium at 27 ° C. The cells were cultured for 2 weeks under the condition of shaking at 160 rpm. The obtained fermented product was heated at 100 ° C. for 10 minutes, freeze-dried and pulverized. In various tests, the garlic fermentation composition was suspended in water and administered orally.
[0033]
(experimental method)
Sarcoma-180 10 to ICR male mice (7 weeks old: CLEA JAPAN)⁶Cell / mouse is transplanted subcutaneously, and the test substance is orally administered 10 times every other day from the next day. The size of the tumor (3/4 × minor axis × minor axis × major axis mm)^Three) Was measured. Thereafter, the spleen was aseptically removed, and a cell suspension was prepared according to a conventional method. Tris buffer solution for removing red blood cells (17 mM Trishydroxymethylaminomethane 0.747%, NH_FourCl, pH 7.65), washed with RPMI 1640, and suspended in RPMI 1640 containing 10% FCS.
[0034]
YAC-1 cells, Salcoma 180 cell labeling
2 x 10 with RPMI 1640 with 10% FCS⁶To YAC-1 cells or salcom-180 cells prepared to cell / 0.1 mL, Na₂ ⁵¹CrO_FourAdd 100 μl (Amersham, 1 μCi / μl in sterile 0.9% NaCl) and add CO₂The cells were cultured for 2 hours in an incubator. After that, it was washed 4 times with RPMI 1640 containing 10% FCS and 3 × 10^FivePrepared to cell / mL.
[0035]
NK activity, killer activity
3 x 10 spleen cells⁷cell / mL (200 times that of YAC-1 cells and Salcoma-180 cells), and 100 μl (3 × 10 6) in a NUNC 96-well U-bottom multi dish.⁶cell) 3 × 10^Fiveprepared to cell / mL⁵¹Cr-labeled YAC-1 cells or Salcoma-180 cells 50 μl (1.5 × 10 5^Fourcell) was added (Exp). Besides this, for maximum liberation,⁵¹Cr-labeled YAC-1 cells or Salcoma-180 cells 50 μl (1.5 × 10 5^Fourcell) to which 100 μl of 1N HCl is added (Tmax)⁵¹Cr-labeled YAC-1 cells or Salcoma-180 cells 50 μl (1.5 × 10 5^Fourcell) to which 100 μl of RPMI 1640 containing 10% FCS was added (Tspon) was prepared. CO in this multi dish₂After culturing for 24 hours in an incubator, 100 μl of the centrifuged supernatant was placed in an RIA tube and released with a gamma counter.⁵¹Cr was measured. Cytotoxicity (%) was calculated from the following formula, and defined as NK activity and killer activity.
[0036]
Cytotoxicity (%) = ((Exp-Tspon) / (Tmax-Tspon)) x 100
[0037]
Statistical processing
After F test, it was tested by Student's-T test or Aspin-Welch method, and a risk rate of less than 5% was regarded as significant and indicated by *. The results are shown below.
[0038]
(result)
(1) Anti-tumor effect
The tumor volume 3 weeks after cancer cell transplantation was 393 mm of the control group.^ThreeOn the other hand, a statistically significant growth inhibitory action was observed in the A-1 and A-6 administration groups. A similar effect was observed with krestin as well.
[0039]
[Table 2]

[0040]
(2) NK activity
NK activity was measured using spleen cells 3 weeks after transplantation of cancer cells. The NK activity was 17.8% and 14.1% in the A-1 and A-6 administration groups compared to 4.6% in the control group, and a statistically significant enhancement effect of NK activity was observed. . A significant potentiating effect was also observed in the krestin administration group.
[0041]
[Table 3]

[0042]
(3) Killer activity
Killer activity was measured using spleen cells 3 weeks after transplantation of cancer cells. Similar to the NK activity, the killer activity showed a significant and statistically significant activity enhancing action in the A-1 and A-6 administration groups.
[0043]
[Table 4]

[0044]
Example 4 Acetaminophen liver injury protective effect
(experimental method)
The same garlic fermentation composition as in Example 3 was used. Six-week-old ddY male mice (Japan SLC) were fasted overnight. The test substance was orally administered twice 2 hours before and 30 minutes before acetaminophen administration, and acetaminophen (400 mg / kg) was intraperitoneally administered. Six hours later, blood was collected from the abdominal vena cava using a heparin-treated syringe, the plasma GPT activity was measured, and used as an indicator of liver damage. Acetaminophen (Wako Pure Chemical Industries, Ltd.) was dissolved in a physiological saline adjusted to pH 11 with tripotassium phosphate aqueous solution (100 mg / mL) in a boiling water bath. Plasma GPT activity was measured using GPT-UV Test Wako (Wako Pure Chemical Industries). The results are shown below.
[0045]
(result)
By administration of acetaminophen, the plasma GPT activity in the control group increased to 468 IU / l, and severe liver damage was induced. On the other hand, the GPT activity in the A-1 and A-6 administration groups was only increased by 146 and 68 IU / l, and a remarkable protective action against liver damage was observed.
[0046]
[Table 5]

[0047]
Example 5 Antidiabetic action
(experimental method)
The same garlic fermentation composition as in Example 3 was used. Six-week-old ddY male mice (Japan SLC) were fasted overnight. 20 μl of blood was collected from the orbital venous plexus using a capillary. A test substance of 4 g / kg was orally administered, 1 hour later, alloxan (Wako Pure Chemical Industries) 50 mg / kg was intravenously administered, and blood was collected similarly after 1 hour and 6 hours. Fasting was released immediately after alloxan administration, the test substance was orally administered once a day from the day before alloxan administration to 3 days later, and blood sampling was performed in the same manner after 1, 2, 4 days. Immediately after blood collection, plasma was separated using a hematocrit centrifuge, and the blood glucose level was measured using Glucose CII-Test Wako (Wako Pure Chemical Industries).
Statistical processing was performed in the same manner as in Example 3. The results are shown in FIG.
[0048]
[Table 6]

[0049]
(result)
The blood glucose level of a normal mouse, which is shown as normal, is about 200 mg / dl, but with the administration of alloxan, the blood glucose level of the control group increased dramatically to 540 mg / dl after 1 hour, and continued to increase thereafter. After 48 hours, it reached 650 mg / dl. On the other hand, in the garlic fermentation composition A-6 administration group, the blood glucose level at 1 hour after alloxan administration remained at 380 mg / dl, and a statistically significant inhibitory effect on the increase in blood glucose was observed with the control group. . Thereafter, a significant low value was observed. A-1 also showed a significant inhibitory effect on blood sugar rise. However, boiled garlic (garlic medium: BG) used as a raw material for fermentation did not show an inhibitory effect on the increase in blood glucose by alloxan administration (FIG. 8).
[0050]
Example 6 Suppressing effect on blood glucose rise
(experimental method)
The same garlic fermentation composition as in Example 3 was used. After 8-week old ddY male mice (Japan SLC) were fasted overnight, 20 μl of blood was collected from the orbital venous plexus using capillaries, and then water was kept until the end of the experiment. The test substance and starch were orally administered simultaneously, and blood was similarly collected after 30, 60, and 120 minutes. Immediately after blood collection, plasma was separated using a hematocrit centrifuge, and the blood glucose level was measured using Glucose CII-Test Wako (Wako Pure Chemical Industries). Statistical processing was performed in the same manner as in Example 3. The results are shown in FIG.
[0051]
[Table 7]

[0052]
(result)
The blood glucose level of the control group to which starch was orally administered increased by 150 mg / dl after 30 minutes. However, in the A-1 administration group, the blood glucose level remained at an increase of 100 mg / dl, and the blood glucose level had a significant inhibitory effect on the increase in blood glucose relative to the control group. Admitted. The subsequent blood glucose level was slightly higher than that of the control group although there was no significant difference, and a delay in the increase in blood glucose was observed. In the A-6 administration group, almost the same result as A-1 was obtained. On the other hand, the boiled garlic (garlic medium: BG) administration group showed the same blood glucose transition as the control, and clearly different results from the garlic fermentation composition (FIG. 9).
[0053]
Example 7 Cholesterol synthesis inhibitory activity of garlic fermentation extract using red koji mold
The same garlic fermentation extract as in Example 2 was used.
(Preparation method of enzyme specimen)
Wistar male rats (6.5 weeks old) were bred for 6 days with powdered EC-2 (manufactured by CLEA Japan, Inc.) under reverse illumination from 17:00 to 5:00 pm tomorrow. 2% cholestyramine The mice were bred for 5 days in CE-2 containing 8% corn oil. At 10:00, which corresponds to midnight, the rats were bled and lethal, and then the liver was removed. A two-fold amount of phosphate buffer was added under ice cooling, and a homogenate was prepared with a Teflon homogenizer. Hereinafter, the microsomal fraction was prepared as follows, and the protein amount was adjusted to 10 mg / mL and then stored at −80 ° C.
[0054]
Rat liver homogenate
↓ Centrifugation (700g, 5 minutes)
Centrifugal supernatant
↓ Centrifugation (12,000g, 30 minutes)
Centrifugal supernatant
↓ Centrifugation (105,000g, 60 minutes)
Sediment (cleaning)
↓ Centrifugation (105,000g, 60 minutes)
Sediment (microsome fraction)
Suspended in phosphate buffer (protein amount 10mg / mL)
[0055]
(Method for measuring HMG-CoA reductase inhibitory activity)
It prepared so that the following reagents might be contained in 50 microliters of enzyme reaction liquid, and it was made to react at 37 degreeC for 30 minutes. The reaction was started by adding an enzyme solution and stopped by adding 20 μl of 2N hydrochloric acid. After addition of hydrochloric acid, the mixture was further incubated at 37 ° C. for 15 minutes. Next, 450 μl of a suspension (1 g / 10 mL) of a cation exchange resin (Bio Rex 5: manufactured by Bio Rad) was added, and after shaking for 1 hour, 400 μl of the supernatant was added to 10 mL of a liquid scintillator (ACS II: manufactured by Amersham). In addition to CoA¹⁴C was counted.
The test substance (fermented extract obtained by the method described in Example 2) was added at 10 μl in 50 μl of the reaction solution, and Litchfild-Wilcoxon was obtained from 3 or more points of data interposing 50% inhibition with respect to the enzyme reaction without addition. IC by law₅₀The value was calculated.
[0056]
Composition of enzyme reaction solution
0.11 mM dl [3-¹⁴C] HMG-CoA (2.25 Ci / mol)
100 mM potassium phosphate buffer (pH 7.4)
10 mM EDTA
10 mM dithiothreitol
5mM NADPH
30-40 μg microsomal protein (in 50 μl of reaction solution)
[0057]
(result)
FIG. 10 shows the HMG-CoA reductase inhibitory activity (IC₅₀(Represented by the inverse of). Red koji mold (A-24 strain) was cultured for 13 to 34 days using a soybean medium, a mixed medium of garlic: soybean (1: 1), and a garlic medium. In the soy medium, almost no inhibitory activity was observed during the study period.₅₀An inhibitory activity of values 0.03 to 0.02 mL / mL was observed. Furthermore, garlic medium has a strong inhibitory activity (IC₅₀Value 0.02-0.007 mL / mL) was observed (FIG. 10).
[0058]
From these results, it was confirmed that the enzyme-inactivated garlic efficiently produced an HMG-CoA reductase inhibitory active substance in the koji mold.
[0059]
Example 8 Formulation Example / Tablet
Lactose, corn starch, crystalline cellulose, carmellose calcium and magnesium stearate were added to the garlic fermentation composition prepared in Example 3 in the following amounts, and mixed for 10 minutes with a Bole container mixer (manufactured by Kotobuki Giken Kogyo). This mixed powder was compression-molded with a tableting machine (collect 19K, manufactured by Kikusui Seisakusho) to produce a tablet having a diameter of 8 mm and a weight of 180 mg. This tablet was excellent in hardness and gastric disintegration.
[0060]
[Table 8]

[0061]
Example 9 Formulation Example / Granule
To the garlic fermentation composition prepared in Example 3, corn starch, crystalline cellulose, hydroxypropyl cellulose and carmellose calcium were added and mixed in the following amounts, and then water was added and kneaded. This kneaded product was formed into a columnar granulated product having a diameter of 0.8 mm by an extrusion granulator (DOME GRAN, manufactured by Fuji Powder), granulated, dried, sieved, and then granulated. This granule is excellent in gastric disintegration and can be provided in a packaging form such as a sachet.
[0062]
[Table 9]

[0063]
Example 10 Formulation Example / Drink
The garlic fermentation composition obtained in Example 2 was sterilized by adding a sweetener, a sour agent, a fragrance, and water according to the following formulation, and then filled into a glass bottle to prepare a drink.
[0064]
[Table 10]

[0065]
【The invention's effect】
The composition of the present invention has no odor and is useful as a preventive / therapeutic agent for diabetes, liver disease, cancer, immune disease, hyperlipidemia and the like.
[Brief description of the drawings]
FIG. 1 is a diagram showing active oxygen scavenging activity.
FIG. 2 is a diagram showing active oxygen scavenging activity.
FIG. 3 is a diagram showing DPPH radical scavenging activity.
FIG. 4 is a diagram showing DPPH radical scavenging activity.
FIG. 5 is a graph showing DPPH radical scavenging activity.
FIG. 6 is a diagram showing DPPH radical scavenging activity.
FIG. 7 is a graph showing DPPH radical scavenging activity.
FIG. 8 is a diagram showing an antialloxan diabetic action.
FIG. 9 is a diagram showing glucose tolerance.
FIG. 10 is a graph showing HMG-CoA reductase inhibitory activity.

Claims (1)

  An antioxidant comprising as an active ingredient a composition obtained by fermenting enzyme-deactivated garlic with koji mold, or a composition obtained by adding beans and / or cereals to enzyme-deactivated garlic and fermenting with koji mold.

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