JP2005154326A - Angiotensin-i converting enzyme inhibitory peptide compound or peptide composition containing the same and method for producing them - Google Patents
Angiotensin-i converting enzyme inhibitory peptide compound or peptide composition containing the same and method for producing them Download PDFInfo
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本発明は、血圧の上昇を調節できるとされるアンジオテンシンＩ変換酵素阻害活性を有するペプチド化合物及びそれを含有する組成物に関するもので、高血圧症を予防したり治療するための医薬品や医薬部外品や食品添加物や機能性食品等に広く利用できるものである。 TECHNICAL FIELD The present invention relates to a peptide compound having an angiotensin I converting enzyme inhibitory activity, which is said to be capable of regulating an increase in blood pressure, and a composition containing the same, and a pharmaceutical or quasi drug for preventing or treating hypertension It can be widely used for food additives and functional foods.
突然死を引き起こす可能性のある心筋梗塞などの冠動脈疾患の危険因子は、高血圧症、高脂血症、耐糖能低下、肥満の４つであり、死の四重奏とも言われている。当該危険因子の内の１つである高血圧症は、日本高血圧学会によれば我が国では３３００万人の患者がいるとされている。高血圧症は治療を受けずに放置しておくと、重症でない限り多くの場合無症状で進行し死に至る事が少なくない事から、サイレントキラ−と呼ばれている。このような高血圧症を改善する要請が強いため、様々な降圧剤や血圧を調節する機能性食品の開発が進められている。 There are four risk factors for coronary artery disease such as myocardial infarction that can cause sudden death: hypertension, hyperlipidemia, impaired glucose tolerance, and obesity, and is also said to be a quartet of death. Hypertension, which is one of the risk factors, is reported to have 33 million patients in Japan according to the Japanese Society of Hypertension. If hypertension is left untreated, it is often called as a silent killer because it often progresses asymptomatic and results in death unless it is severe. Since there is a strong demand for improving such hypertension, various antihypertensive agents and functional foods that regulate blood pressure are being developed.
生体において血圧を調節するメカニズムの１つには、昇圧系であるレニン−アンジオテンシン系と、降圧系であるカリクレイン−キニン系がある。前者のレニン−
アンジオテンシン系では、酵素レニンが腎臓の旁糸球体細胞から循環血液中に分泌され、肝臓で生合成され血液中に存在する基質アンジオテンシノ−ゲンに働いてアンジオテンシンＩ（Asp−Arg−Val−Tyr−Ile−His−Pro−Phe−His−Leu）を生成する。このアンジオテンシンＩをアンジオテンシンＩＩ（Asp−Arg−Val−Tyr−Ile−His−Pro−Phe）に変換する酵素は、主として血管内皮細胞や肺、腎臓近位尿細管に存在するアンジオテンシンＩ変換酵素である。このようにして生じたアンジオテンシンＩＩは、血管平滑筋を収縮させる作用がある。また、当該アンジオテンシンＩＩは副腎皮質に作用してアルドステロンの生成と分泌を促進すると共に、腎臓近位尿細管に働いて腎糸球体で濾過されたナトリウムの再吸収を高める作用がある。その結果、血圧は上昇する。
One mechanism for regulating blood pressure in a living body is a renin-angiotensin system that is a pressor system and a kallikrein-kinin system that is a hypotensive system. The former renin
In the angiotensin system, the enzyme renin is secreted into the circulating blood from the glomerular cells of the kidney and works on the substrate angiotensinogen that is biosynthesized in the liver and present in the blood, thereby causing angiotensin I (Asp-Arg-Val-Tyr). -Ile-His-Pro-Phe-His-Leu). The enzyme that converts this angiotensin I into angiotensin II (Asp-Arg-Val-Tyr-Ile-His-Pro-Phe) is an angiotensin I-converting enzyme that exists mainly in vascular endothelial cells, lungs, and renal proximal tubules. . The angiotensin II produced in this way has the effect of contracting vascular smooth muscle. In addition, the angiotensin II acts on the adrenal cortex to promote the production and secretion of aldosterone, and acts on the renal proximal tubule to increase the reabsorption of sodium filtered through the glomeruli. As a result, blood pressure increases.
一方、後者のカリクレイン−キニン系では、酵素カリクレインが基質キニノ−ゲンに作用してキニンを生じる。そのキニンは血管平滑筋を拡張させて血圧を下げる働きがあるが、アンジオテンシンＩ変換酵素は当該キニンを分解する事が知られている。この様にアンジオテンシンＩ変換酵素は、昇圧系であるレニン−アンジオテンシン系の活性化と、降圧系であるカリクレイン−キニン系の不活性化を同時に行う作用を有しており、結果として血圧を上昇させる作用がある。従って、アンジオテンシンＩ変換酵素の活性を阻害する物質は、血圧の上昇を調節する事が期待できるので、各方面でこれに着目した医薬品及び機能性食品の開発が行われている。 On the other hand, in the latter kallikrein-kinin system, the enzyme kallikrein acts on the substrate kininogen to produce kinin. The kinin works to dilate vascular smooth muscles and lower blood pressure, but angiotensin I converting enzyme is known to degrade the kinin. Thus, the angiotensin I converting enzyme has the action of simultaneously activating the renin-angiotensin system, which is a pressor system, and inactivating the kallikrein-kinin system, which is a hypotensive system, and as a result, increases blood pressure. There is an effect. Therefore, since substances that inhibit the activity of angiotensin I converting enzyme can be expected to regulate an increase in blood pressure, pharmaceuticals and functional foods focusing on this are being developed in various directions.
アンジオテンシンＩ変換酵素阻害物質としては、1977年にOndettiらが発表したカプトプリル（D−3−メルカプト−2−メチルプロパノイル−L−プロリン）に代表される合成化合物が医薬品として実用化されている。又その他、近年、種々の食品中から多数のアンジオテンシンＩ変換酵素阻害ペプチドが見出され、これらの内、牛乳カゼイン、発酵乳、魚肉由来のペプチドを添加した食品が特定保健用食品として実用化されている（特許文献）。
上述のようにアンジオテンシン変換酵素阻害物質は既に多数報告されているが、医薬品にあっては合成法で作られているため高価である。又、合成法で作られている当該アンジオテンシン変換酵素阻害物質は、強力な降圧作用を有するものの用量が不適切であると腎機能障害や低血圧をもたらす事から、医師の管理下において慎重に使用する事が求められている。 As described above, many angiotensin converting enzyme inhibitors have already been reported, but pharmaceuticals are expensive because they are made by a synthetic method. In addition, the angiotensin converting enzyme inhibitor produced by the synthetic method has a strong antihypertensive effect, but it causes renal dysfunction and hypotension if the dose is inappropriate. It is requested to do.
一方、食品由来のアンジオテンシンＩ変換酵素阻害ペプチドは数多く知られているが、これらの中で実用化されているものは、上述の如くごく僅かである。その理由としては、経口摂取時の作用効果が弱かったり、味、臭い、色等にそれぞれ特徴があって、実用に適していない事が多い等が挙げられる。 On the other hand, many food-derived angiotensin I converting enzyme inhibitory peptides are known, but only a few are practically used as described above. The reason for this is that the action and effects at the time of oral ingestion are weak, and the taste, smell, color, etc. are characteristic and are not suitable for practical use.
本発明者らは、血圧の上昇を調節できる食品由来のアンジオテンシンＩ変換酵素阻害物質ペプチドを見つけ出すべく鋭意研究を進めたところ、サメ軟骨を蛋白質分解酵素で処理した蛋白質分解酵素処理物がアンジオテンシンＩ変換酵素を強く阻害することに気付き、当該蛋白質分解酵素処理物中に７種類の強力なアンジオテンシンＩ変換酵素阻害ペプチドを見出した。 The present inventors have conducted extensive research to find a food-derived angiotensin I-converting enzyme inhibitor peptide capable of regulating an increase in blood pressure. As a result, a proteolytic enzyme-treated product obtained by treating shark cartilage with a proteolytic enzyme is converted into angiotensin I. It was noticed that the enzyme was strongly inhibited, and seven kinds of strong angiotensin I converting enzyme inhibitory peptides were found in the proteolytic enzyme-treated product.
それは、下記（１）乃至（７）のアミノ酸配列で示されるアンジオテンシンＩ変換酵素阻害活性を有するペプチド化合物であった。
Tyr−Phe（１）、
Phe−Tyr（２）、
Phe−Ala（３）、
Leu−Val−Gly（４）、
Leu−Ile−Gly（５）、
Leu−Gly−Val（６）、
Ile−Val−Gly（７）。
It was a peptide compound having angiotensin I converting enzyme inhibitory activity represented by the following amino acid sequences (1) to (7).
Tyr-Phe (1),
Phe-Tyr (2),
Phe-Ala (3),
Leu-Val-Gly (4),
Leu-Ile-Gly (5),
Leu-Gly-Val (6),
Ile-Val-Gly (7).
上記７種類のアミノ酸配列で示されるペプチド化合物のうち、特に（５）のLeu−Ile−Glyは、文献未記載の新規なペプチド化合物である。その他の６種類のペプチド化合物は、蛋白質分解酵素処理法やペプチド合成や遺伝子工学手法等により得られることが報告されているが、Phe−Tyrを除いて、それらにアンジオテンシンＩ変換酵素阻害活性があるとの報告はされていない。 Among the peptide compounds represented by the above seven types of amino acid sequences, Leu-Ile-Gly (5) is a novel peptide compound not described in any literature. The other six types of peptide compounds have been reported to be obtained by proteolytic enzyme treatment, peptide synthesis, genetic engineering techniques, etc., except for Phe-Tyr, which have angiotensin I converting enzyme inhibitory activity. No report has been made.
そこで本発明者らは、これらサメ軟骨由来の７種類のアミノ酸配列で示されるペプチド化合物について、そのアンジオテンシンＩ変換酵素阻害活性を測定したところ、それぞれに血管内への吸収性、安定性、安全性が高い強力なアンジオテンシンＩ変換酵素阻害活性を有することを新たに見出した。そこで、発明者らは、このサメ軟骨由来のアンジオテンシンＩ変換酵素阻害ペプチド化合物を利用して、高血圧症を改善したり治療するための医薬品（医薬原料を含む）や医薬部外品や食品添加物や機能性食品等を開発し提供せんとするものである。 Therefore, the present inventors measured the angiotensin I converting enzyme inhibitory activity of these seven shark cartilage-derived peptide compounds represented by amino acid sequences, and found that they were absorbed into blood vessels, stable, and safe. Was newly found to have a high and potent angiotensin I converting enzyme inhibitory activity. Therefore, the inventors have used this shark cartilage-derived angiotensin I converting enzyme inhibitory peptide compound to improve or treat hypertension, including pharmaceuticals (including pharmaceutical raw materials), quasi drugs, and food additives. And develop and provide functional foods.
本発明は、上記課題を解決する為に、サメ軟骨の蛋白質分解酵素処理物中に存在する７種の強力なアンジオテンシンＩ変換酵素
阻害ペプチドを見出し、これを利用してアンジオテンシンＩ変換酵素阻害ペプチド化合物又はそれを含有するペプチド組成物とそれらの製造方法の発明を完成するに至ったものである。
In order to solve the above-mentioned problems, the present invention has found seven kinds of strong angiotensin I converting enzyme inhibitory peptides present in a processed product of shark cartilage proteolytic enzyme, and an angiotensin I converting enzyme inhibitory peptide compound using the same. Or it came to complete invention of the peptide composition containing it, and those manufacturing methods.
本発明は、Tyr−Phe（１）、Phe−Tyr（２）、Phe−Ala（３）、Leu−Val−Gly（４）、Leu−Ile−Gly（５）、Leu−Gly−Val（６）、Ile−Val−Gly（７）のアミノ酸配列で示されるアンジオテンシンＩ変換酵素阻害活性を有するペプチド化合物、及びその薬学的に許容される塩、当該化合物の少なくとも１つを有効成分とする事を特徴とするアンジオテンシンＩ変換酵素阻害剤、少なくとも一つを含有するアンジオテンシンＩ変換酵素阻害活性を有するペプチド化合物を含む組成物、更には当該化合物の少なくとも１つを含有する事を特徴とする食品、医薬品、又は医薬部外品を具現化したものである。 The present invention relates to Tyr-Phe (1), Phe-Tyr (2), Phe-Ala (3), Leu-Val-Gly (4), Leu-Ile-Gly (5), Leu-Gly-Val (6 ), A peptide compound having an angiotensin I converting enzyme inhibitory activity represented by the amino acid sequence of Ile-Val-Gly (7), and a pharmaceutically acceptable salt thereof, comprising at least one of the compounds as an active ingredient Angiotensin I converting enzyme inhibitor characterized by comprising, a composition containing a peptide compound having an angiotensin I converting enzyme inhibitory activity containing at least one, and further a food or pharmaceutical comprising at least one of said compounds Or an quasi-drug.
尚、本明細書中で、Tyrはタイロシン、Pheはフェニルアラニン、Alaはアラニン、Leuはロイシン、Valはバリン、Glyはグリシン、Ileはイソロイシンを意味し、その他のアミノ酸残基を表す各記号もアミノ酸化学における慣用の表示法に基づくものである。又、これらアミノ酸は特に表記しない限りは何れもＬ体である。 In this specification, Tyr is tylosin, Phe is phenylalanine, Ala is alanine, Leu is leucine, Val is valine, Gly is glycine, Ile is isoleucine, and each symbol representing other amino acid residues is also an amino acid. It is based on the conventional notation in chemistry. These amino acids are all in the L form unless otherwise indicated.
即ち、特許を受けようとする第１発明は、下記（１）乃至（７）の１種若しくは２種以上のアミノ酸配列で示されるアンジオテンシンＩ変換酵素阻害活性を有するペプチド化合物である。
Tyr−Phe（１）、
Phe−Tyr（２）、
Phe−Ala（３）、
Leu−Val−Gly（４）、
Leu−Ile−Gly（５）、
Leu−Gly−Val（６）、
Ile−Val−Gly（７）。
That is, the first invention to be patented is a peptide compound having angiotensin I converting enzyme inhibitory activity represented by one or more amino acid sequences of the following (1) to (7).
Tyr-Phe (1),
Phe-Tyr (2),
Phe-Ala (3),
Leu-Val-Gly (4),
Leu-Ile-Gly (5),
Leu-Gly-Val (6),
Ile-Val-Gly (7).
当該第１発明に係る７種類のアミノ酸配列で示されるペプチド化合物は、それぞれに血管内への吸収性、安定性、安全性が高い強力なアンジオテンシンＩ変換酵素阻害活性を有している。従って、そのアンジオテンシンＩ変換酵素阻害活性を有するペプチド化合物の１種でもよいし、２種以上を適宜組み合せたペプチド化合物であってもよい。 The peptide compounds represented by the seven types of amino acid sequences according to the first invention each have a strong angiotensin I converting enzyme inhibitory activity with high absorbability into the blood vessel, stability and safety. Therefore, it may be one type of peptide compound having an angiotensin I converting enzyme inhibitory activity, or may be a peptide compound in which two or more types are appropriately combined.
また、当該第１発明に係る７種類のアミノ酸配列で示されるペプチド化合物は、当該化合物と同じアミノ酸配列を有する蛋白質から上記と同様の蛋白質分解酵素処理にても得られるが、これらのペプチドはアミノ酸を段階的に導入する一般的な有機化学的液相又は固相法によるペプチド合成や遺伝子工学手法等によっても得る事ができる。 The peptide compounds represented by the seven types of amino acid sequences according to the first invention can also be obtained from a protein having the same amino acid sequence as that compound by the same proteolytic enzyme treatment as described above. Can also be obtained by peptide synthesis by a general organic chemical liquid phase or solid phase method in which stepwise introduction is carried out, genetic engineering techniques, or the like.
本発明において用いられる蛋白質分解酵素としては、例えばBacillus属（例えばBacillus subtilis、Bacillus thermoproteolyticus、Bacillus licheniformis等）の産生する酵素、Aspergillus属（例えばAspergillus oryzae、Aspergillus niger、Aspergillus mellens等）の産生する酵素、Rhizopus属（例えばRhizopus niveus、Rhizopus delemar等）の産生する酵素、ペプシン、パンクレアチン、パパイン等が挙げられる。これらの酵素は単独、又は２種以上を組み合わせてもよい。 As the proteolytic enzyme used in the present invention, for example, an enzyme produced by the genus Bacillus (for example, Bacillus subtilis, Bacillus thermoproteolyticus, Bacillus licheniformis, etc.), an enzyme produced by the genus Aspergillus (eg, Aspergillus oryzae, Aspergillus niger, Aspergillus mellens, etc.), Examples include enzymes produced by the genus Rhizopus (for example, Rhizopus niveus, Rhizopus delemar, etc.), pepsin, pancreatin, papain and the like. These enzymes may be used alone or in combination of two or more.
特許を受けようとする第２発明は、第１発明に記載するアンジオテンシンＩ変換酵素阻害活性のペプチド化合物の１種若しくは２種以上と、無機酸若しくは有機酸若しくは無機塩基若しくは有機塩基とで形成してなる薬学的に許容される塩である。 The second invention to be patented is formed of one or more of the angiotensin I converting enzyme inhibitory peptide compounds described in the first invention and an inorganic acid, an organic acid, an inorganic base, or an organic base. A pharmaceutically acceptable salt.
当該第２発明も、由来を問わずに得られた（蛋白質分解酵素処理やペプチド合成や遺伝子工学手法で得られるものを含む。）アンジオテンシンＩ変換酵素阻害活性を有するペプチド化合物とその薬学的に許容される塩である。当該ペプチド化合物には、アンジオテンシンＩ変換酵素阻害活性を有することを、新たに見出し、本発明を完成したものである。 The second invention is also obtained regardless of origin (including those obtained by proteolytic enzyme treatment, peptide synthesis, and genetic engineering techniques). Peptide compounds having angiotensin I converting enzyme inhibitory activity and their pharmaceutically acceptable Salt. The peptide compound was newly found to have angiotensin I converting enzyme inhibitory activity, and the present invention was completed.
本発明で得られるペプチド化合物は必要に応じて無機酸若しくは有機酸との塩や無機塩基若しくは有機塩基との塩を形成させる事ができるが、薬学的に許容される塩が好ましい。酸付加塩としては、例えば、塩酸塩、硝酸塩、硫酸塩、メタンスルホン酸塩、p− トルエンスルホン酸塩、更にはシュウ酸、マロン酸、コハク酸、マレイン酸、又はフマル酸等のジカルボン酸との塩、更に、酢酸、プロピオン酸、又は酪酸等のモノカルボン酸との塩等を挙げる事ができる。又、本発明で得られるペプチド化合物の塩の形成に適した無機塩基は、例えば、アンモニア、ナトリウム、リチウム、カルシウム、マグネシウム、アルミニウム等の水酸化物、炭酸塩及び重炭酸塩等である。有機塩基と
の塩としては、例えば、メチルアミン、ジメチルアミン、トリエチルアミンの様なモノ−、ジ−及びトリ−アルキルアミン塩、モノ−、ジ−及びトリ−ヒドロキシアルキルアミン塩、グアニジン塩、N −メチルグルコサミン塩等を挙げる事ができる。
The peptide compound obtained in the present invention can form a salt with an inorganic acid or an organic acid or a salt with an inorganic base or an organic base, if necessary, but a pharmaceutically acceptable salt is preferred. Examples of the acid addition salt include hydrochloride, nitrate, sulfate, methanesulfonate, p-toluenesulfonate, and dicarboxylic acids such as oxalic acid, malonic acid, succinic acid, maleic acid, and fumaric acid. And salts with monocarboxylic acids such as acetic acid, propionic acid or butyric acid. Inorganic bases suitable for forming a salt of the peptide compound obtained in the present invention are, for example, hydroxides such as ammonia, sodium, lithium, calcium, magnesium and aluminum, carbonates and bicarbonates. Examples of salts with organic bases include mono-, di- and tri-alkylamine salts such as methylamine, dimethylamine and triethylamine, mono-, di- and trihydroxyalkylamine salts, guanidine salts, N- Examples thereof include methyl glucosamine salt.
特許を受けようとする第３発明は、サメ軟骨又はその処理物を蛋白質分解酵素で分解し、分解液から得られる下記（１）乃至（７）の１種若しくは２種以上のアミノ酸配列で示されるアンジオテンシンＩ変換酵素阻害活性を有するペプチド化合物である。
Tyr−Phe（１）、
Phe−Tyr（２）、
Phe−Ala（３）、
Leu−Val−Gly（４）、
Leu−Ile−Gly（５）、
Leu−Gly−Val（６）、
Ile−Val−Gly（７）。
The third invention to be patented is a shark cartilage or a processed product thereof decomposed with a proteolytic enzyme and represented by one or more amino acid sequences of the following (1) to (7) obtained from the decomposition solution A peptide compound having angiotensin I converting enzyme inhibitory activity.
Tyr-Phe (1),
Phe-Tyr (2),
Phe-Ala (3),
Leu-Val-Gly (4),
Leu-Ile-Gly (5),
Leu-Gly-Val (6),
Ile-Val-Gly (7).
当該第３発明から以下第8発明は、サメ軟骨又はその処理物を蛋白質分解酵素で分解し、分解液から得られる（１）乃至（７）のアミノ酸配列で示されるアンジオテンシンＩ変換酵素阻害活性を有するペプチド化合物とその薬学的に許容される塩である。 From the third invention to the eighth invention, the angiotensin I converting enzyme inhibitory activity represented by the amino acid sequence (1) to (7) obtained from a decomposition solution obtained by degrading shark cartilage or a processed product thereof with a proteolytic enzyme And a pharmaceutically acceptable salt thereof.
当該第３発明に係る７種類のアミノ酸配列で示されるペプチド化合物は、それぞれ強力なアンジオテンシンＩ変換酵素阻害活性を有しており、その１種でもよいし、２種以上を適宜組合せた当該ペプチド化合物であってもよいこと勿論である。 The peptide compounds represented by the seven types of amino acid sequences according to the third invention each have strong angiotensin I converting enzyme inhibitory activity, and may be one of them, or the peptide compound in which two or more are appropriately combined Of course, it may be.
ここで当該ペプチド化合物を得るサメの種類は特に限定されないが、ヨシキリザメ(Prionace glauca)、ネズミザメ（Lamna ditropis）、ネコザメ（Heterodontus japonicus）、アオザメ（Isurus oxyrinchus）、アカシュモクザメ（Sphyrna lewini）、メジロザメ（Carcharhinus plumbeus）、ホシザメ（Mustelus manazo）、マオナガ（Alopias valpinus）、ヒラガシラ（Rhizoprionodon acutus）、フジクジラ（Etmopterus lucifer）等が好適に用いられる。 Although the kind of shark which obtains the said peptide compound here is not specifically limited, Blue shark (Prionace glauca), a mouse shark (Lamna ditropis), a cat shark (Heterodontus japonicus), a blue shark (Isurus oxyrinchus), a red shark (Sphyrna lewini), a shark Plumbeus), horse shark (Mustelus manazo), blue-tailed hawk (Alopias valpinus), hiragashira (Rhizoprionodon acutus), whale (Etmopterus lucifer) and the like are preferably used.
また、当該第３発明における蛋白質分解酵素は、第１発明の説明に記載したのと同様の蛋白質分解酵素であり、これらの酵素は単独、又は２種以上を組合わせてもよいこと勿論である。 Further, the proteolytic enzyme in the third invention is the same proteolytic enzyme as described in the explanation of the first invention, and it goes without saying that these enzymes may be used alone or in combination of two or more. .
特許を受けようとする第４発明は、第３発明に記載するアンジオテンシンＩ変換酵素阻害活性を有するペプチド化合物の１種若しくは２種以上と、無機酸若しくは有機酸若しくは無機塩基若しくは有機塩基とで形成してなる薬学的に許容される塩である。 The fourth invention to be patented is formed by one or more of the peptide compounds having angiotensin I converting enzyme inhibitory activity described in the third invention and an inorganic acid, an organic acid, an inorganic base, or an organic base And a pharmaceutically acceptable salt.
当該第４発明は、第３発明に記載する７種類のアミノ酸配列で示されるペプチド化合物の薬学的に許容される塩の１種でもよいし、２種以上を適宜組み合せたペプチド化合物の薬学的に許容される塩である。 The fourth invention may be one of pharmaceutically acceptable salts of peptide compounds represented by the seven types of amino acid sequences described in the third invention, or a pharmaceutically acceptable peptide compound in which two or more are appropriately combined. It is an acceptable salt.
特許を受けようとする第５発明は、第１発明又は第３発明に記載するアンジオテンシンＩ変換酵素阻害活性を有するペプチド化合物の１種若しくは２種以上の有効成分とすることを特徴とするアンジオテンシンＩ変換酵素阻害剤である。 The fifth invention to be patented is an angiotensin I characterized by comprising one or more active ingredients of the peptide compound having angiotensin I converting enzyme inhibitory activity described in the first or third invention. It is a converting enzyme inhibitor.
特許を受けようとする第６発明は、第１発明又は第３発明に記載するアンジオテンシンＩ変換酵素阻害活性を有するペプチド化合物の少なくとも１つを含有する事を特徴とする食品、化粧品、医薬品又は医薬部外品である。 A sixth invention to be patented is a food, cosmetic, pharmaceutical or pharmaceutical comprising at least one peptide compound having an angiotensin I converting enzyme inhibitory activity described in the first or third invention. It is an outside product.
本発明で得られるペプチド化合物は必要に応じて適宜賦形剤等の添加剤と混合して例えば注射剤、経口用液剤、錠剤、顆粒剤、散剤、カプセル剤、坐剤、軟膏、点鼻剤、貼付剤等の形態で製剤化する事ができる。 The peptide compound obtained in the present invention is appropriately mixed with additives such as excipients as necessary, for example, injections, oral solutions, tablets, granules, powders, capsules, suppositories, ointments, nasal drops It can be formulated in the form of a patch.
上記の各種製剤で用いられる添加剤としては、例えばステアリン酸マグネシウム、タルク、乳糖、デキストリン、デンプン類、メチルセルロ−ス、脂肪酸グリセリド類、水、プロピレングリコ−ル、マクロゴ−ル類、アルコ−ル、結晶セルロ−ス、ヒドロキシプロピルセルロ−ス、低置換度ヒドロキシプロピルセルロ−ス、カルメロ−ス類、ポピドン、ポリビニルアルコ−ル、ステアリン酸カルシウム等を挙げる事ができる。この際、必要に応じて、着色剤、安定化剤、抗酸化剤、防腐剤、pH 調節剤、等張化剤、溶解補助剤及び／又は無痛化剤等を添加する事ができる。顆粒剤、錠剤、又はカプセル剤は、コ−ティング基剤、例えばヒドロキシプロピルメチルセルロ−ス、ヒドロキシプロピルメチルセルロ−スフタレ−ト等によってコ−ティングする事もできる。これらの製剤は本発明で得られるペプチド化合物を0.01%以上、好ましくは0.1
〜70%の割合で含有する事ができる。
Examples of additives used in the above various preparations include magnesium stearate, talc, lactose, dextrin, starches, methyl cellulose, fatty acid glycerides, water, propylene glycol, macrogols, alcohol, Crystalline cellulose, hydroxypropyl cellulose, low-substituted hydroxypropyl cellulose, carmelloses, popidone, polyvinyl alcohol, calcium stearate and the like can be mentioned. At this time, a colorant, a stabilizer, an antioxidant, an antiseptic, a pH adjuster, a tonicity agent, a solubilizing agent and / or a soothing agent can be added as necessary. Granules, tablets, or capsules can be coated with a coating base such as hydroxypropylmethylcellulose, hydroxypropylmethylcellulose phthalate and the like. These preparations contain 0.01% or more, preferably 0.1% of the peptide compound obtained in the present invention.
It can be contained in a proportion of ~ 70%.
製剤の調製に際しては必要に応じメント−ル、クエン酸及びその塩類、香料等の矯臭剤を用いる事ができる。更に、本発明で得られるアンジオテンシンＩ変換酵素
阻害剤は治療上有用な他の成分、例えばカプトプリル、エナラプリル等の公知の降圧剤を含有、又は併用する事もできる。
In preparing the preparation, flavoring agents such as menthol, citric acid and salts thereof, and fragrance can be used as necessary. Furthermore, the angiotensin I converting enzyme inhibitor obtained in the present invention may contain other components useful for treatment, for example, known antihypertensive agents such as captopril and enalapril, or may be used in combination.
本発明で得られるペプチド化合物の少なくとも１つを有効成分とする事を特徴とするアンジオテンシンＩ変換酵素
阻害剤はヒトを含めた哺乳動物に経口的又は非経口的（例えば経皮、静脈内、腹腔内等）に投与される。投与量は動物種、対象となる患者の人種、性別、症状、体重、年齢、血圧の程度、投与方法等によって異なり一概には言えないが、一般的なヒトの成人に経口投与する場合は、通常、１日につき体重１kgあたり0.1〜200mg、好ましくは1〜150mgであり、これを通常１日１回又は２〜３回に分けて投与する。しかしながらその投与量は症状の程度に応じ適宜選択する事ができる。
An angiotensin I converting enzyme inhibitor characterized by comprising at least one of the peptide compounds obtained in the present invention as an active ingredient is orally or parenterally (for example, transdermally, intravenously, intraperitoneally) to mammals including humans. Internal). The dose varies depending on the animal species, the race of the target patient, sex, symptoms, weight, age, blood pressure level, method of administration, etc., but it cannot be said unconditionally, but when administered orally to general human adults Usually, it is 0.1 to 200 mg / kg body weight per day, preferably 1 to 150 mg per day, and this is usually administered once a day or divided into 2 to 3 times a day. However, the dose can be appropriately selected according to the degree of symptoms.
本発明のペプチド化合物は優れたアンジオテンシンＩ変換酵素阻害活性を有し、血圧降下作用、ブラジキニン不活性化抑制作用を示す。しかも本発明で得られるペプチド化合物は臭い、味、色に特異な厭味が認められない事から経口摂取が容易である。その為、本発明で得られるペプチド化合物、あるいは組成物を、医薬品としてだけでなく、例えば、ゼリ−、飴、顆粒菓、錠菓、飲料、ス−プ、麺、煎餅、和菓子、洋菓子、冷菓、焼き菓子等の食品に配合、添加し提供する事ができるし、化粧品又は医薬部外品として提供することもできる。 The peptide compound of the present invention has excellent angiotensin I converting enzyme inhibitory activity, and exhibits blood pressure lowering action and bradykinin inactivation inhibiting action. Moreover, the peptide compound obtained by the present invention is easy to be taken orally because no peculiar taste of smell, taste and color is observed. Therefore, the peptide compound or composition obtained in the present invention is not only used as a pharmaceutical, but also, for example, jelly, rice cake, granule confectionery, tablet confectionery, beverage, supermarket, noodle, rice cracker, Japanese confectionery, Western confectionery, frozen confectionery. It can be provided by being added to a food such as baked confectionery, or provided as a cosmetic or quasi-drug.
特許を受けようとする第７発明は、サメ軟骨又はその処理物を蛋白質分解酵素で分解し、分解液から得られる下記（１）乃至（７）の１種若しくは２種以上のアミノ酸配列で示されるアンジオテンシンＩ変換酵素阻害活性を有するペプチド化合物を含む組成物である。
Tyr−Phe（１）、
Phe−Tyr（２）、
Phe−Ala（３）、
Leu−Val−Gly（４）、
Leu−Ile−Gly（５）、
Leu−Gly−Val（６）、
Ile−Val−Gly（７）。
The seventh invention to be patented is represented by one or more amino acid sequences of (1) to (7) below obtained by degrading shark cartilage or a processed product thereof with a proteolytic enzyme and obtained from the degradation solution. A composition comprising a peptide compound having angiotensin I converting enzyme inhibitory activity.
Tyr-Phe (1),
Phe-Tyr (2),
Phe-Ala (3),
Leu-Val-Gly (4),
Leu-Ile-Gly (5),
Leu-Gly-Val (6),
Ile-Val-Gly (7).
特許を受けようとする第８発明は、サメ軟骨又はその処理物を蛋白質分解酵素と反応させて分解させ、分解液から分離処理することを特徴とする下記（１）乃至（７）の１種若しくは２種以上のアミノ酸配列で示されるアンジオテンシンＩ変換酵素阻害活性を有するペプチド化合物又はそれを含有する組成物の製造方法である。
Tyr−Phe（１）、
Phe−Tyr（２）、
Phe−Ala（３）、
Leu−Val−Gly（４）、
Leu−Ile−Gly（５）、
Leu−Gly−Val（６）、
Ile−Val−Gly（７）。
The eighth invention to be patented is one of the following (1) to (7) characterized in that shark cartilage or a processed product thereof is decomposed by reacting with a proteolytic enzyme and separated from the decomposition solution. Or it is a manufacturing method of the peptide compound which has the angiotensin I converting enzyme inhibitory activity shown by 2 or more types of amino acid sequences, or a composition containing it.
Tyr-Phe (1),
Phe-Tyr (2),
Phe-Ala (3),
Leu-Val-Gly (4),
Leu-Ile-Gly (5),
Leu-Gly-Val (6),
Ile-Val-Gly (7).
本発明に係るペプチド化合物を、サメ軟骨又はその処理物より製造する方法を具体的に説明する。サメ軟骨又はその処理物を蛋白質分解酵素にて分解する方法は、常法に従って行う。例えば所望によりサメ軟骨を粉砕後、精製水を加え、必要に応じてｐHと温度を至適値に調整し適当な蛋白質分解酵素を添加してインキュベ−トする。次いで必要に応じて中和した後、酵素を失活させて酵素分解液を得る。分解液を例えば濾紙及び／又は濾過助剤等を用いて濾過する事によって不溶物を除去し、得られた濾液を限外濾過等で処理して分子量５〜１万以下の粗ペプチド画分を得る。得られた粗ペプチド画分は必要に応じて活性炭処理、濾過後濃縮して粗ペプチド混合物を得る。粗ペプチド混合物は所望により液液分配等を経てカラムクロマトグラフィ−にて分画し、優れたアンジオテンシンＩ変換酵素阻害活性を有するペプチド化合物を得ることができる。 A method for producing the peptide compound according to the present invention from shark cartilage or a processed product thereof will be specifically described. A method for degrading shark cartilage or a processed product thereof with a proteolytic enzyme is performed according to a conventional method. For example, after pulverizing shark cartilage, if necessary, purified water is added, and if necessary, pH and temperature are adjusted to optimum values, and an appropriate proteolytic enzyme is added and incubated. Next, after neutralization as necessary, the enzyme is deactivated to obtain an enzyme decomposition solution. The decomposition solution is filtered using, for example, filter paper and / or filter aid to remove insoluble matter, and the resulting filtrate is treated by ultrafiltration to obtain a crude peptide fraction having a molecular weight of 50,000 to 10,000. obtain. The obtained crude peptide fraction is treated with activated carbon as necessary, filtered and concentrated to obtain a crude peptide mixture. The crude peptide mixture can be fractionated by column chromatography through liquid-liquid partitioning or the like, if desired, to obtain a peptide compound having excellent angiotensin I converting enzyme inhibitory activity.
本発明により得られたペプチド化合物は、強いアンジオテンシンＩ変換酵素阻害活性を有し、強い血圧降下作用、ブラジキニン不活性化抑制作用を示す。従って本発明は、例えば本態性高血圧、腎性高血圧、副腎性高血圧等の高血圧症の予防、治療剤、これら疾患の診断薬、各種病態で用いられる降圧剤、心筋梗塞の減少、うっ血性心不全における病態の改善剤等として有用である。 The peptide compound obtained by the present invention has a strong angiotensin I converting enzyme inhibitory activity, and exhibits a strong blood pressure lowering action and a bradykinin inactivation inhibiting action. Therefore, the present invention is useful for preventing or treating hypertension such as essential hypertension, renal hypertension, adrenal hypertension, diagnostic agents for these diseases, antihypertensive agents used in various pathologies, reduction of myocardial infarction, congestive heart failure. It is useful as a disease condition improving agent.
また、本発明で得られるペプチド化合物は、臭い、味、色に特異な厭味が認められない事から経口摂取が容易である。その為、本発明で得られるペプチド化合物、あるいは当該化合物を含有する各種製剤を、例えば、ゼリ−、飴、顆粒菓、錠菓、飲料、ス−プ、麺、煎餅、和菓子、洋菓子、冷菓、焼き菓子等の食品に配合、添加し提供する事ができる。上記の様な有用な作用を有する健康食品や特定保健用食品、機能性食品としての利用が可能である。更に、化粧品や医薬部外品としても提供することもできる。 Moreover, the peptide compound obtained by this invention is easy to ingest orally since the peculiar taste of smell, taste, and color is not recognized. Therefore, the peptide compound obtained in the present invention, or various preparations containing the compound, for example, jelly, rice cake, granule confectionery, tablet confectionery, beverage, supermarket, noodles, rice cracker, Japanese confectionery, Western confectionery, frozen confectionery, It can be formulated and added to foods such as baked goods. It can be used as a health food having a useful action as described above, a food for specified health use, or a functional food. Furthermore, it can also be provided as a cosmetic or quasi-drug.
以下、本発明の実施例について詳細に説明するが、本発明はこれらの例に限定されるものではない。サメ軟骨又はその処理物を蛋白質分解酵素で分解し、分解液から得られるTyr−Phe（１）、Phe−Tyr（２）、Phe−Ala（３）、Leu−Val−Gly（４）、Leu−Ile−Gly（５）、Leu−Gly−Val（６）、Ile−Val−Gly（７）の１種若しくは２種以上のアミノ酸配列で示されるアンジオテンシンＩ変換酵素阻害活性を有するペプチド化合物である。 Examples of the present invention will be described in detail below, but the present invention is not limited to these examples. Tyr-Phe (1), Phe-Tyr (2), Phe-Ala (3), Leu-Val-Gly (4), Leu obtained from proteolytic enzyme by degrading shark cartilage or processed product thereof A peptide compound having an angiotensin I converting enzyme inhibitory activity represented by one or more amino acid sequences of Ile-Gly (5), Leu-Gly-Val (6), and Ile-Val-Gly (7) .
＜製造例１＞アンジオテンシンＩ変換酵素 阻害ペプチド化合物の製造
ヨシキリザメ（Prionace glauca）軟骨3kgを粉砕、精製水9kgを加え55℃に加温した後、パパイン（Carica Papaya）7.5gを添加して14時間攪拌し酵素分解反応を行った。反応液を95℃に加温して酵素活性を失活させ、冷却後、中間孔径7ミクロンのセライトを用いて濾過した。濾液を限外濾過膜（ロミコンHF1.0−43−PM50）を透過させて分子量５万以上の画分を除いた後、噴霧乾燥してアンジオテンシンＩ変換酵素阻害ペプチド混合物260gを得た。
<Production Example 1> Production of angiotensin I converting enzyme inhibitory peptide compound 3 kg of blue shark (Prionace glauca) cartilage was pulverized, 9 kg of purified water was added and heated to 55 ° C, and then 7.5 g of papain (Carica Papaya) was added for 14 hours. The agitation was performed with stirring. The reaction solution was heated to 95 ° C. to deactivate the enzyme activity, cooled, and filtered using Celite having an intermediate pore diameter of 7 microns. The filtrate was passed through an ultrafiltration membrane (Romicon HF1.0-43-PM50) to remove a fraction having a molecular weight of 50,000 or more and then spray-dried to obtain 260 g of an angiotensin I converting enzyme-inhibiting peptide mixture.
上記の方法により得たアンジオテンシンＩ変換酵素阻害ペプチド混合物粗体を下記条件でＨＰＬＣ分析したところ図７に示すようなクロマトグラムを得た。
＜ＨＰＬＣ分析条件＞
カラム Waters μBONDASPHERE C18
3.9×150mm
カラム温度 40℃
移動相Ａ Ｈ2Ｏ(0.1TFA含有)
移動相Ｂ アセトニトリル(0.1TFA含有)
グラジエント 0〜60分にかけてＡ液5％からＢ液40％へのリニアグラジェント60〜75分にかけてＢ液40％
分析時間 75分
流量 0.4ml/min
注入量 15μL
検出 ＵＶ220nm
The crude angiotensin I converting enzyme-inhibiting peptide mixture obtained by the above method was analyzed by HPLC under the following conditions to obtain a chromatogram as shown in FIG.
<HPLC analysis conditions>
Column Waters μBONDASPHERE C18
3.9 × 150mm
Mobile phase A H 2 O (containing 0.1TFA)
Mobile phase B Acetonitrile (containing 0.1TFA)
Gradient From 0 to 60 minutes, A
Analysis time 75 minutes Flow rate 0.4ml / min
Injection volume 15μL
Detection UV220nm
その結果、図７に示すように、サメ軟骨を蛋白質分解酵素で処理した蛋白質分解酵素処理物から得たアンジオテンシンＩ変換酵素阻害ペプチド混合物粗体中に７種類の強力なアンジオテンシンＩ変換酵素阻害ペプチドが含まれていることを確認した。
Tyr−Phe（１）、
Phe−Tyr（２）、
Phe−Ala（３）、
Leu−Val−Gly（４）、
Leu−Ile−Gly（５）、
Leu−Gly−Val（６）、
Ile−Val−Gly（７）。
As a result, as shown in FIG. 7, seven kinds of strong angiotensin I converting enzyme inhibitory peptides were present in the crude angiotensin I converting enzyme inhibitory peptide mixture obtained from the proteolytic enzyme processed product of shark cartilage treated with proteolytic enzyme. Confirmed that it was included.
Tyr-Phe (1),
Phe-Tyr (2),
Phe-Ala (3),
Leu-Val-Gly (4),
Leu-Ile-Gly (5),
Leu-Gly-Val (6),
Ile-Val-Gly (7).
そこで、前記ペプチド混合物粗体中に含有している７種類の強力なアンジオテンシンＩ変換酵素阻害ペプチドの夫々を分離処理することとした。 Therefore, each of the seven types of strong angiotensin I converting enzyme-inhibiting peptides contained in the peptide mixture crude was subjected to separation treatment.
まず、上記ペプチド混合物60.0gを水1200mLに溶解させ1M 塩酸にてｐHを2.0に調整した。この水溶液をｎ−ブタノ−ル1200mLで抽出し、水層を更にｎ−ブタノ−ル600mLで2回抽出した。ｎ−ブタノ−ル層を減圧下濃縮して抽出物10.3gを得た。 First, 60.0 g of the above peptide mixture was dissolved in 1200 mL of water, and the pH was adjusted to 2.0 with 1M hydrochloric acid. This aqueous solution was extracted with 1,200 mL of n-butanol, and the aqueous layer was further extracted twice with 600 mL of n-butanol. The n-butanol layer was concentrated under reduced pressure to obtain 10.3 g of an extract.
この抽出物5.78gを
シリカゲルカラムクロマトグラフィ−（富士シリシア化学、BW−820MH、展開溶媒：n−ブタノ−ル−ピリジン−水−酢酸10:1:2:1）、
シリカゲルカラムクロマトグラフィ−（BW−820MH、展開溶媒：エタノ−ル−水10:1）、
イオン交換樹脂カラムクロマトグラフィ−（アルドリッチ、Amberlite（商標）CG−50、展開溶媒：メタノ−ル）、
シリカゲルカラムクロマトグラフィ−（関東化学、シリカゲル60N フラッシュクロマトグラフィ−用、展開溶媒：n−ブタノ−ル−エタノ−ル−クロロホルム−5％ アンモニア水6：4：2：1）
にて分画し、高いアンジオテンシンＩ変換酵素阻害活性を示す画分A：45mg及び画分B：30mgを得た。
5.78 g of this extract was subjected to silica gel column chromatography (Fuji Silysia Chemical, BW-820MH, developing solvent: n-butanol-pyridine-water-acetic acid 10: 1: 2: 1),
Silica gel column chromatography (BW-820MH, developing solvent: ethanol-water 10: 1),
Ion exchange resin column chromatography (Aldrich, Amberlite ™ CG-50, developing solvent: methanol),
Silica gel column chromatography (for Kanto Chemical, silica gel 60N flash chromatography, developing solvent: n-butanol-ethanol-chloroform-5% aqueous ammonia 6: 4: 2: 1)
Fraction A: 45 mg and fraction B: 30 mg showing high angiotensin I converting enzyme inhibitory activity were obtained.
画分A：29mgを、5％塩化水素−メタノ−ル0.87mLに溶解し、室温にて20時間静置した後、減圧下濃縮乾固して、残留物を32mg得た。 Fraction A: 29 mg was dissolved in 0.87 mL of 5% hydrogen chloride-methanol, allowed to stand at room temperature for 20 hours, and then concentrated to dryness under reduced pressure to obtain 32 mg of a residue.
これをシリカゲルカラムクロマトグラフィ−（BW−820MH、展開溶媒：n−ブタノ−ル−エタノ−ル−クロロホルム−水8:1:2:1）にて分離し、画分A2：6.6mgを得た。画分A2：4.3mgを水0.22mLに溶解した後、炭酸水素ナトリウム4.5mgを加えた。これにベンジルオキシカルボニル（以下Zと略記する）クロリド3.8µLのテトラヒドロフラン溶液43µLを滴加し、室温にて6時間攪拌後、テトラヒドロフランを留去した。酢酸エチル0.2mLで2回抽出し、合わせた有機層を減圧下濃縮乾固して残留物を6.5mg得た。 This was separated by silica gel column chromatography (BW-820MH, developing solvent: n-butanol-ethanol-chloroform-water 8: 1: 2: 1) to obtain fraction A2: 6.6 mg. Fraction A2: 4.3 mg was dissolved in 0.22 mL of water, and then 4.5 mg of sodium bicarbonate was added. To this was added dropwise benzyloxycarbonyl (hereinafter abbreviated as Z) chloride 3.8 µ L in tetrahydrofuran solution 43 µ L, and the mixture was stirred at room temperature for 6 hours, and then tetrahydrofuran was distilled off. Extraction was performed twice with 0.2 mL of ethyl acetate, and the combined organic layers were concentrated to dryness under reduced pressure to obtain 6.5 mg of residue.
これをシリカゲルカラムクロマトグラフィ−（BW−820MH、展開溶媒：トルエン−酢酸エチル2:1及び5:1）にて分離し画分A3：0.8mgを得た。製造例４に記載の合成化合物と1H−NMRを比較した結果、画分A3の構造をZ−Tyr(Z)−Phe−メチルエステル（以下、OMeと略記する）と確定した。当該画分A3から一般的有機化学手法によりTyr−Pheを得る事ができる。このTyr−Pheは、Biochimica et Biophysica Acta(1980),625(2),266−273に記載されたペプチド化合物と同一である事が判明したが、そのアンジオテンシンＩ変換酵素阻害活性は報告されていない。 This was separated by silica gel column chromatography (BW-820MH, developing solvent: toluene-ethyl acetate 2: 1 and 5: 1) to obtain fraction A3: 0.8 mg. As a result of comparing 1 H-NMR with the synthetic compound described in Production Example 4, the structure of fraction A3 was determined to be Z-Tyr (Z) -Phe-methyl ester (hereinafter abbreviated as OMe). Tyr-Phe can be obtained from the fraction A3 by a general organic chemistry method. Although this Tyr-Phe was found to be identical to the peptide compound described in Biochimica et Biophysica Acta (1980), 625 (2), 266-273, its angiotensin I converting enzyme inhibitory activity has not been reported. .
次に、画分B：23mgを5％塩化水素−メタノ−ル0.70mLに溶解し、室温にて16時間静置したのち、減圧下濃縮乾固して、残留物を28mg得た。これをシリカゲルカラムクロマトグラフィ−（BW−820MH、展開溶媒：n−
ブタノ−ル−エタノ−ル−クロロホルム−水8:1:2:1）にて分離し、画分B2：4.6mg、B3：6.1mgを得た。画分B2：3.5mgを水0.25mLに溶解したのち、炭酸水素ナトリウム7.3mgを加えた。これにZクロリド6.2µLのテトラヒドロフラン溶液31µLを滴加し、室温にて5時間攪拌した後、テトラヒドロフランを留去した。その後、酢酸エチル0.3mLで2回抽出し、合わせた有機層を減圧下濃縮乾固して残留物を5.8mg得た。これをシリカゲルカラムクロマトグラフィ−（BW−820MH、展開溶媒：トルエン−酢酸エチル1:1）にて分離し、画分B21：0.4mgを得た。当該合成化合物と1H−NMRを比較した結果、画分B21の構造をZ−Leu−Ile−Gly−OMeと確定した。画分B21から一般的有機化学手法によりLeu−Ile−Glyを得る事ができる。Leu−Ile−Glyは文献未記載の新規ペプチド化合物である。
Next, 23 mg of fraction B was dissolved in 0.70 mL of 5% hydrogen chloride-methanol, allowed to stand at room temperature for 16 hours, and then concentrated to dryness under reduced pressure to obtain 28 mg of residue. This was silica gel column chromatography (BW-820MH, developing solvent: n-
Separation with butanol-ethanol-chloroform-water 8: 1: 2: 1) gave fractions B2: 4.6 mg and B3: 6.1 mg. Fraction B2: 3.5 mg was dissolved in 0.25 mL of water, and then 7.3 mg of sodium bicarbonate was added. Tetrahydrofuran solution 31 µ L of Z chloride 6.2 µ L was added dropwise thereto and stirred at room temperature for 5 hours, and then tetrahydrofuran was distilled off. Thereafter, extraction was performed twice with 0.3 mL of ethyl acetate, and the combined organic layers were concentrated to dryness under reduced pressure to obtain 5.8 mg of residue. This was separated by silica gel column chromatography (BW-820MH, developing solvent: toluene-ethyl acetate 1: 1) to obtain fraction B21: 0.4 mg. As a result of comparing the synthetic compound and 1 H-NMR, the structure of fraction B21 was determined to be Z-Leu-Ile-Gly-OMe. Leu-Ile-Gly can be obtained from fraction B21 by general organic chemistry techniques. Leu-Ile-Gly is a novel peptide compound not described in any literature.
次に、画分B3：5.1mgを水0.25mLに溶解したのち、炭酸水素ナトリウム5.4mgを加えた。これにZクロリド4.6µLのテトラヒドロフラン溶液25µLを滴加し、室温にて4時間攪拌した後、テトラヒドロフランを留去した。その後、酢酸エチル0.2mLで2回抽出し、合わせた有機層を減圧下濃縮乾固して残留物を7.0mg得た。これをシリカゲルカラムクロマトグラフィ−（BW−
820MH、展開溶媒：トルエン−酢酸エチル1:1→トルエン−酢酸エチル1:3）にて分離し、画分B31：0.7mg、画分B32：1.0mgを得た。製造例５に記載の合成化合物と1 H− NMRを比較した結果、画分B31の構造をZ−Phe−Ala−OMeと確定した。当該画分B31から一般的有機化学手法によりPhe−Alaを得る事ができる。当該Phe−AlaはScience，(1981)212(4499),1153−1155に記載されたペプチド化合物と同一である事が判明したが、そのアンジオテンシンＩ変換酵素
阻害活性は報告されていない。、
Next, Fraction B3: 5.1 mg was dissolved in 0.25 mL of water, and then 5.4 mg of sodium bicarbonate was added. To this was added dropwise 25 ml of Z chloride 4.6 µ L in tetrahydrofuran, and the mixture was stirred at room temperature for 4 hours, and then tetrahydrofuran was distilled off. Thereafter, the mixture was extracted twice with 0.2 mL of ethyl acetate, and the combined organic layers were concentrated to dryness under reduced pressure to obtain 7.0 mg of a residue. Silica gel column chromatography (BW-
820MH, developing solvent: toluene-ethyl acetate 1: 1 → toluene-ethyl acetate 1: 3) to obtain fraction B31: 0.7 mg and fraction B32: 1.0 mg. As a result of comparing 1 H-NMR with the synthetic compound described in Production Example 5, the structure of fraction B31 was determined to be Z-Phe-Ala-OMe. Phe-Ala can be obtained from the fraction B31 by a general organic chemistry method. The Phe-Ala was found to be the same as the peptide compound described in Science, (1981) 212 (4499), 1153-1155, but its angiotensin I converting enzyme inhibitory activity has not been reported. ,
また、前記の方法で得た画分B32の構造をZ−Leu−Val−Gly−OMeと確定した。当該画分B32から一般的有機化学手法によりLeu−Val−Glyを得る事ができる。このLeu−Val−GlyはJournal of Experimental Medicine(1985),161(4),805−815に記載されたペプチド化合物と同一である事が判明したが、そのアンジオテンシンＩ変換酵素阻害活性は報告されていない。 In addition, the structure of fraction B32 obtained by the above method was confirmed as Z-Leu-Val-Gly-OMe. Leu-Val-Gly can be obtained from the fraction B32 by a general organic chemistry method. This Leu-Val-Gly was found to be identical to the peptide compound described in Journal of Experimental Medicine (1985), 161 (4), 805-815, but its angiotensin I converting enzyme inhibitory activity has been reported. Absent.
上記と同様の方法により、画分Aと画分Bとの混合物0.12gを得た。これを5％塩化水素−メタノ−ル3.6mLに溶解し、室温にて24 時間静置した後、減圧下濃縮乾固して、残留物を0.13g得た。これを33%ジオキサン水4.0mLに溶解し、炭酸水素ナトリウム0.11gとZクロリド92µLを加えて室温にて16時間攪拌した。溶媒を取り除き残留物を95mg得た。これをシリカゲルカラムクロマトグラフィ−（関東化学、シリカゲル60N フラッシュクロマトグラフィ−用、展開溶媒：トルエン−酢酸エチル2:1→トルエン−酢酸エチル1:3→酢酸エチル→メタノ−ル）にて分離し、Z−Phe−Tyr(Z)−OMeとZ−Tyr(Z)−Phe−OMeの混合物10mg、及びZ−Phe−Tyr−OMeとZ−Tyr−Phe−OMeの混合物2.3mgを得た。尚、これらの構造は製造例３及び製造例４に記載の合成化合物と1H−NMRを比較し確定した。これらから、一般的有機化学手法によりそれぞれPhe−Tyr、Tyr−Pheを得る事ができる。Phe−TyrはJournal of Nutritional Biochemistry(1998),9(7),415−419にニンニク由来のアンジオテンシンＩ変換酵素阻害ペプチドとして報告されている化合物と同一である事が判明した。 In the same manner as above, 0.12 g of a mixture of fraction A and fraction B was obtained. This was dissolved in 3.6 mL of 5% hydrogen chloride-methanol, allowed to stand at room temperature for 24 hours, and then concentrated to dryness under reduced pressure to obtain 0.13 g of residue. This was dissolved in 4.0 mL of 33% aqueous dioxane, 0.11 g of sodium hydrogen carbonate and 92 mL of Z chloride were added, and the mixture was stirred at room temperature for 16 hours. The solvent was removed to obtain 95 mg of residue. This was separated by silica gel column chromatography (for Kanto Chemical, silica gel 60N flash chromatography, developing solvent: toluene-ethyl acetate 2: 1 → toluene-ethyl acetate 1: 3 → ethyl acetate → methanol), Z- 10 mg of a mixture of Phe-Tyr (Z) -OMe and Z-Tyr (Z) -Phe-OMe and 2.3 mg of a mixture of Z-Phe-Tyr-OMe and Z-Tyr-Phe-OMe were obtained. These structures were determined by comparing the synthetic compounds described in Production Example 3 and Production Example 4 with 1 H-NMR. From these, Phe-Tyr and Tyr-Phe can be obtained by general organic chemistry methods, respectively. Phe-Tyr was found to be identical to the compound reported as an angiotensin I converting enzyme inhibitory peptide derived from garlic in Journal of Nutritional Biochemistry (1998), 9 (7), 415-419.
アンジオテンシンＩ変換酵素阻害ペプチド混合物粗体を下記条件でＨＰＬＣ分析したところ図７のクロマトグラムを得た。そこで、ＨＰＬＣ条件にて、Leu−Gly−Valを分取したうえ、これを製造法８で合成した表品と比較して、その構造を決定した。しかるにこのLeu−Gly−ValについてアンジオテンシンＩ変換酵素阻害活性は報告されていない。 The crude product of the angiotensin I-converting enzyme-inhibiting peptide mixture was analyzed by HPLC under the following conditions to obtain the chromatogram of FIG. Therefore, Leu-Gly-Val was fractionated under HPLC conditions, and compared with the surface product synthesized by Production Method 8, the structure was determined. However, no angiotensin I converting enzyme inhibitory activity has been reported for this Leu-Gly-Val.
＜製造例２＞Phe−Tyrの合成
Z−Phe−Tyr 30mgをジオキサン1.2mLに溶解し、水0.6mLを加えた。10％パラジウム−炭素6mgを加えた後、水素雰囲気下４時間攪拌した。綿栓濾過にて触媒を取り除いた後、濾液を減圧下で濃縮乾固して残留物を24mg得た。これをG−25カラムクロマトグラフィ−（1.2g、展開溶媒：水−酢酸20:1、1分画0.3mL）にて精製して、Phe−Tyrを17mg得た（収率81%）。
<Production Example 2> Synthesis of Phe-Tyr
Z-Phe-Tyr 30 mg was dissolved in dioxane 1.2 mL, and water 0.6 mL was added. After adding 6% of 10% palladium-carbon, the mixture was stirred for 4 hours in a hydrogen atmosphere. After removing the catalyst by cotton plug filtration, the filtrate was concentrated to dryness under reduced pressure to obtain 24 mg of a residue. This was purified by G-25 column chromatography (1.2 g, developing solvent: water-acetic acid 20: 1, 1 fraction 0.3 mL) to obtain 17 mg of Phe-Tyr (yield 81%).
＜製造例３＞Z−Phe−Tyr(Z)−OMeの合成
Z−Phe−Tyr 10mgを5 ％塩化水素メタノ−ル溶液0.50mLに溶解し、室温にて2時間静置した後、減圧下濃縮乾固してZ−Phe−Tyr−OMeを10mg得た（収率定量的）。Z−Phe−Tyr−OMe
10mgをテトラヒドロフラン0.50mLに溶解し、0℃にてトリエチルアミン7µLとZクロリド6µLを加えて、室温にて30分間攪拌した。減圧下で濃縮してテトラヒドロフランを留去した後、酢酸エチル2mL、水0.5mL、飽和硫酸水素カリウム水溶液0.2mLを加えて、有機層を分離した。水層を酢酸エチル1mLで1回抽出した後、合わせた有機層を減圧下で濃縮乾固した。残留物をシリカゲルカラムクロマトグラフィ−（BW−
820MH 1.0g 、トルエン−酢酸エチル4:1、1分画0.5mL）にて精製し、Z−Phe−Tyr(Z)−OMeを6.2mg得た（収率48%）。
<Production Example 3> Synthesis of Z-Phe-Tyr (Z) -OMe
10 mg of Z-Phe-Tyr was dissolved in 0.50 mL of 5% hydrogen chloride methanol solution, allowed to stand at room temperature for 2 hours, and then concentrated to dryness under reduced pressure to obtain 10 mg of Z-Phe-Tyr-OMe ( Yield quantitative). Z-Phe-Tyr-OMe
10 mg was dissolved in 0.50 mL of tetrahydrofuran, triethylamine 7 µ L and Z chloride 6 µ L were added at 0 ° C., and the mixture was stirred at room temperature for 30 minutes. After concentration under reduced pressure to distill off tetrahydrofuran, 2 mL of ethyl acetate, 0.5 mL of water, and 0.2 mL of saturated aqueous potassium hydrogensulfate solution were added to separate the organic layer. The aqueous layer was extracted once with 1 mL of ethyl acetate, and then the combined organic layers were concentrated to dryness under reduced pressure. The residue was subjected to silica gel column chromatography (BW-
820MH 1.0 g, toluene-ethyl acetate 4: 1, 1 fraction 0.5 mL) to obtain 6.2 mg of Z-Phe-Tyr (Z) -OMe (yield 48%).
＜製造例４＞Z−Tyr(Z)−Phe−OMeの合成
Tyr−Phe 20mgを5％塩化水素メタノ−ル溶液0.60mLに溶解し、室温にて12
時間静置した後、減圧下濃縮乾固してTyr−Phe−OMe・HClを23mg得た（収率定量的）。当該Tyr−Phe−OMe・HCl 23mgをジクロロメタン0.60mLに溶解し、0℃にてトリエチルアミン23µLとZクロリド21µLを加えて、室温にて3.5 時間攪拌した。トリエチルアミン23µ Lを追加して室温にて30 分間撹拌し、減圧下濃縮してジクロロメタンを留去した後、酢酸エチル2mL、飽和硫酸水素カリウム水溶液0.5mLを加えて、有機層を分離した。水層を酢酸エチル1mLで1回抽出した後、合わせた有機層を減圧下濃縮乾固した。残留物をシリカゲルカラムクロマトグラフィ−（BW−820MH 1.9g、クロロホルム−酢酸エチル10:1、1分画1mL）にて精製し、Z−Tyr(Z)−Phe−OMeを11mg得た（収率30%）。
<Production Example 4> Synthesis of Z-Tyr (Z) -Phe-OMe
Dissolve 20 mg of Tyr-Phe in 0.60 mL of 5% hydrogen chloride methanol solution, and add 12 mg at room temperature.
After standing for a period of time, it was concentrated to dryness under reduced pressure to obtain 23 mg of Tyr-Phe-OMe.HCl (quantitative yield). The Tyr-Phe-OMe · HCl 23 mg was dissolved in 0.60 mL of dichloromethane, triethylamine 23 µ L and
＜製造例５＞Z−Phe−Ala−OMe及びPhe−Alaの合成
Phe 0.31gを水3.1mLに懸濁し、0℃にて炭酸水素ナトリウム0.32gとZクロリド0.27mLを加えた。0℃にて1時間、室温にて18時間攪拌した。ジエチルエ−テル4mLで1回洗浄し、2M塩酸3mLを加えて、酢酸エチル8mLで1回抽出し、減圧下で濃縮乾固して、Z−Pheを0.49g得た（収率88%）。Z−Phe 0.49gをテトラヒドロフラン15mLに溶解し、市販のAla−OMe・HCl 0.22gを加えた。0℃にて1−エチル−3−(3−ジメチルアミノプロピル)カルボジイミド塩酸塩（以下、EDCI・HClと略記する）0.31g、トリエチルアミン0.22mLを加えて、0℃にて2時間、室温にて63時間攪拌した。水2mLを加えた後、減圧下で濃縮してテトラヒドロフランを留去した。酢酸エチル20mL、水1mL、飽和硫酸水素カリウム水溶液0.5mLを加えて、有機層を分離した後、飽和炭酸水素ナトリウム水溶液4mLで1回、飽和塩化ナトリウム水溶液4mLで1回洗浄し、減圧下で濃縮乾固した。
<Production Example 5> Synthesis of Z-Phe-Ala-OMe and Phe-Ala
0.31 g of Phe was suspended in 3.1 mL of water, and 0.32 g of sodium bicarbonate and 0.27 mL of Z chloride were added at 0 ° C. The mixture was stirred at 0 ° C. for 1 hour and at room temperature for 18 hours. The extract was washed once with 4 mL of diethyl ether, added with 3 mL of 2M hydrochloric acid, extracted once with 8 mL of ethyl acetate, and concentrated to dryness under reduced pressure to obtain 0.49 g of Z-Phe (yield 88%). Z-Phe 0.49g was melt | dissolved in tetrahydrofuran 15mL, and commercially available Ala-OMe * HCl 0.22g was added. Add 0.31 g of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (hereinafter abbreviated as EDCI · HCl) and 0.22 mL of triethylamine at 0 ° C, and at 0 ° C for 2 hours at room temperature. Stir for 63 hours. After adding 2 mL of water, the mixture was concentrated under reduced pressure to distill off the tetrahydrofuran. After adding 20 mL of ethyl acetate, 1 mL of water, and 0.5 mL of saturated aqueous potassium hydrogensulfate solution, the organic layer was separated, washed once with 4 mL of saturated aqueous sodium bicarbonate solution, and once with 4 mL of saturated aqueous sodium bicarbonate solution, and concentrated under reduced pressure. Dried to dryness.
残留物をシリカゲルカラムクロマトグラフィ−（BW−820MH 15g、トルエン−酢酸エチル 3:2、1分画4mL）にて精製し、Z−Phe−Ala−OMeを0.34g得た（収率53％）。Z−Phe−Ala−OMe 0.15gをテトラヒドロフラン6mLに溶解した後、水1.5mLを加えた。更に、水酸化リチウム9mgを加えて、室温にて2.5時間攪拌した後、水酸化リチウム1mgを追加して、室温にて6時間攪拌した。水酸化リチウム1mgを追加して、更に室温にて10時間攪拌した後、減圧下で濃縮乾固した。残留物に酢酸エチル5mL、水1mL、飽和硫酸水素カリウム水溶液0.5mLを加えて、有機層を分離した。水層を酢酸エチル2mLで1回抽出した後、合わせた有機層を減圧下で濃縮乾固して、Z−Phe−Alaを0.14g得た（収率定量的）。 The residue was purified by silica gel column chromatography (BW-820MH 15 g, toluene-ethyl acetate 3: 2, 1 fraction 4 mL) to obtain 0.34 g of Z-Phe-Ala-OMe (yield 53%). Z-Phe-Ala-OMe 0.15 g was dissolved in tetrahydrofuran 6 mL, and water 1.5 mL was added. Further, 9 mg of lithium hydroxide was added and stirred at room temperature for 2.5 hours, and then 1 mg of lithium hydroxide was added and stirred at room temperature for 6 hours. After adding 1 mg of lithium hydroxide and further stirring at room temperature for 10 hours, the mixture was concentrated to dryness under reduced pressure. To the residue were added 5 mL of ethyl acetate, 1 mL of water, and 0.5 mL of saturated aqueous potassium hydrogen sulfate solution, and the organic layer was separated. The aqueous layer was extracted once with 2 mL of ethyl acetate, and the combined organic layer was concentrated to dryness under reduced pressure to obtain 0.14 g of Z-Phe-Ala (quantitative yield).
Z−Phe−Ala 50mgをジオキサン2mLに溶解し、水1mLを加えた。10％パラジウム−炭素10mgを加えた後、水素雰囲気下で3時間攪拌した。綿栓濾過にて触媒を取り除いた後、濾液を減圧下で濃縮乾固して残留物を38mg得た。これをG−25カラムクロマトグラフィ−（1.2g、水のみ、1分画0.4mL）にて精製して、Phe−Alaを32mg得た（収率定量的）。 50 mg of Z-Phe-Ala was dissolved in 2 mL of dioxane, and 1 mL of water was added. After adding 10% palladium-carbon (10 mg), the mixture was stirred for 3 hours under a hydrogen atmosphere. After removing the catalyst by cotton plug filtration, the filtrate was concentrated to dryness under reduced pressure to obtain 38 mg of residue. This was purified by G-25 column chromatography (1.2 g, water only, 1 fraction 0.4 mL) to obtain 32 mg of Phe-Ala (quantitative yield).
＜製造例６＞Z−Leu−Val−Gly−OMe及びLeu−Val−Glyの合成
Val 0.25gを水2.5mLに懸濁し、0℃にて炭酸水素ナトリウム0.35gとZクロリド0.30mLを加えた。0℃にて1時間、室温にて18時間攪拌した。ジエチルエ−テル4mLで1回洗浄し、2M塩酸3mLを加えて、酢酸エチル8mLで1回抽出し、減圧下濃縮乾固して、Z−Valを0.54g得た（収率定量的）。
<Production Example 6> Synthesis of Z-Leu-Val-Gly-OMe and Leu-Val-Gly
0.25 g of Val was suspended in 2.5 mL of water, and 0.35 g of sodium bicarbonate and 0.30 mL of Z chloride were added at 0 ° C. The mixture was stirred at 0 ° C. for 1 hour and at room temperature for 18 hours. The extract was washed once with 4 mL of diethyl ether, added with 3 mL of 2M hydrochloric acid, extracted once with 8 mL of ethyl acetate, and concentrated to dryness under reduced pressure to obtain 0.54 g of Z-Val (quantitative yield).
当該Z−Val 0.54gをテトラヒドロフラン16mLに溶解し、市販のGly−OMe・HCl 0.30gを加えた。0℃にてEDCI・HCl 0.49g、トリエチルアミン0.36mLを加えて、0℃にて2時間、室温にて17時間攪拌した。水2mLを加えた後、減圧下濃縮してテトラヒドロフランを留去した。酢酸エチル15mL、水2mL、飽和硫酸水素カリウム水溶液0.5mLを加えて、有機層を分離した後、飽和炭酸水素ナトリウム水溶液3mLで1回、飽和塩化ナトリウム水溶液3mLで1回洗浄し、減圧下濃縮乾固した。残留物をシリカゲルカラムクロマトグラフィ−（BW−820MH 13g、トルエン−酢酸エチル3:2、1分画4mL）にて精製し、Z−Val−Gly−OMeを0.32g得た（収率46
％）。Z−Val−Gly−OMe 73mgを33%臭化水素−酢酸溶液0.51mLに溶解し、室温にて2.5時間静置した後、減圧下濃縮した。
0.54 g of the Z-Val was dissolved in 16 mL of tetrahydrofuran, and 0.30 g of commercially available Gly-OMe · HCl was added. At 0 ° C., 0.49 g of EDCI · HCl and 0.36 mL of triethylamine were added, and the mixture was stirred at 0 ° C. for 2 hours and at room temperature for 17 hours. After adding 2 mL of water, the solution was concentrated under reduced pressure to distill off the tetrahydrofuran. After adding 15 mL of ethyl acetate, 2 mL of water, and 0.5 mL of saturated aqueous potassium hydrogen sulfate solution, the organic layer was separated, washed once with 3 mL of saturated aqueous sodium bicarbonate solution, and once with 3 mL of saturated aqueous sodium chloride solution, and concentrated to dryness under reduced pressure. Solidified. The residue was purified by silica gel column chromatography (BW-820MH 13 g, toluene-ethyl acetate 3: 2, 1 fraction 4 mL) to obtain 0.32 g of Z-Val-Gly-OMe (yield 46
%). Z-Val-Gly-OMe (73 mg) was dissolved in 33% hydrogen bromide-acetic acid solution (0.51 mL), allowed to stand at room temperature for 2.5 hours, and then concentrated under reduced pressure.
残留物にジエチルエ−テル2mLを加え、良く振とうして静置した後、上澄み液を取り除いて、メタノ−ル1mLに溶解した。この溶液のpHは4であった。これを減圧下濃縮乾固して、クル−ドのVal−Gly−OMe・HBrを69mg得た（粗収率113％）。Val−Gly−OMe・HBr 68mgに、Z−Leu 67mgのテトラヒドロフラン溶液2mLを加えた。0℃にてEDCI・HCl
58mg、トリエチルアミン42µLを加えて、0℃にて2時間、室温にて24時間攪拌した。水1mLを加えた後、減圧下濃縮してテトラヒドロフランを留去した。酢酸エチル4mL、水1mL、飽和硫酸水素カリウム水溶液0.2mLを加えて、有機層を分離した後、減圧下で濃縮乾固した。残留物をシリカゲルカラムクロマトグラフィ−（BW−820MH 4.3g、トルエン−酢酸エチル1:1、1分画1.5mL）にて精製し、Z−Leu−Val−Gly−OMeを39mg得た（収率35
％）。
To the residue was added 2 mL of diethyl ether, allowed to stand by shaking well, and then the supernatant was removed and dissolved in 1 mL of methanol. The pH of this solution was 4. This was concentrated to dryness under reduced pressure to obtain 69 mg of crude Val-Gly-OMe.HBr (crude yield 113%). 2 mL of tetrahydrofuran solution of 67 mg of Z-Leu was added to 68 mg of Val-Gly-OMe · HBr. EDCI ・ HCl at 0 ℃
58 mg and triethylamine 42 µ L were added, and the mixture was stirred at 0 ° C. for 2 hours and at room temperature for 24 hours. After adding 1 mL of water, the solution was concentrated under reduced pressure to distill off the tetrahydrofuran. 4 mL of ethyl acetate, 1 mL of water, and 0.2 mL of a saturated aqueous potassium hydrogen sulfate solution were added to separate the organic layer, and then concentrated to dryness under reduced pressure. The residue was purified by silica gel column chromatography (BW-820MH 4.3 g, toluene-ethyl acetate 1: 1, 1 fraction 1.5 mL) to obtain 39 mg of Z-Leu-Val-Gly-OMe (
%).
Z−Leu−Val−Gly−OMe 30mgをテトラヒドロフラン1.2mLに溶解した後、水0.3mLを加えた。水酸化リチウム2mgを加えて、室温にて1.5時間攪拌した後、減圧下濃縮乾固した。残留物に酢酸エチル1mL、水0.3mL、飽和硫酸水素カリウム水溶液0.1mLを加えて、有機層を分離した。水層を酢酸エチル1mLで1回抽出した後、合わせた有機層を減圧下で濃縮乾固して、Z−Leu−Val−Glyを29mg得た（収率定量的）。 After dissolving 30 mg of Z-Leu-Val-Gly-OMe in 1.2 mL of tetrahydrofuran, 0.3 mL of water was added. 2 mg of lithium hydroxide was added, and the mixture was stirred at room temperature for 1.5 hours, and then concentrated to dryness under reduced pressure. To the residue were added 1 mL of ethyl acetate, 0.3 mL of water, and 0.1 mL of saturated aqueous potassium hydrogen sulfate solution, and the organic layer was separated. The aqueous layer was extracted once with 1 mL of ethyl acetate, and then the combined organic layers were concentrated to dryness under reduced pressure to obtain 29 mg of Z-Leu-Val-Gly (quantitative yield).
Z−Leu−Val−Gly 28mgをジオキサン1.2mLに溶解し、水0.6mLを加えた。10％パラジウム−炭素6mgを加えた後、水素雰囲気下で4時間攪拌した。綿栓濾過にて触媒を取り除いた後、濾液を減圧下で濃縮乾固して残留物を23mg得た。これをシリカゲルカラムクロマトグラフィ−（BW−820MH1.2g、n−ブタノ−ル−エタノ−ル−クロロホルム−水を6:4:2:1、1分画0.6mL）にて精製して、Leu−Val−Glyを10mg得た（収率52％）。 28 mg of Z-Leu-Val-Gly was dissolved in 1.2 mL of dioxane, and 0.6 mL of water was added. After adding 6% of 10% palladium-carbon, the mixture was stirred for 4 hours under a hydrogen atmosphere. After removing the catalyst by cotton plug filtration, the filtrate was concentrated to dryness under reduced pressure to obtain 23 mg of a residue. This was purified by silica gel column chromatography (BW-820MH 1.2 g, n-butanol-ethanol-chloroform-water 6: 4: 2: 1, 1 fraction 0.6 mL), and Leu-Val -10 mg of Gly was obtained (52% yield).
＜製造例７＞Z−Leu−Gly−Val−OMe 及びLeu−Gly−Valの合成
タ−シャリ−ブトキシカルボニルオキシ（以下Bocと略記する）−Gly 0.88gをテトラヒドロフラン26mLに溶解し、市販のVal−OMe・HCl 0.84gを加えた。0℃にてEDCI・HCl 0.96g、トリエチルアミン0.69mLを加えて、0℃にて2時間、室温にて20時間攪拌した。水5mLを加えた後、減圧下濃縮してテトラヒドロフランを留去した。酢酸エチル30mL、飽和硫酸水素カリウム水溶液1mLを加えて、有機層を分離した後、飽和炭酸水素ナトリウム水溶液5mLで1回洗浄し、減圧下で濃縮乾固した。残留物をシリカゲルカラムクロマトグラフィ−（BW−820MH
14g、トルエン−酢酸エチル1:1、1分画4mL）にて精製し、Boc−Gly−Val−OMeを1.2g得た（収率86％）。
<Production Example 7> Synthesis of Z-Leu-Gly-Val-OMe and Leu-Gly-Val 0.88 g of tertiary-butoxycarbonyloxy (hereinafter abbreviated as Boc) -Gly was dissolved in 26 mL of tetrahydrofuran, and commercially available Val. -0.84 g of OMe.HCl was added. At 0 ° C., 0.96 g of EDCI · HCl and 0.69 mL of triethylamine were added, and the mixture was stirred at 0 ° C. for 2 hours and at room temperature for 20 hours. After adding 5 mL of water, the solution was concentrated under reduced pressure to distill off the tetrahydrofuran. After adding 30 mL of ethyl acetate and 1 mL of saturated aqueous potassium hydrogen sulfate solution, the organic layer was separated, washed once with 5 mL of saturated aqueous sodium hydrogen carbonate solution, and concentrated to dryness under reduced pressure. The residue was subjected to silica gel column chromatography (BW-820MH
14 g, toluene-ethyl acetate 1: 1, 1 fraction 4 mL) to obtain 1.2 g of Boc-Gly-Val-OMe (yield 86%).
Boc−Gly−Val−OMe 0.50gをトリフルオロ酢酸5.0mLに溶解し、室温にて20分間静置した後、減圧下濃縮乾固して、Gly−Val−OMe・TFAを0.64g得た（粗収率123%）。Gly−Val−OMe・TFA 0.64gにZ−Leu0.43gのテトラヒドロフラン溶液13mLを加えた。0℃にてEDCI・HCl 0.40g
、トリエチルアミン0.29mLを加えて、0℃にて2時間、室温にて14 時間攪拌した。水2mLを加えた後、減圧下で濃縮してテトラヒドロフランを留去した。酢酸エチル15mL、水2mL、飽和硫酸水素カリウム水溶液0.5mLを加えて、有機層を分離した後、飽和炭酸水素ナトリウム水溶液3mLで1回洗浄し、減圧下濃縮乾固した。残留物をシリカゲルカラムクロマトグラフィ−（BW−820MH 23g、トルエン−酢酸エチル1:2、1分画8mL）にて精製し、Z−Leu−Gly−Val−OMeを0.59g得た（収率84
％）。
Boc-Gly-Val-OMe 0.50 g was dissolved in trifluoroacetic acid 5.0 mL, allowed to stand at room temperature for 20 minutes, and then concentrated to dryness under reduced pressure to obtain 0.64 g of Gly-Val-OMe · TFA ( Crude yield 123%). To 0.64 g of Gly-Val-OMe · TFA, 13 mL of tetrahydrofuran solution of 0.43 g of Z-Leu was added. EDCI ・ HCl 0.40g at 0 ℃
Then, 0.29 mL of triethylamine was added, and the mixture was stirred at 0 ° C. for 2 hours and at room temperature for 14 hours. After adding 2 mL of water, the mixture was concentrated under reduced pressure to distill off the tetrahydrofuran. Ethyl acetate (15 mL), water (2 mL), and saturated aqueous potassium hydrogensulfate solution (0.5 mL) were added to separate the organic layer, which was then washed once with saturated aqueous sodium hydrogencarbonate (3 mL) and concentrated to dryness under reduced pressure. The residue was purified by silica gel column chromatography (BW-820MH 23 g, toluene-ethyl acetate 1: 2, 1 fraction 8 mL) to obtain 0.59 g of Z-Leu-Gly-Val-OMe (yield 84).
%).
Z−Leu−Gly−Val−OMe 100mgをテトラヒドロフラン4mLに溶解した後、水1mLを加えた。水酸化リチウム6mgを加えて、室温にて16時間攪拌した後、水酸化リチウム3mgを追加して、室温にて6時間攪拌した。それに水酸化リチウム2mgを追加して、更に室温にて22
時間攪拌した後、減圧下で濃縮乾固した。残留物に酢酸エチル3mL、水1mL、飽和硫酸水素カリウム水溶液0.5mLを加えて、有機層を分離した。水層を酢酸エチル2mLで1回抽出した後、合わせた有機層を減圧下濃縮乾固して、得られた残留物をシリカゲルカラムクロマトグラフィ−（BW−820MH 5.0g、酢酸エチルのみ、1分画1mL）にて精製し、70％程度の純度のZ−Leu−Gly−Valを31mg得た（収率23％）。
After dissolving 100 mg of Z-Leu-Gly-Val-OMe in 4 mL of tetrahydrofuran, 1 mL of water was added. After adding 6 mg of lithium hydroxide and stirring at room temperature for 16 hours, 3 mg of lithium hydroxide was added and stirred at room temperature for 6 hours. Add 2 mg of lithium hydroxide to it and add 22 mg at room temperature.
After stirring for an hour, it was concentrated to dryness under reduced pressure. To the residue were added 3 mL of ethyl acetate, 1 mL of water and 0.5 mL of saturated aqueous potassium hydrogen sulfate solution, and the organic layer was separated. The aqueous layer was extracted once with 2 mL of ethyl acetate, and the combined organic layers were concentrated to dryness under reduced pressure. The obtained residue was subjected to silica gel column chromatography (BW-820MH 5.0 g, ethyl acetate only, 1 fractionation). 1 mg) to obtain 31 mg of Z-Leu-Gly-Val having a purity of about 70% (yield 23%).
更に、70％程度の純度のZ−Leu−Gly−Val 29mgをジオキサン1.2mLに溶解し、水0.6mLを加えた。10％パラジウム−炭素6mgを加えた後、水素雰囲気下で4時間攪拌した。綿栓濾過にて触媒を取り除いた後、濾液を減圧下で濃縮乾固して残留物を22mg得た。これをシリカゲルカラムクロマトグラフィ−（BW−820MH 1.1g、n−ブタノ−ル−エタノ−ル−クロロホルム−水6:4:2:1、1分画0.4mL）にて精製して、純粋なLeu−Gly−Valを8mg得た（収率
36％）。Leu−Gly−Valは特表2003−504062号に記載されたペプチド化合物と同一である事が判明したが、そのアンジオテンシンＩ変換酵素阻害活性は報告されていない。
Furthermore, 29 mg of Z-Leu-Gly-Val having a purity of about 70% was dissolved in 1.2 mL of dioxane, and 0.6 mL of water was added. After adding 6% of 10% palladium-carbon, the mixture was stirred for 4 hours under a hydrogen atmosphere. After removing the catalyst by cotton plug filtration, the filtrate was concentrated to dryness under reduced pressure to obtain 22 mg of residue. This was purified by silica gel column chromatography (BW-820MH 1.1 g, n-butanol-ethanol-chloroform-water 6: 4: 2: 1, 1 fraction 0.4 mL) to obtain pure Leu- 8 mg of Gly-Val was obtained (yield
36%). Leu-Gly-Val was found to be the same as the peptide compound described in JP-T-2003-504062, but its angiotensin I converting enzyme inhibitory activity has not been reported.
＜試験例１＞アンジオテンシンＩ変換酵素阻害物質の阻害活性の測定
アンジオテンシンＩ変換酵素阻害活性を有するペプチド化合物の阻害性の測定は、Cushmann らの方法（Biochemical Pharmacology,20,1637− 1648（1971 ）を一部改変して行った。
<Test Example 1> Measurement of inhibitory activity of angiotensin I converting enzyme inhibitory substance The inhibitory activity of peptide compounds having angiotensin I converting enzyme inhibitory activity was measured by the method of Cushmann et al. (Biochemical Pharmacology, 20, 1637-1648 (1971)). Partly modified.
即ち、1.5mLエッペンドルフチュ−ブに5mMのBenzoyl−Glycyl−L−Histidyl−L−Leucine（ペプチド研究所製）の0.2Mホウ酸−リン酸カリウム緩衝液（0.4MのNaCl含有、pH8.3）を250μL、所定濃度に調製した供試化合物水溶液30μLを加え、37℃で5分間プレインキュベ−ションした。この溶液に対して、アンジオテンシンＩ変換酵素
（ウサギ肺由来、シグマ社製、酵素番号EC3.4.15.1）溶液（60mU/mL 0.2M ホウ酸−リン酸カリウム緩衝液）を添加し、酵素反応を開始した。37℃で60分間、シェ−カ−バス内で100rpmにて反応を行った後、1N塩酸250μLを加え、反応を停止した。これに酢酸エチル580μLを加え、45秒間振とうさせた後、3000rpm
、10分間遠心分離を行い、上清の酢酸エチル層を500μL採取した。水層へ更に520μLの酢酸エチルを加え、２０秒間振とうさせた後、3000rpmで10分間遠心分離を行い、上清の酢酸エチル層を500μL採取した。この操作を再度繰返し、得られた酢酸エチル層合計1.5mL
を遠心エバポレ−タ−にて3000rpmで30分間、減圧条件下にて乾固し、酢酸エチルを完全に除去した。乾固物を高速液体クロマトグラフィ−緩衝液（20 ％アセトニトリル/0.1
％トリフルオロ酢酸水溶液）3mLに溶解した。
That is, 0.2 mM boric acid-potassium phosphate buffer (containing 0.4 M NaCl, pH 8.3) of 5 mM Benzoyl-Glycyl-L-Histidyl-L-Leucine (manufactured by Peptide Institute) in 1.5 mL Eppendorf tube Was added at 30 μL of a test compound aqueous solution prepared to a predetermined concentration, and preincubated at 37 ° C. for 5 minutes. To this solution, an angiotensin I converting enzyme (derived from rabbit lung, Sigma, enzyme number EC3.4.15.1) solution (60mU / mL 0.2M borate-potassium phosphate buffer) was added, and the enzyme reaction was performed. Started. After reaction at 37 ° C. for 60 minutes in a shaker bath at 100 rpm, 250 μL of 1N hydrochloric acid was added to stop the reaction. After adding 580μL of ethyl acetate and shaking for 45 seconds, 3000rpm
The mixture was centrifuged for 10 minutes, and 500 μL of the supernatant ethyl acetate layer was collected. An additional 520 μL of ethyl acetate was added to the aqueous layer, shaken for 20 seconds, centrifuged at 3000 rpm for 10 minutes, and 500 μL of the supernatant ethyl acetate layer was collected. This operation was repeated again and the resulting ethyl acetate layer total 1.5 mL
The solution was dried under reduced pressure using a centrifugal evaporator at 3000 rpm for 30 minutes to completely remove ethyl acetate. The dried product was subjected to high performance liquid chromatography buffer (20% acetonitrile / 0.1
% Trifluoroacetic acid aqueous solution).
生成した馬尿酸を逆相系高速液体クロマトグラフィ−（HPLC）にて分析し、酵素反応で生成した馬尿酸のピ−ク面積を228nmの吸光度を測定する事で求めた。又、酵素反応時に予め1N塩酸を添加して同様の操作を行ったものをブランクとして作成した。HPLCはカラムにμBONDASPHERE C18 φ3.9
×150mm（Waters 社製）を、移動相に20 ％アセトニトリル/0.1 ％トリフルオロ酢酸水溶液を用いた。
The generated hippuric acid was analyzed by reversed-phase high performance liquid chromatography (HPLC), and the peak area of hippuric acid generated by the enzymatic reaction was determined by measuring the absorbance at 228 nm. Further, a blank was prepared by performing the same operation by adding 1N hydrochloric acid in advance during the enzyme reaction. HPLC is μBONDASPHERE C18 φ3.9 on the column
A 150% (Waters) 20% acetonitrile / 0.1% trifluoroacetic acid aqueous solution was used as the mobile phase.
阻害率は次式を用いて算出した。
アンジオテンシンＩ変換酵素 阻害率（％）＝｛1−B−C）÷A｝×100
A：蒸留水添加時のピ−ク面積（228nm）
B：阻害剤添加時のピ−ク面積（228nm）
C：阻害剤添加時のブランクのピ−ク面積（228nm）
The inhibition rate was calculated using the following formula.
Angiotensin I converting enzyme inhibition rate (%) = {1−B−C) ÷ A} × 100
A: Peak area when adding distilled water (228nm)
B: Peak area when an inhibitor is added (228 nm)
C: Peak area of the blank when an inhibitor is added (228 nm)
表１に実施例１及び実施例２に記載した各試料60μg/mLにおけるアンジオテンシンＩ変換酵素 阻害率（％）を示した。 Table 1 shows the inhibition rate (%) of angiotensin I converting enzyme in each sample 60 μg / mL described in Example 1 and Example 2.
＜試験例２＞自然発症高血圧ラットに対する尾静脈投与による降圧効果
試験には体重250±50g、収縮期血圧200±20mmHg、心拍数400±50 回/分の雄性自然発症高血圧ラット（Wister−Okamoto derived Spontaneously Hypertensive Rat、以下SHRと記す）を用いた。試験開始前に明暗周期12時間（午前９時〜午後９時点灯）、室温21〜23℃、湿度50〜70％、飼料（PMI
Nutrition International 社製Lab Diet）及び飲水（水道水質基準適合自家揚水）自由摂取の環境下で45×23×21ｃｍのケ−ジにSHR6匹を入れ1週間馴化飼育した。製造例１で得られたペプチド混合物のｎ−ブタノ−ル抽出液濃縮物をSHR体重1kgあたり30mgの用量で、SHR体重1kgあたり5mLの0.9％食塩水に溶解し、SHR（1群3
匹）に対して尾静脈単回投与して32±1℃の環境下で飼育した。対照群としてはSHR（1群3匹）に同用量の0.9 ％食塩水のみを尾静脈投与した。試料投与直前、及び投与30、60、240分後に血圧を非観血血圧測定装置（Model 59,IITC,CA,USA）を用いて測定した。表２には、その収縮期血圧の経時的変化を示した。
<Test Example 2> Spontaneous hypertensive rats were tested for antihypertensive effect by tail vein administration. Male spontaneously hypertensive rats (Wister-Okamoto derived) with body weight 250 ± 50g, systolic blood pressure 200 ± 20mmHg, heart rate 400 ± 50 times / min. Spontaneously Hypertensive Rat (hereinafter referred to as SHR) was used. 12 hours light-dark cycle before starting the test (lights on from 9:00 am to 9:00 pm), room temperature 21-23 ° C, humidity 50-70%, feed (PMI
Nutrition International Lab Diet) and drinking water (self-pumping water compliant with tap water quality standards) were placed in a 45 x 23 x 21 cm cage and allowed to acclimatize for 1 week. The n-butanol extract concentrate of the peptide mixture obtained in Production Example 1 was dissolved at a dose of 30 mg / kg of SHR body weight in 0.9% saline of 5 mL / kg of SHR body weight.
Mice) and was reared in an environment of 32 ± 1 ° C. after single administration of the tail vein. As a control group, SHR (3 mice per group) was administered with the same dose of 0.9% saline alone via the tail vein. Blood pressure was measured using a non-invasive blood pressure measuring device (Model 59, IITC, CA, USA) immediately before sample administration and 30, 60, and 240 minutes after administration. Table 2 shows changes over time in the systolic blood pressure.
＜試験例３＞SHR に対する強制経口投与による降圧効果
試験例１と同様の方法でSHRを用意し、製造例１で得られたペプチド混合物のｎ−ブタノ−ル抽出液濃縮物をSHR体重1kgあたり300mgの用量で、SHR 体重1kgあたり10mLの0.9
％食塩水に溶解し、SHR （1群3匹）に対して経口単回投与して32±1℃の環境下で飼育した。対照群としてはSHR（1群3匹）に同用量の0.9％食塩水のみを経口投与した。試料投与直前、及び投与30、60、240分後に血圧を非観血血圧測定装置（Model 59,IITC,CA,USA
）を用いて測定した。表２に収縮期血圧の経時的変化を示した。
<Test Example 3> Antihypertensive effect by oral administration to SHR SHR was prepared in the same manner as Test Example 1, and the n-butanol extract concentrate of the peptide mixture obtained in Production Example 1 was used per 1 kg of SHR body weight. At a dose of 300 mg, 0.9 mL of 10 mL per kg of SHR body weight
It was dissolved in% saline, and was orally administered once to SHR (3 mice per group) and reared in an environment of 32 ± 1 ° C. As a control group, the same dose of 0.9% saline was orally administered to SHR (3 mice per group). Non-invasive blood pressure measuring device (Model 59, IITC, CA, USA) immediately before sample administration and 30, 60, 240 minutes after administration
). Table 2 shows changes over time in systolic blood pressure.
本発明により得られたペプチド化合物は、強力なアンジオテンシンＩ変換酵素 阻害活性を有し、強い降圧作用を示すため、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、これら疾患の診断薬、各種病態で用いられる降圧剤、心筋梗塞の減少、うっ血性心不全における病態の改善剤等の医薬品として有用であるとともに、本発明のペプチド化合物は、経口摂取が可能である事から、上記の様な有用な作用を有する健康食品や特定保健用食品や機能性食品としての利用が可能である。 Since the peptide compound obtained by the present invention has a potent angiotensin I converting enzyme inhibitory activity and exhibits a strong antihypertensive action, it prevents or treats hypertension such as essential hypertension, renal hypertension, adrenal hypertension, In addition to being useful as a pharmaceutical for diagnostics of these diseases, antihypertensive agents used in various pathological conditions, reduction of myocardial infarction, pathological conditions in congestive heart failure, etc., the peptide compound of the present invention can be taken orally. Therefore, it can be used as a health food, a specific health food or a functional food having the above-mentioned useful action.
Claims (8)
Tyr−Phe（１）、
Phe−Tyr（２）、
Phe−Ala（３）、
Leu−Val−Gly（４）、
Leu−Ile−Gly（５）、
Leu−Gly−Val（６）、
Ile−Val−Gly（７）。 A peptide compound having an angiotensin I converting enzyme inhibitory activity represented by one or more amino acid sequences of the following (1) to (7).
Tyr-Phe (1),
Phe-Tyr (2),
Phe-Ala (3),
Leu-Val-Gly (4),
Leu-Ile-Gly (5),
Leu-Gly-Val (6),
Ile-Val-Gly (7).
Tyr−Phe（１）、
Phe−Tyr（２）、
Phe−Ala（３）、
Leu−Val−Gly（４）、
Leu−Ile−Gly（５）、
Leu−Gly−Val（６）、
Ile−Val−Gly（７）。 A peptide compound having an angiotensin I converting enzyme inhibitory activity represented by one or more amino acid sequences of the following (1) to (7) obtained by degrading shark cartilage or a processed product thereof with a proteolytic enzyme .
Tyr-Phe (1),
Phe-Tyr (2),
Phe-Ala (3),
Leu-Val-Gly (4),
Leu-Ile-Gly (5),
Leu-Gly-Val (6),
Ile-Val-Gly (7).
Tyr−Phe（１）、
Phe−Tyr（２）、
Phe−Ala（３）、
Leu−Val−Gly（４）、
Leu−Ile−Gly（５）、
Leu−Gly−Val （６）、
Ile−Val−Gly（７）。 A peptide compound having an angiotensin I converting enzyme inhibitory activity represented by one or more amino acid sequences of the following (1) to (7) obtained by degrading shark cartilage or a processed product thereof with a proteolytic enzyme A composition comprising
Tyr-Phe (1),
Phe-Tyr (2),
Phe-Ala (3),
Leu-Val-Gly (4),
Leu-Ile-Gly (5),
Leu-Gly-Val (6),
Ile-Val-Gly (7).
Tyr−Phe（１）、
Phe−Tyr（２）、
Phe−Ala（３）、
Leu−Val−Gly（４）、
Leu−Ile−Gly（５）、
Leu−Gly−Val（６）、
Ile−Val−Gly（７）。
Angiotensin represented by one or more amino acid sequences of the following (1) to (7), wherein shark cartilage or a processed product thereof is decomposed by reacting with a proteolytic enzyme and separated from the decomposition solution A method for producing a peptide compound having I-converting enzyme inhibitory activity or a composition containing the peptide compound.
Tyr-Phe (1),
Phe-Tyr (2),
Phe-Ala (3),
Leu-Val-Gly (4),
Leu-Ile-Gly (5),
Leu-Gly-Val (6),
Ile-Val-Gly (7).
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2007037297A1 (en) * | 2005-09-29 | 2007-04-05 | Maruha Corporation | Composition effective for prevention and treatment of adult disease |
| WO2021059840A1 (en) * | 2019-09-27 | 2021-04-01 | 太陽化学株式会社 | Angiotensin converting enzyme inhibitor |
-
2003
- 2003-11-25 JP JP2003393766A patent/JP4187633B2/en not_active Expired - Fee Related
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2007037297A1 (en) * | 2005-09-29 | 2007-04-05 | Maruha Corporation | Composition effective for prevention and treatment of adult disease |
| JPWO2007037297A1 (en) * | 2005-09-29 | 2009-04-09 | 株式会社マルハニチロ水産 | Composition effective for prevention and treatment of adult diseases |
| US7951782B2 (en) | 2005-09-29 | 2011-05-31 | Maruha Nichiro Seafoods, Inc | Composition effective to prevent or treat adult disease |
| JP4834671B2 (en) * | 2005-09-29 | 2011-12-14 | 株式会社マルハニチロ水産 | Composition effective for prevention and treatment of adult diseases |
| WO2021059840A1 (en) * | 2019-09-27 | 2021-04-01 | 太陽化学株式会社 | Angiotensin converting enzyme inhibitor |
| JPWO2021059840A1 (en) * | 2019-09-27 | 2021-10-07 | 太陽化学株式会社 | Angiotensin converting enzyme inhibitor |
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