JP4651822B2 - SOD-like composition and blood pressure suppressor - Google Patents

Description

上記３群の各ラットを室温２３±１℃、湿度５０〜６０％、１２時間採光（７時〜１９時）の条件下にて４週間（２８日）飼育した。試験期間中、試験群３のラットには、上記（２）で調製されたＳＯＤ様組成物を３．６ｍｇ／匹／日の量で経口投与した。また、水は水道水を自由飲水させ、給与飼料として、オリエンタル酵母工業株式会社製「オリエンタルＭＦ」を自由摂取させた。
そして、飼育最終週（飼育４週目）に、上記３群の各ラットから２４時間の尿を採取した。
上記３群の各ラットは飼育終了後、１０時間絶食させ、エーテル麻酔を施した後、解剖、採血し、肝臓及び腎臓を摘出して湿重量を測定した。組織は脱血処理した後、生理食塩水で洗浄し、−８０℃のフリーザー（三洋電機株式会社製「ＳＡＮＹＯ ＭＤＦ−１９２」）に保存した。
凍結保存しておいた肝臓及び腎臓は解凍した後、３ｍＭ−Ｔｒｉｓ塩酸バッファ（３ｍＭ−Ｔｒｉｓ、０．２５Ｍ−ショ糖、０．１ｍＭ−ＥＤＴＡ ｐＨ７．４）を用いて１０％ホモジネートとした。このホモジネートをエッペン管に分注して−８０℃で保存し、用事解凍し、冷却遠心分離器（久保田商事株式会社製「ＫＵＢＯＴＡ ７９３０」）を用いて、５０００ｒｐｍ、５分間、４℃の条件で遠心分離して上清を得た。
かかる上清を適宜希釈して、以下に示すグルタチオン−Ｓ−トランスフェラーゼ（以下、「ＧＳＴ」という。）活性、グルタチオンペルオキシダーゼ（以下、「ＧＰｘ」という。）活性及びＴＢＡ反応陽性物質（以下、「ＴＢＡＲＳ」という。）の測定を行った。
ＧＳＴ活性は以下の方法により測定した。即ち、上記上清０．２ｍｌを採取し、２０倍に希釈をしてＧＳＴ活性測定用サンプルとした。そして、該ＧＳＴ活性測定用サンプル０．２ｍｌと、第一基質溶液（１０ｍＭ ＧＳＨ溶液、０．２Ｍリン酸カリウム緩衝液〔ｐＨ６．５〕及び蒸留水を１：５：２の割合で調製）を１．６ｍｌとを混和した後、ＣＤＮＢ（１−クロロ２，４ジニトロベンゼン）０．２ｍｌ加えて、３分間反応させ、３４０ｎｍの吸光度を測定した。活性は、モル吸光係数９６００×１０^−３を用いて求めた。この測定結果を図８及び図９に示す。
ＧＰｘ活性は以下の方法により測定した。即ち、上記上清を３０倍に希釈をしてＧＰｘ活性測定用サンプルとした。そして、該ＧＰｘ活性測定用サンプル０．０１ｍｌと、１Ｍ−Ｔｒｉｓ塩酸・ＥＤＴＡ緩衝液（ｐＨ８．０）０．１ｍｌ、０．１Ｍグルタチオン０．０２ｍｌ、１０単位／ｍｌ酵母ＧＳＳＧレダクターゼ０．１ｍｌ、２ｍＭ ＮＡＤＰＨ０．１ｍｌ、及び蒸留水０．６６ｍｌを混合し、３７℃で２分間反応後、７ｍＭ−ｔ１ブチルヒドロペルオキシド０．０１ｍｌを加えて反応を開始し、３分後、３４０ｎｍでの吸光度を測定した。非酵素的反応の吸光度は、酵素に依存しない非酵素的なＮＡＤＰＨの酸化反応が内在しているので、上記ＧＰｘ活性測定用サンプルの代わりに蒸留水を同じ容量（０．０１ｍｌ）加えて測定した。活性値は、ＮＡＤＰＨの３４０ｎｍにおけるミリモル吸光係数εは６．２２×１０Ｍ^−１ｃｍを用いて算出した。この測定結果を図１０及び図１１に示す。
ＴＢＡＲＳは以下の方法により測定した。即ち、上記上清０．３ｍｌに、０．６７％（ｗ／ｖ）ＴＢＡ試薬１．０ｍｌ、及び２０％（ｗ／ｖ）トリクロル酢酸（ＴＣＡ）溶液３ｍｌを順に加え、ボルテックスで十分攪拌した後、１００℃で１０分間加熱した。加熱終了後、氷浴中で１０分間急冷し、ｎ−ブタノール４．０ｍｌを加えて赤色物質を振とう抽出した。抽出後、３０００ｒｐｍで１０分間遠心分離を行った後、ｎ−ブタノール層を採取し、波長５３２ｎｍで比色測定した。定量には１，１，３，３，−ｔｅｔｏｒａｅｔｈｏｘｙｐｒｏｐａｎｅ１１０ｍｇを用い、１％硫酸溶液５０ｍｌを加えて２時間室温に保持した後、１００ｎｍｏｌ／ｍｌに調製し、これを用いて標準溶液（０、１０、２０、３０、４０、５０、６０ｎｍｏｌ／ｍｌ）を作製し、上記上清の代わりに反応させて、上記と同様の操作を行った。これより検量線を作製し、上記上清中のＴＢＡＲＳ量を求めた。この測定結果を図１２、１３及び図１４に示す。
また、上記試験方法における測定結果の統計処理においては、分散分析をおこない、Ｆ値が有意（Ｐ＜０．０５）である場合には、Ｄｕｎｃａｎのｍａｌｔｉｐｌｅ ｒａｎｇｅ ｔｅｓｔを用いて、各群の平均間の有意差（Ｐ＜０．０５＞）検定を行った。
血液は、採血後３０分間室温に静置した後、３０００ｒｐｍで１０分間遠心分離（久保田商事株式会社製「ＫＵＢＯＴＡ ＫＣ−７０」）を行い、上清をエッペン管にとり−８０℃で保存して血清サンプルとした。そして、このサンプルを用いて、以下に示す測定方法により、血清グルコース、総コレステロール（以下、「Ｔ−ｃｈｏｌ」という。）、中性脂肪（以下、「ＴＧ」という。）を測定した。また、採取した尿を用いて、尿中８−ヒドロキシデオキシグアノシン（以下、「尿中８−ＯＨｄＧ」という。）量の測定を行った。
血清グルコースは、測定キットとして和光純薬工業製の「グルコースＣＩＩテストワコー」を用いて、ムタローゼ・ＧＯＤ法により測定した。また、Ｔ−ｃｈｏｌは、測定キットとして和光純薬工業製の「総コレステロールＥテストワコー」を用いて、コレステロールオキシターゼ・ＤＡＯＳ法により測定した。ＴＧは、測定キットとして日本商事製の「ネスコートＴＧキット−ＧＮ」を用いて、Ｌ−グリセロール−３−リン酸オキシダーゼ・酵素法により測定した。尿中８−ＯＨｄＧは、測定キットとして日本老化制御研究所製の「８−ＯＨｄＧ測定用キット」を用いて、ＥＬＩＳＡ法により測定した。血清グルコースの測定結果を図４に、Ｔ−ｃｈｏｌの測定結果を図５に、ＴＧの測定結果を図６に、８−ＯＨｄＧの測定結果を図７にそれぞれ示す。
試験結果
上記〔実施例１〕の結果、表１及び図１より、対照群１、２と、米糠・大豆発酵抽出物にコーヒー豆抽出物を添加した試験群１とを比較すると、まず、従来よりＳＯＤ活性を有することが知られている米糠・大豆発酵抽出物を投与した対照群２では、対照群１に対して、ＳＯＤ活性値が１．８％の割合で上昇していた。そして、この対照群２と試験群１とを比較すると、試験群１では、対照群２よりも米糠・大豆発酵抽出物の含有量が少ないにも関わらず、対照群２のＳＯＤ活性値よりも更に１．８％の割合で上昇していたことが認められた。また、対照群１と試験群１について、ステューデントｔ検定の結果、両群間に有意差（Ｐ＜０．０１）が認められた。同様に、対照群２と試験群１についても、ステューデントｔ検定の結果、両群間に有意差（Ｐ＜０．０５）が認められた。
過酸化脂質は活性酸素障害的疾患の指標となるものであり、その程度がひどければ増加するものである。表２及び図２より、測定の結果、従来より過酸化脂質量を低下させることが知られている米糠・大豆発酵抽出物を投与した対照群２では、対照群１と比べて過酸化脂質量が約５％低下していたが、この対照群２と試験群１とを比較すると、試験群１では、対照群２よりも米糠・大豆発酵抽出物の含有量が少ないにも関わらず、対照群２の過酸化脂質量よりも更に約５％低下していることが認められた。また、対照群１と試験群１について、ステューデントｔ検定の結果、両群間に有意差（Ｐ＜０．０１）が認められた。同様に、対照群２と試験群１についても、ステューデントｔ検定の結果、両群間に有意差（Ｐ＜０．０５）が認められた。
上記〔実施例２〕の結果、表３及び図３より、本実施例のＳＯＤ様組成物を摂取させた結果、試験群２では、対照群３及び対照群４よりも血圧上昇が抑制されていることが認められた。例えば、６週間目での最高血圧値は、対照群３の場合、試験開始時より２１％増加し、従来より血圧低下作用を有することが知られている米糠・大豆発酵抽出物を投与した対照群４の場合は１４％増加しているのに対し、試験群２ではわずか９％増加しているに過ぎない。また、１２週間目では、対照群３の場合、試験開始時より２４％増加し、対照群４の場合は１９％増加しているのに対し、試験群２では１１％増加しているに過ぎない。即ち、経過週数が多くなるごとに、本実施例のＳＯＤ様組成物は顕著な血圧抑制効果を奏することが認められた。また、対照群３と試験群２について、ステューデントｔ検定の結果、４〜１２週間目において、両群間に有意差（Ｐ＜０．０１）が認められた。更に、対照群４と試験群２について、ステューデントｔ検定の結果、６〜１２週間目において、両群間に有意差（Ｐ＜０．０５）が認められた。
上記〔実施例３〕の結果、図４より、血清グルコース濃度は対照群５に比べて、糖尿病ラットである対照群６で有意に増加した。これはＳＴＺ投与による膵β−細胞の破壊に基づくインスリンの絶対不足により、糖代謝が著しく乱れ高血糖をきたしたものと考えられる。しかし、本実施例のＳＯＤ様組成物を投与した試験群３では、対照群６に比べ、血清グルコース濃度は有意に減少した。この結果から、本実施例のＳＯＤ様組成物には、糖代謝を改善し、血清グルコース濃度を低下させる効果を有することが判る。
糖尿病ではインスリンの著しい不足によって、糖代謝をはじめ脂肪の代謝も著しく乱れる。糖尿病に最も関係が深いのは高ＴＧ血症であり、インスリン作用不足によって、ＴＧ−ｒｉｃｈリポタンパクの異化障害による高ＴＧ血症をきたす。図５より、血清Ｔ−ｃｈｏｌは、対照群５に比べて糖尿病ラットである対照群６で著しく高くなるが、本実施例のＳＯＤ様組成物を投与した試験群３では、対照群５のレベルまで有意に低下した。また、図６より、血清ＴＧは、対照群５に比べ、糖尿病ラットである対照群６で顕著に増加したが、試験群３では有意に低下した。これらの結果より、本実施例のＳＯＤ様組成物は、血清脂質の改善効果を有し、糖尿病に起因する高ＴＧ血清等の脂肪の代謝異常を改善する効果を有することが判る。
生体内脂質過酸化反応により生じた活性酵素によるＤＮＡ中の核酸塩基、デオキシグアノシンに対する攻撃の結果、８−ＯＨｄＧが生成する。また、８−ＯＨｄＧは、過剰な酸化ストレスを受けた皮膚細胞や腎細胞中でも蓄積することが明らかにされている。このＤＮＡ中に生じた８−ＯＨｄＧは、通常は修復酵素により切り取られ、血液を経て最終的に酸化障害物として尿中へ排泄される。そして、図７より、尿中８−ＯＨｄＧ量は対照群５に比べて糖尿病ラットである対照群６では有意に増加しているのに対し、本実施例のＳＯＤ様組成物を投与した試験群３では、有意差はみられなかったが低下の傾向を示した。この結果から、糖尿病誘発によるＤＮＡの酸化障害に対して、本実施例のＳＯＤ様組成物が抗酸化効果を示す傾向にあることが判る。
ＧＳＴは動物組織に広く分布し、特に肝組織に多量存在し、生体内における解毒作用等の役割を担っている。図８より、肝組織のＧＳＴ活性は、本実施例のＳＯＤ様組成物の投与による有意差が認められなかったが、図９より、腎組織のＧＳＴ活性は、対照群５に比べて糖尿病ラットである対照群６が有意に高く、本実施例のＳＯＤ様組成物を投与した試験群３でも高い値を示した。この結果より、ＳＴＺ処理による酸化ストレスに対するＧＳＴによる解毒作用は、肝組織よりも腎組織において亢進しているものと考えられる。
ＳＴＺにより細胞内に生成した過酸化物の分解に消費されたグルタチオン（ＧＳＨ）の再生のために、ＧＰｘが増加する考えられている。そして、図１０より、肝組織におけるＧＰｘ活性は、対照群５に比べて、糖尿病ラットである対照群６で減少傾向を示しているのに対し、本実施例のＳＯＤ様組成物を投与した試験群２では有意に増加した。また、図１１より、腎組織でも肝組織と同様にＧＰｘ活性は対照群６に比べて試験群３では有意に増加した。この結果より、本実施例のＳＯＤ様組成物を投与すると、過酸化物の分解に消費されたＧＳＨの再生のためにＧＰｘが増加し、再生したＧＳＨが過酸化物の分解に消費されて抗酸化作用を奏することが判る。
糖尿病の場合、高血糖のためタンパク糖化が亢進し、生じたタンパクからも活性酸素が生成され、血漿中や組織中の過酸化脂質が増加する。その結果、酸性条件下でＴＢＡと反応する性質を有する、過酸化脂質の分解物であるＭＤＡ（マロンジアルデヒド）又はその他のカルボニル化合物等のＴＢＡＲＳが増加する。図１２より、糖尿病ラットである対照群６の肝組織のＴＢＡＲＳは、対照群５に比べて有意に増加した。これに対し、本実施例のＳＯＤ様組成物を投与した試験群３では有意に減少して、対照群５と同レベルまで低下した。また、図１３及び図１４より、腎組織及び血清中のＴＢＡＲＳも肝組織と同様に、糖尿病ラットである対照群６が有意に高いのに対し、本実施例のＳＯＤ様組成物を投与した試験群３では、対照群５とほぼ同レベルまで低下した。この結果より、本実施例のＳＯＤ様組成物を投与することにより、過酸化物の消去効果を奏することが判る。
以上の結果より、従来からＳＯＤ作用及び血圧抑制作用を有することが知られている米糠・大豆発酵抽出物に、コーヒー豆抽出物を添加した組成物を摂取することにより、米糠・大豆発酵抽出物を単独で用いる場合よりも体内のＳＯＤ活性が高められると共に、脂質の酸化が抑制され、血圧上昇が抑制されたことが分かる。即ち、本実施例のＳＯＤ様組成物に含まれる米糠・大豆発酵抽出物とコーヒー豆抽出物の混合物は、互いの相乗効果によって、より高いＳＯＤ活性と、より優れた過酸化脂質抑制効果、血圧抑制効果、糖代謝改善の効果及び血清コレステロールの改善効果等があることが分かる。
また、本発明に係るＳＯＤ様組成物のいずれの成分にも有害成分及び重金属は認められず、特に本発明に係るＳＯＤ様組成物は完全な天然物を原料としているため、安全であるといえる。そして、上記結果に示すように、本発明に係るＳＯＤ様組成物はＳＯＤ作用及び血圧抑制作用に優れるので、血圧抑制効果等のＳＯＤ作用に起因する種々の効果を有するものと考えられる。
産業上の利用可能性
本請求項１乃至４記載の各発明のＳＯＤ様組成物は、従来からＳＯＤ作用を有することが知られている米糠・大豆発酵抽出物単独と比較して、生体内におけるＳＯＤ作用に優れることから、ＳＯＤ作用に起因する効果（例えば、血圧抑制効果等）に有効である。従って、活性酸素種が原因となる各種の病気、例えば、血流障害による病気（心筋梗塞、脳卒中、高血圧、生理痛、肩こり、神経痛、腰痛、二日酔い等）、成人病・内科疾患（癌、腎炎、肝炎、糖尿病等）、美容・皮膚病（しみ、そばかす、肌荒れ、冷え症、便秘、しわ、アトピー性皮膚炎等）等の治療、改善等に優れた効果を発揮するものと考えられる。
また、本請求項５乃至８記載の各発明の血圧抑制剤によれば、近年、特に増加の一途をたどっている高血圧症の予防・改善に対して好適に用いることができる。更に、本請求項１乃至４記載の各発明のＳＯＤ様組成物及び本請求項５乃至８記載の各発明の血圧抑制剤は、いずれも水溶性であることから、吸収効果に優れると共に、経口投与により簡易に摂取でき、水溶性飲料、あるいは食品等の広い分野において利用されることが考えられる。その上、本請求項１乃至４記載の各発明のＳＯＤ様組成物及び本請求項５乃至８記載の各発明の血圧抑制剤は、いずれも完全な天然物を原料としていることから、有害な成分及び重金属は認められず安全である。
【図面の簡単な説明】
第１図は、〔実施例１〕の対照群１、２及び試験群１のＳＯＤ活性値（阻害率％）の平均値を示すグラフである。
第２図は、〔実施例１〕の対照群１、２及び試験群１の過酸化脂質濃度（ｎｍｏｌ／ｄｌ）の平均値を示すグラフである。
第３図は、〔実施例２〕の対照群３、４及び試験群２の最高血圧値（群平均値、ｍｍＨｇ）を示すグラフである。
第４図は、〔実施例３〕の対照群５、６及び試験群３の血清グルコース濃度（ｍｇ／ｄｌ）の平均値を示すグラフである。
第５図は、〔実施例３〕の対照群５、６及び試験群３の血清Ｔ−ｃｈｏｌ（ｍｇ／ｄｌ）の平均値を示すグラフである。
第６図は、〔実施例３〕の対照群５、６及び試験群３の血清ＴＧ（ｍｇ／１００ｍｌ）の平均値を示すグラフである。
第７図は、〔実施例３〕の対照群５、６及び試験群３の尿中８−ＯＨｄＧ量（ｎｇ／２４ｈ）の平均値を示すグラフである。
第８図は、〔実施例３〕の対照群５、６及び試験群３の肝組織におけるＧＳＴ活性（ｕｎｉｔｓ／ｍｇ ｐｒｏｔｅｉｎ）の平均値を示すグラフであり、
第９図は、腎組織におけるＧＳＴ活性（ｕｎｉｔｓ／ｍｇ ｐｒｏｔｅｉｎ）の平均値を示すグラフである。
第１０図は、〔実施例３〕の対照群５、６及び試験群３の肝組織におけるＧＰｘ活性（ｕｎｉｔｓ／ｍｇ ｐｒｏｔｅｉｎ）の平均値を示すグラフであり、
第１１図は、腎組織におけるＧＰｘ活性（ｕｎｉｔｓ／ｍｇ ｐｒｏｔｅｉｎ）の平均値を示すグラフである。
第１２図は、〔実施例３〕の対照群５、６及び試験群３の肝組織におけるチオバルビツール酸反応陽性物質（ＴＢＡＲＳ）（ｎｍｏｌ／ｍｇ ｐｒｏｔｅｉｎ）の平均値を示すグラフであり、
第１３図は、腎組織におけるＴＢＡＲＳ（ｎｍｏｌ／ｍｇ ｐｒｏｔｅｉｎ）の平均値を示すグラフである。
第１４図は、〔実施例３〕の対照群５、６及び試験群３の血清中におけるＴＢＡＲＳ（ｎｍｏｌ／ｍｇ ｐｒｏｔｅｉｎ）の平均値を示すグラフである。Technical field
The present invention relates to an SOD-like composition containing a rice bran / soybean fermented extract and a coffee bean extract and an antihypertensive agent. More specifically, the present invention relates to various diseases caused by active oxygen, such as diseases caused by blood flow disorders (myocardial infarction, stroke, hypertension, menstrual pain, stiff shoulders, neuralgia, low back pain, hangover, etc.), adult diseases / internal medicine SOD-like composition that can be widely used for the treatment and improvement of diseases (cancer, nephritis, hepatitis, diabetes, etc.), beauty and skin diseases (stains, freckles, rough skin, coldness, constipation, wrinkles, atopic dermatitis, etc.) And an antihypertensive agent.
Background art
Superoxide dismutase (hereinafter referred to as SOD) is a superoxide radical (O₂ ⁻) Disproportionation reaction (the following formula) is catalyzed at a rate close to diffusion rate,₂ ⁻It is an enzyme that lowers the concentration.
2O₂ ⁻+ 2H⁺→ H₂O₂+ O₂
O₂ ⁻The reactive oxygen species represented by is usually produced from phagocytic cells such as macrophages activated in vivo, and exhibits bactericidal and tumoricidal effects.
However, it is known that these reactive oxygen species have no selective toxicity and can also act on normal cells, resulting in various obstacles to the living body. For example, as a result of damage to the membrane due to lipid peroxidation, structural change of the protein due to oxidative modification of the protein, DNA cleavage, etc., it has been clarified that it shows a damaging effect on cells and causes various diseases.
Therefore, O₂ ⁻In recent years, the reaction mechanism of SOD is an enzyme that removes SOD, which is present in order to protect the living body from reactive oxygen species and is effective for diseases considered to be caused by the reactive oxygen species. Physiological mechanisms have been studied ["Reactive Oxygen-Molecular Mechanisms of Production, Elimination, and Action in Biology" (New Edition, 2nd edition, published by Kyoritsu Shuppan Co., Ltd., edited by Jun Nakano, pp. 223-230)]. In addition, there is a fact that SOD activity is low in cancer cells, and further, although a direct causal relationship between SOD and carcinogenesis is not clear, there is a report that when SOD or SOD-like substance is injected into cancer cells, proliferation is suppressed ( P. 64). Safe and SOD action (including not only the action of reducing the active oxygen concentration but also the action of prevention and improvement of various diseases thought to be caused by this), and used for foods, etc. Is very useful for human health and beauty, and its need is extremely high. Conventionally, from such a point of view, for example, an active oxygen suppressing composition and a blood pressure suppressing agent obtained by adding a green tea extract to a rice bran / soybean fermented extract as having an action similar to the SOD action and suppressing an increase in blood pressure. Has been developed (Japanese Patent Publication No. 8-40).
Recently, hypertension has been steadily increasing due to the influence of the living environment, particularly the westernization of dietary habits, and has become a major medical problem. Hypertension has primary and secondary, and secondary hypertension has many unclear causes such as Cushing's syndrome and renovascular hypertension, but primary hypertension is also said to be essential hypertension, Unexplained disease. In order to combat lifestyle-related diseases such as hypertension, it is essential to improve lifestyle such as exercise therapy and dietary habits. Therefore, recently, a food material that is excellent in taste, healthy, and capable of preventing / ameliorating lifestyle-related diseases has been desired under the idea of medical food sources.
On the other hand, green coffee beans contain various components such as lipids, carbohydrates, proteins and caffeine in addition to chlorogenic acids which are tannin components. In recent years, it has been clarified that coffee has various effects such as antimutagenicity and antioxidation. As an application example, (1) stabilization of vitamin C by, for example, chlorogenic acids 6-9603), (2) Deterioration prevention of natural fragrance (Japanese Patent Laid-Open No. 4-345893), (3) Dye browning inhibitor (Japanese Patent Laid-open No. 5-32909, Japanese Patent Publication No. 1-28777), (4) Prevention of flavor deterioration (Japanese Patent Laid-open No. Hei 6-38723), (5) Gastric mucosa protecting drug (Japanese Patent Publication No. 63-502349), and (6) N_βAn antioxidant (JP-A-8-157816) characterized by containing -Alkanoyl-5-hydroxytryptamine; (7) a coffee seed extract extract-containing cosmetic containing chlorogenic acid as an essential component (JP-A-8-92057) No.) is known.
Many recent studies on the physiological activity of coffee extract a specific component in coffee beans and examine its physiological activity. However, as described above, since the components in coffee beans contain a large number of substances, it is difficult to separate and identify all of them and to examine their physiological activities. In addition, there may be a case where there is a synergistic effect of not only a specific component but also a plurality of components, but at present, sufficient knowledge about such a synergistic effect has not yet been obtained. Moreover, even if a substance having an effect in vitro is orally administered in vivo, the digestion and absorption processes in various parts of the digestive system are complicatedly involved, and therefore, the same effect is not always achieved. Of the above-mentioned application examples, (1) to (4) and (6) are used outside the living body, so all are only knowledge about the effect outside the body, and the effect when actually administered to the living body. Nothing is stated about. In addition, although the above application example (7) is for use on the human body, it is a cosmetic used on the skin, so there is no description about the pharmacokinetics and effects when it is actually administered to the living body. Absent. Furthermore, in the above application example (5), although chlorogenic acid is administered in vivo, the effect is mainly gastric mucosal protective action due to convergence action, that is, insoluble precipitates that bind to proteins on the gastric mucosa. It is a film that protects the gastric mucosa. Therefore, in this case, it is considered that the administered chlorogenic acid is not so much absorbed from the digestive tract, and it is unclear as to what action effect is exerted on the whole body when the components in the coffee beans are absorbed from the digestive tract.
This invention is made | formed in view of the said present condition, has the effect | action similar to SOD conventionally, and is a rice bran and soybean fermented extract known to show | play the blood pressure suppression effect | action to a coffee bean extract. An object of the present invention is to provide a SOD-like composition that exhibits a safe and excellent SOD action in a living body and a blood pressure suppressor that exerts a blood pressure suppressing action.
Disclosure of the invention
As a result of various studies on the physiological action of coffee beans, the present inventors have previously found that the coffee bean extract has an SOD action and can be widely used for the treatment and improvement of various diseases caused by active oxygen. Has been filed (Japanese Patent Application No. 10-211739). And as a result of continuing examination of the action of the coffee bean extract, by adding the coffee bean extract to the rice bran / soybean fermented extract that has been conventionally known to have an SOD action, the rice bran / soybean fermented extract is obtained. The present invention has been completed by finding that it exhibits a higher SOD effect and superior blood pressure suppressing effect than when administered alone.
The SOD-like composition of the invention described in claim 1 and the blood pressure inhibitor of the invention described in claim 5 are obtained by inoculating a culture medium containing rice bran, soybeans and a carbon source with Bacillus natto or Bacillus subtilis, followed by fermentation. It contains a rice bran / soybean fermented extract obtained in this way and a coffee bean extract.
The “rice bran / soybean fermented extract” is an extract obtained by inoculating Bacillus natto or Bacillus subtilis into a medium containing rice bran, soybeans, and a carbon source, followed by fermentation. The “rice bran / soybean fermented extract” may be a filtered solution of the culture fermentation broth obtained by culturing, or it may be a post-treated solution such as decolorized, or a concentrated concentrate. Liquid may be used. In addition, a solid or powdered powder from which a solvent has been removed by a known method such as spray drying may be used.
The “rice bran” refers to rice germ, defatted rice germ, rice bran, defatted rice bran, etc., and the “soybeans” refers to defatted soybean, quina flour, soybean flour, soybean meal, hydrolysates thereof, etc. Say. In addition, as the “carbon source”, those usually used can be used, and for example, one or more of glucose, dextrin, lactose, starch and the like can be used. Usually, when the rice bran is 100 parts by weight, these addition ratios are 1 to 20 parts by weight of soybeans, preferably 10 to 20 parts by weight, and the carbon source is 20 to 80 parts by weight, preferably 40 to 40 parts by weight. 60 parts by weight. It is because it is most preferable for the growth of bacteria when it is in these ranges.
The above-mentioned “medium” is not particularly limited as long as it contains the rice bran, soybeans and carbon source and can grow Bacillus natto or Bacillus subtilis. Usually, rice bran, soybeans and carbon source are added to water. In addition, a liquid medium obtained by adding rice bran, soybeans, and a carbon source to a coffee bean extract obtained by the method described below may be used. The “medium” is usually a liquid medium, but may be a solid medium.
Further, as the above-mentioned “Natto bacillus” and “Bacillus subtilis”, it is common to use commercially available general Bacillus natto and Bacillus subtilis. However, even natural or chemical mutants such as nitrosoguanidine, X-rays, ultraviolet rays, etc., obtained by artificial mutation means, and mutated strains of Bacillus natto and Bacillus subtilis that have mutated mycological properties can exhibit SOD action. It can be used as long as it does not lose its ability to produce a fermented rice bran / soybean extract.
Usually, fermentation culture is carried out by aeration stirring. The conditions for the fermentation culture are not particularly limited as long as the fermentation is performed, but usually the pH is 7.5 to 10, preferably 8.5 to 10, and the culture temperature is about 40 to 45 ° C. When adjusting the pH of the medium, sodium hydrogen carbonate or the like can be used as an alkaline agent. In addition, protease can be used as a medium raw material. In this case, the soybean peptide is further decomposed, which is useful.
As the “coffee beans”, any varieties such as Arabica, Robusta, and Riberica may be used, and the production area is not particularly limited. In addition, the coffee beans produced for coffee beverages in the cultivation field are separated and removed in large quantities when they are selected for roasting. Can be used as a “bean”. The “coffee beans” are preferably green beans, but can be roasted beans. The “coffee bean extract” may be a liquid obtained by filtering the extract, a liquid obtained by post-treatment such as decolorization, or a concentrated liquid obtained by concentrating this. In addition, a solid or powdered powder from which the solvent has been removed by a known method such as spray drying may be used.
Further, the extraction method for obtaining the “coffee bean extract” is not particularly limited. The coffee beans as the raw material may be unground or ground, and may be subjected to pretreatment such as impurity removal as long as the quality of the extract can be maintained. As an extraction solvent, in addition to water or hot water, an organic solvent such as ethanol, ethyl acetate, or n-hexane, or a mixed solvent of these organic solvents and water or hot water may be used. In addition, since this extract is water-soluble, among these solvents, water (including hot water) or a mixed solvent of ethanol / water is preferable. Extraction conditions are not particularly limited, but usually normal temperature or heat extraction is preferred. The heating temperature and the heating time can also be set to various conditions as long as the extraction can be sufficiently performed and the quality of the extract can be maintained.
Further, in the above extraction, substances that assist the extraction can be added as long as the quality of the extract can be maintained. For example, a reducing substance such as L-ascorbic acid may be added in addition to enzymes such as protease and cellulase. The pH of the extract usually shows 5 to 6 when L-ascorbic acid or the like is added, but this pH is not limited as long as the quality of the extract is not deteriorated.
The content of the coffee bean extract (solid content) in the SOD-like composition of the invention of claim 1 and the antihypertensive agent of claim 5 is the content of the invention of claim 2 and claim 6 of the invention. As shown in the invention, it is usually 0.0003 to 5% by weight, preferably 0.0003 to 1% by weight, more preferably 0.05 to 0.5% by weight. When the concentration of the coffee bean extract is 0.05 to 0.5% by weight, the SOD effect increases particularly remarkably, and even when the concentration of the coffee bean extract exceeds 5% by weight, the SOD effect is increased. This is because the blood pressure suppression action tends to reach a peak.
Further, in the SOD-like composition of the invention of claim 1 and the blood pressure inhibitor of the invention of claim 5, of the coffee bean extract (solid content) relative to the amount of rice bran / soybean fermented extract (solid content) The ratio of the amount is 5 to 70 with respect to 100 parts by weight of rice bran / soybean fermented extract (solid content) in the SOD-like composition as shown in the invention described in claim 3 and the invention described in claim 7. Parts by weight, preferably 5 to 40 parts by weight, more preferably 15 to 30 parts by weight. If it is less than 5 parts by weight, a synergistic action with the rice bran / fermented soybean extract cannot be expected.
Furthermore, the rice bran / soybean fermented extract (solid content) and the coffee bean extract (solid content) in the SOD-like composition of the invention of claim 1 and the antihypertensive agent of the invention of claim 5 As shown in the invention described in claim 4 and the invention described in claim 8, the total amount is 0.0007% by weight or more, preferably 0.015 to 40% by weight, more preferably 0.1 to 15% by weight. %, Most preferably 0.25 to 4% by weight. Less than 0.0007% by weight is not preferable because an SOD-like action cannot be expected.
The SOD-like composition of each of the inventions according to claims 1 to 4 can be used for various diseases caused by active oxygen such as diseases caused by blood flow disorders (myocardial infarction, stroke, hypertension, menstrual pain, stiff shoulders, neuralgia, low back pain. , Hangover, etc., adult diseases / medical diseases (cancer, nephritis, hepatitis, diabetes, etc.), beauty / dermatoses (stains, freckles, rough skin, coldness, constipation, wrinkles, atopic dermatitis, etc.) Can be widely used. Among these, as shown in the respective inventions according to claims 5 to 8, it can be suitably used particularly as a blood pressure suppressant.
In the present invention, various modifications can be made within the scope of the present invention depending on the purpose and application. That is, the form of the above composition is usually a liquid such as an aqueous solution or a stock solution, but is not limited thereto, and a powder product obtained by impregnating the extract with a liquid absorbent powder, a granulated product obtained by granulation, and a bulking agent. It can be set as a tablet or a microcapsule etc. which mix | blended other powder components.
In addition, a product form in which these aqueous solutions and powders are filled in a predetermined container, or these can be used alone or mixed with other agents (whether aqueous solutions, oily liquids or powders). There is no particular limitation on whether or not to perform, for example, it may be a portion type, may be filled in a container of another shape, or may be a powder product filled in a stick-like container (bag).
Furthermore, you may mix | blend and disperse | distribute to the conventional soft drink, a drink agent, a dairy product, an oil-formation product, etc. In addition, this dispersion | distribution does not ask | require water-in-oil type and an oil-in-water type. Moreover, you may mix | blend other nutrient components (For example, various vitamins, a calcium ion component, an iron ion component, etc.), a medicinal component, a seasoning component, an odor component, etc. Of these, water-soluble components are particularly preferable. It is because it can be set as the product melt | dissolved uniformly.
BEST MODE FOR CARRYING OUT THE INVENTION
Hereinafter, the present invention will be described specifically by way of examples.
(1) Preparation of SOD-like composition and blood pressure suppressor used in [Example 1] and [Example 2]
The SOD-like composition used in the following [Example 1] and the blood pressure suppressing agent used in [Example 2] were prepared by the following method.
▲ 1 ▼ Manufacture of coffee bean extract
60 kg of coffee beans are pulverized, and metal foreign matters are removed by passing a magnetic force of 10,000 gauss through the pulverized beans. Next, 5 times the amount of water is mixed into the crushed beans, and 0.02 kg of citric acid, 0.15 kg of L-ascorbic acid, and 0.01 kg of sodium L-ascorbate are added with stirring to pH. 5.6-6.0, and when dissolved, stirring was stopped and extraction was performed at 60-70 ° C. for 3 hours to obtain a coffee bean extract (solid content concentration 3% by weight).
(2) Manufacture of rice bran and soybean fermented extract
30.0 kg of defatted rice bran, 5.0 kg of defatted soybean, 5.0 kg of phytic acid, 15.0 kg of glucose, 10.0 kg of disodium hydrogen phosphate, 2.5 kg of diammonium hydrogen phosphate, Sodium bicarbonate: 45.0 kg, antifoaming agent: 0.25 kg, water: 500 kg were used. The pH is around 9.
The above medium is sterilized at 121 ° C. for 30 minutes, then cooled, then inoculated with 0.05 kg of Natto (manufacturer; Naruse Fermentation Chemical Laboratory), aerated and stirred at 40-45 ° C. for about 48 hours. And cultured to obtain a culture. Thereafter, this culture was subjected to pressure filtration, treated with activated carbon and perlite to deodorize and decolorize to obtain an almost transparent rice bran / soybean fermented extract (solid content concentration 5 wt%). In addition, as this activated carbon, various types of powdered activated carbon (activated carbon S, activated carbon K, etc.) and granular activated carbon (activated carbon SG, etc.) can be used. As the pearlite, “Perlite No. 4180” (manufactured by Daika Line Orient Co., Ltd.). )It was used.
(3) Preparation of SOD-like composition and antihypertensive agent
20 parts by weight (converted to solid content) of the coffee bean extract prepared in (1) above and 80 parts by weight (converted to solid content) of the rice bran / soybean fermented extract manufactured in (2) above were mixed. Then, deodorization and decoloration are performed by activated carbon treatment, and then the mixture is squeezed and filtered to remove solids such as activated carbon. And after vacuum concentrating the mixture after press filtration, it was pulverized by the freeze-drying method. 1 part by weight of the powder produced by the above method is added to 99 parts by weight of “Oriental MF” manufactured by Oriental Yeast Co., Ltd., and the SOD-like composition used in the test method for SOD-like action in [Example 1] Prepared. Moreover, the blood-pressure-inhibitor solution used by the test method of the blood-pressure suppression effect of [Example 2] was prepared by melt | dissolving the powder manufactured by the said method in distilled water as 1% solution.
(2) Preparation of SOD-like composition used in [Example 3]
The SOD-like composition used in [Example 3] was prepared by the following method.
30.0 kg of defatted rice bran, 5.0 kg of defatted soybean, 5.0 kg of phytic acid, 15.0 kg of glucose, 10.0 kg of disodium hydrogen phosphate, 2.5 kg of diammonium hydrogen phosphate, Sodium bicarbonate: 45.0 kg, antifoaming agent: 0.25 kg, water: 500 kg, and 100 kg of the coffee bean extract produced in the above (1) were used. The pH is around 9.
The above medium is sterilized at 121 ° C. for 30 minutes, then cooled, then inoculated with 0.05 kg of Bacillus subtilis (manufacturer; Toyo Fermentation Co., Ltd.), aerated and stirred at 40 to 45 ° C. for about 18 hours. While performing liquid culture. After completion of the fermentation, the solid matter is removed by squeezing filtration and ultrafiltration is performed. Further, the SOD-like composition was prepared by treating with activated carbon, decoloring and deodorizing, finely filtering the finished product, and pulverizing the fermentation broth by freeze drying.
[Example 1]
As test animals, 8 male Wistar rats (purchased from Japan SLC Co., Ltd.) 60 males were used.
The above Wistar rats were preliminarily raised for 3 days, and then divided into 3 groups of 20 controls per group, control groups 1 and 2 and test group 1. And it reared for 30 days on the conditions of room temperature 23 +/- 1 degreeC, humidity 45-55%, and illumination for 12 hours (8 hour-20 hour).
During the test period, test group 1 was allowed to freely ingest the SOD-like composition produced in (3) of (1) above. On the other hand, the control group 1 had the above-mentioned “Oriental MF” as the feed and the control group 2 had 99 parts by weight of the “oriental MF” as the feed, and the rice bran / soybean produced in (2) of (1) above. 1 part by weight of the fermented extract (solid content) was added, and the total amount was 100 parts by weight. During the test period, water was allowed to drink distilled water freely. Then, blood was collected from the heart on the last day of the test and stored immediately at −18 ° C. after blood collection, and the SOD activity and lipid peroxide level in the serum were measured by the following measuring methods.
The SOD activity was measured by the NBT reduction method using “SOD Test Wako” manufactured by Wako Pure Chemical Industries (in-vitro diagnostic drug, approval number (63AM) No. 0285) as a measurement kit. The results are shown in Table 1 and FIG. In addition, lipid peroxide can be obtained by using “Lipid peroxide-Test Wako (Fluorescent Yagi method)” manufactured by Wako Pure Chemical Industries (in-vitro diagnostic drug, approval number (61AM) No. 4641) as a measurement kit. It was measured by an acid (hereinafter referred to as “TBA”) method. The results are shown in Table 2 and FIG.
[Example 2]
As test animals, 60 spontaneously hypertensive rats (SHR) of 3 weeks old resembling human essential hypertension were used.
The spontaneously hypertensive rats were preliminarily bred for 7 weeks, and then divided into 3 groups, 20 groups per group, 3 control groups and 4 test groups. And it was reared as a test period for 12 weeks under conditions of room temperature 23 ± 1 ° C., humidity 45 to 55%, and illumination for 12 hours (8 hours to 20 hours). During the test period, as a daily beverage, 30 g of the antihypertensive agent solution prepared in (3) of (1) above was added to test group 2, 30 g of distilled water was added to control group 3, and the above was applied to control group 4. 30 g of distilled water containing 1% of rice bran / soybean fermented extract produced in (2) of (1) was placed in a water supply bottle and allowed to ingest freely. The water bottle was changed daily during the test period. Moreover, it was set as free intake using "Oriental MF" by Oriental Yeast Co., Ltd. as a feed. The blood pressure was measured at the start of the test and once every two weeks. The measurement results are shown in Table 3 and FIG. In addition, the measured value of Table 3 and FIG. 3 is an average value of the systolic blood pressure value of each group.
Example 3
Twenty-five male Wistar rats (purchased from Nippon SLC Co., Ltd.) were used as test animals.
The Wistar rats were divided into three groups of control groups 5 and 6 and test group 3 as described below after 1 week of preliminary breeding. That is, as control group 5, 5 rats not treated with diabetes-inducing agent streptozotocin (hereinafter referred to as “STZ”), and as control group 4 and test group 3, insulin deficiency in which insulin-deficient diabetes was developed by STZ treatment. Type diabetic rats were divided into 3 groups of 10 animals each.

Each of the three groups of rats was bred for 4 weeks (28 days) under conditions of room temperature 23 ± 1 ° C., humidity 50-60%, and 12-hour lighting (7 o'clock to 19 o'clock). During the test period, rats in test group 3 were orally administered the SOD-like composition prepared in (2) above in an amount of 3.6 mg / animal / day. In addition, tap water was allowed to be freely consumed, and “Oriental MF” manufactured by Oriental Yeast Co., Ltd. was freely ingested as feed feed.
Then, 24 hours of urine was collected from each of the three groups of rats in the last week of breeding (4 weeks of breeding).
The rats of the above three groups were fasted for 10 hours after the breeding was completed, subjected to ether anesthesia, then dissected and collected blood, and the liver and kidney were removed and the wet weight was measured. The tissue was subjected to blood removal treatment, washed with physiological saline, and stored in a freezer (“SANYO MDF-192” manufactured by Sanyo Electric Co., Ltd.) at −80 ° C.
The cryopreserved liver and kidney were thawed and then made 10% homogenate using 3 mM Tris hydrochloride buffer (3 mM Tris, 0.25 M sucrose, 0.1 mM EDTA pH 7.4). This homogenate is dispensed into an Eppendorf tube, stored at −80 ° C., thawed, and then thawed at 5000 rpm for 5 minutes at 4 ° C. using a cooling centrifuge (“KUBOTA 7930” manufactured by Kubota Corporation). The supernatant was obtained by centrifugation.
Such supernatant is appropriately diluted to have the following glutathione-S-transferase (hereinafter referred to as “GST”) activity, glutathione peroxidase (hereinafter referred to as “GPx”) activity, and TBA reaction positive substance (hereinafter referred to as “TBARS”). ”) Was measured.
GST activity was measured by the following method. That is, 0.2 ml of the supernatant was collected and diluted 20 times to obtain a sample for measuring GST activity. Then, 0.2 ml of the GST activity measurement sample and a first substrate solution (10 mM GSH solution, 0.2 M potassium phosphate buffer [pH 6.5] and distilled water prepared at a ratio of 1: 5: 2) After mixing with 1.6 ml, 0.2 ml of CDNB (1-chloro2,4dinitrobenzene) was added and reacted for 3 minutes, and the absorbance at 340 nm was measured. Activity is molar extinction coefficient 9600 × 10^-3Was determined using. The measurement results are shown in FIGS.
GPx activity was measured by the following method. That is, the supernatant was diluted 30 times to obtain a sample for measuring GPx activity. Then, 0.01 ml of the GPx activity measurement sample, 0.1 ml of 1 M-Tris hydrochloric acid / EDTA buffer (pH 8.0), 0.1 M glutathione 0.02 ml, 10 units / ml yeast GSSG reductase 0.1 ml, 2 mM After mixing 0.1 ml of NADPH and 0.66 ml of distilled water and reacting at 37 ° C. for 2 minutes, 0.01 ml of 7 mM t1 butyl hydroperoxide was added to start the reaction. After 3 minutes, the absorbance at 340 nm was measured. . The absorbance of the non-enzymatic reaction was determined by adding the same volume (0.01 ml) of distilled water instead of the sample for GPx activity measurement because the enzyme-independent non-enzymatic NADPH oxidation reaction was inherent. . The activity value is NADPH's millimolar extinction coefficient ε at 340 nm is 6.22 × 10 M.^-1Calculated using cm. The measurement results are shown in FIGS.
TBARS was measured by the following method. That is, 0.6 ml of 0.67% (w / v) TBA reagent and 3 ml of 20% (w / v) trichloroacetic acid (TCA) solution were added in order to 0.3 ml of the above supernatant and vortexed sufficiently. And heated at 100 ° C. for 10 minutes. After completion of the heating, the mixture was rapidly cooled in an ice bath for 10 minutes, 4.0 ml of n-butanol was added, and the red substance was extracted by shaking. After extraction, centrifugation was performed at 3000 rpm for 10 minutes, and then an n-butanol layer was collected and colorimetrically measured at a wavelength of 532 nm. For quantification, 1,1,3,3-tetraethoxypropane 110 mg was used, 50 ml of 1% sulfuric acid solution was added and kept at room temperature for 2 hours, and then adjusted to 100 nmol / ml, and this was used to prepare a standard solution (0, 10, 20, 30, 40, 50, 60 nmol / ml) were prepared and reacted in place of the supernatant, and the same operation as described above was performed. A calibration curve was prepared from this, and the amount of TBARS in the supernatant was determined. The measurement results are shown in FIGS.
In the statistical processing of the measurement results in the above test method, analysis of variance was performed, and when the F value was significant (P <0.05), Duncan's multiple range test was used to calculate the average between each group. Significant difference (P <0.05>) test.
The blood was allowed to stand at room temperature for 30 minutes after blood collection, and then centrifuged at 3000 rpm for 10 minutes (“KUBOTA KC-70” manufactured by Kubota Corporation). The supernatant was collected in an Eppendorf tube and stored at −80 ° C. to obtain serum. A sample was used. Using this sample, serum glucose, total cholesterol (hereinafter referred to as “T-chol”), and neutral fat (hereinafter referred to as “TG”) were measured by the following measurement method. Further, using the collected urine, the amount of urinary 8-hydroxydeoxyguanosine (hereinafter referred to as “urinary 8-OHdG”) was measured.
Serum glucose was measured by the Mutarose GOD method using “Glucose CII Test Wako” manufactured by Wako Pure Chemical Industries, Ltd. as a measurement kit. T-chol was measured by the cholesterol oxidase / DAOS method using “Total Cholesterol E Test Wako” manufactured by Wako Pure Chemical Industries, Ltd. as a measurement kit. TG was measured by the L-glycerol-3-phosphate oxidase / enzyme method using “Nescoat TG kit-GN” manufactured by Nippon Shoji Co., Ltd. as a measurement kit. Urinary 8-OHdG was measured by the ELISA method using “8-OHdG measurement kit” manufactured by Japan Aging Control Laboratory as a measurement kit. The measurement result of serum glucose is shown in FIG. 4, the measurement result of T-chol is shown in FIG. 5, the measurement result of TG is shown in FIG. 6, and the measurement result of 8-OHdG is shown in FIG.
Test results
As a result of the above [Example 1], from Table 1 and FIG. 1, when comparing the control groups 1 and 2 with the test group 1 in which the coffee bean extract was added to the rice bran / soybean fermented extract, In the control group 2 to which the rice bran / soybean fermented extract known to have an activity was administered, the SOD activity value increased at a rate of 1.8% compared to the control group 1. And when this control group 2 and the test group 1 are compared, in the test group 1, although the content of the rice bran / soybean fermented extract is lower than that in the control group 2, it is higher than the SOD activity value of the control group 2. Furthermore, it was recognized that it rose at a rate of 1.8%. Further, as a result of the Student t test, a significant difference (P <0.01) was found between the two groups for the control group 1 and the test group 1. Similarly, for the control group 2 and the test group 1, as a result of the student t test, a significant difference (P <0.05) was recognized between the two groups.
Lipid peroxide is an indicator of active oxygen disorder disease, and increases with increasing severity. From Table 2 and FIG. 2, as a result of the measurement, the amount of lipid peroxide in the control group 2 administered with the rice bran / soybean fermented extract, which has been known to decrease the amount of lipid peroxide as compared with the conventional method, is higher than that of the control group 1. Although the control group 2 and the test group 1 were compared with each other, the test group 1 had a lower rice bran / soybean fermented extract content than the control group 2, but the control It was observed that the amount of lipid peroxide in group 2 was further reduced by about 5%. Further, as a result of the Student t test, a significant difference (P <0.01) was found between the two groups for the control group 1 and the test group 1. Similarly, for the control group 2 and the test group 1, as a result of the student t test, a significant difference (P <0.05) was recognized between the two groups.
As a result of the above [Example 2], from Table 3 and FIG. 3, as a result of ingesting the SOD-like composition of the present Example, the increase in blood pressure was suppressed in Test Group 2 than in Control Group 3 and Control Group 4. It was recognized that For example, in the case of the control group 3, the maximum blood pressure value at the 6th week increased by 21% from the start of the test, and the control to which the rice bran / soybean fermented extract, which is conventionally known to have a blood pressure lowering effect, was administered. In group 4 the increase is 14%, whereas in test group 2 it is only a 9% increase. At the 12th week, the control group 3 increased 24% from the start of the test, the control group 4 increased 19%, while the test group 2 increased only 11%. Absent. That is, it was recognized that the SOD-like composition of this example has a remarkable blood pressure suppression effect as the number of elapsed weeks increases. Further, regarding the control group 3 and the test group 2, as a result of the student t test, a significant difference (P <0.01) was recognized between the two groups at 4 to 12 weeks. Furthermore, regarding the control group 4 and the test group 2, as a result of the Student's t test, a significant difference (P <0.05) was recognized between the two groups in the 6th to 12th weeks.
As a result of the above [Example 3], from FIG. 4, the serum glucose concentration significantly increased in the control group 6 which is a diabetic rat as compared with the control group 5. This is considered to be because glucose metabolism was significantly disturbed and hyperglycemia was caused by an absolute deficiency of insulin based on pancreatic β-cell destruction by STZ administration. However, in the test group 3 to which the SOD-like composition of this example was administered, the serum glucose concentration was significantly reduced as compared with the control group 6. From this result, it can be seen that the SOD-like composition of this example has the effect of improving sugar metabolism and lowering the serum glucose concentration.
In diabetes, due to a remarkable shortage of insulin, the metabolism of fat, including sugar metabolism, is also significantly disturbed. Hyper-TGemia is most closely related to diabetes, and due to insufficient insulin action, hyper-TGemia is caused by catabolism of TG-rich lipoprotein. From FIG. 5, the serum T-chol is significantly higher in the control group 6 which is a diabetic rat than in the control group 5, but in the test group 3 to which the SOD-like composition of this example was administered, the level of the control group 5 Significantly decreased. Further, from FIG. 6, serum TG was significantly increased in the control group 6, which is a diabetic rat, but significantly decreased in the test group 3 as compared with the control group 5. From these results, it can be seen that the SOD-like composition of this example has an effect of improving serum lipids and an effect of improving metabolic abnormalities of fat such as high TG serum caused by diabetes.
As a result of the attack on the nucleobase and deoxyguanosine in the DNA by the active enzyme generated by the lipid peroxidation in vivo, 8-OHdG is produced. 8-OHdG has also been shown to accumulate in skin cells and kidney cells that have undergone excessive oxidative stress. 8-OHdG produced in this DNA is usually excised by a repair enzyme and finally excreted into the urine via blood as an oxidative obstacle. From FIG. 7, the amount of 8-OHdG in urine is significantly increased in the control group 6 which is a diabetic rat compared to the control group 5, whereas the test group to which the SOD-like composition of this example was administered. In No. 3, there was no significant difference, but a tendency to decrease was shown. From this result, it can be seen that the SOD-like composition of the present example tends to exhibit an antioxidant effect against oxidative damage of DNA induced by diabetes.
GST is widely distributed in animal tissues, particularly in liver tissues, and plays a role such as in vivo detoxification. From FIG. 8, the GST activity of the liver tissue was not significantly different depending on the administration of the SOD-like composition of this example. However, from FIG. 9, the GST activity of the kidney tissue was higher than that of the control group 5 in diabetic rats. The control group 6 was significantly higher, and the test group 3 to which the SOD-like composition of this example was administered also showed a high value. From this result, it is considered that the detoxification effect by GST against oxidative stress by STZ treatment is enhanced in kidney tissue rather than liver tissue.
It is considered that GPx increases due to the regeneration of glutathione (GSH) consumed for the decomposition of peroxide generated in cells by STZ. From FIG. 10, the GPx activity in the liver tissue showed a decreasing tendency in the control group 6 which is a diabetic rat as compared with the control group 5, whereas the test in which the SOD-like composition of this example was administered. In group 2, there was a significant increase. Further, from FIG. 11, the GPx activity in the kidney tissue was significantly increased in the test group 3 as compared with the control group 6 as in the liver tissue. From this result, when the SOD-like composition of this example was administered, GPx increased due to the regeneration of GSH consumed for the decomposition of peroxide, and the regenerated GSH was consumed for the decomposition of peroxide. It turns out that there exists an oxidative effect.
In the case of diabetes, protein glycation is promoted due to hyperglycemia, and active oxygen is also generated from the produced protein, and lipid peroxide in plasma and tissues increases. As a result, TBARS such as MDA (malondialdehyde) or other carbonyl compound, which is a degradation product of lipid peroxide, having the property of reacting with TBA under acidic conditions increases. From FIG. 12, TBARS of the liver tissue of the control group 6 which is a diabetic rat increased significantly compared with the control group 5. On the other hand, in test group 3 administered with the SOD-like composition of this example, it significantly decreased and decreased to the same level as in control group 5. Moreover, from FIG.13 and FIG.14, the test which administered the SOD-like composition of a present Example with respect to the TBARS in a renal tissue and serum was significantly high in the control group 6 which is a diabetic rat similarly to a liver tissue. In group 3, the level dropped to almost the same level as in control group 5. From this result, it can be seen that by administering the SOD-like composition of this example, the peroxide scavenging effect is exhibited.
From the above results, the rice bran / soybean fermented extract is obtained by ingesting a composition obtained by adding a coffee bean extract to the rice bran / soybean fermented extract, which has been known to have SOD action and blood pressure suppressing action. It can be seen that the SOD activity in the body is increased as compared with the case of using A, alone, lipid oxidation is suppressed, and an increase in blood pressure is suppressed. That is, the mixture of the rice bran / soybean fermented extract and the coffee bean extract contained in the SOD-like composition of this example has a higher SOD activity, a superior lipid peroxide inhibitory effect, It can be seen that there are an inhibitory effect, an effect of improving sugar metabolism, an effect of improving serum cholesterol, and the like.
Further, no harmful components and heavy metals are observed in any of the components of the SOD-like composition according to the present invention, and in particular, the SOD-like composition according to the present invention is made of a completely natural product, so it can be said to be safe. . And as shown in the said result, since the SOD-like composition which concerns on this invention is excellent in a SOD effect | action and a blood pressure suppression effect, it is thought that it has various effects resulting from SOD effects, such as a blood pressure suppression effect.
Industrial applicability
Since the SOD-like composition of each of the inventions according to claims 1 to 4 is superior in SOD action in vivo as compared with a rice bran / soybean fermented extract alone that has been conventionally known to have SOD action. This is effective for effects caused by the SOD action (for example, blood pressure suppression effect, etc.). Therefore, various diseases caused by reactive oxygen species, such as diseases caused by blood flow disorders (myocardial infarction, stroke, hypertension, menstrual pain, stiff shoulders, neuralgia, back pain, hangover, etc.), adult diseases / medicine diseases (cancer, nephritis) , Hepatitis, diabetes, etc.), beauty / dermatoses (stains, freckles, rough skin, coldness, constipation, wrinkles, atopic dermatitis, etc.), etc.
Moreover, according to the blood pressure suppressant of each invention of claims 5 to 8, it can be suitably used for the prevention and improvement of hypertension, which has been increasing in recent years. Furthermore, since the SOD-like composition of each invention of claims 1 to 4 and the blood pressure inhibitor of each invention of claims 5 to 8 are both water-soluble, they are excellent in absorption effect and oral. It can be easily ingested by administration and can be used in a wide range of fields such as water-soluble beverages and foods. In addition, the SOD-like composition of each invention of claims 1 to 4 and the blood pressure suppressant of each invention of claims 5 to 8 are both harmful because they are made from completely natural products. Components and heavy metals are not recognized and are safe.
[Brief description of the drawings]
FIG. 1 is a graph showing average values of SOD activity values (inhibition rate%) of Control Groups 1 and 2 and Test Group 1 in [Example 1].
FIG. 2 is a graph showing the average value of lipid peroxide concentration (nmol / dl) in Control Group 1 and 2 and Test Group 1 in [Example 1].
FIG. 3 is a graph showing the systolic blood pressure values (group average value, mmHg) of the control groups 3 and 4 and the test group 2 of [Example 2].
FIG. 4 is a graph showing the mean value of serum glucose concentration (mg / dl) in Control Groups 5 and 6 and Test Group 3 in [Example 3].
FIG. 5 is a graph showing the mean values of serum T-chol (mg / dl) in the control groups 5 and 6 and the test group 3 in [Example 3].
FIG. 6 is a graph showing the average value of serum TG (mg / 100 ml) in Control Groups 5 and 6 and Test Group 3 in [Example 3].
FIG. 7 is a graph showing the average values of urinary 8-OHdG amounts (ng / 24h) in Control Groups 5 and 6 and Test Group 3 of [Example 3].
FIG. 8 is a graph showing the average value of GST activity (units / mg protein) in liver tissues of Control Groups 5 and 6 and Test Group 3 of [Example 3];
FIG. 9 is a graph showing the average value of GST activity (units / mg protein) in kidney tissue.
FIG. 10 is a graph showing the average value of GPx activity (units / mg protein) in the liver tissues of the control groups 5 and 6 and the test group 3 of [Example 3];
FIG. 11 is a graph showing the average value of GPx activity (units / mg protein) in kidney tissue.
FIG. 12 is a graph showing the average values of thiobarbituric acid reaction positive substances (TBARS) (nmol / mg protein) in the liver tissues of the control groups 5 and 6 and the test group 3 of [Example 3],
FIG. 13 is a graph showing the average value of TBARS (nmol / mg protein) in kidney tissue.
FIG. 14 is a graph showing the average value of TBARS (nmol / mg protein) in the sera of control groups 5 and 6 and test group 3 of [Example 3].

Claims (8)

SOD characterized by containing a rice bran / soybean fermented extract obtained by inoculating natto or Bacillus subtilis into a medium containing rice bran, soybeans and a carbon source and fermented and cultured, and a coffee bean extract Like composition. The SOD-like composition according to claim 1, wherein the content of the coffee bean extract is 0.0003 to 5% by weight in terms of solid content. The SOD-like composition according to claim 1 or 2, wherein the content of the coffee bean extract (solid content) is 5 to 70 parts by weight with respect to 100 parts by weight of the rice bran / soybean fermented extract (solid content). object. The SOD-like composition according to any one of claims 1 to 3, wherein the total content of the coffee bean extract (solid content) and the rice bran / soybean fermented extract (solid content) is 0.0007 wt% or more. object. A blood pressure characterized by containing a rice bran / soybean fermented extract obtained by inoculating a culture medium containing rice bran, soybeans and a carbon source with Bacillus natto or Bacillus subtilis and fermentation culture, and a coffee bean extract. Inhibitor. The blood pressure inhibitor according to claim 5, wherein the content of the coffee bean extract is 0.0003 to 5% by weight in terms of solid content. The blood pressure inhibitor according to claim 5 or 6, wherein the content of the coffee bean extract (solid content) is 5 to 70 parts by weight with respect to 100 parts by weight of the rice bran / soybean fermented extract (solid content). . The blood pressure suppressant according to any one of claims 5 to 7, wherein the total content of the coffee bean extract (solid content) and the rice bran / soybean fermented extract (solid content) is 0.0007 wt% or more. .

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