JPH07188282A - Novel tripeptide, its production and hypotensor containing the same as an active ingredient - Google Patents

Novel tripeptide, its production and hypotensor containing the same as an active ingredient

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JPH07188282A
JPH07188282A JP18206891A JP18206891A JPH07188282A JP H07188282 A JPH07188282 A JP H07188282A JP 18206891 A JP18206891 A JP 18206891A JP 18206891 A JP18206891 A JP 18206891A JP H07188282 A JPH07188282 A JP H07188282A
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tripeptide
tripeptides
novel
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gel filtration
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Kunio Suetsuna
邦男 末綱
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Suetsuna Yoko
末綱 陽子
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Abstract

PURPOSE:To obtain a novel tripeptide which is useful as a hypotensor with high safety, low toxicity and no anaphylaxy shock. CONSTITUTION:Twenty three kinds of tripeptide having the L-amino acid sequences are represented by the formulas. The tripeptides are obtained by treating sardin muscles with a protease, filtering the product, and fractionating the components passing through the semipermeable membrane by means of a strong acid cation exchange resin, gel filtration, ion-exchange gel filtration, reverse-phase high-performance liquid chromatography in order to collect the fractions containing the components having the activity of inhibiting the enzymes transforming angiotensin.

Description

【発明の詳細な説明】 DETAILED DESCRIPTION OF THE INVENTION

【0001】 [0001]

【産業上の利用分野】本発明は、新規なトリペプチドを有効成分とする血圧降下剤およびその新規なトリペプチドの製法に関するものである。 The present invention relates to relates to a hypotensive agent and preparation of the novel tripeptide as an active ingredient a novel tripeptide.

【0002】 [0002]

【従来の技術】高血圧は、病因的に血圧上昇の原因が明らかなもの(病候性高血圧)と不明なもの(本態性高血圧)とに大別されている。 BACKGROUND OF THE INVENTION Hypertension is classified into a cause of etiologically elevated blood pressure be apparent (disease weathering hypertension) and unknown ones (essential hypertension). 病候性高血圧は原因となる疾患を治癒させることで高血圧を治癒させることができるが、本態性高血圧では原因に対する直接的な治療法は困難である。 Although disease weathering hypertension can cure hypertension by cure of a disease that causes a direct treatment for causing the essential hypertension is difficult. 従来、レニン−アンジオテンシン系(以下、 Traditionally, the renin - angiotensin system (hereinafter,
R・A系と略記する。 Abbreviated as R · A system. )は、本態性高血圧の重要な要因の一つであると考えられており、ここ10年来、R・A ) Is considered to be one of the important factors of essential hypertension, wherein 10 years, R · A
系で中心的な役割を果たしているアンジオテンシン変換酵素(以下、ACEと略記する。)の活性を阻害することによってR. Angiotensin converting enzyme, which plays a central role in the system (hereinafter, abbreviated as ACE.) R. by inhibiting the activity of A系を調節して本態性高血圧を調節する試みが行われてきた。 Adjust the A system is an attempt to regulate the essential hypertension have been made. そのようなACE活性阻害を有する物質としては、合成化合物の場合にはL−プロリン誘導体[M. The substance having such ACE activity inhibition, in the case of a synthetic compound is L- proline derivative [M. A. A. Ondetti,B. Ondetti, B. Rubin et Rubin et
al;Science,196,441(197 al; Science, 196,441 (197
7)]やそれをベースにした化合物が知られており、天然物由来の物質の場合には蛇毒由来のブラディキニン増強因子(C末端がPro)[S. 7)] and it has been known compounds based is, bradykinin potentiator from snake venom in the case of substances derived from natural products (C-terminal Pro) [S. H. H. Ferreia, Ferreia,
D. D. C. C. Bartelt et al;Biochem Bartelt et al; Biochem
istry,9,3583(1970)]、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(C末端がAla−Hyp)[G. istry, 9,3583 (1970)], 6 types of peptides derived from the collagenase digest of gelatin (C-terminal Ala-Hyp) [G. Oshima,H. Oshima, H. Shima Shima
bukuro et al;Biochim. bukuro et al; Biochim. BioP BioP
hs,Acta,566,128(1979)]、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 hs, Acta, 566,128 (1979)], a peptide (C-terminal derived from tryptic digest of bovine casein
Gly−Lys)[S. Gly-Lys) [S. Maruyama,H. Maruyama, H. Suz Suz
uki;Agric. uki; Agric. Biol. Biol. Chem,46,13 Chem, 46,13
93(1982)]などが知られている。 93 (1982)] and the like are known. 食品の場合には鈴木らが大豆、茶類、貝類、果実類などでACE活性阻害を認めている[鈴木健夫、石川宣子ら;農化,5 Soybeans Suzuki et al. In the case of food, tea, shellfish, admits ACE activity inhibition in such as fruits [Takeo Suzuki, Nobuko Ishikawa et al .; Noka, 5
7,1143(1983)]。 7,1143 (1983)]. しかし、これら天然物由来の物質はいずれも静脈内投与で効果が確認されているのみで、経口投与による薬理効果は不明であり、発明されてから長期間経過しているが、未だ医薬品としての開発が進んでいるとの報告はない。 However, only those naturally derived effect any material intravenous administration have been confirmed, the pharmacological effect by oral administration is not known, but has passed a long period of time since the invention, yet as medicaments development There is no report of that proceeding.

【0003】 [0003]

【発明が解決しようとする課題】本発明の目的は、新規なトリペプチド、その製法およびそれを有効成分とする血圧降下剤を提供することである。 OBJECTS OF THE INVENTION It is an object of the present invention is to provide novel tripeptide, the antihypertensive agent and its preparation and its active ingredients.

【0004】 [0004]

【課題を解決するための手段】本発明は、前記の課題を解決するために鋭意研究した結果、イワシ筋肉ならびに大豆のタンパク質分解酵素の分解液から得られた本発明の新規なペプチドが、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。 The present invention SUMMARY OF] As a result of intensive studies to solve the above problems, a novel peptide of the present invention obtained from the decomposition solution of proteolytic enzymes sardine muscle and soybeans, blood pressure It found to have a lowering effect, and have completed the present invention. 即ち、本発明は、 That is, the present invention is, で示されるL体のアミノ酸配列を有する新規な23種のトリペプチド。 In new 23 or tripeptide having the amino acid sequence of the L-form as shown.

【0005】(2)イワシ筋肉をタンパク質分解酵素で処理して得られた生成物を瀘過し、その濾過成分中の半透膜を通過した成分を順次、強酸性陽イオン交換樹脂、 [0005] (2) sardine muscle was filtered and the product obtained was treated with proteolytic enzymes, sequentially component that has passed through the semi-permeable membrane of the filtration component, strongly acidic cation exchange resin,
ゲル濾過、イオン交換性ゲル濾過、逆相高速液体クロマトグラフィーによって分画し、その処理毎に得られた分画からアンジオテンシン変換酵素阻害活性を有する成分を含有する分画を得ることを特徴とする前記の新規な2 Gel filtration, ion exchange gel filtration, fractionated by reversed-phase high performance liquid chromatography, characterized in that to obtain the fractions containing the components having the angiotensin converting enzyme inhibitory activity from fractions obtained for respective processing the novel 2
3種のトリペプチドの製法。 Preparation of 3 kinds of birds peptide. (3)前記の新規なトリペプチドを有効成分とする血圧降下剤。 (3) antihypertensive agent containing as an active ingredient the novel tripeptide. で示されるL体のアミノ酸配列を有する新規な27種のトリペプチド。 In new 27 or tripeptide having the amino acid sequence of the L-form as shown.

【0006】(5)大豆をタンパク質分解酵素で処理して得られた生成物を瀘過し、その濾過成分中の半透膜を通過した成分を順次、強酸性陽イオン交換樹脂、ゲル濾過、イオン交換性ゲル瀘過、逆相高速液体クロマトグラフィーによって分画し、その処理毎に得られた分画からアンジオテンシン変換酵素阻害活性を有する成分を含有する分画を得ることを特徴とする前記の新規な27種のトリペプチドの製法。 [0006] (5) Soybean was filtered and the product obtained was treated with a proteolytic enzyme, a component that has passed through the semi-permeable membrane in the filtration component sequentially, strong acid cation exchange resins, gel filtration, ion exchange gel filtration, fractionated by reversed-phase high performance liquid chromatography, said characterized in that to obtain the fractions containing the components having the angiotensin converting enzyme inhibitory activity from fractions obtained for respective processing preparation of the novel 27 species of birds peptide. (6)前記の新規なトリペプチドを有効成分とする血圧降下剤に関するものである。 (6) it relates to antihypertensive agent containing as an active ingredient the novel tripeptide. 以下、本発明を詳細に説明する。 The present invention will be described in detail. 本発明の新規なトリペプチドは、 The novel tripeptides of the invention, (以上23種、トリペプチドの式中の各記号はペプチド化学におけるアミノ酸配列の各アミノ酸単位を示す。) (Over 23 species, wherein each symbol of the tripeptide denotes each amino acid unit in the amino acid sequences in peptide chemistry.)

【0007】 [0007] (以上27種、トリペプチドの式中の各記号はペプチド化学におけるアミノ酸配列の各アミノ酸単位を示す。) (Above 27 species, wherein each symbol of the tripeptide denotes each amino acid unit in the amino acid sequences in peptide chemistry.)
で示されるL体のアミノ酸配列を有する新規なトリペプチドであり、この常温における性状は白色粉末である。 In a novel tripeptide having the amino acid sequence of the L-form shown, properties in the normal temperature is a white powder.

【0008】前記の新規なトリペプチドの製法としては、そのトリペプチドを化学的に合成する方法またはイワシ筋肉並びに大豆のタンパク質分解酵素の分解液から分離、精製する方法を挙げることができる。 [0008] As preparation of the novel tripeptide may be a method of its isolation tripeptide from decomposition solution of chemically synthesizing methods or sardine muscle and soy proteolytic enzymes and purified. 本発明の新規なトリ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など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トリペプチドのC末端側(カルボキシル末端側)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。 When chemically synthesizing the novel tripeptide of the present invention can be carried out by conventional synthetic methods such as liquid phase method or solid phase method, preferably, the solid support of polymeric by the solid phase method C-terminal side of the tripeptide to the body is good go bound by sequential peptide bond amino acids (carboxyl-terminal) from the L-form corresponding to the amino acid residues.
そして、そのようにして得られた合成トリペプチドは、 Then, the synthetic tripeptide obtained in that way is,
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リ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と略す。)などを用いた通常の方法で精製することができる。 Trifluoromethanesulfonic acid, was cleaved from the polymeric solid support using a hydrogen fluoride to remove the protecting group of the amino acid side chain, high performance liquid chromatography using a column of reverse phase (hereinafter, HPLC abbreviated.) it can be purified by conventional methods using a.

【0009】本発明の新規なトリペプチドを、イワシ筋肉並びに大豆のタンパク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1991年度日本農芸化学会大会(京都)講演要旨集P183 3AP1 [0009] The novel tripeptide of the present invention, can be separated and purified from the decomposition solution of a proteolytic enzyme of the sardine muscle and soy, the Japan Society for Bioscience, Biotechnology, and Agrochemistry tournament in fiscal 1991 in the case (Kyoto) Abstracts P183 3AP1
3の方法に準拠し、例えば以下のようにして行うことができる。 Complies with the third method, for example, can be carried out as follows. 上記の新規なトリペプチドを含有しているイワシ筋肉部分並びに大豆を取り出して、ホモゲナイザーを用いて適当な溶媒(例えば、水、トリスー塩酸緩衝液、 Remove the sardine muscle portion and soybeans containing the above novel tripeptide, a suitable solvent using a homogenizer (e.g., water, Tris-HCl buffer,
リン酸緩衝液などの中性の緩衝液など)中で十分にホモジネートした後、加水分解する。 After sufficient homogenized in a neutral buffer solution, etc.), such as a phosphate buffer, hydrolyzed. 加水分解は常法に従って行う。 Hydrolysis is carried out according to a conventional method. 例えば、ペプシン等タンパク質分解酵素で加水分解する場合は、イワシ筋肉ホモジネート並びに大豆ホモジネー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。 For example, in the case of hydrolysis with pepsin and the like proteolytic enzymes, after further hydrolysis if need sardine muscle homogenates and soybean homogenate, warmed to an optimum temperature of the enzyme, the pH was adjusted to optimum values, incubation with the enzyme. 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。 The filtrate was necessary neutralization, to obtain a hydrolyzed solution to inactivate the enzyme. その加水分解物を濾紙およびセライトなどを用いて濾過することによって不溶性成分を除去し、その得られた瀘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などの中性の緩衝液など) Its hydrolyzate insoluble components were removed by filtration using a filter paper and Celite, the resulting filtrate to using a semipermeable membrane such as cellophane suitable solvent (e.g., water, Tris - HCl buffer such neutral buffer such as liquid phosphoric acid buffer solution)
中で十分に透析し、その瀘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、 Extensively dialyzed at medium, the solution strongly acidic cation exchange resin containing a component which has passed through the semipermeable membrane components in the filtrate (e.g.,
ダウケミカル社製のDowex 50Wなど)にかけ、 Subjected to Dowex 50W, etc.) manufactured by Dow Chemical Company,
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と略す。)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阻害活性分画をゲル瀘過(例えば、ファルマシア製の Sephadex G− As angiotensin-converting enzyme from the adsorption elution fractions (hereinafter, abbreviated as ACE.) To give the fractions containing the component with inhibitory activity, the resulting ACE inhibitory activity fractions gel filtration (e.g., Pharmacia of Sephadex G-
25など)によって分画し、その得られたACE阻害活性分画を陽イオン交換ゲル濾過(例えば、ファルマシア製のSP−Sephadex C−25など)によって分画し、その得られたACE阻害活性分画をさらにHP Fractionated by 25, etc.), the resulting ACE inhibitory active fraction cation exchange gel filtration (e.g., fractionated, such as by) Pharmacia of SP-Sephadex C-25, ACE inhibitory activity fraction thereof obtained In addition HP the field
LC(逆相高速液体クロマトグラフィー)によって分画することによって行うことができる。 It can be carried out by fractionation by LC (reversed phase high performance liquid chromatography).

【0010】本発明の新規なトリペプチドの製法において用いる魚筋肉並びにマメ科植物としては、本発明の目的を達成できる限りいかなる魚筋肉並びにマメ科植物を用いても良いが、好ましくはイワシ並びに大豆を用いるのが良い。 [0010] As the fish muscle and legumes used in preparation of the novel tripeptide of the present invention may be used any fish muscle and legumes as possible achieve the object of the present invention, preferably sardines and soybean It is good to use. 以上のようにして得られた本発明の新規なトリペプチドは、静脈内へ繰り返し投与しても抗体産生を惹起せず、また、アナフィラキシーショックを起こさせない。 The novel tripeptides of the present invention obtained as described above, even after repeated administration intravenously without eliciting antibody production, also does not cause an anaphylactic shock. また、本発明の新規なトリペプチドはL−アミノ酸のみの配列構造からなり、その分子サイズからみて、 Moreover, the novel tripeptide of the present invention consists arrangement of only L- amino acids, as seen from its molecular size,
投与後、生体内のプロテアーゼにより分解されることなく、すみやかに腸管吸収され、その血圧降下作用を発揮するため毒性は極めて低く、安全性は極めて高い(LD After administration, without being degraded by proteases in vivo, are rapidly intestinal absorption, toxicity to exert its hypotensive effect is very low, the safety is very high (LD
50 )5000kg/kg;ラット経口投与)。 50) 5000 kg / kg; rats orally). 本発明に係る新規なトリペプチドは、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、 Novel tripeptide of the present invention, injection with additives of excipients such as commonly used, tablets, capsules, granules,
散剤等に調整することができる。 It can be adjusted to powders, and the like. 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。 As administration methods, typically mammals having a ACE (e.g., human, dog, rat, etc.) is injected into, or the like can be orally administered. 投与量は、例えば、動物体重1kg Dosage, e.g., animal body weight 1kg
当りこのトリペプチドを0.01〜10mgの量である。 Per the tripeptide is an amount of 0.01 to 10 mg. 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。 The frequency of administration, but is usually about 1 to 4 times a day, the route of administration, can be suitably adjusted. 本発明に係る新規なトリ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。 Novel tripeptide of the present invention have angiotensin converting enzyme inhibitory action superior show hypotensive action, bradykinin inactivation inhibitory action. したがって、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、これらの疾患の診断剤や各種の病態において用いられる血圧降下剤として有用であり、更に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。 Therefore, essential hypertension, renal hypertension, prevention of hypertension, such as adrenal hypertension, therapeutic agent, useful as an antihypertensive agent to be used in diagnostics and various disease states of these diseases, organ for further congestive heart failure improvement of normalization of circulating and long-term prognosis has (survival effect) effect and is useful as a heart failure treating agent.

【実施例】以下に実施例として、製造例および試験例を記載し、本発明を更に詳細に説明する。 As EXAMPLE below describes production examples and test examples further illustrate the present invention.

【0011】製造例1 [新規なトリペプチドのイワシ筋肉からの製造]イワシ筋肉500gに脱イオン水1Lを加え、ホモジナイズした後、1N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 [0011] Production Example 1 of deionized water 1L in sardine muscle 500 g [produced from sardine muscle novel tripeptide] In addition, after homogenization, was adjusted to pH 2.0 with 1N hydrochloric acid, pepsin (manufactured by Merck , enzyme number 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。 It was added and subjected to hydrolysis with stirring 37 ° C. 20 hours. 分解反応液を直ちに限外濾過膜(アミコン社製、Y The decomposition reaction immediately ultrafiltration membrane (Amicon, Y
M10型、φ76mm)に通過させ、通過液をDowe M10 type, passed through a φ76mm), Dowe the effluent
x 50W×4[H ]カラム(φ4.5x15cm) x 50W × 4 [H +] column (φ4.5x15cm)
に加えた。 It was added to the. そのカラムを脱イオン水で十分洗浄した後、 After thoroughly washing the column with deionized water,
2N水酸化アンモニウム液2Lを用いて溶出した。 And eluted with 2N ammonium hydroxide solution 2L. 減圧濃縮によりアンモニアを除去し、濃縮液40mlを得た。 Ammonia was removed by concentration under reduced pressure, to obtain a concentrated solution 40 ml. この濃縮液4mlを予め脱イオン水で緩衝化したS S buffered the concentrated solution 4ml advance with deionized water
ephadex G−25カラム(φ2.5x150c ephadex G-25 column (φ2.5x150c
m)に負荷し、流速30ml/hr,各分画量8.6m Loaded in m), a flow rate of 30 ml / hr, each fraction amount 8.6m
lでゲル濾過を行った。 The gel filtration was carried out in l. ゲル瀘過を繰り返して大量分取したACE阻害活性の高い画分を集め凍結乾燥してペプチド粉末とした。 And peptide powder was collected and lyophilized high fractions of mass sorting the ACE inhibitory activity by repeating the gel filtration. このペプチド3gを20mlの脱イオン水に溶解後、予め、脱イオン水で緩衝化したSP−S After dissolving the peptide 3g deionized water 20 ml, previously, SP-S buffered with deionized water
ephadex C−25(H )カラム(φ1.5x ephadex C-25 (H +) column (Fai1.5X
47.2cm)に負荷し、脱イオン水1Lから3%塩化ナトリウム液1Lの濃度勾配法を行い、流速3ml/h Loaded to 47.2cm), carried out the concentration gradient method with 3% sodium chloride solution 1L deionized water 1L, flow rate 3 ml / h
r,各分画量10.0mlでクロマトグラフィーを行った。 r, it was chromatographed in each fraction the amount of 10.0ml. その結果は図1に示すとおりである。 The results are shown in Figure 1. 上記クロマトグラフ中、分画番号22〜28のACE阻害活性分画を集めて凍結乾燥して精製トリペプチド粉末を得た。 In the above chromatograph to obtain the purified tripeptide powder was collected and lyophilized ACE inhibitory activity fractions Fraction numbers 22-28. この精製トリペプチド粉末20mgを60μlの脱イオン水に溶解した後、HPLCを行った。 After dissolving the purified tripeptide powder 20mg of deionized water 60 [mu] l, were HPLC. カラムとしては野村化学(株)製DevelosilODS−5(4.5m The column made by Nomura Chemical (Co., Ltd.) DevelosilODS-5 (4.5m
mIDx25cmL)を使用し、移動相としては0.0 Using mIDx25cmL), as the mobile phase 0.0
5%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220n 5% trifluoroacetic acid (hereinafter abbreviated as TFA.) From performs concentration gradient method of 25% acetonitrile 0.05% TFA, flow rate 1.0 ml / min, detection wavelength 220n
mでクロマトグラフィーを行い、ACE阻害作用を有するトリペプチドを得た。 It was chromatographed m, to obtain a tripeptide having an ACE inhibitory action. その結果は図2に示すとおりであり、23種のトリペプチドの溶出時間は表1のとおりである。 The results are as shown in FIG. 2, the elution time of 23 kinds of tripeptides are shown in Table 1.

【0012】このようにして得られたACE阻害作用を有するトリペプチドのアミノ酸配列は、アプライドバイオシステム社製のプロテインシークエンサー447A型を用いて決定された。 [0012] The amino acid sequence of the tripeptide with ACE inhibitory activity obtained in this manner was determined using an Applied Biosystems, Inc. of protein sequencer 447A type. その結果、23種のトリペプチドはそれぞれ、 Each result, 23 kinds of tripeptides, で示されるL体のアミノ酸残基からなる配列を有するトリペプチドであることが確認された。 It was confirmed in a tripeptide having the sequence consisting of amino acid residues of the L-form as shown. 新規23種のトリペプチドをマススペクトルにより分折した結果、アミノ酸配列およびアミノ酸組成が前記式で示したアミノ酸配列構造を有するトリペプチドであることが確認された。 New 23 kinds of tripeptides the results of partial folding Mass spectrum, it was confirmed amino acid sequence and amino acid composition is a tripeptide having the amino acid sequence structure shown by the formula.
このマススペクトルの結果は表1に示すとおりである。 The result of this mass spectrum is shown in Table 1.

【0013】製造例2 [新規なトリペプチドの大豆からの製造]大豆200g [0013] Production Example 2 [Production of the novel tripeptide of Soybeans Soy 200g
に脱イオン水1Lを加え、ホモジナイズした後、1N塩酸にてpHを2.0に調整し、ペプシン(メルク社製、 Deionized water 1L addition, after homogenization, pH was adjusted to 2.0 with 1N hydrochloric acid, pepsin (Merck,
酵素番号EC3.4.23.1)10gを添加し、37 The addition of enzyme number EC3.4.23.1) 10g, 37
℃20時間撹拌しながら加水分解を行った。 ℃ conduct hydrolysis with stirring for 20 hours. 分解反応液を直ちに限外瀘過膜(アミコン社製、YM10型、φ7 Immediately ultrafiltration membrane (Amicon decomposition reaction, YM10 type, .phi.7
6mm)に通過させ、通過液をDowex 50W×4 Passed through a 6mm), Dowex 50W × 4 the effluent
[H ]カラム(φ4.5x15cm)に加えた。 It was added to the [H +] column (φ4.5x15cm). そのカラムを脱イオン水で十分洗浄した後、2N水酸化アンモニウム液2Lを用いて溶出した。 After thoroughly washing the column with deionized water and eluted with 2N ammonium hydroxide solution 2L. 減圧濃縮によりアンモニアを除去し、濃縮液40mlを得た。 Ammonia was removed by concentration under reduced pressure, to obtain a concentrated solution 40 ml. この濃縮液4 The concentrated solution 4
mlを予め脱イオン水で衝衡化したSephadex ml was 衝衡 of advance with deionized water Sephadex
G−25(φ2.5x150cm)に負荷し、流速30 And loaded onto a G-25 (φ2.5x150cm), flow rate 30
ml/hr,各分画量8.6mlでゲル濾過を行った。 ml / hr, the gel filtration was performed in each fraction volume 8.6 ml.
ゲル濾過を繰り返して大量分取したACE阻害活性の高い分画を集め凍結乾燥してペプチド粉末とした。 And peptide powder was collected and lyophilized higher fraction of large amounts sorting the ACE inhibitory activity by repeating the gel filtration. このペプチド3gを20mlの脱イオン水に溶解後、予め、脱イオン水で緩衝化したSP−Sephadex C−2 After dissolving the peptide 3g deionized water 20 ml, previously, SP-Sephadex C-2 buffered with deionized water
5[H ]カラム(φ1.5x47.2cm)に負荷し、脱イオン水1Lから3%塩化ナトリウム液1Lの濃度勾配法を行い、流速3ml/hr,各分画10.0m 5 [H +] was applied to a column (φ1.5x47.2cm), carried out the concentration gradient method with 3% sodium chloride solution 1L deionized water 1L, flow rate 3 ml / hr, each fraction 10.0m
lでクロマトグラフィーを行った。 Chromatography was performed in l. その結果は図3に示すとおりである。 The results are shown in Figure 3.

【0014】上記クロマトグラフ中、分画番号32〜3 [0014] In the above chromatography, fractions number 32-3
8のACE阻害活性分画を集めて凍結乾燥して精製トリペプチド粉末を得た。 To obtain purified tripeptide powder was collected and lyophilized ACE inhibitory activity fractions 8. この精製トリペプチド粉末20m The purified tripeptide powder 20m
gを60μlの脱イオン水に溶解した後、HPLCを行った。 Was dissolved g of deionized water 60 [mu] l, were HPLC. カラムとしては野村化学(株)製Develos The column Nomura Chemical Co., Ltd. Develos
ilODS−5(4.5mmIDx25cmL)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. ilODS-5 using (4.5mmIDx25cmL), as the mobile phase (hereinafter abbreviated as TFA.) 0.05% trifluoroacetic acid to 25% acetonitrile / 0.
05%TFAの濃度勾配法を行い、流速1.0ml/m Perform concentration gradient method 05% TFA, flow rate 1.0 ml / m
in,検出波長220nmでクロマトグライーを行い、 in, perform chromatography gley over at a detection wavelength of 220nm,
ACE阻害作用を有するリペプチドを得た。 It was obtained Ripepuchido having ACE inhibitory activity. その結果は図4に示すとおりであり、27種のトリペプチドの溶出時間は表2のとおりである。 The results are as shown in FIG. 4, the elution time of 27 kinds of tripeptides are shown in Table 2.

【0015】このようにして得られたACE阻害作用を有するトリペプチドのアミノ酸配列は、アプライドバイオシステム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。 The amino acid sequence of the tripeptide with ACE inhibitory activity obtained in this manner was determined using an Applied Biosystems, Inc. of protein sequencer 477A type. その結果、27種のトリペプチドはそれぞれ、 Each result, 27 kinds of tripeptides, で示されるL体のアミノ酸残基からなる配列を有するトリペプチドであることが確認された。 It was confirmed in a tripeptide having the sequence consisting of amino acid residues of the L-form as shown. 新規27種のトリペプチドをマススペクトルにより分折した結果、アミノ酸配列およびアミノ酸組成が前記式で示したアミノ酸配列構造を有するトリペプチドであることが確認された。 New 27 kinds of tripeptides the results of partial folding Mass spectrum, it was confirmed amino acid sequence and amino acid composition is a tripeptide having the amino acid sequence structure shown by the formula.
このマススペクトルの結果は表2に示すとおりである。 The result of this mass spectrum is shown in Table 2.
精製して得られた本発明に係るイワシ筋肉由来トリペプチド23種より成る分画、並びに大豆由来トリペプチド27種より成る分画は、以下に示す試験によって薬理効果が確認された。 Fractions consisting of sardine muscle-derived tripeptide 23 species of the present invention obtained by purification and fractionation consisting of soy-derived tripeptide 27 species, pharmacological effect was confirmed by the following tests.

【0016】試験例1 [ACE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Hip [0016] Test Example 1 [ACE inhibitory activity assay] ACE (Sigma, enzyme number EC 3.4.15.1) 2.5 mU, synthetic substrate Hip
puryl−L−his−tidyl−L−leuci puryl-L-his-tidyl-L-leuci
ne(ペプチド研究所製)12.5mMを用いLieb Lieb using the ne (manufactured by Peptide Institute, Inc.) 12.5mM
ermanの測定法を改良した山本等の方法(日胸疾会誌,18,297−302(1989))に準じて測定した。 Method of equal Yamamoto, an improvement of the measuring method of the erman (day breast 疾会 magazine, 18,297-302 (1989)) was measured in accordance with. 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。 That is, the resulting hippuric acid extracted with ethyl acetate, was measured at an absorbance of 225 nm. 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E The absorbance at test solution Es, the value when the buffer was added instead of the test solution E
c,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。 c, it was determined inhibition rate from the following equation the value when reacted by adding a pre-stop solution as Eb. 阻害率(%)=(Ec−Es)/(Ec−Eb)×10 Inhibition rate (%) = (Ec-Es) / (Ec-Eb) × 10
0 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。 Inhibitory activity The IC 50 values of 0 ACE inhibitor showed 50% the enzymatic activity of ACE concentration of the sample required to (inhibition rate) Inhibition (M). 本発明に係るイワシ筋肉由来新規23 Sardine muscle-derived new 23 according to the present invention
種のトリペプチドの牛肺血清ACEに対する阻害活性 Inhibitory activity against bovine lung serum ACE species tripeptide
(IC 50 )は表1に示すとおりである。 (IC 50) are shown in Table 1. また、本発明に係る大豆由来新規27種のトリペプチドの牛肺血清A Further, the soybean-derived novel 27 kinds of tripeptides according to the present invention bovine lung serum A
CEに対する阻害活性(IC 50 )は表2に示すとおりである。 Inhibition of CE activity (IC 50) are shown in Table 2.

【0017】試験例2 [新規なトリペプチドのラットへ投与時の降圧の効果] I. [0017] Test Example 2 Effect of antihypertensive upon administration to a novel tripeptide in rats] I. 実験材料 前記製造例1,2で得られた精製トリペプチド粉末。 Purification tripeptide powder obtained in Experimental Materials Preparation Example 1 and 2. すなわちイワシ筋肉由来トリペプチド23種より成る分画(SP−1分画)並びに大豆由来トリペプチド27種より成る分画(SP−1分画)を用いた。 That was used sardine muscle-derived fractions consisting of the tripeptide 23 or (SP-1 fraction) and fraction consisting of soybean-derived tripeptide 27 kinds of (SP-1 fraction). II. II. 実験方法 実験動物は日本チャールズ・リバー(株)より15週令雄性高血圧自然発症ラット(SHR)を購入し、1週間の予備飼育後、収縮期血圧が160mmHg以上(体重280〜330g)の動物6匹1群として用いた。 Experimental Methods Experimental animals were purchased Japan Charles River Co., Ltd. from 15-week-old male spontaneously hypertensive rat (SHR), animal 6 after preliminary breeding for one week, systolic blood pressure more than 160mmHg (body weight 280~330g) It was used as one group mice. ラットは、室温23±25℃、湿度55±10%および12 Rats, room temperature 23 ± 25 ° C., 55 ± 10% humidity and 12
時間明暗(午前6時〜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ケージに1匹ずつ収容し飼育した。 Time dark and housed one by one animal in individual cages stainless wire steel rat breeding room adjusted to (am 6 lighting 6am) breeding. 飼料はオリエンタル酵母工場(株)製M Feed Oriental Yeast Factory Co., Ltd. M
F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。 The F powdery feed, drinking water autologous pumping the water (tap water quality standards conformity) ad libitum, respectively. ラットは4群(1群6匹) Rats four groups (1 group 6 mice)
に分け、第1群には対照として蒸留水を体重100gあたり0.5mlの割合で強制経口投与した、第2群にはトリペプチドの粉末1.0g/kgの用量を蒸留水で調製し、体重100gあたり0.5mlの割合で強制経口投与し、第3群にはトリペプチドの粉末2.0g/k To divide, distilled water was forcibly administered orally at the rate of 100g body weight per 0.5 ml, a dose of powder 1.0 g / kg of the tripeptide was prepared with distilled water in the second group as a control in the first group, oral gavage at a rate of 100g body weight per 0.5 ml, powder 2.0 g / k of the tripeptide in the third group
g、第4群にはトリペプチドの粉末4.0g/kgの用量を、第2群と同様に強制経口投与した。 g, the dose of powder 4.0 g / kg of the tripeptide in the fourth group was administered by gavage as in the second group.

【0018】血圧は非観血的尾動脈血圧測定装置((株)理研開発製、PS−100)を用いtail− [0018] The blood pressure using a non-invasive tail arterial blood pressure measurement device ((Ltd.) manufactured by RIKEN development, PS-100) tail-
cuff法により、投与前、投与後30分、1時間、2 The cuff method, prior to administration, 30 minutes after the administration, 1 hour, 2
時間、4時間および6時間の血圧および心拍数を測定した。 Time was measured blood pressure and heart rate of 4 hours and 6 hours. 血圧は連続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た。 Blood pressure was measured three times consecutively, the difference between the maximum value and the minimum value if it is within 10 mmHg, to obtain an average blood pressure value of the three times. 差が11mmHg以上の場合はさらに2回測定し、 The difference measures two more times in the case of more than 11 mmHg,
最高値および最低値を除き3回の平均血圧値を求めた。 To determine the maximum value and the average blood pressure value of three times except for the minimum value.
また、平均心拍数は平均血圧値を算出したときの測定値を用いて求めた。 The average heart rate was determined by using the measured value when the calculated average blood pressure value. SHRを用いて、イワシ筋肉由来トリペプチド分画(SP−1分画)300,600および1,200mg/kgを単回経口投与した時の、血圧値および心拍数への作用についての結果は、表3,図5に示すとおりである。 Using SHR, result of the action of sardine muscle-derived tripeptide fraction (SP-1 fraction) 300, 600 and 1,200 mg / kg when single oral dose, the blood pressure value and heart rate, Table 3 is as shown in FIG. またSHRを用いて大豆由来トリペプチド分画(SP−1分画)300,600および1, The soybean-derived tripeptide fraction with SHR (SP-1 fraction) 300, 600 and 1,
200mg/kgを単回経口投与した時の、血圧値および心拍数への作用についての結果は、表4、図6に示すとおりである。 When the 200 mg / kg were a single oral dose, the result of the action of the blood pressure value and heart rate, Table 4, it is shown in FIG. 以上の試験の結果、本発明に係るイワシ筋肉由来トリペプチド23種より成る分画並びに大豆由来トリペプチド27種より成る分画は、ACE阻害活性を有し、in vivo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。 Results of the above tests, fractions and fractions consisting of soy-derived tripeptide 27 kinds consisting of sardine muscle-derived tripeptide 23 species of the present invention has an ACE inhibitory activity, also significant hypotensive activity in in vivo it has been confirmed that the show. したがって、本発明に係るイワシ筋肉由来トリペプチド23種並びに大豆由来トリペプチド27種は高血圧症の治療または予防薬として有用である。 Therefore, sardine muscle-derived tripeptide 23 species and soybean-derived tripeptide 27 species of the present invention are useful as therapeutic or prophylactic agent for hypertension. なお、本発明に係るイワシ筋肉由来トリペプチド23種並びに大豆由来トリペプチド27種は、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。 Incidentally, sardine muscle-derived tripeptide 23 species and soybean-derived tripeptide 27 species according to the present invention, in the peptide of structurally partial structure the amino acid sequence, may be employed in the structure.

【0019】 [0019]

【表1】 [Table 1] イワシ筋肉由来トリペプチドのHPLCにおける溶出時間、アミノ酸配列、分子量および阻害活性。 Elution time in HPLC sardine muscle-derived tripeptide amino acid sequence, molecular weight and inhibitory activity.

【0020】 [0020]

【表2】 [Table 2] 大豆由来トリペプチドのHPLCにおける溶出時間、アミノ酸配列、分子量および阻害活性。 Elution time in the HPLC of the soybean-derived tripeptide amino acid sequence, molecular weight and inhibitory activity.

【0021】 [0021]

【表3】 [Table 3] イワシ筋肉由来トリペプチド分画投与におけるSHRの血圧値の経時的変化 Time course of the blood pressure values ​​of SHR in sardine muscle-derived tripeptide fraction administered

【0022】 [0022]

【表4】 [Table 4] 大豆由来トリペプチド分画役与におけるSHRの血圧値の経時的変化 Time course of the blood pressure values ​​of SHR in given soy tripeptide fraction officers

【0023】 [0023]

【図面の簡単な説明】 BRIEF DESCRIPTION OF THE DRAWINGS

【図1】本発明に係るイワシ筋肉由来トリペプチドの、 Sardine muscle-derived tripeptide according to the invention, FIG,
製造例1におけるSP−Sephad−ex C−25 In Production Example 1 SP-Sephad-ex C-25
(H )カラムクロマトグラフィーによるACE阻害ペプチドの分離精製の結果を示す図である。 It shows the results of separation and purification of ACE inhibiting peptides according to (H +) column chromatography.

【図2】本発明に係る大豆由来トリペプチドの、製造例1におけるSP−Sephadex C−25(H [Figure 2] of the soybean-derived tripeptide according to the present invention, SP-Sephadex C-25 in Preparation Example 1 (H +)
カラムクロマトグラフィーによるACE阻害ペプチドの分離精製の結果を示す図である。 It shows the results of separation and purification of ACE inhibiting peptides by column chromatography.

【図3】本発明に係るイワシ筋肉由来トリペプチドの、 Sardine muscle-derived tripeptide according to the present invention; FIG,
製造例1における逆相HPLCによるACE阻害ペプチドの分難精製の結果を示す図である。 It shows the results of partial flame purification of ACE inhibitory peptides by reverse phase HPLC in Production Example 1.

【図4】本発明に係る大豆由来トリペプチドの、製造例1における逆相HPLC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。 Of soybean-derived tripeptide according to Figure 4 the present invention, showing the results of separation and purification of ACE inhibitory peptides by reverse phase HPLC in Production Example 1.

【図5】本発明に係るイワシ筋肉由来トリペプチドの製造例1で得られた23種のトリペプチド分画(SP−1 [5] 23 species obtained in Production Example 1 of sardine muscle-derived tripeptide according to the present invention tripeptide fraction (SP-1
分画)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。 Fractions), showing the change over time in the blood pressure value when administered to SHR.

【図6】本発明に係る大豆由来トリペプチドの製造例2 [6] Production of soybean-derived tripeptide according to the present invention 2
で得られた27種のトリペプチド分画(SP−1分画) 27 kinds of tripeptides fraction obtained in (SP-1 fraction)
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。 The diagrams showing the change over time in the blood pressure value when administered to SHR.

───────────────────────────────────────────────────── フロントページの続き (51)Int.Cl. 6識別記号 庁内整理番号 FI 技術表示箇所 C12N 9/99 ────────────────────────────────────────────────── ─── front page continued (51) Int.Cl. 6 in identification symbol Agency Docket No. FI art display portion C12N 9/99

Claims (6)

    【特許請求の範囲】 [The claims]
  1. 【請求項1】 [Claim 1] で示されるL体のアミノ酸の配列によるペプチド構造を有する新規な23種類のトリペプチド。 In new 23 kinds of tripeptides having a peptide structure according to the sequence of L-amino acids represented.
  2. 【請求項2】 イワシ筋肉をタンパク質分解酵素で処理して得られた生成物を濾過し、その瀘液成分中の半透膜を通過した成分を順次、強酸性陽イオン交換樹脂、ゲル濾過、イオン交換性ゲル濾過、逆相高速液体クロマトグラフィーによって分画し、その処理毎に得られた分画からアンジオテンシン変換酵素阻害活性を有する成分を含有する分画を得ることを特徴とする請求項1の新規な2 Wherein the sardine muscle was filtered The product obtained was treated with a proteolytic enzyme, a component that has passed through the semipermeable membrane of the filtrate in component sequentially, strong acid cation exchange resins, gel filtration, ion exchange gel filtration, according to claim 1, fractionated by reversed-phase high performance liquid chromatography, characterized in that to obtain the fractions containing the components having the angiotensin converting enzyme inhibitory activity from fractions obtained for respective processing of the novel 2
    3種のトリペプチドの製法。 Preparation of 3 kinds of birds peptide.
  3. 【請求項3】 請求項1の新規な23種のトリペプチドから選ばれた1種以上のトリペプチドを有効成分とする血圧降下剤。 3. A hypotensive agent comprising, as an active ingredient, one or more tripeptides selected from the novel 23 kinds of tripeptides according to claim 1.
  4. 【請求項4】 4.] で示されるL体のアミノ酸の配列によるペプチド構造を有する新規な27種のトリペプチド。 In new 27 kinds of tripeptides having a peptide structure according to the sequence of L-amino acids represented.
  5. 【請求項5】 大豆をタンパク質分解酵素で処理して得られた生成物を濾過し、その濾過成分中の半透膜を通過した成分を順次、強酸性陽イオン交換樹脂、ゲル瀘過、 5. soybean product obtained by treating with a proteolytic enzyme was filtered, sequentially component passed through the semi-permeable membrane in the filtration component, strongly acidic cation exchange resin, gel filtration,
    イオン交換性ゲル濾過、逆相高速液体クロマトグラフィーよって分画し、その処理毎に得られた分画からアンジオテンシン変換酵素阻害活性を有する成分を含有する分画を得ることを特徴とする請求項1の新規な27種のトリペプチドの製法。 Ion exchange gel filtration, according to claim 1, fractionated by reversed-phase high performance liquid chromatography, characterized in that to obtain the fractions containing the components having the angiotensin converting enzyme inhibitory activity from fractions obtained for respective processing preparation of the novel 27 species of birds peptide.
  6. 【請求項6】 請求項4の新規な27種のトリペプチドから選ばれた1種以上のトリペプチドを有効成分とする血圧降下剤。 6. The antihypertensive agent comprising as an active ingredient one or more tripeptides selected from the novel 27 kinds of tripeptides according to claim 4.
JP18206891A 1991-04-19 1991-04-19 Novel tripeptide, its production and hypotensor containing the same as an active ingredient Pending JPH07188282A (en)

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JP18206891A JPH07188282A (en) 1991-04-19 1991-04-19 Novel tripeptide, its production and hypotensor containing the same as an active ingredient

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JP18206891A JPH07188282A (en) 1991-04-19 1991-04-19 Novel tripeptide, its production and hypotensor containing the same as an active ingredient

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WO2000044770A1 (en) * 1999-01-28 2000-08-03 Chugai Seiyaku Kabushiki Kaisha Substituted phenethylamine derivatives
WO2000063233A2 (en) * 1999-04-21 2000-10-26 University Of Florida Research Foundation, Inc. Peptides and the use thereof to control pests
WO2000062620A3 (en) * 1999-04-21 2001-07-05 Univ Florida Transformed cells useful for the control of pests
US6593299B1 (en) 1999-04-21 2003-07-15 University Of Florida Research Foundation, Inc. Compositions and methods for controlling pests
US6635265B1 (en) 1999-04-21 2003-10-21 University Of Florida Research Foundation, Inc. Materials and methods useful for the control of insect larvae
WO2004104027A1 (en) * 2003-05-21 2004-12-02 Fuji Oil Company, Limited Composition containing angiotensin converting enzyme inhibitory peptide
US6884878B2 (en) 1999-04-21 2005-04-26 University Of Florida Research Foundation, Inc. Neuropeptides and their use for pest control
WO2004005318A3 (en) * 2002-07-03 2005-11-17 Bio Science International Inc Peptides comprising aromatic d-amino acids and methods of use
US7179793B2 (en) * 2005-02-14 2007-02-20 Ocean Nutrition Canada Limited Anti-hypertensive dietary supplement
JP2008208096A (en) * 2007-02-28 2008-09-11 Kanetoku:Kk New peptide derived from jellyfish protein and use thereof
WO2010082367A1 (en) 2009-01-19 2010-07-22 キッコーマン株式会社 Angiotensin converting enzyme-inhibiting peptide
JP2010248096A (en) * 2009-04-13 2010-11-04 Rheology Kino Shokuhin Kenkyusho:Kk Trp-CONTAINING PEPTIDE
EP2299271A2 (en) 2005-11-09 2011-03-23 Ajinomoto Co., Inc. Kokumi-imparting agent
JP2012046450A (en) * 2010-08-27 2012-03-08 Unitika Ltd Angiotensin-converting enzyme inhibitory peptide, and method for producing the peptide
US8399417B2 (en) 2007-05-08 2013-03-19 Ajinomoto Co., Inc. Food
US8420144B2 (en) 2005-11-09 2013-04-16 Ajinomoto Co., Inc. Kokumi-imparting agent, method of using, and compositions containing same
JP2013116911A (en) * 2007-08-07 2013-06-13 Mg Pharma Kk Hypotensive agent
JP2014138617A (en) * 2014-04-18 2014-07-31 Kikkoman Corp Angiotensin-converting enzyme inhibitor peptide containing composition
EP2990027A1 (en) * 2014-09-01 2016-03-02 Institut Curie Skin whitening peptide agents

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JPS5944324A (en) * 1982-09-04 1984-03-12 Agency Of Ind Science & Technol Angiotensinase inhibitor
JPS62169732A (en) * 1986-01-22 1987-07-25 Lion Corp Hypotensor
JPS63141995A (en) * 1986-12-03 1988-06-14 Agency Of Ind Science & Technol Novel active peptide
JPS63141996A (en) * 1986-12-03 1988-06-14 Agency Of Ind Science & Technol Novel active peptide
JPH0236127A (en) * 1988-07-27 1990-02-06 Agency Of Ind Science & Technol Angiotensinase inhibitor

Patent Citations (5)

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JPS5944324A (en) * 1982-09-04 1984-03-12 Agency Of Ind Science & Technol Angiotensinase inhibitor
JPS62169732A (en) * 1986-01-22 1987-07-25 Lion Corp Hypotensor
JPS63141995A (en) * 1986-12-03 1988-06-14 Agency Of Ind Science & Technol Novel active peptide
JPS63141996A (en) * 1986-12-03 1988-06-14 Agency Of Ind Science & Technol Novel active peptide
JPH0236127A (en) * 1988-07-27 1990-02-06 Agency Of Ind Science & Technol Angiotensinase inhibitor

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WO2000044770A1 (en) * 1999-01-28 2000-08-03 Chugai Seiyaku Kabushiki Kaisha Substituted phenethylamine derivatives
US7553969B1 (en) 1999-01-28 2009-06-30 Chugai Seiyaku Kabushiki Kaisha Substituted phenethylamine derivatives
US7491795B2 (en) 1999-04-21 2009-02-17 University Of Florida Research Foundation, Inc. Neuropeptides and their use for pest control
WO2000063233A2 (en) * 1999-04-21 2000-10-26 University Of Florida Research Foundation, Inc. Peptides and the use thereof to control pests
WO2000063233A3 (en) * 1999-04-21 2001-01-11 Univ Florida Peptides and the use thereof to control pests
US6413530B1 (en) 1999-04-21 2002-07-02 University Of Florida Research Foundation, Inc. Pesticidal peptides
US6566129B1 (en) 1999-04-21 2003-05-20 University Of Florida Research Foundation, Inc. Transformed cells useful for the control of pests
US6593299B1 (en) 1999-04-21 2003-07-15 University Of Florida Research Foundation, Inc. Compositions and methods for controlling pests
US6635265B1 (en) 1999-04-21 2003-10-21 University Of Florida Research Foundation, Inc. Materials and methods useful for the control of insect larvae
WO2000062620A3 (en) * 1999-04-21 2001-07-05 Univ Florida Transformed cells useful for the control of pests
US6884878B2 (en) 1999-04-21 2005-04-26 University Of Florida Research Foundation, Inc. Neuropeptides and their use for pest control
US6562590B1 (en) 1999-04-21 2003-05-13 University Of Florida Research Foundation, Inc. Transformed cells useful for the control of pests
US7714106B2 (en) 1999-04-21 2010-05-11 University Of Florida Research Foundation, Inc. Neuropeptides and their use for pest control
WO2004005318A3 (en) * 2002-07-03 2005-11-17 Bio Science International Inc Peptides comprising aromatic d-amino acids and methods of use
US7655602B2 (en) 2002-07-03 2010-02-02 Bio Science International, Inc. Peptides comprising aromatic D-amino acids and methods of use
JP4797627B2 (en) * 2003-05-21 2011-10-19 不二製油株式会社 Angiotensin converting enzyme inhibitory peptide-containing composition
WO2004104027A1 (en) * 2003-05-21 2004-12-02 Fuji Oil Company, Limited Composition containing angiotensin converting enzyme inhibitory peptide
EP1853290A1 (en) * 2005-02-14 2007-11-14 Ocean Nutrition Canada Limited Anti-hypertensive dietary supplement derived from salmo or oncorhynchus protein hydrolysates
US7179793B2 (en) * 2005-02-14 2007-02-20 Ocean Nutrition Canada Limited Anti-hypertensive dietary supplement
EP1853290A4 (en) * 2005-02-14 2010-07-07 Ocean Nutrition Canada Ltd Anti-hypertensive dietary supplement derived from salmo or oncorhynchus protein hydrolysates
US9395376B2 (en) 2005-11-09 2016-07-19 Ajinomoto Co., Inc. Kokumi-imparting agent
EP2175273B1 (en) * 2005-11-09 2013-09-04 Ajinomoto Co., Inc. Kokumi-imparting agent
EP2299271A2 (en) 2005-11-09 2011-03-23 Ajinomoto Co., Inc. Kokumi-imparting agent
EP2327986A1 (en) 2005-11-09 2011-06-01 Ajinomoto Co., Inc. Kokumi-imparting agent
JP2011115186A (en) * 2005-11-09 2011-06-16 Ajinomoto Co Inc Kokumi-imparting agent
EP2299271A3 (en) * 2005-11-09 2011-07-13 Ajinomoto Co., Inc. Kokumi-imparting agent
US8420144B2 (en) 2005-11-09 2013-04-16 Ajinomoto Co., Inc. Kokumi-imparting agent, method of using, and compositions containing same
JP2008208096A (en) * 2007-02-28 2008-09-11 Kanetoku:Kk New peptide derived from jellyfish protein and use thereof
US8399417B2 (en) 2007-05-08 2013-03-19 Ajinomoto Co., Inc. Food
JP2013116911A (en) * 2007-08-07 2013-06-13 Mg Pharma Kk Hypotensive agent
JP2013144696A (en) * 2007-08-07 2013-07-25 Mg Pharma Kk Anti-hypertensive agent
US9006171B2 (en) 2009-01-19 2015-04-14 Kikkoman Corporation Angiotensin converting enzyme inhibitory peptide
KR20110105761A (en) 2009-01-19 2011-09-27 기꼬만 가부시키가이샤 Angiotensin converting enzyme inhibitory peptide
WO2010082367A1 (en) 2009-01-19 2010-07-22 キッコーマン株式会社 Angiotensin converting enzyme-inhibiting peptide
JP2010248096A (en) * 2009-04-13 2010-11-04 Rheology Kino Shokuhin Kenkyusho:Kk Trp-CONTAINING PEPTIDE
JP2012046450A (en) * 2010-08-27 2012-03-08 Unitika Ltd Angiotensin-converting enzyme inhibitory peptide, and method for producing the peptide
JP2014138617A (en) * 2014-04-18 2014-07-31 Kikkoman Corp Angiotensin-converting enzyme inhibitor peptide containing composition
EP2990027A1 (en) * 2014-09-01 2016-03-02 Institut Curie Skin whitening peptide agents
WO2016034541A1 (en) * 2014-09-01 2016-03-10 Institut Curie Skin whitening peptide agents

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