JP3885214B2

JP3885214B2 - Novel hexapeptide and angiotensin converting enzyme inhibitors

Info

Publication number: JP3885214B2
Application number: JP2003413922A
Authority: JP
Inventors: 邦男末綱; 和晃渡邉
Original assignee: 末綱陽子; オリエンタルバイオ株式会社
Priority date: 2003-11-07
Filing date: 2003-11-07
Publication date: 2007-02-21
Anticipated expiration: 2023-11-07
Also published as: JP2005139158A

Description

本発明は、新規なヘクサペプチド及びアンジオテンシン変換酵素阻害剤に関する。The present invention relates to novel hexapeptides and angiotensin converting enzyme inhibitors.

食品由来のペプチドは、アンジオテンシン変換酵素阻害剤及び血圧降下剤としての利点を持つ。
特開平０６−２５６３８７特開平０６−３４０６９２特開平０７−１８８２８３特開平０９−０５９２９６特開平１０−０３６３９１特開２００３−２６７９９３特開平１１−３３５３９３特開平１１−３４３２９８特開２００１−１０６６９８特開２００１−１０６６９９特開２００１−１０６７００特開２００３−２６７９９５特開２００１−０６４２９９特開２００３−１２８６９５特開２００３−２４６７９６特開２００３−２４６７９７特開２００３−２５２８９７特開２００３−２６７９９４特開平０７−１８８２８２特開平０８−２２５５９３特開平０９−１５７２９２特開平１１−０３５５９８特開平０８−２２５５９４特開平０８−２３１５８９Ｓ．Ｈ．Ｆｅｒｒｅｉａｅｔａｌ．：Ｂｉｏｃｈｅｍｉｓｔｒｙ，９，３５８３（１９７０）Ｇ．Ｏｓｈｉｍａｅｔａｌ．：Ｂｉｏｃｈｉｍ．Ｂｉｏｐｈｓ．Ａｃｔａ，５６６，１２８（１９７９）Ｓ．Ｍａｒｕｙａｍａｅｔａｌ．：Ａｇｒｉｃ．Ｂｉｏｌ．Ｃｈｅｍ．，４６，１３９３（１９８３）山本節子等：日胸疾会誌，１８，２９７−３０２（１９８９） Food-derived peptides have advantages as angiotensin converting enzyme inhibitors and antihypertensive agents.
JP 06-256387 JP-A-06-340692 JP 07-188283 A JP 09-059296 A Japanese Patent Laid-Open No. 10-033691 JP 2003-267993 A JP 11-335393 A JP-A-11-343298 JP 2001-106698 A JP 2001-106699 A JP 2001-106700 A JP 2003-267995 A JP 2001-064299 A JP 2003-128695 A JP2003-246696 JP2003-246797 JP2003-252897 JP 2003-267994 A JP 07-188282 A JP 08-225593 A JP 09-157292 A JP 11-035598 A JP 08-225594 A Japanese Patent Laid-Open No. 08-231589 S. H. Ferreia et al. : Biochemistry, 9, 3583 (1970) G. Oshima et al. : Biochim. Biophs. Acta, 566, 128 (1979) S. Maruyama et al. : Agric. Biol. Chem. 46, 1393 (1983) Setsuko Yamamoto, etc .: Journal of the Japan Breast Society, 18, 297-302 (1989)

レニン−アンジオテンシン系が生体の水・電解質及び血液の調節に重要な役割を果たしていることはよく知られている。このレニン−アンジオテンシン系にはアンジオテンシン変換酵素（以下ＡＣＥと略記する）が存在し、アンジオテンシンＩはＡＣＥによってアンジオテンシンＩＩに変換される。アンジオテンシンＩＩは強力な昇圧物質で、血管、副腎皮質のみならず中枢神経系ならびに末梢神経系に働いて血圧上昇を促す。又、ＡＣＥは生体内降圧物質であるブラジキニンを分解し、不活性化する作用を有し、昇圧系に関与している。従って、ＡＣＥの活性を阻害することによって血圧を降下させることが可能であり、又、そのことは臨床的に高血圧の予防、治療に有効であると考えられている。この目的のためプロリン誘導体であるカプトリルが合成され、その降圧作用が確認されて以来、カプトリルの構造研究に基づく種々のＡＣＥ阻害物質の合成研究が盛んに行われ、最近ではマレイン酸エナラブリルやアラセブリル等の物質が、次々と臨床の場に供されている。現在、ＡＣＥ阻害剤は、高血圧は、本態性高血圧症、病候性高血圧症を問わず、又、軽症、重症を問わず、幅広く用いられ、高血圧症の第一次選択の治療薬中に加えられ、多く優れた点を有することが見出されている。一方、ＡＣＥ阻害物質の作用機序としては、アンジオテンシンＩＩの産生抑制によるアルドステロンやバソプレッシンの分泌抑制、又、腎動脈収縮の解除によるナトリウムや水の排泄促進が考えられている。更に、ＡＣＥ阻害物質については、それがカリクレン−キニン系の不活性化を抑制し、プロスタグランジン系を賦活させることにより末梢血管拡張やナトリウム及び水の排泄を更に促進させると考えられており、心不全の悪循環を断つ上で合目的な治療薬として期待されている。ところで、ＡＣＥ阻害物質としては、上記の合成品の他に天然物又は天然物由来の物質として蛇毒由来のブラジキニン増強因子（Ｃ末端がＰｒｏ）［非特許文献１］、ゼラチンのコラゲナーゼ消化物由来の、６種類のペプチド（いずれもＣ末端がＡｌａ−Ｈｙｐ）［非特許文献２］、牛カゼインのトリプシン消化物由来のペプチド（Ｃ末端がＧｌｙ−Ｌｙｓ）［非特許文献３］等に始まり、本発明者等による、ニンニク由来のジペプチド［特許文献１］、イワシ筋肉由来の５種のヘクサペプチド（いずれもＣ末端から２番目又は３番目がＰｒｏ、Ｎ末端がＬｅｕ）［特許文献２］、ブタプラズマ由来のトリペプチド［特許文献３］、海苔・ヒジキ由来のペプチド［特許文献４、５、６］、朝鮮人参由来のペンタペプチド［特許文献７］、クロレラ由来のペンタペプチド［特許文献８］、スピルリナ由来のテトラペプチド［特許文献９］、アコヤ貝由来のヘクサペプチド［特許文献１０］、アサリ貝由来のペンタペプチド［特許文献１１］、ウロコ由来のヘプタペプチド［特許文献１２］及びワカメ由来のペプチド［特許文献１３〜１８］等が挙げられ、いずれもＡＣＥ阻害剤及び血圧降下剤となり得ることが開示されている。一方，大豆由来の生理活性ペプチドとしても同様に、本発明者等は数多くの特許を開示してきたが、その中で血圧降下ペプチドとしては大豆由来の２７種のトリペプチドＡｌａ−Ｉｌｅ−Ｍｅｔ、Ｔｙｒ−Ａｌａ−Ｖａｌ、Ｇｌｙ−Ｇｌｙ−Ｐｈｅ、Ｇｌｎ−Ｇｌｙ−Ｐｈｅ、Ｌｅｕ−Ｇｌｕ−Ｌｅｕ、Ｔｙｒ−Ａｌａ−Ｐｈｅ、Ｇｌｙ−Ｔｙｒ−Ｉｌｅ、Ｔｙｒ−Ｇｌｕ−Ｐｈｅ、Ａｌａ−Ａｓｐ−Ｔｙｒ、Ｇｌｕ−Ｇｌｙ−Ｇｌｎ、Ｇｌｎ−Ｐｈｅ−Ａｌａ、Ｐｈｅ−Ｍｅｔ−Ｇｌｙ、Ｇｌｙ−Ｐｈｅ−Ｇｌｙ、Ｉｌｅ−Ｇｌｙ−Ｓｅｒ、Ｔｒｐ−Ｔｒｐ−Ｌｅｕ、Ａｌａ−Ａｌａ−Ｌｅｕ、Ｌｅｕ−Ｉｌｅ−Ｐｈｅ、Ａｌａ−Ｌｅｕ−Ａｌａ、Ｐｒｏ−Ｇｌｙ−Ｔｈｒ、Ｐｈｅ−Ｌｅｕ−Ｍｅｔ、Ｔｒｐ−Ａｌａ−Ｐｒｏ、Ｔｙｒ−Ｉｌｅ−Ａｌａ、Ｐｈｅ−Ｓｅｒ−Ｐｒｏ、Ｐｈｅ−Ｐｈｅ−Ｔｙｒ、Ｐｈｅ−Ｖａｌ−Ａｌａ、Ｇｌｙ−Ｐｈｅ−Ｉｌｅ、Ａｌａ−Ａｌａ−Ｖａｌ［特許文献１９］及びＧｌｎ−Ｖａｌ−Ｖａｌ−Ｐｈｅ、Ｉｌｅ−Ｔｈｒ−Ｐｒｏ−Ｌｅｕ、Ｖａｌ−Ｖａｌ−Ｐｈｅ−Ａｓｐ、Ｇｌｙ−Ａｐ−Ａｌａ−Ｐｒｏ−Ａｓｎ、Ｉｌｅ−Ｖａｌ−Ｐｈｅ−Ａｓｐ−Ａｌａ、Ｖａｌ−Ｇｌｎ−Ｖａｌ−Ｖａｌ−Ｐｈｅ、Ｇｌｙ−Ｇｌｕ−Ｌｅｕ−Ｐｈｅ−Ｇｌｕ、Ｖａｌ−Ｔｈｒ−Ｖａｌ−Ｐｒｏ−Ｇｌｎ［特許文献２０］が開示され、活性化酸素阻害ペプチドとしてはＬｅｕ−Ａｓｎ−Ｔｙｒ−Ｃｙｓ−Ｖａｌ−Ａｌａ、Ｌｙｓ−Ｇｌｎ−Ｃｙｓ−Ｇｌｕ−Ｇｌｎ−Ｐｒｏ−Ｐｒｏ−Ｖａｌ−Ｌｅｕ［特許文献２１］及びＬｅｕ−Ｖａｌ−Ｐｒｏ−Ｇｌｎ−Ｇｌｕ−Ｃｙｓ−Ｇｌｎ−Ｌｙｓ［特許文献２２］が開示され、更に、免疫賦活ペプチドとしてはＰｈｅ−Ｔｈｒ−Ｌｙｓ−Ｐｒｏ−Ｇｌｙ、Ｌｅｕ−Ｌｙｓ−Ｐｒｏ−Ａｓｎ、Ｐｈｅ−Ｇｌｙ−Ｐｒｏ−Ｇｌｙ、Ｇｌｕ−Ａｓｐ−Ｌｙｓ−Ｐｒｏ−Ｐｈｅ−Ａｓｎ−Ｌｅｕ、Ａｌａ−Ｇｌｕ−Ｉｌｅ−Ａｓｎ−Ｍｅｔ−Ｐｒｏ−Ａｓｐ−Ｔｙｒ、Ｇｌｕ−Ｇｌｎ−Ｇｌｎ−Ｇｌｙ−Ｌｙｓ−Ｇｌｙ−Ｉｌｅ、Ｓｅｒ−Ｇｌｙ−Ｐｈｅ−Ａｌａ−Ｐｒｏ［特許文献２３］及びＩｌｅ−Ａｌａ−Ｖａｌ−Ｐｒｏ−Ｔｈｒ−Ｇｌｙ−Ｖａｌ−Ａｌａ、Ｉｌｅ−Ｇｌｕ−Ｇｌｕ−Ｇｌｙ−Ａｓｎ［特許文献２４］等が開示されてきた。このような生理活性ペプチドの中で、先に述べたように、これまでＡＣＥ阻害剤としての食品タンパク質由来の血圧降下ペプチドに関して数多くの提案がなされてきたが、規則性を持ったアミノ酸配列を有する鎖長の長いペプチドのＡＣＥ阻害作用並びに経口投与による降圧効果（薬理効果）については未だ不明であり、発見されてから長時間経過しているが、未だ医薬品としての開発が進んでいるとの報告はない。It is well known that the renin-angiotensin system plays an important role in the regulation of biological water, electrolytes and blood. This renin-angiotensin system includes an angiotensin converting enzyme (hereinafter abbreviated as ACE), and angiotensin I is converted to angiotensin II by ACE. Angiotensin II is a powerful vasopressor that works not only in blood vessels and adrenal cortex but also in the central nervous system and peripheral nervous system to promote an increase in blood pressure. ACE also has the action of degrading and inactivating bradykinin, which is a hypotensive substance in vivo, and is involved in the pressor system. Therefore, it is possible to lower blood pressure by inhibiting the activity of ACE, and this is clinically effective for the prevention and treatment of hypertension. Since captoryl, a proline derivative, was synthesized for this purpose and its antihypertensive activity was confirmed, various ACE inhibitors have been actively studied based on the structural study of captolyl. Recently, enalabril maleate, alacebryl, etc. These substances are being used in clinical settings one after another. Currently, ACE inhibitors are widely used for hypertension regardless of whether it is essential hypertension or symptomatic hypertension, and whether it is mild or severe. And have been found to have many excellent points. On the other hand, as the mechanism of action of ACE inhibitors, it is considered that aldosterone and vasopressin secretion are suppressed by suppressing production of angiotensin II, and that sodium and water excretion is promoted by releasing renal artery contraction. Furthermore, for ACE inhibitors, it is believed that it suppresses inactivation of the calicrene-kinin system and further promotes peripheral vasodilation and excretion of sodium and water by activating the prostaglandin system, It is expected to be an appropriate therapeutic agent in breaking the vicious circle of heart failure. By the way, as an ACE inhibitor, in addition to the above-mentioned synthetic product, a snake venom-derived bradykinin enhancing factor (C-terminal is Pro) [Non-patent document 1] as a substance derived from a natural product or a natural product, derived from a collagenase digest of gelatin , 6 types of peptides (all C-terminal is Ala-Hyp) [Non-patent document 2], peptides derived from tryptic digest of bovine casein (C-terminal is Gly-Lys) [Non-patent document 3], etc. According to the inventors, garlic-derived dipeptide [Patent Document 1], five hexapeptides derived from sardine muscle (all are 2nd or 3rd from the C-terminal, Pro is N, and the N-terminal is Leu) [Patent Document 2], pig Plasma-derived tripeptide [Patent Document 3], Nori / Hijiki-derived peptide [Patent Documents 4, 5, 6], Ginseng-derived pentapeptide [Patent Document 7], Black Pentapeptide derived from La [Patent Document 8], Tetrapeptide derived from Spirulina [Patent Document 9], Hexapeptide derived from pearl oyster [Patent Document 10], Pentapeptide derived from clam [Patent Document 11], Hepta derived from scale Peptides [Patent Document 12], wakame-derived peptides [Patent Documents 13 to 18] and the like are mentioned, and it is disclosed that any of them can be an ACE inhibitor and an antihypertensive agent. On the other hand, the present inventors have also disclosed a number of patents as bioactive peptides derived from soybean, and among them, 27 kinds of tripeptides Ala-Ile-Met, Tyr derived from soybean are used as blood pressure lowering peptides. -Ala-Val, Gly-Gly-Phe, Gln-Gly-Phe, Leu-Glu-Leu, Tyr-Ala-Phe, Gly-Tyr-Ile, Tyr-Glu-Phe, Ala-Asp-Tyr, Glu-Gly -Gln, Gln-Phe-Ala, Phe-Met-Gly, Gly-Phe-Gly, Ile-Gly-Ser, Trp-Trp-Leu, Ala-Ala-Leu, Leu-Ile-Phe, Ala-Leu-Ala Pro-Gly-Thr, Phe-Leu-Met, Trp-Ala-Pro, Tyr-I e-Ala, Phe-Ser-Pro, Phe-Phe-Tyr, Phe-Val-Ala, Gly-Phe-Ile, Ala-Ala-Val [Patent Document 19] and Gln-Val-Val-Phe, Ile-Thr -Pro-Leu, Val-Val-Phe-Asp, Gly-Ap-Ala-Pro-Asn, Ile-Val-Phe-Asp-Ala, Val-Gln-Val-Val-Phe, Gly-Glu-Leu-Phe -Glu, Val-Thr-Val-Pro-Gln [Patent Document 20] is disclosed, and as the activated oxygen-inhibiting peptide, Leu-Asn-Tyr-Cys-Val-Ala, Lys-Gln-Cys-Glu-Gln- Pro-Pro-Val-Leu [Patent Document 21] and Leu-Val-Pro-G n-Glu-Cys-Gln-Lys [Patent Document 22] is disclosed, and further, immunostimulatory peptides include Phe-Thr-Lys-Pro-Gly, Leu-Lys-Pro-Asn, and Phe-Gly-Pro-Gly. , Glu-Asp-Lys-Pro-Phe-Asn-Leu, Ala-Glu-Ile-Asn-Met-Pro-Asp-Tyr, Glu-Gln-Gln-Gly-Lys-Gly-Ile, Ser-Gly-Phe -Ala-Pro [Patent Document 23], Ile-Ala-Val-Pro-Thr-Gly-Val-Ala, Ile-Glu-Glu-Gly-Asn [Patent Document 24] and the like have been disclosed. Among such bioactive peptides, as described above, many proposals have been made regarding blood pressure-lowering peptides derived from food proteins as ACE inhibitors, but they have an amino acid sequence with regularity. The ACE inhibitory action of long-chain peptides and the antihypertensive effect (pharmacological effect) by oral administration are still unknown, and it has been a long time since it was discovered, but it has been reported that development as a pharmaceutical is still in progress There is no.

本発明者は、前記の課題を解決するために鋭意研究した結果、大豆蛋白質から得られた本発明に係る新規なヘクサペプチドが血圧降下作用を有することを見出し、本発明を完成するに至った。即ち、大豆蛋白質の蛋白質分解酵素（プロテアーゼ）の分解液から薬理作用を有する物質を検索し、この新規なヘクサペプチドが強いアンジオテンシン変換酵素阻害作用を有することを見出した。そして、この新規なヘクサペプチドを医薬として実用化するための研究を鋭意行い、その結果、この新規なヘクサペプチドが血圧降下作用を有し、天然物由来のアンジオテンシン変換酵素阻害剤としての有用性を見い出した。本発明は係る知見に基づくものである。本発明に係る新規なヘクサペプチドは、次式、
Ｔｈｒ−Ｔｙｒ−Ｇｌｎ−Ａｌａ−Ｐｒｏ−Ｐｈｅ
で示されるＬ体のアミノ酸配列で表される新規なヘクサペプチドであり、常温における性状は白色の粉末である。As a result of diligent research to solve the above-mentioned problems, the present inventor has found that the novel hexapeptide according to the present invention obtained from soybean protein has a blood pressure lowering action, and has completed the present invention. . That is, a substance having a pharmacological action was searched from a protease degrading enzyme (protease) of soybean protein, and the novel hexapeptide was found to have a strong angiotensin converting enzyme inhibitory action. And, as a result of intensive research for practical application of this novel hexapeptide as a medicine, this new hexapeptide has a blood pressure lowering action, and is useful as an angiotensin converting enzyme inhibitor derived from natural products. I found it. The present invention is based on such knowledge. The novel hexapeptide according to the present invention has the following formula:
Thr-Tyr-Gln-Ala-Pro-Phe
Is a novel hexapeptide represented by the amino acid sequence of L-form, and is white powder at room temperature.

本発明に係る新規なヘクサペプチドは、化学的に合成する方法、又は大豆蛋白質の蛋白質分解酵素の分解液から分離精製する方法をあげることができる。本発明に係る新規なヘクサペプチドを化学的に合成する場合には、液相法または固相法等の通常の合成方法によって行うことができるが、好ましくは、固相法によってポリマー性の固相支持体へ前記のヘクサペプチドのカルボキシル末端側（フェニルアラニン）からそのアミノ酸残基に対応したＬ体のアミノ酸を順次ペプチド結合によって結合して行くのが良い。そして、そのようにして得られた合成ヘクサペプチドは、トリフルオロメタンスルホン酸、フッ化水素などを用いてポロマー性の固相支持体から切断した後、アミノ酸側鎖の保護基を除去し、逆相系のカラムを用いた高速液体クロマトグラフィー（以下、ＨＰＬＣと略記する）などを用いた通常の方法で精製することができる。Examples of the novel hexapeptide according to the present invention include a method of chemically synthesizing or a method of separating and purifying from a protein degradation enzyme solution of soybean protein. In the case of chemically synthesizing the novel hexapeptide according to the present invention, it can be carried out by a usual synthesis method such as a liquid phase method or a solid phase method. Preferably, a polymeric solid phase is obtained by a solid phase method. The L-form amino acid corresponding to the amino acid residue from the carboxyl terminal side (phenylalanine) of the hexapeptide may be sequentially bound to the support by peptide bonds. The synthetic hexapeptide thus obtained is cleaved from the porous solid support using trifluoromethanesulfonic acid, hydrogen fluoride, etc., and then the amino acid side chain protecting group is removed and the reverse phase is removed. It can be purified by an ordinary method using high performance liquid chromatography (hereinafter abbreviated as HPLC) using a system column.

上記したように、本発明に係る新規なヘクサペプチドは、大豆蛋白質の蛋白質分解酵素の分解液から分離精製することができるが、その場合には、例えば以下のようにして行うことができる。本発明に係る新規なヘクサペプチドを含有している大豆蛋白質を用いて加水分解する。加水分解方法は常法に従って行う。例えば、ペプシン等の蛋白質分解酵素で加水分解する場合は、大豆蛋白質のホモジネイトを必要とあれば更に加水分解した後、酵素の至適温度まで加温し、ｐＨを至適値に調整し、酵素を加えてインキュベートする。次いで必要に応じ中和した後、酵素を失活させて加水分解液を得る。この加水分解物を濾紙及び／又はセライト等を用いて濾過することによって不溶性成分を除去し、その得られた濾液をセロファンなどの半透膜を用いて適当な溶媒（例えば、水、トリス−塩酸緩衝液、リン酸緩衝液の中性の緩衝液等）中で十分に透析し、その濾液中の成分で半透膜を通過した成分を含む溶液を強酸性陽イオン交換樹脂（例えば、ダウケミカル社製のＤｏｗｅｘ５０Ｗ、アンバーライト社製のＡｍｂｅｒｌｉｔｅＩＲ−１２０等）にかけ、その吸着溶出分画からアンジオテンシン変換酵素（以下、ＡＣＥと略記する）阻害活性を有する成分を含有する分画を得、その得られたＡＣＥ阻害活性画分をゲル濾過（例えば、ファルマシア社製のＳｅｐｈａｄｅｘＧ−２５、バイオラッド社製のＢｉｏ−ＧｅｌＰ−６等）によって分画し、その得られたＡＣＥ阻害活性画分を陽イオン交換ゲル濾過（例えば、ファルマシア社製のＳＰ−ＳｅｐｈａｄｅｘＣ−２５、生化学工業社製のＳＥ−Ｃｅｌｌｕｌｏｓｅ等）によって分画し、最終的に得られたＡＣＥ阻害活性画分を逆相ＨＰＬＣによって分離することによって単離精製を行うことができる。As described above, the novel hexapeptide according to the present invention can be separated and purified from the decomposition solution of soybean protein proteolytic enzyme. In that case, for example, it can be carried out as follows. Hydrolysis is performed using soy protein containing the novel hexapeptide according to the present invention. The hydrolysis method is performed according to a conventional method. For example, when hydrolyzing with a proteolytic enzyme such as pepsin, soy protein homogenate is further hydrolyzed if necessary, then heated to the optimal temperature of the enzyme, pH adjusted to the optimal value, Add and incubate. Next, after neutralization as necessary, the enzyme is deactivated to obtain a hydrolyzed solution. The hydrolyzate is filtered using filter paper and / or celite to remove insoluble components, and the obtained filtrate is filtered using a semipermeable membrane such as cellophane to form a suitable solvent (for example, water, tris-hydrochloric acid). Dialyze sufficiently in a buffer solution or neutral buffer solution of a phosphate buffer solution, and a solution containing a component that has passed through a semipermeable membrane with a component in the filtrate is a strongly acidic cation exchange resin (for example, Dow Chemical). And then a fraction containing an angiotensin converting enzyme (hereinafter abbreviated as ACE) inhibitory activity is obtained from the adsorbed elution fraction. The obtained ACE inhibitory activity fraction was subjected to gel filtration (for example, Sephadex G-25 manufactured by Pharmacia, Bio-Gel P-6 manufactured by Bio-Rad, etc.). The obtained ACE inhibitory activity fraction is fractionated by cation exchange gel filtration (for example, SP-Sephadex C-25 manufactured by Pharmacia, SE-Cellulose manufactured by Seikagaku Corporation), and the like. Isolation and purification can be carried out by separating the obtained ACE inhibitory activity fraction by reverse phase HPLC.

本発明に係る新規なヘクサペプチドの製法において用いる大豆蛋白質としては、本発明の目的を達成できる限りいかなるマメ科植物の蛋白質を用いても良いが、好ましくは大豆蛋白質を用いるのが良い。以上のようにして得られたこの新規なヘクサペプチドは、静脈内へ繰り返し投与を行った場合、抗体産生を惹起せず、アナフィラキシーショックを起こさせない。又、この新規なヘクサペプチドはＬ−アミノ酸のみの配列構造からなり、投与後、生体内のプロテアーゼにより徐々に分解される為、毒性は極めて低く、安全性は極めて高い（ＬＤ_５０＞５０００ｍｇ／ｋｇ；ラット経口投与）。この新規なヘクサペプチドは、通常用いられる賦形剤等の添加物を用いて注射剤、錠剤、カプセル剤、顆粒剤、散剤等に調整することができる。投与方法としては、通常は、ＡＣＥを有している哺乳類（例えば、ヒト、イヌ、ラット等）に注射すること、あるいは経口投与することがあげられる。投与量は、例えば、動物体重当たりこの新規なヘクサペプチドを０．０１〜１０ｍｇの量である。投与回数は、通常１日１〜４回程度であるが、投与経路によって、適宜、調整することができる。As the soy protein used in the method for producing the novel hexapeptide according to the present invention, any legume protein may be used as long as the object of the present invention can be achieved, but soy protein is preferably used. The novel hexapeptide obtained as described above does not cause antibody production and does not cause anaphylactic shock when repeatedly administered intravenously. In addition, since this novel hexapeptide has a sequence structure of only L-amino acids and is gradually degraded by protease in vivo after administration, the toxicity is extremely low and the safety is extremely high (LD ₅₀ > 5000 mg / kg). Rat oral administration). This novel hexapeptide can be prepared into injections, tablets, capsules, granules, powders and the like using commonly used additives such as excipients. The administration method usually includes injection into a mammal having ACE (for example, human, dog, rat, etc.) or oral administration. The dosage is, for example, 0.01-10 mg of this novel hexapeptide per animal body weight. The number of administration is usually about 1 to 4 times a day, but can be appropriately adjusted depending on the administration route.

上記の各種製剤において用いられる賦形剤、結合剤、潤沢剤の種類は、とくに限定されず、通常の注射剤、散剤、顆粒剤、錠剤あるいはカプセル剤に用いられるものを使用することができる。錠剤、カプセル剤、顆粒剤、散剤に用いる添加物としては、下記のものをあげることができる。賦形剤としては、結晶セルロース等の糖類、マンニトール等の糖アルコール類、デンプン類、無水リン酸カルシウム等；結合剤としてはでんぷん類、ヒドロキシプロピルメチルセルローズ等；崩壊剤としてはカルボキシメチルセルロースおよびそのカリウム塩類；潤滑剤としてはステアリン酸およびその塩類、タルク、ワックス類を挙げることができる。又、製剤の調整にあたっては必要に応じメントール、クエン酸およびその塩類、香料等の矯臭剤を用いることができる。注射用の無菌組成物は、常法により、本発明に係る新規なヘクサペプチドを、注射用水、生理食塩水およびキシリトールやマンニトールなどの糖アルコール注射液、プロピレングリコールやポリエチレングリコール等のグリコールに溶解または懸濁させて注射剤とすることができる。この際、緩衝液、防腐剤、酸化防止剤等を必要に応じて添加することができる。この新規なヘクサペプチドを含有する製剤は凍結乾燥品又は乾燥粉末の形とし、用時、通常の溶解剤、例えば水または生理食塩液に溶解して用いることもできる。The types of excipients, binders, and lubricants used in the various preparations are not particularly limited, and those used for ordinary injections, powders, granules, tablets, or capsules can be used. Examples of additives used in tablets, capsules, granules, and powders include the following. Examples of excipients include sugars such as crystalline cellulose, sugar alcohols such as mannitol, starches, anhydrous calcium phosphate and the like; binders such as starches and hydroxypropylmethylcellulose; and disintegrants such as carboxymethylcellulose and potassium salts thereof; Examples of the lubricant include stearic acid and its salts, talc, and waxes. In preparation of the preparation, flavoring agents such as menthol, citric acid and salts thereof, and fragrance can be used as necessary. The sterile composition for injection is prepared by dissolving the novel hexapeptide according to the present invention in water for injection, physiological saline and a sugar alcohol injection solution such as xylitol or mannitol, or a glycol such as propylene glycol or polyethylene glycol according to a conventional method. It can be suspended as an injection. At this time, a buffer solution, a preservative, an antioxidant and the like can be added as necessary. The preparation containing the novel hexapeptide is in the form of a lyophilized product or a dry powder, and can be used by dissolving it in a normal solubilizing agent such as water or physiological saline at the time of use.

本発明に係る新規なヘクサペプチドは、優れたアンジオテンシン変換酵素阻害作用を有し、血圧降下作用、ブラジキニン不活化抑制作用を示す。従って、本態性高血圧、腎性高血圧、副腎性高血圧等の高血圧症の予防、治療剤、これらうっ血性心不全に対する臓器循環の正常化と長期予後の改善（延命効果）作用を有し、心不全の治療剤として有用である。The novel hexapeptide according to the present invention has an excellent angiotensin converting enzyme inhibitory action, and exhibits a blood pressure lowering action and a bradykinin inactivation inhibiting action. Therefore, prevention of hypertension such as essential hypertension, renal hypertension, adrenal hypertension, therapeutic agent, normalization of organ circulation and improvement of long-term prognosis (life extension effect) for these congestive heart failure, treatment of heart failure Useful as an agent.

BEST MODE FOR CARRYING OUT THE INVENTION

本発明は、医薬品としての有用性を有する下記のアミノ酸の配列のペプチド構造を有するヘクサペプチド及びこのヘクサペプチドを有効成分とするアンジオテンシン変換酵素阻害剤に関する。
Ｔｈｒ−Ｔｙｒ−Ｇｌｎ−Ａｌａ−Ｐｒｏ−Ｐｈｅ
（式中、アミノ酸残基を表す各記号は、アミノ酸化学において慣用の表示法によるものである）
以下に実施例として、製造例及び試験例を記載し、本発明を更に詳細に説明するが、本発明はこれら実施例に限定されるものではない。The present invention relates to a hexapeptide having a peptide structure having the following amino acid sequence having utility as a pharmaceutical product and an angiotensin converting enzyme inhibitor containing the hexapeptide as an active ingredient.
Thr-Tyr-Gln-Ala-Pro-Phe
(In the formula, each symbol representing an amino acid residue is based on a conventional display method in amino acid chemistry)
EXAMPLES Examples and test examples will be described below as examples, and the present invention will be described in more detail. However, the present invention is not limited to these examples.

製造例１
大豆２００ｇに脱イオン水１を加えホミジナイズした後、１規定の塩酸にてｐＨを２．０に調整し、ペプシン（メルク社製、酵素番号ＥＣ３．４．２３．１）１０ｇを添加し、３７℃、２０時間撹拌しながら加水分解を行った。反応後、分解液を直ちに限外濾過膜（アミコン社製、ＹＭ１０型、φ７６ｍ）に通過させ、通過液をＤｏｗｅｘ５０Ｗ×４［Ｈ^＋］カラム（φ４．５×１５ｃｍ）に負荷した。そのカラムを脱イオン水で十分洗滌した後、２規定のアンモニウム水２Ｌを用いて溶出した。減圧濃縮操作によりアンモニアを除去して濃縮液４０ｍＬを得た。この濃縮液４ｍＬを予め脱イオン水で緩衝化したＳｅｐｈａｄｅｘＧ−２５（φ２．５×１５０ｃｍ）に負荷し、流速３０ｍＬ／ｈｒ、各分画量８．６ｍＬでゲル濾過操作を行った。ゲル濾過操作を繰り返して大量分取したＡＣＥ阻害活性の高いペプチド画分を集めて凍結乾燥し大豆蛋白質由来の粗ペプチド粉末とした。この大豆蛋白質由来の粗ペプチド粉末３ｇを脱イオン水２０ｍＬに溶解後、予め脱イオン水で緩衝化したＳＰ−ＳｅｐｈａｄｅｘＣ−２５［Ｈ^＋］カラム（φ１．５×４７．２ｃｍ）に負荷し、脱イオン水１Ｌから３％塩化ナトリウム水１Ｌの濃度勾配法を行い、流速１０ｍＬ／ｈｒ、各分画量１０ｍＬでカラムクロマトグラフィー操作を行った。その結果は図１に示すとおりである。ＳＰ−ＳｅｐｈａｄｅｘＣ−２５クロマトグラフ中のＡＣＥ阻害活性画分、分画番号３２〜３８（ＳＰ−１画分）を集めて凍結乾燥し大豆由来の精製ペプチド粉末（以下、大豆ペプチドと略記する）を得た。この大豆ペプチド粉末２０ｍｇを６０μＬの脱イオン水に溶解した後、逆相ＨＰＬＣ操作を行った。カラムとしてはＤｅｖｅｌｏｓｉｌＯＤＳ−５（野村化学社製、４．５ｍｍＩＤ×２５ｍＬ）を使用し、移動相としては０．０５％トリフルオロ酢酸（以下、ＴＦＡと略記する）から２５％アセトニトリル／０．０５％ＴＦＡの濃度勾配法を行い、流速１．０ｍＬ／ｍｉｎ検出波長２２０ｎｍでＨＰＬＣ操作を行った結果、溶出時間；６２．４分にＡＣＥ阻害活性の高いペプチドフラグメントのピークを得た。この逆相ＨＰＬＣ分析の結果は図２に示す通りである。再度、逆相ＨＰＬＣリクロマトグラフィーして得たペプチドフラグメントのアミン酸分析を行ったところ、Ｔｈｒ；１．０２、Ｔｙｒ；１．１１、Ｇｌｎ；１．０９、Ａｌａ；１．１０、Ｐｒｏ；０．９３及びＰｈｅ；１．０６であった。更に、逆相ＨＰＬＣリクロマトグラフィーして得たペプチドフラグメントのＡＣＥ阻害活性を測定したところ、ＩＣ_５０値；１．３４μＭであった。このようにして得られたＡＣＥ阻害作用を有するペプチドフラグメントのアミノ酸配列は、アプライドバイオシステム（ＡＢＩ）社製のプロテインシークエンサー４７７Ａ型を用いて決定された。その結果、本発明に係る新規なペプチドは、次式、
Ｔｈｒ−Ｔｙｒ−Ｇｌｎ−Ａｌａ−Ｐｒｏ−Ｐｈｅ
で示されるＬ体のアミノ酸配列で表される新規なヘクサペプチドであることが確認された。常温における性状は白色の粉末である。尚、本発明に係る新規なヘクサペプチドをＡＣＥ阻害剤として、例えば錠剤に製剤する場合には、常法に従って、例えば次のように処理すればよい：▲１▼ペプチド１５ｇ、▲２▼乳糖８５ｇ、▲３▼コーンスターチ３０ｇ、▲４▼ステアリン酸マグネシウム１．２ｇを原料とし、先ず▲１▼、▲２▼及び１６ｇのコーンスターチを混和し、８．５ｇのコーンスターチから作ったペーストとともに顆粒化し、この顆粒に５．５ｇのコーンスターチと▲４▼とを加え、得られた混合物を圧縮錠剤機で打錠し、錠剤１０００個を製造する。Production Example 1
After demineralized water 1 was added to 200 g of soybean and homogenized, the pH was adjusted to 2.0 with 1N hydrochloric acid, 10 g of pepsin (Merck, enzyme number EC 3.4.23.1) was added, and 37 Hydrolysis was performed with stirring at 20 ° C. for 20 hours. After the reaction, the decomposition solution was immediately passed through an ultrafiltration membrane (Amicon, YM10 type, φ76m), and the passing solution was loaded onto a Dowex 50W × 4 [H ⁺ ] column (φ4.5 × 15 cm). The column was thoroughly washed with deionized water and then eluted with 2 L of 2N ammonium water. Ammonia was removed by a vacuum concentration operation to obtain 40 mL of a concentrated solution. 4 mL of this concentrated solution was loaded on Sephadex G-25 (φ2.5 × 150 cm) previously buffered with deionized water, and gel filtration was performed at a flow rate of 30 mL / hr and each fraction amount of 8.6 mL. Peptide fractions with high ACE inhibitory activity collected by repeated gel filtration were collected and lyophilized to obtain crude peptide powder derived from soybean protein. After dissolving 3 g of this crude protein powder derived from soy protein in 20 mL of deionized water, it was loaded onto an SP-Sephadex C-25 [H ⁺ ] column (φ1.5 × 47.2 cm) previously buffered with deionized water. A concentration gradient method from 1 L of ionic water to 1 L of 3% sodium chloride water was performed, and column chromatography was performed at a flow rate of 10 mL / hr and a fraction amount of 10 mL. The result is as shown in FIG. The ACE inhibitory activity fraction in the SP-Sephadex C-25 chromatograph, fraction numbers 32-38 (SP-1 fraction) were collected, freeze-dried, and purified peptide powder derived from soybean (hereinafter abbreviated as soybean peptide). Obtained. 20 mg of this soy peptide powder was dissolved in 60 μL of deionized water, and then reverse phase HPLC operation was performed. Develosil ODS-5 (manufactured by Nomura Chemical Co., Ltd., 4.5 mm ID × 25 mL) was used as the column, and 0.05% trifluoroacetic acid (hereinafter abbreviated as TFA) to 25% acetonitrile / 0.05 as the mobile phase. As a result of performing a concentration gradient method of% TFA and performing HPLC operation at a flow rate of 1.0 mL / min and a detection wavelength of 220 nm, a peptide fragment peak having a high ACE inhibitory activity was obtained at an elution time of 62.4 minutes. The result of this reverse phase HPLC analysis is as shown in FIG. When the amino acid analysis of the peptide fragment obtained by reverse phase HPLC rechromatography was performed again, Thr; 1.02, Tyr; 1.11, Gln; 1.09, Ala; 1.10, Pro; 0 .93 and Phe; 1.06. Furthermore, when the ACE inhibitory activity of the peptide fragment obtained by reverse phase HPLC rechromatography was measured, the IC ₅₀ value was 1.34 μM. The amino acid sequence of the thus obtained peptide fragment having an ACE inhibitory action was determined using a protein sequencer type 477A manufactured by Applied Biosystems (ABI). As a result, the novel peptide according to the present invention has the following formula:
Thr-Tyr-Gln-Ala-Pro-Phe
It was confirmed that it is a novel hexapeptide represented by the amino acid sequence of L-form represented by The property at room temperature is a white powder. In addition, when the novel hexapeptide according to the present invention is formulated as an ACE inhibitor, for example, into a tablet, it may be treated according to a conventional method, for example, as follows: (1) peptide 15 g, (2) lactose 85 g (3) 30 g of corn starch, (4) 1.2 g of magnesium stearate, and firstly mix (1), (2) and 16 g of corn starch, granulate with paste made from 8.5 g of corn starch, 5.5 g of corn starch and (4) are added to the granules, and the resulting mixture is compressed with a compression tablet machine to produce 1000 tablets.

製造例２
本例は、合成法による製造例である。
Ｔｈｒ−Ｔｙｒ−Ｇｌｎ−Ａｌａ−Ｐｒｏ−Ｐｈｅの合成法
アプライドバイオシステム社製のペプチド自動合成装置４３０Ａ型を用いた固相法によって当該ペプチドを合成した。固相担体としては、スチレンジビニルベンゼン共重合体（ポリスチレン樹脂）をクロロメチル化した樹脂を使用した。まず、当該ヘクサペプチドのアミノ酸配列に従って、常法どおり、そのＣ末端側のフェニルアラニンからクロロメチル樹脂に反応させペプチド結合樹脂を得た。この時のアミノ酸は、ｔ−ブトキシカルボニル（以下、ｔ−Ｂｏｃと略記する）基で保護されたｔ−Ｂｏｃアミノ酸を使用した。次に、このペプチド結合樹脂をエタンジチオールとチオアニソールからなる混合液に懸濁し、室温で１０分間撹拌後、氷冷下でトリフルオロ酢酸を加え、更に１０分間撹拌した。この混合液にトリフルオロメタンスルホン酸を滴下し、室温で３０分間撹拌した後、無水エーテルを加えてその生成物を沈澱させて分離し、その沈澱物を無水エーテルで数回洗浄した後、減圧下で乾燥した。このようにして得られた未精製の合成ペプチドは蒸留水に溶解した後、逆相系のカラムＣ_１８（５μｍ）を用いたＨＰＬＣにより精製した。移動相として（Ａ）０．１％ＴＦＡ含有蒸留水、（Ｂ）０．１％ＴＦＡ含有アセトニトリル溶液を使用し、（Ａ）液が９４分間で６３％→１４％の濃度勾配法により流速１．２ｍＬ／ｍｉｎでクロマトグラフィーを行った。紫外部波長２１７ｎｍで検出し、最大の吸収を示した溶出画分を分取し、これを凍結乾燥することによって目的とする合成ヘクサペプチドを得た。Production Example 2
This example is an example of production by a synthesis method.
Synthesis Method of Thr-Tyr-Gln-Ala-Pro-Phe The peptide was synthesized by a solid phase method using an automatic peptide synthesizer type 430A manufactured by Applied Biosystems. As the solid support, a resin obtained by chloromethylating a styrene divinylbenzene copolymer (polystyrene resin) was used. First, according to the amino acid sequence of the hexapeptide, the C-terminal phenylalanine was reacted with a chloromethyl resin in the usual manner to obtain a peptide-bonded resin. As the amino acid at this time, a t-Boc amino acid protected with a t-butoxycarbonyl (hereinafter abbreviated as t-Boc) group was used. Next, this peptide-bonded resin was suspended in a mixed solution composed of ethanedithiol and thioanisole, stirred at room temperature for 10 minutes, added with trifluoroacetic acid under ice cooling, and further stirred for 10 minutes. Trifluoromethanesulfonic acid was added dropwise to the mixture, and the mixture was stirred at room temperature for 30 minutes. Then, anhydrous ether was added to precipitate the product, and the precipitate was washed several times with anhydrous ether. And dried. The crude synthetic peptide thus obtained was dissolved in distilled water and then purified by HPLC using a reverse phase system column C ₁₈ (5 μm). (A) 0.1% TFA-containing distilled water and (B) 0.1% TFA-containing acetonitrile solution were used as the mobile phase, and (A) the liquid flow rate was 1% by a concentration gradient method of 63% → 14% in 94 minutes. Chromatography was performed at 2 mL / min. The eluate fraction that was detected at the ultraviolet wavelength of 217 nm and showed the maximum absorption was collected and lyophilized to obtain the desired synthetic hexapeptide.

この合成ヘクサペプチドをマススペクトル分析により分析した結果、アミノ酸配列が前記で示したアミノ酸配列構造を有するヘクサペプチドであることが確認された。このマススペクトル分析の結果は図３に示す通りである。更に、この合成ヘクサペプチドのＡＣＥ阻害活性を測定したところ、ＩＣ_５０値；１．０４μＭであった。
このような合成によって得られた本発明に係る新規のヘクサペプチドは、以下に示す試験によって薬理効果が確認された。As a result of analyzing this synthetic hexapeptide by mass spectrum analysis, it was confirmed that the amino acid sequence was a hexapeptide having the amino acid sequence structure shown above. The result of this mass spectrum analysis is as shown in FIG. Furthermore, when the ACE inhibitory activity of this synthetic hexapeptide was measured, the IC ₅₀ value was 1.04 μM.
The pharmacological effect of the novel hexapeptide according to the present invention obtained by such synthesis was confirmed by the following tests.

試験例１（アンジオテンシン変換酵素阻害活性測定法）
ＡＣＥ（シグマ社製、酵素番号ＥＣ３．４．１５．１）２．５ｍＵ、合成基質ヒプリル−Ｌ−ヒスチジル−Ｌ−ロイシン（ペプチド研究所製）１２．５ｍＭを用いＬｉｅｂｅｒｍａｎの測定法を改良した山本等の方法［非特許文献４］に準じて測定した。すなわち、生成した馬尿酸を酢酸エチルにて抽出し２２５ｎｍの吸光度で測定した。被検液での吸光度をＥｓ、被検液の代わりに緩衝液を加えた時の値をＥｃ、予め反応停止液を加えて反応させた時の値をＥｂとして次式から阻害率を求めた。
阻害率（％）＝（Ｅｃ−Ｅｓ）／（Ｅｃ−Ｅｂ）×１００
ＡＣＥ阻害剤の阻害活性ＩＣ_５０値は、ＡＣＥの酵素活性を５０％（阻害率）阻害するために必要な試料の濃度（Ｍ）で示した。Test Example 1 (Angiotensin converting enzyme inhibitory activity measurement method)
Yamamoto improved the measurement method of Lieberman using ACE (manufactured by Sigma, enzyme number EC 3.4.15.1) 2.5 mU, synthetic substrate hipryl-L-histidyl-L-leucine (manufactured by Peptide Institute) 12.5 mM. Measured according to the above method [Non-patent Document 4]. That is, the produced hippuric acid was extracted with ethyl acetate and measured by absorbance at 225 nm. The inhibition rate was calculated from the following equation, where Es is the absorbance in the test solution, Ec is the value when the buffer solution is added instead of the test solution, and Eb is the value when the reaction stop solution is added and reacted in advance. .
Inhibition rate (%) = (Ec−Es) / (Ec−Eb) × 100
The inhibitory activity IC ₅₀ value of the ACE inhibitor was expressed as the concentration (M) of the sample required to inhibit the enzyme activity of ACE by 50% (inhibition rate).

試験例２（高血圧自然発症ラットへ投与時の降圧効果）
実験動物は日本チャールズ・リバー社より１５週令雄性高血圧自然発症ラット（以下、ＳＨＲと略記する）を購入し、１週間の予備飼育後、血圧値（以下、収縮期血圧値；ＳＢＰを表す）が１６０ｍｍＨｇ以上（体重２８０−３３０ｇ）の動物６匹１群として用いた。ラットは、室温２３±２℃、湿度５５±１０％および１２時間明暗（午前６時〜午後６時点灯）に調整された飼育室でステンレスワイヤー製ラット用個別ゲージに１匹ずつ収容し飼育した。飼料はオリエンタル酵母社製ＭＦ粉末飼料を、飲水は自家揚水（水道水質基準適合）をそれぞれ自由に摂取させた。ラットは４群に分け、第１群には対照として蒸留水を体重１００ｇ当たり０．５ｍＬの割合で強制経口投与した。第２群には、大豆ペプチド粉末（ＳＰ−１画分）３００ｍｇ／ｋｇの用量を蒸留水で調製し、体重１００ｇ当たり０．５ｍＬの割合で強制経口投与し、第３群には、大豆ペプチド粉末（ＳＰ−１画分）６００ｍｇ／ｋｇの用量を蒸留水で調製し、体重１００ｇ当たり０．５ｍＬの割合で強制経口投与し、及び、第４群には、大豆ペプチド粉末（ＳＰ−１画分）１，２００ｍｇ／ｋｇの用量を蒸留水で調製し、体重１００ｇ当たり０．５ｍＬの割合で強制経口投与した。血圧値は非観血的尾動脈血圧測定装置（理研開発社製、ＰＳ−１００型）を用いｔａｉｌ−ｃｕｆｆ法により、投与前、投与後３０分、１時間、２時間、４時間及び６時間の血圧値を測定した。血圧値は連続３回測定し、その最高値と最低値の差が１０ｍｍＨｇ以内の場合、その３回の平均血圧値を求め、差が１１ｍｍＨｇ以上の場合には更に２回測定し、最高値及び最低値を除き３回の平均血圧値を求めた。その結果を図４に示した。更に、本発明に係る新規なヘクサペプチド２０ｍｇ／ｋｇをＳＨＲに経口投与した時の収縮期血圧値（ｍｍＨｇ）への作用についての結果は、表１に示す通りである。

以上の試験の結果、本発明に係る新規なヘクサペプチドは、ｉｎｖｉｔｒｏ（試験管内）においてアンジオテンシン変換酵素阻害活性を有し、ｉｎｖｉｖｏ（生体内）においても有意な血圧降下作用を示すことが確認された。従って、この新規なヘクサペプチドは高血圧症の治療又は予防薬として有用である。尚、この新規なヘクサペプチドは、構造的にそのアミノ酸配列を部分構造とするペプチドにおいて、構造中に採用することもできる。Test Example 2 (Antihypertensive effect when administered to spontaneously hypertensive rats)
Laboratory animals purchased 15-week-old male spontaneously hypertensive rats (hereinafter abbreviated as SHR) from Charles River, Japan, and blood pressure values (hereinafter referred to as systolic blood pressure values; SBP) after one week of preliminary breeding. Was used as a group of 6 animals of 160 mmHg or more (weight 280-330 g). Rats were housed individually in stainless steel rat individual gauges in a breeding room adjusted to room temperature 23 ± 2 ° C., humidity 55 ± 10%, and 12 hours light / dark (lights on from 6 am to 6 pm). . The feed was MF powder feed manufactured by Oriental Yeast Co., Ltd., and the drinking water was ingested by private pumping (conforming to tap water quality standards). The rats were divided into 4 groups, and distilled water was administered by gavage to the first group at a rate of 0.5 mL per 100 g body weight as a control. For the second group, a dose of 300 mg / kg of soy peptide powder (SP-1 fraction) was prepared with distilled water and administered by oral gavage at a rate of 0.5 mL per 100 g of body weight. A dose of 600 mg / kg of powder (SP-1 fraction) was prepared with distilled water and administered by oral gavage at a rate of 0.5 mL per 100 g of body weight, and the fourth group contained soybean peptide powder (SP-1 fraction) Min) A dose of 1,200 mg / kg was prepared in distilled water and administered orally by gavage at a rate of 0.5 mL per 100 g body weight. Blood pressure was measured by tail-cuff method using a non-invasive tail artery blood pressure measuring device (manufactured by Riken Development Co., Ltd., model PS-100) before administration, 30 minutes after administration, 1 hour, 2 hours, 4 hours and 6 hours. The blood pressure value was measured. The blood pressure value is measured three times in succession. If the difference between the maximum value and the minimum value is within 10 mmHg, the average blood pressure value for the three times is obtained. If the difference is 11 mmHg or more, the measurement is repeated twice. Three average blood pressure values were determined excluding the lowest value. The results are shown in FIG. Furthermore, the results on the effect on systolic blood pressure (mmHg) when orally administered 20 mg / kg of the novel hexapeptide according to the present invention to SHR are as shown in Table 1.

As a result of the above tests, it was confirmed that the novel hexapeptide according to the present invention has an angiotensin converting enzyme inhibitory activity in vitro (in vitro) and also exhibits a significant blood pressure lowering effect in vivo (in vivo). It was done. Therefore, this novel hexapeptide is useful as a therapeutic or prophylactic agent for hypertension. In addition, this novel hexapeptide can also be employed in the structure of a peptide structurally having its amino acid sequence as a partial structure.

本発明に係る新規なヘクサペプチドの、製造例１におけるＳＰ−ＳｅｐｈａｄｅｘＣ−２５［Ｈ^＋］カラムクロマトグラフィー操作によるＡＣＥ阻害ペプチドの分離精製の結果を示す図である。It is a figure which shows the result of the isolation | separation purification of the ACE inhibitory peptide by SP-SephadexC-25 [H ^<+ >] column chromatography operation in manufacture example 1 of the novel hexapeptide which concerns on this invention. 本発明に係る新規なヘクサペプチドの、製造例１における逆相ＨＰＬＣ操作によるＡＣＥ阻害ペプチドの分離精製の結果を示す図である。It is a figure which shows the result of isolation | separation purification of the ACE inhibitory peptide by the reverse phase HPLC operation in manufacture example 1 of the novel hexapeptide which concerns on this invention. 本発明に係る新規なヘクサペプチドの、製造例２で得られた合成ヘクサペプチドのマススペクトルを示す図である。It is a figure which shows the mass spectrum of the synthetic hexapeptide obtained by manufacture example 2 of the novel hexapeptide which concerns on this invention. 本発明に係る新規なヘクサペプチドの、製造例１におけるＳＰ−ＳｅｐｈａｄｅｘＣ−２５［Ｈ^＋］カラムクロマトグラフィー操作によって得られた大豆ペプチド粉末（ＳＰ−１画分）を、ＳＨＲに投与した場合の血圧値の経時的変化を示す図である。Blood pressure when the soy peptide powder (SP-1 fraction) obtained by the SP-Sephadex C-25 [H ⁺ ] column chromatography operation of the novel hexapeptide according to the present invention in Production Example 1 is administered to SHR It is a figure which shows a time-dependent change of a value.

Claims

The following formula: Thr-Tyr-Gln-Ala-Pro-Phe
A novel hexapeptide having a peptide structure according to the L-amino acid sequence represented by

The following formula: Thr-Tyr-Gln-Ala-Pro-Phe
An angiotensin converting enzyme inhibitor comprising a novel hexapeptide having a peptide structure based on the L-amino acid sequence represented by